SK51398A3 - Polymorphic forms of n-[1(r)-[(1,2-dihydro-1- -methanesulfonylspiro[3h-indole-3,4'-piperidin]-1'-yl)carbonyl]- -2-(phenylmethyloxy)ethyl]-2-amino-2-methyl-propanamide- -methanesulfonate, the preparation thereof, pharmaceutical composition containing them and their use - Google Patents

Abstract

This invention is concerned with polymorphic forms of the compound N-[1(R)-[(1,2-dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate which is a growth hormone secretagogue that is useful in food animals to promote their growth thereby rendering the production of edible meat products more efficient, and in humans, to treat physiological or medical conditions characterized by a deficiency in growth hormone secretion, and to treat medical conditions which are improved by the anabolic effects of growth hormone. The instant polymorphic forms have advantages over the other known forms of N-[1(R)-[(1,2-dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenylmethyloxy)-ethyl]-2-amino-2-methylpropanamide methanesulfonate in terms of thermodynamic stability and suitability for inclusion in pharmaceutical formulations. The present invention is also concerned with processes for preparing these polymorphic forms, pharmaceutical formulations comprising these polymorphic forms as active ingredients and the use of the polymorphic form of the compound and their formulations in the treatment of certain disorders.

Description

Polymorphic forms of N- [1 (R) - [(1,2-dihydro-1-methanesulfonyl-spiro [3H-indole-3,4'-piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] 2-amino-2-methyl-propanamide methanesulfonate, process for their preparation, pharmaceutical composition containing them and their use

Technical field

The invention relates to polymorphic forms of growth hormone secretagogues useful for the production of agents for accelerating the growth of animals and in various combinations for other purposes, for example for the treatment of osteoporosis.

BACKGROUND OF THE INVENTION

Growth hormone, which is secreted by the pituitary gland, promotes the growth of all tissues in the body that are still capable of growth. In addition, growth hormone is known to have the following basic effects on metabolic processes in the body:

1) increases the rate of protein synthesis in cells,

2) reduces the rate of utilization of carbohydrates in cells,

3) Increases the mobilization of free fatty acids and the use of fatty acids to obtain energy.

If growth hormone is deficient, various disorders can arise, one of the most important being dwarf.

A variety of methods are known for releasing growth hormone. For example, it is possible to indirectly induce growth hormone release by chemicals such as arginine, L-3,4-dihydroxyphenylalanine, L-DOPA, glucagon, vasopressin, or insulin hypoglycemia can be induced by insulin, in addition, growth hormone release also increases sleep and exercise. These effects are indirectly induced via the hypothalamus, either by decreasing the secretion of somastatin or by increasing the secretion of the known hormone GRF, a growth hormone releasing hormone or an unknown hormone with a similar effect.

In cases where it is desirable to increase the level of growth hormone in the body, hitherto this problem has usually been solved by administering exogenous growth hormone, by administering GRF or other peptide agents that stimulate the production and / or release of growth hormone. In all cases, due to the peptide nature of the compounds, it is necessary to administer the compounds by injection. The initial source of growth hormone was the extraction of pituitary glands from dead bodies. The product thus obtained was very expensive and, moreover, its use was associated with the risk of transmission of various diseases. Recently, a recombinant growth hormone has been available, with the use of which is now associated with the risk of disease transmission. However, it is still a very expensive product that needs to be injected or sprayed into the nose. In addition, other agents have been developed that promote the release of endogenous growth hormone.

In particular, they are the spiro compounds described in U.S. Pat. No. 5,536,716, PCT Patent Application WO 94/13696 and Proc. Natl. Acad. Sci. USA, 92, 7001-7005, July 1995 as non-peptide agents promoting growth hormone release. The compounds have the ability to stimulate the release of natural endogenous growth hormone and can therefore be used to treat conditions in which growth hormone production or secretion needs to be stimulated, e.g. waves where stimulation of growth hormone production leads to the acquisition of larger and more productive livestock.

Preferred compounds described in the publications include, for example, spiro [3H-indole-3,4'-piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate of formula

-3 ^;

U.S. Pat. No. 5,536,716 and PCT Patent Application WO 94/13696 disclose a method for producing said substance (Examples 18, 19 and 55). In particular, Example 55 states that the compound has a melting point of 166-168 ° C after recrystallization from a mixture of ethyl acetate, ethanol and water. This substance was later identified as one of the polymorphic forms referred to as Form II. In Proc. Natl. Acad. Sci. USA, 92, 7001-7005, July 1995, it is reported that this monohydrate-isolated substance has a melting point of 168-170 ° C, but only a general procedure for the preparation of the substance is given and no indication is given as to how it was obtained. crystallization.

The morphological forms of the active substances may be important in the production of suitable dosage forms, given that, if the morphological form is not stable during clinical and stability studies, it is not possible to compare the substances used from different batches. In the case of the manufacture of a pharmaceutical active substance for further use, it is important to identify its morphological form in each dosage form so that it can be verified that the same form and the same amount of active substance are present in each dosage form. For these reasons, it is first necessary to ascertain whether the active substance is present in a single form or in several morphological forms. In addition, some morphological forms may have higher thermodynamic or hygroscopic stability and may be more suitable for use in pharmaceutical compositions than other forms. The term "polymorphic form" of a chemical compound means the same chemical in different crystalline configurations.

SUMMARY OF THE INVENTION

The present invention provides polymorphic forms of N- [1- (R) - [(1,2-dihydro-1-methanesulfonylspiro [3H-indole-3,4'-piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] 2-amino-2-methylpropanamide methanesulfonate, a process for the preparation of these polymorphic forms, and a pharmaceutical composition containing them.

The pharmaceutical compositions contain the aforementioned substance as its active ingredient and are useful in various diseases. The polymorphic forms of the invention promote the secretion of growth hormone and can therefore be used in the treatment of growth hormone

For example, in humans, these forms can be used in cases where growth hormone secretion is too low and to treat disease states that can be ameliorated by the anabolic action of growth hormone.

The polymorphic forms of the invention have the advantage over other known forms of this compound that they have a higher thermodynamic stability and are therefore more suitable for use in pharmaceutical compositions.

N- [1 (R) - [(1,2-dihydro-1-methanesulfonylspiro [3H-indole-3,4'-piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] - 2-Amino-2-methylpropanamide methanesulfonate can be represented by the formula

It is a substance that promotes the secretion and release of growth hormone in humans and other animals. This feature can be used to promote the growth of livestock and meat animals for the treatment of conditions where growth hormone secretion is reduced and also to improve conditions where the anabolic effects of growth hormone can be utilized.

Polymorphic forms, hereinafter referred to as Form I, Form II, Form III, Form IV, Form V, Form VI, Form VII, Form VIII, Form IX, and Form X, have more favorable properties compared to other crystalline N- [1] forms. - (R) [(1,2-dihydro-1-methanesulfonylspiro [3H-indole-3,4'-piperidin] -r-yl) carbonyl] -2- (phenylmethyloxy) ethyl] -2-amino-2- methylpropanamide methanesulfonate and are therefore more suitable for

- use in pharmaceutical compositions. A preferred crystalline form for pharmaceutical use is Form I because of its thermodynamic stability and that it is not hygroscopic. Another preferred crystalline form for pharmaceutical use is Form IV, which is particularly suitable for use in the manufacture of compressed tablets. Form IV has a higher bulk density than the other forms.

The present invention also provides a process for the preparation of Form I of N- [1- (R) - [(1,2-dihydro-1-methanesulfonylspiro [3H-indole-3,4'-piperidin] -1'-yl) carbonyl] -2- ( phenylmethyloxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate, the process consisting in the formation of a solution of free N- [1- (R) - [(1,2-dihydro-1-methanesulfonylspiro [3H-indole-3,4] 1'-piperidin] 1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] -2-amino-2-methylpropanamide in ethyl acetate containing about 8% by volume of ethanol is treated with about 1.1 equivalents of methanesulfonic acid at about 50 ° C, The mixture is heated to about 55 ° C and then cooled to about 45 ° C.

Alternatively, the temperature can then be raised to about 51 ° C and maintained at this temperature for 2 to 24 hours.

The present invention also provides a further process for the preparation of the above-mentioned Form I, which comprises adding to a solution of free N- [1- (R) - [(1,2-dihydro-1-methanesulfonylspiro [3H-indol-3]). 4'piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] -2-amino-2-methylpropanamide in ethyl acetate containing approximately 8% by volume of ethanol at 50 to 55 ° C alternately adds approximately 1, 1 equivalent methanesulfonic acid and Form I N- [1- (R) - [(1,2-dihydro-1-methanesulfonylspiro [3H-indole-3,4'-piperidin] 1'-yl) carbonyl] -2- ( phenylmethyloxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate, wherein the order of addition is not critical, then the mixture is heated to about 55 ° C for about 2 to 15 hours, then cooled to about 25 to 30 ° C and at this temperature Allow to stand for 2 to 3 hours.

The invention also provides another possible process for the preparation of the above-mentioned Form I by mixing a solution of Form II N- [1- (R) - [(1,2dihydro-1-methanesulfonylspiro [3H-indole-3,4] 1-piperidin] -1'-yl) carbonyl] -2- (phenylmethyl-6oxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate in isopropanol at about 25 ° C for 2 to 24 hours.

The present invention also provides a process for the preparation of Form II of N- [1- (R) - [(1,2-dihydro-1-methanesulfonylspiro [3H-indole-3,4'-piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate, which comprises treating a solution of free N- [1- (R) - [(1,2-dihydro-1-methanesulfonylspiro [3H-indole- 3,4 * -piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] -2-amino-2-methylpropanamide in ethyl acetate containing about 8% by volume of ethanol is treated with about 1.1 equivalents of methanesulfonic acid at temperature about 50 ° C, then the solution is warmed to about 55 ° C, then cooled to room temperature.

The present invention also provides a process for the preparation of Form IV of N- [1- (R) - [(1,2dihydro-1-methanesulfonylspiro [3H-indole-3,4'-piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate by dissolving N- [1- (R) - [(1,2-dihydro-1-methanesulfonylspiro [3H-indole-3,4] '-piperidin] 1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate in one of the morphological forms in a solution in ethanol / water, preferably in a 25:75 v / v ratio solvent The solid is ground to a fine powder and the solid obtained is allowed to stand at a relative humidity of about 75%. Another possible process for the preparation of Form IV of N- [1 (R) - [(1,2-dihydro-1-methanesulfonylspiro [3H-indole-3,4'-piperidin] -1'-yl)] carbonyl] -2- (phenylmethyloxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate by recrystallizing a solution of N- [1- (R) - [(1,2-dihydro-1-methanesulfonylspiro) [3 H -indole-3,4'-piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate of any morphological composition from a mixture of ethyl acetate, ethanol and water, preferably in a volume ratio of 24.8: 1.6: 1.95.

Another aspect of the invention is another method for producing Form IV N- [1- (R) - [(1,2-dihydro-1-methanesulfonylspiro [3H-indole-3,4'-piperidin] -1'-yl) carbonyl] -2 - (phenylmethyloxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate, characterized in that Form I is exposed to a relative humidity of greater than 75%, preferably

By applying a relative humidity of about 88% at room temperature for a sufficiently long time.

The present invention also provides another process for the preparation of Form IV of N- [1- (R) - [(1,2-dihydro-1-methanesulfonylspiro [3H-indole-3,4'-piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate, which is isolated from a suspension of Form I of this substance in a 90:10 mixture of isopropyl acetate and ethanol containing approximately 2.8% by weight of water at about 25 ° C.

The present invention also provides a process for the preparation of Form V of N- [1- (R) - [(1,2-dihydro-1-methanesulfonylspiro [3H-indole-3,4'-piperidin] -1'-yl) carbonyl] -2- ( phenylmethyloxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate by exposing Form IV to a relative humidity of less than 30% at room temperature.

The present invention also provides a process for the preparation of Form VI of N- [1- (R) - [(1,2-dihydro-1-methanesulfonylspiro [3H-indole-3,4'-piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate by drying Form V thereof in the absence of water at about room temperature, for example under nitrogen, dried with molecular sieve at about 25 ° C.

The present invention also provides a process for the preparation of Form VII of N- [1- (R) - [(1,2-dihydro-1-methanesulfonylspiro [3H-indole-3,4'-piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate by isolating it from a suspension of Form I or Form IV thereof in a 90:10 (v / v) isopropyl acetate / ethanol mixture containing: about 1.5% water by weight.

The present invention also provides a process for the preparation of Form VIII of N- [1- (R) - [(1,2dihydro-1-methanesulfonylspiro [3H-indole-3,4'-piperidin] -1'-yl) carbonyl] -2- ( phenylmethyloxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate in which Form VII is dehydrated under an anhydrous inert gas atmosphere such as an anhydrous nitrogen atmosphere for a sufficient period of time.

The present invention also provides a process for the preparation of Form IX of N- [1- (R) [(1,2-dihydro-1-methanesulfonylspiro [3H-indole-3,4'-piperidin] -1'-yl) carbonyl] - 2- (phenylmethyl-oxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate,

If a solution of this substance in water of any morphological composition is prepared, then a solid is isolated which is formed by controlled evaporation of the solution at a relative humidity of 20% at about room temperature.

The present invention also provides a process for the preparation of Form X of N- [1- (R) - [(1,2dihydro-1-methanesulfonylspiro [3H-indole-3,4'-piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate by drying Form IX of said compound at room temperature and ambient humidity for a sufficiently long time.

The present invention also provides another possible process for the preparation of Form X of N- [1 (RH, 4'-dihydro-1-methanesulfonylspiro [1H] indol-5'-piperidin-1'-ylcarbonyl] -1'-phenyl-methyloxy) ethyl] -2-amino- 2-methylpropanamide methanesulfonate, wherein Form I is exposed to 100% relative humidity for about 1 to 4 days.

The present invention also provides a process for the preparation of morphologically homogeneous N- [1- (R) - [(1,2-dihydro-1-methanesulfonylspiro [3H-indole-3,4'-piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate by any of the above methods.

All of the above new polymorphic forms of N- [1- (R) - [(1,2-dihydro-1-methanesulfonylspiro [3H-indole-3,4'-piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) Ethyl] -2-amino-2-methylpropanamide methanesulfonate are growth hormone secretion enhancers that can be used in livestock farmed to support their growth and thereby achieve greater efficiency in meat production. In humans, these agents can be used in conditions where growth hormone secretion is reduced and also in conditions where the anabolic effect of growth hormone can be utilized. The present invention also provides a pharmaceutical composition comprising the above polymorphic forms as its active ingredient.

Data obtained in DSC differential calorimetry chamber

Form I N- [1- (R) - [(1,2-dihydro-1-methanesulfonylspiro [3H-indole-3,4'-piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl ] -2-amino-2-methylpropanamide methane-9sulfonate at 10 ° C / min in an open vial under nitrogen runs as a single endothermic curve with a maximum temperature of about 180 ° C and an extrapolated temperature of about 170 ° C. ° C, the heat associated with this curve is approximately 53 J / g.

Form II curve N- [1- (R) - [(1,2-dihydro-1-methanesulfonylspiro [3H-indole-3,4'-piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl ] -2-Amino-2-methylpropanamide methanesulfonate at 10 ° C / min in open vessel under nitrogen runs as a single endothermic curve at a maximum temperature of about 174 ° C and extrapolated at a melting point of about 165 ° C, heat associated with this curve is approximately 37 J / g.

Form IV N- [1- (R) - [(1,2-dihydro-1-methanesulfonylspiro [3H-indole-3,4'-piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] curve -2-amino-2-methylpropanamide methanesulfonate, as the temperature rises by 10 ° C / min in an open container under nitrogen, occurs as an endothermic curve, associated with water loss at about 45 ° C and then as the endothermic curve with a maximum temperature of about 134 ° C; extrapolated at a melting point of about 129 ° C relative to the melting of Form VI, the heat associated with this curve is about 23 J / g.

The following are summarized data for DSC, in which the sample was heated while increasing the temperature by 10 ° C / min under nitrogen, the extrapolated temperature at the beginning of the melting of the form is given.

Form I 170 ° C (endothermic curve)

Form II 165 ° C (endothermic curve)

Form VI 129 ° C (endothermic curve).

Form I N- [1- (R) - [(1,2-Dihydro-1-methanesulfonylspiro [3H-indole-3,4'-piperidin] 1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] - 2-Amino-2-methylpropanamide methanesulfonate is an anhydrous substance with a melting point of 169 ° C and a solubility in isopropanol of 4.6 mg / ml.

Form II N- [1- (R) - [(1,2-dihydro-1-methanesulfonylspiro [3H-indole-3,4'-piperidin] 1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] -2 -amino-2-methylpropanamide methanesulfonate is an anhydrous substance having a melting point of 158 ° C and an isopropanol solubility of 12.3 mg / ml.

- 10Form III N- [1- (R) - [(1,2-dihydro-1-methanesulfonylspiro [3H-indole-3,4'-piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl -2-Amino-2-methylpropanamide methanesulfonate is a hydrate characterized by an endothermic curve with water loss at a first temperature peak of 46 ° C, followed by an endothermic curve with melting and little decomposition at extrapolated melting point of 123 ° C.

Form IV N- [1- (R) - [(1,2-Dihydro-1-methanesulfonylspiro [3H-indole-3,4'-piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate is a hydrate characterized by an endothermic curve with water loss at a first temperature peak of 45 ° C followed by an endothermic curve for melting and decomposition with an extrapolated melting point of 129 ° C (probably due to melting and decomposition of Form VI) .

Form IV is a hygroscopic hydrate containing 3.5 moles of water per mole of said compound.

Form V N- [1- (R) - [(1,2-Dihydro-1-methanesulfonylspiro [3H-indole-3,4'-piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate is a hygroscopic hydrate containing one mole of water per ml of the above Form V.

Form VI N- [1- (R) - [(1,2-dihydro-1-methanesulfonylspiro [3H-indole-3,4'-piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] -2-Amino-2-methylpropanamide methanesulfonate is an anhydrous substance with a melting point of 129 ° C.

Form VII N- [1- (R) - [(1,2-Dihydro-1-methanesulfonylspiro [3H-indole-3,4'-piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] The 2-amino-2-methylpropanamide methanesulfonate is a hydrate with a broad endothermic curve with water loss at a peak temperature of 60 ° C followed by an endothermic curve for melting and decomposition at extrapolated melting point 144 ° C (probably due to melting and decomposition of Form VIII).

Form VIII N- [1- (R) - [(1,2-dihydro-1-methanesulfonylspiro [3H-indole-3,4'-piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] -2 -amino-2-methylpropanamide methanesulfonate is an anhydrous substance with a melting point of 144 ° C.

Form X N- [1- (R) - [(1,2-Dihydro-1-methanesulfonylspiro [3H-indole-3,4'-piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] The 2-amino-2-methylpropanamide methanesulfonate is characterized by a broad endothermic curve with water loss at a temperature peak of 49 ° C.

X-ray diffraction, XRPD

X-ray diffraction is widely used to illuminate the molecular structure, crystalline form, and polumorphism of various substances. The tests were performed using the Philips APD3720, an automatic device using a Kalfa copper device. The measurement was performed at 2 to 40 ° (2 aunt), the sample was maintained at room temperature.

For Form I, the following major bend values of 6.5, 14.7, 16.9,

17.1, 17.9, 19.5, 21.1, 21.7 and 22.0 ° (2 aunt).

For Form II, the following major bend values of 4.8, 11.8, 17.5, 19.4, 21.6, 21.9, 22.5 and 2.7 ° (2 aunt) were obtained.

For Form III, the following major bend values of 13.8, 14.1, 18.0, 19.5, 20.1, 20.6, 21.8 and 25.7 ° (2 aunt) were obtained.

For Form IV, the following major bend values of 16.0, 16.2, 18.3, 20.1, 21.0 and 24.2 ° (2 aunt) were obtained.

For Form V, the following major bend values of 14.8, 17.1, 17.6, 19.0, 19.1, 19.4, 20.6, 21.5 and 21.8 ° (2 aunt) were obtained.

For Form VI, the following major bend values of 9.8, 14.0, 14.8,

17.1, 17.6, 19.0, 19.5, 20.6 and 21.6 ° (2 aunt).

For Form VII, the following major bend values of 9.1, 11.3,

17.1, 17.4, 20.0, 22.1 and 24.5 ° (2 aunt).

For Form VIII, the following major bend values of 11.5, 11.6,

18.1, 19.6, 22.5, 24.7 and 24.8 ° (2 aunt).

For Form IX, the following major bend values of 8.0, 12.1, 15.3, 15.8, 19.6, 19.7, 21.1, 22.3 and 23.7 ° (aunt) were obtained.

For Form X, the following major bend values of 15.5, 15.8, 18.0, 18.4, 18.6, 19.4, 20.7, 20.8, 23.9 and 24.8 ° were obtained ( 2 aunt).

The above values show that all of the above forms I to X are different crystalline forms.

The above values show that all forms I to X are different crystalline forms.

Microscopic monitoring

Polymorphic forms were observed at 100X magnification in both normal and polarized light. Form I and Form II form needle particles, and both forms are birefringent in polarized light.

hygroscopicity

The total volatile content (as determined by TGA analysis) for solid samples of Forms I, II, III, and IV after exposure to various controlled humidity values is summarized in the following table. Form I contains 0.79% by weight of water, Form II contains 0.56% by weight of water, Form III contains 4.5 to 5.0% by weight of water, and Form IV contains 9.5 to 10.0% by weight of water. water.

Hygroscopicity was monitored by storing solids in an enclosed space with constant relative humidity. Comparing the anhydrous forms I and II at room temperature, Form II was found to be hygroscopic and absorb large amounts of water from a relative humidity of 65%. The uptake of water in large quantities cannot be demonstrated for Form I, except when stored at a relative humidity of more than 76%. The results obtained are shown in Tables 1 and 2.

Table 1

% RH Form I% change Form II% change 0 -0.02 +0.08 11 -0.05 +0.02 33 - +0,33 47 +0.21 +0.39 65 +0.37 > 10.0 ^a 76 +0,12 > 10, o ^and 100 > 12.0 > 10.0 ^ab

13 ^{and the} sample is converted to a gummy semi-solid ^{b by} conversion to form IV by exposure to ambient humidity

The data show that Form I of said substance is substantially anhydrous.

Table 2

% RH Form I,% change 7 +0.4 22 +0.3 47 +0.5 68 0.6 88 + 12.9 * 100 +23.9 ^X

^x material melts RH = relative humidity

Hydrated forms III and IV of N- [1- (R) - [(1,2-dihydro-1-methanesulfonylspiro [3H-indole-3,4'-piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) Ethyl] -2-amino-2-methylpropanamide was also evaluated by storing at constant relative humidity for 48 hours at room temperature. For Form III, water uptake was observed after 48 hours at a relative humidity of 33%. For Form IV, there was no uptake of water up to a relative humidity of 100%, but there was a loss of hydration water when stored at a relative humidity of 11% or less. The results obtained are summarized in Table 3 below.

- 14Table 3

Moisture absorption at room temperature for 48 hours

% RH Form III,% change Form IV,% change 0 - -7.8 11 -0.15 -4.9 33 +2.23 +0.3 47 +3.99 +0.7 66 - +1.4 76 +3.76 +1.5 100 - +5.3

solubility

The solubility of Form I in distilled water at room temperature is greater than 100 mg / ml. The solubility of Form II in water at room temperature in a solution buffered to pH 4-9 is also greater than 100 mg / ml. Furthermore, the solubility of Form I in a mixture of ethanol and water is not disclosed.

ethanol / water,% > 100 25/75 > 100 50/50 > 90 75/25 > 90 100% ethanol

Temperature stability

The stability of said solid forms was monitored by storing in glass vials with screw lids in the dark. Samples were analyzed

by HPLC, the original substance was analyzed quantitatively. Was used the following procedure: column: Beckman Ultrasphere ODS (250 x 4.6mm, 5µ) mobile phase: 0.1% TEA, pH 4.0, H ₃ PO ₄ : acetonitrile (65:35) flow rate: 1.0 ml / min detection at: 228 nm flow time: 14 minutes column temperature: room temperature material volume: 20 μΙ of N- [1- (R) - [(1,2-dihydro-1-methanesulfonylspiro [3H-] solution) indole-3,4‘-piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] -2- amino-2-methylpropanamide methanesulfonate containing 50 pg / ml.

The results for Forms I and II are given below in% by weight of the initial amount.

Form I -% of initial quantity

° C 6 weeks 17 weeks 40 100.1 98.7 60 100.7 101.3 80 100.7 99.4

Form II -% of initial amount after weeks

° C 1 2 4 8 12 24 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 -

The results obtained show that pure solid forms I and II have good thermal stability.

The method of preparation of the active ingredient can be expressed by the following reaction scheme:

Scheme I

O ^ OBn

H I

BnOCOCI- ^{104 eq} · ( ^COCI) gK ₂ CO ₃ , H ₂ O <cat. DMF

O ^ OBn

W Pd / C, H ₂

DIEA thioanisole

COCI

ABOUT

OBn

NaBH ₄ / MeOH

COOH

ABOUT.

COOH

O ^ OBn N

TFA

y ~ OBn

H

CH = NNHPh

1b

- 17 Scheme 1 - continued

l

SO ₂ Me

Ph ⁽ O ⁾ O ^{C 2 H 10} NHBOC

SO ₂ Me

DCC

HOBT iPrOAc: _H2O

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

A solution of 1b in isopropyl acetate and water is treated with commercially available N-BOC-O-benzyl-D-serine in the presence of dicyclohexylcarbodiimide DCC and 1-hydroxybenzotriazole HOBt. After filtering off the dicyclohexylurea DCU as a by-product, the biphasic filtrate is separated and the organic layer is washed successively with 1M aqueous sodium hydroxide, 0.5M aqueous hydrochloric acid and finally with a saturated aqueous sodium bicarbonate solution. Improved coupling results can be achieved by treating a solution of the free amino acid in a mixture of isopropyl acetate and water with DCC, HOBT, and then adding the amino acid at room temperature and allowing the reaction to proceed for 3-5 hours. The mixture was then concentrated in vacuo and the solvent replaced, using ethanol instead of isopropyl acetate. The solvent is usually changed by adding a new solvent in an amount equal to three times the volume of the mixture and then evaporating the mixture to its original volume.

The BOC group is removed from the compound of formula 11 by treatment with three equivalents of methanesulfonic acid MsOH in ethanol at 35-40 ° C. The solution was then partitioned between isopropyl acetate and 1M aqueous sodium hydroxide solution to give the compound of Formula 12.

This material is reacted with N-BOC-alpha-aminobutyric acid, the reaction proceeds well in a two-phase solvent system, for example isopropyl acetate / water 1: 1 in the presence of 1.1 equivalents DCC and HOBt DCU is filtered off, the layers are separated and layer was washed sequentially with 1M sodium hydroxide, 0.5 M aqueous hydrochloric acid, and saturated aqueous sodium bicarbonate to give the compound of Formula 14.

The solvent is replaced with ethanol so that the Boe group can be cleaved with methanesulfonic acid. Removal of the protecting group from the compound of Formula 14 is more difficult than removal from the compound of Formula 11, using a concentrated solution of methanesulfonic acid in ethanol at a temperature of 35-40 ° C. The reaction mixture is then extracted with ethyl acetate containing sodium hydroxide and sodium hydroxide

The free amine of formula 15 is isolated. The organic layer is washed well with 1N sodium hydroxide to ensure complete removal of the methanesulfonic acid.

The ethyl acetate solution of the free base of formula 15 in ethyl acetate was concentrated in vacuo and then dried aesotropically to a KF value of less than 500 mg / ml by adding two volumes of a 90:10 mixture of ethyl acetate and ethanol and then two volumes of ethyl acetate. The resulting anhydrous, slightly cloudy solution of the free base of Formula 16 in ethyl acetate was treated with 25% Darco G-60 activated carbon at room temperature for 10 hours. The activated carbon is then filtered off to give the free base of formula 15.

The formation of the salt of formula 16 with methanesulfonic acid from the compound of formula 15 is carried out in ethyl acetate by treatment with 1.1 equivalents of MsOH at 50 ° C. The free base of formula 15 is treated with 8% by volume ethanol and one equivalent of water. C until complete dissolution. Cooling to room temperature and stirring the resulting suspension for 4 hours yields a crystalline compound of Formula 16, crystalline Form II, having a solubility in isopropyl acetate of 12 g / ml.

Conversion of Form II to Form I is accomplished by forming the salt in a mixture of ethyl acetate and ethanol as described above, but instead of cooling the initial salt solution from 55 ° C to room temperature, the solution is cooled to only 45 ° C. At this temperature, crystals begin to form and the suspension thickens over time. The temperature is then raised to 51 ° C and the suspension is allowed to stand overnight. In this way, complete conversion to Form I of the compound of Formula 16 occurs.

Preferably, the conversion of Form II to Form I is accomplished by adding seed crystals of Form I to a solution of the free base in a mixture of ethyl acetate and ethanol at 50-55 ° C and then allowing the mixture to stand. One may proceed by contacting the free base of Formula 15 with 1.1 equivalents of methanesulfonic acid in 8% ethanol in ethyl acetate at 50-55 ° C. The mixture was then seeded with approximately 2% by weight of Form I of the methanesulfonate of Formula 16 and then allowed to stand overnight at 55 ° C. The mixture was then cooled to room temperature and allowed to stand for about 2-3 hours. The product was collected by filtration at temperature

-20 under nitrogen, dried at 35 ° C under vacuum and sieved to give the methanesulfonate of Formula 16.

The methanesulfonic acid salt of Formula 16 can also be formed by alternately adding 1.1 equivalents of MsOH and seed crystals of Form I to a solution of the free base in a mixture of ethyl acetate and ethanol at a temperature of about 50 ° C, wherein the order of addition of MsOH and seed crystals critical.

The utility of the polymorphic compounds of the invention as growth hormone secretion enhancers can be demonstrated by known methods, for example, see Smith et al., Science, 260, 1640-1643, 1993, in particular Figs. 2 and the corresponding text in this publication, all polymorphic forms of the invention were effective as growth hormone secretagogues. Thus, it can be considered that the polymorphic forms of the invention increase the secretion of natural growth hormone.

The polymorphic compounds of the invention can be used in vitro as a very good tool for understanding the regulation of growth hormone secretion at the pituitary. In these cases, it is necessary to evaluate a number of factors known or likely to affect growth hormone secretion, such as age, sex, nutrition, glucose, amino acid and fatty acid concentrations, and nutritional status such as starvation is also important. In addition, it is possible to use these substances to evaluate the possible modification of growth hormone release by other hormones. For example, somatostatin has already been shown to inhibit the release of growth hormone. Other hormones whose effect on the release of growth hormone is important and still need to be fully understood include sex hormones such as testosterone, estradiol and progesterone, hormones such as testosterone, estradiol and progesterone, adrenal hormones such as cortisol and other corticoids , epinephrine and norepinephrine, pancreatic hormones and other parts of the gastrointestinal tract, such as insulin, glucagon, gastrin and secretin, some vasoactive peptides such as bombesin, neurokinins, and thyroid hormones such as thyroxine and triiodothyronine. The polymorphic compounds of the invention can also be used to monitor for negative or positive feedback of pituitary hormones, such as growth hormone and endorphin-type peptides, on the pituitary gland.

Modification of growth hormone release. Of particular interest to researchers is the use of these substances to illuminate the subcellular mechanism by which growth hormone release is affected.

The polymorphic compounds of the invention can be administered in vivo to animals, including humans, to release growth hormone. For example, they can be administered to livestock such as pigs, cattle, sheep and the like to accelerate their growth rate and increase their final weight, to improve feed utilization and to increase milk production. In addition, the compounds may be used in vivo as a diagnostic means to directly determine whether the pituitary gland is capable of releasing growth hormone. For example, it is possible to administer the compounds in vivo and to take serum samples before and after administration and to determine the concentration of growth hormone in these samples. By comparing the amount of growth hormone in these samples, it is then possible to obtain a direct picture of the ability of the pituitary to release growth hormone.

As already mentioned, the present invention also provides a pharmaceutical composition comprising, as an active ingredient, at least one polymorphic substance of the invention together with a pharmaceutical carrier or diluent. The pharmaceutical composition may optionally contain yet another active agent, for example an anabolic agent, an antibiotic or an agent for the treatment of osteoporosis, or a corticosteroid for reducing catabolic side effects, or other pharmaceutically active substances which increase the effectiveness and reduce the side effects.

Growth and anabolic agents include: TRH, diethylstilbestrol, estrogens, beta-agonists, theophylline, anabolic steroids, enkephalins, prostaglandins of the E series, compounds of U.S. Pat. U.S. Patent No. 3,239,345, such as zeranol; No. 4,036,979, such as sulbenox or else the peptides disclosed in U.S. Pat. 4,411,890.

Another possible combination is the combination of the compounds of the invention with other growth hormone secretagogues such as GHRP-6 and GHRP-1 described in US 4,411,890 and in published applications WO 89/07110, WO 89/07111 and B-HT920 as well as hexarelin and GHRP-2 according to WO 93/04081, it is furthermore possible to use a growth hormone releasing hormone called GHRH or GRF and its analogs or its own growth hormone and its analogs, as well as somatomedins including IGF-1 and IGF- 2 or α-adrenergic

-22agonists such as clonidine or serotonin as well as 5HTID agonists such as sumitriptan or somatostatin inhibiting or releasing agents such as physostigmine and pyridostigmine. Particularly preferred is the use of the compounds of the invention in combination with growth hormone releasing factor or analogues thereof, IGF-1 or IGF-2. For example, the compounds of the invention may be used together with IGF-1 to treat or prevent obesity. In addition, for example, it is possible to use the compounds of the invention together with retinoic acid to improve muscle and skin conditions during aging.

The pharmaceutical compositions of the invention may be prepared by treating the polymorphic substances of the invention with a pharmaceutical carrier or diluent.

It is known that the possible use of growth hormone is very versatile. Administration of the compounds of the invention may have the same effects as administration of endogenous growth hormone itself. Thus, it is possible to summarize the use of these compounds such that they can be used, for example, to stimulate growth hormone release in the elderly, to treat growth hormone deficiency in adults, to prevent catabolic side effects of glucocorticoids, to treat osteoporosis, to stimulate the immune system, to accelerate wound healing or bone fracture healing, for treating growth retardation, for treating acute or chronic renal failure or insufficiency, for treating small stature, including children with growth hormone deficiency, for treating small stature associated with chronic diseases, for obesity and growth retardation, associated with obesity, for the treatment of growth retardation associated with Prader-Willi syndrome and with Turner syndrome, to accelerate convalescence, and to shorten the hospitalization period in patients with burns or major surgery, such as more system, to treat fetal growth retardation within the uterus and bone dysplasia, to treat neuropathies, to replace growth hormone in stressed patients, to treat osteochondrodysplasias, Noonan's syndrome, schizophrenia, depression, Alzheimer's disease, to treat retardation and retardation, for the treatment of pulmonary dysfunction, including artificial respiration, to reduce catabolic response after surgery, to treat malabsorption syndromes, to reduce tachexia and protein loss in chronic diseases such as cancer or AIDS, to accelerate weight gain and to produce protein in patients with parenteral nutrition, in the treatment of hyperinsulinemia, including nesidioblastosis, in auxiliary treatment in attempts to induce ovulation, and in the prevention and treatment of gastric and duodenal ulcers, to stimulate thymus development and to prevent thymus function with age, for the auxiliary treatment of patients with chronic hemodialysis, for the treatment of patients with immune deficiency and for the promotion of antibody production after vaccination, for increasing the lymphocyte count in humans and in particular for increasing the T ₄ / T ₈ cell ratio in patients with decreased this ratio, for example after traumas such as closed head injuries or infections such as bacterial or viral, in particular in HIV infection, to improve muscle strength, mobility, maintain good skin condition, metabolic homeostasis or renal hemeostasis in the elderly, to stimulate osteoblasts , cartilage growth and to stimulate the immune system in a variety of animals, including aging disorders. Furthermore, these substances can be used to stimulate the growth of wool in sheep, to increase feed utilization, to accelerate growth, to increase milk production and to improve the quality of carcass in livestock. In addition, they can be used to treat diseases and conditions that can be controlled by the anabolic effect of growth hormone.

In particular, the compounds of the invention may be useful in the treatment of osteoporosis, catabolic diseases, immune deficiency including T4 / T8 cell disease, hip fractures, bone and muscle disorders in the elderly, reduced growth hormone concentration in adults or in children, obesity, cachexia and protein loss due to a chronic disease, such as AIDS or malignant tumors, and also to treat patients recovering from major surgeries, injuries or burns

In addition, the novel compounds of the invention can be used in the treatment of diseases caused by, or facilitated by, the corticotropin release factor, in the treatment of disorders related to stress and anxiety, including depression and headache, for the treatment of severe disorders

-24gut, immunosuppression, HIV infections, Alzheimer's disease, digestive system disorders, anorexia nervosa, haemorrhagic stress, withdrawal symptoms after drug or alcohol withdrawal, substance abuse and infertility.

It is known that a variety of known agents are currently used to treat the above-mentioned diseases and conditions. It has been mentioned above that by combining such substances, some of which have been mentioned, together with the novel polymorphic substances of the invention, additional and often synergistic effects can be achieved in achieving increased growth rates, anabolic or other desirable effects of these substances. In these combinations, the known active compounds and the novel compounds according to the invention can be present independently in an amount of one tenth to several times the doses in which they are effective when used individually.

The possibility of a combination treatment to inhibit bone resorption, to prevent osteoporosis, and to accelerate bone fracture healing can be illustrated by the combination of bisphosphonates and compounds of the invention that promote growth hormone secretion. The use of bisphosphonates for this purpose is described, for example, in Hamdy N.A.T., Role of Bisphosphonates in Metabolic Bone Diseases, Trends in Endocrinol. Metab., 4, 19-25, 1993. Biphosphonates suitable for use include, for example, alendronate, tiludronate, dimethyl-APD, risedronate, etidronate, YM-175, clodronate, pamidronate, and BM-210995. In relation to the efficacy of the compounds, the daily dose of bisphosphonates for oral administration is in the range of 0.1 mg to 5 g, and the daily dose of the growth hormone secretagogues according to the invention is in the range of 0.01 mg to 20 mg / kg body weight. doses can be administered to patients for the effective treatment of osteoporosis.

In the case of aiendronate, a daily dose of 0.1 mg to 50 mg per oral administration may be combined with a dose of 0.01 mg to 20 mg / kg body weight of growth hormone secretion enhancing compounds for effective treatment of osteoporosis. In the treatment of osteoporosis and other disorders of the bone system, it is also possible to use the compounds of the invention in combination with calcitonin,

Oestrogens, raloxifene, and calcium replenishers such as calcium citrate.

Anabolic effects, particularly in the treatment of male geriatric illnesses, can be ensured by combining the compounds of the invention with anabolic steroids such as oxymethanol, methyltestosterone, fluoxymesterone, and stanozol.

The compounds of the invention may be administered orally, parenterally, for example, intramuscularly, intraperitoneally, intravenously, subcutaneously by injection or in the form of an implant, further into the nose, vagina, rectum, sublingually or even topically. For this purpose, the compounds of the invention are formulated in a dosage form suitable for each mode of administration.

Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active ingredient is admixed with at least one inert pharmaceutically acceptable carrier, such as sucrose, lactose or starch. These dosage forms may also contain, as is customary in pharmaceutical compositions, other substances in addition to inert diluents, for example glidants such as magnesium stearate. In the case of capsules, tablets and pills, the dosage forms may also contain buffers. In addition, the tablets and pills may be enteric coated.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, elixirs, these forms containing conventional inert diluents, for example water. In addition, they may contain other excipients, such as wetting agents, emulsifying agents, suspending agents, sweetening, flavoring and perfuming agents.

Formulations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, or emulsions. Examples of non-aqueous solvents or vehicles include propylene glycol, polyethylene glycol, vegetable oils such as olive or corn oil, gelatin and organic esters suitable for injection, for example ethyl oleate. These dosage forms may also contain adjuvants such as preservatives, wetting agents, emulsifiers and dispersing agents, the compositions may be sterilized, for example, by filtration through a filter, retaining bacteria, addition of a sterilizing agent, irradiation of the composition or heating thereof.

The compositions may also be supplied as sterile solids, for solution in sterile water or other sterile injectable medium just prior to use.

The compositions for rectal or vaginal administration are preferably suppositories containing in addition to the active ingredient excipients such as cocoa butter or other suppository bases.

Formulations for nasal or sublingual administration may also be prepared using conventional excipients.

The compounds of the invention are administered to humans and other animals at dosages that provide optimal efficacy. Obviously, the dosage may vary in each individual case not only depending on the active ingredient or pharmaceutical form selected, but also on the route of administration, the nature of the disease being treated, the age and general condition of the patient, the nutrition and other factors which may evaluate the expert. The resulting daily dose must always be determined by the attending physician in each case.

The dosage of the active ingredient in the pharmaceutical composition of the invention may vary, but the amount of active ingredient in the composition must be such that a suitable dosage form can be prepared. The dosage selected will depend upon the desired therapeutic effect, the mode of administration, and the total time of administration of the composition. For effective release of growth hormone in humans and other animals, for example mammals, daily dosages in the range of 0.001 to 10 mg / kg body weight, preferably 0.001 to 25 mg / kg, and in particular 0.01 to 10 mg / kg per day are usually administered.

Various methods for preparing polymorphic forms of the invention will be explained in the following examples, examples are intended to illustrate these procedures, but are not intended to limit the scope of the invention in any way.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

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

-27Part A: 1,2-dihydro-1-methanesulfonylspiro [3H-indole-3,4‘-piperidine] hydrochloride

To a solution of 1.20 g (5.8 mmol) of 1‘-methyl-1,2-dihydrospiro [3H-indole-3,4‘-piperidine], the preparation of which is described by H. Ong et al. in J. Med. Chem., 23, 981-986 (1983) in 20 mL of dichloromethane at 0 ° C was added 0.90 mL (6.4 mmol) of triethylamine and 0.49 mL (6.35 mmol) of methanesulfonyl chloride. The reaction mixture was stirred for 30 minutes, poured into 15 ml of saturated aqueous sodium carbonate solution and extracted twice with 10 ml of dichloromethane. The combined organic extracts were washed with 20 ml of saturated sodium chloride solution, dried over anhydrous potassium carbonate, filtered and the solvent was removed under reduced pressure. 1.44 g of a light yellow oil of methanesulfonamide are obtained as a derivative which is used as is in the following synthesis without further purification.

To a solution of the crude product in 20 mL of anhydrous 1,2-dichloroethane at 0 ° C was added 1.0 mL (9.3 mmol) of 1-chloroethyl chloroformate. the reaction mixture was stirred at room temperature for 30 minutes and then at reflux temperature for 1 hour. The mixture is concentrated to approximately one third of the volume and then diluted with 20 ml of anhydrous methanol. After heating at reflux for 1.5 hours, the reaction mixture is cooled to room temperature and concentrated to about one-half the volume. The precipitate was collected by filtration and washed with a small amount of cold methanol. 1.0 g of a white solid is obtained as piperidine hydrochloride. The filtrate was concentrated and a small amount of methanol was added first followed by ether. The precipitate is collected again by filtration, washed with cold methanol and dried to give an additional 0.49 g of the desired product. A total of 1.49 g of the title compound was obtained in a yield of 70%.

¹ H NMR (CDCl ₃ , 200 MHz) δ 7.43-7.20 (m, 3H), 7.10 (dd, 1H), 3.98) (bs, 2H), 3.55-3.40 ( bd, 2H), 3.35-3.10 (m, 2H), 2.99 (s, 3H), 2.15 (t, 2H), 2.00 (t, 2H).

Part B: N- [1- (R) - [(1,2-Dihydro-1-methanesulfonylspiro [3H-indole-3,4'-piperidin] -1'yl) carbonyl] -1 '- (phenylmethyloxy) ethyl ] 2- [1,1-dimethylethoxy) carbonyl) amino-2-methylpropanamide

-28Do 0.35 g (1.15 mmol) of (2R) -2 - [(1,1-dimethyethoxy) carbonyl] amino-3- [2 (phenylmethyloxy) ethyl] -1-propanoic acid in 13 ml of dichloromethane is added 0.325 g (1.07 mmol) of 1,2-dihydro-1-methanesulfonylspiro [3H-indole-3,4'-piperidine] hydrochloride, 0.18 ml (1.63 mmol) of N-methylmorpholine, 0.159 g (1, 18 mmol) of 1-hydroxybenzotriazole (HOBT) and the reaction mixture was stirred for 15 minutes. After addition of 0.31 g (1.62 mol) EDC, the mixture is stirred for an additional 1 hour and 60 μΙ of N-methylmorpholine is added. The reaction mixture was stirred for 45 minutes, poured into 5 mL of water. The organic layer was separated, washed with 5 ml of a 0.5N aqueous hydrochloric acid solution and 5 ml of a saturated aqueous sodium carbonate solution. The combined organics were dried over anhydrous magnesium sulfate and concentrated to give 0.627 g of a yellow foam of the title product which was used in the next procedure without further 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 the reaction mixture was stirred at room temperature for 75 min, another 1 mL of trifluoroacetic acid was added, the reaction was stirred for 10 min. then concentrated and diluted with 5.0 ml of dichloromethane. After carefully converting to basic pH by pouring into 10 ml of 10% aqueous sodium carbonate solution, the organic portion was separated and the aqueous portion was further extracted twice with 15 ml of dichloromethane. The combined organics were washed with 5 mL of water, dried over potassium carbonate, filtered and concentrated to give 0.486 g of the amine as a pale yellow foam, which was used for the next synthesis without further purification.

To 0.486 g (1.01 mmol) of the 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) of 1-hydroxybenzotriazole (HOBT) and 0.245 g (1.28 mol) of EDC and the resulting reaction mixture was stirred overnight at room temperature. The reaction mixture is poured into 5.0 ml of water and the organic portion is separated. The aqueous portion was further extracted with 5 mL of dichloromethane. The combined organics were washed with 5.0 mL of a 0.5N aqueous hydrochloric acid solution, 5 mL of a saturated aqueous sodium carbonate solution, dried over anhydrous magnesium sulfate, and concentrated to give 0.751 g of crude product as a yellow foam. A solution of this product in dichloromethane, after absorption on 25 g of silica gel, is purified by chromatography, eluting first with hexanes / acetone / dichloromethane (70: 25: 5) followed by hexanes,

-29-acetone and dichloromethane in a ratio of 65: 30: 5. This afforded 0.63 g of the title compound as a white solid.

¹ H NMR (CDCl 3, 400 MHz) compound is a 3: 2 mixture of 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 (bs, 1H), 4.63 (bd, 1 / 3H), 4.57-4.40 (m, 2 / 3H), 4.10 (bd, 1 / 3H), 4.00 (bd, 1 / 3H), 3.82 (t, 1 H), 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 (bs, 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).

Part C: N- [1- (R) - [(1,2-Dihydro-1-methanesulfonylspiro [3H-indole-3,4'-piperidin] -1'yl) carbonyl] -2- (phenylmethyloxy) ethyl] 2-amino-2-methylpropanamide hydrochloride

To 0.637 g (0.101 mmol) of the intermediate Part B in 5 ml of dichloromethane was added 2.5 ml of trifluoroacetic acid and the reaction mixture was stirred at room temperature for 30 minutes. The mixture was concentrated to an oil, treated with 10 mL of ethyl acetate and washed with 8 mL of 10% aqueous sodium carbonate. The aqueous portion was further extracted with 5 mL of ethyl acetate. The combined organics were washed with 10 mL of water, dried over magnesium sulfate, filtered and concentrated to give 0.512 g of the free bases as a white foam.

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

¹ H NMR (400 MHz, CD ₃ OD): the compound is a 3: 2 mixture of rotamers, δ 7.40-7.28 (m, 4H), 7.25-7.17 (m, 2H), 7 Δ 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 (bs, 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).

-30Example 2

N- [1 (R) - [(1,2-dihydro-1-methanesulfonylspiro [3H-indole-3,4'-piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] -2- amino-2-methylpropanamide

Part A: (2R) - [[[2- (1,1-Dimethylethoxy) carbonyl] amino] -2,2-dimethyl-1-oxoethyl] amino-2- (phenylmethoxy) ethyl ester

The resulting compound is prepared from (2R) -2 - [(1,1-dimethylethoxy) carbonyl] amino-3- (phenylmethyloxy) ethyl-propanoic acid and allyl alcohol by coupling reaction in methylene chloride in the presence of EDC and DMAP.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.25 (s, 5H), 5.8 (m, 1H), 5.2 (dd, 2H), 5.0 (bs, 1H),

4.7 (m, 1H), 4.6 (m, 2H), 4.4 (dd, 2H), 3.9 (dd, 1H), 3.6 (dd, 1H), 1.45 (d, 6H) 1.39 (s, 9H).

Part B: (2R) - [[[2- (1,1-dimethylethoxy) carbonyl] amino] -2,2-dimethyl-1-oxoethyl] amino-2 (phenylmethoxy) ethyl] -1-propanoic acid

To a stirred solution of 6.7 g (15.9 mmol) of the crude intermediate part A,

1.8 g (0.1 equivalents) of tetrakis (triphenylphosphine) palladium and 1.25 g (0.3 equivalents) of triphenylphosphine are added 35 ml of a 0.5 M solution of potassium 2-ethylhexanoate in ethyl acetate. The reaction mixture was stirred at room temperature under nitrogen for 1 hour and then diluted with 100 mL of ether and poured into ice water. The organic portion was separated and the aqueous portion was acidified by the addition of 20% citric acid solution. After extraction with ethyl acetate, the extracts were washed with saturated sodium chloride solution, dried over magnesium sulfate, filtered and evaporated to give the title compound as a solid.

¹ H NMR (400 MHz, CD ₃ OD) δ 7.3 (s, 5H), 4.7 (m, 1H), 4.5 (s, 2H), 4.0 (m, 1H), 3, Δ (m, 1H), 1.4 (d, 6H), 1.3 (s, 9H).

-31 Part C: N- [1 (R) - _[(1> 2-dihydro-1-methanesulfonylspiro [3H-indole-3,4'-piperidin] -1'yl) carbonyl] -2- (phenylmethyloxy) ethyl] -2- (1,1-dimethyl-ethoxy) carbonyl] amino-2-methyl-propanamide

To the reaction solution, 1.0 g (3.44 mmol) of 1-methanesulfonylspiro [indoline-3,4'-piperidine] hydrochloride, 1.44 g (3.78 mmol) of (2R) - [[2- (1,1- dimethylethoxy) carbonyl) amino] -2,2-dimethyl-1-oxoethyl] amino-2- (phenylmethyloxy) ethyl] -1-propanoic acid, 0.58 ml (5.20 mmol) of N-methylmorpholine and 0, 58 g (3.78 mmol) of 1-hydroxybenzotriazole (HOBT) in 50 ml of dichloromethane were added with 1.03 g (5.20 mmol) of EDC and the reaction mixture was stirred at room temperature for 16 hours. The mixture was diluted with 50 mL of dichloromethane and washed with 50 mL of aqueous sodium carbonate solution, dried over anhydrous magnesium sulfate, filtered, and concentrated. The residue was purified by flash chromatography to give 2.148 g of the title compound as a colorless foam (90% yield).

¹ H NMR (400 MHz, CDCl 3) compound is a 3: 2 mixture of 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 (bs, 1H), 4.63 (bd, 1 / 3H), 4.57-4.40 (m, 2 / 3H), 4.10 (bd, 1 / 3H), 4.00 (bd, 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 (bs, 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).

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

To a solution of intermediate Part C intermediate (2.148 g, 3.41 mmol) in dichloromethane (10 mL) was added trifluoroacetic acid (5 mL). The mixture was stirred for 1 hour, concentrated and basified by addition of 100 ml of 5% aqueous sodium carbonate solution and extracted three times with 50 ml of dichloromethane. The combined organic extracts were washed with 50 ml of saturated sodium chloride solution, dried over anhydrous potassium carbonate, filtered and concentrated to give a colorless foam.

To a solution of this foam in 25 mL of ethyl acetate, 4 mL of a 1M solution of hydrochloric acid in ethyl acetate was added at 0 ° C. The precipitate was collected by filtration, washed first with ethyl acetate followed by a 1: 1 mixture of ethyl acetate and ether to give the title compound as a colorless solid (1.79 g, 93%). ¹ H NMR (400 MHz, CD ₃ OD) compound is 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 (bs, 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 3

N- [1 (R) - [(1,2-dihydro-1-methanesulfonylspiro [3H-indole-3,4'-piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] -2- amino-2-metylpropánamidmesylát

The title compound was prepared by treatment with methanesulfonic acid on the free base of the title compound of Part D of Example 5. Crystallization from ethyl acetate / ethanol / water gave the product, m.p. 166-168 ° C. It is a polymorphic form II which was determined by X-ray diffraction on a powder material with major bends at 4.7, 11.6, 17.4, 19.2 and 21.6 ° (2 aunt). Further, differential calorimetry was determined at a temperature rise of 10 ° C / minute in an open vessel under nitrogen. A single endothermic curve with a peak at about 174 ° C and an extrapolated melting onset of about 165 ° C with a heat consumption of about 37 J / g could be observed.

Example 4

OBn .N (2)

CO ₂ H

Isonipecotinic acid -33N-benzylcarbamate (3)

Starting compounds: isonipecotinic acid (2) T.C.I. benzyl chloroformate (Schweitzerhall) potassium carbonate water

4.02 kg (31.1 mol) 6.91 kg (40.5 mol)

10.1 kg (72.9 mol)

40.2 i

Dissolve isonipecotinic acid (2) and potassium carbonate in 40.2 L of water in a 100 L four-necked flask with mechanical stirrer and nitrogen atmosphere and cool to 10 ° C. The temperature of the reaction mixture is maintained at 9-14 ° C during the addition of the benzyl chloroformate. After the addition was complete, the reaction mixture was warmed to 22 ° C and allowed to stand for 58 hours. Addition is terminated after 4 hours at pH 9.0. There was no change in pH after 58 hours.

The reaction mixture was transferred to a 200 L extraction apparatus, washed three times with 13 kg (15 L) isopropyl acetate (IPCA) and once with 12 L ethyl acetate. The aqueous portion was extracted with 8 L of toluene. According to HPLC analysis, the benzyl alcohol content was reduced from 3.8% to 1.4% after washing.

Analytical HPLC: 25 cm Dupont Zorbax RXC8 column, flow rate 1.5 ml / min, detection at 254 nm, isocratic elution mixture containing 35% MeCN methyl cyanide and 65% 0.1% aqueous phosphoric acid, retention time: (3) = 6.9 minutes, benzyl alcohol = 3.3 minutes, toluene = 17.3 minutes.

The aqueous portion was acidified to pH 1.8 by the addition of a 37% aqueous hydrochloric acid solution, causing the development of carbon dioxide which is easy to keep under control. The addition of hydrochloric acid took less than 1 hour and required 10 L of concentrated hydrochloric acid. Extract the aqueous portion three times with 6.6 L of toluene, dry the extracts with 2 kg of sodium sulfate, and filter through a pad of Solka-floc ™. The combined filtrates weighed 17.8 kg. Evaporation to dry weight of the filtrate yielded 7.89 kg of crude carbamate (3) in a yield of 97%. The filtrates are placed in a 100 L container after filtration through a built-in 10 μιτι orifice filter. The extracts are concentrated under pressure at less than 25 ° C

The final concentration of carbamate 3 is 440 g / l. The toluene filtrate concentration serves to azeotropically remove the last traces of water (final KF = 170 mg / ml). The resulting product is 99.1% pure, containing 0.9% of the area under the benzyl alcohol curve as the only impurity present.

Example 5

COCI

Isonipecotinic acid chloride N-benzylcarbamate (4)

Starting compounds:

Isonipecotinic acid N-benzylcarbamate (3) in toluene (MW = 263.3) oxalyl chloride (MW = 126.93)

Ν, Ν-dimethylformamide (MW = 73.10) 10 ml toluene 12 L

7.89 kg (30.0 mol) in 17.91

3.94 kg (31.0 mol)

To a solution of the resulting benzyl carbamate (3) from the previous example in toluene was added 5 mL of Ν, Ν-dimethylformamide and 10 L of toluene. Oxalyl chloride was added over 20 minutes and the reaction was allowed to stand at 18 ° C for 16 hours under a slow stream of nitrogen. Subsequent HPLC analysis showed 1.3% of untreated carboxylic acid (3). The reaction mixture is warmed to 26 ° C, 5 ml of Ν, im-dimethylformamide are added and the mixture is allowed to stand for 2.5 hours. Reaction of 1.0 mL aliquots of reaction mixture was quenched by addition of 5.0 mL of t-butylamine and analyzed after evaporation by HPLC.

Analytical HPLC: 25 cm Dupont Zorbax RXC8 column, 50 ° C, flow rate 1 ml / min, detection at 220 nm, isocratic elution mixture

-35Containing 42% methyl cyanide (MeCN) and 58%, 0.1% aqueous trihydrogen phosphoric acid.

This procedure demonstrates the persistence of less than 0.05% acid (3) of formula A and more than 3% of the area under the curve of the compound of formula B (more than 1 mole% (COCl ₂ )).

N

OBn

O-NH-t-Bu

Y 0 NH-t-Bu

CONH-t-Bu (B)

To remove 5 L of solvent, the reaction mixture was concentrated at a pressure of 1000 Pa and a temperature of 20-25 ° C.

Typical HPLC result of concentrated toluene solution after reaction with t-butylamine:

compound retention time % of the area under the curve carboxylic acid (3) 2.1 <0,5% benzyl 7.8 <0,5% Cbz-t-butylcarboxamide A 11.0 > 99% toluene 12.1 ON THE diterc.butyloxamide B 12.7 <0,5%

Example 6

CHO

-36Piperidine-4-carboxaldehyde-1-benzylcarbamate (5)

Starting compounds:

Isonicotecotinic acid N-benzylcarbamate (4) 3.38 kg (12.0 mol) in toluene (MW = 281.74) in 5.54 kg

DIEA (KF = 18 mg / l)

10% Pd / C (KF <20mg / g)

1.55 kg (15.0 mol) 101 g thioanisole (molecular weight = 124.21, d = 1.058) 0.56 g

DIEA and thioanisole are added to a solution of compound (4) in toluene and the catalyst is suspended in this mixture. The reaction mixture is immediately placed in a 19 L autoclave and hydrogenated at 20 ° C and a hydrogen pressure of 2800 kPa. Within 18 hours, the reaction was treated with 70% of the theoretical amount of hydrogen. The proportion of the mixture in which the reaction was terminated by the addition of tert-butylamine was monitored by analytical HPLC, which showed 14.2% of the area under the acid chloride curve (2). The HPLC conditions correspond to the above conditions. Retention time: (δ) = 8.1 minutes.

A similar suspension of 101 g of catalyst and 0.54 g of thioanisole in 1375 ml of toluene is added to the hydrogenation apparatus. After 23 hours of hydrogenation, HPLC analysis of the portion at which the reaction was terminated by addition of t-butylamine. According to HPLC, 1.8% of the area under the acid chloride curve (2) remains. The reaction mixture was purged with nitrogen and the catalyst and the DIEA.HCl precipitate were removed by Solka-floc ™ filtration. The filter cake was washed with 10 L of toluene. The filtrates are transferred to a 50 L extraction apparatus through a 10 µm orifice filter and washed twice with 7.2 L of a 1M aqueous hydrochloric acid solution and twice with 7.2 L of water. The reaction mixture was concentrated to a 5 L residue at 1000 Pa at 25-30 ° C.

compound retention time (min)% area under the curve carboxylic acid (3) 2.1 <2 dimer (21) 6.6 <1 aldehyde (5) 8.1> 95

According to HPLC, the yield of aldehyde (3) is 94%.

Example 7

(9)

CBZ-spiroindoline (9)

Starting compounds: piperidine-4-carboxaldehyde-1-benzyl carbamate (5) in toluene phenylhydrazine trifluoroacetic acid (TFA) sodium borohydride (NaBH ₄ ) toluene methyl cyanide methanol

1.71 kg (6.89 mol) in 21.4 kg

900 ml, 981 g (9.15 mol) 2.20 l, 3.26 kg (28.6 mol) 300 g (7.93 mol)

34.4 kg

7.0 i

7.0 I

The crude aldehyde solution (5) from the previous section was transferred through a 10 µm filter into a 100 L reaction vessel equipped with a Teflon-coated copper sleeve for cooling or heating and a mechanical stirrer. After addition of 34.4 kg of toluene and 7 L of methyl cyanide, the reaction solution formed is cooled to 0 ° C. Phenylhydrazine is added in portions and the temperature of the reaction mixture is maintained at -1 to 3 ° C during continuous nitrogen bubbling.

Phenylhydrazine is added until TLC and HPLC shows complete consumption of aldehyde (5) and a small excess (<5%) of phenylhydrazine.

-38TLC Conditions: Silica Ocetic, E. Merck Kieselgel G60 F254 0.25 mm; diethyl ether / penate (4/1); developing mixture: 0.5% cerium sulfate, 14% ammonium molybdate in 10% aqueous sulfuric acid followed by heating; Rf: aldehyde (5) = 0.52, phenylhydrazone (7) = 0.61, phenylhydrazine (6) = 0.21. HPLC conditions: 25 cm Dupont Zorbax RXC8 column at 30 ° C, flow rate 1.0 mL / min, detection at 254 nm; Overview of gradient elution mixtures:

time (minutes) acetonitrile: water 0 57:43 10 65: 35 15 75:25 18 75: 25

retention time: phenylhydrazine (6) = 4.5 minutes, toluene = 7.2 minutes, phenylhydrazone (7) = 11.4 minutes.

The reaction mixture was allowed to stand for 30 minutes at 0-2 ° C and trifluoroacetic acid was added while maintaining the temperature at 2-7 ° C. The reaction mixture is heated to 50 ° C for 30 minutes, which is maintained for 17 hours. The nitrogen bubbling of the reaction mixture was stopped and only a slow stream of nitrogen was maintained. The color gradually darkens to dark green over the first hour at 5 ° C. A relatively small amount of white crystalline precipitate (ammonium trifluoroacetate) was formed. After 17 hours, HPLC conditions corresponding to the above indicated that the reaction mixture contained 91.6 area% under indolenine curve and 1.5% raw phenylhydrazone. Standing the mixture over a longer period of time does not increase the indolenine yield (8).

After cooling the reaction mixture to 12 ° C, 7.0 L of methanol are added. Sodium borohydride is added in portions of less than 20 g to maintain the temperature below 15 ° C. This addition takes 30 minutes, during which a slight evolution of hydrogen has occurred which is easy to control. No foaming occurred during the addition. Towards the end of the addition, the color quickly changes from green to brown and then bright orange. Separate a small amount (<200 mL) of the heavier phase (probably aqueous salt solution). HPLC analysis (conditions above) shows consumption of everything

-39 amounts of indolenine (8) (90.4% area under the CBZ-indoline curve (9)); retention time: indoline (8) = 7.5 minutes, indoline (9) = 8.2 minutes.

TLC: ethyl ether solvent, dye spot containing cerium sulfate and ammonium melybdate, or 1% anisaldehyde spot; Rf: indolenine (8) = 0.18, CBZ-indoline (9) = 0.33.

At the end of the reaction, the reaction mixture changes from green to orange. The amount of sodium borohydride required to complete the reaction is significantly dependent on the temperature and amount already added. Ultimately, the quantity and quality of the product is not affected.

The reaction mixture was cooled to 5 ° C for 30 minutes, 8 L of a 3% aqueous ammonium hydroxide solution was added to bring the pH of the aqueous portion to pH 7.4. The mixture was stirred and then allowed to settle, the temperature was raised to 15 ° C and the yellow-cloudy lower aqueous portion was separated. The organic portion was washed once with 4 L of a 3% aqueous ammonium hydroxide solution, twice with 4 L of water and twice with 4 L of saturated sodium chloride solution. After washing, 53.5 kg of organic fraction were obtained with a yield of 94%.

The washed toluene solution was combined with the washed organic portions of two other similarly conducted reactions. The total amount of aldehyde used in the three reactions is 5.06 kg (20.5 mol). The total weight of CBZ-indoline (9) from the combined organic fractions was 5.91 kg (18.3 mol) with a yield of 90%. The combined organic fractions were dried with 5 kg of sodium sulfate and treated with 250 g of charcoal for 30 minutes. Darco G60 and filtered through Solka-floc ™. The filtrates are concentrated under vacuum to a pressure of 1000 Pa and a temperature of less than 25 ° C almost to dryness. Solvent exchange is accomplished by slowly pouring into 30 L of isopropyl acetate and concentrating to 14 L at 2000 Pa and 50-60 ° C. The mixture was heated to reflux to give a clear, homogeneous dark orange solution. After solvent exchange, this ¹ H NMR solution contained approximately 6 mole% residual toluene.

The solution is cooled to 68 ° C and seeded with 4 g of crystalline CBZ-indoline (9). The solution was allowed to cool gradually to 26 ° C over 6 hours and allowed to stand at 20-26 ° C for 9 hours. The suspension was cooled to 2 ° C over 1 hour and allowed to stand at 2 ° C for 1 hour. The product is collected by filtration and

The filter cake was washed twice with 2 L of 5 ° C isopropyl acetate and twice with 2 L of 5 ° C MTBE. The product was dried in a vacuum oven at 30 ° C and under nitrogen atmosphere to give 4.37 kg of the title compound (9) as a light yellow-brown crystalline solid in 74% yield.

According to HPLC, it is a product with a purity of 99.5% of the area under the curve. The washings and 11 L of mother liquor contain an additional 1.15 kg (19%) of the prodrug (9) and about 3% of Cbz-isopecotinic acid phenylhydrazide (retention time = 4.8 minutes).

Example 8

CBZ-spiroindoline methanesulfonamide (1)

Starting compounds: CBZ-spiroindoline (9) methanesulfonyl chloride triethylamine (KF = 151) tetrahydrofuran (KF = 41)

1.69 kg (5.23 mol) 599 g (5.23 mol) 635 g (6.27 mol) 12 L

A 22 L flask was charged with solid CBZ-spiroindoline (9) and then 11.5 L tetrahydrofuran and triethylamine were added to the flask through a 10 µm filter. A homogeneous solution was formed and cooled to 0 ° C. A 1 L addition funnel was charged with methanesulfonyl chloride and 500 mL of tetrahydrofuran. This solution is added to the reaction mixture while maintaining

-41 its temperature in the range of 0 to 4 ° C. The addition, which takes place exothermically, takes 5 hours. A white precipitate, probably triethylammonium hydrochloride, is formed during the addition. According to HPLC analysis, the reaction was complete after the addition was complete and no CBZ-spiroindoline (9) was present. HPLC conditions: 25 cm Dupont Zorbax RXC8 column, flow rate 1.5 ml / min, detection at 254 nm; Overview of gradient elution mixtures:

time (minutes) 0.1% aqueous trihydrogen-

0 phosphorous: methyl cyanide 70: 30 10 70: 30 12 20: 80 25 20: 80

retention time: (9) = 7.6 minutes, (1) = 13.6 minutes.

Upon completion of the addition, the reaction mixture was warmed to 18 ° C and allowed to stand for 16 hours. Comparing the appearance of the reaction mixture and the resulting HPLC after the addition was complete and after 16 hours of standing, it was concluded that there were no changes. The reaction mixture was slowly transferred to a vigorously stirred solution of 30 L of water and 200 mL of a 37% aqueous hydrochloric acid solution placed in a 50 L flask over 1 hour. The vessel temperature is raised from 22 ° C to 28 ° C. the product separated as a light yellow-brown gummy solid which turned into a granular solid. The aqueous suspension was cooled to 22 ° C and allowed to stand for 1 hour. Filter the slurry and wash the filter cake twice with 4 L of a 50:50 mixture of methanol and water. According to HPLC analysis, less than 0.1% CBZ-spiroindoline methanesulfonamide (1) is present in the mother liquor.

The filter cake was washed with 4 L of a 50:50 mixture of methanol and water containing 50 mL of a 28% aqueous ammonium hydroxide solution. The filter cake is washed twice with 4 L of a 50:50 mixture of methanol and water and the solid is dried in a vacuum oven under a nitrogen atmosphere at 50 ° C. 2.03 kg are obtained

-42 off-white powder of the title compound (1) in 97% yield. According to HPLC analysis, the solid contained 93.7% area under the curve of the resultant compound (1).

Example 9

(8)

Alternative procedure for isolation of CBZ-spiroindolenine intermediate (8)

Starting compounds piperidine-4-carboxaldehyde-1-benzyl carbamate (5) phenylhydrazine trifluoroacetic acid (TFA) methylene chloride

12.37 g (0.050 mol)

5.41 g (0.050 mol)

11.56 ml, 17.10 g (0.150 mol) 500 ml

In a 1 L reaction vessel equipped with a Teflon-coated magnetic stirrer, CBZ-aldehyde (5) was dissolved in dichloromethane. The resulting solution was cooled to 0 ° C. Phenylhydrazine is added over 5 minutes via a calibrated syringe and the reaction mixture is maintained at a temperature of -1 to 3 ° C during continuous nitrogen bubbling.

According to TLC and HPLC, the aldehyde (5) was completely consumed and a small excess (<2%) of phenylhydrazine was formed.

TLC conditions: silica, E. Merck Kieselgel G60 F254 0.25 mm; diethyl ether / pentane (4/1); developing mixture: 0.5% cerium sulfate, 14% ammonium molybdate in 10% aqueous sulfuric acid followed by heating; Rf: aldehyde (5) = 0.52, phenylhydrazone (7) = 0.61, phenylhydrazine (6) = 0.21.

HPLC conditions: 25 cm Dupont Zorbax RXC8 column at 30 ° C, flow rate 1.0 ml / min, detection at 254 nm; Gradients report elution mixture concentrations: time (minutes) acetonitrile: water 0 57: 43 10 65: 35 15 75: 25 18 75: 25

retention time: phenylhydrazine (6) = 4.5 minutes, toluene = 7.2 minutes, phenylhydrazone (7) = 11.4 minutes.

The reaction mixture was allowed to stand for 10 minutes at 0-2 ° C and trifluoroacetic acid was added via cannula while maintaining the temperature at 2-7 ° C. The reaction mixture is heated to 35 ° C for 30 minutes, which is maintained for 17 hours. Nitrogen bubbling through the reaction mixture was stopped and only a slow stream of nitrogen was maintained. During the first hour at 35 ° C, the color of the procedure darkens to pink and then dark green. A relatively small amount of white crystalline precipitate (ammonium trifluoroacetate) was formed. After 17 hours, HPLC conditions corresponding to the above indicated that the reaction mixture contained 93% area under the indolenine (8) curve and less than 0.5% of untreated phenylhydrazone. Standing the mixture over an extended period of time does not increase the yield of indolenine (8).

After cooling the reaction mixture to 10 ° C, a mixture containing 60 ml of a 28-30% ammonium hydroxide solution, 90 ml of water and 150 g of crushed ice is added with vigorous stirring. The color of the mixture changes to salmon red. The organic layer was separated and washed twice with 400 ml of water and then with 100 ml of saturated aqueous sodium chloride solution. The organic portion was dried over magnesium sulfate and filtered through a pad of 5 g silica. Evaporation of the filtrate gave 15.84 g of indolenine light orange oil (8) in a yield of 99%.

-44Example 10

Preparation of CBZ-spiroindoline methanesulfonamide (1) without isolation of CBZspiroindoline intermediate (9)

Part 1: CBZ-spiroindoline (9)

Starting compounds:

piperidine-4-carboxaldehyde-1-benzyl carbamate (5) phenylhydrazine (Aldrich) trifluoroacetic acid (TFA) toluene (KF <250 mg / l) methyl cyanide (MeCN) (KF <250 mg / l) sodium borohydride toluene methanol

49.5 g (0.20 mol) 23.7 g (0.22 mol) 75.4 g (0.66 mol) 654 ml

13.3 ml

11.3 g (0.30 mol) 20 ml ml

Using 654 ml of toluene and 13.3 ml of methyl cyanide, a 2% v / v solution of methyl cyanide is prepared. To a 2 L 3-necked flask equipped with a mechanical stirrer was added 617 mL of the above solution, which was degassed by bubbling a gentle stream of nitrogen for 5 minutes. Phenylhydrazine and trifluoroacetic acid were added during bubbling. CBZ-aldehyde (5) was dissolved in the remainder of the above solution (50 mL) and the solution was degassed with nitrogen by bubbling nitrogen through the addition funnel. solution

The vessel was heated to 35 ° C and the aldehyde solution was slowly added to the phenylhydrazine / trifluoroacetic acid mixture over 2 hours. The reaction mixture was allowed to stand at 35 ° C for 15 hours.

HPLC conditions: 25 cm Dupont Zorbax RXC8 column at 50 ° C, flow rate 1 ml / min, detection at 220 nm; isocratic elution mixture of 55% methyl cyanide and 45% 0.1% aqueous trihydrogenphosphoric acid.

Overview of HPLC results after 16 hours of standing:

compound retention time (minutes) % of the area under the curve phenylhydrazine 1.6 0.1 to 0.5 dimer (21) 4.1 <0.1 aldehyde (5) 4.7 <0.1 spiroindoline (9) 5.0 ON THE toluene 6.3 ON THE spiroindoline (8) 6.9 97 phenylhydrazone (7) 10.3 <0.2 other impurities (each <0,2%) a total of 2 to 3

The reaction mixture was cooled to -10 ° C and methanol was added. A suspension of sodium borohydride in 20 ml of toluene was added in small (1 ml) portions over 30 minutes so that the temperature did not rise above -2 ° C.

compound% area under the phenylhydrazine (6) curve 0.1-1

CBZ-spiroindoline (9) 85-90

CBZ-spiroindolenine (8) <0.1 other impurities (each <3%) total 10-15

The temperature of the reaction mixture was raised to 10 ° C over 1 hour before adding 200 mL of 6% aqueous ammonia. The reaction mixture is stirred for 10 minutes, allowed to stand for 10 minutes and the lower aqueous phase is separated. 20 ml are added to the organic phase

-46-acetonitrile and 20 ml of methanol and the solution was washed with 150 ml of 15% sodium chloride solution. The organic fraction contained 92% CBZ-spiroindoline (9).

Part 2: CBZ-spiroindoline methanesulfonamide (1)

Starting compounds:

CBZ-spiroindoline (9) (MW = 322.51) methanesulfonyl chloride

DIEA (KF = 150 mg / L) tetrahydrofuran (THF) (KF = 41 mg / L) (0.184 mol)

21.1 g (0.184 mol) 29.7 g, 40.1 ml (0.230 mol)

150 ml

In a 1 L three-necked flask at 60 to 70 ° C and pressure

24.4 to 33.2 kPa, the crude CBZ-spiroindoline solution (9) from part 1 above is concentrated to a 250 g residue. Tetrahydrofuran and DIEA were added to form a homogeneous solution which was cooled to 0 ° C. A 125 mL addition funnel was charged with methanesulfonyl chloride and 50 mL tetrahydrofuran. This solution was added to the reaction mixture while maintaining its temperature in the range of 0 to 4 ° C and the reaction mixture was allowed to stand at 5 to 8 ° C for 2 hours. The addition is slightly exothermic. A white precipitate, probably DIEA hydrochloride, is formed during the addition. The HPLC conditions are set forth above. According to HPLC analysis, the reaction was complete 1 hour after completion of the addition when no CBZ-spiroindoline (9) was present and the yield based on compound (9) was 94%. Retention time: (1) = 7.8 minutes. A typical HPLC result of the reaction mixture after 2 hours of standing:

compound% area under the curve

CBZ-spiroindoline (9) CBZ-suifonamide (1) other impurities (each <2%) <0,1 90-92 total 8-10

The reaction mixture was warmed to 20 ° C and 200 mL of a 1M aqueous hydrochloric acid solution was added. The mixture is heated to 50 ° C and the aqueous phase is separated. The organic portion is washed successively once with 100 ml of water, once with 100 ml of 5% aqueous sodium carbonate solution and once with 100 ml of water. The organic portion was transferred to a 1 L 3-necked flask equipped with a mechanical stirrer and distillation apparatus. About 400 ml of the mixture was distilled at atmospheric pressure until 150 ml of distillate was collected. The head temperature of the distillation column reaches 107 ° C and the bottom temperature of the distillation column is 110 ° C. The distillation is carried out with constant addition of n-propanol in an amount such that a constant amount of approximately 350 ml remains at the bottom of the distillation column. The distillation was terminated by adding 525 ml of n-propanol with a total of 800 ml of distillate collected.

During the solvent exchange, the temperature of the head and bottom of the distillation column is increased from 94 to 98 ° C. Toluene and n-propanol form an azeotropic with a boiling point of 97.2 ° C, consisting of 47.5% toluene and 52.5% n-propanol. The reaction mixture was allowed to gradually cool to 20 ° C over 3 hours and allowed to stand for 12 hours. The mother liquor contains 2% toluene and 4 mg / ml sulfonamide. The solubility of the sulfonamide in various mixtures of toluene and n-propanol was determined using HPLC.

% toluene in n-propanol solubility (1) in mg / ml

0 2.36 5 3.02 10 4.23 20 7.51 25 10.3

The crystalline suspension is filtered and washed three times with 100 ml of n-propanol. After drying in a vacuum oven at 50 [deg.] C. under a nitrogen atmosphere for 16 hours, 65.5 g of compound (6) are obtained as a yellow-brown substance with a yield of 82% based on aldehyde (5) and a purity of 93.5%.

‘48Typical HPLC result:

compound

CBZ-spiroindoline (9)

CBZ-sulphonamide (1) other impurities (each <0,2%)% of the area under the curve <0,1> 99 total <1

For further purification, a sample of 40.0 g of sulfonamide crystallized from npropanol at 60 ° C was dissolved in 134 ml of ethyl acetate and the solution was treated with 8.0 g of charcoal for 1 hour. Darco G-60 at 60 ° C. After addition of 2.0 g Solka-floc ™, the suspension is filtered through a 4.0 g Solka-floc ™ layer and the layer is washed with 90 ml of ethyl acetate at 60 ° C. It turns into a golden yellow filtrate before adding the active filtrate. The filtrate was distilled at atmospheric pressure in a 500 mL flask and a bottoms temperature of 80-85 ° C to give 100 g (100 mL) of residue. The solution was allowed to cool to 35 ° C over 3 hours. 116 ml of cyclohexane are added to the solution over 1 hour with vigorous stirring at 35 ° C. The reaction mixture was cooled to 20 ° C over 1 hour and allowed to stand at 20 ° C for 12 hours. At 35 ° C, a large amount of sulfonamide crystallized and the mixture thickened. Addition of cyclohexane at 20 ° C makes stirring very difficult. After a period of standing, the amount of compound (s) 2.5 mg / g was determined in the supernatant. The crystalline suspension is filtered and the filter cake is washed with 77 ml of cyclohexane / ethyl acetate 2: 1 and washed twice with 77 ml of cyclohexane. The product was dried in a vacuum oven at 50 ° C for 16 hours. 34.2 g of compound (1) (molecular weight = 400.3) was obtained as a white crystalline solid (85% yield of compound (1), 70% of compound (5)) with a purity higher than 99% by weight.

Example 11

H

I

· HCI

SO ₂ Me

-49Spiroindoline methanesulfonamide sodium (1a)

Starting compounds

CBZ-spiroindoline methanesulfonamide (1) Pearlman's catalyst 20% Pd (OH) ₂ / C THF

MeOH

941 g (2.35 mol) 188g 8 L 7I

The catalyst was suspended in 7 L of MeOH and transferred to a 19 L autoclave containing a solution of compound (1) in 8 L of THF. The mixture was hydrogenated at 25 ° C and 80 psi hydrogen pressure. After 2.5 hours the temperature was raised to 35 ° C for 30 minutes.

HPLC analysis indicated complete consumption of Cbz-spiroindoline methanesulfonamide. HPLC conditions: 25 cm Dupont Zorbax RXC8 column with a flow rate of 1.5 mL / minute and detection at 254 nm.

Gradients report:

time (minutes) 0.1% aqueous phosphoric acid: methyl cyanide

70:30

70:30

20: 80

20: 80

Retention time: spiroindoline = 7.6 minutes, Cbz-spiroindoline methanesulfonamide = 13.6 minutes.

Nitrogen was bubbled through the mixture and the catalyst was removed from the still warm reaction mixture by filtration through Solka-floc ™. The catalyst was washed with 4 L of tetrahydrofuran and 2 L of methanol. The light yellow filtrates are concentrated to a thick oil at a pressure of 1000 Pa and a temperature of less than 25 ° C. Solvent exchange is terminated by slowly pouring into 15 L

-50-ethyl acetate and concentrated to dryness. The residue formed solidified to a hard off-white material, to which 1.5 L of methanol was added and the mixture was heated to 70 ° C to form a homogeneous solution. At a solution temperature of 70 ° C, 10.5 L of ethyl acetate at 20 ° C was added. the temperature is lowered to 40 ° C and the mixture remains homogeneous. In the following experiments, it is believed that a more suitable solvent exchange in the filtrate is a change from a mixture of methanol and tetrahydrofuran to methanol, concentration to the desired volume and subsequent addition of ethyl acetate. this procedure does not solidify the residue during the concentration of the ethyl acetate solution.

Hydrogen chloride is added to the solution with approximately equal volumes of nitrogen. The temperature was raised to 60 ° C over 15 minutes to give a white hydrochloride precipitate. Dilute hydrogen chloride solution only prevents suction of the mixture back and this procedure is not necessary at first.

The reaction mixture was cooled in an ice bath and the addition of hydrogen chloride continued for an additional 1 hour. The temperature is gradually reduced to 20 ° C and the suspension is allowed to stand for 2 hours while the temperature is lowered to 10 ° C. The crystalline product formed is collected by filtration and the filter cake is washed with 3 L of ethyl acetate. After drying in a vacuum oven at 35 ° C, 1.18 kg of the title compound (1a) is obtained in 86% yield as an off-white crystalline solid with HPLC purity greater than 99.5% area under the curve. HPLC conditions: 25 cm Dupont Zorbax RXC8 column, flow rate 1.5 ml / min, detection at 230 nm; isocratic elution mixture of 35% methyl cyanide, 65% 0.1% aqueous ammonium acetate solution. Retention time: (1a) = 5.4 minutes.

Example 12 (1b)

SO ₂ Me

-51 Spiroindoline methanesulfonamide (in free base form) (1b)

Concentrate to about 10 ml a 250 ml portion of the Cbz-hydrogenolysis filtrate containing 4.67 g of compound (1b) (free base). The residue is dissolved in 20 ml of ethyl acetate and the solution is concentrated to about 10 ml. This procedure was repeated once more and then 10 mL of ethyl acetate was added to the residue. When a crystalline precipitate began to form, 20 ml of MTBE were added at once. Further crystallization occurs, but the supernatant still contains a considerable amount of dissolved product from which a precipitate does not form on standing. While stirring vigorously, 70 ml of hexanes are added dropwise over 2 hours. The slow addition of hexanes is necessary to prevent the formation of an amine as an oil.

The reaction mixture was allowed to stand for 1 hour and then filtered. The filter cake is washed with 20 mL of 1: 1 MTBE / hexanes and then with 20 mL of hexanes. The product is dried under a stream of nitrogen to give 3.86 g of the free amines of compound (1b) as off-white crystals with a purity greater than 99.5% area under the curve and a yield of 82%.

HPLC conditions: 25 cm Dupont Zorbax RXC8 column, flow rate 1.5 ml / min, detection at 230 nm; isocratic elution mixture of 35% methyl cyanide, 65% 0.1% aqueous ammonium acetate solution. Retention time: (1b) = 5.4 minutes.

Example 13A

Spiroindoline methanesulfonamide (in free base form) (1b)

Starting compounds: CBZ-spiroindoline-sulfonamide (1)

833.5 g (2.08 mol)

-52 palladium on activated carbon (20% palladium oxide on activated carbon) tetrahydrofuran methanol ammonium hydroxide - concentrated solution

124.5 (15%) 6.5 L 19.51 60 ml.

Due to limited equipment, hydrogenation was performed three times. This synthesis procedure corresponds to a simple procedure. CBZ-spiroindoline sulfonamide (1) is dissolved in 6.5 l of tetrahydrofuran (KF = 53 μg / μl) and then in 4 l of methanol (KF = 18 μg / ml). After addition of the catalyst, the suspension was transferred to a 19 L autoclave. The remainder of 2.5 L of methanol was used for washing. The mixture was heated at 40 psi at 40 ° C for 24 hours. The amount of catalyst present and the reaction time are a function of the purity of the starting compound (1). This material required more than 15% catalyst and a long reaction time. Cleaner doses of spiroindoline required only 5% catalyst and 4-6 hours of reaction.

After completion of the reaction (presence of compound (1) <0.1% area under liquid chromatography), the mixture was filtered through Solka-floc ™ and the carbon filter cake was washed with 13 L of methanol solution containing 60 ml of 0.5% ammonium hydroxide solution. . The combined filtrates (according to assay 1587 g spiroindolinamine 1b) were concentrated in vacuo and the solid formed was partitioned between 40 L of 3: 1 toluene / tetrahydrofuran and 18 L of 0.5 N sodium hydroxide solution and a thick precipitate appeared in the aqueous fraction. The shares are easily separated. The aqueous suspension was extracted with 15 L of methylene chloride. The aqueous and organic fractions were slowly separated. Before addition of methylene chloride, tetrahydrofuran is added to the aqueous portion together with enough sodium chloride to saturate the portion. However, this process does not occur to dissolve the product and methylene chloride is required.

The combined toluene, tetrahydrofuran and methylene chloride fractions were combined and concentrated in a batch concentrator. The residue is rinsed with 7 L

-53metylkyanidu. Finally, 10 L of methyl cyanide is added and the solution is allowed to stand overnight under a nitrogen atmosphere.

Example 13B

Spiroindoline methanesulfonamide (in free base form) (1b)

Starting compounds:

CBZ-spiroindoline sulphonamide (1) 3 kg (7.49 mol) activated carbon brand. Darco G-60 600 g ethyl acetate 361 absolute ethanol 189 l

10% palladium on charcoal 450 g ammonia solution 500 ml

Solka-floc ™ 2.5 kg isopropyl acetate 651

Mixture of 1 kg of CBZ-spiroindoline (1) and 200 g of activated charcoal Darco G-60 in 9 L of ethyl acetate was heated at 60-65 ° C under nitrogen atmosphere for 8 hours with stirring. The Darco was removed by filtration at 60-65 ° C, the solid was washed with 3 L of hot ethyl acetate and the filtrate and washings were combined. Liquid chromatography confirms a negligible loss due to the use of activated charcoal grade. Darco G-60 The ethyl acetate solution was evaporated to dryness in vacuo using a 20 L Buchi apparatus, then rinsed twice with 5 L absolute ethanol, heated to 65 to 70 ° C and placed in

The reaction mixture was washed with 1 L of absolute ethanol. A suspension of 75 g of 10% palladium on charcoal (7.5% by weight) in 750 ml of absolute ethanol is then added to the auctolate, followed by a further 250 ml of absolute ethanol.

The reaction mixture was hydrogenated under vigorous stirring for 3 hours at a hydrogen pressure of 2800 Pa and a temperature of 65 ° C. An additional 75 g of palladium on charcoal was added, the reaction mixture was hydrogenated for a further two hours and left in a closed vessel overnight. After transfer of the reaction mixture, which is still at a temperature of 60-65 ° C, into a 20 L Buchi apparatus, the batch is degassed with a formic acid-removing method of feeding and bleeding, i. adding and rinsing a total of 18 L of absolute ethanol.

this procedure is repeated two more times and the three batches are combined in a 36 L glass-lined vessel. The combined batches are again degassed by repeating the addition of 10 L absolute ethanol each time followed by distillation under vacuum. 0.5 kg of Solka-floc ™ is added to the batch and the mixture is washed with 10 L of ethanol. A suspension of Solka-floc ™ in 20 L of ethanol is filled with an Estrella filter and the resulting mixture is heated to 60-65 ° C. At this temperature, the reaction mixture is filtered through a heated filter by means of a pump into two tinned stainless steel vessels. The first reaction vessel, filter, pump and tube were washed with a hot mixture of 500 mL of aqueous ammonia in 25 L of absolute ethanol at 60-65 ° C.

The batch is filtered into a reaction vessel using a built-in filter containing a preformed column having a 10 μιτι orifice size and then concentrated in vacuo to a volume of approximately 15 L. Ethanol is replaced with a total of 45 L of isopropyl acetate by feeding and bleeding. adding and rinsing three times with 15 L of isopropyl acetate. According to gas chromatography, the reaction mixture contained less than 1% ethanol upon completion of the solvent exchange. The reaction mixture is diluted to a volume of approximately 33 L by addition of 20 L of isopropyl acetate. this solution of spiroindolinamine (1b), which is 1.855 kg by liquid chromatography, isopropyl acetate is used in the following synthesis.

55Example 14A

Boc-O-benzycserine-spiroindoline (11)

Starting compounds: spiroindolinamine (1b) amino acid (10)

1587 g (5.966 mol) 1938 g (6.563 mol)

Ph. CO ₂ H

NHBOC dicyclohexylcarbodiimide (DCC)

N-hydroxybenztriazole (HOBt) methyl cyanide

0.5N sodium hydroxide solution

0.5N hydrochloric acid saturated sodium bicarbonate solution isopropyl acetate

1334.5 g (6.563 mol) 884 g (6.563 mol)

I 18 I 18 I 18 I 28 I

A solution of spiroindolinamine (1b) in 25 L of methyl cyanide or aqueous isopropyl acetate is treated successively at room temperature and under nitrogen with 884 g (1.1 equivalents) of HOBT as a solid, 1334.5 g DCC (1.1 equivalents) as a melt ( prepared by heating for 1 hour in hot water at 60 ° C) and

Finally, 1938 g of amino acid (10) as a solid. The reaction mixture was stirred for three hours to form a thick precipitate of DCU. According to liquid chromatography, 0.5% of the area under the amine curve (1b) remains in the reaction mixture. 9 L of isopropyl acetate was added, the suspension was filtered through Solka-floc ™, and the filter cake was washed with 19 L of isopropyl acetate. The combined organic solution was washed successively with 18 L of a 0.5 N sodium hydroxide solution, 18 L of a 0.5 N hydrochloric acid solution, and 18 L of a saturated sodium bicarbonate solution.

Washing with water gave an emulsion which was discarded. The organic portion is concentrated in vacuo and the residue is dissolved in methanol or ethanol to a final volume of 10 L of solution. The yield is 3026 g i.e. 89%.

The yield of compound 11 or 14 from a reaction in which alternative peptide binding agents such as carbonyldiimidazole is used. or a mixture of anhydrides such as sec-butyl carbonate is lower. Compound (11) is epimerized to a high degree. Other peptide binding agents are financially unavailable.

Example 14b

O (11)

SO ₂ Me

Boc-O-benzylserine-spiroindoline (11)

Starting compounds: spiroindoline-amine (1b) isopropyl acetate dicyclohexylcarbodiimide (DCC)

1.855 kg (6.96 mol) 29 l

1.58 kg (7.65 mol)

- 571-hydroxybenzotriazole (HOBt)

N-Boc-O-benzyl-D-serine

1M aqueous sodium hydroxide solution saturated aqueous sodium bicarbonate solution absolute ethanol

1.03 kg (7.62 mol) 2.26 kg (7.65 mol) 26 L

I

I

To a stirred solution of 1.855 kg of spiroindolinamine (1b) in 33 L of isopropyl acetate was added 20 L of water in the reaction vessel. 1.58 kg (1.1 equivalents) of DCC, 1.03 kg (1.1 equivalents) of HOBt, and finally (1.1 equivalents) 2.26 kg of N-Boc-O are added sequentially at room temperature and under a nitrogen atmosphere. benzyl-Dserínu. The reagents were washed with 7 L of isopropyl acetate. After stirring at room temperature under nitrogen for 5 hours, the ratio of product to starting compound in the reaction mixture was 99.4: 0.6 by liquid chromatography. The reaction mixture is filtered into another vessel through an Estrella filter using only a cloth mounted in a cardboard and a pump. The first vessel is rinsed with 22 L of isopropyl acetate and this material is rinsed with the filter, pump and tubing connecting the vessel. The mixture of the two phases was stirred in the vessel for 10 minutes and then left to stand overnight at room temperature for 15 minutes.

The next day, the organic solution was washed with 26 L of a 1M aqueous sodium hydroxide solution, 26 L of a 0.5 M aqueous hydrochloric acid solution and finally 26 L of a saturated aqueous sodium bicarbonate solution. The yield of the title compound according to liquid chromatography is 3.787 kg, i.e. 93% of 3 kg (7.49 mol) CBZ-spiroindoline (1) as starting material. The reaction mixture is concentrated in vacuo (internal temperature -13 to 15 ° C, cuff temperature = 40 ° C, vacuum = 3857 Pa) to approximately 15 L. The solvent is converted to ethanol by slowly adding and rinsing 50 L of ethanol to a final volume of approximately I. According to gas chromatography, less than 1% isopropyl acetate is present in the solution. The solution was used in the following synthesis.

Example 58A

O-benzylserine-spiroindoline (in the form of free bases) (12)

Starting compounds: Boc-O-benzylserine-spiroindoline (11) methanesulfonic acid (MsOH) methanol isopropyl acetate (iPrOAc)

0.5N sodium hydroxide solution

3026 g (5.57 mol) 1.161 (17.9 mol) 10 L 24 L 35 L

A solution of Boc-O-benzylserine-spiroindoline (11) in 10 L of methanol (or ethanol) was treated with 1.16 L of methanesulfonic acid as a pure substance, which was added for about 30 to 40 minutes (initial temperature 16 ° C, final temperature 28). C). The dark red solution was allowed to stand under nitrogen overnight. The reaction mixture was transferred to a 100 L extraction apparatus containing 24 L of isopropyl acetate and 35 L of 0.5 N sodium hydroxide solution. The initial pH of the aqueous portion was 7. Upon addition of 6M sodium hydroxide solution, the pH was raised to or reached = 10.5. As the pH of the solution increases, its color changes from red to yellow. The aliquots were separated and 24 L of the organic portion by NMR contained 13 mole% methanol in isopropanol (5% v / v). Liquid chromatographic assay yield is 2.48 kg.

O-benzylserine-spiroindoline (in the form of free bases) (12)

Starting compounds: Boc-O-benzylserine-spiroindoline (11) methanesulfonic acid isopropyl acetate

1M aqueous sodium hydroxide solution 50% aqueous sodium hydroxide solution

3.87 kg (6.96 mol) 2.006 kg (20.87 mol) 38 L

I

1,6 1

To a stirred solution of 3.87 kg of Boc-O-benzylserine-spiroindoline (11) in ethanol (15 L) in ethanol was added 2.006 kg (1.355 L, approximately 3 equivalents) of methanesulfonic acid in a reaction vessel. The reaction mixture is heated to 35-40 ° C. After 7 hours, the starting material disappears by liquid chromatography and the reaction is cooled to room temperature overnight. The following day, 44 L of water was added with stirring. After cooling to about 5 ° C and 30 minutes of stirring, the mixture is filtered into a second vessel through a built-in 10 µm filter and pumped back into the reaction vessel. The vessels, filter devices and finally the solution in the reaction vessel are rinsed with 10 L of water, followed by the sequential addition of 38 L of isopropyl acetate and 16 L of 1M aqueous sodium hydroxide solution. The reaction mixture was cooled to 10-15 ° C. The pH of the lower phase is approximately 7 and 1.6 L of 50% aqueous hydroxide solution is added

After stirring for 15 minutes at 10-15 ° C, the mixture is allowed to settle for 10-15 minutes. 78.1 kg of the lower aqueous phase are separated, which, according to liquid chromatography, contains 28.4 g of compound (12) with a theoretically calculated yield of 0.85%. The 51 L organic fraction contained 3.057 kg of the title compound according to liquid chromatography with a yield of 92% based on 3 kg (7.49 mol) of CBZ spiroindoline sulfonamide (1). The resulting solution was used in the following synthesis.

Example 16A

(14)

Boc-aminoisobutyryl-O-benzylserine-spiroindoline (14)

Starting compounds: spiroindolinamine (12) amino acid peptide (13) dicyclohexylcarbodiimide (DCC)

1-hydroxybenzotriazole (HOBt) isopropyl acetate (IPAc) water

0.5N sodium hydroxide solution 0.5N hydrochloric acid solution saturated sodium bicarbonate solution

2481 g (5.57 mol) 1247.1 g (6.16 mol)

1266.7 g (6.16 mol) 827 g (6.16 mol)

I 37 I 36 I 36 I 36 I

The solution of amine (12) in isopropyl acetate is diluted to 39 L with isopropyl acetate (IPAc) and 37 L of water is added. The two-phase mixture is treated successively with 827 g of HOBT as a solid at room temperature and under nitrogen,

1266.7 g of DCC as melt and amino acid (13). The reaction mixture was stirred for 2 hours and the disappearance (<0.3% area under the curve) of the starting compound (12) was detected by liquid chromatography. The reaction mixture was filtered through Solka-floc ™ and the solid was washed with 13 L of isopropyl acetate. The reaction mixture can thus be stored overnight.

After transfer of the reaction mixture to a 100 L extraction apparatus, the aqueous portion was separated and the organic portion was washed successively with 36 L of 0.5 N sodium hydroxide solution, 0.5 N hydrochloric acid solution and saturated sodium bicarbonate solution. The assay yield is 3160 g (81% of spiroindoline with a ± 5% volume measurement error). The solution is concentrated to a small volume and washed twice with 4 L of ethanol. Intermediate (14) can be separated by crystallization by addition of water.

The yield of compound (14) from the reaction using alternative peptide reagents such as carbonyldiimidazole or a mixture of anhydrides (sec-butyl carbonate) is lower and with a high degree of epimerization. Other peptide binding agents are financially unavailable.

Example 16B

-62Boc-aminoisobutyryl-O-benzylserine-spiroindoline (14)

Starting compounds: spiroindolinamine (12) dicyclohexylcarbodiimide (DCC) 1-hydroxybenzotriazole (HOBt) Boc-2-aminoisobutyric acid (13) isopropyl acetate

1M aqueous sodium hydroxide solution

3.057 kg (6.89 mol) 1.56 kg (7.56 mol) 1.02 kg (7.55 mol) 1.54 kg (7.58 mol) 32 I

I 38 I

0.5 M aqueous hydrochloric acid solution 38 L saturated aqueous sodium bicarbonate solution 38 L absolute ethanol

I

To an approximately 51 L stirred solution of 3.057 kg of spiroindolinamine (12) in isopropyl acetate was added 49 L of water under a nitrogen atmosphere at room temperature. Gradually, 1.56 kg of DCC, 1.02 kg (approximately 1.1 equivalent) of HOBt, and finally 1.54 kg (approximately 1.1 equivalent) are added to the reaction vessel at room temperature and under nitrogen atmosphere. N-Boc-O-benzyl-D-serine. After stirring vigorously for 2 hours at room temperature under nitrogen atmosphere, completion of the reaction was confirmed by liquid chromatography. The reaction mixture was filtered through an Estrella filter using a pump. The first vessel, filter, pump and connecting tube are rinsed with 22 L of isopropyl acetate. The two-phase mixture was stirred in the vessel for 5 minutes and the fractions were separated. Bottom aqueous portion with weight

51.1 kg are separated. The organic solution was washed successively with 38 L of a 1M aqueous sodium hydroxide solution, 38 L of a 0.5 M aqueous hydrochloric acid solution and finally 38 L of a saturated aqueous sodium bicarbonate solution.

The organic solution is transferred via a built-in filter (containing a preformed column with a 10 µm aperture) to another vessel where the solvent is changed to ethanol. The vessel is rinsed with 10 L of isopropyl acetate and this solution is used to wash the pump, filter and tube into the reaction vessel. The filtrate and washings were combined. The total volume measured by the dipstick is 75 L. According to liquid chromatography, 4.395 kg is obtained

-63Boc-aminoisobutyryl-O-benzylserine-spiroindoline (14) with a total yield of 93% (from 7.49 ml of the starting CBZ-spiroindoline-sulfonamide (1)).

The reaction mixture was concentrated in vacuo to a volume of approximately 15 L. The solvent was changed to ethanol by slowly adding and rinsing a total of 5 L of absolute ethanol. According to gas chromatography, less than 1% of isopropyl acetate remains in the batch. The 25 L solution contains 4.395 kg of compound (14), which is used in the next synthesis. If necessary, compound (14) can be crystallized by adding water.

Example 17A

Aminoisobutyryl-O-benzylserine-spiroindoline (15)

Starting compounds: Boc-spiroindoline (14) methanesulfonic acid (MsOH) ethanol water

1N sodium hydroxide solution ethyl acetate activated carbon Darco 60

3160 g (5.03 mol)

979 ml (15.1 mol)

6.2 l 30 l 11 l 26 l 1 kg

A solution of Boc-spiroindoline (14) in 6.2 L of ethanol was treated with 979 mL of methanesulfonic acid. The temperature is then raised from 20 to 30 ° C and the reaction is allowed to proceed

-64the night. After 12 hours at 20 ° C, 15% of the area under the curve of the starting compound remains in the mixture. The mixture was therefore heated at 35 ° C for 6 hours. After completion of the reaction (less than 0.1 area% under the curve of compound (14)), the reaction mixture was cooled to 20 ° C and 30 L of water was added. The solution is filtered through a glass funnel and polypropylene filter to remove residual DCU. The mixture was transferred to a 100 L extraction apparatus and 26 L of ethyl acetate was added. The aqueous portion was brought to basic pH by the addition of 11 L of cooled 1N sodium hydroxide solution and 1 L of 50% sodium hydroxide solution. Ice must be added to keep the temperature below 14 ° C. A higher temperature leads to the formation of an emulsion.

The organic portion is distilled at 50 ° C and 2793 Pa to a KF value of less than 1000 pg / ml. Lower KF is the result of more effective action of activated carbon and its better isolation after salt formation. A KF = 160 pg / ml was obtained to give 700 g of product. The solution was diluted with ethyl acetate to a total volume of 31 L (the solution contained 2.40 kg of product by liquid chromatography). Darco G-60 activated charcoal is added and the mixture is stirred for 24 hours. After filtration through Solkafloc ™, the filter cake was washed with 16 L of ethyl acetate to give 2.34 kg of product.

Example 17B ··

(15)

Aminoisobutyryl-O-benzylserine-spiroindoline (15)

-65Substance compounds: Boc-spiroindoline (14) methanesulfonic acid ethyl acetate

1M aqueous sodium hydroxide solution 50% aqueous sodium hydroxide solution Darco G-60

Solka floc ™

4.395 kg (6.99 mol) 2.017 kg (20.99 mol)

1851 161 2.6 1,900 g 2.5 kg

To approximately 25 L of a stirred solution of 4.395 kg of Boc-spiroindoline (14) in ethanol placed in the reaction vessel was added 2.017 kg (1.36 L, approximately 3 equivalents) of methanesulfonic acid at room temperature. The reaction mixture was warmed to 35-40 ° C and stirred overnight. The next day, the reaction mixture contained approximately 1.1% area under the curve of the starting material and therefore the reaction was continued for a further 4 hours. According to liquid chromatography, the ratio of product to starting material was 99.6: 0.4. The reaction mixture was concentrated in vacuo to a volume of about 15 L and then diluted with 44 L of water. The reaction mixture is cooled to 5 ° C, stirred for 30 minutes and filtered using a pump through a built-in Sparkler filter (containing a pre-made 10 µm hole column) into a second vessel to remove a small amount of residual DCU.

The vessel, pump, filter and tube are washed with 10 L of water, which is then added to the reaction vessel. 36 L of ethyl acetate was added and the stirred mixture was cooled to 10 ° C. 16 L of cold 1 M aqueous sodium hydroxide solution at 5-10 ° C and 2.6 L of cold 50% aqueous sodium hydroxide solution at 5-10 ° C were added at this temperature. The temperature was raised to 14 ° C. The resulting mixture was stirred at less than 14 ° C for 15 minutes and the lower aqueous portion was separated.

The reaction mixture was concentrated in vacuo to a volume of approximately 20 L and a mixture of 35 L of ethyl acetate and 5 L of ethanol was added to a final volume of 20 L. At the end of the distillation, KF was 9160 mg / ml. The solvent was replaced with ethyl acetate at a slow rate

Adding a rinse with a total of 40 L of ethyl acetate. At the end of this distillation, the KF was 446 mg / ml. The reaction mixture was diluted with 10 L of ethyl acetate.

900 g of activated charcoal are added to the mixture. Darco G-60 and washed with 6 L of ethyl acetate. The mixture was stirred overnight at room temperature, and 0.5 kg of Solkafloc ™ was added to the reaction mixture with stirring the following day. 2.0 kg of Solka-floc ™ was stirred in a small amount of ethyl acetate to fill the Estrella filter. Excess solvent is pumped off through the built-in Estrella filter. Excess solvent is pumped through a built-in Sparkler filter containing a 10 μίτι aperture column. The suspension is filtered twice by means of a pump into two 40 L stainless steel storage vessels. The liquid is clear and clear. The reaction vessel was washed with 22 L of ethyl acetate and this material was washed with other equipment and poured into storage vessels. The contents of both storage vessels are transferred to the reaction vessels and the solution is mixed thoroughly.

The 58 L reaction mixture had a KF value of 2950 mg / mL and was then dried again in vacuo to a volume of 20-25 L. After diluting the reaction mixture to 46 L (measured by dipping) by adding 25 L of ethyl acetate, the KF value was 363 mg / ml. The reaction mixture was diluted to a volume of 62 L by addition of 17 L of ethyl acetate and used in the next final synthesis procedure.

Example 18A

(16)

-67Spiro [3H-indole-3,4'-piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate (16)

Starting compounds: amine (15) methanesulfonic acid (MsOH) ethyl acetate ethanol

8% ethanol in ethyl acetate solution

2340 g (4.43 mol)

316 ml (4.88 mol)

I

4.8 I 20 I.

A volume of the solution of compound (15) from the previous part is brought by adding ethyl acetate and 4.8 L of ethanol to a volume of 60 L. At 45 ° C, a solution of 316 mL of methanesulfonic acid in 3 L of ethyl acetate is added. 496 g of the title compound of formula I (10% by weight of free amine as seeding material) were added to the dark red homogeneous solution for seeding. The temperature is raised to about 48 ° C and the reaction mixture is allowed to stand at 52 ° C for 1.5 hours. Analysis showed complete formation of the resultant compound of Formula I. By using less than 10% of the inoculum material, the stand time (greater than 3 hours) was prolonged. The suspension is allowed to cool to 20 ° C overnight and filtered under a nitrogen centrifuge. The filter cake was washed with 20 L of 8% ethanol in ethyl acetate. The presence of nitrogen is very important because wet crystals are very hygroscopic. The reaction mixture was dried under vacuum at 35 ° C to give

2.7 kg of the resultant compound of formula I with a total yield of 56% and a purity of 99.9% of the area under the curve and in the presence of less than 0.1% enantiomer.

The conversion of the compound of formula II to the compound of formula I is accomplished by salt formation in a mixture of ethyl acetate and ethanol by the addition of methanesulfonic acid 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 thickens. The temperature was raised to 51 ° C and the suspension was allowed to stand overnight. The compound (16) is converted to the compound of formula (I)

By the procedure, it is possible to form the seed crystals of the compound (16) of the general formula

Example 18B

Starting compounds: amine (15) methanesulfonic acid ethyl acetate absolute ethanol

SpirofSH-indole-5'-piperidin-1'-ylcarbonyl] 2- (phenylmethyloxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate (Formula I)

Spiro [3H-indole-3,4'-piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate (16)

3.1 kg (5.86 mol)

620 g (6.45 mol)

I

8.7 I g (0.11 mol)

To a solution of approximately 62 L of 3.1 kg of amine (15) in ethyl acetate is added 6.4 L of absolute ethanol in the reaction vessel. The reaction mixture was heated to 50 ° C in the reaction vessel and a solution of 620 g (412 mL, 1.1 equivalents) of methanesulfonic acid in 11 L of ethyl acetate was added over a period of about 5 minutes at 50-54 ° C. The reaction mixture was seeded with 70 g of spiro [3H-indole-3,4'-piperidin] -1-yl) carbonyl] -2- (phenylmethyloxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate

-69 (Formula I). The resulting suspension was stirred overnight and heated to 55 ° C under nitrogen.

The next day, the suspension is cooled to 15-20 ° C for 2 hours and then dropped through a 50 cm polypropylene filter dropwise in a nitrogen atmosphere, washing the product as a solid with a mixture of 2.3 L absolute ethanol in 26 L ethyl acetate. The white solid product was collected and dried in an Apex oven at 35 ° C for about two days. The dried spiro [3H-indole-3,4'-piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate weighing 3.352 kg was passed through a Jackson-Crockatt sieve to give 3.357 kg of the title compound, including 70 g of the vaccine. Total yield is 3.277 kg.

HPLC conditions:

Liquid chromatography:

Retention time on a Zorbax RX-C8 column (4.6 mm x 25 cm), detection at 210 nm, flow rate = 1.5 mL / minute.

Compound 1: 60:40 CH3CN-H2O (1% H3PO4) RT = 5.0 minutes Compound 1b: 35:65 CH3CN-H2O (0.1% by volume NH ₄ OAc) RT = 6.2 minutes 10 Compound 60: 40 CH3CN-H2O (0.1% H3PO4) RT = 2.9 min Compound 11: 60:40 CH3CN-H2O (1% H3PO4) RT = 5.4 min Compound 12: 40:60 CH3CN-H2O (pH 5, 25 NaH ₂ PO ₄ ) (6.9 g / l water) (pH achieved by addition of sodium hydroxide) RT = 5.6 minutes compound 14: 60:40 CH 3 CN-H 2 O (0.1% H 3 PO 4) RT = 4.65 minutes Compound 15: 40:60 CH ₃ CN-H ₂ O (pH = 5.25 NaH ₂ PO ₄ ) (6.9 g / l water) (pH achieved by addition of sodium hydroxide) RT = 4.9 min

Liquid chromatography:

Retention time on a Zorbax RX-C8 column (4.6 mm x 25 cm), detection at 210 nm, flow rate = 1.2 ml / min, column temperature = 48 ° C.

Solvent A = 0.05% phosphoric acid solution + 0.01% triethylamine in water.

-70Solvent B = acetonitrile.

Solvent Concentration Gradient Overview:

time % A % B 0 minutes 95 5 35 minutes 10 90 38 minutes 95 5 40 minutes 95 5

compound 1 compound 1b compound 10 compound 11 compound 12 compound 14 compound 15

Retention time (minutes)

25.2

8.5

20.5

26.3

14.8

25.6

15.7

Example 19

Preparation of a compound of formula I, N- [1 (R) - [(1,2-dihydro-1-methanesulfonylspiro [3H-indole-3,4'-piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] -2-amino-2-methyl-propanamide methanesulfonate

The conversion of the compound of formula II to the compound of formula I is carried out according to the procedure of Example 18A. A salt is formed in a mixture of ethyl acetate and ethanol, cooled to 45 ° C by addition of methanesulfonic acid, and the initial salt solution at 55 ° C. Upon reaching this temperature, crystals begin to form and the suspension becomes dense. After raising the temperature to 51 ° C, the suspension was allowed to stand overnight to complete the conversion of the compounds.

-71 Example 20

Preparation of N- [1 (R) - [(1,2-dihydro-1-methanesulfonyl-spiro ^{[3H-indole-3,4'-piperidine] -1} yl) carbonyl] -2- (phenylmethyloxy) ethyl] -2-amino-2-methyl-propanamide methanesulfonate (Formula I)

Conversion of a compound of formula II to a compound of formula I is accomplished by stirring for 2 to 24 hours with a solution of N- [1 (R) - [(1,2dihydro-1-methanesulfonyl-spiro [3H-indole-3,4'-piperidine]] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] -2-amino-2-methylpropanamide of formula II in isopropanol at about 25 ° C.

Example 21

Preparation of N-Fluoro-1 H -dihydro-1-methanesulfonyl-spiroOH-indole-5H-piperidin-1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] -2-amino-2-methyl-propanamide methanesulfonate (Formula IV)

In a mixture of 24.8 ml of ethyl acetate, 1.6 ml of ethanol and 1.95 ml of water, 8.4 g of N- [1 (R) - [(1,2-dihydro- 1-Methanesulfonylspiro [3H-indole-3,4'-piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate in a suitable morphological form. At 40 ° C the solvent is evaporated and the solid formed is ground to a fine powder which is exposed to approximately 75% relative humidity to give the title compound of formula IV.

Example 22

Preparation of N- [1 (R) - [(1,2-Dihydro-1-methanesulfonyl-spiro [3H-indole-3,4'-piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] - 2-amino-2-methylpropanamide methanesulfonate (Formula IV)

-72 From a solution of ethyl acetate, ethanol and water in a volume ratio of 24.8: 1.6: 1.95, N- [1 (R) - [(1,2-dihydro-1-methanesulfonyl-spiro) is crystallized in a suitable morphological form [3H-Indole-3,4'-piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate to give compound of formula IV.

Example 23

Preparation of N- [1 (R) - [(1,2-Dihydro-1-methanesulfonyl-spiro [3H-indole-3,4'-piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] - 2-Amino-2-methyl-propanamide methanesulfonate of formula IV

A suspension of N- [1 (R) [(1,2-dihydro-1-methanesulfonyl-spiro [3H-indole-3,4'-piperidin] -1'-yl) carbonyl] is stirred at approximately 25 ° C overnight. ] -2- (phenylmethyloxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate of formula I in a 90:10 mixture of isopropyl acetate and ethanol containing approximately

2.8% water by volume. The title compound is obtained as a solid.

In the description and the example section, the subject matter has been described in connection with some particular and preferred embodiments, but it is understood that one skilled in the art could suggest various modifications, changes, modifications to the above procedures, particularly as regards deleting, replacing or adding some parts of the procedures. in doing so, depart from the spirit of the invention.

For example, it would be possible to use other effective dosages than those mentioned, in particular due to changes in the mammal's response to treatment or due to the disease in which the active ingredients are used. Indeed, the specific reactions to the active ingredient may vary depending on the type of active ingredient or the pharmaceutical carrier chosen, the chosen pharmaceutical form and the route of administration, and such expected changes must be taken into account. Thus, the above data is not intended to limit the invention.

Claims (27)

PATENT CLAIMS A polymorphic form of N- [1 (R) - [(1,2-dihydro-1-methanesulfonyl-spiro [3H-indole-3,4'-piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] 2-amino-2-methyl-propanamide methanesulfonate, referred to as Form I, in which X-ray diffraction baseline bending values of 6.5, 14.7, 16.9, 17.1, 17.9, 19.5, 21.1, 21.7 and 22.0 ° (2 aunt). 2. Polymorph form of N- [1 (R) - [(1,2-dihydro-1-methanesulfonyl-spiro [3H-indol-3,4'-piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl 2-Amino-2-methyl-propanamide methanesulfonate according to claim 1, wherein the curve when heated in a differential calorimeter at 10 ° C / minute under nitrogen is an endothermic melting curve with an extrapolated melting onset temperature of 170 ° C. 3. Polymorphic form of N- [1 (R) - [(1,2-dihydro-1-methanesulfonyl-spiro [3H-indol-3,4'-piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl 2-Amino-2-methyl-propanamide methanesulfonate according to claim 1, wherein the solubility in isopropanol is 4.6 mg / ml. 4. Polymorph form of N- [1 (R) - [(1,2-dihydro-1-methanesulfonyl-spiro [3H-indol-3,4'-piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl 2-Amino-2-methyl-propanamide methanesulfonate according to claim 1, which appears to be birefringent in polarized light. 5. Polymorphic form of N- [1 (R) - [(1,2-dihydro-1-methanesulfonyl-spiro [3H-indole-3,4'-piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] Of 2-amino-2-methylpropanamide methanesulfonate, referred to as Form IV, in which X-ray diffraction baseline bending values of 16.0, 16.2, 18.3, 20.1, 21.0 and 24 can be observed , 2 ° (2 aunt). 6. Polymorph form of N- [1 (R) - [(1,2-dihydro-1-methanesulfonyl-spiro [3H-indol-3,4'-piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl 2-Amino-2-methyl-propane-74 amide methanesulfonate according to claim 5, wherein the curve under heating at 10 ° C / minute under nitrogen is an endothermic curve for water loss at 45 ° C, followed by an endothermic curve for melting and with an extrapolated melting point of 129 ° C. 7. Form of N- [1 (R) - [(1,2-dihydro-1-methanesulfonyl-spiro [3H-indol-3,4'-piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] polymorphic form 2-amino-2-methyl-propanamide methanesulfonate according to claim 5, comprising 3.5 moles of water per 1 mol of N- [1 (R) [(1,2-dihydro-1-methanesulfonyl-spiro [3H-indole- 3,4 &apos; -piperidine] -r-yl) carbonyl] -2- (phenylmethyloxy) ethyl] -2-amino-2-methyl-propanamide methanesulfonate. A process for preparing the polymorphic form of N- [1 (R) - [(1,2-dihydro-1-methanesulfonylspiro [3H-indole-3,4'-piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) Ethyl] -2-amino-2-methyl-propanamide methanesulfonate according to claim 1, referred to as Form I, characterized in that the solution of Form II N- [1 (R) - [(1 2-Amino-2-methyl-propanamide methanesulfonate in isopropanol at about 25 ° C for 2 hours up to 24 hours. A pharmaceutical composition comprising a pharmaceutical carrier and an effective amount of a polymorphic form of N- [1 (R) - [(1,2-dihydro-1-methanesulfonyl-spiro [3H-indole-3,4'-piperidine]] - 1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] -2-amino-2-methyl-propanamide methanesulfonate according to claim 1. 10. A pharmaceutical composition for the treatment of osteoporosis comprising a combination of a bisphosphonate and a polymorphic form of N- [1 (R) - [(1,2dihydro-1-methanesulfonyl-spiro [3H-indole-3,4'-]]. piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate according to claim 1. -7511. Pharmaceutical composition according to claim 10, characterized in that it contains alendronate as the bisphosphonate. A pharmaceutical composition comprising a pharmaceutical carrier and an effective amount of the polymorphic form of N- [1 (R) - [(1,2-dihydro-1-methanesulfonyl-spiro [3H-indole-3,4'-piperidine]] - 1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] -2-amino-2-methyl-propanamide methanesulfonate according to claim 5. 13. A pharmaceutical composition for the treatment of osteoporosis comprising a combination of a bisphosphonate and a polymorphic form of N- [1 (R) - [(1,2dihydro-1-methanesulfonyl-spiro [3H-indole-3,4'-]]. piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate according to claim 5. 14. The pharmaceutical composition of claim 13, wherein the bisphosphonate is alendronate. Use of the polymorphic form of N- [1 (R) - [(1,2-dihydro-1-methanesulfonylspiro [3H-indole-3,4'-piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl 2-Amino-2-methyl-propanamide methanesulfonate according to claim 1, for the manufacture of a medicament for increasing the concentration of endogenous growth hormone in a human or other animal. Use of the polymorphic form of N- [1 (R) - [(1,2-dihydro-1-methanesulfonylspiro [3H-indole-3,4'-piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl 2-Amino-2-methyl-propanamide methanesulfonate according to claim 1 in combination with another growth hormone secretagogue for the manufacture of a medicament for increasing endogenous growth hormone secretion in a human or other animal. The use of claim 16, wherein the further growth hormone secretion enhancer is selected from the group of growth hormone releasing GHRP-6 peptide, growth hormone releasing GHRP-2 peptide, GHRP-1 peptide, -76 growth hormone releasing factor, B-HT920, growth hormone release factor, growth hormone analog release factor, IGF-1 and IGF-2. Use of the polymorphic form of N- [1 (R) - [(1,2-dihydro-1-methanesulfonylspiro [3H-indole-3,4'-piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl 2-Amino-2-methyl-propanamide methanesulfonate according to claim 1 for the manufacture of a medicament for increasing feed utilization, promoting growth, increasing milk production and improving meat quality in livestock. Use of the polymorphic form of N- [1 (R) - [(1,2-dihydro-1-methanesulfonylspiro [3H-indole-3,4'-piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl 2-Amino-2-methyl-propanamide methanesulfonate according to claim 1 for the manufacture of a medicament for the treatment or prevention of osteoporosis, catabolic diseases, immune system deficiency including conditions with reduced T ₄ / T ₈ cells, hip fractures, muscle and skeletal disorders in the elderly, growth hormone deficiency in adults or children, obesity, cachexia and protein loss in chronic diseases such as AIDS or malignant tumors, treatment of patients recovering from major surgery, injuries or burns. Use of the polymorphic form of N- [1 (R) - [(1,2-dihydro-1-methanesulfonylspiro [3H-indole-3,4'-piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl 2-Amino-2-methyl-propanamide methanesulfonate according to claim 1 in combination with a bisphosphonate for the manufacture of a medicament for the treatment of osteoporosis. The use according to claim 20, wherein alendronate is administered as the bisphosphonate. Use of the polymorphic form of N- [1 (R) - [(1,2-dihydro-1-methanesulfonylspiro [3H-indole-3,4'-piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl 2-Amino-2-methyl-propanamide methanesulfonate according to claim 5, for the manufacture of a medicament for increasing the concentration of endogenous growth hormone in a human or other animal. -77 Use of the polymorphic form of N- [1 (R) - [(1,2-dihydro-1-methanesulfonylspiro [3H-indole-3,4'-piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl 2-Amino-2-methyl-propanamide methanesulfonate according to claim 5 in combination with another growth hormone secretagogue for the manufacture of a medicament for increasing endogenous growth hormone secretion in a human or other animal. The use of claim 23, wherein the further growth hormone secretion enhancer is selected from the group of growth hormone releasing peptide GHRP-6, growth hormone releasing peptide GHRP-2, growth hormone releasing peptide GHRP-1, B-HT920, growth hormone release factor, growth hormone analog release factor, IGF-1 and IGF-2. Use of the polymorphic form of N- [1 (R) - [(1,2-dihydro-1-methanesulfonylspiro [3H-indole-3,4'-piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] -2-amino-2-methyl-propanamide methanesulfonate according to claim 5 for the manufacture of a medicament for increasing feed utilization, promoting growth, increasing milk production and improving meat quality in livestock. Use of the polymorphic form of N- [1 (R) - [(1,2-dihydro-1-methanesulfonylspiro [3H-indole-3,4'-piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl -2-Amino-2-methyl-propanamide methanesulfonate according to claim 5 for the manufacture of a medicament for the treatment or prevention of osteoporosis, catabolic diseases, immune system deficiency including conditions with reduced T4 / T8 cell ratio, hip fractures, disorders musculoskeletal system in the elderly, growth hormone deficiency in adults or children, obesity, cachexia and protein loss in chronic diseases such as AIDS or malignant tumors, treatment of patients recovering from major surgery, injuries or burns. Use of the polymorphic form of N- [1 (R) - [(1,2-dihydro-1-methanesulfonylspiro [3H-indole-3,4'-piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl 2-Amino-2-me-78-ethyl-propane-amide-methanesulfonate according to claim 5 in combination with a bisphosphonate for the manufacture of a medicament for the treatment of osteoporosis. The use of claim 27, wherein alendronate is administered as the bisphosphonate.