FLUORINE-LABELLED HALICHONDRIN DERIVATIVES AND RELATED METHODS OF SYNTHESIS

Background of the invention

Halichondrin B is a structurally complex, macrocyclic compound that was originally isolated from the marine sponge Halichondria okadai, and subsequently was found in Axinella sp., Phakellia carteri, and Lissodendoryx sp. Eribulin is a synthetic analog of halichondrin B. The mesylate salt of eribulin (eribulin mesylate, which is marketed under the trade name HALAVEN®) is approved for the treatment of patients with breast cancer who have previously received at least two chemotherapeutic regimens for the treatment of metastatic disease that included an anthracycline and a taxane in either the adjuvant or metastatic setting.

Summary of the invention

The invention provides halichondrin derivatives and diastereomers thereof containing fluorine, e.g., ¹⁸ F, as well as methods of synthesizing these compounds.

In a first aspect, the invention provides a compound according to formula (I):

In another aspect, the invention provides a compound according to formula (II):

or a salt thereof, e.g., a pharmaceutically acceptable salt thereof. In some embodiments, the compound is the mesylate salt of formula (II).

In an additional aspect, the invention provides a compound according to formula (III):

(Hi) or a salt thereof, e.g., a pharmaceutically acceptable salt thereof, wherein n is an integer from 1 to 10. In some embodiments, the compound is the mesylate salt of formula (III).

In another aspect, the invention provides a compound according to formula (IV):

or a salt thereof, e.g., a pharmaceutically acceptable salt thereof, wherein n is an integer from 1 to 10. In some embodiments, the compound is the mesylate salt of formula (IV).

In some embodiments of the compounds according to formula (III) or (IV), n is 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, n is 2.

In another aspect, the invention provides a compound according to formula (V):

In embodiments of the above aspects of the invention, the invention features a diastereomer of a compound according to formula (I), (II), (III), or (IV) at position 34. The invention also features a mixture of a compound according to formula (I), (II), (III), or (IV) with its diastereomer at position 34. In some embodiments, the fluorine within any of the above compounds is fluorine-18.

In an additional aspect, the invention provides a method of synthesizing a compound according to formula (I) by reacting an epoxide according to formula (VI):

with a fluoride source to form a compound according to formula (I).

In some embodiments, the method includes synthesizing the epoxide according to formula (VI) by reacting a compound according to formula (XII):

with a base, e.g., sodium hydroxide, sodium hydride, sodium t-butoxide, potassium t-butoxide, or DBU. In some embodiments, the method includes synthesizing the compound according to claim (XII) by reacting a diol according to formula (XIII):

with a tosylating agent.

In another aspect, the invention provides a method of synthesizing a compound according to formula (II) or a salt thereof, e.g., a pharmaceutically acceptable salt thereof, by reacting a compound according to formula (XIV):

with a phosphine, e.g., triphenylphosphine, under Staudinger reaction conditions to form a compound according to formula (II) or salt thereof.

In some embodiments, the method includes synthesizing the compound according to formula (XIV) by reacting a compound according to formula (XV):

with a fluoride source, such as cesium fluoride (e.g., Cs ¹⁸ F).

In another aspect, the invention provides a method of synthesizing a compound according to formula (III) or a salt thereof, e.g., a pharmaceutically acceptable salt thereof, by reacting a compound according to formula (XVI):

(XVI) or a salt thereof with an azide according to formula (XVII):

to form a compound according to formula (III) or salt thereof, wherein n is an integer from 1 to 10. In some embodiments, n is 2. In some embodiments, the reaction is performed in the presence of a Cu(l) salt, such as Cul.

In another aspect, the invention provides a method of synthesizing a compound according to formula (IV) or a salt thereof, e.g., a pharmaceutically acceptable salt thereof, by reacting a compound according to formula (XVIII):

(XVIII) or a salt thereof with an azide according to formula (XVII) to form a compound according to formula (IV) or salt thereof, wherein n is an integer from 1 to 10. In some embodiments, n is 2. In some embodiments, the reaction is performed in the presence of a Cu(l) salt, such as Cul.

In some embodiments, the azide according to formula (XVII) is synthesized by reacting a compound according to formula (XIX):

with a fluoride source, such as cesium fluoride (e.g., Cs ¹⁸ F), wherein n is an integer from 1 to 10. In some embodiments, n is 2.

In another aspect, the invention provides a method of synthesizing a compound according to formula (V) by reacting a compound according to formula (XX):

with a fluoride source to form a compound according to formula (V). In some embodiments, the fluoride source is bis(2-methoxyethyl)aminosulfur trifluoride (compound (IX)).

In another aspect, the invention provides a compound according to formula (XV) or (XIV). In another aspect, the invention provides a compound according to formula (XVI) or a salt thereof. In yet another aspect, the invention provides a compound according to formula (XVIII) or a salt thereof. The invention also features a diastereomer at position 34 of a compound according to formula (XIV), (XV), (XVI), or (XVIII), or a salt thereof. The invention also features a mixture of a compound according to formula (XIV), (XV), (XVI), or (XVIII), or a salt thereof, with its diastereomer at position 34.

The compound of any of formulas (I), (II), (III), (IV), and (V), or a salt thereof, e.g., a

pharmaceutically acceptable salt thereof, may also be in the form of an isotopically enriched composition, i.e., in fluorine-18. Compounds of formulas (XIV) and (XVII) may also be in the form of an isotopically enriched composition, i.e., in fluorine-18. The term "isotopically enriched," as used herein, refers to a composition including an isotope, e.g., ¹⁸ F, at a position in the compound in an abundance greater than other isotopes, e.g., ¹⁹ F, at that same position. Typically and depending on the isotope, compositions enriched in a particular isotope may have an isotopic enrichment factor of at least 5, at least 10, at least 50, at least 500, at least 2000, at least 3000, at least 6000, or at least 6600, e.g., relative to ¹⁹ F.

In another aspect, the invention provides a pharmaceutical composition containing an effective amount of a compound of any of formulas (I), (II), (III), (IV), and (V), or a salt thereof, e.g., a

pharmaceutically acceptable salt thereof, (e.g., a ¹⁸ F-containing compound of any of formulas (I), (II), (III), (IV), and (V), or a salt thereof, e.g., a pharmaceutically acceptable salt thereof, or an isotopically enriched composition of a compound of any of formulas (I), (II), (III), (IV), and (V), or a salt thereof, e.g., a pharmaceutically acceptable salt thereof) and a pharmaceutically acceptable carrier.

In an additional aspect, the invention provides a method of using a compound of any of the above-described embodiments to image a subject, e.g., a human subject. In some embodiments, the method includes administering the compound to the subject and detecting the presence of the compound. In some embodiments, the detecting includes analyzing the subject by positron emission tomography.

Brief Description of the Figures

Figure 1 is a graph showing the percent radiochemical yield (RCY) of compound (I) as a function of reaction time using the epoxide ring opening protocol described in Example 1 , below.

Figures 2a-d are a series of high pressure liquid chromatography (HPLC) traces showing the conversion of epoxide (VI) to compound (I) as described in Example 1 , below.

Detailed Description

The invention provides halichondrin derivatives and diastereomers thereof represented by formulas (l)-(V), below, as well as methods of preparing these compounds by, e.g., an epoxide ring opening process, azide-alkyne cycloaddition, or by nucleophilic substitution.

In formulas (III) and (IV) above, n is an integer from 1 to 10, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments of the invention, n is 2. The invention further features pharmaceutically acceptable salts of the compounds of formulas (II), (III), and (IV), e.g., the mesylate salt of these compounds.

The compounds produced by the methods of the invention, such as compounds containing fluorine-18, can be used for a variety of purposes. Eribulin is a known chemotherapeutic agent and is a synthetic analog of halichondrin B. Fluorinated compounds of the invention can be administered to a human patient suffering from cancer, optionally in combination with additional chemotherapeutic agents, to treat the cancer. Additionally, as fluorine-1 8 is a well-established radiolabel for positron emission tomography, compounds of the invention containing fluorine-18 can be administered to a patient to visualize a sample within a subject, such as a particular organ or tissue within the subject. For instance, compounds of the invention containing fluorine-18 may be administered to a patient to image one or more solid tumors within a subject, e.g., that is undergoing chemotherapeutic treatment.

Using the methods of the invention, fluorinated compound (I) can be synthesized under epoxide ring opening conditions, e.g., as shown in Scheme 1 , below.

Scheme 1 .

Epoxide (VI) can be synthesized by tosylation of diol (XIII), e.g., using p-toluenesulfonic anhydride in basic media, followed by an intramolecular nucleophilic substitution reaction in which the tosyl group at C-35 is displaced by the oxygen at position 34 of the molecule as is known in the art. Diol (XIII) can be synthesized, e.g., according to methods described in US 6,214,865 or US 7,982,060.

Epoxide (VI) can subsequently undergo a ring opening process upon reaction with a fluoride source to produce a compound containing a fluorine substituent at C-35.

Fluoride sources useful in conjunction with the methods of the invention include fluoride salts, such as cesium fluoride (e.g., Cs ¹⁸ F). Additional fluoride sources include salen-coordinated cobalt complexes, for example, CAS 1587724-18-2 described in Graham et al. J. Am. Chem. Soc. 136:5291 - 5294 (2014) and Kalow et al. J. Am. Chem. Soc. 133:16001 -16012 (201 1 ).

The methods of the invention can be used to synthesize a compound according to formula (II), e.g., using a Staudinger azide reduction as shown in Scheme 2 below. Mesylate (XV), in which the halichondrin C-35 amine is masked as an azide substituent, can be synthesized by mesylation of an azido alcohol precursor using mesylating reagents know in the art, such as methanesulfonyl chloride in the presence of a base. The azido alcohol precursor (VII) can be synthesized as described, e.g., in US 6,214,865. The resulting mesylate (XV) can be converted to compound (XIV) by nucleophilic substitution upon reaction with a fluoride source, such as cesium fluoride. The azide functionality can in turn be reduced upon treatment with a phosphine (e.g., triphenylphosphine), producing an iminophosphorane that yields compound (II) in aqueous media.

Scheme 2.

The methods of the invention can be used to synthesize a compound according to formula (III) or (IV) by an azide-alkyne cycloaddition process, e.g., as shown in Schemes 3 and 4, below. Compounds containing an alkynyl substituent, e.g., at the C-35 amine (compound (XVI)) or the C-34 hydroxyl (compound (XVIII)) can be conjugated to fluorinated azido tags, such as compound (XVII) by a Cu(l)- catalyzed cycloaddition reaction. Exemplary Cu(l) salts useful for the catalysis of this reaction include copper (I) iodide. The fluorinated azide (XVII) can be synthesized, e.g., by nucleophilic displacement of a mesylate precursor, such as compound (XIX).



The invention additionally provides methods of synthesizing fluorinated compound (V). As shown in Scheme 5, below, compound (V) can be synthesized in a one-step nucleophilic substitution process by reaction of precursor (XX) with Deoxofluor™ (N,N-bis(2-methoxyethyl)aminosulfur trifluoride) (Compound (IX))). Compound (XX) can be synthesized by reduction of an aldehyde precursor, e.g., as described in US 6,214,865. Scheme 5.

The invention additionally features salts of compounds (ll)-(IV), e.g., pharmaceutically acceptable salts thereof. Salification reaction conditions are known in the art. Salification of fluorinated compounds of the invention, such as compounds (ll)-(IV), above, can afford a salt of the compound (e.g., a mesylate salt of compound (II), (III), or (IV)). In particular, the salification reaction can involve contacting a fluorinated compound of the invention with a Bronsted acid (e.g., a pharmaceutically acceptable Bronsted acid (e.g., methanesulfonic acid)) to afford a salt of the fluorinated compound (e.g., Handbook of Pharmaceutical Salts: Properties, Selection and Use, ed.: Stahl and Wermuth, Wiley-VCH/VHCA, Weinheim/Zurich, 2008). Salts, e.g., pharmaceutically acceptable salts, of fluorinated compounds of the invention, e.g., a mesylate salt of compound (II), (III), or (IV), can be formed by methods known in the art, e.g., in situ during the final isolation and purification of the compound or separately by reacting the free base group with a suitable organic acid. Certain intermediates may also be in salt form, as described herein. In one example, the fluorinated compound is treated with a solution of methanesulfonic acid (MsOH) and Nh OH in water and acetonitrile. The mixture is concentrated, the residue subsequently dissolved in DCM-pentane, and the solution is added to anhydrous pentane. The resulting precipitate is filtered and dried under high vacuum to provide a mesylate salt of the fluorinated compound.

C-34 diastereomers of the fluorinated compounds described herein can be produced from starting materials having the opposite stereochemistry at position 34 in the schemes described herein.

Formulations

Compounds and compositions (e.g., isotopically enriched compositions) of the invention can be formulated, e.g., as pharmaceutically acceptable salts, which are a salt within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic response and the like and commensurate with a reasonable benefit/risk ratio.

Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in: Berge et al., J. Pharmaceutical Sciences 66:1 -19, 1977 and in Pharmaceutical Salts: Properties, Selection, and Use, (Eds. P.H. Stahl and C.G. Wermuth), Wiley-VCH, 2008.

Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate,

benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2- hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts and the like. A preferred salt is the mesylate salt.

Compounds and compositions (e.g., isotopically enriched compositions) of the invention can also be formulated as pharmaceutical compositions, e.g., by combining an effective amount of the fluorinated compound, such as compound (I) or (II), with a pharmaceutically acceptable carrier. Compounds and compositions of the invention may be formulated for treatment of a patient (e.g., a human patient suffering from cancer). Compounds and compositions of the invention may be formulated for administration to a patient to visualize a sample within the patient, such as a tissue of interest, e.g., by positron emission tomography. In these instances, an effective amount is typically the amount needed to image a subject by positron emission tomography.

Pharmaceutical compositions can be prepared using standard methods known in the art, e.g., using materials from commercial sources. A compound of any of formulas (l)-(V), for instance, may be provided in liquid form, for intravenous administration to a patient.

Pharmaceutical compositions used in the invention can be prepared by, for example, mixing or dissolving the active ingredient(s), having the desired degree of purity, in a physiologically acceptable carrier (see, e.g., Remington The Science and Practice of Pharmacy, 22 ^nd edition), 2013 Pharmaceutical Press). Acceptable carriers include water and saline, optionally including buffers such as phosphate, citrate, or other organic acids; antioxidants including butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), ascorbic acid; low molecular weight (less than about 1 0 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as

polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagines, arginine or lysine;

monosaccharides, disaccharides, or other carbohydrates including glucose, mannose, or dextrins;

chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium ; and/or nonionic surfactants such as TWEEN™, PLURONICS™, or PEG.

Optionally, the formulations of the invention contain a pharmaceutically acceptable preservative.

In some embodiments the preservative concentration ranges from 0.1 to 2.0%, typically v/v. Suitable preservatives include those known in the pharmaceutical arts, such as benzyl alcohol, phenol, m-cresol, methylparaben, and propylparaben. Further, the formulations of a compound of any of formulas (ll)-(IV) can optionally include a pharmaceutically acceptable salt, such as sodium chloride at, for example, about physiological concentrations. Thus, in one example, a compound of any of formulas (ll)-(IV) is formulated in 0.9% Sodium Chloride Injection (USP).

The formulations noted above (and others) can be used for parenteral administration of the drugs. Thus, the drugs can be administered by routes including intravenous, intra-tumoral, peri-tumoral, intra-arterial, intra-dermal, intra-vesical, ophthalmic, intramuscular, intradermal, intraperitoneal, pulmonary, subcutaneous, and transcutaneous routes. Other routes can also be used including, for example, transmucosal, transdermal, inhalation, intravaginal, rectal, and oral administration routes.

The dosage of compound of formulas (l)-(V) administered can differ markedly depending on the type of target disease, the choice of delivery method, as well as the age, sex, and weight of the patient, the severity of the symptoms, along with other factors. Examples

Example 1. Synthesis of compound (l-A) by epoxide ring opening

[ ¹⁸ F] fluoride was purchased from PetNet solutions (Woburn, MA). (R, R, R, /=?)-(linked salen)C020Ts2 (CAS 1587724-18-2) was supplied by Princeton University. Light QMA Carbonate ion exchange resin was preconditioned, in turn, with deionized water (10 ml), 1 M NaHC03 (10 ml), and deionized water (10ml). The cartridge was then loaded with aqueous [ ¹⁸ F] fluoride. A solution of (R, R, R, /=?)-(linked salen)Co20Ts2 (30 mg) in methanol (1 ml), which was prepared immediately before use and sonicated briefly to ensure complete dissolution, was applied to the cartridge using a 5-ml syringe, and the resulting fluorinated cobalt complex was eluted with methanol (0.5 ml). The combined dark green solution was concentrated to dryness under a stream of nitrogen at room temperature. To the dark solid residue was added acetonitrile (0.5 ml), the mixture was briefly sonicated to ensure completely dissolution and again concentrated to dryness at room temperature. To the dark green residue was added a solution of epoxide (VI) (10 mg) in acetonitrile (1 ml). The mixture was then maintained at 50°C on a heating block. Reaction progress was periodically monitored by removing a 1 μΙ aliquot from the reaction mixture and analyzing the sample by RP-HPLC. Figure 1 illustrates the increase in radiochemical yield (RCY) as a function of reaction time over the course of 0-120 minutes. Figures 2a-d show the results of analytical HPLC analysis of 1 -μΙ aliquots of the reaction mixture at reaction times of 0, 40, 80, and 120 minutes. Example 2. Purification of compound (l-A) by high pressure liquid chromatography

Compound (l-A) was obtained as described in Example 1 and was subsequently purified on a reverse-phase HPLC system using a semi-preparative column. Compound (l-A) was eluted isocratically with a 50:50 mixture (v/v) of water (40 mM NhUOAc) and acetonitrile at a flow rate of 5 ml/min. The product peak corresponding to compound (l-A) exhibited a retention time of between 15 and 17 minutes. This peak was collected, and the HPLC solvent was subsequently removed by evaporation. The product was dissolved in a 90:10 mixture (v/v) of saline and ethanol. The solution was sterile filtered through 0.22 μιτι membrane filter. Quality control was performed on an analytical HPLC system using a reverse phase analytical column. Under analytical conditions, compound (l-A) exhibited a retention time of

approximately 1 7 minutes. UV detection was performed at 205 nm. The radiochemical purity of the product was found to be greater than 95%, and no apparent chemical impurity was detected at 205 nm.

Example 3. Synthesis of compound (I) by CsF-mediated epoxide ring opening

Compound (VI) (10 mg, 0.014 mmol) was mixed in t-amyl alcohol (400 μΙ, 3.653 mmol) with 10 mg 4-angstrom molecular sieve powder. CsF (21 .31 mg, 0.14 mmol) was added. The resulting suspension was heated to 100°C, and the progress was monitored by LCMS. After 48 hours of stirring, the mixture was filtered through a Celite pad and rinsed with DCM 3 x 1 ml. The filtrate was concentrated and purified by flash chromatography, eluting with EtOAc/heptane followed by MeOH/DCM to yield the fluorinated product (I) (2.3 mg, yield 22%). ¹ HNMR (400 MHz, CDC ) δ ppm 5.07 (s, 1 H), 4.94 (s, 1 H), 4.89 (s, 1 H), 4.81 (s, 1 H), 4.69 (t, J = 4.8 Hz, 1 H), 4.61 (t, J = 4.8 Hz, 1 H), 4.47 (dd, J = 5.6, 9.6 Hz, 1 H), 4.41 (dd, J = 4.8, 9.6 Hz, 1 H), 4.37-4.28 (m, 4H), 4.19 (dd, J = 4.8, 6.4 Hz, 1 H), 4.16-4.02 (m, 2H), 4.04 (dd, J = 4.8, 6.4 Hz, 1 H), 3.98-3.90 (m, 2H), 3.88-3.84 (m, 1 H), 3.64-3.59 (m, 2H), 3.44 (s, 3H), 3.31 (d, J = 2.8 Hz, 1 H), 2.88 (dd, J = 2.0, 9.6 Hz, 1 H), 2.87-2.84 (m, 1 H), 2.72 (dd, J = 9.6, 16.0 Hz, 1 H), 2.53-2.42 (m , 3H), 2.34-2.14 (m , 8H), 2.14-2.07 (m, 1 H), 2.01 -1 .84 (m, 4H), 1 .77-1 .64 (m , 2H), 1 .62-1 .30 (m, 6H) , 1 .1 1 (d, J = 6.4 Hz, 3H), 1 .14-1 .05 (m , 1 H). LCMS (M+H)=733.7.

Example 4. Synthesis of compound (II) by Staudinger azide reduction

Compound (VI) (98 mg, 0.1 37 mmol) was dissolved in a mixture of DMF (3 mL) and water (400 μΙ_), sodium azide (134 mg, 2.062 mmol) was added and heated at 45 °C for 24 h . UPLC/MS showed desired mass and the starting material was consumed. The crude mixture was diluted with EtOAc (40 mL), washed with ammonium chloride (sat. solution, 2x1 0 mL), dried over sodium sulfate and

concentrated under vacuum. The crude was purified on Biotage flash purification (Heptane/EtOAc: 20- 1 00%) to obtain 92 mg of the product (compound (VII)). ¹ H NMR (400 MHz, CDC ) δ 5.05 (d, J = 2.0 Hz, 1 H), 4.91 (d, J = 2.0 Hz, 1 H), 4.87 (s, 1 H), 4.80 (d, J = 2.0 Hz, 1 H), 4.68 (dd, J = 4.8, 4.8 Hz, 1 H), 4.59 (dd, J = 4.4, 4.4 Hz, 1 H), 4.36-4.30 (m, 2H), 4.29 (ddd, J = 4.4, 4.4, 1 0.4 Hz, 1 H), 4.1 7 (dd, J = 4.8, 6.8 Hz, 1 H), 4.14-4.07 (m , 1 H), 4.02 (dd, J = 4.1 , 6.4 Hz, 1 H), 3.99-3.88 (m, 3H), 3.81 (ddd, J = 3.6, 3.6, 7.2 Hz, 1 H), 3.63-3.56 (m , 2H), 3.44 (d, J = 2.2 Hz, 1 H), 3.42 (s, 3H), 3.34-3.25 (m, 3H), 2.89-2.81 (m, 2H), 2.70 (dd, J = 1 0.0, 1 6.4 Hz, 1 H), 2.51 -2.41 (m, 3H) , 2.32-2.31 (m , 7H), 2.1 0-2.058 (m , 1 H), 2.01 -1 .88 (m, 4H), 1 .79-1 .64 (m , 4H), 1 .63-1 .53 (m, 2H), 1 .49-1 .29 (m , 4H), 1 .08 (d, J = 6.8 Hz, 3H).

Compound (VII) (20 mg, 0.026 mmol) was dissolved in DCM (2 mL) and cooled to -5 °C, and then triethylamine (0.01 8 mL, 0.132 mmol) was added followed by Ms-CI (0.01 mL, 0.1 1 9 mmol). The reaction mixture was stirred for 0.5 h at -5 to 0 °C, then warmed to room temperature, and stirring continued for 1 h. The reaction mixture was diluted with DCM (1 0 mL), water (5 mL) was added, the organic layer was separated, and the aqueous layer was extracted with DCM (2x5 mL).The organic layer was dried with sodium sulfate, concentrated, and purified on Biotage flash purification (EtOAc/Heptane: 30-1 00%) to obtain 1 9 mg of product (86%, compound (XV)). ¹ H NMR (400 MHz, CDC ) δ 5.09 (s, 1 H), 4.95 (s, 1 H), 4.88 (s, 1 H), 4.88-4.82 (m, 1 H),4.81 (s, 1 H), 4.69 (dd, J = 4.4, 4.4 Hz, 1 H), 4.60 (dd, J = 4.2, 4.2 Hz, 1 H), 4.36-4.29 (m, 2H), 4.29 (ddd, J = 4.0, 4.0, 6.4 Hz, 1 H), 4.1 8 (dd, J = 4.4, 6.4 Hz, 1 H), 4.1 1 (dd, J = 9.0, 9.0 Hz, 1 H), 4.02 (dd, J = 4.4, 6.4 Hz, 1 H), 3.95 (dd, J = 1 0.2, 1 0.2 Hz, 1 H), 3.88-3.83 (m, 1 H), 3.80 (ddd, J = 3.2, 6.8, 6.8 Hz, 1 H), 3.68-3.54 (m , 4H), 3.42 (s, 3H), 3.31 (d, J = 2.8 Hz, 1 H), 3.1 1 (s, 3H), 2.89-2.82 (m , 2H), 2.70 (dd, J = 1 0.0, 1 6.4 Hz, 1 H), 2.54-2.45 (m , 2H), 2.24-2.04 (m , 1 1 H), 2.02-1 .85 (m , 4H), 1 .78- 1 .68 (m, 3H), 1 .64-1 .1 52 (m , 3H), 1 .48-1 .25 (m, 6H) , 1 .09 (d, J = 6.8 Hz, 3H).

CsF (273 mg, 1 .795 mmol) was added to a dry vial , followed by dry 2-methylbutan-2-ol (4 mL). Compound (XV) in dry 2-methylbutan-2-ol (1 mL) was added and heated for 1 h at 90-95 °C, and the ensuing reaction was monitored by UPLC/MS. The reaction mixture was cooled to room temperature, diluted with EtOAc (1 0 mL), and washed with NH4CI (saturated solution, 2 mL). The organic layer was separated, dried, and concentrated to isolate 25 mg of crude product, which was taken to the next step without further purification.

The crude material (25 mg, 0.033 mmol) was dissolved in a mixture of THF (4 mL) and water (0.4 mL). Triphenylphosphine (1 00 mg, 0.33 mmol) was added, and the reaction mixture was subsequently heated at 65 °C for 30 minutes. After cooling to room temperature, the solvent was removed under vacuum , and the crude material was dissolved in DMF (6 mL) and purified by H PLC (C1 8 column, run time 1 0 minutes, acetonitrile/water) to obtain 6.5 mg of product. Residual DMF was removed by dissolving in DCM (2 mL) and washing with aqueous NaHCCb (3x1 mL). The solution was dried over sodium sulfate and concentrated under vacuum to obtain 5.5 mg of compound (II). ¹ H NMR (400 MHz, CDCb) δ 5.08 (s, 1 H), 4.95 (s, 1 H), 4.91 (s, 1 H), 4.81 (s, 1 H), 4.70 (dd, J = 4.4, 4.4 Hz, 1 H), 4.61 (dd, J = 4.4, 4.4 Hz, 1 H), 4.35-4.27 (m, 3H), 4.19 (dd, J = 5.4, 5.4 Hz, 1 H), 4.15-4.10 (m, 1 H), 4.40 (dd, J = 4.4, 6.4 Hz, 1 H), 3.97 (dd, J = 10.0, 10.0 Hz, 1 H), 3.89-3.84 (m, 1 H), 3.78 (ddd, J = 3.0, 6.8, 6.8 Hz, 1 H), 3.65- 3.58 (m, 2H), 3.44 (s, 3H), 3.30 (d, J = 3.2 Hz, 1 H), 2.97-2.84 (m, 3H), 2.72 (dd, J = 10.0, 16.4 Hz, 1 H), 2.57-2.39 (m, 4H), 2.35-1 .85 (m, 14H), 1 .78-1 .54 (m, 8H), 1 .49-1 .30 (m, 3H), 1 .1 1 (d, J = 6.4 Hz, 3H).

Example 5. Synthesis of compound (III) by Cu(l)-catalyzed azide-alkyne cycloaddition

Compound (VI) (40 mg, 0.056 mmol) was placed in a vial, dissolved in DMF (2 mL), and prop-2- yn-1 -amine (155 mg, 2.806 mmol) was added. The vial was sealed and heated at 80 °C for 18 h. The reaction was monitored by UPLC/MS. The reaction was quenched by adding NaHCC (10 mL, sat.

solution), stirred for 10 minutes, and extracted with DCM (2x20 mL), and then the organic layer was washed with brine (2x1 0 mL), dried, and concentrated under vacuum. The crude material was purified on Biotage eluting with EtOAc/Heptane (60-90%), followed by DCM/Methanol (4-15% with 0.1 % Et ₃ N) to isolate 22 mg of compound (XVI) (51 %). Ή NMR (400 MHz, CDCb) δ 5.06 (s, 1 H), 4.93 (s, 1 H), 4.89 (s, 1 H), 4.80 (s, 1 H), 4.68 (dd, J = 4.4, 4.4 Hz, 1 H), 4.59 (dd, J = 4.4, 4.4 Hz, 1 H), 4.36-4.30 (m, 2H), 4.29 (ddd, J = 4.4, 4.4, 1 0.4 Hz, 1 H), 4.1 7 (dd, J = 4.8, 6.8 Hz, 1 H), 4.14-4.04 (m, 1 H), 4.02 (dd, J = 4.4, 6.4 Hz, 1 H), 3.98-3.81 (m, 4H), 3.63-3.56 (m, 2H), 3.44 (d, J = 2.0 Hz, 2H), 3.42 (s, 3H), 3.27 (d, J = 2.8 Hz, 1 H), 2.89-2.82 (m, 2H), 2.78 (dd, J = 3.4, 1 1 .8 Hz, 1 H), 2.74-266 (m, 2H), 2.52-2.38 (m, 3H), 2.33-2.13 (m, 8H), 2.10-1 .65 (m, 1 1 H), 1 .63-1 .51 (m, 2H), 1 .47-1 .32 (m, 4H), 1 .08 (d, J = 6.8Hz, 3H).

Cesium fluoride (82 mg, 0.538 mmol) and dry 2-methylbutan-2-ol (1 .5 mL) were added to a dry vial and sonicated for 5 minutes. Compound (XIX) in dry 2-methylbutan-2-ol (0.5 mL) was added and heated for 25 minutes at 1 00 °C. NH4CI (saturated solution, 0.5 ml) was added and diluted with EtOAc (0.5 mL), and the organic layer was subsequently separated and dried over sodium sulfate. The resulting solution of product (2 mL) was used for the next step. 500μί of this solution was concentrated to yield 5.5 mg of product (XVII) (74%) for checking purity by NMR. ¹ H NMR (400 MHz, CDCb) δ 4.56 (dt, J = 4.4, 47.6 Hz, 2H), 3.78 (dd, J = 4.2, 4.2 Hz, 2H), 3.72-3.65 (m, 12H), 3.39 (dd, J = 5.2, 5.2 Hz, 2H).

The above-described crude solution (105 μί) of compound (XVII) (1 .152 mg, 5.209 μιηοΙ) was added to a dry vial containing a solution of compound (XVI) (2 mg, 2.604 μιτιοΙ) in acetonitrile (200 μί) and Hunig's Base (22.74 μί, 13 mmol). Copper iodide (0.496 mg, 2.604 μιτιοΙ) in acetonitrile (50 μί) was added, and the mixture was heated at 60 °C for 5 minutes. UPLC/MS indicated the reaction was complete within 5 minutes at 60 °C. Solvent was removed under vacuum, and the residue was dissolved in DMF and purified by HPLC (C18 column, run time 10 minutes, acetonitrile/water) to isolate 1 .75 mg of compound (III) (69%). ¹ H NMR (400 MHz, CD3OD) δ 8.20 (s, 1 H), 5.16 (s, 1 H), 5.04 (s, 1 H), 4.73 (dd, J = 4.2, 4.2 Hz, 1 H), 4.65-4.59 (m, 4H), 4.51 (br. s, 1 H), 4.84 (dd, J = 3.6, 3.6 Hz, 1 H), 4.38-4.27 (m, 4H), 4.20 (dd, J = 4.8, 4.8 Hz, 1 H), 4.16-4.04 (m, 3H), 4.00 (dd, J = 10.0, 10.0 Hz, 1 H), 3.95-3.84 (m, 5H), 3.79-3.60 (m, 1 1 H), 3.43 (s, 3H), 3.37 (br. s, 1 H), 3.19 (d, J = 1 0.8, 1 H), 3.07 (dd, J = 10.0, 10.0 Hz, 1 H), 2.94 (dd, J = 10.6, 1 .0 Hz, 1 H), 2.93-2.84 (m, 1 H), 2.74 (dd, J = 10.0, 16.8 Hz, 1 H), 2.70 (dd, J = 1 1 .0, 19.0 Hz, 1 H), 2.51 -2.31 (m, 5H), 2.23-1 .71 (m, 13H), 1 .61 -1 .30 (m, 6H), 1 .15 (d, J = 6.4 Hz, 3H). Example 6. Synthesis of compound (IV) by Cu(l)-catalyzed azide-alkyne cycloaddition

Compound (VII) (65 mg, 0.086 mmol) was dissolved in THF (3 ml_) and NaH (5.50 mg, 0.138 mmol) was added, stirred for 30 minutes at 0 °C. 3-Bromoprop-1 -yne (63.9 mg, 0.43 mmol) in toluene was added and stirred for 14 h at room temperature. The reaction mixture was diluted with EtOAc (30 mL), washed with ammonium chloride (sat. solution, 2X10 mL), dried over sodium sulfate and concentrated under vacuum. The crude was purified on Biotage (Heptane/EtOAc: 20-100%) to obtain 24 mg of compound (VIII) (35%) and 8 mg of starting Compound (VII). ¹ H NMR (400 MHz, CDCb) δ 5.09 (s, 1 H), 4.94 (s, 1 H), 4.90 (s, 1 H), 4.80 (s, 1 H), 4.69 (dd, J = 4.4, 4.4 Hz, 1 H), 4.60 (dd, J = 4.4, 4.4 Hz, 1 H), 4.36-4.29 (m, 3H), 4.26 (d, J = 2.4 Hz, 1 H), 4.18 (dd, J = 5.6, 5.6 Hz, 1 H), 4.14-4.09 (m, 1 H), 4.03 (dd, J = 5.2, 5.2 Hz, 1 H), 3.95 (dd, J = 9.4, 9.4 Hz, 1 H), 3.88-3.81 (m, 2H), 3.70 (ddd, J = 8.0, 5.6, 2.8 Hz, 1 H), 3.63-3.58 (m, 2H), 3.44-3.40 (m, 1 H), 3.43 (s, 3H), 3.29 (d, J = 3.2 Hz, 1 H), 2.89-2.82 (m, 2H), 2.76-2.69 (m, 1 H), 2.51 -2.39 (m, 3H), 2.33-1 .89 (m, 13H), 1 .68-1 .54 (m, 2H), 1 .76-1 .67 (m, 3H), 1 .48-1 .24 (m, 6H), 1 .09 (d, J = 6.8 Hz, 3H).

Compound (VIII) (22 mg, 0.028 mmol) was dissolved in a mixture of THF (2.5 mL) and water (0.25 mL), triphenylphosphine (145 mg, 0.554 mmol) was added and heated at 64 °C for 1 h. The reaction mixture was concentrated under vacuum. Crude was purified on Biotage to obtain 13 mg of compound (XVIII) (DCM/Methanol: 100:5-6% with 0.1 % ammonium in methanol). ¹ H NMR (400 MHz, CDCb) δ 5.40-5.32 (m, 1 H),5.07 (s, 1 H), 4.95 (s, 1 H), 4.90 (s, 1 H), 4.80 (s, 1 H), 4.68 (dd, J = 4.6, 4.6 Hz, 1 H), 4.60 (dd, J = 4.4, 4.4 Hz, 1 H), 4.38-4.25 (m, 3H), 4.23-4.15 (m, 3H), 4.1 1 (dd, J = 9.2, 9.2 Hz, 1 H), 4.03 (dd, J = 4.4, 6.0 Hz, 1 H), 4.03 (dd, J = 10.4, 10.4 Hz, 1 H), 4.82 (d, J = 10.4 Hz, 1 H), 3.72-3.64 (m, 2H), 3.64-3.54 (m, 2H), 3.43 (s, 3H), 3.27 (d, J = 3.2 Hz, 1 H), 2.95-2.76 (m, 3H), 2.72 (dd, J = 10.0, 1 5.2 Hz, 1 H), 2.52-2.10 (m, 14H), 2.02-1 .86 (m, 6H), 1 .76-1 .148 (m, 6H), 1 .48-1 .38 (m, 3H), 1 .09 (d, J = 6.4 Hz, 3H).

The crude solution (734 μί) of compound (XVII) was added to a dry vial containing a solution of compound (XVIII) (14 mg, 18 μιτιοΙ) in acetonitrile (950 μΐ) and Hunig's Base (159 μΐ, 0.912 mmol).

Copper iodide (3.47 mg, 18 μιηοΙ) in acetonitrile (950 μΙ) was added, and the mixture was heated at 60 °C for 15 minutes. UPLC/MS indicated the reaction was complete within 15 minutes. Solvent was removed under vacuum, and the residue was dissolved in DMF and purified by HPLC (C18 column, run time 10 minutes, acetonitrile/water) to isolate 7.5 mg of compound (IV) (42%). ¹ H NMR (400 MHz, CDCb) δ 7.89 (s, 1 H), 5.09 (s, 1 H), 4.97 (s, 1 H), 4.88 (s, 1 H), 4.79 (s, 1 H), 4.79-4.70 (m, 1 H), 4.68 (dd, J = 4.6, 4.6 Hz, 1 H), 4.63-4.58 (m, 3H), 4.54-4.47 (m, 3H), 4.37-4.21 (m, 2H), 4.29 (ddd, J = 4.0, 4.0, 10.0 Hz, 1 H), 4.18 (dd, J = 5.0, 5.0 Hz, 1 H), 4.10 (dd, J = 9.2, 9.2 Hz, 1 H), 4.03 (dd, J = 5.5, 5.5 Hz, 1 H), 3.96 (dd, J = 10.0, 10.0 Hz, 1 H), 3.90-3.80 (m, 4H), 3.80-3.57 (m, 20H), 3.42 (s, 3H), 3.38 (dd, J = 5.0, 5.0 Hz, 1 H), 3.32 (s, 1 H), 3.1 5-2.81 (m, 2H), 2.72 (dd, J = 10.2, 16.2, 1 H), 2.56 (dd, J = 12.0, 1 7.6 Hz, 1 H), 2.50-2.42 (m, 1 H), 2.41 -2.12 (m, 8H), 2.12-1 .81 (m, 5H), 1 .79-1 .64 (m, 2H), 1 .62-1 .52 (m, 1 H), 1 .48-1 .32 (m, 3H), 1 .15 (d, J = 6.0 Hz, 3H).

Example 7. Synthesis of compound (V) by Bis(2-methoxyethyl)aminosulfur trifluoride-mediated nucleophilic substitution

Compound (XX) was dissolved in DCM (268 μΙ, 4.165 mmol) and cooled to 0°C. Bis(2- methoxyethyl)aminosulfur trifluoride (Compound (IX), 5 μΙ, 0.027 mmol) was added, and the reaction was subsequently monitored by TLC analysis (1 :4 heptane:EtOAc by volume). When the reaction was complete, water was added, and the product was extracted with DCM. Concentration provided a residue that was purified by preparative TLC (1 :4 heptane:EtOAc by volume) to provide 0.7 mg compound (V) (5%). ¹ H NMR (400 MHz, CDC ) δ 5.03 (br s, 1 H), 4.89 (br s, 1 H), 4.87 (br s, 1 H), 4.78-4.80 (m, 1 H), 4.78 (br s, 1 H), 4.73-4.65 (m, 2H), 4.65-4.55 (m, 3H), 4.55-4.47 (m, 1 H), 4.39-4.25 (m, 2H), 4.21 -4.1 7 (m, 2H), 4.1 1 -4.00 (m, 3H), 3.72-3.64 (m, 1 H), 3.57-3.51 (m, 1 H), 3.44-3.37 (m, 1 H), 3.34 (s, 3H), 2.87 (dd, J = 12.0, 4.0 Hz, 1 H), 2.76 (dd, J = 16.0, 12.0, 1 H), 2.90-2.82 (m, 1 H), 2.38-2.1 6 (m, 2H), 2.16-1 .98 (m, 6H), 1 .98-1 .79 (m, 4H), 1 .79-1 .59 (m, 3H), 1 .49-1 .39 (m, 6H), 1 .39-1 .32 (m, 3H), 1 .14-1 .00 (m, 3H).

Other embodiments are within the claims.