RU2838992C2 - Crystalline forms of janus kinase inhibitor - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to a method of producing a crystalline form of 3-((3S,4R)-3-methyl-6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1,6-diazaspiro[3.4]octane-1-yl)-3-oxopropane nitrile, having Form β, having a powder X-ray diffraction spectrum containing characteristic peaks with values of 2θ (°): 10.5 ± 0.2, 11.8 ± 0.2, 19.3 ± 0.2 and 22.0 ± 0.2. Method is carried out by crystallisation 3-((3S,4R)-3-methyl-6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1,6-diazaspiro[3.4]octan-1-yl)-3-oxopropane nitrile from a solvent, in which solvent contains 1:3 (vol./vol.) of 1-butanol and acetonitrile, respectively. Mixture of the compound and the solvent is stirred at 40 °C for 10 days, with subsequent cooling to room temperature.

EFFECT: obtaining a stable crystalline form of 3-((3S,4R)-3-methyl-6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1,6-diazaspiro[3.4]octan-1-yl)-3-oxopropane nitrile.

1 cl, 17 dwg, 14 tbl, 10 ex

Description

Field of technology to which the invention relates

The present invention relates to crystalline forms of the Janus kinase (JAK) inhibitor 3-((3S,4R)-3-methyl-6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1,6-diazaspiro[3.4]octan-1-yl)-3-oxopropanenitrile (Compound A), as well as to their compositions, methods for their preparation, methods for their use and methods for quantitative analysis.

State of the art

Janus kinase (JAK) inhibitors are of interest for the treatment of various diseases, including autoimmune diseases, inflammatory diseases, and cancer. To date, two JAK inhibitors have received approval from the U.S. Food and Drug Administration (FDA). Ruxolitinib has been approved for the treatment of primary myelofibrosis and polycythemia vera (PV), and tofacitinib has been approved for the treatment of rheumatoid arthritis. Other JAK inhibitors have been described in the literature. The compound 3-((3S,4R)-3-methyl-6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1,6-diazaspiro[3.4]octan-1-yl)-3-oxopropanenitrile (Compound A) (see structure below) is an example of a spirocyclic JAK inhibitor described in U.S. Patent Application Publication No. 2011/0136778 and International Application No. PCT/JP2016/070046.

[Chem. 1]

Drug compounds, such as, for example, Compound A, are typically combined with other pharmaceutically acceptable ingredients to form compositions suitable for administration to patients. Solid formulations often require that the drug compound have appropriate characteristics in the solid state, such as resistance to heat and moisture, ease of handling, and other characteristics that facilitate the preparation of solid dosage forms. Accordingly, there is a continuing need for solid forms of existing drug molecules. The crystalline forms of Compound A described herein address this need.

Brief description of the essence of the invention

The present invention relates, inter alia, to crystalline Forms β and γ of 3-((3S,4R)-3-methyl-6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1,6-diazaspiro[3.4]octan-1-yl)-3-oxopropanenitrile (Compound A).

The present invention also relates to compositions comprising Form α together with Form β or Form γ, or both.

The present invention also relates to pharmaceutical compositions containing one or more crystalline forms or compositions of the present invention.

The present invention also relates to methods for producing the crystalline forms of the present invention.

The present invention also relates to methods for estimating the amount of crystalline forms of the present invention.

The present invention also relates to methods for inhibiting Janus kinase comprising contacting the crystalline form of the present invention with Janus kinase.

The present invention also relates to a method of treating or preventing a disease in a patient, comprising administering to the patient a therapeutically effective amount of a crystalline form or composition of the present invention.

The present invention also relates to a crystalline form or composition of the present invention for use in prophylaxis or therapy.

The present invention also relates to the use of a crystalline form or composition according to the present invention for the preparation of a medicament for use in therapy or prophylaxis.

Brief description of the drawings

Fig. 1 shows the powder X-ray diffraction spectrum corresponding to Form α.

Fig. 2 shows the X-ray powder diffraction spectrum corresponding to Form β.

Fig. 3 shows the powder X-ray diffraction spectrum corresponding to Form γ.

Fig. 4 shows the DSC thermogram corresponding to Form α.

Fig. 5 shows the DSC thermogram corresponding to Form β.

Fig. 6 shows the DSC thermogram corresponding to Form γ.

Fig. 7 shows the thermogravimetric differential thermal analysis (TG-DTA) data corresponding to Form α.

Fig. 8 shows the thermogravimetric differential thermal analysis (TG-DTA) data corresponding to Form β.

Fig. 9 shows the thermogravimetric differential thermal analysis (TG-DTA) data corresponding to Form γ.

Fig. 10 shows solid-state ^13C NMR spectroscopy data corresponding to Form α.

Fig. 11 shows solid-state ^13C NMR spectroscopy data corresponding to Form β.

Fig. 12 shows solid-state ¹³ C NMR spectroscopy data corresponding to Form γ.

Figure 13 shows superimposed X-ray powder diffraction spectra obtained for a standard sample of Compound A containing 0, 2, 5, and 10% Form β (see Example 4).

Figure 14 shows superimposed X-ray powder diffraction spectra obtained for a standard sample of Compound A containing 0, 1, 2, 5, and 10% Form γ (see Example 4).

Fig. 15 shows (from bottom to top) the powder X-ray diffraction spectra of Example 6 for the used Form α of Compound A, the used Form β of Compound A, a 1:1 by weight mixture of Forms α and β before mixing, and the crystals after mixing.

Fig. 16 shows the powder X-ray diffraction spectra of Example 7 for the used Form α of Compound A, the used Form γ of Compound A, a 1:1 by weight mixture of Forms α and γ before mixing, and the crystals after mixing.

Fig. 17 depicts the powder X-ray diffraction spectra of Example 8 for the Form γ of Compound A used, the Form β of Compound A used, a 1:1 by weight mixture of Forms γ and β before mixing, crystals obtained from formamide, crystals from N,N-dimethylformamide, and crystals from dimethyl sulfoxide.

Embodiments of the invention

The present invention describes, inter alia, crystalline forms of 3-((3S,4R)-3-methyl-6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1,6-diazaspiro[3.4]octan-1-yl)-3-oxopropanenitrile, which are Janus kinase (JAK) inhibitors and can be used to treat various diseases associated with an increase in the number or overexpression of Janus kinase receptors. In some embodiments, the crystalline forms of the present invention inhibit JAK3. In some embodiments, the crystalline forms of the present invention inhibit JAK2.

One aspect of the present invention is as follows.

[Item 1] Crystalline form of 3-((3S,4R)-3-methyl-6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1,6-diazaspiro[3.4]octan-1-yl)-3-oxopropanenitrile having Form β.

[Item 2] The crystalline form according to Item 1, having a powder X-ray diffraction spectrum containing a characteristic peak with a 2θ (°) value of about 11.8.

[Item 3] The crystalline form of Item 1 having a powder X-ray diffraction spectrum comprising two or more characteristic peaks with 2θ (°) values selected from about 10.5, about 11.8, about 19.3, and about 22.0.

[Item 4] The crystalline form of Item 1, having a powder X-ray diffraction spectrum comprising three or more characteristic peaks with 2θ (°) values selected from about 7.8, about 10.5, about 11.8, about 13.4, about 13.9, about 17.8, about 19.3, about 22.0, about 23.6, and about 28.0.

[Item 5] The crystalline form according to any one of Items 1 to 4, having a DSC thermogram characterized by having an endothermic peak at about 186°C.

[Item 6] The crystalline form according to any one of Items 1 to 5, having a solid-state ^13C NMR spectrum which is characterized by having at least one peak with a chemical shift value of about 165.1 ppm.

[Item 7] The crystalline form according to any one of Items 1 to 5, having a solid-state ^13C NMR spectrum which is characterized by having at least 5 peaks selected from peaks with chemical shift values of about 16.5, about 25.8, about 26.5, about 33.1, about 34.8, about 36.7, about 38.8, about 48.2, about 53.4, about 77.7, about 79.5, about 101.2, about 102.6, about 117.5, about 120.6, about 151.1, about 154.3, and about 165.1 ppm.

[Item 8] A crystalline form according to any one of Items 1-7, having space group P1 with the following unit cell parameters:

Table 1

a (Å) 8,043 b (Å) 11,371 c (Å) 16,522 α (°) 97,537 β (°) 94,541 γ (°) 90,294

[Item 9] A crystalline form according to any one of Items 1-8 having a purity of at least about 50%.

[Item 10] The crystalline form of any one of Items 1-8 having a purity of at least about 75%.

[Item 11] The crystalline form of any one of Items 1-8 having a purity of at least about 85%.

[Item 12] The crystalline form according to any one of Items 1-8, having a purity of at least about 90%.

[Item 13] A crystalline form according to any one of Items 1-8 having a purity of at least about 95%.

[Item 14] Crystalline form of 3-((3S,4R)-3-methyl-6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1,6-diazaspiro[3.4]octan-1-yl)-3-oxopropanenitrile having Form γ.

[Item 15] The crystalline form of Item 14 having an X-ray powder diffraction spectrum comprising a characteristic peak with a 2θ (°) value of about 16.5.

[Item 16] The crystalline form of Item 14 having an X-ray powder diffraction spectrum comprising two or more characteristic peaks with 2θ (°) values selected from about 16.5, about 17.7, about 21.4, about 21.8, and about 23.1.

[Item 17] The crystalline form of Item 14, having an X-ray powder diffraction spectrum comprising three or more characteristic peaks with 2θ (°) values selected from about 7.7, about 10.6, about 13.3, about 13.9, about 15.5, about 16.5, about 17.7, about 17.9, about 19.0, about 21.4, about 21.8, about 23.1, about 23.7, and about 28.1.

[Item 18] A crystalline form according to any one of Items 14 to 17, having a DSC thermogram characterized by having an endothermic peak at about 196°C.

[Item 19] The crystalline form according to any one of Items 14 to 18, having a solid-state ^13C NMR spectrum which is characterized by having at least one peak with a chemical shift value of about 162.9 ppm.

[Item 20] The crystalline form according to any one of Items 14 to 19, having a solid-state ^13C NMR spectrum which is characterized by having at least 5 peaks selected from peaks with chemical shift values of about 16.9, about 26.5, about 32.9, about 36.4, about 48.1, about 53.7, about 78.6, about 102.6, about 116.4, about 117.9, about 121.5, about 151.8, about 154.6, and about 162.9 ppm.

[Item 21] A crystalline form according to any one of Items 14 to 20, having a space group P2 ₁ with the following unit cell parameters:

Table 2

a (Å) 8,095 b (Å) 8,017 c (Å) 11,459 α (°) 90 β (°) 95,663 γ (°) 90

[Item 22] A crystalline form according to any one of Items 14-21 having a purity of at least about 50%.

[Item 23] A crystalline form according to any one of Items 14-21 having a purity of at least about 75%.

[Item 24] A crystalline form according to any one of Items 14-21 having a purity of at least about 85%.

[Item 25] A crystalline form according to any one of Items 14-21 having a purity of at least about 90%.

[Item 26] A crystalline form according to any one of Items 14-21 having a purity of at least about 95%.

[Item 27] A method for producing a crystalline form according to any one of Items 1 to 13, comprising crystallizing 3-((3S,4R)-3-methyl-6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1,6-diazaspiro[3.4]octan-1-yl)-3-oxopropanenitrile from a solvent.

[Item 28] The method of Item 27, wherein the solvent comprises 1-butanol and acetonitrile.

[Item 29] The method of Item 28, wherein the ratio of 1-butanol to acetonitrile is about 1:3 (v/v).

[Item 30] Crystalline form β obtained by the method according to any of Items 27 to 29.

[Item 31] A process for preparing a crystalline form according to any one of Items 14 to 26, comprising converting Form α of 3-((3S,4R)-3-methyl-6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1,6-diazaspiro[3.4]octan-1-yl)-3-oxopropanenitrile into Form γ.

[Item 32] The method of Item 31 comprising stirring Form α of 3-((3S,4R)-3-methyl-6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1,6-diazaspiro[3.4]octan-1-yl)-3-oxopropanenitrile in dimethylformamide (DMF).

[Item 33] The method of Item 31 comprising stirring Form α of 3-((3S,4R)-3-methyl-6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1,6-diazaspiro[3.4]octan-1-yl)-3-oxopropanenitrile in formamide.

[Item 34] Crystalline form γ obtained by the method according to any one of Items 31 to 33.

[Item 35] A composition of crystalline 3-((3S,4R)-3-methyl-6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1,6-diazaspiro[3.4]octan-1-yl)-3-oxopropanenitrile comprising Form α and Form β.

[Item 36] The composition of Item 35, consisting essentially of Form α and Form β.

[Item 37] The composition of Item 35 or 36, wherein Form β is present in an amount of from about 1 to about 50% w/w relative to Form α.

[Item 38] The composition of Item 35 or 36, wherein Form β is present in an amount of from about 1 to about 20% w/w relative to Form α.

[Item 39] The composition of Item 35 or 36, wherein Form β is present in an amount of from about 1 to about 10% w/w relative to Form α.

[Item 40] The composition of Item 35 or 36, wherein Form β is present in an amount of from about 1 to about 5% w/w relative to Form α.

[Item 41] The composition according to any one of Items 35-40, further comprising Form γ.

[Item 42] A composition of crystalline 3-((3S,4R)-3-methyl-6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1,6-diazaspiro[3.4]octan-1-yl)-3-oxopropanenitrile comprising Form β and Form γ.

[Item 43] The composition of Item 42, consisting essentially of Form β and Form γ.

[Item 44] The composition of Item 42 or 43, wherein Form γ is present in an amount of from about 1 to about 50% w/w relative to Form β.

[Item 45] The composition of Item 42 or 43, wherein Form γ is present in an amount of from about 1 to about 20% w/w relative to Form β.

[Item 46] The composition of Item 42 or 43, wherein Form γ is present in an amount of from about 1 to about 10% w/w relative to Form β.

[Item 47] The composition of Item 42 or 43, wherein Form γ is present in an amount of from about 1 to about 5% w/w relative to Form β.

[Item 48] The composition according to any one of Items 42-47, further comprising Form α.

[Item 49] A pharmaceutical composition comprising the crystalline form or composition according to any one of Items 1 to 48 and a pharmaceutically acceptable carrier.

[Item 50] The pharmaceutical composition of Item 49, further comprising a second therapeutic agent.

[Item 51] A pharmaceutical composition according to Item 49 or 50, which is suitable for oral, parenteral, pulmonary, topical or external use.

[Item 52] A pharmaceutical composition according to Item 49 or 50, which is suitable for external use.

[Item 53] The pharmaceutical composition according to Item 49 or 50 in the form of a tablet, capsule, pill, powder or ointment.

[Item 54] A pharmaceutical composition according to Item 49 or 50 in the form of a powder suitable for reconstitution in liquid for intravenous, intramuscular or subcutaneous administration.

[Item 55] A pharmaceutical composition according to Item 49 or 50, comprising white soft paraffin, hard paraffin, squalene or a mixture thereof.

[Item 56] A method of inhibiting Janus kinase comprising contacting Janus kinase with a crystalline form or composition according to any one of Items 1-55.

[Item 57] The method of Item 56, wherein the Janus kinase is Janus kinase 3 (JAK3).

[Item 58] The method of Item 56, wherein the Janus kinase is Janus kinase 2 (JAK2).

[Item 59] A method for treating or preventing a disease selected from organ transplant rejection, graft-versus-host disease after transplant, autoimmune disease, allergic diseases, and chronic myeloproliferative disease, comprising administering to a mammal a therapeutically effective amount of the crystalline form or composition of any one of Items 1-55.

[Item 60] A method for treating or preventing rheumatoid arthritis, psoriasis, alopecia areata, dry eye, atopic dermatitis, eczema or hand eczema, comprising administering to a mammal a therapeutically effective amount of the crystalline form or composition of any one of Items 1-55.

[Item 61] A crystalline form or composition according to any one of Items 1 to 55 for use as a pharmaceutically active ingredient.

[Item 62] The crystalline form or composition according to any one of Items 1 to 55 for use in the treatment or prevention of organ rejection after transplantation, graft-versus-host disease after transplantation, autoimmune disease, allergic diseases, or chronic myeloproliferative disease.

[Item 63] The crystalline form according to any one of Items 1 to 55 for use in the treatment or prevention of rheumatoid arthritis, psoriasis, alopecia areata, dry eye, atopic dermatitis, eczema or hand eczema.

[Item 64] A method of measuring the amount of Form β present in the preparation of Form α of 3-((3S,4R)-3-methyl-6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1,6-diazaspiro[3.4]octan-1-yl)-3-oxopropanenitrile, comprising measuring the area of a peak in an X-ray powder diffraction spectrum that is characteristic of Form β and comparing the area of that peak with a standard.

[Item 65] The method of Item 64, wherein the peak characteristic of Form β is at about 10.6° 2-theta.

[Item 66] A method of measuring the amount of Form γ present in the preparation of Form α of 3-((3S,4R)-3-methyl-6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1,6-diazaspiro[3.4]octan-1-yl)-3-oxopropanenitrile, comprising measuring the area of a peak in an X-ray powder diffraction spectrum that is characteristic of Form γ and comparing the area of that peak with a standard.

[Item 67] The method of Item 66, wherein the peak characteristic of Form γ is at about 16.6° 2-theta.

As used herein, “crystalline form” means a specific crystal lattice configuration of a crystalline compound. Different crystalline forms of the same compound typically have different crystal lattices (e.g., unit cell size) and different physical properties associated with differences in the crystal lattices, and in some cases have different water or solvent contents. Different crystal lattices can be identified by solid-state characterization methods such as powder X-ray diffraction. Other characterization methods such as differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), dynamic vapor sorption (DVS), solid-state NMR, etc. further assist in identifying the crystalline form and also assist in assessing its stability and water/solvent content.

Different crystalline forms of a single compound may include both anhydrous forms of the compound and solvated/hydrated forms of the compound, where each of the anhydrous forms and solvated/hydrated forms is distinguished from the others by differences in their X-ray powder diffraction spectra, thereby indicating differences in the crystal lattices. In some cases, a single crystalline form (e.g., identified by a unique X-ray powder diffraction spectrum) may have different water or solvent contents, with the lattice remaining substantially unchanged (as is the X-ray powder diffraction spectrum) despite variations in water and/or solvent content.

A powder X-ray diffraction spectrum, consisting of reflections (peaks), is commonly regarded as a fingerprint of a given crystalline form. It is well known that the relative intensities of peaks in a powder X-ray diffraction spectrum may vary depending on, among other things, the sample preparation technique, the crystal size distribution, the sample mounting technique, and the instrument used for analysis. In some cases, new peaks may be observed or existing peaks may disappear depending on the type of instrument or its settings (e.g., whether a Ni filter is used or not). In addition, differences in instruments and other factors may affect 2-theta values. Thus, peak values such as those reported herein may vary by ±0.2°, ±0.1°, or ±0.04° (2-theta), and the term “substantially” when used in the context of powder X-ray diffraction herein encompasses the above-listed variations.

Similarly, temperature values when discussing the results of DSC, TGA, or other thermal experiments may vary by approximately ±3°C depending on the instrument, its settings, sample preparation, etc. Thus, temperature values such as those given herein may vary by ±3°C, and a crystalline form described herein having a DSC thermogram or other thermogram “substantially” the same as in any of the figures encompasses such variations.

Furthermore, chemical shift values can vary up to ±0.2 ppm in ^13C NMR spectra, and the term “substantially” when used in the context of NMR data in this text covers such variations.

Form β

The present invention describes a crystalline form of 3-((3S,4R)-3-methyl-6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1,6-diazaspiro[3.4]octan-1-yl)-3-oxopropanenitrile (Compound A) having Form β. The crystalline form corresponding to Form β is anhydrous, as evidenced, for example, by DSC and TG-DTA data (see Example 2). In some embodiments, Form β has an X-ray powder diffraction spectrum comprising a characteristic peak with a 2θ (°) value of about 11.8. In other embodiments, Form β has an X-ray powder diffraction spectrum comprising two or more characteristic peaks with 2θ (°) values selected from about 10.5, about 11.8, about 19.3, and about 22.0. In other embodiments, Form β has an X-ray powder diffraction spectrum comprising three or more characteristic peaks with 2θ (°) values selected from about 7.8, about 10.5, about 11.8, about 13.4, about 13.9, about 17.8, about 19.3, about 22.0, about 23.6, and about 28.0. In other embodiments, Form β has an X-ray powder diffraction spectrum comprising three or more characteristic peaks with 2θ (°) values selected from the peaks listed in Table 6 (see Example 2). In other embodiments, Form β has an X-ray powder diffraction spectrum substantially as in Fig. 2.

In some embodiments, Form β has a DSC thermogram that features an endothermic peak at about 186°C. In some embodiments, Form β has a DSC thermogram that features an extrapolated transition onset temperature of about 185°C. In other embodiments, Form β has a DSC thermogram substantially as in Fig. 5.

In some embodiments, Form β has a solid-state ¹³ C NMR spectrum that is characterized by having at least one peak with a chemical shift value of about 165.1 ppm. In other embodiments, Form β has a solid-state ¹³ C NMR spectrum that is characterized by having at least 5 peaks selected from peaks with chemical shift values of about 16.5, about 25.8, about 26.5, about 33.1, about 34.8, about 36.7, about 38.8, about 48.2, about 53.4, about 77.7, about 79.5, about 101.2, about 102.6, about 117.5, about 120.6, about 151.1, about 154.3, and about 165.1 ppm. In other embodiments, Form β has a solid state ^13C NMR spectrum substantially as depicted in Fig. 11.

In some embodiments, Form β has space group P1 with the following unit cell parameters:

Table 3

a (Å) 8,043 b (Å) 11,371 c (Å) 16,522 α (°) 97,537 β (°) 94,541 γ (°) 90,294

according to the data of X-ray structural analysis of a single crystal (see Example 5).

Form β may have a purity of at least about 50%, at least about 75%, at least about 85%, at least about 90%, or at least about 95%. In some embodiments, Form β is substantially pure.

Form β can be prepared by crystallizing 3-((3S,4R)-3-methyl-6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1,6-diazaspiro[3.4]octan-1-yl)-3-oxopropanenitrile (Compound A) from a solvent. For example, Compound A (e.g., as Form α) can be combined with a solvent to form a solution or suspension from which crystals of Form β are obtained. In some embodiments, the solvent includes a nitrile, such as acetonitrile, or a mixture of 1-butanol and acetonitrile. In some embodiments, the solvent comprises about 1:3 (v/v) 1-butanol and acetonitrile, respectively. In some embodiments, Compound A can be combined with a solvent and heated to a temperature between about 30 and about 50°C. In some embodiments, the mixture may be heated to a temperature between about 35 and about 45°C, or to about 40°C. After heating, the mixture may be cooled, such as to about room temperature (e.g., about 23°C), to obtain the crystalline product, Form β.

The present invention also describes Form β prepared by any of the methods described above.

Form γ

The present invention also provides a crystalline form of 3-((3S,4R)-3-methyl-6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1,6-diazaspiro[3.4]octan-1-yl)-3-oxopropanenitrile (Compound A) having Form γ. The crystalline form corresponding to Form γ is anhydrous, as demonstrated, for example, by DSC and TG-DTA data (see Example 3). In some embodiments, Form γ has an X-ray powder diffraction spectrum comprising a characteristic peak with a 2θ (°) value of about 16.5. In other embodiments, Form γ has an X-ray powder diffraction spectrum comprising two or more characteristic peaks with 2θ (°) values selected from about 16.5, about 17.7, about 21.4, about 21.8, and about 23.1. In other embodiments, Form γ has an X-ray powder diffraction spectrum comprising three or more characteristic peaks with 2θ (°) values selected from about 7.7, about 10.6, about 13.3, about 13.9, about 15.2, about 16.5, about 17.7, about 17.9, about 19.0, about 21.4, about 21.8, about 23.1, about 23.7, and about 28.1. In other embodiments, Form γ has an X-ray powder diffraction spectrum comprising three or more characteristic peaks with 2θ (°) values selected from the peaks listed in Table 7 (see Example 3). In other embodiments, Form γ has an X-ray powder diffraction spectrum substantially as in Fig. 3.

In some embodiments, Form γ has a DSC thermogram that features an endothermic peak at about 196°C. In some embodiments, Form γ has a DSC thermogram that features an extrapolated transition onset temperature of about 196°C. In other embodiments, Form γ has a DSC thermogram substantially as in Fig. 6.

In some embodiments, Form γ has a solid-state ¹³ C NMR spectrum that is characterized by having at least one peak with a chemical shift value of about 162.9 ppm. In other embodiments, Form γ has a solid-state ¹³ C NMR spectrum that is characterized by having at least 5 peaks selected from peaks with chemical shift values of about 16.9, about 26.5, about 32.9, about 36.4, about 48.1, about 53.7, about 78.6, about 102.6, about 116.4, about 117.9, about 121.5, about 151.8, about 154.6, and about 162.9 ppm. In other embodiments, Form γ has a solid-state ¹³ C NMR spectrum substantially as depicted in Fig. 11.

In some embodiments, Form γ has space group P2 ₁ with the following unit cell parameters:

Table 4

a (Å) 8,095 b (Å) 8,017 c (Å) 11,459 α (°) 90 β (°) 95,663 γ (°) 90

according to the data of X-ray structural analysis of a single crystal (see Example 5).

Form γ may have a purity of at least about 50%, at least about 75%, at least about 85%, at least about 90%, or at least about 95%. In some embodiments, Form γ is substantially pure.

Form γ can be prepared by converting Form α of 3-((3S,4R)-3-methyl-6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1,6-diazaspiro[3.4]octan-1-yl)-3-oxopropanenitrile to Form γ. This conversion can be carried out, for example, by stirring Form α in dimethylformamide (DMF). Stirring can be carried out, for example, at room temperature. Alternatively, Form γ can be prepared by combining Compound A with formamide and adding a seed crystal of Form γ. This preparation can be carried out at room temperature and optionally under an inert atmosphere such as nitrogen.

The present invention also describes Form γ prepared by any of the methods described above.

Compositions

In addition to Forms β and γ, Compound A can be obtained as anhydrous crystalline Form α, which is described in Example 1. Accordingly, the present invention describes mixtures of two or more of Forms α, β, and γ of Compound A.

In some embodiments, the present invention features a crystalline 3-((3S,4R)-3-methyl-6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1,6-diazaspiro[3.4]octan-1-yl)-3-oxopropanenitrile composition (Compound A) comprising Form α and Form β. In some embodiments, Form β is present in an amount of about 1 to about 50% w/w relative to Form α. In other embodiments, Form β is present in an amount of about 1 to about 20% w/w relative to Form α. In other embodiments, Form β is present in an amount of about 1 to about 10% w/w relative to Form α. In other embodiments, Form β is present in an amount of about 1 to about 5% w/w relative to Form α. In some embodiments, the composition comprises Form α and Form β and is substantially free of other crystalline forms of Compound A. In other embodiments, the composition comprising Form α and Form β further comprises Form γ. In some embodiments, the composition consists essentially only of Form α and Form β.

Additionally, the present invention features a composition of crystalline 3-((3S,4R)-3-methyl-6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1,6-diazaspiro[3.4]octan-1-yl)-3-oxopropanenitrile (Compound A) comprising Form β and Form γ. In some embodiments, Form γ is present in an amount of from about 1 to about 50% w/w relative to Form β. In other embodiments, Form γ is present in an amount of from about 1 to about 20% w/w relative to Form β. In other embodiments, Form γ is present in an amount of from about 1 to about 10% w/w relative to Form β. In other embodiments, Form γ is present in an amount of from about 1 to about 5% w/w relative to Form β. In some embodiments, the composition comprises Form β and Form γ and is substantially free of other crystalline forms of Compound A. In other embodiments, the composition comprising Form β and Form γ further comprises Form α. In some embodiments, the composition consists essentially only of Form β and Form γ.

The present invention also provides compositions comprising Form β or Form γ and one or more other substances. In some embodiments, the composition comprises at least about 50%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% by weight of Form β or Form γ.

Analytical methods

The present invention describes a method of determining the amount of Form β present in the preparation of Form α, comprising measuring the area of a peak in an X-ray powder diffraction spectrum characteristic of Form β and comparing the area of the peak to a standard. In some embodiments, the peak characteristic of Form β is at about 10.6° 2-theta. The standard can be a standard curve calculated from the peak areas of known amounts of Form β in Form α. Similarly, the present invention describes a method of measuring the amount of Form γ present in the preparation of Form α, comprising measuring the area of a peak in an X-ray powder diffraction spectrum characteristic of Form γ and comparing the area of the peak to a standard. In some embodiments, the peak characteristic of Form γ is at about 16.6° 2-theta. A standard curve calculated from the peak areas of known amounts of Form γ in Form α can serve as a standard.

Pharmaceutical compositions and applications

The crystalline forms of the present invention can be formulated into pharmaceutical compositions that comprise a crystalline form of the present invention or a composition of the present invention together with at least one pharmaceutically acceptable carrier (or excipient). In one embodiment, the pharmaceutical composition is suitable for oral, parenteral, pulmonary, topical or external use. In some embodiments, the pharmaceutical composition is in the form of an oral preparation, such as a tablet, capsule, granule, powder, molded tablet, syrup, emulsion, suspension, or a parenteral preparation, such as an external preparation, suppository, injection, drops, nasal preparation, pulmonary preparation. In some embodiments, the pharmaceutical composition is suitable for external use, such as an ointment.

The pharmaceutical compositions of the present invention can be prepared by mixing one or more crystalline forms of the present invention or a composition of the present invention with at least one or more pharmaceutically acceptable carriers in suitable amounts according to techniques known in the art of medical preparations. The amount of Compound A in the pharmaceutical composition depends on the dosage forms, dosages, etc., and can be, for example, from about 0.1 to 100% by weight of the composition. The pharmaceutical compositions of the present invention can be, for example, in the form of a tablet, capsule, pill, powder or ointment. In some embodiments, the pharmaceutical compositions of the present invention can be in the form of a powder suitable for reconstitution in a liquid for intravenous (IV), intramuscular (IM) or subcutaneous (SC) administration. In addition, the pharmaceutical compositions of the present invention can further comprise a second therapeutic agent.

The term “pharmaceutically acceptable carrier” includes various conventional organic or inorganic carriers for pharmaceutical compounds, such as a diluent, disintegrating agent, binder, flow additive and lubricant for solid preparations, a solvent, solubilizer, suspending agent, tonicity regulator, buffer additive and soothing agent for liquid preparations, and a base, emulsifier, humectant, stabilizer, stabilizing agent, dispersant, plasticizer, pH regulator, absorption promoter, gelling agent, antiseptic, filler, anti-inflammatory agent, solubilizer and suspending agent for semi-solid preparations. In addition, additives such as a preservative, antioxidant, coloring agent and sweetener can be used if necessary.

Examples of diluents include lactose, sucrose, D-mannitol, D-sorbitol, corn starch, dextrin, microcrystalline cellulose, crystalline cellulose, carmellose, carmellose calcium, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, acacia, etc.

Examples of disintegrants include carmellose, carmellose calcium, carmellose sodium, sodium carboxymethyl starch, croscarmellose sodium, crospovidone, low-substituted hydroxypropyl cellulose, hydroxypropyl cellulose, crystalline cellulose, etc.

Examples of binders include hydroxypropyl cellulose, hydroxypropyl methylcellulose, povidone, crystalline cellulose, sucrose, dextrin, starch, gelatin, sodium carmellose, acacia, etc.

Examples of flow aids include light anhydrous silicic acid, magnesium stearate, etc.

Examples of lubricants include magnesium stearate, calcium stearate, talc, etc.

Examples of solvents include purified water, ethanol, propylene glycol, macrogol, sesame oil, corn oil, olive oil, etc.

Examples of solubilizers include propylene glycol, D-mannitol, benzyl benzoate, ethanol, triethanolamine, sodium carbonate, sodium citrate, etc.

Examples of suspending agents include benzalkonium chloride, carmellose, hydroxypropyl cellulose, propylene glycol, povidone, methylcellulose, glyceryl monostearate, etc.

Examples of tonicity regulators include glucose, D-sorbitol, sodium chloride, D-mannitol, etc.

Examples of buffering additives include sodium hydrogen phosphate, sodium acetate, sodium carbonate, sodium citrate, etc.

Examples of sedatives include benzyl alcohol, etc.

Examples of preservatives include ethyl paraoxybenzoate, chlorobutanol, benzyl alcohol, sodium dehydroacetate, sorbic acid, etc.

Examples of antioxidants include sodium sulfite, ascorbic acid, etc.

Examples of coloring agents include food colorings (e.g. food coloring red #2 or 3, food coloring yellow #4 or 5, etc.), β-carotene, etc.

Examples of sweeteners include sodium saccharin, dipotassium glycyrrhizinate, aspartame, etc.

The pharmaceutical compositions of the present invention can be administered orally or parenterally (e.g., locally, rectally, intravenously, etc.) to a patient, such as non-human mammals (e.g., mice, rats, rodents, guinea pigs, rabbits, cats, dogs, pigs, cows, horses, sheep, monkeys, etc.) or humans, in a therapeutically effective amount for treating or preventing a disease. The term "mammal" includes both humans and non-human mammals. The dose of the pharmaceutical composition depends on the subjects, diseases, pathological conditions, dosage form, administration route. The dose for oral administration to adult patients (body weight about 60 kg) suffering from organ rejection after transplantation, graft-versus-host disease after transplantation, autoimmune disease or allergic disease, etc. can be, for example, about 1 mg to 1 gram per day. The entire dose may be administered at one time or in several separate doses.

For external pharmaceutical compositions, the carrier (or diluent) may be white soft paraffin, hard paraffin, squalene, or a combination thereof. White soft paraffin, hard paraffin, and squalene may be combined in a ratio of 70-90 wt.%, 5-10 wt.%, and 5-20 wt.%, respectively. An example of such a preparation contains Compound A, white soft paraffin, 5±2 wt.% hard paraffin, and 10±2 wt.% squalene.

The external agent can be applied, for example, by simple application, smearing or spraying, depending on the dosage form, etc. The amount of the external agent applied to the affected area can be selected depending on the content of the active substance, etc., and the external agent can be applied, for example, once or in several doses during the day. In some embodiments, the application is carried out once a day or twice a day.

The pharmaceutical compositions can be prepared according to conventional pharmaceutical techniques, such as, for example, those described in Remingtons Pharmaceutical Sciences, 17th edition, edited by Alfonoso R. Gennaro, Mack Publishing Company, Easton, PA (1985), which is incorporated herein by reference in its entirety.

The crystalline forms of the present invention can be used in a method of inhibiting Janus kinase, such as inhibiting JAK3, JAK2, or both, comprising contacting the crystalline form of the present invention with a Janus kinase inhibitor. The contact can occur in vitro or in vivo.

The crystalline forms or compositions of the present invention can be used as an active ingredient in a method of treating or preventing one or more of the following diseases in a patient:

(a) organ rejection after transplantation or graft-versus-host disease after transplantation;

(b) autoimmune diseases including rheumatoid arthritis, psoriasis, psoriatic arthritis, multiple sclerosis, ulcerative colitis, Crohn's disease, systemic lupus erythematosus, type 1 diabetes, myasthenia gravis, Castleman's disease, juvenile idiopathic arthritis, dry eye; and

(c) allergic diseases, including asthma, atopic dermatitis, rhinitis.

In some embodiments, the crystalline forms of the present invention can be used as an active ingredient in a therapeutic or prophylactic agent for rheumatoid arthritis, psoriasis, focal alopecia, dry eye, atopic dermatitis, eczema or hand eczema.

The crystalline forms of the present invention can be used as an active substance in a therapeutic or prophylactic agent for chronic myeloproliferative diseases including polycythemia vera, primary myelofibrosis, idiopathic thrombocythemia, etc.

The term “therapeutically effective amount” of a compound, as used herein, means an amount sufficient to cure, alleviate, or partially arrest the clinical manifestations of a particular disease and its complications. An amount sufficient to accomplish such a task is called a “therapeutically effective amount.” The effective amount for each purpose depends on the severity of the disease or lesion, as well as the body weight and general health of the subject.

The term “treatment” as used herein includes alleviation of a symptom, prevention of development of a complication, maintenance of remission, prevention of exacerbation, and prevention of recurrence. The term “treatment” may also include slowing the progression of a disease, disorder, or condition, alleviating or eliminating symptoms and complications, and/or curing or eliminating a disease, disorder, or condition.

The term "treatment" can also mean the care of a patient with the aim of combating a disease, disorder, or pathological condition.

The term “prevention” means suppressing the occurrence of a symptom.

Combination therapy

The crystalline forms of the present invention can be administered to a patient in combination with a therapeutically effective amount of one or more additional therapeutic agents. The crystalline form of the present invention can be administered simultaneously (e.g., together) with the additional therapeutic agent (e.g., as a single dosage form or as separate dosage forms). Likewise, the crystalline form of the present invention and the additional therapeutic agent can be administered to a patient sequentially. For example, the additional therapeutic agent is administered when the crystalline form of the present invention already exhibits its therapeutic effect, or vice versa.

Some examples are provided below to aid in understanding the invention described herein. It should be understood that these examples are provided for illustrative purposes only and do not limit the scope of the present invention in any way.

Examples

Example 1

Preparation of 3-((3S,4R)-3-methyl-6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1,6-diazaspiro[3.4]octan-1-yl)-3-oxopropanenitrile (Compound A) and characterization of Crystal Form α.

Obtaining Compound A

Compound A was prepared by the synthesis method presented below and described in PCT/JP2016/070046 (Examples 1-15). A seed crystal was used in the crystallization steps, but crystals of each compound can be obtained according to the described procedures without using a seed crystal.

The abbreviations used in this text have the following meanings:

S-BAPO: (S)-2-(benzylamino)propan-1-ol

S-BBMO: tert-butyl (S)-N-benzyl-N-(1-hydroxypropan-2-yl)glycinate

R-BCAB: tert-butyl (R)-N-benzyl-N-(2-chloropropyl)glycinate

S-MABB: tert-butyl (3S)-1-benzyl-3-methylazetidine-2-carboxylate

S-MABB-HC: tert-butyl (3S)-1-benzyl-3-methylazetidine-2-carboxylate hydrochloride

S-MACB-HC: tert-butyl (3S)-3-methylazetidine-2-carboxylate hydrochloride

S-ZMAB: 1-benzyl 2-(tert-butyl) (3S)-3-methylazetidine-1,2-dicarboxylate

RS-ZMBB: 1-benzyl 2-(tert-butyl) (2R,3S)-2-(2-(tert-butoxy)-2-oxoethyl)-3-methylazetidine-1,2-dicarboxylate

RS-ZMAA: (2R,3S)-1-((benzyloxy)carbonyl)-2-(carboxymethyl)-3-methylazetidine-2-carboxylic acid

RS-ZMAA-DN⋅2H ₂ O: disodium (2R,3S)-1-((benzyloxy)carbonyl)-2-(carboxymethyl)-3-methylazetidine-2-carboxylate dihydrate

RS-ZMOO: benzyl (2R,3S)-2-(2-hydroxyethyl)-2-(hydroxymethyl)-3-methylazetidine-1-carboxylate

RS-ZMSS: benzyl (2R,3S)-3-methyl-2-(2-((methylsulfonyl)oxy)ethyl)-2-(((methylsulfonyl)oxy)methyl)azetidine-1-carboxylate

SR-ZMDB: benzyl (3S,4R)-6-benzyl-3-methyl-1,6-diazaspiro[3.4]octane-1-carboxylate

SR-MDOZ: benzyl (3S,4R)-3-methyl-1,6-diazaspiro[3.4]octane-1-carboxylate

SR-MDOZ-OX: benzyl (3S,4R)-3-methyl-1,6-diazaspiro[3.4]octane-1-carboxylate oxalate

SR-MDPZ: benzyl (3S,4R)-3-methyl-6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1,6-diazaspiro[3.4]octane-1-carboxylate

SR-MDOP: 4-[(3S,4R)-3-methyl-1,6-diazaspiro[3.4]-octan-6-yl]-7H-pyrrolo[2,3-d]pyrimidine

Compound A: 3-[(3S,4R)-3-methyl-6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1,6-diazaspiro[3.4]octan-1-yl]-3-oxopropanenitrile

CPPY: 4-chloro-7H-pyrrolo[2,3-d]pyrimidine

DPCN: 1-cyanoacetyl-3,5-dimethyl-1H-pyrazole

TBBA: tert-butyl bromoacetic acid

THF: tetrahydrofuran.

The instruments used and the conditions for recording the spectra are indicated below.

¹ H-NMR spectra were recorded in CDCl ₃ or DMSO-d ₆ using tetramethylsilane as an internal standard, and all δ values are reported in parts per million (ppm). Spectra were recorded on a 400 MHz NMR instrument unless otherwise stated.

The symbols in the Examples have the meanings indicated below.

c: singlet

d: doublet

t: triplet

kv: quartet

dd: doublet of doublets

ddd: doublet doublets doublets

broad.s: broadened singlet

m: multiplet

J: spin-spin interaction constant

The ion content of the sample was determined as the average of three measurements for the sample.

Device: Ion exchange chromatograph LC-20 (Shimadzu Corporation)

Analysis conditions: SHIMADZU CDD-10A VP conductivity detector

SHIMADZU SHIM-PAC IC-A3 Anion Analysis Column

SHIMADZU SHIM-PAC IC-C1 Cation Analysis Column

The water content of the sample was determined using the Karl Fischer titration method.

Device: Titrator for determination of water content CA-06 (Mitsubishi Chemical)

Analysis conditions:

Sample quantity: about 20mg

Reagent: Aquamicron AX anode solution (API Corporation)

Aquamicron CXU Cathode Solution (API Corporation)

The mass percentages of carbon, hydrogen and nitrogen in the samples were determined by elemental analysis.

Step A. Preparation of S-MABB-HC (Compound [5])

[Chem. 2]

Stage 1

[Chem. 3]

S-BAPO[1] (35.0 g, 212 mmol) was added into water (175 mL) at room temperature under nitrogen atmosphere. Toluene (53 mL) and potassium carbonate (32.2 g, 233 mmol) were added to the resulting suspension at room temperature. TBBA (434.4 g, 223 mmol) was added dropwise to the resulting solution at room temperature, then the used dropping funnel was washed with toluene (17 mL), and the washings were added to the reaction mixture. The reaction mixture was stirred at 65 °C for 21 h, then cooled to room temperature. Toluene (105 mL) was added to the reaction mixture, then the mixture was stirred, and the organic layer was separated. The organic layer was washed with water (175 mL), the aqueous layer was separated, and the solvent was removed from the organic layer in vacuo. Toluene (105 mL) was added to the residue and the resulting toluene solution was evaporated. This operation was repeated two more times to obtain a toluene solution of S-BBMO [2] (74.0 g, 212 mmol theoretically). The resulting toluene solution of S-BBMO was used in the next step, considering the yield to be 100%.

The crude product S-BBMO, which was obtained according to the above procedure, was evaporated to dryness and then characterized by NMR and MS methods.

^1H -NMR (DMSO- _d6 ) δ: 7.36-7.13 (5H, m), 4.26 (1H, dd, J = 6.8, 3.9 Hz), 3.72 (2H, dd, J = 14.2, 6.8 Hz), 3.47-3.38 (1H, m), 3.30-3.08 (3H, m), 2.79 (1H, sextet, J = 6.8 Hz), 1.35 (9H, s), 0.96 (3H, d, J = 6.8 Hz).

MS: m/z = 280 [M+H] ⁺

Stage 2

[Chem. 4]

Toluene (200 mL), tetrahydrofuran (35 mL), and triethylamine (25.7 g, 254 mmol) were added to a toluene solution of S-BBMO [2] (74.0 g, 212 mmol) at room temperature under nitrogen atmosphere. Methanesulfonyl chloride (26.7 g, 233 mmol) was added dropwise to the resulting mixture at 0 °C, then the used dropping funnel was washed with toluene (10 mL), and the washings were added to the reaction mixture. The reaction mixture was stirred at room temperature for 2 h, then at 65 °C for 22 h, and cooled to room temperature. Aqueous sodium bicarbonate solution (105 mL) was added to the reaction mixture, and the mixture was stirred, and the organic layer was separated. The organic layer was washed with water (105 mL), the aqueous layer was separated and the solvent was removed from the organic layer in vacuo. Toluene (105 mL) was added to the residue and the toluene solution was evaporated. This operation was repeated two more times to obtain a toluene solution of R-BCAB [3] (75.3 g, 212 mmol theoretically). The obtained toluene solution of R-BCAB was used in the next step, considering the yield to be 100%.

The crude product R-BCAB, which was obtained according to the above-described procedure, was evaporated to dryness and then analyzed by NMR and MS.

^1H -NMR (DMSO- _d6 ) δ: 7.28-7.11 (5H, m), 4.24-4.11 (1H, m), 3.80 (2H, d, J = 3.6 Hz), 3.24 (2H, d, J = 3.6 Hz), 2.98-2.78 (2H, m), 1.46-1.37 (12H, m).

MS: m/z = 298 [M+H] ⁺

Stage 3

[Chem. 5]

Tetrahydrofuran (88.0 mL) and 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (42.0 mL) were added to a toluene solution of R-BCAB [3] (75.3 g, 212 mmol) under nitrogen atmosphere at room temperature. Lithium bis(trimethylsilyl)amide/tetrahydrofuran solution (195 mL, 233 mmol) was added dropwise to the resulting solution at 0 °C, then the used dropping funnel was washed with tetrahydrofuran (17.0 mL), and the washings were added to the reaction mixture. The reaction mixture was stirred at 0 °C for 1 h and then warmed to room temperature. Water (175 mL) and toluene (175 mL) were added to the reaction mixture, then the mixture was stirred, and the organic layer was separated. The resulting organic layer was washed with aqueous ammonium chloride (175 mL) and then with water (175 mL), and the solvent was removed from the organic layer in vacuo. Ethyl acetate (175 mL) was added to the residue, and the resulting ethyl acetate solution was evaporated. This operation was repeated two more times to obtain an ethyl acetate solution of S-MABB [4] (66.5 g, 212 mmol theoretically). The resulting ethyl acetate solution of S-MABB was used in the next step, considering the yield to be 100%.

The crude product S-MABB, which was obtained according to the above procedure, was evaporated to dryness and then characterized by NMR and MS.

¹ H-NMR (DMSO-d ₆ ) δ: 7.28-7.25 (10H, m), 3.75 (1H, d, J = 12.7 Hz), 3.68 (1H, d, J = 1.4 Hz), 3.66 (1H, d, J = 6.7 Hz), 3.46 (2H, d, J = 12.7 Hz), 3.30-3.17 (2H, m), 2.95 (1H, dd, J = 6.2, 1.2 Hz), 2.77 (1H, dd, J = 6.1, 2.2 Hz), 2.65-2.55 (1H, m), 2.48-2.40 (2H, m), 1.35 (9H, s), 1.35 (9H, s), 1.12 (3H, d, J = 7.2 Hz), 1.09 (3H, d, J = 6.2 Hz).

MS: m/z = 262 [M+H] ⁺

Stage 4

[Chem. 6]

To the obtained ethyl acetate solution of S-MABB [4] (66.5 g, 212 mmol theoretically) were added ethyl acetate (175 mL) and activated carbon (3.5 g) under nitrogen atmosphere, then the mixture was stirred at room temperature for 2 h. The activated carbon was removed by filtration, and the precipitate on the filter was washed with ethyl acetate (175 mL). The washings were added to the filtrate. To the solution at 0 °C was added a crystal of S-MABB-HC (17.5 mg), which was prepared according to the procedure described in this text, then 4 M hydrogen chloride solution in ethyl acetate (53.0 mL, 212 mmol) was added dropwise at 0 °C. The reaction mixture was stirred at 0 °C for 17 h, and the precipitated solid was collected on the filter and washed with ethyl acetate (70 mL). The resulting wet solid was dried under vacuum to give S-MABB-HC [5] (48.3 g, 162 mmol, yield: 76.4%).

S-MABB-HC, which was obtained according to the above-described method, was studied by NMR and MS methods and the Cl content was determined.

¹ H-NMR (DMSO-d ₆ ) δ: 11.08 (1H, br.s), 10.94 (1H, br.s), 7.52-7.42 (10H, m), 5.34 (1H, t, J = 8.4 Hz), 4.90 (1H, br.s), 4.45-4.10 (5H, m), 3.92-3.49 (3H, br.m), 3.10-2.73 (2H, br.m), 1.35 (9H, s), 1.29 (9H, s), 1.24 (3H, d, J = 6.7 Hz), 1.17 (3H, d, J = 7.4 Hz).

MS: m/z = 262 [M+H-HCl] ⁺

Cl content (ion exchange chromatography): 11.9% (theoretical: 11.9%).

Step B. Preparation of S-MACB-HC (Compound [6])

[Chem. 7]

To a solution of S-MABB-HC [5] (5.0 g, 16.8 mmol) in methanol (15.0 mL) was added 5% palladium carbon (manufactured by Kawaken Fine Chemicals Co., Ltd., PH type, 54.1% water content 1.0 g) under a nitrogen atmosphere at room temperature. The reaction vessel was filled with hydrogen, the mixture was stirred under a hydrogen pressure of 0.4 MPa at room temperature for 12 hours, the hydrogen in the reaction vessel was replaced with nitrogen, and then 5% palladium carbon was removed by filtration. The reaction vessel and 5% palladium carbon were washed with methanol (10 mL). The washings were added to the filtrate to obtain a methanol solution of S-MACB-HC [6] (24.8 g, 16.8 mmol theoretically). The resulting methanol solution of S-MACB-HC was used in the next step, considering the yield to be 100%.

The crude product S-MACB-HC, which was obtained according to the above-described procedure, was evaporated to dryness and studied by NMR and MS methods.

¹ H-NMR (DMSO-d ₆ ) δ: 9.60 (bbs, 1H), 4.97 (d, 1H, J = 9.2 Hz), 4.61 (d, 1H, J = 8.4 Hz), 4.01 (dd, 1H, J = 10.0, 8.4 Hz), 3.78-3.74 (m, 1H), 3.54 (dd, 1H, J = 9.6, 8.4 Hz), 3.35 (dd, 1H, J = 10.0, 6.0 Hz), 3.15-3.03 (m, 1H), 3.00-2.88 (m, 1H), 1.49 (s, 9H), 1.47 (s, 9H), 1.22(d, 3H, J = 6.8 Hz), 1.14 (d, 3H, J = 7.2 Hz).

MS: m/z = 172 [M+H] ⁺ (free form).

Step C. Preparation of S-ZMAB (Compound [7])

[Chem. 8]

Into a methanol solution of S-MACB-HC [6] (24.8 g, 16.8 mmol theoretically), N,N-diisopropylethylamine (4.8 g, 36.9 mmol) was added dropwise at room temperature under nitrogen atmosphere, then the used dropping funnel was washed with tetrahydrofuran (2.5 mL), and the washings were added to the reaction mixture. To the resulting reaction mixture, benzyl chloroformate (3.0 g, 17.6 mmol) was added dropwise at 0 °C, then the used dropping funnel was washed with tetrahydrofuran (2.5 mL), and the washings were added to the reaction mixture. The reaction mixture was stirred at 0 °C for 1 hour, and then the solvent was removed in vacuo. Toluene (25.0 mL) and aqueous citric acid solution (25.0 mL) were added to the residue, the mixture was stirred, and the organic layer was separated. The resulting organic layer was washed with aqueous sodium bicarbonate solution (25.0 mL) and then with water (25.0 mL), and the solvent was removed from the organic layer in vacuo. Toluene (15.0 mL) was added to the residue, and the resulting toluene solution was evaporated. This operation was repeated once more to obtain a toluene solution of S-ZMAB [7] (6.9 g, 16.8 mmol theoretically). The resulting toluene solution of S-ZMAB was used in the next step, considering the yield to be 100%.

The crude product S-ZMAB, which was obtained according to the above procedure, was evaporated to dryness and then characterized by NMR and MS methods.

^1H -NMR ( _CDCl3 ) δ: 7.38-7.28 (m, 10H), 5.16-5.04 (m, 4H), 4.60 (d, 1H, J = 9.2 Hz), 4.18-4.12 (m, 2H), 4.04 (t, 1H, J = 8.6 Hz), 3.66 (dd, 1H, J = 7.6, 7.2 Hz), 3.50 (dd, 1H, J = 8.0, 5.2 Hz), 3.05-2.94 (m, 1H), 2.60-2.50 (m, 1H), 1.43 (bs, 18H), 1.33 (d, 3H, J = 6.5 Hz), 1.15 (d, 3H, J = 7.2 Hz).

MS: m/z = 328 [M+Na] ⁺ .

Step D. Preparation of RS-ZMBB (Compound [8])

[Chem. 9]

Tetrahydrofuran (15.0 mL) was added to a toluene solution of S-ZMAB [7] (6.9 g, 16.8 mmol) at room temperature under nitrogen atmosphere. Lithium bis(trimethylsilyl)amide/tetrahydrofuran solution (14.7 mL, 17.6 mmol) was added dropwise to the toluene solution at -70 °C. The used dropping funnel was washed with tetrahydrofuran (2.5 mL), and the washings were added to the reaction mixture. The reaction mixture was stirred at -70 °C for 6 h, then a solution of TBBA (3.4 g, 17.6 mmol) in tetrahydrofuran (2.5 mL) was added dropwise to the reaction mixture at -70 °C. The used dropping funnel was washed with tetrahydrofuran (2.5 ml), and the washings were added to the reaction mixture. The reaction mixture was stirred at -70 °C for 1 hour, then warmed to room temperature. Aqueous ammonium chloride solution (25 ml) and toluene (25 ml) were added to the reaction mixture, the mixture was stirred, and the organic layer was separated. The resulting organic layer was washed with aqueous citric acid solution (25 ml, × 2), aqueous sodium bicarbonate solution (25 ml), and water (25 ml), then the solvent was removed from the organic layer in vacuo. Acetonitrile (15 ml) was added to the residue, and the resulting acetonitrile solution was evaporated. This operation was repeated two more times. Acetonitrile (15 ml) and activated carbon (0.25 g) were added to the residue, and the mixture was stirred at room temperature for 2 hours. The activated carbon was removed by filtration, and the reaction flask and filter cake were washed with acetonitrile (10 mL). The washings were added to the filtrate and evaporated in vacuo to give an acetonitrile solution of RS-ZMBB [8] (13.2 g, 16.8 mmol theoretically). The resulting acetonitrile solution of RS-ZMBB was used in the next step, considering the yield to be 100%.

The crude product RS-ZMBB, which was obtained according to the above procedure, was evaporated to dryness and then characterized by NMR and MS methods.

^1H -NMR (DMSO- _d6 ) δ: 7.38-7.29 (m, 5H), 5.09-4.96 (m, 2H), 3.91 (t, 0.4H, J=8.0 Hz), 3.79 (t, 0.6H, J=8.0 Hz), 3.55 (t, 0.4H, J= 7.2 Hz), 3.46 (t, 0.6H, J = 7.5 Hz), 3.14-3.04 (m, 1H), 2.83-2.72 (m, 2H), 1.38 (bs, 9H), 1.37 (brs, 3.6H), 1.34 (brs, 5.4H), 1.12-1.09 (m, 3H).

MS: m/z = 420 [M+H] ⁺ .

Step E. Preparation of RS-ZMAA-DN ^,2 H ₂ O (Compound [9])

[Chem. 10]

To the obtained acetonitrile solution of RS-ZMBB [8] (13.2 g, 16.8 mmol theoretically), acetonitrile (15 mL) was added at room temperature under a nitrogen atmosphere. p-Toluenesulfonic acid monohydrate (6.4 g, 33.6 mmol) was added to the solution at room temperature. The reaction mixture was stirred at 50 °C for 12 hours, then cooled to room temperature, and water (7.5 mL) was added dropwise to the reaction mixture. The reaction mixture was cooled to 0 °C, and 4 mol/L aqueous sodium hydroxide solution (17.6 mL, 70.5 mmol) was added dropwise. After the reaction mixture was stirred at room temperature for 1 hour, acetonitrile (75 mL) was added dropwise at room temperature, and the mixture was stirred for 3 hours. The resulting solid precipitate was collected on a filter and washed with a mixture of acetonitrile:water = 4:1 (10 mL) and then with acetonitrile (10 mL). The resulting wet solid was dried in vacuo to give RS-ZMAA-DN ^,2 H ₂ O [9] (5.2 g, 13.4 mmol, yield: 85.4%).

RS-ZMAA-DN ^,2 H ₂ O, which was obtained according to the method described above, was analyzed by NMR and MS methods, the Na content and water content were determined.

^1H -NMR (DMSO- _d6 ) δ: 7.32-7.22 (m, 5H), 4.97 (d, 1H, J = 12.7 Hz), 4.84 (d, 1H, J = 12.7 Hz), 3.79 (t, 1H, J = 8.0 Hz), 3.29 (d, 1H, J = 14.8 Hz), 3.16-3.12 (m, 1H), 2.17-2.09 (m, 2H), 1.07 (d, 3H, J = 6.9 Hz).

MS: m/z = 352 [M+H] ⁺ (anhydrous).

Na content (ion exchange chromatography): 13.3% (after correction for water content) (13.1% theoretical).

Water content (Karl Fischer method): 9.8% (9.3% theoretical).

Step F. Preparation of RS-ZMAA (Compound [10])

[Chem. 11]

To 1 mol/L hydrochloric acid (180 mL) were added RS-ZMAA-DN ^,2 H ₂ O [9] (30 g, 77.5 mmol) and acetonitrile (60 mL), and the mixture was stirred at room temperature for about 15 minutes. Ethyl acetate (240 mL) was added to the reaction mixture, the mixture was stirred, and the organic layer was separated. The organic layer was washed with 10% sodium chloride solution (60 mL x 2). The organic layer was stirred with magnesium sulfate (6 g), magnesium sulfate was removed by filtration, and the filter cake was washed with ethyl acetate (60 mL). The filtrate and washings were combined, and the solvent was removed in vacuo. Tetrahydrofuran (240 mL) was added to the residue, and the resulting tetrahydrofuran solution was evaporated. This operation was repeated two more times. Tetrahydrofuran (60 ml) was added to the residue to obtain a tetrahydrofuran solution of RS-ZMAA [10]. The obtained tetrahydrofuran solution of RS-ZMAA was used in the next step, considering the yield to be 100%.

RS-ZMAA, which was obtained according to the above-described method, was studied by NMR and MS methods.

¹ H-NMR (DMSO-D ₆ ) δ: 7.35-7.28 (m, 5H), 5.06-4.94 (m, 2H), 3.86 (dt, 1H, J = 48.4, 7.9 Hz), 3.50 (dt, 1H, J = 37.9, 7.4 Hz), 3.16-3.02 (bm, 1H), 2.91-2.77 (bm, 2H), 1.08 (d, 3H, J = 6.9 Hz).

MS: m/z = 308 [M+H] ⁺ .

Step G. Preparation of RS-ZMOO (Compound [11])

[Chem. 12]

Tetrahydrofuran (50 mL) was added to a tetrahydrofuran solution of RS-ZMAA [10] (25.8 mmol theoretically) under a nitrogen atmosphere. Boron trifluoride complex etherate (4.40 g) was added dropwise at a temperature of 0°C to 5°C. The used dropping funnel was washed with tetrahydrofuran (5 mL), and the washing solutions were added to the reaction mixture. A 1.2 mol/L borane-tetrahydrofuran complex solution (43.0 mL) was added dropwise to the reaction mixture at a temperature of 0°C to 5°C, the reaction mixture was stirred at 0°C to 5°C for about 30 minutes, and then further stirred at room temperature overnight. A 1.2 mol/L borane-tetrahydrofuran complex solution (21.1 mL) was added dropwise to the reaction mixture at a temperature of 0°C to 5°C, and the reaction mixture was stirred at room temperature overnight. After stirring, water (40 mL) was added dropwise to the reaction mixture at a temperature of 0°C to 15°C. Sodium bicarbonate (5.42 g) was added to the reaction mixture at a temperature of 0°C to 15°C. The remaining sodium bicarbonate in the vessel was washed with water (10 mL), and the washings were added to the reaction mixture. The reaction mixture was stirred at room temperature for 2 hours, then toluene (50 mL) was added, and the reaction mixture was further stirred. The organic layer was separated. The obtained organic layer was washed with 10% sodium chloride solution (20 ml x 1), a mixture (x 3) of 5% aqueous sodium bicarbonate solution (20 ml) and 10% sodium chloride solution (20 ml), a mixture (x 1) of 5% aqueous potassium hydrogen sulfate solution (10 ml) and 10% sodium chloride solution (10 ml), and then with 10% sodium chloride solution (20 ml x 2). The organic layer was stirred with magnesium sulfate (8.9 g), magnesium sulfate was removed by filtration, and the filter cake was washed with toluene (20 ml). The washings were added to the filtrate, and the filtrate was evaporated in vacuo. Toluene (80 ml) was added to the evaporation residue. The solution was evaporated in vacuo and toluene (15 ml) was added to obtain a toluene solution of RS-ZMOO [11]. The obtained toluene solution of RS-ZMOO was used in the next step, considering the yield to be 100%.

RS-ZMOO, which was obtained according to the above-described method, was studied by NMR and MS methods.

¹ H-NMR (CDCl ₃ ) δ: 7.39-7.30 (m, 5H), 5.10 (s, 2H), 4.15-4.01 (br.m, 2H), 3.83-3.73 (br.m, 3H), 3.48 (dd, 1H, J = 8.3, 6.4 Hz), 2.59-2.50 (bm, 1H), 2.46-2.40 (bm, 1H), 2.07-1.99 (m, 1H), 1.14 (d, 3H, J = 7.2 Hz).

MS: m/z = 280 [M+H]+.

Stage H. Preparation of RS-ZMSS (Compound [12])

[Chem. 13]

Toluene (55 mL) was added to a toluene solution of RS-ZMOO [11] (23.7 mmol theoretically) under a nitrogen atmosphere. Triethylamine (5.27 g) was added dropwise at a temperature of from -10 °C to 10 °C, the used dropping funnel was washed with toluene (1.8 mL), and the washings were added to the reaction mixture. Methanesulfonyl chloride (5.69 g) was added dropwise to the resulting reaction mixture at a temperature of from -10 °C to 10 °C, then the used dropping funnel was washed with toluene (1.8 mL), and the washings were added to the reaction mixture. The reaction mixture was stirred at a temperature of from 0 °C to 10 °C for about 2 hours, and then water (28 mL) was added dropwise at a temperature of from 0 °C to 20 °C. The reaction mixture was stirred at 0°C to 20°C for about 30 minutes, and the organic layer was separated. The resulting organic layer was washed twice with 10% sodium chloride solution (18 mL). The organic layer was stirred with magnesium sulfate (2.75 g), magnesium sulfate was removed by filtration, and the filter cake was washed with toluene (18 mL). The washings were added to the filtrate, and then the solvent was removed from the filtrate in vacuo. Toluene was added to the evaporation residue to 18 mL, obtaining a toluene solution of RS-ZMSS [12]. The resulting toluene solution of RS-ZMSS was used in the next step, considering the yield to be 100%.

RS-ZMSS, which was obtained according to the above-described method, was analyzed by NMR and MS methods.

¹ H-NMR (DMSO-D ₆ ) δ: 7.37-7.27 (br.m, 5H), 5.10-4.98 (m, 2H), 4.58-4.22 (br.m, 4H), 3.84 (dt, 1H, J = 45.6, 8.1 Hz), 3.48-3.33 (br.m, 1H), 3.17-3.10 (m, 6H), 2.81-2.74 (b.m, 1H), 2.22-2.12 (m, 2H).

MS: m/z = 436 [M+H] ⁺ .

Step I. Preparation of SR-ZMDB (Compound [13])

[Chem. 14]

Toluene (55 mL) was added to a toluene solution of RS-ZMSS [12] (23.7 mmol theoretically) under a nitrogen atmosphere. Benzylamine (17.8 g) was added dropwise at room temperature, the used dropping funnel was washed with toluene (9.2 mL), and the washings were added to the reaction mixture. The reaction mixture was stirred at room temperature for about 1 hour, at a temperature of 55°C to 65°C for about 3 hours, and then at a temperature of 70°C to 80°C for 6 hours. After cooling the reaction mixture to room temperature, 10% NaCl solution (28 mL) was added dropwise, and the reaction mixture was stirred at room temperature for about 30 minutes. Toluene (37 mL) was added to the reaction mixture, the mixture was stirred, and the organic layer was separated. The obtained organic layer was washed with a mixture (x 2) of 10% sodium chloride solution (18 ml) and acetic acid (2.84 g), and then with 10% sodium chloride solution (11 ml, x 1). The solvent from the organic layer was removed in vacuo to half the volume, and acetic anhydride (1.45 g) was added to the evaporation residue at room temperature. The mixture was stirred for about 3 hours. Potassium hydrogen sulfate solution (3.87 g) and water (92 ml) were added dropwise to the reaction mixture at room temperature. The reaction mixture was stirred, and the aqueous layer was separated. The obtained aqueous layer was washed with toluene (18 ml), and toluene (73 ml) and then sodium bicarbonate (6.56 g) were added to the aqueous layer at room temperature, and the mixture was stirred. The organic layer was separated and washed with 10% sodium chloride solution (11 ml). The organic layer was stirred with magnesium sulfate (2.75 g), and magnesium sulfate was removed by filtration. The filter cake was washed with toluene (18 ml), and the washings were added to the filtrate, and the filtrate was evaporated in vacuo. Toluene (44 ml) was added to the evaporation residue to give a toluene solution of SR-ZMDB [13]. The resulting toluene solution of SR-ZMDB was used in the next step, considering the yield to be 100%.

¹ H-NMR (CDCl ₃ ) δ: 7.35-7.20 (m, 10H), 5.08 (d, 2H, J = 23.6 Hz), 3.94 (q, 1H, J = 7.9 Hz), 3.73-3.42 (brm, 2H), 3.30-3.23 (m, 1H), 3.05 (dd, 1H, J = 19.7, 9.5 Hz), 2.79 (dt, 1H, J = 69.6, 6.1 Hz), 2.57-2.32 (brm, 4H), 1.96-1.89 (m, 1H), 1.09 (d, 3H, J = 6.9 Hz).

MS: m/z = 351 [M+H] ⁺ .

Step J. Preparation of SR-MDOZ (Compound [14])

[Chem. 15]

To a solution of 1-chloroethyl chloroformate (3.72 g) in toluene (28 ml), a toluene solution of SR-ZMDB [13] (23.7 mmol theoretically) was added dropwise at a temperature of 0°C to 10°C under a nitrogen atmosphere, then the used dropping funnel was washed with toluene (4.6 ml), and the washings were added to the reaction mixture. Triethylamine (718 mg) was added to the reaction mixture at a temperature of 0°C to 10°C, and the reaction mixture was stirred at a temperature of 15°C to 25°C for about 2 hours. Then, methyl alcohol (46 ml) was added to the reaction mixture, and the mixture was stirred at a temperature of 50°C to 60°C for another about 2 hours. The solvent from the reaction mixture was removed in vacuo until the volume was less than about 37 ml. To the evaporation residue, 2 mol/L hydrochloric acid (46 ml) was added dropwise at a temperature of 15°C to 20°C, the mixture was stirred, and the aqueous layer was separated. The resulting aqueous layer was washed with toluene (28 ml, × 2). To the aqueous layer, 20% sodium chloride solution (46 ml) and tetrahydrofuran (92 ml) were added, and then 8 mol/L aqueous sodium hydroxide solution (18 ml) was added dropwise at a temperature of 0°C to 10°C. The organic layer was separated, washed with 20% sodium chloride solution (18 ml, × 2), and then the solvent was removed in vacuo from the organic layer. Tetrahydrofuran (92 ml) was added to the evaporation residue, and the solution was evaporated in vacuo. This operation was repeated once more. The evaporation residue was dissolved in tetrahydrofuran (92 ml). The solution was stirred with magnesium sulfate (2.75 g) and magnesium sulfate was removed by filtration. The filter cake was washed with tetrahydrofuran (28 ml), the washings were added to the filtrate, and the filtrate was evaporated in vacuo. The volume of the evaporation residue was adjusted to about 20 ml with tetrahydrofuran to obtain a tetrahydrofuran solution of SR-MDOZ [14] (net weight: 4.01 g, 15.4 mol, yield: 65.0%).

SR-MDOZ, which was obtained according to the above-described method, was evaporated to dryness and then studied by NMR and MS methods.

^1H -NMR ( _CDCl3 ) δ: 7.37-7.28 (m, 5H), 5.08 (dd, 2H, J = 16.8, 12.8 Hz), 4.00 (dd, 1H, J = 17.1, 8.3 Hz), 3.40-3.31 (m, 1H), 3.24 (d, 1H, J = 12.7 Hz), 3.00 (dd, 1H, J = 54.9, 12.4 Hz), 2.87-2.57 (m, 3H), 2.47-2.27 (m, 1H), 1.91-1.80 (m, 1H), 1.14 (d, 3H, J = 7.2 Hz).

MS: m/z = 261 [M+H] ⁺ .

Step K. Preparation of SR-MDOZ-OX (Compound [15])

[Chem. 16]

Under a nitrogen atmosphere, oxalic acid (761 mg) was dissolved in tetrahydrofuran (40 mL), and a tetrahydrofuran solution of SR-MDOZ [14] (3.84 mmol theoretically) was added dropwise to the resulting oxalic acid solution at room temperature. A crystal of SR-MDOZ-OX (1 mg), which was obtained by the procedure described in this text, was added to the solution at room temperature, and the mixture was stirred at room temperature for about 3.5 hours to observe the precipitation of a crystalline precipitate. A tetrahydrofuran solution of SR-MDOZ (3.84 mmol) was added dropwise to the resulting suspension at room temperature, and the mixture was stirred at room temperature for about 1 hour. The suspension was heated and stirred at 50°C to 60°C for about 2 hours, and then stirred at room temperature overnight. The suspension was filtered and the wet crystals on the filter were washed with tetrahydrofuran (10 ml), dried in vacuo to give SR-MDOZ-OX [15] (2.32 g, 6.62 mol, yield: 86.2%).

SR-MDOZ-OX, which was obtained according to the above-described method, was analyzed by NMR, MS, and elemental analysis was performed.

¹ H-NMR (DMSO-D ₆ ) δ: 7.37-7.30 (m, 5H), 5.15-5.01 (m, 2H), 3.92 (dt, 1H, J = 43.5, 8.4 Hz), 3.48-3.12 (br.m, 5H), 2.67-2.56 (m, 1H), 2.46-2.35 (m, 1H), 2.12-2.05 (m, 1H), 1.13 (d, 3H, J = 6.9 Hz).

MS: m/z = 261 [M+H] ⁺

Elemental analysis: C 58.4 wt.%, H 6.4 wt.%, N 7.9 wt.% (theoretical: C 58.3 wt.%, H 6.3 wt.%, N 8.0 wt.%).

Step L. Preparation of SR-MDPZ (Compound [16])

[Chem. 17]

To SR-MDOZ-OX [15] (12.0 g, 34.2 mmol) were added ethanol (36 mL), water (72 mL), CPPY [20] (5.36 g, 34.9 mmol) and then K ₃ PO ₄ (21.8 g, 103 mmol) under nitrogen atmosphere. The reaction mixture was stirred at 80 °C for 5 h and then cooled to 40 °C. Toluene (120 mL) was added at 40 °C and the organic layer was separated. The resulting organic layer was washed with 20% aqueous potassium carbonate solution (48 mL) and then washed twice with water (48 mL). The solvent was removed from the organic layer in vacuo. tert-Butanol (60 mL) was added to the residue and the resulting tert-butanol solution was evaporated. This operation was repeated two more times. Tert-butanol (36 ml) was added to the evaporation residue to obtain a solution of SR-MDPZ [16] in tert-butanol (61.1 g, 34.2 mmol theoretically). The resulting tert-butanol solution of SR-MDPZ was used in the next step, considering the yield to be 100%.

SR-MDPZ, which was obtained by the above-described procedure, was isolated as a solid from a mixture of ethyl acetate and n-heptane and then characterized by NMR and MS.

¹ H-NMR (DMSO-d ₆ ) δ: 11.59 (br.s, 1H), 8.08 (s, 1H), 7.41-7.26 (br.m, 3H), 7.22-7.08 (br.m, 3H), 6.64-6.51 (br.m, 1H), 5.07-4.91 (br.m, 2H), 4.09-3.67 (b.m, 5H), 3.47-3.32 (b.m, 1H), 2.67-2.55 (b.m, 2H), 2.21-2.15 (b.m, 1H), 1.11 (d, 3H, J = 6.9 Hz).

MS: m/z = 378 [M+H] ⁺

Step M: Preparation of SR-MDOP (Compound [17])

[Chem. 18]

To a solution of SR-MDPZ [16] in tert-butanol (34.2 mmol theoretically) were added ammonium formate (10.8 g, 171 mmol), water (60 mL), and 10% palladium carbon (manufactured by Kawaken Fine Chemicals Co., Ltd., M type, 52.6% water content, 1.20 g) under a nitrogen atmosphere. The reaction mixture was stirred at 40°C for 13 hours, then cooled to room temperature, and the precipitated precipitate was removed by filtration. The reaction vessel and the filter cake were washed with tert-butanol (24 mL), the washings were added to the filtrate, and 8M aqueous sodium hydroxide solution (25.7 mL, 205 mmol) and sodium chloride (13.2 g) were added to the filtrate. The reaction mixture was stirred at 50°C for 2 hours, then toluene (84 ml) was added at room temperature, and the organic layer was separated. The resulting organic layer was washed with 20% sodium chloride solution (60 ml), stirred with anhydrous sodium sulfate, and then sodium sulfate was removed by filtration. The filter cake was washed with a mixture of toluene : tert-butanol = 1 : 1 (48 ml), the washings were added to the filtrate, and the filtrate was evaporated in vacuo. Toluene (60 ml) was added to the evaporation residue, and the resulting solution was stirred at 50°C for 2 hours, then the solvent was removed in vacuo. Toluene (60 ml) was again added to the evaporation residue, and the resulting solution was evaporated. Toluene (48 ml) was added to the evaporation residue and the solution was stirred at room temperature for 1 h, then under ice cooling for 1 h. The separated solid was collected on a filter and washed with toluene (24 ml). The resulting wet solid was dried in vacuo to give SR-MDOP [17] (7.07 g, 29.1 mmol, yield: 84.8%).

SR-MDOP, which was obtained according to the above-described method, was studied by NMR and MS methods.

¹ H-NMR (DMSO-d ₆ ) δ: 11.57 (brs, 1H), 8.07 (s, 1H), 7.10 (d, 1H, J = 3.2 Hz), 6.58 (d, 1H, J = 3.2 Hz), 3.92-3.59 (brs, 4H), 3.49 (dd, 1H, J = 8.3, 7.2 Hz), 2.93 (dd, 1H, J = 7.2, 6.1 Hz), 2.61-2.53 (m, 2H), 2.12-2.01 (brm, 2H), 1.10 (d, 3H, J = 6.9 Hz).

MS: m/z = 244 [M+H] ⁺ .

Step N. Preparation of Compound A mono-ethanolate (Compound [18])

[Chem. 19]

Under a nitrogen atmosphere, acetonitrile (60 mL) and triethylamine (416 mg, 4.11 mmol) were added to SR-MDOP [17] (5.00 g, 20.5 mmol), and DPCN [21] (3.69 g, 22.6 mmol) in acetonitrile (35 mL) was added dropwise to the resulting solution at 45 °C, then the used dropping funnel was washed with acetonitrile (5.0 mL), and the washings were added to the reaction mixture. The reaction mixture was stirred at 45 °C for 3 hours and then cooled to room temperature. 5% aqueous sodium bicarbonate solution (25 mL), 10% sodium chloride solution (25 mL), and ethyl acetate (50 mL) were added to the reaction mixture, then the mixture was stirred and the organic layer was separated. The solvent was removed from the organic layer in vacuo. Tetrahydrofuran (50 ml) was added to the residue, and the resulting tetrahydrofuran solution was evaporated. This operation was repeated three more times. Tetrahydrofuran (50 ml) and water were added to the evaporation residue, bringing the water content to 5.5%. The formed precipitate was removed by filtration. The reaction vessel and the precipitate on the filter were washed with tetrahydrofuran (15 ml), the washings were added to the filtrate, and the solvent was removed from the filtrate in vacuo. Ethanol (50 ml) and a crystal of Compound A (5.1 mg), which was obtained by the method described below in Example 15, were added to the evaporation residue. The mixture was stirred at room temperature for 1 hour and evaporated in vacuo. Ethanol (50 ml) was added to the residue, and the solution was evaporated again. Ethanol (15 ml) was added to the evaporation residue and the solution was stirred at room temperature for 1 h. The precipitated solid was collected on a filter and washed with ethanol (20 ml). The resulting wet solid was dried in vacuo to give Compound A mono-ethanolate [18] (6.26 g, 17.6 mmol, yield: 85.5%).

Compound A mono-ethanolate, which was obtained according to the above-described method, was analyzed by NMR and MS methods.

¹ H NMR (DMSO-d ₆ ) δ: 11.59 (br s, 1H), 8.08 (s, 1H), 7.11 (dd, 1H, J = 3.5, 2.3 Hz), 6.58 (dd, 1H, J = 3.5, 1.8 Hz), 4.34 (t, 1H, J = 5.1 Hz), 4.16 (t, 1H, J = 8.3 Hz), 4.09-3.92 (m, 3H), 3.84-3.73 (m, 1H), 3.71 (d, 1H, J = 19.0 Hz), 3.65 (d, 1H, J = 19.0 Hz), 3.58 (dd, 1H, J = 8.2, 5.9 Hz), 3.44 (dkv, 2H, J = 6.7, 5.1 Hz), 2.69-2.60 (m, 2H), 2.23-2.13 (brm, 1H), 1.12 (d, 3H, J = 7.1 Hz), 1.06 (t, 3H, J = 6.7 Hz).

MS: m/z = 311 [M+H] ⁺

Step O. Purification of Compound A (Compound [19])

[Chem. 20]

Compound A mono-ethanolate [18] (4.00 g, 11.2 mmol) and n-butanol (32 ml) were mixed under nitrogen atmosphere and the mixture was dissolved at 110°C. The mixture was cooled to 85°C and a crystal of Compound A (4.0 mg), which was obtained by the procedure described in this text, was added, then the mixture was stirred at 85°C for 2 h, at 75°C for 1 h and at room temperature for 16 h. The precipitated solid was collected on a filter and washed with n-butanol (8.0 ml) and then ethyl acetate (8.0 ml). The resulting wet solid was dried in vacuo to give compound A [19] (3.18 g, 10.2 mmol, yield: 91.3%).

Compound A, which was obtained according to the above-described procedure, was analyzed by NMR and MS methods.

¹ H-NMR (DMSO-d ₆ ) δ: 11.59 (bbs, 1H), 8.08 (s, 1H), 7.11 (dd, 1H, J = 3.5, 2.5 Hz), 6.58 (dd, 1H, J = 3.5, 1.8 Hz), 4.16 (t, 1H, J = 8.3 Hz), 4.09-3.93 (m, 3H), 3.84-3.73 (m, 1H), 3.71 (d, 1H, J = 19.0 Hz), 3.65 (d, 1H, J = 19.0 Hz), 3.58 (dd, 1H, J = 8.2, 5.9 Hz), 2.69-2.59 (m, 2H), 2.23-2.13 (m, 1H), 1.12 (d, 3H, J = 7.2 Hz).

MS: m/z = 311 [M+H] ⁺

Using Compound A, which was obtained by a similar method, X-ray structural analysis of a single crystal was performed.

(1) Obtaining a single crystal

To 10 mg of Compound A in a 2.0 ml wide-neck LaPha ROBO Vial® was added 0.5 ml of chloroform. The vial was capped and Compound A was completely dissolved. To slowly evaporate the solvent, a hole was made in the septum mounted in the cap using a TERUMO® syringe needle and the vial was left to stand at room temperature. The resulting single crystal was used for X-ray structural analysis.

(2) Device

Radiation source: SPring-8 BL32B2

Detector: Rigaku R-AXIS V diffractometer

(3) Measurement method

The single crystal was exposed to 0.71068 radiation , recording the diffraction of X-rays

(4) Analysis

Using the anomalous X-ray scattering effect of the chlorine atom in the obtained chloroform solvate of Compound A, the absolute configuration of Compound A was determined to be (3S,4R). Based on the determined absolute configuration of Compound A, the absolute configuration of each intermediate in the synthesis was identified.

Description of Form α

Powder X-ray diffraction

Form α was characterized by powder X-ray diffraction using a Spectris Co., Ltd. PANalytical instrument Model Number: X’Pert Pro with the following parameters:

Radiation : Cu-Kα ₁ / 45 kV / 40 mA Monochromator : Graphite monochromator Scanning range : from 3.0 to 40.0° Soller's Gap : 0.02 rad Divergence gap : 1 / 2 Anti-scattering gap : 1 / 2 Mask Fixed mask 4mm Mode : Transmission Optics : Focusing mirror Wobble scan Wobble axis:omega
Number of steps: 7
Stage size: 1,000
Range:6 Sample holder : 96 well plate Scan time : 20 sec Scanning step : 0.00334°

The X-ray powder diffraction spectrum for Form α is shown in Fig. 1 and the resulting data are summarized below in Table 5.

Table 5 Diffraction angle
[2θ (°)] Relative intensity
[%] Intensity of the diffraction pattern
[imp/s] 7.8 33.39 3282.99 10.2 19.13 1880,69 11.4 11.24 1105,05 12.3 2.82 277.49 12.7 11.01 1082.97 13.4 12.90 1268,20 14.7 100,00 9832,97 15.9 20.81 2046.10 16.2 8.44 829.65 16.4 14.61 1436.83 17.1 8.78 863.45 17.9 61.50 6047,48 18.9 9.37 920,90 19.5 1.55 152.43 20.2 7.58 745.51 20.8 2.87 282.31 21.2 1.59 156.25 22.2 3.66 360.04 22.6 67.73 6659,41 22.9 17.99 1768.98 23.5 2.82 277.10 24.3 10.67 1048.90 24.8 5.71 561.67 25.6 10.68 1049.68 26.5 2.48 243.62 27.0 47,44 4664,60 27.7 1.63 160.56 28.3 1.19 117.03 29.6 1.28 125.94 30.1 1.15 113.57 30.9 1.71 168.29 31.7 1.06 104,00 32.6 1.63 159.85 33.4 3.41 335.16 34.0 1.52 149.80 35.4 0.94 92.36 36.1 1.45 142.86 36.9 1.28 125.60 37.9 0.61 59,60 39.5 1.38 136.12

Differential Scanning Calorimetry (DSC)DSC data were measured on a TA Instruments Model Number Q2000 with the following parameters:

Sample size: approximately 3 mg

Cell: Hermetically sealed aluminum cell

Range: 25-230°C

Heating rate: 2°C/min in nitrogen flow.

The DSC thermogram for Form α is shown in Fig. 4, showing a single endothermic peak at 188°C with an onset at 185°C.

Thermogravimetric differential thermal analysis (TG-DTA)

The TG-DTA data were measured on a Mettler Toledo Model Number: TGA/SDTA851e/SF instrument with the following parameters:

Sample: approximately 5 mg

Range: 25-250°C

Heating rate: 5°C/min in nitrogen flow.

The extrapolated transition onset temperature was 186.29°C, and no weight loss was observed in the range from 25 to 220°C. The TG-DTA data for Form α are shown in Fig. 7.

Solid State ¹³ C NMR

Solid-state ^13C NMR data were recorded on a Bruker BioSpin Corporation AVANCE III 400 instrument with the following parameters:

Sensor: 4mm CP/MAS

Spectrum recording temperature: room temperature

Standard: Glycine (External standard: The downfield peak was assigned a value of 176.03 ppm)

Core: ^13C (100.6228303 MHz)

Rotation speed: 14.3 kHz

Delay time: 5 seconds

Number of scans: 3072

Sensor mode: CP/MAS

The solid state ^13C NMR spectrum for Form α is shown in Fig. 10 and contains peaks at 14.8, 17.0, 24.5, 27.2, 35.0, 35.9, 38.6, 48.8, 49.5, 53.6, 78.3, 101.4, 103.0, 116.2, 118.0, 122.0, 122.7, 151.2, 154.8, 162.4, and 163.3 ppm.

Example 2

Preparation and description of Form β of Compound A

Receiving

Compound A (3992.3 g, crystalline form α), previously passed through a sieve with a mesh size of 500 μm, was ground using a Spiral Jet Mill 100AS (manufactured by Hosokawa Micron Corporation) under the following conditions to obtain a ground product (3473.6 g), and Compound A (361.0 g) adhered to the nozzle ring.

Grinding conditions

Gas used: Nitrogen

Grinding nozzle: 1.5mm diameter x 4

Ejector nozzle: 1.3mm in diameter

Grinding air pressure: 0.50-0.60 MPa

Ejector air pressure: 0.50-0.60 MPa

Feed rate of Compound A: 8 g/min

Compound A (50.0 g, 161 mmol) adhered to the nozzle ring was added to 1-butanol/acetonitrile (1:3 v/v) (250 mL), and the mixture was stirred at 40 °C for 10 days. The resulting suspension was cooled to room temperature and further stirred at room temperature for 24 h. The precipitated crystalline material was collected on a filter. The resulting crystalline product was washed with ethyl acetate (50 mL) twice and dried under reduced pressure at 40 °C to give Compound A, crystalline form β (35.5 g, yield 71%).

Description

Powder X-ray diffraction

The X-ray powder diffraction spectrum for Form β is shown in Fig. 2 and the resulting data are given below in Table 6. The apparatus and experimental parameters are the same as described above in Example 1 for the study of Form α.

Table 6 Diffraction angle
[2θ (°)] Relative intensity
[%] Intensity of the diffraction pattern
[imp/s] 7.8 20.40 699.45 10.5 18.12 621.23 11.8 6.39 218.98 12.3 9.47 324.65 13.4 47.83 1639.59 13.9 31.01 1063,00 14.3 14.01 480.22 14.7 18.95 649.62 15.6 20.90 716.30 17.1 6.64 227.53 17.8 100,00 3428,04 19.0 7.07 242.48 19.3 8.25 282.66 20.0 2.45 84.08 21.2 11.00 377.11 22.0 19.74 676.56 23.6 64.54 2212,58 24.7 6.14 210.37 26.0 4.10 140.49 26.9 6.97 238.82 28.0 33.58 1151.04 32.3 1.40 47.83 35.9 0.92 31.62 38.7 0.95 32.55

Differential scanning calorimetry (DSC)

DSC data were measured on a TA Instruments Model Number Q2000 with the following parameters:

Sample size: approximately 3 mg

Cell: Hermetically sealed aluminum cell

Range: 25-230°C

Heating rate: 2°C/min in nitrogen flow.

The DSC thermogram for Form β is shown in Fig. 5, showing a single endothermic peak at 187°C with an onset at 185°C.

Thermogravimetric differential thermal analysis (TG-DTA)

TG-DTA data were obtained as described above in Example 1, except that the measurement range for Form β was from 25 to 230°C. An endothermic peak was detected at 188.59°C, the extrapolated transition onset temperature was 186.68°C, and no weight loss was observed in the range from 25 to 220°C. The data for Form β are shown in Fig. 8.

Solid State ¹³ C NMR

Solid state ^13C NMR data for Form β were obtained as described above in Example 1. ^{The 13C} NMR spectrum is shown in Fig. 11 and contains peaks at 16.5, 25.8, 26.5, 33.1, 34.8, 36.7, 38.8, 48.2, 53.4, 77.7, 79.5, 101.2, 102.6, 117.5, 120.6, 151.1, 154.3, and 166.1 ppm.

Example 3

Preparation and description of Form γ of Compound A

Getting 1

Compound A (2491.5 g, crystalline form α), previously passed through a sieve with a mesh size of 500 μm, was milled using a Spiral Jet Mill 100AS (manufactured by Hosokawa Micron Corporation) under the following conditions to obtain a milled product (2106.5 g), and Compound A (247.9 g) adhered to the nozzle ring.

Grinding conditions

Gas used: Nitrogen

Grinding nozzle: 1.5mm diameter x 4

Ejector nozzle: 1.3mm in diameter

Grinding air pressure: 0.50-0.60 MPa

Ejector air pressure: 0.50-0.60 MPa

Feed rate of Compound A: 4 g/min

Compound A (100.0 g, 322 mmol) adhered to the nozzle ring was added to formamide (330 mL) under nitrogen atmosphere, and a seed crystal (1.00 g of Crystal Form γ, which may also contain some Form β of Compound A) was added to the mixture. The mixture was stirred at room temperature for 30 days. The crystalline precipitate was collected on a filter and washed with ethyl acetate (150 mL) twice. The resulting wet crystalline material was added to ethyl acetate (300 mL) under nitrogen atmosphere, and the mixture was stirred at room temperature for 30 min. The crystalline precipitate was collected on a filter, washed with ethyl acetate (150 mL) twice, and dried under reduced pressure at 40 °C to give Compound A, Crystal Form γ (74.1 g, yield 74%).

Getting 2

Compound A, Form α (containing a small but unspecified amount of Form β) (100 mg, 0.322 mmol) was added to N,N-dimethylformamide (0.2 mL) and the mixture was stirred at room temperature for 6 days. The crystalline precipitate was isolated by filtration. The resulting wet crystalline material was dried under reduced pressure at room temperature for 18 h to afford compound A, Form γ (13.7 mg, 13.7% yield).

Description

Powder X-ray diffraction

The X-ray powder diffraction spectrum for Form γ is shown in Fig. 3 and the resulting data are given below in Table 7. The apparatus and experimental parameters are the same as described above in Example 1 for the study of Form α.

Table 7 Diffraction angle
[2θ (°)] Relative intensity
[%] Intensity of the diffraction pattern
[imp/s] 7.7 10.32 692.73 10.6 7.64 512.67 13.3 100,00 6709,91 13.9 14.17 951.02 15.2 79.93 5363,01 15.6 16.12 1081.78 16.5 14.81 993.47 17.7 52.89 3549,06 17.9 45.66 3063,89 19.0 26.13 1753.04 19.9 2.07 139.16 21.0 10.34 693.77 21.4 7.31 490.71 21.8 36.03 2417.29 22.8 3.79 254.11 23.1 27.25 1828,29 23.7 51.32 3443.62 24.1 9.91 665.12 24.3 6.86 460.35 24.7 2.39 160.44 25.1 9.88 663,01 26.0 1.44 96.76 27.1 5.51 369.82 27.4 2.94 197.55 28.1 34.39 2307.33 29.7 4.91 329.16 30.2 5.33 357.79 30.7 1.40 93,96 31.6 1.81 121.53 32.4 1.65 110.79 33.2 1.40 93.71 34.0 3.22 216.08 34.3 2.79 187.09 34.7 2.99 200.46 35.3 2.19 146.78 35.9 2.71 181.75 38.0 1.21 80.94 38.7 1.36 91.15

Differential Scanning Calorimetry (DSC) DSC data were measured on a TA Instruments Model Number Q2000 with the following parameters:

Sample size: approximately 3 mg

Cell: Hermetically sealed aluminum cell

Range: 25-230°C

Heating rate: 2°C/min in nitrogen flow.

The DSC thermogram for Form γ is shown in Fig. 6, showing a single endothermic peak at 196°C with an onset at 196°C.

Thermogravimetric differential thermal analysis (TG-DTA)

TG-DTA data were obtained as described above in Example 1. The endothermic peak was detected at 198.68°C, the extrapolated transition onset temperature was 197.38°C, and no weight loss was observed in the range from 25 to 220°C. Data for Form γ are shown in Fig. 9.

Solid State ¹³ C NMR

Solid state ^13C NMR data for Form γ were obtained as described above in Example 1. ^{The 13C} NMR spectrum is shown in Fig. 12 and contains peaks at 16.9, 26.5, 32.9, 36.4, 48.1, 53.7, 78.6, 102.6, 116.4, 117.9, 121.5, 151.8, 154.6 and 162.9 ppm.

Example 4

Quantitative assessment of Form β and γ content in Form α samples

Production lots of Compound A, Form α, as well as an ointment formulation containing Compound A, Form α, were tested for the presence of Forms β and γ. Using powder X-ray diffraction, the content of Forms β and γ can be quantified to an accuracy of 1 wt%.

The batches of Compound A, Form α used for analysis are listed in Table 8.

Table 8 Party Size P 22.40 kg V 19.15 kg

The batches of ointment containing Form α Compound A (3%) used for the analysis are listed in Table 9. RH refers to relative humidity. M refers to duration in months.

Table 9 Party Active ingredient batch Size Storage conditions 223-1A P 60.00 kg 25°C/60% RH 18M 239-1A P 60.00 kg Head. 266-1 V 4.00 kg 40°C/75% RH 6M 320 V 4.00 kg Head.

The content of Forms β and γ was quantitatively assessed in the above batches and ointments using a PANalytical X’Pert PRO powder X-ray diffractometer with the following operating parameters:

Radiation : Cu-Kα ₁ / 45 kV / 40 mA Monochromator : Graphite monochromator Scan Range (Shape β) : 9.5-12.5° Scanning Range (Gamma Shape) : 15.0-17.5° Soller's Gap : 0.02 rad Divergence gap : 1 / 2 Anti-scattering gap : 1 / 2 Mask Fixed mask 20mm Mode : Transmission Optics : Focusing mirror Sample holder : Insert into the holder
(Product No. 9430 018 18251)
Holder
(Product No. 9430 018 18401) Scan time : 3000 sec Scanning step : 0.0008° or 0.0016° Film : Mylar ^TM film

Form β Test

Preparation of test sample (Compound A, Form α, drug substance)

A portion of the test sample of Compound A was ground into a fine powder in an agate mortar. Approximately 10 mg was fixed in a sample holder.

Preparation of test sample (ointment)

Approximately 1 gram of the ointment sample to be tested was suspended in 80 ml of n-hexane. The flask was stoppered and shaken vigorously for 30 seconds. The resulting mixture was suction filtered onto quantitative filter paper (5B), washed with 5 ml of n-hexane five times. Approximately 10 mg of the precipitate was fixed in the sample holder.

Obtaining a standard sample

Lot P-3273-58 (Form β) was added to Compound A, Lot U (Form α) to give final concentrations of 2, 5, and 10 w/w% and mixed in a mortar. Approximately 10 mg of product was fixed in a sample holder.

Methodology

X-ray powder diffraction analysis was performed on the test samples and standard samples according to the instrument settings and data recording parameters specified above (see X-ray Powder Diffraction Method, General Tests, Processes and Apparatus, The Japanese Pharmacopoeia, 17th edition, April 1, 2016, English edition, pp. 79-83). The diffraction peak areas (10.6°) obtained for the standard samples and the test sample were determined by automatic integration or manually. The amount (%) of Form β in the test sample was calculated using a calibration curve constructed from the peak areas of the standard samples.

Powder X-ray diffraction spectra obtained for a standard sample containing 0%, 2%, 5% and 10% w/w of Form β are shown in Fig. 13.

Form γ Test

Preparation of test sample (Compound A, Form α, drug substance)

A portion of the test sample of Compound A was ground into a fine powder in an agate mortar. Approximately 10 mg was fixed in a sample holder.

Preparation of test sample (ointment)

Approximately 1 gram of the ointment sample to be tested was suspended in 80 ml of n-hexane. The flask was stoppered and shaken vigorously for 30 seconds. The resulting mixture was suction filtered onto quantitative filter paper (5B), washed with 5 ml of n-hexane five times. Approximately 10 mg of the precipitate was fixed in the sample holder.

Obtaining a standard sample

Compound A Lot YKF56-28G (Form γ) was added to Batch U (Form α) to give final concentrations of 1, 2, and 5 w/w% and mixed in a mortar. Approximately 10 mg of product was fixed in a sample holder.

Methodology

X-ray powder diffraction analysis was performed on the test samples and standard samples according to the instrument settings and data recording parameters specified above (see X-ray Powder Diffraction Method, General Tests, Processes and Apparatus, The Japanese Pharmacopoeia, 17th edition, April 1, 2016, English edition, pp. 79-83). The diffraction peak areas (16.6°) obtained for the standard samples and the test sample were determined by automatic integration or manually. If the peak area of the test sample was not greater than that of the 1% standard sample, the value was recorded as “not greater than 1%”. If the peak area of the test sample was greater than that of the 1% standard sample and not greater than that of the 2% standard sample, the value was recorded as “not greater than 2%”. If the peak area in the test sample was greater than that of the 2% standard sample and not greater than that of the 5% standard sample, the value was recorded as “not greater than 5%”.

The powder X-ray diffraction spectra obtained for the standard sample containing 0%, 1%, 2% and 5% w/w of the γ Form are shown in Fig. 14.

Results

The results of the quantitative determinations are summarized in Table 10. Compound A, drug substance Lot P, JTE-052 Ointment Lot 223-1A, and Lot 239-1A, drug substance Manufacturing Lot P all contained measurable amounts of Form β.

Table 10 Party Pharmaceutical substance, Batch Storage conditions Form β Form γ P -- -- 9.3% ≤ 1% V -- -- ≤ QL ^a ≤ 1% 223-1A P 25°C/60% RH 18M 6.4% ≤ 1% 239-1A P Head. 7.9% ≤ 1% 266-1 V 40°C/75% RH 6M ≤ QL ≤ 1% 320 V Head. ≤ QL ≤ 1% QL: Measurement limit = 1.12% calculated according to QL=10 (σ/tilt angle).

Example 5

X-ray diffraction analysis of a single crystal

The single crystal X-ray structures were determined for Forms α, β and γ according to the parameters given below. The structural data are given in Table 11.

For Form α, the X-ray structure of the single crystal was determined using a Rayonix MX225HE detector and a SPring-8 BL41XU radiation source with an emitter wavelength of 0.71068 Å.

For Forms β and γ, the X-ray structure of single crystals was determined using a DECTRIS PILATUS3 6M detector and a SPring-8 BL41XU radiation source with an emitter wavelength of 0.71068 Å.

Table 11 Crystalline form β γ α Temp. (°C) 100 100 100 Space group P1 (#1) P2 ₁ (#4) P1 (#1) Z 4 2 2 Z' 4 1 2 a (Å) 8,043 8,095 7.8 b (Å) 11,371 8,017 8.6 c (Å) 16,522 11,459 11.5 α (°) 97,537 90 95.2 β (°) 94,541 95,663 99.0 γ (°) 90,294 90 93.9 D _calc. (g/cm ³ ) 1,381 1,393 1,356 Volume (Å ₃ ) 1493.1 740 760 R ₁ (%) (<10%) 7.49 6.28 9.51 R _wp (%) (<25%) 23.01 17.29 24.72 GOF
(0.8-1.3) 1,036 0.984 1,068

Example 6

Comparative Experiment between Forms α and β of Compound A

Forms α and β of Compound A were mixed in a ratio of 1:1 by weight, and 1-butanol (800 μl) was added to the resulting mixture (80 mg). The resulting suspension was stirred at room temperature for 12 days. The precipitate was filtered off at the same temperature and then dried under reduced pressure at room temperature for 19 hours. The diffraction angles 2θ and the intensities of the diffraction peaks were determined by powder X-ray diffraction for the Forms α and β of Compound A used, for a 1:1 mixture by weight of Forms α and β of Compound A before stirring, and for the obtained crystals. The apparatus and parameters for recording the spectra in this case are the same as those described above for the study of Form α in Example 1. The spectra obtained are shown in Fig. 15. This Figure shows the spectra of the Form α of Compound A used, the Form β of Compound A used, a 1:1 mixture by weight of Forms α and β of Compound A before mixing, and the crystals obtained after mixing, from bottom to top.

The intensity ratio of the diffraction peaks before and after stirring was compared: the peak with a diffraction angle of 2θ 10.2° for Form α and the peak with a diffraction angle of 2θ 10.6° for Form β. As shown in the table below, the intensity ratio of the diffraction peak of Form β and the peak of Form α after stirring increased compared to the ratio before stirring.

Table 12

Diffraction peak intensity 10.2°
(Form α) [imp/s] Diffraction peak intensity 10.6°
(Form β) [imp/s] Ratio of diffraction peak intensities (Form β/Form α) Before mixing 601,66 225.88 0.38 After mixing 819.03 673.08 0.82

Example 7

Comparative Experiment between Forms α and γ of Compound A

Forms α and γ of Compound A were mixed in a ratio of 1:1 by weight, and 1-butanol (1 ml) was added to the resulting mixture (100 mg). The resulting suspension was stirred at room temperature for 11 days. The precipitate was filtered off at the same temperature. The diffraction angles 2θ and the intensities of the diffraction peaks were determined by powder X-ray diffraction for the Forms α and γ of Compound A used, for a 1:1 mixture by weight of Forms α and γ of Compound A before stirring, and for the crystals obtained. The apparatus and parameters for recording the spectra in this case are the same as those described above for the study of Form α in Example 1. The spectra obtained are shown in Fig. 16. This Figure shows the spectra of the Form α of Compound A used, the Form γ of Compound A used, a 1:1 mixture by weight of Forms α and β of Compound A before mixing, and the crystals obtained after mixing, from bottom to top.

The intensity ratio of the diffraction peaks before and after stirring was compared: the peak with a diffraction angle of 2θ 10.2° for Form α and the peak with a diffraction angle of 2θ 10.7° for Form γ. As shown in the table below, the intensity ratio of the diffraction peak of Form γ and the peak of Form α after stirring increased compared with the ratio before stirring.

Table 13

Diffraction peak intensity 10.2°
(Form α) [imp/s] Diffraction peak intensity 10.7°
(Form γ) [imp/s] Ratio of diffraction peak intensities (Form γ/Form α) Before mixing 1116.69 371.13 0.33 After mixing 418.12 208.04 0.50

Example 8

Comparative Experiment between Forms β and γ of Compound A

(1) Forms β and γ of Compound A were mixed in a ratio of 1:1 by weight, and formamide (1 ml) was added to the resulting mixture (150 mg). The resulting suspension was stirred at room temperature for 1 day. The precipitate was filtered off at the same temperature.

(2) Forms β and γ of Compound A were mixed in a ratio of 1:1 by weight, and N,N-dimethylformamide (1 ml) was added to the resulting mixture (150 mg). The resulting suspension was stirred at room temperature for 1 day. The precipitate was filtered off at the same temperature.

(3) Forms β and γ of Compound A were mixed in a ratio of 1:1 by weight, and dimethyl sulfoxide (1 ml) was added to the resulting mixture (800 mg). The resulting suspension was stirred at room temperature for 1 day. The precipitate was filtered off at the same temperature.

The diffraction angles 2θ and the intensities of the diffraction peaks were determined by powder X-ray diffraction for the used Forms β and γ of Compound A, a 1:1 mixture by weight of Forms β and γ of Compound A before stirring, and for the crystals obtained as described above in (1), (2) and (3). The apparatus and parameters for recording the spectra in this case are the same as those described above for the study of Form α in Example 1. The spectra obtained are shown in Fig. 17. This Figure shows the spectra of the used Form γ of Compound A, the used Form β of Compound A, a 1:1 mixture by weight of Forms β and γ of Compound A before stirring, and the crystals obtained after stirring in formamide, in N,N-dimethylformamide and in dimethyl sulfoxide, from bottom to top.

According to the results for (1), (2) and (3) described above, the mixtures of crystalline forms formed a single crystalline form γ.

Example 9

Solubility testing

Each sample (i.e., Forms α, β, and γ of Compound A) was weighed into a 4 mL glass vessel. A test solution (3 mL) was added to the vessel, and the mixture was suspended. About 150 mg of the sample was weighed for the test solution, which was Japanese Pharmacopoeia (JP 1) dissolution test liquid No. 1, and about 15 mg of the sample was weighed for the test solution, which was Japanese Pharmacopoeia (JP 2) dissolution test liquid No. 2, and for water. Each mixture was shaken on a precision temperature shaker (manufactured by TAITEC) at 20°C for 3 hours. After shaking, the supernatant solution was filtered through a polytetrafluoroethylene filter disc (Millex-LG; manufactured by Millipore) with a particle size of 0.2 μm and a diameter of 4 mm. Further analysis was performed using high performance liquid chromatography (HPLC). The results are shown in the table below.

Table 14

Solvent Form α
(anhydrous form) Form β Form γ Water 2.6 mg/ml 2.0 mg/ml 1.5 mg/ml JP 1 Liquid 45.0 mg/ml 39.1 mg/ml 36.4 mg/ml JP 2 liquid 2.7 mg/ml 2.1 mg/ml 1.5 mg/ml JP 1 Liquid (Japanese Pharmacopoeia): Dissolve 2.0 g sodium chloride in 7.0 ml hydrochloric acid and add water to 1000 ml. The resulting solution is clear and colorless, the pH value is about 1.2.
JP 2 Liquid (Japanese Pharmacopoeia): Mix one volume of water with one volume of phosphate buffered saline solution with pH = 6.8.

Form α showed the highest solubility in each of the solvents tested, and Form γ showed the lowest solubility in each of the solvents tested.

Example 10

Preparations

Examples of preparations containing Compound A (e.g., as Form α, Form β, Form γ, or a mixture of any of these) are provided below and are not intended to be limiting.

Preparation 1 (Preparation of capsule)

1) Compound A 30 mg

2) Microcrystalline cellulose 10 mg

3) Lactose 19 mg

4) Magnesium stearate 1 mg

1), 2), 3) and 4) are mixed to fill into a gelatin capsule.

Preparation 2 (Preparation of tablet)

1) Compound A 10 g

2) Lactose 50 g

3) Corn starch 15 g

4) Calcium carmellose 44 g

5) Magnesium stearate 1 g

The entire amount of 1), 2) and 3) and 30 grams of 4) were combined with water, dried in a vacuum and then granulated. The resulting granules were mixed with 14 grams of 4) and 1 gram of 5) and tabletted on a tablet press. In this way, 1000 tablets were obtained, each containing 10 mg of Compound A.

Various modifications of the present invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also within the scope of the present invention. Each literature reference, including all patents, patent applications, and journal publications cited in this application, is incorporated herein by reference in their entirety.

Claims (1)

A method for producing a crystalline form of 3-((3S,4R)-3-methyl-6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1,6-diazaspiro[3.4]octan-1-yl)-3-oxopropanenitrile having Form β, having an X-ray powder diffraction spectrum containing characteristic peaks with 2θ (°) values of 10.5±0.2, 11.8±0.2, 19.3±0.2 and 22.0±0.2, comprising crystallizing 3-((3S,4R)-3-methyl-6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1,6-diazaspiro[3.4]octan-1-yl)-3-oxopropanenitrile from a solvent, wherein the solvent contains 1:3 (v/v) 1-butanol and acetonitrile, respectively, and the mixture of the compound and solvent is stirred at 40°C for 10 days, followed by cooling to room temperature.