Crystalline intermediate

Abstract

Provided herein are crystalline intermediates useful in the manufacture of 6,7-epoxytigliane compounds. Also provided are methods of making 6,7-epoxytigliane compounds using the crystalline intermediates, as well as high purity 6,7-epoxytigliane compounds that can be produced using the crystalline intermediates.

Description

[Technical field]

[0001] This application claims priority from Australian Provisional Patent Application No. 2021904153, filed on December 21, 2021, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to crystalline intermediates useful in the manufacture of 6,7-epoxytigliane compounds. Methods for making 6,7-epoxytigliane compounds using the crystalline intermediates are also disclosed, as well as high purity 6,7-epoxytigliane compounds that can be produced using the crystalline intermediates. [Background technology]

[0003] The 6,7-epoxytiglianes are a class of diterpene esters that have been shown to have human and veterinary therapeutic applications in diseases such as solid tumor cancer and bacterial infections, as well as in promoting wound healing (WO2007070985, WO2014169356). Several 6,7-epoxytigliane compounds, such as tigilanol tiglate (compound 1, 12-tigloyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one, WO2007070985), occur naturally in plants belonging to the Fontainea and Hylandia species. Other epoxytigliane compounds, such as 12,13-dihexanoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tigliane-3-one (compound 2, WO2014169356), are derived by semi-synthetic processes from naturally occurring 6,7-epoxytigliane compounds.

[0004] Although at least some epoxytigliane compounds are available in therapeutic quantities from plant materials, the resulting extract contains multiple closely related epoxytigliane compounds that may be difficult to separate. Because the variation in the extracted 6,7-epoxytigliane compounds occurs in the ester groups at the C12 and C13 positions of the 6,7-epoxytigliane structure, it has previously been proposed to treat the extract to deacylate the C12 and C13 esters to obtain a single 6,7-epoxytigliane compound with hydroxy groups at the C12 and C13 positions. This intermediate compound can then be synthetically manipulated to produce a single desired natural or unnatural 6,7-epoxytigliane compound (WO2014169356).

[0005] However, the 6,7-epoxytigliane compounds thus produced still require purification to ensure the removal of undesirable solvents and impurities that may be considered harmful. In some synthesis processes, a small portion of the 6,7-epoxytigliane compounds undergoes ring opening of the 6,7-epoxide ring to produce hydrochloride adducts such as: [ka] It has been found to form

[0006] These compounds are considered toxic impurities and are difficult to remove from the desired 6,7-epoxytigliane compound, resulting in losses and low yields of the desired compound during the chromatographic purification steps.

[0007] Purification of the product 6,7-epoxytigliane compounds by crystallization has been attempted, but these compounds appear to be particularly resistant to crystallization.

[0008] There is a need for methods of producing 6,7-epoxytigliane compounds that result in high yields and purity of therapeutic products.

[0009] It should be understood that where any prior art publication is referred to in this specification, such reference is not an acknowledgement that the publication forms part of the common general knowledge in the art in Australia or any other country. Summary of the Invention

[0010] The present invention is based, at least in part, on the discovery that a key intermediate in the synthetic preparation of therapeutically useful 6,7-epoxytigliane compounds crystallizes to form a highly pure product, allowing for the preparation of the desired 6,7-epoxytigliane compounds in high yield and purity.

[0011] In a first aspect of the present invention, a compound of formula (I): [ka] or a stereoisomer, pharma- ceutically acceptable salt or solvate thereof.

[0012] In another aspect of the invention there is provided a method of making a crystalline form of a compound of formula (I), comprising the steps of: i) Formula (II): [ka] wherein each R is independently selected from H and -C(O)R1, and when only one compound of formula (II) is present in the composition, at least one R group is not hydrogen, and R1 is C1~C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 Alkynyl, cycloalkyl, aryl, C 1~10 Alkylcycloalkyl, C 2~10 Alkenylcycloalkyl, C 2~10 Alkynylcycloalkyl, C 1~10 Alkylaryl, C 2~10 Alkenylaryl, C 2~10 Alkynylaryl, C 1~10AlkylC(O)R2, C 2~10 Alkenyl C(O)R2, C 2~10 Alkynyl C(O)R2, C 1~10 AlkylCH(OR2)(OR2), C(O)C 2~10 Alkenyl CH(OR2)(OR2), C 2~10 AlkynylCH(OR2)(OR2), C 1~10 AlkylSR2, C 2~10 Alkenyl SR2, C 2~10 AlkynylSR2, C 1~10 AlkylC(O)OR2, C 2~10 Alkenyl C(O)OR2, C 2~10 Alkynyl C(O)OR2, C 1~10 AlkylC(O)SR2, C 2~10 Alkenyl C(O)SR2, C 2~10 AlkynylC(O)SR2, [ka] is selected from R2 is hydrogen, -C 1~10 Alkyl, -C 2~10 Alkenyl, -C 2~10 alkynyl, cycloalkyl or aryl; wherein each alkyl, alkenyl, alkynyl, cycloalkyl or aryl group is optionally substituted; ii) treating the compound of formula (II) with 2,2-dimethoxypropane and a weak acid catalyst to give the compound of formula (III): [ka] forming a 5,20-acetonide of iii) deesterifying the esters at C12 and C13 of formula (III) to give a compound of formula (I): [ka] providing a compound of formula (I); and iv) crystallizing the compound of formula (I).

[0013] In yet a further aspect of the present invention, there is provided 12-tigloyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (Compound 1) or 12,13-dihexanoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (Compound 2) in substantially pure form.

[0014] In yet a further aspect of the present invention, a compound of formula (I): [ka] The present invention provides a method for making a crystalline form of a compound of formula (I), comprising crystallizing a compound of formula (I) from acetonitrile or methanol solvent. [Brief description of the drawings]

[0015] [Figure 1] 1 shows the X-ray powder diffraction pattern of anhydrous crystalline Form A of compound of formula (Ia).

[0016] [Diagram 2] 1 shows the X-ray powder diffraction pattern of anhydrous crystalline Form B of compound of formula (Ia).

[0017] [Diagram 3] Figure 2 shows the X-ray powder diffraction pattern of the methanol solvate of compound of formula (Ia).

[0018] [Figure 4] 1 shows the X-ray powder diffraction pattern of the dihydrate of compound of formula (Ia). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] Detailed Description of the Invention definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For purposes of the present invention, the following terms are defined below.

[0020] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0021] As used herein, the term "about" refers to a quantity, level, value, dimension, size, or amount that varies by as much as 30%, 25%, 20%, 15%, or 10% relative to a reference quantity, level, value, dimension, size, or amount. When used in reference to the location of a peak in an X-ray powder diffraction (XRPD) pattern, the term "about" includes peaks within ±0.2 degrees 2θ of the stated location, particularly within ±0.1 degrees 2θ of the stated location. For example, as used herein, an XRPD peak at "about 10.0 degrees 2θ" means that the stated peak occurs between 9.8 and 10.2 degrees 2θ.

[0022] Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" are understood to mean the inclusion of a stated step or element or group of steps or elements but not to the exclusion of any other step or element or group of steps or elements.

[0023] The term "alkyl" refers to optionally substituted straight and branched chain saturated hydrocarbon groups having 1 to 20 carbon atoms. Where appropriate, the alkyl group may have a specific number of carbon atoms, such as -C1-C6 alkyl, including alkyl groups having 1, 2, 3, 4, 5 or 6 carbon atoms in a straight or branched arrangement. Non-limiting examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, s- and t-butyl, pentyl, 2-methylbutyl, 3-methylbutyl, hexyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2-ethylbutyl, 3-ethylbutyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl and pentadecyl.

[0024] The term "alkenyl" refers to an optionally substituted unsaturated straight or branched chain hydrocarbon having 2 to 20 carbon atoms and having at least one double bond. Where appropriate, the alkenyl group may have a specific number of carbon atoms, such as C2-C6 alkenyl, including alkenyl groups having 2, 3, 4, 5 or 6 carbon atoms in a straight or branched arrangement. Non-limiting examples of alkenyl groups include ethenyl, propenyl, isopropenyl, butenyl, s- and t-butenyl, pentenyl, hexenyl, hepta-1,3-diene, hexa-1,3-diene, non-1,3,5-triene, and the like.

[0025] The term "alkynyl" refers to an optionally substituted unsaturated straight or branched chain hydrocarbon having 2 to 20 carbon atoms and having at least one triple bond. Where appropriate, the alkynyl group may have a specific number of carbon atoms, such as C2-C6 alkynyl, which includes alkynyl groups having 2, 3, 4, 5 or 6 carbon atoms in a straight or branched arrangement. Non-limiting examples include ethynyl, propynyl, butynyl, pentynyl and hexynyl.

[0026] The terms "cycloalkyl" and "carbocyclic" refer to an optionally substituted saturated or unsaturated, but not aromatic, monocyclic, bicyclic, or tricyclic hydrocarbon group. Where appropriate, cycloalkyl groups may have a specific number of carbon atoms, e.g., C3-C6 cycloalkyl is a carbocyclic group having 3, 4, 5, or 6 carbon atoms. Non-limiting examples may include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, and the like.

[0027] "Aryl" refers to a C6-C aryl group having up to seven atoms in each ring, where at least one ring is aromatic. 14 Aryl means a 1-membered monocyclic, bicyclic or tricyclic carbocyclic ring system. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl and biphenyl. Aryl may contain 1 to 3 benzene rings. When more than one aromatic ring is present, the rings may be fused together such that adjacent rings share a common bond.

[0028] Each alkyl, alkenyl, alkynyl, cycloalkyl, or aryl, whether as an individual entity or as part of a larger entity, is 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C 3~6 Cycloalkyl, oxo (=O), -OH, SH, OC 1~6 Alkyl, OC 2~6 Alkenyl, OC 2~6 Alkynyl, OC 3~6 Cycloalkyl, SC 1~6 Alkyl, SC 2~6 Alkenyl, SC 2~6 Alkynyl, SC 3~6 Cycloalkyl, CO2H, CO2C 1~6 Alkyl, NH2, NH(C 1~6 alkyl), N(C 1~6alkyl)2, NH(phenyl), N(phenyl)2, CN, NO2, halogen, CF3, -OCF3, SCF3, CHF2, OCHF2, SCHF2, phenyl, C 1~6 Alkylphenyl, Ophenyl, C(O)phenyl and C(O)C 1~6 and optionally substituted with one or more optional substituents selected from the group consisting of alkyl. Examples of suitable substituents include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, vinyl, methoxy, ethoxy, propoxy, isopropoxy, butoxy, methylthio, ethylthio, propylthio, isopropylthio, butylthio, hydroxy, hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl, fluoro, chloro, bromo, iodo, cyano, nitro, -CO2H, -CO2CH3, -C(O)CH3, trifluoromethyl, trifluoromethoxy, trifluoromethylthio, difluoromethyl, difluoromethoxy, difluoromethylthio, morpholino, amino, methylamino, dimethylamino, phenyl, phenoxy, phenylcarbonyl, benzyl, and acetyl.

[0029] The epoxytigliane compounds may be in the form of pharma-ceutically acceptable salts. However, it is understood that non-pharma-ceutically acceptable salts are also included within the scope of the invention, as these may be useful as intermediates in the preparation of pharma-ceutically acceptable salts, or may be useful during storage or transport. Suitable pharma- ceutically acceptable salts include, but are not limited to, salts of pharma- ceutically acceptable inorganic acids, such as hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, carbonic acid, boric acid, sulfamic acid, and hydrobromic acid, or salts of pharma- ceutically acceptable organic acids, such as acetic acid, propionic acid, butyric acid, tartaric acid, maleic acid, hydroxymaleic acid, fumaric acid, maleic acid, citric acid, lactic acid, mucic acid, gluconic acid, benzoic acid, succinic acid, oxalic acid, phenylacetic acid, methanesulfonic acid, toluenesulfonic acid, benzenesulfonic acid, salicylic acid, sulfanilic acid, aspartic acid, glutamic acid, edetic acid, stearic acid, palmitic acid, oleic acid, lauric acid, pantothenic acid, tannic acid, ascorbic acid, and valeric acid.

[0030] Base salts include, but are not limited to, those formed with pharma- ceutically acceptable cations, such as sodium, potassium, lithium, calcium, magnesium, ammonium and alkylammonium.

[0031] Basic nitrogen-containing groups may be quaternized with such reagents as lower alkyl halides, e.g., methyl, ethyl, propyl, and butyl chloride, bromide, and iodide; dialkyl sulfates, e.g., dimethyl and diethyl sulfate.

[0032] It will also be appreciated that epoxytigliane compounds may have asymmetric centers and therefore may exist in two or more stereoisomeric forms. Thus, the present invention also relates to compounds that are substantially in one stereoisomeric form at one or more asymmetric centers, for example, compounds with greater than 90% ee, for example, about 95% or 97% ee, or greater than 99% ee, and mixtures thereof, for example racemic mixtures. Such stereoisomers can be obtained by isolation from natural sources, by asymmetric synthesis, such as using chiral intermediates, or by chiral resolution. The compounds of the present invention may also exist as geometric isomers. The present invention also relates to compounds in substantially pure cis (Z) or trans (E) form or mixtures thereof.

[0033] The term "6,7-epoxytigliane compound" refers to the following basic carbocyclic structure: [ka] It refers to a compound having the formula:

[0034] This compound has a tricyclo[9.3.0.0]tetradecane system with a fused cyclopropane ring attached to a six-membered ring. An epoxide is fused to the seven-membered ring at the 6,7 positions.

[0035] An example of a 6,7-epoxytigliane compound is a 6,7-epoxytiglien-3-one compound. The term "epoxytiglien-3-one compound" refers to a compound having the epoxytigliane structure defined above, in which the five-membered ring has a 1,2-ene-3-one structure: [ka] Refers to...

[0036] The term "substantially pure form" refers to a product having a chemical purity of more than 96%, particularly more than 97%, more than 98%, more than 99% or 100% and / or an ee of more than 97%, particularly more than 98% or more than 99% and more particularly 100%. In some embodiments, a substantially pure form refers to when impurity compounds A and / or B are present at less than 0.5%, less than 0.1%, less than 0.05%, and most particularly when they are absent.

[0037] The present invention relates to a compound of formula (I) in crystalline form, in particular to a compound of formula (Ia) in crystalline form: [ka]

[0038] In some embodiments, the crystalline form is an anhydrous crystalline form. In other embodiments, the crystalline form is a methanol solvate. In another embodiment, the crystalline form is a dihydrate.

[0039] In embodiments, when the crystalline form is an anhydrous crystalline form of the compound of formula (Ia), the crystalline form may be one of two forms.

[0040] The anhydrous crystalline form, Form A, exhibits an X-ray powder diffraction (XRPD) pattern comprising at least one peak at about 10.5 degrees 2θ. In another embodiment, the anhydrous crystalline form A exhibits an XRPD pattern comprising a peak at about 10.5 degrees 2θ and at least one peak selected from the group consisting of about 6.2 degrees 2θ and about 7.6 degrees 2θ. In another embodiment, the anhydrous crystalline form A exhibits an XRPD pattern comprising a peak at about 10.5 degrees 2θ, at least one peak selected from about 6.2 degrees 2θ and about 7.6 degrees 2θ, and at least one peak selected from about 12.5 degrees 2θ and about 15.2 degrees 2θ. In yet a further embodiment, the anhydrous crystalline form A exhibits an XRPD pattern including a peak at about 10.5 degrees 2θ, at least one peak selected from about 6.2 and about 7.6 degrees 2θ, at least one peak selected from about 12.5 and about 15.2 degrees 2θ, and at least one peak selected from about 16.2, about 16.7, about 18.3, about 19.9, about 23.3, and about 27.8 degrees 2θ. In one embodiment, the XRPD pattern is substantially the same as Figure 1. In another embodiment, the anhydrous crystalline form A has an XRPD pattern with peaks at 6.2, 7.6, 10.5, 12.5, 15.2, 16.2, 16.7, 18.3, 19.9, 21.5, 23.3, 25.2, and 27.8 degrees 2θ±0.2.

[0041] Additionally, the anhydrous crystalline form A has the following molecular weights: about 3458.4, 3345.6, 3056.9, 2984.6, 2968.6, 2941.1, 2882.4, 1708.3, 1636.1, 1458.5, 1376.1, 1319.0, 1223.5, 1085.7, 1025.4, 1000.3, 973.7, 930.3, 902.1, 880.3, 832.1, and 769.0 cm -1 Including absorption frequencies in the attenuated total reflectance (ATR) mode, 4000-550 cm -1 Characterize by Fourier transform infrared (FTIR) spectra in the spectral range.

[0042] In some embodiments, the anhydrous crystalline form A has a water content of less than 0.5%, particularly less than 0.3%, for example about 0.28%.

[0043] In some embodiments, the anhydrous crystalline form A has a level of residual organic solvent less than 50 ppm, particularly less than 40 ppm, more particularly less than 25 ppm, for example, about 34 ppm.

[0044] A second anhydrous crystalline form (form B) can be formed from anhydrous crystalline form A under high humidity conditions, for example at a temperature of 25° C. and a relative humidity of 80% over a period of 68 hours. Anhydrous crystalline form B is stable upon storage and can be present upon storage of anhydrous crystalline form A.

[0045] The anhydrous crystalline form, Form B, exhibits an X-ray powder diffraction (XRPD) pattern comprising at least one peak at about 11.4 degrees 2θ. In another embodiment, the anhydrous crystalline form B exhibits an XRPD pattern comprising a peak at about 11.4 degrees 2θ and at least one peak selected from the group consisting of about 8.5 degrees 2θ and about 9.8 degrees 2θ. In another embodiment, the anhydrous crystalline form B exhibits an XRPD pattern comprising a peak at about 11.4 degrees 2θ, at least one peak selected from about 8.5 degrees 2θ and about 9.8 degrees 2θ, and at least one peak selected from about 4.9 degrees 2θ and about 14.6 degrees 2θ. In yet a further embodiment, the anhydrous crystalline form B exhibits an XRPD pattern including a peak at about 11.4 degrees 2θ, at least one peak selected from about 8.5 and about 9.8 degrees 2θ, at least one peak selected from about 4.9 and about 14.6 degrees 2θ, and at least one peak selected from about 16.5, about 17.5, about 19.5, about 21.4, about 27.7, and about 28.7 degrees 2θ. In one embodiment, the XRPD pattern is substantially the same as Figure 2. In another embodiment, the anhydrous crystalline form B has an XRPD pattern with peaks at 4.9, 8.5, 9.8, 11.4, 14.6, 16.5, 17.5, 19.5, 21.4, 27.7, 28.7 ± 0.2 degrees 2θ.

[0046] In some embodiments, the anhydrous crystalline form B has a water content of less than 0.5%, in particular less than 0.3%, for example about 0.28%.

[0047] In some embodiments, the anhydrous crystalline form B has a level of residual organic solvent less than 50 ppm, particularly less than 40 ppm, more particularly less than 25 ppm, for example, about 34 ppm.

[0048] In embodiments where the crystalline form is a methanol solvate crystalline form of the compound of Formula (Ia), the crystalline form exhibits an X-ray powder diffraction (XRPD) pattern comprising at least one peak at about 9.5 degrees 2θ. In another embodiment, the crystalline form exhibits an XRPD pattern comprising a peak at about 9.5 degrees 2θ and at least one peak selected from the group consisting of about 7.2 degrees 2θ and about 13.1 degrees 2θ. In another embodiment, the crystalline form exhibits an XRPD pattern comprising a peak at about 9.5 degrees 2θ, at least one peak selected from about 7.2 degrees 2θ and about 13.1 degrees 2θ, and at least one peak selected from about 11.5 degrees 2θ, about 14.5 degrees 2θ, and about 17.9 degrees 2θ. In still further embodiments, the crystalline form exhibits an XRPD pattern including a peak at about 9.5 degrees 2θ, at least one peak selected from about 7.2 and about 13.1 degrees 2θ, at least one peak selected from about 11.5, about 14.5, and about 17.9 degrees 2θ, and at least one peak selected from about 20.3, about 21.2, about 22.8, and about 32.6 degrees 2θ. In one embodiment, the XRPD pattern is substantially the same as FIG. In another embodiment, the methanol solvate crystalline form has an XRPD pattern with peaks at 7.2, 9.5, 11.5, 12.6, 13.1, 14.0, 14.5, 17.0, 17.7, 17.9, 19.2, 20.3, 21.2, 22.8, 23.7, 24.7, 27.1, 28.3, 29.7, and 32.6±0.2 degrees two-theta.

[0049] Further, the crystalline methanol solvate has a molecular weight of 4000-550 cm in attenuated total reflectance (ATR) mode, including absorption frequencies at about 3443.1, 3374.1, 3189.1, 3002.6, 2975.7, 2944.4, 2863.6, 1703.3, 1635.7, 1461.9, 1372.9, 1290.7, 1221.1, 1077.1, 1025.1, 992.0, 973.9, 929.7, 905.8, 876.7, 829.0, and 769.1. -1 Characterize by Fourier transform infrared (FTIR) spectra in the spectral range.

[0050] In some embodiments, the crystalline methanol solvate has a differential scanning calorimetry (DSC) profile characterized by an endothermic peak with an onset at about 145.33°C, and in some embodiments, one or two weaker endothermic peaks at about 221.12°C and / or 234.22°C.

[0051] In some embodiments, the crystalline methanol solvate contains 4-8%, particularly 5-6%, residual methanol as determined by thermogravimetric analysis.

[0052] In an embodiment where the crystalline form is a dihydrate crystalline form of the compound of Formula (Ia), the crystalline form exhibits an X-ray powder diffraction (XRPD) pattern comprising at least one peak at about 10.5 degrees 2θ. In another embodiment, the crystalline form exhibits an XRPD pattern comprising a peak at about 10.5 degrees 2θ and at least one peak selected from the group consisting of about 7.4 degrees 2θ and about 12.4 degrees 2θ. In another embodiment, the crystalline form exhibits an XRPD pattern comprising a peak at about 10.5 degrees 2θ, at least one peak selected from about 7.4 degrees 2θ and about 12.4 degrees 2θ, and at least one peak selected from about 6.2 degrees 2θ, about 9.9 degrees 2θ, and about 15.3 degrees 2θ. In yet a further embodiment, the crystalline form exhibits an XRPD pattern including a peak at about 10.5 degrees 2θ, at least one peak selected from about 7.4 and about 12.4 degrees 2θ, at least one peak selected from about 6.2, about 9.9, and about 15.3 degrees 2θ, and at least one peak selected from about 8.9, about 18.6, about 20.2, about 22.4, about 22.9, about 26.0, about 27.0, about 27.3, and about 33.7 degrees 2θ. In one embodiment, the XRPD pattern is substantially the same as FIG. In another embodiment, the dihydrate crystalline form has an XRPD pattern with peaks at 6.2, 7.4, 8.9, 9.9, 10.5, 12.4, 15.3, 18.6, 20.2, 21.1, 22.4, 22.9, 26.0, 27.0, 27.3, and 33.7±0.2 degrees two-theta.

[0053] The dihydrate crystalline form may form upon storage at low temperatures, for example at about 5° C., and may convert back to anhydrous Form A or anhydrous Form B upon storage at room temperature and 80% humidity.

[0054] In some embodiments, the crystalline form is substantially pure, e.g., having a chemical purity of greater than 96%, 97%, 98% or 99%, particularly a chemical purity of 99% to 100%, more particularly a chemical purity of 99.5% to 100%, and optionally an ee of greater than 97%, particularly an ee of greater than 98% or an ee of greater than 99%, more particularly an ee of 100%.

[0055] In some embodiments, the crystalline form is a single crystalline form, such as an anhydrous crystalline form, Form A or Form B. In other embodiments, the crystalline form is a mixture of crystalline forms, such as a mixture of Form A and Form B, or a mixture of Form A and a dihydrate crystalline solvate. However, even when multiple crystalline forms are present, the compound of formula (Ia) may still be substantially pure, having a chemical purity of more than 96%, 97%, 98% or 99%, particularly a chemical purity of 99% to 100%, more particularly a chemical purity of 99.5% to 100%, and optionally an ee of more than 97%, particularly an ee of more than 98% or an ee of more than 99%, more particularly an ee of 100%.

[0056] In certain embodiments, the crystalline form of the compound of formula (I) is an anhydrous crystalline form, in particular the compound of formula (Ia): [ka] It is an anhydrous crystalline form of

[0057] In some embodiments, the anhydrous crystalline form is Form A. In other embodiments, the anhydrous crystalline form is Form B.

[0058] The crystalline forms of the present invention can be prepared by crystallization of the compound of formula (I) from a suitable solvent. Suitable solvents were investigated as shown in Example 7.

[0059] Crystallization from methanol gave a crystalline methanol solvate. Crystallization occurred after dissolving the compound in methanol at 20-25° C., preferably 2-10 volumes (2V-10V) relative to the weight of the compound, particularly 3V-8V, more particularly 3V-7V, more particularly about 3V-6V. Crystallization occurred spontaneously after dissolution.

[0060] Crystallization from acetonitrile afforded Form A, a crystalline anhydrous form, via a 1:1 acetonitrile (MeCN) solvate. Dissolution of the compound of formula (I) was obtained under reflux and crystallization occurred upon cooling to about 68-70° C. Crystallization occurred at ratios of 3V to 15V, particularly 4V to 12V, more particularly 5V to 10V of solvent compared to the weight of the compound. The initial MeCN solvate was collected by filtration and dried to afford the anhydrous crystalline Form A.

[0061] Advantageously, Form A, being an anhydrous crystalline form, is solvent-free and gave a higher yield of 85-95% compared to 70-80% yield for the crystalline methanol solvate.

[0062] The anhydrous crystalline form A was stable at 60% relative humidity, but at higher humidities, such as 80% relative humidity, it could convert to a dihydrate (stored at about 5° C.) or a second anhydrous crystalline form, form B (stored at about 25° C.).

[0063] Preparation of the Compound of Formula (I) in Anhydrous or Solvated Form Compounds of formula (I) can be prepared as shown in Scheme 1 and as described in WO 2014 / 169356 (Example 3). [ka]

[0064] In one aspect of the invention there is provided a method of making a crystalline form of a compound of formula (I), comprising the steps of: i) Formula (II): [ka] wherein each R is independently selected from H and -C(O)R1, and when only one compound of formula (II) is present in the composition, at least one R group is not hydrogen, and R1 is C1~C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20Alkynyl, cycloalkyl, aryl, C 1~10 Alkylcycloalkyl, C 2~10 Alkenylcycloalkyl, C 2~10 Alkynylcycloalkyl, C 1~10 Alkylaryl, C 2~10 Alkenylaryl, C 2~10 Alkynylaryl, C 1~10 AlkylC(O)R2, C 2~10 Alkenyl C(O)R2, C 2~10 Alkynyl C(O)R2, C 1~10 AlkylCH(OR2)(OR2), C(O)C 2~10 Alkenyl CH(OR2)(OR2), C 2~10 AlkynylCH(OR2)(OR2), C 1~10 AlkylSR2, C 2~10 Alkenyl SR2, C 2~10 AlkynylSR2, C 1~10 AlkylC(O)OR2, C 2~10 Alkenyl C(O)OR2, C 2~10 Alkynyl C(O)OR2, C 1~10 AlkylC(O)SR2, C 2~10 Alkenyl C(O)SR2, C 2~10 AlkynylC(O)SR2, [ka] is selected from R2 is hydrogen, -C 1~10 Alkyl, -C 2~10 Alkenyl, -C 2~10 alkynyl, cycloalkyl or aryl; wherein each alkyl, alkenyl, alkynyl, cycloalkyl or aryl group is optionally substituted; ii) treating the compound of formula (II) with 2,2-dimethoxypropane and a weak acid catalyst to give the compound of formula (III): [ka] forming a 5,20-acetonide of iii) deesterifying the esters at C12 and C13 of formula (III) to give a compound of formula (I): [ka] and crystallizing the compound of formula (I).

[0065] In some embodiments, the composition comprising one or more compounds of formula (II) is a mixture of compounds of formula (II), particularly of formula (IIa): [ka] The present invention includes a mixture of compounds.

[0066] For example, a composition comprising one or more compounds of formula (II) or (IIa) may comprise one or more compounds selected from the following: [Table 1] or stereoisomers thereof, particularly stereoisomers having a stereocenter as shown in formula (IIa).

[0067] In some embodiments, the weak acid catalyst is, for example, a weak Bronsted acid catalyst, such as pyridinium p-toluenesulfonate or camphorsulfonic acid (CSA), cation exchange resins, p-toluenesulfonic acid, H2SO4, zinc chloride and other Lewis acids, and BF3 etherate. In certain embodiments, the weak acid catalyst is pyridinium p-toluenesulfonate.

[0068] The solvent used in the formation of the acetonide is any suitable solvent capable of solubilizing the reactants. Suitable solvents include acetone, dimethylformamide, toluene, dichloromethane, tetrahydrofuran (THF), 2-methyltetrahydrofuran, and acetonitrile.

[0069] Deesterification of C12 and C13 esters occurs in the presence of a base. Suitable bases include carbonates such as Cs2CO3, Na2CO3, K2CO3 and Li2CO3. In a particular embodiment, the base used is Cs2CO3 in an aqueous or alcoholic solvent or in dimethylformamide (DMF), particularly Cs2CO3 in methanol.

[0070] In some embodiments, the 5,20-acetonide compound of formula (I) is purified by column chromatography prior to crystallization. In other embodiments, the compound of formula (I) is crystallized without purification by chromatography.

[0071] The crystallization of the compound of formula (I) can be carried out from a suitable solvent such as methanol or acetonitrile. From methanol solvent, the methanol solvate crystal form is obtained. From acetonitrile, the crystalline acetonitrile solvate, the crystalline anhydrous form, form A and form B, and the dihydrate crystal form are obtained. In certain embodiments, the crystal form is the crystal form of the compound of formula (Ia).

[0072] One or more crystalline forms of the compound of formula (I) can be used to prepare therapeutically active 6.7-epoxytigliane compounds, such as Compound 1 and Compound 2, in high yield and purity.

[0073] The use of one or more crystalline forms of the compound of formula (I) in the synthesis of 6,7-epoxytigliane compounds may improve the overall yield obtained by up to 50% and / or reduce impurities such as solvents and / or hydrochlorides A and B to therapeutically acceptable levels.

[0074] In some embodiments, the method of the present invention further comprises the step of esterifying one or both of the C12 hydroxy group and the C13 hydroxy group of the compound of formula (I), as shown in Scheme 2, where the starting material is a crystalline compound of formula (I). [ka]

[0075] The reaction depicted in Scheme 2 may be carried out in a single step in which esterification of both the C12 hydroxy group and the C13 hydroxy group occurs with a single activated carboxylic acid, in which embodiment, each R3 is the same.

[0076] In other embodiments, the C12 hydroxy group and the C13 hydroxy group are esterified in separate sequential reactions to produce a 6,7-epoxytigliane compound having different C12 and C13 esters, where each R3 is different. During the reaction, the initial esterification occurs at the C13 hydroxy group, and the reaction may be monitored to see when the reaction at C13 is complete.

[0077] The carboxylic acid may be activated for reaction with a hydroxy group by any means known in the art. In some embodiments, the activated carboxylic acid is an acid chloride or anhydride. In some embodiments, the activated carboxylic acid may be a symmetrical anhydride, such as tiglic anhydride. In other embodiments, the activated carboxylic acid may be an asymmetrical anhydride, such as tiglic-pivalic anhydride. In still other embodiments, the carboxylic acid is activated with a carbodiimide, for example, suitable carbodiimides include N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC), or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).

[0078] Each R3 is the same or different and is independently selected from the group consisting of H and -C(O)R1, and R1 is any one of C1 to C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 Alkynyl, cycloalkyl, aryl, C 1~10 Alkylcycloalkyl, C 2~10 Alkenylcycloalkyl, C 2~10 Alkynylcycloalkyl, C 1~10 Alkylaryl, C 2~10Alkenylaryl, C 2~10 Alkynylaryl, C 1~10 AlkylC(O)R2, C 2~10 Alkenyl C(O)R2, C 2~10 Alkynyl C(O)R2, C 1~10 AlkylCH(OR2)(OR2), C(O)C 2~10 Alkenyl CH(OR2)(OR2), C 2~10 AlkynylCH(OR2)(OR2), C 1~10 AlkylSR2, C 2~10 Alkenyl SR2, C 2~10 AlkynylSR2, C 1~10 AlkylC(O)OR2, C 2~10 Alkenyl C(O)OR2, C 2~10 Alkynyl C(O)OR2, C 1~10 AlkylC(O)SR2, C 2~10 Alkenyl C(O)SR2, C 2~10 AlkynylC(O)SR2, [ka] is selected from R2 is hydrogen, -C 1~10 Alkyl, -C 2~10 Alkenyl, -C 2~10 alkynyl, cycloalkyl or aryl; wherein each alkyl, alkenyl, alkynyl, cycloalkyl or aryl group is optionally substituted. In certain embodiments, at least one R3 is other than hydrogen, and in particular both R3 are other than hydrogen.

[0079] Once the C12 and C13 esters have been formed in the desired manner, the 5,20-acetonide protecting group can be removed to form the epoxy tigliane compound of formula (IV), as shown in Scheme 3. [ka]

[0080] Acetonide protecting groups can be removed under any suitable condition known in the art, for example, under aqueous acidic conditions. Suitable acids that can be used to provide acidic conditions include hydrochloric acid (HCl), trifluoroacetic acid, perchloric acid, sulfuric acid, Amberlite™ resin or other strong ion exchange resins, and Lewis acids, such as BF3 etherate. Suitable solvents include aqueous and alcoholic solvents, such as water, methanol, aqueous methanol, aqueous tetrahydrofuran, aqueous acetonitrile, and the like. Examples of suitable conditions include HCl in aqueous methanol or aqueous tetrahydrofuran, trifluoroacetic acid in aqueous acetonitrile, or aqueous perchloric acid (HClO4).

[0081] In one embodiment, the method produces 12,13-dihexanoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 2) in high yield and / or in substantially pure form.

[0082] In another embodiment, the method produces 12-tigloyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (Compound 1) in high yield and / or in substantially pure form.

[0083] In one aspect of the invention, there is provided 12,13-dihexanoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (Compound 2) or 12-tigloyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (Compound 1) produced by the above method.

[0084] In yet a further aspect of the present invention, a compound of formula (I): [ka] The present invention provides a method for making a crystalline form of a compound of formula (I), comprising crystallizing a compound of formula (I) from acetonitrile or methanol solvent. EXAMPLES

[0085] Example 1 - Extraction of biomass 9.49 kg of ground seeds of Fontainea picrosperma (analysis: total tiglianone compounds: 6.7%, 635.8 g) were suspended in methanol (24 L) and the resulting suspension was maintained under stirring at room temperature for 4 h. The suspension was left for 2.5 h and then the methanol phase was removed and collected. The remaining biomass was re-extracted two more times following the same procedure and then washed once with 10 L of methanol with a settling time of 10 min. The combined methanol extracts were concentrated under vacuum at a temperature below 40° C. to a final volume of 20 L and the crude methanol solution was analyzed by high performance liquid chromatography (HPLC) and Karl Fischer (KF) titration. HPLC was performed using a reversed phase amide column (Halo P / N 92814-707) and a guard column (Halo P / N 92814-107) with a linear gradient of solvent A: water / 0.1% formic acid and solvent B: acetonitrile / 0.1% formic acid. [Table 2]

[0086] The flow rate was 1 mL / min, injection volume was 5.0 μL, column temperature was 40° C., and detection was at 249 nm.

[0087] The analysis is shown in Table 1. [Table 3]

[0088] Example 2 - Purification of crude extract The crude concentrated methanol solution from Example 1 was extracted twice with n-hexane at room temperature (1st extraction 30 L, 2nd extraction 15 L). Each extract was mixed for about 10 minutes, then the phases were allowed to separate for 10 minutes before the hexane phase was collected. The collected hexane phases were combined and re-extracted twice with a methanol / water (90 / 10) mixture and then drained. The combined water-methanol phase was pooled with the previous methanol extract and diluted with 10% NaCl (aq, 5 L). The aqueous methanol solution was extracted three times with dichloromethane (1st extraction 10 L, 2nd and 3rd extractions 5 L). The combined organic phases were dried over Na2SO4 and then concentrated under vacuum at a temperature below 40°C until an oil was obtained with a water content of less than 0.1% as measured by KF.

[0089] The combined aqueous methanol phase was pooled with the previous methanol extract and a further batch of purified crude extract was prepared using the method described above, except diluted with water rather than 10% NaCl to reduce the formation of chloro adducts.

[0090] Example 3 - Preparation of epoxytigliane-5,20 acetonide ester The oil from Example 2 was dissolved in acetone (4 L) and then 2,2-dimethoxypropane (1.125 L) and pyridinium p-toluenesulfonate (0.338 Kg) were added. The mixture was heated under stirring at 40° C. for 25 hours. A sample was then checked by HPLC analysis as described in Example 1 to confirm the completion of the reaction. Ethyl acetate was added to the reaction mixture and then the reaction mixture was washed twice with water. The collected organic phase was dried under vacuum to give the 5,20-acetonide ester as an oil.

[0091] Example 4 - Deesterified Epoxytigliane-5,20-acetonide: Preparation of the compound of formula (Ia) The oil from Example 3 was dissolved in methanol (10 L) and 1 kg of Cs2CO3 was added. The mixture was kept under stirring at 25° C. for 22 hours, after which water (2 L) was added. The resulting mixture was washed with n-hexane three times (3×3 L). Ethyl acetate (10 L), water (8 L) and H2SO42N solution (5 L) were added to the remaining water-methanol phase. The organic phase was collected and the water-methanol solution was re-extracted with ethyl acetate (10 L×4 times) and all the ethyl acetate organic solutions were pooled together. The ethyl acetate solution was then washed twice (2 L×2) with 10% Na2SO4 aqueous solution, draining the aqueous phase each time and then concentrating under vacuum until a two-phase mixture was obtained. After phase separation, the aqueous phase was drained and the organic phase was concentrated again to obtain the 12,13-dihydroxyacetonide compound of formula (II) as an oil. The oil was purified by column chromatography on silica gel (n-hexane / ethyl acetate 20:80 as eluent) to give 163 g of compound with a purity of 81.3% as determined by HPLC.

[0092] Example 5 - Preparation of 12,13-dihexanoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one-5,20-acetonide To the dichloromethane solution of the product of Example 4 (5 L), 27.8 g of 4-DMAP and 132.0 g of hexanoic acid were added, and the resulting mixture was stirred until dissolved. 216.8 g of ethylcarbodiimide hydrochloride (EDC HCl) was added, and the mixture was kept stirring at 25° C. for 24 hours. The reaction mixture was worked up by adding 3.3 L of H2SO42N dropwise, and after phase separation, the aqueous phase was removed. The organic residual solution was washed with water (3.3 L x 3), and then concentrated to dryness to obtain the dihexanoyl product.

[0093] Further batches of 12,13-dihexanoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one-5,20-acetonide were prepared using the method described above, except that a preliminary water wash step was performed prior to the addition of H2SO4 to reduce the formation of chloro adducts.

[0094] Example 6 - Preparation of 12,13-dihexanoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (Compound 2) To the acetonitrile solution of the product of Example 5 (3.1 L), an acidic aqueous solution containing HClO4 70% (300 mL water + 12.4 g HClO4 70%) was added, the resulting mixture was stirred at 25°C for 47 hours, and the reaction completion was monitored by thin layer chromatography (eluent MeCN / H2O 85:15 v / v, silica gel, 60 RP18F 254S). The reaction was worked up by adding 5 L of dichloromethane and 900 mL of 10% aqueous sodium acetate solution. The aqueous phase was drained, and the organic solution was washed twice with water (2 L x 2), then concentrated to dryness to obtain crude 12,13-dihexanoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one.

[0095] The crude product was purified by column chromatography on a reversed-phase RP18 (acetonitrile / water 50:50 + 20 ppm H3PO4 as eluent). Only the central fractions containing the majority of the desired compound were pooled. [ka] The presence of the hydrochloride adduct impurity, believed to be

[0096] The impurity compounds were analyzed by LC-HRMS (sodium salt). 32 H 49 O 10 Cl Na=651.2906.

[0097] The pooled fractions were diluted with water and extracted three times with dichloromethane. The combined organic phases were concentrated to dryness to give 93.7 g (57% from 10 kg wet seeds) of pure 12,13-dihexanoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one.

[0098] A further batch of 12,13-dihexanoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (Compound 2) was prepared using the method described above, except that a water washing step was performed on the concentrated 12,13-dihexanoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one prior to final concentration to dryness of the 12,13-dihexanoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one to prevent the formation of impurities.

[0099] Example 7 - Crystallization studies Early intermediates in the synthetic route to produce compound 2, such as the compound of formula (I), were examined for crystallization potential.

[0100] An aliquot (0.83 L containing approximately 10 g of compound) of the purified compound of formula (Ia) obtained from Example 4 was used for crystallization studies. The results of the preliminary screening are shown in Table 2. [Table 4]

[0101] Crystallization in methanol and acetonitrile was further investigated.

[0102] Example 8 - Crystalline Compound (Ia) as MeOH solvate. A sample of the product of Example 4 (amorphous solid, about 80-85% pure by HPLC) was dissolved in methanol at room temperature. Complete dissolution occurred within seconds. Uncontrolled crystallization was induced immediately after dissolution. The resulting product was in the solvate crystalline form with a MeOH content of about 5-6%. Table 3 below summarizes the crystallization trials that were performed. [Table 5]

[0103] The crystalline methanol solvate of compound of formula (Ia) was characterized by an X-ray powder diffraction (X-RPD) pattern, obtained using copper wavelengths λ1 and λ2 of 1.54056 Å and 1.54439 Å, which was indicative of a crystalline structure and contained unique reflections, expressed as 2θ degree values, at: 2θ 7.2, 9.5, 11.5, 12.6, 13.1, 14.0, 14.5, 17.0, 17.7, 17.9, 19.2, 20.3, 21.2, 22.8, 23.7, 24.7, 27.1, 28.3, 29.7, 32.6.

[0104] 4000~500cm in ATR mode -1 The Fourier transform infrared (FTIR) spectrum of the crystalline methanol solvate in the spectral range is approximately 3443.1, 3374.1, 3189.1, 3002.6, 2975.7, 2944.4, 2863.6, 1703.3, 1635.7, 1461.9, 1372.9, 1290.7, 1221.1, 1077.1, 1025.1, 992.0, 973.9, 929.7, 905.8, 876.7, 829.0, 769.1 cm -1 The absorption frequency was characteristic at

[0105] The differential scanning calorimetry (DSC) profile of the crystalline methanol solvate is characterized by an endothermic peak with onset at about 145.33° C., as well as two other weaker endothermic peaks with onsets at about 221.12° C. and 234.22° C., respectively.

[0106] The crystalline methanol solvate was further characterized by a thermogravimetric profile (TG) showing a weight loss of 5.5%, consistent with the presence of 5-6% residual MeOH.

[0107] Example 9 - Anhydrous crystalline compound of formula (Ia) A mixture of the product of Example 4 and acetonitrile was stirred under reflux until a clear solution was obtained, then the solution was allowed to cool and crystallization occurred spontaneously upon cooling at about 68-70° C. The following table summarizes the crystallization trials that were performed. [Table 6]

[0108] Crystallization from acetonitrile formed a solid which was a 1:1 MeCN solvate. The acetonitrile solvate was collected and dried to obtain Form A of the anhydrous crystalline compound of formula (I). The MeCN solvate had a loss on drying (LoD) of 7.8% by weight. The MeCN solvate was characterized by an X-ray powder diffraction (X-RPD) pattern obtained using copper wavelengths λ1 and λ2 of 1.54056 Å and 1.54439 Å, which indicated a crystalline structure and contained unique reflections, expressed as 2θ degree values, at 6.2, 7.5, 10.4, 12.2, 14.9, 16.0, 17.7, 18.5, 19.5, 20.9, 22.4, 24.7, 26.8, and 33.4±0.2 degrees 2θ.

[0109] Form A of the crystalline anhydrous compound of formula (Ia) was characterized by an X-ray powder diffraction (X-RPD) pattern obtained using copper wavelengths λ1 and λ2 of 1.54056 Å and 1.54439 Å, which was indicative of a crystalline structure and contained unique reflections, expressed as 2θ degree values, at 6.2, 7.6, 10.5, 12.5, 15.2, 16.2, 16.7, 18.3, 19.9, 21.5, 23.3, 25.2, and 27.8±0.2 degrees 2θ.

[0110] Form A of the anhydrous crystalline compound of formula (Ia) was further characterized by a water content of about 0.28% by coulometric titration and 34 ppm of residual organic solvents determined by headspace gas chromatography with flame ionization detection (HS GC FID).

[0111] Form A of the crystalline anhydrous compound of formula (Ia) was stable for at least 9 months in industrial packaging (10 mL type III amber glass vials closed by black plastic screw caps with polyethylene stoppers) at 25° C. and 60% relative humidity, and was stable as a free crystalline powder at 80° C. and 60% humidity for 24 hours.

[0112] Upon storage for 5 days at 5° C., Form A of the anhydrous crystalline compound converted to the dihydrate form of the compound with a loss on drying (LoD) of 9.3% by weight, which was able to convert back to the anhydrous crystalline form, Form A, upon storage for 5 days at 25° C. and 80% relative humidity.

[0113] The dihydrate crystalline form was characterized by an X-ray powder diffraction (X-RPD) pattern obtained using copper wavelengths λ1 and λ2 of 1.54056 Å and 1.54439 Å, which was indicative of a crystalline structure and contained unique reflections, expressed as 2θ degree values, at 6.2, 7.4, 8.9, 9.9, 10.5, 12.4, 15.3, 18.6, 20.2, 21.1, 22.4, 22.9, 26.0, 27.0, 27.3, and 33.7±0.2 degrees 2θ.

[0114] Storage of the anhydrous crystalline form of Form A at 25° C. and 80% relative humidity for 68 hours or at 40° C. and 80% relative humidity for 7 days resulted in the formation of a new stable anhydrous crystalline form, Form B. Form B had an LoD of 0.3% by weight and was stable unchanged for at least an additional 7 days at 25° C. and 80% relative humidity. The anhydrous crystalline form B was characterized by an X-ray powder diffraction (X-RPD) pattern obtained using copper wavelengths λ1 and λ2 of 1.54056 Å and 1.54439 Å, which indicated a crystalline structure and contained unique reflections, expressed as 2θ degree values, at 4.9, 8.5, 9.8, 11.4, 14.6, 16.5, 17.5, 19.5, 21.4, 27.7, 28.7±0.2 degrees 2θ.

[0115] Example 10: Comparison of purification by crystallization with and without purification Using the methods described above for preparing compound 2, analyses were performed to compare the preparation of compound 2 via the compound of formula (Ia) with and without crystallization.

[0116] From a batch of 9.5 kg wet seeds, following the above procedure, approximately 165-176 g of compound of formula Ia was obtained after chromatography.

[0117] Without the crystallization step, after removing about 10 g of compound of formula Ia for other tests, processing of compound of formula Ia gave compound 2 in the amount of 92 g, and analysis by HPLC showed that the product had a purity of 97.5%.

[0118] With crystallization of the compound of formula (Ia), after removing about 4.7 g of the compound of formula Ia for other tests, processing of the compound of formula Ia afforded compound 2 in the amount of 152 g, which was shown to have a purity of 98.7% as analyzed by HPLC.

[0119] Example 11: Preparation of anhydrous crystalline compound of formula (Ia) Selected fractions from the silica gel purification of compound of formula (Ia) (160-190 g of compound of formula Ia) were solvent exchanged into acetonitrile (12 volumes used) with the bath temperature set at 40-45° C. and a vacuum of approximately 100 mbar. The mixture of compound of formula Ia was then heated to reflux (82° C.) in acetonitrile (10 volumes) until dissolved, then cooled to 20±5° C. with stirring. The resulting slurry was stored at 20±5° C. overnight, then cooled to 0±5° C. for at least 1 hour. The mixture was then filtered through a sintered glass filter under vacuum. The filter cake was washed with acetonitrile (1 volume). The solids were kept under vacuum while on the filter, then placed in a steel tray and dried in a dryer at approximately 50° C. under vacuum. The yield of compound of formula Ia ranged from 138-167 g.

Claims

1. Formula (I): 【Chemistry 1】 or a stereoisomer, pharma- ceutically acceptable salt or solvate thereof.

2. Formula (Ia): 【Chemistry 2】 2. The crystalline form of the compound of formula (I) according to claim 1, which is a compound of the formula:

3. 3. The crystalline form of claim 1 or 2, which is an anhydrous form.

4. 4. The crystalline form of claim 3, exhibiting an X-ray powder diffraction pattern including a peak at about 10.5 degrees 2θ.

5. 5. The crystalline form of claim 3 or 4, which exhibits an X-ray powder diffraction pattern comprising peaks at about 6.2 degrees 2θ, about 7.6 degrees and about 10.5 degrees 2θ.

6. 6. The crystalline form of any one of claims 3 to 5, exhibiting an X-ray powder diffraction pattern comprising peaks at about 6.2 degrees, about 7.6 degrees, about 10.5 degrees, about 12.5 degrees and about 15.2 degrees 2θ.

7. 7. The crystalline form of any one of claims 3 to 6, exhibiting an X-ray powder diffraction pattern comprising peaks at about 6.2 degrees, about 7.6 degrees, about 10.5 degrees, about 12.5 degrees, about 15.2 degrees, about 16.2 degrees, about 18.3 degrees, about 19.9 degrees, about 23.2 degrees, and about 27.8 degrees two theta.

8. 4. The crystalline form of claim 3, exhibiting an X-ray powder diffraction pattern including a peak at about 11.4 degrees 2θ.

9. 9. The crystalline form of claim 8, exhibiting an X-ray powder diffraction pattern comprising peaks at about 8.5 degrees 2θ, about 9.8 degrees, and about 11.4 degrees 2θ.

10. 10. The crystalline form of claim 8 or 9, exhibiting an X-ray powder diffraction pattern comprising peaks at about 4.9 degrees 2θ, about 8.5 degrees, about 9.8 degrees, about 11.4 degrees and about 14.6 degrees 2θ.

11. 7. The crystalline form of any one of claims 3 to 6, exhibiting an X-ray powder diffraction pattern comprising peaks at about 4.9 degrees, about 8.5 degrees, about 9.8 degrees, about 11.4 degrees, about 14.6 degrees, about 16.5 degrees, about 17.5 degrees, about 19.5 degrees, about 21.4 degrees, about 27.7 degrees and about 28.7 degrees 2θ.

12. 3. The crystalline form of claim 1 or 2 in the form of a methanol solvate.

13. 13. The crystalline form of claim 12, exhibiting an X-ray powder diffraction pattern including a peak at about 9.5 degrees 2θ.

14. 14. The crystalline form of claim 12 or 13, exhibiting an X-ray powder diffraction pattern comprising peaks at about 7.2 degrees 2θ, about 9.5 degrees and about 13.1 degrees 2θ.

15. 15. The crystalline form of any one of claims 12 to 14, exhibiting an X-ray powder diffraction pattern comprising peaks at about 7.2 degrees, about 9.5 degrees, about 11.5 degrees, about 13.1 degrees and about 17.9 degrees two theta.

16. 16. The crystalline form of any one of claims 12 to 15, exhibiting an X-ray powder diffraction pattern comprising peaks at about 7.2 degrees, about 9.5 degrees, about 11.5 degrees, about 12.6 degrees, about 13.1 degrees, about 14.0 degrees, about 17.9 degrees, about 20.3 degrees, about 21.2 degrees, about 22.8 degrees, and about 32.6 degrees two theta.

17. 3. The crystalline form of claim 1 or 2, which is a dihydrate crystalline form.

18. 20. The crystalline form of claim 17, exhibiting an X-ray powder diffraction (XRPD) pattern comprising at least one peak at about 10.5 degrees 2θ.

19. 19. The crystalline form of claim 17 or 18, exhibiting an XRPD pattern comprising peaks at about 10.5 degrees 2θ, about 7.4 degrees, and about 12.4 degrees 2θ.

20. 20. The crystalline form of any one of claims 17 to 19, exhibiting an XRPD pattern comprising peaks at about 10.5 degrees 2θ, about 7.4 degrees, about 12.4 degrees, about 6.2 degrees, about 9.9 degrees, and about 15.3 degrees 2θ.

21. 22. The crystalline form of any one of claims 17-21, exhibiting an XRPD pattern having peaks at about 6.2 degrees 2θ, about 7.4 degrees, about 8.9 degrees, about 9.9 degrees, about 10.5 degrees, about 12.4 degrees, about 15.3 degrees, about 18.6 degrees, about 20.2 degrees, about 21.1 degrees, about 22.4 degrees, about 22.9 degrees, about 26.0 degrees, about 27.0 degrees, about 27.3 degrees, and about 33.7 degrees.

22. 22. The crystalline form of any one of claims 1 to 21, wherein the compound of formula (I) or formula (Ia) is in substantially pure form.

23. A method of making a crystalline form of a compound of formula (I), comprising the steps of: i) Formula (II): 【Chemistry 3】 wherein each R is H and -C(O)R １ and when only one compound or formula (II) is present in the composition, at least one R group is not hydrogen; and R １ is C １ ~C ２０ Alkyl, C ２ ~C ２０ Alkenyl, C ２ ~C ２０ Alkynyl, cycloalkyl, aryl, C １～１０ Alkylcycloalkyl, C ２～１０ Alkenylcycloalkyl, C ２～１０ Alkynylcycloalkyl, C １～１０ Alkylaryl, C ２～１０ Alkenylaryl, C ２～１０ Alkynylaryl, C １～１０ Alkyl C(O)R ２ , C ２～１０ Alkenyl C(O)R ２ , C ２～１０ AlkynylC(O)R ２ , C １～１０ Alkyl CH(OR ２ ) (OR ２ ), C(O)C ２～１０ Alkenyl CH(OR ２ ) (OR ２ ), C ２～１０ AlkynylCH(OR ２ ) (OR ２ ), C １～１０ Alkyl SR ２ , C ２～１０ Alkenyl SR ２ , C ２～１０ AlkynylSR ２ , C １～１０ AlkylC(O)OR ２ , C ２～１０ AlkenylC(O)OR ２ , C ２～１０ AlkynylC(O)OR ２ , C １～１０ AlkylC(O)SR ２ , C ２～１０ AlkenylC(O)SR ２ , C ２～１０ AlkynylC(O)SR ２ , 【Chemistry 4】 is selected from R ２ is hydrogen, -C １～１０ Alkyl, -C ２～１０ Alkenyl, -C ２～１０ alkynyl, cycloalkyl or aryl; wherein each alkyl, alkenyl, alkynyl, cycloalkyl or aryl group is optionally substituted; ii) treating said compound of formula (II) with 2,2-dimethoxypropane and a weak acid catalyst to obtain a compound of formula (III): 【Chemistry 5】 and forming a 5,20-acetonide of iii) de-esterifying the esters at C12 and C13 of formula (III) and treating the compound of formula (III) with a base to give formula (I): 【Chemistry 6】 providing a compound of formula (I); and iv) crystallizing said compound of formula (I).

24. The compound of formula (II) has the formula (IIa): 【Chemistry 7】 wherein each R is independently as defined in claim 23.

25. 25. The method of claim 24, wherein the stereochemistry is maintained to produce a compound of formula (Ia).

26. 26. The method of any one of claims 23 to 25, wherein the compound of formula (I) or formula (Ia) is crystallized from methanol solvent to form a methanol solvate.

27. 26. The method of any one of claims 23 to 25, wherein the compound of formula (I) or formula (Ia) is crystallized from acetonitrile to form an anhydrous crystalline form.

28. 28. The method of any one of claims 23 to 27, further comprising esterifying one or both of the C12 hydroxy group and the C13 hydroxy group of said compound of formula (I) or said compound of formula (Ia) by reaction with an activated carboxylic acid.

29. Removal of the 5,20-acetonide group under oxidation conditions affords the compound of formula (IV): 【Chemistry 8】 [wherein each R ３ is H and -C(O)R １ are independently selected from, and R １ is C １ ~C ２０ Alkyl, C ２ ~C ２０ Alkenyl, C ２ ~C ２０ Alkynyl, cycloalkyl, aryl, C １～１０ Alkylcycloalkyl, C ２～１０ Alkenylcycloalkyl, C ２～１０ Alkynylcycloalkyl, C １～１０ Alkylaryl, C ２～１０ Alkenylaryl, C ２～１０ Alkynylaryl, C １～１０ Alkyl C(O)R ２ , C ２～１０ Alkenyl C(O)R ２ , C ２～１０ AlkynylC(O)R ２ , C １～１０ Alkyl CH(OR ２ ) (OR ２ ), C(O)C ２～１０ Alkenyl CH(OR ２ ) (OR ２ ), C ２～１０ AlkynylCH(OR ２ ) (OR ２ ), C １～１０ Alkyl SR ２ , C ２～１０ Alkenyl SR ２ , C ２～１０ AlkynylSR ２ , C １～１０ AlkylC(O)OR ２ , C ２～１０ AlkenylC(O)OR ２ , C ２～１０ AlkynylC(O)OR ２ , C １～１０ AlkylC(O)SR ２ , C ２～１０ AlkenylC(O)SR ２ , C ２～１０ AlkynylC(O)SR ２ , 【Chemistry 9】 is selected from R ２ is hydrogen, -C １～１０ Alkyl, -C ２～１０ Alkenyl, -C ２～１０ alkynyl, cycloalkyl or aryl; wherein each alkyl, alkenyl, alkynyl, cycloalkyl or aryl group is optionally substituted. or a stereoisomer or a pharma- ceutically acceptable salt thereof.

30. 24. 12-Tigloyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one produced by the method of claim 23.

31. 24. 12,13-Dihexanoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one produced by the method of claim 23.

32. 12-tigloyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one or 12,13-dihexanoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one in substantially pure form.

33. Formula (I): 【Chemistry 10】 20. A method of making a crystalline form of a compound of formula (I), comprising crystallizing said compound of formula (I) from acetonitrile or methanol solvent.

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