Triprostinil monohydrate crystal and preparation method thereof

Abstract

The invention relates to a treprostinil monohydrate crystal and a preparation method thereof. The present invention provides novel crystalline forms of treprostinil monohydrate, mixtures comprising the same, and methods of making the same.

Description

[0001] This application is a divisional application of the application for patent with the application date of October 28, 2021, the application number of 202111259365.2, and the invention name of “Treprostinil monohydrate crystal and its preparation method”. TECHNICAL FIELD

[0002] The present invention relates generally to solid forms of benzindene prostacyclin derivatives, and in particular, to novel crystalline forms of Treprostinil monohydrate and methods of preparation thereof. BACKGROUND

[0003] Treprostinil (UT15) is a synthetic analog of benzindene prostacyclin having the following structural formula:

[0004] .

[0005] Treprostinil is used to treat patients with pulmonary arterial hypertension (PAH) to improve their exercise capacity and can be prepared in various dosage forms and can be administered orally, inhaled or injected. US 2015 / 148414 discloses Tyvaso® (treprostinil) for infusion is a long-acting pulmonary vasodilator for the treatment of pulmonary hypertension to provide significantly lower plasma concentrations. Remodulin® (treprostinil sodium) injection is another formulation approved by the Food and Drug Administration (FDA) for the treatment of PAH, which is a sterile sodium salt formulated for subcutaneous or intravenous administration. US 8,232,316 discloses oral administration of treprostinil diethanolamine, which can increase oral bioavailability and circulating concentrations of treprostinil. US 6,521,212 and US 6,756,033 also disclose the treatment of pulmonary hypertension by inhalation of treprostinil.

[0006] Trapidil is a highly polar compound containing one carboxylic acid (-COOH) and two hydroxyl (-OH) functional groups, and is very susceptible to esterification with alcohols, including trapidil itself, to form its dimer or ester. WO 2009 / 137066 discloses the preparation of comparative anhydrous trapidil crystals (Lot No. 01A07002). The anhydrous trapidil crystals initially contained 0.5% trapidil dimer (0.2% 750W93 + 0.3% 751W93), which can have been generated during the high-temperature production process. WO 2009 / 137066 also discloses that, according to stability testing, the dimer continuously forms in anhydrous trapidil at 25°C, and at higher temperatures, the dimer formation increases, while at 5°C it is negligible. Thus, WO 2009 / 137066 provides monohydrate trapidil crystals. Compared to anhydrous trapidil crystals, monohydrate trapidil crystals are more stable and can be stored at room temperature for a long period of time. According to accelerated stability testing at 25°C, 30°C, and 40°C for 6 months, the dimer formation of monohydrate trapidil crystals is almost negligible (Lot No. 01M07033). However, the use of alcoholic solvents during monohydrate trapidil crystallization generates trapidil esterification impurities. As disclosed in WO 2009 / 137066, monohydrate trapidil crystals with about 99.5% purity prepared using an ethanol-water system (Lot No. D-1007-089, Vial 2) initially contained 0.2% trapidil ethyl ester or UT-15 ethyl ester, 0.1% 750W93, 0.04% 751W93, and 0.05% Impurity 1 and <0.05% Impurity 2. WO 2009 / 137066 further discloses that monohydrate trapidil crystals with about 99.6% purity (Lot No. 01M07033) initially contained 0.1% 3AU90, 0.2% trapidil ethyl ester or UT-15 ethyl ester, 0.08% 750W93, and <0.05% 751W93.

[0007] Based on the above problems, US 9,822,057 and US 10,167,247 disclose another method for preparing monohydrate trapidil, which uses a solvent other than ethanol to avoid generating trapidil ethyl ester. US 9,822,057 and US 10,167,247 further disclose two crystalline forms of monohydrate trapidil (Form A and Form B), respectively as Figure 1 and Figure 3However, the preparation methods of the monohydrate treprostinil crystalline Form A and Form B are complicated. The monohydrate treprostinil crystalline Form A was prepared from a slurry sample (containing 500 mg of treprostinil and 3.0 ml of 1,4-dioxane / water 1:1 v / v solution) which required long time rotation (about three days) at room temperature on a turntable and filtration to isolate the solid. The solid was then ground into smaller pieces stepwise for further drying (about one day) to obtain the monohydrate treprostinil crystalline Form A. The monohydrate treprostinil crystalline Form B was also prepared from a slurry sample (containing 1,019 mg of treprostinil and 3.5 ml of methanol and 3.5 ml of water) which required long time standing (about three days) at room temperature in a capped vial and filtration to isolate the solid. The solid was then ground into smaller pieces stepwise for further drying (about 44 hours) to obtain the monohydrate treprostinil crystalline Form B. The water content of the monohydrate treprostinil crystalline Form B was still 12.24% after drying for about two days. The water content of the monohydrate treprostinil crystalline Form B does not seem to be easily reduced to about 4.41% (one mole of water in treprostinil is calculated to be 4.41 wt%). The preparation methods disclosed in US 9,822,057 and US 10,167,247 can obtain monohydrate treprostinil crystalline Form A and Form B with less impurities; however, these complicated preparation methods are not suitable for industrial scale operation.

[0008] Therefore, there is a need to prepare monohydrate treprostinil crystals in an efficient and economical manner in which the formation of undesired impurities during the preparation process can also be effectively avoided. SUMMARY

[0009] SUMMARY

[0010] In view of this, the present inventors conducted a series of experiments and surprisingly found that the addition of phosphoric acid to a basic aqueous solution of treprostinil can obtain monohydrate treprostinil crystals in an accurate and simple manner. The method can be performed at room temperature with relatively short time (four to six hours) and without the use of organic solvents (e.g. alcohol), thereby significantly reducing the possibility of the formation of treprostinil dimer and / or undesired esterified impurities. The resulting monohydrate treprostinil crystals with high yield (>90%) and high purity (>99%) are also easy to filter and dry.

[0011] One aspect of the present application provides two novel crystalline forms of monohydrate treprostinil, “Form I and Form II”, and the preparation methods thereof.

[0012] In one embodiment, the present application provides a method for preparing crystalline treprostinil monohydrate Form I, comprising: providing a basic aqueous solution of treprostinil; adding phosphoric acid to the basic aqueous solution of treprostinil until the aqueous solution becomes an acidic aqueous solution (pH of about 2 to about 6), wherein the rate of decrease in pH is less than about 0.2 per minute; and stirring until a precipitate forms. The method further comprises the steps of filtering the precipitate and / or adding water to rinse the precipitate, thereby isolating crystalline treprostinil monohydrate Form I, and optionally drying the crystalline treprostinil monohydrate Form I.

[0013] In one embodiment, the present application provides crystalline treprostinil monohydrate Form I, which exhibits two most intense characteristic peaks in its X-ray powder diffraction (XRPD) pattern at the following 2Θ reflection angles: 5.43 ± 0.2° and 10.87 ± 0.2°. The crystalline treprostinil monohydrate Form I is substantially free of any other crystalline form of treprostinil.

[0014] In one embodiment, the present application provides crystalline treprostinil monohydrate Form I, which exhibits two most intense characteristic peaks in its X-ray powder diffraction (XRPD) pattern at the following 2Θ reflection angles: 5.43 ± 0.2° and 10.87 ± 0.2°. The crystalline treprostinil monohydrate Form I is substantially free of any other crystalline form of treprostinil.

[0015] In one embodiment, the present application provides a method for preparing crystalline treprostinil monohydrate Form II, comprising: providing a basic aqueous solution of treprostinil; adding phosphoric acid to the basic aqueous solution of treprostinil until the aqueous solution becomes an acidic aqueous solution (pH of about 2 to about 6), wherein the rate of decrease in pH is more than about 0.6 per minute; and stirring until a precipitate forms. The method further comprises the steps of filtering the precipitate and / or adding water to rinse the precipitate, thereby isolating crystalline treprostinil monohydrate Form II, and optionally drying the crystalline treprostinil monohydrate Form II.

[0016] In one embodiment, the present application provides crystalline treprostinil monohydrate Form II, which exhibits two most intense characteristic peaks in its XRPD pattern at the following 2Θ reflection angles: 5.19 ± 0.2° and 10.40 ± 0.2°. The crystalline treprostinil monohydrate Form II is substantially free of any other crystalline form of treprostinil.

[0017] In one embodiment, the present application provides crystalline treprostinil monohydrate Form II, which exhibits two most intense characteristic peaks in its XRPD pattern at the following 2Θ reflection angles: 5.19 ± 0.2° and 10.40 ± 0.2°. The crystalline treprostinil monohydrate Form II is substantially free of any other crystalline form of treprostinil. In one embodiment, the present application provides crystalline treprostinil monohydrate Form II, which exhibits two most intense characteristic peaks in its XRPD pattern at the following 2Θ reflection angles: 5.19 ± 0.2° and 10.40 ± 0.2°. The crystalline treprostinil monohydrate Form II is substantially free of any other crystalline form of treprostinil.

[0018] According to another aspect, the present application provides a mixture comprising both crystalline Form I and Form II of treprostinil monohydrate, the XRPD pattern of which exhibits common characteristic peaks at 2Θ reflection angles of 5.26 ± 0.2°, 13.20 ± 0.2° and 16.25 ± 0.2°, as well as separate characteristic peaks belonging to Form I at 10.65 ± 0.2° and 12.20 ± 0.2° and to Form II at 10.36 ± 0.2°, 11.61 ± 0.2° and 12.60 ± 0.2°. The mixture of treprostinil monohydrate crystals comprises at least about 10% of Form I or at least about 10% of Form II. The mixture of treprostinil monohydrate crystals is substantially free of any other crystalline form of treprostinil.

[0019] In one embodiment, the present application provides a method of preparing a mixture comprising both crystalline Form I and Form II of treprostinil monohydrate, comprising: providing a basic aqueous solution of treprostinil; adding phosphoric acid to the basic aqueous solution of treprostinil until the aqueous solution becomes acidic (pH of about 2 to about 6), wherein the rate of decrease in pH is more than about 0.2 and less than about 0.6 per minute; and stirring until a precipitate forms. The method further comprises the steps of filtering out the precipitate and / or adding water to rinse the precipitate, thereby isolating a mixture comprising both crystalline Form I and Form II of treprostinil monohydrate.

[0020] The drawings illustrate

[0021] Figure 1 X-ray powder diffraction (XRPD) pattern showing crystalline Form A of treprostinil monohydrate.

[0022] Figure 2 Differential scanning calorimetry (DSC) thermogram showing crystalline Form A of treprostinil monohydrate.

[0023] Figure 3 X-ray powder diffraction (XRPD) pattern showing crystalline Form B of treprostinil monohydrate.

[0024] Figure 4 Differential scanning calorimetry (DSC) thermogram showing crystalline Form B of treprostinil monohydrate.

[0025] Figure 5 X-ray powder diffraction (XRPD) pattern showing crystalline Form I of treprostinil monohydrate of the present application.

[0026] Figure 6 Differential scanning calorimetry (DSC) thermogram showing crystalline Form I of treprostinil monohydrate of the present application.

[0027] Figure 7 Thermogravimetric analysis (TGA) pattern showing crystalline Form I of treprostinil monohydrate of the present application.

[0028] Figure 8 X-ray powder diffraction (XRPD) pattern showing crystalline Form II of treprostinil monohydrate of the present application.

[0029] Figure 9 Differential scanning calorimetry (DSC) thermogram pattern showing crystalline Form II of treprostinil monohydrate of the present application.

[0030] Figure 10 Thermogravimetric analysis (TGA) pattern showing crystalline Form II of treprostinil monohydrate of the present application.

[0031] Figure 11 X-ray powder diffraction (XRPD) pattern showing a mixture of crystalline Form I and crystalline Form II of treprostinil monohydrate of the present application. DETAILED DESCRIPTION

[0032] The term "substantially free of any other crystalline form of treprostinil" or similar terms, as used herein, means that the compound or mixture in question contains no more than about 0.5%, about 0.4%, about 0.3%, about 0.2%, or about 0.1% of any other crystalline form of treprostinil. The term "about" is used herein to denote the inherent deviations or variations in the method used to determine a value or the variations that exist among study individuals. The values of the 2Θ reflection angles of the XRPD patterns described herein can include a deviation of ±0.2°.

[0033] Preparation of treprostinil basic aqueous solution

[0034] A treprostinil basic aqueous solution can be prepared by any suitable method. A typical synthesis of treprostinil is shown in Scheme A below:

[0035] Scheme A

[0036] .

[0037] Trapidil can be obtained from hydrolysis of trapidil ester or trapidil nitrile by adding base (such as potassium hydroxide, sodium hydroxide and lithium hydroxide) and water into an organic solvent (such as methanol, ethanol, isopropanol, tetrahydrofuran and acetone), or by adding hydrolytic enzyme into an aqueous base buffer. After the hydrolysis reaction, the produced trapidil is dissolved in an aqueous phase, and the residual organic solvent can be removed by extraction or concentration. When hydrolytic enzyme is used, the hydrolytic enzyme can be removed by filtration. The produced aqueous solution without organic solvent and hydrolytic enzyme is referred to as "trapidil basic aqueous solution" herein. In some embodiments, the pH value of the trapidil basic aqueous solution is not limited to a value in the range of about 7 to about 14, about 8 to about 13, about 9 to about 12, about 10 to about 11, or about 8 to about 12.

[0038] In one embodiment, the trapidil basic aqueous solution can be obtained by dissolving trapidil alkali metal salt (such as trapidil sodium, trapidil potassium and trapidil lithium) or trapidil amine salt (such as trapidil diethanolamine and trapidil edetate) in water.

[0039] In one embodiment, the trapidil basic aqueous solution can be obtained by dissolving trapidil in an organic solvent (such as ethyl acetate) and then extracting with an aqueous base (such as aqueous sodium bicarbonate solution), and then collecting the separated aqueous phase as the trapidil basic aqueous solution.

[0040] Acidifying the trapidil basic aqueous solution to obtain trapidil solid

[0041] In prior art (such as WO 2009 / 137066 and J. Org. Chem. 69, 1890-1902 (2004)), trapidil obtained by adding hydrochloric acid into trapidil basic aqueous solution is a yellow gummy solid or a viscous liquid. Therefore, prior art has to use organic solvent extraction of gummy or viscous trapidil suspended in aqueous solution. The present inventors surprisingly found that trapidil obtained by adding phosphoric acid into trapidil basic aqueous solution is a white crystalline solid, which can be easily filtered and dried compared to gummy solid or viscous liquid. The white crystalline solid of trapidil is in monohydrate form, as evidenced by thermogravimetric analysis (TGA) and Karl Fisher titration. In addition, the monohydrate trapidil of the present invention shows two novel crystalline forms (Form I and Form II described below), as evidenced by X-ray powder diffraction (XRPD). The inventors also found that slow addition of phosphoric acid helps the formation of Form I, while fast addition helps the formation of Form II.

[0042] Crystalline Form I of monohydrate trapidil and preparation thereof

[0043] In some embodiments, a method for preparing crystalline form I of treprostinil monohydrate (form I treprostinil monohydrate crystals) comprises the steps of:

[0044] (a) providing a basic aqueous solution of treprostinil;

[0045] (b) slowly adding phosphoric acid dropwise to the basic aqueous solution of treprostinil until the aqueous solution becomes an acidic aqueous solution having a pH of about 2 to about 6, wherein the pH of the aqueous solution decreases at a rate of less than about 0.2 per minute;

[0046] (c) stirring until a precipitate forms;

[0047] (d) filtering out the precipitate and / or adding water to rinse the precipitate, thereby isolating form I treprostinil monohydrate crystals; and

[0048] (e) optionally drying the form I treprostinil monohydrate crystals.

[0049] In some embodiments, the concentration of treprostinil in the basic aqueous solution of treprostinil can range from, but is not limited to, about 0.01 g / ml to about 0.10 g / ml, preferably about 0.02 g / ml to about 0.07 g / ml, and more preferably about 0.03 g / ml to about 0.05 g / ml. The basic aqueous solution of treprostinil can be prepared at a temperature ranging from about 0 °C to about 60 °C, preferably about 10 °C to about 50 °C, and more preferably at room temperature to about 40 °C, but the present application is not limited thereto.

[0050] In some embodiments, the phosphoric acid can be prepared in the form of an aqueous solution of phosphoric acid. In some embodiments, the aqueous solution of phosphoric acid added to the aqueous basic solution of treprostinil is prepared by adding phosphoric acid to water. The concentration of the aqueous solution of phosphoric acid can range from, but is not limited to, about 0.1 N to about 20 N, preferably about 1 N to about 15 N, and more preferably about 5 N to about 10 N, but the present application is not limited thereto. In some embodiments, the pH of the aqueous solution of treprostinil is adjusted by adding the aqueous solution of phosphoric acid, but is not limited to, about 2 to about 6, preferably about 2 to about 5, and more preferably about 2 to about 4. The aqueous solution of phosphoric acid can be added to the aqueous basic solution of treprostinil at a temperature ranging from about 0 °C to about 60 °C, preferably about 10 °C to about 50 °C, and more preferably room temperature to about 40 °C, but the present application is not limited thereto. The rate of decrease of the pH of the aqueous solution of treprostinil for preparing Form I should be controlled to be less than about 0.2 per minute. The lower limit of the rate of decrease of the pH of the aqueous solution of treprostinil for preparing Form I is not limited herein and can be any suitable value as predetermined or determined. In some embodiments, the aqueous solution of phosphoric acid is added to the aqueous basic solution of treprostinil over a period of more than about 30 minutes.

[0051] In some embodiments, the precipitation of treprostinil monohydrate, crystalline Form I, can be performed at a temperature ranging from about 0 °C to about 60 °C, preferably about 5 °C to about 50 °C, and more preferably about 10 °C to about 40 °C, but the present application is not limited thereto.

[0052] In some embodiments, the step of filtering out the precipitate comprises washing the precipitate with water. The volume of the washing water can range from, but is not limited to, about 50 ml to about 400 ml, preferably about 75 ml to about 350 ml, and more preferably about 100 ml to about 300 ml, per 1 g of the precipitate, but the present application is not limited thereto. Since no organic solvent is used, the resulting treprostinil monohydrate Form I crystals do not contain any residual organic solvent. The treprostinil monohydrate Form I crystals without residual organic solvent are very easy to filter compared to the slurry samples disclosed in US 9,822,057 and US 10,167,247 or the gummy solid or viscous liquid prepared as disclosed in WO 2009 / 137066 and J. Org. Chem. 69, 1890-1902 (2004). The filtered treprostinil monohydrate Form I crystals have a lower viscosity and can be easily stripped from the drum and dried due to their compact solid characteristics.

[0053] In some embodiments, the drying step of the monohydrate treprostinil Form I crystals can be performed under a reduced pressure of about 0.001 Torr to about 20 Torr, preferably about 0.01 Torr to about 10 Torr, and more preferably about 0.01 Torr to about 1 Torr, but the present application is not limited thereto. The drying step of the monohydrate treprostinil Form I crystals can be performed at a temperature ranging from about 0 °C to about 40 °C, preferably about 5 °C to about 30 °C, and more preferably about 10 °C to room temperature, but the present application is not limited thereto.

[0054] In comparison to the monohydrate treprostinil preparation methods disclosed in US 9,822,057 and US 10,167,247, which take about four days and are complicated with a trituration step, the present method, which takes about four to six hours, is not only simpler but also more time efficient. The method described in the present application can also significantly reduce the possibility of forming treprostinil dimer and treprostinil esterization impurities. In addition, the monohydrate treprostinil Form I crystals having a granular feature obtained in the present application are more convenient for filtration, drying, and weighing for industrial processing.

[0055] Characteristics of monohydrate treprostinil crystalline Form I

[0056] The present application provides a novel monohydrate treprostinil crystalline Form I. The characteristics of the monohydrate treprostinil crystalline Form I have been verified by Fourier transform infrared (FTIR) spectroscopy, Karl-Fischer titration, and TGA. In one embodiment, the monohydrate treprostinil crystalline Form I exhibits an FTIR characteristic peak at about 3513 ± 4 cm-1, highlighting the characteristics of a hydrate, which indicates that the hydrated form of treprostinil is a unique molecular entity. In addition, one mole of water molecule in treprostinil is calculated to be 4.41 wt%. The water content of about 4.41 ± 1% measured by Karl-Fischer titration and TGA (as shown in Figure 7

[0057] ​In one embodiment, the XRPD pattern of crystalline Treprostinil monohydrate Form I exhibits two most intense characteristic peaks at the following 2Θ reflection angles: 5.43 ± 0.2° and 10.87 ± 0.2°. In some embodiments, the XRPD pattern is essentially free of characteristic peaks at a 2Θ reflection angle of 21.71 ° (the most intense peak of Form A) or 21.56 ° (the most intense peak of Form B). In preferred embodiments, the XRPD pattern of Form I further comprises characteristic peaks at the following 2Θ reflection angles: 12.30 ± 0.2°, 16.34 ± 0.2° and 20.39 ± 0.2°. More preferably, the XRPD pattern of crystalline Treprostinil monohydrate Form I is in accordance with that shown in Figure 1. Figure 5 Specific data for crystalline Treprostinil monohydrate Form I is shown in Table 1.

[0058] Table 1

[0059]

[0060] In one embodiment, the present application provides crystalline Treprostinil monohydrate Form I having an XRPD pattern substantially as shown in Figure 5 Figure 1.

[0061] From the XRPD characteristics, it is known that the crystalline Form I of treprostinil monohydrate is different from the crystalline Form A or Form B of treprostinil monohydrate disclosed in US 9,822,057 and US 10,167,247. The two strongest characteristic peaks of Form I are located at 5.43° and 10.87°, which are significantly different from the two strongest characteristic peaks of Form A located at 10.36° and 21.71° and Form B located at 20.56° and 21.56°. The crystalline Form I of treprostinil monohydrate does not have the strongest characteristic peak located at 21.71° or 21.56°, which means that Form I is substantially free of Form A and Form B. In the present invention, the crystalline Form I of treprostinil monohydrate is substantially free of the characteristic peak having a 2Q reflection angle of 21.71° or 21.56°. As used herein, the term "substantially free of the characteristic peak" means that in the XRPD pattern of the crystalline Form I of treprostinil monohydrate, the peak intensity located at 21.71° or 21.56° is about 10% less and preferably about 3% less than the strongest peak intensity located at 10.87±0.2° but is not limited thereto. In addition, Form A includes two peaks located at 5.17° and 5.88° in the range of 5 to 7°, but Form I includes three different peaks located at 5.43°, 6.05° and 6.61° in the range of 5 to 7°. Form A further includes four peaks located at 10.36°, 11.62°, 12.59° and 13.15° in the range of 10 to 14°, but Form I includes three different peaks located at 10.87°, 12.30° and 13.19° in the range of 10 to 14°. Furthermore, Form B includes four peaks located at 10.66°, 12.10°, 12.90° and 13.10° in the range of 10 to 14°, but Form I includes three different peaks located at 10.87°, 12.30° and 13.19° in the range of 10 to 14°. Form B further includes ten peaks located at 19.45°, 19.80°, 20.17°, 20.56°, 20.99°, 21.22°, 21.56°, 22.26°, 22.91° and 23.10° in the range of 19 to 23.5°, but Form I includes seven different peaks located at 19.65°, 20.39°, 20.74°, 21.32°, 21.66°, 22.42° and 23.26° in the range, which have significant (and relative) intensity differences. In addition, the 10.36° peak belonging to Form A and the 10.66° peak belonging to Form B are not present in the XRPD pattern of Form I, indicating that Form I is an independent crystalline form not including Form A and / or Form B, and Form I is different from Form A or Form B. Based on the differences in XRPD peaks in position and relative intensity compared to the crystalline Form A and Form B of treprostinil monohydrate, although the present invention is not limited by any theory, it is believed that the peak differences are due to structural differences, rather than due to sample conditions such as particle size.This confirms that the crystalline form I of treprostinil monohydrate is a novel crystalline form.

[0062] In one embodiment, the present application provides crystalline form I of treprostinil monohydrate having a differential scanning calorimetry (DSC) thermogram comprising two major endothermic peaks: one having a peak onset temperature of 65.9 ± 2 °C and a peak maximum of 79.2 ± 2 °C, and another having a peak onset temperature of 123.2 ± 2 °C and a peak maximum of 125.1 ± 2 °C. In a preferred embodiment, the present application provides crystalline form I of treprostinil monohydrate having a DSC thermogram substantially as shown in FIG. 2. Figure 6

[0063] In one embodiment, the DSC characteristics of crystalline form I of treprostinil monohydrate are different from the DSC characteristics of crystalline form A ( Figure 2 ) and form B ( Figure 4 ) of treprostinil monohydrate disclosed in US 9,822,057 and US 10,167,247. Form A comprises two peaks at about 61.94 °C and 78.30 °C and one peak at about 126.26 °C, and form B comprises two peaks at about 60.08 °C and 74.79 °C and one peak at about 125.18 °C. However, form I comprises only one peak at about 79.24 °C and one peak at about 125.11 °C. The onset temperature of the 79.24 °C peak of form I (65.85 °C) is higher than the peak maximum temperatures of the 61.94 °C peak of form A and the 60.08 °C peak of form B, indicating that crystalline form I of treprostinil monohydrate lacks the 61.94 °C and 60.08 °C peaks. The different temperature of the peak at about 79.24 °C and the absence of the peak at about 60.08 °C to 61.94 °C indicate that crystalline form I of treprostinil monohydrate comprises a unique crystalline form compared to crystalline form A and form B of treprostinil monohydrate, and form I is different from form A or form B. In some embodiments, crystalline form I of treprostinil monohydrate is substantially free of any other crystalline form of treprostinil.

[0064] ​In one embodiment, the monohydrate treprostinil Form I crystals have superior filterability over the slurry samples of monohydrate treprostinil crystalline Form A and Form B disclosed in US 9,822,057 and US 10,167,247 or the gelatinous solid or viscous liquid of crystalline monohydrate treprostinil prepared as described in WO 2009 / 137066 and J. Org. Chem. 69, 1890-1902 (2004) due to its crystal characteristics. The benefits of monohydrate treprostinil Form I crystals having good filterability are: (1) non-desired impurities dissolved in the filtrate can be easily removed while filtering and rinsing the monohydrate treprostinil Form I crystals; (2) impurities resulting from residual solvents can be avoided due to the shorter filtration time of the monohydrate treprostinil Form I crystals; and (3) the filtered monohydrate treprostinil Form I crystals can be easily dried. Based on the above advantages, esterification impurities of treprostinil can be easily excluded and treprostinil dimer formation can be avoided.

[0065] Monohydrate treprostinil crystalline Form II and preparation thereof

[0066] In one embodiment, the method for preparing monohydrate treprostinil crystalline Form II (monohydrate treprostinil Form II crystals) comprises the following steps:

[0067] (a) providing a basic aqueous solution of treprostinil;

[0068] (b) rapidly adding phosphoric acid to the basic aqueous solution of treprostinil until the aqueous solution becomes an acidic aqueous solution having a pH value of about 2 to about 4, wherein the pH value of the aqueous solution decreases at a rate of more than about 0.6 per minute;

[0069] (c) stirring until a precipitate forms;

[0070] (d) filtering out the precipitate and / or adding water to rinse the precipitate, thereby isolating monohydrate treprostinil crystalline Form II; and

[0071] (e) optionally drying the monohydrate treprostinil crystalline Form II.

[0072] In some embodiments, the concentration of treprostinil in the basic aqueous solution of treprostinil can range from, but is not limited to, about 0.01 g / ml to about 0.10 g / ml, preferably about 0.02 g / ml to about 0.07 g / ml, and more preferably about 0.03 g / ml to about 0.05 g / ml. The basic aqueous solution of treprostinil can be prepared at a temperature ranging from about 0 °C to about 60 °C, preferably about 10 °C to about 50 °C, and more preferably room temperature to about 40 °C, but the present application is not limited thereto.

[0073] In some embodiments, the phosphoric acid can be prepared in the form of an aqueous solution of phosphoric acid. In some embodiments, the aqueous solution of phosphoric acid added to the aqueous basic solution of treprostinil is prepared by adding phosphoric acid to water. The concentration of the aqueous solution of phosphoric acid can range from, but is not limited to, about 0.1 N to about 20 N, preferably about 1 N to about 15 N, and more preferably about 5 N to about 10 N, but the present application is not limited thereto. In some embodiments, the pH of the aqueous solution of treprostinil is adjusted by adding the aqueous solution of phosphoric acid, but is not limited to, about 2 to about 6, preferably about 2 to about 5, and more preferably about 2 to about 4. The aqueous solution of phosphoric acid can be added to the aqueous basic solution of treprostinil at a temperature ranging from about 0 °C to about 60 °C, preferably about 10 °C to about 50 °C, and more preferably room temperature to about 40 °C, but the present application is not limited thereto. The rate of decrease of the pH of the aqueous solution of treprostinil for preparing Form II should be controlled to be more than about 0.6 per minute. The upper limit of the rate of decrease of the pH of the aqueous solution of treprostinil for preparing Form II is not limited herein and can be any suitable value as predetermined or determined. In some embodiments, the aqueous solution of phosphoric acid is added to the aqueous basic solution of treprostinil in about 1 to about 10 minutes.

[0074] In some embodiments, the precipitation of treprostinil monohydrate Form II crystals can be carried out at a temperature ranging from about 0 °C to about 60 °C, preferably about 5 °C to about 50 °C, and more preferably about 10 °C to about 40 °C, but the present application is not limited thereto.

[0075] In some embodiments, the step of filtering out the precipitate comprises washing the precipitate with water. The volume of the washing water can range from, but is not limited to, about 50 ml to about 400 ml, preferably about 75 ml to about 350 ml, and more preferably about 100 ml to about 300 ml, per 1 g of precipitate, but the present application is not limited thereto. Since no organic solvent is used, the resulting treprostinil monohydrate Form II crystals do not contain any residual organic solvent. The treprostinil monohydrate Form II crystals free of residual organic solvent are very easy to filter compared to the slurry samples disclosed in US 9,822,057 and US 10,167,247 and the gummy solid or viscous liquid prepared as disclosed in WO 2009 / 137066 and J. Org. Chem. 69, 1890-1902 (2004). The filtered treprostinil monohydrate Form II crystals have a lower viscosity and can be easily stripped from the drum and dried due to their compact solid characteristics.

[0076] In some embodiments, the drying step of the monohydrate treprostinil Form II crystals can be performed under a reduced pressure of about 0.001 Torr to about 20 Torr, preferably about 0.01 Torr to about 10 Torr, and more preferably about 0.01 Torr to about 1 Torr, but the present application is not limited thereto. The drying step of the monohydrate treprostinil Form II crystals can be performed at a temperature ranging from about 0 °C to about 40 °C, preferably about 5 °C to about 30 °C, and more preferably about 10 °C to room temperature, but the present application is not limited thereto.

[0077] In comparison to the monohydrate treprostinil preparation methods disclosed in US 9,822,057 and US 10,167,247, which take about four days and have a complicated trituration step, the novel method of the present application, which takes about four to six hours, is not only simpler but also more time efficient. In addition, the method of the present application can also significantly reduce the possibility of forming treprostinil dimer and treprostinil esterization impurities. Furthermore, the monohydrate treprostinil Form II crystals having a granular feature obtained by the present application are more convenient for filtration, drying, and weighing for industrial processing.

[0078] Characteristics of monohydrate treprostinil crystalline Form II

[0079] The present application also provides a novel monohydrate treprostinil crystalline Form II. The characteristics of the monohydrate treprostinil crystalline Form II have been verified by FTIR spectroscopy, Karl-Fischer titration, and TGA. In one embodiment, the monohydrate treprostinil crystalline Form II exhibits an FTIR characteristic peak at about 3513 ± 4 cm-1, highlighting the characteristics of the hydrate, which indicates that the hydrated form of treprostinil is a unique molecular entity. In addition, one mole of water molecule in treprostinil is calculated to be 4.41 wt%. In some embodiments, the water content of about 4.41 ± 1% measured by Karl-Fischer titration and TGA (as shown in Figure 10 The monohydrate treprostinil crystalline Form II obtained by the method of the present application is essentially in monohydrate form. The monohydrate treprostinil crystalline Form II has a purity of at least about 99.5%, at least about 99.6%, at least about 99.7%, at least about 99.8%, or at least about 99.9%, in addition to residual solvents.

[0080] In one embodiment, the XRPD pattern of crystalline treprostinil monohydrate Form II exhibits two most intense characteristic peaks at the following 2Θ reflection angles: 5.19 ± 0.2° and 10.40 ± 0.2°. In some embodiments, the XRPD pattern is substantially free of characteristic peaks at a 2Θ reflection angle of 21.71 ° (the most intense peak of Form A) or 21.56 ° (the most intense peak of Form B). In a preferred embodiment, the XRPD pattern of Form II further comprises characteristic peaks at the following 2Θ reflection angles: 11.62 ± 0.2°, 16.19 ± 0.2°, and 20.14 ± 0.2°. More preferably, the XRPD pattern of crystalline treprostinil monohydrate Form II is in accordance with that shown in Figure 2. Figure 8 Specific data for crystalline treprostinil monohydrate Form II is shown in Table 2.

[0081] Table 2

[0082]

[0083] In one embodiment, the present application provides crystalline treprostinil monohydrate Form II having an XRPD pattern substantially as shown in Figure 8 Figure 2.

[0084] Form II of treprostinil monohydrate is substantially free of characteristic peaks at 21.71° or 21.56° which means that Form II is substantially free of Form A and Form B. As used herein, the term "substantially free of characteristic peaks" means that the intensity of the peak at 21.71° or 21.56° is less than about 10% and preferably less than about 3% of the intensity of the strongest peak at 10.40 ± 0.2° in the XRPD pattern of treprostinil monohydrate crystalline Form II, but is not limited thereto. In addition, Form II includes five major peaks at 5.19°, 10.40°, 11.62°, 16.19°, and 20.14°; however, Form A includes five different major peaks at 5.17°, 10.36°, 11.62°, 19.95°, and 21.71° in the range. Form A includes very strong peaks in the range of 19 to 25°, but Form II includes only one major peak at 20.14° in the range of 19 to 25°. Notably, Form II includes a peak at 21.13° which is not present in the XRPD pattern of Form A. Furthermore, Form B includes three peaks at 5.32°, 5.92°, and 6.44° in the range of 5 to 7°, but Form II includes only two peaks at 5.19° and 5.93° in the range of 5 to 7°. Form B further includes four peaks at 10.66°, 12.10°, 12.90°, and 13.10° in the range of 10 to 14°, but Form II includes four different peaks at 10.40°, 11.62°, 12.61°, and 13.19° in the range of 10 to 14°. In addition, the 23.06° peak belonging to Form A and the 19.45° peak belonging to Form B are not present in the XRPD pattern of Form II, indicating that Form II is an independent crystalline form that does not include Form A and / or Form B, and that Form II is different from Form A or Form B. Based on the differences in the positions and relative intensities of the XRPD peaks of Form A and Form B of treprostinil monohydrate crystalline forms, although the present application is not bound by any theory, it is believed that the peak differences arise from structural differences, rather than sample conditions such as particle size, which confirms that treprostinil monohydrate crystalline Form II is a novel crystalline form.

[0085] In one embodiment, the present application provides crystalline treprostinil monohydrate Form II having a DSC thermogram pattern comprising two major endothermic peaks: one having a peak onset temperature of 58.8 ± 2 °C and a peak maximum of 73.8 ± 2 °C, and another having a peak onset temperature of 123.9 ± 2 °C and a peak maximum of 125.1 ± 2 °C. In a preferred embodiment, the present application provides crystalline treprostinil monohydrate Form II having a DSC thermogram pattern substantially as shown in FIG. 1. Figure 9

[0086] In one embodiment, the DSC characteristics of crystalline treprostinil monohydrate Form II are different from the DSC characteristics of crystalline treprostinil monohydrate Form A Figure 2 ) and Form B Figure 4 ) disclosed in US 9,822,057 and US 10,167,247. Form A comprises two peaks at about 61.94 °C and 78.30 °C and one peak at about 126.26 °C, and Form B comprises two peaks at about 60.08 °C and 74.79 °C and one peak at about 125.18 °C. However, Form II comprises only one peak at about 73.79 °C and one peak at about 125.10 °C. The onset temperature of the 73.79 °C peak of Form II is 58.76 °C. But the DSC thermogram pattern of Form II does not show the 61.94 °C or 60.08 °C peaks, indicating that crystalline treprostinil monohydrate Form II lacks the 61.94 °C peak of Form A and the 60.08 °C peak of Form B. The different temperature of the peak at about 73.8 °C and the disappearance of the peak at about 60.08 °C to 61.94 °C indicate that crystalline treprostinil monohydrate Form II comprises a unique crystalline form compared to crystalline treprostinil monohydrate Form A and Form B, and Form II is different from Form A or Form B. In some embodiments, crystalline treprostinil monohydrate Form II is substantially free of any other crystalline form of treprostinil.

[0087] ​In one embodiment, the monohydrate treprostinil Form II crystals have superior filterability over the slurry samples of monohydrate treprostinil crystalline Form A and Form B disclosed in US 9,822,057 and US 10,167,247 or the gelatinous solid or viscous liquid of crystalline monohydrate treprostinil prepared as described in WO 2009 / 137066 and J. Org. Chem. 69, 1890-1902 (2004) due to their crystal characteristics. The benefits of monohydrate treprostinil Form II crystals having good filterability are: (1) non-desired impurities dissolved in the filtrate can be easily removed while filtering and rinsing the monohydrate treprostinil Form II crystals; (2) impurities resulting from residual solvents can be avoided due to the short filtration time of the monohydrate treprostinil Form II crystals; and (3) the filtered monohydrate treprostinil Form II crystals are easily dried. Based on the above advantages, esterified impurities of treprostinil can be easily excluded and the formation of treprostinil dimer can be avoided.

[0088] Preparation of a mixture of monohydrate treprostinil Form I and Form II crystals

[0089] In the present invention, the rate of pH decrease adjusted by the addition of phosphoric acid during the preparation of monohydrate treprostinil crystals is the key to whether monohydrate treprostinil crystalline Form I or Form II can be obtained. When the rate of pH decrease is controlled to be more than about 0.6 per minute, Form II preferentially precipitates. However, when the rate of pH decrease is controlled to be less than about 0.2 per minute, Form I preferentially precipitates. When the rate of pH decrease is controlled between about 0.2 and about 0.6 per minute, a mixture of monohydrate treprostinil Form I and Form II crystals can be obtained. Other operating conditions for preparing a mixture of monohydrate treprostinil Form I and Form II crystals are similar to those for preparing Form I or Form II, and are therefore omitted. The method of preparing a mixture of monohydrate treprostinil Form I and Form II crystals is shown in Example 7, and the resulting XRPD pattern is shown in Figure 11 . Specific data for a mixture of monohydrate treprostinil Form I and Form II crystals are shown in Table 3.

[0090] Table 3

[0091]

[0092] In some embodiments, a mixture of the monohydrate treprostinil crystalline Form I and the monohydrate treprostinil crystalline Form II has an XRPD pattern comprising the Form I and Form II common peaks at 5.26 ± 0.2°, 13.20 ± 0.2°, and 16.25 ± 0.2°, and separate peaks at 10.65 ± 0.2° and 12.20 ± 0.2° belonging to Form I and at 10.36 ± 0.2°, 11.61 ± 0.2°, and 12.60 ± 0.2° belonging to Form II. In one embodiment, the peaks at 5.26 ± 0.2°, 13.20 ± 0.2°, and 16.25 ± 0.2° are common peaks for Form I and Form II, which are produced by the adjacent region overlap of the peaks at 5.43 ± 0.2°, 13.20 ± 0.2°, and 16.34 ± 0.2° for Form I and 5.19 ± 0.2°, 13.20 ± 0.2°, and 16.19 ± 0.2° for Form II. However, in the range of 10 to 14°, separate peaks at 10.65° and 12.20° appear to be associated with Form I, and the peaks at 10.36°, 11.61°, and 12.60° are associated with Form II. Based on these results, it is believed that the sample is a mixture of monohydrate treprostinil Form I and Form II crystals. The absence of the most intense characteristic peak at 21.71° or 21.56° indicates that the mixture of monohydrate treprostinil Form I and Form II crystals is substantially free of monohydrate treprostinil Form A or Form B crystals. The mixture of monohydrate treprostinil Form I and Form II has a purity of at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% in addition to residual solvent.

[0093] In some embodiments, the mixture of monohydrate treprostinil Form I and Form II crystals comprises at least about 10% of Form I or Form II. In some embodiments, the mixture of monohydrate treprostinil Form I and Form II crystals comprises about 10% to 90% of Form I and about 90% to 10% of Form II, or about 20% to 80% of Form I and about 80% to 20% of Form II, although the application is not limited in this respect. In some embodiments, the mixture of monohydrate treprostinil Form I and Form II crystals is substantially free of any other crystalline form of treprostinil.

[0094] In one embodiment, the mixture of Form I and Form II crystals of monohydrate treprostinil has superior filterability over the slurry samples of crystalline Form A and Form B of monohydrate treprostinil disclosed in US 9,822,057 and US 10,167,247 or the gelatinous solid or viscous liquid of crystalline monohydrate treprostinil prepared as described in WO 2009 / 137066 and J. Org. Chem. 69, 1890-1902 (2004) due to its crystal characteristics. The benefits of the mixture of Form I and Form II crystals of monohydrate treprostinil with good filterability are: (1) while filtering and rinsing the mixture of Form I and Form II crystals of monohydrate treprostinil, the undesirable impurities dissolved in the filtrate can be easily removed; (2) since the filtration time of the mixture of Form I and Form II crystals of monohydrate treprostinil is shorter, the impurities generated by residual solvent can be avoided; and (3) the filtered mixture of Form I and Form II crystals of monohydrate treprostinil is easy to dry. Based on the above advantages, the esterification impurities of treprostinil can be easily excluded and the treprostinil dimer formation can be avoided.

[0095] Although the word "about" precedes the minimum and maximum values in the ranges recited, the use of numerical values in the specification and claims of this application, unless otherwise specifically indicated, are not intended to be an absolute restriction. In this way slight variations on the stated values are intended to be within the scope of the stated values. Further, the ranges of the present disclosure are intended to be continuous ranges, including every value between the minimum and maximum values and any range derivable therein. Also disclosed herein are any and all ratios which can be formed from the stated values by dividing any of the stated values by any other of the stated values (and ranges of any such ratios). Accordingly, it is intended that all such alternative ratios, ranges and ranges of ratios are to be explicitly added to the description and claims. Those skilled in the art will appreciate that the values recited herein can be easily modified to arrive at a number of such alternative ratios, ranges and ranges of ratios, and that all such alternative ratios, ranges and ranges of ratios represent embodiments of the present invention.

[0096] All matter and / or methods disclosed and taught herein can be made and executed without undue experimentation in light of the present disclosure. While the matter and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those with ordinary skill in the art that variations can be applied to the matter and / or methods described and / or in the steps of the method or in the order of the steps of the method without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications against which are considered within the spirit, scope and concept of the invention as defined by the following claims.

[0097] Examples

[0098] X-ray powder diffraction (XRPD) analysis: XRPD patterns were collected on a Bruker D2 PHASER diffractometer with fixed divergence slits and 1D LYNXEYE detector. The sample (about 100 mg) was placed flat on the sample holder. The prepared sample was analyzed using Cu Ka radiation at a power of 10 mA and 30 kV, using a step size of 0.02 degrees and a step time of 1 second, in the 2 theta range of 4 to 40 degrees. Cu K beta radiation was removed by a divergence beam nickel filter.

[0099] Differential scanning calorimetry (DSC) analysis: DSC thermograms were collected on a TA DISCOVERY DSC25 instrument. The sample was weighed into an aluminum pan with crimped lid closure. The prepared sample was analyzed from 25 °C to 200 °C at a scan rate of 10 °C / min under a nitrogen flow of about 50 ml / min. The melting temperature and heat of fusion were calibrated prior to measurement by indium (In).

[0100] Thermogravimetric analysis (TGA): TGA thermograms were collected on a TA Q500 instrument. The sample was weighed onto a platinum pan. The prepared sample was analyzed from ambient temperature to 500 °C at a scan rate of 10 °C / min under nitrogen. Both temperature and weight calibrations were performed prior to measurement.

[0101] Ultra performance liquid chromatography (UPLC) analysis: UPLC spectra were collected on a Waters ACQUITY UPLC instrument. Conditions are as follows: samples were diluted to 1 mg / ml with 50 / 50 (v / v) acetonitrile / H20. The column was a Waters BEH C18, 1.7 μm, . The mobile phase was 60 / 40 (v / v) buffer / acetonitrile: 0 to 10 minutes; gradient change of buffer / acetonitrile 60 / 40 to 5 / 95 (v / v) (curve 6): 10 to 20 minutes; 5 / 95 (v / v) buffer / acetonitrile: 20 to 25 minutes; gradient change of buffer / acetonitrile 5 / 95 to 0 / 100 (v / v) (curve 6): 25 to 30 minutes; 0 / 100 (v / v) buffer / acetonitrile: 30 to 35 minutes. The buffer solution was an aqueous solution adjusted to pH 3.0 with trifluoroacetic acid. The flow rate was set to 0.42 ml / min. The column temperature was set to 45 °C and the sample temperature was set to 25 °C. 1.5 μL was injected. The run time was 35 minutes. The UV detector was set to 210 nm.

[0102] Example 1

[0103] Preparation of crystalline treprostinil monohydrate Form I

[0104] Dissolve 2-(((lR,2R,3aS,9aS)-2-hydroxy-l-((S)-3-hydroxyoctyl)-2,3,3a,4,9,9a- hexahydro-lH-cyclopenta[b]naphthalen-6-yl)oxy)acetonitrile (1.00 g, 2.7 mmol) in 10 ml 2-propanol, then add 4 ml potassium hydroxide solution (16% w / v) and stir at 80°C for 2 hours. Slowly cool the reaction mixture to room temperature, quench with hydrochloric acid solution and concentrate to remove 2-propanol, then add 30 ml saturated aqueous sodium bicarbonate solution and 30 ml ethyl acetate for extraction. The resulting treprostinil is then extracted with sodium bicarbonate solution (pH ~ 8.3) at 20°C to form a homogeneous solution. Afterwards, the treprostinil basic aqueous solution is acidified by slowly adding 9 N aqueous phosphoric acid solution at a rate of ~ 0.15 per minute to adjust the pH to ~ 3, and then stir at 20°C for 1 hour until most of the crystals precipitate. The resulting precipitated crystals are then filtered and rinsed with 180 ml water, then dried under high vacuum (~ 0.01 torr) at 20°C for 2 hours to give 1.01 g of monohydrate treprostinil Form I crystals (yield: 91.9%). XRPD and DSC results are the same as shown in Figure 5 and Figure 6 UPLC analysis of the product shows a purity of 100.0%, no treprostinil ethyl ester and treprostinil dimer are detected, and no other impurities are found.

[0105] Example 2

[0106] Preparation of monohydrate treprostinil Form I crystals

[0107] Dissolve 2-(((lR,2R,3aS,9aS)-2-hydroxy-l-((S)-3-hydroxyoctyl)-2,3,3a,4,9,9a- hexahydro-lH-cyclopenta[b]naphthalen-6-yl)oxy)acetonitrile (1.00 g, 2.7 mmol) in 10 ml of 2-propanol and then add 4 ml of potassium hydroxide solution (16% w / v) and stir at 80 °C for 2 hours. Slowly cool the reaction mixture to room temperature, quench with hydrochloric acid solution and concentrate to remove 2-propanol, add 25 ml of saturated aqueous sodium bicarbonate solution and 25 ml of ethyl acetate for extraction. The resulting treprostinil is then extracted with sodium bicarbonate solution (pH ~ 8.3) at 30 °C to form a homogeneous solution. Afterwards, the treprostinil basic aqueous solution is acidified by slowly adding 7 N aqueous phosphoric acid solution at a decreasing rate of about 0.17 per minute to adjust the pH to about 2.5, and then stir at 30 °C for 1 hour until most of the crystals precipitate. The resulting precipitated crystals are then filtered and rinsed with 200 ml of water, then dried under high vacuum (about 0.01 torr) at 30 °C for 2 hours to give 1.03 g of treprostinil monohydrate Form I crystals (yield: 93.7%). The XRPD and DSC results are the same as shown in Figure 5 and Figure 6 The UPLC analysis of the product shows a purity of 100.0%, no treprostinil ethyl ester and treprostinil dimer are detected, and no other impurities are found.

[0108] Example 3

[0109] Preparation of treprostinil monohydrate Form I crystals

[0110] Dissolve 2,2'-azanediyl bisethanol 2-(((lR,2R,3aS,9aS)-2-hydroxy-l-((S)-3- hydroxyoctyl)-2,3,3a,4,9,9a-hexahydro-lH-cyclopenta[b]naphthalen-5-yl)oxy)acetate (1.00 g, 2.0 mmol) in 20 ml of saturated aqueous sodium bicarbonate solution (pH ~ 8.3) to form a homogeneous solution. Then, the treprostinil basic aqueous solution is acidified by slowly adding 9 N aqueous phosphoric acid solution at a decreasing rate of about 0.16 per minute to adjust the pH to about 2, and then stir at 25 °C for 1 hour until most of the crystals precipitate. The resulting precipitated crystals are filtered and rinsed with 180 ml of water, and then dried under high vacuum (about 0.1 torr) at 25 °C for 3 hours to give 0.80 g of treprostinil monohydrate Form I crystals (yield: 97.2%). The XRPD and DSC results are the same as shown in Figure 5 and Figure 6The results shown in Table 1 were the same for the results shown in Table 2. UPLC analysis of the product showed a purity of 100.0%, no detectable treprostinil ethyl ester and treprostinil dimer, and no other impurities.

[0111] Example 4

[0112] Preparation of treprostinil dihydrate Form II crystals

[0113] (1R,2R,3aS,9aS)-2-hydroxy-l-((S)-3-hydroxyoctyl)-2,3,3a,4,9,9a-hexahydro-lH- cyclopenta[b]naphthalen-6-yl)oxy)acetonitrile (1.00 g, 2.7 mmol) was dissolved in 10 ml of 2-propanol, followed by the addition of 4 ml of potassium hydroxide solution (16% w / v) and stirring at 80 °C for 2 hours. The reaction mixture was slowly cooled to room temperature and quenched with hydrochloric acid solution and concentrated to remove 2-propanol, 30 ml of potassium hydroxide solution and 30 ml of ethyl acetate were added for extraction. The resulting treprostinil was then extracted with aqueous potassium hydroxide solution (pH ~ 14) at 20 °C to form a homogeneous solution. Subsequently, the aqueous treprostinil base solution was acidified by rapidly adding 9 N aqueous phosphoric acid solution at a rate of about 1.2 per minute to adjust the pH to about 2, and stirred at 20 °C for 1 hour until most of the crystals precipitated. The resulting precipitated crystals were filtered and rinsed with 200 ml of water, and then dried under high vacuum (about 0.01 torr) at 20 °C for 2 hours to obtain 1.03 g of treprostinil dihydrate Form II crystals (yield: 93.7%). The XRPD and DSC results were the same as those shown in Table 1. Figure 8 and Figure 9 The results shown in Table 1 were the same for the results shown in Table 2. UPLC analysis of the product showed a purity of 100.0%, no detectable treprostinil ethyl ester and treprostinil dimer, and no other impurities.

[0114] Example 5

[0115] Preparation of treprostinil dihydrate Form II crystals

[0116] Dissolve 2-(((lR,2R,3aS,9aS)-2-hydroxy-l-((S)-3-hydroxyoctyl)-2,3,3a,4,9,9a- hexahydro-lH-cyclopenta[b]naphthalen-6-yl)oxy)acetonitrile (1.00 g, 2.7 mmol) in 10 ml of 2-propanol, then add 4 ml of potassium hydroxide solution (16% w / v) and stir at 80°C for 2 hours. Slowly cool the reaction mixture to room temperature and quench with hydrochloric acid solution and concentrate to remove 2-propanol, add 20 ml of sodium hydroxide solution and 20 ml of ethyl acetate for extraction. The resulting treprostinil is then extracted with aqueous sodium hydroxide (pH ~ 12) at 10°C to form a homogeneous solution. Subsequently, the treprostinil basic aqueous solution is acidified by rapidly adding 10 N aqueous phosphoric acid solution at a decreasing rate of about 1.9 per minute to adjust the pH to about 2.5, and stir at 10°C for 1 hour until most of the crystals precipitate. Filter the resulting precipitated crystals and rinse with 250 ml of water, and then dry under high vacuum (about 0.01 torr) at 25°C for 2 hours to obtain 1.02 g of treprostinil monohydrate Form II crystals (yield: 92.8%). The XRPD and DSC results are the same as shown in Figure 8 and Figure 9 The UPLC analysis of the product shows a purity of 100.0%, no detection of treprostinil ethyl ester and treprostinil dimer, and no other impurities are found.

[0117] Example 6

[0118] Preparation of treprostinil monohydrate Form II crystals

[0119] Dissolve 2-(((lR,2R,3aS,9aS)-2-hydroxy-l-((S)-3-hydroxyoctyl)-2,3,3a,4,9,9a- hexahydro-lH-cyclopenta[b]naphthalen-6-yl)oxy)acetonitrile (1.00 g, 2.7 mmol) in 10 ml of 2-propanol, then add 4 ml of potassium hydroxide solution (16% w / v) and stir at 80°C for 2 hours. Slowly cool the reaction mixture to room temperature and quench with hydrochloric acid solution and concentrate to remove 2-propanol, add 20 ml of sodium hydroxide solution and 20 ml of ethyl acetate for extraction. The resulting treprostinil is then extracted with aqueous sodium hydroxide (pH ~ 12) at 10°C to form a homogeneous solution. Subsequently, the treprostinil basic aqueous solution is acidified by rapidly adding 10 N aqueous phosphoric acid solution at a decreasing rate of about 1.9 per minute to adjust the pH to about 2.5, and stir at 10°C for 1 hour until most of the crystals precipitate. Filter the resulting precipitated crystals and rinse with 250 ml of water, and then dry under high vacuum (about 0.01 torr) at 25°C for 2 hours to obtain 1.02 g of treprostinil monohydrate Form II crystals (yield: 92.8%). The XRPD and DSC results are the same as shown in Figure 8 and Figure 9 The UPLC analysis of the product shows a purity of 100.0%, no detection of treprostinil ethyl ester and treprostinil dimer, and no other impurities are found.

[0120] Example 7

[0121] Preparation of a mixture of Form I and Form II crystals of treprostinil monohydrate

[0122] Sodium 2-(((lR,2R,3aS,9aS)-2-hydroxy-l-((S)-3-hydroxyoctyl)-2,3,3a,4,9,9a- hexahydro-lH-cyclopenta[b]naphthalen-5-yl)oxy)acetate (1.00 g, 2.4 mmol) was dissolved in 20 ml of sodium hydroxide solution (pH ~ 8.3) at 20 °C to form a homogeneous aqueous solution of treprostinil base. Subsequently, the aqueous solution of treprostinil base was acidified by adding 9 N aqueous phosphoric acid at a rate of ~ 0.32 per minute to adjust the pH to ~ 2, and stirred at 20 °C for 1 hour until most of the crystals precipitated. The resulting precipitated crystals were filtered and rinsed with 200 ml of water, and then dried at 20 °C under high vacuum (~ 0.1 torr) for 2 hours to obtain 0.99 g of treprostinil monohydrate crystals (yield: 90.1 %) comprising ~ 60% of Form I and 40% of Form II and substantially free of any other crystalline form of treprostinil. The XRPD results are shown in Figure 11

[0123] While the present application has been described with reference to illustrative examples, it is understood that the present application can be carried out in many ways, and that modifications or substitutions can be made by persons skilled in the art without departing from the application in its broader aspects. Therefore, the true scope of the application should be determined by the following claims.​

Claims

1. A monohydrated trastanocycline crystal form I, characterized in that... Its X-ray powder diffraction (XRPD) pattern contains two strongest characteristic peaks located at the following 2θ reflection angles: 5.43±0.2° and 10.87±0.2°.

2. The monohydrated cycloprostacyclin crystal form I as described in claim 1, wherein the XRPD pattern further comprises characteristic peaks located at the following 2θ reflection angles: 12.30±0.2°, 16.34±0.2°, and 20.39±0.2°.

3. The monohydrated cycloprostacyclin crystal form I as described in claim 1, wherein the XRPD pattern is shown in Figure 5.

4. The monohydrate treprostacyclin crystalline form I as described in claim 1, further comprising a differential scanning calorimetry (DSC) thermogram pattern, the thermogram pattern comprising two main endothermic peaks: one with a peak onset temperature of 65.9±2℃ and a peak maximum temperature of 79.2±2℃, and the other with a peak onset temperature of 123.2±2℃ and a peak maximum temperature of 125.1±2℃.

5. The monohydrated cycloprostacyclin crystalline form I as described in claim 4, wherein the DSC thermogram pattern is shown in Figure 6.

6. The monohydrate treprostacyclin crystalline form I as described in claim 1, having a purity of at least 99.8% excluding residual solvent.

7. The monohydrate treprostacyclin crystalline form I as described in claim 6, having a purity of at least 99.9% excluding residual solvent.

8. A monohydrated cycloprostacyclin crystal form II, characterized in that... Its XRPD pattern contains two strongest characteristic peaks located at the following 2θ reflection angles: 5.19±0.2° and 10.40±0.2°.

9. The monohydrated cycloprostacyclin crystal form II as claimed in claim 8, wherein the XRPD pattern further comprises characteristic peaks located at the following 2θ reflection angles: 11.62±0.2°, 16.19±0.2°, and 20.14±0.2°.

10. The monohydrated cycloprostacyclin crystal form II as described in claim 8, wherein the XRPD pattern is shown in FIG8.

11. The monohydrate treprostacyclin crystalline form II as described in claim 8, further comprising a DSC thermographic pattern, the thermographic pattern comprising two main endothermic peaks: one with a peak onset temperature of 58.8±2℃ and a peak maximum temperature of 73.8±2℃, and the other with a peak onset temperature of 123.9±2℃ and a peak maximum temperature of 125.1±2℃.

12. The monohydrated cycloprostacyclin crystalline form II as described in claim 11, wherein the DSC thermogram pattern is shown in Figure 9.

13. The monohydrate treprostacyclin crystalline form II as described in claim 8, having a purity of at least 99.8% excluding residual solvent.

14. The monohydrate treprostacyclin crystalline form II as described in claim 13, having a purity of at least 99.9% excluding residual solvent.

15. A mixture of monohydrate treprostacyclin crystalline form I and monohydrate treprostacyclin crystalline form II, characterized in that the XRPD pattern of the mixture comprises a common characteristic peak at 5.26±0.2°, 13.20±0.2° and 16.25±0.2°, and separate characteristic peaks belonging to form I at 10.65±0.2° and 12.20±0.2° and belonging to form II at 10.36±0.2°, 11.61±0.2° and 12.60±0.2°, and the mixture comprises at least 10% of form I or form II.

16. The mixture of claim 15, having a purity of at least 99.8% except for residual solvent.

17. The mixture of claim 16, having a purity of at least 99.9% except for residual solvent.

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