Patent Application
20130143884
The present invention relates to some 4-[2-[[5-methyl-1-(2-naphthalenyl)-1H-pyrazol-3-yl]oxy]ethyl]morpholine salts, to pharmaceutical compositions comprising them, and to their use in therapy and/or prophylaxis of sigma receptor associated diseases.
The search for new therapeutic agents has been greatly aided in recent years by better understanding of the structure of proteins and other biomolecules associated with target diseases. One important class of these proteins is the sigma (a) receptor, a cell surface receptor of the central nervous system (CNS) which may be related to the dysphoric, hallucinogenic and cardiac stimulant effects of opioids. From studies of the biology and function of sigma receptors, evidence has been presented that sigma receptor ligands may be useful in the treatment of psychosis and movement disorders such as dystonia and tardive dyskinesia, and motor disturbances associated with Huntington's chorea or Tourette's syndrome and in Parkinson's disease (Walker, J. M. et al, Pharmacological Reviews, 1990, 42, 355). It has been reported that the known sigma receptor ligand rimcazole clinically shows effects in the treatment of psychosis (Snyder, S. H., Largent, B. L. J. Neuropsychiatry 1989, 1, 7). The sigma binding sites have preferential affinity for the dextrorotatory isomers of certain opiate benzomorphans, such as (+)SKF 10047, (+)cyclazocine, and (+)pentazocine and also for some narcoleptics such as haloperidol.
The sigma receptor has at least two subtypes, which may be discriminated by stereoselective isomers of these pharmacoactive drugs. SKF 10047 has nanomolar affinity for the sigma 1 (σ-1) site, and has micromolar affinity for the sigma 2 (σ-2) site. Haloperidol has similar affinities for both subtypes. Endogenous sigma ligands are not known, although progesterone has been suggested to be one of them. Possible sigma-site-mediated drug effects include modulation of glutamate receptor function, neurotransmitter response, neuroprotection, behavior, and cognition (Quirion, R. et al. Trends Pharmacol. Sci., 1992, 13:85-86). Most studies have implied that sigma binding sites (receptors) are plasmalemmal elements of the signal transduction cascade. Drugs reported to be selective sigma ligands have been evaluated as antipsychotics (Hanner, M. et al. Proc. Natl. Acad. Sci., 1996, 93:8072-8077). The existence of sigma receptors in the CNS, immune and endocrine systems have suggested a likelihood that it may serve as link between the three systems.
In view of the potential therapeutic applications of agonists or antagonists of the sigma receptor, a great effort has been directed to find selective ligands. Thus, the prior art discloses different sigma receptor ligands. 4-[2-[[5-methyl-1-(2-naphthalenyl)-1H-pyrazol-3-yl]oxy]ethyl]morpholine is one of such promising sigma receptor ligands. The compound and its synthesis are disclosed and claimed in WO 2006/021462.
4-[2-[[5-methyl-1-(2-naphthalenyl)-1H-pyrazol-3-yl]oxy]ethyl]morpholine is a highly selective sigma-1 (σ-1) receptor antagonist. It has displayed strong analgesic activity in the treatment and prevention of chronic and acute pain, and particularly, neuropathic pain. The compound has a molecular weight 337.42 uma. The structural formula of the compound is:
To carry out its pharmaceutical development and realize its potential, there is a need in the art for additional forms of 4-[2-[[5-methyl-1-(2-naphthalenyl)-1H-pyrazol-3-yl]oxy]ethyl]morpholine that will facilitate the preparation of better formulations of this active pharmaceutical ingredient. Furthermore, new forms of the compound may also improve its production, handling and storage characteristics and its therapeutic effects such as pharmacological properties.
In this regard, alternative forms of the compound may have widely different properties such as, for example, enhanced thermodynamic stability, higher purity or improved bioavailability (e.g. better absorption, dissolution patterns). Specific compound forms could also facilitate the manufacturing (e.g. enhanced flowability), handling and storage (e.g. non-hygroscopic, long shelf life) of the compound formulations or allow the use of a lower dose of the therapeutic agent, thus decreasing its potential side effects. Thus it is important to provide such forms, having desirable properties for pharmaceutical use.
The inventors of the present invention, after an extensive research on different forms of 4-[2-[[5-methyl-1-(2-naphthalenyl)-1H-pyrazol-3-yl]oxy]ethyl]morpholine (herein referred as “compound 63”), have surprisingly found and demonstrated that some of its salts and specifically its hydrochloride salt provides advantageous production, handling, storage and/or therapeutic properties.
Thus, in a first aspect the present invention relates to a 4-[2-[[5-methyl-1-(2-naphthalenyl)-1H-pyrazol-3-yl]oxy]ethyl]morpholine salt selected from the group consisting of ethanesulfonate, fumarate, hydrochloride, malate, maleate, malonate and methanesulfonate.
In a preferred embodiment, the present invention is directed to the hydrochloride salt of 4-[2-[[5-methyl-1-(2-naphthalenyl)-1H-pyrazol-3-yl]oxy]ethyl]morpholine (herein referred as “P027” or “example 1”).
The P027 compound has a molecular weight 373.88 uma, a pKa of 6.73 and a melting point of 194.2° C. The compound is very soluble in water and freely soluble in methanol, 1N hydrochloric acid and dimethyl sulphoxide. It is sparingly soluble in ethanol, slightly soluble in acetone and practically insoluble in ethyl acetate and in 1N sodium hydroxide. The product exhibits a better dissolution and absorption profile in vivo than its related base.
In another aspect, the present invention is directed to a process for the preparation of the hydrochloride salt of 4-[2-[[5-methyl-1-(2-naphthalenyl)-1H-pyrazol-3-yl]oxy]ethyl]morpholine which comprises:
A further aspect of the present invention includes pharmaceutical compositions comprising 4-[2-[[5-methyl-1-(2-naphthalenyl)-1H-pyrazol-3-yl]oxy]ethyl]morpholine hydrochloride and a pharmaceutically acceptable carrier, adjuvant or vehicle.
In a further aspect the invention is directed to 4-[2-[[5-methyl-1-(2-naphthalenyl)-1H-pyrazol-3-yl]oxy]ethyl]morpholine hydrochloride for use as medicament, preferably as sigma ligand, i.e., for use the treatment and/or prophylaxis of a sigma receptor mediated disease or condition.
Another aspect of this invention relates to a method of treating and/or preventing a sigma receptor mediated disease which method comprises administering to a patient in need of such a treatment a therapeutically effective amount of a compound as above defined or a pharmaceutical composition thereof.
These aspects and preferred embodiments thereof are additionally also defined in the claims.
The inventors have found that the compound P027, which is the HCl salt of 4-[2-[[5-methyl-1-(2-naphthalenyl)-1H-pyrazol-3-yl]oxy]ethyl]morpholine, has advantages due to the fact, among others, that it is a crystalline solid, which simplifies isolation, purification and handling.
Indeed, after an extensive screening of salts, the inventors have observed that a large number of acids (e.g. sulphuric acid or L-tartaric acid) did not afford a solid when mixing with the 4-[2-[[5-methyl-1-(2-naphthalenyl)-1H-pyrazol-3-yl]oxy]ethyl]morpholine but an oil. Further, among the acids suitable for obtaining a salt in solid form, hydrochloric acid was the one that provided better results in terms of easiness of preparation, physical stability, scaling-up, solubility, etc.
Thus, the present invention relates to a 4-[2-[[5-methyl-1-(2-naphthalenyl)-1H-pyrazol-3-yl]oxy]ethyl]morpholine salt selected from the group consisting of ethanesulfonate, fumarate, hydrochloride, malate, maleate, malonate and methanesulfonate. These salts were able to provide crystalline solids.
Preferably, the present invention is directed to 4-[2-[[5-methyl-1-(2-naphthalenyl)-1H-pyrazol-3-yl]oxy]ethyl]morpholine hydrochloride (P027).
The hydrochloride salt of 4-[2-[[5-methyl-1-(2-naphthalenyl)-1H-pyrazol-3-yl]oxy]ethyl]morpholine can be prepared by adding an hydrochloric acid solution to its corresponding base dissolved in the appropriate solvent. In a particular embodiment, the P027 compound may be conveniently obtained by dissolving the free base compound in ethanol saturated with HCl.
As noted previously, it has been reported that 4-[2-[[5-methyl-1-(2-naphthalenyl)-1H-pyrazol-3-yl]oxy]ethyl]morpholine is a highly selective sigma-1 (σ-1) receptor antagonist, displaying strong analgesic activity in the treatment and prevention of chronic and acute pain, and particularly, neuropathic pain (see WO 2006/021462). It has now been found that the hydrochloride salt of 4-[2-[[5-methyl-1-(2-naphthalenyl)-1H-pyrazol-3-yl]oxy]ethyl]morpholine is particularly suitable for use as medicament.
The present invention therefore further provides medicaments or pharmaceutical compositions comprising 4-[2-[[5-methyl-1-(2-naphthalenyl)-1H-pyrazol-3-yl]oxy]ethyl]morpholine hydrochloride together with a pharmaceutically acceptable carrier, adjuvant, or vehicle, for administration to a patient.
More particularly, the P027 compound is useful in the treatment and/or prophylaxis of a sigma receptor mediated disease or condition.
In a more preferred embodiment the P027 compound is used in the manufacture of a medicament for the treatment and/or prophylaxis of a disease selected from the group consisting of diarrhoea; lipoprotein disorders; migraine; obesity; arthritis; hypertension; arrhythmia; ulcer; learning, memory and attention deficits; cognition disorders; neurodegenerative diseases; demyelinating diseases; addiction to drugs and chemical substances including cocaine, amphetamine, ethanol and nicotine; tardive diskinesia; ischemic stroke; epilepsy; stroke; stress; cancer; psychotic conditions, in particular depression, anxiety or schizophrenia; inflammation; or autoimmune diseases.
The auxiliary materials or additives of a pharmaceutical composition according to the present invention can be selected among carriers, excipients, support materials, lubricants, fillers, solvents, diluents, colorants, flavour conditioners such as sugars, antioxidants, binders, adhesives, disintegrants, anti-adherents, glidants and/or agglutinants. In the case of suppositories, this may imply waxes or fatty acid esters or preservatives, emulsifiers and/or carriers for parenteral application. The selection of these auxiliary materials and/or additives and the amounts to be used will depend on the form of application of the pharmaceutical composition.
The medicament or pharmaceutical composition according to the present invention may be in any form suitable for the application to humans and/or animals, preferably humans including infants, children and adults and can be produced by standard procedures known to those skilled in the art. Therefore, the formulation in accordance with the invention may be adapted for topical or systemic application, particularly for dermal, transdermal, subcutaneous, intramuscular, intra-articular, intraperitoneal, intravenous, intra-arterial, intravesical, intraosseous, intracavernosal, pulmonary, buccal, sublingual, ocular, intravitreal, intranasal, percutaneous, rectal, vaginal, oral, epidural, intrathecal, intraventricular, intracerebral, intracerebroventricular, intracisternal, intraspinal, perispinal, intracranial, delivery via needles or catheters with or without pump devices, or other application routes.
The mentioned formulations will be prepared using standard methods such as those described or referred to in the Spanish and US Pharmacopoeias and similar reference texts.
In one embodiment of the invention it is preferred that the P027 compound is used in therapeutically effective amounts. The physician will determine the dosage of the present therapeutic agent which will be most suitable and it will vary with the form of administration and the particular compound chosen, and furthermore, it will vary with the patient under treatment, the age of the patient, the type of disease or condition being treated. When the composition is administered orally, larger quantities of the active agent will be required to produce the same effect as a smaller quantity given parenterally. The compound is useful in the same manner as comparable therapeutic agents and the dosage level is of the same order of magnitude as is generally employed with these other therapeutic agents. This active compound will typically be administered once or more times a day for example 1, 2, 3 or 4 times daily, with typical total daily doses in the range of from 0.1 to 1000 mg/kg/day.
The following examples are merely illustrative of certain embodiments of the invention and cannot be considered as restricting it in any way.
The following techniques have been used in this invention for identifying the different salts of compound 63 obtained:
Differential Scanning Calorimetry Analysis (DSC)
Thermogravimetric Analysis (TGA)
Proton Nuclear Magnetic Resonance (1H-NMR)
Fourier Transformed Infrared Spectroscopy (FTIR)
Compound 63 can be can be prepared as disclosed in the previous application WO2006/021462. Its hydrochloride can be obtained according the following procedure:
Compound 63 (6.39 g) was dissolved in ethanol saturated with HCl, the mixture was stirred then for some minutes and evaporated to dryness. The residue was crystallized from isopropanol. The mother liquors from the first crystallization afforded a second crystallization by concentrating. Both crystallizations taken together yielded 5.24 g (63%) of the corresponding hydrochloride salt (m.p.=197-199° C.).
1H-NMR (DMSO-d5) δ ppm: 10.85 (bs, 1H), 7.95 (m, 4H), 7.7 (dd, J=2.2, 8.8 Hz, 1H), 7.55 (m, 2H), 5.9 (s, 1H), 4.55 (m, 2H), 3.95 (m, 2H), 3.75 (m, 2H), 3.55-3.4 (m, 4H), 3.2 (m, 2H), 2.35 (s, 3H).
HPLC purity: 99.8%.
With this method, the hydrochloride salt is obtained as a crystalline solid with a very good yield. Further, its high melting point is particularly convenient from a pharmaceutical standpoint since it implies that the product shows a good physical stability.
Extraction of Compound 63 from its Hydrochloride Salt (Example 1)
The sample used in this invention is the Example 1. The base (compound 63) was extracted with CH2Cl2 from a basic aqueous solution (pH>10, using a 0.5 M aqueous solution of NaOH) of example 1, rendering orange oil.
General Method to Crystallize Other Salts of Compound 63
Salts were prepared initially mixing 1 mL of a 0.107 M solution of compound 63, as the orange oil previously obtained (see Example 1), in methanol with 1 mL of a 0.107 M solution of the corresponding counterion in methanol. The mixtures were stirred for one hour and the solvent evaporated under vacuum (Genevac, 8 mm Hg), obtaining oil or a white solid depending on the salt.
The product obtained in the initial preparation was solved in the minimum amount of crystallization solvent at its boiling temperature or at a maximum of 75° C. If after the addition of 4 mL of solvent, the salt did not dissolve completely, the suspension was stirred at high temperature for 30 minutes and the residue was separated by hot filtration or centrifugation. The mother liquors were cooled to room temperature and kept for 24 hours.
When solid was formed, it was separated (filtration or centrifugation). If not, the solution was kept in the refrigerator (4° C.) for a few days. If solid was formed, it was separated from the solution. If not, the solution was kept in the freezer (−21° C.) for a few days. If solid was formed, it was separated from the solution. In case that after all these manipulations no solid was obtained the solution was left evaporating up to dryness.
All obtained solids were dried in the vacuum drying oven at 40° C. (10 mm Hg) for 4 hours and, if enough quantity was available, were analysed. The initial characterisation was done by 1H-NMR to confirm the synthesis of the salt. The solvents used in this invention are listed in table 1.
The acids used to investigate the crystalline salts of compound 63 were selected according to the following criteria (Table 2):
Although several of the acids selected have two or even three (citric acid) acidic positions, in principle, only sulfuric acid has a second proton acidic enough to form the disalt with compound 63. So in total there are eleven different salts that could be formed.
The general strategy performed to study the crystalline salts of compound 63 can be divided into three steps:
Initially, a crystallization screening was performed using the selected counterions shown in Table 2, to seek for promising crystalline salts. The screening was performed at a small scale (40 mg of compound 63), using a large range of crystallization solvents (Table 1) and different crystallization methodologies. In the screening, crystallization conditions were not strictly monitored, and the solids obtained were characterized by 1H-NMR. NMR spectroscopy gives a good indication of salt formation, since the 1H-NMR spectrum of the salt differs substantially from that of the acid and base mixture. A clear shift of the signals associated to the hydrogens close to the protonated nitrogen is observed. Moreover, when the acid counterion has characteristic signals in the 1H-NMR, these can be identified, allowing to determine the salt stoichiometry and to have a qualitative idea of the salt purity.
In a second step, all crystalline salts were scaled-up at 100-500 mg scale in the solvents that gave the best result in the screening procedure. Moreover, a crystallization methodology appropriate for industrial production was used. The salts obtained were fully characterized by 1H-NMR, DSC, TGA and FTIR. The aim of this step was, first to design a scalable procedure to prepare the selected salts with an optimized yield, and second to fully characterize them.
Finally, a group of selected crystalline salts, with adequate solid state properties (crystallinity and thermal stability) were prepared at a scale of 2-3 g starting from compound 63.
From Salt Crystallization Screening to Large Scale Preparation (Steps 1-3)
Initially, a crystallization screening of compound 63 with the ten counterions depicted in table 2 was performed, at a 40 mg scale, in the following ten solvents: acetone, ethyl acetate, chloroform, N,N-dimethylformamide, methanol, ethanol, isopropanol, 2-butanol, acetonitrile and tetrahydrofuran. The procedure started with the preparation of equimolar mixtures, from known concentration methanol dissolutions, of compound 63 and the different acid counterions. The resulting crude, after the methanol evaporation, was crystallized from the hot solvents formerly mentioned. Different crystallization strategies were used depending on the solubility of each acid and compound 63 mixture, and therefore the solids were obtained using different procedures. For some acids, the mixture was not soluble in the hot crystallization solvent, obtaining a slurry solid. In other cases, the solid crystallized during room temperature cooling of the solution, or after several days at 4° C. or at −18° C. Finally, in some crystallization attempts, the solid was obtained after slow evaporation of the solvent at room temperature. In several cases, more than one solid per crystallization attempt were obtained.
From this first crystallization screening (table 3), the following observations could be drawn:
Taking into account these results, a second crystallization screening was performed in nine additional solvents. Less polar solvents (isobutyl acetate, dimethyl carbonate, chlorobenzene, cyclohexane, 3-pentanone, toluene, methyl tert-butyl ether, diisopropyl ether) and water were selected in order to decrease the solubility of the salts (Table 4).
From this second crystallization screening, the following observations could be drawn:
Taking into account the results of the two crystallization screenings described, we optimize the generation of the best characterized non solvated salts of compound 63 with fumaric, maleic, methanesulfonic, ethanesulfonic, L-malic, and malonic acids. The optimization scale-up experiments were performed starting from 100 mg of compound 63. The scale-up procedure was also optimized for the salts with fumaric, maleic, methanesulfonic, ethanesulfonic, L-malic and malonic acids.
Finally, the preparation of the salts for the six selected counterions was scale-up at 2-3 g and they were fully characterized. The overall process in this invention is summarized in the following table.
During the initial screening the crystallization of the fumarate salt was attempted in 10 different solvents. Crystalline solids corresponding to the salt were obtained in all solvents, except DMF and chloroform, using different crystallization techniques: slurry, cooling a saturated solution or after complete evaporation of the solvent. In chloroform the initial acid was recovered, whereas in DMF the salt separated as orange oil. Two non-solvated solids were obtained, the first one in methanol, isopropanol and butanol, and the second one only in ethanol. Finally, solvates were obtained in acetone, ethyl acetate and THF, and a mixture of the two solids was generated in acetonitrile.
A non-solvated crystalline solid, in principle any of the ones obtained in the screening, was chosen for the scale-up. Initially, the scale up was attempted in acetonitrile, since it was the solvent that rendered a crystalline product in which the salt was less soluble. Although the salt was obtained in very good yield (83%), the process was not optimal for scale-up since the acid is not soluble in acetonitrile and the final salt precipitated from a mixture of compound 63 as an oil and fumaric acid as a solid, both suspended in the solvent. The crystallization was then attempted in ethanol to generate pure solid S5. Very disappointingly, in the scale-up in ethanol, a new, poorly crystalline solid was generated in low yield. Finally, the crystallization was performed in acetonitrile, adding the acid dissolved in an alcohol (ethanol or isopropanol). Slightly better results are obtained when fumaric acid is dissolved in ethanol and the addition is performed at room temperature (Table 6). On the other hand, a mixture of phases was obtained when the suspension was kept at 4° C. for two days (Table 6, entry 4).
1Referred to starting example 1.
2Solvent used to dissolve compound 63.
3Solvent used to dissolve the fumaric acid.
4Temperature at which the acid and base are mixed.
5Temperature at which the final solid is harvested.
6All experiments were seeded.
The experimental procedure used to prepare the fumarate salt at 0.5 g scale (entry 2 in table 6) was as follows:
The formation of the salts can be easily characterized by the 1H-NMR spectrum which changes substantially compared to the free base. In the case of the fumarate salt, signals coming from hydrogen atoms close to the basic nitrogen (hydrogens 1 and 2 in the formula below) are clearly shifted downfield (table 7). Smaller shifts can also be observed on signals coming from hydrogen atoms further away from the nitrogen (hydrogens 3 and 4 in Figure C). Moreover, the signal from the fumarate appears on the expected chemical shift (δ: 6.72 ppm). The integrations of signals corresponding to the anion and the cation unambiguously confirm that the equimolecular salt, and not the disalt, is formed (
Molecular formula of compound 63 with indication of hydrogens that shift in the 1H-NMR spectrum after forming the salt.
The DSC analysis at a heating rate of 10° C./min presents a small endothermic peak, followed by a small exothermic peak and an intense endothermic signal (
On the TG analysis a small weight loss of 0.3% at temperatures between 120 and 150° C. and a dramatic weight loss starting at 190° C. due to decomposition are observed.
The characterisation of the fumarate salt is the following (
1H-NMR (400 MHz, d4-methanol) δ: 2.35 (s, 3H), 2.92-3.00 (m, 4H), 3.17 (t, J=5 Hz, 2H), 3.80 (t, J=5 Hz, 4H), 4.44 (t, J=5 Hz, 2H), 5.83 (s, 1H), 6.72 (s, 2H), 7.52-7.62 (m, 3H), 7.89-7.96 (m, 3H), 8.00 (d, J=9 Hz, 1H).
Residual solvents from 1H-NMR: 0.2% w/w of acetonitrile.
FTIR (ATR) υ: 3435, 3148, 3037, 2943, 2855, 1876, 1731, 1664, 1650, 1559, 1509, 1488, 1446, 1394, 1372, 1314, 1236, 1186, 1166, 1133, 1098, 1081, 1047, 1014, 981, 932, 917, 859, 816, 787, 769 and 748 cm−1.
DSC (10° C./min): Two endothermic fusion peaks with an onset at 131 and 142° C.
TGA (10° C./min): A weight loss of 0.3% between 120 and 150° C. The decomposition process starts at 190° C.
During the initial screening the crystallization of the maleate salt was attempted in 10 different solvents. The salt was very soluble in all the solvents assayed. Solubilities between 50 and 200 mg/mL were observed, except for ethyl acetate, in which the salt had a solubility of 20 mg/mL. Crystalline solids were obtained in all solvents after cooling the solution to room temperature or, for chloroform, methanol and DMF, after complete evaporation of the solvent. Four different solids were detected. A non solvated crystalline phase was obtained in the majority of the crystallizations. Moreover, a solvate was generated in THF and two other not completely characterized solids were generated in three of the experiments.
Taking into account the boiling point and the amount of solvent needed for the crystallization (66 mg/mL), isopropanol was the solvent chosen for the scale-up and synthesis of the crystalline salt. An initial attempt cooling a mixture of maleic acid and compound 63 in isopropanol from 60° C. to room temperature rendered the salt as oil (Table 7). This oil crystallized after stirring again the mixture at 60° C. for several hours. A similar methodology in more diluted conditions rendered the salt directly as a solid. Finally, the process was optimized generating the direct precipitation of the salt after adding an isopropanol solution of the acid over an isopropanol solution of compound 63 at room temperature.
1Refered to starting example 1.
The experimental procedure used to prepare the maleate salt at 2.5 g scale was as follows:
The maleate salt can be easily characterized by the 1H-NMR spectrum (
The DSC analysis (
The characterisation of the maleate salt is the following (
1H-NMR (400 MHz, d-chloroform) δ: 2.35 (s, 3H), 3.02-3.64 (m, 6H), 3.99 (t, J=5 Hz, 4H), 4.61-4.66 (m, 2H), 5.70 (s, 1H), 6.30 (s, 2H), 7.50-7.58 (m, 3H), 7.79-7.82 (m, 1H), 7.84-7.95 (m, 3H).
Residual solvents from 1H-NMR: 1.1% w/w of isopropanol.
FTIR (ATR) υ: 3043, 2853, 1707, 1619, 1599, 1557, 1487, 1445 1374, 1357, 1340, 1302, 1237, 1163, 1135, 1096, 1041, 1022, 930, 919, 861, 817, 762 and 750 cm−1.
DSC (10° C./min): Endothermic fusion peak with an onset at 139° C.
TGA (10° C./min): A weight loss of 1.0% between 110-150° C. The decomposition process starts at 150° C.
During the initial screening with the first set of ten solvents, the methanesulfonate salt could not be crystallized. The salt was very soluble in all the solvents assayed (>200 mg/mL), rendering oils after complete evaporation of the solvent. When the crystallization was attempted in the second set of nine more apolar solvents, oils were also recovered in the vast majority of the experiments, either after evaporation of the solvent, or because the oily salt did not dissolve. Nevertheless, a crystalline solid corresponding to the salt was obtained from the toluene solution cooled at −18° C. after separating the excess of salt as oil. Thus, toluene was chosen for the optimization and scale-up of the synthesis of the salt.
In the first scale-up attempt, methanesulfonic acid was added directly to a toluene solution of compound 63, but the salt rapidly separated as an oil. This oil crystallized after being stirred together with the solvent for several hours at room temperature. In order to provoke the direct crystallization of the solid salt, the same process was repeated in the presence of seed crystals of the salt. Moreover, in order to improve the salt colour, the methanesulfonic acid was distilled just before use (180° C., 1 mBar).
The experimental procedure used to prepare the methanesulfonate salt at 2.5 g scale was as follows:
The methanesulfonate salt can be easily characterized by the 1H-NMR spectrum (
The DSC analysis (
The characterisation of the methanesulfonate salt is the following (
1H-NMR (400 MHz, d-chloroform) δ: 2.36 (s, 3H), 2.84 (s, 3H), 3.03-3.15 (m, 2H), 3.54-3.61 (m, 2H), 3.63-3.71 (m, 2H), 3.97-4.05 (m, 2H), 4.10-4.20 (m, 2H), 4.71-4.76 (m, 2H), 5.75 (s, 1H), 7.50-7.59 (m, 3H), 7.79-7.82 (m, 1H), 7.84-7.95 (m, 3H).
Residual solvents from 1H-NMR: 0.58% w/w of toluene.
FTIR (ATR) υ: 3018, 2957, 2920, 2865, 2693, 2627, 1634, 1602, 1562, 1509, 1485, 1435, 1392, 1376, 1265, 1221, 1164, 1131, 1098, 1049, 1033, 1007, 934, 914, 862, 822, 772 and 759 cm−1.
DSC (10° C./min): Endothermic fusion peak with an onset at 145° C.
TGA (10° C./min): A weight loss of 0.5% between 120 and 160° C. The decomposition process starts at 260° C.
During the initial screening with the first set of ten solvents, the ethanesulfonate salt could only be crystallized in acetonitrile. But, since the salt was very soluble in all the solvents assayed (>200 mg/mL) this solid was obtained only after complete evaporation of the solvent. In the remaining experiments, oil was generated after complete evaporation of the solvent. When the crystallization was attempted in the second set of nine more apolar solvents, three solids where obtained in methyl tert-butyl ether, isobutyl acetate, and toluene mixed with oily salt. In these experiments, the oily salt did not completely dissolve. Toluene was chosen to optimize and scale-up the synthesis of the salt.
In the initial scale up of the ethanesulfonate, the oily salt was suspended in hot toluene and allowed to cool. The salt did not crystallize and it remained as oil. In a second attempt, in which the ethanesulfonic acid was slowly added to a solution of compound 63 in toluene, a brown solid separated on cooling. When repeating this same procedure at room temperature, oil readily appeared which slowly crystallized after being stirred together with the solvent for several days. In order to provoke the direct crystallization of the salt, the same process was repeated at room temperature in the presence of seed crystals of the salt. Moreover, in order to improve the salt colour, the ethanesulfonic acid was distilled just before use (200° C., 1 mBar).
The experimental procedure used to prepare the ethanesulfonate salt at 2.5 g scale was as follows:
The formation of the ethanesulfonate salt can be easily deduced from the 1H-NMR spectrum (
The DSC analysis (
The characterisation of the ethanesulfonate salt is the following (
1H-NMR (400 MHz, d-chloroform) δ: 1.37 (t, J=7 Hz, 3H), 2.36 (s, 3H), 2.93 (q, J=7 Hz, 2H), 3.03-3.15 (m, 2H), 3.55-3.62 (m, 2H), 3.64-3.72 (m, 2H), 3.96-4.04 (m, 2H), 4.11-4.21 (m, 2H), 4.71-4.77 (m, 2H), 5.75 (s, 1H), 7.50-7.59 (m, 3H), 7.79-7.83 (m, 1H), 7.84-7.95 (m, 3H).
Residual solvents from H-NMR: 0.35% w/w of toluene.
FTIR (ATR) υ: 3021, 2958, 2924, 2863, 2625, 2488, 1633, 1603, 1565, 1508, 1485, 1470, 1437, 1391, 1376, 1353, 1334, 1265, 1242, 1210, 1160, 1149, 1131, 1098, 1027, 1008, 978, 934, 916, 856, 819, 776, and 739 cm−1.
DSC (10° C./min): Endothermic fusion peak with an onset at 133° C.
TGA (10° C./min): A weight loss of 0.3% between 110 and 160° C. The decomposition process starts at 280° C.
During the initial screening with the first set of ten solvents, the malate salt could be crystallized in acetonitrile and isopropanol. Nevertheless, the salt was very soluble in both solvents (>200 mg/mL) and the two solids were obtained only after complete evaporation. In the remaining experiments, oil was generated after complete evaporation of the solvent. When the crystallization was attempted in the second set of nine more apolar solvents, although the salt was less soluble, a crystalline solid was obtained only in 3-pentanone. The other experiments rendered oil. Taking into account these results, 3-pentanone was chosen to optimize and scale-up the synthesis of the salt.
The initial scale-up attempts for the preparation of the salt were performed adding a solution of L-malic acid in 3-pentanone to a solution of compound 63 also in 3-pentanone at temperatures between 50 and 70° C. Using this procedure the salt separated sometimes as oil on cooling. This oil easily crystallized after being stirred together with the solvent at 50° C. for some hours. Direct production of the crystalline salt could be induced by seeding, as it is described in the procedure used to prepare the malate salt at 2.5 g scale that follows:
The formation of the malate salt can be easily deduced from the 1H-NMR spectrum (
On the DSC analysis (
The characterisation of the malate salt is the following (
1H-NMR (400 MHz, d4-methanol) δ: 2.35 (s, 3H), 2.59 (dd, J1=16 Hz, J2=7 Hz, 1H), 2.79 (dd, J1=16 Hz, J3=5 Hz, 1H), 2.89-2.97 (m, 4H), 3.13 (t, J=5 Hz, 2H), 3.80 (t, J=5 Hz, 4H), 4.39 (dd, J2=7 Hz, J3=5 Hz, 1H), 4.43 (t, J=5 Hz, 2H), 5.83 (s, 1H), 7.52-7.61 (m, 3H), 7.89-7.96 (m, 3H), 8.00 (d, J=9 Hz, 1H).
FTIR (ATR) υ: 3171, 3003, 2874, 1718, 1597, 1556, 1487, 1468, 1440, 1360, 1268, 1142, 1126, 1097, 1050, 1022, 1010, 986, 950, 920, 902, 863, 822, 797, 770, 746 and 742 cm−1.
DSC (10° C./min): Endothermic fusion peak with an onset at 125° C.
TGA (10° C./min): A weight loss starting at 150° C. due to decomposition.
During the initial screening with the first set of ten solvents, the malonate salt could only be crystallized in isopropanol. Nevertheless, the salt was very soluble in this solvent (>200 mg/mL) which anticipated problems on scaling-up. For this reason, the crystallization was attempted in the second set of nine more apolar solvents. In this second set of experiments, a crystalline solid was obtained only from methyl tert-butyl ether on cooling a saturated solution to −18° C. after separating, at high temperature, an abundant part of the salt as oil.
Taking into account these results, the scale-up of the malonate salt was first attempted in isopropanol. Very disappointingly, the oil separated right after mixing the acid and compound 63. The oil crystallized in a poor yield after being stirred for several hours together with the solvent. Yield could be improved when methyl tert-butyl ether was added during the crystallization process after the oiling out. To avoid the generation of the salt initially as oil and to improve the yield, the crystallization process was modified. A solution of malonic acid in isopropanol was added to a solution of compound 63 in methyl tert-butyl ether. Using this procedure, the salt was generated directly as a solid but still some oiling out could be observed. Finally, direct and complete crystallization of the salt could be obtained with seeding as it is described in the following procedure:
The formation of the malonate salt can be easily deduced from the 1H-NMR spectrum (
The DSC analysis (
The characterization of the malonate salt is the following (
1H-NMR (400 MHz, d-chloroform) δ: 2.35 (s, 3H), 3.10-3.40 (m, 4H), 3.23 (s, 2H), 3.40-3.46 (m, 2H), 3.97 (t, J=5 Hz, 4H), 4.59-4.64 (m, 2H), 5.70 (s, 1H), 7.49-7.58 (m, 3H), 7.79-7.82 (m, 1H), 7.84-7.95 (m, 3H).
Residual solvents from 1H-NMR: 0.2% w/w of isopropanol and 0.2% of methyl tert-butyl ether.
FTIR (ATR) υ: 3148, 3027, 2942, 2857, 1718, 1621, 1599, 1561, 1488, 1443, 1374, 1343, 1308, 1260, 1165, 1135, 1097, 1080, 1046, 1022, 1011, 932, 918, 863, 819 and 752 cm−1.
DSC (10° C./min): Endothermic fusion peak with an onset at 90° C.
TGA (10° C./min): Weight loss starting at 100° C. due to decomposition.
Summary of Salt Crystallization Screening
Attempts to form salts of compound 63 with sulphuric acid and L-tartaric acid were unsuccessful and only oils were obtained.
Other salts, although in solid form, were only obtained by a complex synthetic process on comparing it with the experimental part for the hydrochloride synthesis, or under unique experimental conditions. Further, a non crystalline solid was frequently obtained instead of the crystalline form obtained for the hydrochloride. All these drawbacks imply that the scale-up for the associated synthetic process will be very complicated.
In the following table 8 a summary of key data referred to each solid salt prepared in large scale in this invention is shown: grade of crystallinity, crystallization solvent, yield and melting temperature.
As may be observed from the above, the hydrochloride salt is always obtained as a crystalline solid with a very good yield (including crystallization) and has a melting point over 50° C. among the other salts which clearly implies an advantage relating to the physical stability. Additionally, on comparing the TGA analysis the hydrochloride has a clean profile and no solvent loses are detected.
Further, some additional experiments (thermodynamic solubility, pharmacokinetic) were performed for example 1 (P027) in order to confirm the suitability of this compound for pharmaceutical purposes.
General protocol for thermodynamic solubility at pH 7.4 and pH 2 is described below.
A) Thermodynamic Solubility at pH 7.4
Buffer pH 7.4 (50 mM)
Buffer phosphates pH 7.4 was prepared as follows:
Samples Equilibrium
Samples were equilibrated using:
Procedure
Problem Compound
2 mg in an HPLC vial (by duplicate) was weight and 1 ml of buffer was added. The vial was maintained at 25° C., in the stirrer Thermomixer Comfort., during 24 hours. Centrifugation at 4000 rpm followed during 15 min.
The resulting upper layer was collected with a glass pipette and transferred to the HPLC vials. Again centrifuged and the injector programmed at 2.7 mm high.
Standards (by Duplicate)
Sol.A: 2 mg in 5 ml methanol (400 ug/ml)
Sol.B: 1 ml Sol.A to 10 ml with methanol (40 ug/ml)
Sol.C: 5 ml Sol.B to 50 ml with methanol (4 ug/ml)
Sol.D: 4 ml Sol.C to 10 ml with methanol (1.6 ug/ml)
Sol.E: 5 ml Sol.D to 25 ml with methanol (0.32 ug/ml)
10 μl of all prepared solutions were injected, beginning with the more diluted standard. Blanks were also injected, for checking the absence of contamination.
The standard calibration curve was done (see
10 μl of problem compound solution were injected, by duplicate and the average peak area (if quantifiable) interpolated in the standard curve (see Tables 9, 10 and 11 and example below).
Chromatographic Conditions
B) Thermodynamic Solubility at pH 2
The previous procedure was executed with HCl 0.01N.
Thermodinamical Solubility for Example 1
According to the described protocol it was obtained 227 μg/ml (pH=7.4). See associated graphic in
The pharmacokinetics of Example 1 in Wistar Hannover rats following a single oral administration of 25 mg/kg (expressed as compound 63) was tested. For this purpose, plasma samples were collected at different time points and analyzed using HPLC (High pressure liquid chromatography) method with fluorescence detection.
Sample Obtention
Two groups were used in this test. Group 1 received vehicle and Group 2 received Example 1 at 25 mg/kg with an administration volume of 10 mL/kg.
Blood samples were extracted from the retro-orbital zone at the following time points: pre-dose, 15 min, 30 min, 1 h, 1.5 h, 2 h, 3 h, 4 h, 5 h, 6 h, 8 h and 24 h. Blood was then transferred into heparin-containing plastic tubes. Plasma was obtained by centrifugation at approximately 3000 rpm for 10 min at 4° C. These plasma samples were labeled and frozen at a temperature of approximately −65° C. until analysis.
Analysis of Samples
Samples were analyzed by a previously validated analytical method. Briefly, rat plasma samples were thawed at room temperature and centrifuged at 3000 rpm for 10 min at approximately 4° C. 300 μl of plasma samples were placed into vials and spiked with 30 μl of internal standard working solution. The vials were capped and mixed thoroughly.
The following solid-phase extraction method was used for the extraction of Example 1.
Samples were then chromatographied using as mobile phase a mixture of 20 mM potassium phosphate monobasic adjusted at pH 3, and acetonitrile (70-73%) A and (30-27%) B (v/v) at room temperature. The flow rate used was 0.5 ml/min and analysis time was around 17 min.
The peaks corresponding to Example 1 and its internal standard were quantified by fluorescence detection at an excitation wavelength of 260 nm and an emission wavelength of 360 nm. The rest of parameters were: Response time: >0.2 min (4 s standard) and PMT gain 8.
Pharmacokinetic Parameters
The pharmacokinetic parameters were obtained from the mean plasma level curves by means of non-compartmental kinetics using the software program WinNonlin Professional version 5.0.1.
The peak plasma concentration values (Cmax) and the time to reach such concentration (tmax) were obtained directly from the experimental data. The elimination constant (kel) was calculated by linear regression of the last phase of the curve (log concentration vs. time). The elimination half-life (t1/2) was determined with the expression t1/2=0.693/kel. The area under the curve of plasma levels vs. time from zero to the last time determined (AUC0-t) was calculated be means of the trapezoidal method. The area under the curve of plasma levels vs time from zero to infinity (AUC0-∞) was calculated with the expression: AUC0-∞=AUC0-t+Clast/kel, where Clast is the plasma concentration at the last time measured.
Pharmacokinetic Parameters Cmax and AUC of Example 1
According to the described protocol it was obtained Cmax: 1152.8 ng/ml, AUC0-t: 1218.4 ng·h/ml and AUC0-∞: 1249.6 ng·h/ml. See associated graphics in
The results obtained in the last two tests (solubility and pharmacokinetic) enforce the hydrochloride as the better salt for compound 63 for related formulations and clinical studies.
| Number | Date | Country | Kind |
|---|---|---|---|
| 09382261.7 | Nov 2009 | EP | regional |
| 10382025.4 | Feb 2010 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2010/068256 | 11/25/2010 | WO | 00 | 12/3/2012 |
