Patent Description:
STING (stimulator of interferon genes) is a signaling molecule in the innate response to dsDNA in the cytosol. STING deletion has been reported in multiple human cancers. In addition, deregulation of STING signaling in human cancers also has been reported in melanoma (<NPL>) and colon cancer. Interestingly, in those studies, genomic analysis results showed loss expression of STING is not due to gene deletion or mutation, but through epigenetic changes. (<NPL>; <NPL>). STING's cancer protection activity is also supported by evidence obtained from mouse model studies. STING knockout mice have shown defective tumor control.

In addition, STING's role in protecting ontogenesis has been demonstrated in several mouse spontaneous models, including glioma (<NPL>), and colon cancer (<NPL>). This anti-tumor effect may be due to its ability to counter over-activation of NF-kB and STAT3. (Ohkuri <NUM>). Activation of STING pathway also showed potent activity in preclinical mouse tumor models. (Woo <NUM>;<NPL>;<NPL>; <NPL>;<NPL>). This anti-tumor activity is likely due to disruption of tumor vasculature and followed by induction of adaptive immune response. Accordingly, direct stimulation of STING in a tumor microenvironment by an agonist may represent a novel approach for treating multiple cancer types.

A compound represented by the formula (I), namely, (1R,3R,15E,28R,29R,30R,31R,34R,36R,<NUM>,41R)-<NUM>,<NUM>-Difluoro-<NUM>,<NUM>-bis(sulfanyl)-<NUM>,<NUM>,<NUM><NUM>,<NUM>,<NUM>,<NUM>-hexaoxa-<NUM>,<NUM>,<NUM>,<NUM>,<NUM>,<NUM>,<NUM>,<NUM>,<NUM>,<NUM>-decaaza-34λ<NUM>,39λ<NUM> -diphosphaoctacyclo[<NUM>. <NUM><NUM>,<NUM>. <NUM><NUM>,<NUM>. <NUM><NUM>,<NUM>. <NUM><NUM>,<NUM>. <NUM><NUM>,<NUM>. <NUM><NUM>,<NUM>]dotetraconta-<NUM>,<NUM>,<NUM>,<NUM>,<NUM>,<NUM>,<NUM>,<NUM>,<NUM>-nonaene-<NUM>,<NUM>-dione (hereafter referred to as Compound (I)), suppresses the growth of tumors. <CHM>
Further cyclic di-nucleotide compounds for use as STING modulators are known from <CIT>.

Generally, the physical properties of a compound, salts thereof, and their crystals used as a pharmaceutical product largely influence on the bioavailability of a drug, the purity of an active pharmaceutical ingredient, prescription of a preparation and the like. An object of the present invention is therefore to provide salts of Compound (I) or crystals thereof with a potential to be used as drug substance in pharmaceuticals.

The present inventor has found salts of Compound (I) or crystals thereof with a potential to be used as drug substance in pharmaceuticals, thereby completing the invention.

Specifically, the present invention provides the following <<NUM>> to <<NUM>>.

The salts of Compound (I) and the crystals thereof provided by the present invention possess properties such as hygroscopicity as shown in the examples described in later and a potential to be used as drug substance in pharmaceuticals.

A salt of the Compound (I) of the present invention, a crystal thereof, and production methods thereof will be described in detail.

As used herein, a "salt" refers to a chemical entity made up of the Compound (I) as the acidic component and a specific number of equivalents of a base to the Compound (I). Here, the term " a salt of
(1R,3R,15E,28R,29R,30R,31R,34R,36R,<NUM>,41R)-<NUM>,<NUM>-Difluoro-<NUM>,<NUM>-bis(sulfanyl) -<NUM>,<NUM>,<NUM>,<NUM>,<NUM>,<NUM>-hexaoxa-<NUM>,<NUM>,<NUM>,<NUM>,<NUM>,<NUM>,<NUM>,<NUM>,<NUM>,<NUM>-decaaza-34λ<NUM>,39λ<NUM>-diphosphaoc tacyclo[<NUM>. <NUM><NUM>,<NUM>. <NUM><NUM>,<NUM>. <NUM><NUM>,<NUM>. <NUM><NUM>,<NUM>. <NUM><NUM>,<NUM>. <NUM><NUM>,<NUM>]dotetraconta-<NUM>,<NUM>,<NUM>,<NUM>,<NUM>,<NUM>,<NUM>,<NUM>,<NUM>-nonae ne-<NUM>,<NUM>-dione (Compound (I)) represented by the formula (I), and a base selected from the group consisting of sodium hydroxide, sodium carbonate, ammonia in ethanol and ammonium hydroxide etc." is used for the same meaning as " a salt of (1R,3R,15E,28R,29R,30R,31R,34R,36R,<NUM>,41R)-<NUM>,<NUM>-Difluoro-<NUM>,<NUM>-bis(sulfanyl) -<NUM>,<NUM>,<NUM>,<NUM>,<NUM>,<NUM>-hexaoxa-<NUM>,<NUM>,<NUM>,<NUM>,<NUM>,<NUM>,<NUM>,<NUM>,<NUM>,<NUM>-decaaza-34λ<NUM>,39λ<NUM>-diphosphaoc tacyclo[<NUM>. <NUM><NUM>,<NUM>. <NUM><NUM>,<NUM>. <NUM><NUM>,<NUM>. <NUM><NUM>,<NUM>. <NUM><NUM>,<NUM>. <NUM><NUM>,<NUM>]dotetraconta-<NUM>,<NUM>,<NUM>,<NUM>,<NUM>,<NUM>,<NUM>,<NUM>,<NUM>-nonae ne-<NUM>,<NUM>-dione (Compound (I)) represented by the formula (I) formed with a base selected from the group consisting of sodium hydroxide, sodium carbonate, ammonia in ethanol and ammonium hydroxide etc.".

Examples of a "salt" used herein include salts with inorganic bases, and in particular, pharmaceutically acceptable salts are preferred.

A salt of the Compound (I) may also be a solvate or a hydrate. As used herein, a solvate or a hydrate of the salt of Compound (I) means a solid formed from the salt of the Compound (I) together with solvent molecules or water molecule. Examples of the solvent in the solvate include: a ketone solvent such as acetone, methyl ethyl ketone or cyclohexanone; an ester solvent such as ethyl acetate or methyl acetate; an ether solvent such as <NUM>, <NUM>-dimethoxyethane or methyl-tert-butyl ether; an alcohol solvent such as methanol, ethanol, <NUM>-propanol or isopropanol; a polar solvent such as N-methyl-<NUM>-pyrrolidone, N,N-dimethylformamide or dimethyl sulfoxide.

As used herein, a "crystal" refers to a crystal of the salt of Compound (I) or a crystal of the Compound (I). Accordingly, a crystal of Compound (I) ammonium salt, for example, means a crystal of the salt formed between Compound (I) and ammonia (or ammonium hydroxide). In addition, a crystal of Compound (I) di-ammonium salt, for example, means a crystal of the salt formed between one molecule of Compound (I) and two molecules of ammonia (or ammonium hydroxide).

Examples of crystals disclosed herein include:.

The peaks in a powder X-ray diffraction, described above, are characteristic for each of the crystal (Form <NUM>) of Compound (I) di-ammonium salt, the crystal (Form <NUM>) of Compound (I) di-ammonium salt, the crystal (Form <NUM>) of Compound (I) di-ammonium salt, the crystal (Form <NUM>) of Compound (I) di-ammonium salt, the crystal (Form <NUM>) of Compound (I) di-ammonium salt, the crystal (Form <NUM>) of Compound (I) mono-ammonium salt, the crystal of Compound (I) sodium salt and the crystal of Compound (I).

Generally, errors in diffraction angles (2θ) within the range of ± <NUM>° may arise in powder X-ray diffraction, and thus the above-described values of diffraction angles need to be considered to include values within the range of approximately ± <NUM>°. Included in the present invention are, therefore, not only crystals with peaks at exactly the same diffraction angles in powder X-ray diffraction, but also crystals with peaks within an error range of approximately ± <NUM>° of the diffraction angles. Hence, "having a diffraction peak at a diffraction angle (<NUM> ± <NUM>°) of <NUM>° as used herein, for example, means "having a diffraction peak at a diffraction angle (2θ) of <NUM>° to <NUM>°. The same is also applied to other diffraction angles.

Generally, peak intensities and half-value widths of diffraction angles (2θ) in powder X-ray diffraction are different for each measurement because of differences in measurement conditions and dispersions of size and shape of each particle of powder crystal and not always stable even though forms of crystals are same. Therefore, in case of comparing a powder X-ray diffraction pattern, when diffraction angles (2θ) are the same but peak intensities, relative peak intensities and half-value widths are different, those differences does not imply that the measured forms of crystals differ from each other. Thus, a crystal of salt having a powder X-ray diffraction pattern, which has aforementioned differences with respect to characteristic diffraction peaks of a certain crystal of salt according to the present invention, means that the crystal has the same crystal form of the crystal of salt according to the present invention.

As used herein, "having a powder X-ray diffraction pattern substantially the same as the powder X-ray diffraction pattern shown in <FIG>" means it includes not only the case of having exactly the same powder X-ray diffraction pattern as shown in <FIG>, but also the case that peak intensities, relative peak intensities and half-value widths are different, or the case of having the characteristic peaks within an error range of approximately ± <NUM>° of the diffraction angles. Thus every crystal having such the powder X-ray diffraction pattern means that the crystal is identical to the crystal according to the present invention.

Methods for producing a salt of the Compound (I) and a crystal thereof will be described in detail.

Compound (I) can be synthesized as described specifically in Production Example <NUM> or in Production Example <NUM> below.

A salt of the Compound (I) can be obtained by a conventional method for producing a salt. Specifically, it can be produced, for example, by suspending or dissolving Compound (I) in a solvent, with heating if necessary, then by adding a base (for example, sodium hydroxide, sodium carbonate for sodium salt; ammonia in ethanol and ammonium hydroxide for mono- or di-ammonium salt) to the obtained suspension or solution and by stirring or leaving the resultant suspension or solution for several minutes to several days at room temperature or with ice-bath cooling. A salt of the Compound (I) may be obtained as crystals or amorphous substances according to the production methods. Examples of the solvents to be used in these methods include alcohol solvents such as ethanol, <NUM>-propanol and isopropanol; acetonitrile; ketone solvents such as acetone and <NUM>-butanone; ester solvents such as ethyl acetate; saturated hydrocarbon solvents such as hexane and heptane; ether solvents such as t-butyl methyl ether or water. Each of these solvents may be used alone, or two or more may be mixed and used.

A crystal of the salt of Compound (I) may be produced by the above-mentioned methods for producing a salt of the Compound (I), or by heat-dissolving a salt of the Compound (I) in a solvent and crystallizing it through cooling with stirring.

A salt of the Compound (I) to be used in the crystallization may be in any form: it may be a solvate, a hydrate, an anhydrate, an amorphous substance, a crystalline substance (including those consisting of a plurality of crystalline polymorphs) or a combination thereof.

Examples of the solvents to be used in the crystallization include alcohol solvents such as methanol, ethanol, isopropanol and <NUM>-propanol; acetonitrile; amide solvents such as N, N-dimethylformamide; ester solvents such as ethyl acetate; saturated hydrocarbon solvents such as hexane and heptane; ketone solvents such as acetone and <NUM>-butanone; ether solvents such as t-butyl methyl ether or water. Furthermore, each of these solvents may be used alone, or two or more may be mixed and used.

The amount of the solvent to be used may be suitably selected, provided that the lower limit is the amount with which the free form of Compound (I) or the salt thereof is dissolved by heating or the suspension can be stirred, and that the upper limit is the amount with which the yield of the crystal is not significantly reduced.

A seed crystal (e.g., the crystal of the desired salt of Compound (I)) may be added or may not be added during the crystallization. The temperature at which the seed crystal is added is not particularly limited, but is preferably <NUM> to <NUM>.

As the temperature to be employed when the salt of Compound (I) is dissolved by heating, that at which Compound (I) dissolves may be suitably selected depending on the solvent, but it is preferably within the range between the temperature at which the recrystallization solvent starts to reflux and <NUM>, and more preferably <NUM> to <NUM>.

Cooling during the crystallization could give substances containing different forms of crystals (polymorphism) in the case of rapid cooling. It is therefore desirable to perform the cooling while controlling the cooling rate as appropriate based on the consideration of its effect on the quality, grain size and the like of the crystal. Preferred is, for example, cooling at a cooling rate of <NUM> to <NUM>/hour. More preferred is cooling at a cooling rate of, for example, <NUM> to <NUM>/hour.

Furthermore, the final crystallization temperature may be selected suitably for the yield, quality and the like of the crystal, but is preferably <NUM> to -<NUM>.

The target crystal can be obtained by isolating the formed crystal through a conventional filtration procedure, washing the filtered-off crystal with a solvent if necessary, and further drying it. As the solvent to be used for washing the crystal, the same solvent as in the crystallization can be used. Furthermore, each of these solvents may be used alone, or two or more may be mixed and used. Preferably, it is, for example, acetone, <NUM>-butanone, ethyl acetate, t-butyl methyl ether, hexane or a mixed solvent of hexane/<NUM>-butanone.

The crystal isolated through the filtration procedure may be dried appropriately by leaving it in air or under nitrogen flow, or by heating.

As the drying time, the time until the amount of residual solvent becomes less than the predefined amount may be selected as appropriate depending on the amount of production, the drying apparatus, the drying temperature and the like. Furthermore, drying may be performed under airflow or under reduced pressure. The degree of pressure reduction may be selected as appropriate depending on the amount of production, the drying apparatus, the drying temperature and the like. The obtained crystal may be left in air as required after drying.

A crystal of Compound (I) can be obtained by a conventional method for producing a crystal as shown above.

A pharmaceutical composition of the present invention could be prepared by mixing pharmaceutically acceptable additives with the salt of Compound (I) or the crystal thereof. A pharmaceutical composition of the present invention could be prepared according to the known method such as a method described in the general rules for preparations of the<NPL>on.

A pharmaceutical composition of the present invention could be administered to patients appropriately depending on the dosage form.

A pharmaceutical composition of the present invention has applicability as a therapeutic agent for treating cancers since the salt of Compound (I) or the crystal thereof can potently activate STING pathway and show potent antitumor activities. Examples of cancers include glioma, melanoma and colon cancer.

The dosage of the salt of Compound (I) or the crystal thereof varies depending on the extent of the symptom, age, gender, body weight, dosage form, the type of the salt, the specific type of the disease and the like. In the case of adults, typically, about 30µg to <NUM>, preferably 100µg to <NUM>, and more preferably 100µg to <NUM> per day is orally administered, or about 30µg to <NUM>, preferably 100µg to <NUM>, and more preferably 100µg to <NUM> per day is administered by injection, in each case, in a single dose or in divided doses.

Hereinafter, the present invention will be described in detail with the production examples and examples. However, the present invention is not intended to be limited by these examples.

The following abbreviations may be used throughout the examples. DMT: <NUM>,<NUM>'-Dimethoxytrityl.

Each crystalline sample was placed on the sample stage of a powder X-ray diffractometer and the analysis was performed under one of the following conditions.

The coupling constant is recorded in hertz (Hz). The abbreviations of splitting patterns are as follows:
s: singlet, d: doublet, t: triplet, q: quartet, m: multiplex, bs: broad singlet, br s: broad singlet, dd: doublet of doublets, dt: doublet of triplets, br d: broad doublet, br t: broad triplet.

Unless indicated otherwise, <NUM>H NMR spectra were taken on a Bruker <NUM> or <NUM> NMR.

The obtained solid was weighed into a sampling cup and the sampling cup was placed inside an isothermal chamber at <NUM>. The relative humidity (RH) was controlled from <NUM>% to <NUM>% using a gravimetric vapor sorption system and the sample weight at each RH stage was measured within a predetermined interval of time (e.g. every <NUM> minutes). The weight change at each RH stage was evaluated in a stepwise manner, and then was finally determined under the following criteria. The maximum weight change for each measurement is less than <NUM>% (w/w) in <NUM> minutes or <NUM>% (w/w) in <NUM> minute.

To a mixture of (2R,3R,4R,5R)-<NUM>-(<NUM>-benzamido-<NUM>-purin-<NUM>-yl)-<NUM>-((bis(<NUM>-methoxyphenyl) (phenyl)methoxy)methyl)-<NUM>-fluorotetrahydrofuran-<NUM>-yl (<NUM>-cyanoethyl) diisopropy-lphosphoramidite (Compound (<NUM>)) (mixture of phosphorous diastereomers; <NUM>, <NUM>. 332mmol, <NUM> eq. , ChemGenes Corporation catalog # ANP-<NUM>), allyl alcohol (<NUM>, 142mmol, <NUM>. 55eq) and triphenylphosphine (<NUM>, 146mmol, <NUM>. ) in THF (<NUM>) was added DEAD (40wt% solution in toluene; <NUM>, 137mmol, <NUM>. ) at ambient temperature. Stirring was continued at ambient temperature and the reaction was monitored by LC/MS. Upon completion (<NUM>), the mixture was concentrated in vacuo (<NUM>) and resultant mixture was purified by silica gel column chromatography (<NUM> x <NUM> columns, <NUM> to <NUM>% EtOAc in n-heptane buffered with <NUM>% triethylamine) to give Compound (<NUM>) as a white foam (<NUM>, quantitative yield, mixture of phosphorous diastereomers).

<NUM>H NMR (<NUM>:<NUM> mixture of phosphorous diastereomers, <NUM>, CDCl<NUM>) δ1. <NUM>-<NUM> (m, <NUM>), <NUM> (t, J=<NUM>, <NUM>), <NUM> (t, J=<NUM>, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s <NUM>), <NUM> (s <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (br d, J=<NUM>, <NUM>), <NUM> (br d, J=<NUM>, <NUM>), <NUM> (br d, J=<NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>).

To a solution of Compound (<NUM>) (<NUM>, <NUM>. 28mmol, 1eq. ) in acetonitrile (<NUM>) was added water (<NUM>, <NUM>. 55mmol, <NUM>. ) and pyridine trifluoroacetate salt (<NUM>, <NUM>. 93mmol, <NUM>. After stirring at ambient temperature for <NUM> minute, tert-butylamine (<NUM>, <NUM>, <NUM>. 20mol, 60eq. ) was added. Upon complete cleavage of cyanoethyl group (monitored by LC/MS), the reaction mixture was concentrated in vacuo and azeotroped twice with acetonitrile. The crude mixture was dissolved in DCM (<NUM>) and treated with water (<NUM>, <NUM>. 55mmol, <NUM>. ) and NaHSO<NUM>-SiO<NUM> (<NUM>, <NUM>. 55mmol, 2eq. ) at ambient temperature. Upon complete cleavage of DMT group (monitored by LC/MS, approximately <NUM> hour), the reaction mixture was filtered and rinsed twice with DCM/MeOH (<NUM>/<NUM>, <NUM>). The combined filtrates were concentrated in vacuo and treated with <NUM>:<NUM> mixture of n-heptane/toluene (~<NUM>). The top layer was removed by decantation. The same operation was repeated once more with n-heptane/toluene (<NUM>/<NUM>, <NUM>) and the bottom layer was azeotroped twice with acetonitrile to give Compound (<NUM>) (<NUM>% theoretical yield assumed). The product was used in the next step without further purification.

To a mixture of Compound (<NUM>) (<NUM>, <NUM>. 27mmol, 1eq. ) and Compound (<NUM>) (<NUM>, <NUM>. 28mmol, 1eq. ) in acetonitrile (<NUM>) was added pyridine trifluoroacetate salt (azeotropically dried with pyridine; <NUM>, <NUM>. 94mmol, <NUM>. After <NUM> minutes, DDTT (<NUM>, <NUM>. 09mmol, <NUM>. , ChemGenes Corporation catalog # RN-<NUM>) was added and, upon complete sulfurization (monitored by LC/MS), the reaction mixture was concentrated in vacuo. The residue was dissolved in DCM (<NUM>) and treated with water (<NUM>, 32mmol, 10eq. ) and <NUM>% dichloroacetic acid (<NUM>, <NUM>. 9mmol, <NUM>. ) in DCM (<NUM>). After <NUM> minutes, the reaction was quenched with pyridine (<NUM>) and concentrated in vacuo. The residue was azeotroped with pyridine to give Compound (<NUM>) (<NUM>, <NUM>% theoretical yield assumed). The product was used in next the step without further purification.

To a solution of Compound (<NUM>) (<NUM>, <NUM>. 15mmol, 1eq. ) in pyridine (<NUM>) was added DMOCP (<NUM>, <NUM>. 88mmol, <NUM>. ) at ambient temperature. Upon complete macrocyclization (monitored by LC/MS), water (<NUM>, <NUM>. 5mmol, x10 fold relative to DMOCP) was added followed by <NUM>-benzo[c][<NUM>,<NUM>]dithiol-<NUM>-one (<NUM>, <NUM>. 73mmol, <NUM>. Upon complete sulfurization (approximately <NUM> minutes), the reaction mixture was partially concentrated in vacuo to approximately <NUM> and poured into a mixture of saturated aqueous NaHCO<NUM> (<NUM>) and water (<NUM>). After <NUM> stirring at ambient temperature, the mixture was extracted with <NUM>:<NUM> mixture of EtOAc/MTBE (<NUM> × <NUM> times). The organic layers were combined, washed with brine (<NUM>), dried over MgSO<NUM> and concentrated in vacuo. The residue was purified by silica gel column chromatography (<NUM>-<NUM>% MeOH in DCM) to give Compound (<NUM>) (<NUM>, <NUM>. 20mmol, <NUM>% theoretical yield assumed) as a brown oil. The product was used in the next step without further purification.

To a solution of Compound (<NUM>) (<NUM>, <NUM>. 20mmol, 1eq. ) in acetonitrile (<NUM>) was added <NUM>-nitrobenzyl bromide (<NUM>, <NUM>. 2mmol, <NUM>. ) and triethylamine (<NUM>, <NUM>. 8mmol, <NUM>. Upon complete reaction (monitored by LC/MS, approximately <NUM> hours at ambient temperature), the reaction mixture was concentrated in vacuo and purified by silica gel column chromatography (<NUM>% ethyl acetate/n-heptane to <NUM>% ethyl acetate) to give <NUM> product as a mixture of phosphorous diastereomers. Preparative HPLC separation of the diastereomers gave Compound (<NUM>) (SR isomer; <NUM>, <NUM>. 180mmol, <NUM>% overall yield from Compound (<NUM>)) and Compound (<NUM>) (RR isomer; <NUM>, <NUM>. 149mmol, <NUM>% overall yield from Compound (<NUM>)).

Compound (<NUM>) (SpRp) <NUM>H NMR (<NUM>, CDCl<NUM>) δ(ppm): <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (ddd, J = <NUM>, <NUM>, <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>). Compound (<NUM>) (RpRp) <NUM>H NMR (<NUM>, CDCl<NUM>) δ(ppm): <NUM> (s, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>).

To a heated (<NUM>) solution of Compound (<NUM>) (<NUM>, <NUM>. 414mmol, 1eq. ) in toluene (<NUM>) was added Hoveyda-Grubbs Catalyst™ 2nd generation ((<NUM>,<NUM>-Bis-(<NUM>,<NUM>,<NUM>-trimethylphenyl)-<NUM>-imidazolidinylidene)dichloro (o-isopropoxyphenylmethylene)ruthenium; available at SIGMA-ALDRITCH (registered trademark) Catalog No. <NUM>; <NPL>; <NUM>, <NUM>. 15mmol, <NUM>. ) and quinone (<NUM>, <NUM>. 243mmol, <NUM>. The mixture was heated to reflux and reaction progress was monitored by LC/MS. After <NUM> hours an additional catalyst was added (<NUM>, <NUM>. 15mmol, <NUM>. ) and the reaction was continued for additional <NUM> hours. After cooling down, the mixture was treated with DMSO (<NUM>, <NUM>. 3mmol, 20eq. ) at ambient temperature for <NUM> hours, concentrated in vacuo and purified by silica gel column chromatography (SiO<NUM> <NUM>, <NUM>% ethyl acetate in n-heptane to <NUM> % ethyl acetate) to give Compound (<NUM>) (<NUM>, <NUM>. 163mmol, <NUM>% yield) as a brown dry foam.

<NUM>H NMR (<NUM>, CDCl<NUM>) δ (ppm): <NUM> (s, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (br d, J = <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (dt, J = <NUM>, <NUM>, <NUM>), <NUM> (dt, J = <NUM>, <NUM>, <NUM>), <NUM> (dd, J = <NUM>, <NUM>. <NUM>), <NUM> (dd, J = <NUM>, <NUM>. <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>).

To a solution of Compound (<NUM>) (<NUM>, <NUM>. 072mmol, 1eq. ) in <NUM>,<NUM>-dioxane (<NUM>) was added thiophenol (<NUM>, <NUM>. 55mmol, 119eq. ) and triethylamine (<NUM>, <NUM>. 31mmol, 88eq. The resulting mixture was stirred at ambient temperature. Upon complete reaction (monitored by LC/MS, <NUM> hours), methanol (<NUM>) and <NUM>% ammonium hydroxide (<NUM>) were added and resultant mixture was heated to <NUM>. Upon complete reaction (monitored by LC/MS, <NUM> hours), the mixture was cooled to ambient temperature and the resultant brownish slurry was filtered and rinsed with water (<NUM>). The filtrate was filtered again to remove additional solids. The final filtrate was extracted twice with a <NUM>:<NUM> mixture of toluene and n-heptane (<NUM>). The aqueous layer was concentrated in vacuo and then re-suspended in water (<NUM>). The resulting solid was filtered off and the filtrate was subjected to preparative HPLC to give di-ammonium salt of Compound (I) (also referred to as Compound (1a)) (<NUM>, <NUM>. 050mmol, <NUM>% yield) as a white solid.

Compound (<NUM>) (<NUM>, <NUM>. 53mol, 1wt, 1vol, 1eq. ) was dissolved in pyridine (<NUM>, <NUM>. 2mol, <NUM>. 89wt, <NUM>. 0vols, 23eq. The mixture was cooled to <NUM> and treated with <NUM>,<NUM>'-dimethoxytrityl chloride (DMTCl; <NUM>, <NUM>. 60mol, <NUM>. 953wt, <NUM>. The mixture was stirred at <NUM> to <NUM> for <NUM> and then allowed to warm to ambient temperature. The reaction was monitored by LC/MS and complete conversion was confirmed after overnight stirring. The reaction mixture was cooled to below <NUM> and quenched by treatment with MeOH (<NUM>, <NUM>. 05mol, <NUM>. 172wt, <NUM>. 217vol, <NUM>. ) for <NUM> minutes. The mixture was co-evaporated with toluene (<NUM>, <NUM>. 04wt, <NUM>. 51vol) under vacuum and then diluted with a mixture of EtOAc (<NUM>, <NUM>. 5wt, <NUM>. 0vol) and n-heptane (<NUM>, <NUM>. 42wt, <NUM>. The organic layer was washed with saturated NaHCO<NUM> (9wt% solution in water; <NUM>, <NUM>. An additional EtOAc (<NUM>, <NUM>. 5wt, <NUM>. 0vol) was added to completely dissolve the crude product. After stirred for <NUM> minutes, the two layers were separated. The organic layer was washed with water (<NUM>, <NUM>. 5wt, <NUM>. Solid began slowly precipitating out of the organic layer. The water layer was separated. The organic layer was then concentrated to approx. The crude product was slurried with a mixture of n-heptane (<NUM>, <NUM>. 40wt, <NUM>. 51vol) and toluene (<NUM>, <NUM>. 76wt, <NUM>. After stirring for <NUM> minutes, the pale yellow solid was collected by vacuum filtration. The filter cake was sequentially rinsed with: (<NUM>) a mixture of n-heptane (<NUM>, <NUM>. 72wt, <NUM>. 05vol) and toluene (<NUM>, <NUM>. 46wt, <NUM>. 53vol), and then (<NUM>) n-heptane (<NUM>, <NUM>. 6wt, <NUM>. The solid was dried with no heat for <NUM> minutes and then transferred to trays for drying at <NUM> in a vacuum oven overnight to give Compound (<NUM>) as pale yelllow solid (<NUM>, <NUM>. 47mol, <NUM>. 75wt, <NUM>% yield).

<NUM>H NMR (<NUM>, CHLOROFORM-d) δ(ppm): <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (ddd, J = <NUM>, <NUM>, <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (br s, <NUM>).

Compound (<NUM>) (<NUM>, <NUM>. 15mol, 1wt, 1vol, 1eq. ) and imidazole (<NUM>, <NUM>. 73mol, <NUM>. 274wt, <NUM>. ) were dissolved in DMF (<NUM>, <NUM>. 78wt, <NUM>. 0vol) and the resultant mixture was cooled to <NUM>. TBS-Cl (<NUM>, <NUM>. 27mol, <NUM>. 444wt, <NUM>. ) was added. The mixture was stirred at <NUM> to <NUM> for <NUM>, allowed to slowly warm to ambient temperature (progress monitored by LCMS). The reaction was complete <NUM> after TBS-Cl addition, yet allowed to stir at ambient temperature for an additional <NUM>. The mixture was cooled to <NUM> and treated with methanol (<NUM>, <NUM>, <NUM>. 3mol, <NUM>. 17wt, <NUM>. 22wt, <NUM>. ) for <NUM> minutes. The reaction mixture was diluted with a mixture of MTBE (<NUM>, <NUM>, <NUM>. 96wt, <NUM>. 0vol) and EtOAc (<NUM>, <NUM>, <NUM>. 60wt, <NUM>. 0vol) followed by saturated NH<NUM>Cl (28wt% solution in water; <NUM>, <NUM>. Solids began slowly falling out of solution. The mixture was allowed to warm to <NUM> and water (<NUM>, <NUM>, <NUM>. 5wt, <NUM>. 5vol) was added to the (T-internal = <NUM>). More solids began precipitating out of the mixture. An additional water (<NUM>, <NUM>, <NUM>. 5wt, <NUM>. 5vol) and MTBE (<NUM>, <NUM>, <NUM>. 4wt, <NUM>. 3vol) were added to the mixture. The off-white solid was collected by vacuum filtration. The reactor was rinsed with water (<NUM>, <NUM>. 74vol) and then MTBE (<NUM>, <NUM>, <NUM>. 10wt, <NUM>. 19vol) to transfer any remaining solid to the filter. The filter cake was rinsed sequentially with: (<NUM>) water (<NUM>, <NUM>, <NUM>. 2wt, <NUM>. 2vol), (<NUM>) water (<NUM>, <NUM>, <NUM>. 2wt, <NUM>. 2vol), (<NUM>) a mixture of MTBE (<NUM>, <NUM>, <NUM>. 5wt, <NUM>. 1vol) and n-heptane (<NUM>, <NUM>, <NUM>. 4wt, <NUM>. 1vol), (<NUM>) a mixture of MTBE (<NUM>, <NUM>, <NUM>. 5wt, <NUM>. 1vol) and n-heptane (<NUM>, <NUM>, <NUM>. 4wt, <NUM>. The recovered solid was dried under vacuum at <NUM> over <NUM> days to give Compound (<NUM>) as white solid (<NUM>, <NUM>. 991mol, <NUM>. 12wt, <NUM>% yield).

<NUM>H NMR (<NUM>, CHLOROFORM-d) δ (ppm): <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (ddd, J = <NUM>, <NUM>, <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>).

Compound (<NUM>) (<NUM>, <NUM>. 47mol, 1wt, 1vol, 1eq. ) and imidazole (<NUM>, <NUM>. 20mol, <NUM>. 151wt, <NUM>. ) were dissolved in DMF (<NUM>, <NUM>, <NUM>. 3wt, <NUM>. 5vol) and the mixture was cooled to <NUM>. TBS-Cl (<NUM>, <NUM>. 62mol, <NUM>. 245wt, <NUM>. ) was added. The reaction was stirred at <NUM> to <NUM> for <NUM>, allowed to slowly warm to ambient temperature and monitored by LCMS. After <NUM>, an additional imidazole (<NUM>, <NUM>. 47mol, <NUM>. 10wt, <NUM>. ) and TBS-Cl (<NUM>, 735mmol, <NUM>. 112wt, <NUM>. ) were added and stirring was continued at ambient temperature for <NUM> and at <NUM> for <NUM>. The resulting mixture was cooled to <NUM> and treated with MeOH (<NUM>, <NUM>. 94mol, 2eq. ) for <NUM> minutes. In a separate reactor was added ice (<NUM>, 5wt) and saturated NH<NUM>Cl (28wt% solution in water; <NUM>, 5vol). The reaction mixture was added to the ice/NH<NUM>Cl mixture. An off white solid began precipitating out of solution immediately. An additional <NUM> of ice (<NUM>, 2wt) and water (<NUM>, 3vol) were added to the mixture. The reaction flask was rinsed with water (<NUM>, <NUM>. 5vol) and the rinsate was added to the mixture. n-Heptane (<NUM>, 2vol) was added to the mixture and stirring was continued for <NUM> minutes. The off white solid was collected by vacuum filtration. The filter cake was rinsed with: (<NUM>) water (<NUM>, <NUM>. 0vol), (<NUM>) water (<NUM>, <NUM>. 0vol), (<NUM>) n-heptane (<NUM>, <NUM>. 0vol), (<NUM>) n-heptane (<NUM>, <NUM>. The recovered solid was dried under vacuum at <NUM> for <NUM> days to give Compound (<NUM>) as off-white solid (<NUM>, <NUM>. 39mol, <NUM>. 10wt, <NUM>% yield). <NUM>H NMR (<NUM>, CHLOROFORM-d) δ (ppm): <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (ddd, J = <NUM>, <NUM>, <NUM>, <NUM>), <NUM> (ddd, J = <NUM>. <NUM>, <NUM>, <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>).

Compound (<NUM>) (<NUM>, <NUM>. 27mol, 1wt, 1vol, 1eq. ) and trans-<NUM>-butene-<NUM>,<NUM>-diol (olefin geometry confirmed by <NUM>H-NMR; <NUM>, <NUM>. 335wt, <NUM>. ) were azeotroped twice with THF (<NUM>, <NUM>. The residue was dissolved in a mixture of THF (<NUM>, 10vol) and toluene (<NUM>, 15vol). Triphenylphosphine (<NUM>, <NUM>. 65mol, <NUM>. 432wt, <NUM>. ) was added and then the reaction mixture was cooled to -<NUM>. DIAD (<NUM>, <NUM>. 65mol, <NUM>, <NUM>. 333wt, <NUM>. 320vol, <NUM>. ) was added slowly over <NUM> minutes while keeping T-internal below <NUM>. The reaction was stirred at <NUM> - <NUM> for <NUM> and monitored by LCMS. The ice bath was removed and the mixture was allowed to warm up to rt. After overnight stirring (<NUM>), triphenylphosphine (<NUM>, <NUM>. 32mol, <NUM>. 083wt, <NUM>. 25eq) and DIAD (<NUM>, <NUM>. 32mol, <NUM>, <NUM>. 064wt, <NUM>. 062vol, <NUM>. ) were added. After additional <NUM> at rt, the reaction mixture was diluted with MTBE (<NUM>, 10vol), washed twice with half-saturated NaCl (18wt% solution in water; <NUM> x <NUM>) and concentrated in vacuo to a thick oil. The mixture was re-dissolved in a mixture of MTBE (<NUM>, 4vol) and n-heptane (<NUM>, <NUM>. 5vol) and then cooled to <NUM>. A seed crystal of triphenylphosphine oxide was added to the solution. Solids slowly began precipitating out of solution and was stirred overnight. The white solid was collected by vacuum filtration and rinsed with MTBE (<NUM>, 2vol) to isolate <NUM> of triphenylphosphine oxide. The filtrate was concentrated and purified via Biotage <NUM> KP-Sil (SiO<NUM> <NUM>; preteated with <NUM>% TEA in heptane/ EtOAc; eluents: n-heptane/EtOAc (<NUM> of <NUM>% EtOAc with <NUM>% TEA, <NUM> of <NUM>% EtOAc with <NUM>% TEA, <NUM> of <NUM>% EtOAc with <NUM>% TEA) → <NUM>% EtOAc with <NUM>% TEA). The column was monitored by TLC (<NUM>:<NUM> EtOAc/n-heptane). The clean product fractions were combined and concentrated under vacuum to give Compound (<NUM>) as pale white foam solid (<NUM>, contained <NUM> wt% DIAD derived co-product, net <NUM>, <NUM>. 63mol, <NUM>% adjusted yield). The mixture fractions were combined and concentrated under vacuum to give pale yellow foam solid (<NUM>), which was subjected to repurification via Biotage <NUM> HP-Sphere (<NUM> SiO<NUM>; pretreated with <NUM>% TEA in n-heptane/EtOAc; loaded sample with toluene eluents: n-heptane/EtOAc/<NUM>%TEA (<NUM> of <NUM>% EtOAc with <NUM>% TEA,<NUM> <NUM>% EtOAc with <NUM>% TEA) → EtOAc with <NUM>% TEA). The column was monitored by TLC (<NUM>/<NUM>/<NUM> EtOAc/n-heptane/ TEA). The clean product fractions were combined and concentrated under vacuum to give additional Compound (<NUM>) as pale white foam solid (<NUM>, <NUM>. 24mol, <NUM>% yield).

<NUM>H NMR (<NUM>, CHLOROFORM-d) δ (ppm): <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (td, J = <NUM>, <NUM>, <NUM>), <NUM> (td, J = <NUM>, <NUM>, <NUM>), <NUM> (ddd, J = <NUM>, <NUM>, <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (ddd, J = <NUM>, <NUM>, <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>).

Compound (<NUM>) (<NUM>, <NUM>. 930mol, 1wt, 1vol, 1eq. ) and Compound (<NUM>) (<NUM>, <NUM>. 07mol, <NUM>. 652wt, <NUM>. ) were azeotropically dried with THF (<NUM> x <NUM>, <NUM> x <NUM>. 8vol) and re-dissolved in THF (<NUM>, <NUM>, <NUM>. 0vol) at rt. Triphenylphosphine (<NUM>, <NUM>. 21mol, <NUM>. 396wt, <NUM>. ) was added and the mixture was cooled below -<NUM>. DIAD (<NUM>, <NUM>. 16mol, <NUM>, <NUM>. 294wt, <NUM>. 283vol, <NUM>. ) was added T-internal below <NUM>. The reaction was allowed to warm to rt slowly. The reaction was monitored by LCMS. After <NUM>, the reaction mixture was concentrated in vacuo to a thick oil, azeotroped with n-heptane (<NUM>, <NUM>, <NUM>. 71wt, <NUM>. 50vol) and then re-dissolved in a mixture of MTBE (<NUM>, <NUM>, <NUM>. 2wt, <NUM>. 0vol) and n-heptane (<NUM>, <NUM>, <NUM>. 68wt, <NUM>. The solution was seeded with triphenylphosphine oxide and cooled to <NUM>, diluted with n-heptane (<NUM>, <NUM>, <NUM>. 34wt, <NUM>. 50vol) and stirred at <NUM> for <NUM> minutes. The white solid precipitate was collected by vacuum filtration and rinsed with <NUM>:<NUM> (v/v) mixture of MTBE and n-heptane (<NUM>) to give triphenylphosphine oxide (<NUM>). The filtrate was concentrated under vacuum and purified via Biotage <NUM> KP-Sil (SiO2 <NUM>; pretreated with <NUM>% TEA; loaded sample by dissolving in toluene eluents: <NUM>:<NUM> n-heptane/EtOAc (<NUM>) and <NUM> TEA, <NUM>:<NUM> (<NUM>), <NUM>:<NUM> (<NUM>) and <NUM>% TEA, <NUM>:<NUM> (<NUM>) and <NUM>% TEA, and <NUM>% EtOAc (<NUM>) and <NUM>% TEA). The combined clean product fractions were concentrated under vacuum to give Compound (<NUM>) as off white solid foam (<NUM>). The mixture fractions were combined and concentrated under vacuum (<NUM>). A white insoluble solid formed by dilution with toluene (<NUM>) prior to loading on Biotage <NUM> was removed by vacuum filtration. The material soluble in toluene was purified via Biotage <NUM> HP-Sphere (SiO<NUM> <NUM> (pretreated with <NUM>% TEA); sample loading with toluene; eluents: <NUM>:<NUM> n-heptane/ EtOAc (<NUM>) w/ <NUM>% TEA, <NUM>:<NUM> (<NUM>) w/ <NUM>% TEA, <NUM>:<NUM> (<NUM>) w/ <NUM>% TEA). The column was monitored by TLC (<NUM>:<NUM> n-heptane/ EtOAc). The combined clean product fractions were concentrated under vacuum to give additional Compound (<NUM>) as off white solid foam (<NUM>. Total <NUM>+<NUM>,<NUM> = <NUM>, 930mmol, <NUM>. 03wt, <NUM>% yield).

<NUM>H NMR (<NUM>, CHLOROFORM-d) δ (ppm): <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (ddd, J = <NUM>, <NUM>, <NUM>, <NUM>), <NUM> (ddd, J = <NUM>, <NUM>, <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>).

To a solution of Compound (<NUM>) (<NUM>, 309mmol, 1wt, 1vol, 1eq. ) in pyridine (<NUM>, <NUM>, <NUM>. 2mol, <NUM>. 9wt, <NUM>. 0vol, 49eq. ) was added diphenyl phosphite (<NUM>, <NUM>, <NUM>. 46mol, <NUM>. 26wt, <NUM>. 22vol, <NUM>. The reaction was stirred at rt and was monitored by LCMS. After <NUM> (<NUM>% conversion) an additional diphenyl phosphite (<NUM>, <NUM>, 155mmol, <NUM>. 088wt, <NUM>. 073vol, <NUM>. ) was added. After an additional <NUM> an extra diphenyl phosphite (<NUM>, <NUM>, 31mmol, <NUM>. 018wt, <NUM>. 015vol, <NUM>. ) was added and the reaction was continued for an additional <NUM> (<NUM>% conversion). The reaction mixture was added to a mixture of saturated NaHCO<NUM> (9wt% solution in water; <NUM>, 5vol) and water (<NUM>, <NUM>. 5vol) while keeping T-internal <NUM> to <NUM>. The reactor was rinsed with a small volume of EtOAc. Stirring was continued at rt for <NUM> minutes and monitored the reaction by LCMS (<NUM>% conversion). The reaction mixture was extracted twice with <NUM>:<NUM> mixture of EtOAc and MTBE (<NUM> x <NUM>, <NUM> x <NUM> vol). The combined organic layers were washed with water (<NUM>, 10vol), concentrated in vacuo and azetroped with toluene (<NUM> x <NUM>, <NUM> x 10vol; continuous feeding) for removal of pyridine to give Compound (<NUM>) (<NUM>. pyridine remained).

The crude Compound (<NUM>) was dissolved in dichloromethane (<NUM>, <NUM>, <NUM>. 9wt, <NUM>. 5vol) at ambient temperature. Water (<NUM>, <NUM>. 136vol, 10eq. ) was added followed by a solution of dichloroacetic acid (<NUM>, <NUM>, <NUM>. 93mol, <NUM>. 29wt, <NUM>. 19vol, <NUM>. ) in DCM (<NUM>, <NUM>. 5vol) while keeping the internal T below <NUM>. (Turned into an orange solution). After <NUM>, triethylsilane (Et<NUM>SiH; <NUM>, <NUM>, <NUM>. 09mol, <NUM>. 875wt, <NUM>. 20vol, <NUM>. ) (T-internal went from <NUM> to <NUM>) was added and stirring was continued for <NUM>. Triethylamine (<NUM>, <NUM>, <NUM>. 09mol, <NUM>. 762wt, <NUM>. 05vol, <NUM>. ) was added (T-internal went from <NUM> to <NUM>). The mixture was concentrated to <NUM> (<NUM>. 8wt), redissolved in EtOAc (<NUM>, <NUM>, 14wt, 15vol), sequentially washed with: (<NUM>) water (<NUM>, <NUM>. 5vol) and saturated NaHCO<NUM> (9wt% solution in water, <NUM>, <NUM>. The crude product EtOAc solution was stored at - <NUM> overnight. ; <NUM>, <NUM>. 0vol) and in next day, the solution was concentrated in vacuo at <NUM>. The crude mixture thus obtained (<NUM>) was triturated with: (<NUM>) n-heptane (<NUM>, <NUM>. 5vol), (<NUM>) a mixture of n-heptane (<NUM>, <NUM>. 0vol) and toluene (<NUM>, <NUM>. The solution part (supernatant) was decanted off and the solid remained at the bottom was dissolved in acetonitrile (<NUM>, 10vol). The mixture was concentrated in vacuo at <NUM> and azeotroped with acetonitrile twice to give Compound (<NUM>). The product was used for the subsequent stage without purification (theoretical <NUM>% yield assumed).

Compound (<NUM>) (<NUM>, 309mmol, 1wt, 1vol, 1eq. ) was dissolved in anhydrous pyridine (<NUM>, <NUM>, 39wt, 40vol) at rt. Triethylamine (<NUM>, 927mmol, <NUM>, <NUM>. 28wt, <NUM>. 38vol, <NUM>. ) was added followed by <NUM>-chloro-<NUM>,<NUM>-dimethyl-<NUM>,<NUM>,<NUM>-dioxaphosphinane <NUM>-oxide (DMOCP; <NUM>, 556mmol, <NUM>. 31wt, <NUM>. The resultant mixture was stirred at ambient temperature for <NUM> minutes and monitored by LCMS (<NUM>% conversion) to generate Compound (<NUM>).

TEA (<NUM>, 927mmol, <NUM>, <NUM>. 28wt, <NUM>. 38vol, <NUM>. ), water (<NUM>, <NUM>. 56mol, <NUM>. 30wt, <NUM>. 30wt, 18eq) and sulfur (<NUM>, <NUM>. 08mol, <NUM>. 10wt, <NUM>. 5eq) were added to the above mixture of Compound (<NUM>). After <NUM> minutes (<NUM>% conversion), NaHCO<NUM> (9wt% solution in water; <NUM>, 10vol) was added while keeping T-internal below <NUM> (<NUM> to <NUM>). The resultant mixture was filtered for removal of salts. The filtrate was concentrated the mixture in vacuo, diluted with MTBE (<NUM>, 15vol), and wash twice with NaCl (30wt% solution in water; <NUM> x <NUM>, <NUM> x 4vol). Insoluble solids were filtered off and the filtrate was concentrated in vacuo and azeotroped with toluene (<NUM>, 12vol). The resulting solid was removed by filtration and the crude mixture was dissolved in toluene and purified via Biotage <NUM> KP-Sil (SiO<NUM> <NUM>; pretreated with n-heptane/EtOAc/TEA (<NUM>/<NUM>/<NUM> CV); eluted with: EtOAc/TEA (<NUM>/<NUM> CV), EtOAc/MeOH/TEA (<NUM>/<NUM>/<NUM> CV), EtOAC/MeOH/TEA (<NUM>/<NUM>/<NUM>. The column was monitored by TLC (EtOAC/MeOH/TEA=<NUM>/<NUM>/<NUM>). Fractions containing the Sp isomer were combined and concentrated under vacuum to give Compound (<NUM>) as light pink foam solid (Sp isomer; <NUM>, 128mmol, <NUM> wt, <NUM>% yield). Fractions containing the Rp isomer were combined and concentrated under vacuum to give Compound (<NUM>) as light pink foam solid (Rp isomer; <NUM>, 53mmol, <NUM>. 19wt, <NUM>% yield).

<NUM>H NMR (<NUM>, CHLOROFORM-d) δ (ppm): <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (t, J = <NUM>, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (td, J = <NUM>, <NUM>, <NUM>), <NUM> (td, J = <NUM>, <NUM>, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (t, J = <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>).

<NUM>H NMR (<NUM>, CHLOROFORM-d) δ(ppm): <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (ddd, J = <NUM>, <NUM>, <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (t, J = <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>).

Compound (<NUM>) (<NUM>, 183mmol, 1wt, 1vol, 1eq) was dissolved in a mixture of pyridine (<NUM>, <NUM>. 56mol, <NUM>, <NUM>. 3wt, <NUM>. 4vol) and TEA (<NUM>, <NUM>. 0mol, <NUM>, <NUM>. 7wt, 12vol, 104eq. Triethylamine trihydrofluoride (<NUM>, <NUM>. 62mol, <NUM>, <NUM>. 2wt, <NUM>. 2vol, <NUM>. as complex, 27eq. HF) was added and the mixture was stirred at rt while the conversion was monitored by LCMS. After <NUM> (<NUM>% conversion), methoxytrimethylsilane (TMSOMe; <NUM>, <NUM>. 2mol, <NUM>, <NUM>. 8wt, <NUM>. 3vol, 55eq. ) was added and stirring was continued for <NUM> minutes. A sticky solid coated the reactor. The solution part (supernatant) was decanted off. The solid was triturated twice with toluene (<NUM> × <NUM>, <NUM> × 10vol; supernatant decanted off). The crude solid remained in the reactor was dissolved in dichloromethane (<NUM>, 10vol) and washed with NH<NUM>Cl (28wt% solution in water; <NUM>, 10vol). The aqueous layer was back-extracted with dichloromethane (<NUM>, 10vol). The combined organic layers were washed with a mixture of NaCl (36wt% solution in water; <NUM>, 5vol) and water (<NUM>, 5vol), and then concentrated under vacuum to give Compound (<NUM>) as tan dry foam (<NUM>, 155mmol, <NUM>. 70wt, <NUM>% yield). The crude product was taken onto the next step without purification.

Compound (<NUM>) (<NUM>, 153mmol, 1wt, 1vol, 1eq. ) was azeotroped with acetonitrile (<NUM>, 27vol) and then re-dissolved in acetonitrile (<NUM>, <NUM>, <NUM>. 5wt, <NUM>. 0vol) at rt. <NUM>-Nitrobenzyl bromide (<NUM>, 205mmol, <NUM>. 30wt, <NUM>. 34eq) was added at rt and the reaction was monitored by LCMS. After <NUM> (<NUM>% conversion), EtOAc (<NUM>, 10vol), NH<NUM>Cl (28wt% solution in water; <NUM>, 2vol) and water (<NUM>, 2vol) were added (pH = <NUM>) and the resultant mixture was partially concentrated under vacuum at <NUM> to a weight of <NUM>. EtOAc (<NUM>, 15vol) was added and the mixture was stirred for <NUM> minutes. The two layers were separated. The aqueous layer was extracted with ethyl acetate (<NUM>, 5vol). The combined organic layers were sequentially washed with: (<NUM>) a mixture of NaCl (36wt% solution in water; <NUM>, 2vol) and water (<NUM>, 2vol) and (<NUM>) water (<NUM>, 4vol). The organic layer was then concentrated under vacuum and azeotroped with n-heptane (<NUM>, 10vol). MTBE (<NUM>, <NUM>. 3vol) was added to the crude solid and the mixture was heated at <NUM>. The mixture was diluted with EtOAc (<NUM>, 2vol) and slowly cooled to <NUM>. The dense solid was allowed to settle and the supernatant was pumped off through a filter frit tube. The solid was rinsed twice with MTBE (<NUM> × <NUM>, <NUM> × 2vol; supernatant pumped off through the filter frit tube each time) and dried under vacuum at <NUM> overnight to give Compound (<NUM>) as pale yellow solid (<NUM>). The filtrate was concentrated under vacuum yielding a brown oil (<NUM>), which was subjected to purification via Biotage Snap-Ultra <NUM> (eluents: <NUM> to <NUM>% MeOH in EtOAc) to give additional Compound (<NUM>) as pale yellow solid (<NUM>). Total <NUM> + <NUM> = <NUM> (net 152mmol, <NUM>% pure, <NUM>% yield).

<NUM>H NMR (<NUM>, CHLOROFORM-d) δ (ppm); <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>).

Compound (<NUM>) (<NUM>% pure, net <NUM>, <NUM>. 3mmol, 1wt, 1vol, 1eq. ) and <NUM>-cyanoethyl N, N, N', N' - tetraisopropylphosphorodiamidite (<NUM>, <NUM>. 5mmol, <NUM>. 33wt, <NUM>. 35vol, <NUM>. ) were azeotroped with anhydrous acetonitrile three times (<NUM> × <NUM>), re-dissolved in dichloromethane (<NUM>, 10vol) and cooled to <NUM>-<NUM>. Diisopropylammonium tetrazolide (<NUM>, <NUM>. 1mmol, <NUM>. 085wt, <NUM>. ) was added. The resulting reaction mixture was stirred at <NUM> for <NUM>, warmed to <NUM> over <NUM>, held at <NUM> for <NUM> and warmed up to rt over <NUM>. The reaction was monitored by LCMS and TLC (EtOAc with <NUM>% TEA). After <NUM>, anhydrous acetonitrile (<NUM>, 10vol) was added and the mixture was stored at -<NUM> over <NUM> days.

The mixture from Stage <NUM> was warmed to ambient temperature and added via a dropping funnel in portions (<NUM> every <NUM> minutes, over <NUM>) into a mixture of pyridine trifluoroacetate salt (azetroped in advance with pyridine twice; <NUM>, 217mmol, <NUM>. 57wt, <NUM>. ) and acetonitrile (<NUM>, 80vol). The reaction was monitored by LCMS. After <NUM>, a solution of <NUM>-cyanoethyl N, N, N', N' - tetraisopropylphosphorodiamidite (<NUM>, 18mmol, <NUM>. ) in acetonitrile (<NUM>) was added over <NUM>. Amount of the additional reagent was determined based on the remaining Compound (<NUM>) (~<NUM>% based on LCMS). More conversion of the diol was observed after <NUM>.

((Dimethylaminomethylidene)amino)-<NUM>-<NUM>,<NUM>,<NUM>-dithiazoline-<NUM>-thione (DDTT; <NUM>, 101mmol, <NUM>. 28wt, <NUM>. ) was added and stirring was continued for <NUM>. The reaction mixture was partially concentrated to ~<NUM> and diluted with MTBE (<NUM>, 20vol), NaHCO<NUM> (9wt% solution in water; <NUM>, 15vol) and water (<NUM>, 5vol). pH = <NUM>. The layers were separated and the aqueous layer was extracted with a mixture of MTBE (<NUM>, 20vol) and EtOAc (<NUM>, 15vol). The combined organic layers were washed twice with <NUM>% aq. NaCl (<NUM> x <NUM>, <NUM> x 10vol), concentrated under vacuum at <NUM> and azeotroped with toluene (<NUM>, 20vol). LCMS and TLC (EtOAc) indicated Compound (<NUM>) (SpRp, desired): Compound (<NUM>) (SpSp) = <NUM>: <NUM>.

The crude product was purified via Biotage <NUM> KP-Sil, (SiO<NUM> <NUM>; eluents: EtOAc/ n-heptane: <NUM>:<NUM> (<NUM> CV), <NUM>:<NUM> (<NUM> CV), <NUM>:<NUM> (<NUM> CV), <NUM>% EA (<NUM> CV), <NUM> - <NUM>% MeOH in EA <NUM> CV) to give Compound (<NUM>) (<NUM>, <NUM>. 5mmol, <NUM>% yield).

Compound (<NUM>) (SpRp): <NUM>H NMR (<NUM>, CHLOROFORM-d) δ(ppm): <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (td, J = <NUM>, <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (td, J = <NUM>, <NUM>, <NUM>), <NUM> (td, J = <NUM>, <NUM>, <NUM>).

Compound (<NUM>) (SpSp) <NUM>H NMR (<NUM>, CHLOROFORM-d) δ (ppm): <NUM> (s, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (t, J = <NUM>, <NUM>).

Compound (<NUM>) (<NUM>, <NUM>. 6mmol, 1wt, 1vol, 1eq. ) was dissolved in <NUM>,<NUM>-dioxane (<NUM>, 6vol). Thiophenol (<NUM>, <NUM>. 09mol, <NUM>, <NUM>. 2wt, 3vol, >30eq. ) was added followed by triethylamine (<NUM>, <NUM>. 54mol, <NUM>, <NUM>. 2wt, 3vol). Some exotherm was observed (T-internal increased by ~<NUM>), therefore, water/ice bath was used to cool and control T-internal below <NUM>. The reaction was monitored by LCMS. After <NUM>, MeOH (<NUM>, 8vol) and NH<NUM>OH (28wt%; 15mol, <NUM>, 8vol, >200eq. ) were added. The resulting mixture was heated at <NUM> for <NUM>, cooled to rt and stirred overnight. After <NUM>, water (<NUM>, 10vol) was added (no solid observed) and the mixture was extracted three times with <NUM>:<NUM> (v/v) mixture of n-heptane and toluene (<NUM> x <NUM>, <NUM> x 12vol), followed by with toluene (<NUM>, 8vol). The aqueous layer was concentrated in vacuo at <NUM>-<NUM> and diluted with water (<NUM>, 15vol). The resulting slurry was kept overnight at rt. The resulting solid was filtered off, rinsing with water (<NUM>, 5vol). The filtrate was still cloudy and filtered through celite and a Kuno filter. Cloudiness was still present. HCl (<NUM> solution in water; <NUM>, 132mmol, <NUM>. ) was added over <NUM> and pH was checked (pH <<NUM>). Stirring was continued at rt for <NUM> and the mixture was filtered. The filter cake was rinsed with water (<NUM> × <NUM>), dried in a vacuum oven at <NUM> for <NUM> days and with no heat for <NUM> day to give Compound (I) as pale orange solid (<NUM>, <NUM>. 1mmol, <NUM>. 63wt, <NUM>% yield).

To the free acid Compound (I) (<NUM>, <NUM>. 03mmol, 1wt, 1vol, 1eq. ) was added ammonia (<NUM> solution in MeOH; <NUM>, 440mmol, 10vol, 15eq. EtOH (<NUM>, <NUM>. 5vol) was added and the resulting solution was filtered through a Kuno filter (<NUM> micron; PTFE), rising with <NUM>:<NUM> (v/v) mixture of MeOH and EtOH (<NUM>, 4vol). The filtrate was concentrate in vacuo at <NUM> yielding an off white solid, which was dried at rt overnight, grinded with a spatular (easy to break) and dried further in vacuum at rt. The isolated solid was then suspended in toluene (<NUM>) and stirred at rt for <NUM> minutes. The solid was then collected by vacuum filtration and rinsed with toluene twice (<NUM> × <NUM>). The solid was then dried under vacuum in a vacuum oven to give <NUM> of Compound (1a) (Compound (I) di-ammonium salt).

Compound (1a) (Compound (I) di-ammonium salt) (<NUM>, <NUM>. 36mmol, 1wt, 1vol, 1eq. ) was dissolved in a mixture of water (<NUM>, 30vol) and ammonium hydroxide (28wt%; <NUM>, 18mmol, <NUM>. ) (pH = <NUM> - <NUM>) and extracted with toluene three times (<NUM> × <NUM>, <NUM> × 14vol), EtOAc three times (<NUM> × <NUM>, <NUM> × <NUM> vol) and toluene three times (<NUM> × <NUM>, <NUM> × 14vol). The resulting aqueous layer was treated with HCl (<NUM> solution in water; <NUM>, 90mmol, <NUM>. ) over a period of <NUM> hours (pH <NUM> or less). The mixture stirred for <NUM> minutes and then the solid precipitate was collected by vacuum filtration. The filter cake was washed with water three times (<NUM> × <NUM>, <NUM> × 9vol) and dried in vacuo overnight. Ammonia (<NUM> solution in MeOH; <NUM>, 500mmol, <NUM>. ) and ethanol (<NUM>) were added to the solid and the resulting mixture was concentrated in vacuo until crystals appeared (~<NUM>), at which time concentration was stopped and the mixture was stirred for <NUM> minutes. Ethanol (<NUM>) was added and the mixture was partially concentrated (<NUM> removed). The same operation was repeated two more times, and then the mixture was cooled to <NUM> and stirred for <NUM>. The white solid was collected by vacuum filtration and washed with cold ethanol (<NUM>) followed by ethyl acetate (<NUM> × <NUM>). The white solid was dried under vacuum at rt for <NUM> days to give Compound (1a) (Compound (I) di-ammonium salt) as white solid (<NUM>, <NUM>. 3mmol, <NUM>. 75wt, <NUM>% yield). The filtrate was concentrated under vacuum and dried under vacuum at rt for <NUM> days to give Compound (1a) (Compound (I) di-ammonium salt) as off white solid (<NUM>, <NUM>. 3mmol, <NUM>% yield).

An <NUM>H NMR spectrograph of Compound (I) ammonium salt is shown in <FIG>. The resulting spectrum was:.

About <NUM> of Compound (I) ammonium salt was dissolved in 600µL of water. 120µL of this solution was put in another glass vial and then this vial was stored in fixed container with <NUM> of MeCN at room temperature for <NUM> week. This is the H<NUM> O/ MeCN vapor diffusion method of sample preparation.

A colorless block single crystal (<NUM> × <NUM> × <NUM>) found in crystallization solution was dispersed in liquid Parabar <NUM> and was mounted on a Dual - Thickness MicroMounts™ (MiTeGen). Diffraction data was collected at -<NUM> on XtaLAB PRO P200 MM007HF (Rigaku) with ω axis oscillation method using multi-layer mirror monochromated Cu-Kα radiation.

<FIG> shows an ORTEP figure of a crystal of Compound (I) ammonium salt, where two molecules are present in an asymmetric unit, along with a number of disordered water molecules. <FIG> shows an ORTEP figure of one of the two molecules in the asymmetric unit from <FIG>. <FIG> shows an ORTEP figure of the other molecule in the asymmetric unit from <FIG>.

The crystal structure of Compound (I) ammonium salt was solved with a final R-factor of <NUM>. The Flack parameter was nearly zero (<NUM>(<NUM>)), indicating that the absolute configuration of Compound (I) ammonium salt is (R, S). The crystal structure analysis also indicated that several water molecules were present in the large channel of Compound (I) ammonium salt, which indicated that water molecules were able to easily slip out from the channel. The analysis also confirmed that the conformations of both crystallographically independent molecules in the asymmetric unit were almost the same.

To a suspension of Compound (I) (<NUM>) in isopropyl acetate (<NUM>) was added a mixture of <NUM>% ammonia aqueous solution (62µL), isopropyl acetate (<NUM>) and <NUM>-propanol (<NUM>), and the resulting slurry was stirred at room temperature overnight. The precipitates were cropped by filtration, rinsed with isopropyl acetate (<NUM>), and the solid obtained was dried under reduced pressure at room temperature for <NUM> hours to give the titled crystal (<NUM>). The titled crystal was identified as a hydrate.

To a suspension of Compound (I) (<NUM>) in isopropyl acetate (<NUM>) was added a mixture of <NUM>% ammonia aqueous solution (<NUM>µL), isopropyl acetate (<NUM>) and <NUM>-propanol (<NUM>), and the resulting slurry was stirred at room temperature overnight. The precipitates were cropped by filtration, rinsed with isopropyl acetate (<NUM>), and the solid obtained was dried under reduced pressure at room temperature for <NUM> hours and successively absorbed moisture by keeping under <NUM>% relative humidity at room temperature overnight to give the titled crystal (<NUM>). The titled crystal was identified as a hydrate.

To Compound (I) (<NUM>) were added <NUM>% ammonia aqueous solution (<NUM>) and ethanol (<NUM>), and the resulting solution was concentrated down to about <NUM> under reduced pressure at <NUM>. To the residue was added ethanol (<NUM>), and the solution was concentrated down to about <NUM> under reduced pressure at <NUM>. To the residue was added ethanol (<NUM>), and the resulting solution was concentrated down to <NUM> under reduced pressure at <NUM>. The residue solution was stirred at room temperature for <NUM> hours and the precipitates fell out of the solution. To the slurry was gradually added isopropyl acetate (<NUM>) dropwise for <NUM> minutes, and the resulting slurry was stirred at room temperature overnight. The precipitates were cropped by filtration and the solid obtained was dried under reduced pressure at room temperature for <NUM> hours to give the titled crystal (<NUM>). The titled crystal was identified as a hydrate.

Approximately <NUM> of crystal (Form <NUM>) of Compound (I) di-ammonium salt was placed in a desiccator inside a <NUM> incubator, where the humidity condition was controlled at <NUM>% RH using a saturated potassium nitrate solution. The solid sample was stored for <NUM> days to give the titled crystal (Approximately <NUM>). The titled crystal was identified as a hydrate.

To a solution of Compound (I) (<NUM>) in 2mol/L ammonia in methanol (<NUM>) was added ethanol (<NUM>), and the solution was concentrated down to about <NUM> under reduced pressure at <NUM>. The residue was stirred at room temperature for <NUM> hours and the precipitates fell out of the solution. To the slurry was added ethanol (<NUM>), and the mixture was concentrated down to about <NUM> under reduced pressure at <NUM>. To the residue was added ethanol (<NUM>), and the mixture was concentrated down to about <NUM> under reduced pressure at <NUM>. To the resulting slurry was added water (40µL), and the mixture was stirred at room temperature for <NUM> hour and with ice-cooling for <NUM> hours. The precipitates were cropped by filtration, rinsed successively with ethanol (<NUM>) and ethyl acetate (<NUM>), and the solid obtained was dried under reduced pressure at room temperature for <NUM> hour to give the titled crystal (<NUM>).

To a suspension of Compound (I) (<NUM>) in <NUM>-propanol (<NUM>) was added <NUM>% ammonia aqueous solution (36µL), and the resulting solution was stirred at <NUM>. To the solution was added <NUM>% ammonia aqueous solution (108µL) and the precipitates fell out of the solution. After the temperature was spontaneously decreased to room temperature, the precipitates were cropped by filtration to give the titled crystal (<NUM>).

To Compound (I) di-ammonium salt obtained in Example <NUM> (<NUM>) was added ethanol (<NUM>), and the resulting slurry was stirred at room temperature overnight. Then, the precipitates were cropped by filtration to give the titled crystal (<NUM>).

To Compound (I) (<NUM>) was added methanol (<NUM>), and the resulting slurry was stirred at room temperature. To the slurry was added 1mol/L aqueous sodium hydroxide solution (<NUM>), and the resulting solution was stirred at <NUM> for around an hour. After the temperature was spontaneously decreased to room temperature, the solution was concentrated down under nitrogen gas flow with stirring. Crystallization was initiated as the solvent was gradually removed. The solid obtained was dried under reduced pressure at room temperature to give the titled crystal (<NUM>).

To a solution of Compound (I) (<NUM>) in a mixture of ethanol (<NUM>), 2mol/L ammonia in ethanol (<NUM>) and water (<NUM>) was added acetic acid (<NUM>) dropwise, and the solution was stirred at room temperature. To the solution was added a seed crystal of compound (I) obtained in a similar way to the following Example <NUM>-<NUM>. Then the mixture was stirred at room temperature for <NUM> minutes and the precipitates fell out of the solution. To the slurry was gradually added acetic acid (<NUM>) dropwise for <NUM> minutes, and the resulting slurry was stirred at room temperature overnight. The precipitates were cropped by filtration, and rinsed successively with cold <NUM>% aqueous ethanol (<NUM>) and tert-butyl methyl ether (<NUM>). The solid obtained was dried under reduced pressure at room temperature for <NUM> hours to give the titled crystal (<NUM>) in <NUM>% yield.

To Compound (I) (<NUM>) were added ethanol (<NUM>) and 2mol/L ammonia in ethanol (<NUM>), and the mixture was stirred at room temperature and dissolved. The resulting solution was filtered through a cotton pad and the pad was rinsed with 2mol/L ammonia in ethanol (<NUM>) and ethanol (<NUM>), and the solution was added water (<NUM>). To the resulting solution was gradually added acetic acid (<NUM>) dropwise at room temperature for <NUM> minutes and the precipitates fell out of the solution, and the slurry was stirred at room temperature for <NUM> minutes. To the slurry was gradually added acetic acid (<NUM>) dropwise for <NUM> minutes, and the resulting slurry was stirred at room temperature overnight. The precipitates were cropped by filtration, and rinsed successively with cold <NUM>% aqueous ethanol (<NUM>) and water (<NUM>). The solid obtained was dried under reduced pressure at room temperature overnight and at <NUM> for <NUM> hours to give the titled crystal (<NUM>) in <NUM>% yield.

Approximately <NUM> of the crystal (Form <NUM>) sample was accurately weighed into an aluminum pan and then the analysis was performed under the following conditions. In the TG thermogram, weight loss due to dehydration was observed in the range from room temperature to <NUM>.

The crystal (Form <NUM>) sample was placed on the sample stage of a powder X-ray diffractometer and the analysis was performed at room temperature and above <NUM>, where significant weight loss due to dehydration was observed in the TG-DTA thermogram of the crystal (Form <NUM>). The measurement conditions are as follows.

In the TG-DTA thermogram of the crystal (Form <NUM>) (<FIG>), significant weight loss due to dehydration was observed in the temperature range up to <NUM>, accompanied with endothermic peaks. When the crystal (Form <NUM>) is heated, a significant change was found in the powder X-ray diffraction (PXRD) pattern above <NUM> (<FIG>). As shown in <FIG>, the changed PXRD pattern was comparable to that of the crystal (Form <NUM>). Then, the PXRD pattern of the crystal (Form <NUM>) was further changed to another pattern at or above <NUM> (<FIG>). These findings indicated that the crystal (Form <NUM>) and the crystal (Form <NUM>) would be hydrates.

In hygroscopicity measurement of the crystal (Form <NUM>) in the RH range from <NUM>% to <NUM>% at <NUM>, a hysteresis loop between adsorption and desorption processes was observed as shown in <FIG>. In the adsorption process, the weight change level was gradually increased to <NUM>% up to <NUM>% RH, and then finally reached approximately <NUM>% at <NUM>% RH. No change was observed in the PXRD patterns before and after the hygroscopicity measurement. In contrast, the PXRD pattern of the crystal (Form <NUM>) sample stored at <NUM> and <NUM>% RH for <NUM> days was different from the initial pattern as described in <FIG>. The crystal form of the changed PXRD pattern was defined as Form <NUM>. These findings prove that the crystal (Form <NUM>) is a hydrate.

The following test examples were carried out to examine the pharmacological effects of the Compound (1a).

THP1-Dual™ Cells (InvivoGen, Cat# thpd-nfis) were applied for EC<NUM> determination. THP1 Dual™ Cells have been characterized to carry the HAQ STING genotype by the vendor Invivogen (Insight <NUM>-<NUM>). Cells were grown and maintained under conditions as recommended by manufacturer. The interferon regulatory factor (IRF) pathway induction described in manufacturer's manual was followed for EC<NUM> determination. In brief, cells were seeded and treated with different concentrations of compound for 20hrs while incubated at <NUM>, <NUM>% CO<NUM>. Cells were resuspended and QUANTI-Luc™ solution (Cat. #: rep-qlc1) was added. Resulting light emission was measured by luminometer (Envision, Perkin Elmer). Obtained signals were plotted and EC<NUM> was calculated with GraphPad Prism7 software. EC<NUM> value is reported in Table <NUM> below.

Human STING has <NUM> major variants, including WT, HAQ, REF, and AQ variants. REF-STING, also referred to as R232H, for example, occurs in about <NUM>% of the human population. Compared to the wild-type allele, R232H has decreased response to bacterial and metazoan cyclic dinucleotides. Details of these <NUM> major variants as well as other rare variants are reported by<NPL>. STING variant specific reporter cell lines were established by using THP1-Dual™ KO-STING cells (InvivoGen, Cat# thpd-kostg) and three STING variant protein expression vectors. The expression vector map for WT STING is shown in <FIG>. For the other two expression vectors, different STING variant sequences were used in that vector, with the WT STING replaced by the appropriate nucleotide sequence.

STING variant-expressing vectors for WT-STING, REF-STING, and AQ-STING were prepared and stably transfected into THP1-Dual™ KO-STING cells to prepare STING variant-specific reporter assays for WT-STING, REF-STING and AQ-STING, respectively. EC<NUM> values were determined as described above in Pharmacological Test Example <NUM> for the HAQ STING agonist activity reporter assay. Results are shown below in Table <NUM>. The DNA sequences used for these STING variants are shown in SEQ ID NO: <NUM> (Nucleotide Sequence of WT Human STING), SEQ ID NO: <NUM> (Nucleotide Sequence of REF Human STING), and SEQ ID NO: <NUM> (Nucleotide Sequence of AQ Human Sting).

RAW-Lucia™ ISG Cells (InvivoGen, Cat# rawl-isg) were used for a mouse STING agonist reporter assay. EC<NUM> values were determined as described above in Pharmacological Test Example <NUM> in the HAQ STING agonist activity reporter assay. Results are shown below in Table <NUM>.

A DSF assay was employed to measure the physical interaction between compound and recombinant STING protein. Truncated recombinant STING protein (a. <NUM>-<NUM>) (SEQ ID NO: <NUM>) was expressed in E. coli and isolated for the assay, as described below. Assay matrix was prepared in <NUM>-well plates to a final volume of 10µL per well consisting of <NUM> recombinant STING protein (a. <NUM>-<NUM>) (SEQ ID NO: <NUM>), <NUM> PBS pH <NUM>, supplemented with <NUM> KCl, 5X SYPRO orange dye and <NUM> compound (final DMSO conc. <NUM>-<NUM>%). Assays were performed on a QuantStudio <NUM> Flex Real-Time PCR System using a temperature gradient from <NUM> to <NUM> at a rate of <NUM>/min, and excitation and emission filters at <NUM> and <NUM>, respectively. According to the fluorescence derivative curves assigned by the Applied Biosystems (registered trademark) Protein Thermal Shift software (algorithm version <NUM>. ), the thermal melt (Tm) of the unbound and ligand bound recombinant STING protein and the difference in thermal melt (dTm D) was calculated.

In general, compounds with ΔTm values larger than <NUM> are considered to have a physical interaction with the tested protein, and the value of ΔTm is positively associated with compound binding affinity. Here, Compound (1a) showed the ΔTm of <NUM> (Table <NUM> shown above), indicating physical interaction with STING protein.

Human blood from <NUM> healthy donors was collected using <NUM> BD Vacutainer Sodium heparin tubes (cat# <NUM>). Peripheral blood mononuclear cell (PBMC) isolation was done using SIGMA ACCUSPIN <NUM> Tubes (cat# A2055) and sigma ACCUSPIN System-HISTOPAQUE-<NUM> (cat# A7054) using protocol provided by manufacturer. PBMC layer was harvested and washed with <NUM>× Phosphate Buffered Saline (PBS) as suggested by Sigma. PBMC were counted and finally suspended at 1x10e6/ml in RPMI (corning cat# <NUM>-<NUM>-CV) supplemented with <NUM>% fetal bovine serum (FBS) (Gibco cat# <NUM>). <NUM> of cell (1x10e6) were transferred into Falcon <NUM> Round Bottom Polypropylene Test Tube (cat# <NUM>) and stimulated with different concentrations (<NUM>, <NUM>, <NUM>, <NUM>) for <NUM> hours in <NUM>% CO<NUM> incubator at <NUM>. After <NUM> hours of incubation the tubes were centrifuged at 1400rpm for <NUM> minutes and supernatants were harvested. Supernatant were stored in -<NUM> for subsequent IFNβ measurement. IFNβ measurement was done using Human IFN-β Base Kit (Meso Scale Diagnostics cat# K151ADA) and protocol provided by manufacturer was used. IFN-beta estimation was done by reading assay plate at MESO SECTOR Imager <NUM> and using MSD Discovery Workbench <NUM> program. After <NUM> hours IFNβ protein was analyzed. The results showed that Compound (1a) can induce primary human PBMC IFNβ protein production in a dose-dependent manner. Results shown in Table <NUM> reflect an average of measurements conducted using five different donors.

For IFNβ mRNA quantification, total RNA was isolated using the RNeasy Mini Kit (Qiagen, Germany) according to the manufacturer's protocol. IFNβ mRNA was quantified by qPCR assay. In brief, total RNA (400ng to 1000ng) was converted to cDNA in a 60µl reaction volume using SuperScript VILO MasterMix (Life Technologies, USA). Obtained cDNAs (<NUM> ng) were subsequently amplified using Applied Biosystems TaqMan expression assays using RNA-specific primers for IFNB <NUM> (Hs01077958_s1), and GAPDH (Hs99999905_m1). A qPCR analysis was performed with TaqMan Fast Advanced Master Mix (Life Technologies, USA) on the Applied Biosystems Quantstudio <NUM> Flex Real-Time PCR System, with an initial <NUM>-min step at <NUM> followed by <NUM> for <NUM> and <NUM> cycles of <NUM> for <NUM> and <NUM> for <NUM>. Relative gene expression was calculated after normalization against the reference gene GAPDH using the <NUM>-ΔΔCT method. Calculations were done using the Applied Biosystems Quantstudio <NUM> Flex software v1. IFNβ mRNA fold changes vs. vehicle treated samples are summarized in Table <NUM>. The results showed that Compound (1a) can induce IFNβ mRNA in primary PBMC in a dose- and time-dependent manner. Table <NUM> shows an average calculated from five different donors.

Compound (1a) was tested for its anti-cancer activity in CT26 dual tumor model, which is a mouse colon cancer model. Female of <NUM>-<NUM> week old Balb/cJ mice (Jackson Labs, Bar Harbor, Maine) were implanted subcutaneously with CT26 tumor cells on both sides of each animal, <NUM><NUM> cells for each side. For study A, treatment was started <NUM> days (<NUM>/kg, <NUM>/kg and <NUM>/kg) after the tumor implantation, when the average tumors reached approximately <NUM><NUM>. For study B, treatment was started <NUM> days (<NUM>/kg, and <NUM>/kg) after the tumor implantation, when the average tumors reached approximately <NUM><NUM>. The treatment scheme is described in Table <NUM> and Table <NUM>.

All the mice in the study have two subcutaneous CT26 tumors. The "treated tumor" indicates the tumor with compound direct administration, while "untreated tumor" indicates the tumor without direct compound administration. Tumor volume was followed throughout the experiment. Tumor volume is measured two times weekly after the start of treatment. Tumor burden is calculated from caliper measurements by the formula for the volume of a prolate ellipsoid (L×W<NUM>)/<NUM> where L and W are the respective orthogonal length and width measurements (mm). Compound (1a) showed potent and curative activity in CT26 dual tumor model (<FIG> and <FIG>). For treated tumors, a cure rate of <NUM>% was detected even at the lowest dose tested in the study (<FIG>, <NUM>/kg dose). At the same time, the highest dose (<NUM>/kg) cured <NUM>% of animals of that tumor at the end of study. For the untreated tumors, a dose-dependent anti-tumor effect was also evident. The top dose group (<NUM>/kg) showed <NUM>% curative effects; all the lower doses also showed tumor growth inhibition activity. Hence, a therapeutic window of <NUM>/kg to <NUM>/kg for Compound (1a) was observed, with anti-tumor activity seen not only locally but also systemically, based on effects at the non-injected distal tumor site. In conclusion, these results indicate that local administration of Compound (1a) can induce both local and systemic (abscopal) anti-cancer activity.

Compound (1a) was tested for its anti-cancer activity in a CT26 liver metastatic model. Anesthetized female <NUM>-<NUM> week-old BALB/cJ mice (Jackson Labs, Bar Harbor, Maine) were implanted intra-splenically with luciferase-expressing CT26 tumor cells (<NUM> × <NUM><NUM> cells per mouse). A subsequent ten minutes waiting period allowed tumor cells to circulate into the animals' livers. Spleens were then removed and animals were sutured and allowed to recover. Three days later, CT26 tumor cells (<NUM><NUM> cells per mouse) were again implanted, this time subcutaneously (sc) under the right forelimb area, to enable development of a tumor mass for compound administration. Nine days after intra-splenic injection, compound (<NUM>/kg) was administered intratumorally, a single time, into the sc tumor.

The local anti-cancer effect of compound was measured through its effect on the sc tumor, while the compound's abscopal effect was assessed by the overall survival of treated mice compared with vehicle-treated control mice, based on the detrimental effect of the growing tumor mass in each mouse liver. Compound (1a) showed both potent activity towards the local sc tumors and also curative systemic activity in <NUM> of <NUM> treated animals (<FIG>). These results indicate that local administration of Compound (1a) can induce both local and systemic (abscopal) anti-cancer activity including deep lesion such as in the liver.

Compound (1a) was tested for its anti-cancer activity in a GL261 brain orthotopic model. GL261 is a murine glioma cell line. Luciferase expressing GL261 mouse glioma cells (<NUM>×<NUM><NUM> cells/mouse) were intra-cranially implanted into female <NUM>-<NUM> week-old B6 albino mice (Jackson Labs, Bar Harbor, Maine). Three to <NUM> days later, GL261 cells were implanted subcutaneously (<NUM><NUM>cells/mouse) under the right forelimb area to allow development of a tumor mass for compound administration. Ten days after intracranial tumor cell implantation, compound (<NUM>/kg) was administered intratumorally, a single time, into the sc tumor.

Claim 1:
A crystal of (1R,3R,15E,28R,29R,30R,31R,34R,36R,<NUM>,41R)-<NUM>,<NUM>-Difluoro-<NUM>,<NUM>-bis(sulfanyl)-<NUM>,<NUM>,<NUM><NUM>,<NUM>,<NUM>,<NUM>-hexaoxa-<NUM>,<NUM>,<NUM>,<NUM>,<NUM>,<NUM>,<NUM>,<NUM>,<NUM>,<NUM>-decaaza-34λ<NUM>,39λ<NUM>-diphosphaoctacyclo[<NUM>.<NUM> .<NUM><NUM>,<NUM>.<NUM><NUM>,<NUM>.<NUM><NUM>,<NUM>.<NUM><NUM>,<NUM>.<NUM><NUM>,<NUM>.<NUM><NUM>,<NUM>]dotetraconta-<NUM>,<NUM>,<NUM>,<NUM>,<NUM>,<NUM>,<NUM>,<NUM>,<NUM>-nonaene-<NUM>,<NUM>-dione (Compound (I)) di-ammonium salt (Form <NUM>), having diffraction peaks at diffraction angles (2θ ± <NUM>°) of <NUM>°, <NUM>°, <NUM>°, <NUM>° and <NUM>° in a powder X-ray diffraction.
<CHM>