Patent Grant
8183280
The present invention relates to compounds, which act as FAP inhibitors, pharmaceutical products comprising the compounds, and methods of treatment using the compounds.
The enzyme fibroblast activation protein alpha (FAPα), herein abbreviated FAP, is a serine protease that possesses dipeptidyl-peptidase activity specific for N-terminal Xaa-Pro sequences. In addition to the dipeptidyl peptidase activity FAP also possesses collagenolytic activity capable of degrading gelatin and type I collagen. FAP is a type II transmembrane serine protease which is expressed as a homodimer. The 95-kDa protein exhibits 48% amino acid identity with DPIV and displays structural similarity to other members of the dipeptidyl peptidase family including DP8 and DP9. Unlike DPIV, FAP has also been reported to possess endopeptidase activity (Aertgeerts, J. Biol. Chem., 2005, 280, 19441). FAP overexpression has been shown to potentiate tumour growth (Cheng, Mol. Cancer. Ther., 2005, 4, 351), and this potentiation is dependent upon its enzymatic activity. Natural substrates for FAP have not yet been identified. FAP is not expressed by normal mature somatic tissues and benign epithelial tumours but is expressed on stromal fibroblasts in more than 90% of carcinomas including breast, colon, ovarian, bladder and pancreas (Garin-Chesa et al., Proc. Natl. Acad. Sci., 1990, 87: 7235) as determined by immunohistochemistry, making this a very specific target for potential anti-tumour agents. Expression of FAP has also been correlated with invasive potential of melanoma cells (Aoyama et al., Proc. Natl. Acad. Sci., 1990, 87: 8296) and its presence has been associated with metastasis of colorectal tumours (Iwasa et al., Cancer Letts., 2003, 199: 91). Its presence has been shown to increase tumour formation in animal models (Cheng et al., Cancer Res., 2002, 62: 4767).
The catalytic action of FAP expressed in stromal fibroblasts in carcinomas has been utilised in the conversion of prodrugs of cytotoxic or cytostatic drugs to active drugs as a potential treatment for cancer (EP 1333033).
In addition to its activity in tumour tissue FAP is also expressed in organs undergoing non-malignant remodelling, for example liver fibrosis (Levy et al., Hepatol., 1999, 29: 1768). In patients with fibrosis as a result of alcohol abuse or viral challenge, the cells responsible for the excessive scarring and constriction of the liver express large quantities of FAP. There is evidence to suggest that FAP plays an important role in the formation of the fibrotic scar.
Normal tissue is normally FAP negative, however, it is found transiently in healing wounds (Ramirez-Montagut et al., Oncogene, 2004, 23, 5435) and it is possible FAP plays a role in tissue remodelling and repair. Other potential indications for FAP inhibitors include blood cell disorders such as anemia and chemotherapy-induced neutopenia (Rosenblum et al., Curr. Opin. Chem. Biol., 2003, 7, 496).
The only FAP inhibitor reported to date is ValboroPro (PT-100, Talobostat) which has been reported to show potent anti-tumour activity in mice, slowing growth of syngeneic tumours and causing regression and rejection of tumours after oral administration (Adams et al., Cancer Res., 2004, 64: 5471). This compound is currently undergoing clinical trials as a treatment for cancer (McIntyre, Drugs of the Future, 2004, 29, 882). However, this compound, which is a non-specific inhibitor of dipeptidyl peptidases, has been shown to be a potent inhibitor of DPIV (Coutts et. al., J. Med. Chem., 1996, 39, 2087). It has been demonstrated (Cheng, Mol. Cancer Ther., 2005, 4, 351) that FAP-driven tumour growth can be attenuated using ValboroPro; this effect is unlikely to be due to DPIV inhibition suggesting that FAP plays an important role in the promotion of tumour growth. ValboroPro has also been shown to stimulate the growth of hematopoietic progenitorcells and to accelerate neutrophil and erythrocyte regeneration, this regeneration was observed in DPIV knock-out mice (Jones, Blood, 2003, 102, 150).
Inhibitors of DPIV are at present in clinical trials for treatment of type II diabetes. Of the many reported inhibitors of DPIV one class of compound reported are the aminoacyl pyrrolidine nitriles (Evans, IDrugs, 2002, 5, 577). However, these fall into two distinct classes; those with an unsubstituted amino group at the N-terminus (WO 9515309, WO 0181337, WO 0181304), and the N-allylglycine derivatives (WO 0196295). No data has been reported for the activity of these compounds as FAP inhibitors. There are no reports of DPIV inhibitors with an N-acyl or N-carbamoyl N-terminus.
Whilst inhibitors of prolyl ologopeptidases (which have endopeptidase activity) are known, they appear not to have been reported as showing any activity against cancer in clinical testing.
According to an aspect, the present invention aims at providing active, selective FAP inhibitors, preferably without DPIV inhibition. Preferably, the compounds are easy to synthesize. More preferably, they are orally available.
According to an aspect, the present invention relates to the compounds of claim 1.
The term “alkyl” includes saturated hydrocarbon residues including:
The term “alkenyl” includes monounsaturated hydrocarbon residues including:
The term “aryl” includes optionally substituted phenyl and optionally substituted naphthyl. Examples of such aryl groups include, but are not limited to, phenyl, 2-tolyl, 3-chlorophenyl, 4-chlorophenyl, 3-fluorophenyl, 2-methoxyphenyl, 3-methoxyphenyl, 4-methoxyphenyl, 2,5-difluorophenyl, 1-naphthyl and 2-naphthyl.
The term “heteroaryl” includes optionally substituted heterocycles. Such heteroaryl groups include, but are not limited to, pyridyl, 2-chloropyridyl, 4-methylpyridyl, thienyl, 3-chlorothienyl, 2,3-dimethylthiophenyl, furyl and 2-methylfuryl.
Further aspects of the invention are provided according to the claims.
According to an aspect the compounds of the invention are inhibitors of FAP. According to an aspect the compounds of the invention are useful for the treatment of cancer and/or liver cirrhosis. According to an aspect of the present invention it is envisionaged that at least one FAP inhibitor may be co-administered with at least one known cancer agent. Such anti-cancer agents include the compounds mentioned in claim 12 of WO2004045593.
According to an aspect, the present invention relates to the particularly preferred compounds of claim 25. The particularly preferred compounds are inhibitors of FAP of high activity.
According to an aspect, the invention is further related to pharmaceutical compositions incorporating a compound according to the invention used as a FAP inhibitor; these compositions are particularly useful for medical indications such as the treatment of cancer and liver cirrhosis.
According to an aspect, the invention concerns the use of a compound of the invention for the manufacture of a medicament for the treatment of a condition selected among cancer, liver cirrhosis, malignant and benign tumours, ectopic tissue growth, wound healing, organ fibrosis, cirrhosis, metastasis, blood cell disorders, anemia, and chemotherapy-induced neutropenia.
The compounds according to the present invention are useful for treatment of several diseases, disorders or conditions. The term “treatment” used herein relates to both treatment in order to cure or alleviate a disease, disorder or a condition, and to treatment in order to prevent the development of a disease, disorder or a condition. The treatment may either be performed in an acute or in a chronic way. The human or animal to be treated, i.e. the patient, may be any human or non-human mammal in need of treatment according to the invention.
The term “therapeutically effective amount” relates to an amount that will lead to the desired therapeutical effect. The therapeutically effective amount will be determined by the attending physician taking into consideration all appropriate factors. Generally a single dose will comprise between 0.1 mg and 1000 mg, preferably between 1 mg and 250 mg, of the active compound according to the invention. The dose may be given on a single occasion or repeatedly. When given repeatedly, it may be given at regular intervals, such as once, twice or three times daily, or on demand, according to the condition being treated.
According to an aspect, the invention concerns a pharmaceutical composition comprising a compound according to the invention as an active agent. Any excipients used will depend on the intended nature of the formulation, which will, in turn, depend on the intended route of administration. Administration may be oral, transmucosal (such as sublingual, buccal, intranasal, vaginal and rectal), transdermal or by injection (such as subcutaneous, intramuscular and intravenous). Oral administration is generally preferred. For oral administration, the formulation may be a tablet, a capsule or a sachet.
The pharmaceutical composition according to an aspect of the present invention may be presented in any form that is known in the art. For example, the formulation may be presented as a tablet, capsule, powder, suppository, cream, solution or suspension, or in a more complex form such as an adhesive patch. The formulation will generally include one or more excipients, such as diluents, bulking agents, binding agents, dispersants, solvents, preservatives, flavouring agents and the like.
The pharmaceutical composition according to the present invention may optionally comprise at least one further additive such as a disintegrating agent, binder, lubricant, flavoring agent, preservative, colorant and a mixture thereof. Representative examples can be found in “Handbook of Pharmaceutical Excipients”; Ed. A. H. Kibbe, 3rd Ed., American Pharmaceutical Association, USA and Pharmaceutical Press UK, 2000.
The compounds of the present invention can be prepared by methods generally known in the art and illustrated in the following non-limiting examples.
The compounds according to general formula 1 can be prepared using conventional synthetic methods.
In a first step (Scheme 1) a nitrogen containing heterocycle (5) is coupled using standard peptide coupling conditions to an alpha amino acid (6) suitably amino-protected with a standard protecting group such as tert-butyloxycarbonyl (BOC), benzyloxycarbonyl (Z) or 9-fluorenylmethyloxycarbonyl (Fmoc). The use of such groups is well known in the art. Where R1 has a reactive functional group such as an amine or a carboxylic acid, this group will also be protected. The principles of functional group protection are well known in the art and are described in, for example, J. F. W. McOmie, “Protective Groups in Organic Chemistry”, Plenum Press, 1973; T. W. Greene and P. G. M. Wuts, “Protective Groups in Organic Synthesis”, 3rd edition, John Wiley, 1999; and P. J. Kocienski, “Protecting groups”, Georg Thieme Verlag, 1994.
In a second step when the group A of 7 is a carbamoyl group this is dehydrated to the corresponding nitrile using standard dehydrating conditions such as trifluoroacetic anhydride, or phosphorous oxychloride in pyridine or DMF (Scheme 2). Deprotection using standard methods followed by reaction with an acid chloride or isocyanate in the presence of base gives the compounds of the invention. Alternatively such compounds may be prepared from the deprotected compound by coupling the amine with a carboxylic acid using standard coupling conditions or reacting the amine with a further amine in the presence of CDI or phosgene or diphosgene.
Alternatively in a second step the intermediate 7 can be deprotected using standard methods and reacted with an acid chloride or isocyanate in the presence of base (Scheme 3). Alternatively such compounds may be prepared from the deprotected compound by coupling the amine with a carboxylic acid using standard coupling conditions or reacting the amine with a further amine in the presence of CDI or phosgene or diphosgene.
When the group A of 8 is a carbamoyl group this is dehydrated to a nitrile using standard dehydrating conditions such as trifluoroacetic anhydride, or phosphorous oxychloride in pyridine or DMF giving the compounds of the invention (Scheme 4).
When the group A of 8 is a hydroxymethyl group this is oxidised to an aldehyde 9, using an oxidising agent such as Dess Martin Periodinane giving the compounds of the invention (Scheme 5).
The aldehyde 9 can be further manipulated to give ketones (Scheme 6). This involves reaction of the aldehyde with organometallic reagents such as Grignard reagents or lithiated aromatics and heteroaromatics and the subsequent oxidation of the resulting alcohol using an oxidising agent such as Dess Martin Periodinane giving the compounds of the invention.
An alternative route to the ketones starts from a nitrogen containing heterocycle-2-carboxaldehyde suitably amino-protected with a standard protecting group such as tert-butyloxycarbonyl (BOC), benzyloxycarbonyl (Z) or 9-fluorenylmethyloxycarbonyl (Fmoc). The use of such groups is well known in the art. In a first step the aldehyde is reacted with organometallic reagents such as Grignard reagents or lithiated aromatics and heteroaromatics (Scheme 7). The protecting group is removed by standard methods giving compound 10.
An alpha amino acid suitably carboxyl-protected with a standard protecting group is reacted with an acid chloride or isocyanate in the presence of base (Scheme 8). Alternatively such compounds may be prepared from the deprotected compound by coupling the amine with a carboxylic acid using standard coupling conditions or reacting the amine with a further amine in the presence of CDI or phosgene or diphosgene. The carboxylic acid is protected as an ester such as methyl, benzyl or tert-butyl. The use of such groups is well known in the art. Where R1 has a reactive functional group such as an amine or a carboxylic acid, this group will also be protected. After deprotection using standard methodology the resulting acid is coupled with the amine 10 using standard coupling methodology. Subsequent oxidation of the resulting alcohol using an oxidising agent such as Dess Martin Periodinane gives the compounds of the invention.
When the A group of 8 is a boronic acid this is protected as an ester by protecting groups described in the literature such as pinanediol esters. This protecting group is removed by methods described in the literature such as sodium metaperiodate/ammonium acetate or phenylboronic acid giving the compounds of the invention (Scheme 9).
When the R3 group of the alpha amino acid 6 is not hydrogen the required amino acid is prepared from the carboxyl protected amino acid by reductive amination of the free amino group with an aldehyde and either sodium cyanoborohydride or sodium triacetoxyborohydride or similar reagent (Scheme 10) This is followed by protection of the amino function and deprotection of the carboxyl function using standard methods to give the required amino acids. The carboxylic acid is protected as an ester such as methyl, benzyl or tert-butyl. The amine is protected with a standard protecting group such as tert-butyloxycarbonyl (BOC), benzyloxycarbonyl (Z) or 9-fluorenylmethyloxycarbonyl (Fmoc). The use of such groups is well known in the art. Where R1 has a reactive functional group such as an amine or a carboxylic acid, this group will also be protected
The nitrogen containing heterocycle derivatives are either known compounds or can be prepared by simple modification of published synthetic routes.
The general methods are further illustrated in the following, non-limiting examples. These examples shall not be construed as a limitation of how the invention may be practiced.
Examples of Q being CN are provided in Examples E1-E24, E34-E61 and Tables 1-23. Examples of Q being B(OH)2 are provided in Examples E29-E30. Examples of Q being C(═O)X1 are provided in Examples E31-E33 and E62-E66. Examples of Q being H are provided in Examples E25-E28 and Tables 24-25.
All citations are incorporated by reference.
The compounds were tested for protease (FAP, DPIV, DP8 and DP9) inhibitory activity using a fluorogenic assay utilising dipeptide-AFC substrates. For example, DPIV inhibitory activity was measured using H-Ala-Pro-AFC as a substrate using a method described in WO 9515309. The particularly preferred compounds were competitive inhibitors with IC50 of <1 μM for FAP and IC50>1 μM for DPIV, DP8 and DP9.
The following abbreviations have been used:
Nα-(tert-Butyloxycarbonyl)-L-isoleucine (4.2 g, 18.2 mmol) was dissolved in CH2Cl2/DMF (9:1, 100 ml). The solution was cooled to 0° C., L-prolinamide (2.5 g, 21.7 mmol), 1-hydroxybenzotriazole hydrate (4.9 g, 36.2 mmol) and water soluble carbodiimide (4.4 g, 22.0 mmol) were added and after 15 min the pH adjusted to pH9 with triethylamine. After 18 h at 0° C. to room temperature the solvent was removed in vacuo and the residue was taken up in ethyl acetate (200 ml). The solution was washed with 0.3M KHSO4, sat. NaHCO3, water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 2% methanol, 98% chloroform) to give a colourless oil identified as the title compound (4.8 g, 14.58 mmol, 80%).
(2S)-1-((2S)-2-(tert-Butyloxycarbonylamino)-3-methylpentanoyl)pyrrolidine-2-carboxamide (4.8 g, 14.58 mmol) was dissolved in dry THF (100 ml). The solution was cooled to 0° C., triethylamine (2.8 g, 28 mmol) was added followed by the slow addition of trifluoroacetic anhydride (6.8 g, 32.4 mmol). The pH was adjusted to pH9 with triethylamine. After 30 min the reaction mixture was diluted with ethyl acetate (100 ml). The solution was washed with 0.3M KHSO4, sat. NaHCO3, water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 60% Pet. ether, 40% ethyl acetate) to give a colourless oil identified as the title compound (3.4 g, 11.0 mmol, 75%).
(2S)-1-((2S)-2-(tert-Butyloxycarbonylamino)-3-methylpentanoyl)pyrrolidine-2-carbonitrile (1.51 g, 4.88 mmol) was treated with trifluoroacetic acid (50 ml). After 1 h at room temperature the solvent was removed in vacuo and the residue dissolved in CH2Cl2 (100 ml). The solution was cooled to 0° C., triethylamine (980 mg, 9.8 mmol) was added followed by 2-anisoyl chloride (920 mg, 5.4 mmol). After 18 h at 0° C. to room temperature the solvent was removed in vacuo and the residue dissolved in ethyl acetate (200 ml). This solution was washed with 0.3M KHSO4, sat. NaHCO3, water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 60% Pet. ether, 40% ethyl acetate) to give a colourless oil identified as the title compound (1.53 g, 4.46 mmol, 91%).
(2S)-1-((2S)-2-(tert-Butyloxycarbonylamino)-3-methylpentanoyl)pyrrolidine-2-carbonitrile (3.28 g, 10.2 mmol) was treated with trifluoroacetic acid (50 ml). After 1 h at room temperature the solvent was removed in vacuo and the residue dissolved in CH2Cl2 (100 ml). The solution was cooled to 0° C., triethylamine (2.5 g, 25 mmol) was added followed by ethylisocyanate (810 mg, 11.4 mmol). After 18 h at 0° C. to room temperature the solvent was removed in vacuo and the residue dissolved in ethyl acetate (200 ml). This solution was washed with 0.3M KHSO4, sat. NaHCO3, water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was recrystallised from Pet. ether and ethyl acetate to give a white solid identified as the title compound (2.58 g, 9.21 mmol, 87%).
(2S)-1-((2S)-2-Amino-3-methylpentanoyl)pyrrolidine-2-carbonitrile hydrochloride (50 mg, 0.2 mmol) was dissolved in CH2Cl2 (100 ml). The solution was cooled to 0° C., triethylamine (50 mg, 0.5 mmol) was added followed by 3-anisoyl chloride (38 mg, 0.22 mmol). After 18 h at 0° C. to room temperature the solvent was removed in vacuo and the residue dissolved in ethyl acetate (200 ml). This solution was washed with 0.3M KHSO4, sat. NaHCO3, water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 60% Pet. ether, 40% ethyl acetate) to give a colourless oil identified as the title compound (59 mg, 0.17 mmol, 86%).
(2S)-1-((2S)-2-Amino-3-methylpentanoyl)pyrrolidine-2-carbonitrile hydrochloride (50 mg, 0.2 mmol) was dissolved in CH2Cl2 (100 ml). The solution was cooled to 0° C., triethylamine (50 mg, 0.5 mmol) was added followed by 4-anisoyl chloride (38 mg, 0.22 mmol). After 18 h at 0° C. to room temperature the solvent was removed in vacuo and the residue dissolved in ethyl acetate (200 ml). This solution was washed with 0.3M KHSO4, sat. NaHCO3, water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 60% Pet. ether, 40% ethyl acetate) to give a colourless oil identified as the title compound (46 mg, 0.13 mmol, 67%).
(2S)-1-((2S)-2-Amino-3-methylpentanoyl)pyrrolidine-2-carbonitrile hydrochloride (50 mg, 0.2 mmol) was dissolved in CH2Cl2 (100 ml). The solution was cooled to 0° C., triethylamine (50 mg, 0.5 mmol) was added followed by pivaloyl chloride (24 mg, 0.20 mmol). After 18 h at 0° C. to room temperature the solvent was removed in vacuo and the residue dissolved in ethyl acetate (200 ml). This solution was washed with 0.3M KHSO4, sat. NaHCO3, water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 60% Pet. ether, 40% ethyl acetate) to give a colourless oil identified as the title compound (37 mg, 0.13 mmol, 63%).
Nα-(tert-Butyloxycarbonyl)-L-proline (5.0 g, 23.26 mmol) was dissolved in CH2Cl2/DMF (9:1, 100 ml). The solution was cooled to 0° C., L-prolinamide (2.9 g, 25.2 mmol) 1-hydroxybenzotriazole hydrate (6.3 g, 46.7 mmol) and water soluble carbodiimide (5.6 g, 28.0 mmol) were added and after 15 min the pH adjusted to pH9 with triethylamine. After 18 h at 0° C. to room temperature the solvent was removed in vacuo and the residue was taken up in ethyl acetate (200 ml). This solution was washed with 0.3M KHSO4, sat. NaHCO3, water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 9% methanol, 91% chloroform) to give a colourless oil identified as the title compound (5.36 g, 17.2 mmol, 74%).
(2S)-1-((2S)-1-(tert-Butyloxycarbonylamino) pyrrolidine-2-carbonyl)pyrrolidine-2-carboxamide (5.26 g, 16.4 mmol) was dissolved in dry THF (100 ml). The solution was cooled to 0° C., triethylamine (3.3 g, 33 mmol) was added followed by the slow addition of trifluoroacetic anhydride (7.8 g, 37.1 mmol). The pH was adjusted to pH9 with triethylamine. After 30 min the reaction mixture was diluted with ethyl acetate (100 ml), washed with water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 4% methanol, 96% chloroform) to give a colourless oil identified as the title compound (3.9 g, 13.3 mmol, 79%).
(2S)-1-((2S)-1-(tert-Butyloxycarbonylamino)pyrrolidine-2-carbonyl)pyrrolidine-2-carbonitrile (3.8 g, 12.9 mmol) was treated with trifluoroacetic acid (50 ml). After 1 h at room temperature the solvent was removed in vacuo and the residue dissolved in CH2Cl2 (100 ml). The solution was cooled to 0° C., triethylamine (2.7, 27 mmol) was added followed by 3-anisoyl chloride (2.51 g, 14.7 mmol). After 18 h at 0° C. to room temperature the solvent was removed in vacuo and the residue dissolved in ethyl acetate (200 ml). This solution was washed with 0.3M KHSO4, sat. NaHCO3, water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 3% methanol, 97% chloroform) to give an orange oil identified as the title compound (3.6 g, 11.6 mmol, 85%).
(2S)-1-((2S)-1-(tert-Butyloxycarbonylamino)pyrrolidine-2-carbonyl)pyrrolidine-2-carbonitrile (1.43 g, 4.88 mmol) was treated with trifluoroacetic acid (50 ml). After 1 h at room temperature the solvent was removed in vacuo and the residue dissolved in CH2Cl2 (100 ml). The solution was cooled to 0° C., triethylamine (980 mg, 9.8 mmol) was added followed by isopropylisocyanate (515 mg, 6.65 mmol). After 18 h at 0° C. to room temperature the solvent was removed in vacuo and the residue dissolved in ethyl acetate (200 ml). This solution was washed with 0.3M KHSO4, sat. NaHCO3, water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 4% methanol, 96% chloroform) to give a colourless oil identified as the title compound (1.29 g, 4.65 mmol, 95%).
(2S)-1-((2S)-2-Amino-3,3-dimethylbutanoyl)pyrrolidine-2-carbonitrile hydrochloride (150 mg, 0.61 mmol; prepared according to Jenkins et al., WO 9515309) was dissolved in CH2Cl2 (40 ml). The solution was cooled to 0° C., triethylamine (120 mg, 1.2 mmol) was added followed by 2-anisoyl chloride (120 mg, 0.7 mmol). After 18 h at 0° C. to room temperature the solvent was removed in vacuo and the residue dissolved in ethyl acetate (200 ml). This solution was washed with 0.3M KHSO4, sat. NaHCO3, water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 60% Pet. ether, 40% ethyl acetate) to give a white solid identified as the title compound (172 mg, 0.5 mmol, 82%).
Fmoc-Sieber resin (1 g, 0.6 mmol/g substitution) was swollen with CH2Cl2 for 10 min. The resin was further washed with DMF three times. The Fmoc-group was removed with 20% piperidine in DMF for 30 min. Further repeated washings were done with DMF (3×2 min), CH2Cl2 (3×2 min), and DMF (1×1 min). Then Fmoc-L-proline (1.5 mmol, 2.5 equivalents with respect to resin loading), PyBop® (1.5 mmol) and diisopropylethylamine (3.0 mmol) together in DMF (10 ml) were added to the resin. The resin was shaken at room temperature for 60 min. A small sample of resin was subjected to Kaiser test for the completion of reaction. The resin was filtered and washed with DMF (3×2 min), CH2Cl2 (2×2 min), MeOH (2×2 min), CH2Cl2 (2×2 min) and MeOH (3×2 min).
Resin from step A was swollen with CH2Cl2 (1×10 min) then further washed with DMF three times. The Fmoc group was removed with 20% piperidine in DMF for 30 min. Further resin washings were done with DMF (3×2 min), CH2Cl2 (3×2 min) and DMF (1×1 min). Then Fmoc-L-Proline (1.5 mmol, 2.5 equivalents with respect to initial resin loading), PyBop® (1.5 mmol) and diisopropylethylamine (3 mmol) together in DMF (10 ml) were added to the resin. The resin was shaken at room temperature for 60 min. A small sample of resin was submitted for isatin test for completion of reaction. The resin was filtered and washed with DMF (3×2 min), CH2Cl2 (2×2 min), MeOH (2×2 min), CH2Cl2 (2×2 min) and MeOH (3×2 min).
Resin from step B was swollen with CH2Cl2 (1×10 min). The resin was further washed with DMF three times. The Fmoc group was removed with 20% piperidine in DMF for 30 min. Further resin washings were done with DMF (3×2 min) and CH2Cl2 (3×2 min). Then 3-chlorobenzoyl chloride (1.5 mmol, 2.5 equivalents with respect to original resin loading) and diisopropylethylamine (3 mmol) were added in CH2Cl2 (10 ml) were added to the resin. The resin was shaken at room temperature for 18 h. A small sample was subjected to isatin test for completion of reaction. The resin was filtered and washed with CH2Cl2 (3×2 min), MeOH (2×2 min), CH2Cl2 (2×2 min), and MeOH (2×2 min).
Resin from step C was swollen with CH2Cl2 (12 ml). Then trifluoroacetic anhydride (3 mmol, 5 equivalents with respect to initial resin) and pyridine (6 mmol) were added to the resin. The resin shaken at room temperature for 18 h. Resin filtered, washed CH2Cl2. The solvent was removed in vacuo and the residue dissolved in ethyl acetate (70 ml). This solution was washed with 0.3M KHSO4, water, 1M NaHCO3, water and brine, dried (Na2SO4) and evaporated in vacuo, the residue was lyophilised from acetonitrile/water to yield an orange oil identified as the title compound (150 mg, 0.45 mmol).
2 g of resin synthesised as described for step A of Example E9 was swollen with CH2Cl2 (1×10 min) then further washed with DMF three times. The Fmoc group was removed with 20% piperidine in DMF for 30 min. Further resin washings were done with DMF (3×2 min), CH2Cl2 (3×2 min) and DMF (1×1 min). Then Boc-L-leucine (3 mmol, 2.5 equivalents with respect to initial resin loading), PyBop® (3 mmol) and diisopropylethylamine (6 mmol) together in DMF (20 ml) were added to the resin. The resin was shaken at room temperature for 60 min. A small sample of resin was submitted for isatin test for completion of reaction. The resin was filtered and washed with DMF (3×2 min), CH2Cl2 (2×2 min), MeOH (2×2 min), CH2Cl2 (2×2 min) and MeOH (3×2 min).
Resin from step C was swollen with CH2Cl2 (6 ml). Then trifluoroacetic anhydride (6 mmol, 5 equivalents with respect to initial resin) and 12 mmol of pyridine were added to the resin. Resin shaken at room temperature for 18 h. Resin filtered, washed CH2Cl2. The solvent was removed in vacuo and the residue dissolved in ethyl acetate (70 ml). This solution was washed with 0.3M KHSO4, water, 1M NaHCO3, water and brine, dried (Na2SO4) and evaporated in vacuo, the residue was lyophilised from acetonitrile/water to yield an orange oil identified as the title compound (220 mg, 0.72 mmol, 60%).
(2S)-1-((2S)-2-(tert-Butyloxycarbonylamino)-4-methylpentanoyl)pyrrolidine-2-carbonitrile (50 mg, 0.16 mmol) was treated with trifluoroacetic acid (5 ml). After 1 h at room temperature the solvent was removed in vacuo and the residue dissolved in CH2Cl2 (5 ml). The solution was cooled to 0° C., triethylamine (32 mg, 0.32 mmol) was added followed by 3-anisoyl chloride (30 mg, 0.176 mmol). After 18 h at 0° C. to room temperature the solvent was removed in vacuo and the residue dissolved in ethyl acetate (70 ml). This solution washed with 0.3M KHSO4, sat. NaHCO3, water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 60% Pet. ether, 40% ethyl acetate) to give a colourless oil identified as the title compound (35 mg, 0.10 mmol, 64%).
(2S)-1-((2S)-2-(2-Amino)-3-methylpentanoyl)pyrrolidine-2-carbonitrile hydrochloride (75 mg, 0.3 mmol) was dissolved in CH2Cl2 (20 ml). To this solution was added triethylamine (100 mg, 1.0 mmol) followed by 2-phenylethylisothiocyanate (50 mg, 0.31 mmol). After 18 h at room temperature the solvent was removed in vacuo and the residue dissolved in ethyl acetate (70 ml). This solution was washed with 0.3M KHSO4, sat. NaHCO3, water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 20% Pet. ether, 80% ethyl acetate) to give a white solid identified as the title compound (86 mg, 0.23 mmol, 76%).
(2S)-1-((2S)-1-(2-tert-Butyloxycarbonylamino)pyrrolidine-2-carbonyl)pyrrolidine-2-carbonitrile (160 mg, 0.55 mmol) was treated with trifluoroacetic acid (50 ml). After 1 h at room temperature the solvent was removed in vacuo and the residue dissolved in CH2Cl2 (20 ml). Triethylamine (150 mg, 1.5 mmol) was added followed by n-propylisothiocyanate (55 mg, 0.5 mmol). After 18 h at room temperature the solvent was removed in vacuo and the residue dissolved in ethyl acetate (70 ml). This solution was washed with 0.3M KHSO4, sat. NaHCO3, water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 20% Pet. ether, 80% ethyl acetate) to give a white solid identified as the title compound (55 mg, 0.19 mmol, 34%).
Glycine methylester hydrochloride (2.0 g, 16.0 mmol) was dissolved in MeOH/AcOH (9:1, 25 ml). Triethylamine (1.8 g, 18.0 mmol) was added followed by 2-butanone (1.3 g, 18.0 mmol). After 2 h at room temperature sodium triacetoxyborohydride (3.81 g, 18.0 mmol) was added. After 18 h at room temperature the solvent was removed in vacuo and the residue was taken up in chloroform (200 ml). This solution was washed with sat. NaHCO3, water and brine, dried (Na2SO4) and evaporated in vacuo to give a colourless oil identified as the title compound (646 mg, 4.46 mmol, 28%).
N)-(2-Methylpropyl)glycine methyl ester (646 mg, 4.45 mmol) was dissolved in CH2Cl2 (25 ml). Triethylamine (560 mg, 5.6 mmol) was added followed by di-tertbutyl dicarbonate (1.07 g, 4.90 mmol). After 18 h at room temperature N,N-dimethylethylenediamine (1 ml) was added, the solvent was removed in vacuo and the residue was taken up in chloroform (100 ml). This solution was washed with 0.3M KHSO4, sat. NaHCO3, water and brine, dried (Na2SO4) and evaporated in vacuo to give an amber oil identified as the title compound (960 mg, 3.92 mmol, 88%).
Nα-(tert-Butyloxycarbonyl)-Nα-(2-methylpropyl) glycine methyl ester (960 mg, 3.91 mmol) was dissolved in THF/H2O (9:1, 25 ml). Lithium hydroxide monohydrate (492 mg, 11.7 mmol) was added. After 18 h at room temperature the reaction mixture was diluted with ethyl acetate (100 ml). This solution was washed with 1M HCl, water and brine, dried (Na2SO4) and evaporated in vacuo to give a colourless oil identified as the title compound (360 mg, 1.56 mmol, 40%).
Nα-(tert-Butyloxycarbonyl)-Nα-(2-methylpropyl) glycine (360 mg, 1.56 mmol) was dissolved in CH2Cl2/DMF (9:1, 20 ml). The solution was cooled to 0° C., L-prolinamide hydrochloride (282 mg, 1.87 mmol), 1-hydroxybenzotriazole hydrate (295 mg, 2.18 mmol) and water soluble carbodiimide (358 mg, 1.87 mmol) were added and after 15 min the pH adjusted to pH9 with triethylamine. After 18 h at 0° C. to room temperature the solvent was removed in vacuo and the residue was taken up in ethyl acetate (100 ml). This solution was washed with 0.3M KHSO4, sat. NaHCO3, water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 4% methanol, 94% chloroform, 2% triethylamine) to give a colourless oil identified as the title compound (330 mg, 1.01 mmol, 65%).
(2S)-1-(2-(N-tert-Butyloxycarbonyl-N-(2-methylpropyl)amino)acetyl)pyrrolidine-2-carboxamide (330 mg, 1.01 mmol) was dissolved in dry THF (20 ml). The solution was cooled to 0° C., triethylamine (210 mg, 2.1 mmol) was added followed by the slow addition of trifluoroacetic anhydride (460 g, 2.2 mmol). The pH was adjusted to pH9 with triethylamine. After 30 min the reaction mixture was diluted with ethyl acetate (100 ml), washed with water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 3% methanol, 97% chloroform) to give an orange oil identified as the title compound (156 mg, 0.50 mmol, 50%).
(2S)-1-(2-(N-tert-Butyloxycarbonyl-N-(2-methylpropyl)amino)acetyl)pyrrolidine-2-carbonitrile (25 mg, 0.081 mmol) was treated with trifluoroacetic acid (50 ml). After 1 h at room temperature the solvent was removed in vacuo and the residue dissolved in CH2Cl2 (100 ml). The solution was cooled to 0° C., triethylamine (30 mg, 0.3 mmol) was added followed by 2-anisoyl chloride (15 mg, 0.088 mmol). After 18 h at 0° C. to room temperature the solvent was removed in vacuo and the residue dissolved in ethyl acetate (200 ml). This solution was washed with 0.3M KHSO4, sat. NaHCO3, water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 2% methanol, 98% chloroform) to give a colourless oil identified as the title compound (18 mg, 0.052 mmol, 66%).
(2S)-1-((2S)-2-(2-Methoxybenzoylamino)-5-(9-fluorenylmethyloxycarbonyl carbonylamino) pentanoyl)pyrrolidine-2-carbonitrile (200 mg, 0.35 mmol) prepared by the method described for Example E10 was dissolved in acetonitrile (10 ml), diethylamine (10 ml) was added. After 90 min at room temperature the solvent was removed in vacuo. The residue was purified by flash chromatography (eluant: 1% triethylamine, 5% methanol, 94% chloroform) to give a colourless oil identified as the title compound (99 mg, 0.28 mmol, 80%).
(2S)-1-((2S)-2-(2-Methoxybenzoylamino)-6-(9-fluorenylmethyloxycarbonyl carbonylamino) hexanoyl)pyrrolidine-2-carbonitrile (176 mg, 0.3 mmol) prepared by the method described for Example E10 was dissolved in acetonitrile (10 ml), diethylamine (10 ml) was added. After 90 min at room temperature the solvent was removed in vacuo. The residue was purified by flash chromatography (eluant: 1% triethylamine, 5% methanol, 94% chloroform) to give a colourless oil identified as the title compound (78 mg, 0.22 mmol, 73%).
(4R)-3-(tert-Butyloxycarbonyl)thiazolidine-4-carboxylic acid (12.5 g, 54.1 mmol) was dissolved in CH2Cl2/DMF (9:1, 150 ml). To this solution at 0° C. was added 1-hydroxybenzotriazole hydrate (14.6 g, 108 mmol) and water-soluble carbodiimide (13.0 g, 65 mmol). After 1 h at 0° C. ammonia (35%, 50 ml) was added. After 18 h at 0° C. to room temperature the solvent was removed in vacuo and the residue was taken up in ethyl acetate (500 ml). This solution was washed with 0.3M KHSO4, sat. NaHCO3, water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 2% methanol, 98% chloroform) to give a colourless oil identified as the title compound (8.9 g, 38.4 mmol, 71%).
(4R)-3-(tert-Butyloxycarbonyl)thiazolidine-4-carboxamide (8.6 g, 37.1 mmol) was dissolved in 4M HCl/dioxan (50 ml). After 1 h at room temperature the solvent was evaporated in vacuo to give a white solid identified as the title compound (6.2 g, 36.8 mmol, 99%).
Nα-(tert-Butyloxycarbonyl)-L-isoleucine (1.37 g, 5.9 mmol) was dissolved in CH2Cl2/DMF (9:1, 100 ml). The solution was cooled to 0° C., (4R)-thiazolidine-4-carboxamide hydrochloride (1 g, 5.9 mmol), 1-hydroxybenzotriazole hydrate (920 mg, 6.8 mmol) and water soluble carbodiimide (1.4 g, 7.0 mmol) were added and after 15 min the pH adjusted to pH9 with triethylamine. After 18 h at 0° C. to room temperature the solvent was removed in vacuo and the residue was taken up in ethyl acetate (200 ml). This solution was washed with 0.3M KHSO4, sat. NaHCO3, water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 4% methanol, 96% chloroform) to give a colourless oil identified as the title compound (1.65 g, 4.8 mmol, 82%).
((4R)-1-((2S)-2-(2-tert-Butyloxycarbonylamino)-3-methylpentanoyl)thiazolidine-4-carboxamide (1.3 g, 3.76 mmol) was dissolved in dry THF (100 ml). The solution was cooled to 0° C., triethylamine (750 mg, 7.5 mmol) was added followed by the slow addition of trifluoroacetic anhydride (1.7 g, 8.1 mmol). The pH was adjusted to pH9 with triethylamine. After 30 min the reaction mixture was diluted with ethyl acetate (100 ml), washed with water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 80% Pet. ether, 20% ethyl acetate) to give a yellow oil identified as the title compound (321 mg, 0.98 mmol, 26%).
(4R)-1-((2S)-2-(2-tert-Butyloxycarbonylamino)-3-methylpentanoyl)thiazolidine-4-carbonitrile (50 mg, 0.15 mmol) was treated with trifluoroacetic acid (50 ml). After 1 h at room temperature the solvent was removed in vacuo and the residue dissolved in CH2Cl2 (100 ml). The solution was cooled to 0° C., triethylamine (40 mg, 0.4 mmol) was added followed by 3-anisoyl chloride (26 mg, 0.15 mmol). After 18 h at 0° C. to room temperature the solvent was removed in vacuo and the residue dissolved in ethyl acetate (200 ml). This solution was washed with 0.3M KHSO4, sat. NaHCO3, water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 60% Pet. ether, 40% ethyl acetate) to give a colourless oil identified as the title compound (20 mg, 0.055 mmol, 37%).
N-(tert-Butyloxycarbonyl)-L-4-trans-hydroxyproline methyl ester (2.5 g, 10.2 mmol) was dissolved in CH2Cl2 (70 ml). Dess-Martin periodinane (5.0 g, 12.1 mmol) was added. After 3 h at room temperature the solvent was removed in vacuo and the residue was taken up in ethyl acetate (300 ml). This solution was washed with sat. NaHCO3, water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 10% ethyl acetate, 90% Pet. ether ° C.) to give a colourless oil identified as the title compound (2.4 g, 9.7 mmol, 95%).
Methyl (2S)—N-(tert-butyloxycarbonyl)pyrrolidin-4-one-2-carboxylate (2.3 g, 9.3 mmol) was dissolved in CH2Cl2 (70 ml). (Diethylamino)sulphur trifluoride (4.5 g, 27.9 mmol) was added to this solution at 0° C. After 18 hs at 0° C. to room temperature the reaction mixture was carefully poured into sat. NaHCO3 (100 ml) and stirred for 15 min and extracted with CH2Cl2. The organic extract was washed with water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 10% ethyl acetate, 90% Pet. ether ° C.) to give a colourless oil identified as the title compound (2.4 g, 8.9 mmol, 96%).
Methyl (2S)—N-(tert-butyloxycarbonyl)-4,4-difluoropyrrolidine-2-carboxylate (2.2 g, 8.3 mmol) was dissolved in THF (100 ml). Aqueous lithium hydroxide (1M, 10.6 ml, 10.6 mmol) was added. After 3 h at room temperature the reaction mixture was diluted with ethyl acetate (150 ml), washed with 1M HCl, water, and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 95% chloroform, 4% methanol, 1% acetic acid) to give an orange oil identified as the title compound (2.1 g, 8.3 mmol 100%).
(2S)—N-(tert-Butyloxycarbonyl)-4,4-difluoropyrrolidine-2-carboxylic acid (1.0 g, 4.0 mmol) was dissolved in CH2Cl2/DMF (9:1, 50 ml). To this solution at 0° C. was added 1-hydroxybenzotriazole hydrate (1.1 g, 8.1 mmol) and water soluble carbodiimide (960 mg, 4.8 mmol). After 1 h at 0° C. ammonia (35%, 5 ml) was added. After 18 h at 0° C. to room temperature the solvent was removed in vacuo and the residue was taken up in ethyl acetate (200 ml). This solution was washed with 0.3M KHSO4, sat. NaHCO3, water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 85% ethyl acetate, 15% Pet. ether ° C.) to give a colourless oil identified as the title compound (945 mg, 3.8 mmol, 95%).
(2S)—N-(tert-Butyloxycarbonyl)-4,4-difluoropyrrolidine-2-carboxamide (530 mg, 2.12 mmol) was dissolved in 4M HCl/dioxan (30 ml) after 1 h at room temperature the solvent was removed in vacuo and the residue dissolved in CH2Cl2/DMF (9:1, 20 ml). To this solution at 0° C. was added e-(tert-butyloxycarbonyl)-L-valine (461 mg, 2.12 mmol), 1-hydroxybenzotriazole hydrate (401 mg, 2.97 mmol) and water-soluble carbodiimide (487 mg, 2.54 mmol). After 15 min at 0° C. the pH was adjusted to pH 8 with N,N-disopropylethylamine. After 18 h at 0° C. to room temperature the reaction mixture was diluted with CHCl3 (70 ml). This solution was washed with 0.3M KHSO4, sat. NaHCO3, water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (Eluant: 97% chloroform, 2% methanol, 1% triethylamine) to give a white solid identified as the title compound (430 mg, 1.23 mmol, 58%).
(2S)-1-((2S)-2-(2-tert-Butyloxycarbonylamino)-3-methylbutanoyl)-4,4-difluoropyrrolidine-2-carboxamide (430 mg, 1.23 mmol) was dissolved in dry THF (100 ml). The solution was cooled to 0° C., triethylamine (246 mg, 2.46 mmol) was added followed by the slow addition of trifluoroacetic anhydride (520 mg, 2.46 mmol). The pH was adjusted to pH9 with triethylamine. After 30 min the reaction mixture was diluted with ethyl acetate (100 ml), washed with water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 80% Pet. ether, 20% ethyl acetate) to give an orange oil identified as the title compound (323 mg, 0.98 mmol, 79%).
(2S)-1-((2S)-2-(2-tert-Butyloxycarbonylamino)-3-methylbutanoyl)-4,4-difluoropyrrolidine-2-carbonitrile (31 mg, 0.09 mmol) was treated with trifluoroacetic acid (10 ml). After 1 h at room temperature the solvent was removed in vacuo and the residue dissolved in CH2Cl2 (20 ml). The solution was cooled to 0° C., triethylamine (30 mg, 0.3 mmol) was added followed by 2-anisoyl chloride (18 mg, 0.11 mmol). After 18 h at 0° C. to room temperature the solvent was removed in vacuo and the residue dissolved in ethyl acetate (200 ml). This solution was washed with 0.3M KHSO4, sat. NaHCO3, water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 80% Pet. ether, 20% ethyl acetate) to give a colourless oil identified as the title compound (14 mg, 0.04 mmol, 43%).
(2S)-1-((2S)-2-(2-tert-Butyloxycarbonylamino)-3-methylbutanoyl)-4,4-difluoropyrrolidine-2-carbonitrile (30 mg, 0.091 mmol) was treated with trifluoroacetic acid (10 ml). After 1 h at room temperature the solvent was removed in vacuo and the residue dissolved in CH2Cl2 (100 ml). The solution was cooled to 0° C., triethylamine (30 mg, 0.3 mmol) was added followed by ethylisocyanate (8 mg, 0.1 mmol). After 18 h at 0° C. to room temperature the solvent was removed in vacuo and the residue dissolved in ethyl acetate (200 ml). The solution was washed with 0.3M KHSO4, sat. NaHCO3, water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 1% methanol, 99% chloroform) to give a colourless oil identified as the title compound (18 mg, 0.060 mmol, 66%).
(2S)—N-(tert-Butyloxycarbonyl)-4,4-difluoropyrrolidine-2-carboxamide (1.16 g, 4.65 mmol) was dissolved in 4M HCl/dioxan (30 ml) after 1 h at room temperature the solvent was removed in vacuo and the residue dissolved in CH2Cl2/DMF (9:1, 20 ml). To this solution at 0° C. was added Nα-(tert-butyloxycarbonyl)-L-proline (1.0 g, 4.65 mmol), 1-hydroxybenzotriazole hydrate (880 mg, 6.65 mmol) and water soluble carbodiimide (1.07 g, 5.35 mmol). After 15 min at 0° C. the pH was adjusted to pH 9 with triethylamine. After 18 h at 0° C. to room temperature the reaction mixture was diluted with chloroform (70 ml). This solution was washed with 0.3M KHSO4, sat. NaHCO3, water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (Eluant: 97% chloroform, 2% methanol, 1% triethylamine) to give a white solid identified as the title compound (698 mg, 2.01 mmol, 43%).
(2S)-1-((2S)-1-(2-tert-Butyloxycarbonylamino)pyrrolidine-2-carbonyl)-4,4-difluoropyrrolidine-2-carboxamide (698 mg, 2.01 mmol) was dissolved in dry THF (50 ml). The solution was cooled to 0° C., triethylamine (200 mg, 2.0 mmol) was added followed by the slow addition of trifluoroacetic anhydride (844 mg, 4.02 mmol). The pH was adjusted to pH9 with triethylamine. After 30 min the reaction mixture was diluted with ethyl acetate (100 ml), washed with water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 80% Pet. ether, 20% ethyl acetate) to give an off-white identified as the title compound (232 mg, 0.708 mmol, 35%).
(2S)-1-((2S)-1-(2-tert-Butyloxycarbonylamino)pyrrolidine-2-carbonyl)-4,4-difluoropyrrolidine-2-carbonitrile (30 mg, 0.09 mmol) was treated with trifluoroacetic acid (50 ml). After 1 h at room temperature the solvent was removed in vacuo and the residue dissolved in CH2Cl2 (100 ml). The solution was cooled to 0° C., triethylamine (30 mg, 0.3 mmol) was added followed by ethylisocyanate (0.8 mg, 0.01 mmol). After 18 h at 0° C. to room temperature the solvent was removed in vacuo and the residue dissolved in ethyl acetate (200 ml). This solution was washed with 0.3M KHSO4, sat. NaHCO3, water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 60% Pet. ether, 40% ethyl acetate) to give a colourless oil identified as the title compound (25 mg, 0.083 mmol, 92%).
(2S)—N-(tert-Butyloxycarbonyl)-3,4-dehydropyrrolidine-2-carboxylic acid (2.0 g, 9.38 mmol) was dissolved in CH2Cl2/DMF (9:1, 50 ml). To this solution at 0° C. was added 1-hydroxybenzotriazole hydrate (1.77 g, 13.1 mmol) and water soluble carbodiimide (2.16 g, 11.26 mmol). After 1 h at 0° C. ammonia (35%, 7 ml) was added. After 18 h at 0° C. to room temperature the reaction mixture was diluted with chloroform (150 ml). This solution was washed with 0.3M KHSO4, sat. NaHCO3, water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 2% methanol, 98% chloroform) to give a white solid identified as the title compound (1.58 g, 7.4 mmol, 79%).
(2S)—N-(tert-Butyloxycarbonyl)-3,4-dehydropyrrolidine-2-carboxamide (1.58 g, 7.44 mmol) was dissolved in 4M HCl/dioxan (50 ml). After 1 h at room temperature the solvent was evaporated in vacuo to give a white solid identified as the title compound (830 mg, 5.58 mmol, 75%).
Nα-(tert-Butyloxycarbonyl)-L-isoleucine (645 mg, 2.79 mmol) was dissolved in CH2Cl2/DMF (9:1, 100 ml). The solution was cooled to 0° C., (2S)-3,4-dehydropyrrolidine-2-carboxamide hydrochloride (415 mg, 2.79 mmol), 1-hydroxybenzotriazole hydrate (528 mg, 3.91 mmol) and water soluble carbodiimide (642 mg, 3.35 mmol) were added and after 15 min the pH adjusted to pH9 with triethylamine. After 18 h at 0° C. to room temperature the reaction mixture was diluted with chloroform (100 ml). This solution was washed with 0.3M KHSO4, sat. NaHCO3, water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 4% methanol, 96% chloroform) to give an amber solid identified as the title compound (648 mg, 1.98 mmol, 71%).
(2S)-1-((2S)-2-(2-tert-Butyloxycarbonylamino)-3-methylpentanoyl)-3,4-dehydropyrrolidine-2-carboxamide (648 mg, 1.99 mmol) was dissolved in dry THF (100 ml). The solution was cooled to 0° C., triethylamine (398 mg, 3.98 mmol) was added followed by the slow addition of trifluoroacetic anhydride (836 mg, 3.98 mmol). The pH was adjusted to pH9 with triethylamine. After 30 min the reaction mixture was diluted with ethyl acetate (100 ml), washed with water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 100% ethyl acetate) to give a pale yellow solid identified as the title compound (408 mg, 1.33 mmol, 67%).
(2S)-1-((2S)-2-(2-tert-Butyloxycarbonylamino)-3-methylpentanoyl)-3,4-dehydropyrrolidine-2-carbonitrile (15 mg, 0.05 mmol) was treated with trifluoroacetic acid (50 ml). After 1 h at room temperature the solvent was removed in vacuo and the residue dissolved in CH2Cl2 (100 ml). The solution was cooled to 0° C., diisopropylethylamine (15 mg, 0.15 mmol) was added followed by 2-anisoyl chloride (9 mg, 0.059 mmol). After 18 h at 0° C. to room temperature the solvent was removed in vacuo and the residue dissolved in ethyl acetate (200 ml). This solution was washed with 0.3M KHSO4, sat. NaHCO3, water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 60% Pet. ether, 40% ethyl acetate) to give a colourless oil identified as the title compound (12 mg, 0.035 mmol, 71%).
(2S)—N-(tert-Butyloxycarbonyl)pipecolinic acid (3.0 g, 13.0 mmol) was dissolved in CH2Cl2/DMF (9:1, 150 ml). To this solution at 0° C. was added 1-hydroxybenzotriazole hydrate (2.0 g, 14.8 mmol) and water soluble carbodiimide (2.74 g, 13.7 mmol). After 1 h at 0° C. ammonia (35%, 25 ml) was added. After 18 h at 0° C. to room temperature the solvent was removed in vacuo and the residue was taken up in ethyl acetate (500 ml). This solution was washed with 0.3M KHSO4, sat. NaHCO3, water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 4% methanol, 96% chloroform) to give a white solid identified as the title compound (1.7 g, 7.4 mmol, 57%).
(2S)—N-(tert-Butyloxycarbonyl)piperidine-2-carboxamide (1.7 g, 7.4 mmol) was dissolved in 4M HCl/dioxan (50 ml). After 1 h at room temperature the solvent was evaporated in vacuo to give a white solid identified as the title compound (1.1 g, 6.7 mmol, 89%).
Nα-(tert-Butyloxycarbonyl)-L-isoleucine (772 mg, 3.3 mmol) was dissolved in CH2Cl2 (50 ml). The solution was cooled to 0° C., (2S)-piperidine-2-carboxamide hydrochloride (550 mg, 3.3 mmol) and PyBop® (1.74 g, 3.5 mmol) were added and the pH adjusted to pH9 with triethylamine. After 18 h at 0° C. to room temperature the solvent was removed in vacuo and the residue was taken up in ethyl acetate (200 ml). This solution was washed with 0.3M KHSO4, sat. NaHCO3, water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 2% methanol, 98% chloroform) to give a yellow oil identified as the title compound (654 mg, 1.9 mmol, 58%).
(2S)-1-((2S)-2-(2-tert-Butyloxycarbonylamino)-3-methylpentanoyl)piperidine-2-carboxamide (654 mg, 1.9 mmol) was dissolved in dry THF (50 ml). The solution was cooled to 0° C., triethylamine (400 g, 4.0 mmol) was added followed by the slow addition of trifluoroacetic anhydride (880 mg, 4.2 mmol). The pH was adjusted to pH9 with triethylamine. After 30 min the reaction mixture was diluted with ethyl acetate (100 ml), washed with water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 60% Pet. ether, 40% ethyl acetate) to give a colourless oil identified as the title compound (300 g, 0.93 mmol, 49%).
(2S)-1-((2S)-2-(2-tert-Butyloxycarbonylamino)-3-methylpentanoyl)piperidine-2-carbonitrile (50 mg, 0.15 mmol) was treated with trifluoroacetic acid (50 ml). After 1 h at room temperature the solvent was removed in vacuo and the residue dissolved in CH2Cl2 (100 ml). The solution was cooled to 0° C., triethylamine (43 mg, 0.43 mmol) was added followed by 2-anisoyl chloride (30 mg, 0.18 mmol). After 18 h at 0° C. to room temperature the solvent was removed in vacuo and the residue dissolved in ethyl acetate (200 ml). This solution was washed with 0.3M KHSO4, sat. NaHCO3, water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 60% Pet. ether, 40% ethyl acetate) to give a colourless oil identified as the title compound (20 mg, 0.056 mmol, 38%).
(2S)—N-(tert-Butyloxycarbonyl)-L-azetidine-2-carboxylic acid (3.87 g 19.23 mmol) was dissolved in CH2Cl2/DMF (9:1, 150 ml). To this solution at 0° C. was added 1-hydroxybenzotriazole hydrate (3.64 g, 26.92 mmol) and water-soluble carbodiimide (4.42 g, 23.08 mmol). After 1 h at 0° C. ammonia (35%, 15 ml) was added. After 18 h at 0° C. to room temperature the solvent was removed in vacuo and the residue was taken up in ethyl acetate (500 ml). This solution was washed with 0.3M KHSO4, sat. NaHCO3, water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 2% methanol, 98% chloroform) to give a colourless oil identified as the title compound (1.14 g, 5.77 mmol, 30%).
(2S)—N-(tert-Butyloxycarbonyl)azetidine-2-carboxamide (1.14 g, 5.69 mmol) was dissolved in 4M HCl/dioxan (50 ml). After 1 h at room temperature the solvent was evaporated in vacuo to give a white solid identified as the title compound (775 mg, 5.63 mmol, 99%).
Nα-(tert-Butyloxycarbonyl)-L-isoleucine (657 mg, 2.84 mmol) was dissolved in CH2Cl2/DMF (9:1, 100 ml). The solution was cooled to 0° C., (2S)-azetidine-2-carboxamide hydrochloride (388 mg, 2.84 mmol), 1-hydroxybenzotriazole hydrate (538 mg, 3.98 mmol) and water soluble carbodiimide (654 mg, 3.41 mmol) were added and after 15 min the pH adjusted to pH9 with triethylamine. After 18 h at 0° C. to room temperature the reaction mixture was diluted with chloroform (200 ml). This solution was washed with 0.3M KHSO4, sat. NaHCO3, water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 4% methanol, 96% chloroform) to give an orange oil identified as the title compound (620 mg, 1.99 mmol, 70%).
(2S)-1-((2S)-2-(2-tert-Butyloxycarbonylamino)-3-methylpentanoyl)azetidine-2-carboxamide (620 mg, 1.98 mmol) was dissolved in dry THF (100 ml). The solution was cooled to 0° C., triethylamine (390 mg, 3.9 mmol) was added followed by the slow addition of trifluoroacetic anhydride (832 mg, 3.96 mmol). The pH was adjusted to pH9 with triethylamine. After 30 min the reaction mixture was diluted with ethyl acetate (100 ml), washed with water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 3% methanol, 97% chloroform) to give a colourless oil identified as the title compound (354 mg, 1.2 mmol, 61%).
(2S)-1-((2S)-2-(2-tert-Butyloxycarbonylamino)-3-methylpentanoyl)azetidine-2-carbonitrile (50 mg, 0.168 mmol) was treated with trifluoroacetic acid (10 ml). After 1 h at room temperature the solvent was removed in vacuo and the residue dissolved in CH2Cl2 (100 ml). The solution was cooled to 0° C., diisopropylethylamine (50 mg, 0.50 mmol) was added followed by 2-anisoyl chloride (28 mg, 0.185 mmol). After 18 h at 0° C. to room temperature the solvent was removed in vacuo and the residue dissolved in ethyl acetate (200 ml). This solution was washed with 0.3M KHSO4, sat. NaHCO3, water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 50% Pet. ether, 50% ethyl acetate) to give a colourless oil identified as the title compound (10 mg, 0.03 mmol, 18%).
(4R)-1-((2S)-2-(3-Methoxybenzoylamino)-3-methylpentanoyl)thiazolidine-4-carbonitrile (70 mg, 0.2 mmol) was dissolved in CH2Cl2 (20 ml). The solution was cooled to 0° C., 3-chloroperoxybenzoic acid (33 mg, 0.19 mmol) was added. After 30 mins at 0° C. the solvent was evaporated in vacuo. The residue was purified by flash chromatography (eluant: 20% Pet. ether, 80% ethyl acetate) to give an orange oil identified as the title compound (14 mg, 0.037 mmol, 19%).
(4R)-1-((2S)-2-(3-Methoxybenzoylamino)-3-methylpentanoyl)thiazolidine-4-carbonitrile (70 mg, 0.2 mmol) was dissolved in CH2Cl2 (20 ml). The solution was cooled to 0° C., 3-chloroperoxybenzoic acid (170 mg, 1.0 mmol) was added. After 18 h at room temperature the reaction mixture was diluted with chloroform (70 ml). This solution was washed with sat. NaHCO3, water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 20% Pet. ether, 80% ethyl acetate) to give an orange oil identified as the title compound (18 mg, 0.045 mmol, 23%).
L-Isoleucine tertbutyl ester hydrochloride (500 mg, 2.23 mmol) was dissolved in CH2Cl2 (10 ml). Triethylamine (600 g, 6.0 mmol) and 2-anisoyl chloride (456 mg, 2.68 mmol) were added. After 18 h at room temperature the solvent was removed in vacuo and the residue was taken up in ethyl acetate (100 ml). This solution was washed with 0.3M KHSO4, sat. NaHCO3, water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 60% Pet. ether, 40% ethyl acetate) to give a colourless oil identified as the title compound (341 mg, 1.06 mmol, 48%).
(2S)-2-(2-Methoxybenzoylamino)-3-methylpentanoic acid tertbutyl ester (341 mg, 1.06 mmol) was dissolved in trifluoroacetic acid/CH2Cl2 (1:1, 10 ml). After 2 h at room temperature the solvent was removed in vacuo and the residue was purified by flash chromatography (eluant: 1% acetic acid, 2% methanol, 97% chloroform) to give a colourless oil identified as the title compound (242 mg, 0.91 mmol, 86%).
(2S)-2-(2-Methoxybenzoylamino)-3-methylpentanoic acid (25 mg, 0.94 mmol) was dissolved in CH2Cl2/DMF (9:1, 10 ml). The solution was cooled to 0° C., pyrrolidine (8 mg, 0.113 mmol), 1-hydroxybenzotriazole hydrate (18 mg, 0.132 mmol) and water soluble carbodiimide (22 mg, 0.132=1) were added and after 15 min the pH adjusted to pH9 with triethylamine. After 18 h at 0° C. to room temperature the solvent was removed in vacuo and the residue was taken up in ethyl acetate (70 ml). This solution was washed with 0.3M KHSO4, sat. NaHCO3, water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 20% Pet. ether, 80% ethyl acetate) to give an amber oil identified as the title compound (10 mg, 0.031 mmol, 33%).
(2S)-2-(2-Methoxybenzoylamino)-3-methylpentanoic acid (25 mg, 0.94 mmol) was dissolved in CH2Cl2/DMF (9:1, 10 ml). The solution was cooled to 0° C., 3,3-difluoropyrrolidine hydrochloride (16 mg, 0.113 mmol), 1-hydroxybenzotriazole hydrate (18 mg, 0.132 mmol) and water soluble carbodiimide (22 mg, 0.132 mmol) were added and after 15 min the pH adjusted to pH9 with triethylamine. After 18 h at 0° C. to room temperature the solvent was removed in vacuo and the residue was taken up in ethyl acetate (70 ml). This solution was washed with 0.3M KHSO4, sat. NaHCO3, water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 50% Pet. ether, 50% ethyl acetate) to give an amber oil identified as the title compound (10 mg, 0.028 mmol, 30%).
L-Proline benzyl ester hydrochloride (500 mg, 2.073 mmol) was dissolved in CH2Cl2 (10 ml). Triethylamine (600 mg, 6.0 mmol) and 3-anisoyl chloride (422 mg, 2.48 mmol) were added. After 18 h at room temperature the solvent was removed in vacuo and the residue was taken up in ethyl acetate (100 ml). This solution was washed with 0.3M KHSO4, sat. NaHCO3, water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 3% methanol, 97% chloroform) to give a colourless oil identified as the title compound (654 mg, 1.93 mmol, 93%).
(2S)-1-(3-Methoxybenzoylamino)pyrrolidine-2-carboxylic acid benzyl ester (654 mg, 1.93 mmol) was dissolved in THF/H2O (9:1, 10 ml). Lithium hydroxide monohydrate (243 mg, 5.79 mmol) was added. After 18 h at room temperature the reaction mixture was diluted with ethyl acetate (50 ml). This solution was washed with 1M KHSO4, water and brine, dried (Na2SO4) and evaporated in vacuo to give a colourless oil identified as the title compound (470 mg, 1.89 mmol, 98%).
(2S)-1-(3-Methoxybenzoylamino)pyrrolidine-2-carboxylic acid (61 mg, 0.245 mmol) was dissolved in CH2Cl2/DMF (9:1, 10 ml). To this solution was added thiazolidine (8 mg, 0.113 mmol), PyBrop® (126 mg, 0.271 mmol) and diisopropylethylamine (73.5 mg, 0.735 mmol). After 18 h at room temperature the solvent was removed in vacuo and the residue was taken up in ethyl acetate (70 ml). This solution was washed with 0.3M KHSO4, sat. NaHCO3, water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 3% methanol, 97% chloroform) to give an amber oil identified as the title compound (27 mg, 0.084 mmol, 34%).
L-Proline benzyl ester hydrochloride (500 mg, 2.073 mmol) was dissolved in CH2Cl2 (10 ml). Triethylamine (600 mg, 6.0 mmol) and isopropylisocyanate (210 mg, 2.48 mmol) were added. After 18 h at room temperature the solvent was removed in vacuo and the residue was taken up in ethyl acetate (100 ml). This solution was washed with 0.3M KHSO4, sat. NaHCO3, water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 3% methanol, 97% chloroform) to give a colourless oil identified as the title compound (528 mg, 1.82 mmol, 88%).
(2S)-1-(Isopropylcarbamoyl)pyrrolidine-2-carboxylic acid benzyl ester (528 mg, 1.82 mmol) was dissolved in THF/H2O (9:1, 10 ml). Lithium hydroxide monohydrate (229 mg, 5.46 mmol) was added. After 18 h at room temperature the reaction mixture was diluted with ethyl acetate (50 ml). This solution was washed with 1M KHSO4, water and brine, dried (Na2SO4) and evaporated in vacuo to give a colourless oil identified as the title compound (148 mg, 0.74 mmol, 41%).
1-((2S)-1-(Isopropylcarbamoyl)pyrrolidine-2-carboxylic acid (25 mg, 0.125 mmol) was dissolved in CH2Cl2 (10 ml). To this solution was added pyrrolidine (7 mg, 0.138 mmol), HBTU (52 mg, 0.138 mmol) and diisopropylethylamine (32.3 mg, 0.323 mmol). After 18 h at 0° C. to room temperature the solvent was removed in vacuo and the residue was taken up in ethyl acetate (70 ml). The solution was washed with 0.3M KHSO4, sat. NaHCO3, water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 1% triethylamine, 2% methanol, 97% chloroform) to give an amber oil identified as the title compound (11 mg, 0.043 mmol, 35%).
(2R)-1-((2S)-2-(1,1-Dimethylethoxycarbonylamino)-3-methylbutanoyl)pyrrolidine-2-boronate-(1S,2S,3R,5S)-pinanediol ester (synthesised as described in WO 0310127) (70 mg, 0.16 mmol) was dissolved in 4M HCl/dioxin (30 ml). After 1 h at room temperature the solvent was removed in vacuo and the residue dissolved in CH2Cl2 (25 ml). The solution was cooled to 0° C., triethylamine (36 mg, 0.36 mmol) was added followed by 2-anisoyl chloride (30 mg, 0.18 mmol). After 18 h at 0° C. to room temperature the solvent was removed in vacuo and the residue dissolved in ethyl acetate (70 ml). This solution was washed with 0.3M KHSO4, sat. NaHCO3, water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 60% Pet. ether, 40% ethyl acetate) to give a colourless oil identified as the title compound (65 mg, 0.13 mmol, 83%).
(2R)-1-((2S)-2-(2-Methoxybenzoylamino)-3-methylbutanoyl)pyrrolidine-2-boronate-(1S,2S,3R,5S)-pinanediol ester (70 mg, 0.16 mmol) was dissolved in acetone (10 ml). Sodium iodide (106 mg, 0.71 mmol) and 0.1M ammonium acetate (5 ml) were added. After 18 h at room temperature the acetone was removed in vacuo and 2M sodium hydroxide (10 ml) was added to the residue which was washed with CH2Cl2. The aqueous layer was acidified to pH 7 with 1M HCl and extracted with chloroform (3×50 ml). The combined organic extracts were washed with water and brine, dried (Na2SO4) and evaporated in vacuo to give a colourless oil identified as the title compound (28 mg, 0.08 mmol, 66%).
(2R)-1-((2S)-2-(1,1-Dimethylethoxycarbonylamino)-3-methylbutanoyl)pyrrolidine-2-boronate-(1S,2S,3R,5S)-pinanediol ester (synthesised as described in WO 0310127) (70 mg, 0.16 mmol) was dissolved in 4M HCl/dioxin (30 ml). After 1 h at room temperature the solvent was removed in vacuo and the residue dissolved in CH2Cl2 (25 ml). The solution was cooled to 0° C., triethylamine (36 mg, 0.36 mmol) was added followed by ethylisocyanate (15 mg, 0.21 mmol). After 18 h at 0° C. to room temperature the solvent was removed in vacuo and the residue dissolved in ethyl acetate (70 ml). This solution was washed with 0.3M KHSO4, sat. NaHCO3, water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 10% Pet. ether, 90% ethyl acetate) to give a colourless oil identified as the title compound (48 mg, 0.11 mmol, 73%).
(2R)-1-((2S)-2-(Ethylcarbamoylamino)-3-methylbutanoyl)pyrrolidine-2-boronate-(1S,2S,3R,5S)-pinanediol ester (43 mg, 0.10 mmol) was dissolved in acetone (10 ml). Sodium iodide (87 mg, 0.40 mmol) and 0.1M ammonium acetate (5 ml) were added. After 18 h at room temperature the acetone was removed in vacuo and 2M sodium hydroxide (10 ml) was added to the residue which was washed with CH2Cl2. The aqueous layer was acidified to pH 7 with 1M HCl and extracted with chloroform (3×50 ml). The combined organic extracts were washed with water and brine, dried (Na2SO4) and evaporated in vacuo to give a colourless oil identified as the title compound (9 mg, 0.032 mmol, 20%).
Nα-(tert-Butyloxycarbonyl)-L-isoleucine (5.0 g, 21.6 mmol) was dissolved in CH2Cl2/DMF (9:1, 100 ml). The solution was cooled to 0° C., (S)-(+)-2-pyrrolidinemethanol (2.5 g, 24.7 mmol), 1-hydroxybenzotriazole hydrate (5.9 g, 43.7 mmol) and water soluble carbodiimide (5.2 g, 26.0 mmol) were added and after 15 min the pH adjusted to pH9 with triethylamine. After 18 h at 0° C. to room temperature the solvent was removed in vacuo and the residue was taken up in ethyl acetate (300 ml). This solution was washed with 0.3M KHSO4, sat. NaHCO3, water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 30% Pet. ether, 70% ethyl acetate) to give a colourless oil identified as the title compound (6.8 g, 21.6 mmol, 100%).
(2S)-1-((2S)-2-(tert-Butyloxycarbonylamino)-3-methylpentanoyl)-2-hydroxymethylpyrrolidine (2.0 g, 6.36 mmol) was treated with 4M HCl/dioxan (50 ml). After 1 h at room temperature the solvent was removed in vacuo and the residue dissolved in CH2Cl2 (100 ml). The solution was cooled to 0° C., triethylamine (1.4 g, 14 mmol) was added followed by 2-anisoyl chloride (1.2 g, 7.04 mmol). After 18 h at 0° C. to room temperature the solvent was removed in vacuo and the residue dissolved in ethyl acetate (200 ml). This solution was washed with 0.3M KHSO4, sat. NaHCO3, water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 10% Pet. ether, 90% ethyl acetate) to give a colourless oil identified as the title compound (1.53 g, 4.46 mmol, 91%).
(2S)-2-Hydroxymethyl-1-((2S)-2-(2-methoxybenzoylamino)-3-methylpentanoyl)pyrrolidide (1.3 g, 3.73 mmol) was dissolved in CH2Cl2 (100 ml). Dess Martin Periodinane (2.0 g, 4.8 mmol) was added. After 3 h at room temperature the solvent was removed in vacuo and the residue dissolved in ethyl acetate (200 ml), washed with sat. NaHCO3, water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 30% Pet. ether, 70% ethyl acetate) to give a colourless oil identified as the title compound (1.05 g, 3.0 mmol, 80%).
(2S)-2-Formyl-1-((2S)-2-(2-methoxybenzoylamino)-3-methylpentanoyl)pyrrolidide (100 mg, 0.28 mmol) was dissolved in dry THF (30 ml). Methyl magnesium bromide (1.4M solution in THF/toluene, 4:1, 0.3 ml, 0.42 mmol) was added to this solution at 0° C. After 4 h at 0° C. to room temperature 0.3M KHSO4 (20 ml) was added and the reaction mixture extracted with ethyl acetate. The organic extract was washed with water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 40% Pet. ether, 60% ethyl acetate) to give a colourless oil identified as the title compound (36 mg, 0.099 mmol, 35%).
(2S)-2-(1-(1R,S)-Hydroxyethyl)-1-((2S)-2-(2-methoxybenzoylamino)-3-methylpentanoyl)pyrrolidide (32 mg, 0.088 mmol) was dissolved in CH2Cl2 (100 ml). Dess Martin Periodinane (42 mg, 0.099 mmol) was added. After 3 h at room temperature the solvent was removed in vacuo and the residue dissolved in ethyl acetate (70 ml). This solution was washed with sat. NaHCO3, water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 20% Pet. ether, 80% ethyl acetate) to give a colourless oil identified as the title compound (12 mg, 0.033 mmol, 38%).
(2S)-2-Formyl-1-((2S)-2-(2-methoxybenzoylamino)-3-methylpentanoyl)pyrrolidide (150 mg, 0.28 mmol) was dissolved in dry THF (30 ml). Ethyl magnesium bromide (1M solution in THF, 0.56 ml, 0.56 mmol) was added to this solution at 0° C. After 18 h at 0° C. to room temperature 0.3M KHSO4, (20 ml) was added and the reaction mixture extracted with ethyl acetate. The organic extract was washed with water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 30% Pet. ether, 70% ethyl acetate) to give a colourless oil identified as the title compound (62 mg, 0.16 mmol, 38%).
(2S)-2-(1-(1R,S)-Hydroxypropyl)-1-((2S)-2-(2-methoxybenzoylamino)-3-methylpentanoyl)pyrrolidide (52 mg, 0.138 mmol) was dissolved in CH2Cl2 (100 ml). Dess Martin Periodinane (70 mg, 0.17 mmol) was added. After 3 h at room temperature the solvent was removed in vacuo and the residue dissolved in ethyl acetate (70 ml). This solution was washed with sat. NaHCO3, water and brine, dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 20% Pet. ether, 80% ethyl acetate) to give a colourless oil identified as the title compound (24 mg, 0.064 mmol, 47%).
Examples E34-E61 were synthesised according to methods described for Examples E1, E2 and E10, while Examples E62-E66 were synthesised according to methods described in Example E31.
The 1H NMR data for Examples E1-E66 are as follows
1H NMR: δ(ppm)
| Number | Date | Country | Kind |
|---|---|---|---|
| 05108049 | Sep 2005 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IB2006/003512 | 8/31/2006 | WO | 00 | 9/15/2009 |
| Publishing Document | Publishing Date | Country | Kind |
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| WO2007/085895 | 8/2/2007 | WO | A |
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| 20100081701 A1 | Apr 2010 | US |
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| 60713324 | Sep 2005 | US |
