Indian Patents. 222670:PROCESS FOR PREPARING BICYCLIC PEPTIDE COMPOUNDS OF FORMULA I

Title of Invention	PROCESS FOR PREPARING BICYCLIC PEPTIDE COMPOUNDS OF FORMULA I
Abstract	The present invention relates to a new process carried out entirely in solution, for the preparation in high yields of high purity bicyclic peptide compounds of formula (I), useful as intermediates for preparing compounds with pharmacological activity.

Full Text	Field of the invention The present invention relates to a new process for the preparation of bicyclic peptide compounds of formula (I) hereinafter reported, useful as intermediates in the preparation of pharmacologically active compounds, and in particular in the preparation of bicyclic glycopeptides of formula (l-A) hereinafter reported, which possess antagonist activity of the tachykinin NK2 receptor. State of the art Compounds of formula (l-A) and in particular the compound [N-4-(2-acetylamino-2-deoxy-p-D-glucopyranosyl)-L-asparaginyl-L-a-aspartyl-L-triptophyl-L-phenylalanyl-L-2,3-diaminopropionyl-L-leucyl]-C-4,2-N-3,5-lactam-C-1,6-N-2,1-lactam (compound of formula (l-A) hereinafter reported, in which P^ = R2 = R3 = H, known with the trade name "Nepadutant") are compounds having a strong antagonist activity of the tachykinin NK2 receptor, and can therefore be used for preparing pharmaceutical compounds for treating diseases, useful in the treatment and prevention of diseases where tachykinins are implicated as neuromodulators. This compound and some of its intermediates are described in the European Patent No. 815 126 B1, particularly in Example 4. This document describes, on pages 4 and 5, the methods, already known in the literature, of synthesis in solution or in solid phase of linear peptides by sequential coupling of suitably protected amino acids and their subsequent final cyclization, in order to obtain compounds of general formula (I). These methods have been described in a very general way, while more details have been provided for preparing the compounds in Examples 1 and 2. In these examples, the synthesis used was the coupling of Fmoc amino acids in solid phase until a linear peptide was obtained which, after detachment from the resin, is cyclized, purified by HPLC and cyclized again. It is important to note that, following to this path of synthesis, the glycosidic pendant is introduced at the stage of synthesis in solid phase of the linear peptide on the resin, as a side chain suitably protected of Asparagine. Summary of the invention The Applicant has now surprisingly found a new and more efficient process for the preparation of bicyclic peptide compounds of formula (I) hereinafter reported, useful as intermediates for preparing compounds with pharmacological activity. The new process is carried out entirely in solution rather than in solid phase and allows products with high purity and high yields to be obtained. It is therefore subject of the present invention a process for the preparation of bicyclic peptide compounds of formula (I) (SEQ. ID. 1) wherein Ai and A2 are two nitrogen protecting groups different from each other, and R5 and R6, different from each other, are chosen from benzy\|oxy and lower alkyloxy groups in which the alkyl part comprises a linear; or branched C1-C4 group; 2) intramolecular cyclisation of the compound of formula (III) coming from step 1) in .the presence of a solvent and of a suitable coupling agent to give the compound of formula (IV) (SEQ. ID. 3): wherein R5 is as defined above; 3) deprotection of the compound of formula (IV) coming from step 2) in the presence of a solvent to give the compound of formula (V) wherein R5 is as defined above; 4) coupling between the compound of formula (V) coming from step 3) and a protected amino-acid of formula (Via) in the presence of a solvent, to give compounds of formula (VII) (SEQ. ID. 4): wherein A3 is a nitrogen protecting group; R7 is chosen from benzyloxy and lower alkyloxy groups, in which the alky! part comprises a linear or branched C1-C4 group; R$ is a residual group deriving from an activation procedure on the carboxyl group; 5) deprotection of the compound of formula (VII) coming from step 4) in the presence of a solvent to give a compound of formula (VIII) wherein R7 is as defined above; 6) intramolecular cyclisation, in the presence of a solvent and of a suitable coupling agent, of the compound of formula (VIII) coming from step 5) to give a wherein R7 is as defined above. Compound of formula (III) is represented by SEQ. ID. 2 wherein The compounds of formula (I) can be used for example for the preparation of bicyclic glycopeptide compounds of formula (l-A) hereinafter reported, which possess a powerful antagonist activity towards the tachykinin NK2 receptor; the Applicant has found a new preparation process, whereby a glycosidic pendant is introduced into compounds of formula (I) by a reaction carried out in solution, and the purification of the final product by HPLC is not necessary, so that large scale production of these compounds can be achieved at decidedly lower costs than those of the current production process. A further subject of the present invention is a process for the preparation of bicyclic glycopeptide compounds of formula (l-A) (SEQ. ID. 5) wherein Ri, R2 and R3, equal or different from each other, can be hydrogen or an oxygen protecting group, comprising the following steps: 1A) activation of bicyclic peptide compounds of formula (I) with a suitable coupling agent to obtain a derivative of formula (ll-A) wherein R is selected from the group consisting of benzotriazole, possibly substituted with a halogen, azabenzotriazole and succinimidyl; 2A) reaction of the compound of formula (ll-A) coming from step 1A) in the presence of a solvent with a glycosidic derivative of formula (lll-A) wherein R, Ri, R2 and R3 are as defined above. A further subject of the invention is a process for preparing the compound of formula (1-A) starting from the compounds of formula (II) and formula (III), passing via the formation of the compound of formula (I) as described in the two aforementioned processes. The processes of the invention, carried out entirely by means of reactions in solution rather than in solid phase, show unexpectedly high yields and do not require the use of HPLC purification processes, thus allowing a significant reduction of the production costs and enabling large scale preparation to be achieved. Detailed description of the invention The nitrogen protecting groups used in the present processes can be chosen from any of the protecting groups that can be used for peptide synthesis such as those repprtpH in M Rnrfanskv "P^nZ/Vte Chemistry" Snrinner \A=>rlaa 1988 or in J. Jones, "The Chemical Synthesis of Peptides", Clarendon Press. Oxford 1994. According to the invention, the nitrogen protecting groups are preferably selected from the group consisting of benzyloxycarbonyl and alkoxy carbonyl in which the alkyl part comprises a linear or branched C1-C4 group; more preferably they are chosen from t-butoxycarbonyl (Bcc) and benzyloxycarbonyl (Z). R8 is a residual group deriving from an activation procedure, preferably chosen from the group consisting of benzyloxycarbonyl, alkoxycarbonyl comprising in the- alkyl part a linear or branched C1-C4 group, succinimidyl, benzotriazole possibly substituted by a halogen, and azabenzotriazole. The linear peptides of formula (II) can be prepared by one of the following strategies: a) Stepwise strategy: with this strategy the amino acids necessary for obtaining the peptide of formula (II) are sequentially coupled starting from a derivative of the amino acid Dpr of formula (X), protected on nitrogen and prepared separately or generated in situ wherein: A2 and A4, different from each other, are nitrogen protecting groups, as defined above; R9 is a residual group deriving from an activation procedure, preferably chosen from the group consisting of benzyloxycarbonyl, alkoxycarbonyl comprising in the alkyl part a linear or branched C1-C4 group, and succinimidyl; the derivative of formula (X) above reported is reacted with a Leu ester (XI) in the presence of a solvent wherein R5 is defined as above, thus' obtaining the dipeptide A4-Dpr(A2)-Leu-R5, which is then deprotected by a suitable method depending on the protecting group on nitrogen to be removed, and compatible with the protecting group to be maintained. The dipeptide thus deprotected is subsequently coupled with the activated ester of the amino acid Phe, and so on in sequence with Trp and Asp until the compounds of formula (II) are obtained. b)- Strategy 2+2+1: this strategy consists of coupling the monodeprotected dipeptide H-Dpr(A2)-Leu-R5l obtained as described above according to strategy a), with an activated derivative of the dipeptide having the following formula (XII) A5-Trp-Phe-OH (XII) wherein A2 and A5, different from each other, are nitrogen protecting groups, as defined above; prepared separately or generated in situ by coupling an activated ester of a Trp protected on nitrogen prepared separately or generated in situ, with a Phe ester and subsequent hydrolysis of the ester group. The resulting tetrapeptide A5-Trp-Phe-Dpr(A2)-Leu-R5 is suitably deprotected from the group attached to the nitrogen of Trp and coupled with a compound of formula (Vlb) wherein A-i, Re and R8 are defined as above. c) Strategy 3+2: according to this strategy the tripeptide A:-Asp(R6)-Trp-Phe-OH, obtained by removing the nitrogen protecting group from the compounds of formula (XII) above reported, and subsequent coupling with a compound of formula (Vlb) above reported, is then coupled with the monodeprotected dipeptide H-ppr-(A2)-Leu-R5 prepared as described according to the procedure of strategy a). As used in the present invention, the term "lower alkoxyl groups" refers to those alkoxyl groups in which the alkyl part comprises a linear or branched C1-C4 group, preferably selected from the group consisting of methyl, ethyl, propyl, butyl, isopropyl and t-butyl. This should be meant also for the alkyloxycarbonyl groups of the invention, in which the alkyl part comprises a linear or branched C1-C4 group, preferably selected from the group consisting of methyl, ethyl, propyl, butyl, isopropyl and t-butyl. The coupling agent can be chosen from any one of those more commonly used in peptide synthesis, so as to generate an activated amino acid derivative such as those reported for example in M. Bodansky, "Peptide Chemistry", Springer Verlag 1988 or in J. Jones, "The Chemical Synthesis of Peptides", Clarendon Press. Oxford 1994. The activated derivatives, if not commercially available, can be prepared separately or in situ by reaction between an amino acid or a peptide and one or more of the numerous known coupling agents, such as isobutyl chloroformate (IBCF), a carbodiimide selected from dicyclohexylcarbodiimide (DCC) and 1-ethyl- 3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC.HCI) possibly in combination with a hydroxyderivative selected from 1-hydroxybenzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole (HOAt), 6-chloro-1-hydroxybenzotriazole (CI-HOBt) and hydroxysuccinimide (HOSu); a phosphonium salt, N-oxide guanidine salt or uronium salt, such as (Benzotriazol-1- yloxy)tri(dimethylamino)phosphonium hexafluorophosphate (BOP), (Benzotriazol- 1-yloxy)tripyrrplidine phosphonium hexafluorophosphate (PyBOP), 1- [bis(dimethylamino)methylene]-1H-benzotriazolium-3-oxide hexafluorophosphate (HBTU), 1-[bis(dimethylamino)methylene]-5-chloro-1H-benzotriazolium-3-oxide hexafluorophosphate (HCTU), 1-[bis(dimethylamino)methylene]-1H- benzotriazolium-3-oxide tetrafluoroborate (TBTU), 1- [bis(dimethylamino)methylene]-1H-1,2,3-triazole[4,5-b]pyridinium-3-oxide hexafluorophosphate (HATU), 1-[bis(dimethylamino)methylene]-5-chloro-1 H- benzotriazolium-3-oxide tetrafluoroborate (TCTU), O- [(ethoxycarbonyOcyanomethylenaminoj-N.N.N'.N'-tetramethyluronium tetrafluoroborate (TOTU), 0-(bicycIo[2.2.1]hept-5-ene-2,3-dicarboximido)- N.N.N'.N'-tetramethyluronium tetrafluoroborate (TNTU), or O-(N-succinimidyl)- N.N.N'.N'-tetramethyluronium tetrafluoroborate (TSTU). Where the derivative is generated in situ the coupling reaction is carried out immediately afterwards by adding the other reagent, which obviously, in the case of intramolecular cyclizations, corresponds to the free amine end present in the molecule itself. The coupling reaction is usually carried out in the presence of a tertiary amine such as N-methylmorpholine (NMM), triethylamine (TEA) or diisopropylethylamine (DIPEA) in an organic solvent chosen from those generally used for peptide synthesis. Preferred solvents for the coupling reaction are ethyl acetate (AcOEt), dimethylformamide (DMF) and N-methylpyrrolidone (NMP). The coupling reactions can be carried out at a temperature that would not cause degradations or render the reaction too slow, the temperature being preferably comprised between -20 and +50°C. The deprotections in the processes of the invention are achieved by the appropriate methods for groups to be removed and compatible with the groups to be retained; generally the present deprotection reactions are carried out by means of catalytic hydrogenation or by acid or base treatments. For hydrogenations, the catalyst can be chosen from those varieties of catalysts which are available and suitable for this purpose; 5% or 10% Palladium are preferred. The solvent for the deprotection reactions by catalytic hydrogenation can be chosen from those that dissolve the compounds in reaction, excluding ketones such as acetone, the solvents which poison the catalyst and those that react with the components of the reaction themselves. DMF, NMP, organic acids such as acetic acid and p-toluene sulfonic acid (PTSA), and alcohols such as methanol, ethanol, and isopropanol, or mixtures thereof, are the preferred reaction solvents. The hydrogenation reaction temperature is comprised between -20 and +50°C. For deprotections by acid treatment, mineral acids are preferably used, such as hydrochloric acid, or organic acids, such as trifluoroacetic acid or formic acid, which can be used alone or mixed with other solvents. The temperature is between -20 and +50°C. For deprotections by basic treatment, hydroxides of alkali metals and alkaline earth metals are preferably used in the presence of a solvent such as water, dioxane, acetonitrile, methanol, ethanol, isopropanol, or mixtures thereof; the temperature is comprised between -20 and +50°C. The term "oxygen protecting group" as used in the present invention refers to a protecting group selected from those commonly used for the protection of -OH groups and well known to any person skilled in the art, selected for example from the group consisting of-COR4 wherein R4 is a linear or branched alkyl group, with from 1 to 4 carbon atoms, the phenyl being possibly substituted by a halogen atom, benzyl or benzoyl; the oxygen protecting group is preferably acetyl. According to the invention the glycopeptide compounds of formula (l-A) can be obtained by reacting a glycosidic derivative of formula (IIl-A) with an activated peptide derivative of formula (ll-A), obtained by an activation reaction or generated in situ by a compound of formula (I). Therefore, in the preparation process of bicyclic glycopeptide compounds of formula (1-A), the glycosidic group is introduced not in the linear peptide, but in the bicyclic peptide compound. If compounds of formula (lll-A) are reacted in which R1, R2 and R3 are not hydrogen, the compounds of formula (l-A) obtained can be transformed into the corresponding compounds in which Ri=R2=R3=H, by means of catalytic hydrogenation or by an acid or base treatment according to the nature of the protecting groups Ri, R2 and R3. The glycosidic compounds of formula (lll-A) preferably used in the process of the invention are selected from the group consisting of 2-acetamide-2-deoxy-(3-D- glucopyranosylamine and 2-acetamide-3,4,6-tri-0-acetyl-2-deoxy-(3-D- glucopyranosylamine, which are known in the literature and can be prepared for example as described respectively in I. Shin et al., Tetrahedron Letters, 42 (2001) 1325-1328 and D. Macmillan et al., Organic Letters, Vol. 4, Ns 9, 2002. The following examples and schemes of synthesis are given to provide a non- imiting illustration of the invention. Scheme 1 indicates the synthesis path which, starting from the compounds of formula (II) leads to those of formula (l-A), whereas schemes 2-4 show the three different strategies for preparing the compounds of formula (II). The protecting groups shown as examples are t-butoxycarbonyl (BOC) and benzyloxycarbonyl (Z) for the amino extremities and methylester and t-butylester for the carboxyl extremities. The numbers given beside each compound in the following schemes corresponds to the numbers attributed to the compounds in the examples. The identification and evaluation of purity for the compounds prepared has been established by elemental analysis, HPLC, 1H-NMR, IR and mass analysis. 4 EXAMPLE 1 Preparation of Z-Asp(OHVTrp-Phe-DprfH)-Leu-OMe (SEQ. ID. 1) A 72 mmol/l solution of Z-Asp(OtBu)-Trp-Phe-Dpr(BOC)-Leu-OMe, prepared as described in Example 15, in 95% formic acid is heated to 40°C under vacuum for 4 hours. The reaction mixture is evaporated under reduced pressure and the residue is redissolved with a 8:2 CH3CN - H20 mixture. The suspension is cooled to 15-20°C and the pH is corrected to 6 by adding a 20% aqueous NMM solution. The acetonitrile is evaporated under reduced pressure and the resulting suspension is filtered. The whitish solid obtained is washed with H20 and dried under vacuum at 30-40°C to provide a yield equal to 96.4%. 1H-NMR dimethylsulfoxide-d6 (DMSO-d6) 5: 0.86 (2d; 6H); 1.47-1.75 (m; 3H); 2.32-2.68 (m; 2H); 2.79-3.55 (m; 6H); 3.63. (s; 3H); 4.25-4.65 (m; 5H); 4.99 (AB-Syst; 2H); 6.91-7.43 (m; 14H); 7.48-7.60 (2d; 2H); 7.82 (b; 2H); 8.03-8.43 (4d; 4H); 10.83 (s; 1H); 12.35 (b; 1H). EXAMPLE 2 Preparation of Z-Asp-Trp-Phe-Dpr-Leu-Ome (SEQ. ID. 2) 2.2 equivalents of NMM are added to a 24 mmol/l solution of Z-Asp(OH)-Trp-Phe- Dpr(NH2)-Leu-Ome in DMF and after 5-10 minutes 1.2 equivalents of PyBOP are added. After 2-3 hours of stirring at room temperature the solution is evaporated under reduced pressure until a fluid residue is obtained which is dropped into a 0.5 M aqueous solution of NaHC03. The resulting suspension is filtered and the solid obtained is washed with a 4:6 DMF - H20 mixture and then with H20 until neutral pH is achieved and dried under vacuum at 30-50°C, providing a yield equal to 84.2%. 1H-NMR(DMSOd6)5: 0.83 (2d; 6H); 1.34-1.69 (m; 3H); 2.31-2.92 (m; 4H); 3.03-3.91 (m; 4H); 3.61 (s; 3H); 4.17-4.63 (m; 5H); 5.01 (AB-Syst.; 2H); 6.84-7.48 (m; 16H); 7.60 (d; 1H); 7.87 (d;2H); 8.01 (t; 1H); 8.27 (d; 1H); 10.81 (s; 1H). EXAMPLE 3 Preparation of Z-Asp-Trp-Phe-Dpr-Leu-OH (SEQ. ID. 3) A cloudy solution containing 77 mmol/l of Z-Asp-Trp-Phe-Dpr-Leu-Ome in a 8:2 dioxane - H20 mixture is -heated to 35°C and maintained at pH 12.0 - 12.5 by slowly and continuously adding 1.5 N NaOH. At the end of the reaction the cloudy solution is brought to pH 9 by adding 6N HCI, clarified by filtration on a co-adjuvant filtration bed and acidified to pH 3 by again adding 6N HCI. The solution is concentrated under reduced pressure until a filterable solution is obtained. The whitish filtered solid is washed with a 1:1 dioxane - H20 mixture and then with H20 and dried under vacuum at 30-40°C, providing a yield equal to 97.7%. 1H-NMR (DMSO-d6) 5: 0.84 (2d; 6H); 1.42-1.76 (m; 3H); 2.29-3.48 (m; 7H); 3.85 (m; 1H); 4,10-4.65 (m; 5H); 5.00 (AB-Syst; 2H); 6.86-7.47 (m; 16H); 7.55-8.36 (4d+m; 5H); 10.80 (d; 1H); 12.65 (b;1H). EXAMPLE 4 Preparation of Z-Asp(OtBu)-Asp-Trp-Phe-Dpr-Leu-OH (SEQ. ID. 4) L I A 66 mmol/l solution of Z-Asp-Trp-Phe-Dpr-Leu-OH in DMF is hydrogenated at room temperature in the presence of 1 equivalent of NMM and catalytic quantities of 10% Pd/C, at 50% wetness. After reacting for 6 hours the suspension is filtered to remove catalyst and filtrate is diluted with DMF to obtain a 53 mmol/l solution of H-Asp-Trp-Phe-Dpr- Leu-OH, to which 4 equivalents of NMM and 1.05 equivalents of Z-Asp(OtBu)Osu are added. After stirring for 5 hours at room temperature the mixture is evaporated under reduced pressure until a residue is obtained which is dropped into 0.05 N H2SO4. The resulting suspension is filtered and the solid obtained is washed with a 1:1 DMF - H20 mixture and then with H20 and dried under vacuum at 30-40°C, to provide a yield equal to 93.7%. 1H-NMR(DMSO-d6)6: 0.84 (2d; 6H), 1.35 (s; 9H); 1.40-1.70 (m; 3H); 2.20-3.94 (m; 10H); 4.10-4.81 (m; 6H); 4.92-5.12 (AB-Syst.; 2H); 6.74-7.57 (m; 17H); 7.71-8.35 (4d+1t; 5H); 10.70 (s;1H); 12.70 (b;1H). EXAMPLE 5 Preparation of Cyclo[Asp(OtBu)-Asp-Trp-Phe-Dpr-Leu] (SEQ. ID. 5) A 47 mmol/l solution of Z-Asp(ptBu)-Asp-Trp-Phe-Dpr-Leu-QH in DMF is hydrogenated at room temperature, in the presence of 1 equivalent of DIPEA and catalytic quantities of 10% Pd/C at 50% wetness. After reacting for about 2 hours the suspension is filtered to remove the catalyst and diluted with DMF until a 19 mmol/l solution of H-Asp(OtBu)-Asp-Trp-Phe-Dpr- ! I Leu-OH is obtained to which 1.4 equivalents of DIPEA and 1.2 equivalents of HATU are added. After stirring for 30-60 minutes at room temperature the solution is evaporated under reduced pressure until a residue is obtained which is dropped into a 0.5 M aqueous solution of NaHC03. The resulting suspension is filtered and the solid obtained is washed with abundant H20 until the pH is neutral, and dried under vacuum at 30-50°C, obtaining a yield equal to 94.1%. 1H-NMR(DMSO-d6)6: 0^88 (2d; 6H); 1.38 (s; 9H); 1.31-1.72 (m; 3H); 2.33-2.99 (m; 6H); 3.20-3.63 (m; 3H); 3.87-4.62 (m; 7H); 6.75-7.50 (m; 13H); 8.04 (b; 1H); 8.56 (d; 1H); 8.76 (d; 1H);9.18(b;1H);10.84(s;1H). EXAMPLE 6 Preparation of Cyclo[Asp(OH)-Asp-Trp-Phe-Dpr-Leu] (SEQ. ID. 6) I I A 83 mmoi/l solution of cyclo[Asp(OtBu)-Asp-Trp-Phe-Dpr-Leu] in 90% formic acid is heated at 40°C under vacuum for 2 hours. The reaction mixture is evaporated under reduced pressure until a dense residue is obtained which is redissolved in H2O. The resulting suspension is filtered and the solid obtained is washed with H20, dried under vacuum at 30-40°C and finally purified by means of a Sephadex® LH- 20 column, eluting with methanol. 314 g of a white solid are obtained (titre 95.2%, yield 82.0%). 1H-NMR (DMSO-d6) 5: 0.88 (2d; 6H); 1.31-1.77 (m; 3H); 2.32-3.73 (m; 9H); 3.80-4.65 (m; 7H); 6.82-7.51 (m; 13H); 7.94-9.19 (2d; 2b; 4H); 10.85 (s; 1H); 12.20 (s; 1H). EXAMPLE 7 Preparation of Cyclo[As,p-Asp-Trp-Phe-Dpr-Leu] (SEQ. ID. 7) 1 1 Glc(triOAc)NHAc 3 equivalents of NMM, 1.2 equivalents of HATU and 2-acetamide-3,4,6-tri-0-acetyl-2-deoxy-p-D-glucopyranosylamine are added to a 0.24 mol/l solution of Cyclo[Asp(OH)-Asp-Trp-Phe-Dpr-Leu] in DMF at 10 minute intervals. After stirring for 1 hour at 0-4°C the reaction mixture is evaporated under reduced pressure until a fluid residue is obtained which is dropped into a 1% aqueous solution of NaHC03. The resulting suspension is filtered and the solid obtained is washed with H2O, dried under vacuum at 30-40°C and purified by crystallization from a EtOH - H20 mixture. 117 g of a white solid are obtained (titre 96.0%, yield 87.0%). 1H-NMR (DMSO-d6) 5: 10.80 (d;1H); 8.90 (b; 1H); 8.72 (d; 1H); 8.47 (d; 1H); 8.46 (d; 1H); 8.08 (b; 1H); 7.84 (d; 1H); 7.43 (dd; 1H); 7.33 (dd; 1H); 7.24 (b; 1H); 7.23 (m; 2H); 7.16 (m; 3H); 7.14 (d; 1H); 7.06 (dt; 1H); 7.00 (d; 1H); 6.98 (dt; 1H); 6.90 (t; 1H); 5.18 (dd; 1H); 5.12 (dd; 1H); 4.82 (dd; 1H), 4.18 (dd; 1H); 3.96 (dd; 1H); 3.85 (ddd; 1H); 3.80 (ddd; 1H); 4.53 (m, 1H); 4.47 (m; 1H); 4.43 (m; 1H); 4.39 (m; 1H); 4.16 (m; 1H); 4.08 (m; 1H), 3.58 (m; 1H); 3.30 (m; TK); 2.98 (m; 1H); 2.88 (m; 1H); 2.86 (m; 1H); 2.70 (m; 1H); 2.65 (m; 1H); 2.60 (m; 1H); 2.19 (m; 1H); 2.00 (s, 3H); 1.96 (s; 3H); 1.90 (s; 3H), 1.73 (s; 3H); 1.65 (m; 1H); 1.52 (m; 1H); 1.37 (m; 1H); 0.92 (d; 3H); 0.85 (d; 3H). EXAMPLE 8 Preparation of CyclofAsp-Asp-Trp-Phe-Dpr-Leu] (Nepadutant) (SEQ. ID. 8) GlcNHAc Method a) 2 equivalents of NMM and 1.3 equivalents of TBTU and 2-acetamide-2-deoxy-(3-D-glucopyranosylamine are added at 10 minute intervals to a 83 mmol/l solution in DMF of cyclo[Asp(OH)-Asp-Trp-Phe-Dpr-Leu] (prepared as described in Example 6). ' ! After stirring for 1 hour at room temperature, the reaction mixture is evaporated under reduced pressure until a dense oily residue is obtained which is redissolved with a 2:8 acetonitrile - t-butoxymethane (TBME) mixture. The resulting suspension is vigorously stirred for 30 minutes at room temperature and then filtered. The solid obtained is washed with TBME, dried under vacuum at 25-30°C and finally purified by preparative HPLC using eluent mixtures composed of acetonitrile and water. 151 g of a white solid are obtained (titre 93.0%, yield 89.3%). 1H-NMR(DMSO-d6)6: 0.85 (d; 3H); 0.92 (d; 3H); 1.36 (m; 1H); 1.51 (m; 1H); 1.65 (m; 1H); 1.76 (s; 3H); 2.16 (dd; 1H); 2.57 (dd; 1H); 2.63 (dd; 1H); 2.67 (dd; 1H); 2.83 (dd; 1H); 2.88 (dd; 1H); 2.93 (m; 1H); 3.04-3.09 (m; 2H); 3.27-3.32 (m; 2H); 3.42 (m; 1H); 3.50 (ddd + b; 2H); 3.65 (dd; 1H); 3.96 (b; 1H); 4.09 (m; 1H); 4.12 (m; 1H); 4.35 (m; 1H); 4.43 (m; 1H); 4.50 (m; 1H); 4.53 (m + t; 2H); 4.81 (dd; 1H); 4.94 (d; 1H); 4.98 (d; 1H); 3.91 (b; 1H); 6.98 (t + b; 2H); 7.06 (t; 1H); 7.14-7.17 [m; 4H); 7.24 (t; 2H); 7.27 (b; 1H); 7.33 (d; 1H); 7.42 (d; 1H); 7.77 (d; 1H); 8.05 (b; IH); 8.10 (d; 1H); 8.51 "(cf; 1H); 8.77 (d; 1H); 9.00 (b; 1H); 10.84 (d; 1H). Method b) ).04 equivalents of 0.1 N NaOMe in MeOH are added to a 0.89 mol/l solution in MeOH of cyclo[Asp-Asp-Trp-Phe-Dpr-Leu] Glc(triOAc>NHAc prepared as described in Example 7. After stirring for 3 hours at room temperature the pH is corrected to 6.5-7 and Amberlyst® 15 is added. Following removal of the resin, the solution is concentrated under reduced pressure until a residue is obtained which is diluted with TBME. The resulting suspension is filtered and the white solid obtained is washed with TBME and dried under vacuum at 35-40°C, providing a yield equal to 94.8%. EXAMPLE 9 Preparation of Z-Dpr(BOC)-Leu-OMe Method a) 1.2 equivalents of NMM are added io'a 0.66 mol/l solution of Z-Dpr(BOC)-OH in DMF. The solution is cooled to -25°C and 1 equivalent of IBCF is dropped in while maintaining the temperature below -20°C. After about 10 minutes a 0.78 mol/l pre-coofed solution containing 1 equivalent of H-Leu-OME HCI and NMM in DMF is dropped in, always maintaining the temperature below -15°C. After stirring for one hour the reaction mixture is dropped into a 0.5 M aqueous solution of NaHC03. The resulting suspension is filtered and the solid obtained is washed sequentially with H20, 0.05 M H2SO4 and H20 until the pH is neutral and dried under vacuum at 30-50°C, providing a yield equal to 89.0%. melting point 122-125°C; 1H-NMR (DMSO-d6) 5: 0.85 (2d; 6H>; 1.37 (s; 9H); 140-1.71 (m; 3H); 3.01-3.36 (m; 2H); 3.61 (s; 3H); 4.06-4.37 (m; 2H); 5.03 (s; 2H); 7.35 (s; 5H); 6.66 (t; 1H); 7.20 (d; 1H); 8.29 (d; 1H). Method b) 1 equivalent of DCC is added to a 0.35 mol/l solution of Z-Dpr(BOC)-OH in DMF containing 1 equivalent of HOSu, cooling to 0-5°C. The mixture is brought to room temperature and stirred for 1 hour. The DCC is removed by filtration and to the clear filtrate are added 1.2 equivalents of H-Leu-Ome HCI and 2.6 equivalents of NIMM. After stirring for 2-3 hours at room temperature the mixture is diluted with 0.5 N NaHC03 then cooled to -5°C. The resulting suspension is filtered and the solid obtained is washed sequentially with 0.5 N NaHC03i a 2:1 H20 - DMF mixture and water, then dried under vacuum at 30-40°C, providing a yield equal to 93%. EXAMPLE 10 Preparation of H-DprfBOC)-Leu-OMe A 0.14 mol/l solution of Z-Dpr(BOC)-Leu-OMe in MeOH containing 1 equivalent of PTSA is hydrogenated at room temperature in the presence of catalytic quantities of 10% Pd/C, 50% wetness. After reacting for about 2 hours the suspension is filtered to remove the catalyst and the filtrate is diluted with DMF. The MeOH and the H2O are completely evaporated under reduced pressure and the residual DMF solution, containing the dipeptide, is used for the subsequent coupling. EXAMPLE 11 Preparation of Z-Phe-DprfBOCVLeu-OMe The compound was prepared from the dipeptide H-Dpr(BOC)-Leu-OMe from Example 10, according to the method described in Example 9 using Z-Phe-OH. 1H-NMR (DMSO-de) 6: 0.86 (2d; 6H); 1.38 (s; 9H); 1.40-1.74 (m; 3H); 2.73-3.02 (m; 2H); 3.10-3.41 (m; 2H); 3.62 (s; 3H); 4.17-4.46 (m; 3H); 4.94 (AB-Syst.; 2H); 7.18-7.39 (m; 10H); 6.52 (t; 1H); 7.52 (d; 1H); 8.13 (d; 1H); 8.25 (d, 1H). EXAMPLE 12 Preparation of H-Phe-Dpr(BOC)-Leu-OMe The compound was obtained from the protected derivative from Example 11, according to the method in Example 10, using DMF as the solvent. EXAMPLE 13 Preparation of Z-Trp-Phe-Dpr(BOC)-Leu-OMe (SEQ. ID. 9) The compound was prepared using the method in Example 9 from the tripeptide of Example 12 and using Z-Trp-OH or by coupling the two dipeptides Z-Trp-Phe- OH and H-Dpr(BOC)-Leu-OMe, obtained as described in Examples 17 and 10 respectively. 1H-NMR(DMSO-d6)5: 0.86 (2d; 6H); 1.37 (s; 9H); 1.40-1.76 (m; 3H); 2.73-3.41 (m; 6H); 3.62 (s; 3H), 4.16-4.67 (m; 4H); 4.93 (AB-Syst.; 2H); 6.89-7.65 (m; 16H); 6.55 (t; 1H); 8.07 (d; 1H); 8.11 (d; 1H); 8.29 (d; 1H);10.79 (s; 1H). EXAMPLE 14 Preparation of H-Trp-Phe-Dpr(BOC)-Leu-OMe (SEQ. ID. 10) The compound was obtained from the protected derivative of Example 13, according to the method given in Example 10, using NMP as solvent. EXAMPLE 15 Preparation of Z-Asp(OtBu)-Trp-Phe-DprfBOC)-Leu-OMe CSEQ. ID. 11) Method a) 1 volume of CH3CN, 1.5 equivalents^ of DIPEA and 1.15 equivalents of Z- Asp(OtBu)-OSu are added to a 0.16 mol/l solution of H-Trp-Phe-Dpr(BOC)-Leu- OMe in NMP, derived from the hydrogenation reaction. After stirring for 3-4 hours at room temperature the reaction mixture is cooled to 5°C and is diluted with H20. The resulting suspension is filtered and the solid obtained is washed with a 3:7 CH3CN - H20 mixture and with H20 and then dried under vacuum at 30-50°C, providing a yield equal to 90%. Method b) 1 equivalent of DIPEA, 1.1 equivalents of TBTU and after 5 minutes 1 equivalent of the 0.25mol/l H-Dpr(BOC)-Leu-OMe solution in DMF derived from the hydrogenation reaction (example 10), are added to a 0.22 mol/l solution of Z-Asp- (OtBu)-Trp-Phe-OH in DMF cooled to -5°C, maintaining the temperature below - 5°C. After stim'ng for about 2 hours the reaction mixture is diluted with a 0.5 M aqueous solution of NaHC03. The resulting suspension is filtered and the solid obtained is washed sequentially with H20, a 3:4 DMF - 0.5 M NaHC03 mixture in H20, H20 and then dried under vacuum at 30-40°C providing a yield of 84.4%. melting point 215-218°C; 1H-NMR (DMSO-d6) 5: 0.86 (2d; 6H); 1.34 (s; 9H); 1.37 (s; 9H), 1.40-1.72 (m; 3H); 2.23-2.67 (m; 2H); 2.71-3.39 (m; 6H); 3.62 (s; 3H); 4.23-4.58 (m; 5H); 5.01 (AB-Syst., 2H); 6.89-7.58 (m; 16H); 6.50 (t; 1H); 7.87-8.29 (4d; 4H); 10.78 (s; 1H). EXAMPLE 16 Preparation of Z-Trp-Phe-QMe The compound was prepared according to the method of Example 9, coupling the two amino acids Z-Trp-OH and H-Phe-OMe. 1H-NMR(CDCI3)5: 2.88-2.98 (m; 2H); 3.11 (dd; 1H); 3.32 (dd; 1H); 3.62 (s; 3H); 4.40-4.58 (m; 1H); 4.16-4.30 (m; 1H); 5.11 (s; 2H); 5.45 (d; 1H), 6.11 (d; 1H); 6.72-6.85 (m; 2H), 6.92-7.46 (m; 12H); 7.67 (d; 1H); 8.03 (s; 1H). EXAMPLE 17 Preparation of Z-Trp-Phe-OH The compound was prepared from the methylester of Example 16, according to the method described in Example 3. 1H-NMR(DMSO-d6)5: 2.70-3.15 (m; 4H); 4.20-4.36 (m; 1H); 4.38-4.55 (m; 1H); 4.92 (s; 2H); 6.85-7.42 (m; 15H); 7.63 (d; 1H); 8.26 (d; 1H); 10.81 (s; 1H); 12.30 (b; 1H). EXAMPLE 18 Preparation of H-Trp-Phe-OH The compound was prepared from the protected derivative of Example 17, in accordance with the method of Example 10, using acetic acid as solvent. EXAMPLE 19 Preparation of Z-Asp(OtBu)-Trp-Phe-OH The compound was prepared in accordance with the method of Example 15 (method a) from the dipeptide of Example 18. 1H-NMR(DMSO-d6)5: 1.35 (s; 3H); 2.21-2.67 (m; 2H); 2.71-3.18 (m; 4H); 4.22-4.53 (m; 3H); 5.00 (AB-Syst.; 2H); 6.87-7.43 (m; 14H); 7.55 (m; 2H); 7.94 (d; 1H); 8.17 (d; 1H); 10.80 (s; 1H); 12.25 (b;1H). WE CLAIM: 1. Process for preparing bicydic peptide compounds of formula (f) comprising the following steps: 1) deprotection of the linear pentapeptide of formula (II) in the presence of a solvent to give the compound of formula (III): wherein Ai and A2 are two nitrogen protecting groups different from each other, and R5 and R6, different from each other, are chosen from benzyloxy and lower alkyloxy groups in which the alkyl part comprises a linear or branched C1-C4 group; 2) intramolecular cyclisation of the compound of formula (III) coming from step 1) in the presence of a solvent and of a suitable coupling agent to give the compound of formula (IV) wherein R5 is as defined above; 3) deprotection of the compound of formula (IV) coming from step 2) in the presence of a solvent to give the compound of formula (V) wherein R5 is as defined above; 4) coupling between the compound of formula (V) coming from step 3) and a protected smino-acid of formula (Via) in the presence of a solvent, to give compounds of formula (VII) wherein A3 is a nitrogen protecting group; R7 is chosen from benzyloxy and lower alkyloxy groups, in which the alkyl part comprises a linear or branched C1-C4 group; R8 is a residual group deriving from an activation procedure on the carboxyl group; 5) deprotection of the compound of formula (VII) coming from step 4) in the. presence of a solvent to give a compound of formula (VIII) wherein R7 is as defined above; 6) intramolecular cyclisation, in the presence of a solvent and of a suitable coupling agent, of the compound of formula (VIII) coming from step 5) to give a bicyclic compound of formula (IX) wherein R7 is as defined above; 7) deprotection of the bicyclic compound of formula (IX) coming from step 6) in the presence of a solvent, to obtain the compound of formula (I) wherein R7 is as defined above. 2. Process according to claim 1, wherein the linear peptides of formula (II) are obtained by means of a sequential coupling strategy of suitable amino acids starting from a derivative of the amino acid Dpr of formula (X), protected on nitrogen and prepared separately or generated in situ wnerem A2and A4, different from each other, are nitrogen protecting groups; Rg is a residual group deriving from an activation procedure, preferably chosen from the group consisting of benzyloxycarbonyl, alkoxycarbonyl comprising a linear or branched C1-C4 group in the alkyl part, and succinimidyl; according to the following steps: .- reaction of the derivative of formula (X) above reported in the presence of a solvent with a Leu ester of formula (XI) wherein R5 is defined as in claim 1, to obtain the dipeptide Ai-Dpr^-Leu-Rs, - deprotection of the dipeptide A4-Dpr(A2)-Leu-R5, to obtain the monodeprotected dipeptide H-Dpr(A2)-Leu-R5; - coupling the monodeprotected dipeptide H-Dpr(A2)-Leu-R5 with the activated ester of the subsequent amino acid Phe and then successively with Trp and Asp, until the compounds of formula (II) are obtained. 3. Process according to claims 1 and 2, wherein the linear peptides of formula (II) are obtained by means of a synthesis strategy comprising the following steps: - coupling of the monodeprotected dipeptide H-Dpr(A2)-Leu-R5, obtained as described in claim 2, with an activated derivative of the dipeptide of the following formula (XII) A5-Trp-Phe-OH (XII) wherein A2 and As, different from each other, are nitrogen protecting groups, prepared separately or generated in situ by coupling an activated ester of a Trp protected on nitrogen prepared separately or generated in situ, with a Phe ester and subsequent hydrolysis of the ester group, to obtain the tetrapeptide As-Trp-Phe-Dpr(A2)-Leu-R5; - suitable deprotection of the tetrapeptide A5-Trp-Phe-Dpr(A2)-Leu-R5 from the group attached to the nitrogen of Trp; - coupling of the deprotected tetrapeptide with a compound of formula (VI b) wherein A-i, R6 and R8 are defined as in claim 1. 4. Process according to claims 1-3, wherein the linear peptides of formula (II) are obtained by means of a synthesis strategy of the 3+2 type that involves coupling the tripeptide ArAsp(R6)-Trp-Phe-OH, obtained by removing the nitrogen protecting group from the compounds of formula (XII) above reported, subsequent coupling with a compound of formula (Vlb) above reported and then further coupling with the monodeprotected dipeptide H-Dpr-(A2)-Leu-R5 prepared as described in claim 2. 5. Process according to claim 1, wherein said nitrogen protecting groups are selected from the group consisting of benzyloxycarbonyl and alkoxy carbonyls in which the alkyl part comprises a linear or branched C1-C4 group. 6. Process according to claim 5, wherein said nitrogen protecting groups are selected from t-butoxycarbonyl and benzyloxycarbonyl. 7. Process according to claim 1, wherein said Rs group is selected from the group consisting of benzyloxycarbonyl, alkyloxycarbonyl comprising a linear or branched C1-C4 group in the alkyl part, succinimidyl, benzotriazole possibly substituted by a halogen and azabenzotriazole. 8. Process according to claims 1-7, wherein said linear or branched C1-C4 group is selected from the group consisting of methyl, ethyl, propyl, butyl, isopropyl and t-butyl. 9. Process for preparing a bicyclic glycopeptide compound of formula (l-A) wherein Ri, R2 and R3, equal or different from each other, can be hydrogen or an oxygen protecting group, comprising the following steps: 1A) activation of the bicyclic peptide compounds of formula (I) with a suitable coupling agent to obtain a derivative of formula (ll-A) (I) (H-A) wherein R is a group selected from benzotriazole, possibly substituted with a halogen, azabenzotriazole and succinimidyl; 2A) reaction of the compound of formula (fl-A) deriving from step 1A) in the presence of a solvent with the glycosidic derivative of formula (Ill-A) wherein R, R-i, R2, R3 are defined as above. 10. Process according to claim 9, wherein the compounds of formula (l-A) wherein R-,, R2 and R3 are different from H, are transformed into the corresponding compounds of formula (I-A) wherein Ri=R2=R3=H, by a deprotection reaction in the presence of a solvent. 11. Process according to claim 9, wherein said oxygen protecting groups are selected from the group consisting of -COR4 wherein R4 is a linear or branched C1-C4 alkyl group, phenyl possibly substituted with a halogen atom, benzyl or benzoyl. 12. Process according to claim 11, wherein said C1-C4 alkyl group is selected from the group consisting of methyl, ethyl, propyl, butyl, isopropyl and t-butyl. 13. Process according to claim 12, wherein said C1-C4 alkyl group is methyl. 14. Process according to claim 9, wherein said glycosidic derivatives of formula (lll-A) are selected from the group consisting of 2-acetamide-2-deoxy-(3-D- gjucopyranosylamine and 2-acetamide-3,4,6-tri-0-acetyl-2-deoxy-£-D- glucoDyanosylamine, 15. Process according to claim 9, wherein said bicyclic peptide compounds of formula (I) are prepared as described in claim 1. 16. Process according to claims 1 or 9, wherein said coupling agent is selected from the group consisting of isobutyl chloroformate, a carbodiimide possibly in combination with a hydroxyderivative, phosphonium salts, N-oxide guanidine salts and uranium salts. 17. Process according to claim 16, wherein said carbodiimides are selected from dicyclohexylcarbodiimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; said hydroxyderivative is selected from 1-hydroxybenzotriazole, 6-chloro-1-hydroxybenzotriazole, hydroxysuccinimide and 1-hydroxy-7-azabenzotriazole; said phosphonium salts, N-oxide guanidine salts and uranium salts are selected from (Benzotriazol-1-yloxy)tri(dimethylamino)phosphonium hexafluorophosphate, (Benzotriazol-1 -yloxy)tripyrrolidine phosphonium hexafluorophosphate, 1-[bis(dimethylamino)methylene]-1H-benzotriazolium-3-oxide hexafluorophosphate, 1-[bis(dimethylamino)methylene]-5-chloro-1 H-benzotriazolium-3-oxide hexafluorophosphate, 1 -[bis(dimethyIamino)methyleneJ-1 H-benzotriazolium-3-oxide tetrafluoroborate, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazole[4,5-b]pyridinium-3-oxide hexafluorophosphate, 1-[bis(dimethylamino)methylene]-5-chloro-1H-benzotriazolium-3-oxide tetrafluoroborate, 0-[(ethoxycarbonyl)cyanomethylenamino]-N,N,N',N'-tetramethyluronium tetrafluoroborate, 0-(bicyclo[2.2.1]hept-5-ene-2,3-dicarboximidoJ-N.N.N'.N'-tetramethyluronium tetrafluoroborate, and 0-(N-succinimidyO-N.N.N'.N'-tetramethyluronium tetrafluoroborate. 18. Process according to claims 1 or 9, wherein said coupling reactions are carried out in the presence of a tertiary amine in an organic solvent at a temperature comprised between -20 and +50°C. 19. Process according to claim 18, wherein said tertiary amine is selected from the group consisting of N-methylmorpholine, triethylamine and diisopropylethylamine, and said organic solvent is selected from the group consisting of ethyl acetate, dimethylformamide and N-methylpyrrolidone. , 20. Process according to claims 1 or 10, wherein said deprotection reactions are carried out by means of hydrogenation in the presence of a catalyst in a solvent selected from solvents which dissolve the components of the reaction without reacting with them, excluding ketones and solvents which poison the catalyst, at a temperature comprised between -20 and +50°C. 21. Process according to claim 20, wherein said catalyst is selected from 5% and 10% Palladium and said solvent is selected from dimethylformamide, N- methylpyrrolidone, acetic acid, p-toluenesulfonic acid, methanol, ethanol, isopropanol, and mixtures thereof. 22. Process according to claims 1 or 10, wherein said deprotection reactions are carried out by means of acid treatment with pure acids or with acids mixed with other solvents, at a temperature comprised between -20 and +50°C. 23 Process according to claim 22, wherein said acids are selected from hydrochloric acid, trifluoroacetic acid and formic acid. 24. Process according to claims 1 or 10, wherein said deprotection reactions are carried out by means of treatment with a base compound in the presence of a solvent, at a temperature comprised between -20 and +50°C. 25. Process according to claim 24, wherein said base compound is selected from hydroxides of alkali metals or alkaline earth metals, and said solvent is selected from the group consisting of water, dioxane, acetonitrile, methanol, ethanol, isopropanol, and mixtures thereof.

Full Text

Field of the invention
The present invention relates to a new process for the preparation of bicyclic peptide compounds of formula (I) hereinafter reported, useful as intermediates in the preparation of pharmacologically active compounds, and in particular in the preparation of bicyclic glycopeptides of formula (l-A) hereinafter reported, which possess antagonist activity of the tachykinin NK2 receptor. State of the art
Compounds of formula (l-A) and in particular the compound [N-4-(2-acetylamino-2-deoxy-p-D-glucopyranosyl)-L-asparaginyl-L-a-aspartyl-L-triptophyl-L-phenylalanyl-L-2,3-diaminopropionyl-L-leucyl]-C-4,2-N-3,5-lactam-C-1,6-N-2,1-lactam (compound of formula (l-A) hereinafter reported, in which P^ = R2 = R3 = H, known with the trade name "Nepadutant") are compounds having a strong antagonist activity of the tachykinin NK2 receptor, and can therefore be used for preparing pharmaceutical compounds for treating diseases, useful in the treatment and prevention of diseases where tachykinins are implicated as neuromodulators.
This compound and some of its intermediates are described in the European Patent No. 815 126 B1, particularly in Example 4. This document describes, on pages 4 and 5, the methods, already known in the literature, of synthesis in solution or in solid phase of linear peptides by sequential coupling of suitably protected amino acids and their subsequent final cyclization, in order to obtain compounds of general formula (I).
These methods have been described in a very general way, while more details have been provided for preparing the compounds in Examples 1 and 2. In these examples, the synthesis used was the coupling of Fmoc amino acids in solid phase until a linear peptide was obtained which, after detachment from the resin, is cyclized, purified by HPLC and cyclized again. It is important to note that, following to this path of synthesis, the glycosidic pendant is introduced at the stage of synthesis in solid phase of the linear peptide on the resin, as a side chain suitably protected of Asparagine. Summary of the invention

The Applicant has now surprisingly found a new and more efficient process for the preparation of bicyclic peptide compounds of formula (I) hereinafter reported, useful as intermediates for preparing compounds with pharmacological activity. The new process is carried out entirely in solution rather than in solid phase and allows products with high purity and high yields to be obtained. It is therefore subject of the present invention a process for the preparation of bicyclic peptide compounds of formula (I) (SEQ. ID. 1)

wherein Ai and A2 are two nitrogen protecting groups different from each other,
and R5 and R6, different from each other, are chosen from benzy|oxy and lower
alkyloxy groups in which the alkyl part comprises a linear; or branched C1-C4
group;
2) intramolecular cyclisation of the compound of formula (III) coming from step 1)

in .the presence of a solvent and of a suitable coupling agent to give the compound of formula (IV) (SEQ. ID. 3):

wherein R5 is as defined above;
3) deprotection of the compound of formula (IV) coming from step 2) in the
presence of a solvent to give the compound of formula (V)

wherein R5 is as defined above;

4) coupling between the compound of formula (V) coming from step 3) and a
protected amino-acid of formula (Via) in the presence of a solvent, to give
compounds of formula (VII) (SEQ. ID. 4):

wherein A3 is a nitrogen protecting group; R7 is chosen from benzyloxy and lower alkyloxy groups, in which the alky! part comprises a linear or branched C1-C4 group; R$ is a residual group deriving from an activation procedure on the carboxyl group;
5) deprotection of the compound of formula (VII) coming from step 4) in the
presence of a solvent to give a compound of formula (VIII)

wherein R7 is as defined above;
6) intramolecular cyclisation, in the presence of a solvent and of a suitable
coupling agent, of the compound of formula (VIII) coming from step 5) to give a

wherein R7 is as defined above.
Compound of formula (III) is represented by SEQ. ID. 2 wherein
The compounds of formula (I) can be used for example for the preparation of
bicyclic glycopeptide compounds of formula (l-A) hereinafter reported, which
possess a powerful antagonist activity towards the tachykinin NK2 receptor; the
Applicant has found a new preparation process, whereby a glycosidic pendant is
introduced into compounds of formula (I) by a reaction carried out in solution, and
the purification of the final product by HPLC is not necessary, so that large scale

production of these compounds can be achieved at decidedly lower costs than those of the current production process.
A further subject of the present invention is a process for the preparation of bicyclic glycopeptide compounds of formula (l-A) (SEQ. ID. 5)

wherein Ri, R2 and R3, equal or different from each other, can be hydrogen or an oxygen protecting group, comprising the following steps:
1A) activation of bicyclic peptide compounds of formula (I) with a suitable coupling agent to obtain a derivative of formula (ll-A)

wherein R is selected from the group consisting of benzotriazole, possibly substituted with a halogen, azabenzotriazole and succinimidyl; 2A) reaction of the compound of formula (ll-A) coming from step 1A) in the presence of a solvent with a glycosidic derivative of formula (lll-A)

wherein R, Ri, R2 and R3 are as defined above.
A further subject of the invention is a process for preparing the compound of
formula (1-A) starting from the compounds of formula (II) and formula (III), passing
via the formation of the compound of formula (I) as described in the two
aforementioned processes.
The processes of the invention, carried out entirely by means of reactions in
solution rather than in solid phase, show unexpectedly high yields and do not
require the use of HPLC purification processes, thus allowing a significant
reduction of the production costs and enabling large scale preparation to be
achieved.
Detailed description of the invention
The nitrogen protecting groups used in the present processes can be chosen from
any of the protecting groups that can be used for peptide synthesis such as those
repprtpH in M Rnrfanskv "P^nZ/Vte Chemistry" Snrinner \A=>rlaa 1988 or in J.

Jones, "The Chemical Synthesis of Peptides", Clarendon Press. Oxford 1994.
According to the invention, the nitrogen protecting groups are preferably selected
from the group consisting of benzyloxycarbonyl and alkoxy carbonyl in which the
alkyl part comprises a linear or branched C1-C4 group; more preferably they are
chosen from t-butoxycarbonyl (Bcc) and benzyloxycarbonyl (Z).
R8 is a residual group deriving from an activation procedure, preferably chosen
from the group consisting of benzyloxycarbonyl, alkoxycarbonyl comprising in the-
alkyl part a linear or branched C1-C4 group, succinimidyl, benzotriazole possibly
substituted by a halogen, and azabenzotriazole.
The linear peptides of formula (II) can be prepared by one of the following
strategies:
a) Stepwise strategy: with this strategy the amino acids necessary for obtaining
the peptide of formula (II) are sequentially coupled starting from a derivative of the
amino acid Dpr of formula (X), protected on nitrogen and prepared separately or
generated in situ
wherein:
A2 and A4, different from each other, are nitrogen protecting groups, as defined
above;
R9 is a residual group deriving from an activation procedure, preferably chosen
from the group consisting of benzyloxycarbonyl, alkoxycarbonyl comprising in the
alkyl part a linear or branched C1-C4 group, and succinimidyl;
the derivative of formula (X) above reported is reacted with a Leu ester (XI) in the
presence of a solvent

wherein R5 is defined as above,

thus' obtaining the dipeptide A4-Dpr(A2)-Leu-R5, which is then deprotected by a
suitable method depending on the protecting group on nitrogen to be removed,
and compatible with the protecting group to be maintained.
The dipeptide thus deprotected is subsequently coupled with the activated ester of
the amino acid Phe, and so on in sequence with Trp and Asp until the compounds
of formula (II) are obtained.
b)- Strategy 2+2+1: this strategy consists of coupling the monodeprotected
dipeptide H-Dpr(A2)-Leu-R5l obtained as described above according to strategy a),
with an activated derivative of the dipeptide having the following formula (XII)
A5-Trp-Phe-OH
(XII) wherein A2 and A5, different from each other, are nitrogen protecting groups, as
defined above;
prepared separately or generated in situ by coupling an activated ester of a Trp
protected on nitrogen prepared separately or generated in situ, with a Phe ester
and subsequent hydrolysis of the ester group.
The resulting tetrapeptide A5-Trp-Phe-Dpr(A2)-Leu-R5 is suitably deprotected from
the group attached to the nitrogen of Trp and coupled with a compound of formula
(Vlb)

wherein
A-i, Re and R8 are defined as above.
c) Strategy 3+2: according to this strategy the tripeptide A:-Asp(R6)-Trp-Phe-OH,
obtained by removing the nitrogen protecting group from the compounds of
formula (XII) above reported, and subsequent coupling with a compound of
formula (Vlb) above reported, is then coupled with the monodeprotected dipeptide

H-ppr-(A2)-Leu-R5 prepared as described according to the procedure of strategy a).
As used in the present invention, the term "lower alkoxyl groups" refers to those alkoxyl groups in which the alkyl part comprises a linear or branched C1-C4 group, preferably selected from the group consisting of methyl, ethyl, propyl, butyl, isopropyl and t-butyl. This should be meant also for the alkyloxycarbonyl groups of the invention, in which the alkyl part comprises a linear or branched C1-C4 group, preferably selected from the group consisting of methyl, ethyl, propyl, butyl, isopropyl and t-butyl.
The coupling agent can be chosen from any one of those more commonly used in peptide synthesis, so as to generate an activated amino acid derivative such as those reported for example in M. Bodansky, "Peptide Chemistry", Springer Verlag 1988 or in J. Jones, "The Chemical Synthesis of Peptides", Clarendon Press. Oxford 1994.
The activated derivatives, if not commercially available, can be prepared
separately or in situ by reaction between an amino acid or a peptide and one or
more of the numerous known coupling agents, such as isobutyl chloroformate
(IBCF), a carbodiimide selected from dicyclohexylcarbodiimide (DCC) and 1-ethyl-
3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC.HCI) possibly in
combination with a hydroxyderivative selected from 1-hydroxybenzotriazole
(HOBt), 1-hydroxy-7-azabenzotriazole (HOAt), 6-chloro-1-hydroxybenzotriazole
(CI-HOBt) and hydroxysuccinimide (HOSu); a phosphonium salt, N-oxide
guanidine salt or uronium salt, such as (Benzotriazol-1-
yloxy)tri(dimethylamino)phosphonium hexafluorophosphate (BOP), (Benzotriazol-
1-yloxy)tripyrrplidine phosphonium hexafluorophosphate (PyBOP), 1-
[bis(dimethylamino)methylene]-1H-benzotriazolium-3-oxide hexafluorophosphate
(HBTU), 1-[bis(dimethylamino)methylene]-5-chloro-1H-benzotriazolium-3-oxide
hexafluorophosphate (HCTU), 1-[bis(dimethylamino)methylene]-1H-
benzotriazolium-3-oxide tetrafluoroborate (TBTU), 1-
[bis(dimethylamino)methylene]-1H-1,2,3-triazole[4,5-b]pyridinium-3-oxide
hexafluorophosphate (HATU), 1-[bis(dimethylamino)methylene]-5-chloro-1 H-
benzotriazolium-3-oxide tetrafluoroborate (TCTU), O-

[(ethoxycarbonyOcyanomethylenaminoj-N.N.N'.N'-tetramethyluronium
tetrafluoroborate (TOTU), 0-(bicycIo[2.2.1]hept-5-ene-2,3-dicarboximido)-
N.N.N'.N'-tetramethyluronium tetrafluoroborate (TNTU), or O-(N-succinimidyl)-
N.N.N'.N'-tetramethyluronium tetrafluoroborate (TSTU).
Where the derivative is generated in situ the coupling reaction is carried out
immediately afterwards by adding the other reagent, which obviously, in the case
of intramolecular cyclizations, corresponds to the free amine end present in the
molecule itself.
The coupling reaction is usually carried out in the presence of a tertiary amine
such as N-methylmorpholine (NMM), triethylamine (TEA) or diisopropylethylamine
(DIPEA) in an organic solvent chosen from those generally used for peptide
synthesis. Preferred solvents for the coupling reaction are ethyl acetate (AcOEt),
dimethylformamide (DMF) and N-methylpyrrolidone (NMP).
The coupling reactions can be carried out at a temperature that would not cause
degradations or render the reaction too slow, the temperature being preferably
comprised between -20 and +50°C.
The deprotections in the processes of the invention are achieved by the
appropriate methods for groups to be removed and compatible with the groups to
be retained; generally the present deprotection reactions are carried out by means
of catalytic hydrogenation or by acid or base treatments.
For hydrogenations, the catalyst can be chosen from those varieties of catalysts
which are available and suitable for this purpose; 5% or 10% Palladium are
preferred. The solvent for the deprotection reactions by catalytic hydrogenation
can be chosen from those that dissolve the compounds in reaction, excluding
ketones such as acetone, the solvents which poison the catalyst and those that
react with the components of the reaction themselves. DMF, NMP, organic acids
such as acetic acid and p-toluene sulfonic acid (PTSA), and alcohols such as
methanol, ethanol, and isopropanol, or mixtures thereof, are the preferred reaction
solvents. The hydrogenation reaction temperature is comprised between -20 and
+50°C.
For deprotections by acid treatment, mineral acids are preferably used, such as
hydrochloric acid, or organic acids, such as trifluoroacetic acid or formic acid,

which can be used alone or mixed with other solvents. The temperature is
between -20 and +50°C.
For deprotections by basic treatment, hydroxides of alkali metals and alkaline
earth metals are preferably used in the presence of a solvent such as water,
dioxane, acetonitrile, methanol, ethanol, isopropanol, or mixtures thereof; the
temperature is comprised between -20 and +50°C.
The term "oxygen protecting group" as used in the present invention refers to a
protecting group selected from those commonly used for the protection of -OH
groups and well known to any person skilled in the art, selected for example from
the group consisting of-COR4 wherein R4 is a linear or branched alkyl group, with
from 1 to 4 carbon atoms, the phenyl being possibly substituted by a halogen
atom, benzyl or benzoyl; the oxygen protecting group is preferably acetyl.
According to the invention the glycopeptide compounds of formula (l-A) can be
obtained by reacting a glycosidic derivative of formula (IIl-A) with an activated
peptide derivative of formula (ll-A), obtained by an activation reaction or generated
in situ by a compound of formula (I). Therefore, in the preparation process of
bicyclic glycopeptide compounds of formula (1-A), the glycosidic group is
introduced not in the linear peptide, but in the bicyclic peptide compound.
If compounds of formula (lll-A) are reacted in which R1, R2 and R3 are not
hydrogen, the compounds of formula (l-A) obtained can be transformed into the
corresponding compounds in which Ri=R2=R3=H, by means of catalytic
hydrogenation or by an acid or base treatment according to the nature of the
protecting groups Ri, R2 and R3.
The glycosidic compounds of formula (lll-A) preferably used in the process of the
invention are selected from the group consisting of 2-acetamide-2-deoxy-(3-D-
glucopyranosylamine and 2-acetamide-3,4,6-tri-0-acetyl-2-deoxy-(3-D-
glucopyranosylamine, which are known in the literature and can be prepared for
example as described respectively in I. Shin et al., Tetrahedron Letters, 42 (2001)
1325-1328 and D. Macmillan et al., Organic Letters, Vol. 4, Ns 9, 2002.
The following examples and schemes of synthesis are given to provide a non-
imiting illustration of the invention.
Scheme 1 indicates the synthesis path which, starting from the compounds of

formula (II) leads to those of formula (l-A), whereas schemes 2-4 show the three
different strategies for preparing the compounds of formula (II).
The protecting groups shown as examples are t-butoxycarbonyl (BOC) and
benzyloxycarbonyl (Z) for the amino extremities and methylester and t-butylester
for the carboxyl extremities.
The numbers given beside each compound in the following schemes corresponds
to the numbers attributed to the compounds in the examples.
The identification and evaluation of purity for the compounds prepared has been
established by elemental analysis, HPLC, 1H-NMR, IR and mass analysis.
4

EXAMPLE 1
Preparation of Z-Asp(OHVTrp-Phe-DprfH)-Leu-OMe (SEQ. ID. 1)
A 72 mmol/l solution of Z-Asp(OtBu)-Trp-Phe-Dpr(BOC)-Leu-OMe, prepared as
described in Example 15, in 95% formic acid is heated to 40°C under vacuum for
4 hours.
The reaction mixture is evaporated under reduced pressure and the residue is
redissolved with a 8:2 CH3CN - H20 mixture.
The suspension is cooled to 15-20°C and the pH is corrected to 6 by adding a
20% aqueous NMM solution.
The acetonitrile is evaporated under reduced pressure and the resulting
suspension is filtered.
The whitish solid obtained is washed with H20 and dried under vacuum at 30-40°C
to provide a yield equal to 96.4%.
1H-NMR dimethylsulfoxide-d6 (DMSO-d6) 5:
0.86 (2d; 6H); 1.47-1.75 (m; 3H); 2.32-2.68 (m; 2H); 2.79-3.55 (m; 6H); 3.63. (s;
3H); 4.25-4.65 (m; 5H); 4.99 (AB-Syst; 2H); 6.91-7.43 (m; 14H); 7.48-7.60 (2d;
2H); 7.82 (b; 2H); 8.03-8.43 (4d; 4H); 10.83 (s; 1H); 12.35 (b; 1H).
EXAMPLE 2
Preparation of Z-Asp-Trp-Phe-Dpr-Leu-Ome (SEQ. ID. 2)
2.2 equivalents of NMM are added to a 24 mmol/l solution of Z-Asp(OH)-Trp-Phe-
Dpr(NH2)-Leu-Ome in DMF and after 5-10 minutes 1.2 equivalents of PyBOP are
added.
After 2-3 hours of stirring at room temperature the solution is evaporated under
reduced pressure until a fluid residue is obtained which is dropped into a 0.5 M
aqueous solution of NaHC03.
The resulting suspension is filtered and the solid obtained is washed with a 4:6
DMF - H20 mixture and then with H20 until neutral pH is achieved and dried under
vacuum at 30-50°C, providing a yield equal to 84.2%.
1H-NMR(DMSOd6)5:
0.83 (2d; 6H); 1.34-1.69 (m; 3H); 2.31-2.92 (m; 4H); 3.03-3.91 (m; 4H); 3.61 (s;
3H); 4.17-4.63 (m; 5H); 5.01 (AB-Syst.; 2H); 6.84-7.48 (m; 16H); 7.60 (d; 1H); 7.87

(d;2H); 8.01 (t; 1H); 8.27 (d; 1H); 10.81 (s; 1H).
EXAMPLE 3
Preparation of Z-Asp-Trp-Phe-Dpr-Leu-OH (SEQ. ID. 3)
A cloudy solution containing 77 mmol/l of Z-Asp-Trp-Phe-Dpr-Leu-Ome in a 8:2
dioxane - H20 mixture is -heated to 35°C and maintained at pH 12.0 - 12.5 by
slowly and continuously adding 1.5 N NaOH.
At the end of the reaction the cloudy solution is brought to pH 9 by adding 6N HCI,
clarified by filtration on a co-adjuvant filtration bed and acidified to pH 3 by again
adding 6N HCI.
The solution is concentrated under reduced pressure until a filterable solution is
obtained.
The whitish filtered solid is washed with a 1:1 dioxane - H20 mixture and then with
H20 and dried under vacuum at 30-40°C, providing a yield equal to 97.7%.
1H-NMR (DMSO-d6) 5:
0.84 (2d; 6H); 1.42-1.76 (m; 3H); 2.29-3.48 (m; 7H); 3.85 (m; 1H); 4,10-4.65 (m;
5H); 5.00 (AB-Syst; 2H); 6.86-7.47 (m; 16H); 7.55-8.36 (4d+m; 5H); 10.80 (d; 1H);
12.65 (b;1H).
EXAMPLE 4
Preparation of Z-Asp(OtBu)-Asp-Trp-Phe-Dpr-Leu-OH (SEQ. ID. 4)
L I
A 66 mmol/l solution of Z-Asp-Trp-Phe-Dpr-Leu-OH in DMF is hydrogenated at
room temperature in the presence of 1 equivalent of NMM and catalytic quantities of 10% Pd/C, at 50% wetness.
After reacting for 6 hours the suspension is filtered to remove catalyst and filtrate is diluted with DMF to obtain a 53 mmol/l solution of H-Asp-Trp-Phe-Dpr- Leu-OH,
to which 4 equivalents of NMM and 1.05 equivalents of Z-Asp(OtBu)Osu are
added.
After stirring for 5 hours at room temperature the mixture is evaporated under

reduced pressure until a residue is obtained which is dropped into 0.05 N H2SO4.
The resulting suspension is filtered and the solid obtained is washed with a 1:1
DMF - H20 mixture and then with H20 and dried under vacuum at 30-40°C, to
provide a yield equal to 93.7%.
1H-NMR(DMSO-d6)6:
0.84 (2d; 6H), 1.35 (s; 9H); 1.40-1.70 (m; 3H); 2.20-3.94 (m; 10H); 4.10-4.81 (m;
6H); 4.92-5.12 (AB-Syst.; 2H); 6.74-7.57 (m; 17H); 7.71-8.35 (4d+1t; 5H); 10.70
(s;1H); 12.70 (b;1H).
EXAMPLE 5
Preparation of Cyclo[Asp(OtBu)-Asp-Trp-Phe-Dpr-Leu] (SEQ. ID. 5)
A 47 mmol/l solution of Z-Asp(ptBu)-Asp-Trp-Phe-Dpr-Leu-QH
in DMF is hydrogenated at room temperature, in the presence of 1 equivalent of DIPEA and catalytic quantities of 10% Pd/C at 50% wetness. After reacting for about 2 hours the suspension is filtered to remove the catalyst and diluted with DMF until a 19 mmol/l solution of H-Asp(OtBu)-Asp-Trp-Phe-Dpr-
! I
Leu-OH is obtained to which 1.4 equivalents of DIPEA and 1.2 equivalents of
HATU are added.
After stirring for 30-60 minutes at room temperature the solution is evaporated
under reduced pressure until a residue is obtained which is dropped into a 0.5 M
aqueous solution of NaHC03.
The resulting suspension is filtered and the solid obtained is washed with
abundant H20 until the pH is neutral, and dried under vacuum at 30-50°C,
obtaining a yield equal to 94.1%.
1H-NMR(DMSO-d6)6:
0^88 (2d; 6H); 1.38 (s; 9H); 1.31-1.72 (m; 3H); 2.33-2.99 (m; 6H); 3.20-3.63 (m;
3H); 3.87-4.62 (m; 7H); 6.75-7.50 (m; 13H); 8.04 (b; 1H); 8.56 (d; 1H); 8.76 (d;
1H);9.18(b;1H);10.84(s;1H).
EXAMPLE 6
Preparation of Cyclo[Asp(OH)-Asp-Trp-Phe-Dpr-Leu] (SEQ. ID. 6)
I I

A 83 mmoi/l solution of cyclo[Asp(OtBu)-Asp-Trp-Phe-Dpr-Leu] in 90% formic acid
is heated at 40°C under vacuum for 2 hours.
The reaction mixture is evaporated under reduced pressure until a dense residue
is obtained which is redissolved in H2O.
The resulting suspension is filtered and the solid obtained is washed with H20,
dried under vacuum at 30-40°C and finally purified by means of a Sephadex® LH-
20 column, eluting with methanol.
314 g of a white solid are obtained (titre 95.2%, yield 82.0%).
1H-NMR (DMSO-d6) 5:
0.88 (2d; 6H); 1.31-1.77 (m; 3H); 2.32-3.73 (m; 9H); 3.80-4.65 (m; 7H); 6.82-7.51
(m; 13H); 7.94-9.19 (2d; 2b; 4H); 10.85 (s; 1H); 12.20 (s; 1H).
EXAMPLE 7
Preparation of Cyclo[As,p-Asp-Trp-Phe-Dpr-Leu] (SEQ. ID. 7)
1 1
Glc(triOAc)NHAc 3 equivalents of NMM, 1.2 equivalents of HATU and 2-acetamide-3,4,6-tri-0-acetyl-2-deoxy-p-D-glucopyranosylamine are added to a 0.24 mol/l solution of Cyclo[Asp(OH)-Asp-Trp-Phe-Dpr-Leu] in DMF at 10 minute intervals. After stirring for 1 hour at 0-4°C the reaction mixture is evaporated under reduced pressure until a fluid residue is obtained which is dropped into a 1% aqueous solution of NaHC03.
The resulting suspension is filtered and the solid obtained is washed with H2O, dried under vacuum at 30-40°C and purified by crystallization from a EtOH - H20 mixture.
117 g of a white solid are obtained (titre 96.0%, yield 87.0%). 1H-NMR (DMSO-d6) 5:
10.80 (d;1H); 8.90 (b; 1H); 8.72 (d; 1H); 8.47 (d; 1H); 8.46 (d; 1H); 8.08 (b; 1H); 7.84 (d; 1H); 7.43 (dd; 1H); 7.33 (dd; 1H); 7.24 (b; 1H); 7.23 (m; 2H); 7.16 (m; 3H); 7.14 (d; 1H); 7.06 (dt; 1H); 7.00 (d; 1H); 6.98 (dt; 1H); 6.90 (t; 1H); 5.18 (dd; 1H); 5.12 (dd; 1H); 4.82 (dd; 1H), 4.18 (dd; 1H); 3.96 (dd; 1H); 3.85 (ddd; 1H); 3.80 (ddd; 1H); 4.53 (m, 1H); 4.47 (m; 1H); 4.43 (m; 1H); 4.39 (m; 1H); 4.16 (m; 1H);

4.08 (m; 1H), 3.58 (m; 1H); 3.30 (m; TK); 2.98 (m; 1H); 2.88 (m; 1H); 2.86 (m; 1H);
2.70 (m; 1H); 2.65 (m; 1H); 2.60 (m; 1H); 2.19 (m; 1H); 2.00 (s, 3H); 1.96 (s; 3H);
1.90 (s; 3H), 1.73 (s; 3H); 1.65 (m; 1H); 1.52 (m; 1H); 1.37 (m; 1H); 0.92 (d; 3H);
0.85 (d; 3H).
EXAMPLE 8
Preparation of CyclofAsp-Asp-Trp-Phe-Dpr-Leu] (Nepadutant) (SEQ. ID. 8)
GlcNHAc Method a)
2 equivalents of NMM and 1.3 equivalents of TBTU and 2-acetamide-2-deoxy-(3-D-glucopyranosylamine are added at 10 minute intervals to a 83 mmol/l solution in DMF of cyclo[Asp(OH)-Asp-Trp-Phe-Dpr-Leu] (prepared as described
in Example 6). ' !
After stirring for 1 hour at room temperature, the reaction mixture is evaporated
under reduced pressure until a dense oily residue is obtained which is redissolved
with a 2:8 acetonitrile - t-butoxymethane (TBME) mixture. The resulting
suspension is vigorously stirred for 30 minutes at room temperature and then
filtered.
The solid obtained is washed with TBME, dried under vacuum at 25-30°C and
finally purified by preparative HPLC using eluent mixtures composed of
acetonitrile and water.
151 g of a white solid are obtained (titre 93.0%, yield 89.3%).
1H-NMR(DMSO-d6)6:
0.85 (d; 3H); 0.92 (d; 3H); 1.36 (m; 1H); 1.51 (m; 1H); 1.65 (m; 1H); 1.76 (s; 3H);
2.16 (dd; 1H); 2.57 (dd; 1H); 2.63 (dd; 1H); 2.67 (dd; 1H); 2.83 (dd; 1H); 2.88 (dd;
1H); 2.93 (m; 1H); 3.04-3.09 (m; 2H); 3.27-3.32 (m; 2H); 3.42 (m; 1H); 3.50 (ddd +
b; 2H); 3.65 (dd; 1H); 3.96 (b; 1H); 4.09 (m; 1H); 4.12 (m; 1H); 4.35 (m; 1H); 4.43
(m; 1H); 4.50 (m; 1H); 4.53 (m + t; 2H); 4.81 (dd; 1H); 4.94 (d; 1H); 4.98 (d; 1H);
3.91 (b; 1H); 6.98 (t + b; 2H); 7.06 (t; 1H); 7.14-7.17
[m; 4H); 7.24 (t; 2H); 7.27 (b; 1H); 7.33 (d; 1H); 7.42 (d; 1H); 7.77 (d; 1H); 8.05 (b;
IH); 8.10 (d; 1H); 8.51 "(cf; 1H); 8.77 (d; 1H); 9.00 (b; 1H); 10.84 (d; 1H).
Method b)
).04 equivalents of 0.1 N NaOMe in MeOH are added to a 0.89 mol/l solution in

MeOH of cyclo[Asp-Asp-Trp-Phe-Dpr-Leu]
Glc(triOAc>NHAc prepared as described in Example 7.
After stirring for 3 hours at room temperature the pH is corrected to 6.5-7 and Amberlyst® 15 is added. Following removal of the resin, the solution is concentrated under reduced pressure until a residue is obtained which is diluted with TBME.
The resulting suspension is filtered and the white solid obtained is washed with TBME and dried under vacuum at 35-40°C, providing a yield equal to 94.8%. EXAMPLE 9
Preparation of Z-Dpr(BOC)-Leu-OMe Method a)
1.2 equivalents of NMM are added io'a 0.66 mol/l solution of Z-Dpr(BOC)-OH in DMF. The solution is cooled to -25°C and 1 equivalent of IBCF is dropped in while maintaining the temperature below -20°C.
After about 10 minutes a 0.78 mol/l pre-coofed solution containing 1 equivalent of H-Leu-OME HCI and NMM in DMF is dropped in, always maintaining the temperature below -15°C.
After stirring for one hour the reaction mixture is dropped into a 0.5 M aqueous solution of NaHC03.
The resulting suspension is filtered and the solid obtained is washed sequentially with H20, 0.05 M H2SO4 and H20 until the pH is neutral and dried under vacuum at 30-50°C, providing a yield equal to 89.0%. melting point 122-125°C; 1H-NMR (DMSO-d6) 5:
0.85 (2d; 6H>; 1.37 (s; 9H); 140-1.71 (m; 3H); 3.01-3.36 (m; 2H); 3.61 (s; 3H); 4.06-4.37 (m; 2H); 5.03 (s; 2H); 7.35 (s; 5H); 6.66 (t; 1H); 7.20 (d; 1H); 8.29 (d; 1H).
Method b)
1 equivalent of DCC is added to a 0.35 mol/l solution of Z-Dpr(BOC)-OH in DMF containing 1 equivalent of HOSu, cooling to 0-5°C. The mixture is brought to room temperature and stirred for 1 hour. The DCC is removed by filtration and to the clear filtrate are added 1.2 equivalents of H-Leu-Ome HCI and 2.6 equivalents of

NIMM. After stirring for 2-3 hours at room temperature the mixture is diluted with
0.5 N NaHC03 then cooled to -5°C.
The resulting suspension is filtered and the solid obtained is washed sequentially
with 0.5 N NaHC03i a 2:1 H20 - DMF mixture and water, then dried under vacuum
at 30-40°C, providing a yield equal to 93%.
EXAMPLE 10
Preparation of H-DprfBOC)-Leu-OMe
A 0.14 mol/l solution of Z-Dpr(BOC)-Leu-OMe in MeOH containing 1 equivalent of
PTSA is hydrogenated at room temperature in the presence of catalytic quantities
of 10% Pd/C, 50% wetness.
After reacting for about 2 hours the suspension is filtered to remove the catalyst
and the filtrate is diluted with DMF.
The MeOH and the H2O are completely evaporated under reduced pressure and
the residual DMF solution, containing the dipeptide, is used for the subsequent
coupling.
EXAMPLE 11
Preparation of Z-Phe-DprfBOCVLeu-OMe
The compound was prepared from the dipeptide H-Dpr(BOC)-Leu-OMe from
Example 10, according to the method described in Example 9 using Z-Phe-OH.
1H-NMR (DMSO-de) 6:
0.86 (2d; 6H); 1.38 (s; 9H); 1.40-1.74 (m; 3H); 2.73-3.02 (m; 2H); 3.10-3.41 (m;
2H); 3.62 (s; 3H); 4.17-4.46 (m; 3H); 4.94 (AB-Syst.; 2H); 7.18-7.39 (m; 10H); 6.52
(t; 1H); 7.52 (d; 1H); 8.13 (d; 1H); 8.25 (d, 1H).
EXAMPLE 12
Preparation of H-Phe-Dpr(BOC)-Leu-OMe
The compound was obtained from the protected derivative from Example 11,
according to the method in Example 10, using DMF as the solvent.
EXAMPLE 13
Preparation of Z-Trp-Phe-Dpr(BOC)-Leu-OMe (SEQ. ID. 9)
The compound was prepared using the method in Example 9 from the tripeptide
of Example 12 and using Z-Trp-OH or by coupling the two dipeptides Z-Trp-Phe-
OH and H-Dpr(BOC)-Leu-OMe, obtained as described in Examples 17 and 10

respectively.
1H-NMR(DMSO-d6)5:
0.86 (2d; 6H); 1.37 (s; 9H); 1.40-1.76 (m; 3H); 2.73-3.41 (m; 6H); 3.62 (s; 3H),
4.16-4.67 (m; 4H); 4.93 (AB-Syst.; 2H); 6.89-7.65 (m; 16H); 6.55 (t; 1H); 8.07 (d;
1H); 8.11 (d; 1H); 8.29 (d; 1H);10.79 (s; 1H).
EXAMPLE 14
Preparation of H-Trp-Phe-Dpr(BOC)-Leu-OMe (SEQ. ID. 10)
The compound was obtained from the protected derivative of Example 13,
according to the method given in Example 10, using NMP as solvent.
EXAMPLE 15
Preparation of Z-Asp(OtBu)-Trp-Phe-DprfBOC)-Leu-OMe CSEQ. ID. 11)
Method a)
1 volume of CH3CN, 1.5 equivalents^ of DIPEA and 1.15 equivalents of Z-
Asp(OtBu)-OSu are added to a 0.16 mol/l solution of H-Trp-Phe-Dpr(BOC)-Leu-
OMe in NMP, derived from the hydrogenation reaction. After stirring for 3-4 hours
at room temperature the reaction mixture is cooled to 5°C and is diluted with H20.
The resulting suspension is filtered and the solid obtained is washed with a 3:7
CH3CN - H20 mixture and with H20 and then dried under vacuum at 30-50°C,
providing a yield equal to 90%.
Method b)
1 equivalent of DIPEA, 1.1 equivalents of TBTU and after 5 minutes 1 equivalent
of the 0.25mol/l H-Dpr(BOC)-Leu-OMe solution in DMF derived from the
hydrogenation reaction (example 10), are added to a 0.22 mol/l solution of Z-Asp-
(OtBu)-Trp-Phe-OH in DMF cooled to -5°C, maintaining the temperature below -
5°C.
After stim'ng for about 2 hours the reaction mixture is diluted with a 0.5 M aqueous
solution of NaHC03.
The resulting suspension is filtered and the solid obtained is washed sequentially
with H20, a 3:4 DMF - 0.5 M NaHC03 mixture in H20, H20 and then dried under
vacuum at 30-40°C providing a yield of 84.4%.
melting point 215-218°C; 1H-NMR (DMSO-d6) 5:
0.86 (2d; 6H); 1.34 (s; 9H); 1.37 (s; 9H), 1.40-1.72 (m; 3H); 2.23-2.67 (m; 2H);

2.71-3.39 (m; 6H); 3.62 (s; 3H); 4.23-4.58 (m; 5H); 5.01 (AB-Syst., 2H); 6.89-7.58 (m; 16H); 6.50 (t; 1H); 7.87-8.29 (4d; 4H); 10.78 (s; 1H). EXAMPLE 16
Preparation of Z-Trp-Phe-QMe
The compound was prepared according to the method of Example 9, coupling the two amino acids Z-Trp-OH and H-Phe-OMe. 1H-NMR(CDCI3)5:
2.88-2.98 (m; 2H); 3.11 (dd; 1H); 3.32 (dd; 1H); 3.62 (s; 3H); 4.40-4.58 (m; 1H); 4.16-4.30 (m; 1H); 5.11 (s; 2H); 5.45 (d; 1H), 6.11 (d; 1H); 6.72-6.85 (m; 2H), 6.92-7.46 (m; 12H); 7.67 (d; 1H); 8.03 (s; 1H). EXAMPLE 17
Preparation of Z-Trp-Phe-OH
The compound was prepared from the methylester of Example 16, according to the method described in Example 3. 1H-NMR(DMSO-d6)5:
2.70-3.15 (m; 4H); 4.20-4.36 (m; 1H); 4.38-4.55 (m; 1H); 4.92 (s; 2H); 6.85-7.42 (m; 15H); 7.63 (d; 1H); 8.26 (d; 1H); 10.81 (s; 1H); 12.30 (b; 1H). EXAMPLE 18
Preparation of H-Trp-Phe-OH
The compound was prepared from the protected derivative of Example 17, in accordance with the method of Example 10, using acetic acid as solvent. EXAMPLE 19
Preparation of Z-Asp(OtBu)-Trp-Phe-OH
The compound was prepared in accordance with the method of Example 15 (method a) from the dipeptide of Example 18. 1H-NMR(DMSO-d6)5:
1.35 (s; 3H); 2.21-2.67 (m; 2H); 2.71-3.18 (m; 4H); 4.22-4.53 (m; 3H); 5.00 (AB-Syst.; 2H); 6.87-7.43 (m; 14H); 7.55 (m; 2H); 7.94 (d; 1H); 8.17 (d; 1H); 10.80 (s; 1H); 12.25 (b;1H).

WE CLAIM:
1. Process for preparing bicydic peptide compounds of formula (f)

comprising the following steps:
1) deprotection of the linear pentapeptide of formula (II) in the presence of a
solvent to give the compound of formula (III):

wherein Ai and A2 are two nitrogen protecting groups different from each other, and R5 and R6, different from each other, are chosen from benzyloxy and lower alkyloxy groups in which the alkyl part comprises a linear or branched C1-C4 group;
2) intramolecular cyclisation of the compound of formula (III) coming from step 1)
in the presence of a solvent and of a suitable coupling agent to give the
compound of formula (IV)

wherein R5 is as defined above;
3) deprotection of the compound of formula (IV) coming from step 2) in the
presence of a solvent to give the compound of formula (V)

wherein R5 is as defined above;
4) coupling between the compound of formula (V) coming from step 3) and a protected smino-acid of formula (Via) in the presence of a solvent, to give compounds of formula (VII)

wherein A3 is a nitrogen protecting group; R7 is chosen from benzyloxy and lower alkyloxy groups, in which the alkyl part comprises a linear or branched C1-C4 group; R8 is a residual group deriving from an activation procedure on the carboxyl group;
5) deprotection of the compound of formula (VII) coming from step 4) in the.
presence of a solvent to give a compound of formula (VIII)

wherein R7 is as defined above;
6) intramolecular cyclisation, in the presence of a solvent and of a suitable
coupling agent, of the compound of formula (VIII) coming from step 5) to give a
bicyclic compound of formula (IX)

wherein R7 is as defined above;
7) deprotection of the bicyclic compound of formula (IX) coming from step 6) in the
presence of a solvent, to obtain the compound of formula (I)

wherein R7 is as defined above.
2. Process according to claim 1, wherein the linear peptides of formula (II) are obtained by means of a sequential coupling strategy of suitable amino acids starting from a derivative of the amino acid Dpr of formula (X), protected on nitrogen and prepared separately or generated in situ

wnerem
A2and A4, different from each other, are nitrogen protecting groups;
Rg is a residual group deriving from an activation procedure, preferably chosen
from the group consisting of benzyloxycarbonyl, alkoxycarbonyl comprising a
linear or branched C1-C4 group in the alkyl part, and succinimidyl;
according to the following steps:
.- reaction of the derivative of formula (X) above reported in the presence of a
solvent with a Leu ester of formula (XI)

wherein R5 is defined as in claim 1, to obtain the dipeptide Ai-Dpr^-Leu-Rs,
- deprotection of the dipeptide A4-Dpr(A2)-Leu-R5, to obtain the monodeprotected dipeptide H-Dpr(A2)-Leu-R5;
- coupling the monodeprotected dipeptide H-Dpr(A2)-Leu-R5 with the activated ester of the subsequent amino acid Phe and then successively with Trp and Asp, until the compounds of formula (II) are obtained.
3. Process according to claims 1 and 2, wherein the linear peptides of formula (II) are obtained by means of a synthesis strategy comprising the following steps:
- coupling of the monodeprotected dipeptide H-Dpr(A2)-Leu-R5, obtained as
described in claim 2, with an activated derivative of the dipeptide of the following
formula (XII) A5-Trp-Phe-OH
(XII)
wherein A2 and As, different from each other, are nitrogen protecting groups,
prepared separately or generated in situ by coupling an activated ester of a Trp protected on nitrogen prepared separately or generated in situ, with a Phe ester and subsequent hydrolysis of the ester group, to obtain the tetrapeptide As-Trp-Phe-Dpr(A2)-Leu-R5;
- suitable deprotection of the tetrapeptide A5-Trp-Phe-Dpr(A2)-Leu-R5 from the group attached to the nitrogen of Trp;
- coupling of the deprotected tetrapeptide with a compound of formula (VI b)

wherein A-i, R6 and R8 are defined as in claim 1.
4. Process according to claims 1-3, wherein the linear peptides of formula (II) are obtained by means of a synthesis strategy of the 3+2 type that involves coupling the tripeptide ArAsp(R6)-Trp-Phe-OH, obtained by removing the nitrogen protecting group from the compounds of formula (XII) above reported, subsequent coupling with a compound of formula (Vlb) above reported and then further coupling with the monodeprotected dipeptide H-Dpr-(A2)-Leu-R5 prepared as described in claim 2.
5. Process according to claim 1, wherein said nitrogen protecting groups are selected from the group consisting of benzyloxycarbonyl and alkoxy carbonyls in which the alkyl part comprises a linear or branched C1-C4 group.
6. Process according to claim 5, wherein said nitrogen protecting groups are selected from t-butoxycarbonyl and benzyloxycarbonyl.
7. Process according to claim 1, wherein said Rs group is selected from the group consisting of benzyloxycarbonyl, alkyloxycarbonyl comprising a linear or branched C1-C4 group in the alkyl part, succinimidyl, benzotriazole possibly substituted by a halogen and azabenzotriazole.
8. Process according to claims 1-7, wherein said linear or branched C1-C4 group is selected from the group consisting of methyl, ethyl, propyl, butyl, isopropyl and t-butyl.
9. Process for preparing a bicyclic glycopeptide compound of formula (l-A)

wherein Ri, R2 and R3, equal or different from each other, can be hydrogen or an oxygen protecting group, comprising the following steps:
1A) activation of the bicyclic peptide compounds of formula (I) with a suitable coupling agent to obtain a derivative of formula (ll-A)

(I) (H-A)
wherein R is a group selected from benzotriazole, possibly substituted with a
halogen, azabenzotriazole and succinimidyl;

2A) reaction of the compound of formula (fl-A) deriving from step 1A) in the presence of a solvent with the glycosidic derivative of formula (Ill-A)

wherein R, R-i, R2, R3 are defined as above.
10. Process according to claim 9, wherein the compounds of formula (l-A) wherein R-,, R2 and R3 are different from H, are transformed into the corresponding compounds of formula (I-A) wherein Ri=R2=R3=H, by a deprotection reaction in the presence of a solvent.
11. Process according to claim 9, wherein said oxygen protecting groups are selected from the group consisting of -COR4 wherein R4 is a linear or branched C1-C4 alkyl group, phenyl possibly substituted with a halogen atom, benzyl or benzoyl.
12. Process according to claim 11, wherein said C1-C4 alkyl group is selected from the group consisting of methyl, ethyl, propyl, butyl, isopropyl and t-butyl.
13. Process according to claim 12, wherein said C1-C4 alkyl group is methyl.
14. Process according to claim 9, wherein said glycosidic derivatives of formula (lll-A) are selected from the group consisting of 2-acetamide-2-deoxy-(3-D-

gjucopyranosylamine and 2-acetamide-3,4,6-tri-0-acetyl-2-deoxy-£-D-
glucoDyanosylamine,
15. Process according to claim 9, wherein said bicyclic peptide compounds of formula (I) are prepared as described in claim 1.
16. Process according to claims 1 or 9, wherein said coupling agent is selected from the group consisting of isobutyl chloroformate, a carbodiimide possibly in combination with a hydroxyderivative, phosphonium salts, N-oxide guanidine salts and uranium salts.
17. Process according to claim 16, wherein said carbodiimides are selected from dicyclohexylcarbodiimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; said hydroxyderivative is selected from 1-hydroxybenzotriazole, 6-chloro-1-hydroxybenzotriazole, hydroxysuccinimide and 1-hydroxy-7-azabenzotriazole; said phosphonium salts, N-oxide guanidine salts and uranium salts are selected from (Benzotriazol-1-yloxy)tri(dimethylamino)phosphonium hexafluorophosphate, (Benzotriazol-1 -yloxy)tripyrrolidine phosphonium hexafluorophosphate, 1-[bis(dimethylamino)methylene]-1H-benzotriazolium-3-oxide hexafluorophosphate, 1-[bis(dimethylamino)methylene]-5-chloro-1 H-benzotriazolium-3-oxide hexafluorophosphate, 1 -[bis(dimethyIamino)methyleneJ-1 H-benzotriazolium-3-oxide tetrafluoroborate, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazole[4,5-b]pyridinium-3-oxide hexafluorophosphate, 1-[bis(dimethylamino)methylene]-5-chloro-1H-benzotriazolium-3-oxide tetrafluoroborate, 0-[(ethoxycarbonyl)cyanomethylenamino]-N,N,N',N'-tetramethyluronium tetrafluoroborate, 0-(bicyclo[2.2.1]hept-5-ene-2,3-dicarboximidoJ-N.N.N'.N'-tetramethyluronium tetrafluoroborate, and 0-(N-succinimidyO-N.N.N'.N'-tetramethyluronium tetrafluoroborate.

18. Process according to claims 1 or 9, wherein said coupling reactions are carried out in the presence of a tertiary amine in an organic solvent at a temperature comprised between -20 and +50°C.
19. Process according to claim 18, wherein said tertiary amine is selected from the group consisting of N-methylmorpholine, triethylamine and diisopropylethylamine, and said organic solvent is selected from the group consisting of ethyl acetate, dimethylformamide and N-methylpyrrolidone.

, 20. Process according to claims 1 or 10, wherein said deprotection reactions are carried out by means of hydrogenation in the presence of a catalyst in a solvent selected from solvents which dissolve the components of the reaction without reacting with them, excluding ketones and solvents which poison the catalyst, at a temperature comprised between -20 and +50°C.
21. Process according to claim 20, wherein said catalyst is selected from 5% and
10% Palladium and said solvent is selected from dimethylformamide, N-
methylpyrrolidone, acetic acid, p-toluenesulfonic acid, methanol, ethanol,
isopropanol, and mixtures thereof.
22. Process according to claims 1 or 10, wherein said deprotection reactions are
carried out by means of acid treatment with pure acids or with acids mixed with
other solvents, at a temperature comprised between -20 and +50°C.
23 Process according to claim 22, wherein said acids are selected from hydrochloric acid, trifluoroacetic acid and formic acid.
24. Process according to claims 1 or 10, wherein said deprotection reactions are
carried out by means of treatment with a base compound in the presence of a
solvent, at a temperature comprised between -20 and +50°C.
25. Process according to claim 24, wherein said base compound is selected from
hydroxides of alkali metals or alkaline earth metals, and said solvent is selected
from the group consisting of water, dioxane, acetonitrile, methanol, ethanol,
isopropanol, and mixtures thereof.

Documents:

1484-chenp-2005 abstract duplicate.pdf

1484-chenp-2005 abstract.pdf

1484-chenp-2005 claims.pdf

1484-chenp-2005 claism duplicate.pdf

1484-chenp-2005 correspondence-others.pdf

1484-chenp-2005 correspondence-po.pdf

1484-chenp-2005 description (complete) duplicate.pdf

1484-chenp-2005 description (complete).pdf

1484-chenp-2005 form-1.pdf

1484-chenp-2005 form-18.pdf

1484-chenp-2005 form-26.pdf

1484-chenp-2005 form-3.pdf

1484-chenp-2005 form-5.pdf

1484-chenp-2005 pct search report.pdf

1484-chenp-2005 pct.pdf

1484-chenp-2005 petition.pdf

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Patent Number

222670

Indian Patent Application Number

1484/CHENP/2005

PG Journal Number

47/2008

Publication Date

21-Nov-2008

Grant Date

20-Aug-2008

Date of Filing

01-Jul-2005

Name of Patentee

MENARINI RICERCHE S.P.A

Applicant Address

VIA TITO SPERI, 10, I-00040 POMEZIA,

Inventors:

#	Inventor's Name	Inventor's Address
1	SALIMBENI, ALDO	VIA GAETANO STRAMBIO, 11, I20133 MILAN,
2	POMA, DAVIDE	VIA MARTIRI DI CEFALONIA, 2, I-20059 VIMERCATE,
3	TUROZZI, DAMIANO	VIA ALEGOSA, 2, I-20029 TURBIGO,
4	MANZINI, STEFANO	VIA DELLA MATTONAIA, 25,I-50121 FIRENZE,
5	MAGGI, CARLO, ALBERTO	VIA L. MICHELAZZI, 43, I-50141 FIRENZE,

PCT International Classification Number

C07K7/56

PCT International Application Number

PCT/EP03/13696

PCT International Filing date

2003-12-04

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	FI2002A000239	2002-12-06	Italy