SK51198A3 - Method for producing n-[1(r)-[(1,2-dihydro-1- -methanesulfonylspiro[3h-indole-3,4'-piperidine-1'-yl)carbonyl]- -2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamide - Google Patents

Abstract

The present invention is directed to a novel process for the preparation of the compound N-[1(R)-[(1,2-dihydro-1-methanesulfonyl-spiro[3H-indole-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenylmethyl-oxy)ethyl]-2-amino-2-methyl-propanamide, and salts thereof, which has structure (1) and which has the ability to stimulate the release of natural or endogenous growth hormone. This compound may be used to treat conditions which require the stimulation of growth hormone production or secretion such as in humans with a deficiency of natural growth hormone or in animals used for food or wool production where the stimulation of growth hormone will result in a larger, more productive animal.

Description

Process for the preparation of N- [1 (R) - [(1,2-dihydro-1-methanesulfonylspiro [OH] -indole-5'-piperidin] 1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] -2-amino- 2-methylpropanamide

J

Technical field

The invention relates to a process for the production of a substance which increases the secretion of growth hormone from the pituitary.

BACKGROUND OF THE INVENTION

Growth hormone is a substance secreted from the pituitary and stimulates the growth of all tissues that are capable of growth. In addition, growth hormone is known to have the following basic effects on metabolic processes in the body: 1) increases protein synthesis in all cells of the organism, 2) decreases the rate of carbohydrate utilization in cells of the organism, 3) increases mobilization of free fatty acids and utilization of fatty acids for getting energy. Growth hormone deficiency causes various disorders, such as dwarf.

A variety of procedures for the release of growth hormone are known. For example, chemicals such as arginine, L-3,4-dihydroxyphenylalanine, L-DOPA, glucagon, vasopressin, insulin-induced hypoglycaemia, as well as various activities such as sleep and physical exercise directly increase the release of growth hormone from the pituitary, probably through the hypothalamus, by decreasing the secretion of somatostatin or by increasing the secretion of the known GRF to release growth hormone or by acting through a hitherto unknown endogenous hormone that promotes the release of growth hormone.

If it is desired to increase the concentration of growth hormone, this problem is usually solved by administering exogenous growth hormone or GRF or peptide compounds stimulating growth hormone production and / or release. In any case, the peptide nature of these substances requires their injection. Originally, the source of growth hormone was only pituitary extraction from the dead. It was therefore a very costly product which was also associated with the risk of transmission of various diseases from the source from which the glands were obtained.

Recently, there is a recombinant growth hormone that is no longer associated with this risk, but it is still a very expensive product that must also be given as an injection or spray for nasal administration. Other endogenous growth hormone release stimulating agents have also been developed.

In particular, some of the spiro compounds described in PCT Patent Application WO 94/13 696 and Proc. Natl. Acad. Sci., USA, 92, 7001-7005, July 1995. They are non-peptide in nature, promoting hormone release. These substances have the ability to stimulate the secretion of natural growth hormone and can therefore be used in the treatment of conditions in which growth hormone production or secretion needs to be stimulated. For example, in humans, there is a deficiency of natural growth hormone, while in animals raised for wool or meat production, the administration of growth hormone can improve the growth of livestock and hence their performance.

Among the preferred compounds disclosed in the above-described international patent application are in particular N- [1 (R) - [(1,2-dihydro-1-methanesulfonylspiro [3H-indole-3,4'-piperidin] -1'-yl) carbonyl -2- (phenylmethyl-oxy) -ethyl] -2-amino-2-methyl-propanamide of the formula

PCT Patent Application WO 94/13 696 describes a number of processes for the preparation of this compound in Examples 18, 19 and 55. However, a very expensive EDC-type binder has been used to produce this compound, costing 1 kg of this compound is $ 1,100. In addition, it is necessary to use multiples of trifluoroacetic acid equivalent as a catalyst to cleave the BOC protecting groups and process.

-3 still requires complicated chromatographic purification, in addition, the entire process results in a gummy product.

The invention therefore proposes a process which is carried out in six stages without isolation between the stages, in this way a material with a purity equal to or higher than 99.9% can be obtained at high yield. In addition, costs are falling due to the fact that DCC is used instead of EDC, kg of this substance costs only $ 40. Another advantage is less environmental pollution due to the use of methanesulfonic acid instead of trifluoroacetic acid as a catalyst, moreover, it is necessary to use a much smaller number of equivalents of this acid when cleaving the protecting groups. Isolation of the resulting product is easy.

SUMMARY OF THE INVENTION

The present invention provides a process for the 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 of formula

and salts thereof, especially methanesulfonates.

This compound has the ability to stimulate the release of natural or endogenous growth hormone and can therefore be used to treat conditions that require stimulation of growth hormone production or secretion. For example, in humans, it is a deficiency of natural growth hormone, in animals and an increase in meat or wool production.

The process according to the invention makes it possible to obtain the desired substance from inexpensive and environmentally acceptable starting materials, reagents and solvents. The process does not require chromatographic purification, and the resulting product can be obtained from spiroindoline sulfonamide as an intermediate without isolation of any intermediate.

The individual reactions during the general process of the invention are summarized in the following Scheme I

Scheme I protection: amino groups preparation of chloride reduction;

acid chloride

COOH

COOH COCI

-5 Scheme I - continued

wherein L is an amino protecting group and X 'is a corresponding negatively charged ion.

In this general production method, the first reaction relates to the preparation of a compound of formula I

where L is an amino protecting group, the preparation consists in reacting the amino acid of the formula with a compound of the formula

in the presence of an acid activating agent in an inert solvent in the presence of a catalyst to form a compound of formula I.

An acidic activating agent suitable for use in this process may be, for example, DCC, EDC, ECAC, and BOP, with suitable activating agent being DCC, N, Nlohex-dicyclohexylcarbodiimide.

The catalyst suitable for use in this process may be HOBT and the like, a preferred catalyst is HOBT, hydroxybenzotriazole.

Inert solvents suitable for use during the reaction include acetonitrile, isopropyl acetate, ethyl acetate, propionitrile, water, chlorinated hydrocarbons such as dichloromethane, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene, o-dichlorobenzene, benzene, toluene, xylenes and the like, as well as mixtures of these, the preferred solvent is in particular acetonitrile, isopropyl acetate and water.

the reaction may preferably be carried out at a temperature range of -40 to 150 ° C, most preferably in the range of 20 to 35 ° C.

The amino protecting groups include, for example, benzyl, benzyloxymethyl, benzyloxycarbonyl, (carbobenzyl), benzylsulfonyl, 2-bromoethyloxycarbonyl, tert-butoxycarbonyl, 2-chlorobenzyloxycarbonyl, 2-chloroethyloxycarbonylcarbonyl, 4-tert-amyloxy, di-tert-amyloxy. -methoxybenzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 2-nitrophenylsulfonyl, phthaloyl, 2,2,2-trichloro-tert-butyloxycarbonyl, trifluoroacetyl, triphenylmethane, allyloxycarbonyl and vinyloxycarbonyl and the like, and especially preferred groups are tert-butyloxycarbonyl; butoxycarbonyl.

From the standpoint of economy, it is advantageous to carry out this reaction in situ without isolating the compound of formula I which can be used directly for the next reaction.

In the general process of the invention, the second reaction consists in the preparation of a compound of formula II

(H)

In carrying out this procedure, a compound of formula (I) is reacted

wherein L is an amino protecting group, with a reagent for removing the protecting group to form a compound of formula II.

The amino protecting groups include, for example, benzyl, benzyloxymethyl, benzyloxycarbonyl (carbobenzyloxy), benzylsulfonyl, 2-bromomethyloxycarbonyl, tert-butoxycarbonyl, 2-chlorobenzyloxycarbonyl, 2-chloroethoxycarbonyl, methoxy-9-methoxycarbonyl, di-tert-amyloxy, 4-nitrobenzyloxycarbonyl, 2-nitrophenylsulfonyl, phthaloyl, 2,2,2-trichloro-tert-butyloxycarbonyl, trifluoroacetyl, triphenylmethane, allyloxycarbonyl and vinyloxycarbonyl and the like, particularly preferred groups are benzyloxycarbonyl and tert-butoxycarbonyl; .

In this procedure, it is possible to remove the amino protecting group using the appropriate catalyst. The tert-butoxycarbonyl group may be removed in a solvent such as methanol, ethanol, methylene chloride, ethyl acetate or isopropyl acetate by treatment with a strong acid. The strong acids include methanesulfonic acid, trifluoroacetic acid, hydrochloric acid, hydrogen chloride, hydrogen bromide, hydrogen iodide, trifluoromethanesulfonic acid, camphorsulfonic acid, sulfuric acid, phosphoric acid, and also arylsulfonic acids, for example benzenesulfonic acid, p-benzenesulfonic acid, p-benzenesulfonic acid, p-benzenesulfonic acid, p-benzenesulfonic acid. Preferred catalysts are, for example, trifluoroacetic acid, methanesulfonic acid, camphorsulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid and p-chlorobenzenesulfonic acid. The most preferred

The catalyst is methanesulfonic acid. A preferred solvent is methanol or ethanol, especially ethanol.

The preferred reaction temperature range is -40 to 150 ° C, most preferably the range is 10 to 40 ° C.

The benzyloxycarbonyl group can be removed in a number of ways, for example by hydrogenation with hydrogen in the presence of a noble metal catalyst or an oxide thereof, for example palladium on activated carbon in a protic solvent such as ethanol. If catalytic hydrogenation is not possible due to the presence of other potentially reactive functional groups, the latter can also be removed by treatment with a solution of hydrogen bromide in acetic acid or with a mixture of TFA and dimethylsulfide.

For reasons of economy, it is advantageous to carry out this acid-catalyzed deprotection without isolating the compound of formula II.

A third reaction of this general procedure is to prepare a compound of formula III

ph

N — L H (III)

SO ₂ Me wherein L represents an amino protecting group by reaction of an amino acid of the general formula

wherein L is an amino protecting group with a compound of formula II

in the presence of an acidic activating agent in an inert solvent and in the presence of a catalytic agent to form a compound of formula III.

Acid activating agents suitable for use in the present process include DCC, EDC, ECAC, and BOP, with DCC, N, N‘-dicyclohexylcarbodiimide being the preferred activating agent.

Catalysts suitable for use herein include HOBT and the like, a preferred catalyst agent is HOBT, hydroxybenzotriazole.

Inert solvents suitable for carrying out this process include acetonitrile, isopropyl acetate, ethyl acetate, propionyl, water, chlorinated hydrocarbons such as dichloromethane, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene, o-dichlorobenzene, benzene, toluene, xylenes and the like, as well as mixtures thereof. the preferred solvent is a mixture of isopropyl acetate and water, preferably in a volume ratio of about 40:60 to 60:40, in particular a volume ratio of about 50:50. The preferred reaction temperature range is -40 to 150 ° C, most preferably the reaction temperature is 20 to 50 C.

Examples of amino protecting groups include benzyl, benzyloxymethyl, benzyloxycarbonyl (carbobenzyloxy), benzylsulfonyl, 2-bromomethyloxycarbonyl, tert-butoxycarbonyl, 2-chlorobenzyloxycarbonyl,

2-chloroethyloxycarbonyl, di-tert-amyloxycarbonyl, 9-fluorenylmethyloxycarbonyl, isopropoxycarbonyl, 4-methoxybenzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 2-nitrophenylsulfonyl, phthaloyl, 2,2,2-trichloro-carbonyl, tert-butyloxycarbonyl, tri-tert-butyloxyethoxy vinyloxycarbonyl and the like, particularly preferred groups are benzyloxycarbonyl and tert-butoxycarbonyl groups, most preferably tert-butoxycarbonyl.

From an economic standpoint, it is preferred to carry out the reaction in situ without isolating the compound of formula III. In another embodiment, the compound may be isolated as a separate intermediate.

A fourth reaction during the process of the invention is the preparation of a compound of formula IV and pharmaceutically acceptable salts thereof

(IV) in which a compound of formula III is reacted

(III) wherein L is an amino protecting group, with a protecting group cleavage reagent to form a compound of formula IV.

-12Z amino-protecting groups include, for example, benzyl, benzyloxymethyl, benzyloxycarbonyl (carbobenzyloxy), benzylsulfonyl, 2-bromomethyloxycarbonyl, tert-butoxycarbonyl, 2-chlorobenzyloxycarbonyl, 2-chloroethyloxycarbonylcarbonyl, di-tert-butoxycarbonyloxycarbonyl, di-tert-butoxycarbonyl, -methoxybenzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 2-nitrophenylsulfonyl, phthaloyl, 2,2,2-trichloro-tert-butyloxycarbonyl, trifluoroacetyl, triphenylmethane, allyloxycarbonyl and vinyloxycarbonyl and the like, and especially preferred groups are tert-butyloxycarbonyl; a butoxycarbonyl group.

In this procedure, the amino protecting group can be removed using the appropriate catalyst, the t-butoxycarbonyl group can be removed in a solvent such as methanol, ethanol, methylene chloride, ethyl acetate or isopropyl acetate by treatment with a strong acid. Strong acids include methanesulfonic acid, trifluoroacetic acid, hydrochloric acid, gaseous hydrogen chloride, hydrogen bromide, hydrogen iodide, trifluoromethanesulfonic acid, camphorsulfonic acid, sulfuric acid, phosphoric acid, and also arylsulfonic acids, for example benzenesulfonic acid, penzenesulfonic acid, p-toluenesulfonic acid, p-toluenesulfonic acid. Preferred catalysts are, for example, trifluoroacetic acid, methanesulfonic acid, camphorsulfonic acid, benzenesulfonic acid, ptoluenesulfonic acid and p-chlorobenzenesulfonic acid. The most preferred catalyst is methanesulfonic acid. A preferred solvent is methanol or ethanol, especially ethanol.

The preferred reaction temperature range is -40 to 150 ° C, most preferably the range is 10 to 40 ° C.

The benzyloxycarbonyl group can be removed in a number of ways, for example by hydrogenation with hydrogen in the presence of a noble metal catalyst or an oxide thereof, for example palladium on activated carbon in a protic solvent such as ethanol. If it is not possible to use catalytic hydrogenation due to the presence of other potentially reactive functional groups, the acid can also be removed by treatment with a solution of hydrogen bromide in acetic acid or by a mixture of TFA and dimethylsulfide.

From an economic standpoint, it is preferred to carry out this in situ catalyzed deprotection without isolating the compound of formula IV.

During the general process of the invention, the fifth reaction relates to the preparation of a pharmaceutically acceptable salt of a compound of formula IV, in particular a methanesulfonate of formula V

the process consists in reacting the compound of formula IV

with an acid, preferably methanesulfonic acid, to form a compound of formula V. The compound of formula V is preferably isolated in the form of the methanesulfonate.

The preferred solvent is ethyl acetate and ethanol, most preferably a mixture of ethyl acetate and ethanol.

From an economic point of view, it is preferable to form the salt in situ without isolating the compound of formula V after its preparation as described above.

In a preferred embodiment of the process according to the invention, the individual steps of Scheme II are carried out

-14 Scheme II (\ OBn ° <y ^0Bn _N Pd / c, h ₂

1.04 eq. (COCI) ₂

BnOCOCI.

K 2 CO 3, h ₂ o

cat. DMF

DIEA thioanisole

COOH

1b

-15 Scheme II - continued

DCC

HOBT / -x ^ / CO ₂ H Ph O y ²

NHBOC

SO ₂ Me

SO ₂ Me iPrOAc: H ₂ O

In this preferred embodiment, prior to hydrogenation, CBZspiroindoline of formula I is treated with Darco (20% by weight). Hydrogenation is carried out in ethanol at 65 ° C in the presence of 10% palladium on charcoal with vigorous stirring.

A solution of 1b in isopropyl acetate mixed with water is treated with commercially available N-BOC-benzyl-D-serine in the presence of dicyclohexylcarbodiimide DCC and 1-hydroxybenzotriazole HOBt. After filtration to remove the by-product, the dicyclohexylurea BCU is separated and the organic phase is washed successively with 1M aqueous sodium hydroxide solution, 0.5M aqueous hydrochloric acid solution and finally with saturated aqueous sodium bicarbonate solution. Very good results can be obtained by treating a solution of the free amino acid in a mixture of isopropyl acetate and water, at the same time DCC and HOBT, and then allowing the mixture to react at room temperature after the addition of the second reagent for 3-5 hours. Then the reaction mixture was evaporated in vacuo and the solvent was changed from isopropyl acetate to ethanol. This solvent exchange is generally carried out by repeatedly adding a new solvent and evaporating the mixture, adding in total three volumes of the ethanol reaction mixture to remove all isopropyl acetate.

The BOC protecting 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 mixture was then partitioned between isopropyl acetate and 1M aqueous sodium hydroxide solution to give the compound of Formula 12.

The coupling of the compound of formula 12 and N-COC-alpha-aminoisobutyric acid is preferably carried out in a biphasic solvent system which is a 1: 1 mixture of isopropyl acetate and water, in the presence of DCC and HOBt, using 1.1 equivalents of each. Then, the DCU is filtered off, the layers are separated and the organic layer is washed successively with 1M aqueous sodium hydroxide solution, 0.5 M aqueous hydrochloric acid solution and saturated aqueous sodium bicarbonate solution to give the compound of formula 14.

Then the solvent is exchanged with ethanol for subsequent cleavage of the Boe group by treatment with methanesulfonic acid. Removal of the protecting group from the compound of Formula 14 is more difficult than that of the compound of Formula 11 and requires

17 using a concentrated solution of methanesulfonic acid in ethanol with heating to 35-40 ° C. After extraction of the mixture with a mixture of ethyl acetate and sodium hydroxide, the free amine of formula 15 is isolated. The organic layer is then washed thoroughly with 1M NaOH to completely remove the methanesulfonic acid.

The ethyl acetate solution of the free base of formula 15 is concentrated in vacuo and then dried in an azeotropic manner (KF < 500 mg / ml) by adding two volumes of the reaction solvent mixture, using a 90:10 ethyl acetate / ethanol mixture, then two more volumes of ethyl acetate. The resulting anhydrous slightly cloudy solution of the free base of Formula 15 in ethyl acetate was treated with Darco G-60 (25% by weight) at room temperature for about 10 hours. The Darco is then removed by filtration to give the free base of Formula 15.

The methanesulfonic acid salt of formula 16 is obtained from the free base of formula 15 in ethyl acetate by treatment with 1.1 equivalents of MsOH at about 50 ° C. The free base of Formula 15 is treated with 8% (v / v) ethanol and one equivalent of water, and the mixture is heated to 55 ° C until the components are completely dissolved. Then the mixture was cooled to room temperature and the resulting suspension was stirred for 4 hours to give the methanesulfonate of formula 16 in crystalline form II, solubility in IPA = 12 mg / ml.

Crystalline Form II can be converted to Form I by forming the salt in a mixture of ethyl acetate and ethanol as described above, but instead of cooling the salt solution at 55 ° C to room temperature, it is cooled to only 45 ° C. Crystals also begin to form at this temperature over time. The temperature is raised to 51 ° C and the suspension is allowed to stand overnight at this temperature to completely convert the compound of Formula 16 to crystalline Form I.

Preferably, however, the conversion of crystalline Form II to Form I is accomplished by adding Form I crystals as seeding material to a solution of the free base in a mixture of ethyl acetate and ethanol at a temperature of 50-55 ° C followed by standing. The free base of formula 15 was treated with 1.1 equivalents of methanesulfonic acid in 8% ethanol in ethyl acetate at 50-55 ° C. Approximately 2% by weight of the methanesulfonate of formula 16 in crystalline form I is then added, then the mixture is allowed to stand overnight at 55 ° C. The mixture was then cooled to room temperature and allowed to stand

- 18 about 2 to 3 hours. The product is filtered at room temperature under nitrogen, dried at 35 ° C under vacuum and passed through a sieve to give the methanesulfonate of formula 16.

The methanesulfonate of Formula 16 can also be formed by sequentially adding to the solution of the free base in a mixture of ethyl acetate and ethanol at 50 ° C 1.1 equivalents of MsOH and Form I crystals as seeding material, the order of addition of acid and crystals being not critical.

Abbreviations will be used throughout the application, the meaning of which is given below

explained: • Bu butyl Bn benzyl BOC Boe t -butyloxy BOP benzotriazol-1-yloxy-tris (dimethylamino) phosphonium hexafluorophosphate CBZ, Cbz benzyloxycarbonyl DCC N, N'-dicyclohexylcarbodiimide DIEA diisopropylethylamine DMF N, N-dimethylformamide DMAP 4-dimethylaminopyridine EDC 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide. hydrochloride EDAC ethyl-3- (3-dimethylamino) propyl carbodiimide EI-MS electron mass spectroscopy et ethyl eq. FAB-MS eq fast atom bombardment mass spectroscopy h lesson

HOBT, HOBt hydroxybenzotriazole

HPLC high pressure liquid chromatography

iPrOAc isopropyl KHMDS potassium bis (trimethylsilyl) amide LAH lithium aluminum hydride LHMDS lithium bis (trimethylsilyl) amide Me methyl

MF molecular formula MHz megacycle MPLC medium pressure liquid chromatography MsOH methanesulfonic acid NMM N-methylmorpholine NMR nuclear magnetic resonance imaging ph phenyl pr propyl TFA " trifluoroacetic acid THF tetrahydrofuran TLC thin layer chromatography TMS tetramethylsilane.

Some terms are used throughout the application and will be explained below.

The term "peptide coupling reactions" refers to the coupling of a carboxylic acid with an amine using an acidic activating agent such as EDC, DCC and BOP in an inert solvent in the presence of a catalyst such as HOBT. Inert solvents suitable for this bond include acetonitrile, isopropyl acetate, ethyl acetate, propionyl, water, chlorinated hydrocarbons such as dichloromethane, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene, o-dichlorobenzene, benzene, toluene, xylenes, and the like. .

The term "L and" amino protecting group "refers to the presence of a suitable amino protecting group, such as those summarized in Greene TW, Wuts PGM, Protective Groups in Organic Synthesis, 2nd Edition, John Wiley and Sons , Inc., New York, 1991. A suitable protecting group is selected such that it is not cleaved under the selected reaction conditions for the desired cleavage. These protecting groups are independently selected for each reaction of the process.

The amino protecting groups include, for example, benzyl, benzyloxymethyl, benzyloxycarbonyl (carbobenzyloxy), benzylsulfonyl, 2-bromoethyloxycarbonyl, tert-butoxycarbonyl, 2-chlorobenzyloxycarbonyl, 2-chloroethyloxycarbonylcarbonyl, di-tert-amyloxy, tert-butyloxy, methoxybenzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 2-nitrophenylsulfonyl, phthaloyl, 2,2,2-trichloro-tert-butyloxycarbonyl, trifluoroacetyl, triphenylmethane, allyloxycarbonyl and vinyloxycarbonyl and the like, and especially preferred groups are benzyloxycarbonyl; a t-butoxycarbonyl group.

In this procedure, it is possible to remove the amino protecting group using the appropriate catalyst. The tert-butoxycarbonyl group may be removed in a solvent such as methanol, ethanol, methylene chloride, ethyl acetate or isopropyl acetate by treatment with a strong acid. Strong acids include methanesulfonic acid, trifluoroacetic acid, hydrochloric acid, gaseous hydrogen chloride, hydrogen bromide, hydrogen iodide, trifluoromethanesulfonic acid, gafrosulfonic acid, sulfuric acid, phosphoric acid, and also arylsulfonic acids, for example benzenesulfonic acid, pensenesulfonic acid, pensenesulfonic acid, pensenesulfonic acid and pensenesulfonic acid. Preferred catalysts are, for example, trifluoroacetic acid, methanesulfonic acid, gafrosulfonic acid, benzenesulfonic acid, ptoluenesulfonic acid and p-chlorobenzenesulfonic acid. The most preferred catalyst is methanesulfonic acid. A preferred solvent is methanol or ethanol, especially ethanol.

The benzyloxycarbonyl group can be removed in a number of ways, for example by hydrogenation with hydrogen in the presence of a noble metal catalyst or an oxide thereof, for example palladium on activated carbon in a protic solvent such as ethanol. If catalytic hydrogenation is not possible due to the presence of other potentially reactive functional groups, the latter can also be removed by treatment with a solution of hydrogen bromide in acetic acid or a mixture of TFA and dimethylsulfide.

The amino compounds which are the starting materials for the process according to the invention can be used in the form of salts with acids derived from inorganic and organic acids. By way of example, hydrochloric, nitric, sulfuric, phosphoric, formic, acetic, trifluoroacetic, propionic, maleic, succinic, malic, methanesulfonic and the like can be mentioned. Such resulting products obtained by the process of the invention may be isolated in the form of pharmaceutically acceptable acid salts. In addition, some groups

Sodium, potassium, lithium, calcium, magnesium and the like salts may be formed with an acid function, for example a carboxyl group, as well as salts with organic bases.

In the preparation of the compounds according to the invention, it may be necessary in some cases to reverse the order of the reactions so as to facilitate the reaction or to avoid the formation of undesirable by-products. In general, however, the process of the invention is carried out in the order of reactions described above.

A variety of starting materials are commercially available or known from the literature, other materials can be prepared by procedures described in the literature for analogs, and the products can be purified, for example, by crystallization; by conventional or reverse-phase chromatography.

The invention is illustrated by the following examples, which are not intended to limit the scope of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Preparation of isonicotecotinic acid N-benzylcarbamate, i. 4-Piperidinecarboxylic acid N-benzylcarbamate Compound 3

CO ₂ H (2)

Material isonipecotinic acid (T.C.I.) (4-piperidinecarboxylic acid) benzyl chloroformate (Schveitzerhall) potassium carbonate water

4.02 kg (31.10 mol) 6.91 kg (30.5 mol) 10.10 kg (72.90 mol) 40.20 L

A -22 V four-necked 100 L flask equipped with a mechanical stirrer was dissolved in 40.20 L of water with isonipecotinic acid and potassium carbonate (under a potassium atmosphere) and the resulting reaction mixture was cooled to 10 ° C.

Benzyl chloroformate was then added to the reaction mixture at a temperature ranging from 9 to 14 ° C, and after this operation was completed, the mixture was heated to 22 ° C and then allowed to "stand" for 58 hours. . The addition of benzyl chloroformate was complete after 4 hours, the pH of the mixture being 9, which remained unchanged for 58 hours.

The reaction mixture was then transferred to a 200 L extractor and washed with 3 x 13 kg (15 L) isopropyl acetate and 1 x 12 L ethyl acetate. After separation of the aqueous and organic phases, the aqueous layer was extracted with 8 L of toluene. After subsequent washing, the benzyl alcohol content was reduced with 3.80% to 1.40% as determined by high performance liquid chromatography (HPLC) analysis.

Analytical equipment of high performance liquid chromatography (HPLC):

Refill Dupont Zorbax RXC8; 25.0 cm column; at a flow rate of 1.50 ml / min and detection 254 nm.

Isocratic mixture:

% methyl cyanide, 65% aqueous 1% phosphoric acid.

Retention times:

Compound 3 = 6.90 minutes; benzyl alcohol = 3.30 minutes; toluene = 17.30 minutes.

The aqueous phase was acidified to pH 1.8 with 37% hydrochloric acid, evolving carbon dioxide as the acid was added. The evolution of this gas could be easily controlled. Hydrochloric acid was added for less than 1 hour and the concentrated hydrochloric acid consumed was 10 liters.

The aqueous phase was then extracted 3 times with 6 L of toluene. The combined toluene extracts were dried with 2 kg of sodium sulfate and then filtered through a pad of Solka-Floc filter material ^R.

The weight of the combined filtrates was 17.8 kg.

Evaporation of the weighed aliquot of filtrate to dryness gave 7.89 kg (97%) of the crude carbamate (compound 3). The filtrates were pumped through a pre-filter (filter pore size 10.0 µm), concentrated into a reaction vessel containing 1001a extracts at 21 kPa and below 25 ° C to a volume of 18 liters.

The final carbamate concentration (compound 3) was 440 g / l. Concentration of the toluene filtrate was used to azeotropically remove the final traces of water (final KF = 170.0 mg / l _; KF- = analytical evaluation of the water content according to the Karl Fischer method).

The percent area of purity of the desired compound 3 is

99.1% and 0.90% for benzyl alcohol as the only impurity.

Example 2

Preparation of Isonipecotinic Chloride N-Benzylcarbamate Compound 4 (4)

COCI

material:

Isonipecotinic acid N-benzylcarbamate (compound 3) in toluene (MW = 263.30) oxalyl chloride (MW = 126.93) dimethylformamide (MW = 73.10) toluene

7.89 kg (30.0 mol)

17,90 I

3.94 kg (31.0 mol)

10.0 ml 12.0 L

To a toluene solution of the benzyl carbamate (compound 3) obtained in Example 1 above was added 5.0 mL of dimethylformamide and 10.0 L of toluene.

-24K of the resulting reaction mixture was added over 20 minutes oxalyl chloride and the mixture was left at 18 ° C while bubbling a weak stream of nitrogen to "mature" for 16 hours.

Analysis of the reaction mixture by HPLC showed that 1.30% of the carboxylic acid (compound 3) was still unreacted. The reaction mixture was then warmed to 26 ° C and left to stand for 2.5 hours after addition of 5.0 ml of dimethylformamide.

An aliquot of the reaction mixture (1.0 mL) was mixed with 5.0 mL of t-butylamine and after evaporation analyzed by HPLC.

Zorbax RXC8 cartridge; Dupont; 25.0 cm column, at 50 ° C and 1.0 mL / minute and detection at 220 nm.

Isocratic mixture:

% methyl cyanide and 58% aqueous 0.1% phosphoric acid.

By this method, <0.05% of the acid residue (compound 3), (as apparent from the "A" evaluation and> 3% area "B"),> 1 mol% ((COCI) 2 = oxalyl chloride) was found.

The crude aldehyde solution (compound 5) obtained in the previous Example 3 above was pumped through a pre-filter (filter pore size = 10 µm) to a 100 liter reactor equipped with a Teflon-coated copper coil allowing cooling or heating reactor and mechanical stirrer.

To said solution of compound 5 was added 34.4 kg of toluene and 7.0 L of methyl cyanide and the resulting reaction mixture was cooled to 0 ° C. Phenylhydrazine was then added in one portion and the temperature of the reaction mixture was maintained at a temperature ranging from -1 ° C to 3 ° C while bubbling nitrogen through the reaction mixture.

Phenylhydrazine was added until complete consumption of the aldehyde, i.e. compound 5 in the reaction, and a slight excess (< 5%) of phenylhydrazine was confirmed by TLC and HPLC analysis. TLC analysis conditions:

-25Silicon dioxide; E. Merck Kieselgel G60-F254; 0.25 mm, diethyl ether; pentane (4: 1) and a developing agent; 0.5% cerium sulphate; 14% ammonium molybdate in 10% aqueous sulfuric acid (after heating).

Rf:

Compound 5, i. piperidine-4-carboxaidehyd-1-benzylcarbamate = 0.52, phenylhydrazone = 0.51, phenylhydrazine = 0.21.

Conditions of HPLC analysis:

Dupont Zorbax cartridge, RXC-8, column 25.0 cm, at 30 ° C with a flow rate of 1.0

O ^ NH-tBu

CT NH-t-Bu

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

A typical high performance liquid chromatography (HPLC) profile, the concentration of a toluene solution when mixed with tert-butylamine as above, is as follows:

ml / min and detection at 254 nm:

CONH-t-Bu

Retention time (minutes) Area (%) identification 2.10 <0,5% carboxylic acid (compound 3) 7.80 <0,5% benzyl 11.0 > 99,0% benzyloxycarbonyl butylcarboxamide (structural formula A) 12.10 undetectable number toluene 12.70 <0,5% diterc.butyloxyamid (structural formula B)

-26Example 3

Preparation of piperidine-4-carboxaldehyde-1-benzylcarbamate Compound 5

M

(5)

CHO

material:

Isonipecotinic chloride N-benzylcarbamate (compound 4) in toluene (molecular weight = 281.74) di-isopropylethylamine (DIEA) (KF = 18.0 mg / l) palladium on charcoal (10%) (KF = 20 0 mg / g) thioanisole (h = 1.058) (molecular weight = 124.21)

3.38 kg (12.0 mol)

5.54 kg

1.55 kg (15.0 mol)

101 Og

0.56 g

To the solution of compound 4, i. The isonipecotinic chloride N-benzylcarbamate obtained in the previous Example 2 was added with diisopropylethylamine and thioanisole and the catalyst was suspended in the reaction mixture thus prepared.

The resulting reaction mixture was immediately pumped into an autoclave of approximately 19 liters and hydrogenated at 20 ° C and 50 psi hydrogen.

After 18 hours, 70% of the theoretical amount of hydrogen was consumed in the reaction.

After mixing an aliquot with tert-butylamine, HPLC analysis showed a 14.2 area% residual acid chloride.

The HPLC analysis conditions were the same as those described above. Retention time; Compound 5 = 8.10 minutes

A second batch of catalyst was added to the autoclave, i. 101.0 g and 0.54 g of thioanisole slurry in 1.375 ml of toluene.

After 23 hours after mixing an aliquot of the reaction mixture with tert-butylamine, HPLC analysis showed that 1.80 area% acid chloride remained.

The reaction mixture was then purged with nitrogen and the di-isopropylethylamine hydrochloride precipitated catalyst was removed by filtration through a filter, Solka-Floc ^R material. of a 50 L extractor and then washed with 2 x 7.2 L of aqueous 1 M hydrochloric acid solution followed by 2 x 7.2 L of water.

The reaction mixture was then concentrated at 10 mbar (25-30 ° C) until a residue of 5 liters remained.

Retention time (minutes) Area (%) identification 2.10 <2.0 carboxylic acid (compound 3) 6.60 <1.0 dimer 8.10 > 95 aldehyde (compound 5)

The yield of the aldehyde, i. of the title compound 5 as judged by HPLC analysis was 94%.

Example 4

Preparation of Benzyloxycarbonyl-spiroindoline Compound 9

material:

piperidin-4-karboxaldehys-1-benzyl carbamate

(compound 5) 1.71 kg (6.89‘mol) in toluene 21,4 kg phenylhydrazine 900 ml, 981 g trifluoroacetic acid (9.15 mol) 2.20 l, 3.26 kg sodium borohydride (28.6 mol) 300.0 g (7.93 mol) toluene 34.40 kg MeCN 7.0 I methanol 7.0 I Gradient scheme: time (minutes) acetonitrile: Water 0 57 43 10 65 35 15 75 25 18 75 25

Retention times: phenylhydrazine = 4.50 minutes toluene = 7.20 minutes phenylhydrazine = 11.4 minutes

The reaction mixture was aged for 30 minutes at 0 ° C to 2 ° C and then trifluoroacetic acid was added over a temperature range of 0 ° C to 2 ° C. The reaction mixture was then heated to 50 ° C for 30 minutes and held at this temperature for 17 hours. The bubbling was stopped and only a slight stream of nitrogen was passed through the reaction mixture.

During the first hour, the color of the reaction mixture gradually darkened to 5 ° C at 5 ° C, forming a small amount of (relatively) white crystalline precipitate (ammonium trifluoroacetate).

After 17 hours, HPLC analysis showed (under the same conditions described above) that the reaction mixture contained 91.60 area% indolenine and 1.50% residual unreacted phenylhydrazone.

The aging of the reaction mixture for a long time does not increase the yield of indolenine in this process.

After cooling the reaction mixture to 12 ° C, 7 liters of methanol were added thereto, and then at a temperature maintained below 15 ° C, sodium borohydride was added in small amounts (<20.0 g). Said operation was carried out for 30 minutes, while a slight hydrogen evolution was observed but was easily controlled and in fact caused no foaming.

At the end of the addition of sodium borohydride, the color of the reaction mixture rapidly changed from green to brown and then to a bright orange color.

A small amount (<200 ml) of the heavier phase was separated (mainly water-soluble salts).

HPLC analysis carried out under the same conditions described above showed that all indolenine was consumed (90.4 area% benzyloxycarbonylindoline, i.e. compound 9).

Retention times:

indolenine = 7.50 minutes, indoline = 8.20 minutes.

Thin Layer Chromatography (TLC) analysis:

-30 Ethyl ether as solvent, cerium sulfate-ammonium molybdate as a detection reagent or detection with 1% anisaldehyde.

Retention factors:

indolenine = 0.18; benzyloxycarbonyl-indoline = 0.33.

The change in color of the reaction mixture from green to orange corresponds very closely to the completion of the reaction. The amount of sodium borohydride required to complete the reaction is very strongly dependent on the temperature and rate of addition of sodium borohydride, but the yield and product quality are not actually affected, provided the reaction is complete.

The reaction mixture was cooled to 5 ° C over 30 minutes and then 8 L of an aqueous 3% ammonium hydroxide solution was added to adjust the pH of the aqueous phase to pH 7.40. The reaction mixture was then stirred and allowed to settle. The temperature of the mixture was raised to 15 ° C and the cloudy, yellow colored bottom phase was separated.

The organic phase was washed with 4 L aqueous 3% ammonium hydroxide solution and then 2 x 4 L water followed by 4 L brine.

The weight of the organic phase after washing was 53.3 kg and the yield as described was 94%.

The washed toluene solution was combined with the washed organic phases from two other, similar reaction procedures. The total amount of aldehyde (used in 3 reactions) was 5.06 kg (20.5 mol).

The total amount of benzyloxycarbonyl-spiroindoline (compound 9) found in the combined organic phases was 5.91 kg (18.3 mol, 90% yield).

The combined organic phases were dried with 5 kg of sodium sulfate, treated with 250 g of activated carbon (Darco-G60) for 30 minutes and were filtered through a filter material Solka_floc ^R. The filtrates were concentrated in vacuo at 1.0 kPa at below 25 ° C until the residue was almost dry.

Further treatment with the solvent was carried out by slowly mixing in 30 L of isopropyl acetate and re-concentrating to 14 L at 20 kPa at 50 to 60 ° C. The reaction mixture was then heated to reflux to obtain a clear, homogeneous, strongly orange solution.

Nuclear Magnetic Resonance Analysis ( ¹ H-NMR) showed the solution to contain about 6 mol% residual toluene after solvent removal.

The solution was then cooled to 68 ° C and seeded with 4 g of crystalline benzyloxycarbonyl-spiroindoline (compound 9) and then allowed to "stand" at 20 to 26 ° C for 9 hours.

The slurry was then cooled to 2 ° C over 1 hour and allowed to "ripen" at that temperature for 1 hour.

The product was isolated by filtration and the filter cake was washed twice at 5 ° C with 2 liters of isopropyl acetate and 2 x 2 L (also at 5 ° C) of tert-butyl methyl ether (MTBE).

The product was then dried in a vacuum oven at 30 ° C under a nitrogen atmosphere to give 4.37 kg (74% yield) of the title compound 9, i. benzyloxycarbonyl-spiroindoline as a light brown colored substance in the form of a crystalline powder.

HPLC analysis showed 99% purity.

The mother liquor (11 L) and the washings contained an additional 1.15 kg (19%) of compound 9 and about 3% benzyloxycarbonyl-isonipecotinic acid phenylhydrazide (retention time = 4.8 minutes).

Example 5

Preparation of Benzyloxycarbonyl-spiroindoline-methanesulfonamide Compound 1

(1)

-32Materiál:

benzyloxycarbonyl-spiroindoline (compound 9) methanesulfonyl chloride triethylamine (KF = 151 tetrahydrofuran (KF = 41))

1.69 kg (5.23 mol) 599.0 g (5.23 mol) 635.0 g (6.27 mol) 12.01

A 22 L reaction vessel was charged with benzyloxycarbonylspiroindoline (Compound 9) followed by 11.5 L of tetrahydrofuran. Triethylamine was then added to the resulting reaction mixture through a prefilter (filter pore size = 10 μίτι).

The resulting homogeneous reaction mixture was cooled to 0 ° C and 1 L of methanesulfonyl chloride and 500 mL of tetrahydrofuran were added via an addition funnel. A solution of methanesulfonyl chloride in tetrahydrofuran was then added to the reaction mixture over a period of 5 hours at 0 ° C to 4 ° C. The reaction was exothermic.

During this operation a white precipitate formed, predominantly triethylamine hydrochloride.

HPLC analysis showed that the reaction at the end of the addition was complete and no compound 9 was detected anymore.

HPLC Chromatography Conditions:

Dupont Zorbax RXC8 cartridge, 25 cm column at 1.5 min flow rate and detection at 254 nm.

Gradient scheme:

time (minutes) 0.1% aqueous solution: phosphoric acid MeCN 0 70 30 3 70 30 12 20 80 25 20 80

I

-33Relation times:

compound 9 = 7.6 minutes, compound 1 = 13.6 minutes.

After the addition was complete, the reaction mixture was warmed to 18 ° C and allowed to "mature" for 16 hours.

There were no changes in the HPLC profile between the end of the addition and the 16 hours 'maturation'.

The reaction mixture was then slowly pumped over 1 hour with a vigorously stirred solution of 30 liters of water and 200 ml of 37% hydrochloric acid, placed in a 50 liter reaction vessel. The temperature in the reaction vessel rose from 22 ° C to 28 ° C.

The product was isolated as a light yellowish-brown gummy consistency that turned into a granular solid. The aqueous suspension was then cooled to 22 ° C and allowed to "mature" for 1 hour. Then, the suspension was filtered and the filter cake was washed with 2 x 4 L of methanol / water (50:50).

Less than 0.1% of Compound 1, i.e. CBZspiroindoline methanesulfonamide.

The filter cake was washed with 4 L of a 50:50 mixture of methanol and water, to which was added 50 mL of a 28% aqueous ammonium hydroxide solution (ammonia water). The filter cake was washed 2 times with 4 L of a 50:50 mixture of methanol and water, and the solid was dried under a nitrogen atmosphere in a vacuum oven at 50 ° C.

2.03 kg (97% yield) of the title compound 1 were obtained as an off-white powder.

HPLC analysis showed 93.7 area% Compound 1.

Example 6

Optional procedure for isolation of the intermediate benzyloxycarbonyl-spiroindolenine compound 8

(8)

material:

piperidine-4-carboxaldehyde-1-benzylcarbamate (compound 5) phenylhydrazine trifluoroacetic acid dichloromethane

12.37 g (0.050 mol)

5.41 g (0.050 mol) 11.56 ml, 17.10 g, (0.0150 mol)

500 ml

Compound 5, i. piperidine-4-carboxaldehyde-1-benzylcarbamate was dissolved in dichloromethane submitted in a 1.0 L reaction flask equipped with a Teflon coated magnetic stir bar. The resulting solution was cooled to 0 ° C, and phenylhydrazine was added over 5 minutes via a weighed syringe, maintaining the temperature of the reaction mixture with nitrogen, ranging from -1 ° C to 3 ° C.

Analysis by thin layer chromatography (TLC) and HPLC showed complete consumption of compound 5, i. piperidine-4-carboxaldehyde-1-benzylcarbamate and a slight excess (less than 2%) of phenylhydrazine.

Thin-layer chromatography conditions:

Silicon dioxide; E. Merck Kieselgel G60-F254, 0.25 mm, diethyl ether: pentane (4: 1) and detection reagent = 0.5% cerium sulfate, 14% ammonium molybdate in 10% sulfuric acid (subsequently heated).

Rf:

Compound 5, i. piperidine-4-carboxaldehyde-1-benzylcarbamate = 0.52, phenylhydrazone = 0.61, phenylhydrazine = 0.21.

HPLC Chromatography Conditions:

-35 Dupont Zorbax RXC8 cartridge, 25 cm column with a flow rate of 1.0 mL / minute at 30 ° C and detection at 254 nm.

Gradient scheme:

Time (minutes) acetonitrile water 0 57 43 • 10 65 35 -. ¹⁵ 75 25 « 18 75 25

Retention times:

phenylhydrazine = 4.5 minutes, toluene 7.20 minutes, phenylhydrazone = 11.4 minutes.

After the reaction mixture was allowed to "mature" at 0 ° C to 2 ° C for 10 minutes, trifluoroacetic acid was added via syringe at a temperature ranging from 2 to 7 ° C. The reaction mixture was then heated to 35 ° C for 30 minutes and held at that temperature for 17 hours. The nitrogen was bubbled into the reaction mixture and only a small stream of nitrogen was passed through the mixture.

During the first hour of heating at 35 ° C, the color of the reaction mixture gradually darkened to a pink color and then to a green color, leaving only a relatively small amount of white crystalline precipitate (ammonium trifluoroacetate).

After 17 hours 'aging', HPLC (under the same conditions described above) revealed 93.0 area% indolenine (compound 8) and less than 0.5% residual unreacted phenylhydrazone. "Aging" of the mixture for a long time had no effect on the increase in indolenine yield.

After the reaction mixture was cooled to 10 ° C, a mixture containing 60 ml of 28-30% ammonium hydroxide, 90 ml of water and 150 g of crushed ice was added with vigorous stirring. The color of the reaction mixture changed to salmon pink.

After separation, the organic phase was washed with 2 x 400 ml of water and then with 100 ml of saturated aqueous sodium chloride solution. Subsequently, the organic phase was dried with magnesium sulfate and filtered through a 5 g silica pad.

The filtrate was evaporated to give 15.84 g (99% yield) of the title indolenine, i. benzyloxycarbonyl-spiroindolenine (compound 8) in the form of a light orange, oily consistency.

Example 7

Process for the preparation of benzyloxycarbonyl-spiroindoline-methanesulfonamide, isolation of the benzyloxycarbonyl-spiroindoline intermediate, i. compound 9 compound 1

Stage 1

Preparation of Benzyloxycarbonyl-spiroindoline Compound 9

material:

piperidine-4-carboxaldehyde-1-benzylcarbamate (compound 5) phenylhydrazine (Aldrich) trifluoroacetic acid toluene (KF <250 mg / l) methyl cyanide (KF <250 mg / liter) sodium borohydride toluene methanol

49.50 g (0.20 mol) 23.70 g (0.22 mol) 75.40 g (0.66 mol) 654.0 ml

13.3 ml

11.3 g (0.30 mol) 20.0 ml

50.0 ml

A solution (2% by volume) of methyl cyanide in toluene was prepared by mixing 654 ml of toluene and 13.3 ml of methyl cyanide.

In a 2 L reaction three-neck flask equipped with a mechanical stirrer, 617 mL of the above solution was degassed by bubbling a gentle stream of nitrogen through the solution for 5 minutes. Phenylhydrazine and trifluoroacetic acid were then added to the mixture while degassing.

Simultaneously, piperidine-4-carboxaldehyde-1-benzylcarbamate (compound 5) was placed in the addition funnel and degassed with bubbling nitrogen in the residue (50.0 mL) of the above solution. The solution in the reaction flask was then heated to 35 ° C and the aldehyde solution (compound 5) was slowly added over 2 hours to a mixture of phenylhydrazine and trifluoroacetic acid.

The reaction mixture was aged at 35 ° C for 16 hours.

HPLC Chromatography Conditions:

Dupont Zorbax RXC8 cartridge, 25 cm column, flow rate 1.0 mL / min at 50 ° C and detection at 220 nm.

Isocratic mixture:

55% methyl cyanide, 45% aqueous 0.1% phosphoric acid.

Typical HPCL profile after 16 hours of “maturation” of the reaction mixture:

Retention time (minutes) Area (%) identification 1.60 0,1 -0,50 phenylhydrazine 4.10 <0,10 dimer 4.70 <0,10 aldehyde (compound 5) 5.00 undetectable number Spiroindoline (Compound 9) 6.30 undetectable number toluene 6.90 97 spiroindolenín (compound 8) 10,30 <0,20 phenylhydrazone

-382 - 3 total other impurities (<0.2% each)

After the reaction mixture was cooled to -10 ° C, methyl cyanide was added thereto, followed by addition of 20 mL of sodium borohydride in toluene over 30 minutes, followed by small portions (1.0 mL) with careful monitoring of the temperature. , which was not allowed to exceed -2 ° C.

Area (%) identification 0.10 to 1.0 phenylhydrazine 85-90 CBZspiroindoline (Compound 9) <0,10 benzyloxycarbonyl-spiroindolenín (compound 8) 10-15 total other impurities (<3% each)

After the temperature of the reaction mixture was raised to 10 ° C over 1 hour, 200 ml of aqueous 6% ammonia solution was added thereto, and the reaction mixture was then stirred for 10 minutes.

After separating the two phases for 10 minutes, the bottom phase was drained and discarded. 20 ml of acetonitrile and 20 ml of methanol were added to the organic phase and the phases were washed with 150 ml of 15% brine solution.

The organic phase was found to contain benzyloxycarbonyl-spiroindoline (compound 9) in 92% yield.

Stage 2

Preparation of Benzyloxycarbonyl-spiroindoline-methanesulfonamide Compound 1

-39Benzyloxycarbonyl-spiroindoline material (compound 9, molecular weight = 322.51) methanesulfonyl chloride _with di-isopropylethylamine (KF = 150 mg / l) '(0.184 mol)

21.10 g (0.184 mol) 29.70 g, 40.10 ml, tetrahydrofuran (KF = 41 mg / l), (0.230 mol) 150.0 ml

The crude benzyloxycarbonyl-spiroindoline solution (Compound 9) was obtained under Step 1 above, and concentrated in a 1 liter 3-neck flask at 60-70 ° C and a vacuum of 26.7 kPa until a residue of 250 was left. g to which tetrahydrofuran and diisopropylethylamine were added.

After cooling to 0 ° C, a mixture of methanesulfonyl chloride in 50.0 mL of tetrahydrofuran was added over 2 hours via a 125 mL addition funnel while maintaining the reaction temperature between 0 ° C and 4 ° C. C.

The reaction mixture was then allowed to "mature" at 5 to 8 ° C for 2 hours. A slight exothermic reaction was observed during the addition and a white precipitate formed, predominantly diisopropylethylamine hydrochloride.

HPLC chromatography conditions were the same as those described above.

Analysis by HPLC showed that the reaction was complete 1 hour after the addition (compound 9 was undetectable) and the yield from compound 9 was 94%.

Retention time:

Compound 1 = 7.80 minutes

Typical HPLC profile after 2 hours of “maturation” of the reaction mixture:

Area (%) identification <0,10 CBZspiroindoline (Compound 9) 90 to 92 , benzyloxycarbonylsulfonamide (Compound 1) 8 to 10 in total other impurities (<2% each)

To the reaction mixture heated to 20 ° C was added 200 mL of a 1M aqueous hydrochloric acid solution and the resulting mixture was heated to 5 ° C.

After separation of the organic and aqueous phases, the organic phase was washed successively with 100 ml of water, 100 ml of 5% aqueous sodium bicarbonate solution and 100 ml of water. The organic phase was then transferred to a 1 liter three-necked reaction flask equipped with a mechanical stirrer and distillation head.

The reaction mixture (ca. 400 mL) was then distilled at atmospheric pressure until 150 mL of distillate was collected. The temperature at the top of the distillation column was 107 ° C, the temperature in the reaction zone was 110 ° C. Thereafter, distillation was continued with regular addition of n-propanol so that the volume (ca. 350 ml) in the reaction cell remained constant.

Distillation was discontinued after a total of 525 ml of n-propanol was added and a total of 800 ml of distillate was collected.

The temperature of both the head of the distillation column and the flask increased from 94 ° C to 98 ° C during the solvent exchange. Toluene and n-propanol form an azeotrope that boils at 97.20 ° C and is composed of 47.5% toluene and 52.5% n-propanol.

The reaction mixture was then allowed to gradually cool to 20 ° C over 3 hours and then stirred for 12 hours.

2% toluene and 4.0 mg / ml sulfonamide were found in the mother liquors.

The solubility of the sulfonamide in various mixtures of toluene and n-propanol was determined by HPLC:

-41% toluene in n-propanol

ABOUT

Solubility of Compound 1 in mg / ml 2? 36

3.02

4.23

7.51

10,30

The crystalline slurry was filtered and washed 3 times with 100 ml n-propanol. The product was dried under a nitrogen atmosphere in a vacuum oven at 50 ° C for 16 hours.

65.5 g of compound 6 (yield from aldehyde i.e. compound 5 was 82%) was obtained as a yellow-brown solid with a purity of 93.50% (w / w).

Typical HPLC profile of a solid:

Area (%)

T o / i> 99 <1.0 total

Identification of benzyloxycarbonyl-siproindoline (Compound 9) Benzyloxycarbonyl-sulfonamide (Compound 1) Other impurities (<0.2% each)

For further purification, a 40 g sample of crystallized sulfonamide n-propanol was dissolved at 60 ° C in 134 mL of ethyl acetate and then treated with 8 g of activated carbon (Darco-G60) at 60 ° C for 1 hour. . After addition of 2 g of filter material Solka Floc ^R slurry was filtered through a 4 g of the material Solka-Floc ^R and the filter cake at 60 DEG C. washed with 90 ml of ethyl acetate.

The reaction mixture was brown before the addition of activated carbon. The filtration was carried out satisfactorily without blockage of the gold-yellow filtrate. The filtrate was then distilled at atmospheric pressure

-42 in the reaction flask (filtrate temperature in the distillation flask was 80-85 ° C) until there was no residue in the flask weighing 100 g (100 ml).

This solution was cooled to 35 ° C over 3 hours, after which the first hour was added 116.0 ml of cyclohexane and stirred vigorously at 35 ° C. After cooling the mixture to 20 ° C for 1 hour, the reaction mixture was aged at 20 ° C for 12 hours.

Even at a temperature of 35 ° C, most of the sulfonamide crystallized from the mixture and the mixture showed a thick consistency. After addition of cyclohexane, stirring was made more difficult at 20 ° C. After maturation, 2.50 mg / g of Compound 1 was detected in the supernatant. The crystalline slurry was filtered and the filter cake was washed with 77.0 mL of cyclohexane / ethyl acetate (2: 1) and 2 x 77.0 mL of cyclohexane.

The product was dried in a vacuum oven under a nitrogen atmosphere at 50 ° C for 16 hours.

34.20 g of the title compound 1 (MW = 400.30) was obtained as a white crystalline solid (85% yield from crude compound 1 and 70% yield from compound 5, reaching 99%). 9% purity (% by weight).

Example 8

Preparation of the hydrochloride salt of spiroindoline methanesulfonamide Compound 1a (h) (1a)

SO ₂ Me

-43Materiál:

benzyloxycarbonyl-spiroindoline-methanesulfonamide (compound 1)

A Pearlman catalyst, i. 20% palladium hydroxide on activated carbon tetrahydrofuran methanol

941.0 g (2.35 mol)

188.0 g 8 liters 7 liters

The catalyst was suspended in 7 liters of methanol and transferred to an autoclave containing approximately 19 liters, to which was added a solution of compound 1 in 8 liters of tetrahydrofuran.

The resulting mixture was hydrogenated at 25 ° C and 50 psi hydrogen. After 2.5 hours, the temperature was raised to 35 ° C for 30 minutes.

HPLC analysis indicated complete consumption of benzyloxycarbonylspiroindoline methanesulfonamide.

HPLC Chromatography Conditions:

Dupont Zorbax RXC8 cartridge, 25 cm column at a flow rate of 1.50 mL / min and detection at 254 nm.

Gradient scheme:

Time (minutes) 0.1% aqueous phosphoric acid solution and methyl cyanide 0 70 30 3 70 30 12 20 80 25 20 80

Retention times:

spiroindoline = 7.6 minutes, benzyloxycarbonyl-spiroindoline methanesulfonamide = 13.6 minutes.

The reaction mixture was bubbled with nitrogen and the catalyst was removed while still hot through the Solka-Floc ^R filter material.

Then, the catalyst was washed with 4 L tetrahydrofuran and 2 L methanol. The light yellow filtrates were concentrated to a dense oily consistency at <25 ° C and 1.0 kPa. The solvent was removed by slow addition of 15 L of ethyl acetate followed by concentration to dryness.

The residue solidified to a hard off-white mass, to which 1.5 L of methanol was added and the resulting mixture was heated to 70 ° C until a homogeneous solution was formed. Still at 70 ° C, 10.5 L of ethyl acetate heated to 20 ° C was added to the solution. in this operation, the temperature of the mixture was lowered to 40 ° C and the reaction mixture remained homogeneous.

Subsequent experiments indicated that it is much more convenient to convert the methanoltetrahydrofuran mixture of filtrates to methanol, concentrate to the desired volume, and then add ethyl acetate. This operation eliminates solidification of the residue after concentration of the ethyl acetate solution.

An equal volume of hydrogen chloride and nitrogen gas was then injected into the solution. The temperature of the mixture was raised to 60 ° C over 15 minutes and a white precipitate of the hydrochloride salt formed.

Injection of hydrogen chloride only prevents back-suction of the reaction mixture and may not be necessary.

The reaction mixture was then cooled in an ice bath, and hydrogen chloride gas was continued for an additional 1 hour while the temperature of the mixture was gradually reduced to 20 ° C. The suspension was then allowed to "ripen" for 2 hours, lowering the temperature to 10 ° C. The crystalline product was isolated by filtration, and the filter cake was washed with 3 L of ethyl acetate and then dried in a vacuum oven at 35 ° C.

1.18 kg (yield 86%) of the title compound 1a was obtained as an off-white, crystalline solid, which was found to be > 99.5% pure by HPLC analysis.

-45 HPLC Conditions:

Zorbax RXC8 cartridge, 25 cm column with a flow rate of 1 °, 5 mL / minute and detection at 230 nm.

Isocratic mixture:

% methyl cyanide, 65% aqueous 0.1% ammonium acetate solution.

Retention time: Compound 1a = 5.40 minutes.

Example 9

Preparation of Spiroindoline Methanesulfonamide (free base form) Compound 1b

An aliquot of 250 ml benzyloxycarbonyl hydrogenolysis filtrate containing 4.67 g of compound 1b (free base) was concentrated to about 10 ml.

The residue was dissolved in ethyl acetate (20 mL) and the solution was concentrated again to a volume of 10 mL. This operation was repeated once more and 10 ml of ethyl acetate was added to the residue, whereupon a crystalline precipitate began to form. After a single addition of 20 ml of tert-butyl methyl ether, an additional solid crystalline material precipitated, the supernatant still containing a substantial amount of dissolved product which did not precipitate on standing.

Then 70 ml of hexane was added dropwise to the reaction mixture with vigorous stirring over 2 hours.

The slow addition of hexane is necessary to eliminate the formation of an oily form of the amine.

The reaction mixture was then allowed to "ripen" with stirring for 1 hour and then filtered. The filter cake was washed with 20 mL of a 1: 1 mixture of tert-butyl methyl ether and hexane and then with 20 mL of hexane.

The product was dried under a stream of nitrogen to give 3.86 g (yield 82%) of the free amine of compound 1b as an off-white crystalline substance with a purity of> 99.50%.

HPLC conditions:

Dupont Zorbax RXC8 cartridge, 25 cm column with a flow rate of 1.50 ml / min and detection at 230 nm.

Isocratic mixture:

% methyl cyanide, 65% aqueous 0.1% ammonium acetate solution.

Retention time: Compound 1b = 5.40 minutes.

Example 10A

Preparation of Spirpindoline Methanesulfonamide (as the complete base) Compound 1b

(1b)

material:

benzyloxycarbonyl-spiroindoline sulfonamide (compound 1) palladium hydroxide on activated carbon (20% palladium hydroxide by weight) tetrahydrofuran methanol ammonium hydroxide (concentrated)

833.50 g (2.80 mol)

124.50 g (14%)

6.50 I

19.50 1 60.0 ml

-47 Due to limited apparatus equipment, hydrogenation was performed 3 times. The following description applies to only one of the 3 operations.

Benzyloxycarbonyl-spiroindoline sulfonamide (compound 1) was dissolved in

6.5 L (KF = 53 pg / μΙ) of tetrahydrofuran and 4 L (KF = 18.0 pg / ml) of methanol was added to the resulting solution, followed by a catalyst,

The resulting slurry was transferred to an autoclave containing approximately 19 liters. The remainder (2.50 L) of methanol was used for rinsing.

The reaction mixture was heated at 40 ° C and 0.3 MPa for 24 hours. The catalyst loading and reaction time are a function of the purity of the starting material, i. of compound 1.

This material was unique in terms of catalyst consumption > 15% and reaction time. Cleaner batches of spiroindoline consume only 5% of the catalyst and the reaction time is 4 to 6 hours.

After completion of the reaction (<0.1 area% of compound 1 as determined by liquid chromatography), the reaction mixture was filtered through SolkaFloc ^R filter material and the activated carbon filter cake was washed with 13 L of methanol containing 60 ml of 0.5% ammonium hydroxide. The combined filtrates (analysis showed 1.587 g of spiroindolinamine (compound 1b)) were concentrated in vacuo and the resulting solids were shaken between 40 L of a 3: 1 mixture of toluene and tetrahydrofuran and 18 L of 0.5% aqueous sodium hydroxide.

Although the respective layers (organic and aqueous) were easily separated, a large precipitate was observed in the aqueous layer. The aqueous suspension was then extracted with 15 L of dichloromethane, both phases, i. organic and aqueous were separated only slowly.

Before addition of dichloromethane, tetrahydrofuran was added to the aqueous layer, along with enough sodium chloride to saturate the aqueous layer. However, dissolution of the product was not achieved and required the use of dichloromethane.

The combined toluene, tetrahydrofuran and dichloromethane layers were concentrated in an appropriate batch processing apparatus. The residue was rinsed with 7 liters of methyl cyanide.

-48 In the final phase, 10 liters of methyl cyanide was added to the mixture and the mixture was allowed to stand under a nitrogen atmosphere overnight.

Example 10B

Preparation of Spiroindoline Methanesulfonamide (free base form) Compound 1b

material:

benzyloxycarbonyl-spiroindoline sulfonamide (compound 1) activated carbon (Darco G-60) ethyl acetate ethanol absolute palladium on activated carbon (10%) ammonia solution

Solka-Floc ^R (filter material) isopropyl acetate

3.0 kg (7.49 mol) 0.6 kg 36.0 liters 189.0 liters

0.45 kg 0.5 liters 2.5 kg 65.0 liters

The mixture, prepared by mixing 1 kg of benzyloxycarbonyl-spiroindoline (Compound 1) and 200 g of activated carbon (Darco G-60) in 9 liters of ethyl acetate, was stirred under a nitrogen atmosphere at 60-65 ° C for 8 hours.

Thereafter, activated carbon (Darco G-60) was removed from the reaction mixture by filtration at 60-65 ° C and the solid was washed with 3 L of hot ethyl acetate and the filtrate and washings were combined.

Liquid chromatography analysis (weight ratios) confirmed only negligible losses of activated carbon (Darco G-60).

The ethyl acetate solution was evaporated to dryness in vacuo using a 20 liter Buchi apparatus, which was then rinsed with 2 x 5 liter absolute ethanol. The material was slurried in 8 liters of absolute ethanol, heated to 65-70 ° C and transferred to an autoclave containing 20 liters.

Rinse was. made with 1 liter of absolute ethanol. Then, a slurry prepared from 75 g of 10% palladium on charcoal (7.5% w / w) in 750 ml of absolute ethanol was added to the autoclave, and the rinse was performed with another portion of 250 ml of absolute ethanol.

The whole batch was hydrogenated with vigorous stirring at 65 ° C and 20 psi of hydrogen for 3 hours. After this time, a second portion (75 g) of 10% palladium on charcoal was added and the whole batch was hydrogenated for an additional 2 hours. It was then sealed overnight.

Subsequently, the batch (still at 50-65 ° C) was transferred to a Buchi apparatus (20 liters content) and degassed under vacuum to remove formic acid by the "add and remove" procedure, absolute ethanol (total 18 liters).

The above operation was repeated 2 more times and the three combined batches were placed in a container equipped with an insert of approximately 37 liters. The combined batches were degassed again after addition and distillation (under vacuum) of absolute ethanol (2 x 10 liters).

After adding Solka-Floc ^R filter material as a slurry (after stirring in 20 liters of ethanol).

The resulting mixture was heated to 60-65 ° C and was then transferred at that temperature through a heated filter using a pump to 2 stainless steel containers. The original vessel, filter, pump and piping were rinsed with a hot mixture (60-65 ° C) of aqueous ammonia (500 mL) in 25 L of absolute ethanol. The filtrate and washings were combined and placed in 2 stainless steel containers.

The batch was then transferred to a suitable container using a prefilter with a filter cartridge (10 µm pore size) and then concentrated to a smaller volume (about 15 liters) under vacuum.

Ethanol was replaced with isopropyl acetate, an "addition and collection" system with isopropyl acetate (45 liters total), corresponding to three times the batch volume, maintaining the brew volume at approximately 15 liters. After this solvent operation was completed, less than 1% residual ethanol was detected by gas chromatography.

The batch was then diluted to approximately 33 liters by addition of 20 liters of isopropyl acetate and this solution of spiroindolinamide (i.e. compound 1b) (1.855 kg by liquid chromatography data) in isopropyl acetate was used in the next step of the process.

Example 11A

Preparation of t-butyloxycarbonyl-O-benzylserine-spiroindoline Compound 11

O (11)

SO ₂ Me

Material spiroindolinamine (compound 1b) amino acid (according to structural formula below)

1587 g (5.966 mol)

1938 g (6.563 mol)

-51 CO ₂ H

NHBOC

Ν, Ν'-dicyclohexylcarbodiimide (DCC) hydroxybenzotriazole methyl cyanide

0.5N sodium hydroxide solution 0.5N hydrochloric acid solution saturated aqueous sodium bicarbonate solution isopropyl acetate

1334.5 g (6.563 mol)

884.0 g (6.563 mol), 0 liters 18.0 liters 18.0 liters

18.0 liters 28.0 liters

The solution, prepared under a nitrogen atmosphere at room temperature by dissolving spiroindolinamine (compound 1b) in methyl cyanide or isopropyl acetate / water (25 liters), was mixed sequentially with 884.0 g (1.10 equivalents) of hydroxybenzotriazole (as a solid) and further with 1334.5 g (1.10 equivalents) of Ν, d'-dicyclohexylcarbodiimide (in the form of a melt, prepared in water hot at about 60 ° C for 1 hour) and finally with 1938 g of amino acid (according to the formula in "Materials" ") In the form of a solid.

The resulting reaction mixture was stirred for 3 hours, during which time a thick precipitate of DCU, Ν, Ν-dichloroethane, was formed. Analysis by liquid chromatography revealed approximately 0.5 area% of residual amine, ie compound 1b. Then 9.0 L of isopropyl acetate was added to the mixture and the resulting slurry was filtered through Solka-Floc ^R filter material and the filter cake was washed with 19 L of isopropyl acetate.

The combined organic filtrates were washed successively with 18 liters of 0.5N aqueous sodium hydroxide solution, 18 liters of 0.5N aqueous hydrochloric acid, and 18 liters of saturated aqueous sodium bicarbonate.

Since a final water wash at this point resulted in the formation of an emulsion, it was therefore omitted.

The organic layer was concentrated in vacuo and the residue was dissolved in methanol or ethanol (final volume 10 liters). Analysis yielded 3.026 g (89%).

The use of alternative peptide coupling agents such as carbonylimidazole, or the formation of mixed anhydrides such as secondary butyl carbonate, yields poorer yields of compound 11 and / or compound 14 with a higher degree of epimerization for the first mentioned compound 11. Other peptide coupling agents were costly.

Example 11B

Preparation of t-butyloxycarbonyl-O-benzylserine-spiroindoline Compound 11

N-BOC

SO ₂ Me

material:

spiroindolinamine (compound 1b) isopropyl acetate dicyclohexylcarbodiimide (DCC) 1-hydroxybenzotriazole (HOBt) N-t-butyloxycarbonyl-O-benzyl-D-serine 1M aqueous sodium hydroxide solution 0.5M aqueous hydrochloric acid solution saturated aqueous bicarbonate solution

1.855 kg (6.96 mol) 29.0 liters

1.580 kg (7.65 mol) 1.030 kg (7.62 mol) 2.260 kg (7.65 mol) 26.0 liters

26,0 liters

-53 sodium absolute ethanol

26.0 liters 50.0 liters

To the solution prepared by dissolving 1.855 kg of spiroindolinamine (compound 1b) in 33 liters of isopropyl acetate and charged to the reaction vessel, the following chemicals were added sequentially at room temperature and under a nitrogen atmosphere:

1.58 kg (1.10 equivalent) of dicyclohexylcarbodiimide (DCC),

1.03 kg (1.10 equivalent) of 1-hydroxybenzotriazole, and v

2.26 kg (1.10 equivalents) of N-t-butyloxycarbonyl-O-benzyl-D-serine.

The reagents were rinsed with 7 liters of isopropyl acetate. The batch was then stirred under a nitrogen atmosphere at room temperature for 5 hours, at which point the product / starting material ratio was already 99.4: 0.60 by liquid chromatography.

The reaction mixture was then filtered through an Estrella-type filter fitted only with fabric and hard filter paper using a pump suitable for transferring the mixture to another reaction vessel. The reaction vessel from which the reaction mixture was pumped was rinsed with 22 liters of isopropyl acetate, which was also used to pierce the liter and the conduit leading to the reaction vessel into which the reaction mixture was transferred.

The biphasic mixture in the reaction vessel was stirred for 10 minutes and then left for 15 minutes to separate the two phases. After separation of the lower aqueous phase, the organic layer was allowed to stand overnight at room temperature.

The next day, the organic layer was washed with 26 L of 1M aqueous sodium hydroxide solution, then with 26 L of 0.5 M aqueous hydrochloric acid solution and finally with 26 L of saturated aqueous sodium bicarbonate.

Analysis by liquid chromatography showed a total yield of 3.777 kg of the desired compound 11 (93% yield based on 3.0 kg (7.49 mol of starting material, i.e. benzyloxycarbonyl-spiroindoline) (compound 1).

Then the whole batch was concentrated under vacuum (internal temperature = 13-15 ° C, jacket temperature 40 ° C, vacuum = 2.96 kPa to approximately 15 liters volume) and the solvents were replaced with ethanol (50 liters) while maintaining the volume. 15 liter liters.

Gas chromatography showed a residue of < 1% isopropyl acetate.

The above solution was used at other stages of the process.

Example 12A

Preparation of O-benzylserine-spiroindoline (as the free base) Compound 12

material:

t-butyloxycarbonyl-O-benzylserine spiroindoline (free base) methanesulfonic acid methanol isopropyl acetate

0.5N aqueous sodium hydroxide solution

3026 g (5.57 mol) 1.161 (17.90 mol) 10.00 liters 24.00 liters 35.00 liters t-butyloxycarbonyl-benzylserine-spiroindoline (compound 11) dissolved in 10 L of methanol (or ethanol) was mixed for about 30 to 40 minutes with 1.16 L of methanesulfonic acid (initial temperature was 16 ° C, final temperature 28 ° C).

The dark red reaction mixture was allowed to "mature" under a nitrogen atmosphere overnight and was then pumped into a 100 liter extractor containing 24 liters of isopropyl acetate and 35 liters of 0.5N aqueous sodium hydroxide solution. The aqueous layer had a pH of 7.0.

An amount of 6M aqueous sodium hydroxide solution was then added to the mixture until a pH of 10.5 was reached. At increasing pH, the color of the reaction mixture went from red to yellow.

Then, the organic and aqueous phases were separated and the organic layer (24 liters was analyzed by nuclear magnetic resonance (NMR) and found to be 13 mol% methanol in isopropyl acetate (5% v / v).

Analysis by liquid chromatography showed a yield of 2.48 kg.

Example 12B

Preparation of O-benzylserine-spiroindoline (as the free base) Compound 12

material:

t-butyloxycarbonyl-O-benzylserine-spiroindoline (compound 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) 38.0 liters 16.0 liters 1.6 liters

-56K of the solution prepared in the reaction vessel by mixing 3.777 kg of t-butyloxycarbonyl-O-benzylserine-spiroindoline (compound 11) in ethanol (total volume approximately 15 liters) was added with stirring 2.006 kg (1.355 L) approximately 3 equivalents) methanesulfonic acid and this the batch was heated to 35-40 ° C.

After 7 hours, the absence of starting material was detected by liquid chromatography and the reaction was allowed to stand overnight to warm to room temperature. The next day, 44 liters of water was added to the batch with stirring, and the whole batch was cooled to about 5 ° C. It was then stirred for 30 minutes and filtered through a pre-filter equipped with a 10 µιτι filter cartridge into the cartridge.

The entire batch was then pumped back into the reaction vessel and after rinsing the reaction vessel and the pipeline leading to the container with 10 liters of water, the water was used to rinse the reaction vessel. Then 38 liters of isopropyl acetate were added to the batch followed by 16 liters of 1M aqueous sodium hydroxide solution.

The batch was then cooled to 10-15 ° C. The lower layer was found to have a pH of approximately 7. Then, after addition of 1.6 L of 50% aqueous sodium hydroxide solution, the pH was adjusted to > 10. The mixture was then stirred at 10-15 ° C for 25 minutes and allowed to settle for 10 to 15 minutes and the bottom layer was separated (78 liters).

Liquid chromatography showed that the aqueous liquid contained 28.4 g (0.85% of theory) of the title compound 12. The volume of the organic solution was 51 liters.

Liquid chromatography showed an overall yield of 92%, i. 3.057 kg of 12, based on 3.0 kg (7.49 mol) of the starting benzyloxycarbonyl-spiroindoline sulfonamide, i. of compound 1.

This solution was used for the next step.

Example 57A

Preparation of t-butyloxycarbonyl-aminobutyryl-O-benzylserine-spiroindoline Compound 14

material:

spiroindolinamine (compound 12) amino acid peptide structural formula dicyclohexylcarbodiimide (DCC)

-hydroxybenzotriazole isopropyl acetate water

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

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

1266.7 g (6.16 mol) 827.0 g (6.16 mol) 52.0 liters 37.0 liters 36.0 liters 36.0 liters

36,0 liters

The solution prepared by mixing spiroindolinamine (compound 12) with isopropyl acetate was diluted with additional isopropyl acetate to a total volume of 37 liters and 37 L of water was added.

The resulting biphasic mixture was then sequentially mixed with 827.0 g of solid form of 1-hydroxybenzotriazole and 1266.7 g of dicyclohexylcarbodiimide (in the form of a melt) and an amino acid peptide (see Structural Formula in the Material section) so that the addition took place under nitrogen. at room temperature.

The reaction mixture was stirred for 2 hours and then detected by the absence of starting material, ie compound 12 (<0.3 area%) by liquid chromatography. Then the reaction mixture was filtered through Solka-Floc ^R filter material and the solids were washed with 13 L of isopropyl acetate. The material was stored overnight as a biphasic mixture.

The reaction mixture was then pumped into a 100-L extractor and after separation of the aqueous and organic phases, the organic layer was washed successively with 36 L of a 0.5N aqueous sodium hydroxide solution and a 0.5N aqueous hydrochloric acid solution and finally with a saturated aqueous sodium bicarbonate solution.

An analytical analysis of 81% yield of compound 14, i. 3160 g based on the starting spiroindoline (Compound 12), considering a ± 5% volumetric error.

The reaction mixture was then concentrated to a small volume and washed twice with 4 L of ethanol. If desired, the intermediate compound 14 can be isolated by addition of water by crystallization.

The use of alternative peptide coupling agents such as carbodiimidazole or mixed anhydride forming agents such as sec-butyl carbonate provides worse yields of compound 14 with a higher degree of epimerization.

Other peptide coupling agents were unbearably expensive.

Example 59B

Preparation of t-butyloxycarbonyl-aminoisobutyryl-O-benzylserine-spiroindoline Compound 14

material:

spiroindolinamine (compound 12) dicyclohexylcarbodiimide (DCC)

1-hydroxybenzotriazole (HOBt) t-butyloxycarbonyl-2-aminobutyric acid, i. t-butyloxycarbonyl-2-aminoisobutanoic acid isopropyl acetate

1M aqueous sodium hydroxide solution 0.5M aqueous hydrochloric acid solution saturated aqueous sodium bicarbonate ethanol absolute

3.057 kg (6.89 mol) 1.560 kg (7.56 mol) 1.020 kg (7.55 mol)

1.540 kg (7.58 mol) 32 liters 38 liters 38 liters liters 45 liters

To a solution prepared in the reaction vessel by mixing 3.057 kg of spiroindolinamine (compound 12) with isopropyl acetate (total volume of approximately 51 liters) was added under nitrogen atmosphere at room temperature while stirring with 49.0 liters of water.

The following chemicals were added sequentially to the resulting reaction mixture: 1.56 kg of dicyclohexylcarbodiimide (about 1.10 equivalents)

1.02 kg of 1-hydroxybenzotriazole (approx. 1.10 equivalents) a

1.54 kg of N-butyloxycarbonyl-2-aminobutyric acid (about 1.10 equivalents).

The resulting reaction mixture was stirred vigorously at room temperature for 2 hours, at which time liquid phase analysis showed complete reaction. The reaction mixture is then filtered using a pump through an Estrella-type filter into another reaction vessel.

Then the reaction vessel, filter, pump and piping leading to the next reaction vessel were flushed with 22 liters of isopropyl acetate.

The biphasic reaction mixture was then stirred for 5 minutes and the organic and aqueous phases were allowed to separate. The lower aqueous phase was separated without problems (aqueous liquid weight = 51.10 kg).

The organic phase was then washed successively, without problems, with 38 liters of a 1M aqueous sodium hydroxide solution, then with 38 liters of a 0.5M aqueous hydrochloric acid solution and finally with 38 liters of a saturated aqueous sodium bicarbonate solution.

The organic mixture was then pumped through a pre-filter (10 µm filter cartridge) into a further reaction vessel for subsequent treatment with ethanol. The reaction vessel was then flushed with 10 liters of isopropyl acetate, which was used to flush the pump, filter, and piping leading to the next reaction vessel.

The filtrate and washings were combined. The total volume was 75 liters as determined by the measuring rod.

Liquid chromatography showed a recovery of 93%, i. 4.395 kg of t-butyloxycarbonyl-aminoisobutyryl-O-benzylserine-spiroindoline (compound 14) from 7.49 mol of the starting benzyloxycarbonyl-spiroindoline sulfonamide (compound DV) was concentrated in vacuo to a smaller volume (approximately 15 liters) and worked up by exchanging isopropyl acetate by sequential and withdrawal for absolute ethanol (total amount 45 liters).

At the end of the above operation, it was determined by gas chromatography that it remained in a mixture of <1% isopropyl acetate.

This solution (25 L) containing 4.395 g of the title compound 14 was used in the next step.

If desired, the intermediate compound 14 can be isolated by addition of water by crystallization.

Example 14A 'Preparation of aminoisobutyryl-O-benzylserine-spiroindoline Compound 15

N (15)

SO ₂ Me

material:

t-butyloxycarbonyl-spiroindoline methanesulfonic acid ethanol water

1N aqueous sodium hydroxide ethyl acetate active (Darco G-60

3160 g (5.03 mol) 979 ml (15.0 mol)

6.20 liters 30.00 liters 11.00 liters 26.00 liters 1.0 kg

The solution prepared by dissolving t-butyloxycarbonyl-spiroindoline (compound 14) in 6.2 liters of ethanol was mixed with 979.0 ml of methanesulfonic acid, whereby

The temperature of the mixture rose from 20 ° C to 30 ° C and the reaction was allowed to proceed overnight.

After 12 hours, a mixture of 15% of the starting material was found at 20 ° C and then heated at 35 ° C for 6 hours.

After completion of the reaction (<0.1% compound), the reaction mixture was cooled to 20 ° C and after addition of 30 liters of water was filtered through a glass funnel equipped with a polypropylene filter to remove N, N-dichloroethane residues.

The reaction mixture was then pumped into a 100 L extractor and 26.0 L of ethyl acetate was added. The aqueous layer was then basified by the addition of 11.0 liters of cooled 1N aqueous sodium hydroxide solution and 1.0 liters of 50% aqueous sodium hydroxide solution.

In order to maintain the temperature below 14 ° C, it was necessary to add ice to the reaction mixture, as higher temperatures resulted in significant emulsion problems.

The organic layer was distilled at a temperature of 50 ° C and approximately 2,80 kPa until a KF <1000 pg / ml was achieved. Lower KF values result in a much more efficient activated carbon process and better utilization in the step involving salt formation. KF values of 160.0 pg / ml were achieved with a batch of 700 g. The solution was diluted with ethyl acetate up to a total volume of 31.0 liters (2.40 kg by liquid chromatography). Activated carbon (Darco G-60) was then added to the reaction mixture, and the reaction mixture was stirred for 24 hours. Then the mixture was filtered using Solka-Floc ^R filter material and the filter cake was washed with 16 L of ethyl acetate.

2.34 kg of the title compound 15 were obtained.

Example 14B

Preparation of aminoisobutyryl-O-benzylserine-spiroindoline

Compound 15

-63 (15)

material:

t-butyloxycarbonyl-spiroindoline (compound 14) methanesulfonic acid ethyl acetate

1M aqueous sodium hydroxide solution 50% aqueous sodium hydroxide activated carbon (Darco G-60)

Solka-Floc ^R (filter material)

4.395 kg (6.99 mol)

2.017 kg (20.99 mol) 185.00 liters 16.00 liters 2.60 liters 0.90 kg 2.50 kg

To the solution prepared in the reaction vessel by dissolving 4.395 kg of t-butyloxycarbonyl-spiroindoline (compound 14) in approximately 25 liters of ethanol was added 2.017 kg (1.36 liters) of methanesulfonic acid at room temperature with stirring. The batch was heated to 35 ° C to 40 ° C and then stirred overnight.

The next day the reaction mixture contained approximately 1.10% of the starting material and the reaction was therefore prolonged for a further 4 hours, when the ratio of product to starting material was determined by liquid chromatography at a value of 99.6: 0.4. The entire batch was then concentrated in vacuo to a volume of approximately 15 liters and subsequently diluted with 44 liters of water. The batch was then cooled to 5 ° C, stirred for 30 minutes and then filtered using a Sparkler pre-filter (filter cartridge with pores).

(64 µm size) and pumped into another reaction vessel to remove a small amount of residual N, N-dichloroethane.

Reaction vessel: pump, filter and piping were rinsed with 10 liters of water added to the batch in the reaction vessel. After adding 36 L of ethyl acetate to the reaction mixture, the mixture in the reaction vessel was cooled to 10 ° C with stirring.

To this mixture, cooled in the reaction vessel, was added 16 liters of 1M aqueous sodium hydroxide solution, cooled to 5-10 ° C, and

2.60 liters of 50% aqueous sodium hydroxide solution, also cooled to 10 ° C.

During this operation, the temperature of the reaction mixture rose to 14 ° C. The resulting mixture was stirred at less than 14 ° C for 15 minutes, and then the lower aqueous layer was separated.

The whole batch was then concentrated in vacuo to a volume of approximately 20 liters, and a mixture obtained by mixing 35 liters of ethyl acetate and 5 liters of ethanol was added to the batch so that the batch volume remained at 20 liters.

At the end of the distillation, KF values were 9.160 mg / ml ^-1 . Then, the solvent in the batch was replaced by an addition and collection system with ethyl acetate (total consumption of ethyl acetate = 40 liters). At the end of the distillation, KF values were 446 mg / ml ^-1 . The batch was then diluted with 10 L of ethyl acetate.

In the next operation, 900 g of activated carbon (Darco G-60) was added to the turbid mixture, which was rinsed with 6 liters of ethyl acetate. The reaction mixture was then stirred at room temperature overnight.

The next day, 0.500 kg of Solka-Floc ^R filter material (while stirring the mixture) and then 2 kg of Solka-Floc ^R in a small amount of ethyl acetate were added to the mixture and applied to an Estralla filter. Excess solvent was pumped through a Sparkler prefilter (10 µm filter cartridge).

The reaction mixture, in the form of a slurry, was pumped through the filter through the filter and then through another filter into two 40 L stainless steel containers (each).

Visual inspection showed the mixture to be clean and clear. The reaction vessel was rinsed with 22 liters of ethyl acetate, which were used to rinse the entire system mentioned above, with the above-mentioned reservoir.

The contents of both containers were then pumped into the reaction vessel and the solution was then mixed together.

The batch (58 l) were determined value KF = 2950 mg / ml @ ^1, and therefore, the mixture was concentrated in vacuo to approximately 20-25 liters and the batch was diluted to a volume of 46.0 liters (detected density bar), the addition of 25, 0 liters of ethyl acetate. Then the whole batch was diluted by adding 17.0 liters to a total volume of 62.0 liters and the whole mixture was ready for use in the final stage of the process.

Example 15A

Preparation of spiro [3H-indole-3,4'-piperidin] -1'-yl) carbonyl] -2- (phenylmethoxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate

Compound 16

(16)

material:

Aminoisobutyryl-O-benzylserine-spiroindoline (compound 15) methanesulfonic acid ethyl acetate ethanol

8% ethanol in ethyl acetate

2340 g (4.43 mol) 316 ml (4.88 mol) 60.0 liters 4.8 liters 20.0 liters

The entire batch of compound 15 obtained in Example 14B above was adjusted to 60 liters by the addition of 4.80 liters of ethanol.

316 mL of methanesulfonic acid in 3.0 L of ethyl acetate was added to the solution at 45 ° C. 496.0 g of title compound 16 (configuration I) was added to the resulting deep red homogeneous solution, i. 10% vaccine, by weight of the free amine used). The temperature of the reaction mixture was raised to about 48 ° C and the mixture was then stirred at 52 ° C for 1.5 hours.

A complete conversion to the title compound 16 (configuration I) was detected analytically.

If less than 10% of the vaccine dose is used, the reaction time should be extended to more than 3 hours.

The reaction mixture in a slurry was allowed to warm to 20 ° C overnight and then centrifuged under a nitrogen atmosphere. The filter cake was then washed with 20 L of 8% ethanol in ethyl acetate.

note:

The presence of nitrogen is essential during centrifugation because the wet crystalline substance is strongly hygroscopic.

The batch was then dried under vacuum at 35 ° C to give 2.70 kg (56% yield) of the title compound 16 (configuration I) at a purity of 99.9% (less than 0.1% enantiomer). .

Conversion of configuration II to configuration I can also be carried out when a salt (similar to that described above) is formed in a mixture of ethyl acetate and ethanol by addition of methanesulfonic acid and the starting salt solution (temperature 55 ° C) is cooled to 45 ° C when crystals and mash start to form at this time.

Then the temperature of the reaction mixture is raised to 51 ° C and the slurry is allowed to "ripen" overnight.

Compound 16 is expected to be completely converted to configuration I.

This procedure can be used to prepare seed crystals of configuration I for compound 16.

Example 15B

Preparation of spiro [3H-indole-3,4‘-piperidin] -1‘-yl) carbonyl] -2- (phenylmethyloxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate

Compound 16

(16)

material:

Aminoisobutyryl-O-benzylserine-spiroindoline (compound 15) methanesulfonic acid ethyl acetate ethanol absolute spiro [3H-indole-3,4'-piperidin] -1'-yl) carbonyl] -2 (phenylmethyloxy) ethyl] -2-amino -2-methylpropanamide methanesulfonate (configuration I)

3.10 kg (5.86 mol) 0.62 kg (6.45 mol) 37.0 liters 8.7 liters

70,0 g

To a solution prepared by dissolving 3.10 kg of aminoisobutyryl-O-benzylserine-spiroindoline (compound 15) in ethyl acetate presented in the reaction vessel was added 6.40 liters of absolute ethanol.

To this 50 ° C batch was added over approximately 5 minutes at 50-54 ° C, a mixture prepared by mixing 620 g (412 mL, 1.10 equivalents) of methanesulfonic acid in 11 L of ethyl acetate. Then the batch was seeded with 70 g of spiro [3H-indole-3,4'-piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate (configuration I) and the resulting reaction mixture was stirred and heated under nitrogen overnight.

The next day, the slurry was cooled to 15-20 ° C and held at this temperature for 2 hours and then dropped onto a polypropylene filter (50 cm) under nitrogen. The solid product was washed with a mixture prepared by mixing 2.30 L of absolute ethanol in 26 L of ethyl acetate.

The white solid product was removed and dried at 35 ° C in a vacuum oven (Apex) at the appropriate time (approximately <2 days).

Dried spiro [3H-indole-3,4'-piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate (3.352 kg) was sieved using a sieve. Jackson-Crockatt devices.

3,347 kg (including vaccine dose) were obtained, i. yield 3.277 kg, desired product.

Configuration I: 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 is in the form of an anhydrous polymorph, characterized by the following values:

Melting point: 168-171 ° C

Solubility in isopropanol: 4.60 mg / ml

Differential Scanning Calorimetry (DSC) curve for configuration I of N- [1 (R) - [(1,2dihydro-1-methanesulfonyl-spiro- [3H-indol-3,4'-piperidin] -1-yl) -carbonyl] -2- (phenylmethyloxy) ethyl] -2-amino-2-methylpropanamide methanesulfonate, exhibits at 10 ° C / minute in an open crucible under a stream of nitrogen, during melting by a simple endothermal reaction with a temperature maximum of about 180 ° C and an extrapolated temperature threshold (melting point) about 170 ° C, associated with a heating of 53 J / g. Configuration I was investigated using X-ray diffraction values with a reflection of approximately:

-696.50, 14.70, 16.90, 17.10, 17.90, 19.50, 21.10, 21.70 and 22.0 ° (2 aunt).

Data were obtained using an automatic diffraction apparatus (Philips and APD3720, with Kalfa copper radiation. Registration was performed (from 2 to 40 ° (2 aunt) with a sample maintained at room temperature).

HPLC Chromatography Conditions:

Liquid chromatographic retention times were registered using a Zorbax-RXC8 cartridge (4.6 mm x 25 cm), lambda = 210 nm, flow rate = 1.50 ml / min.

Compound 1: -60: 40, methyl cyanide: water (1% phosphoric acid), retention time = 5 minutes.

Compound 1b: 35:65, methyl cyanide: water (0.1% ammonium acetate, weight ratios), retention time = 6.20 minutes.

Compound 10: 60:40, methyl cyanide: water (0.1% phosphoric acid), retention time = 2.90 minutes.

Compound 11: 60:40, methyl cyanide: water (0.1% phosphoric acid), retention time = 5.40 minutes.

Compound 12: 40:60, methyl cyanide: water, pH = 5.25, sodium dihydrogen phosphate (6.90 g / liter of water, pH adjusted with sodium hydroxide), retention time = 5.60 minutes. Compound 14 60:40, methyl cyanide: water (0.1% phosphoric acid), retention time = 4.65 minutes.

Compound 15 40:60, methyl cyanide: water (pH = 5.25, sodium dihydrogen phosphate (6.90 g / l water), pH adjusted with sodium hydroxide), retention time = 4.9 minutes.

Liquid chromatography retention times were registered using a Zorbax-C8 cartridge (4.6 mm x 25 cm), lambda = 210 nm, flow rate 1.20 ml / min, column temperature = 48 ° C.

Solvent A = 0.05% phosphoric acid + 0.01% triethylamine in water Solvent B = acetonitrile

-70Gradient system:

Time % A % B 0 minutes 95 5 35 minutes 10 90 38 minutes 95 5 40 minutes 95 5 Retention time (minutes) Compound 1 25.20 Compound 1b 8.50 Compound 10 20.50 Compound 11 26.30 Compound 12 14.80 Compound 14 25.60 Compound 15 15.70

The invention has been described in connection with some of its embodiments, but it will be apparent to one skilled in the art that various modifications, changes, modifications may be made, particularly to the extent that certain procedures may be omitted, added or modified without it has departed from the spirit and scope of the invention. For example, it would be possible to use reaction conditions other than those described in the description and example section, in particular due to the use of other reagents of the described reagent group and the like. Changes may also occur with respect to the specific reactivity of the starting materials, which may vary according to some substituents or depending on the processing method. It is to be understood that all such contemplated modifications and modifications also fall within the scope of the present invention.

Claims (4)

PATENT CLAIMS A process for the preparation of N- [1- (R) - [(1,2-dihydro-1-methanesulfonylspiro [3H-indole-3,4'-piperidin] -1'-yl) carbonyl] -2- (phenylmethyloxy) ethyl 2-Amino-2-methylpropanamide of the general formula V, characterized in that it is: 1) reacting an amino acid of the general formula Ph ^ o y N-L wherein L is an amino protecting group, with a compound of formula -72 in the presence of a first acid activating agent in a first inert solvent in the presence of a first catalyst reagent to form a compound of formula I wherein L is an amino protecting group, then 2) reacting a compound of formula I with a first amino-protecting group cleavage reagent to form a compound of formula II SO ₂ Me then take 3) an amino acid of the general formula -73h ₃ c ch ₃ HOOC ^ NL ² H wherein L is an amino protecting group, with a compound of formula II in the presence of a second acid activating agent in a second inert solvent in the presence of a second catalytic agent, to give a compound of formula III wherein L is an amino protecting group. 4) reacting a compound of formula III with a second amino-protecting group cleavage reagent to form a compound of formula IV or a pharmaceutically acceptable salt thereof; -74a is reacted with methanesulfonic acid to form a compound of formula V. A process for the preparation of a compound of formula I wherein L is an amino-protecting group, which comprises reacting an amino group of formula I with a compound of formula I in the presence of an acid activating agent in an inert solvent in the presence of a catalytic agent to form a compound I. -753. The method according to claim 2, characterized in that DCC is used as the acid activating agent. The process according to claim 2, characterized in that HOBT is used as the catalytic agent. Process according to claim 2, characterized in that the reaction is carried out in a solvent selected from acetonitrile, isopropyl acetate, propionyl, water, chlorinated hydrocarbons such as dichloromethane, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene, o-dichlorobenzene, benzene , toluene, xylenes and the like, and mixtures of these solvents. Process according to claim 5, characterized in that the reaction is carried out in a solvent selected from the group of isopropyl acetate, water and acetonitrile. Process according to claim 2, characterized in that the reaction is carried out at a temperature of 20 to 35 ° C. Process according to claim 2, characterized in that tert-butoxycarbonyl is used as the amino-protecting group. Process according to claim 2, characterized in that the reaction is carried out in situ without isolation of the resulting formula I. A process for the preparation of a compound of formula II N SO ₂ Me -76 characterized by reacting a compound of the general formula wherein L is an amino protecting group with an amino-deprotecting agent to form a compound of formula II. Process according to Claim 10, characterized in that a tert-butoxycarbonyl group is used as the amino-protecting group in the starting material of the formula I. 12. A process according to claim 10, wherein methanesulfonic acid is used as the amino protecting group cleavage agent. Process according to Claim 10, characterized in that it is carried out in situ without isolation of the product of formula II. A process for the preparation of a compound of formula III SO ₂ Me (III) -77where L is an amino-protecting group characterized in that an amino acid of the formula H ₃ c ch ₃ HOOC ^ NL ² H wherein L is an amino protecting group, with a compound of formula II in the presence of an activating agent in an inert solvent in the presence of a catalytic agent to form a compound of formula III. 15. The method of claim 14, wherein DCC is used as the acid activating agent. 16. The process according to claim 14, wherein the catalyst agent is HOBT. 17. A process according to claim 14 wherein the solvent is a mixture of isopropyl acetate and water. -7818. The process of claim 17 wherein the solvent is a mixture of isopropyl acetate and water in a volume ratio of about 40:60 to 60:40. 19. The process of claim 14, wherein the reaction is carried out at a temperature in the range of 20 to 35 ° C. 20. The process according to claim 14, wherein tert-butoxycarbonyl is used as the amino-protecting group. A process according to claim 14, characterized in that it is carried out in situ without isolation of the resulting product of formula III. 22. A process for the preparation of a compound of formula IV or a pharmaceutically acceptable salt thereof, wherein the compound of formula III is reacted SO ₂ Me Oli) -79where L is an amino protecting group, with an amino protecting group cleavage reagent to form a compound of formula IV. 23. The process according to claim 22, wherein t-butoxycarbonyl is used as the protecting group in the starting material of formula III. 24. The process according to claim 22, wherein methanesulfonic acid is used as the amino protecting group cleavage agent. The method of claim 22, wherein the process is carried out in a solution comprising ethanol. The process according to claim 22, characterized in that it is carried out in situ without isolation of the compound of formula IV after its preparation. is that it is 27. A process for producing a compound of formula V, comprising reacting a compound of formula IV with methanesulfonic acid to form a compound of formula V. 28. The process of claim 27 wherein the process is carried out in solution in a mixture of ethyl acetate and ethanol.