HRP960494A2 - Process for the preparation of a growth hormone secretagogue - Google Patents

Description

Stanje tehnike State of the art

Hormon rasta, koji izlučuje hipofiza, potiče rast svih tjelesnih tkiva koja imaju sposobnost rasta. Pored toga, poznato je da hormon rasta ima slijedeće osnovne učinke na metaboličke procese u tijelu: (1) povećanje brzine sinteze proteina u svim stanicama tijela; (2) smanjenje brzine korištenja ugljikohidrata u stanicama tijela; (3) povećanje mobilizacije slobodnih masnih kiselina i iskorištavanje masnih kiselina za dobivanje energije. Manjak sekrecije hormona rasta može dovesti do različitih medicinskih poremećaja, kao što je patuljast rast. Growth hormone, secreted by the pituitary gland, stimulates the growth of all body tissues that have the ability to grow. In addition, growth hormone is known to have the following basic effects on metabolic processes in the body: (1) increasing the rate of protein synthesis in all cells of the body; (2) reducing the rate of utilization of carbohydrates in body cells; (3) increasing the mobilization of free fatty acids and utilization of fatty acids for energy. Lack of growth hormone secretion can lead to various medical disorders, such as dwarfism.

Poznato je više puteva oslobađanja hormona rasta. Na primjer, kemijske tvari poput arginina, L-3,4-dihidroksifenilalanina (L-DOPA), glukagona, vazopresina i hipoglikemije uzrokovane inzulinom, kao i aktivnosti poput spavanja ili vježbanja, neizravno uzrokuju otpuštanje hormona rasta iz hipofize djelujući na neki način na hipotalamus; možda smanjivanjem sekracije somatostatina ili povećanjem sekrecije poznatih sekretagoga čimbenika otpuštanja hormona rasta (GRF) ili nepoznatih endogenih hormona oslobađanja hormona rasta ili na više spomenutih načina istodobno. Several pathways of growth hormone release are known. For example, chemicals such as arginine, L-3,4-dihydroxyphenylalanine (L-DOPA), glucagon, vasopressin, and insulin-induced hypoglycemia, as well as activities such as sleep or exercise, indirectly cause the release of growth hormone from the pituitary gland by acting in some way on the hypothalamus. ; perhaps by decreasing somatostatin secretion or increasing secretion of known growth hormone-releasing factor (GRF) secretagogues or unknown endogenous growth hormone-releasing hormones or in the aforementioned ways simultaneously.

U slučajevima kada su bile poželjne povišene količine hormona rasta, problem je rješavan uvođenjem egzogenog hormona rasta ili primjenom GRF ili peptidnog spoja koji pospješuje stvaranje i/ili otpuštanje hormona rasta. U oba slučaja peptidni sastav spoja uvjetuje primjenu putem injekcije. Ranije se kao izvor hormona rasta koristila ekstrakcija hipofize kadavera. Takav je postupak dovodio do znatne skupoće proizvoda, a bio je i povezan s rizikom prijenosa bolesti koje zahvaćaju hipofizu na primatelja hormona rasta. Kasnije je postao dostupan hormon rasta dobiven rekombinacijom koji je, iako je uklonjen rizik prijenosa bolesti, zadržao svojstvo znatne skupoće i nužnosti primjene injekcijom ili sprejem za nos. Razvijeni su i drugi spojevi koji pospješuju otpuštanje endogenog hormona rasta. In cases where increased amounts of growth hormone were desired, the problem was solved by the introduction of exogenous growth hormone or the administration of GRF or a peptide compound that enhances the formation and/or release of growth hormone. In both cases, the peptide composition of the compound requires administration by injection. Previously, cadaver pituitary extraction was used as a source of growth hormone. Such a procedure led to a considerable cost of the product, and was also associated with the risk of transmission of diseases affecting the pituitary gland to the recipient of the growth hormone. Later, recombinant growth hormone became available which, although the risk of disease transmission was removed, retained the characteristic of considerable cost and the necessity of administration by injection or nasal spray. Other compounds have been developed that promote the release of endogenous growth hormone.

Poglavito se to odnosi na neke spiro spojeve koji su opisani u PCT patentnoj objavi WO 94/13696 i u Proc. Natl. Acad. Sci. USA, 92, 7001-7005 (srpanj 1995.) kao nepeptidni sekretagogi hormona rasta. Ovi spojevi pospješuju otpuštanje prirodnog ili endogenog hormona rasta, pa se mogu koristiti u liječenju stanja koja zahtijevaju poticanje stvaranja ili sekrecije hormona rasta, kao što je to slučaj kod ljudi s manjkom prirodnog hormona rasta ili kod životinja koje se koriste u proizvodnji hrane ili vune, gdje će poticanje hormona rasta imati za posljedicu nastajanje veće, produktivnije životinje. This mainly applies to some spiro compounds which are described in PCT patent publication WO 94/13696 and in Proc. Natl. Acad. Sci. USA, 92, 7001-7005 (July 1995) as non-peptide growth hormone secretagogues. These compounds promote the release of natural or endogenous growth hormone, so they can be used in the treatment of conditions that require the stimulation of the formation or secretion of growth hormone, such as in humans with a deficiency of natural growth hormone or in animals used for food or wool production, where growth hormone stimulation will result in a larger, more productive animal.

Medu preporučenim spojevima koji su tamo opisani nalazi se spiro[3H-indol-3,4'-piperdin]-1'-il) karbonil]-2- (fenilmetil- oksi)etil]-2-amino-2-metilpropanamid čija je struktura: Among the recommended compounds described there is spiro[3H-indol-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenylmethyl-oxy)ethyl]-2-amino-2-methylpropanamide whose structure:

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PCT patentna objava WO 94/13696 uključuje postupke priprave ovog spoja (vidi Primjere 18, 19 i 55). Međutim, sinteza spoja postiže se uporabom vrlo skupog aminokiselinskog sredstva za vezanje EDC (1100 USD/kg); uporabom brojnih ekvivalenata trifluoroctene kiseline kao katalizatora deprotekcije BOC skupine; opsežnim kromatografskim pročišćavanjem; i "gumiranjem" konačnog proizvoda. PCT patent publication WO 94/13696 includes procedures for the preparation of this compound (see Examples 18, 19 and 55). However, synthesis of the compound is achieved using a very expensive amino acid binding agent EDC ($1100/kg); using numerous equivalents of trifluoroacetic acid as a catalyst for the deprotection of the BOC group; extensive chromatographic purification; and "rubbing" the final product.

Prednosti ovog izuma uključuju: neizolacijski postupak u šest koraka koji omogućuje visok prinos i kojim se dobije tvar čistoće > 99.9%; smanjenje troškova zahvaljujući uporabi DCC (40 USD/kg) umjesto EDC (1100 USD/kg); smanjenje utjecaja na okoliš zahvaljujući uporabi metansulfonske kiseline umjesto trifluoroctene kiseline kao katalizatora (također i manje ekvivalenata katalizatora) kod deprotekcije; i lakoću izolacije konačnog proizvoda. The advantages of this invention include: a non-isolating six-step process that provides a high yield and obtains a substance of purity > 99.9%; cost reduction thanks to the use of DCC ($40/kg) instead of EDC ($1100/kg); reduction of environmental impact due to the use of methanesulfonic acid instead of trifluoroacetic acid as a catalyst (also less catalyst equivalents) in deprotection; and ease of isolation of the final product.

Sažetak izuma Summary of the invention

Ovaj izum opisuje postupak priprave spoja N-[1(R)-[(1,2-dihidro-1-metansulfonil-spiro [3H-indol-3,4'- piperdin]-1'-il) karbonil]-2-(fenilmetil-oksi)etil]-2-amino-2-metilpropanamida čija je struktura: This invention describes a process for preparing the compound N-[1(R)-[(1,2-dihydro-1-methanesulfonyl-spiro [3H-indol-3,4'-piperdin]-1'-yl)carbonyl]-2- (phenylmethyl-oxy)ethyl]-2-amino-2-methylpropanamide whose structure is:

[image] [image]

kao i njegovih soli, osobito metansulfonantne soli. as well as its salts, especially methanesulfonate salts.

Ovaj spoj ima svojstvo poticanja otpuštanja prirodnog ili endogenog hormona rasta, pa se može koristiti u liječenju stanja koja zahtijevaju poticanje stvaranja ili sekrecije hormona rasta, kao što je to slučaj kod ljudi s manjkom prirodnog hormona rasta ili kod životinja koje se koriste u proizvodnji hrane ili vune, gdje će poticanje hormona rasta imati za posljedicu nastajanje veće, produktivnije životinje. This compound has the property of stimulating the release of natural or endogenous growth hormone, so it can be used in the treatment of conditions that require the stimulation of the creation or secretion of growth hormone, such as in humans with a deficiency of natural growth hormone or in animals used in food production or wool, where growth hormone stimulation will result in a larger, more productive animal.

Opis izuma Description of the invention

Ovaj izum opisuje novi postupak priprave spoja N-[1(R)-[(1,2-dihidro-1-metansulfonil-spiro[3H-indol-3,4'- piperdin] -1'-il)karbonil]-2-(fenilmetil-oksi)etil]-2-amino-2-metilpropanamida čija je struktura: This invention describes a new process for the preparation of the compound N-[1(R)-[(1,2-dihydro-1-methanesulfonyl-spiro[3H-indol-3,4'-piperdin]-1'-yl)carbonyl]-2 -(phenylmethyl-oxy)ethyl]-2-amino-2-methylpropanamide whose structure is:

[image] [image]

kao i njegovih soli, osobito metansulfonantne soli. as well as its salts, especially methanesulfonate salts.

Ovim se postupkom dobiva željeni spoj iz lako dostupnih, jeftinih i ekološki prihvatljivih početnih reagensa i otapala. Postupak ne zahtijeva uporabu nikakvih kromatografskih pročišćavanja, a konačni proizvod je moguće proizvesti iz međuspojeva spiroindolin sulfonamida bez izolacije bilo kojih međuspojeva. This process yields the desired compound from readily available, inexpensive and environmentally friendly starting reagents and solvents. The procedure does not require the use of any chromatographic purifications, and the final product can be produced from spiroindoline sulfonamide intermediates without isolating any intermediates.

Pojedini postupci u okviru cjelovitog postupka sažeti su na slijedećoj shemi: Individual procedures within the overall procedure are summarized in the following diagram:

[image] [image]

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(gdje je L prikladna amino zaštitna skupina, a X- je prikladan protuion). (where L is a suitable amino protecting group and X- is a suitable counterion).

Unutar cjelovitog postupka, prvi postupak sadrži pripravu spoja formule I: Within the complete process, the first process comprises the preparation of the compound of formula I:

[image] [image]

gdje je L amino zaštitna skupina, spajanjem aminokiseline čija je formula: where L is an amino protecting group, by connecting an amino acid whose formula is:

[image] [image]

sa spojem formule: with a compound of the formula:

[image] [image]

u nazočnosti kiselog aktivirajućeg sredstva u inertnom otapalu u nazočnosti katalitičkog sredstva, čime se dobije spoj formule I. in the presence of an acidic activating agent in an inert solvent in the presence of a catalytic agent, thereby obtaining a compound of formula I.

Kisela aktivirajuća sredstva pogodna za ovaj postupak uključuju: DCC, EDC, ECAC I BOP, a preporučeno kiselo aktivirajuće sredstvo je DCC (N,N'-dicikloheksilkarbodiimid). Acid activators suitable for this process include: DCC, EDC, ECAC and BOP, and the recommended acid activator is DCC (N,N'-dicyclohexylcarbodiimide).

Katalizirajuća sredstva pogodna za ovaj postupak uključuju: HOBT i druga, a preporučeno katalitičko sredstvo je HOBT (hidroksibenzotriazol). Catalyzing agents suitable for this process include: HOBT and others, and the recommended catalytic agent is HOBT (hydroxybenzotriazole).

Inertna otapala prikladna za ovaj postupak uključuju: acetonitril; izo-propil acetat; etil acetat; propionitril; vodu; klorirane ugljikovodike kao što su: diklorometan, kloroform, tetraklorugljik, dikloroetan, klorobenzen, orto-diklorobenzen; benzen; toluen; ksilen i drugi, kao i njihove mješavine, a preporučeno otapalo je ili acetonitril ili izopropil acetat i voda. Inert solvents suitable for this procedure include: acetonitrile; iso-propyl acetate; ethyl acetate; propionitrile; water; chlorinated hydrocarbons such as: dichloromethane, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene, ortho-dichlorobenzene; benzene; toluene; xylene and others, as well as their mixtures, and the recommended solvent is either acetonitrile or isopropyl acetate and water.

Preporučeni raspon temperature reakcije je između -40 i 150°C, najbolje između 20 i 35°C. The recommended reaction temperature range is between -40 and 150°C, preferably between 20 and 35°C.

Prikladne amino zaštitne skupine uključuju: benzil, benziloksimetil, benziloksikarbonil (karbobenziloksi), benzilsulfonil, 2-bromo-etiloksikarbonil, t-butoksi-karbonil, 2-kloro-benziloksi-karbonil, 2-kloroetiloksikarbonil, di-t-amiloksikarbonil, 9-fluoroenil-metiloksikarbonil, izopropoksikarbonil, 4-metoksi-benziloksikarbonil, 4-nitrobenziloksikarbonil, 2-nitrofenil-sulfonil, ftaloil, 2,2,2-trikloro-t-butiloksikarbonil, trifluoroacetil, trifenilmetan, aliloksikarbonil, viniloksikarbonil skupine i druge, preporučene su skupine benziloksikarbonil (karbobenziloksi), t-butoksi-karbonil, a najviše se preporučuje t-butoksi-karbonil skupina. Suitable amino protecting groups include: benzyl, benzyloxymethyl, benzyloxycarbonyl (carbobenzyloxy), benzylsulfonyl, 2-bromo-ethyloxycarbonyl, t-butoxy-carbonyl, 2-chloro-benzyloxy-carbonyl, 2-chloroethyloxycarbonyl, di-t-amyloxycarbonyl, 9-fluoroenyl -methyloxycarbonyl, isopropoxycarbonyl, 4-methoxy-benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 2-nitrophenyl-sulfonyl, phthaloyl, 2,2,2-trichloro-t-butyloxycarbonyl, trifluoroacetyl, triphenylmethane, allyloxycarbonyl, vinyloxycarbonyl groups and others, benzyloxycarbonyl groups are recommended (carbobenzyloxy), t-butoxy-carbonyl, and the t-butoxy-carbonyl group is most recommended.

U cilju učinkovitosti, preporučuje se da se vezanje provodi in situ, bez izoliranja spoja formule I nakon njegove priprave gore spomenutim postupkom. For the sake of efficiency, it is recommended that the binding be carried out in situ, without isolating the compound of formula I after its preparation by the above-mentioned process.

Unutar cjelovitog postupka, drugi postupak sadrži pripravu spoja formule II: Within the complete process, the second process comprises the preparation of the compound of formula II:

[image] [image]

koja obuhvaća reagiranje spoja formule I: which comprises reacting the compound of formula I:

[image] [image]

gdje je L amino zaštitna skupina, s amino deprotekcijskom skupinom čime se dobije spoj formule II. where L is an amino protecting group, with an amino deprotecting group to give a compound of formula II.

Prikladne amino zaštitne skupine uključuju: benzil, benziloksimetil, benziloksikarbonil (karbobenziloksi), benzilsulfonil, 2-bromo-etiloksikarbonil, t-butoksi-karbonil, 2-kloro-benziloksi-karbonil, 2-kloroetiloksikarbonil, di-t-amiloksikarbonil, 9-fluoroenil-metiloksikarbonil, izopropoksikarbonil, 4-metoksi-benziloksikarbonil, 4-nitrobenziloksikarbonil, 2-nitrofenil-sulfonil, ftaloil, 2,2,2-trikloro-t-butiloksikarbonil, trifluoroacetil, trifenilmetan, aliloksikarbonil, viniloksi karbonil skupine i druge, preporučene su skupine benziloksikarbonil (karbobenziloksi), t-butoksi-karbonil, a najviše se preporučuje t-butoksi-karbonil skupina. Suitable amino protecting groups include: benzyl, benzyloxymethyl, benzyloxycarbonyl (carbobenzyloxy), benzylsulfonyl, 2-bromo-ethyloxycarbonyl, t-butoxy-carbonyl, 2-chloro-benzyloxy-carbonyl, 2-chloroethyloxycarbonyl, di-t-amyloxycarbonyl, 9-fluoroenyl -methyloxycarbonyl, isopropoxycarbonyl, 4-methoxy-benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 2-nitrophenyl-sulfonyl, phthaloyl, 2,2,2-trichloro-t-butyloxycarbonyl, trifluoroacetyl, triphenylmethane, allyloxycarbonyl, vinyloxycarbonyl groups and others are recommended groups benzyloxycarbonyl (carbobenzyloxy), t-butoxy-carbonyl, and the most recommended is the t-butoxy-carbonyl group.

U ovom postupku, uklanjanje amino zaštitne skupine može se postići uporabom prikladnog katalizirajućeg sredstva. Uklanjanje t-butoksikarbonil zaštitne skupine može se provesti u otapalu kao što je metanol, etanol, metilen klorid, etilacetat ili izopropilacetat, pomoću jake kiseline. Takve jake kiseline uključuju metansulfonsku kiselinu, trifluoroctenu kiselinu, klorovodičnu kiselinu, klorovodik kao plin, bromovodik, jodovodik, trifluormetansulfonsku kiselinu, kamforsulfonsku kiselinu, sumpornu kiselinu, fosfornu kiselinu i arilsulfonsku kiselinu poput benzensulfonske kiseline, p-toluensulfonske kiseline i p-klorbenzensulfonske kiseline. Preporučljiva katalizirajuća sredstva uključuju: trifluoroctenu kiselinu, metansulfonsku kiselinu, kamforsulfonsku kiselinu, benzensulfonsku kiselinu, p-toluensulfonsku kiselinu i p-klorbenzensulfonsku kiselinu. Najpreporučljivije katalitičko sredstvo je metansulfonska kiselina. Preporučljivo otapalo je metanol ili etanol, najbolje etanol. In this process, removal of the amino protecting group can be achieved by using a suitable catalyzing agent. Removal of the t-butoxycarbonyl protecting group can be carried out in a solvent such as methanol, ethanol, methylene chloride, ethyl acetate or isopropyl acetate, using a strong acid. Such strong acids include methanesulfonic acid, trifluoroacetic acid, hydrochloric acid, hydrogen chloride gas, hydrogen bromide, hydrogen iodide, trifluoromethanesulfonic acid, camphorsulfonic acid, sulfuric acid, phosphoric acid, and arylsulfonic acids such as benzenesulfonic acid, p-toluenesulfonic acid, and p-chlorobenzenesulfonic acid. Recommended catalyzing agents include: trifluoroacetic acid, methanesulfonic acid, camphorsulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid and p-chlorobenzenesulfonic acid. The most recommended catalytic agent is methanesulfonic acid. The recommended solvent is methanol or ethanol, preferably ethanol.

Preporučljivi raspon temperature reakcije je između -40 i 150°C, najbolje između 10 i 40°C. The recommended reaction temperature range is between -40 and 150°C, preferably between 10 and 40°C.

Uklanjanje benziloksikarbonil (karbobenziloksi) skupine može se postići brojnim postupcima, na primjer katalitičkom hidrogenacijom vodikom u nazočnosti plemenite kovine ili njenog oksida, kao što je paladij na aktiviranom ugljiku u protonskom otapalu kao što je etanol. U slučajevima gdje je katalitička hidrogenacija onemogućena nazočnošću druge potencijalno reaktivne funkcijske skupine, uklanjanje benziloksikarbonil (karbobenziloksi) skupine može se također postići djelovanjem otopine bromovodika u octenoj' kiselini, ili djelovanjem smjese TFA i dimetilsulfida. Removal of the benzyloxycarbonyl (carbobenzyloxy) group can be achieved by a number of methods, for example by catalytic hydrogenation with hydrogen in the presence of a noble metal or its oxide, such as palladium on activated carbon in a protic solvent such as ethanol. In cases where catalytic hydrogenation is prevented by the presence of another potentially reactive functional group, removal of the benzyloxycarbonyl (carbobenzyloxy) group can also be achieved by the action of a solution of hydrogen bromide in acetic acid, or by the action of a mixture of TFA and dimethylsulfide.

U cilju učinkovitosti, preporučuje se da se ova deprotekcija katalizirana kiselinom provede in situ bez izoliranja spoja formule II nakon njegove priprave gore spomenutim postupkom. For efficiency, it is recommended that this acid-catalyzed deprotection be carried out in situ without isolating the compound of formula II after its preparation by the above-mentioned process.

Unutar cjelovitog postupka, treći postupak sadrži pripravu spoja formule III: Within the complete process, the third process comprises the preparation of the compound of formula III:

[image] [image]

gdje je L amino zaštitna skupina, spajanjem aminokiseline čija je formula: where L is an amino protecting group, by connecting an amino acid whose formula is:

[image] [image]

gdje je L amino zaštitna skupina, sa spojem formule II: where L is an amino protecting group, with a compound of formula II:

[image] [image]

u nazočnosti kiselog aktivirajućeg sredstva u inertnom otapalu u nazočnosti katalitičkog sredstva, čime se dobije spoj formule III. in the presence of an acidic activating agent in an inert solvent in the presence of a catalytic agent, thereby obtaining the compound of formula III.

Kisela aktivirajuća sredstva pogodna za ovaj postupak uključuju: DCC, EDC, ECAC i BOP, a preporučeno kiselo aktivirajuće sredstvo je DCC (N,N'-dicikloheksilkarbodiimid). Acid activators suitable for this process include: DCC, EDC, ECAC and BOP, with the recommended acid activator being DCC (N,N'-dicyclohexylcarbodiimide).

Katalizirajuća sredstva pogodna za ovaj postupak uključuju: HOBT i druga, a preporučeno katalitičko sredstvo je HOBT (hidroksibenzotriazol). Catalyzing agents suitable for this process include: HOBT and others, and the recommended catalytic agent is HOBT (hydroxybenzotriazole).

Inertna otapala prikladna za ovaj postupak uključuju: acetonitril; izo-propil acetat; etil acetat; propionitril; vodu; klorirane ugljikovodike kao što su: diklorometan, kloroform, tetraklorugljik, dikloroetan, klorobenzen, orto-diklorobenzen; benzen; toluen; ksilen i drugi, kao i njihove mješavine, a preporučeno otapalo je smjesa izopropil acetata i vode, preporučljivo u omjeru približno 40:60 do 60:40 (volumni udjeli), najbolje oko 50:50 (volumni udjeli). Inert solvents suitable for this procedure include: acetonitrile; iso-propyl acetate; ethyl acetate; propionitrile; water; chlorinated hydrocarbons such as: dichloromethane, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene, ortho-dichlorobenzene; benzene; toluene; xylene and others, as well as their mixtures, and the recommended solvent is a mixture of isopropyl acetate and water, preferably in a ratio of approximately 40:60 to 60:40 (volume fractions), preferably around 50:50 (volume fractions).

Preporučeni raspon temperature reakcije je između -40 i 150°C, najbolje između 20 i 50°C. The recommended reaction temperature range is between -40 and 150°C, preferably between 20 and 50°C.

Prikladne amino zaštitne skupine uključuju: benzil, benziloksimetil, benziloksikarbonil (karbobenziloksi), benzilsulfonil, 2-bromo-etiloksikarbonil, t-butoksi-karbonil, 2-kloro-benziloksi-karbonil, 2-kloroetiloksikarbonil, di-t-amiloksikarbonil, 9-fluoroenil-metiloksikarbonil, izopropoksikarbonil, 4-metoksi-benziloksikarbonil, 4-nitrobenziloksikarbonil, 2- nitrofenil-sulfonil, ftaloil, 2,2,2-trikloro-t-butiloksikarbonil, trifluoroacetil, trifenilmetan, aliloksikarbonil, viniloksi karbonil skupine i druge, a preporučene su skupine benziloksikarbonil (karbobenziloksi), t-butoksi-karbonil, a najviše se preporučuje t-butoksi-karbonil skupina. Suitable amino protecting groups include: benzyl, benzyloxymethyl, benzyloxycarbonyl (carbobenzyloxy), benzylsulfonyl, 2-bromo-ethyloxycarbonyl, t-butoxy-carbonyl, 2-chloro-benzyloxy-carbonyl, 2-chloroethyloxycarbonyl, di-t-amyloxycarbonyl, 9-fluoroenyl -methyloxycarbonyl, isopropoxycarbonyl, 4-methoxy-benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 2-nitrophenyl-sulfonyl, phthaloyl, 2,2,2-trichloro-t-butyloxycarbonyl, trifluoroacetyl, triphenylmethane, allyloxycarbonyl, vinyloxycarbonyl groups and others, and others are recommended benzyloxycarbonyl (carbobenzyloxy), t-butoxy-carbonyl, and the most recommended t-butoxy-carbonyl group.

U cilju učinkovitosti, preporučuje se da se vezanje provodi in situ bez izoliranja spoja formule III nakon njegove priprave gore spomenutim postupkom. Na drugi način, spoj formule III može se izolirati kao posebni međuspoj. For the sake of efficiency, it is recommended that the binding be carried out in situ without isolating the compound of formula III after its preparation by the above-mentioned process. Alternatively, the compound of formula III can be isolated as a separate intermediate.

Unutar cjelovitog postupka, četvrti postupak sadrži pripravu spoja formule IV, ili njegovih farmaceutski prikladnih soli: Within the complete process, the fourth process comprises the preparation of the compound of formula IV, or its pharmaceutically acceptable salts:

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koja obuhvaća reagiranje spoja formule III: which comprises reacting the compound of formula III:

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gdje je L amino zaštitna skupina, s amino deprotekcijskim sredstvom čime se dobije spoj formule IV. where L is an amino protecting group, with an amino deprotecting agent to give a compound of formula IV.

Prikladne amino zaštitne skupine uključuju: benzil, benziloksimetil, benziloksikarbonil (karbobenziloksi), benzilsulfonil, 2-bromo-etiloksikarbonil, t-butoksi-karbonil, 2-kloro-benziloksi-karbonil, 2-kloroetiloksikarbonil, di-t-amiloksikarbonil, 9-fluoroenil-metiloksikarbonil, izopropoksikarbonil, 4-metoksi-benziloksikarbonil, 4-nitrobenziloksikarbonil, 2-nitrofenil-sulfonil, ftaloil, 2,2,2-trikloro-t-butiloksikarbonil, trifluoroacetil, trifenilmetan, aliloksikarbonil, viniloksi karbonil skupine i druge, a preporučene su skupine benziloksikarbonil (karbobenziloksi), t-butoksi-karbonil, a najviše se preporučuje t-butoksi-karbonil skupina. Suitable amino protecting groups include: benzyl, benzyloxymethyl, benzyloxycarbonyl (carbobenzyloxy), benzylsulfonyl, 2-bromo-ethyloxycarbonyl, t-butoxy-carbonyl, 2-chloro-benzyloxy-carbonyl, 2-chloroethyloxycarbonyl, di-t-amyloxycarbonyl, 9-fluoroenyl -methyloxycarbonyl, isopropoxycarbonyl, 4-methoxy-benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 2-nitrophenyl-sulfonyl, phthaloyl, 2,2,2-trichloro-t-butyloxycarbonyl, trifluoroacetyl, triphenylmethane, allyloxycarbonyl, vinyloxycarbonyl groups and others, and others are recommended benzyloxycarbonyl (carbobenzyloxy), t-butoxy-carbonyl, and the most recommended t-butoxy-carbonyl group.

U ovom postupku, uklanjanje amino zaštitne skupine može se postići uporabom prikladnog katalizirajućeg sredstva. Uklanjanje t-butoksikarbonil zaštitne skupine može se provesti u otapalu kao što je metanol, etanol, metilen klorid, etil acetat ili izopropilacetat, pomoću jake kiseline. Takve jake kiseline uključuju metansulfonsku kiselinu, trifluoroctenu kiselinu, klorovodičnu kiselinu, klorovodik kao plin, bromovodik, jodovodik, trifluormetansulfonsku kiselinu, kamforsulfonsku kiselinu, sumpornu kiselinu, fosfornu kiselinu i arilsulfonsku kiselinu poput benzensulfonske kiseline, p-toluensulfonske kiseline i p-klorbenzensulfonske kiseline. Preporučljiva katalizirajuća sredstva uključuju: trifluoroctenu kiselinu, metansulfonsku kiselinu, kamforsulfonsku kiselinu, benzensulfonsku kiselinu, p-toluensulfonsku kiselinu i p-klorbenzensulfonsku kiselinu. Najpreporučljivije katalitičko sredstvo je metansulfonska kiselina. Preporučuje se da se spoj formule V izolira u obliku metansulfonatne soli. Preporučljivo otapalo je metanol ili etanol, najbolje etanol. In this process, removal of the amino protecting group can be achieved by using a suitable catalyzing agent. Removal of the t-butoxycarbonyl protecting group can be carried out in a solvent such as methanol, ethanol, methylene chloride, ethyl acetate or isopropyl acetate, using a strong acid. Such strong acids include methanesulfonic acid, trifluoroacetic acid, hydrochloric acid, hydrogen chloride gas, hydrogen bromide, hydrogen iodide, trifluoromethanesulfonic acid, camphorsulfonic acid, sulfuric acid, phosphoric acid, and arylsulfonic acids such as benzenesulfonic acid, p-toluenesulfonic acid, and p-chlorobenzenesulfonic acid. Recommended catalyzing agents include: trifluoroacetic acid, methanesulfonic acid, camphorsulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid and p-chlorobenzenesulfonic acid. The most recommended catalytic agent is methanesulfonic acid. It is recommended that the compound of formula V be isolated in the form of the methanesulfonate salt. The recommended solvent is methanol or ethanol, preferably ethanol.

Preporučljivi raspon temperature reakcije je između -40 i 150°C, najbolje između 10 i 40°C. The recommended reaction temperature range is between -40 and 150°C, preferably between 10 and 40°C.

Uklanjanje benziloksikarbonil (karbobenziloksi) skupine može se postići brojnim postupcima, na primjer katalitičkom hidrogenacijom vodikom u nazočnosti plemenite kovine ili njenog oksida, kao što je paladij na aktiviranom ugljiku u protonskom otapalu kao što je etanol. U slučajevima gdje je katalitička hidrogenacija onemogućena nazočnošću druge potencijalno / reaktivne funkcijske skupine, uklanjanje benziloksikarbonil (karbobenziloksi) skupine može se također postići djelovanjem otopine bromovodika u octenoj kiselini, ili djelovanjem smjese TFA i dimetilsulfida. Removal of the benzyloxycarbonyl (carbobenzyloxy) group can be achieved by a number of methods, for example by catalytic hydrogenation with hydrogen in the presence of a noble metal or its oxide, such as palladium on activated carbon in a protic solvent such as ethanol. In cases where catalytic hydrogenation is prevented by the presence of another potentially/reactive functional group, removal of the benzyloxycarbonyl (carbobenzyloxy) group can also be achieved by the action of a solution of hydrogen bromide in acetic acid, or by the action of a mixture of TFA and dimethylsulfide.

U cilju učinkovitosti, preporučuje se da se ova deprotekcija katalizirana kiselinom provede in situ bez izoliranja spoja formule IV nakon njegove priprave gore spomenutim postupkom. For efficiency, it is recommended that this acid-catalyzed deprotection be carried out in situ without isolating the compound of formula IV after its preparation by the above-mentioned process.

Unutar cjelovitog postupka, peti postupak sadrži pripravu farmaceutski prikladne soli spoja formule IV, poglavito metansulfonske soli, tj. spoja formule V: Within the complete process, the fifth process contains the preparation of a pharmaceutically suitable salt of the compound of the formula IV, mainly the methanesulfonic salt, i.e. the compound of the formula V:

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koja obuhvaća reagiranje spoja formule IV: which comprises reacting the compound of formula IV:

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s kiselinom, preporučljivo metansulfonskom kiselinom, čime se dobije spoj formule V. with an acid, preferably methanesulfonic acid, to give a compound of formula V.

Preporučuje se da se spoj formule V izolira u obliku metansulfonatne soli. Preporučena otapala uključuju etilacetat i etanol, najbolje smjesu etil acetata i etanola. It is recommended that the compound of formula V be isolated in the form of the methanesulfonate salt. Recommended solvents include ethyl acetate and ethanol, preferably a mixture of ethyl acetate and ethanol.

U cilju učinkovitosti, preporučuje se da se stvaranje soli provede in situ bez izoliranja spoja formule V nakon njegove priprave gore spomenutim postupkom. For the sake of efficiency, it is recommended that the salt formation be carried out in situ without isolating the compound of formula V after its preparation by the above-mentioned process.

U preporučenim oblicima ovog izuma, pojedini postupci kao dijelovi cjelovitog postupka prikazani su slijedećom shemom: In the recommended forms of this invention, individual procedures as parts of a complete procedure are shown in the following scheme:

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U ovom preporučenom obliku, CBZ-spiroindolin 1 se izlaže djelovanju Darco (20% tež. udjela) prije hidrogeniranja. Hidrogeniranje se provodi u etanolu na 65°C preko 10%-tnog Pd/C snažnim miješanjem. In this recommended form, CBZ-spiroindoline 1 is exposed to Darco (20% by weight) prior to hydrogenation. Hydrogenation is carried out in ethanol at 65°C over 10% Pd/C with vigorous stirring.

Otopina 1b u izopropil acetatu i vodi spaja se s komercijalno dostupnim N-BOC-O-benzil-D-serinom u nazočnosti dicikloheksilkarbodiimida (DCC) i 1-hidroksibenzotriazola (HOBt). Nakon filtriranja dicikloheksilurea (DCU) sporednog proizvoda, dvofazni se filtrat izdvoji i organski sloj se uzastopno ispire 1M vodenom otopinom natrij hidroksida, 0.5M vodenom klorovodičnom kiselinom i, na kraju, zasićenim vodenim natrij hidrogenkarbonatom. Bolji rezultati ovog spajanja postižu se kada se otopina slobodnog amino u PrOAc/H2O izloži djelovanju DCC, HOBT, a potom provede dodavanje aminokiseline na temperaturi okoline, nakon čega slijedi reakcija u trajanju od 3-5 sati. Smjesa se potom koncentrira in vacuo i vrši se izmjena otapala iz izopopil acetata u etanol. Ova se izmjena otapala obično izvodi brzo, na "feeding and bleeding" način, pomoću trostrukog volumena smjese da bi se uklonio izopropil acetat. A solution of 1b in isopropyl acetate and water is coupled with commercially available N-BOC-O-benzyl-D-serine in the presence of dicyclohexylcarbodiimide (DCC) and 1-hydroxybenzotriazole (HOBt). After filtering off the dicyclohexylurea (DCU) byproduct, the biphasic filtrate was separated and the organic layer was washed sequentially with 1M aqueous sodium hydroxide solution, 0.5M aqueous hydrochloric acid, and finally saturated aqueous sodium hydrogencarbonate. Better results of this coupling are obtained when the solution of the free amino in PrOAc/H2O is exposed to the action of DCC, HOBT, and then the addition of the amino acid is carried out at ambient temperature, followed by a reaction lasting 3-5 hours. The mixture is then concentrated in vacuo and the solvent is changed from isopropyl acetate to ethanol. This solvent exchange is usually performed rapidly, in a "feeding and bleeding" fashion, using three times the volume of the mixture to remove the isopropyl acetate.

BOC skupina 11 uklanja se djelovanjem metansulfonske kiseline (MsOH) (3 ekv.) u etanolu na 35-40°C. Razdjeljivanjem između izopropil acetata i vodene otopine 1M natrij hidroksida dobije se spoj 12. The BOC group 11 is removed by the action of methanesulfonic acid (MsOH) (3 eq.) in ethanol at 35-40°C. Partitioning between isopropyl acetate and aqueous 1M sodium hydroxide solution gives compound 12.

Vezanje 12 i N-BOC-α-aminoizomaslačne kiseline najbolje se provodi u dvofaznom sustavu otapala - izopropil acetat/voda (1:1) u nazočnosti DCC i HOBt (1.1 ekv. svakog). Uklanjanjem DCU filtracijom, razdvajanjem slojeva i ispirajem organskog sloja 1M vodenim natrij hidroksidom, 0.5M vodenom klorovodičnom kiselinom i zasićenim vodenim natrij hidrogenkarbonatom dobije se spoj 14. The binding of 12 and N-BOC-α-aminoisobutyric acid is best carried out in a biphasic solvent system - isopropyl acetate/water (1:1) in the presence of DCC and HOBt (1.1 eq each). By removing DCU by filtration, separating the layers and washing the organic layer with 1M aqueous sodium hydroxide, 0.5M aqueous hydrochloric acid and saturated aqueous sodium hydrogencarbonate, compound 14 is obtained.

Smjesi se izmijeni otapalo u etanol u svrhu posljedičnog cijepanja Boe skupine metansulfonskom kiselinom. Deprotekcija spoja 14 je teža nego spoja 11 i zahtijeva koncentriranu otopinu etanol/metansulfonske kiseline i zagrijavanje do 35-40°C. Nakon ekstrakcijskog postupka (EtOAc-NaOH), izolira se slobodni amin 15. Organski se sloj dobro ispere s 1N NaOH čime se omogući potpuno uklanjanje metansulfonske kiseline. The solvent of the mixture was changed to ethanol in order to subsequently cleave the Boe group with methanesulfonic acid. Deprotection of compound 14 is more difficult than compound 11 and requires a concentrated ethanol/methanesulfonic acid solution and heating to 35-40°C. After the extraction procedure (EtOAc-NaOH), the free amine 15 is isolated. The organic layer is washed well with 1N NaOH, which enables the complete removal of methanesulfonic acid.

Etil acetatna otopina slobodne baze 15 koncentrira se do malog obujma in vacuo i azeotropno se suši (KF<500 mg/mL) "feeding and bleeding" postupkom pomoću trostrukog volumena smjese 90/10 etil acetat/etanola, a nakon toga dvostrukim volumenom smjese etil acetata. Na dobivenu suhu, lagano magličastu otopinu slobodne baze 15 u etil acetatu djeluje se otopinom Darco G-60 (25 mas.%) na sobnoj temperaturi tijekom približno 10 sati. Uklanjanjem Darco filtracijom pomoću filtracijskog sredstva dobije se slobodna baza 15. The ethyl acetate solution of the free base 15 is concentrated to a small volume in vacuo and azeotropically dried (KF<500 mg/mL) by the "feeding and bleeding" process using a triple volume of a 90/10 ethyl acetate/ethanol mixture, followed by a double volume of an ethyl acetate/ethanol mixture. of acetate. The resulting dry, slightly hazy solution of free base 15 in ethyl acetate is treated with a solution of Darco G-60 (25 wt.%) at room temperature for approximately 10 hours. Removal by Darco filtration using a filtration agent yields the free base 15.

Stvaranje soli metansulfonske kiseline 16 iz 15 provodi se u EtOAc s 1.1 ekv. MsOH na oko 50°C. Slobodna baza 15 izloži se djelovanju 8 vol.%-tnog EtOH i jednog ekv. H2O i zagrijava na 55°C do potpunog otapanja. Hlađenje do temperature okoline i miješanje dobivene smjese tijekom 4 sata daje kristalnu tvar 16 označenu kao kristalni Oblik II (topljivost u IPA=12 mg/mL). Formation of the methanesulfonic acid salt 16 from 15 was carried out in EtOAc with 1.1 eq. MsOH at about 50°C. The free base 15 is exposed to the action of 8 vol.% EtOH and one equiv. H2O and heated to 55°C until complete dissolution. Cooling to ambient temperature and stirring the resulting mixture for 4 hours gives crystalline substance 16 designated as crystalline Form II (solubility in IPA=12 mg/mL).

Prevođenje Oblika II u Oblik I postiže se oblikovanjem soli u EtOAc-EtOH kao što je gore opisano, ali se - umjesto hlađenja početne otopine soli (na 55°C) na temperaturu okoline - vrši hlađenje na 45°C. Pri toj temperaturi počinju se javljati kristali i smjesa s vremenom postaje gušća. Potom se temperatura podiže na 51°C i smjesa se ostavi stajati preko noći. Očekuje se potpuna pretvorba u Oblik I spoja 16. Conversion of Form II to Form I is achieved by forming the salt in EtOAc-EtOH as described above, but - instead of cooling the initial salt solution (at 55°C) to ambient temperature - cooling to 45°C is performed. At this temperature, crystals begin to appear and the mixture becomes thicker over time. Then the temperature is raised to 51°C and the mixture is left to stand overnight. Complete conversion to Form I of compound 16 is expected.

Preporučuje se pretvorba Oblika II u Oblik I dodavanjem kristala Oblika I otopini slobodne baze u EtOAc-EtOH na 50-55°C nakon čega smjesa odstoji. Prema tome, slobodna baza 15 može se izložiti djelovanju 1.1 ekv. metansulfonske kiseline u 8%-tnom etanolu u etil acetatu na 50-55°C. Smjesi se potom doda klica približno 2 mas.% Oblika I metansulfonatne soli 16 i smjesa se ostavi stajati na 55°C preko noći. Smjesa se hladi na sobnu temperaturu i ostavi stajati približno 2-3 sata. Proizvod se izolira filtracijom na sobnoj temperaturi u atmosferi dušika, suši na 35°C in vacuo i prosijava, čime se dobije metansulfonatna sol 16. It is recommended to convert Form II to Form I by adding crystals of Form I to a solution of the free base in EtOAc-EtOH at 50-55°C, after which the mixture is allowed to stand. Therefore, the free base 15 can be exposed to 1.1 eq. of methanesulfonic acid in 8% ethanol in ethyl acetate at 50-55°C. Approximately 2 wt.% of Form I methanesulfonate salt 16 germ is then added to the mixture and the mixture is left to stand at 55°C overnight. The mixture is cooled to room temperature and left to stand for approximately 2-3 hours. The product is isolated by filtration at room temperature under a nitrogen atmosphere, dried at 35°C in vacuo and sieved, thereby obtaining the methanesulfonate salt 16.

Sol metansulfonske kiseline 16 također se može dobiti naizmjeničnim provođenjem postupnog dodavanja MsOH (1.1 ekv.) i dodavanja klice kristala Oblika I otopini slobodne baze u EtOAc-EtOH na oko 50°C, pri čemu redoslijed adicije MsOH i dodavanja nije presudan. Methanesulfonic acid salt 16 can also be obtained by alternating the stepwise addition of MsOH (1.1 eq) and the addition of a seed of Form I crystals to a solution of the free base in EtOAc-EtOH at about 50°C, the order of addition of MsOH and addition not being critical.

U opisu ove primjene, korištene su slijedeće kratice slijedećih značenja: In the description of this application, the following abbreviations with the following meanings are used:

Bu butil Bu butyl

Bn benzil Bn benzyl

BOC, Boc t-butiloksikarbonil BOC, Boc t-butyloxycarbonyl

BOP benzotriazol-1-iloksi tris(dimetilamino)-fosfonij heksafluorofosfat BOP Benzotriazol-1-yloxy tris(dimethylamino)-phosphonium hexafluorophosphate

izrač. izračunato calc. calculated

CBZ, Cbz benziloksikarbonil CBZ, Cbz benzyloxycarbonyl

DCC N,N'-dicikloheksilkarbodiimid DCC N,N'-dicyclohexylcarbodiimide

DIEA di-izopropiletilamin DIEA di-isopropylethylamine

DMF N,N-dimetilformamid DMF N,N-dimethylformamide

DMAP 4-dimetilaminopiridin DMAP 4-dimethylaminopyridine

EDC 1-(3-dimetilaminopropil)-3-etilkarbodiimid hidroklorid EDC 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride

EDAC etil-3-(3-dimetilamino)-propilkarbodiimid EDAC ethyl-3-(3-dimethylamino)-propylcarbodiimide

EI-MS elektronska ionsko masena spektroskopija EI-MS electron ion mass spectroscopy

Et etil Et ethyl

ekv. ekvivalent(i) eq. equivalent(s)

FAB-MS masena spktroskopija bombardiranjem brzim atomima FAB-MS mass spectroscopy by fast atom bombardment

h sat(i) h hour(s)

HOBT, HOB t hidroksibenzotriazol HOBT, HOB t hydroxybenzotriazole

HPLC visokotlačna tekućinska kromatografija HPLC high-pressure liquid chromatography

iPrOAc izo-propil acetat iPrOAc iso-propyl acetate

KHMDS kalij bis(trimetilsilil)amid KHMDS potassium bis(trimethylsilyl)amide

LAH litij aluminij hidrid LAH lithium aluminum hydride

LHMDS litij bis(trimetilsilil)amid LHMDS lithium bis(trimethylsilyl)amide

Me metil Me methyl

MF molekularna formula MF molecular formula

MHz megaherc MHz megahertz

MPLC srednjetlačna tekućinska kromatografija MPLC medium pressure liquid chromatography

MsOH metan sulfonska kiselina MsOH methane sulfonic acid

NMM N-metilmorfolin NMM N-methylmorpholine

NMR nuklearna magnetska rezonanca NMR nuclear magnetic resonance

Ph fenil Ph phenyl

Pr propil Pr propyl

pripr. pripravljen ex. ready

TFA trifluoroctena kiselina TFA trifluoroacetic acid

THF tetrahidrofuran THF tetrahydrofuran

TLC tankoslojna kromatografija TLC thin layer chromatography

TMS tetrametilsilan TMS tetramethylsilane

U gore spominjanim strukturnim formulama i u ovoj specifikaciji, slijedeći izrazi imaju navedena značenja: In the structural formulas mentioned above and in this specification, the following terms have the following meanings:

Izraz "reakcija peptidnog vezanja", u značenju u kojem se ovdje koristi, označuje spajanje karboksilne kiseline s aminom uz korištenje kiselog aktivirajućeg sredstva kao što je EDC, DCC ili BOP u inertnom otapalu i u nazočnosti katalizatora kao što je HOBT. Inertna otapala prikladna za ovakvo vezanje uključuju: acetonitril, izopropil acetat, etil acetat, propionitril, vodu, klorirane ugljikovodike poput diklormetana, kloroforma, ugljik tetraklorida, dikloretana, klorbenzena ili orto-diklorbenzena, benzen, toluen, ksilene, kao i kombinacije navedenih i slično. The term "peptide coupling reaction", as used herein, refers to the coupling of a carboxylic acid with an amine using an acidic activator such as EDC, DCC or BOP in an inert solvent and in the presence of a catalyst such as HOBT. Inert solvents suitable for such bonding include: acetonitrile, isopropyl acetate, ethyl acetate, propionitrile, water, chlorinated hydrocarbons such as dichloromethane, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene or ortho-dichlorobenzene, benzene, toluene, xylenes, as well as combinations of the foregoing and the like. .

Varijabla "L" i izraz "amino zaštitna skupina" označava nazočnost odgovarajuće zaštitne skupine za amino, kao što su one opisane u Green, T.W., Wuts, P.G.M. Protective Groups in Organic Synthesis, 2. izd., John Wiley & Sons, Inc., New York, 1991. Odgovarajuća zaštitna skupina mora odolijevati reakcijskim uvjetima međupostupaka prije nego što se, kada se to želi, ukloni. Amino zaštitna skupina neovisno je odabrana za svaki pojedini postupak unutar cjelovitog postupka. The variable "L" and the term "amino protecting group" indicates the presence of an appropriate amino protecting group, such as those described in Green, T.W., Wuts, P.G.M. Protective Groups in Organic Synthesis, 2nd ed., John Wiley & Sons, Inc., New York, 1991. The appropriate protecting group must withstand the reaction conditions of the intermediate before it can be removed, when desired. The amino protecting group is independently selected for each individual procedure within the complete procedure.

Prikladne amino zaštitne skupine uključuju: benzil, benziloksimetil, benziloksikarbonil (karbobenziloksi), benzilsulfonil, 2-bromo-etiloksikarbonil, t-butoksi-karbonil, 2-kloro-benziloksi-karbonil, 2-kloroetiloksikarbonil, di-t-amiloksikarbonil, 9-fluoroenil-metiloksikarbonil, izopropoksikarbonil, 4-metoksi-benziloksikarbonil, 4-nitrobenziloksikarbonil, 2-nitro fenil-sulfonil, ftaloil, 2,2,2-trikloro-t-butiloksikarbonil, trifluoroacetil, trifenilmetan i viniloksikarbonil skupine i druge, a preporučene su skupine benziloksikarbonil (karbobenziloksi) i t-butoksi-karbonil, a najviše se preporučuje t-butoksi-karbonil skupina. Suitable amino protecting groups include: benzyl, benzyloxymethyl, benzyloxycarbonyl (carbobenzyloxy), benzylsulfonyl, 2-bromo-ethyloxycarbonyl, t-butoxy-carbonyl, 2-chloro-benzyloxy-carbonyl, 2-chloroethyloxycarbonyl, di-t-amyloxycarbonyl, 9-fluoroenyl -methyloxycarbonyl, isopropoxycarbonyl, 4-methoxy-benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 2-nitrophenyl-sulfonyl, phthaloyl, 2,2,2-trichloro-t-butyloxycarbonyl, trifluoroacetyl, triphenylmethane and vinyloxycarbonyl groups and others, and benzyloxycarbonyl groups are recommended (carbobenzyloxy) and t-butoxy-carbonyl, and the t-butoxy-carbonyl group is most recommended.

U ovom postupku, uklanjanje amino zaštitne skupine može se postići uporabom prikladnog katalizirajućeg sredstva. Uklanjanje t-butoksikarbonil zaštitne skupine može se provesti u otapalu kao što je metanol, etanol, metilen klorid, etil acetat ili izopropilacetat, pomoću jake kiseline. Takve jake kiseline uključuju metansulfonsku kiselinu, trifluoroctenu kiselinu, klorovodičnu kiselinu, klorovodik kao plin, bromovodik, jodovodik, trifluormetansulfonsku kiselinu, kamforsulfonsku kiselinu, sumpornu kiselinu, fosfornu kiselinu i arilsulfonsku kiselinu poput benzensulfonske kiseline, p-toluensulfonske kiseline i p-klorbenzensulfonske kiseline. Preporučljiva katalizirajuća sredstva uključuju: trifluoroctenu kiselinu, metansulfonsku kiselinu, kamforsulfonsku kiselinu, benzensulfonsku kiselinu, p-toluensulfonsku kiselinu i p-klorbenzensulfonsku kiselinu. Najpreporučljivije katalitičko sredstvo je metansulfonska kiselina. Preporučljivo otapalo je metanol ili etanol. In this process, removal of the amino protecting group can be achieved by using a suitable catalyzing agent. Removal of the t-butoxycarbonyl protecting group can be carried out in a solvent such as methanol, ethanol, methylene chloride, ethyl acetate or isopropyl acetate, using a strong acid. Such strong acids include methanesulfonic acid, trifluoroacetic acid, hydrochloric acid, hydrogen chloride gas, hydrogen bromide, hydrogen iodide, trifluoromethanesulfonic acid, camphorsulfonic acid, sulfuric acid, phosphoric acid, and arylsulfonic acids such as benzenesulfonic acid, p-toluenesulfonic acid, and p-chlorobenzenesulfonic acid. Recommended catalyzing agents include: trifluoroacetic acid, methanesulfonic acid, camphorsulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid and p-chlorobenzenesulfonic acid. The most recommended catalytic agent is methanesulfonic acid. The recommended solvent is methanol or ethanol.

Uklanjanje benziloksikarbonil (karbobenziloksi) skupine može se postići brojnim postupcima, na primjer katalitičkom hidrogenacijom vodikom u nazočnosti plemenite kovine ili njenog oksida, kao što je paladij na aktiviranom ugljiku u protonskom otapalu kao što je etanol. U slučajevima gdje je katalitička hidrogenacija onemogućena nazočnošću druge potencijalno reaktivne funkcijske skupine, uklanjanje benziloksikarbonil (karbobenziloksi) skupine može se također postići djelovanjem otopine bromovodika u octenoj kiselini, ili djelovanjem smjese TFA i dimetilsulfida. Removal of the benzyloxycarbonyl (carbobenzyloxy) group can be achieved by a number of methods, for example by catalytic hydrogenation with hydrogen in the presence of a noble metal or its oxide, such as palladium on activated carbon in a protic solvent such as ethanol. In cases where catalytic hydrogenation is prevented by the presence of another potentially reactive functional group, removal of the benzyloxycarbonyl (carbobenzyloxy) group can also be achieved by the action of a solution of hydrogen bromide in acetic acid, or by the action of a mixture of TFA and dimethylsulfide.

Amino spojevi korišteni kao početne tvari u postupcima ovog izuma mogu biti u obliku svojih kiselih soli, kao što su soli dobivene pomoću anorganskih i organskih kiselina. Primjeri takvih kiselina uključuju klorovodičnu, nitratnu, sumpornu, fosfornu, mravlju, octenu, trifluoroctenu, propionsku, maleinsku, sukcinilnu, malonsku, metansulfonsku i druge kiseline. Analogno tome, spojevi dobiveni postupcima ovog izuma mogu se izolirati u obliku svojih farmaceutski prikladnih kiselih soli. Pored toga, neki spojevi koji imaju funkciju zakiseljavanja, kao što je karboksi, mogu biti u obliku svojih anorganskih soli s protuionom odabranim između natrija, kalija, litija, kalcija, magnezija i drugih, kao i od organskih baza. Amino compounds used as starting materials in the processes of this invention can be in the form of their acid salts, such as salts obtained using inorganic and organic acids. Examples of such acids include hydrochloric, nitric, sulfuric, phosphoric, formic, acetic, trifluoroacetic, propionic, maleic, succinic, malonic, methanesulfonic and other acids. Analogously, the compounds obtained by the methods of this invention can be isolated in the form of their pharmaceutically acceptable acid salts. In addition, some compounds having an acidifying function, such as carboxy, may be in the form of their inorganic salts with a counterion selected from sodium, potassium, lithium, calcium, magnesium and others, as well as organic bases.

Priprava spojeva postupkom ovog izuma može se izvesti sekvencijskim ili konvergentnim putevima sinteze. Bilježi se da, u pojedinim slučajevima, slijed provođenja prethodnih shema može biti promijenjen da bi se pospješila reakcija ili da bi se izbjegli neželjeni proizvodi reakcije. Općenito, postupak iz ovog izuma provodi se u sekvencijskom obliku, kao što je ovdje i izloženo. The preparation of the compounds by the process of the present invention can be carried out by sequential or convergent synthesis routes. It is noted that, in some cases, the sequence of carrying out the preceding schemes can be changed to speed up the reaction or to avoid unwanted reaction products. In general, the process of the present invention is carried out in a sequential manner, as set forth herein.

Mnoge početne tvari su ili komercijalno dostupne ili opisane u literaturi, dok se druge mogu pripraviti prema postupcima iz literature koji su iznijeti za odgovarajuće spojeve. Vještine potrebne za izvođenje reakcije i pročišćavanje dobivenih proizvoda reakcije poznate su upućenima u struku. Postupci pročišćavanja uključuju kristalizaciju, normalnu i obrnuto faznu kromatografiju. Many starting materials are either commercially available or described in the literature, while others can be prepared according to literature procedures outlined for the corresponding compounds. The skills required to perform the reaction and to purify the resulting reaction products are known to those skilled in the art. Purification procedures include crystallization, normal and reverse phase chromatography.

Slijedeći se primjeri iznose u svrhu daljnje ilustracije, a nije im namjena ograničavati opisani izum. The following examples are presented for the purpose of further illustration, and are not intended to limit the described invention.

Primjer 1 Example 1

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Izonipekotični kiseli-N-benzil karbamat (3) Isonipecotic acid-N-benzyl carbamate (3)

Tvari: Substances:

izonipekotična kiselina (2) T.C.I. 4.02 kg (31.1 mol) isonipecotic acid (2) T.C.I. 4.02 kg (31.1 mol)

benzil kloroformat (Schweitzerhall) 6.91 kg (40.5 mol) benzyl chloroformate (Schweitzerhall) 6.91 kg (40.5 mol)

K2CO3 10,1 kg (72.9 mol) K2CO3 10.1 kg (72.9 mol)

voda 40.2 L water 40.2 L

Izonipekotična kiselina (2) i K2SO4 otope se u 40.2 L vode u 100-litrenoj posudi s četiri grla i mehaničkim miješalom u dušiku i otopina se ohladi na 10°C. Doda se benzil kloroformat uz održavanje temperature između 9 i 14°C, smjesa se zagrijava do 22°C nakon što je dodavanje završeno i ostavi se stajati 58 sati. Dodavanje je završeno unutar 4 sata i pH u tom trenutku iznosio je 9.0. Nakon stajanja od 58 sati nije bilo promjene pH. Isonipecotic acid (2) and K2SO4 are dissolved in 40.2 L of water in a 100-liter four-necked vessel with a mechanical stirrer under nitrogen and the solution is cooled to 10°C. Benzyl chloroformate is added while maintaining the temperature between 9 and 14°C, the mixture is heated to 22°C after the addition is complete and allowed to stand for 58 hours. The addition was complete within 4 hours and the pH at that time was 9.0. After standing for 58 hours, there was no change in pH.

Reakcijska se smjesa premjesti u 200-litreni ekstraktor i ispire s 3 x 13 kg (15 L) IPAC i 1 x 12 L EtOAc. Vodeni se sloj ekstrahira s 8 L toluena. Nakon ispiranja sadržaj benzil alkohola se smanji s 3.8% na 1.4% pomoću HPLC analize. HPLC analiza: Dupont Zorbax 25 cm RXC8 stupac sa protokom 1.5 mL/min i mjerenjem pri 254 nm; izokratična smjesa s 35% MeCN, 65% 0.1%-tnog vod. H3PO4; vremena retencije: 3 = 6.9 min., benzil alkohol = 3.3 min., toluen = 17.3 min. The reaction mixture was transferred to a 200 L extractor and washed with 3 x 13 kg (15 L) IPAC and 1 x 12 L EtOAc. The aqueous layer is extracted with 8 L of toluene. After washing, the content of benzyl alcohol is reduced from 3.8% to 1.4% using HPLC analysis. HPLC analysis: Dupont Zorbax 25 cm RXC8 column with a flow rate of 1.5 mL/min and measurement at 254 nm; isocratic mixture with 35% MeCN, 65% 0.1% aq. H3PO4; retention times: 3 = 6.9 min., benzyl alcohol = 3.3 min., toluene = 17.3 min.

Vodena se faza zakiseli s 37%-tnom vodenom HCl do pH 1.8. Tijekom dodavanja HCl razvija se ugljični dioksid, ali razvijanje se lako kontrolira. Dodavanje HCl traje manje od 1 sat i zahtijeva 10 L konc. HCl. Vodena se faza ekstrahira s 3 x 6.6 L toluena. Toluenski ekstrakti suše se s 2 kg natrij sulfata i filtriraju kroz Solka-floc™. Težina kombiniranih filtrata iznosila je 17.8 kg. Sirovi prinos karbamata 3 bio je 7.89 kg (97%) (dobiveno isparavanjem vaganih alikvota filtrata do suhoće). Filtrati se prenesu kroz 10 u inline filter u 100-litrenu posudu. Ekstrakti se koncentriraju pri 10 mbar na <25°C do volumena od 18 L. The aqueous phase is acidified with 37% aqueous HCl to pH 1.8. During the addition of HCl, carbon dioxide is evolved, but the evolution is easily controlled. The addition of HCl takes less than 1 hour and requires 10 L conc. HCl. The aqueous phase is extracted with 3 x 6.6 L of toluene. The toluene extracts are dried with 2 kg of sodium sulfate and filtered through Solka-floc™. The weight of the combined filtrates was 17.8 kg. The crude yield of carbamate 3 was 7.89 kg (97%) (obtained by evaporating weighed aliquots of the filtrate to dryness). The filtrates are transferred through a 10 in-line filter to a 100-liter container. The extracts are concentrated at 10 mbar at <25°C to a volume of 18 L.

Konačna koncentracija karbamata 3 iznosi 440 g/L. Koncentracija toluenskog filtrata služi da bi se azeotropno uklonili konačni ostaci vode (konačni KF=170 mg/L). Proizvod ima 99.1 površ.% čistoću s 0.9 površ.% benzil alkohola kao jedinim onečišćenjem. The final concentration of carbamate 3 is 440 g/L. The concentration of the toluene filtrate serves to azeotropically remove the final water residues (final KF=170 mg/L). The product has 99.1 surface% purity with 0.9 surface% benzyl alcohol as the only impurity.

Primjer 2 Example 2

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Izonipekotični kiseli klorid-N-benzil karbamat (4) Isonipecotic acid chloride-N-benzyl carbamate (4)

Tvari: Substances:

izonipekotični kiseli N-benzil karbamat (3) 7.89 kg (30.0 mol) u isonipecotic acid N-benzyl carbamate (3) 7.89 kg (30.0 mol) in

u toluenu (MT = 263.30) 17.9 L in toluene (MT = 263.30) 17.9 L

oksalil klorid (MT = 126.93) 3.94 kg (31.0 mol) oxalyl chloride (MT = 126.93) 3.94 kg (31.0 mol)

DMF (MT = 73.10) 10 mL DMF (MT = 73.10) 10 mL

toluen 12 L toluene 12 L

U toluensku otopinu benzil karbamata 3 iz prethodnog koraka doda se 5 mL DMF i 10 L toluena. Dodaje se oksalil klorid tijekom razdoblja od 20 min. Reakcijska se smjesa ostavi stajati tijekom 16 h na 18°C uz lagano strujanje dušika. HPLC analiza reakcijske smjese pokazala je da je 1.3% karboksilne kiseline 3 ostalo nereagirano. Reakcijska se smjesa zagrijava na 26°C i doda se 5 mL DMF. Smjesa se ostavi stajati tijekom 2.5 h. 5 mL of DMF and 10 L of toluene are added to the toluene solution of benzyl carbamate 3 from the previous step. Oxalyl chloride is added over a period of 20 min. The reaction mixture was left to stand for 16 h at 18°C with a gentle stream of nitrogen. HPLC analysis of the reaction mixture showed that 1.3% of carboxylic acid 3 remained unreacted. The reaction mixture is heated to 26°C and 5 mL of DMF is added. The mixture is left to stand for 2.5 hours.

Alikvot od 1.0 mL reakcijske smjese pomiješa se s 5.0 mL terc-butilamina i, nakon isparavanja, analizira pomoću HPLC: 25 cm Dupont Zorbax RXC8 stupac, na 50°C, sa protokom 1 mL/min i mjerenjem pri 220 nm; izokratični 42% MeCN, 58% 0.1%-tnog vod. H3PO4. Postupak je pokazao da je preostalo <0.05% kiseline 3 (prosudba prema A) i > 3 površ.% B (>1 mol% (COCl)2). An aliquot of 1.0 mL of the reaction mixture was mixed with 5.0 mL of tert-butylamine and, after evaporation, analyzed by HPLC: 25 cm Dupont Zorbax RXC8 column, at 50°C, with a flow rate of 1 mL/min and measurement at 220 nm; isocratic 42% MeCN, 58% 0.1% aq. H3PO4. The procedure showed that <0.05% of acid 3 remained (judgment according to A) and > 3 surface% of B (>1 mol% (COCl)2).

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Smjesa se koncentrira pri 10 mbar i temperaturi od 20-25°C dok se ne ukloni 5 L otapala. The mixture is concentrated at 10 mbar and a temperature of 20-25°C until 5 L of solvent is removed.

Tipični HPLCHPLC profil koncentrirane toluenske otopine nakon gore opisanog dodavanja t-BuNH2 je slijedeći: A typical HPLCHPLC profile of a concentrated toluene solution after the addition of t-BuNH2 as described above is as follows:

vrijeme retencije (min.) površ.% spoj retention time (min.) surface% compound

2.1 <0.5% karboksilna kiselina 3 2.1 <0.5% carboxylic acid 3

7.8 <0.5% benzil klorid 7.8 <0.5% benzyl chloride

11.0 >99% Cbz-t-butilkarboksamid A 11.0 >99% Cbz-t-butylcarboxamide A

12.1 NA toluen 12.1 NA toluene

12.7 <0.5% diterc-butiloksamid B 12.7 <0.5% di-tert-butyloxamide B

Primjer 3 Example 3

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Piperidin-4-karboksaldehid-l-benzil karbamat (5) Piperidine-4-carboxaldehyde-l-benzyl carbamate (5)

Tvari: Substances:

izonipekotični kiseli klorid N-benzil karbamat (4) 3.38 kg (12.0 mol) isonipecotic acid chloride N-benzyl carbamate (4) 3.38 kg (12.0 mol)

u toluenu (MW = 281.74) u 5.54 kg in toluene (MW = 281.74) in 5.54 kg

DIEA (KF=18 mg/L) 1.55 kg (15.0 mol) DIEA (KF=18 mg/L) 1.55 kg (15.0 mol)

10%-tni Pd/C (KF<20 mg/L) 101 g 10% Pd/C (KF<20 mg/L) 101 g

tioanisol (MT= 124.21, d = 1.058) 0.56 g thioanisole (MT= 124.21, d = 1.058) 0.56 g

Otopini spoja 4 u toluenu iz prethodnog koraka dodaju se DIEA i tioanisol i u smjesu se suspendira katalizator. Smjesa se odmah stavi u 5-galonski autoklav i hidrogenira na 20°C i 40 psi H2. Nakon 18 sati reakcija dostigne 70% teorijske količine vodika, a provedena HPLC analiza alikvota pomiješanog s terc-butilaminom pokazuje da je preostalo 14.2 površ.% kiselog klorida 2. Uvjeti HPLC isti kao i gore opisani. Vrijeme retencije: 5 = 8.1 min. DIEA and thioanisole are added to the solution of compound 4 in toluene from the previous step and the catalyst is suspended in the mixture. The mixture was immediately placed in a 5-gallon autoclave and hydrogenated at 20°C and 40 psi H 2 . After 18 hours, the reaction reaches 70% of the theoretical amount of hydrogen, and the HPLC analysis of an aliquot mixed with tert-butylamine shows that 14.2 surface% of acid chloride 2 remains. The HPLC conditions are the same as described above. Retention time: 5 = 8.1 min.

Slijedeća količina katalizatora (101 g) i tioanisola (0.54 g) doda se u hidrogenator u obliku smjese u 1375 mL toluena. Nakon 23 sata, provedena HPLC analiza alikvota pomiješanog s terc-butilaminom pokazuje da je preostalo 1.8 površ.% kiselog klorida 2. Smjesa se pročisti dušikom, a katalizator i precipitirani DIEA●HCl uklone se filtracijom kroz Solka-floc™. Filterski se kolač ispire s 10 L toluena. Filtrati se kroz 10 u. inline filter provedu u 50-litreni ekstraktor i ispiru s 2 x 7.2 LIM vodene HCl i 2 x 7.2 L vode. Smjesa se koncentrira pri 10 mbar i temperaturi od 25-30°C dok ne preostane 5 L ostatka. The next amount of catalyst (101 g) and thioanisole (0.54 g) was added to the hydrogenator in the form of a mixture in 1375 mL of toluene. After 23 hours, HPLC analysis of an aliquot mixed with tert-butylamine shows that 1.8 surface% of acid chloride 2 remains. The mixture is purged with nitrogen, and the catalyst and precipitated DIEA·HCl are removed by filtration through Solka-floc™. The filter cake is washed with 10 L of toluene. The filtrate is passed through a 10 u. inline filter into a 50-liter extractor and washed with 2 x 7.2 L aqueous HCl and 2 x 7.2 L water. The mixture is concentrated at 10 mbar and a temperature of 25-30°C until 5 L of the residue remains.

Vrijeme retencije (min.) površ.% spoj Retention time (min.) surface% compound

2.1 <2 karboksilna kiselina 3 2.1 <2 carboxylic acid 3

6.6 <1 dimer 21 6.6 <1 dimer 21

8.1 >95 aldehid 5 8.1 >95 aldehyde 5

Ispitivani prinos aldehida 3 bio je 94% prema HPLC analizi. The tested yield of aldehyde 3 was 94% according to HPLC analysis.

Primjer 4 Example 4

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CBZ-spiroindolin (9) CBZ-spiroindoline (9)

Tvari: Substances:

piperidin-4-karboksaldehid-1-benzilkarbamat (5) 1.71 kg (6.89 mol) Piperidine-4-carboxaldehyde-1-benzylcarbamate (5) 1.71 kg (6.89 mol)

u toluenskoj otopini u 21.4 kg in toluene solution in 21.4 kg

fenilhidrazin 900 mL, 981 g (9.15 mol) phenylhydrazine 900 mL, 981 g (9.15 mol)

trifluoroctena kiselina (TFA) 2.20 L, 3.26 kg (28.6 mol) trifluoroacetic acid (TFA) 2.20 L, 3.26 kg (28.6 mol)

NaBH4 300 g (7.93 mol) NaBH4 300 g (7.93 mol)

toluen 34.4 kg toluene 34.4 kg

MeCN 7.0 L5 MeCN 7.0 L5

MeOH 7.0 L MeOH 7.0 L

Sirova otopina aldehida 5 iz prethodnog koraka provodi se kroz 10 μ inline filter u 100-litreni reaktor opremljen bakrenom spiralom za hlađenje ili grijanje obloženom s teflonom, i mehaničkim miješalom. Dodaju se toluen (34.4 kg) i MeCN (7 L) i dobivena se smjesa hladi na 0°C. Postupno se doda i fenilhidrazin i temperatura se održava na -1 do 3°C uz neprestani prolaz mjehurića dušika kroz reakcijsku smjesu. The crude solution of aldehyde 5 from the previous step is passed through a 10 μ inline filter into a 100-liter reactor equipped with a copper spiral for cooling or heating coated with Teflon, and a mechanical stirrer. Toluene (34.4 kg) and MeCN (7 L) were added and the resulting mixture was cooled to 0°C. Phenylhydrazine is gradually added and the temperature is maintained at -1 to 3°C with the continuous passage of nitrogen bubbles through the reaction mixture.

Dodaje se fenilhidrazin dok TLC i HPLC analize ne pokažu potpuno nestajanje aldehida 5 i pojavu laganog viška (<5%) fenilhidrazina. Uvjeti TLC: Silica, E. Merck Kieselgel G60 F254 0.25 mm; dietileter/pentan (4/1); sredtvo za razvijanje 0.5%-tni cerij sulfat, 14%-tni amonij molibdat u 10%-tnoj vodenoj sumpornoj kiselini, potom zagrijavanje; Rf: aldehid 5 = 0.52, fenilhidrazon 7 = 0.61, fenilhidrazin 6 = 0.21. Uvjeti HPLC: 25 cm Dupont Zorbax RXC8 stupac, na 30°C, sa protokom 1 mL/min i mjerenjem pri 254 nm; tablica gradijenata: Phenylhydrazine is added until TLC and HPLC analyzes show the complete disappearance of aldehyde 5 and the appearance of a slight excess (<5%) of phenylhydrazine. TLC conditions: Silica, E. Merck Kieselgel G60 F254 0.25 mm; diethylether/pentane (4/1); developer 0.5% cerium sulfate, 14% ammonium molybdate in 10% aqueous sulfuric acid, then heating; Rf: aldehyde 5 = 0.52, phenylhydrazone 7 = 0.61, phenylhydrazine 6 = 0.21. HPLC conditions: 25 cm Dupont Zorbax RXC8 column, at 30°C, with a flow rate of 1 mL/min and measurement at 254 nm; gradient table:

vrijeme (min.) acetonitril: voda time (min.) acetonitrile: water

0 57:43 0 57:43

10 65:35 10 65:35

15 75:25 15 75:25

18 75:25 18 75:25

vremena retencije: fenilhidrazin 6 = 4.5 min, toluen = 7.2 min, fenilhidrazon 7 = 11.4 min. retention times: phenylhydrazine 6 = 4.5 min, toluene = 7.2 min, phenylhydrazone 7 = 11.4 min.

Reakcijska se smjesa ostavi stajati tijekom 30 minuta na 0-2°C, nakon čega se doda TFA uz održavanje temperature između 2 i 7°C. Reakcijska se smjesa zagrijava na 50°C tijekom 30 min. i održava na toj temperaturi 17 sati. Zaustavi se protok dušika kroz reakcijsku smjesu i uspostavi se slabo strujanje dušika iznad reakcijske smjese. Tijekom prvog sata na 5°C boja smjese postupno tamni do tamnozelene i oblikuje se relativno mala količina bijelog kristalnog precipitata (amonij trifluoroacetat). Nakon 17 sati, HPLC analiza (uz iste uvjete kao gore) pokazuje da reakcijska smjesa sadrži 91.6 površ.% indolenina 8 i 1.5% nereagiranog ostatka fenilhidrazona. Stajanje smjese tijekom duljeg razdoblja ne povećava prinos indolenina 8. The reaction mixture was allowed to stand for 30 minutes at 0-2°C, after which TFA was added while maintaining the temperature between 2 and 7°C. The reaction mixture is heated to 50°C for 30 min. and keeps it at that temperature for 17 hours. Stop the flow of nitrogen through the reaction mixture and establish a weak flow of nitrogen above the reaction mixture. During the first hour at 5°C, the color of the mixture gradually darkens to dark green and a relatively small amount of white crystalline precipitate (ammonium trifluoroacetate) forms. After 17 hours, HPLC analysis (with the same conditions as above) shows that the reaction mixture contains 91.6 surface% of indolenine 8 and 1.5% of unreacted phenylhydrazone residue. Standing the mixture for a longer period does not increase the yield of indolenine 8.

Reakcijska se smjesa hladi na 12°C i dodaje se 7.0 L MeOH. Potom se u malim obrocima (<20 g) dodaje NaBH4 uz održavanje temperature ispod 15°C. Dodavanje traje 30 minuta. Tijekom dodavanja zapaženo je umjereno razvijanje vodika koje se lako kontrolira, tako da pjenjenja praktički nema. Pri kraju dodavanja dolazi do nagle promjene boje iz zelene u smeđu, pa u jasno narančastu. Dolazi do odvajanja manje količine (<200 mL) teže faze (vjerojatno vodenih soli). HPLC analiza (uz uvjete kao ranije) pokazuje da je sav indolenin 8 potrošen (90.4 površ.% CBZ indolina 9); retencijska vremena: indolenin 8 = 7.5 min, indolin 9 = 8.2 min. TLC: etileter kao otapalo, cerij sulfat-amonij molibdat bojanje ili bojanje 1%-tnim anisaldehidom; retencijski faktori: indolenin 8 = 0.18, CBZ-indolin 9 = 0.33. The reaction mixture is cooled to 12°C and 7.0 L of MeOH is added. NaBH4 is then added in small portions (<20 g) while maintaining the temperature below 15°C. Adding takes 30 minutes. During the addition, a moderate evolution of hydrogen was observed, which is easily controlled, so that there is practically no foaming. At the end of the addition, there is a sudden color change from green to brown, then to a clear orange. A smaller amount (<200 mL) of the heavier phase (probably aqueous salts) is separated. HPLC analysis (with conditions as before) shows that all indolenine 8 is consumed (90.4 surface% of CBZ indole 9); retention times: indolenine 8 = 7.5 min, indoline 9 = 8.2 min. TLC: ethyl ether as solvent, cerium sulfate-ammonium molybdate staining or staining with 1% anisaldehyde; retention factors: indolenin 8 = 0.18, CBZ-indoline 9 = 0.33.

Promjena boje iz zelene u narančastu u tijesnoj je vezi sa završnom točkom reakcije. Količina NaBH4 potrebna za okončanje reakcije izravno je ovisna o temperaturi i brzini dodavanja NaBH4, dok su prinos i kakvoća proizvoda praktički nepromjenjivi, uz uvjet da je reakcija dovršena. Reakcijska smjesa se ohladi na 5°C tijekom 30 minuta. Potom se doda 8 L 3%-tnog vodenog NH4OH da bi se omogućio porast pH vodene faze na 7.4, smjesa se potom promućka i ostavi se da se slegne. Temperatura naraste na 15°C. Magličasto žuta donja vodena faza potom se odijeli. Organska se faza ispire s 4 L 3%-tnog NH4OH, 2 x 4 L vode i 2 x 4 L slane vode. Težina organske faze nakon ispiranja bila je 53.5 kg, a prinos 94%. The color change from green to orange is closely related to the end point of the reaction. The amount of NaBH4 required to complete the reaction is directly dependent on the temperature and rate of addition of NaBH4, while the yield and quality of the product are practically unchanged, provided that the reaction is complete. The reaction mixture was cooled to 5°C over 30 minutes. 8 L of 3% aqueous NH 4 OH are then added to allow the pH of the aqueous phase to rise to 7.4, the mixture is then shaken and allowed to settle. The temperature rises to 15°C. The hazy yellow lower aqueous phase is then separated. The organic phase is washed with 4 L of 3% NH4OH, 2 x 4 L of water and 2 x 4 L of brine. The weight of the organic phase after washing was 53.5 kg, and the yield was 94%.

Isprana toluenska otopina kombinira se s ispranim organskim fazama dvije druge reakcije sličnih tijekova. Ukupni aldehid korišten u tri reakcije iznosio je 5.06 kg (20.5 mol). Ukupna težina CBZ-indolina 9 u kombiniranim organskim fazama iznosila je 5.91 kg (18.3 mol, 90% prinosa). Kombinirane organske faze sušene su s 5 kg natrij sulfata, izložene djelovanju 250 g Darco G60 ugljika tijekom 30 minuta i filtrirane kroz Solka-floc™. Filtrati se koncentriraju u vakuumu pri 10 mbar na <25°C dok ostatak ne bude gotovo suh. Izmjena otapala upotpuni se laganim ukapavanjem u 30 L IPAC i ponovnim koncentriranjem do 14 L pri 200 mbar na 50-60°C. Smjesa se grije do refluksa u svrhu dobivanja bistre homogene tamnonarančaste otopine. 1H NMR analiza pokazala je da otopina sadrži oko 6 mol% ostanog toluena nakon izmjene otapala. The washed toluene solution is combined with the washed organic phases of two other reactions of similar flows. The total aldehyde used in the three reactions was 5.06 kg (20.5 mol). The total weight of CBZ-indoline 9 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, exposed to 250 g of Darco G60 carbon for 30 minutes and filtered through Solka-floc™. The filtrates are concentrated in vacuo at 10 mbar at <25°C until the residue is almost dry. Solvent exchange is completed by slowly dropping into 30 L of IPAC and re-concentrating to 14 L at 200 mbar at 50-60°C. The mixture is heated to reflux to obtain a clear homogeneous dark orange solution. 1H NMR analysis showed that the solution contains about 6 mol% of remaining toluene after the solvent exchange.

Otopina se hladi do 68°C i cijedi kroz 4 g kristalnog CBZ-indolina 9. Otopina se pusti postupno hladiti do 26°C tijekom 6 h i potom stoji 9 h na 20-26°C. Smjesa se hladi do 2°C tijekom 1 sat i potom stoji na 2°C tijekom 1 h. Proizvod se izdvoji filtriranjem, a filterski se kolač ispire s 2 x 2 L IPAC temperature 5°C i 2 x 2 L MTBE temperature 5°C. Proizvod se zatim suši u vakuumskoj peći na 30°C uz protok dušika, čime se dobije 4.37 kg (74%) naslovnog spoja 9 u obliku blijedo žutosmeđeg kristalnog praška. HPLC analiza spoja pokazala je 99.5 površ.%-tnu čistoću. Matična tekućina (11 L) i ispirci sadržavali su 1.15 kg (19%) dodatnog proizvoda 9 i približno 3% hidrazida CBZ-izonipekotične kiseline (retencijsko vrijeme = 4.8 min.). The solution is cooled to 68°C and strained through 4 g of crystalline CBZ-indoline 9. The solution is allowed to gradually cool to 26°C for 6 h and then stands for 9 h at 20-26°C. The mixture is cooled to 2°C for 1 hour and then stands at 2°C for 1 hour. The product is separated by filtration, and the filter cake is washed with 2 x 2 L IPAC temperature 5°C and 2 x 2 L MTBE temperature 5°C. The product is then dried in a vacuum oven at 30°C with nitrogen flow, which gives 4.37 kg (74%) of the title compound 9 in the form of a pale tan crystalline powder. HPLC analysis of the compound showed 99.5 surface% purity. The mother liquor (11 L) and washings contained 1.15 kg (19%) of additional product 9 and approximately 3% of CBZ-isonipecotic acid hydrazide (retention time = 4.8 min.).

Primjer 5 Example 5

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CBZ-spiroindolin-metansulfonamid (1) CBZ-spiroindoline-methanesulfonamide (1)

Tvari: Substances:

CBZ-spiroindolin (9) 1.69 kg (5.23 mol) CBZ-spiroindoline (9) 1.69 kg (5.23 mol)

metansulfonil klorid 599 g (5.23 mol) methanesulfonyl chloride 599 g (5.23 mol)

Et3N (KF = 151) 635 g (6.27 mol) Et3N (KF = 151) 635 g (6.27 mol)

THF (KF = 41) 12 L THF (KF = 41) 12 L

Posuda od 22 litre napuni se krutim CBZ-spiroindolinom 9 i potom se u posudu prenesu 11.5 L THF i Et3N kroz 10 μ inline filter. Dobivena homogena otopina ohladi se na 0°C. Lijevak s nakapnicom od jedne litre napuni se s metansulfonil kloridom i s 500 mL THF. Otopina MsCl u THF doda se reakcijskoj smjesi uz održavanje temperature između 0 i 4°C. Dodavanje traje 5 h i egzotermično je. Bijeli precipitat, vjerojatno trietilamonij hidroklorid, nastaje tijekom dodavanja. HPLC analiza pokazala je dovršenost reakcije na kraju dodavanja (spoj 9 nije se mogao dokazati). A 22 liter vessel was filled with solid CBZ-spiroindoline 9 and then 11.5 L of THF and Et3N were transferred to the vessel through a 10 μ inline filter. The obtained homogeneous solution is cooled to 0°C. A one-liter stoppered funnel was charged with methanesulfonyl chloride and 500 mL of THF. A solution of MsCl in THF was added to the reaction mixture while maintaining the temperature between 0 and 4°C. The addition lasts 5 h and is exothermic. A white precipitate, probably triethylammonium hydrochloride, forms during the addition. HPLC analysis showed completion of the reaction at the end of the addition (compound 9 could not be demonstrated).

Uvjeti HPLC: 25 cm Dupont Zorbax , RXC8 stupac sa protokom 1.5 mL/min i mjerenjem pri 254 nm. Tablica gradijenata: HPLC conditions: 25 cm Dupont Zorbax, RXC8 column with a flow rate of 1.5 mL/min and measurement at 254 nm. Table of gradients:

vrijeme (min.) 0.1%-tni vod. H3PO4:MeCN time (min.) 0.1% lead. H3PO4:MeCN

0 70:30 0 70:30

3 70:30 3 70:30

12 20:80 12 20:80

25 20:80 25 20:80

Vremena retencije: 9 = 7.6 min., 1 = 13.6 min. Retention times: 9 = 7.6 min., 1 = 13.6 min.

Nakon završetka dodavanja, reakcijska se smjesa zagrije do 18°C i ostavi stajati tijekom 16 sati. Nije bilo promjene u izgledu reakcijske smjese i HPLC profilu između stanja nakon završetka dodavanja i nakon 16 sati stajanja. Reakcijska se smjesa polako, tijekom jednog sata, prenese u snažno miješanu otopinu 30 L vode i 200 mL 37%-tne HCl u 50-litrenoj posudi. Temperatura u 50-litrenoj posudi naraste pritom s 22 na 28°C. Proizvod se izdvoji kao blijedo žutosmeđa gumasta kruta tvar koja prijeđe u zrnatu krutu tvar. Vodena suspenzija hladi se na 22°C i ostavi stajati 1 h. Suspezija se filtrira i filterski se kolač ispire s 2 x 4 L MeOH/vode (50/50). HPLC aanaliza pokazala je nazočnost <0.1% CBZ-spiroindolin-metansulfonamida 1 u matičnim otopinama. After the addition is complete, the reaction mixture is heated to 18°C and left to stand for 16 hours. There was no change in the appearance of the reaction mixture and the HPLC profile between the conditions after the addition was complete and after 16 hours of standing. The reaction mixture is slowly, over the course of one hour, transferred into a vigorously stirred solution of 30 L of water and 200 mL of 37% HCl in a 50-liter container. The temperature in the 50-liter container rises from 22 to 28°C. The product separates out as a pale tan gummy solid that turns into a granular solid. The aqueous suspension is cooled to 22°C and left to stand for 1 h. The suspension was filtered and the filter cake was washed with 2 x 4 L MeOH/water (50/50). HPLC analysis showed the presence of <0.1% CBZ-spiroindoline-methanesulfonamide 1 in the mother solutions.

Filterski kolač se ispire s 4 L MeOH/vode (50/50) kojoj je dodano 50 mL 28%-tnog vodenog NH4OH. Filterski kolač ispire se s 2 x 4 L MeOH/vode (50/50), a kruta tvar se suši u vakuumskoj peći na 50°C u strujanje dušika, čime se dobije 2.03 kg (97%) naslovnog proizvoda 1 u obliku prljavobijelog praška. HPLC analiza krute tvari dala je vrijednost od 93.7 površ.% 1. The filter cake was washed with 4 L of MeOH/water (50/50) to which 50 mL of 28% aqueous NH4OH was added. The filter cake is washed with 2 x 4 L MeOH/water (50/50) and the solid is dried in a vacuum oven at 50°C in a stream of nitrogen to give 2.03 kg (97%) of the title product 1 as an off-white powder. . HPLC analysis of the solid substance gave a value of 93.7 surface% 1.

Primjer 6 Example 6

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Mogući postupak izolacije međuspoja CBZ-spiroindolenina (8) A possible procedure for the isolation of the CBZ-spiroindolenine intermediate (8)

Tvari: Substances:

piperidin-4-karboksaldehid-l-benzil 12.37 g (0.050 mol) Piperidine-4-carboxaldehyde-1-benzyl 12.37 g (0.050 mol)

karbamat (5) carbamate (5)

fenilhidrazin 5.41 g (0.050 mol) phenylhydrazine 5.41 g (0.050 mol)

trifluoroctena kiselina (TFA) 11.56 mL, 17.10 g (0.150 mol) trifluoroacetic acid (TFA) 11.56 mL, 17.10 g (0.150 mol)

metilen klorid 500 mL methylene chloride 500 mL

CBZ-aldehid 5 otopi se u diklormetanu u posudi od jedne litre opremljenoj magnetskim miješalom obloženim teflonom. Dobivena se otopina hladi na 0°C. Putem izvagane štrcaljke dodaje se fenilhidrazin tijekom 5 minuta uz održavanje temperature na -1 do 3°C uz stalni protok mjehurića dušika kroz reakcijsku smjesu. CBZ-aldehyde 5 was dissolved in dichloromethane in a one-liter vessel equipped with a Teflon-lined magnetic stirrer. The resulting solution is cooled to 0°C. Phenylhydrazine is added via a weighed syringe for 5 minutes while maintaining the temperature at -1 to 3°C with a constant flow of nitrogen bubbles through the reaction mixture.

TLC i HPLC analize pokazale su potpunu potrošenost CBZ-aldehida 5 i pojavu laganog viška (<2%) fenilhidrazina. Uvjeti TLC: Silica, E. Merck Kieselgel G60 F254 0.25 mm; dietileter/pentan (4/1); 0.5%-tni cerij sulfat kao sredstvo za razvijanje, 14%-tni amonij molibdat u 10%-tnoj vodenoj sumpornoj kiselini, potom zagrijavanje; Rf: aldehid 5 = 0.52, fenilhidrazon 7 = 0.61, fenilhidrazin 6 = 0.21. Uvjeti HPLC: 25 cm Dupont Zorbax RXC8 stupac, na 30°C, sa protokom 1 mL/min i mjerenjem pri 254 nm; tablica gradijenata: TLC and HPLC analyzes showed the complete consumption of CBZ-aldehyde 5 and the appearance of a slight excess (<2%) of phenylhydrazine. TLC conditions: Silica, E. Merck Kieselgel G60 F254 0.25 mm; diethylether/pentane (4/1); 0.5% cerium sulfate as developer, 14% ammonium molybdate in 10% aqueous sulfuric acid, then heating; Rf: aldehyde 5 = 0.52, phenylhydrazone 7 = 0.61, phenylhydrazine 6 = 0.21. HPLC conditions: 25 cm Dupont Zorbax RXC8 column, at 30°C, with a flow rate of 1 mL/min and measurement at 254 nm; gradient table:

vrijeme (min.) acetonitril:voda time (min.) acetonitrile:water

0 57:43 0 57:43

10 65:35 10 65:35

15 75:25 15 75:25

18 75:25 18 75:25

vremena retencije: fenilhidrazin 6-4.5 min, toluen = 7.2 min, fenilhidrazon 7 = 11.4 min. retention times: phenylhydrazine 6-4.5 min, toluene = 7.2 min, phenylhydrazone 7 = 11.4 min.

Reakcijska se smjesa ostavi stajati tijekom 10 minuta na 0-2°C, nakon čega se štrcaljkom doda TFA uz održavanje temperature između 2 i 7°C. Reakcijska se smjesa zagrijava na 35°C tijekom 30 min. i održava na toj temperaturi 17 sati. Zaustavi se protok dušika kroz reakcijsku smjesu i uspostavi se slabo strujanje dušika iznad reakcijske smjese. Tijekom prvog sata na 35°C boja smjese postupno tamni, od ružičaste do tamnozelene, i oblikuje se relativno mala količina bijelog kristalnog precipitata (amonij trifluoroacetat). Nakon 17 sati, HPLC analiza (uz iste uvjete kao gore) pokazuje da reakcijska smjesa sadrži 93 površ.% indolenina 8 i <0.5% nereagiranog ostatka fenilhidrazona. Stajanje smjese tijekom duljeg razdoblja ne povećava prinos indolenina 8. Reakcijska se smjesa hladi na 10°C i dodaje se smjesa koja se sastoji od 60 mL 28-30%-tnog amonij hidroksida, 90 mL vode i 150 g smrvljenog leda; tijekom dodavanja smjesa se dobro miješa. Boja smjese mijenja se u boju lososa (narančastoljubičasta). Organska se faza odijeli i dvaput ispere s 400 mL vode, potom sa 100 mL zasićenog vodenog NaCl. Organska se faza suši preko magnezij sulfata i filtrira kroz "jastučić" od 5 g silike. Filtrat se ispari i time se dobije 15.84 g (99%) indolenina 8 u obliku blijedonarančastog ulja. The reaction mixture is left to stand for 10 minutes at 0-2°C, after which TFA is added with a syringe while maintaining the temperature between 2 and 7°C. The reaction mixture is heated to 35°C for 30 min. and keeps it at that temperature for 17 hours. Stop the flow of nitrogen through the reaction mixture and establish a weak flow of nitrogen above the reaction mixture. During the first hour at 35°C, the color of the mixture gradually darkens, from pink to dark green, and a relatively small amount of white crystalline precipitate (ammonium trifluoroacetate) forms. After 17 hours, HPLC analysis (with the same conditions as above) shows that the reaction mixture contains 93 surface% of indolenine 8 and <0.5% of unreacted phenylhydrazone residue. Standing the mixture for a longer period does not increase the yield of indolenine 8. The reaction mixture is cooled to 10°C and a mixture consisting of 60 mL of 28-30% ammonium hydroxide, 90 mL of water and 150 g of crushed ice is added; while adding the mixture is mixed well. The color of the mixture changes to the color of salmon (orange-purple). The organic phase is separated and washed twice with 400 mL of water, then with 100 mL of saturated aqueous NaCl. The organic phase is dried over magnesium sulfate and filtered through a "pad" of 5 g of silica. The filtrate was evaporated to give 15.84 g (99%) of indolenine 8 in the form of a pale orange oil.

Primjer 7 Example 7

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Postupak priprave CBZ-spiroindolin-metansulfonamida (1) bez izolacije međuspoja CBZ-spiroindolina (9) Procedure for the preparation of CBZ-spiroindoline-methanesulfonamide (1) without isolation of the intermediate compound CBZ-spiroindoline (9)

Korak 1: CBZ-spiroindolin (9) Step 1: CBZ-spiroindoline (9)

Tvari: Substances:

piperidin-4-karboksaldehid-l-benzil karbamat (5) 49.5 g (0.20 mol) Piperidine-4-carboxaldehyde-1-benzyl carbamate (5) 49.5 g (0.20 mol)

fenilhidrazin (Aldrich) 23.7 g (0.22 mol) phenylhydrazine (Aldrich) 23.7 g (0.22 mol)

trifluoroctena kiselina (TFA) 75.4 g (0.66 mol) trifluoroacetic acid (TFA) 75.4 g (0.66 mol)

toluen (KF<250 mg/L) 654 mL toluene (KF<250 mg/L) 654 mL

MeCN (KF<250 mg/L) 13.3 mL MeCN (KF<250 mg/L) 13.3 mL

NaBH4 11.3 g (0.30 mol) NaBH4 11.3 g (0.30 mol)

toluen 20 mL toluene 20 mL

MeOH 50 mL MeOH 50 mL

2%-tna (vol. udio) otopina MeCN u toluenu dobije se miješanjem 654 mL toluena i 13.3 mL MeCN. U dvolitrenoj posudi s tri grla, opremljenoj mehaničkim miješalom, iz 617 mL gore spomenute otopine ukloni se plin pomoću finog strujanja dušika kroz otopinu tijekom 5 min. Fenilhidrazin i TFA dodaju se u smjesu još za vrijeme uklanjanja plina. A 2% (vol. fraction) solution of MeCN in toluene is obtained by mixing 654 mL of toluene and 13.3 mL of MeCN. In a two-liter, three-necked vessel equipped with a mechanical stirrer, 617 mL of the above-mentioned solution is degassed using a fine flow of nitrogen through the solution for 5 min. Phenylhydrazine and TFA are added to the mixture during degassing.

CBZ-aldehid 5 otopi se u ostatku gore pripremljene otopine (50 mL) i vrši se uklanjanje plina puštanjem mjehurića dušika kroz otopinu dok se ona nalazi u lijevku za dodavanje. Otopina u posudi zagrije se do 35°C, pa se u fenilhidrazin/TFA tijekom dva sata polako dodaje otopina aldehida. Smjesa se ostavi stajati na 35°C tijekom 16 h. CBZ-aldehyde 5 is dissolved in the rest of the solution prepared above (50 mL) and the gas is removed by blowing nitrogen bubbles through the solution while it is in the addition funnel. The solution in the vessel is heated to 35°C, and the aldehyde solution is slowly added to the phenylhydrazine/TFA over two hours. The mixture is left to stand at 35°C for 16 h.

Uvjeti HPLC: 25 cm Dupont Zorbax RXC8 stupac, na 50°C, na 50°C, sa protokom 1 mL/min i mjerenjem pri 220 nm; izokratični 55% MeCN, 45% 0.1%-tnog vod. H3PO4. Tipični HPLC profil nakon 16 sati stajanja: HPLC conditions: 25 cm Dupont Zorbax RXC8 column, at 50°C, at 50°C, with a flow rate of 1 mL/min and measurement at 220 nm; isocratic 55% MeCN, 45% 0.1% aq. H3PO4. Typical HPLC profile after 16 hours of standing:

vrijeme retencije (min.) površ.% spoj retention time (min.) surface% compound

1.6 0.1-0.5 fenilhidrazin 6 1.6 0.1-0.5 phenylhydrazine 6

4.1 <0.1 dimer 21 4.1 <0.1 dimer 21

4.7 <0.1 aldehid 5 4.7 <0.1 aldehyde 5

5.0 NA spiroindolin 9 5.0 NA spiroindoline 9

6.3 NA toluen 6.3 NA toluene

6.9 97 spiroindolenin 8 6.9 97 spiroindolenine 8

10.3 <0.2 fenilhidrazon 7 10.3 <0.2 phenylhydrazone 7

2-3 uk. druga onečišćenja <0.2%ea. 2-3 incl. other impurities <0.2%ea.

Smjesa se hladi na -10°C i dodaje se MeOH. Potom se, u malim obrocima, tijekom 30 minuta dodaje suspenzija natrij borohidrida u 20 mL toluena, pazeći pritom da temperatura ne prijeđe -2°C. The mixture is cooled to -10°C and MeOH is added. Then, in small portions, a suspension of sodium borohydride in 20 mL of toluene is added over 30 minutes, making sure that the temperature does not exceed -2°C.

površ.% spoj surface % compound

0.1-1 fenilhidrazin 6 0.1-1 phenylhydrazine 6

85-90 CBZ-spiroindolin 9 85-90 CBZ-spiroindoline 9

<0.1 CBZ-spiroindolenin 8 <0.1 CBZ-spiroindolenine 8

10-15 uk. druga onečišćenja (<3%ea.) 10-15 incl. other impurities (<3%ea.)

Temperatura naraste na 10°C tijekom 1h, pa se doda 6%-tni vodeni amonijak (200 mL). Smjesa se miješa 10 minuta, ostavi se da se slegne slijedećih 10 minuta, nakon čega se donja vodena faza odstranjuje. Organskoj se fazi dodaju acetonitril (20 mL) i MeOH (20 mL), potom se ona ispire sa 150 mL 15%-tne otopine soli. Za organsku se fazu nađe da sadrži 92% prinosa CBZ-spiroindolina 9. The temperature rises to 10°C for 1 hour, then 6% aqueous ammonia (200 mL) is added. The mixture is stirred for 10 minutes, allowed to settle for another 10 minutes, after which the lower aqueous phase is removed. Acetonitrile (20 mL) and MeOH (20 mL) are added to the organic phase, then it is washed with 150 mL of 15% salt solution. The organic phase was found to contain 92% yield of CBZ-spiroindoline 9.

Korak 2: CBZ-spiroindolin-metansulfonamid (1) Step 2: CBZ-spiroindoline-methanesulfonamide (1)

Tvari: Substances:

CBZ-spiroindolin (9) (MT = 322.51) (0.184 mol) CBZ-spiroindoline (9) (MT = 322.51) (0.184 mol)

metansulfonil klorid 21.1 g (0.184 mol) methanesulfonyl chloride 21.1 g (0.184 mol)

DIEA (KF=150 mg/L) 29.7 g, 40.1 mL (0.230 mol) DIEA (KF=150 mg/L) 29.7 g, 40.1 mL (0.230 mol)

THF (KF = 41 mg/L) 150 mL THF (KF = 41 mg/L) 150 mL

Sirova otopina CBZ-spiroindolina 9 iz Koraka 1 koncentrira se u jednolitrenoj posudi s tri grla (60-70°C, 150-200 Torr) dok ne ostane 250 g ostatne tvari. Tada se dodaju THF i DIEA, a dobivena homogena otopina ohladi se na 0°C. Lijevak s kapaljkom od 125 mL napuni se metansulfonil kloridom i 50 mL THF. Tijekom 2 sata u reakcijsku se smjesu dodaje MsCl u THF uz održavanje temperature između 0 i 4°C, nakon čega se smjesa ostavi stajati tijekom 2 sata na 5-8°C. Dodavanje je blago egzotermno. Bijeli precipitat, vjerojatno DIEA-hidroklorid, nastaje tijekom dodavanja. Uvjeti dodavanja bili su isti kao i ranije opisani. HPLC analiza pokazala je dovršenost reakcije 1 sat nakon završetka dodavanja (spoj 9 nije se mogao dokazati) uz prinos spoja 9 od 94%. Vrijeme retencije: 1 = 7.8 min. Tipični HPLC profil reakcijske smjese nakon dvosatnog stajanja: The crude solution of CBZ-spiroindoline 9 from Step 1 is concentrated in a one-liter three-necked vessel (60-70°C, 150-200 Torr) until 250 g of residue remains. Then THF and DIEA are added, and the resulting homogeneous solution is cooled to 0°C. A 125 mL dropper funnel was filled with methanesulfonyl chloride and 50 mL THF. During 2 hours, MsCl in THF is added to the reaction mixture while maintaining the temperature between 0 and 4°C, after which the mixture is left to stand for 2 hours at 5-8°C. The addition is slightly exothermic. A white precipitate, probably DIEA-hydrochloride, forms during the addition. Addition conditions were the same as previously described. HPLC analysis showed completion of the reaction 1 hour after the end of the addition (compound 9 could not be demonstrated) with a yield of compound 9 of 94%. Retention time: 1 = 7.8 min. Typical HPLC profile of the reaction mixture after standing for two hours:

površ.% spoj surface % compound

<0.1 CBZ-spiroindolin 9 <0.1 CBZ-spiroindoline 9

90-92 CBZ-sulfonamid 1 90-92 CBZ-sulfonamide 1

8-10 uk. druga onečišćenja (<2% ea.) 8-10 incl. other impurities (<2% ea.)

Smjesa se zagrije na 20°C i doda se 200 mL 1M vodene HCl. Smjesa se zagrije do 50°C i vodena se faza odvoji. Organska se faza ispire redom' sa: 100 mL vode, 100 mL 5%-tnog vodenog natrij bikarbonata i 100 mL vode. Organska se faza prenosi u jednolitrenu posudu s tri grla, opremljenu mehaničkim miješalom i destilatorom. Smjesa (oko 400 mL) se destilira pri atmosferskom tlaku dok se ne prikupi 150 mL destilata. Temperatura glave pritom dostiže 107°C, uz temperaturu posude od 110°C. Destilacija se nastavi uz stalno dodavanje n-propanola brzinom koja će omogućiti stalan volumen (oko 350 mL) u posudi. Destilacija se prekida kada se doda ukupno 525 mL n-PrOH i prikupi ukupno 800 mL destilata. The mixture is heated to 20°C and 200 mL of 1M aqueous HCl is added. The mixture is heated to 50°C and the aqueous phase is separated. The organic phase is washed in order with: 100 mL of water, 100 mL of 5% aqueous sodium bicarbonate and 100 mL of water. The organic phase is transferred to a one-liter vessel with three necks, equipped with a mechanical stirrer and distiller. The mixture (about 400 mL) is distilled at atmospheric pressure until 150 mL of distillate is collected. The temperature of the head reaches 107°C, with a bowl temperature of 110°C. Distillation continues with the constant addition of n-propanol at a rate that will allow a constant volume (about 350 mL) in the vessel. Distillation is stopped when a total of 525 mL of n-PrOH is added and a total of 800 mL of distillate is collected.

Temperatura glave i posude naraste tijekom izmjene otapala sa 94 na 98°C. Toluen i n-PrOH dovode do azeotropnog vrenja na 97.2°C, koje se sastoji od 47.5% toluena i 52.5% n-PrOH. Smjesa se ostavi postupno hladiti do 20°C tijekom 3 h, a potom stoji 12 h. U matičnoj tekućini nađen je 2%-tni sadržaj toluena i 4 mg/mL sulfonamida. Topljivost sulfonamida u različitim smjesama toluena i n-PrOH određena je HPLC analizom: The temperature of the head and vessel rises during the solvent exchange from 94 to 98°C. Toluene and n-PrOH lead to an azeotropic boiling at 97.2°C, which consists of 47.5% toluene and 52.5% n-PrOH. The mixture is allowed to gradually cool to 20°C for 3 hours, and then it stands for 12 hours. A 2% content of toluene and 4 mg/mL sulfonamide was found in the mother liquor. The solubility of sulfonamides in different mixtures of toluene and n-PrOH was determined by HPLC analysis:

%-tak toluena u n-PrOH topljivost spoja 1 u mg/mL % of toluene in n-PrOH solubility of compound 1 in mg/mL

0 2.36 0 2.36

5 3.02 5 3.02

10 4.23 10 4.23

20 7.51 20 7.51

25 10.3 25 10.3

Kristalna se smjesa filtrira i ispire s 3 x 100 mL n-PrOH. Proizvod se suši u vakuumskoj peći na 50°C uz strujanje dušika tijekom 16 sati čime se dobije 65.5 g (82% od aldehida 5) spoja 6 kao žutosmeđe krute tvari čistoće oko 93.5 mas.%. The crystalline mixture is filtered and washed with 3 x 100 mL of n-PrOH. The product is dried in a vacuum oven at 50°C with a stream of nitrogen for 16 hours, which gives 65.5 g (82% of aldehyde 5) of compound 6 as a tan solid with a purity of about 93.5 wt.%.

Tipični HPLC profil krute tvari: A typical HPLC profile of a solid:

površ.% spoj surface % compound

<0.1 CBZ-spiroindolin 9 <0.1 CBZ-spiroindoline 9

>99 CBZ-sulfonamid 1 >99 CBZ-sulfonamide 1

<1 uk. druga onečišćenja (<0.2% ea.) <1 incl. other impurities (<0.2% ea.)

Radi dodatnog pročišćavanja, uzorak od 40.0 g n-PrOH kristaliziranog sulfonamida otopi se u 134 mL EtOAc na 60°C i izloži djelovanju 8.0 g Darco G-60 ugljika tijekom 1 sat na 60°C. Nakon dodavanja 2.0 g Solkafloc™, smjesa se filtrira kroz "jastučić" od 4.0 g Solkafloc™ i "jastučić" se ispire s 90 mL EtOAc na 60°C. Prije dodavanja ugljika, boja otopine je smeđa. Filtriranje je prošlo dobro i bez zastoja, a njime se dobio zlatnožuti filtrat. Filtrat se destilira pri atmosferskom tlaku u posudi od 500 mL (uz temperaturu posude od 80-85°C) dok ne preostane 100 g (100 mL) ostatne tvari. Ta se otopina ostavi ohladiti do 35°C tijekom 3 sata. Nakon jednosatnog razdoblja, dodaje se 116 mL cikloheksana, uz dobro miješanje, na 35°C. Smjesa se hladi na 20°C tijekom 1 h i ostavi stajati na 20°C tijekom 12 h. Na temperaturi od 35°C kristalizirao je velik dio sulfonamida, pa smjesa postaje gusta. Dodavanje cikloheksana na 20°C otežava miješanje. Nakon razdoblja stajanja, nađeno je da nadtalog sadrži 2.5 mg spoja l/g. Kristalna se smjesa filtrira i kolač se ispire sa 77 mL cikloheksan-EtOAc u omjeru 2:1 i s 2 x 77 mL cikloheksana. Proizvod se suši u vakuumskoj peći na 50°C uz strujanje dušika tijekom 16 sati, čime se dobije 34.2 g spoja 1 (MT = 400.3) u obliku bijele kristalne krute tvari (85%-tni povrat iz sirovog spoja 1, 70%-tni iz spoja 5, uz >99.9 mas.%-tnu čistoću). For additional purification, a 40.0 g sample of n-PrOH crystallized sulfonamide was dissolved in 134 mL of EtOAc at 60°C and treated with 8.0 g of Darco G-60 carbon for 1 hour at 60°C. After addition of 2.0 g Solkafloc™, the mixture is filtered through a 4.0 g Solkafloc™ "pad" and the "pad" is washed with 90 mL EtOAc at 60°C. Before adding carbon, the color of the solution is brown. The filtration went well and without any interruptions, and a golden yellow filtrate was obtained. The filtrate is distilled at atmospheric pressure in a 500 mL vessel (at a vessel temperature of 80-85°C) until 100 g (100 mL) of the residual substance remains. This solution is allowed to cool to 35°C for 3 hours. After a one-hour period, 116 mL of cyclohexane is added, with good stirring, at 35°C. The mixture is cooled to 20°C for 1 h and left to stand at 20°C for 12 h. At a temperature of 35°C, a large part of the sulfonamide crystallized, so the mixture becomes thick. Addition of cyclohexane at 20°C makes mixing difficult. After a standing period, the supernatant was found to contain 2.5 mg of the compound l/g. The crystalline mixture is filtered and the cake is washed with 77 mL of cyclohexane-EtOAc in a ratio of 2:1 and with 2 x 77 mL of cyclohexane. The product is dried in a vacuum oven at 50°C with nitrogen flow for 16 hours, which gives 34.2 g of compound 1 (MT = 400.3) in the form of a white crystalline solid (85% recovery from crude compound 1, 70% from compound 5, with >99.9 wt.% purity).

Primjer 8 Example 8

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HCl sol spiroindolin-metansulfonamida (1a) HCl salt of spiroindoline-methanesulfonamide (1a)

Tvari: Substances:

CBZ-spiroindolin-metansulfonamid (1) 941 g (2.35 mol) CBZ-spiroindoline-methanesulfonamide (1) 941 g (2.35 mol)

Pearlmanov katalizator 20% 188 g Pearlman catalyst 20% 188 g

Pd(OH)2/C Pd(OH)2/C

THF 8 L THF 8 L

MeOH 7 L MeOH 7 L

Katalizator se suspendira u 7 L MeOH i prenese u 5-galonski autoklav, a nakon njega se doda otopina spoja 1 u 8 L THF. Smjesa se lizira vodikom na 25°C pri 80 psi H2. Nakon 2.5 h temperatura se digne na 35°C tijekom 30 min. The catalyst was suspended in 7 L MeOH and transferred to a 5-gallon autoclave, followed by the addition of a solution of compound 1 in 8 L THF. The mixture is lysed with hydrogen at 25°C at 80 psi H 2 . After 2.5 h the temperature rises to 35°C for 30 min.

HPLC analiza pokazala je potpuni nestanak CBZ-spiroindolin-metansulfonamida. Uvjeti HPLC: : 25 cm Dupont Zorbax RXC8 stupac sa protokom 1.5 mL/min i mjerenjem pri 254 nm. Tablica gradijenata: HPLC analysis showed complete disappearance of CBZ-spiroindoline-methanesulfonamide. HPLC conditions: : 25 cm Dupont Zorbax RXC8 column with a flow rate of 1.5 mL/min and measurement at 254 nm. Table of gradients:

vrijeme (min.) 0.1%-tni vod. H3PO4: MeCN time (min.) 0.1% lead. H3PO4: MeCN

0 70:30 0 70:30

3 70:30 3 70:30

12 20:80 12 20:80

25 20:80 25 20:80

Vremena retencije: spiroindolin = 7.6 min., Retention times: spiroindoline = 7.6 min.,

CBZ-spiroindolin-metansulfonamid = 13.6 min. CBZ-spiroindoline-methanesulfonamide = 13.6 min.

Smjesa se pročisti dušikom, a katalizator se ukloni filtracijom kroz Solka-floc™ dok je smjesa još topla. Katalizator se ispire s 4 L THF i 2 L MeOH. Blijedožuti filtrati se koncentriraju pri 10 mbar i <25°C dok ne dobiju izgled gustog ulja. Izmjena otapala dovrši se polaganim ukapavanjem 15 L EtOAc i ponovnim koncentriranjem do suhoće. Ostatna se tvar skrutne do tvrde prljavobijele mase. Doda se MeOH (1.5 L) i smjesa se zagrije do 70°C da bi nastala homogena otopina. Dok je temperatura otopine 70°C, doda se 10.5 L EtOAc čija je temperatura 20°C. Temperatura pada do 40°C i smjesa ostaje homogena. The mixture is purged with nitrogen, and the catalyst is removed by filtration through Solka-floc™ while the mixture is still warm. The catalyst is washed with 4 L THF and 2 L MeOH. The pale yellow filtrates are concentrated at 10 mbar and <25°C until they have the appearance of a thick oil. The solvent exchange was completed by slowly dropping 15 L of EtOAc and reconcentrating to dryness. The remaining substance solidifies to a hard off-white mass. MeOH (1.5 L) was added and the mixture was heated to 70°C to form a homogeneous solution. While the temperature of the solution is 70°C, 10.5 L of EtOAc whose temperature is 20°C is added. The temperature drops to 40°C and the mixture remains homogeneous.

Naknadni pokusi ukazuju da je pogodnije provesti izmjenu otapala MeOH-THF filtrata u MeOH, koncentrirati željeni volumen i onda dodati EtOAc. Ovim se redoslijedom izbjegava skrućivanje ostatne tvari nakon koncentriranja EtOAc otopine. Subsequent experiments indicate that it is more convenient to carry out a solvent exchange of the MeOH-THF filtrate to MeOH, concentrate to the desired volume and then add EtOAc. This order avoids solidification of the residual material after concentration of the EtOAc solution.

U otopinu se pušta klorovodik razrijeđen približno jednakim volumenom dušika. Temperatura raste na 60°C tijekom 15 minuta i oblikuje se bijeli precipitat klorovodične soli. Razrjeđivanje HCl dušikom služi samo da bi se izbjeglo povratno usisavanje reakcijske smjese i nije neophodno. Hydrogen chloride diluted with an approximately equal volume of nitrogen is released into the solution. The temperature rises to 60°C during 15 minutes and a white precipitate of the hydrochloric salt is formed. Dilution of HCl with nitrogen serves only to avoid back-suction of the reaction mixture and is not necessary.

Smjesa se hladi u ledenoj kupelji, a dodavanje vodika nastavlja se tijekom 1 h. Temperatura postupno pada do 20°C. Suspenzija se ostavi stajati 2 h uz pad temperature na 10°C. Kristalni proizvod izolira se filtriranjem, a filterski se kolač ispire s 3L EtOAc. Potom se suši u vakuumskoj peći na 35°C čime se dobije 1.18 kg (86%) proizvoda la kao prljavobijele kristalne krute tvari čistoće >99.5 površ.% prema HPLC analizi. Uvjeti HPLC: Dupont Zorbax 25 cm RXC8 stupac sa protokom 1.5 mL/min i mjerenjem pri 230 nm; izokratična smjesa s 35% MeCN, 65% 0.1%-tnog vodenog amonij acetata. Vrijeme retencije: 1a = 5.4 min. The mixture is cooled in an ice bath, and the addition of hydrogen continues for 1 h. The temperature gradually drops to 20°C. The suspension is left to stand for 2 h with the temperature dropping to 10°C. The crystalline product is isolated by filtration, and the filter cake is washed with 3 L of EtOAc. It is then dried in a vacuum oven at 35°C, resulting in 1.18 kg (86%) of product la as an off-white crystalline solid with purity >99.5 surface% according to HPLC analysis. HPLC conditions: Dupont Zorbax 25 cm RXC8 column with a flow rate of 1.5 mL/min and measurement at 230 nm; isocratic mixture with 35% MeCN, 65% 0.1% aqueous ammonium acetate. Retention time: 1a = 5.4 min.

Primjer 9 Example 9

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Spiroindolin-metansulfonamid (oblik slobodne baze) (1b) Spiroindoline-methanesulfonamide (free base form) (1b)

Alikvot od 250 mL filtrata CBZ-hidrogenolize koji sadrži 4.67 g 1b (slobodne baze) koncentrira se do približno 10 mL. Ostatak se otopi u 20 mL EtOAc i otopina se ponovno koncentrira do približno 10 mL. Postupak se još jedamput ponovi, i ostatnoj se tvari doda 10 mL EtOAc. Počinje se oblikovati kristalni precipitat. Odjednom se doda MTBE (20 mL). Precipitira se dodatna kristalna kruta tvar, ali nadtalog i dalje sadrži znatnu količinu otopljenog proizvoda koji stajanjem ne precipitira. Smjesi se ukapavanjem , dodaju heksani (70 mL) tijekom 2 sata, uz snažno miješanje. Postupno dodavanje heksana je nužno da bi se izbjeglo zauljivanje amina. An aliquot of 250 mL of CBZ-hydrogenolysis filtrate containing 4.67 g of 1b (free base) is concentrated to approximately 10 mL. The residue was dissolved in 20 mL of EtOAc and the solution was re-concentrated to approximately 10 mL. The procedure is repeated once more, and 10 mL of EtOAc is added to the residue. A crystalline precipitate begins to form. MTBE (20 mL) was added at once. An additional crystalline solid is precipitated, but the supernatant still contains a considerable amount of dissolved product which does not precipitate on standing. The mixture is added dropwise, hexanes (70 mL) are added over 2 hours, with vigorous stirring. The gradual addition of hexane is necessary to avoid oiling of the amine.

Promiješana smjesa ostavi se stajati 1 sat i potom se filtrira. Filterski se kolač ispire s 20 mL 1:1 MTBE-heksana i potom s 20 mL heksana. Proizvod se suši uz strujanje dušika i tako se dobije 3.86 g (82%) slobodnog amina lb u obliku prljavobijele kristalne čvrste tvari čistoće >99.5 površ.%. Uvjeti HPLC: Dupont Zorbax 25 cm RXC8 stupac sa protokom 1.5 mL/min i mjerenjem pri 230 nm; izokratična smjesa s 35% MeCN, 65% 0.1%-tnog vodenog amonij acetata. Vrijeme retencije: 1b = 5.4 min. The mixed mixture is left to stand for 1 hour and then filtered. The filter cake is washed with 20 mL of 1:1 MTBE-hexane and then with 20 mL of hexane. The product is dried under a stream of nitrogen and thus 3.86 g (82%) of free amine lb is obtained in the form of an off-white crystalline solid with a purity of >99.5 surface %. HPLC conditions: Dupont Zorbax 25 cm RXC8 column with a flow rate of 1.5 mL/min and measurement at 230 nm; isocratic mixture with 35% MeCN, 65% 0.1% aqueous ammonium acetate. Retention time: 1b = 5.4 min.

Primjer 10A Example 10A

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Spiroindolin-metansulfonamid (oblik slobodne baze) (1b) Spiroindoline-methanesulfonamide (free base form) (1b)

Tvari: Substances:

CBZ-spiroindolin-sulfonamid (1) 833.5 g (2.08 mol) CBZ-spiroindoline-sulfonamide (1) 833.5 g (2.08 mol)

Pd(OH)2/C (20% tež. Pd(OH)2) 124.5 (15%) Pd(OH)2/C (20% wt. Pd(OH)2) 124.5 (15%)

THF 6.5 L THF 6.5 L

MeOH 19.5 L MeOH 19.5 L

NH4OH (konc.) 60 mL NH4OH (conc.) 60 mL

Hidrogenacija se provodi tri (3) puta zbog ograničene opreme; ovaj se postupak odnosi na jedan ciklus. CBZ-spiroindolin sulfonamid 1 otopi se u THF (6.5 L, KF = 53 μg/uL), potom se doda MeOH (KF=18 μg/mL, 4 L), nakon njega katalizator, i smjesa se prenese u autoklav od 5 galona. Ostatak MeOH (2.5 L) koristi se za ispiranje. Smjesa se zagrijava na 40°C pri 50 psi tijekom 24 sata. Učinak katalizatora i vrijeme reakcije funkcije su čistoće početne tvari 1. Upotrijebljena tvar bila je jedinstvena, koristeći >15% katalizatora uz dugo vrijeme reakcije. Čišća punjenja spiroindolina koristila su samo 5% katalizatora uz vrijeme reakcije od 4-6 sati. Hydrogenation is performed three (3) times due to limited equipment; this procedure refers to one cycle. CBZ-spiroindoline sulfonamide 1 was dissolved in THF (6.5 L, KF = 53 μg/uL), then MeOH (KF=18 μg/mL, 4 L) was added, followed by the catalyst, and the mixture was transferred to a 5-gallon autoclave . The remaining MeOH (2.5 L) is used for washing. The mixture is heated to 40°C at 50 psi for 24 hours. Catalyst performance and reaction time are functions of the purity of starting material 1. The material used was unique, using >15% catalyst with a long reaction time. Cleaner charges of spiroindoline used only 5% catalyst with a reaction time of 4-6 hours.

Nakon dovršetka reakcije (<0.1 A% spoja 1 prema LC), smjesa se filtrira kroz Solka Floc™ i ugljični se kolač ispire s MeOH (13 L) koji sadrži NH4OH (0.5%, 60 mL). Kombinirani filtrati (test je pokazao 1587 g spiroindolin amina 1b) koncentriraju se in vacuo, a dobivene se krute tvari razdjeljuju između 40 L toluen:THF (3:1) i 0.5N NaOH (18 L). Unatoč laganom razdvajanju slojeva, u vodenom se sloju vidi obilan precipitat. Zbog toga se vodena suspenzija ekstrahira s CH2Cl2 (15 L). Vodeni i organski sloj odvajaju se polagano. Prije dodavanja CH2Cl2 vodenom se sloju dodao THF i dovoljno NaCl da se sloj zasiti. Unatoč tome, nije postignuto otapanje proizvoda, što je naložilo potrebu korištenja CH2Cl2. Upon completion of the reaction (<0.1 A% compound 1 by LC), the mixture was filtered through Solka Floc™ and the carbon cake was washed with MeOH (13 L) containing NH 4 OH (0.5%, 60 mL). The combined filtrates (assay showed 1587 g of spiroindoline amine 1b) were concentrated in vacuo, and the resulting solids were partitioned between 40 L of toluene:THF (3:1) and 0.5N NaOH (18 L). Despite the slight separation of the layers, an abundant precipitate can be seen in the aqueous layer. Therefore, the aqueous suspension is extracted with CH2Cl2 (15 L). The aqueous and organic layers separate slowly. Before adding CH2Cl2, THF and enough NaCl to saturate the aqueous layer were added. Despite this, no dissolution of the product was achieved, necessitating the use of CH2Cl2.

Kombinirani slojevi toluena, THF i CH2Cl2 kombiniraju se i koncentriraju u koncentratoru. Ostatak se zalije sa 7 L CH3CN. Na kraju se doda 10 L CH3CN i otopina stoji preko noći u atmosferi dušika. The combined layers of toluene, THF and CH2Cl2 are combined and concentrated in a concentrator. The residue is poured with 7 L of CH3CN. Finally, 10 L of CH3CN is added and the solution is left overnight in a nitrogen atmosphere.

Primjer 10B Example 10B

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Spiroindolin-metansulfonamid (oblik slobodne baze) (1b) Spiroindoline-methanesulfonamide (free base form) (1b)

Tvari: Substances:

CBZ-spiroindolin-sulfonamid (1) 3 kg (7.49 mol) CBZ-spiroindoline-sulfonamide (1) 3 kg (7.49 mol)

Darco G-60 600 g Darco G-60 600 g

etil acetat 36 L ethyl acetate 36 L

apsolutni etanol 189 L absolute ethanol 189 L

10%-tni Pd/C 450 g 10% Pd/C 450 g

otopina amonijaka 500 mL ammonia solution 500 mL

Solka Floc™ 2.5 kg Solka Floc™ 2.5 kg

izopropil acetat 65 L isopropyl acetate 65 L

Smjesa CBZ-spiroindolina (1) (1 kg) i Darco G-60 (200 g) u etil acetatu (9 L) miješa se i grije na 60-65°C u atmosferi dušika tijekom 8 sati. Darco se uklanja filtracijom na 60-65°C, kruta tvar se ispire vrućim etil acetatom (3 L) nakon čega se filtrat i ispirci kombiniraju. LC mas/mas test potvrdio je beznačajan gubitak Darco. Otopina etil acetata ispari se do suhoće in vacuo pomoću 20-litrenog Buchi uređaja, potom se zalijeva apsolutnim etanolom (2x5 L). Dobivena se masa miješa s apsolutnim etanolom (8 L) zagrijanim na 65-70°C i smješta u 20-litreni autoklav. Masa se potom ispire apsolutnim etanolom (1L). Nakon toga se u autoklav doda 10%-tni paladij na drvenom ugljenu (75 g, 7.5 mas.%) u apsolutnom etanolu (750 mL) i vrši se ispiranje s daljnjom količinom apsolutnog etanola (250 mL). A mixture of CBZ-spiroindoline (1) (1 kg) and Darco G-60 (200 g) in ethyl acetate (9 L) is stirred and heated to 60-65°C under a nitrogen atmosphere for 8 hours. Darco is removed by filtration at 60-65°C, the solid is washed with hot ethyl acetate (3 L), after which the filtrate and washings are combined. LC mass/mass test confirmed insignificant loss of Darco. The ethyl acetate solution is evaporated to dryness in vacuo using a 20-liter Buchi apparatus, then diluted with absolute ethanol (2x5 L). The resulting mass is mixed with absolute ethanol (8 L) heated to 65-70°C and placed in a 20-liter autoclave. The mass is then washed with absolute ethanol (1 L). After that, 10% palladium on charcoal (75 g, 7.5 wt.%) in absolute ethanol (750 mL) is added to the autoclave and washing is carried out with a further amount of absolute ethanol (250 mL).

Sadržaj autoklava se hidrogenira na 65°C, uz snažno miješanje, pri 40 psi tlaka vodika, tijekom 3 sata, nakon čega se dodaje slijedeća količina 10%-tnog paladija na drvenom ugljenu (75 g); slijedi hidrogenacija slijedeća 2 sata i zatvaranje autoklava preko noći. Smjesa se (još vruća, 60-65°C) prenosi u 20-litarski Buchi uređaj radi uklanjanja plinova in vacuo, čime se uklanja mravlja kiselina iz apsolutnog etanola "feeding and bleeding" postupkom (18 L uk.). The contents of the autoclave are hydrogenated at 65°C, with vigorous stirring, at 40 psi of hydrogen pressure, for 3 hours, after which the following amount of 10% palladium on charcoal (75 g) is added; followed by hydrogenation for the next 2 hours and closing the autoclave overnight. The mixture (still hot, 60-65°C) is transferred to a 20-liter Buchi device for in vacuo degassing, which removes formic acid from absolute ethanol by the "feeding and bleeding" process (18 L incl.).

Ovaj se postupak ponavlja još dvaput, potom se tri tako dobivene mase pomiješaju u 10-galonskoj staklom obloženoj cijevi, te se - pomoću dodavanja i destilacije {in vacuo) apsolutnog etanola (2 x 10 L) - vrši ponovno uklanjanje plina. Smjesi se doda Solkafloc™ (0.5 kg) i vrši se ispiranje etanolom (10 L). Estrella filter puni se sa Solkafloc™ (2 kg) rastopljenim u etanolu (20 L). This procedure is repeated two more times, then the three masses obtained in this way are mixed in a 10-gallon glass-lined tube, and - by means of the addition and distillation (in vacuo) of absolute ethanol (2 x 10 L) - the gas is again removed. Solkafloc™ (0.5 kg) is added to the mixture and washed with ethanol (10 L). The Estrella filter is filled with Solkafloc™ (2 kg) dissolved in ethanol (20 L).

Dobivena smjesa zagrije se na 60-65°C i pri toj temperaturi pomoću crpke prenese preko grijanog filtera do dva spremnika od nerđajućeg čelika obložena katranom. Početna cijev, filter, crpka i vodovi ispiru se vrućom (60-65°C) smjesom vodenog amonijaka (500 mL) u apsolutnom etanolu (25 L). Filtrat i ispirci stavljaju se zajedno u dva spremnika od nerđajućeg čelika. The resulting mixture is heated to 60-65°C and at that temperature it is transferred by means of a pump through a heated filter to two stainless steel tanks lined with tar. The starter tube, filter, pump and lines are flushed with a hot (60-65°C) mixture of aqueous ammonia (500 mL) in absolute ethanol (25 L). The filtrate and washings are placed together in two stainless steel containers.

Masa se potom prenosi u cijev koja koristi in-line filter koji sadrži 10-mikronski uložak, gdje se koncentrira in vacuo do postizanja malog obujma (-15 L). Etanol se zamijenjuje izopropil acetatom "feeding and bleeding" postupkom trostrukog volumena izopropil acetata (45 L uk.) uz održavanje volumena smjese na oko 15 L. Nakon završene izmjene otapala, sadržaj ostatnog etanola bio je <1% prema GC. Smjesa se potom razrijedi na približno 33 L dodavanjem izpopropil acetata (20 L), i takva se otopina spiroindolin-amina lb (1.855 kg prema LC analizi) u izopropil acetatu koristi se u slijedećem koraku postupka. The mass is then transferred to a tube using an in-line filter containing a 10-micron cartridge, where it is concentrated in vacuo to a small volume (-15 L). Ethanol is replaced by isopropyl acetate "feeding and bleeding" using a triple volume of isopropyl acetate (45 L incl.) while maintaining the volume of the mixture at about 15 L. After the solvent change was completed, the remaining ethanol content was <1% by GC. The mixture is then diluted to approximately 33 L by adding isopropyl acetate (20 L), and such a solution of spiroindolinamine lb (1,855 kg by LC analysis) in isopropyl acetate is used in the next step of the process.

Primjer 11A Example 11A

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Boc-O-benzilserin spiroindolin (11) Boc-O-benzylserine spiroindoline (11)

Tvari: Substances:

spiroindolin-amin (1b) 1587 g (5.966 mol) spiroindoline-amine (1b) 1587 g (5.966 mol)

aminokiselina (10) 1938 g (6.563 mol) amino acid (10) 1938 g (6.563 mol)

[image] [image]

DCC 1334.5 g (6.563 mol) DCC 1334.5 g (6.563 mol)

HOBT 884 g (6.563 mol) HOBT 884 g (6.563 mol)

CH3CN 25 L CH3CN 25 L

0.5N NaOH 18 L 0.5N NaOH 18 L

0.5N HCl 18 L 0.5N HCl 18 L

NaHCO3 zasić. 18 L NaHCO3 sat. 18 L

iPrOAc 28 L iPrOAc 28 L

Spiroindolin-amin 1b u CH3CN ili iPrOAciH2O (25 L), na temperaturi okoline, u dušiku, uzastopno se izlaže djelovanju HOBT (884 g; 1.1 ekv.) u obliku krute tvari, DCC (1334.5 g, 1.1 ekv.) u obliku otopine (grijanjem u vrućoj vodi na 60°C tijekom 1 sat) i aminokiselini 10 (1938 g) u obliku krute tvari. Smjesa se miješa tijekom 3 sata i za to vrijeme dolazi do obilne precipitacije DCU; LC analiza pokazala je oko 0.5 A% ostatnog amina 1b. Potom se doda IPAc (9 L), smjesa se filtrira kroz Solka Floc™ i kolač se ispire s IPAc (19 L). Kombinirana organska otopina redom se ispire s 0.5N NaOH (18 L), 0.5N HCl (18 L) i zasićenim NaHCO3 (18 L). Konačnim ispiranjem vodom u ovoj se fazi reakcije dobije emulzija koja se ukloni. Spiroindolinamine 1b in CH3CN or iPrOAciH2O (25 L), at ambient temperature, under nitrogen, is successively treated with HOBT (884 g; 1.1 eq.) as a solid, DCC (1334.5 g, 1.1 eq.) as a solution (by heating in hot water at 60°C for 1 hour) and amino acid 10 (1938 g) as a solid. The mixture is stirred for 3 hours, during which time copious precipitation of DCU occurs; LC analysis showed about 0.5 A% of the remaining amine 1b. IPAc (9 L) is then added, the mixture is filtered through Solka Floc™ and the cake is washed with IPAc (19 L). The combined organic solution was washed sequentially with 0.5N NaOH (18 L), 0.5N HCl (18 L) and saturated NaHCO3 (18 L). The final rinsing with water in this phase of the reaction results in an emulsion which is removed.

Organski se sloj koncentrira in vacuo i ostatak se otopi u MeOH ili, EtOH (10 L konačnog volumena). Ispitivanjem se nađe prinos od 3026 g (89%). The organic layer was concentrated in vacuo and the residue was dissolved in MeOH or EtOH (10 L final volume). The test found a yield of 3026 g (89%).

Uporaba alternativnih sredstava za spajanje peptida, kao što je karbonildiimidazol, ili stvaranje miješanih anhidrida, kao što je sek-butil karbonat, dat će manje prinose spojeva 11 i/ili 14 s višim stupnjem epimerizacije u slučaju spomenutih spojeva. Ostala sredstva za spajanje peptida su i skuplja. The use of alternative peptide coupling agents, such as carbonyldiimidazole, or the formation of mixed anhydrides, such as sec-butyl carbonate, will give lower yields of compounds 11 and/or 14 with a higher degree of epimerization in the case of said compounds. Other agents for joining peptides are also more expensive.

Primjer 11B Example 11B

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Boc-O-benzilserin spiroindolin (11) Boc-O-benzylserine spiroindoline (11)

Tvari: Substances:

spiroindolin-amin (1b) 1.855 kg (6.96 mol) spiroindoline-amine (1b) 1.855 kg (6.96 mol)

izopropil acetat 29 L isopropyl acetate 29 L

dicikloheksilkarbodiimid (DCC) 1.58 kg (7.65 mol) dicyclohexylcarbodiimide (DCC) 1.58 kg (7.65 mol)

1-hidroksibenzotriazol (HOBt) 1.03 kg (7.62 mol) 1-hydroxybenzotriazole (HOBt) 1.03 kg (7.62 mol)

N-Boc-O-benzil-D-serin 2.26 kg (7.65 mol) N-Boc-O-benzyl-D-serine 2.26 kg (7.65 mol)

1M vodeni natrij hidroksid 26 L 1M aqueous sodium hydroxide 26 L

0.5M vodena klorovodična kiselina 26 L 0.5M aqueous hydrochloric acid 26 L

zasić. vodeni natrij hidrogenkarbonat 26 L satiated. aqueous sodium hydrogencarbonate 26 L

apsolutni etanol 50 L absolute ethanol 50 L

Voda (20 L) se doda miješanoj otopini spiroindolin-amina 1b (1.855 kg) u izopropil acetatu (33 L) u reakcijskoj cijevi. Potom se, na sobnoj temperaturi, u atmosferi dušika, redom dodaju slijedeće kemikalije: DCC (1.58 kg, 1.1 ekv.), HOBt (1.03 kg, 1.1 ekv.) i na kraju N-Boc-O-benzil-D-serin (2.26 kg, 1.1 ekv.). Reagensi se ispiru izopropil acetatom (7 L). Smjesa se miješa na sobnoj temperaturi u atmosferi dušika tijekom 5 sati, a LC pokazuje omjer proizvod/početna tvar 99.4/0.6. Smjesa se potom filtrira, kroz Estrella filter samo od tkanine i kartona, uporabom crpke u drugu cijev. Ta se cijev ispire izopropil aceatom (22 L) koji je korišten i za ispiranje filtera, crpke i vodova u primajuću cijev. Dvofazna smjesa u cijevi miješa se tijekom 10 minuta i potom se ostavi slegnuti 15 minuta. Donji vodeni sloj se odvoji, a organski se ostavi stajati na sobnoj temperaturi preko noći. Water (20 L) was added to a stirred solution of spiroindolinamine 1b (1.855 kg) in isopropyl acetate (33 L) in a reaction tube. Then, at room temperature, in a nitrogen atmosphere, the following chemicals are added in order: DCC (1.58 kg, 1.1 equiv.), HOBt (1.03 kg, 1.1 equiv.) and finally N-Boc-O-benzyl-D-serine ( 2.26 kg, 1.1 eq.). The reagents are washed with isopropyl acetate (7 L). The mixture is stirred at room temperature under a nitrogen atmosphere for 5 hours, and the LC shows a product/starting substance ratio of 99.4/0.6. The mixture is then filtered, through an Estrella filter made only of fabric and cardboard, using a pump into another tube. This pipe is flushed with isopropyl acetate (22 L) which was also used to flush the filter, pump and lines into the receiving pipe. The two-phase mixture in the tube is stirred for 10 minutes and then allowed to settle for 15 minutes. The lower aqueous layer is separated, and the organic matter is left to stand at room temperature overnight.

Slijedećeg dana se organska otopina ispire s 1M vodenom otopinom natrij hidroksida (26 L), potom s 0.5M vodenom klorovodičnom kiselinom (26 L) i na kraju sa zasićenim vodenim natrij hidrogen karbonatom (26 L). LC analiza pokazala je prinos od 3.787 kg, 93% ukupni prinos od 7.49 mol (3 kg) početnog CBZ-spiroindolina (1). Smjesa se koncentrira in vacuo (unutrašnja temperatura=13-15°C, temperatura obloge = 40°C, vakuum = 29") da se smanji obujam (oko 15 L) i otapalo se promijeni u etanol postupkom "feeding and bleeding" etanola (50 L) uz održavanje volumena na oko 15 L. GC pokazala je <1% ostatnog izopropil acetata. Ova se otopina koristi u slijedećem koraku postupka. The next day, the organic solution is washed with 1 M aqueous sodium hydroxide solution (26 L), then with 0.5 M aqueous hydrochloric acid (26 L) and finally with saturated aqueous sodium hydrogen carbonate (26 L). LC analysis showed a yield of 3.787 kg, 93% overall yield of 7.49 mol (3 kg) of starting CBZ-spiroindoline (1). The mixture is concentrated in vacuo (internal temperature = 13-15°C, jacket temperature = 40°C, vacuum = 29") to reduce the volume (about 15 L) and the solvent is changed to ethanol by the "feeding and bleeding" process of ethanol ( 50 L) while maintaining the volume at about 15 L. GC showed <1% isopropyl acetate remaining.This solution is used in the next step of the procedure.

Primjer 12A Example 12A

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O-benzilserin spiroindolin (oblik slobodne baze) (12) O-benzylserine spiroindoline (free base form) (12)

Tvari: Substances:

Boc-O-benzilserin spiroindolin (11) 3026 g (5.57 mol) Boc-O-benzylserine spiroindoline (11) 3026 g (5.57 mol)

metan sulfonska kiselina (MsOH) 1.16 L (17.9 mol) methane sulfonic acid (MsOH) 1.16 L (17.9 mol)

MeOH 10 L MeOH 10 L

iPrOAc 24 L iPrOAc 24 L

0.5N NaOH 35 L 0.5N NaOH 35 L

Boc-O-bezilserin spiroindolin 11 u 10 L MeOH (ili EtOH) izlaže se čistoj MsOH (1.16 L) koja se dodaje oko 30-40 minuta (početna temperatura 16°C, konačna temperatura 28°C). Tamnocrvena otopina ostavi se stajati preko noći u dušiku. Potom se smjesa crpkom prenosi u 100-litreni ekstraktor koji sadrži 24L iPrOAc i 35 L 0.5 N NaOH. pH vodenog sloja iznosi 7. Slijedi dodavanje NaOH (6M) do porasta pH na >10.5. S porastom pH dolazi do promjene boje iz crvene u žutu. Slojevi se razdvoje i organski se sloj (24 L) pomoću NMR odredi sadržaj 13 mol% MeOH u iPrOAc (5 vol.%). LC test pokazao je 2.48 kg. Boc-O-benzylserine spiroindoline 11 in 10 L MeOH (or EtOH) is exposed to pure MsOH (1.16 L) which is added over about 30-40 minutes (initial temperature 16°C, final temperature 28°C). The dark red solution was left to stand overnight in nitrogen. The mixture is then pumped into a 100-liter extractor containing 24 L of iPrOAc and 35 L of 0.5 N NaOH. The pH of the aqueous layer is 7. Followed by the addition of NaOH (6M) until the pH rises to >10.5. As the pH increases, the color changes from red to yellow. The layers were separated and the organic layer (24 L) was determined by NMR to contain 13 mol% MeOH in iPrOAc (5 vol.%). The LC test showed 2.48 kg.

Primjer 12B Example 12B

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O-benzilserin spiroindolin (oblik slobodne baze) (12) O-benzylserine spiroindoline (free base form) (12)

Tvari: Substances:

Boc-O-benzilserin spiroindolin (11) 3.787 kg (6.96 mol) Boc-O-benzylserine spiroindoline (11) 3.787 kg (6.96 mol)

metansulfonska kiselina 2.006 kg (20.87 mol) methanesulfonic acid 2.006 kg (20.87 mol)

izopropil acetat 38 L isopropyl acetate 38 L

1M vodeni natrij hidroksid 16 L 1M aqueous sodium hydroxide 16 L

50%-tni vodeni natrij hidroksid 1.6 L 50% aqueous sodium hydroxide 1.6 L

Metansulfonska kiselina (2.006 kg, 1.355 L, oko 3 ekv.) doda se u miješanu otopinu Boc-O-benzilserin spiroindolina (11) (3.787 kg) u etanolu (ukupni volumen oko 15 L) u reakcijskoj cijevi. Smjesa se zagrijava na 35-40°C. Nakon 7 sati, LC pokazuje odsutnost početne tvari, pa se smjesa ostavi hladiti na sobnu temperaturu preko noći. Slijedećeg dana, smjesi se uz miješanje doda voda (44 L). Smjesa se ohladi na oko 5°C, miješa tijekom 30 minuta i potom filtrira kroz inline filter (ispunjen s 10μ punjenjem) u spremnik. Smjesa se potom isisava natrag u cijev. Vodom (10L) se ispiru cijev i vodovi u spremnik, a koristi se i za povratno ispiranje u cijev. Doda se izopropil acetat (38 L) i 1M vodeni natrij hidroksid (16 L). Smjesa se ohladi na 10-15°C, provjeri se pH donjeg vodenog sloja koji iznosi oko 7, i doda se 50%-tna vodena otopina natrij hidroksida (1.6 L) (pH>10). Smjesa se miješa na 10-15°C tijekom 25 minuta i potom se ostavi slegnuti 10-15 minuta. Donji vodeni sloj se izdvoji (78.1 kg). LC test pokazao je nazočnost 28.4 g tvari 12 (0.85% teorijskog) u tekućinama. Volumen organske otopine = 51 L. LC pokazuje 3.057 kg, 92%-tni ukupni prinos od 3 kg, 7.49 mol CBZ-spiroindolin sulfonamida (1). Ova se otopina koristi za slijedeći stupanj. Methanesulfonic acid (2,006 kg, 1,355 L, about 3 eq.) was added to a stirred solution of Boc-O-benzylserine spiroindoline (11) (3,787 kg) in ethanol (total volume about 15 L) in a reaction tube. The mixture is heated to 35-40°C. After 7 hours, LC shows the absence of starting material, so the mixture is allowed to cool to room temperature overnight. The next day, water (44 L) is added to the mixture while stirring. The mixture is cooled to about 5°C, stirred for 30 minutes and then filtered through an inline filter (filled with a 10μ charge) into a container. The mixture is then sucked back into the pipe. Water (10L) is used to flush the pipe and lines into the tank, and it is also used for backwashing into the pipe. Isopropyl acetate (38 L) and 1M aqueous sodium hydroxide (16 L) were added. The mixture is cooled to 10-15°C, the pH of the lower aqueous layer is checked, which is about 7, and a 50% aqueous solution of sodium hydroxide (1.6 L) is added (pH>10). The mixture is stirred at 10-15°C for 25 minutes and then left to settle for 10-15 minutes. The lower aqueous layer is separated (78.1 kg). The LC test showed the presence of 28.4 g of substance 12 (0.85% of theory) in the liquids. Volume of organic solution = 51 L. LC shows 3.057 kg, 92% overall yield of 3 kg, 7.49 mol of CBZ-spiroindoline sulfonamide (1). This solution is used for the next step.

Primjer 13A Example 13A

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Boc-aminoizobutiril O-benzilserin spiroindolin (14) Boc-aminoisobutyryl O-benzylserine spiroindoline (14)

Tvari: Substances:

spiroindolin amin (12) 2481 g (5.57 mol) spiroindoline amine (12) 2481 g (5.57 mol)

aminokiselinski peptid (13) 1247.1 g (6.16 mol) amino acid peptide (13) 1247.1 g (6.16 mol)

[image] [image]

DCC 1266.7 g (6.16 mol) DCC 1266.7 g (6.16 mol)

HOBT 827 g (6.16 mol) HOBT 827 g (6.16 mol)

IPAc 52 L IPAc 52 L

H2O 37 L H2O 37 L

0.5N NaOH 36 L 0.5N NaOH 36 L

0.5N HCl 36 L 0.5N HCl 36 L

zasić. NaHCO3 36 L satiated. NaHCO3 36 L

Otopina amina 12 u IPAc razrijedi se s IPAc na ukupni volumen 39 L i doda se 37L H2O. Dvofazna se smjesa uzastopno izlaže HOBT (827 g) u obliku krute zvari, DCC (1266.7 g) u obliku otopine i aminokiselini 13, sve na sobnoj temperaturi, u dušiku. Reakcijska se smjesa miješa 2 h, za koje vrijeme LC analiza pokazuje iščezavanje početne tvari 12 (<0.3 A%). Smjesa se filtrira kroz Solka Floc™ i krute se tvari ispiru s 13L IPAc. Tvar se u ovoj fazi može pohraniti preko noći kao dvofazna smjesa. A solution of amine 12 in IPAc is diluted with IPAc to a total volume of 39 L and 37 L of H2O is added. The biphasic mixture is successively exposed to HOBT (827 g) in the form of a solid weld, DCC (1266.7 g) in the form of a solution and amino acid 13, all at room temperature, in nitrogen. The reaction mixture is stirred for 2 h, during which time LC analysis shows the disappearance of starting substance 12 (<0.3 A%). The mixture is filtered through Solka Floc™ and the solids are washed with 13L IPAc. In this phase, the substance can be stored overnight as a two-phase mixture.

Smjesa se prenese u 100-litreni ekstraktor, vodeni se sloj odvoji, a organski uzastopno ispire s 36 L 0.5N NaOH, 0.5N HCl i zasićenim NaHCO3. Prinos = 3160 g (81% iz spiroindolina ±5% za pogrešku mjerenja volumena). Otopina se koncentrira do malog obujma i dolijeva se etanol (2 x 4 L). Po želji, meduspoj 14 može se izdvojiti dodavanjem vode da bi se kristalizirao. The mixture is transferred to a 100-liter extractor, the aqueous layer is separated, and the organics are successively washed with 36 L of 0.5N NaOH, 0.5N HCl and saturated NaHCO3. Yield = 3160 g (81% from spiroindoline ±5% for volume measurement error). The solution is concentrated to a small volume and ethanol (2 x 4 L) is added. If desired, intermediate compound 14 can be isolated by adding water to crystallize it.

Uporaba alternativnih sredstava za spajanje peptida, kao što je karbonildiimidazol, ili stvaranje miješanih anhidrida, kao što je sek-butil karbonat, dat će manje prinose spoja 14 s višim stupnjem epimerizacije. Ostala sredstva za spajanje peptida su i skuplja. The use of alternative peptide coupling agents, such as carbonyldiimidazole, or the formation of mixed anhydrides, such as sec-butyl carbonate, will give lower yields of compound 14 with a higher degree of epimerization. Other agents for joining peptides are also more expensive.

Primjer 13B Example 13B

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Boc-aminoizobutiril O-benzilserin spiroindolin (14) Boc-aminoisobutyryl O-benzylserine spiroindoline (14)

Tvari: Substances:

spiroindolin amin (12) 3.057 kg (6.89 mol) spiroindoline amine (12) 3.057 kg (6.89 mol)

dicikloheksilkarbodiimid (DCC) 1.56 kg (7.56 mol) dicyclohexylcarbodiimide (DCC) 1.56 kg (7.56 mol)

1-hidroksibenzotriazol (HOBt) 1.02 kg (7.55 mol) 1-hydroxybenzotriazole (HOBt) 1.02 kg (7.55 mol)

Boc-2-aminoizomaslačna kiselina (13) 1.54 kg (7.58 mol) Boc-2-aminoisobutyric acid (13) 1.54 kg (7.58 mol)

izopropil acetat 32 L isopropyl acetate 32 L

1M vodeni natrij hidroksid 38 L 1M aqueous sodium hydroxide 38 L

0.5M vodena klorovodična kiselina 38 L 0.5M aqueous hydrochloric acid 38 L

zasić. vodeni natrij hidrogenkarbonat 38 L satiated. aqueous sodium hydrogencarbonate 38 L

apsolutni etanol 45 L absolute ethanol 45 L

Voda (49 L) se, uz miješanje, doda otopini spiroindolin amina 12 (3.057 kg) u izopropil acetatu (ukupni volumen oko 51L) u reakcijskoj cijevi na sobnoj temperaturi, u atmosferi dušika. Zatim se redom dodaju slijedeće kemikalije: DCC (1.56 kg, oko 1.1 ekv.), HOBt (1.02 kg, oko 1.1 ekv.) i N-Boc-2-aminoizomaslačna kiselina 13 (1.54 kg, oko l.lekv.). Smjesa se snažno miješa tijekom 2 sata na sobnoj temperaturi, kada LC pokazuje dovršenost reakcije. Smjesa se filtrira u drugu cijev putem Estrella filtera i pomoću crpke. Izopropil acetat (22 L) koristi se za ispiranje cijevi, filtera, crpke i vodova do crpke. Dvofazna se smjesa potom miješa 5 minuta i ostavi da se slojevi razdvoje. Donji vodeni sloj odvojen je bez teškoća (težina vodenog dijela = 51.1 kg). Organska se otopina potom uzastopno ispire s 1M vodenim natrij hidroksidom (38 L), 0.5M vodenom klorovodičnom kiselinom (38 L), te zasićenim vodenim natrij hidrogenkarbonatom (38 L) bez teškoća. Water (49 L) was added, with stirring, to a solution of spiroindoline amine 12 (3,057 kg) in isopropyl acetate (total volume about 51 L) in a reaction tube at room temperature, under a nitrogen atmosphere. Then the following chemicals are added in order: DCC (1.56 kg, about 1.1 equiv.), HOBt (1.02 kg, about 1.1 equiv.) and N-Boc-2-aminoisobutyric acid 13 (1.54 kg, about 1. equiv.). The mixture was stirred vigorously for 2 hours at room temperature, when LC indicated completion of the reaction. The mixture is filtered into another pipe through an Estrella filter and by means of a pump. Isopropyl acetate (22 L) is used to flush the pipes, filter, pump and lines to the pump. The two-phase mixture is then stirred for 5 minutes and allowed to separate the layers. The lower water layer was separated without difficulty (weight of the water part = 51.1 kg). The organic solution is then successively washed with 1M aqueous sodium hydroxide (38 L), 0.5M aqueous hydrochloric acid (38 L), and saturated aqueous sodium hydrogencarbonate (38 L) without difficulty.

Organska otopina se, pomoću crpke, preko inline filtera (koji sadrži 10μ punjenje) prenosi u drugu cijev da bi se otapalo izmijenilo u etanol. Cijev se ispire izopropil acetatom (10 L), kao i crpka, filter i vodovi do cijevi. Filtrat i ispirci se kombiniraju. Ukupni volumen = 75 L (mjereno sondom). LC testom odredi se količina od 4.395 kg Boc-aminoizobutiril O-benzilserin spiroindolina (14), tj. 93%-tni ukupni prinos od 7.49 mol početnog CBZ-spiroindolin sulfonamida (1). The organic solution is pumped through an inline filter (containing a 10μ charge) into another tube to convert the solvent into ethanol. The pipe is washed with isopropyl acetate (10 L), as well as the pump, filter and lines to the pipe. The filtrate and washings are combined. Total volume = 75 L (measured with probe). The LC test determined the amount of 4,395 kg of Boc-aminoisobutyryl O-benzylserine spiroindoline (14), i.e. 93% total yield of 7.49 mol of initial CBZ-spiroindoline sulfonamide (1).

Smjesa se koncentrira in vacuo do manjeg obujma (oko 15 L) i izopropil acetat se izmijeni u etanol "feeding and bleeding" postupkom apsolutnog etanola (ukupno 45 L). Na kraju izmjene otapala, GC je pokazala <1% ostatnog izopropil acetata. Ova otopina (25 L), koja sadrži 4.395 kg spoja 14 koristi se u slijedećem koraku. Po želji, međuspoj 14 može se izdvojiti dodavanjem vode da bi se kristalizirao. The mixture is concentrated in vacuo to a smaller volume (about 15 L) and the isopropyl acetate is converted to ethanol by the "feeding and bleeding" process of absolute ethanol (45 L in total). At the end of the solvent change, GC showed <1% isopropyl acetate remaining. This solution (25 L), which contains 4,395 kg of compound 14, is used in the next step. If desired, intermediate 14 can be isolated by adding water to crystallize it.

Primjer 14A Example 14A

[image] [image]

Aminoizobutiril O-benzilserin spiroindolin (15) Aminoisobutyryl O-benzylserine spiroindoline (15)

Tvari: Substances:

Boc spiroindolin (14) 3160 g (5.03 mol) Boc spiroindoline (14) 3160 g (5.03 mol)

metansulfonska kiselina (MsOH) 979 mL (15.1 mol) methanesulfonic acid (MsOH) 979 mL (15.1 mol)

EtOH 6.2 L EtOH 6.2 L

H2O 30 L H2O 30 L

1N NaOH 11 L 1N NaOH 11 L

EtOAc 26 L EtOAc 26 L

Darco 60 aktivirani ugljik 1 kg Darco 60 activated carbon 1 kg

Boc spiroindolin 14 otopi se u 6.2 L EtOH i doda se MsOH (979 mL). Temperatura pritom naraste s 20 na 30°C i smjesa se ostavi preko noći da bi se reakcija dovršila. Nakon 12 h na 20°C zaostalo je još 15 A% početne tvari, pa se smjesa grije na 35°C daljnjih 6 sati. Nakon dovršetka reakcije(<0.1 A% 14), smjesa se ohladi na 20°C, doda se 30 L H2O i otopina se filtrira kroz stakleni lijevak s polipropilenskim filterom da bi se uklonio ostatni DCU. Boc spiroindoline 14 was dissolved in 6.2 L EtOH and MsOH (979 mL) was added. The temperature rises from 20 to 30°C and the mixture is left overnight to complete the reaction. After 12 h at 20°C, 15 A% of the starting substance remained, so the mixture was heated at 35°C for a further 6 hours. After completion of the reaction (<0.1 A% 14), the mixture was cooled to 20°C, 30 L of H2O was added and the solution was filtered through a glass funnel with a polypropylene filter to remove the remaining DCU.

Smjesa se potom prenese u 100-litreni ekstraktor i doda se 26 L EtOAc. Vodeni se sloj zaluži dodavanjem ohlađenog 1N NaOH (11 L) i 1 L 50%-tnog NaOH. Dodavanje leda potrebno je da bi se temperatura održala ispod 14°C. Više temperature imale bi kao posljedicu znatne probleme s emulgiranjem. The mixture was then transferred to a 100 L extractor and 26 L EtOAc was added. The aqueous layer is made alkaline by adding cooled 1N NaOH (11 L) and 1 L of 50% NaOH. Adding ice is necessary to keep the temperature below 14°C. Higher temperatures would result in significant emulsification problems.

Organski se sloj destilira na 50°C pri ~21" Hg dok se ne postigne KF<1000 μg/mL. Niže vrijednosti KF dovode do boljeg učinka izlaganja ugljiku i boljeg prinosa u koraku stvaranja soli. Vrijednost KF 160 ug/mL postignuta je za mjeru od 700 g. Otopina se razrijedi etil acetatom do ukupnog volumena od 31 L (LC test 2.40 kg). Doda se aktivirani ugljik (Darco G-60) i smjesa se miješa tijekom 24 h. Zatim se smjesa filtrira kroz Solka Floc™ i filterski se kolač ispire etil acetatom (16 L); vrijednost testa 2.34 kg. The organic layer is distilled at 50°C at ~21" Hg until KF<1000 μg/mL is reached. Lower KF values lead to better carbon exposure performance and better yield in the salt formation step. A KF value of 160 ug/mL was achieved for 700 g measure. The solution is diluted with ethyl acetate to a total volume of 31 L (LC test 2.40 kg). Activated carbon (Darco G-60) is added and the mixture is stirred for 24 h. The mixture is then filtered through Solka Floc™ and filter cake is washed with ethyl acetate (16 L), test value 2.34 kg.

Primjer 14B Example 14B

[image] [image]

Aminoizobutiril O-benzilserin spiroindolin (15) Aminoisobutyryl O-benzylserine spiroindoline (15)

Tvari: Substances:

Boc spiroindolin (14) 4.395 kg (6.99 mol) Boc spiroindoline (14) 4.395 kg (6.99 mol)

metansulfonska kiselina 2.017 kg (20.99 mol) methanesulfonic acid 2.017 kg (20.99 mol)

etil acetat 185 L ethyl acetate 185 L

1M vodeni natrij hidroksid 16 L 1M aqueous sodium hydroxide 16 L

50%-tni vodeni natrij hidroksid 2.6 L 50% aqueous sodium hydroxide 2.6 L

Darco G-60 900 g Darco G-60 900 g

Solka Floc™ 2.5 kg Solka Floc™ 2.5 kg

Metansulfonska kiselina (2.017 kg, 1.36 L, oko 3 ekv.) doda se, uz miješanje, u otopinu Boe spiroindolina 14 (4.395 kg) u etanolu (ukupni volumen oko 25 L) u reakcijskoj cijevi, na sobnoj temperaturi. Smjesa se zagrije na 35-40°C i miješa preko noći. Slijedećeg dana, smjesa je sadržavala oko 1.1 A% početne tvari, pa je reakcija nastavljena još 4 sata, nakon čega je pomoću LC ustanovljen omjer proizvod/početna tvar 99.6/0.4. Potom se smjesa koncentrira in vacuo do volumena oko 15 L i razrijedi s vodom (44 L). Zatim se smjesa ohladi na 5°C, miješa tijekom 30 minuta i, pomoću crpke, filtrira kroz Sparkler inline filter (koji sadrži 10(i punjenje) u drugu cijev, da bi se uklonila manja količina ostatnog DCU. Methanesulfonic acid (2,017 kg, 1.36 L, about 3 eq.) was added, with stirring, to a solution of Boe spiroindoline 14 (4,395 kg) in ethanol (total volume about 25 L) in a reaction tube at room temperature. The mixture is heated to 35-40°C and stirred overnight. The next day, the mixture contained about 1.1 A% of the starting substance, so the reaction was continued for another 4 hours, after which the product/starting substance ratio of 99.6/0.4 was determined by LC. The mixture is then concentrated in vacuo to a volume of about 15 L and diluted with water (44 L). The mixture is then cooled to 5°C, stirred for 30 minutes and, using a pump, filtered through a Sparkler inline filter (containing 10(i charge) into another tube, to remove a small amount of residual DCU.

Cijev, crpka, filter i vodovi isperu se vodom (10 L) koja se doda u cijev. U cijev se potom doda etil acetat (36 L) i smjesa se, uz miješanje, ohladi na 10°C. Otopina hladne (5-10°C) 1M vodene otopine natrij hidroksida (16 L) i hladne (5-10°C) 50%-tne vodene otopine natrij hidroksida (2.6 L) dodaju se na 10°C pri čemu temperatura naraste na 14°C. Dobivena se smjesa miješa tijekom 15 minuta na <14°C i potom se donji vodeni sloj izdvoji. The pipe, pump, filter and lines are washed with water (10 L) which is added to the pipe. Ethyl acetate (36 L) was then added to the tube and the mixture was cooled to 10°C with stirring. A solution of cold (5-10°C) 1M aqueous sodium hydroxide solution (16 L) and cold (5-10°C) 50% aqueous sodium hydroxide solution (2.6 L) are added at 10°C, the temperature rising to 14°C. The resulting mixture is stirred for 15 minutes at <14°C and then the lower aqueous layer is separated.

Smjesa se koncentrira in vacuo do oko 20 L volumena, nakon čega se doda smjesa etil acetata (35 L) i etanola (5 L) uz održavanje volumena na oko 20 L. Na kraju destilacije KF iznosi 9160 mg/mL. Otapalo smjese izmijeni se u etil acetat postupkom "feeding and bleeding" etil acetata (ukupno 40 L). Na kraju ove destilacije, KF iznosi 446 mg/mL. Smjesa se nakon toga razrijedi s etil acetatom (10 L). The mixture is concentrated in vacuo to a volume of about 20 L, after which a mixture of ethyl acetate (35 L) and ethanol (5 L) is added while maintaining the volume at about 20 L. At the end of the distillation, the KF is 9160 mg/mL. The solvent of the mixture is changed to ethyl acetate by the "feeding and bleeding" process of ethyl acetate (40 L in total). At the end of this distillation, the KF is 446 mg/mL. The mixture is then diluted with ethyl acetate (10 L).

Magličastoj se smjesi doda Darco G-60 (900 g). Slijedi ispiranje etil acetatom (6 L). Ova se smjesa miješa preko noći na sobnoj temperaturi. Slijedećeg dana, smjesi u cijevi se - uz miješanje - dodaje Solka Floc™ (0.5 kg); zatim se Solka Floc™ (2.0 kg) miješa u malo etil acetata i puni u Estrella filter. Višak otapala otcrpi se kroz Sparkler inline filter koji sadrži 10|o. punjenje. Otopina se prenese iz cijevi kroz filter pomoću crpke, a potom kroz drugi filter u spremnike od nerđajućeg čelika od 2 x 40 L. Promatranjem se ustanovi da su tekućine prozirne i čiste. Cijev se ispire etil acetatom (22 L), koji se koristi i za ispiranje gore opisanog puta do spremnika od nerđajućeg čelika. Sadržaj oba spremnika prenese se u reakcijsku cijev i dobivena se otopina izmiješa. Darco G-60 (900 g) is added to the hazy mixture. Followed by washing with ethyl acetate (6 L). This mixture is stirred overnight at room temperature. The next day, Solka Floc™ (0.5 kg) is added to the mixture in the tube - with stirring; then Solka Floc™ (2.0 kg) is mixed with a little ethyl acetate and filled into the Estrella filter. Excess solvent is drained through a Sparkler inline filter containing 10|o. charge. The solution is transferred from the pipe through the filter using a pump, and then through another filter into 2 x 40 L stainless steel tanks. The liquid is observed to be clear and clean. The tube is flushed with ethyl acetate (22 L), which is also used to flush the above-described path to the stainless steel tank. The contents of both containers are transferred to the reaction tube and the resulting solution is mixed.

KF smjese (58 L) iznosio je 2950 mg/mL, pa je ponovno sušena koncentriranjem in vacuo do volumena od 20-25 L. Zatim se smjesa razrijedi do volumena od 46 L (sonda) dodavanjem etil acetata (25 L). KF iznosi 363 mg/mL. Smjesa se zatim razrijedi na volumen od 62 L dodavanjem etil acetata (17 L) i takva se koristi u završnom koraku postupka. The KF of the mixture (58 L) was 2950 mg/mL, so it was dried again by concentrating in vacuo to a volume of 20-25 L. Then the mixture was diluted to a volume of 46 L (probe) by adding ethyl acetate (25 L). KF is 363 mg/mL. The mixture was then diluted to a volume of 62 L by adding ethyl acetate (17 L) and used as such in the final step of the procedure.

Primjer 15A Example 15A

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Spiro[3H-indol-3,4'-piperdin]-1'-il)karbonil]-2-(fenilmetiloksi)etil]-2-amino-2-metilpropanamid metansulfonat (16) Spiro[3H-indol-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate (16)

Tvari: Substances:

amin (15) 2340 g (4.43 mol) amine (15) 2340 g (4.43 mol)

metan sulfonska kiselina (MsOH) 316 mL (4.88 mol) methane sulfonic acid (MsOH) 316 mL (4.88 mol)

EtOAc 60 L EtOAc 60 L

EtOH 4.8 L EtOH 4.8 L

8%-tni EtOH u EtOAc 20 L 8% EtOH in EtOAc 20 L

Volumen otopine 15 iz prethodnog koraka podešen je na 60 L dodavanjem etil acetata i EtOH (4.8 L). MsOH (316 mL) se doda u 3 L EtOAc na 45°C. Tamnocrvenoj homogenoj otopini doda se 496 g klice Oblika I naslovnog spoja (10%-tni udio klice prema težini slobodnog amina je iskorišten). Temperatura pritom naraste do oko 48°C i reakcijska se smjesa ostavi stajati na 52°C tijekom 1.5 sati. Ispitivanje pokazuje potpunu' pretvorbu naslovnog spoja (Oblik I). (Dulje razdoblje (>3 h) bilo je potrebno za manje od 10% udjela klice). Smjesa se ostavi hladiti na 20°C preko noći i filtrira se u centrifugi u dušiku. Kolač se ispire s 20 L 8%-tnog EtOH u EtOAc. Tijekom filtracije bitna je nazočnost dušika zbog velike higroskopnosti vlažnih kristala. Smjesa se suši na 35°C u vakuumu čime se dobije 2.7 kg (56%-tni ukupni prinos) naslovnog spoja (Oblika I)(99.9 A%-tna čistoća; <0.1% enantiomera). The volume of solution 15 from the previous step was adjusted to 60 L by adding ethyl acetate and EtOH (4.8 L). MsOH (316 mL) was added to 3 L EtOAc at 45°C. 496 g of germ Form I of the title compound are added to the dark red homogeneous solution (a 10% proportion of the germ by weight of free amine was used). The temperature rises to about 48°C and the reaction mixture is left to stand at 52°C for 1.5 hours. The assay shows complete conversion of the title compound (Form I). (A longer period (>3 h) was required for less than 10% germination). The mixture is left to cool at 20°C overnight and is filtered in a centrifuge under nitrogen. The cake was washed with 20 L of 8% EtOH in EtOAc. During filtration, the presence of nitrogen is important due to the high hygroscopicity of wet crystals. The mixture is dried at 35°C under vacuum to give 2.7 kg (56% overall yield) of the title compound (Form I) (99.9 A% purity; <0.1% enantiomer).

Prevođenje Oblika II u Oblik I također se postiže kada se sol oblikuje u EtOAc-EtOH dodavanjem MsOH na gore opisani način i hlađenje početne otopine soli (na 55°C) do 45°C. Na ovoj se temperaturi počinju javljati kristali, a otopina vremenom postaje gušća. Potom se temperatura podigne na 51°C i otopina se ostavi stajati preko noći. Očekuje se potpuna pretvorba u Oblik I spoja 16. Navedeni je postupak također moguće provesti u svrhu dobivanja klice kristala Oblika I spoja 16. Conversion of Form II to Form I is also achieved when the salt is formed in EtOAc-EtOH by adding MsOH as described above and cooling the initial salt solution (at 55°C) to 45°C. At this temperature, crystals begin to appear, and the solution becomes thicker over time. Then the temperature is raised to 51°C and the solution is left to stand overnight. A complete conversion to Form I of compound 16 is expected. The mentioned procedure can also be carried out in order to obtain a crystal seed of Form I of compound 16.

Primjer 15B Example 15B

[image] [image]

Spiro[3H-indol-3,4'-piperdin]-1'-il)karbonil]-2-(fenilmetiloksi)etil]-2-amino-2-metilpropanamid metansulfonat (16) Spiro[3H-indol-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate (16)

Tvari: Substances:

amin (15) 3.1 kg (5.86 mol) amine (15) 3.1 kg (5.86 mol)

metansulfonska kiselina 620 g (6.45 mol) methanesulfonic acid 620 g (6.45 mol)

etil acetat 37 L ethyl acetate 37 L

apsolutni etanol 8.7 L absolute ethanol 8.7 L

spiro[3H-indol-3,4'-piperidin]-1'-il)karbonil]-2-(fenilmetil- spiro[3H-indol-3,4'-piperidin]-1'-yl)carbonyl]-2-(phenylmethyl-

oksi)etil]-2-amino-2-metilpropanamid metansulfonat 70 g (0.11 mol) oxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate 70 g (0.11 mol)

(Oblik I) (Form I)

Apsolutni etanol (6.4 L) doda se otopini amina (15) (3.1 kg) u etil acetatu (ukupni volumen oko 62 L) u reakcijskoj cijevi. Smjesa se zagrije na 50°C i tijekom približno 5 minuta na temperaturi od 50-54°C dodaje se metansulfonska kiselina (620 g, 412 mL, 1.1 ekv.) u etil acetatu (11 L). Smjesi se doda spiro[3H-indol-3,4'-piperdin}-1'-il) karbonil]- 2-(fenilmetiloksi)etil]-2-amino-2-metilpropanamid metansulfonat (Oblik I) (70 g) i dobivena se smjesa miješa i grije na 55°C u atmosferi dušika preko noći. Slijedećeg dana, smjesa se hladi na 15-20°C, održava tako tijekom 2 sata i potom ukapava na polipropilenski filter od 50 cm u atmosferi dušika. Kruti proizvod se ispire smjesom apsolutnog etanola (2.3 L) u etil acetatu (26 L). Bijeli, kruti proizvod se odgrabi i suši u Apex peći in vacuo na 35°C tijekom odgovarajućeg razdoblja (otprilike dva dana). Suhi spiro[3H-indol-3,4'- piperdin]-1'-il)karbonil]- 2-(fenilmetiloksi)etil]-2- amino-2- metilpropan amid metansulfonat (3.352 kg) prosijava se kroz Jackson-Crockatt sito čime se dobije 3.347 kg (uključujući sito, 70 g); prinos = 3.277 kg. Absolute ethanol (6.4 L) was added to a solution of amine (15) (3.1 kg) in ethyl acetate (total volume about 62 L) in a reaction tube. The mixture is heated to 50°C and methanesulfonic acid (620 g, 412 mL, 1.1 eq.) in ethyl acetate (11 L) is added over approximately 5 minutes at a temperature of 50-54°C. Spiro[3H-indol-3,4'-piperidin}-1'-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate (Form I) (70 g) was added to the mixture and the resulting mixture is stirred and heated to 55°C in a nitrogen atmosphere overnight. The next day, the mixture is cooled to 15-20°C, kept like that for 2 hours and then dripped onto a 50 cm polypropylene filter in a nitrogen atmosphere. The solid product is washed with a mixture of absolute ethanol (2.3 L) in ethyl acetate (26 L). The white, solid product is scraped off and dried in an Apex oven in vacuo at 35°C for an appropriate period (approximately two days). Dry spiro[3H-indol-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropane amide methanesulfonate (3,352 kg) is sieved through a Jackson-Crockatt sieve resulting in 3,347 kg (including sieve, 70 g); yield = 3,277 kg.

Oblik I N-[1(R)-[1,2-dihidro-1-metansulfonil-spiro[3H-indol-3,4'-piperdin]-1'-il)karbonil]-2-(fenilmetiloksi)etil]-2- Form I N-[1(R)-[1,2-dihydro-1-methanesulfonyl-spiro[3H-indol-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenylmethyloxy)ethyl] -2-

amino-2-metilpropanamid metansulfonat je bezvodni polimorf slijedećih svojstava: amino-2-methylpropanamide methanesulfonate is an anhydrous polymorph with the following properties:

talište na 168-171°C i topljivost u izopropanolu 4.6 mg/mL. melting point at 168-171°C and solubility in isopropanol 4.6 mg/mL.

DSC krivulja Oblika I N-[1(R)-[(1,2-dihidro-1-metansulfonil-spiro[3H-indol-3,4'-piperdin]-1'-il)karbonil]-2-(fenilmetiloksi)etil]-2-amino-2-metilpropanamid metansulfonata na 10°C/min u otvorenoj posudi, uz strujanje dušika ima oblik jednostavne endoterme, zbog taljenja, s vršnom vrijednosti temperature oko 180°C i ekstrapoliranom porastu temperature (talište) oko 170°C, uz odgovarajuću toplinu od približno 53 J/g. DSC curve of Form I N-[1(R)-[(1,2-dihydro-1-methanesulfonyl-spiro[3H-indol-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenylmethyloxy )ethyl]-2-amino-2-methylpropanamide methanesulfonate at 10°C/min in an open vessel, with nitrogen flow, has the form of a simple endotherm, due to melting, with a peak temperature value of around 180°C and an extrapolated rise in temperature (melting point) around 170 °C, with a corresponding heat of approximately 53 J/g.

Oblik I opisan je prašinastim ogibom rendgenskih zraka s refleksijama kod približno 6.5, 14.7, 16.9, 17.1, 17.9, 19.5, 21.1, 21.7 i 22.0° (2 theta). Podaci su prikupljeni uporabom Philips APD3720 automatskog uređaja za prašinasti ogib s bakrenim Ka zračenjem. Mjerenja su provedena od 2° do 40° (2 theta) na uzorku održavanom na sobnoj temperaturi. Form I is described by a dusty X-ray bend with reflections at approximately 6.5, 14.7, 16.9, 17.1, 17.9, 19.5, 21.1, 21.7, and 22.0° (2 theta). Data were collected using a Philips APD3720 automatic dust bender with copper Ka radiation. Measurements were made from 2° to 40° (2 theta) on a sample maintained at room temperature.

Uvjeti HPLC: LC vremena retencije na Zorbax RX-C8 (4.6 mm x 25 cm), λ = 210nm, brzina protoka = 1.5 mL/min. HPLC conditions: LC retention time on Zorbax RX-C8 (4.6 mm x 25 cm), λ = 210nm, flow rate = 1.5 mL/min.

Spoj 1: 60:40 CH3CN-H2O (1% H3PO4) RT = 5.0 min. Compound 1: 60:40 CH3CN-H2O (1% H3PO4) RT = 5.0 min.

Spoj 1b: 35:65 CH3CN-H2O (0.1 mas.% NH4OAc) RT = 6.2 min. Compound 1b: 35:65 CH3CN-H2O (0.1 wt.% NH4OAc) RT = 6.2 min.

Spoj 10: 60:40 CH3CN-H2O (0.1 H3PO4) RT = 2.9 min. Compound 10: 60:40 CH3CN-H2O (0.1 H3PO4) RT = 2.9 min.

Spoj 11: 60:40 CH3CN-H2O (0.1% H3PO4) RT = 5.4 min. Compound 11: 60:40 CH3CN-H2O (0.1% H3PO4) RT = 5.4 min.

Spoj 12: 40:60 CH3CN-H2O [pH = 5.25 NaH2PO4 (6.9 g/L H2O) Compound 12: 40:60 CH3CN-H2O [pH = 5.25 NaH2PO4 (6.9 g/L H2O)

(podešavanje pH pomoću NaOH)] RT = 5.6 min. (pH adjustment with NaOH)] RT = 5.6 min.

Spoj 14: 60:40% CH3CN-H2O (0.1% H3PO4) RT = 4.65 min. Compound 14: 60:40% CH3CN-H2O (0.1% H3PO4) RT = 4.65 min.

Spoj 15: 40:60% CH3CN-H2O [pH = 5.25 NaH2PO4 (6.9 g/L H2O) Compound 15: 40:60% CH3CN-H2O [pH = 5.25 NaH2PO4 (6.9 g/L H2O)

(podešavanje pH pomoću NaOH)] RT = 4.9 min. (pH adjustment with NaOH)] RT = 4.9 min.

LC vremena retencije na Zorbax RX-C8 (4.6 mm x 25 cm), λ = 210 nm, brzina protoka = 1.2 mL/min., temperatura stupca = 48cC, Otapalo A = 0.05% fosforne kiseline + 0.01% trietilamina u vodi; Otapalo B = acetonitril Sustav gradijenata: LC retention times on Zorbax RX-C8 (4.6 mm x 25 cm), λ = 210 nm, flow rate = 1.2 mL/min., column temperature = 48cC, Solvent A = 0.05% phosphoric acid + 0.01% triethylamine in water; Solvent B = acetonitrile Gradient system:

vrijeme %A %B time %A %B

0 min. 95 5 0 min. 95 5

35 min. 10 90 35 min. 10 90

38 min. 95 5 38 min. 95 5

40 min. 95 5 40 min. 95 5

vrijeme retencije (min.) retention time (min.)

Spoj 1 25.2 Connection 1 25.2

Spoj 1b 8.5 Compound 1b 8.5

Spoj 10 20.5 Compound 10 20.5

Spoj 11 26.3 Compound 11 26.3

Spoj 12 14.8 Compound 12 14.8

Spoj 14 25.6 Connection 14 25.6

Spoj 15 15.7 Compound 15 15.7

Iako je izum opisan i ilustriran kroz nekoliko njegovih pojedinih oblika, stručnjaci će uvidjeti da su moguće brojne adaptacije, izmjene, modifikacije, zamjene, oduzimanja ili dodavanja postupaka i protokola, bez iskoraka izvan zamisli i okvira izuma. Na primjer, mogu se primijeniti uvjeti reakcije različiti od gore opisanih, i to kao rezultat promjena reagensa ili metodologije priprave spojeva procesa ovog izuma. Slično tome, specifična reaktivnost početnih tvari može varirati ovisno o pojedinim nazočnim supstituentima ili uvjetima izrade, pa se očekivane varijacije ili razlike u rezultatima moraju promatrati u odnosu na objekte i postupke ovog izuma. Potrebno je, dakle, shvatiti izum prema području slijedećih zahtjeva uz razumnu širinu njihove interpretacije. Although the invention is described and illustrated through several of its individual forms, experts will see that numerous adaptations, changes, modifications, substitutions, subtractions or additions of procedures and protocols are possible, without stepping outside the imagination and scope of the invention. For example, reaction conditions other than those described above may be applied as a result of changes in the reagents or the methodology of preparing the compounds of the process of this invention. Similarly, the specific reactivity of the starting materials may vary depending on the particular substituents present or manufacturing conditions, so expected variations or differences in results must be viewed in relation to the objects and processes of this invention. It is therefore necessary to understand the invention according to the scope of the following requirements with a reasonable breadth of interpretation.

Claims (30)

1. Postupak priprave spoja formule V: [image] naznačen time, što se sastoji od: (1) spajanja aminokiseline formule: [image] sa spojem formule: [image] u nazočnosti prvog kiselog aktivirajućeg sredstva u prvom inertnom otapalu u nazočnosti prvog katalizirajućeg sredstva, čime se dobije spoj formule I: [image] gdje je L amino zaštitna skupina, nakon čega slijedi: (2) reagiranje spoja formule I s prvim amino deprotekcijskim sredstvom, čime se dobije spoj formule II: [image] nakon čega slijedi: (3) spajanje aminokiseline formule: [image] gdje je L amino zaštitna skupina, sa spojem formule II u nazočnosti drugog kiselog aktivirajućeg sredstva u drugom inertnom otapalu u nazočnosti drugog katalizirajućeg sredstva, čime se dobije spoj formule III: [image] gdje je L amino zaštitna skupina, nakon čega slijedi: (4) reagiranje spoja formule III s drugim amino deprotekcijskim sredstvom, čime se dobije spoj formule IV, ili njegove farmaceutski prikladne soli: [image] nakon čega slijedi: reagiranje spoja formule IV s metansulfonskom kiselinom, čime se dobije spoj formule V.1. Procedure for preparing the compound of formula V: [image] characterized by consisting of: (1) joining amino acids of the formula: [image] with a compound of the formula: [image] in the presence of a first acidic activating agent in a first inert solvent in the presence of a first catalyzing agent, thereby obtaining a compound of formula I: [image] where L is an amino protecting group, followed by: (2) reacting the compound of formula I with the first amino deprotection agent, thereby obtaining the compound of formula II: [image] followed by: (3) joining the amino acid of the formula: [image] where L is an amino protecting group, with a compound of formula II in the presence of a second acidic activating agent in a second inert solvent in the presence of a second catalyzing agent to give a compound of formula III: [image] where L is an amino protecting group, followed by: (4) reacting the compound of formula III with another amino deprotection agent, thereby obtaining the compound of formula IV, or its pharmaceutically suitable salts: [image] followed by: reacting the compound of formula IV with methanesulfonic acid to obtain the compound of formula V. 2. Postupak priprave spoja formule I: [image] gdje je L amino zaštitna skupina, naznačen time, što se sastoji od spajanja aminokiseline formule: [image] sa spojem formule: [image] u nazočnosti kiselog aktivirajućeg sredstva u inertnom otapalu u nazočnosti katalizirajućeg sredstva, čime se dobije spoj formule I.2. Procedure for preparing the compound of formula I: [image] where L is an amino protecting group, indicated by the fact that it consists of joining an amino acid of the formula: [image] with a compound of the formula: [image] in the presence of an acidic activating agent in an inert solvent in the presence of a catalyzing agent, thereby obtaining a compound of formula I. 3. Postupak iz Zahtjeva 2, naznačen time, što je kiselo aktivirajuće sredstvo DCC.3. The method of claim 2, characterized in that the acid activating agent is DCC. 4. Postupak iz Zahtjeva 2, naznačen time, što je katalizirajuće sredstvo HOBT.4. The method from Claim 2, characterized in that the catalyzing agent is HOBT. 5. Postupak iz Zahtjeva 2, naznačen time, što je otapalo odabrano iz skupine koja se sastoji od: acetonitrila; izopropil acetata; etil acetata; propionitrila; vode; kloriranih ugljikovodika poput diklormetana, kloroforma, ugljik tetraklorida, dikloretana, klorbenzena, orto-diklorbenzena; benzena; toluena; ksilena; i drugih; kao i njihovih smjesa.5. The method of claim 2, characterized in that the solvent is selected from the group consisting of: acetonitrile; isopropyl acetate; ethyl acetate; propionitrile; water; chlorinated hydrocarbons such as dichloromethane, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene, ortho-dichlorobenzene; benzene; toluene; xylene; and others; as well as their mixtures. 6. Postupak iz Zahtjeva 5, naznačen time, što je otapalo odabrano iz skupine koja se sastoji od: izopropil acetata; vode; i acetonitrila.6. The method of claim 5, characterized in that the solvent is selected from the group consisting of: isopropyl acetate; water; and acetonitrile. 7. Postupak iz Zahtjeva 2, naznačen time, što je temperatura reakcije između 20 i 35°C.7. The process from Claim 2, characterized in that the reaction temperature is between 20 and 35°C. 8. Postupak iz Zahtjeva 2, naznačen time, što je za spoj formule I odabrana amino zaštitna skupina: t-butoksi-karbonil.8. The method from Claim 2, characterized in that the amino protecting group selected for the compound of formula I is: t-butoxy-carbonyl. 9. Postupak iz Zahtjeva 2, naznačen time, što se odvija in situ bez izoliranja spoja formule I nakon njegove priprave.9. The process from Claim 2, characterized in that it takes place in situ without isolating the compound of formula I after its preparation. 10. Postupak priprave spoja formule II: [image] naznačen time, što se sastoji od reagiranja spoja formule I: [image] gdje je L amino zaštitna skupina, s amino deprotekcijskim sredstvom, čime se dobije spoj formule II.10. Procedure for preparing the compound of formula II: [image] characterized by the reaction of the compound of formula I: [image] where L is an amino protecting group, with an amino deprotecting agent, giving a compound of formula II. 11. Postupak iz Zahtjeva 10, naznačen time, što je za spoj formule I odabrana amino zaštitna skupina: t-butoksi-karbonil.11. The method from Claim 10, characterized in that the amino protecting group selected for the compound of formula I is: t-butoxy-carbonyl. 12. Postupak iz Zahtjeva 10, naznačen time, što je amino deprotekcijsko sredstvo metansulfonska kiselina.12. The method from Claim 10, characterized in that the amino deprotecting agent is methanesulfonic acid. 13. Postupak iz Zahtjeva 10, naznačen time, što se odvija in situ bez izoliranja spoja formule II nakon njegove priprave.13. The process from Claim 10, characterized in that it takes place in situ without isolating the compound of formula II after its preparation. 14. Postupak priprave spoja formule III: [image] gdje je L amino zaštitna skupina, naznačen time, što se sastoji od spajanja aminokiseline formule: [image] gdje je L amino zaštitna skupina, sa spojem formule II, [image] u nazočnosti kiselog aktivirajućeg sredstva u inertnom otapalu u nazočnosti katalizirajućeg sredstva, čime se dobije spoj formule III.14. Procedure for preparing the compound of formula III: [image] where L is an amino protecting group, indicated by the fact that it consists of joining an amino acid of the formula: [image] where L is an amino protecting group, with a compound of formula II, [image] in the presence of an acidic activating agent in an inert solvent in the presence of a catalyzing agent, thereby obtaining a compound of formula III. 15. Postupak iz Zahtjeva 14, naznačen time, što je kiselo aktivirajuće srdstvo DCC.15. The method of Claim 14, characterized in that the acid activating core is DCC. 16. Postupak iz Zahtjeva 14, naznačen time, što je katalizirajuće sredstvo HOBT.16. The method of Claim 14, characterized in that the catalyzing agent is HOBT. 17. Postupak iz Zahtjeva 14, naznačen time, što je otapalo izopropil acetat:voda.17. The method of Claim 14, characterized in that the solvent is isopropyl acetate:water. 18. Postupak iz Zahtjeva 17, naznačen time, što je otapalo izopropil acetat:voda u omjeru od približno 40:60 do 60:40 (vol. udjeli).18. The method of Claim 17, characterized in that the solvent is isopropyl acetate:water in a ratio of approximately 40:60 to 60:40 (volume fractions). 19. Postupak iz Zahtjeva 14, naznačen time, što je temperatura reakcije između 20 i 35°C19. The process from Claim 14, characterized in that the reaction temperature is between 20 and 35°C 20. Postupak iz Zahtjeva 14, naznačen time, što je za spoj formule III odabrana amino zaštitna skupina: t-butoksi-karbonil.20. The method from Claim 14, characterized in that the amino protecting group selected for the compound of formula III is: t-butoxy-carbonyl. 21. Postupak iz Zahtjeva 14, naznačen time, što se odvija in situ bez izoliranja spoja formule III nakon njegove priprave.21. The process from Claim 14, characterized in that it takes place in situ without isolating the compound of formula III after its preparation. 22. Postupak priprave spoja formule IV, ili njegove farmaceutski prikladne soli: [image] naznačen time, što se sastoji od reagiranja spoja formule III: [image] gdje je L amino zaštitna skupina, sa amino deprotekcijskim sredstvom, čime se dobije spoj formule IV.22. Process for the preparation of the compound of formula IV, or its pharmaceutically suitable salt: [image] characterized by the reaction of the compound of formula III: [image] where L is an amino protecting group, with an amino deprotecting agent, giving a compound of formula IV. 23. Postupak iz Zahtjeva 22, naznačen time, što je za spoj formule III odabrana amino zaštitna skupina: t-butoksi-karbonil.23. The method from Claim 22, characterized in that the amino protecting group selected for the compound of formula III is: t-butoxy-carbonyl. 24. Postupak iz Zahtjeva 22, naznačen time, što je amino deprotekcijsko sredstvo metansulfonska kiselina.24. The method of claim 22, characterized in that the amino deprotecting agent is methanesulfonic acid. 25. Postupak iz Zahtjeva 22, naznačen time, što se odvija u otopini koja je etanol.25. The method from Claim 22, characterized in that it takes place in a solution that is ethanol. 26. Postupak iz Zahtjeva 22, naznačen time, što se odvija in situ bez izoliranja spoja formule IV nakon njegove priprave.26. The process from Claim 22, characterized in that it takes place in situ without isolating the compound of formula IV after its preparation. 27. Postupak priprave spoja formule V: [image] naznačen time, što se sastoji od reagiranja spoja formule IV: [image] s metansulfonskom kiselinom, čime se dobije spoj formule V.27. Procedure for preparing the compound of formula V: [image] characterized in that it consists of reacting a compound of formula IV: [image] with methanesulfonic acid to give the compound of formula V. 28. Postupak iz Zahtjeva 27, naznačen time, što se odvija u otopini koja se sastoji od etil acetata i etanola.28. The process from Claim 27, characterized in that it takes place in a solution consisting of ethyl acetate and ethanol. 29. Postupak priprave polimorfnog oblika, označenog kao Oblik I N-[1(R)-[(1,2-dihidro-1- metan sulfonil- spiro[3H-indol-3,4'-piperdin]-1'-il)karbonil]-2-(fenilmetiloksi)etil]-2-amino-2-metilpropanamida, naznačen time, što se sastoji od: djelovanja na otopinu slobodne baze N-[1(R)-[(1,2-dihidro-1- metansulfonil- spiro[3H- indol-3,4'- piperdin]-1'-il)karbonil]-2-(fenilmetiloksi)etil]-2-amino-2-metilpropanamida u etil acetatu koji sadrži oko 8 vol.% etanola 1.1 ekvivalentom metansulfonske kiseline na temperaturi od približno 50°C, nakon čega slijedi zagrijavanje na približno 55°C, te hlađenje na približno 45°C.29. Procedure for the preparation of the polymorphic form, designated as Form I N-[1(R)-[(1,2-dihydro-1-methane sulfonyl-spiro[3H-indol-3,4'-piperdin]-1'-yl )carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamide, characterized in that it consists of: acting on a solution of the free base N-[1(R)-[(1,2-dihydro-1 - methanesulfonyl-spiro[3H-indol-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamide in ethyl acetate containing about 8 vol.% of ethanol 1.1 equivalent of methanesulfonic acid at a temperature of approximately 50°C, followed by heating to approximately 55°C, and cooling to approximately 45°C. 30. Postupak iz Zahtjeva 29, naznačen time, što se temperatura postupno povećava do približno 51°C i na toj vrijednosti održava 2-24 sata.30. The method from Claim 29, characterized in that the temperature is gradually increased to approximately 51°C and maintained at that value for 2-24 hours.