EA000528B1 - Polymorphic forms of a growth hormone secretagogue - Google Patents

Abstract

1. A polymorphic form of the compound N-[1(R)-[(1,2-dihydro-1-methanesulfonyl-spiro[3H-indole-3,4'-piperidin]-1'-yl)-carbonyl]-2-(phenylmethyl-oxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate designated Form I, characterized by an X-ray powder diffraction pattern with principal reflections at approximately: 6.5, 14.7, 16.9, 17.1, 17.9, 19.5, 21.1, 21.7, and 22.0 degree (2 theta). 2. The polymorphic form of Claim 1 characterized by an endothern of melting with an extrapolated onset temperature of about 170 degree C when heated in a differential scanning calorimetric cell at a rate of 10 degree C/min under a nitrogen atmosphere. 3. The polymorphic form of Claim 1 characterized in that solubility in isopropanol 4,6 mg/ml. 4. The polymorphic form of Claim 1 characterized by a double refraction in polirized light. 5. The polymorphic form of the compound N-[1(R)-[(1,2-dihydro- l-methanesulfonyl-spiro[3H-indole-3,4'-piperidin]-1'-yl)-carbonyl]-2-(phenylmethyl-oxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate designated Form IV, characterized by an X-ray powder diffraction pattern with principal reflections at approximately: 16.0, 16.2, 18.3, 20.1, 21.0, and 24.2 degree (2 theta). 6. The polymorphic form of Claim 5 characterized by a water loss endothenn at a temperature of about 45 degree C followed by an endothern with an extrapolated onset temperature of about 129 degree C when heated in a differential scanning calorimetric cell at a rate of 10 degree C/min under a nitrogen atmosphere. 7. The polymorphic form of Claim 5 which contains approximately 3.5 moles of water per mole of N-[l(R)-[(l,2-dihydro-l-methanesulfonyl-spiro[3H-indole-3,4'-piperidin]- 1'-yl)carbonyl]-2-(phenylmethyl-oxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate. 8. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of the polymorphic form of Claim 1. 9. The pharmaceutical composition useful for the treatment of osteoporosis which comprises a combination of a bisphosphonate compound and the polymorphic form of Claim 1. 10. The pharmaceutical composition of Claim 9 wherein the bisphosphonate compound is alendronate. 11. The pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of the polymorphic form of Claim 5. 12. A pharmaceutical composition useful for the treatment of osteoporosis which comprises a combination of a bisphosphonate compound and the polymorphic form of Claim 5. 13. The pharmaceutical composition of Claim 12 wherein the bisphosphonate compound is alendronate. 14. A method for increasing levels of endogenous growth hormone in a human or an animal which comprises administering to such human or animal an effective amount of the polymorphic form of Claim 1. 15. A method for increasing levels of endogenous growth hormone in a human or an animal which comprises administering to such human or animal an effective amount of the polymorphic form of Claim 1 and an additional growth hormone secretagogue. 16. The method of Claim 15 wherein the additional growth hormone secretagogue is selected from the group consisting of: growth hormone releasing peptide GHRP-6; growth hormone releasing peptide GHRP-2; growth hormone releasing peptide GHRP-1; B-HT920; growth hormone releasing factor; an analog of growth hormone releasing factor; IGF-1 and IGF-2. 17. A method for increasing feed efficiency, promoting growth, increasing milk production and improving the carcass quality of livestock which comprises administering to such livestock an effective amount of the polymorphic form of Claim 1. 18. A method of treating or preventing a condition selected from the group consisting of: osteoporosis; catabolic illness; immune deficiency, including that in individuals with a depressed T4/T8 cell ratio; hip fracture; musculoskeletal impairment in the elderly; growth hormone deficiency in adults or in children; obesity; cachexia and protein loss due to chronic illness such as AIDS or cancer; and treating patients recovering from major surgery, wounds or bums, in a patient in need thereof which comprises the administration to the patient of an effective amount of the polymorphic form of Claim 1. 19. A method for the treatment of osteoporosis which comprises administering to a patient with osteoporosis a combination of a bisphosphonate compound and the polymorphic form of Claim 1. 20. The method of Claim 19 wherein the bisphosphonate compound is alendronate. 21. A method for increasing levels of endogenous growth hormone in a human or an animal which comprises administering to such human or animal an effective amount of the polymorphic form of Claim 5. 22. A method for increasing levels of endogenous growth hormone in a human or an animal which comprises administering to such human or animal an effective amount of the polymorphic form of Claim 1 and an additional growth hormone secretagogue. 23. The method of Claim 22 wherein the additional growth hormone secretagogue is selected from the group consisting of: growth hormone releasing peptide GHRP-6; growth hormone releasing peptide GHRP-2; growth hormone releasing peptide GHRP-1; B-HT920; growth hormone releasing factor; an analog of growth hormone releasing factor; IGF-1 and IGF-2. 24. A method for increasing feed efficiency, promoting growth, increasing milk production and improving the carcass quality of livestock which comprises administering to such livestock an effective amount of the polymorphic form of Claim 5. 25. A method of treating or preventing a condition selected from the group consisting of: osteoporosis; catabolic illness; immune deficiency, including that in individuals with a depressed T4/T8 cell ratio; hip fracture; usculoskeletal impairment in the elderly; growth hormone deficiency in adults or in children; obesity; cachexia and protein loss due to chronic illness such as AIDS or cancer; and treating patients recovering from major surgery, wounds or bums, in a patient in need thereof which comprises the administration to the patient of an effective amount of the polymorphic form of Claim 5. 26. A method for the treatment of osteoporosis which comprises administering to a patient with osteoporosis a combination of a bisphosphonate compound and the polymorphic form of Claim 5. 27. The method of Claim 26 wherein the bisphosphonate compound is alendronate. 28. A process for the preparation of the polymorphic form of Claim 1 designated Form I which comprises: stirring a solution of Form II of N-[l(R)-[(l,2-dihydro-1 -methanesulfonyl-spiro[3H-indole-3,4'-piperidin]-1'-yl)carbonyl]-2-(phenylmethyl-oxy)ethyl]-2-amino-2-methylpropanamide methane-sulfonate in isopropanol at approximately 25 degree C for about 2-24 hours.

Description

Growth hormone, which is secreted by the pituitary gland, stimulates the growth of all body tissues capable of growth. In addition, it is known that growth hormone has the following main effects on the metabolic processes of the body: (1) an increased rate of protein synthesis in all cells of the body; (2) a reduced rate of carbohydrate utilization in the cells of the body; (3) increased mobilization of free fatty acids and the use of fatty acids for energy. Lack of growth hormone secretion can lead to various medical conditions, such as dwarfism.

Various ways of releasing growth hormone are known. For example, chemicals such as arginine, L-3,4, -dihydroxyphenylalanine (L-DOPA), glucagon, vasopressin, as well as insulin-dependent hypoglycemia and such active processes as sleep and exercise indirectly cause the release of growth hormone from the pituitary gland [pituitary gland ], acting in some way on the hypothalamus, possibly reducing the secretion of somatostatin, or increasing the secretion of the known release factor of the growth hormone secretion stimulator (GRF) or an unknown endogenous hormone that releases growth hormone or all of them.

In cases where an increase in growth hormone levels is desired, the problem is usually solved by administering exogenous growth hormone or by administering GRF or a peptide compound that stimulates the production and / or release of growth hormone. In any case, the peptide nature of the compound requires that it be administered in the form of injections. Initially, the sources of growth hormone were extracts from the pituitary glands of the [pituitary] corpses. This, as a result, provided a very expensive product and there was a risk of transferring to the recipient growth hormone of the disease associated with the source of the pituitary gland. Recombinant growth hormone is available, which, despite the absence of the risk of transmission of the disease, nevertheless remains, despite the absence of the risk of transmission of the disease, a very expensive product that must be administered by injection or nasal spray. Other compounds have been developed that stimulate the release of endogenous growth hormone.

In particular, certain spiro compounds are disclosed in U.S. Patent No. 5,536,716, PCT Patent Publication WO

94/13696 and in Rgos. Natl. Acad. Sci. USA, 92, 70017005 (July 1995) as non-peptide growth hormone secretion stimulants. These compounds have the ability to stimulate the release of natural or endogenous growth hormone and can thus be used to treat conditions requiring stimulation of the production or secretion of growth hormone, as is the case in people with a deficiency of natural growth hormone or in animals intended for the production of products food or wool, where the result of stimulation of growth hormone will be a larger and more productive animal.

Among the preferred substances disclosed in the above works, spiro [3H-indole-3, 4'-piperidine] -1'-yl) carbonyl] -2 (phenylmethyl-hydroxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate, which has a structure ^{so uru:}

US Patent U.S. Patent No. 5,536,716 and PCT Patent Publication WO 94/1 3696 disclose methods for preparing this substance (see Examples 18.19 and 55). In particular, in example 55, it was determined that the compound obtained by recrystallization from a mixture of ethyl acetate - ethanol - water has a melting point of 1 66168 ° C. This compound was subsequently identified as having a polymorphic form, designated here as Form II. In the publication Proc. Natl. Acad.Sci. USA, 92, 70017005 (July 1995), it is noted that the compound isolated as monohydrate has a melting point of 168-170 ° C, however, only general methods for preparing the substance are disclosed, and it is not disclosed how the substance was crystallized.

The morphological forms of pharmaceutical compounds may be of interest in terms of developing a suitable dosage form, because if the morphological form is not kept constant during clinical or stability studies, the exact dose used or measured cannot be comparable from one batch to another. Since the pharmaceutical compound is made for use, it is important to know the morphological form contained in each dosage form in order to be sure that the same form is used in the preparation process and the same amount of preparation is included in each dose. Therefore, it is necessary to know for sure whether a single morphological form or any known combination of morphological forms is present. In addition, certain morphological forms may have enhanced thermodynamic or hydroscopic stability and may be more suitable than other morphological forms for incorporation into pharmaceutical compositions. The term polymorphic form of a chemical compound, as used here, means the same chemical nature, but with a different crystal lattice.

Summary of invention

The present invention relates to polymorphic forms of the compound: N- [1 (R) - (1,2-dihydro-1-methanesulfonyl-spiro [3H-indol-3, 4'-piperidine] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate, as well as a method for producing such polymorphic forms.

The present invention also relates to pharmaceutical compositions comprising these polymorphic forms as an active ingredient and the use of these polymorphic forms and compositions for treating certain disorders.

The polymorphic forms of this invention, which are a stimulator of growth hormone secretion, are useful for administration to meat animals to initiate their growth, which leads to more efficient production of meat food products, as well as people for the treatment of physiological or medical conditions characterized by insufficient secretion of growth hormone, as well as for the treatment of medical conditions that improve due to the anabolic effects of growth hormone.

These polymorphic forms have advantages over other known forms of N- [1 (R) - (1,2-dihydro-1-methanesulfonylspiro [3H-indole-3,4'-piperidine] -1'-yl) carbonyl] -2 - (phenylmethyloxy (ethyl) -2-amino-2-methylpropanamide methanesulfonate in terms of thermodynamic stability and convenience for inclusion in pharmaceutical compositions.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to new polymorphic forms of the compound N- [1 (R) - (1,2-dihydro-1-methanesulfonyl-spiro [3H-indole-3, 4'-piperidine] -1'-yl) carbonyl] -2 - (phenylmethyloxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate and methods for preparing these polymorphic forms.

Compound N- [1 (R) - (1,2-dihydro-1-methanesulfonyl-spiro [3H-indole-3,4'-piperidine] -1'yl) carbonyl] -2- (phenylmethyl-hydroxy) - ethyl] -2amino-2-methylpropanamide methanesulfonate has the structure:

and is a growth hormone secretion stimulator that induces the release of growth hormone in humans and animals. This property can be used to initiate the growth of meat animals in order to more efficiently produce meat food products, as well as to treat physiological and medical conditions in humans characterized by insufficient secretion of growth hormone and to treat medical conditions that are improved due to the anabolic effects of growth hormone.

Specific polymorphic forms (indicated here as Form I, Form II, Form III, Form IV, Form V, Form VI, Form VII, Form VIII, Form IX, Form X) have advantageous properties over other crystalline forms of the compound in that they are more convenient for inclusion in pharmaceutical compositions. The preferred crystalline form for pharmaceutical development is Form I, due to its thermodynamic stability and non-hygroscopic properties. Another preferred crystalline form for pharmaceutical development is Form IV, due to its preparative properties, in particular compressibility in the manufacture of tablets. Form IV was found to have a higher bulk density than other forms.

The present invention also relates to a process for the preparation of Form I N- [1 (R) - (1,2-dihydro-1-methanesulfonyl-spiro [3H-indole3,4'-piperidine] -1'-yl) carbonyl] -2- (phenylmethyloxy ) ethyl] -2-amino-2-methylpropanamide methanesulfonate, which includes the treatment of a solution of the free base N- [1 - (R) - (1,2-dihydro-1-methanesulfonyl-spiro [3H-indole-3, 4'- piperidine] -1'-yl) carbonyl] -2 (phenylmethyloxy) ethyl] -2-amino-2-methylpropanamide in ethyl acetate containing ethanol (about 8 volume%) methanesulfonic acid (about 1, 1 equivalent) at about 50 ° C heating approximately to 55 ° C and cooling to approximately 45 ° C.

Optionally, the temperature can be further increased to approximately 51 ° C, at which it is maintained for 2-24 hours.

The present invention further relates to an alternative method for preparing the form IN [1 (R) - (1,2-dihydro-1-methanesulfonylethyl] -2amino-2-methylpropanamide methanesulfonate, which comprises the alternative addition of N- [1 (R) free base to the solution - (1,2-dihydro-1-methanesulfonyl-spiro [3H-indole-3,4'-piperidine] -1'-yl) carbonyl] -2- (phenylmethyl-hydroxy) ethyl] -2-amino-2-methyl- propanamide in ethyl acetate containing ethanol (about 8 volume%) at about 50-55 ° C, methanesulfonic acid (about 1.1 equivalents) and form I N- [1 (R) - (1,2-di1tscro-1-methanesulfonyl -spiro [3H- indole-3,4'-piperidine] -1'5 yl) carbonyl] -2- (phenylmethyl-hydroxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate (where the relative order of addition is not critical), followed by heating approximately to 55 ° C in about 2-15 hours, cooling to about 25-30 ° C and aging for about 2-3 hours

The present invention further relates to an alternative method of obtaining the form IN [1 (R) - (1,2-dihydro-1-methanesulfonyl-spiro [3H-indole-3,4'-piperidine] -1'-yl) carbonyl] -2 (phenylmethyl-hydroxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate, which involves stirring a solution of Form II N [1 (R) - (1,2-dihydro-1-methanesulfonylspiro [3Nindole-3,4'-piperidine ] -1'-yl) carbonyl] -2- (phenylmethyl-hydroxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate in isopropanol at about 25 ° C for about 2-24 hours

The present invention also relates to a process for preparing Form II N- [1 (R) - (1,2-dihydro-1-methanesulfonyl-spiro [3H-indol-3, 4'-piperidine] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate, which includes treating the solution of the free base N- [1 (R) - (1,2-dihydro-1-methanesulfonyl-spiro [3H-indole-3,4 ' -piperidine] -1'-yl) carbonyl] -2 (phenylmethyl-hydroxy) ethyl] -2-amino-2-methylpropanamide in ethyl acetate containing ethanol (about 8 volume%), methanesulfonic acid (about 1.1 equivalents) approximately at 50 ° C, heating approx. about 55 ° С and cooling to ambient temperature.

The present invention further relates to a process for the preparation of Form IV N- [1 (R) - (1,2-dihydro-1-methanesulfonyl-spiro [3H-indol-3, 4'-piperidine] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate, which includes the dissolution of N- [1 (R) - (1,2-dihydro-1-methanesulfonyl-spiro [3H-indole-3,4'-piperidine] -1'yl) carbonyl] -2- (phenylmethyl-hydroxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate of undetermined morphological composition in an ethanol / water solution (in the preferred 25:75 v / v);

evaporation of the solvent from the solution, preferably at a temperature of 40 ° C;

grinding the resulting solid into a fine powder; and maintaining the fine powder at a relative humidity of approximately 75%.

The present invention further relates to an alternative process for the preparation of Form IV N- [1 (R) - (1,2-dihydro-1-methanesulfonyl-spiro 3H-indole-3,4'-piperidine] -1'-yl) carbonyl] - 2 (phenylmethyl-hydroxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate, which involves the recrystallization of N- [1 (R) - (1,2-dihydro 1-methanesulfonyl-spiro [3H-indole-3,4'- piperidine] -1'-yl) carbonyl] -2 - (phenylmethyl-hydroxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate of unspecified morphological composition from an ethyl acetate / ethanol / water solution (preferably 24.8 / 1.6 / 1 95 v / v / v).

The present invention further relates to an alternative process for the preparation of Form IV H- | (L) - (1.2-dihydro-1-methanesulfonyl-spiro [3H-indol-3,4'-piperidine] -1'-yl) carbonyl] -2 ( phenylmethyl-hydroxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate, which includes the aging of Form I N- [1 (R) - (1,2-dihydro-1-methanesulfonyl-spiro [3H-indole3,4'-piperidine] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate at a relative humidity of more than about 75%, preferably about 88%, at ambient temperature, over enough time.

The present invention further relates to an alternative process for the preparation of Form IV N- [1 (R) - (1,2-dihydro-1-methanesulfonyl-spiro [3H-indol-3,4'-piperidine] -1'-yl) carbonyl] -2 (phenylmethyl-hydroxy) -ethyl] -2-amino-2-methylpropanamide methanesulfonate, which involves the isolation from the suspension of Form I N- [1 (R) (1,2-dihydro-1-methanesulfonyl-spiro [3H-indole -3,4'-piperidine] -1'-yl) carbonyl] -2- (phenylmethyl-hydroxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate in a mixture of isopropyl acetate / ethanol (volume ratio 90:10) containing approximately 2.8 wt.% water at approximately 25 ° C.

The present invention further relates to a process for preparing Form V N- [1 (R) - (1,2-dihydro-1-methanesulfonyl-spiro [3H-indole-3, 4'-piperidine] -1'-yl) carbonyl] -2 - (phenylmethyloxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate, which includes the aging of Form IV N- [1 (R) - (1,2-dihydro-1-methanesulfonyl-spiro [3H-indole-3, 4'-piperidine] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate at a relative humidity lower than about 30% at ambient temperature.

The present invention further relates to a process for the preparation of Form VI N- [1 (R) - (1,2-dihydro-1-methanesulfonyl-spiro [3H-indole3,4'-piperidine] -1'-yl) carbonyl] -2- (amino -2 methyl propanamide methanesulfonate, which includes:

drying form V N- [I (R) - (1,2-dihydro-3-methanesulfonyl-spiro [3H-indol-3, 4'-piperidine] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] 2-amino-2-methylpropanamide methanesulfonate in the absence of moisture at approximately room temperature, such as, for example, in an atmosphere of nitrogen dried over molecular sieves at about 25 ° C.

The present invention further relates to a process for the preparation of Form VII N- [1 (R) - (1,2-dihydro-1-methanesulfonyl-spiro [3H-indole-3, 4'-piperidine] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate, which comprises isolating from a suspension of Form I or Form IV N- [1 (R) - (1,2-dihydro-1-methanesulfonylspiro [3H-indole-3, 4'-piperidine] -1'-yl) carbonyl] -2 (phenylmethyloxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate in isopropyl acetate / ethanol (volume ratio 90:10) containing approximately 1.5 wt.% water.

The present invention further relates to a process for the preparation of Form VIII N- [1 (R) - (1,2-dihydro-1-methanesulfonyl-spiro [3H-indol-3, 4'-piperidine] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate, which includes the dehydration of form VII N- [1 (R) - (1,2-dihydro-1-methanesulfonyl-spiro [3H-indole-3, 4'-piperidine] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate in an atmosphere of dry inert gas, such as dry nitrogen, for a sufficient time.

The present invention further relates to a process for the preparation of Form IX N- [1 (R) - (1,2-dihydro-1-methanesulfonyl-spiro [3H-indol-3, 4'-piperidine] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate, which includes obtaining a solution of ^ [1 Щ) - (1,2-dihydro1-methanesulfonyl-spiro [3H-indole-3,4'-piperidine] -1'- il) carbonyl] -2- (phenylmethyl-hydroxy) ethyl] 2-amino-2-methylpropanamide methanesulfonate of an undetermined morphological composition in water, followed by isolation of the resulting solid by controlled evaporation at 20%, moisture at approximately room temperature.

The present invention further relates to a method for preparing Form X N- [1 (R) - (1,2-dihydro-1-methanesulfonyl-spiro [3H-indol-3, 4'-piperidine] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate, which includes drying the form IX of N- [1 (R) - (1,2-dihydro-1-methanesulfonyl-spiro [3H-indole-3, 4'-piperidine] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate at approximately room temperature and humidity for a sufficient time.

The present invention further relates to an alternative process for the preparation of Form X N- [1 (R) - (1,2-dihydro-1-methanesulfonyl-spiro [3H-indol-3,4'-piperidine] -1'-yl) carbonyl] -2 (phenylmethyl-hydroxy) -ethyl] -2-amino-2methylpropanamide methanesulfonate, which includes the aging of Form I N- [1 (R) - (1,2-dihydro-1-methanesulfonyl-spiro [3H-indole-3, 4 ' piperidine] -1'-yl) carbonyl] -2 - (phenylmethyloxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate at 10% relative humidity for approximately 1 to 4 days.

In addition, the present invention also relates to a method for the preparation of morphologically homogeneous ^ [1CH) - (1,2-dihydro-1 methanesulfonyl-spiro [3H-indole-3,4'-piperidine] -1'-yl) carbonyl] -2- (phenylmethyl-hydroxy) ethyl] 2-amino-2-methylpropanamide methanesulfonate, which includes any of the methods mentioned here.

Compounds of the present invention, new polymorphic forms ^ [1 Щ) - (1,2-dihydro1-methanesulfonyl-spiro [3H-indole-3,4'-piperidine] -1'-yl) carbonyl] -2- (phenylmethyl-hydroxy) ] ethyl] -2-amino-2-methylpropanamide methanesulfonate, are growth hormone secretion stimulants that are useful for administration to meat animals to initiate their growth, thereby contributing to more efficient production of meat food products, as well as to people for treating physiological and medical conditions characterized by growth hormone secretion deficiency as well as for the treatment of medical conditions that improve due to the anabolic effects of growth hormone. Accordingly, the present invention further relates to pharmaceutical compositions comprising a polymorphic form as an active ingredient and to the use of such polymorphic forms and compositions containing them for the treatment of certain disorders.

Differential Scanning Calorimentic Cell (DSC)]

DSC curve for Form I N- [1 (R) - (1,2-dihydro-1-methanesulfonyl-spiro [3H-indole-3, 4'-piperidine] -1'-yl) carbonyl] -2 - (phenylmethyloxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate at 10 ° C / min in an open cell in a stream of nitrogen represents a single melting endotherm, with a peak temperature of approximately 180 ° C and an extrapolated initial temperature (melting point) of approximately 170 ° C, corresponding to heat of approximately 53 J / g.

DSC curve for Form II N- [1- (R) - (1,2-dihydro-1-methanesulfonyl-spiro [3H-indol-3, 4'-piperidine] -1'-yl) carbonyl] -2- (phenylmethyloxy ) ethyl] -2-amino-2-methylpropanamide methanesulfonate at 10 ° C / min in an open cell in a stream of nitrogen represents a single melting endotherm, with a peak temperature of approximately 1 74 ° C and an extrapolated initial temperature (melting point) of approximately 165 ° C corresponding to a heat of approximately 37 J / g.

DSC curve for Form IV N- [1- (R) - (1,2-dihydro-1-methanesulfonyl-spiro [3H-indole-3, 4'-piperidine] -1'-yl) carbonyl] -2- (phenylmethyloxy ) -ethyl] -2-amino-2-methylpropanamide methanesulfonate at 10 ° C / min in an open cell in a nitrogen stream represents the endotherm of water loss at approximately 45 ° C, followed by an endotherm with a peak temperature of approximately 134 ° C and an extrapolated initial temperature (melting point) approximately 129 ° C, melting form VI corresponding heat of approximately 23 J / g

DSC data [samples are heated at a rate of 10 ° C / min in a nitrogen atmosphere (extrapolated initial temperature)]:

Form I: 170 ° C (melting endotherm)

Form II: 165 ° C (melting endotherm)

Form VI: 129 ° C (melting endotherm)

Form I N- | 1 (L) - (1.2-dihydro-1-methanesulfonyl-spiro [3H-indole-3,4'-piperidine] -1'-yl) carbonyl] -2- (phenylmethyl-hydroxy) ethyl] -2-amino 2-methylpropanamide methanesulfonate is a relatively anhydrous polymorph. characterized by the following properties: so pl. 169 ° C and solubility in isopropanol 4.6 mg / ml.

Form II N ₄ 1 (R) - (1.2-dihydro-1-methanesulfonyl-spiro [3H-indole-3. 4'-piperidine] -1'carbonyl] -2- (phenylmethyl-hydroxy) ethyl] -2-amino -2-methylpropanamide methanesulfonate is an anhydrous polymorph characterized by the following properties; mp 158 ° C and solubility in isopropanol 12.3 mg / ml.

Form III N - [(R) - (1.2-dihydro-1-methanesulfonyl-spiro [3H-indole-3,4'-piperidine] -1 'yl) carbonyl] -2- (phenylmethyl-hydroxy) ethyl] -2 amino-2-methylpropanamide methanesulfonate is a hydrate. characterized by the following properties: endoteroma water loss with a peak temperature of 46 ° C. followed by a slight melting / decomposition endotherm with an extrapolated initial temperature of 123 ° C.

Form IV N ₄ 1- (R) - (1.2-dihydro-1-methanesulfonyl-spiro [3H-indole-3,4'-piperidine] -1 'yl) carbonyl] -2- (phenylmethyl-hydroxy) ethyl] - 2-amino-2-methyl-propanamide methanesulfonate is a hydrate. characterized by the following properties: a water loss endotherm with a peak temperature of 45 ° C, followed by a melting / decomposition endotherm with an extrapolated initial temperature of 129 ° C (presumably, melting / decomposition of form VI).

Form IV N ₄ 1 (R) - (1.2-dihydro-1-methanesulfonyl-spiro [3H-indole-3,4'-piperidine] -1 'yl) carbonyl] -2- (phenylmethyl-hydroxy) ethyl] -2 -amino-2-methylpropanamide methanesulfonate manifests itself as a hygroscopic hydrate. containing 3.5 moles of water per mole of N- [1 (R) - (1.2 dihydro-1-methanesulfonyl-spiro [3H-indol-3. 4'-piperidine] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl ] -2-amino-2-methylpropanamide methanesulfonate.

Form VN ₄ 1 (R) - (1.2-dihydro-1-methanesulfonyl-spiro [3H-indole-3.4'-piperidine] -1'yl) carbonyl] -2- (phenylmethyl-hydroxy) ethyl] -2-amino 2-methylpropanamide methanesulfonate manifests itself as a hygroscopic hydrate. containing 1 mol of water per mole of N- [1 (R) - (1.2 dihydro-1-methanesulfonyl-spiro [3H-indole-3. 4'-piperidine] -1'-yl) carbonyl] -2 - (phenylmethyloxy) - 2-amino-2-methylpropanamide methanesulfonate.

Form VI N ₄ 1 (L) - (1.2-dihydro-1-methanesulfonyl-spiro [3H-indole-3.4'-piperidine] -1'yl) carbonyl] -2- (phenylmethyl-hydroxy) ethyl] -2-amino -2-methylpropanamide methanesulfonate manifests itself as an anhydrous polymorph and is characterized by a melting point of 129 ° C.

Form VII ^ [1CH) - (1.2-dihydro-1 methanesulfonyl-spiro [3H-indole-3.4'-piperidine] -1'-yl) carbonyl] -2- (phenylmethyl-hydroxy) ethyl] 2-amino-2-methylpropanamide methanesulfonate is a hydrate. characterized by the following properties: wide endotherm of water loss with a peak temperature of 60 ° C. followed by a melting / decomposing endotherm with an extrapolated initial temperature of 144 ° C (presumably. melting / decomposing form VIII).

Form VIII N ₄ 1 (R) - (1.2-dihydro-1-methanesulfonyl-spiro [3H-indole-3.4'-piperidine] -1'yl) carbonyl] -2- (phenylmethyl-hydroxy) ethyl] -2-amino -2-methylpropanamide methanesulfonate is an anhydrous polymorph. characterized by so pl. 1 44 ° C.

Form X N ₄ 1 (R) - (1.2-dihydro-1-methanesulfonyl-spiro [3H-indole-3.4'-piperidine] -1'yl) carbonyl] -2- (phenylmethyl-hydroxy) ethyl] -2-amino -2-methylpropanamide methanesulfonate is characterized by a wide endotherm of water loss with a peak temperature of 49 ° C.

X-ray Powder Diffraction [XRay Powder Diffraction (XRPD)]

X-ray powder diffraction studies are widely used to establish molecular structure. crystallinity and polymorphism. X-ray powder diffraction (XRPD) diagrams were recorded using a Philips APD3720 Automated Powder Diffraction instrument with a copper source of Ka radiation. The measurements were carried out from 2 to 40 ° (2 theta) with the sample. maintained at ambient temperature.

Form I was characterized by an X-ray powder diffraction pattern with basic reflections at approximately: 6.5; 14.7; 16.9; 17.1; 17.9; 19.5; 21.1; 21.7 and 22.0 ° (2 theta).

Form II was characterized by an X-ray powder diffraction pattern with basic reflections at approximately: 4.8; 11.8; 17.5; 19.4; 21.6; 21.9; 22.5 and 22.7 ° (2 theta).

Form III was characterized by an X-ray powder diffraction pattern with basic reflections at approximately: 13.8; 14.1; 18.0; 18.8; 19.5; 20.1; 20.6; 21.8 and 25.7 ° (2 theta).

Form IV was characterized by an X-ray powder diffraction pattern with basic reflections at approximately: 16.0; 16.2; 18.3; 20.1; 21.0 and 24.2 ° (2 theta).

Form V was characterized by an X-ray powder diffraction pattern with basic reflections at approximately: 14.8; 17.1; 17.6; 19.0; 19.1; 19.4; 20.6; 21.5 and 21.8 (2 theta).

Form VI was characterized by an X-ray powder diffraction pattern with basic reflections at approximately: 9.8; 14.0; 14.8; 17.1; 17.6; 19.0; 19.5; 20.6 and 21.6 ° (2 theta).

Form VII was characterized by an X-ray powder diffraction chart with basic reflections at approximately: 9.1; 11.3; 17.1; 17.4; 20.0; 21.1 and 24.5 ° (2 theta).

Form VIII was characterized by an X-ray powder diffraction chart with basic reflections at approximately: 11.5; 11.6; 18.1; 19.6; 22.5; 24.7 and 24.8 ° (2 theta).

Form IX was characterized by an X-ray powder diffraction chart with basic reflections at approximately: 8.0; 12.1; 15.3; 15.8; 19.6; 19.7; 21.1; 22.3 and 23.7 ° (2 theta).

Form X was characterized by an X-ray powder diffraction pattern with basic reflections at approximately: 15.5; 15.8; 18.0; 18.4; 18.6; 19.4; 20.7; 20.8; 23.9 and 24.8 ° (2 theta).

These XRPD diagrams confirm that all forms IX are different crystalline forms.

Microscopy

The study of polymorphic forms was carried out at 100x magnification in ordinary and polarized light. Form I and form II were needle-shaped particles. Both Form I and Form II were birefringent in polarized light.

Hygroscopicity

The total content of volatile components (established by TGA analysis) in solid samples of forms I, II, III and IV after exposure to various controlled humidity conditions is shown in the table below. It was found that Form I contains 0.79 wt.% Water; Form II contains 0.56 wt.% water; Form III contains 4.5-5.0 wt.% water; and Form IV contains 9.510.0 wt.% water.

Hygroscopicity was evaluated by storing the solid compound in chambers with constant relative humidity. A comparison of anhydrous forms I and II at room temperature showed that form II is hygroscopic and exhibits a large increase in humidity, starting from 65% Ob [relative humidity]. Form I does not show any significant increase in humidity, except when stored at a humidity above 76% Ob. The results are presented below in tables 1 and 2.

Table 1

Room temperature (48 h)

Form I Form II % OB % gain / loss % gain / loss 0 -0.02 +0.08 eleven -0.05 +0.02 33 - +0.33 47 +0.21 +0.39 65 +0.37 > 10.0 ^a 76 +0.12 > 10.0 ^a one hundred > 12.0 > 10.0 ^a ' ^b

(^ the sample becomes semi-solid, like rubber) ( ^{1 :,} = goes into form IV after aging at ambient humidity)

table 2

Room temperature (96 h)

Form I % OB % gain / loss 7 +0.4 22 +0.3 47 +0.5 68 +0.6 88 + 12.9 * one hundred + 23.9 *

(* = moisture absorbing and liquefying material)

Hydrated Forms III and IV VIII N [1 (R) - (1,2-dihydro-1-methanesulfonyl-spiro [3H-indole-3,4'-piperidine] -1'-yl) carbonyl] -2 (phenylmethyl- hydroxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate was also evaluated when stored in relative humidity chambers for 48 hours at room temperature. Form III shows an increase in humidity after 48 hours storage at 33% Ob. Form IV, although it does not add a significant amount of moisture before storing it at 1 00% RH, loses its hydration water when stored at 11% RH or lower. The results are presented below in table 3.

Table 3

Room temperature (48 h)

Form III Form IV % OB - -7.8 eleven -0.15 -4.9 33 +2.23 +0.3 47 +3.99 +0.7 66 - +1.4 76 +3.76 +1.5 one hundred - +5.3

These data indicate that Form I is relatively anhydrous.

Solubility

The solubility of Form I in distilled water at room temperature is> 100 mg / ml. Solubility in water (room temperature) of form II in buffered solutions (pH 4-9):> 100g / ml. The solubility of form I in ethanol / water mixtures is shown below:

Solubility % Ethanol / N ₂ O (mg / ml) 25/75 > 100 50/50 > 100 75/25 > 90 100% ethanol > 90

Thermal Stability - Pure Compound

The stability of the solid state of the pure compound was evaluated during storage of the drug in test tubes with a screw cap in the dark. Samples were analyzed by HPLC and the base material was quantified. The following are the conditions of the applied isocratic method:

Column: Beckman Ultrasphere ODS (250x4.6 mm, 5 μ)

Mobile phase 0.1% TEA, pH 4.0 s HzPO4: Acetonitrile (65:35) Flow rate 1.0 ml / min Detection Wavelength: 228 nm Travel time 14 min Column temperature Ambient temperature Injection volume 20 μl N- [1 (R) - (1,2-dihydro-1 - methanesulfonyl-spiro [3H-indol-3,4'-piperidine] -1'-yl) carbonyl] -2- (phenylmethyl-hydroxy) ethyl-2amino-2-methylpropanamide methanesulfonate (50 μg / ml)

The results below for forms I and II are calculated in weight percent.

Form I

% source ° C 6 weeks 17 weeks 40 100.1 98.7 60 100.7 101.3 80 100.7 99,4

Form II

% Original ° C Week 1 2 weeks 4 weeks 8 weeks 12 weeks 24 weeks 40 99.7 100.7 99.5 - 100.1 100.1 60 99,4 99.6 100.3 100.0 101.0 100.8 80 99,2 100.3 99,2 99.8 100,4 -

These results show that pure solid form I and form II have good thermal stability.

Methods of obtaining the compounds of the present invention are presented below.

Scheme I

As shown in Scheme I, before hydrogenation, CBZ-spiroindoline 1 is treated with Darco (20% by weight). Hydrogenation is carried out in ethanol at 65 ° C over 10% Pd / C with vigorous stirring.

A solution of 1 b in isopropyl acetate and water is combined with commercially available H-BOC-0 benzyl ^ -serine in the presence of dicyclohexylcarbodiimide (DCC) and 1-hydroxybenzotriazole (HOBt). After filtering the by-product of dicyclohexylurea (DCU), the 2-phase filtrate was separated and the organic layer was washed successively with a 1 M aqueous solution of sodium hydroxide, a 0.5 M aqueous solution of hydrochloric acid and, finally, a saturated aqueous solution of sodium bicarbonate. The best results of this combination are achieved when a solution of the free amine in iPrOAc / H ₂ O is treated with DCC, HOBT, followed by the addition of amino acids at ambient temperature and subsequent reaction for 3-5 hours. The reaction mass is then concentrated in vacuo and the solvent is replaced with sisopropyl acetate ethanol. The replacement of the solvent is carried out quickly by feeding and flowing out three times the volume of the reaction mixture to remove isopropyl acetate.

The BOC group of compound 11 is removed by treatment with methanesulfonic acid (MsOH) (3 equiv.) In ethanol at 35-40 ° C. The separation between isopropyl acetate and an aqueous 1M sodium hydroxide solution gives 1 2.

The combination of 12 with N-BOC-a-aminoisobutyric acid is preferably carried out in a two-phase solvent system of isopropyl acetate / water (1: 1) in the presence of DCC and HOBt (1.1 eq. Each). Removal of DCU by filtration, separation of the layers, and washing of the organic layer sequentially with a 1 M aqueous solution of sodium hydroxide, 0.5 M aqueous solution of hydrochloric acid, and saturated aqueous sodium bicarbonate gave 1 4.

In the mixture, the solvent is replaced with ethanol for subsequent cleavage of the BOC group with methanesulfonic acid. Removal of protection 1 4 is more difficult than that of 11 and requires a concentrated solution of ethanol / methanesulfonic acid and heating to 35-40 ° C. After extraction (EtOAc-NaOH), free amine 15 is isolated. The organic layer is washed well with 1 N NaOH to ensure complete removal of methanesulfonic acid.

A solution of ethyl acetate free base 1 5 is concentrated to a small volume in vacuum and azeotroped to dryness (KF <500 mg / ml ^-1 ) by the method of feeding and flowing with 2 loading volumes of a mixture of ethyl acetate / ethanol in a ratio of 90/10 and then with 2 loading volumes of ethyl acetate. The resulting dry, slightly hazy solution of free base 1 5 in ethyl acetate was treated with Darco G-60 (25% by weight) at room temperature for about 10 hours. Removal of Darco by filtration with a filtering agent gave free base 15.

The formation of methanesulfonic acid salt 16 of 15 is carried out in EtOAc with 1.1 equiv. MsOH at approximately 50 ° C. The free base 15 is treated with 8 vol.% EtOH and 1 equiv. H ₂ O and heated to 55 ° C until complete dissolution. Cooling to ambient temperature and stirring the resulting suspension for 4 h gives crystalline material 1 6, designated crystalline form II [solubility in IPA = 12 μg / ml].

The conversion of form II to form I is achieved when a salt is formed in EtOAc-EtOH as described above, but instead of cooling the initial salt solution (at 55 ° C) to ambient temperature, it is cooled to 45 ° C. At this temperature, crystals should begin to appear, and the suspension should become thicker over time. The temperature is then raised to 51 ° C. and the suspension is aged overnight. It is assumed that a complete conversion of 1 6 into Form I must undergo.

Preferably, the conversion of form II to form I is achieved by adding seed crystals of form I to a solution of the free base in EtOAc-EtOH at 50-55 ° C. followed by aging. Accordingly, free base 15 can be treated with 1.1 equivalents of methanesulfonic acid in 8% ethanol in ethyl acetate at 50-55 ° C. The reaction mass is then affected with approximately 2% by weight of Form I of the methanesulfonic salt 16, and then aged at 55 ° C. overnight. The reaction mass is cooled to room temperature and aged for approximately 2-3 hours. The product is isolated by filtration at room temperature in a nitrogen atmosphere, dried at 35 ° C in vacuo, sieved and methanesulfonic salt 16 is obtained.

The methanesulfonic acid salt 1-6 can also be formed by alternative stepwise addition of MsOH (1.1 equiv.) And seed crystals of Form I to a solution of the free base in EtOAc-EtOH at about 50 ° C, where the order of addition of MsOH and seed is not critical.

The usefulness of the polymorphic compounds of the present invention as stimulators of growth hormone secretion can be demonstrated by methods known in the art, such as, for example, the studies described by Smith et al., Science, 260, 1640-1643 (1993) (see text to Fig. 2 in this work). In particular, all polymorphic forms of the present invention had activity as growth hormone secretion stimulators in the above text. This result is an indicator of the truly existing activity of the polymorphic forms of the present invention as stimulators of growth hormone secretion.

Growth hormone releasing compounds of the present invention can be used in vitro as unique tools for understanding how growth hormone secretion is regulated at the pituitary [pituitary] level. This analysis involves the use in their assessment of many factors that are studied or known as affecting the secretion of growth hormone, such as age, gender, nutrition factors, glucose, amino acids, fatty acids, as well as conditions during fasting and non-fasting. In addition, the compounds of the present invention can be used to evaluate how other hormones modulate growth hormone release activity. For example, it has already been established that somatostatin inhibits the release of growth hormone. Other hormones that are important and need to be studied with respect to their effects on the release of growth hormone include gonadal hormones such as testosterone, estradiol and progesterone; hormones of the adrenal cortex, for example cortisol and other corticoids, epinephrine and norepinephrine; pancreatic and gastrointestinal hormones, for example, insulin, glucagon, gastrin, secretin; vasoactive peptides, such as bombesin, neurokinins; thyroid hormones, for example thyroxine and triiodothyronine. The compounds of the present invention can also be used to study the possible effects of the negative or positive feedback of certain pituitary [pituitary] hormones, for example, growth hormone and endorphin peptides, on the pituitary gland to modulate the release of growth hormone. Of particular scientific importance is the use of these compounds to elucidate subcellular mechanisms mediating the release of growth hormone.

The compounds of the present invention can be administered to animals, including humans, to release growth hormone in vivo. For example, the compounds may be administered to commercially significant animals, such as pigs, cattle, sheep, and the like, to increase growth rate and increase their size, to improve the efficiency of feeding and increase the milk productivity of such animals. In addition, these compounds can be administered to humans in vivo as diagnostic tools to directly determine whether the pituitary gland releases growth hormone. For example, the compounds of the present invention may be administered in vivo to children. Serum samples taken before and after such administration can be tested for growth hormone content. Comparison of the amounts of growth hormone in each of these samples allows you to directly determine the ability of the patient's pituitary to release growth hormone.

Accordingly, the present invention includes its scope in pharmaceutical compositions comprising, as an active ingredient, at least one of the compounds of the present invention in combination with a pharmaceutical carrier or solvent.

Optionally, the active ingredient in the pharmaceutical compositions may include an anabolic agent in addition to at least one of the compounds of the present invention or another composition that has a different activity, such as an antibiotic growth factor or an agent for treating osteoporosis, or used in combination with corticosteroids to minimize catabolic side effects or with other pharmaceutically active agents in cases where this combination increases effectiveness and minimizes side effects you.

Growth stimulants and anabolic agents include, but are not limited to, TRH, diethylstilbesterol, estrogens, (β-agonists, theophylline, anabolic steroids, enkephalins, E-series prostaglandins, compounds disclosed in US patent No. 3,239,345, for example, zeranol and compounds, disclosed in US Pat. No. 4,036,979, for example, sulbenox or peptides disclosed in US Pat. No. 4,411,890.

Another use of the compounds of this invention is their use in combination with other growth hormone secretion stimulants, such as peptides releasing growth hormone GHRP-6, GHRP-1, described in US patent Nos 4,411,890 and in publications WO 89/07110, WO 89 / 07111 and B-HT920, as well as hexarelin and GHRP-2, as described in WO 93/04081 or growth hormone releasing hormone (GHRH, denoted as GRF) and its analogues, either growth hormone and its analogues, or somatomedins, including IGF-1 and IGF-2, or α-adrenergic agonists, such as clonidine or serotonin 5HTID agonists, such as umitriptan, or agents that inhibit somatostatin or its release, such as physostigmine and pyridostigmine. In particular, the compounds of this invention can be used in combination with a growth hormone release factor, an analogue of a growth hormone release factor, IGF-1 and IGF-2. For example, the compound of the present invention can be used in combination with IGF-1 to treat or prevent obesity. In addition, the compound of the present invention can be used in combination with retinoic acid to improve the muscles and skin associated with the aging process.

The present invention also relates to a method for producing a medicament for stimulating the release of growth hormone in humans and animals, comprising combining a polymorphic form of the present invention with a pharmaceutical carrier or solvent.

As is well known to specialists, the known and potential possibilities of using growth hormone are diverse and numerous. Administration of the compounds of this invention in order to stimulate the release of endogenous growth hormone can have the same results and beneficial effects as those of growth hormone itself. The various uses of the present compounds can be summarized as follows: stimulating the release of growth hormone in the elderly; treatment of adults with growth hormone deficiency; prevention of catabolic side effects of glucocorticoids; treatment of osteoporosis; stimulation of the immune system, accelerating wound healing; acceleration of fusion of bone fractures; growth retardation treatment; treatment of acute and chronic renal failure; treatment of physiological short stature, including children with growth hormone deficiency; treatment of short stature associated with a chronic disease; treatment of obesity and stunting associated with obesity; treatment of growth retardation associated with Prader-Willi syndrome and Turner'a syndrome; accelerated recovery and reduced hospitalization of patients with burns or after surgical operations such as gastrointestinal surgery; treatment of intrauterine growth retardation and skeletal dysplasia, treatment of peripheral neuropathies; replenishment of growth hormone in patients with stressful conditions; treatment of osteochondrodysplasias, Noonan's syndrome, schizophrenia, depression, Alzheimer's disease, delayed wound healing, as well as psychosocial deprivation; treatment of pulmonary dysfunction and ventilatory dependence; attenuation of a protein catabolic reaction after major operations; treatment of malabsorption syndromes; reduced cachexia and protein loss due to chronic diseases such as cancer or AIDS; acceleration of weight gain and protein buildup in patients with full parenteral nutrition [TPN (total parenteral nutrition)]; treatment of hyperinsulinemia, including nezidioblastosis (pancreatic islet hyperplasia); induced ovulation and the prevention and treatment of gastric and duodenal ulcers; stimulating the development of thymus and preventing age-related decline in thymic function; additional therapy in the treatment of patients with chronic hemodialysis; treatment of immunocompromised patients and activation of antibody response after vaccination; an increase in the total number of lymphocytes in people, in particular, an increase in the ratio of T ₄ / T ₈ cells in the body of people with an inhibited ratio of T4 / T8 cells as a result of, for example, physical trauma, such as a closed head injury, or as a result of a bacterial infection or viral, especially in the case of infection with the human immunodeficiency virus; increase in muscle strength, mobility, maintenance of skin thickness, metabolic homeostasis, renal homeostasis in decrepit elderly people; stimulation of osteoblasts, bone reconstruction, cartilage growth; stimulation of the immune system in pets and treatment of aging disorders in pets; stimulation of livestock growth; stimulating the growth of wool in sheep. Further, the compounds are useful for increasing feed efficiency, enhancing growth, increasing milk production and improving the quality of carcasses. Similarly, the compounds of the present invention are useful in a method of treating diseases or conditions that are improved by the anabolic effects of elevated levels of growth hormone, which includes the administration of the compounds of the present invention.

In particular, the compounds are useful for preventing or treating conditions including osteoporosis; catabolic disease; immunodeficiency, including that in individuals with a suppressed ratio of T4 / T8 cells; hip fracture; musculoskeletal disorders in the elderly; growth hormone deficiency in adults and children; obesity; cachexia and protein loss associated with chronic diseases such as AIDS or cancer; for the treatment of patients recovering from major surgeries, wounds or burns.

In addition, the compounds of the present invention may be useful for the treatment of diseases caused or made possible by the action of corticotropin-releasing factor or disorders associated with stress or anxiety, including stress-induced depression and headache, abdominal intestinal syndrome, immunosuppression, HIV infection, Alzheimer's disease, gastrointestinal disease, anorexia nervosa, hemorrhagic stress, symptoms of alcohol and drug withdrawal, drug dependence, problems f rtilnosti [ability to bear children].

It is well known to those skilled in the art that there are numerous compounds currently used to treat diseases or with the therapeutic indications listed above. Combinations of these therapeutic agents, some of which have also been mentioned above together with the growth hormone secretion stimulants of the present invention, will introduce additional, complementary and often synergistic properties that enhance the growth promoting, anabolic and desirable properties of these various therapeutic agents. In these combinations, therapeutic agents and growth hormone secretion stimulators of the present invention may independently be present in a dose ranging from one hundredth to one dose, which are effective when these compounds and secretion stimulants are used separately.

Combination therapy aimed at inhibiting bone resorption, preventing osteoporosis and accelerating the healing of bone fractures can be illustrated by combinations of bisphosphonates and growth hormone secretion stimulators of the present invention. The use of bisphosphonates for these purposes is described, for example, in Hamdy, N.A.T., Role of Biphosphonates in Metabolic Bone Diseases, Trends in Endocrinol. Metab., 4, 19-25 (1993). Bisphosphonates useful for these purposes include alendronate, tilundronate, dimethylAPD, risedronate, ethidronate, YM-175, clodronate, pamidronate and BM-210995. Accordingly, their activities, oral daily doses of bisphosphonate, comprising from 0.1 to 5 g and oral daily doses of growth hormone secretion stimulants of the present invention, comprising from 0.01 mg / kg to 20 mg / kg of body weight, are administered to patients for the effective treatment of osteoporosis.

In the case of alendronate, oral daily doses of 0.1 mg to 50 mg are combined, for the effective treatment of osteoporosis, with growth hormone secretion stimulants of the present invention in an amount of 0.01 mg / kg to 20 mg / kg. Osteoporosis and other bone disorders can also be treated with the compounds of this invention in combination with calcitonin, estrogens, raloxifene, and calcium sources such as calcium citrate.

Anabolic effects, especially in the treatment of male geriatric patients, are obtained using the compounds of this invention in combination with anabolic steroids such as oxymetholone, methyltestosterone, fluoxymesterone and stanozolol.

The compounds of this invention can be administered orally, parenterally (for example, in the form of intramuscular, intraperitoneal, intravenous or subcutaneous injection, or by implantation), intranasal, intravaginal, intrarectal, sublingual or local route and can be formulated in dosage forms suitable for each method of administration.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound is mixed with at least one inert, pharmacologically acceptable carrier, such as sucrose, lactose or starch. Such dosage forms may also include commonly used additional substances that are not inert solvents, for example, lubricants, such as magnesium stearate. In the case of capsules, tablets and pills, dosage forms may also include buffering agents. Tablets and pills can also be made with an enteric coating.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, elixirs containing inert solvents commonly used in practice, such as water. In addition to such inert solvents, the compositions may also include adjuvants, such as wetting agents, emulsifying and suspending agents, as well as sweetening, flavoring and flavoring agents.

According to this invention, compositions for parenteral administration include sterile aqueous and non-aqueous solutions, suspensions or emulsions. Examples of non-aqueous solvents or carriers are propylene glycol, polyethylene glycol, vegetable oils such as olive and corn oil, gelatin, as well as injectable organic esters such as ethyl oleate. Such dosage forms may also contain adjuvants, such as preserving, wetting, emulsifying and dispersing agents. They can be sterilized, for example, by filtration through a bacteria-retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating them. They can also be made in the form of sterile solid compositions which can be dissolved in sterile water or in another sterile injectable medium immediately before use.

Compositions for rectal or vaginal administration are predominantly suppositories, which may contain, in addition to the active substance, excipients such as cocoa butter or suppository wax.

Compositions for intranasal and sublingual administration are also prepared with standard excipients well known in the art.

The compound of this invention may be administered to a patient (animal or human) in need of such treatment in doses that will provide optimal pharmaceutical efficacy. It is desirable that the dose required for use in any particular application varies from patient to patient, depending not only on the choice of a particular compound or composition, but also on the route of administration, the nature of the condition being treated, the age and condition of the patient, concomitant treatment or a special diet followed by the patient, as well as other factors known to those skilled in the art, so that the attending physician will ultimately be able to choose the appropriate dose.

The dose of the active ingredient in the compositions of this invention may vary; however, it is necessary that the amount of the active ingredient be such that a suitable dosage form is obtained. The dose chosen depends on the desired therapeutic effect, the route of administration and the duration of treatment. Typically, levels from 0.0001 to 10 mg / kg body weight per day are administered to patients and animals, such as mammals, to obtain effective release of growth hormone. Preferably, the level is from about 0.001 to 25 mg / kg per day; more preferably about 0.01 to 10 mg / kg per day.

Methods for producing polymorphic forms of the present invention are illustrated in the following examples. The following examples are given to illustrate the present invention and should not be construed as limiting the scope of this invention.

Example 1

N- [1 (R) - (1,2-Dihydro-1-methanesulfonylspiro [3H-indole-3,4'-piperidine] -1'-yl) carbonyl] -2 - (phenylmethyl-hydroxy) ethyl] -2 amino-2 methylpropanamide.

Stage A. 1,2-Dihydro-1-methanesulfonylspiro [3H-indole-3,4'-piperidine] hydrochloride.

To a solution of 1.20 g (5.8 mmol) of 1'-methyl1,2-dihydro-spiro [3H-indole-3,4'-piperidine] (prepared as described by H. O ng, et al., J. Med. Chem., 23, 981-986 (1983), triethylamide (0.90 ml; 6.4 mmol) and methanesulfonyl chloride (0.49 ml; 6.35 mmol) were added to 20 ml of dry dichloromethane at 0 ° C. and stirred for 30 minutes, the Reaction mixture was poured into 15 ml of a saturated aqueous solution of sodium bicarbonate and was extracted with dichloromethane (2 × 10 ml). The combined organic layers were washed with brine (20 ml), dried over anhydrous potassium carbonate, filtered and the solvent removed under reduced pressure to obtain 1.44 g of the methanesulfonamide derivative as pale yellow oil which was used without further purification.

To a solution of the crude product in 20 ml of 1,2-dichloroethane at 0 ° C was added 1.0 ml (9.30 mmol) of 1-chloroethyl chloroformate and then stirred at room temperature for 30 minutes and finally at reflux for 1 hour The reaction mixture was concentrated to approximately one third of the volume and then diluted with 20 ml of dry methanol and refluxed for 1.5 hours. The reaction mixture was cooled to room temperature and concentrated to approximately half the volume. The precipitate was filtered and washed with a small volume of cold methanol. 1.0 g of HO piperidine salt was obtained as a white solid. The filtrate was concentrated and a small volume of methanol was added, followed by ether. The precipitated material was again filtered, washed with cold methanol and dried. Additionally received 0.49 g of the desired product. The total yield was 1.49 g (70%).

¹ H NMR (CDCl ₃ , 200 MHz) δ 7.43-7.20 (m, 3H), 7.10 (dd 1H), 3.98 (br s, 2H), 3.55-3.40 (broad dd, 2H), 3.55-3.10 (m, 2H), 2.99 (s, 3H), 2.15 (t, 2H), 2.00 (t, 2H).

Stage B. M1SC) - [(1,2-Dihydro-1-methanesulfonyl-spiro [3-indole-3,4'-piperidine] -1'yl) carbonyl] -2- (phenylmethyl-hydroxy) ethyl] -2 - [(1,1dimethylethoxy) carbonyl] amino-2-methylpropanamide.

To 0.35 g (1.15 mmol) ^) - 2 - [(1,1-dimethylethoxy) carbonyl] amino-3- [2- (phenylmethyloxy) ethyl] -1-propanoic acid in 13 ml of dichloromethane was added 1, 2-dihydro-1-methanesulfonylspiro [3H-indole-3,4'-piperidine] hydrochloride (0.325 g, 1.07 mmol), 0.18 ml (1.63 mmol) of N-methylmorpholine, 0.159 g (1.18 mmol) 1hydroxybenzotriazole (HOBT) and stirred for 15 minutes. EDC (0.31 g, 1.62 mol) was added and stirring was continued for 1 hour. An additional 60 μl of N-methylmorpholine was added and stirred for 45 minutes. The reaction mixture was poured into 5 ml of water and the organic layer was separated. The organic layer was washed with 5 ml of a 0.5N aqueous hydrochloric acid aqueous solution and 5 ml of a saturated aqueous sodium bicarbonate solution. The combined organic layers were dried over anhydrous magnesium sulfate and concentrated to obtain 0.627 g of the product as a yellow foam, which was used further without purification.

To 0.627 g (1.07 mmol) of the above product in 5 ml of dichloromethane was added 1.0 ml of trifluoroacetic acid and stirred at RT for 75 minutes. An additional 1.00 ml of trifluoroacetic acid was added and stirred for 10 minutes. The reaction mixture was concentrated, diluted with 5 ml of dichloromethane and carefully made alkaline by pouring in 10 ml of a 10% aqueous solution of sodium carbonate. The organic layer was separated and then the aqueous layer was extracted with 2x15 ml of dichloromethane. The combined organic layers were washed with 5 ml of water, dried over potassium carbonate, filtered and concentrated to give 0.486 g of the amine as a light yellow foam, which was used further without purification.

To 0.486 g (1.01 mmol) of amine and 10 ml of dichloromethane was added 0.26 g (1.28 mmol) of 2 [(1,1-dimethylethoxy) carbonyl] amino-2-methylpropanoic acid, 0.173 g (1.28 mmol) ) 1hydroxy-benzotriazole (HOBT) and EDC (0.245 g, 1.28 mol) and stirred at RT overnight. The reaction mixture was poured into 5.0 ml of water and the organic layer was separated. The aqueous layer was back extracted with 5 ml of dichloroethane. The combined organic layers were washed with 5.0 ml of a 0.5N aqueous hydrochloric acid solution, 5 ml of a saturated aqueous sodium bicarbonate solution, dried over anhydrous magnesium sulfate and concentrated to obtain 0.751 g of a crude product as a yellow foam. A solution of the crude product in dichloromethane was chromatographed over 25 g of silica gel and eluted first with hexane / acetone / dichloromethane (65/30/5). 0.63 g of the title compound was obtained as a white solid.

¹ H NMR (СОС1 ₃ , 400 MHz). The compound exists as a mixture of 3: 2 rotamers δ 7.40-7.10 (m, 6H), 7.06 (d, 1 / 3H), 7.02 (t, 1 / 3H), 6.90 (t, 1 / 3H) ^- , 6.55 (d, 1 / 3H), 5.15 (m, 1H), 4.95 (br.s., 1H), 4.63 (br.d, 1 / 3H) 4.57-4.40 (m, 2 2 / 3H), 4.10 (br d, 1 / 3H), 4.00 (br d, 1 / 3H), 3.82 (t, 1H ), 3.78-3.62 (m, 2H), 3.60-3.50 (m, 1H), 3.04 (q, 1H), 2.87 (s, 1H), 2.86 ( s, 2H), 2.80-2.60 (m, 1H), 1.90 (br s, 1H), 2.85-2.75 (m, 1H), 1.82-1.60 ( m, 3H), 1.55-1.45 (m, 1H), 1.45 (s, 4H), 1.42 (s, 2H), 1.39 (s, 9H).

Step C. N- [1 (R) - (1,2-Dihydro-1-methanesulfonylspiro [3H-indole-3,4'-piperidine] -1'yl) carbonyl] -2- (phenylmethyl-hydroxy) ethyl] -2-amino-2-methylpropanamide hydrochloride.

To 0.637 g (0.101 mmol, intermediate of step B in 5 ml of dichloromethane, 2.5 ml of trifluoroacetic acid was added and stirred at RT for 30 minutes, the reaction mixture was concentrated to oil, placed in 10 ml of ethyl acetate and washed with 8 ml of 10%) an aqueous solution of sodium carbonate. The aqueous layer was then extracted with 5 ml of ethyl acetate. The combined organic layers were washed with 10 ml of water, dried over magnesium sulfate, filtered and concentrated to give 0.512 g of a free base as a white foam.

To 0.512 g of the free base in 5 ml of ethyl acetate at 0 ° C, 0.2 ml of a saturated solution of hydrochloric acid in ethyl acetate was added and stirred for 1.5 hours. The white precipitate was filtered under nitrogen, washed with ether and dried, yielding 0.50 g the title compound as a white solid.

'H NMR (400 MHz, SE ₃ , OO). The compound exists as a mixture of 3: 2 rotamers δ 7.40-7.28 (m, 4H), 7.25-7.17 (m, 2H), 7.08 (t, 1 / 3H), 7.00 ( t, 1 / 3H), 6.80 (d, 1 / 3H), 5.16 (ddd, 1H), 4.60-4.42 (m, 3H), 4.05 (t, 1H), 3 90 (br s, 2H), 3.83-3.70 (m, 2H), 3.30-3.15 (m, 1H), 2.97 (s, 1H), 2.95 (s , 2H), 2.90-2.78 (m, 1H), 1.96 (t, 1 / 3H), 1.85-1.65 (m, 4H), 1.63 (s, 2H), 1.60 (s, 4H).

Example 2

N- [1 (R) - (1,2-Dihydro-1-methanesulfonylspiro [3H-indole-3,4'-piperidine] -1 '-carbonyl] 2- (phenylmethyl-hydroxy) ethyl] -2-amino 2-methylpropanamide.

Stage A. (2R) - [[[- 2 - [(1,1-dimethylethoxy) carbonyl] amino] 2,2-dimethyl-1-oxoethyl] -1-propanoic acid allyl ether.

Prepared from (2R) -2 - [(1,1-dimethylethoxy) carbonyl] amino-3- [phenylmethyloxy) ethylpropanoic acid and allyl alcohol by coupling in CH ₂ Cl ₂ in the presence of EDC and DMAR.

'H NMR (400 MHz, CDCl3) δ 7.25 (s, 5H), 5.8 (m, 1H), 5.2 (dd, 2H), 5.0 (br s, 1H), 4, 7 (m, 1H), 4.6 (m, 2H), 4.4 (dd, 1H), 3.9 (dd, 1H), 3.6 (dd, 1H), 1.45 (d, 6H ), 1.39 (s, 9H).

Stage B. (2R) - [[[- 2 - [(1,1-dimethylethoxy) carbonyl] amino] -2,2-dimethyl-1-oxoethyl] amino-2- (phenylmethyloxy) ethyl) -1-propanoic acid .

To a stirred solution of the crude intermediate of step A (6.7 g, 15.9 mmol), tetrakis (triphenylphosphine) palladium (1.8 g, 0.1 eq.) And triphenylphosphine (1.25 g, 0.3 eq. ) potassium 2-ethylhexanoate solution (35 ml 0.5M solution in EtOAc) was added. The reaction mixture was stirred at room temperature under nitrogen for 1 h and then diluted with ether (100 ml) and poured into ice water. The organic layer was separated and the aqueous fraction was acidified with citric acid (20%), then extracted with EtOAc. The EtOAc extracts were washed with brine, dried over magnesium sulfate, filtered and evaporated to give the title compound as a solid.

¹ H NMR (400 MHz, CD3OD) δ 7.3 (s, 5H), 4.7 (m, 1H), 4.5 (s, 2H), 4.0 (m, 1H), 3.6 ( m, 1H), 1.4 (d, 6H), 1.3 (s, 9H).

Step C. ^ [1CH) - (1,2-dihydro-1 methanesulfonyl-spiro [3H-indole-3,4'-piperidine] -1'-carbonyl] -2-phenylmethoxy) ethyl] -2-amino-2- methylpropanamide hydrochloride.

To a solution of 1.0 g (3.44 mmol) of 1-methanesulfonylspiro [3H-indoline-3,4'-piperidine] hydrochloride, 1.44 g (3.78 mmol) (2R) [[- 2- (1, 1 dimethylethoxy) carbonyl] amino-2,2-dimethyl-1-oxoethyl] amino-2- (phenylmethyloxy) ethyl) -1-propanoic acid, N-methyl morpholine (0.58 ml, 5.20 mmol) and 1-hydroxybenzotriazole (HOBT) (0.58 g, 3.78 mol) in 50 ml of dichloromethane was added EDC (1.03 g, 5.20 mol) and stirred at RT for 1 6 hours. The reaction mixture was diluted with additional 50 ml of dichloromethane and washed with aqueous bicarbonate sodium (50 ml), dried over anhydrous magnesium sulfate, filtered and conc ntrirovali. Flash chromatography (50 g of silica gel) of a crude oily residue gave 2.148 g (90%) of the desired product as a colorless foam.

'll NMR (ODOls, 400 MHz). The compound exists as a mixture of 3: 2 rotamers δ 7.40-7.10 (m, 6H), 7.06 (d, 1 / 3H), 7.02 (t, 1 / 3H), 6.90 (t, 1/3 H), 6.55 (d, 1 / 3H), 5.15 (m, 1H), 4.95 (br s,

1H), 4.63 (br d, 1 / 3H), 4.57-4.40 (m, 2 2 / 3H), 4.10 (br d, 1 / 3H), 4.00 (br .d, 1 / 3H), 3.82 (t, 1H), 3.78-3.62 (m, 2H), 3.60-3.50 (m, 1H), 3.04 (q, 1H ), 2.87 (s, 1H), 2.86 (s, 2H), 2.80-2.60 (m, 1H), 1.90 (br s, 1H), 2.85-2, 75 (m, 1H), 1.82-1.60 (m, 3H), 1.55-1.45 (m, 1H), 1.45 (s, 4H), 1.42 (s, 2H) 1.39 (s, 9H).

Step D. N- [1 (R) - (1,2-Dihydro-1-methanesulfonylspiro [3H-indole-3,4'-piperidine] -1 'yl) carbonyl] -2- (phenylmethyloxy) ethyl] -2 amino-2-methylpropanamide hydrochloride.

To a solution of 2.148 g (3.41 mmol) of step C intermediate in 10 ml of dichloromethane was added 5 ml of trifluoroacetic acid and stirred for 1 h. The reaction mixture was concentrated and made basic with 100 ml of 5% aqueous sodium carbonate solution and extracted with dichloromethane (3x50 ml). The combined organic layers were washed with brine (50 ml), dried over anhydrous potassium carbonate, filtered and the colorless foam was concentrated. To a solution of foam in 25 ml of ethyl acetate at 0 ° C. was added 4 ml of a 1M solution of hydrochloric acid in ethyl acetate. The precipitate was filtered and washed first with ethyl acetate and then with ethyl acetate (1: 1), dried, and 1.79 g (93%) of the title compound was obtained as a colorless solid.

'H NMR (400 MHz, CDzOD). The compound exists as a 3: 2 mixture of rotamers. δ 7.40-7.28 (m, 4H), 7.25-7.17 (m, 2H), 7.08 (t, 1 / 3H), 7.00 (t, 1 / 3H), 6 80 (d, 1 / 3H), 5.16 (ddd, 1H), 4.60-4.42 (m, 3H), 4.05 (t, 1H), 3.90 (br s, 2H ), 3.83-3.70 (m, 2H), 3.30-3.15 (m, 1H), 2.97 (s, 1H), 2.95 (s, 2H), 2.90- 2.78 (m, 1H), 1.96 (t, 1 / 3H), 1.85-1.65 (m, 4H), 1.63 (s, 2H), 1.60 (C, 4H) .

Example 3

N- [1 (R) - (1,2-Dihydro-1-methanesulfonylspiro [3H-indole-3,4'-piperidine] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] -2-amino -2-methylpropanamide mesylate.

This compound was obtained by treating the free base obtained in Example 5, Step D, with methanesulfonic acid. The title compound was obtained by recrystallization from a mixture of ethyl acetate-ethanol-water. Mp 166168 ° C. This sample was then identified as polymorphic form II. It was characterized by an X-ray powder diffraction pattern with basic reflections at approximately 4.7, 11.6, 17.4, 19.2 and 21.6 ° (2 theta). Further, it was characterized by a DSC at a gradient of 10 ° C / min in an open cell in a nitrogen flow and had one melting endotherm with a temperature peak of about 1 74 ° C and an extrapolated initial temperature (melting point) of approximately 165 ° C, corresponding to a heat of approximately 37 J / g .

Example 4. O ^ SE

from ₂ n

Isonipecotic acid-L-benzyl carbamate (3).

Materials:

Isonipecotic Acid (2) T.C.I. 4.02 kg (31.1 moles Benzyl chloroformate (Schweitzerhall) 6.91 kg (40.5 moles) K2CO3 10.1 kg (72.9 moles) Water 40.2 L

Isonipecotic acid (2) and K ₂ CO _{3 were} dissolved in 40.2 L of water in a 100 L 4-necked flask with mechanical stirring in an N ₂ atmosphere and the solution was cooled to 10 ° C. Benzyl chloroformate was added, maintaining the temperature between 9 and 14 ° C and after completion of the addition and standing for 58 h, the mixture was heated to 22 ° C. The addition was completed after 4 hours when the pH was 9.0. After standing for 58 hours, there was no change in pH.

The reaction mixture was transferred to a 200 L extractor and washed with 3x13 kg (15 L) IP AC and 1x12 L EtOAc. The aqueous layer was extracted with 8 L of toluene. After washing, the benzyl alcohol content decreased from 3.8% to 1.4% according to HPLC analysis. HPLC analysis: 25 cm RXC8 Dupont Zorbax column with a flow of 1.5 ml / min and detection at 254 nm; isocratic mixture with 35% MeCN, 65% with 0.1% aqueous НзРО ₄ ; retention time: 3 = 6.9 min, benzyl alcohol = 3.3 min, toluene = 17.3 min.

The aqueous phase was acidified with a 37% aqueous HO solution to a pH of 1.8.

Carbon dioxide was released during the addition, but gas evolution was easily controlled. Addition of HO took <1 h and required 10 L conc. BUT. The aqueous phase was extracted with 3x6.6 L of toluene. The toluene extracts were dried with 2 kg of sodium sulfate and filtered through a layer of Solka-floc ™. The weight of the combined filtrates was 17.8 kg. The yield of crude carbamate 3 was 7.89 kg (97%) obtained by evaporation of dry weight aliquots of filtrates). The filtrates were passed through a 10 μm in-line filter into a 1,00 L flask. The extracts were concentrated at 10 mbar at <25 ° C to a volume of 1 8 L. The final concentration of carbamate 3 was 440 g / L. The concentration of toluene filtrate served to azeotropically remove the last traces of water (finally KF = 170 mg / L). The product was 99.1% pure with 0.9% benzyl alcohol as the sole impurity.

Example 5

coci

Chlorohydride-N-benzyl carbamate of isonipecotic acid (4).

Materials:

Isonipecotic acid -N-benzyl carbamate (3) in toluene (M.V. = 263.30) 7.89 kg (30.0 moles) in 17.9 liters Oxalyl chloride (M.V. = 126.93) 3.94 kg (31.0 moles) DMF (M.V. = 73.10) 10 ml Toluene 12 l

To the toluene solution of benzyl carbamate 3 from the previous step was added 5 ml of DMF and 10 l of toluene. After 20 minutes, oxalyl chloride was added. The reaction mixture was kept for 16 hours at 18 ° C in a slow stream of nitrogen. HPLC analysis of the reaction mixture showed that 1.3% of carboxylic acid 3 remained unreacted. The reaction mixture was heated to 26 ° C and 5 ml of DMF was added. The mixture was kept for 2.5 hours, a 1.0 ml aliquot of the reaction mixture was quenched with 5.0 ml of tert-butylamine and, after evaporation, analyzed with HPLC: 25 cm RXC8 Dupont Zorbax column at 50 ° C, flow rate of 1 ml / min and detection at 220 nm; isocratic 42% MeCN, 58% with 0.1% aqueous. This method showed that <0.05% acid 3 remained (judging by A), and showed> 3% B (> 1 mol% (COCl) ₂ ).

The mixture was concentrated at 10 mbar and a temperature of 20-25 ° C until 5 l of solvent was removed.

A typical HPLC picture of a concentrated toluene solution after quenching of T-BuNH ₂ described above is as follows:

Retention Time (min) % surface Identity 2.1 <0.5% Carboxylic Acid 3 7.8 <0.5% Benzyl chloride 11.0 > 99% Cbz-Butylcarboxamide A 12.1 NA Toluene 12.7 <0.5 di-tert-butyloxamide B

Example 6

sleep

Piperidin-4-carboxaldehyde-1-benzylcarbamate (5).

Materials:

Hydrochloride - \ - isonipecotic acid carbamate (4) in toluene (M.V. = 281.74) 3.38 kg (12.0 moles) in 5.54 kg DIEA (KF = 18 mg / L) 1.55 kg (15.0 moles) 10% Pd / C (KF <20 mg / g) 101 g thioanisole (M.V. = 124.21, d = 1,058) 0.56 g

DIEA and thioanisole were added to the solution (4) in toluene from the previous step, and the catalyst was suspended in this mixture. The mixture was immediately placed in an 18.9 L autoclave and hydrogenated at 20 ° C and 2.8 kg / cm ² H ₂ . After 18 hours, the reaction mixture absorbed 70% of the theoretical amount of hydrogen, and HPLC analysis of an aliquot that was quenched with tert-butylamine showed that 1 4.2% of acid chloride 2 remained. The conditions of HPLC were the same as above. Retention time: 5 = 8.1 min.

A second charge of catalyst (101 g) and thioanisole (0.54 g) was added to the hydrogenerator as a suspension in 1,375 ml of toluene. After 23 h, HPLC analysis of an aliquot that was quenched with tert-butylamine showed that 1.8% of the acid chloride remained.

The mixture was purged with nitrogen and the catalyst and precipitated DIEA · Hd were removed by filtration through Solka-floc ™. The filter cake was washed with 10 L of toluene. The filtrates were passed through a 10 μm in-line filter into a 50 L extractor and washed with 2 × 7.2 L of a 1M aqueous Hd solution and 2 × 7.2 L of water. The mixture was concentrated at 10 mbar and a temperature of 25-30 ° C to obtain a residue of 5 l.

Retention time Surface% Identity 2.1 <2 carboxylic acid 3 6.6 <1 dimer 21 8.1 > 95 aldehyde 5

The experimental yield of aldehyde 3 was 94% according to HPLC analysis.

Example 7

CDZ-Spiroindoline (9). Materials:

Solution of piperidine-4-carboxaldehyde-1-benzyl carbamate (5) in toluene 1.71 kg (6.89 moles) 21.4 kg Phenylhydrazine 900 ml, 981 g (9.15 moles) Trifluoroacetic Acid (TFA) 2.20 L, 3.26 kg (28.6 moles) NaBH4 300 g, (7.93 moles) Toluene 34.4 kg Mecn 7.0 L Meon 7.0 L

A solution of crude aldehyde 5 from the previous step was passed through a 10 μm in-line filter into a 100 L reactor equipped with Teflon-lined copper coils for cooling or heating and a mechanical stirrer. Toluene (34.4 kg) and MeCN (7 L) were added and the resulting solution was cooled to 0 ° C. Phenylhydrazine was added in portions and the temperature was maintained at -1 to 3 ° C while continuously bubbling nitrogen into the reaction mixture.

Phenylhydrazine was added until TLC and HPLC analysis showed complete consumption of aldehyde 5 and the appearance of a slight excess (<5%) of phenylhydrazine. TLC conditions: Silica, E. Merck Kieselgel G60 F254 0.25 mm; diethyl / pentane (4/1); developing agent 0.5% cerium sulfate, 14% ammonium molybdate in 10% aqueous sulfuric acid and then heating; Rf: aldehyde 5 = 0.52, phenylhydrazone 7 = 0.61, phenylhydrazine 6 = 0.21.

HPLC conditions: 25 cm RXC8 Dupont Zorbax column at 30 ° C with a flow of 1.0 ml / min and detection at 254 nm; gradient scale:

Time (min) Acetonitrile: water 0 57:43 10 65:35 fifteen 75:25 eighteen 75:25

Retention time: phenylhydrazine 6 = 4.5 min, toluene = 7.2 min, phenylhydrazone 7 = 11.4.

The reaction mixture was kept for 30 minutes at 0-2 ° C. TFA was added while maintaining the temperature between 2 and 7 ° C. The reaction mixture was heated to 50 ° C. for 30 minutes and maintained for 17 hours. The nitrogen sparging through the reaction mixture was stopped and a slow stream of nitrogen was maintained over the reaction mixture. During the first hour at 5 ° C, the solution gradually darkened to dark green and a relatively small amount of a white crystalline precipitate (ammonium trifluoroacetate) formed. After 17 hours, HPLC analysis (the conditions are the same as above) showed that the reaction mixture contained 91.6% of indolenin 8 and that 1.5% of unreacted phenylhydrazone remained. Standing the mixture for longer periods of time did not increase the yield of indolenin 8.

The reaction mixture was cooled to 12 ° C and 7.0 L of MeOH was added. NaBN ₄ was added in small portions (<20 g), keeping the temperature below 15 ° C. Adding took 30 minutes. Moderate hydrogen evolution was observed during the addition, but it was easy to control and there was practically no foaming. Shortly before the end of the addition, the color quickly changed from green to brown, and then to bright orange. A small amount (<200 ml) of the heavier phase (presumably aqueous salts) has separated. An analysis of HPLC (the conditions are the same as before) showed that all indolenyl 8 was consumed (90.4% CBZindoline); retention time: indolenin 8 = 7.5 min, indoline 9 = 8.2 min. TLC: ethyl ether as a solvent, the manifestation of ammonium by molybdate, cerium sulfate or 1% anisaldehyde; retention factors: indolenin 8 = 0.18, CBZ-indoline 9 = 0.33.

The moment of color change from green to orange is very close to the end point of the reaction. The amount of NaBH ₄ required to complete the reaction strongly depends on the temperature and rate of addition of NaBH ₄ , but does not affect the yield and quality of the product, provided that the reaction is complete. The reaction mixture was cooled to 5 ° C. over 30 minutes. Then 8 L of 3% aqueous NH ₄ OH (8 L) was added to adjust the pH of the aqueous phase to 7.4, the mixture was stirred and allowed to precipitate. The temperature rose to 15 ° C. The turbid yellow lower aqueous phase was separated. The organic phase was washed with 4 L of 3% aqueous NH ₄ OH, 2x4 L of water and 2x4 L of saline. The weight of the organic phase after washing was 53.5 kg, and the experimental yield was 94%.

The washed toluene solution was combined with the washed organic phases of two other similar reactions. The total amount of aldehyde used in the three reactions was 5.06 kg (20.0 moles). The total weight of CBZindoline 9 found in the combined organic phases was 5.91 kg (18.3 moles, experimental entry 90%). The combined organic phases were dried with 5 kg of sodium sulfate, treated with 250 g of Darco G-60 coal for 30 minutes, and filtered through Solkafloc ™. The filtrates were concentrated in vacuo at 10 mbar at <25 ° C to an almost dry residue. The solvent change was completed slowly, feeding 30 l of IPAC and re-concentrating to 1 4 l at 200 mbar at 50-60 ° C. The mixture was heated under reflux and a pure homogeneous dark orange solution was obtained. MmP analysis showed that the solution contained about 6 mol% of residual toluene after replacing the solvent.

The solution was cooled to 68 ° C and seeded with 4 g of crystalline CBZ-indoline 9. The solution was allowed to gradually cool to 26 ° C over 6 hours and stood for 9 hours at 20-26 ° C. The suspension was cooled to 2 ° C for 1 h and kept at 2 ° C for 1 h. The product was isolated by filtration, and the filter cake was washed with 2x2 L 5 ° C IP AC and 2x2 L 5 ° C MTBE. The product was dried in a vacuum oven at 30 ° C in a weak stream of nitrogen and received 4.37 kg (74%) of the title compound 9 in the form of a light tan crystalline powder. HPLC analysis of the product showed 99.5% purity. The mother liquor (11 L) and washes contained 1.15 kg (19%) of additional product 9 and about 3% of isonipecotic acid Cbz-phenylhydrazide (retention time = 4.8 min).

CBZ-Spiroindoline-methanesulfonamide (1).

Materials:

CBZ-Spiroindoline (9) 1.69 kg (5.23 moles) Methanesulfonyl chloride 599 g (5.23 moles) Et3N (KF = 151) 635 g (6.27 moles) THF (KF = 41 mg / g) 12 l

Solid CBZspiroindoline 9 was loaded into a 22 L flask, and then through a 10 μm in-line filter, 11.5 L THF and Et ₃ N were passed into the flask. The resulting homogeneous solution was cooled to 0 ° C. Methanesulfonyl chloride and 500 ml THF were charged into a 1 L dropping funnel. A solution of MsCl in THF was added to the reaction mixture, maintaining the temperature between 0 and 4 ° C. The addition took 5 hours and was exothermic. A white precipitate formed during the addition, presumably triethylammonium hydrochloride. HPLC analysis showed that the reaction was completed at the end of the addition (9 was not detected).

HPLC conditions: 25 cm Dupont Zorbax RXC8 column at 30 ° C with a flow of 1.5 ml / min and detection at 254 nm. Gradient Scale:

Time (min) 0.1% aq. H ₃ PO ₄ : MeCN 0 70:30 3 70:30 12 20:80 25 20:80

Retention time: 9 = 7.6 min, 1 = 13.6 min.

After completion of the addition, the reaction mixture was heated to 18 ° C and held for 16 hours. There were no changes in the reaction mixture in the HPLC picture between the end of the addition and after 16 hours of aging. The reaction mixture was slowly transferred over 1 h into a vigorously stirred solution of 30 L of water and 200 ml of 37% aqueous HO in a 50 L flask. The temperature in a 50 L flask rose from 22 to 28 ° C. The product was isolated as a pale tan gummy solid which turned into a granular solid. The aqueous suspension was cooled to 22 ° C and held for 1 h. The suspension was filtered and the filter cake was washed with 2x4 L MeOH / water (50/50). HPLC analysis showed that uterine fluids contained <0.1% CBZ-spiroindolinol of tansulfonamide 1.

The filter cake was washed with 4 L of MeOH / water (50/50), to which 50 ml of 28% aqueous NH ₄ OH was added. The filter cake was washed with 2x4 L MeOH / water (50/50) and the solid was dried in a vacuum oven at 50 ° C. under a nitrogen stream to obtain 2.03 kg (97%) of title product 1 as an off-white powder. HPLC analysis of solids showed 93.7% of product 1.

Example 9

An optional step for isolating the intermediate CBZ-spiroindolenin intermediate (8). Materials:

Piperidin-4-carboxaldehyde 1-benzyl carbamate (5) 12.37 g (0.050 mol) Phenylhydrazine 5.41 g (0.050 mol) Trifluoroacetic Acid (TFA) 11.56 ml, 17.10 g (0.150 mol) Methylene chloride 500 ml

CBZ-aldehyde 5 was dissolved in dichloromethane in a 1 L flask equipped with a Teflon-coated magnetic stirrer. The resulting solution was cooled to 0 ° C. Phenylhydrazine was added via a suspended syringe over 5 minutes and the temperature was maintained at -1 to 3 ° C, while nitrogen was continuously bubbled through the reaction mixture. TLC and HPLC analyzes showed complete consumption of CDZaldehyde 5 and the appearance of a slight excess (<2%) of phenylhydrazine. TLC conditions: Silica, E. Merck Kieselgel G-60 F254 0.25 mm; diethyl ether / pentane (4/1); developing agent 0.5% cerium sulfate, 14% ammonium molybdate in 10% aqueous sulfuric acid with heating; Rf: aldehyde 5 = 0.52, phenylhydrazone 7 = 0.61, phenylhydrazine 6 + 0.21. Conditions HPLC 25 cm RXC8 Dupont Zorbax column at 30 ° C with a flow of 1.0 ml / min and detection at 254 nm; gradient scale:

Time (min) Acetonitrile: water 0 57:43 10 65:35 fifteen 75:25 eighteen 75:25

Retention time: phenylhydrazine 6 = 4.5 minutes, toluene = 7.2 minutes, phenylhydrazone 7 = 11.4 minutes.

The reaction mixture was kept for 10 minutes at 0-2 ° C and TFA was added via syringe while maintaining a temperature of 2 and 7 ° C. The reaction mixture was heated for 30 minutes to a temperature of 35 ° C, which was maintained for 1 to 7 hours. Nitrogen sparging through the reaction mixture was stopped and a slow stream of nitrogen was maintained over the reaction mixture. During the first hour at 35 ° C, the solution gradually darkened to pink-violet, and then to dark green and a relatively small amount of white crystalline precipitate (ammonium trifluoroacetate) formed. After 17 h, the analysis of HPLC (the conditions are the same as above) showed that the reaction mixture contained 93% indolenin 8 and <0.5% unreacted phenylhydrazone remained. Holding the mixture for longer periods of time did not increase the experimental yield of indolenin 8. The reaction mixture was cooled to 10 ° C and a mixture containing 60 ml of 28-30% ammonium hydroxide 90 ml of water and 1 50 g of crushed ice was added with good stirring. The color of the mixture changed to salmon. The organic phase was separated and washed twice with 400 ml of water, and then with 100 ml of saturated aqueous HC1. The organic phase was dried over magnesium sulfate and filtered through a layer of 5 g of silica. The filtrate was evaporated to obtain 15.87 g (99%) of indolenin 8 as a pale orange oil.

Example 10

Method for the preparation of CBZ Spiroindoline methanesulfonamide (1) without isolation of the intermediate CBZ Spiroindoline (9).

Stage 1. CBZ-Spiroindoline (9).

Materials:

Piperidin-4-carboxaldehyde-1 benzyl carbamate (5) 49.5 g (0.20 mol) Phenylhydrazine (Aldrich) 23.7 g (0.22 mol Trifluoroacetic Acid (TFA) 75, 4 g (0.66 mol Toluene (KF <250 ml / g) 654 ml MESA (KF <250 ml / L) 13.3 ml NaBN ₃ 11.3 g (0.20 mol) Toluene 20 ml Meon 50 ml

A 2% (v / v) solution of MeOH in toluene was prepared using 654 ml of toluene and 13.3 ml of MeCA. In a 2 L 3-necked flask equipped with a mechanical stirrer, 617 ml of the above solution was degassed by passing a thin stream of nitrogen through the solution for 5 minutes. Phenylhydrazine and TFA were added to the solution even during degassing.

CBZ-aldehyde 5 was dissolved in the remaining solution prepared above (50 ml) and degassed by sparging nitrogen through a solution in a dropping funnel. The solution in the flask was heated to 35 ° C and the aldehyde solution was slowly added to phenylhydrazine-TFA for 2 hours. The mixture was allowed to stand at 35 ° C for 16 hours.

HPLC conditions: 25 cm RXC8 Dupont Zorbax column at 50 ° C with a flow of 1 ml / min and detection at 220 nm; isocratic 55% MeCN, 45% 0.1% aqueous H ₃ PO ₄ . Typical HPLC picture after 1 6 hours of standing:

Retention Time (min) Surface% Identity 1,6 0.1-0.5 Phenylhydrazine 6 4.1 <0.1 Dimer 21 4.7 <0.1 Aldehyde 5 5,0 NA Spiroindoline 9 6.3 NA Toluene 6.9 97 Spiroindolenin 8 10.3 <0.2 Phenylhydrazone 7 2-3 total Other impurities <0.2% ea.

The mixture was cooled to −10 ° C. and MeOH was added. A suspension of sodium borohydride in 20 ml of toluene was added in small portions (1 ml) over 30 minutes, making sure that the temperature did not exceed -2 ° C.

Surface% Identity 0.1-1 Phenylhydrazine 6 85-90 CBZ-Spiroindoline 9 <0.1 CDZ-Spiroindolenin 8 10-15 total Other impurities (<3% ea.)

The temperature was raised to 1 0 ° C in 1 h and 6% aqueous ammonia (200 ml) was added. The mixture was stirred for 10 minutes, allowed to settle for another 10 minutes, and the lower aqueous phase was removed. Acetonitrile (20 ml) and MeOH (20 ml) were added to the organic phase and washed with 150 ml of 15% (saline). It was found that the organic phase contained CBZspiroindoline 9 with 92% experimental yield.

Stage 2. CBZ-Spiroindolinemethanesulfonamide (1).

Materials:

CBZ-Spiroindolin (9) (m.v. = 322.51) (0.184 mol) Methanesulfonyl chloride 21.1 g (0.184 mol) DIEA (KF = 150 mg / L) 29.7 g, 40 ml (0.230 mol) THF (KF = 41 mg / L) 150 ml

A crude solution of the CBZ solution of spiroindoline 9 from step 1 above was concentrated in a 1 L 3-necked flask (60-70 ° C, 150-200 Torr) to give 250 g of a residue. TNF and DIEA were added and the resulting homogeneous solution was cooled to 0 ° C. 1 25 ml dropping funnel was charged with methanesulfonyl chloride and 50 ml of THF. A solution of MsCl in THF was added over 2 hours to the reaction mixture, maintaining the temperature from 0 to 4 ° С, and the mixture was kept for 2 hours at 5-8 ° С. The addition was slightly exothermic. A white precipitate formed, presumably DIEA hydrochloride, during the addition. The HPLC conditions were the same as above. The analysis of HPLC showed that the reaction was completed 1 h after completion of the addition (9 was not detected) and the experimental yield of 9 was 94%. Retention time: 1 = 7.8 min.

Typical HPLC picture of the reaction mixture after 2 h of aging:

Surface% Identity <0.1 CBZ-Spiroindoline 9 90-92 CDZ-Sulfonamide 1 8-10 total Other impurities (<2% ea.)

The mixture was heated to 20 ° C and 200 ml of 1M aqueous HC1 was added. The mixture was heated to 50 ° C and the aqueous phase was separated. The organic phase was washed successively with 100 ml of water, 100 ml of an aqueous solution of sodium bicarbonate and 1 00 ml of water. The organic phase was transferred to a 1L 3-necked flask equipped with a mechanical stirrer and a distillation device. The mixture (about 400 ml) was distilled at atmospheric pressure to obtain 150 ml of distillate. The temperature of the top reached 107 ° C; the temperature in the cube was 110 ° C. The distillation continued with the continuous addition of npropanol at such a rate as to maintain a constant volume (about 350 ml) in the cube. The distillation was terminated when a total of 525 ml of n-RGON was added and a total of 800 ml of distillate was collected.

During the replacement of the solvent, the temperature of both the top and the cube increased from 94 ° C to 98 ° C. Toluene and n-RgON formed an azeotrope boiling at 97 ° C, consisting of 47% toluene and 52.5% n-RgON. The mixture was allowed to cool gradually to 20 ° C. over 3 hours and held for 1 to 2 hours. The mother liquor was found to contain 2% toluene and 4 mg / ml sulfonamide. The solubility of sulfonamide in various mixtures of toluene and n-RgON was determined by HPLC analysis:

% toluene in n-RgON Solubility 1 in mg / ml 0 2,36 5 3.02 10 4.23 twenty 7.51 25 10.3

The crystalline suspension was filtered and washed with 3x10 ml of n-RgON. The product was dried in a vacuum oven at 50 ° C with nitrogen passing for 16 hours to obtain 65.6 g (82% of aldehyde 5) of product 6 as a tan solid with 93.5% by weight purity.

Typical HPLC solid picture:

Surface% Identity <0.1 CBZ-spiroindoline > 99 CBZ-sulfonamide 1 <1 total other impurities (<0.2% ea.)

For further purification, a 40.0 g sample of a sulfonamide crystallized from n-PrHON in 134 ml EtOAc at 60 ° C was dissolved and treated with 8.0 g Darco G-60 for 1 h at 60 ° C. After adding 2.0 g of So1kafloc ™, the suspension was filtered through a 4.0 g layer of Solka-floc ™ and the layer was washed with 90 ml EtOAc at 60 ° C. Before adding coal, the solution was brown. The filtration proceeded well, without clogging, and gave a golden yellow filtrate. The filtrate was distilled at atmospheric pressure in a 500 ml flask (cube temperature 80-85 ° C) to 100 g (100 ml) of the residue. This solution was allowed to cool to 35 ° C. over 3 hours. 116 ml of cyclohexane was added over 1 hour with good stirring at 35 ° C. The mixture was cooled to 20 ° C for 1 h and kept at 20 ° C for 12 h. At 35 ° C, a greater amount of sulfonamide crystallized and the mixture became thick. The addition of cyclohexane at 20 ° C makes mixing difficult. After standing, it was found that the supernatant contains 2.5 mg 1 / g. The crystalline suspension was filtered and the filter cake was washed with 77 ml of cyclohexane / EtOAc (2: 1) and 2x77 ml of cyclohexane. The product was dried in a vacuum oven at 50 ° C under a stream of nitrogen for 1 6 hours, obtaining 34.2 of product 1 (MV = 400.3) as a white crystalline solid (85% isolation from crude 1, 70% out of 5 with> 99.9 wt.% purity).

Example 11

BUT the salt of spiroindoline-methanesulfonamide (1A).

Materials:

CBZ-Spiroindoline-methanesulfonamide (one) 941 g (2.35 moles) Pearlman catalyst 20% Pd (OH) 2 / C 188 g Thf 8 l Meon 7 l

The catalyst was suspended in 7 L of MeOH and transferred to an 18.9 L autoclave, followed by the addition of a solution of 1 in 8 L of THF. The mixture was hydrogenated at 25 ° C. at 5.62 kg / cm ² H ₂ . After 2.5 hours, the temperature was raised to 35 ° C over 30 minutes. HPLC analysis showed complete utilization of CBZ-spiroindolin-methanesulfonamide. HPLC conditions: 25 cm RXC8 Dupont Zorbax column with a flow of 1.5 ml / min and detection at 254 nm.

Gradient Scale:

Time (min) 0.1% aq. PzRO ₄ : MeCN 0 70:30 3 70:30 12 20:80 25 20:80

Spiroindoline retention time = 7.6 min; CB2-spiroindoline-methanesulfonamide = 3.6.

The mixture was purged with nitrogen and the catalyst was removed by filtration through Solka-floc ™ while the mixture was still warm. The catalyst was washed with 4 L of THF and 2 L of MeOH. The pale yellow filtrates were concentrated to a thick oil at 10 mbar and <25 ° C. The solvent was replaced slowly by adding EtOAc in 15 L and reconcentrating to dryness. The residue solidified to give a solid off-white mass. MeOH (1.5 L) was added and the mixture was heated to 70 ° C. to obtain a homogeneous solution. While the solution was at a temperature of 70 ° C, 10.5 L EtOAc was added at 20 ° C. The temperature dropped to 40 ° C, and the mixture remained homogeneous.

Subsequent experiments gave reason to believe that it is more convenient for us to replace the solvent of MeOH-THF filtrates with MeOH, concentrate to the desired volume, and then add EtOAc. This avoids hardening of the residue after concentration of the EtOAc solution.

Hydrogen chloride diluted with an approximately equal volume of nitrogen was passed into the solution. The temperature rose to 60 ° C for 1 5 min, and a white precipitate of hydrochloride salt formed. Dilution of BUT with nitrogen only avoids the suction of the reaction mixture back and is not necessary.

The mixture was cooled in an ice bath and continued for 1 h by addition of hydrogen chloride. The temperature gradually dropped to 20 ° C. The suspension was kept for 2 hours, while the temperature decreased to 10 ° C. The crystalline product was isolated by filtration and the filter cake was washed with 3 L EtOAc. It was dried in a vacuum oven at 35 ° C, yielding 1.18 kg (86%) of the title product 1a as an off-white crystalline solid> 99% pure, according to HPLC analysis. HPLC conditions: 25 cm RXC8 Dupont Zorbax column with a flow of 1.5 ml / min and detection at 230 nm; isocratic 35% MeCN, 65% with 0.1% aqueous ammonium acetate. Retention time: 1a = 5.4 min.

Example 12

Spiroindolin-methanesulfonamide (free base form) (1 b).

A 250 ml aliquot of the filtrate from Cbz hydrogenolysis containing 4.67 substances of 1b (free base) was concentrated to approximately 10 ml. The residue was dissolved in 20 ml EtOAc, and the solution was concentrated again to approximately 10 ml. This operation was repeated once more and 10 ml EtOAc was added to the residue. A crystalline precipitate began to form. MTBE (20 ml) was added in one portion. An additional crystalline solid precipitated, but the supernatant still contained a significant amount of the dissolved product, which did not precipitate upon settling. Hexanes (70 ml) were added dropwise to the mixture over 2 hours with vigorous stirring. Slow addition of hexanes is necessary in order to avoid oiling the amine.

The stirred mixture was kept for 1 h and filtered. The filter cake was washed with 20 ml of MTBE-hexanes and then with 20 ml of hexanes. The product was dried under a stream of nitrogen to obtain 3.86 g (82%) of the free amine of substance 1 b as an off-white crystalline solid> 99.5% pure. HPLC conditions: 25 cm RXC8 Dupont Zorbax column with a flow of 1.5 ml / min and detection at 230 nm; isocratic 35% MeCN, 65% with 0.1% aqueous ammonium acetate. Retention time: 1b = 5.4 min.

Example 13 A.

Spiroindolin-methanesulfonamide (free base form) (1 b). Materials:

CDZ-Spiroindoline-sulfonamide (1) 833.5 g (2.08 mol) Pd (OH) 2 (20% by weight of Pd (OH) 2 124.5 (15%) Thf 6.5l Meon 19.5 L NH4OH (conc.) 60 ml

Hydrogenations were performed three (3) times due to equipment limitations; This method is designed for one run. CBZ-spiroindolin sulfonamide 1 was dissolved in THF (6.5 L, KF = 53 μg / μl) and then MeOH (KF = 1 8 μg / μl, 4 L) was added, followed by the addition of a catalyst, and the suspension was transferred to 18.9 L autoclave. The remainder of MeOH (2.5 L) was used for irrigation. The mixture was heated to 40 ° C. at 3.5 kg / cm ² for 24 hours. The catalyst loading and reaction time were a function of the purity of the starting material 1. The material was unique, requiring> 15% catalyst and a long reaction time. Pure batches of spiroindoline required only 5% of the catalyst and 4-6 hours of reaction time.

Upon completion (<0.1 A% 1 by LC), the mixture was filtered through Solka-floc ™ and the carbon cake was washed with MeOH (13 L) containing NH ₄ OH (0.5%, 60 ml). The combined filtrates (analysis shows 1587 g of the amine spiroindolin 1 b) were concentrated in vacuo and the resulting solids were partitioned between 40 L (toluene: TNF (3: 1) and 0.5 N NaOH (18 L)). Although the layers separated easily, a heavy precipitate could be seen in the aqueous layer. The aqueous suspension was extracted with CH ₂ Cl ₂ (15 L). The aqueous and organic layers were separated slowly. Prior to the addition of CH ₂ Cl _2, TNF was added to the aqueous layer together with a sufficient amount of NaCl to saturate the layer. However, the dissolution of the product was not achieved, which necessitated the use of CH ₂ Cl ₂ .

The combined layers of toluene, TNF and CP ₂ Cl _{2 were} combined and concentrated in a continuous concentrator. The residue was washed with 7 L of CH3CN. At the end, 1 0 L of CH3CN was added, and the solution was allowed to stand overnight in an N2 atmosphere.

Example 13B

Spiroindolin-methanesulfonamide (free base form) (1 b). Materials:

CDZ-Spiroindoline-sulfonamide (1) 3 kg (7.49 moles) Darco G-60 600 kg Ethyl acetate 36 l Absolute ethanol 189 l 10% Pd / C 450 g Ammonia solution 500 ml Solka-floc ™ 2.5 kg Isopropyl acetate 65 l

A mixture of CBZ-Spiroindoline (1) (1 kg) and Darco G-60 (200 g) in ethyl acetate (9 L) was stirred and heated at 60-65 ° C under nitrogen for 8 hours. Darco was removed by filtration at 60-65 ° C, the solid was washed with ethyl acetate (3 L), and the filtrate and washes were combined. LC weight / weight analysis confirmed a slight loss of Darco. The ethyl acetate solution was evaporated to dryness in vacuo using a 20 L Buchi apparatus, and then washed with absolute ethanol (2 x 5 L). This material was then suspended in absolute ethanol (8 L), heated to 65-70 ° C and placed in a 20 L autoclave. The batch was then irrigated with absolute ethanol (1 L). A suspension of 10% palladium on charcoal (75 g, 7.5% by weight) in absolute ethanol (750 ml) was then added to the autoclave and irrigated inside with an additional portion of absolute ethanol (250 ml).

The batch was then hydrogenated at 65 ° C with vigorous stirring at a pressure of hydrogen of 2.8 kg / cm ² for 3 h, a second portion of 10% palladium on charcoal (75 g) was added, the batch was hydrogenated for an additional 2 h and hermetically sealed closed for the night. The batch was transferred (still hot, 60-65 ° C) to a Buchi apparatus and degassed in vacuo, removing formic acid by feeding and draining absolute ethanol (total 1 8 L).

The procedure was repeated twice and three loading batches were combined in a 38-liter reactor with an inner glass coating and the combined batch was again degassed by adding and distilling (in vacuum) absolute ethanol (2x10 L). Solkafloc ™ (0.5 kg) was added to the batch and internally irrigated with ethanol (10 L). The Estrella Solkafloc ™ filter (2 kg) was loaded as a suspension in ethanol (20 ml). The resulting mixture was heated to 60-65 ° C and then passed at this temperature through a heated filter using a pump into two stainless steel hoppers. The initial reactor, filter, pump and pipelines were washed with a hot (60-65 ° C) mixture of aqueous ammonia (500 ml) in absolute ethanol (25 L). The filtrate and washes were combined in two stainless steel hoppers.

The batch was then transferred to the vessel using an in-line filter, with a 10 micron cartridge inserted, then concentrated in vacuo to a small volume (~ 1 5 L). Ethanol was replaced with isopropyl acetate by feeding and flowing out 3 loading volumes of isopropyl acetate (45 l in total), maintaining a loading volume of ~ 1 5 l. After completion of the solvent replacement, upon completion, the charge contained <1% according to GC. The batch was then diluted to ~ 33 L by adding isopropyl acetate (20 L) and this solution of spiroindoline-amine 1 b (1, 855 kg by LC analysis) in isopropyl acetate was used in the next stage of the process.

Boc-0-Benzylserine Spiroindoline (11).

Materials:

Spiroindoline (1b) 1587 g (5.966 moles)

Amino acid (10) 1938 g (6.563 moles

P _h ^ oY ^C ° ^2H

NHBOC

DCC 1334.5 g (6.563 moles) Hobt 884 g (6.563 moles) SNsN 25 l 0.5N NaOH 18 l 0.5N HCl 18 l sat. Nil 1CHO 18 l from Prgoas 28 l

Spiroindolin-amine 1b in CH3CN or from PrOAc: H ₂ O (25 L) at ambient temperature under N _{2 was} sequentially treated with HOBT (884 g, 1.1 eq.) As a solid, DCC (1334.5 g, 1.1 equiv.) As a melt (heating in hot water at 60 ° C for about 1 hour) and, finally, amino acid 10 (1938 g) as a solid. The mixture was stirred for 3 hours, after which a heavy precipitate of DCU formed, and LC analysis showed approximately 0.5 A% of the remaining amine lb. IP Ac (9 L) was added, the slurry was filtered through Solka-floc ™ and washed with IPAc (19 L). The combined organic solution was washed successively with 0.5 N NaOH (18 L), 0.5 N HCl (18 L) and saturated HNO (O (18 L). The last wash with water resulted in an emulsion and was therefore excluded.

The organic layer was concentrated in vacuo, and the residue was dissolved in MeOH or EtOH (10 L final volume). Experimental yield 3026 g (89%).

The use of alternative coupling peptide agents, such as carbonyldiimidazole, or the formation of mixed anhydrides such as sec-butyl carbonate, gave a lower yield of substance 11 and / or 1 4 with a high degree of epimerization in the case of the first compound. Other peptide coupling agents were not available due to the high cost.

Boc-0-Benzylserine Spiroindoline (11). Materials:

Spiroindolin-amine (1b) 1,855 kg (6.96 moles) Isopropyl acetate 29 l Dicyclohexylcarbodiimide (DCC) 1.58 kg (7.65 moles) 1-Hydroxybenzotriazole (HOBt) 1.03 kg (7.62 moles) ^ Boc-0-benzyl-O-Serine 2.26 kg (7.65 moles) 1M aqueous solution of sodium hydroxide 26 l 0.5 M aqueous hydrochloric acid 26 l Sat. aqueous solution of sodium bicarbonate 26 l Absolute ethanol 50 l

Water (20 L) was added to a stirred solution of spiroindolin-amine 1 b (1, 855 kg) in isopropyl acetate (33 L) in a reaction vessel, then the following chemicals were successively added at room temperature under a nitrogen atmosphere: DCC (1.58 kg, 1.1 eq.), HOBt (1.03 kg, 1.1 eq.) And, at the end, N-Boc-0 benzyl-O-Serine (2.26 kg, 1.1 eq.). The reagents were washed off with isopropyl acetate (7 L). The batch was stirred at room temperature under a nitrogen atmosphere for 5 hours when LC showed a product / starting material ratio of 99.4 / 0.6. The mixture was then filtered into another vessel using a pump through an Estrella filter using only cloth and cardboard. The vessel from which the mixture was removed was rinsed with isopropyl acetate (22 L) and the filter, pump and hoses connecting to the receiving vessel were rinsed with it. The 2-phase mixture was stirred in a vessel for 10 minutes and then allowed to settle for 15 minutes. The lower aqueous layer was separated and the organic layer was allowed to settle overnight at room temperature.

The next day, the organic layer was washed with a 1M aqueous sodium hydroxide solution (26 L), then with a 0.5M aqueous hydrochloric acid solution (26 L), and finally a saturated aqueous sodium hydrogen carbonate solution (26 L). LC analysis showed an experimental yield of 3.787 kg, 93% of the total yield, based on 7.49 moles (3 kg) of the starting CBZspiroindoline (1). The batch was concentrated in vacuo (internal temperature = 13-15 ° C, casing temperature = 40 ° C, vacuum = 36 mm) to a small volume (~ 15 L) and the solvent was replaced with ethanol by feeding and flowing ethanol (50 L) while keeping the volume at ~ 15 L, GC showed <1% of the remaining isopropyl acetate. This solution was used in the next step of the process.

0-Benzylserine spiroindoline (free base form) (1 2). Materials:

Boc-0-Benzylserine Spiroindoline (11) 3026 g (5.57 moles) Methanesulfonic acid ^ sOH) 1.16 L (17.9 mol) Meon 10 l from Prgoas 24 l 0.5N NaOH 35 l

Boc-0-Benzylserine spiroindoline 11 in 10 L of MeOH (or EtOH) was treated with pure Ms OH (1.16 L), which was added at 45 for approximately 30-40 minutes (initial temperature 16 ° C, final temperature 28 ° FROM). The dark red solution was kept overnight in an atmosphere of N ₂ . The mixture was then pumped into a 100 L extractor containing 24 L of PrOAc and 35 L of 0.5N NaOH. The pH of the aqueous layer is 7. NaOH (6M) was added until the pH became> 10.5. As the pH increased, the solution changed color from red to yellow. The layers were separated, and the organic layer, according to NMR, contained 13 mol% MeOH in iso PrOAc [5 vol.%]. LC analysis of 2.48 kg.

0-Benzylserine spiroindoline (free base form) (1 2). Materials:

Boc-0-Benzylserine Spiroindoline (11) 3,787 kg (6.96 moles) Methanesulfonic acid 2.006 kg (20.87 moles) Isopropyl acetate 38 l 1M aqueous solution of sodium hydroxide 16 l 50% aqueous solution of sodium hydroxide 1,6 l

Methanesulfonic acid (2.006 kg, 1.355 L ~ 3 equivalents) was added to a stirred solution of Boc-0-benzylserine spiroindoline (11) (3.787 kg) in ethanol (total volume ~ 15 L) in the reaction vessel. The loading batch was heated to 35-40 ° C. After 7 hours, LC showed the absence of starting material and the reaction mass was allowed to cool to room temperature overnight. The next day, water (44 L) was added to the batch while stirring. The batch was cooled to ~ 5 ° C, stirred for 30 min, and then filtered through an in-line filter (with a 10 μm cartridge inserted) into the hopper. Then the batch was pumped back into the vessel. The vessel and tubes leading to the hopper were rinsed with water (10 L), and the liquid was pumped with the same volume after rinsing back into the vessel. Isopropyl acetate (38 L) was added, followed by a 1M aqueous sodium hydroxide solution (16 L). The batch was cooled to 10-15 ° C, it was established that the pH of the lower aqueous layer was ~ 7 and 50% aqueous sodium hydroxide solution (1.6 L) was added (pH> 10). The batch was stirred at 10-1 5 ° C for 25 minutes and then allowed to settle for 10-15 minutes. The lower aqueous layer (78.1 kg) was separated. LC sample showed that in aqueous solutions contained 28.4 g of substance 1 2 (0.85% of theory). The volume of the organic solution was 51 L. An LC sample showed 3.057 kg, 92% of the total yield of 3 kg, 7.49 moles of CBZspiroindoline sulfonamide (1). This solution was used in the next step.

Boc-aminoisobutyryl 0-benzylserine spiroindoline (1 4).

Materials:

Spiroindoline amine (12) 2481 g (5.57 moles)

Amino acid peptide (13) 1247.1 g (6.16 mol) CH ₃ CH ₃ HOgC ^ SjH-BOC

DCC 1266.7 g (6.16 moles novt 827 g (6.16 moles) IPAc 52 l H2O 37 l 0.5N NaOH 36 l 0.5N HC1 36 l sat. No. 11CO ₃ 36 l

A solution of amine 1 2 in IPAc was diluted to a total volume of 39 L using IPAc and 37 L of H ₂ O was added. The two-phase mixture was then treated sequentially with HOBT (827 g) as a solid, DCC (1266.7 g) as a melt and amino acid 1 3 at ambient temperature in the atmosphere. The reaction mixture was stirred for 2 hours, after which LC analysis showed the disappearance of starting material 1 2 (<0.3 A%). The mixture was filtered with Solka-floc ™ and the solids were washed with 1 3 L IPAc. The material can at this stage be stored as a two-phase mixture overnight.

The mixture was transferred to a 1 00 L extractor, the aqueous layer was separated, and the organic layer was washed sequentially with 36 L of 0.5 N NaOH, 0.5 N HCl and saturated NO. HX) ₃ . The experimental yield of 3160 g (81% of spiroindoline ± 5% per volume measurement error). The solution was concentrated to a small volume and washed with ethanol (2x4 L). If desired, intermediate 1-4 can be isolated by adding water to crystallize.

The use of alternative coupling peptide agents, such as carbonyldiimidazole, or the formation of mixed anhydrides, such as sec-butyl carbonate, gave a lower yield of substance 1 with a high degree of epimerization. Other peptide coupling reagents were not available due to the high cost.

Vos-aminoisobutyryl 0-benzylserine spiroindoline (14) Materials:

Spiroindolin-amine (12) 3,057 kg (6.89 moles) Dicyclohexylcarbdiimide (DCC) 1.56 kg (7.56 moles) 1-Hydroxybenzotriazole (HOBt) 1.02 kg (7.55 moles) Boc-2-aminoisobutyric acid (13) 1.54 kg (7.58 moles) Isopropyl acetate 32 l 1M aqueous solution of sodium hydroxide 38 l 0.5 M aqueous hydrochloric acid 38 l Sat. aqueous solution of sodium bicarbonate 38 l Absolute ethanol 45 l

Water (49 L) was added to a stirred solution of spiroindoline amine 12 (3.057 kg) in isopropyl acetate (total volume ~ 51 L) in a reaction vessel at room temperature under a nitrogen atmosphere. The following chemicals were then used sequentially: DCC (1.56 kg, ~ 1.1 eq.), HOBt (1.02 kg, ~ 1.1 eq.), And finally N-Boc-2-aminoisobutyric acid 13 ( 1.54 kg, ~ 1.1 equiv.). The mixture was vigorously stirred at room temperature for 2 hours until LC showed the completion of the reaction. The mixture was filtered into another vessel through an Estrella filter using a pump. Isopropyl acetate (22 L) was used to rinse the vessel, filter, pump and hoses connecting to the receiving vessel. The 2-phase mixture was then stirred for 5 minutes and the layers allowed to separate. The lower aqueous layer was separated without difficulty (weight of aqueous solutions = 51.1 kg). The organic solution was then washed successively with a 1 M aqueous solution of sodium hydroxide (38 L), 0.5 M aqueous hydrochloric acid (38 L) and, finally, without difficulty, a saturated aqueous solution of sodium hydrogen carbonate (38 L).

The organic solution was then transferred via a pump through an in-line filter (containing a 10 μm cartridge) to another vessel to replace the solvent with ethanol. The vessel was rinsed with isopropyl acetate (10 L) and it was also used to rinse the pump, filter and tubes connecting with the receiving vessel. The filtrate and washes were combined. Total volume = 75 l (using a measuring rod). An LC sample yielded 4.395 kg of Vosaminoisobutyril 0-benzylserine spiroindoline (14), i.e. 93% of the total 7.49 moles of the starting CBZ-spiroindoline sulfonamide (1).

The batch was concentrated in vacuo to a small volume (~ 1 5 L) and isopropyl acetate was replaced with ethanol by feeding and draining absolute ethanol (45 l in total). At the end of the solvent change, GC analysis showed <1% residual isopropyl acetate. This solution (25 L) containing 4.395 kg of substance 1 4 was used in the next step. Optionally, intermediate 1-4 can be isolated by adding water for crystallization.

Amino isobutyryl 0-benzylserine spiroindoline (15).

Materials:

Voe Spiroindolin (14) 3160 g (5.03 moles) Methanesulfonic Acid (MsOH) 979 ml (15.1 moles EtOH 6.2 L H2O 30 l IN NaOH 11 l EtOAc 26 g Activated Carbon Darco 60 1 kg

Boc-Spiroindoline 14 was dissolved in 6.2 L EtOH and treated with MsOH (979 ml). The temperature increased from 20 to 30 ° C and the reaction was allowed to proceed overnight. After 1 2 hours at 20 ° C, 1 5 A% of the starting material remained, and the mixture was heated to 35 ° C for 6 hours. Upon completion (<0.1 A% 1 4), the reaction was cooled to 20 ° C and 30 l of H ₂ O, and the solution was filtered through a glass funnel with a polypropylene filter and the DCU residue was filtered off. The mixture was transferred to a 1 00 L extractor and 26 L EtOA was added. The aqueous layer was made basic by the addition of chilled 1 N NaOH (11 L) and 1 L 50% NaOH. To confirm temperatures below 1 ° C, ice was required. Higher temperatures led to greater difficulties associated with emulsification.

The organic layer was distilled at 50 ° C. at approximately 533 Hg to KF <1000 μg / ml. Lower KFs result in more efficient coal treatments and better recovery at the salt formation stage. A KF of 1 60 μg / ml was achieved with a loading of 700 g. The solution was diluted with ethyl acetate to a total volume of 31 L (LC analysis 2.40 kg). Activated carbon was added (Darco G-60) and the mixture was stirred for 24 hours. The mixture was filtered through Solka-floc ™ and the filter cake was washed with ethyl acetate (16 L), assay

2.34 kg.

Amino isobutyryl 0-benzylserine spiroindoline (15).

Materials:

Boc Spiroindoline (14) 4.395 kg (6.99 moles) Methanesulfonic acid 2.017 kg (20.99 moles Ethyl acetate 185 l 1M aqueous solution of sodium hydroxide 16 l 50% aqueous solution of sodium hydroxide 2.6l Darco G-60 900 g Solka-floc ™ 2.5 kg

Methanesulfonic acid (2.017 kg, 1.36 L, ~ 3 eq.) Was added to a stirred solution of Boc Spiroindoline 14 (4.395 kg) in ethanol (total volume ~ 25 L) in a reaction vessel at room temperature. The batch was heated to 35-40 ° C and stirred overnight. The next day, the batch contained ~ 1.1 A of starting material, and the reaction was continued for another 4 hours, after which LC showed a product / starting ratio of 99.6 / 0.4. The batch was concentrated in vacuo to a volume of ~ 15 L and then diluted with water (44 L). The batch was cooled to 5 ° C, stirred for 30 min, and then filtered through a Sparkler in-line filter (containing a 10 μm cartridge) using a pump into another vessel and a small amount of residual DCU was removed.

The vessel, pump, filter and hoses were rinsed with water (10 L), and the same water was added to the vessel. Ethyl acetate (36 L) was added to the vessel and, while stirring the mixture, it was cooled to 10 ° C. A solution of cold (5-10 ° C) 1M aqueous solution of sodium hydroxide (16 L) and cold (5-10 ° C) of a 50% aqueous solution of sodium hydroxide (2.6 L) was added at 10 ° C and the temperature was raised to 1 4 ° C. The resulting mixture was stirred for 15 min at <14 ° C, and then the lower aqueous layer was separated.

The batch was concentrated in vacuo to a volume of ~ 20 L, and then a mixture of ethyl acetate (35 L) and ethanol (5 L) was introduced inside while maintaining the volume at ~ 20 L. At the end of this distillation, KF was 9160 mg / ml ^-1 . Replace the solvent with ethyl acetate by feeding and flowing out ethyl acetate (total 40 L). At the end of the distillation, KF was 446 mg / ml ^-1 . The batch was diluted with ethyl acetate (10 L).

Darco G-60 (900 g) was added to the cloudy mixture. It was washed off with ethyl acetate (6 L). The mixture was stirred at room temperature overnight. The next day, Solka-floc ™ (0.5 kg) was added to the stirred vessel, and then Solka-floc ™ (0.5 kg) in a small amount of ethyl acetate was mixed and loaded into an Estrella filter. Excess solvent was pumped through a Sparkler in-line filter containing a 1.0 μm cartridge. The suspension was removed from the vessel through a filter using a pump, and then through another filter in 2 x 40 L stainless steel bins. A visual inspection showed that the liquids were clear and clean. The vessel was rinsed with ethyl acetate (22 L) and used to rinse the pipe over stainless steel hoppers. The contents of both bins were transferred to a reaction vessel and the solution was thoroughly mixed.

A loading batch (58 L), which had a KF of 2950 mg / ml ^-1 , was again dried, concentrating in vacuo to a volume of 20-25 L. The batch was diluted to a volume of 46 L (measuring rod) by the addition of ethyl acetate (25 L). KF was 363 mg / ml ^-1 . The batch was diluted to a volume of 62 L by adding ethyl acetate (1 7 L) and used in the final stage of the process.

Spiro [3H-indole-3,4'-piperidine] -1'-yl) carbonyl] -2- (phenylmethyl-hydroxy) -2-amino-2methylpropanamide methanesulfonate (1 6).

Materials:

Amine (15) 2340 g (4.43 moles) Methanesulfonic acid (MsOH) 316 g (4.88 moles) EtOAc 60 l EtOH 4.8 L 8% EtOH and EtOAc 20 l

The volume of solution 15 from the previous step was adjusted to 60 L with ethyl acetate and EtOH (4.8 L) was added. MsOH (316 ml) was added in 3 L EtOAc at 45 ° C. To a dark red homogeneous solution, 496 g of seed was added to the title compound of Form I (10% seed was used, by weight of free amine). The temperature rose to about 48 ° C., and the reaction mixture was kept at 52 ° C. for 1.5 hours. Analysis showed complete conversion to the title compound (Form I). (For less than 10% seed, longer (> 3 hours) aging was required. Suspensions were allowed to cool to 20 ° C. overnight and centrifuged under N _{2. The} cake was washed with 20 L of 8% EtOH in EtOAc. the atmosphere is N ₂ during filtration because the wet crystals are very hygroscopic.The batch was dried at 35 ° C in vacuo to give 2.7 kg (56% total yield) of the title compound (Form I) (99.9 A% purity; < 0.1% enantiomer).

The conversion of form II to form I is also carried out, obtaining a salt in EtOAc-EtOH with the addition of M ^ OH, as described above, and the initial salt solution (at 55 ° C) is cooled to 45 ° C. At this temperature, crystals begin to appear, and the suspension becomes thicker over time. The temperature is then raised to 51 ° C. and the suspension is maintained overnight. Complete conversion of compound 16 to Form I is expected. This method can also be used to obtain seed crystals of Form I of Product 1-6.

Spiro [3H-indol-3,4'-piperidine) -1'-yl) carbonyl] -2- (phenylmethyl-hydroxy) ethyl] -2-amino2-methylpropanamide methanesulfonate (1 6).

Materials:

Amine (15) 3.1 kg (5.86 moles) Methanesulfonic acid 620 g (6.45 moles) Ethyl acetate 37 l Absolute ethanol 8.7 L Spiro [3H-indole-3,4'-piperidine] -1 'yl) carbonyl] -2-phenylmethyl-hydroxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate (form I) 70 g (0.11 mol)

Absolute ethanol (6.4 L) was added to a solution of amine (15) (3.1 kg) in ethyl acetate (total volume ~ 62 L) in the reaction vessel. The batch was heated to 50 ° C and a solution of methanesulfonic acid (620 g, 41 2 ml, 1.1 eq.) In ethyl acetate (11 L) was added over ~ 5 min at 50-54 ° C. The batch was seeded with spiro [3H-indole-3,4'-piperidine] -1'-carbonyl] -2- (phenylmethyloxy) ethyl] 2-amino-2-methyl-propanamide methanesulfonate (form I) (70 g) and obtained in as a result, the suspension was stirred and heated at 55 ° C. under nitrogen overnight.

The next day, the suspension was cooled to 15-20 ° C, kept for 2 hours, and then a 50 cm polyethylene filter was added dropwise in a nitrogen atmosphere. The solid was washed with a mixture of absolute ethanol (2.3 L) in ethyl acetate (26 L). The white, solid product was scraped off and dried in an Apex oven in vacuo at 35 ° C for an appropriate period of time (approximately two days). Dried spiro [3H-indole-3,4'-piperidine] - 1'-yl) carbonyl] -2 (phenylmethyloxy) ethyl] -2-amino-2methylpropanamide methanesulfonate (3.352 kg) was sieved using a Jackson-Crockatt sieve to give 3.347 kg (including seed, 70 g), yield = 3.277 kg.

HPLC conditions.

LC retention time on Zorbax RX-C8 (4.6 mm x 25 cm), λ = 210 nm, flow rate = 1.5 ml / min.

Compound 1: 60:40 CH _; C \ '- P _: O (1% H ^ ₄ ) RT = 5.0 min;

Compound 1b: 35:65 CH _; C \ '- P _: O (0.1 wt.% NH4OAc) RT = 6.2 min;

Compound 10: 60:40 CH _; C \ '- H _: O (0.1 H ^ ₄ ) RT = 2.9 min;

Compound 11: 60:40 CH3CH-H2O (0.1% HzPO4) RT = 5.4 min;

Compound 12: 40:60 CH _; C \ '- H _: O [pH 5.25 NaH ₂ PO ₄ (6.9 g / L H ₂ O) (adjusting pH with NaOH)] RT = 5.6 min;

Compound 14: 60:40 CH _; CX-P _: O (0.1% HzPO4) RT = 4.65 min;

Compound 15: 40:60 CH3CN-H2O [pH 5.25 NaH ₂ PO ₄ (6.9 g / L H ₂ O)] pH adjustment with NaOH) RT = 4.9 min;

LC retention time on Zorbax RX-C8 (4.6 mm x 25 cm), λ = 210 nm, flow rate = 1.2 ml / min, column temperature = 48 ° C. Solvent A = 0.05%. Phosphoric acid + 0.01%. Triethylamine in water.

Solvent B - Acetonitrile. Gradient system:

Time min % BUT % IN 0 95 5 35 10 90 38 95 5 40 95 5

Retention time (min.).

Compound 1 25,2 Compound 1 b 8.5 Compound 1 0 20.5 Compound 11 26.3 Compound 1 2 14.8 Compound 1 4 25.6 Compound 1 5 15.7 Example 1 9.

Obtaining the form IN- [1 (R) - (1, 2-dihydro-1-methanesulfonyl-spiro [3H-indole-3,4'-piperidine] - 1'-yl) carbonyl] -2- (phenylmethyl-hydroxy) ethyl] 2-amino-2-methylpropanamide methanesulfonate.

The conversion of form II to form I can be carried out according to the method of example 18A, where a salt is formed in EtOAc-EtOH with the addition of MOH, and the initial salt solution (at 55 ° C) is cooled to 45 ° C. At this temperature, crystals should begin to appear and the suspension will become thicker over time. The temperature is then raised to 51 ° C. and the suspension is maintained overnight. One should expect complete conversion to form 1.

Example 20

Obtaining form I N- | 1 (%) - (1.2-dihydro1-methanesulfonyl-spiro [3H-indol-3,4'-piperidine] - 1'-yl) carbonyl] -2- (phenylmethyl-hydroxy) ethyl] 2-amino-2-methylpropanamide methanesulfonate.

The conversion of form II to form I is carried out by mixing the solution of form II N- | 1 (R) (1,2-dihydro-1-methanesulfonyl-spiro [3H-indole-3,4'-piperidine] -1'-yl) carbonyl] -2 - (phenylmethyl-hydroxy) ethyl-2-amino 2-methylpropanamide methanesulfonate in isopropanol at approximately 25 ° C for approximately 2-24

hours

Example 21

Preparation of Form IV No. [1SH) - (1,2-dihydro-1-methanesulfonyl-spiro | 3H-indol-3,4'-piperidine] -1'-yl] carbonyl] -2- (phenylmethyl-hydroxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate.

Sample 8.4 g ^ [1CH) - (1,2-dihydro-1-methanesulfonyl-spiro [3H-indole-3,4'-piperidine] 1'-yl) carbonyl] -2- (phenylmethyl-hydroxy) - morphological ethyl] -2-amino-2-methylpropanamide methanesulfonate is dissolved in a mixture of 24.8 ml of ethyl acetate, 1.6 ml of ethanol and 1.95 ml of water with stirring at 42 ° C. The solvent was evaporated from the solution at a temperature of 40 ° C., the resulting solid was triturated in a mortar to a fine powder and the fine powder was exposed to a relative humidity of approximately 75% to obtain the title form IV.

Example 22

Preparation of Form IV ^ [1CH) - (1,2-dihydro-1-methanesulfonyl-spiro [3H-indole-3,4'-piperidine] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] -2- amino-2-methylpropanamide methanesulfonate.

Sample N- [1 (R) - (1,2-dihydro-1-methanesulfonyl-spiro [3H-indole-3,4'-piperidine] -1'-yl) carbonyl] -2- (phenylmethyl-hydroxy) ethyl ] Unsolved morphological] -2-amino-2-methyl-propanamide methanesulfonate is recrystallized from a solution of ethyl acetate-ethanol-water (in a volume ratio of 24.8 / 1.6 / 1.95) to obtain the title form IV.

Example 23

Obtaining the form IV ^ [1 Щ) - (1,2-dihydro-1-methanesulfonyl-spiro [3Н-indole-3,4'-piperidine] -1'-yl) carbonyl] -2- (phenylmethyl-hydroxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate.

Suspension of the form I ^ [1 Щ) - (1,2-dihydro1-methanesulfonyl-spiro [3H-indole-3,4'-piperidine] -1'-yl) carbonyl] -2- (phenylmethyl-hydroxy) ethyl] - 2-amino-2-methylpropanamide methanesulfonate in an isopropyl acetate / ethanol mixture (90:10 by volume) containing approximately 2.8% by weight of water was stirred at approximately 25 ° C. overnight and the resulting solid was isolated.

Specialists in this field it is clear that, despite the description of the present invention and its illustration with the help of specific specific inclusions and references, various changes to modifications, substitutions, deletions or additions can be made in methods ways. For example, depending on the various reactions of a mammal being treated for any of the indications using the above compounds of the invention, effective doses different from those given here may be used. Likewise, the observed specific pharmacological reactions may vary, respectively, depending on the particular particular active compound or pharmaceutical carrier available, as well as on the type of composition and route of administration used, and such expected variants or differences in results are considered in accordance with the objects and practical methods of applying the present invention. It is desirable, therefore, that the invention be considered in the range of the claimed claims that follow below and that within reason these points are construed as broadly as possible.

Claims (28)

CLAIM one . The polymorphic form of the compound N [1 (R) - (1,2-dihydro-1-methanesulfonyl-spiro [3H-indole-3,4'-piperidine] -1'-yl) carbonyl] -2 (phenylmethyl-hydroxy) ethyl ] -2-amino-2-methylpropanamide methanesulfonate, designated as Form I, which is characterized by an X-ray powder diffraction pattern with basic reflections at approximately: 6.5; 14.7; 16.9; 17.1; 17.9; 19.5; 21.1; 21.7 and 22.0 ° (2 theta). 2. The polymorphic form according to claim 1, characterized by a melting endotherm with an extrapolated initial temperature of approximately 170 ° C when heated in a differential scanning calorimeter chamber at a heating rate of 10 ° C / min in a nitrogen atmosphere. 3. The polymorphic form according to claim 1, which has a solubility in isopropanol of 4.6 mg / ml 4. The polymorphic form according to claim 1, which has a birefringence in polarized light. 5. Polymorphic form of the compound N [1 (R ') - (1,2-dihydro-1-methanesulfonyl-spiro [3H-indole-3,4'-piperidine] -1'-yl) carbonyl] -2 (phenylmethyl- hydroxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate, designated as Form IV, which is characterized by an X-ray powder diffraction pattern with basic reflections at about: 16.0; 16.2; 18.3; 20.1; 21.0 and 24.2 ° (theta). 6. The polymorphic form according to claim 5, characterized by an endotherm of water loss at a temperature of approximately 45 ° C followed by an endotherm with an extrapolated initial temperature of approximately 129 ° C when heated in a differential scanning calorimeter chamber at a heating rate of 1 ° C / min in a nitrogen atmosphere. 7. The polymorphic form according to claim 5, which contains approximately 3.5 moles of water per mole of N- [1 (R) - (1,2-dihydro-1-methanesulfonyl-spiro [3H-indole-3,4'-piperidine ] -1'-yl) carbonyl] -2 (phenylmethyl-hydroxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate. 8. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of a polymorphic form according to claim 1. 9. A pharmaceutical composition useful for the treatment of osteoporosis, which comprises a combination of a bisphosphonate compound and a polymorphic form according to claim 1. 10. The pharmaceutical composition according to claim 9, where the bisphosphonate compound is alendronate. 11. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of a polymorphic form according to claim 5. 1 2. A pharmaceutical composition useful for the treatment of osteoporosis, which comprises a combination of a bisphosphonate compound and a polymorphic form according to claim 5. 13. The pharmaceutical composition of claim 12, wherein the bisphosphonate compound is alendronate. 1 4. A method for increasing levels of endogenous growth hormone in a human or animal, which comprises administering to such a human or animal an effective amount of a polymorphic form according to claim 1. 1 5. A method for increasing levels of endogenous growth hormone in a human or animal, which comprises administering to such a human or animal an effective amount of a polymorphic form according to claim 1 and an additional growth hormone secretion stimulator. 16. The method according to clause 15, where the additional stimulator of secretion of growth hormone is selected from the group comprising a peptide that releases growth hormone GHRP-6; growth hormone peptide GHRP-2; growth hormone peptide GHRP-1; BHT920; growth hormone release factor; growth hormone release factor analogue; IGF-1 and IGF-2. 1 7. A method of increasing feed efficiency, enhancing growth, increasing milk production and improving the quality of carcasses of livestock, comprising administering to such pets an effective amount of a polymorphic form according to claim 1. 1 8. A method of treating or preventing conditions selected from the group including: osteoporosis; catabolic disease; immunodeficiency, including that in individuals with depression, the ratio of T ₄ / T ₈ cells; hip fracture; musculoskeletal disorders in the elderly, growth hormone deficiency in adults and children; obesity; cachexia and protein loss associated with a chronic disease such as AIDS or cancer; treatment of patients recovering from extensive surgical interventions, wounds and burns in patients in need of such treatment, which includes the introduction to the patient an effective amount of the polymorphic form according to claim 1. 19. A method of treating osteoporosis, which comprises administering to a patient with osteoporosis a combination of a bisphosphonate compound and a polymorphic form according to claim 1. 20. The method according to claim 19, where the bisphosphonate compound is alendronate. 21. A method of increasing levels of endogenous growth hormone in a human or animal, which comprises administering to such a human or animal an effective amount of a polymorphic form according to claim 5. 22. A method of increasing levels of endogenous growth hormone in a human or animal, which comprises administering to such a human or animal an effective amount of a polymorphic form according to claim 1 and an additional growth hormone secretion stimulator. 23. The method according to item 22, where the additional stimulator of secretion of growth hormone is selected from the group comprising a peptide that releases growth hormone GHRP-6; growth hormone peptide GHRP-2; growth hormone peptide GHRP-1; BHT920; growth hormone release factor; growth hormone release factor analogue; IGF-1 and IGF-2. 24. A method of increasing feed efficiency, enhancing growth, increasing milk production and improving the quality of carcasses of livestock, comprising administering to such pets an effective amount of a polymorphic form according to claim 5. 25. A method for treating or preventing conditions selected from the group including: osteoporosis; catabolic disease; immunodeficiency, including that in individuals with depression, the ratio of T4 / T8 cells; hip fracture; musculoskeletal disorders in the elderly, growth hormone deficiency in adults and children; obesity; cachexia and protein loss associated with a chronic disease such as AIDS or cancer; treating patients recovering from extensive surgical interventions, wounds and burns in patients in need of such treatment, which includes administering to the patient an effective amount of a polymorphic form according to claim 5. 26. A method of treating osteoporosis, which comprises administering to a patient with osteoporosis a combination of a bisphosphonate compound and a polymorphic form according to claim 5. 27. The method according to p, where the bisphosphonate compound is alendronate. 28. The method for producing the polymorphic form according to claim 1, designated as Form I, which comprises stirring a solution of Form II N [1 (R) - (1,2-dihydro-1-methanesulfonyl-spiro [3H-indole-3,4 ' -piperidine] -1'-yl) carbonyl] -2 (phenylmethyl-hydroxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate in isopropanol at about 25 ° C for about 2-24 hours