Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
  • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
  • C07D471/10—Spiro-condensed systems
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P5/00—Drugs for disorders of the endocrine system
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/06—Dipeptides
  • C07K5/06008—Dipeptides with the first amino acid being neutral
  • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
  • C07K5/06034—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• Growth hormone which is secreted from the pituitary, stimulates growth of all tissues of the body that are capable of growing.
• growth hormone is known to have the following basic effects on the metabolic processes of the body: (1) Increased rate of protein synthesis in all cells of the body; (2) Decreased rate of carbohydrate utilization in cells of the body; (3) Increased mobilization of free fatty acids and use of fatty acids for energy.
• a deficiency in growth hormone secretion can result in various medical disorders, such as dwarfism.
• Various ways are known to release growth hormone.
• chemicals such as arginine, L-3,4-dihydroxyphenyl- alanine (L-DOPA), glucagon, vasopressin, and insulin induced hypoglycemia, as well as activities such as sleep and exercise, indirectly cause growth hormone to be released from the pituitary by acting in some fashion on the hypothalamus perhaps either to decrease somatostatin secretion or to increase the secretion of the known secretagogue growth hormone releasing factor (GRF) or an unknown endogenous growth hormone-releasing hormone or all of these.
• GRF growth hormone releasing factor
• the problem was generally solved by providing exogenous growth hormone or by administering GRF or a peptidal compound which stimulated growth hormone production and/or release.
• the peptidyl nature of the compound necessitated that it be administered by injection.
• the source of growth hormone was the extraction of the pituitary glands of cadavers. This resulted in a very expensive product and carried with it the risk that a disease associated with the source of the pituitary gland could be transmitted to the recipient of the growth hormone.
• Recombinant growth hormone has become available which, while no longer carrying any risk of disease transmission, is still a very expensive product which must be given by injection or by a nasal spray.
• Other compounds have been developed which stimulate the release of endogenous growth hormone.
• certain spiro compounds are disclosed in U.S. Patent No. 5,536,716, PCT Patent Publication WO 94/13696 and Proc. Natl. Acad. Sci. USA. 92, 7001-7005 (July 1995) as being non-peptidal growth hormone secretagogues. These compounds have the ability to stimulate the release of natural or endogenous growth hormone and thus may be used to treat conditions which require the stimulation of growth hormone production or secretion such as in humans with a deficiency of natural growth hormone or in animals used for food or wool production where the stimulation of growth hormone will result in a larger, more productive animal.
• Example 55 states that the compound prepared by recrystalhzation from ethyl acetate-ethanol-water had a melting point of "166-168°C". This compound was subsequently identified as being of the polymorphic form designated "Form II" herein.
• Proc. Natl. Acad. Sci. USA. 92, 7001-7005 notes that the compound isolated as a monohydrate had a melting point of 168-170°C, but only discloses very general methods for preparing the compound and does not disclose how the compound was crystallized.
• Morphological forms of pharmaceutical compounds may be of interest to those involved in the development of a suitable dosage form because if the morphological form is not held constant during clinical and stability studies, the exact dosage used or measured may not be comparable from one lot to the next.
• a pharmaceutical compound is produces for use, it is important to recognize the morphological form delivered in each dosage form to assure that the production process use the same form and that the same amount of drug is included in each dosage. Therefore, it is imperative to assure that either a single morphological form or some known combination of morphological forms is present.
• certain morphological forms may exhibit enhanced thermodynamic or hydroscopic stability and may be more suitable than other morphological forms for inclusion in pharmaceutical formulations.
• a "polymorphic form" of a chemical compound is the same chemical entity, but in a different crystalline arrangement.
• the present invention is concerned with polymorphic forms of the compound: N-[l(R)-[(l,2-dihydro-l-methanesulfonyl- spiro[3H-indole-3,4'-piperdin]- -yl)carbonyl]-2-(phenylmethyl- oxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate, as well as processes for the preparation of such polymorphic forms.
• the present invention is also concerned with pharmaceutical formulations comprising these polymorphic forms as an active ingredient and the use of these polymorphic forms and their formulations in the treatment of certain disorders.
• the polymorphic forms of this invention are growth hormone secretagogue that are useful in food animals to promote their growth thereby rendering the production of edible meat products more efficient, and in humans, to treat physiological or medical conditions characterized by a deficiency in growth hormone secretion, and to treat medical conditions which are improved by the anabolic effects of growth hormone.
• the present invention is directed to novel polymorphic forms of the compound N-[l(R)-[(l,2-dihydro-l-methanesulfonyl- spiro[3H-indole-3,4'-piperdin]- -yl)carbonyl]-2-(phenylmethyl- oxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate and the processes for the preparation of these polymorphic forms.
• This property can be utilized to promote the growth of food animals to render the production of edible meat products more efficient, and in humans, to treat physiological or medical conditions characterized by a deficiency in growth hormone secretion, and to treat medical conditions which are improved by the anabolic effects of growth hormone.
• the present invention is also concerned with a process for the preparation of Form I of N-[l(R)-[(l,2-dihydro-l-methanesulfonyl- spiro[3H-indole-3,4'-piperdin]-l'-yl)carbonyl]-2-(phenylmethyl- oxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate which comprises: treating a solution of the free base of N-[1(R)-[(1,2- dihydro-l-methanesulfonyl-spiro[3H-indole-3,4'-piperdin]-l'- yl)carbonyl]-2-(phenylmethyl-oxy)ethyl]-2-amino-2-methylpropanamide in ethyl acetate containing ethanol (about 8 volume %) with methanesulfonic acid (about 1.1 equivalents) at approximately 50°C, heating
• the temperature subsequently may be raised to approximately 51°C where it is maintained for 2-24 hours.
• the present invention is further concerned with an alternate process for the preparation of Form I of N-[1(R)-[(1,2- dihydro-l-methanesulfonyl-spiro[3H-indole-3,4'-piperdin]-l'- yl)carbonyl]-2-(phenylmethyl-oxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate which comprises: alternatively adding to a solution of the free base of N- [l(R)-[(l,2-dihydro-l-methanesulfonyl-spiro[3H-indole-3,4'-piperdin]- r-yl)carbonyl]-2-(phenylmethyl-oxy)ethyl]-2-amino-2-methylpropan- amide in ethyl acetate containing ethanol (about 8
• the present invention is further concerned with an alternate process for the preparation of Form I of N-[1(R)-[(1,2- dihydro-l-methanesulfonyl-spiro[3H-indole-3,4'-piperdin]- - yl)carbonyl]-2-(phenylmethyl-oxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate which comprises: stirring a solution of Form II of N-[l(R)-[(l,2-dihydro-l- methanesulfonyl-spiro[3H-indole-3,4'-piperdin]- -yl)carbonyl]-2- (phenylmethyl-oxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate in isopropanol at approximately 25°C for about 2-24 hours.
• the present invention is also concerned with a process for the preparation of Form II of N-[l(R)-[(l,2-dihydro-l-methane- sulfonyl-spiro[3H-indole-3,4'-piperdinl- -yl)carbonyl]-2-(phenyl- methyl-oxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate which comprises: treating a solution of the free base of N-[1(R)-[(1,2- dihydro- l-methanesulfonyl-spiro[3H-indole-3,4'-piperdin]- 1 '- yl)carbonyl]-2-(phenylmethyl-oxy)ethyl]-2-amino-2-methylpropanamide in ethyl acetate containing ethanol (about 8 volume %) with methanesulfonic acid (about 1.1 equivalents) at approximately 50°
• the present invention is further concerned with a process for the preparation of Form IV of N-[l(R)-[(l,2-dihydro-l- methanesulfonyl-spiro[3H-indole-3,4'-piperdin]- -yl)carbonyl]-2- (phenylmethyl-oxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate which comprises: dissolving N-[ 1 (R)-[( 1 ,2-dihydro- 1 -methanesulfonyl- spiro[3H-indole-3,4'-piperdin]-l'-yl)carbonyl]-2-(phenylmethyl- oxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate of optional mo ⁇ hological composition in a solution of ethanol/water (preferably 25:75 v/v); evaporating the solvent from the solution, preferably
• the present invention is further concerned with an alternate process for the preparation of Form IV of N-[1(R)-[(1,2- dihydro-l-methanesulfonyl-spiro[3H-indole-3,4'-piperdin]- - yl)carbonyll-2-(phenylmethyl-oxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate which comprises: recrystalhzation of N-[l(R)-[(l,2-dihydro-l- methanesulfonyl-spiro[3H-indole-3,4'-piperdin]- -yl)carbonyl]-2- (phenylmethyl-oxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate of optional mo ⁇ hological composition from a solution of ethylacetate/ethanol/water (preferably 24.8/1.6/1.95 v/v/v).
• the present invention is further concerned with an alternate process for the preparation of Form IV of N-[1(R)-[(1,2- dihydro-l-methanesulfonyl-spiro[3H-indole-3,4'-piperdin]-l'- yl)carbonyl]-2-(phenylmethyl-oxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate which comprises: exposing Form I of N-[l(R)-[(l,2-dihydro-l- methanesulfonyl-spiro[3H-indole-3,4'-piperdin]- -yl)carbonyl]-2- (phenylmethyl-oxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate to a relative humidity of greater than about 75%, more preferably a relative humidity of about 88%, at ambient temperature for a sufficient time.
• the present invention is further concerned with an alternate process for the preparation of Form IV of N-[1(R)-[(1,2- dihydro- l-methanesulfonyl-spiro[3H-indole-3,4'-piperdin]- 1'- yl)carbonyl]-2-(phenylmethyl-oxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate which comprises: isolation from a slurry of Form I of N-[1(R)-[(1,2- dihydro-l-methanesulfonyl-spiro[3H-indole-3,4'-piperdin]- - yl)carbonyl]-2-(phenylmethyl-oxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate in isopropyl acetate/ethanol (90:10 v/v) containing approximately 2.8 wt% water at approximately 25°C
• the present invention is further concerned with a process for the preparation of Form V of N-[l(R)-[(l,2-dihydro-l- methanesulfonyl-spiro[3H-indole-3,4'-piperdin]- -yl)carbonyl]-2- (phenylmethyl-oxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate which comprises: exposing Form IV of N-[l(R)-[(l,2-dihydro-l- methanesulfonyl-spiro[3H-indole-3,4'-piperdin]- 1 '-yl)carbonyl]-2- (phenylmethyl-oxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate to below 30% relative humidity at ambient temperature.
• the present invention is further concerned with a process for the preparation of Form VI of N-[l(R)-[(l,2-dihydro-l- methanesulfonyl-spiro[3H-indole-3,4'-piperdin]- -yl)carbonyl]-2- (phenylmethyl-oxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate which comprises: drying Form V of N-[l(R)-[(l,2-dihydro-l-methane- sulfonyl-spiro[3H-indole-3,4'-piperdin]- 1 '-yl)carbonyl]-2-(phenyl- methyl-oxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate in the absence of moisture at approximately room temperature, such as under an atmosphere of sieve dried nitrogen at approximately 25°C.
• the present invention is further concerned with a process for the preparation of Form VII of N-[l(R)-[(l,2-dihydro-l- methanesulfonyl-spiro[3H-indole-3,4'-piperdin]-r-yl)carbonyl]-2- (phenylmethyl-oxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate which comprises: isolation from a slurry of Form I or Form IV of N-[1(R)- [( 1 ,2-dihydro- 1 -methanesulfonyl-spiro[3H-indole-3,4'-piperdin]- 1 '- yl)carbonyl]-2-(phenylmethyl-oxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate in isopropyl acetate/ethanol (90: 10 v/v) containing approximately 1.5 wt
• the present invention is further concerned with a process for the preparation of Form VIII of N-[l(R)-[(l,2-dihydro-l- methanesulfonyl-spiro[3H-indole-3,4'-piperdin]-r-yl)carbonyl]-2- (phenylmethyl-oxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate which comprises: dehydrating Form VII of N-[l(R)-[(l,2-dihydro-l- methane-sulfonyl-spiro[3H-indole-3,4'-piperdin]- -yl)carbonyl]-2- (phenyl-methyl-oxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate under a dry inert gas, such as dry nitrogen, for a sufficient time.
• a dry inert gas such as dry nitrogen
• the present invention is further concerned with a process for the preparation of Form IX of N-[l(R)-[(l,2-dihydro-l- methanesulfonyl-spiro[3H-indole-3,4'-piperdin]-l'-yl)carbonyl]-2- (phenylmethyl-oxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate which comprises: preparing a solution of N-[l(R)-[(l ,2-dihydro-l-methane- sulfonyl-spiro[3H-indole-3,4'-piperdin]-r-yl)carbonyl]-2-(phenyl- methyl-oxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate of optional mo ⁇ hological composition in water, followed by isolating the solid formed by controlled evaporation at 20% relative humidity at approximately room temperature
• the present invention is further concerned with a process for the preparation of Form X of N-[l(R)-[(l,2-dihydro-l- methanesulfonyl-spiro[3H-indole-3 ,4'-piperdin]- 1 '-yl)carbonyl]-2- (phenylmethyl-oxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate which comprises: drying Form IX of N-[l(R)-[(l,2-dihydro-l-methane- sulfonyl-spiro[3H-indole-3,4'-piperdin]- -yl)carbonyl]-2-(phenyl- methyl-oxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate at ambient room temperature and humidity for a sufficient time.
• the present invention is further concerned with an alternate process for the preparation of Form X of N-[1(R)-[(1,2- dihydro-l-methanesulfonyl-spiro[3H-indole-3,4'-piperdin]-l'- yl)carbonyl]-2-(phenylmethyl-oxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate which comprises: exposing Form I of N-[l(R)-[(l,2-dihydro-l-methane- sulfonyl-spiro[3H-indole-3,4'-piperdin]-l'-yl)carbonyl]-2-(phenyl- methyl-oxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate to 100% relative humidity for approximately 1 to 4 days.
• the present invention is also directed to a process for the preparation of mo ⁇ hologically homogeneous N-[1(R)-[(1,2- dihydro-l-methanesulfonyl-spiro[3H-indole-3,4'-piperdin]-r- yl)carbonyl]-2-(phenylmethyl-oxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate comprising any of the processes mentioned herein.
• the compounds of this invention are growth hormone secretagogues that are useful in food animals to promote their growth thereby rendering the production of edible meat products more efficient, and in humans, to treat physiological or medical conditions characterized by a deficiency in growth hormone secretion, and to treat medical conditions which are improved by the anabolic effects of growth hormone.
• the present invention is further concerned with pharmaceutical formulations comprising a polymo ⁇ hic form as an active ingredient, and the use of this polymo ⁇ hic form and its formulations in the treatment of certain disorders.
• Differential Scanning Calorimeteric Cell TDSC1 Differential Scanning Calorimeteric Cell TDSC1
• Form I 170°C (melting endotherm)
• Form II 165°C (melting endotherm)
• Form VI 129°C (melting endotherm)
• Form I of N-[l(R)-[(l,2-dihydro-l-methanesulfonyl- spiro[3H-indole-3,4'-piperdin]-r-yl)carbonyl]-2-(phenylmethyl- oxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate is a relatively anhydrous polymo ⁇ h characterized by the following properties: a melting point of 169°C and solubility in isopropanol of 4.6 mg/mL.
• Form II of N-[l(R)-[(l,2-dihydro-l-methanesulfonyl- spiro[3H-indole-3,4'-piperdin]- -yl)carbonyl]-2-(phenylmethyl- oxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate is an anhydrous polymo ⁇ h characterized by the following properties: a melting point of 158°C and solubility in isopropanol of 12.3 mg/mL.
• Form III of N-[l(R)-[(l,2-dihydro-l-methanesulfonyl- spiro[3H-indole-3,4'-piperdin]- -yl)carbonyl]-2-(phenylmethyl- oxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate is a hydrate characterized by the following properties: a water loss endotherm at a peak temperature of 46°C, followed by a minor melting/decomposition endotherm with an extrapolated onset temperature of 123°C.
• Form IV of N-[l(R)-[(l,2-dihydro-l-methanesulfonyl- spiro[3H-indole-3,4'-piperdin]-r-yl)carbonyl]-2-(phenylmethyl- oxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate is a hydrate characterized by the following properties: a water loss endotherm at a peak temperature of 45°C, followed by a melting/decomposition endotherm with an extrapolated onset temperature of 129°C (presumably due to the melting/ decomposition of Form VI).
• N-[l(R)-[(l,2-dihydro-l-methanesulfonyl- spiro[3H-indole-3,4'-piperdin]-l'-yl)carbonyl]-2-(phenylmethyl- oxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate appears to be a hygroscopic hydrate containing 3.5 moles of water per mole of N-[l (R)-[( 1 ,2-dihydro- l-methanesulfonyl-s ⁇ iro[3H-indole-3,4'- piperdin]-l'-yl)carbonyl]-2-(phenylmethyl-oxy)ethyl]-2-amino-2- methylpropanamide methanesulfonate.
• Form VII of N-[l(R)-[(l,2-dihydro-l-methanesulfonyl- spiro[3H-indole-3,4'-piperdin]- 1 '-yl)carbonyl]-2-(phenylmethyl- oxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate is a hydrate characterized by the following properties: a broad water loss endotherm at a peak temperature of 60°C, followed by a melting/decomposition endotherm with an extrapolated onset temperature of 144°C (presumably due to the melting/decomposition of Form VIII).
• Form VIII N-[l(R)-[(l,2-dihydro-l-methanesulfonyl- spiro[3H-indole-3,4'-piperdin]-r-yl)carbonyl]-2-(phenylmethyl- oxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate is an anhydrous polymo ⁇ h characterized by a melting point of 144°C.
• Form X of N-[l(R)-[(l,2-dihydro-l-methanesulfonyl- spiro[3H-indole-3,4'-piperdin]- -yl)carbonyl]-2-(phenylmethyl- oxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate is characterized by a broad water loss endotherm at a peak temperature of 49°C.
• X-Ray powder diffraction studies are widely used to elucidate molecular structures, crystallinity and polymo ⁇ hism.
• X-ray powder diffraction (XRPD) patterns were collected using a Philips APD3720 Automated Powder Diffraction instrument with copper K ⁇ radiation. Measurements were made from 2° to 40° (2 theta) with the sample maintained at ambient room temperature.
• Form I was characterized by an X-ray powder diffraction pattern with principal reflections at approximately: 6.5, 14.7, 16.9, 17.1, 17.9, 19.5, 21.1, 21.7, and 22.0° (2 theta).
• Form II was characterized by an X-ray powder diffraction pattern with principal reflections at approximately: 4.8, 11.8, 17.5, 19.4, 21.6, 21.9, 22.5, and 22.7° (2 theta).
• Form III was characterized by an X-ray powder diffraction pattern with principal reflections at approximately: 13.8, 14.1, 18.0, 18.8, 19.5, 20.1, 20.6, 21.8, and 25.7° (2 theta).
• Form IV was characterized by an X-ray powder diffraction pattern with principal reflections at approximately: 16.0, 16.2, 18.3, 20.1, 21.0, and 24.2° (2 theta).
• Form V was characterized by an X-ray powder diffraction pattern with principal reflections at approximately: 14.8, 17.1, 17.6, 19.0, 19.1, 19.4, 20.6, 21.5, and 21.8° (2 theta).
• Form VI was characterized by an X-ray powder diffraction pattern with principal reflections at approximately: 9.8, 14.0, 14.8, 17.1, 17.6, 19.0, 19.5, 20.6, and 21.6° (2 theta).
• Form VII was characterized by an X-ray powder diffraction pattern with principal reflections at approximately: 9.1,
• Form VIII was characterized by an X-ray powder diffraction pattern with principal reflections at approximately: 11.5, 11.6, 18.1, 19.6, 22.5, 24.7, and 24.8° (2 theta).
• Form IX was characterized by an X-ray powder diffraction pattern with principal reflections at approximately: 8.0, 12.1, 15.3, 15.8, 19.6, 19.7, 21.1, 22.3, and 23.7° (2 theta).
• Form X was characterized by an X-ray powder diffraction pattern with principal reflections at approximately: 15.5, 15.8, 18.0,
• Solubility The solubility of Form I in distilled water at room temperature is > 100 mg/mL.
• the solubility of Form I in ethanol/water mixtures is shown below:
• the CBZ-Spiroindoline 1 is treated with Darco (20% by weight) prior to hydrogenation.
• the hydrogenation is carried out in ethanol at 65°C over 10% Pd/C with vigorous stirring.
• a solution of lb 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).
• DCC dicyclohexylcarbodiimide
• HOBt 1-hydroxybenzotriazole
• the BOC-group of JJ_ is removed by treatment with methanesulfonic acid (MsOH) (3 eq) in ethanol at 35-40°C. Partitioning between isopropyl acetate and aqueous IM sodium hydroxide solution affords 12.
• MsOH methanesulfonic acid
• the coupling of 12 with N-BOC- ⁇ -aminoisobutyric acid is best conducted in a two-phase solvent system, isopropyl acetate/water ( 1 : 1 ) in the presence of DCC and HOBt (1.1 eq. each). Removal of the DCU by filtration, separation of the layers and washing the organic layer successively with IM aqueous sodium hydroxide, 0.5M aqueous hydrochloric acid and saturated aqueous sodium hydrogen carbonate affords 14. The mixture is solvent switched to ethanol for the subsequent methanesulfonic acid cleavage of the Boc group. Deprotection of 14 is more difficult than that of H and requires a concentrated solution of ethanol methanesulfonic acid and heating to 35- 40°C. After extractive workup (EtOAc-NaOH), the free amine 15 is isolated. The organic layer is washed well with IN NaOH to ensure complete removal of methanesulfonic acid.
• the ethyl acetate solution of the free base JJ> is concentrated to low bulk in vacuo and is azeotroped dry (KF ⁇ 500 mgml " ) by "feeding and bleeding" 2x batch volumes of 90/10, ethyl acetate/ethanol followed by 2x batch volumes of ethyl acetate.
• the resulting dry, slightly hazy solution of the free base 15 in ethyl acetate is treated with Darco G-60 (25 weight %) at room temperature for about 10 hours. Removal of the Darco by filtration with a filtration agent gives the free base 15.
• Formation of the methanesulfonic acid salt 16 from 15 is carried out in EtOAc with 1.1 eq of MsOH at about 50°C.
• Form II The conversion of Form II to Form I is accomplished where the salt is formed in EtOAc-EtOH as above, but instead of cooling the initial solution of the salt (at 55°C) to ambient temperature, it is cooled to 45°C. Crystals should start appearing at that temperature and the slurry should become thicker with time. The temperature is then raised to 51 °C and the slurry is aged overnight. Complete conversion to Form I of 16 should be expected.
• the conversion of Form II to Form I is achieved by adding seed crystals of Form I to a solution of the free base in EtOAc-EtOH at 50-55°C followed by aging.
• the free base 15 may be treated with 1.1 equivs. of methanesulfonic acid in 8% ethanol in ethyl acetate at 50-55 °C.
• the batch is then seeded with approximately 2% by weight of Form I of the methanesulfonate salt 16, and then aged at 55°C overnight.
• the batch is cooled to room temperature and aged 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 to give the methanesulfonate salt 16.
• the methanesulfonic acid salt 16 may also be formed by alternating the stepwise addition of MsOH (1.1 eq) and seed crystals of Form I to a solution of the free base in EtOAc-EtOH at about 50°C, wherein the order of addition of the MsOH and the seed is not critical.
• the utility of the polymo ⁇ hic compounds of the present invention as growth hormone secretagogues may be demonstrated by methodology known in the art, such as an assay described by Smith , et al., Science. 260. 1640-1643 (1993) (see text of Figure 2 therein).
• all of the polymo ⁇ hic forms of the present invention had activity as growth hormone secretagogues in the aforementioned assay. Such a result is indicative of the intrinsic activity of the polymo ⁇ hic forms of the present invention as growth hormone secretagogues.
• the growth hormone releasing compounds of the present invention are useful in vitro as unique tools for understanding how growth hormone secretion is regulated at the pituitary level. This includes use in the evaluation of many factors thought or known to influence growth hormone secretion such as age, sex, nutritional factors, glucose, amino acids, fatty acids, as well as fasting and non- fasting states. In addition, the compounds of this invention can be used in the evaluation of how other hormones modify growth hormone releasing activity. For example, it has already been established that somatostatin inhibits growth hormone release.
• hormones that are important and in need of study as to their effect on growth hormone release include the gonadal hormones, e.g., testosterone, estradiol, and progesterone; the adrenal hormones, e.g., cortisol and other corticoids, epinephrine and norepinephrine; the pancreatic and gastrointestinal hormones, e.g., insulin, glucagon, gastrin, secretin; the vasoactive peptides, e.g., bombesin, the neurokinins; and the thyroid hormones, e.g., thyroxine and triiodothyronine.
• gonadal hormones e.g., testosterone, estradiol, and progesterone
• the adrenal hormones e.g., cortisol and other corticoids, epinephrine and norepinephrine
• the pancreatic and gastrointestinal hormones e.g., insulin, glucagon,
• the compounds of the present invention may also be employed to investigate the possible negative or positive feedback effects of some of the pituitary hormones, e.g., growth hormone and endo ⁇ hin peptides, on the pituitary to modify growth hormone release.
• some of the pituitary hormones e.g., growth hormone and endo ⁇ hin peptides
• the compounds of the present invention may also be employed to investigate the possible negative or positive feedback effects of some of the pituitary hormones, e.g., growth hormone and endo ⁇ hin peptides, on the pituitary to modify growth hormone release.
• some of the pituitary hormones e.g., growth hormone and endo ⁇ hin peptides
• the compounds of the present invention may be administered to animals, including man, to release growth hormone in vivo.
• the compounds can be administered to commercially important animals such as swine, cattle, sheep and the like to accelerate and increase their rate and extent of growth, to improve feed efficiency and to increase milk production in such animals.
• these compounds can be administered to humans in vivo as a diagnostic tool to directly determine whether the pituitary is capable of releasing growth hormone.
• the compounds of the present invention can be administered in vivo to children. Serum samples taken before and after such administration can be assayed for growth hormone. Comparison of the amounts of growth hormone in each of these samples would be a means for directly determining the ability of the patient's pituitary to release growth hormone.
• the present invention includes within its scope pharmaceutical compositions comprising, as an active ingredient, at least one of the compounds of the present invention in association with a pharmaceutical carrier or diluent.
• the active ingredient of the pharmaceutical compositions can comprise an anabolic agent in addition to at least one of the compounds of the present invention or another composition which exhibits a different activity, e.g., an antibiotic growth permittant or an agent to treat osteoporosis or in combination with a corticosteroid to minimize the catabolic side effects or with other pharmaceutically active materials wherein the combination enhances efficacy and minimizes side effects.
• Growth promoting and anabolic agents include, but are not limited to TRH, diethylstilbesterol, estrogens, ⁇ -agonists, theophylline, anabolic steroids, enkephalins, E series prostaglandins, compounds disclosed in U.S. Patent No. 3,239,345, e.g., zeranol, and compounds disclosed in U.S. Patent No. 4,036,979, e.g., sulbenox or peptides disclosed in U.S. Patent No. 4,411,890.
• a still further use of the compounds of this invention is in combination with other growth hormone secretagogues such as the growth hormone releasing peptides GHRP-6, GHRP- 1 as described in U.S. Patent Nos.
• the compounds of this invention may be used in combination with growth hormone releasing factor, an analog of growth hormone releasing factor, IGF-1, or IGF-2.
• a compound of the present invention may be used in combination with IGF-1 for the treatment or prevention of obesity.
• a compound of this invention may be employed in conjunction with retinoic acid to improve the condition of musculature and skin that results from intrinsic aging.
• the present invention is further directed to a method for the manufacture of a medicament for stimulating the release of growth hormone in humans and animals comprising combining a polymo ⁇ hic form of the present invention with a pharmaceutical carrier or diluent.
• growth hormone As is well known to those skilled in the art, the known and potential uses of growth hormone are varied and multitudinous.
• the administration of the compounds of this invention for pu ⁇ oses of stimulating the release of endogenous growth hormone can have the same effects or uses as growth hormone itself.
• These varied uses of the present compounds thus may be summarized as follows: stimulating growth hormone release in elderly humans; treating growth hormone deficient adults; prevention of catabolic side effects of glucocorticoids; treatment of osteoporosis; stimulation of the immune system, acceleration of wound healing; accelerating bone fracture repair; treatment of growth retardation; treating acute or chronic renal failure or insufficiency; treatment of physiological short stature, including growth hormone deficient children; treating short stature associated with chronic illness; treatment of obesity and growth retardation associated with obesity; treating growth retardation associated with Prader-Willi syndrome and Turner's syndrome; accelerating the recovery and reducing hospitalization of burn patients or following major surgery such as gastrointestinal surgery; treatment of intrauterine growth retardation, and skeletal dysplasia, treatment of peripheral neuropath
• the instant compounds are useful for increasing feed efficiency, promoting growth, increasing milk production and improving the carcass quality of livestock.
• the instant compounds are useful in a method of treatment of diseases or conditions which are benefited by the anabolic effects of enhanced growth hormone levels that comprises the administration of an instant compound.
• the instant compounds are useful in the prevention or treatment of a condition selected from the group consisting of: osteoporosis; catabolic illness; immune deficiency, including that in individuals with a depressed T4/T8 cell ratio; hip fracture; musculoskeletal impairment in the elderly; growth hormone deficiency in adults or in children; obesity; cachexia and protein loss due to chronic illness such as AIDS or cancer; and treating patients recovering from major surgery, wounds or burns, in a patient in need thereof.
• a condition selected from the group consisting of: osteoporosis; catabolic illness; immune deficiency, including that in individuals with a depressed T4/T8 cell ratio; hip fracture; musculoskeletal impairment in the elderly; growth hormone deficiency in adults or in children; obesity; cachexia and protein loss due to chronic illness such as AIDS or cancer; and treating patients recovering from major surgery, wounds or burns, in a patient in need thereof.
• the instant compounds may be useful in the treatment of illnesses induced or facilitated by corticotropin releasing factor or stress- and anxiety-related disorders, including stress-induced depression and headache, abdominal bowel syndrome, immune suppression, HIV infections, Alzheimer's disease, gastrointestinal disease, anorexia nervosa, hemorrhagic stress, drug and alcohol withdrawal symptoms, drug addiction, and fertility problems.
• corticotropin releasing factor or stress- and anxiety-related disorders including stress-induced depression and headache, abdominal bowel syndrome, immune suppression, HIV infections, Alzheimer's disease, gastrointestinal disease, anorexia nervosa, hemorrhagic stress, drug and alcohol withdrawal symptoms, drug addiction, and fertility problems.
• the therapeutic agents and the growth hormone secretagogues of this invention may be independently present in dose ranges from one one-hundredth to one times the dose levels which are effective when these compounds and secretagogues are used singly.
• Combined therapy to inhibit bone reso ⁇ tion, prevent osteoporosis and enhance the healing of bone fractures can be illustrated by combinations of bisphosphonates and the growth hormone secretagogues of this invention.
• the use of bisphosphonates for these utilities has been reviewed, for example, by Hamdy, N.A.T., Role of Bisphosphonates in Metabolic Bone Diseases, Trends in Endocrinol. Metab.. 4, 19-25 (1993).
• Bisphosphonates with these utilities include alendronate, tiludronate, dimethyl-APD, risedronate, etidronate, YM- 175, clodronate, pamidronate, and BM-210995.
• oral daily dosage levels of the bisphosphonate of between 0.1 mg and 5 g and daily dosage levels of the growth hormone secretagogues of this invention of between 0.01 mg/kg to 20 mg/kg of body weight are administered to patients to obtain effective treatment of osteoporosis.
• osteoporosis and other bone disorders may also be treated with compounds of this invention in combination with calcitonin, estrogens, raloxifene and calcium supplements such as calcium citrate.
• Anabolic effects especially in the treatment of geriatric male patients are obtained with compounds of this invention in combination with anabolic steroids such as oxymetholone, methyltesterone, fluoxymesterone and stanozolol.
• the compounds of this invention can be administered by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous or subcutaneous injection, or implant), nasal, vaginal, rectal, sublingual, or topical routes of administration and can be formulated in dosage forms appropriate for each route of administration.
• parenteral e.g., intramuscular, intraperitoneal, intravenous or subcutaneous injection, or implant
• nasal, vaginal, rectal, sublingual, or topical routes of administration and can be formulated in dosage forms appropriate for each route of administration.
• Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules.
• the active compound is admixed with at least one inert pharmaceutically acceptable carrier such as sucrose, lactose, or starch.
• Such dosage forms can also comprise, as is normal practice, additional substances other than inert diluents, e.g., lubricating agents such as magnesium stearate.
• the dosage forms may also comprise buffering agents. Tablets and pills can additionally be prepared with enteric coatings.
• Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, the elixirs containing inert diluents commonly used in the art, such as water. Besides such inert diluents, compositions can also include adjuvants, such as wetting agents, emulsifying and suspending agents, and sweetening, flavoring, and perfuming agents.
• Preparations according to this invention for parenteral administration include sterile aqueous or non- aqueous solutions, suspensions, or emulsions.
• non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate.
• Such dosage forms may also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. They may be sterilized by, for example, filtration through a bacteria-retaining filter, by inco ⁇ orating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions. They can also be manufactured in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use.
• compositions for rectal or vaginal administration are preferably suppositories which may contain, in addition to the active substance, excipients such as cocoa butter or a suppository wax.
• Compositions for nasal or sublingual administration are also prepared with standard excipients well known in the art.
• the compound of this invention may be administered to patients (animals and humans) in need of such treatment in dosages that will provide optimal pharmaceutical efficacy. It will be appreciated that the dose required for use in any particular application will vary from patient to patient, not only with the particular compound or composition selected, but also with the route of administration, the nature of the condition being treated, the age and condition of the patient, concurrent medication or special diets then being followed by the patient, and other factors which those skilled in the art will recognize, with the appropriate dosage ultimately being at the discretion of the attendant physician.
• the dosage of active ingredient in the compositions of this invention may be varied; however, it is necessary that the amount of the active ingredient be such that a suitable dosage form is obtained.
• the selected dosage depends upon the desired therapeutic effect, on the route of administration, and on the duration of the treatment. Generally, dosage levels of between 0.0001 to 10 mg/kg. of body weight daily are administered to patients and animals, e.g., mammals, to obtain effective release of growth hormone. Preferably, the dosage level will be about 0.001 to about 25 mg/kg per day; more preferably about 0.01 to about 10 mg/kg per day.
• Step A 1,2-Dihydro- l-methanesulfonylspiro[3H-indole-3,4'- piperdinel hy drochloride
• Step B N-[l(R)-[(l,2-Dihydro-l-methanesulfonylspiro[3H-indole- 3,4'-piperdin]- 1 '-yl)carbonyl]-2-(phenylmethyloxy)ethyl]- 2-[( 1 , 1 -dimethylethoxy)carbonyl]amino-2-methyl- propanamide
• Step C N-[ 1 (R)- [( 1 ,2-Dihydro- 1 -methanesulfonylspiro[3H-indole- 3,4'-piperidin]-r-yl)carbonyl]-2-(phenylmemyloxy)ethyl]-
• Step A (2R)-[[[-2-(l,l-dimethylethoxy)carbonyl]amino]-2,2- dimethyl- l-oxoethyl]amino-2-(phenylmethoxy)ethyl]-l- propanoic acid allyl ester
• Step B (2R)-[[[-2-(l,l-dimethylethoxy)carbonyl]amino]-2,2- dimethyl- l-oxoethyl]amino-2-(phenylmethyloxy)ethyl)- 1 - propanoic acid
• Step C N-[ 1 (R)- [( 1 ,2-Dihydro- 1 -methanesulfonylspiro[3H-indole-
• reaction mixture was diluted with an additional 50 mL of dichloromethane and washed with aqueous sodium bicarbonate solution (50 mL), dried over anhydrous magnesium sulfate, filtered, and concentrated. Flash chromatography (50 g silica gel) of the crude oily residue gave 2.148 g (90%) of the desired material as a colorless foam.
• Step D N- [ 1 (R)- [( 1 ,2-Dihydro- 1 -methanesulfonylspiro[3H-indole- 3,4'-piperdin]-r-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-
• This compound was prepared by the treating the free base obtained in Example 5, Step D, with methane sulfonic acid.
• This sample was subsequently identified as being polymo ⁇ hic Form II. It was characterized by an X-ray powder diffraction pattern with principal reflections at approximately: 4.7, 11.6, 17.4, 19.2, and 21.6° (2 theta).
• Isonipecotic acid (2) and K2CO3 were dissolved in 40.2 L of water in a 100 L 4 neck flask with mechanical stirring under N2 and the solution was cooled to 10°C.
• Benzyl chloroformate was added, maintaining the temperature between 9 and 14°C, and the mixture was warmed up to 22°C after the addition was complete and aged for 58 h.
• the addition was completed in 4 h at which point the pH was 9.0. After aging for 58 h there was no change in the pH.
• the aqueous phase was acidified with 37% aqueous HCI to pH 1.8. Carbon dioxide was evolved during the addition of HCI, but gas evolution was easily controlled. The addition of HCI took ⁇ 1 h and required 10 L of cone. HCI.
• the aqueous phase was extracted with 3 x
• the product was 99.1 area % pure with 0.9 area % benzyl alcohol as the only impurity.
• Toluene 12 L To the toluene solution of benzyl carbamate 3 from the preceding step was added 5 mL of DMF and 10 L of toluene. The oxalyl chloride was added over a period of 20 min. The reaction mixture was aged for 16 h at 18°C under a slow stream of nitrogen. HPLC analysis of the reaction mixture showed that 1.3% of the carboxylic acid 3 still remained unreacted. The reaction mixture was warmed to 26°C, and 5 mL of DMF were added. The mixture was aged for 2.5 h.
• the assay yield of aldehyde 3 was 94% by HPLC analysis.
• the crude aldehyde 5 solution from the previous step was transferred through a 10 ⁇ inline filter to a 100 L reactor equipped with Teflon coated copper coils for cooling or heating and a mechanical stirrer. Toluene (34.4 kg) and MeCN (7 L) were added, and the resulting solution was cooled to 0°C. Phenylhydrazine was added in portions and the temperature was maintained at -1 to 3°C while nitrogen was continuously bubbled through the reaction mixture. The phenylhydrazine was added until TLC and HPLC analysis indicated complete consumption of the aldehyde 5 and the appearance of a slight excess ( ⁇ 5%) of phenylhydrazine. TLC conditions: Silica, E.
• HPLC conditions 25 cm Dupont Zorbax RXC8 column at 30°C with 1.0 mL/min flow and detection at 254 nm; gradient schedule:
• the reaction mixture was aged for 30 min at 0-2°C, and TFA was added maintaining the temperature between 2 and 7°C.
• the reaction mixture was warmed to 50°C over 30 min, and maintained for 17 h.
• the nitrogen sparge through the reaction mixture was stopped and a slow stream of nitrogen was maintained over the reaction mixture.
• the color gradually darkened to a deep green, and a relatively small amount of a white crystalline precipitate (ammonium trifluoroacetate) formed.
• HPLC analysis (same conditions as above) indicated that the reaction mixture contained 91.6 area % indolenine 8 and 1.5% of unreacted phenylhydrazone remained.
• the color change from green to orange corresponds very closely to reaction end point.
• the quantity of NaBH4 required to complete the reaction is heavily dependent on the temperature and rate of addition of NaBH4, but the yield and quality of the product is virtually unaffected provided that the reaction is complete.
• the reaction mixture was cooled to 5°C over a period of 30 min.
• 8 L of 3% aqueous NH4OH (8 L) were added to bring the pH of the aqueous phase to 7.4, the mixture was agitated, and allowed to settle.
• the temperature rose to 15°C.
• the cloudy yellow lower aqueous phase was separated.
• the organic phase was washed with 4 L of 3% aqueous NH4OH, 2 x 4 L of water, and 2 x 4 L of brine.
• the weight of the organic phase after the washings was 53.5 kg, and the assay yield was 94%.
• the washed toluene solution was combined with the washed organic phases of two other similarly processed reactions.
• the total aldehyde used in the three reactions was 5.06 kg, (20.5 mol).
• the total weight of CBZ-indoline 9 assayed in the combined organic phases was 5.91 kg, (18.3 mol, 90% assay yield).
• the combined organic phases were dried with 5 kg of sodium sulfate, treated with 250 g of Darco G60 carbon for 30 min, and filtered through Solka-flocTM.
• the filtrates were vacuum concentrated at 10 mbar at ⁇ 25°C until the residue was near dryness.
• the solvent switch was completed by slowly bleeding in 30 L of IPAC and reconcentrating to 14 L at 200 mbar at 50-60°C.
• the mixture was heated to reflux in order to obtain a clear homogeneous deep orange solution. ⁇ H NMR analysis indicated that the solution contained ca. 6 mol% of residual toluene after solvent switch.
• the solution was cooled to 68 °C and seeded with 4 g of crystalline CBZ-indoline 9.
• the solution was allowed to gradually cool to 26°C over 6 h and aged for 9 h at 20-26°C.
• the slurry was cooled to 2°C over 1 h and aged at 2°C for lh.
• the product was isolated by filtration, and the filter cake was washed with 2 x 2 L of 5°C IPAC and 2 x 2 L of 5°C MTBE.
• the product was dried in the vacuum oven at 30°C under a nitrogen bleed to give 4.37 kg (74%) of the title compound 9 as a light tan crystalline powder.
• HPLC analysis of the product indicated 99.5 area % purity.
• reaction mixture was warmed to 18°C and aged for 16 h. There was no change in the appearance of the reaction mixture, and HPLC profile between the end of the addition and after the 16 h age.
• the reaction mixture was slowly transferred over lh into a vigorously stirred solution of 30 L of water and 200 mL of 37% aqueous HCI in a 50 L flask. The temperature in the 50 L flask rose from 22 to 28°C. The product separated as a pale tan gummy solid which changed to a granular solid.
• the aqueous suspension was cooled to 22°C and aged for 1 h. The suspension was filtered, and the filter cake was washed with 2 x 4 L of MeOH/water (50/50).
• HPLC analysis indicated that ⁇ 0.1% of the CBZ-Spiroindoline- methanesulfonamidel was in the mother liquors.
• the filter cake was washed with 4 L of MeOH/water (50/50) to which 50 mL of 28% aqueous NH4OH had been added.
• the filter cake was washed with 2 x 4 L of MeOH water (50/50), and the solid was dried in the vacuum oven at 50°C under a nitrogen bleed to give 2.03 kg (97%) of the title product 1 as an off-white powder.
• HPLC analysis of the solids indicated 93.7 area % 1.
• Trifiuoroacetic acid 11.56 mL,17.10 g
• the CBZ-aldehyde 5 was dissolved in dichloromethane in a 1 L flask equipped with Teflon coated magnetic stirring bar. The resulting solution was cooled to 0°C. Phenylhydrazine was added via a weighed syringe over 5 min and the temperature was maintained at -1 to 3°C while nitrogen was continuously bubbled through the reaction mixture. TLC and HPLC analysis indicated complete consumption of the CBZ-aldehyde 5 and the appearance of a slight excess ( ⁇ 2%) of phenylhydrazine. TLC conditions: Silica, E.
• TFA was added by syringe maintaining the temperature between 2 and 7°C.
• the reaction mixture was warmed to 35°C over 30 min, and maintained for 17 h.
• the nitrogen sparge through the reaction mixture was stopped and a slow stream of nitrogen was maintained over the reaction mixture.
• the color gradually darkened to a rosy pink then to a deep green, and a relatively small amount of a white crystalline precipitate (ammonium trifluoroacetate) formed.
• HPLC analysis (same conditions as above) indicated that the reaction mixture contained 93 area % indolenine 8 and ⁇ 0.5% of unreacted phenylhydrazone remained.
• TFA Trifiuoroacetic acid
• a 2% (by volume) solution of MeCN in toluene was made up using 654 mL of toluene and 13.3 mL of MeCN.
• a 2 L 3 neck flask equipped with a mechanical stirrer 617 ml of the above solution were degassed by passing a fine stream of nitrogen through the solution for 5 min. Phenylhydrazine and TFA were added to the mixture while still degassing.
• the CBZ-aldehyde 5 was dissolved in the rest of the solution prepared above (50 mL) and degassed by bubbling nitrogen through the solution while in the addition funnel.
• the solution in the flask was heated to 35°C, and the aldehyde solution was slowly added to the phenylhydrazine-TFA over 2 h. The mixture was aged at 35°C for 16h.
• HPLC conditions 25 cm Dupont Zorbax RXC8 column at 50°C with 1 mL/min flow and detection at 220 nm; isocratic 55% MeCN, 45% of 0.1% aqueous H3PO4.
• the mixture was warmed to 20°C, and 200 mL of IM aqueous HCI was added.
• the mixture was warmed to 50°C, and the aqueous phase was separated.
• the organic phase was washed sequentialy with 100 mL water, 100 mL 5% aqueous sodium bicarbonate, and 100 mL water.
• the organic phase was transferred to a 1 L 3 neck flask equipped for mechanical stirring and distillation.
• the mixture (ca 400 mL) was distilled at atmospheric pressure until 150 mL of distillate had been collected.
• the head temperature reached 107°C; the pot temperature was 110°C.
• the distillation was continued with continuous addition of n-propanol at such a rate as to maintain a constant volume (ca 350 mL) in the pot.
• the distillation was stopped when a total of 525 mL of n-PrOH had been added and a total of 800 mL of distillate had been collected.
• the crystalline slurry was filtered and washed with 3 x 100 mL of n-PrOH.
• the product was dried in a vacuum oven at 50°C with a nitrogen bleed for 16 h to furnish 65.5g (82 % from aldehyde 5) of 6 as a tan solid with 93.5 wt% purity.
• 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 h at 60°C. After the addition of 2.0 g SolkaflocTM, the slurry was filtered through a pad of 4.0 g SolkaflocTM, and the pad was washed with 90 mLof EtOAc at 60°C. Prior to the addition of the carbon the solution was a brown color. The filtration proceeded well without plugging to give a golden yellow filtrate.
• the filtrate was distilled at atmospheric pressure in a 500 mL flask (pot temperature 80-85°C) until 100 g (100 mL) of residue remained. This solution was allowed to cool to 35°C over 3 h. Over a lh period, 116 mL of cyclohexane was added with good agitation at 35°C. The mixture was cooled to 20°C over 1 h and aged at 20°C for 12 h. At 35°C much of the sulfonamide has crystallized out and the mixture was thick. Addition of cyclohexane at 20°C makes agitation difficult. After the aging period, the supernatant was found to contain 2.5 mg 1/g.
• the crystalline slurry was filtered and the cake was washed with 77 mL of 2: 1 cyclohexane-EtOAc and 2 x 77 mL of cyclohexane.
• the catalyst was suspended in 7 L of MeOH and transferred into the 5 gal autoclave followed by the solution of 1 in 8 L of THF.
• the mixture was hydrogenolyzed at 25 °C at 80 psi of H2- After 2.5 h the temperature was raised to 35°C over 30 min.
• HPLC analysis indicated complete consumption of Cbz- spiroindoline-methanesulfonamide.
• HPLC conditions 25 cm Dupont Zorbax RXC8 column with 1.5 mL/min flow and detection at 254 nm.
• Gradient Schedule Time (min) 0.1% aq.
• H ⁇ P ⁇ 4 MeCN
• the mixture was purged with nitrogen and the catalyst was removed by filtration through Solka-flocTM while still warm.
• the catalyst was washed with 4 L of THF and 2 L of MeOH.
• the pale yellow filtrates were concentrated to a thick oil at 10 mbar and ⁇ 25°C.
• the solvent switch was completed by slowly bleeding in 15 L of EtOAc and reconcentrating to dryness.
• the residue solidified to a hard off- white mass.
• MeOH (1.5 L) was added and the mixture was heated to 70°C to give a homogenous solution. While the solution was at 70°C, 10.5 L of EtOAc at 20°C was added. The temperature fell to 40°C, and the mixture remained homogenous.
• the hydrogenation was run three (3) times due to equipment limitations; this procedure refers to a single run.
• the catalyst loading and reaction time are a function of the purity of starting material 1. This material was unique requiring > 15% catalyst and long reaction time.
• the batch was hydrogenated at 65 °C with vigorous stirring under 40 psi hydrogen pressure for 3 hours, a second portion of 10% palladium on charcoal (75 g) was added, the batch was hydrogenated for a further 2 hours and then sealed overnight.
• the batch was transferred (still hot, 60-65°C) to a 20 L Buchi apparatus and degassed in vacuo to remove formic acid by "feeding and bleeding" absolute ethanol (18 L total).
• the initial vessel, the filter, the pump and the lines were rinsed with a hot (60-65 °C) mixture of aqueous ammonia (500 ml) in absolute ethanol (25 L).
• the filtrate and washings were combined in the two stainless-steel bins.
• the batch was then transferred to a vessel using an in-line filter containing a 10 micron cartridge, and then concentrated in vacuo to low bulk (-15 L).
• the ethanol was replaced by isopropyl acetate by the "feeding and bleeding" of 3x batch volumes of isopropyl acetate (45 L total), while maintaining a batch volume of -15 L.
• the solvent switch when complete, contained ⁇ 1% residual ethanol by GC.
• the batch was then diluted to -33 L by the addition of isopropyl acetate (20 L), and this solution of spiroindoline-amine lb (1.855 kg by LC analysis) in isopropyl acetate was used for the next stage of the process.
• the mixture was then filtered through an Estrella filter using cloth and cardboard only and utilizing a pump into another vessel.
• the sending vessel was rinsed with isopropyl acetate (22 L) and this was used to rinse the filter, the pump and the lines into the receiving vessel.
• Methanesulphonic acid (2.006 kg, 1.355 L, -3 equivs.) was added to the stirred solution of Boc-O-benzylserine spiroindoline (11) (3.787 kg) in ethanol (total volume -15 L) in a reaction vessel.
• the batch was warmed to 35-40°C. After 7 hours, LC showed the absence of starting material and the reaction was allowed to cool to room temperature overnight.
• water (44 L) was added to the batch with stirring.
• the batch was cooled to -5°, stirred for 30 minutes and then filtered through an in-line filter (loaded with a lO ⁇ cartridge) into a bin. The batch was then sucked back into the vessel.
• the solution of the amine 12 in IPAc was diluted to a total volume of 39 L with IPAc and 37 L of H2 ⁇ was added.
• the biphasic mixture was then treated in sequence with HOBT (827 g) as a solid, DCC (1266.7 g) as a melt, and amino acid 13 at ambient temperature under nitrogen.
• the reaction mixture was stirred for 2 h upon which time LC analysis indicated dissappearance of the starting material 12 ( ⁇ 0.3 A%).
• the mixture was filtered through Solka FloeTM and the solids were washed with 13 L of IPAc. The material may be stored at this point as a biphasic mixture overnight.
• IM aqueous sodium hydroxide 38 L
• 0.5M aqueous hydrochloric acid 38 L
• saturated aqueous sodium hydrogen carbonate 38 L
• the Boc spiroindoline 14 was dissolved in 6.2 L of EtOH and treated with MsOH (979 mL). The temperature rose from 20 to 30°C and the reaction was allowed to proceed overnight. After 12 hours at 20°C there was still 15 A% of starting material left so the mixture was heated to 35°C for 6 hours. Upon completion ( ⁇ 0.1 A% 14) the reaction was cooled to 20°C and 30 L of H2 ⁇ were added and the solution was filtered through a glass funnel with a polypropylene filter to filter off residual DCU. The mixture was transferred to a 100 L extractor and 26 L of EtOAc were added. The aqueous layer was basified via addition of chilled IN NaOH (11 L) and 1 L of 50% NaOH. Addition of ice was required to keep the temperature below 14°C. Higher temperatures resulted in significant emulsion problems.
• the organic layer was distilled at 50°C at ca. 21 " of Hg until KF ⁇ 1000 ⁇ g/mL. Lower KF's result in more efficient carbon treatments and better recovery at the salt formation step. KF's of 160 ⁇ g/mL were achieved at the 700 g scale.
• the solution was diluted with ethyl acetate to a total volume of 31 L (LC assay 2.40 kg).
• Activated carbon (Darco G-60) was added and the mixture was stirred for 24 h.
• the mixture was filtered through Solka FloeTM and the filter cake was washed with ethyl acetate (16 L), assay 2.34 Kg.
• Methanesulfonic acid (2.017 kg, 1.36 L, -3 equivs.) was added to the stirred solution of the Boc spiroindoline 14 (4.395 kg) in ethanol (total volume -25 L) in a reaction vessel at room temperature.
• the batch was warmed to 35-40°C, and stirred overnight.
• the batch contained -1.1 A% of starting material and so the reaction was continued for a further 4 hours, then LC showed ratio of product/ starting material to be 99.6/0.4.
• the batch was concentrated in vacuo to -15 L volume and then diluted with water (44 L).
• the batch was cooled to 5°C, stirred for 30 minutes and then filtered through a Sparkler in-line filter (containing a lO ⁇ cartridge) using a pump to another vessel to remove a small amount of residual DCU.
• the vessel, the pump, the filter and the lines were rinsed with water (10 L), and this was added to the vessel.
• Ethyl acetate (36 L) was added to the vessel and the stirred mixture was cooled to 10°C.
• a solution of cold (5-10°C) IM aqueous sodium hydroxide solution (16 L) and cold (5-10°C) 50% aqueous sodium hydroxide solution (2.6 L) were added at 10°C and the temperature rose to 14°C. The resulting mixture was stirred for 15 minutes at ⁇ 14°C and then the lower aqueous layer separated off.
• the batch was concentrated in vacuo to -20 L volume and then a mixture of ethyl acetate (35 L) and ethanol (5 L) was fed in while maintaining the volume at -20 L. At the end of this distillation the KF was 9160 mgml " .
• the batch was solvent switched to ethyl acetate by "feeding and bleeding" ethyl acetate (40 L total). At the end of this distillation, KF was 446 mgml " .
• the batch was diluted with ethyl acetate (10 L).
• the batch (58 L) had a KF of 2950 mgml " 1 and so was redried by concentrating in vacuo to 20-25 L volume.
• the batch was diluted to 46 L volume (dipstick) by the addition of ethyl acetate (25 L).
• the KF was 363 mgml " .
• the batch was diluted to 62 L volume by the addition of ethyl acetate (17 L) and was used for the final stage of the process.
• the volume of the solution of 15 from the previous step was adjusted to 60 L with ethyl acetate and EtOH (4.8 L) was added.
• the MsOH (316 mL) was added in 3 L of EtOAc at 45°C.
• To the deep red homogeneous solution was added 496 g of the title compound Form I seed (10% seed based on the weight of the free amine was employed). The temperature rose to ca. 48°C and the reaction was aged at 52°C for 1.5 hours. Analysis indicated complete conversion to the title compound (Form I). (At less than 10% seed longer age (> 3 hours) was required). The slurry was allowed to cool to 20°C overnight and was filtered in a centrifuge under N2.
• Form I (99.9 A% purity; ⁇ 0.1% enantiomer).
• the conversion of Form II to Form I is also accomplished where the salt is formed in EtOAc-EtOH by addition of MsOH as above and the initial solution of the salt (at 55°C) is cooled to 45°C. Crystals start appearing at that temperature and the slurry becomes thicker with time. The temperature is then raised to 51°C and the slurry is aged overnight. Complete conversion to Form I of 16 should be expected. This procedure may also be employed to prepare seed crystals of Form I of 16.
• Absolute ethanol (6.4 L) was added to the solution of the amine (15) (3.1 kg) in ethyl acetate (total volume -62 L) in a reacttion vessel. The batch was warmed to 50°C and a solution of methanesulfonic acid (620 g, 412 ml, 1.1 equivs.) in ethyl acetate (1 1 L) was added over -5 minutes at 50-54°C.
• Form II to Form I may be accomplished by the procedure of Example 18A where the salt is formed in EtOAc- EtOH by addition of MsOH and the initial solution of the salt (at 55°C) is cooled to 45°C. Crystals should start appearing at that temperature and the slurry should become thicker with time. The temperature is then raised to 51°C and the slurry is aged overnight. Complete conversion to Form I should be expected.
• Form II The conversion of Form II to Form I is accomplished by stirring a solution of Form II of N-[l(R)-[(l,2-dihydro-l- methanesulfonyl-spiro[3H-indole-3,4'-piperdin]-l'-yl)carbonyl]-2- (phenylmethyl-oxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate in isopropanol at approximately 25 °C for about 2-24 hours.

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