HK1178159B - 9e-15-(2-pyrrolidin-1-yl-ethoxy)-7,12,25-trioxa-19,21,24-triaza- tetracyclo[18.3.1.1(2,5).1(14)18)]hexacosa-1(24),2,4,9,14,16,18(26),20,22-nonaene citrate salt - Google Patents

Description

9E-15- (2-pyrrolidin-1-yl-ethoxy) -7,12, 25-trioxa-19, 21, 24-triaza-tetracyclo [18.3.1.1(2,5).1(14,18) ] hexacosan-1 (24),2,4,9,14,16,18(26),20, 22-nonene citrate

Technical Field

The present invention relates to the citrate salt of 9E-15- (2-pyrrolidin-1-yl-ethoxy) -7,12, 25-trioxa-19, 21, 24-triaza-tetracyclo [18.3.1.1(2,5).1(14,18) ] hexacosan-1 (24),2,4,9,14,16,18(26),20, 22-nonene. The invention also relates to pharmaceutical compositions containing the citrate salt and methods of using the salt in the treatment of certain medical conditions.

Background

Patent application PCT/SG2006/000352 for the first time describes the compound 9E-15- (2-pyrrolidin-1-yl-ethoxy) -7,12, 25-trioxa-19, 21, 24-triaza-tetracyclo [18.3.1.1(2,5).1(14,18) ] hexacosan-1 (24),2,4,9,14,16,18(26),20, 22-nonene (compound 1), which compound has great promise as a pharmaceutically active agent for the treatment of a variety of medical conditions. Based on the demonstrated activity profile of the compound, drug development studies of the compound are ongoing.

In pharmaceutical research adapted for mass production and eventual commercial applications, an acceptable level of pharmaceutical activity for the considered object is only one of the important variables that must be considered. For example, in the formulation of pharmaceutical compositions, it is absolutely essential that the form of the pharmaceutically active substance be reliably reproducible in an industrial manufacturing process and be sufficiently durable to withstand the conditions to which the pharmaceutically active substance is subjected.

From a manufacturing perspective, it is important that the industrial manufacturing process of the pharmaceutically active substance is such that the same material can be produced when the same manufacturing conditions are used. Furthermore, it is desirable that the pharmaceutically active substance is present in solid form, and minor changes in manufacturing conditions do not result in major changes in the solid form of the pharmaceutically active substance produced. For example, it is important that the manufacturing method produces materials with the same crystallinity on a reliable basis, and that the method also produces materials with the same degree of hydration.

Furthermore, it is important that the pharmaceutically active substance is stable to degradation, hygroscopicity and subsequent alteration to its solid form. It is important to facilitate incorporation of the pharmaceutically active ingredient into a pharmaceutical formulation. If the pharmaceutically active substance is hygroscopic (viscous), meaning that it absorbs water over time, it is almost impossible to reliably formulate the pharmaceutically active substance into a drug because the amount of substance to be added to provide the same dose will vary greatly depending on the degree of hydration. Furthermore, changes in hydrated or solid forms (polymorphism) can lead to changes in physicochemical properties, such as solubility or dissolution rate, which in turn can lead to inconsistent oral absorption by the patient.

Thus, the chemical stability, solid state stability, and "shelf life" of the pharmaceutically active agent are all important factors. Ideally, the pharmaceutically active agent and any composition comprising the pharmaceutically active agent should be capable of being effectively stored for an appreciable period of time without the active component exhibiting significant changes in its physicochemical properties, e.g., its activity, moisture content, solubility characteristics, solid form, etc.

Initial studies of 9E-15- (2-pyrrolidin-1-yl-ethoxy) -7,12, 25-trioxa-19, 21, 24-triaza-tetracyclo [18.3.1.1(2,5).1(14,18) ] hexacosan-1 (24),2,4,9,14,16,18(26),20, 22-nonene (nonaene) were performed on the hydrochloride salt and studies showed that the polymorph was prevalent and the compound was found to take more than one crystal form depending on the manufacturing conditions. Furthermore, it was observed that the ratio of the polymorphs varied from batch to batch even though the manufacturing conditions remained constant. From an industrial point of view, these batch-to-batch inconsistencies make the hydrochloride salt less desirable.

Accordingly, it would be desirable to develop salts of 9E-15- (2-pyrrolidin-1-yl-ethoxy) -7,12, 25-trioxa-19, 21, 24-triaza-tetracyclo [18.3.1.1(2,5).1(14,18) ] hexacosan-1 (24),2,4,9,14,16,18(26),20, 22-nonene to overcome or ameliorate one or more of the above problems.

Disclosure of Invention

The present invention provides the citrate salt (salt of citric acid) of 9E-15- (2-pyrrolidin-1-yl-ethoxy) -7,12, 25-trioxa-19, 21, 24-triaza-tetracyclo [18.3.1.1(2,5).1(14,18) ] hexacosan-1 (24),2,4,9,14,16,18(26),20, 22-nonene.

In some embodiments, the salt is crystalline.

In some embodiments, the salt is a 1:1 citrate salt. In some embodiments, the citrate salt exhibits an X-ray diffraction peak at 22.4 ° ± 0.5 ° on the 2 θ scale.

In some embodiments, the citrate salt also displays X-ray diffraction peaks on the 2 theta scale at 10.0 ° ± 0.5 °, 15.6 ° ± 0.5 ° and 17.2 ° ± 0.5 °.

In some embodiments, the citrate salt exhibits at least four X-ray diffraction peaks on a 2 Θ scale selected from the group consisting of: 7.9 ° ± 0.5 °, 10.0 ° ± 0.5 °, 15.6 ° ± 0.5 °, 15.9 ° ± 0.5 °, 16.8 ° ± 0.5 °, 17.2 ° ± 0.5 °, 21.1 ° ± 0.5 ° and 22.4 ° ± 0.5 °.

In some embodiments, the citrate salt exhibits at least 6X-ray diffraction peaks on a 2 Θ scale selected from the group consisting of: 7.9 ° ± 0.5 °, 10.0 ° ± 0.5 °, 15.6 ° ± 0.5 °, 15.9 ° ± 0.5 °, 16.8 ° ± 0.5 °, 17.2 ° ± 0.5 °, 21.1 ° ± 0.5 ° and 22.4 ° ± 0.5 °.

In some embodiments, the citrate salt exhibits X-ray diffraction peaks on the following 2 Θ scale: 7.9 ° ± 0.5 °, 10.0 ° ± 0.5 °, 15.6 ° ± 0.5 °, 15.9 ° ± 0.5 °, 16.8 ° ± 0.5 °, 17.2 ° ± 0.5 °, 21.1 ° ± 0.5 ° and 22.4 ° ± 0.5 °.

In some embodiments, the citrate salt also displays X-ray diffraction peaks on the following 2-theta scale: 11.1 ° ± 0.5 °, 18.1 ° ± 0.5 °, 21.8 ° ± 0.5 °, 23.2 ° ± 0.5 ° and 27.6 ° ± 0.5 °.

In some embodiments, the citrate salt also displays X-ray diffraction peaks on the following 2-theta scale: 7.0 ° ± 0.5 °, 14.0 ° ± 0.5 °, 19.0 ° ± 0.5 °, 19.8 ° ± 0.5 °, 23.6 ° ± 0.5 °, 24.3 ° ± 0.5 °, 25.2 ° ± 0.5 °, 25.7 ° ± 0.5 °, 26.1 ° ± 0.5 °, 26.5 ° ± 0.5 ° and 32.1 ° ± 0.5 °.

The invention also provides a pharmaceutical composition comprising the above salt.

In another embodiment, the present invention provides a method of treating or preventing a proliferative disease comprising administering to a patient in need thereof a therapeutically effective amount of a salt of the present invention. In some embodiments, the proliferative disease is cancer.

In another embodiment, the invention provides the use of a salt of the invention for the treatment of a proliferative disease. In some embodiments, the proliferative disease is cancer.

In another embodiment, the invention provides the use of a salt of the invention in the manufacture of a medicament for the treatment of a proliferative disease. In some embodiments, the proliferative disease is cancer.

Brief description of the drawings

FIG. 1 shows the diffraction pattern of the high resolution X-ray powder diffraction (XRPD) of the hydrochloride salt prepared in batch 1, THF.

Figure 2 shows the diffraction pattern of the high resolution X-ray powder diffraction (XRPD) of the hydrochloride salt prepared in batch 2, MeCN.

Figure 3 shows the diffraction pattern of batch 3, a high resolution X-ray powder diffraction (XRPD) of the hydrochloride salt prepared in acetone.

Figure 4 shows the diffractogram of high resolution X-ray powder diffraction (XRPD) of citrate prepared in batch 4, THF.

Figure 5 shows the diffractogram of the high resolution X-ray powder diffraction (XRPD) of the citrate salt prepared in batch 5, MeCN.

Figure 6 shows the diffraction pattern of batch 6, a high resolution X-ray powder diffraction (XRPD) of citrate salt prepared in acetone.

Figure 7 shows the diffractogram of high resolution X-ray powder diffraction (XRPD) of batch 7, citrate prepared in acetone (20 gram scale).

Figure 8 shows the diffraction pattern of high resolution X-ray powder diffraction (XRPD) of batch 8, citrate prepared in acetone (20 gram scale).

FIG. 9 shows the diffraction patterns of high resolution X-ray powder diffraction (XRPD) for batches 4-6.

FIG. 10 shows the low resolution X-ray powder diffraction patterns of batches 4-6.

Figure 11 shows overlapping high resolution and low resolution X-ray powder diffraction patterns for batch 4.

Figure 12 shows the X-ray powder diffraction curves of batch 4 before storage and after one week of storage at 40 ℃ and 75% relative humidity.

Figure 13 shows the X-ray powder diffraction curves of batch 5 before storage and after one week of storage at 40 ℃ and 75% relative humidity.

Figure 14 shows the X-ray powder diffraction curves of batch 6 before storage and after one week of storage at 40 ℃ and 75% relative humidity.

Figure 15 shows differential scanning calorimetry (DSC, top) and thermogravimetric analysis (TGA) data for batch 4.

FIG. 16 shows overlapping DSC curves for batches 4-6.

Figure 17 shows overlapping TGA curves for batches 4-6.

Fig. 18 shows the kinetics of gravimetric vapor sorption for batch 4.

Fig. 19 shows the isotherm of gravimetric vapor sorption for batch 4.

Fig. 20 shows X-ray powder diffraction curves for batch 4 before and after the gravimetric vapor sorption experiment was performed.

Figure 21 shows the X-ray powder diffraction profile of the samples from the solubility screen.

FIG. 22 shows batch 4 in d6-DMSO¹H NMR spectrum.

FIG. 23 shows batch 4 at D₂Of O¹H NMR spectrum.

Detailed Description

As noted above, it has now been found that certain salts of 9E-15- (2-pyrrolidin-1-yl-ethoxy) -7,12, 25-trioxa-19, 21, 24-triaza-tetracyclo [18.3.1.1(2,5).1(14,18) ] hexacosan-1 (24),2,4,9,14,16,18(26),20, 22-nonene exist as a single, durable polymorph. In particular, the applicant has found that the citrate salt of this compound (salt of citric acid) can exist as a single polymorph.

At the same time, it is considered that the structure of citric acid is clear to the skilled person, and in order to avoid any uncertainty, the structure is as follows:

citric acid

The comparative studies described herein for the hydrochloride and citrate salts were performed on the batches shown in table 1.

TABLE 1-batch List of HCl and citrate salts for comparative studies

Initial studies on compound I involved the hydrochloride salt thereof. The findings are summarized below, i.e. the initial preparation of the hydrochloride solid form was found to be inconsistent with significant changes in X-ray powder diffraction (XRPD) data.

Compound 1 was prepared as the hydrochloride salt in 3 different solvents, giving batch 1 (prepared in THF), batch 2 (prepared in acetonitrile) and batch 3 (prepared in acetone) as crystalline materials. Figures 1,2 and 3 show significant changes in XRPD diffractograms between these batches, indicating that the crystal structures of these hydrochloride salts are generally not consistent in different solvents, even when prepared under similar conditions.

Due to the unacceptable variability observed with the above-mentioned hydrochloride salts, there is a need for an alternative durable solid form. It was further found that an attempt was made to identify citrate as one such durable solid form.

Five batches of citrate salts of 9E-15- (2-pyrrolidin-1-yl-ethoxy) -7,12, 25-trioxa-19, 21, 24-triaza-tetracyclo [18.3.1.1(2,5).1(14,18) ] hexacosan-1 (24),2,4,9,14,16,18(26),20, 22-nonene were characterized. The analytical results are shown in the following examples.

X-ray powder diffraction (XRPD) was used to characterize the citrate salt of compound 1. A significant series of X-ray diffraction peaks of the citrate salt of the present invention were collected at high resolution and are shown in table 2.

Table 2-significant X-ray diffraction peak list for citrate salt

Peak position (2-theta deg. + -. 0.5 deg.)	Relative strength
		7.0	Weak (weak)
7.9	High strength
		10.0	High strength
11.1	In
		14.0	In
15.6	High strength
		15.9	High strength
16.8	High strength
		17.2	High strength
18.1	High strength
		19.0	In
19.8	In
		21.1	High strength
21.8	In
		22.4	High strength
23.2	In
		23.6	In

Peak position (2-theta deg. + -. 0.5 deg.)	Relative strength
		24.3	In
25.2	Weak (weak)
		25.7	In
26.1	In
		26.5	Weak (weak)
27.6	High strength
		32.1	Weak (weak)

It can be seen that the citrate salt can be characterised as showing an X-ray diffraction peak at 22.4 ° ± 0.5 ° on the 2 θ scale.

The citrate salt may also be characterized as exhibiting X-ray diffraction peaks at 10.0 ° ± 0.5 °, 15.6 ° ± 0.5 ° and 17.2 ° ± 0.5 ° on the 2 θ scale.

In some embodiments, the citrate salt may be further characterized as exhibiting at least four X-ray diffraction peaks on a 2 Θ scale selected from the group consisting of: 7.9 ° ± 0.5 °, 10.0 ° ± 0.5 °, 15.6 ° ± 0.5 °, 15.9 ° ± 0.5 °, 16.8 ° ± 0.5 °, 17.2 ° ± 0.5 °, 21.1 ° ± 0.5 ° and 22.4 ° ± 0.5 °.

In some embodiments, the citrate salt may be further characterized as exhibiting at least 6X-ray diffraction peaks on a 2 Θ scale selected from the group consisting of: 7.9 ° ± 0.5 °, 10.0 ° ± 0.5 °, 15.6 ° ± 0.5 °, 15.9 ° ± 0.5 °, 16.8 ° ± 0.5 °, 17.2 ° ± 0.5 °, 21.1 ° ± 0.5 ° and 22.4 ° ± 0.5 °.

In some embodiments, the citrate salt may be further characterized as exhibiting X-ray diffraction peaks on the following 2 Θ scale: 7.9 ° ± 0.5 °, 10.0 ° ± 0.5 °, 15.6 ° ± 0.5 °, 15.9 ° ± 0.5 °, 16.8 ° ± 0.5 °, 17.2 ° ± 0.5 °, 21.1 ° ± 0.5 ° and 22.4 ° ± 0.5 °.

In some embodiments, the citrate salt also displays X-ray diffraction peaks on the following 2-theta scale: 11.1 ° ± 0.5 °, 18.1 ° ± 0.5 °, 21.8 ° ± 0.5 °, 23.2 ° ± 0.5 ° and 27.6 ° ± 0.5 °.

In some embodiments, the citrate salt may be further characterized as exhibiting X-ray diffraction peaks on the following 2 Θ scale: 7.9 ° ± 0.5 °, 10.0 ° ± 0.5 °, 11.1 ° ± 0.5 °, 15.6 ° ± 0.5 °, 15.9 ° ± 0.5 °, 16.8 ° ± 0.5 °, 17.2 ° ± 0.5 °, 18.1 ° ± 0.5 °, 21.8 ° ± 0.5 °, 21.1 ° ± 0.5 °, 22.4 ° ± 0.5 °, 23.2 ° ± 0.5 ° and 27.6 ° ± 0.5 °.

Although the above peaks are characteristic peaks, the citrate salt also shows X-ray diffraction peaks on the following 2 θ scale: 7.0 ° ± 0.5 °, 14.0 ° ± 0.5 °, 19.0 ° ± 0.5 °, 19.8 ° ± 0.5 °, 23.6 ° ± 0.5 °, 24.3 ° ± 0.5 °, 25.2 ° ± 0.5 °, 25.7 ° ± 0.5 °, 26.1 ° ± 0.5 °, 26.5 ° ± 0.5 ° and 32.1 ° ± 0.5 °.

Those skilled in the art will appreciate that the relative intensity of diffraction may vary depending on factors such as the sample preparation method and the type of instrument used. Furthermore, in some cases, some of the peaks described above may not be detectable.

The salts of the invention can be produced by the following process: the free base of compound (I) is reacted with citric acid in a suitable solvent and recovered from the reaction mixture of the salt obtained after crystallization, precipitation or evaporation.

The reaction to form the salt may be carried out in any non-interfering solvent or mixture of solvents in which the free base has suitable solubility. Examples of such suitable solvents include acetonitrile, tetrahydrofuran and acetone. The process generally involves dissolving the free base in a suitable solvent at elevated temperature, for example above 20 ℃. In some embodiments, the free base is dissolved in a solvent (e.g., acetone) at a temperature of about 56 ℃. In some embodiments, the free base is dissolved in a solvent (e.g., acetonitrile) at a temperature of about 82 ℃.

When the free base is dissolved in a suitable solvent, the process comprises adding a suitable amount of the acid. The acid is typically added as a solution in a suitable solvent, typically the same solvent is used to dissolve the free base. The amount of acid may vary, although typically the amount of acid is the same as the stoichiometric amount or in a slight excess. After the acid is added, the process typically further comprises stirring the reaction mixture at the addition temperature for 1 hour, followed by cooling the reaction mixture to a temperature below the reaction temperature to promote crystallization. When the desired level of crystal formation is achieved, the crystals are isolated by filtration and dried using methods conventional in the art.

Another embodiment of the invention provides the use of a salt of the invention for the treatment of a proliferative disease. PCT/SG2006/000352 discloses formulations and methods for the use of such compounds and diseases that can be treated.

The invention will now be described by way of the following non-limiting examples. The hydrochloride salt of the comparative example was prepared as described above and analyzed in a similar manner.

Example 1-formation of HCl salt in THF solvent (batch 1):

to 15 ml of THF was added the free base of compound 1(0.200 g, 0.432 mmol, 1 eq). The solution was heated to reflux until complete dissolution was observed and held for 1 hour. 1N HCl (0.518 mL, 0.518 mmol, 1.2 equiv) was then added slowly at reflux. The mixture was refluxed for a further 15 minutes and then cooled. Crystallization was observed during the gradual cooling. The crystals were stirred at room temperature for 12 hours and vacuum filtered. The product was dried under vacuum to give 165 mg of product.

Example 2-in CH₃Formation of HCl salt in CN solvent (batch 2):

to 70 ml of CH₃To CN was added the free base of compound 1 (0.300 g, 0.648 mmol, 1 eq). The solution was heated to reflux until complete dissolution was observed and held for 1 hour. 1N HCl (0.778 mL, 0.778 mmol, 1.2 equiv.) was then added slowly under reflux. The mixture was refluxed for a further 15 minutes and then cooled. Crystallization was observed during the gradual cooling. The crystals were stirred at room temperature for 12 hours and vacuum filtered. The product was dried under vacuum to give 190 mg of product.

Example 3-formation of HCl salt in acetone solvent (batch 3):

to 50ml of acetone was added the free base of compound 1(0.200 g, 0.432 mmol, 1 eq). The solution was heated to reflux until complete dissolution was observed and held for 1 hour. 1N HCl (0.518 mL, 0.518 mmol, 1.2 equiv) was then added slowly at reflux. The mixture was refluxed for a further 15 minutes and then cooled. Crystallization was observed during the gradual cooling. The crystals were stirred at room temperature for 12 hours and vacuum filtered. The product was dried under vacuum to give 180 mg of product.

Example 4-formation of citrate salt in THF solvent (batch 4):

to 12 ml of THF was added the free base of compound 1 (0.300 g, 0.648 mmol, 1 eq). The solution was heated to reflux until complete dissolution was observed and held for 1 hour. A solution of citric acid (0.149 g, 0.778 mmol, 1.2 eq) dissolved in 12 ml THF was then added slowly under reflux. The mixture was refluxed for a further 15 minutes and then cooled. Crystallization was observed during the gradual cooling. The crystals were stirred at room temperature for 12 hours and vacuum filtered. The product was dried under vacuum to give 250 mg of product.

Example 5-in CH₃Citrate salt formation in CN solvent (batch 5):

to 45 ml of CH₃To CN was added the free base of compound 1(0.200 g, 0.432 mmol, 1 eq). The solution was heated to reflux until complete dissolution was observed and held for 1 hour. Then slowly adding the mixture dissolved in 12 ml of CH under the reflux condition₃Citric acid solution in CN (0.099 g, 0.518 mmol, 1.2 eq). The mixture was refluxed for a further 15 minutes and then cooled. Crystallization was observed during the gradual cooling. The crystals were stirred at room temperature for 12 hours and vacuum filtered. The product was dried under vacuum to give 220 mg of product.

Example 6-formation of citrate salt in acetone solvent (batch 6):

to 50ml of acetone was added compound 1(0.200 g, 0.432 mmol, 1 eq.) as the free base. The solution was heated to reflux until complete dissolution was observed and held for 1 hour. Citric acid solution (0.099 g, 0.518 mmol, 1.2 eq) dissolved in 12 ml of acetone was then added slowly under reflux. The mixture was refluxed for a further 15 minutes and then cooled. Crystallization was observed during the gradual cooling. The crystals were stirred at room temperature for 12 hours and vacuum filtered. The product was dried under vacuum to give 198 mg of product.

Example 7X-ray powder diffraction study

Condition 1a (high resolution)

X-ray powder diffraction (XRPD) patterns were collected with an α Siemens D5000 diffractometer (α Siemens D5000 diffractometer) using 30 consecutive scan patterns of Cu K radiation (1.54A), 40kV, step size (step size)0.03 ° and step time (step time) -0.5 seconds, i.e., θ - θ mA (using Cu K radiation (1.54A), 40kV, 30continuous scan mode with step size of 0.03 ° and step time-0.5 seconds, wa θ - θ mA). The sample-detector distance used gives an effective theta range of 22 deg. -50 deg.. The sample analysis time (exposure to X-ray beam) was 13 minutes 33 seconds. The software used for data collection was DIFFRACplus-D5000#1, which was analyzed and represented data using Diffrac Plus-D5000# 1.

The samples tested under ambient conditions were flat plate samples prepared using powder (not milled). Approximately 100-200mg of the sample was lightly pressed on a slide to obtain a flat surface.

Condition 1b (high resolution)

X-ray powder diffraction (XRPD) patterns were collected with a Bruker AXS C2GADDS diffractometer using cuka radiation (40kV, 40mA), an automatic XYZ stage, a laser video microscope with automatic sample positioning and a HiStar two-dimensional area detector. The X-ray optical component is composed of a single bodyThe multilayer mirror is connected with a pinhole collimator of 0.3 mm. The beam divergence, i.e. the effective size of the X-ray beam on the sample, is about 4 mm. Using a theta-theta continuous scan mode, the sample-detector distance is 20cm, resulting in an effective 2 theta range of 3.2 deg. -29.7 deg.. The sample is typically exposed to the X-ray beam for 120 seconds. The software for data collection isGADDS for WNT 4.1.16, data were analyzed and presented using Diffrac Plus EVA v 9.0.0.2 or v 13.0.0.2. The samples tested under ambient conditions were flat plate samples prepared using powder (not milled). Approximately 1-2mg of the sample was lightly pressed on a glass slide to obtain a flat surface.

Condition 2 (Low resolution)

Similarly, X-ray powder diffraction patterns were collected using a Bruker D8 diffractometer using Cu Ka radiation (40kV, 40mA), a theta-2 theta goniometer, divergent V4 and receiving slits (divergence of V4 and receiving slits), a Ge monochromator and a Lynxeye detector. Instrument performance was checked using certified corundum standards (NIST 1976). The software used for data collection was Diffrac Plus XRD Commander v2.5.0, data analyzed and represented using Diffrac Plus EVA v 11.0.0.2 or v 13.0.0.2. The samples tested under ambient conditions were flat plate samples prepared using powder (as received). Approximately 15mg of the sample was slowly loaded into a hole cut in a polished zero background (510) silicon wafer. The sample is rotated in its own plane during analysis. The data collected are specifically as follows:

● angular range: 2-42 degree 2 theta

● Step size (Step size): 0.05 degree 2 theta

● Collection time: 0.5 second step^-1

A high resolution XRPD curve was obtained for each sample (condition 1a) and the results are shown in figures 4-8, showing that five samples of citrate all have the same crystalline phase. Similarly, batches 4-6 were collected under condition 1b and FIG. 9 shows the curves overlaid to show that the patterns are very similar, demonstrating that they all have the same crystalline phase.

Low resolution XRPD curves were also collected using a Bruker GADDS diffractometer (condition 2), thus giving reference patterns for polymorphic screening analysis. Fig. 10 shows the curve overlap for batches 4-6, and fig. 11 shows a comparison of the high resolution curve and the low resolution curve for batch 4.

Samples prepared for collection of low resolution XRPD curves were placed in a chamber maintained at 40 ℃ and 75% relative humidity. After one week, the sample was reanalyzed with low resolution XRPD (condition 2) to check for phase transition. Fig. 12-14 show results compared to the original XRPD curves. It can be seen that the citrate salt of the present invention is stable for at least one week under such conditions without undergoing a phase transition.

Example 8 Nuclear Magnetic Resonance (NMR) study

Collection was carried out using a Bruker400MHz instrument (Bruker 400MHz instrument)¹H NMR spectra, Bruker400MHz instrument equipped with autosampler and controlled by DRX400 console. Automated experiments were performed using ICON-NMR v4.0.4 (type 1 (build 1)), run with Topspin v 1.3 (Patch level 8) and standard Bruker loading experiments (using the standard Bruker loaded experiments). In D6-DMSO or D₂Samples were prepared in O. Offline analysis was performed using ACD SpecManager v 9.09 (model 7703).

¹H NMR showed that all three samples were the same compound. The stoichiometric ratio of citrate was determined by integrating the counterion signal. However, these signals appear below the DMSO signal in the resulting spectra (batch 4, fig. 22), and as a result, integration of the citric acid signal was not possible. FIG. 23 shows batch 4 at D₂Of O¹HNMR. In this solvent, integration of the citric acid signal showed a stoichiometric ratio of 1:1 as expected.

Example 9 Differential Scanning Calorimetry (DSC) and thermogravimetric analysis (TGA)

Differential Scanning Calorimetry (DSC) data was collected using a Mettler DSC 823e equipped with a 34-bit autosampler. Qualified indium was used to correct the energy and temperature of the instrument. Typically, 0.5-3 mg of each sample was heated from 25 ℃ to 350 ℃ in a pinhole aluminum dish at 10 ℃ min-1. The nitrogen purge on the sample was maintained at 50 ml.min-1. The instrument control and data analysis software was STARe v 9.10.

Thermogravimetric analysis (TGA) data was collected using a Mettler TGA/SDTA 851e equipped with a 34-bit autosampler. Certified indium was used to correct instrument temperature. Typically, 5-30 mg of each sample was loaded into a pre-weighed aluminum crucible at 10 ℃ for min^-1Heating from room temperature to 350 ℃. The nitrogen purge on the sample was maintained at 50ml.min^-1. The instrument control and data analysis software was STARe v 9.10.

The DSC curve (figure 15) for batch 4 shows a significant thermal change at 176 ℃. A corresponding weight loss of about 20% was observed in TGA (figure 15). This weight loss and the complex shape of the DSC endotherm indicate that gross decomposition (gross degradation) occurs at temperatures greater than 176 ℃. Without wishing to be bound by theory, this may indicate dissociation of the salt and subsequent decomposition of the citric acid.

Batches 5 and 6 show similar DSC and TGA profiles (figures 16 and 17 show overlapping DSC and TGA data, respectively).

Example 10 Gravimetric Vapor Sorption (GVS)

Sorption isotherms were obtained using an SMS DVS Intrinsic moisture sorption analyzer (SMS DVS Intrinsic moisture sorption analyzer), controlled by the SMS Analysis software package (SMS Analysis Suite software). The sample temperature was maintained at 25 ℃ by instrument control. Humidity control with mixed dry and wet nitrogen flow at a total flow rate of 200 ml/min^-1. The relative humidity (dynamic range of 1.0-100% RH) near the sample was measured with a calibrated Rotronic probe. The change in weight of the sample (mass relaxation) was continuously monitored with a microbalance (precision. + -. 0.005 mg) as a function of% RH. Typically, 5-20 mg of sample is placed in a tared mesh stainless steel basket (tared mesh stainless steel basket) under ambient conditions. The samples were loaded and removed at 40% RH and 25 ℃ conditions (typical room temperature conditions). Moisture sorption isotherm analysis (2 scans resulted in 1 complete cycle) was performed as described below. 2Standard isothermal analyses were performed at 5 ℃ in the range of 0.5-90% RH at 10% RH intervals.

TABLE 3 method parameters for SMS DVS intrinsic experiments

Parameter(s)	Numerical value
		Adsorption-scanning 1	40-90

Desorption/adsorption-Scan 2	90-drying, drying-40
		Interval (% RH)	10
Number of scans	2
		Flow rate (ml. min.)-1)	200
Temperature (. degree.C.)	25
		Stability (. degree.C. min.)-1)	0.2
Sorption time (hours)	Completion in 6 hours (time out)

The hygroscopicity of citrate was investigated by performing a gravimetric vapour sorption experiment on batch 4. Approximately 20 mg of the sample was held at 25 ℃ while varying the ambient humidity over two complete cycles. The kinetic curves shown in fig. 18 show that the samples of batch 4 reached weight equilibrium in each% RH step. In the early stages of the experiment, the samples took longer to reach equilibrium. This may be due to displacement of residual solvent.

The isotherms shown in figure 19 show that the samples absorb < 0.6% water between 40% RH and 90% RH. The maximum weight difference (between 0% RH and 90% RH) is less than 1% w/w, indicating that the citrate salt is not hygroscopic. Furthermore, there is no evidence of the presence of the hydrated form of the citrate salt.

TABLE 4 isothermal weight values of GVS for batch 4

At the end of the GVS experiment, the sample was withdrawn and analyzed by XRPD to check for the presence of any gross phase changes. The results (fig. 20) show no overall phase change.

Example 11 determination of chemical purity by High Performance Liquid Chromatography (HPLC)

Purity analysis was performed on an Agilent HP1100 series system (Agilent HP1100 series system) equipped with a diode array detector and using the software ChemStation vb.02.01-SR 1. The parameters used are summarized in table 5.

TABLE 5 HPLC METHOD PARAMETERS FOR DETERMINING CHEMICAL PURITY

The chemical purity of the citrate batches 4-6 was determined using the HPLC procedure. The numerical results are shown in Table 6.

TABLE 6 purity measurement results

It can be seen that the measured purity of each sample was greater than 98.1%.

Example 12 solubility and polymorphism evaluation

For each solvent studied, approximately 8 mg of compound 1 was weighed into an 8 ml screw-top glass bottle. Solvent was added in 10 volumes of aliquots and the mixture was sonicated and heated (with a hot air gun) to facilitate dissolution. If dissolution is not achieved after addition of 100 volumes of solvent, an additional 100 volumes are added. The details and observations of each experiment (table 7) show that complete dissolution is achieved only in water.

TABLE 7 specific solubility evaluation

The vial was then placed in a humidity chamber and cycled at 25 deg.C/50 deg.C (8 hour cycle) for 24 hours. At the end of this time the sample was examined and then the lid was released to allow the solvent to evaporate. The dried samples were then transferred to a quartz array while the samples with solvent still present were vacuum filtered onto the sinter. The samples were then analyzed by XRPD to assess their crystalline state and crystalline form. XRPD analysis results (fig. 21) showed that all samples (with the only exception of water) were of form 1. The sample obtained from the aqueous solution was amorphous (fig. 21, the uppermost curve does not show a sharp peak).

The solubility of compound 1 citrate in organic solvents has proven to be extremely limited. The lack of color in the solvent in contact with the yellow crystals indicates minimal solubility. All residual crystals in the organic solvent of the screened samples were form 1. The citrate content dissolved in water was found to be 100 mg.ml^-1. The solid recovered by evaporating the solution was found to be amorphous. Solubility screening did not show the presence of any solvate or polymorph of the citrate salt.

The results of examples 1-12 are summarized in the following table.

TABLE 8 batch characterization summary

The details of the described embodiments of the invention should not be interpreted as limiting the invention. Various equivalent embodiments and changes may be made without departing from the spirit and scope of the invention, and it is to be understood that such equivalent embodiments are also part of the invention.

Claims (5)

1. A crystalline citrate salt of E-15- (2-pyrrolidin-1-yl-ethoxy) -7,12, 25-trioxa-19, 21, 24-triaza-tetracyclo [18.3.1.1(2,5).1(14,18) ] hexacosan-1 (24),2,4,9,14,16,18(26),20, 22-nonene, wherein said salt is in the form of a polymorph showing x-ray diffraction peaks on the 2 theta scale as shown in figure 8.
2. 2. The salt of claim 1, wherein the salt is a 1:1 salt.
3. 3. A pharmaceutical composition comprising the salt of claim 1 or 2.
4. 4. Use of a salt according to claim 1 or 2 in the manufacture of a medicament for the treatment of a proliferative disease.
5. 5. The use of claim 4, wherein the proliferative disease is cancer.