The invention describes specific polymorphic forms of Ingenol 3-(3,5-diethylisoxazole-4- carboxylate), and a process for preparation of the polymorphic forms.

Background of the invention

Ingenol 3-(3,5-diethylisoxazole-4-carboxylate) is described in PCT/DK2011/000154. Presently, the compound is investigated for its effects in treating actinic keratosis in clinical trials. In the prior disclosure, the compound has not been described in any solid state. The present invention discloses novel crystalline forms with beneficial

characteristics as well as methods for their preparation.

Description of drawings

Figure 1 : the amount of residual heptane in the crystals.

Figure 2:The DSC (solid) and the TGA (dash) curve of polymorph F of compound 4. Figure 3 : The DSC (solid) and the TGA (dash) curve of polymorph B of compound 4. Figure 4: Comparison of the DSC curves for polymorph F (solid) and B (dash).

Figure 5 : The m-ATR FTIR spectrum of polymorph F of compound 4.

Figure 6: The m-ATR FTIR spectrum of polymorph B of compound 4.

Figure 7: Comparison of the m-ATR FTIR spectre of polymorph F (solid) vs B (dash) of compound 4.

Figure 8: The XRPD pattern of polymorph F of compound 4.

Figure 9: The XRPD pattern of the polymorph B of compound 4.

Figure 10 : Comparison of the XRPD pattern of polymorph F (solid) and B (dash) of compound 4.

Figure 11 :XRPD for form A of compound 4

Figure 12 : The DSC (solid) and the TGA (dash) curve of Form A of compound 4. Summary of the invention

The present invention provides novel crystalline forms of Ingenol 3-(3,5- diethylisoxazole-4-carboxylate). The present invention provides methods for their preparation. The characteristics of the crystalline forms are provided by the present invention.

Detailed description of the invention:

In the context of the present invention "the compound" refers to Ingenol 3-(3,5- diethylisoxazole-4-carboxylate). Also in the context of the present disclosure, the compound is named compound 4.

In the context of the present invention "about" in the context of temperature refers to ±20 °C. In embodiments the term refers to ± 10°C. In preferred embodiments the term refers to ±5°C. The term "about" is mentioned in the context of time and has the meaning of up to ±20%.

Prior art discloses Ingenol 3-(3,5-diethylisoxazole-4-carboxylate) being prepared from the starting material, ingenol, by protection using acetonide. From this intermediate, Ingenol-5.20-acetonide-3-(3,5-diethylisoxazole-4-carboxylate ) was prepared by using 3,5-Diethylisoxazole-4-carbonyl chloride, which together with the ingenol-5,20- acetonide, were stirred in a microwave oven at 150 °C in acetonitrile for 10-30 min. Deprotection was performed in tetrahydrofuran (0.47 ml_) under argon with HCI, followed by chromatography. This process was not suitable for larger scale production facilities, and therefore improvements of the process was required to achieve a commercial process, which was robust, using acceptable solvents and providing good yields. Also, it is of importance that the compound and its intermediates have handling properties, which makes it possible to handle as well the intermediates, as the highly potent compound end product. Prior art does not mention the physical characteristics of the isolated product.

The present invention provides a number of improvements to the above process. An improvement is in the preparation of the intermediate acid chloride (compound E below). The process disclosed by the present invention provides a product, which leads to a high purity end-product as the intermediate compound, 3, obtained using this acid chloride, is crystalline and with the crystalline properties allows for simple purification. Another improvement in the process eliminates the need for microwave oven in the process as disclosed in the prior art. The present invention also provides crystallization processes for the final compound, which controls the crystalline structure of the compounds and the preferred form of the final product can be obtained.

The intermediate acid chloride was repared by the following reaction scheme:

The starting material was dissolved in an inert organic solvent. In an embodiment the solvent is an ether. In a preferred embodiment the solvent is methyl- tert-butyl-ether (MTBE). A base is added together with propionylchloride. In embodiments of the invention the base is selected from calcium hydroxide, Potassium hydroxide, Sodium hydroxide or similar. In a specific embodiment the base is calcium hydroxide. The reaction was quenched by addition of acid, and worked up to afford B as an oil.

Process description, Step 2a :

Compound B, water and hydroxylamine hydrochloride is added to the reaction flask and the reaction is heated to about 40 °C. Organic solvent was added and the mixture was worked up to afford C as an oil.

Process description, Step 3a :

Compound C, water and base was mixed to hydrolyse the ester. The product, D, is a solid, which can be isolated on a filter after crystallisation in heptane. Process description, Step 4a :

Compound D, organic solvent and thionyl chloride is heated to about 95°C for about 60 minutes. In preferred embodiments the solvents are heptane and toluene. The product is isolated as a crude liquid which can be distilled under vacuum while heating to provide a colourless to pale yellow liquid.

The intermediate, E, is used in step 2 of the process below:

As to the protection of the 5- and 20- positions in ingenol by acetonide in step 1, the following has been performed : Ingenol was dissolved in acidified acetone. In an embodiment of the invention the acid used was MSA (methane sulfonic acid). In another embodiment of the invention the acid was p-toluene sulfonic acid. In an embodiment of the invention the reaction was performed at about 45 °C. Then base was added . In an embodiment the base was triethylamine. The reaction mixture was then worked up and the compound isolated as a white solid from ethylacetate/heptane.

The protected ingenol as prepared above, compound 2, was dissolved in organic solvent, such as THF, and cooled to about -10 °C . Then strong base in the form of LiHMDS (Lithium HexaMethylDiSilazide) in a surplus and compound E was added. After end reaction the mixture was worked up and crystallied in isopropylalcohol/Water. In an embodiment MeOH/water can be used. The product was isolated on a filter as a white solid.

The optimized reaction eliminates the use of the microwave oven of the prior art process. The yield of the present process provides excellent yield of a highly purified product.

Step 3 as outlined below provides the different polymorphs of the present invention. Form A, Form B or Form F

3 Step 3 4

Reaction i :

The deprotection of compound 3 was performed in organic solvent, water and MSA under elevated temperatures. MSA (methanesulfonic acid) is a preferred embodiment, however other acids may be useful such as p-toluenesulfonic acid. In embodiments the organic solvent is THF.

Reaction ii :

From reaction ii, the polymorphic form A can be isolated, which can then be converted into either form B or form F dependent on the conditions. One form (hereinafter called form B) is a fluffy crystalline form containing some heptane. The fluffy character of the polymorphic form presents some safety issues in the production process since the material is easily spread into the surroundings when the reaction flask/tank is opened after isolation and drying. The present invention therefore discloses a crystalline form, which is safer to work with, and in addition is a very stable form of the compound. The polymorphic form (hereinafter known as form "F") has characteristics which allows the crystalline structure to pack more tightly, and therefore it volume is about half of the corresponding Form B. The more tightly packed structure of the material provides a volume of final product which is less fluffy and easier to handle after isolation and drying. This provides easier and safer handling of the product. Further, form F contains less heptane, which is also an advantage since heptane is not desirable to have in a pharmaceutical product. And additionally, form F is more stable than form B.

In very small amounts the following compound was isolated as a by-product:

Examples:

Step 1, Synthesis of compound 2 :

Step 1 was performed using a Flowreactor. Compound A (lOg) was dissolved in acetone (600 ml_) and protected under the presence of methanesulfonic acid (MSA)(2.11g dissolved in THF 113ml_) at about 45 °C. Reaction mixture was quenched with triethylamine when the conversion is preferably above 95%, to afford the crude reaction mixture which was further processed in a reactor. The mixture was concentrated under reduced pressure at temperatures of below 55 °C, and ethyl acetate (EtOAc) 200ml_ was added. The organic phase was washed two times with water (25ml_), concentrated under reduced pressure at temperature below 50°C, and EtOAc 100 ml_ was added. The organic phase was evaporated to a slurry under vacuum (Temperature on reactor jacket < 50°C ) . EtOAc (66 ml_) was added to the slurry and the mixture was heated to reflux. Heptane (132 ml_) was added while maintaining reflux. The reactor jacket was cooled to 60°C and then to -1°C during 2 ± 0,5 hours and age for 12-96 hours. The product was isolated on a filter, and the filter cake was washed with a mixture of EtOAc and Heptane (16 & 32 ml_). The product was dried under vacuum at 60 °C to afford compound 2. The crude product was purified by recrystallisation from EtOAc/Heptane to afford compound 2 as a white solid. Yield : 40-80 %.

Step la-4a : preparation of compound E

Process description, Step la, compound B:

Compound A and MTBE were mixed and calcium hydroxide and propionylchloride was added. The reaction was cooled and aqueous HCI was added. The reaction was worked up using NaHC0 ₃ . The organic layer was concentrated under vacuum to afford compound B as an oil. Step 2a, compound C:

Compound B, water and hydroxylamine hydrochloride was added to the reaction flask and the reaction was heated to about 40 °C. Add organic solvent and work up the reaction mixture to afford C as an oil. Process description, Step 3a :

Compound C, water and base was mixed to hydrolyse the ester. The product, D, was a solid, which can be isolated on a filter. Process description, Step 4a :

Compound D, heptane and thionyl chloride was heated to about 95°C for about 60 minutes. The product was isolated as a crude liquid which can be distilled under vacuum while heating to provide a colourless to pale yellow liquid.

Step 2 : Synthesis of Compound 3 :

Compound 2 (lOg) was dissolved in THF (100 mL) and cooled to about -10 °C. Then LiHMDS (29.7 mL) in a small surplus was charged to reactor over 5-50 min. Compound E (0.58g) was dissolved in THF (25 mL) in a suitable container and transferred to the reactor over 10 min. The mixture was stirred for about 15 minutes, and an IPC was taken. If the conversion was 95% complete or beyond that, then NaHC0 ₃ (aq.sat.) (25mL) and water (75-100mL) was added and the reaction allowed to heat to 20°C. MTBE (200-300mL) was added and phases separated. The organic phase was

evaporated to a slurry under vacuum with a temperature below 30-50°C.

Isopropylalcohol (100 mL) was added and the mixtures heated to reflux. Purified water (150 mL) was added while maintaining reflux. The reaction mixtures was cooled to -1°C during 6 hours and aged for 1-96 hours. The product was isolated on a filter, and washed with a mixture of isopropylalcohol and water (50 and 50 mL) and dried under vacuum at 40-50°C. Compound 3 was isolated as a white solid (80-95 % yield).

Step 3 : Synthesis of compound 4 Compound 3 (lOg) was dissolved in THF(100 mL) and cooled to -5 °C. A solution of MSA (14.2g), THF (67mL) and purified water (18.5mL) was added, and the reaction was heated to 50 °C. When the conversion was above 99%, the reaction was cooled to 15 °C and NaHCOs (aq. Sat.) ( 106mL) and MTBE (175-225mL) was added and the phases separated. Organic phase was washed with water (75mL) two times, added EtOAc (150- 200 mL) and concentrated under vacuum with a temperature below 50°C. EtOAc (30 mL) was added to the slurry and the mixture was heated to reflux. When all material was dissolved, Heptane (175 mL) was added while maintaining reflux. The reactor jacket was cooled to -1°C over 10-12 hours and then cooled to -10°C for minimum 4 hours. The product was isolated on a filter and washed with Heptane (30 mL). The product was transferred from filter to reactor and Heptane (150 mL) and seeding crystals (LEO

43204, form F) was added. The mixture was heated to reflux for 1-2 hours. The reactor jacket was cooled to -1°C over 10-12 hours and aged for 0-96 hours at -1°C. The product was isolated on a filter, washed with Heptane (30 ml_) and dried under vacuum at 60 °C for minimum 16 hours to afford compound 4 as a white solid (80-85 % yield).

The last step of the present reaction offers the options of obtaining different polymeric forms of the final compound. Presently, forms A, B and F has been identified. Form A was a solvate with ethylacetate and in one embodiment of the invention it was not isolated as such but converted to form F by boiling form A in heptane. In an embodiment where A was isolated, the compound has a melting point of 112°C (± 2 °C).

Form F (from form B)

200 mg of polymorph B were suspended in 5 ml_ of Heptane in a round bottom flask equipped with a condenser and a magnetic stirring unit. The suspension was seed with 2% of polymorph F and refluxed until all of polymorph B was transformed to polymorph F. This happens usual within one hour.

Form F (from A) :

200 mg of solid form A were suspended in 5 ml_ of Heptane in a round bottom flask equipped with a condenser and a magnetic stirring unit. The suspension was seed with 2% of polymorph F and refluxed until all of form A was transformed to polymorph F. This happens usual within one hour.

Form B offers excellent stability of the crystalline form. However, the crystal does not pack its crystals tight and therefore the volume of a crystalline form B was large.

Initially, the form was describes as having potential electrostatic properties, which indeed was problematic from a production perspective, since the crystals were unwilling to settle on the bottom of the reactor. With very high potency of the compound this provided a health problem for the employees working with the process.

Form F solved the problem completely. The polymorphic form provides excellent stability while still dissolved easily in the formulation, but importantly, the crystals pack to a very small size, and they are not fluffy after isolation and drying.

In addition, form F also allows for heptane to be removed much more readily from the final product. Since heptane is not a part of the final formulation, it is preferred to be able to remove the solvent from the final compound. This is much easier done with form F than with the corresponding form B.

Examples: Polymorph B and polymorph F have both passed the 4 week stress stability test at 60 C, 60°C/75% RH and 80°C/75% RH without any solid state transformation.

Stability testing:

Approximately 20 mg compound 4 was placed in each of three brown glasses (3 x 20 mg) and closed with a screw cap. One glass (time zero) was placed in a refrigerator at the beginning of the study. Two glasses were placed in an oven at 80°C. One glass was pulled at each time point and stored in a refrigerator protected from light until analysis. The analysis was performed by reversed phase UPLC with UV-detection at 220 nm on HSS C18 column at 40 °C in water-acetonitrile 95 : 5 to acetonitrile.

Result:

Based on these data, it can be concluded that polymorphic form F is stable at 80°C for 14 days. Form B is slightly less stable than form F, as a minor decrease in assay and organic impurities were observed. However, all results for form B were well within the specification limits after storage for 14 days at 80°C.

Instrumentation

X-rav powder diffraction (XRPD) : The diffractogram was obtained on a conventional

X'pert PRO MPD diffracto meter from PANalytical configured with transmission geometry and equipped with a PIXcel detector. A continuous 2Θ scan range of 3-30° was used with a CuKa radiation λ = 1.5418 A source and a generator power of 40KV and 45mA. A 2Θ step size of 0.0070°/step with a step time of 148.92 s was used. Samples were gently flattened onto a well in a 96-well plate for transmission measurements. The well plate was moved forward and backward in the x direction and all experiments were performed at room temperature.

FTIR-ATR spectroscopy (attenuated total reflectance fourier transform infrared spectroscopy) : The spectrum was recorded on a FTIR instrument, Equinox 55 or Tensor 27 from Bruker equipped with a GoldenGate ATR unit from SPECAC. A spectral resolution of 3 cm ¹ was used.

Differential scanning calorimetry (DSC) : DSC experiments were carried out using a Perkin Elmer DSC8500 system. About 0.5-3 mg of sample was used for the

measurements. An aluminium pan was used for the analysis and was sealed by applying pressure by hand and pushing each part of the pan together. The temperature was ramped from -60 to 200°C at 20°C/min. Nitrogen was used as the purge gas with a flow rate of 20 mL/min.

Thermo gravimetric analysis (TGA) : TGA experiments were conducted using a Perkin Elmer Pyris 1 TGA instrument. About 10-20 mg of sample was loaded into a ceramic pan for the measurements. The sample temperature was ramped from 25 to 500°C at 20°C/min. Nitrogen was used as the purge gas at a flow rate of 40 mL/min.

The given error ranges in this application for the spectroscopic characteristics, including those in the claims, may be more or less depending of factors well known to a person skilled in the art of spectroscopy and may for example depend on sample preparation, such as particle size distribution, or if the crystal form is part of a formulation, on the composition of the formulation, as well as instrumental fluctuations, and other factors. An error range of ±5 includes, but is not limited to variations of ±5, ±4, ±3, ±2, ± 1, ±0.5, ±0.4, ±0.3, ±0.2, and ±0.1; an error range of ±3 includes, but is not limited to variations of ±3, ±2, ± 1, ±0.5, ±0.4, ±0.3, ±0.2, and ±0.1 ; an error range of ± 1 includes, but is not limited to variations of ±0.9, ±0.8, ±0.7, ±0.6, ±0.5, ±0.4, ±0.3, ±0.2, and ±0.1; and an error range of ±0.2 includes, but is not limited to variations of ±0.2, ±0.15, ±0.1, ±0.09, ±0.08, ±0.07, ±0.06, ±0.05, ±0.04, ±0.03, ±0.02, and ±0.01.

Characterisation

DSC:

Polymorph F has a differential scanning calorimetry (DSC) curve comprising an endo thermo event with an onset at about 166°C (± 2 °C) see figure 2.

Polymorph B has a differential scanning calorimetry (DSC) curve comprising an endo thermo event with an onset at about 131°C (± 2 °C). No endo thermo event with an onset at about 166°C (± 2 °C) is observed, see Figure 3 and Figure 4.

FTIR-ATR:

In one embodiment the polymorph F of compound 4 has an attenuated total reflectance fourier transform infrared (FTIR-ATR) spectrum essentially similar as shown in Figure 5. The polymorph F of compound 4 is characterized by an attenuated total reflectance fourier transform infrared (FTIR-ATR) spectrum exhibiting one or more attenuated total reflectance peaks at approximately 1721, 1592, 1097, 1034 and/or 947 (± 3 cm ^"1 ), respectively.

In one embodiment the polymorph B of compound 4 has an attenuated total reflectance fourier transform infrared (FTIR-ATR) spectrum essentially similar as shown in Figure 6 . The polymorph B of compound 4 is characterized by an attenuated total reflectance fourier transform infrared (FTIR-ATR) spectrum exhibiting one or more attenuated total reflectance peaks at approximately 1698 and/or 1366 (± 3 cm ^"1 ), respectively.

XRPD :

Polymorph F: In one embodiment the polymorph F of compound 4 has an XRPD pattern essentially similar as shown in Figure 8. The polymorph F of compound 4 is characterized by an XRPD pattern exhibiting one or more reflection peaks at approximately 20= 8.2, 9.4, 16.4, 19.0 and/or 22.3 (±0.1 degrees) respectively . Polymorph B of compound 4 has an XRPD pattern essentially similar as shown in Figure 9. Polymorph B of compound 4 is characterized by an XRPD pattern exhibiting one or more reflection peaks at approximately 20= 6.0, 10.6, 11.4, 12.6, 16.0 and/or 22.5 (±0.1 degrees) respectively. Form A: In one embodiment the form A of compound 4 has an XRPD pattern essentially similar as shown in Figure 11. In an embodiment the form A of compound 4 is characterized by an XRPD pattern exhibiting one or more reflection peaks at

approximately 20= 5.8, 8.7, 13.6, and/ 19.3 (±0.1 degrees) respectively.