Title of Invention

"PROCESS FOR THE PRODUCTION OF N-METHYL-HOMOCYSTEINES COMPOUNDS"

Abstract Process for the production of N-methyl-homocysteines compounds of general formula I in which X1 stands for benzyl (Bn), 4-methoxybenzyl (Mob), diphenylmethyl, bis(4-methoxyphenyl)methyl, 4,4"-dimethoxytriphenylmethyl (DMT), triphenylmethyl (trityl), methoxymethyl (MOM), 9H-fluoren-9-ylmethyl or tert-butylsulfide, and X2 stands for 9H-fluoren-9-ylmethyloxycarbonyl (Fmoc), 2,7-dibromo-9H-fluoren-9-ylmethyloxycarbonyl, tert-butyloxycarbonyl (Boc), benzyl oxycarbonyl (Cbz or Z), trifluoroacetyl, 2,2,2-trichloroethyloxycarbonyl (Troc), 2-trimethylsilylethyloxycarbonyl (Teoc) or 2-trimethylsilylethylsulfonyl wherein the oxazolidinone of formula II is opened with a reducing agent of the kind such as herein described in the presence of an acid at a temperature of -20°C to + 100°C to form the N-methylmethionine of formula III and then a sulfur protective group and a nitrogen protective group are introduced in a way that is known in the art.
Full Text The present invention relates to process for the production of n-methyl-homocysteines compounds.
Description
The invention relates to the subjects that are characterized in the claims, namely N-meihyl-homocyteines of general formulas I and II, their use, and process for their production.
N-Methyl-amino acids are valuable intermediate products in synthetic chemistry. Primarily pharmaceutical chemistry uses such components very frequently, since many highly potent and selective pharmaceutical agents contain N-methyl-amino acids.
Optically active N-methyl-amino acids occur naturally as single compounds (e.g., N-methyl-tryptophans; Liehigs Ann. Chem. 1935, 520, 31-34), but are also found as components in a number of biologically active natural substances, such as, e.g., the dolastatins (G. R. Pettit et a.l...J. Org. Chan. 1990. 55, 2989-2990; Tetrahedron 1993, 41, 9151-9170) or the didemnins (K. L Rinchart et al,.J. Nat. Prod. 1988, 51, 1-21; J. Am. Chem. Soc. 1987, 109, 6846-6848; J. Am. Chem. Soc. 1995. 117. 3734-3748). Other examples are jasplakinolide (J. Org. Chem. 1991, 56, 5196-5202} and other cytotoxic peptides (J. Org. Chem., 1989, 54, 617-627; Tetrahedron 1995, 57, 10053-10062; J. Org. Chem. 1989, 54,736-738). The cyclosponn that has an immunosuppressive action also contains N-methyl-amino acids (see, for example, R. M. Wenger, Hevy. Chun Ada 1983, 66, 2672-2702; S. L. Schreiber et al., Tetrahedron Lett. 1988, 29, 6577-0580). N-Methyl-nmino acids can also have neuropharmacological activity (J. C. Watkius,J. Med. Pharm. Chem. 1962, 5, 1187-1199).
The incorporation of N-melhyl-amino acids in biologically active peptides is often used, on the one hand. in pharmaceutical research to study conformation and biological activity (see, e,g.,.J. Org. Chem. 1981. 46, 3436-3440; Int. J. Pept. Protein Res. 1995, 46, 47-55; Int. J. Pe.pt.
Protein Res. 1986, 27, 617-632). H. Kessler, Angew. Chem. [Applied Chemistry] 1982, 94, 509-520, and G. R. Marshall et al., Ann. Rep. Med. Chem. 1978, 13, 221-23% provide an overview on conformation studies of peptides and the connection with biological activity.
On the other hand, the substitution of amino acids in peptides by the corresponding N-mcthyl compounds is used to increase the activity or selectivity of the peptide ligands (see, e.g., ./. Med. Chem. 1994, 37, 769-780; Int. J. Pept. Protein Res. 1995, 46, 47-55). N-Methyl-peptide bonds often show higher stability compared to proteolysis than the non-methylated bonds, which can result in an increased oral availability and extended action (R. H. Mazur et al., J. Med. Chem. 1980, 23. 758-763). Regarding the design of peptides by incorporation of N-methyl-arnino acids and by other modifications, see W. F. Degrado, Adv. Protein Chem. 1988, 39, 51-124 and J. Rizo, L. M. Gierasch, Annu. Rev. Biochem 1993, 61, 387-418. For overviews regarding peptidomimetics, see A. Giannis et al., Angew. Chem. 1993,105, 1303-1326 and J. Gante, Angew. Chem. 1994, 106, 1780-1802.
In WO 01 /44177, a peptide that contains an N-methyl-homocysteine in a cyclic 6-mer partial sequence is described. The binding of a metal ion-binding sequence is carried out via the sulfur atom of the homocysteine. By the chelation of radionuclides such as 99nTc or l86Re, use as radiodi agnostic agents or radiotherapeutic agents in the diagnosis or therapy of carcinoses is possible (see also US 6,056,940, US 6,022,857, US 6,017,509).
The synthesis of the peptide is carried out in WO 01/44177 by N-methylation in the step-by-step course of the synthesis (see, e.g., J. Am. Chem. Soc. 1997, 119, 2301-2302). A 4-mer peptide, which is C-terminally bonded to a solid phase, is reacted with the amino acid N-Fmoc-S-tritylhomocysteine (Fmoc = 9H-fluoren-9-ylmethyloxycarbonyl), which is accessible from methionine in three steps (J. Med. Chem. 1996, 39, 1361-1371) to form 5-mer peptide. After the protective group is cleaved off, the terminal amino group of the homocysteine, is reacted with 2-nitrobenzenesulfonic acid chloride to form sulfonamide. The methylation with MTBD (1,3,4,6,7,8-hexahydro-l-memyl-2H-pyrimido[l,2-a]pyrimidene) and subsequently the cleavage of the sulfonyl group with 2-mercaptoethanol are then carried out. The peptide sequence is then

further built up. This synthesis, however, has the drawback of an incomplete reaction and
requires an additional purification of the peptide.
The creation of peptide sequences that contain N-methyl-amino acids is usually carried
out by the reaction of a partial sequence with an Fmoc-protected N-methyl-amino acid (see, e.g.,
./. Med. Chem. 1996, 39, 1361-1371). The amino acid N-Fmoc-N-methyl-S-tritylhomocysteine
that is required for the peptide sequence in WO 01/44177 therefore could not previously be
produced.
In the literature, a number of methods for producing N-methyl-amino acids, which
usually start from the corresponding non-methylated amino acids that are commercially available
in large amounts as chiral natural substances, are provided.
The first synthesis of non-racemic N-methyl-amino acids comes from E. Fischer. In this case, the N-tosyi derivatives of the amino acids are methylated by reaction with sodium hydroxide solution and methyl iodide. After that, the cleavage of the tosyl group is carried out with boiling hydrochloric acid (Ann. Chem. 1913, 398, 96-125; Ber. dt chem. Ges. 1915, 48, 360-378). It is described that partial racemization occurs with this method (A. H. Cook et al., J. Chem. ,Soc. 1949, 1022-1028).
Another method of Fischer is the reaction of 2-bromocarboxylic acids with methylamine (Ber dt. chem. Ges. 1916, 49, 1355-1366; for an application, also see A. H. Cook et al., J. Chem. Soc. 1.949, 1022-1028). In this case, partially racemized products are also produced (see P. Quitt et al,Heivv. Chim Acta 1963, 46, 327-333). We could confirm this observation with a reaction of 2-broomo-4-methylsuifaayi-butanoic acid with methylamine. In the literature, the production of N-methyl-L-methionine is described with the diazotization of D-methionine with subsequent substitution by bromide under retention, followed by a substitution by methylamine under inversion (N. Izumiya, A. Nagamatsu, Kyushu. Mem. Med. Sci. 1953, 4, 1-16). The enantiomeric purity of the product was not determined. We could show that even with a diazotization method of Ellman that is described as mild (Synthesis 1999, 583-585), the reaction sequence proceeds with onlv 75% enantiomeric excess. This method therefore is not suitable for the production of

enantiomer-pure N-methyl-homocysteines.
A method that is used very frequently is tha direct alkylation according to Benoiton with sodium hydride and methyl iodide in THF or DMF (Can. J. Chem. 1973, 51, 1915-1919. Can. J. ('hem. 1977. 55. 906-910). This method is used with respect to the standard for the production of Z- and Boc-protected N-methyl-amino acids (see, e.g., Tetrahedron 1995, 51, 10653-10662, D. L. Boger et al., J. Am. Chem. Soc. 1999, 121, 6197-6205, H. Waldmann et al., Chem. Eur. J. 1999, 5, 227-236). The reaction of Boc-protected methionine amide with methylating agents, such as metliyl iodide, in our case results in the methylation of the sulfur atom with the formation of sulfonium ions (see also S. J. F. Macdonald et al., J. Med. Chem. 1999, 64, 5166-5175). This method therefore cannot be used for the production of N-methyl-homocysteines.
There are a number of methods for the production of N-methyl-amino acids that start from amino acid esters. These include the selective reduction of N-formyl amino acid esters with borane [Tetrahedron Lett. 1982, 23, 3315-3318,/. Org. Chem. 1991, 56, 5196-5202) and the reaction of Schiff bases of amino acid esters with dimethyl sulfate or methyl triflate with subsequent hydrolysis (M. J. O'Donnell et al., Tetrahedron Lett. 1984, 25, 3651-3654). With the saponification of amino acid esters to form amino acids, however, partial racemization occurs (S. T. Clieung, N. L. Benoiton, Can. J. Chem, 1977, 55, 906-910). These methods therefore also are not suitable.
Amino acids can also be reacted in a reductive alkylation with an aldehyde. The Schiff base that is intermediately formed is reduced in situ in the presence of hydrogen and a catalyst (R. E. Bowman et al. J. Chem. Soc. 1950, 1342-1345 and 1346-1349) or with sodium borohydride (K. A. Schellenberg, J. Org. Chem. 1963, 28, 3259-3261). This method is performed both in solution (see, e.g., J. Org. Chem. 1996, 61, 3849-3862; J. Org Chem. 1995, 60, 6776-6784) and in solid phase (see, e.g., J. Org. Chem. 1996, 61, 6720-6722, Tetrahedron Lett. 1997. 38. 4943-4946, Bioorg Med. Chem. Lett. 1995, 5, 47-50). Di- and tripeptides were also already permethylated by Bowman according to this method (J. Chem. Soc. 1950, 1349-1351). A selective monomethylation with formaldehyde is not possible according to this method

but rather results in mixtures that consist of unmethylated, monomethylated and dimethylated amino acid, which cannot be separated (Quitt et al., Helv. Chim Acta 1963, 46, 327-333; see also R. E. Bowman. H. H. Stroud,./. Chem. Soc. 1950, 1342-1345).
For the synthesis of Fmoc-protected N-methyl-amino acids, the method of Freidinger (./. Org. Chem. 1983, 48, 77-81) is very popular. This method consists of two stages: in' the first step, an oxazolidinone is fonned by reaction of the amino acid with formaldehyde, which then is reduced to the N-methyl compound in the second step by means of triethylsilane under acidic conditions. The reaction of Fmoc-L-methionine with fomialdehyde to form oxazolidinone with a yield of 88% was described by Friedinger, but the reduction with triethylsilane to Fmoc-N-methyl-L-methionine is still incomplete even after five days and has only a 22% yield (J. Org. Chem. 1983, 48, 77-81).
The object of the invention was therefore to find compounds that are used for the creation of peptide sequences, which contain the N-methylated homocysteines, and to avoid the solid phase N-methylation, as well as a method for production thereof.
The object of the invention is achieved by the N-methyl-homocysteines, which can be both enantiomer-pure and racemic, of general formula I,
(Formula Removed)
in which X l stands for a sulfur protective group and X2 stands for a hydrogen atom or for a nitrogen protective group. X1 preferably stands for benzyl (Bn), 4-methoxybenzyl (Mob), diphenylmetliyl, Bis(4-methoxyphenyl)methyl, 4,4'-dimethoxytriphenylmethyl (DMT), triphenylmethyl (trityl), methoxymethyl (MOM), 9H-fluoren-9-ylmethyl or tert-butylsulfide (T. W. Greene, P. G. M. Wuts, Protective Groups in Organic Synthesis, 2nd Edition, J. Wiley & Sons, New York 1991), preferably for benzyl (Bn), 4-methoxybenzyl (Mob), diphenylmethyl,

bis(4-methoxyphenyl)methyl, 4,4'-dimetlioxytriphenylmethyl (DMT) or triphenylmethyl (ti'ityl), especially preferably for triphenylmethyl (trityl), and X2 in the case of a protective group preferably for 9H-fluoren-9-ylmefhyloxycarbonyl (Fmoc), 2,7-dibromo-9H-fluoren-9-ylmethyloxycarbonyl, tert-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz or Z), tritluoroacetyl, 2,2,2-trichloroefhyloxycarbonyl (Troc), 2-trimethylsilylethyl-oxycarbonyl (Teoc) or 2-trimethylsilylethylsulfonyl (T. W. Greene, P. G. M. Wuts, see above), preferably for tert-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz or Z) or 9H-fluoren-9-ylmethyloxy-carbonyl (Fmoc), especially preferably for 9H-fluoren-9-ylmethyloxycarbonyl (Fmoc).
The production of the compounds of general formula I according to the invention is carried out in that the oxazolidinone of formula II



(Formula Removed)
which is produced from methionine (see Example 1) according to literature methods (Ben-Ishai, J. Am. Chem. Soc. 1957, 79, 5736-5738; M. A. Blaskovich, M. Kahn, Synthesis 1998, 379-380; G. V. Reddy et al., Synth. Commun. 1999, 29, 4071-4077) is opened with a reducing agent in the presence of an acid to form the N-methylmethionine of formula III
(Formula Removed)
and then a sulfur protective group and optionally a nitrogen protective group are introduced in a

way that is known in the art (see T. W. Greene, P. G. M. Wuts, see'above). Alkylsilanes, preferably triethylsilane, are preferably used as reducing agents. Trifluoroacetic acid, pentafluoropropionic acid, trifluoromethanesulfonic acid or methanesulfonic acid, preferably trifluoroacetic acid, are preferably used as acids. The reaction is preferably carried.out in chlorinated solvents, dioxane, THF, dimethoxyethane, preferably in dichloromethane or chloroform. The reaction is performed at a temperature of-20°C to +100°C, preferably at 0 to 40"C. The reaction times are at five minutes up to ten hours, preferably 0.5 to 2 hours. N-Methylmethionine is obtained with yields of 70 to 95%.
The compounds of general formula I are used according to methods that are known to one skilled in the art (see M. Gooodman (editors), Houben-Weyl, Vol. E22, Synthesis of Peptides and Peptidomimetics. Thieme, 2001) for the creation of peptides and peptide intermediate stages that contain N-methyl-homocysteine, preferably for the production of Fmoc-(N-CH3)Hcy(Trf)-Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-OH. cyclo-Tyr-D-Trp-Lys-Thr-Phe-(N-CH3)Hcy and cyclo-Tyr-D-Trp-Lys-Thr--Phe(N-CH3)HcyCH2CO-ß-Dap-Phe(4-NH2)-Cys-Thr-Ser).
Example I
a) tert-Butyl-(S)-4-[2-(methylsulfanyl)ethyl)]-5-oxooxazolidinone-3-carboxylate
A mixture of 100 g of N-(tert-butoxycarbonyl)-L-methionine (0.4 mol), 100 g of paraformaldehyde (3.3 mol), 200 g of dried magnesium sulfate (1.7 mol) and 4 g of paratoluenesulfonic acid (0.02 mol) in 1 1 of toluene is heated for 3 hours to 90°C. It is allowed to cool to 20"C, and 800 ml of a saturated sodium bicarbonate solution is added to it wliile being cooled with ice. Tt is filtered off, and the residue is washed.with 400 ml of ethyl acetate. The organic phase is extracted with 300 ml of water, dried on sodium sulfate and evaporated to the dry state in a vacuum. The crude product is dissolved in 150 ml of hexane/ethyl acetate 1:3 and filtered on silica gel, and the silica gel is rewashed with 500 ml of hexane/ethyl acetate 1:3. It is evaporated to the dry state in a vacuum, and 85.3 g (0.33 mmol, 83% of theory) of a yellow oil is obtained.

Cld.: C 50.56 H 7.33 N 5.36 S 12.27 Fad.: C 50.38 H 7.24 N 5.43 S 12.10
1R: 2980, 2920. 1800, 1715, 1390, 1170, 1050
NMR (CDC13): 1.5 (9H), 2.1 (3H), 2.15-2.35 (2H), 2.5-2.68 (2H), 4.33 (1H), 5.21 (1H), 5.48 (1H)
MS (EI): m/z 261, 205, 188, 116, 100, 57
b) N-Methyl-L-methionine
45 ml of trifluoroacetic acid and 40 ml of triethylsilane (0.25 mol) are added in drops at 0"C to a solution of 19.4 g of tert-butyl-(S)-4-[2-(methylsulfanyl)ethyl)]-5-oxooxazolidinone-3-carboxyiate (0.074 mol) in 65 ml of dichloromethane at 0°C. It is stirred for one more hour while being cooled with ice and for one more hour at 20°C. Then, the reaction solution is evaporated to the dry state in a vacuum. The residue is again taken up three times with 100 ml of dichloromethane and again evaporated to the dry state. The residue is then taken up in 100 ml of water and extracted three times with 50 ml of MTBE. The aqueous phase is evaporated to the dry state in a vacuum. The residue is again taken up three times with 50 ml of ethanol and again evaporated to the dry state. The residue is mixed with 150.ml of MTBE and stirred for 2 hours at 20°C. It is filtered off and rewashed with 50 ml of MTBE. After drying, 9.6 g (0.059 mol, 80% of theory) of a colorless solid is obtained.
Fnd.: C 44.1 5 H 8.03 N 8.58 S 19.64
Cld.: C 44.01 H 7.83 N 8.72 S 19.80
TR: 3440, 3010, 2920, 2830, 2420, 1735, 1675, 1580, 1205, 1140, 800, 720
NMR(DMSO): 2.01-2.08 (5H), 2.5-2.65 (5H), 3.74-3.78 (1H)
MS (FAB): m/z 164 .
CD (water): 200 nm, Δε 1.64

c) N-Methyl-S-trityl-L-homocysteine
7.5 g (0.046 mol) of N-methyl-L-methionine is introduced,.and 200 ml of ammonia is condensed while being cooled with MeOH/dry ice. At -35°C, 5.2 g (0.23 mol) of sodium is added in portions over one hour and stirred for two more hours. Then, 9.7 g (0.18 mol) of ammonium chloride is added in portions, the cooling is withdrawn, and the ammonia is driven off overnight with nitrogen. 14.0 g (0.054 mol) of triphenylrnethanol is added. While being cooled with ice, 50 ml of dichloromethane and 90 ml of trifluoroacetic acid are then added. It is stirred for one hour at room temperature and evaporated to the dry state. The residue is suspended in 200 ml of water and brought to pH 13 with sodium hydroxide solution. After one more hour of stirring, it is suctioned off, and the solid is suspended in 500 ml of water. By adding citric acid, it is set at pH 4. 600 ml of MTBE is added, and it is stirred for 30 minutes. It is suctioned off and washed twice with 100 ml each of MTBE. The residue is mixed with 100 ml of dichloromethane, 20 ml of ethanol is added, and then 500 ml of MTBE is added in drops. It is stirred lor one more hour, suctioned off, and washed with MTBE. After drying, 12.6 g (0.032 mol, 70% of theory) of a colorless solid is obtained.
Cld.: C 73.62 H 6.44 N3.58 S 8.19
Fnd.: C 73.40 H 6.30 N 3.71 S 8.02
IR: 3440. 3055. 2850, 2400. 1615, 1485, 1445, 1395, 740, 700
NMR (CDCl3): 1.28-1.62 (2H), 2.12 (3H), 2.25-2.45 (2H), 2.84-2.92 (1H), 7.12-7.41 (15H)
MS (EI): m/z 243, 165
d) N-(9H-Fluoren-9-ylmethyloxycarbonyl)-N-methyl-S-trityl-L-homocysteine
16.6 g (0.042 mol) of N-methyl-S-trityl-L-homocysteine is suspended in 90 ml of water and 100 ml of THF, and 12.58 g (0.12 mol) of sodium carbonate is added. At 10°C, 14.88 g (0.044 mol) of fluorenylmethylsuccinimidylcarbonate in 50 ml of THF is added in drops, and it

is stiired for three more hours. Then, it is mixed with 80 ml of water and 130 ml of ethyl acetate, stirred for ten minutes, and brought to pH 4 with citric acid. The organic phase is washed twice with 100 ml each-of water and once with 100 ml of common salt solution. The aqueous phases are subsequently re-extracted once with 100 ml of ethyl acetate. The combined organic phase is concentrated by evaporation in a rotary evaporator, mixed with 260 ml of ethyl acetate and concentrated by evaporation in a rotary evaporator to 29.2 g of foam, which is chromatographed with dichloromethane/acetone 6:4. 22.4 g (0.036 mol, 86% of theory) of product is obtained.
Cld.: C 76.32 H 5.75 N 2.28 S 5.22
Fnd.: C 76.11 H 5.52 N2.40 S 5.38
1R: 3430, 3060, 2950, 1740, 1710, 1175, 740, 700
NMR (CDC13): 1.46-2.35 (4H), 2.66-2.73 (3H), 4.13-4.6 (4H), 7.13-7.79 (23H)
MS (FAB): m/z 614, 636
Example 2
The enantiomeric compound in the title compound of Example Id can be obtained analogously if, as described above, N-(tert-butoxycarbonyl)-D-metliionine is used instead of N-. (tert-butoxycarbonyi)-L-methionine. a) tert-Butyl-(R)-4-[2-(methylsulfanyl)ethyl)]-5-oxooxazolidinone-3-carboxylate
Cld.: C 50.56 H 7.33 N 5.36 S 12.27
End.: C 50.30 H 7.51 N 5.50 S 12.41
1R: 2980, 2920, 1800, 1715, 1390, 1175, 1050
NMR (CDC13): 1.5 (9H), 2.08 (3H), 2.15-2.35 (2H), 2.5-2.68 (2H), 4.33 (1H), 5.21 (1H),
5.48 (1H)
MS (El): m/z 261, 205, 188, 116, 100, 57

b) N-Methyl-D-methionine
Cld.: C 44.15 H 8.03 N 8.58 S 19.64 End.: C 43.98 Li 7.81 N 8.70 S 19.82
TR: 3430, 3010, 2920, 2830, 2420, 1630, 1580, 1395
NMR (D20): 2.13 (3H), 2.12-2.22 (2H), 2.55-2.68 (2H), 2.74 (3H), 3.68-3.73 (IH)
MS (El): m/z 163, 145, 118, 88, 70, 61
CD (water): 200 nm, As 1.87
c) N-Methyl-S-trityl-D-homocysteine
Cld.: C 73.62 H 6.44 N 3.58 S 8.19 Fad.: C 73.45 li 6.25 N3.78 S 8.15
IR: 3440, 3055, 2850, 2400, 1615, 1485, 1445, 1395, 740, 700 NMR (CDC13): 1.28-1.62 (2H), 2.12 (3H), 2.25-2.45 (2H), 2.84-2.92 (IH), 7.12-7.41 (15H)
MS (EI); m/z 243, 165
d) N-(9H-F1uoren-9-y]methyloxycarbonyl)-N-methyl-S-trityl-D-homocysteine
('Id.: C 76.32 1-1. 5.75 N 2.28 S 5.22 Fad.: C 76.15 115.58 N2.41 S 5.30
TR: 3440, 3055, 2950, 2600, 1740, 1705, 1445, 1170, 740, 700 .
NMR (CDC13): 1.45-2.35 (4H), 2.65-2.73 (3H), 4.14-4.62 (4H), 7.13-7.81 (23H)
MS (FAB): m/z 636, 614

Example 3
N-Benzyloxycarbonyl-N-methyl-S-trityl-L-homocysteine
16.6 g (0.042 mol) of N-methyl-S-trityl-L-homocysteine (title compound of Example lc) is suspended in 90 ml of water and 100 ml of THF, and 12.58 g (0.12 mol) of sodium carbonate is added. At 10°C. 1.0.97 g (0.044 mol) of N-benzyloxycarbonyl-oxysuccinimide, dissolved in 50 ml of THF, is added in drops and stirred for three more hours at 10°C. Then, it is mixed with 80 ml of water and 130 ml of ethyl acetate, stirred for ten minutes at room temperature and brought to pH 4 with citric acid. The organic phase is washed twice with 100 ml each of water and once with 100 ml of common salt solution. The aqueous phases are subsequently re-extracted once with 100 ml of ethyl acetate. The combined organic phases are evaporated-to the dry state in a vacuum; the residue is chromatographed on silica gel (mobile solvent: dichloromethane/acetone 6:4). 22.08 g (87% of theory) of product is obtained as a solid, colorless foam.
Elementary analysis:
Cld.: C.73.12 H 5.94 N 2.66 S 6.10
Fnd.: C 72.87 H 6.08 N 2.60 S 5.98
Example 4
N-tert-Butyloxycarbonyl-N-methyl-S-trityl-L-homocysteine
I 6.6 g (0.042 mol) of N-methyl-S-trityl-L-homocysteine (title compound of Example lc) is suspended in 90 ml of water and 100 ml of THF, and 12.58 g (0.12 mol) of sodium carbonate is added. At 10"C, 9.60 g (0.044 mol) of Di-tert-butyl-dicarbonate, dissolved in 50 ml of THF, is added in drops and stirred for three more hours at 10°C. Then, it is mixed with 80 ml of water and 130 ml of ethyl acetate, stirred for ten minutes at room temperature and brought to pH 4 with citric acid. The organic phase is washed twice with 100 ml each of water and once with 100 ml of common salt solution. The aqueous phases are subsequently re-extracted once with 100 ml of ethyl acetate. The combined organic phases are evaporated to the dry state in a vacuum; the

residue is chroraatographed on silica gel (mobile solvent: dichloromethane/acetone 6:4). 17.14 g (83% of theory) of a product is obtained as a solid, colorless foam.
Elementary analysis:
Cld.:'C 70.85 H 6.77 N2.85 S 6.52
Fnd.: C 70.68 H 6.91 N2.74 S 6.42
Example 5
a) N-Methyl-S-(4,4'-dimethoxy-trityl)-L-homocysteine
7.5 g (0.046 mol) of N-methyl-L-methionine (title compound of Example lb) is introduced, and 200 ml of ammonia is condensed while being cooled with MeOH/dry ice. At -35"C, 5.2 g (0.23 mol) of sodium is added in portions over one hour and stirred for two more hours. Then, 9.7 g (0.18 mol) of ammonium chloride is added in portions, the cooling is withdrawn, and the ammonia is driven off overnight with nitrogen. 17.3 g (0.054 mol) of bis(4-methoxyphenyl)-phenyl-methanol (DMT) is added. While being cooled with ice, 50 ml of dichloromethane and 90 ml of trifluoroacetic acid are then added. It is stirred for one hour at room temperature and evaporated to the dry state. The residue is suspended in 200 ml of water and brought to pH 13 with sodium hydroxide solution. After one more hour of stirring, it is suctioned off, and the solid is suspended in 500 ml of water. By adding citric acid, it is set at pH 4. 600 ml of MTBE is added and stirred for 30 minutes. It is suctioned off and washed twice with I 00 ml each of MTBE. The residue is mixed with 100 ml of dichloromethane, 20 ml of ethanol is added, and then 500 ml of MTBE is added in drops. It is stirred for one more hour, suctioned off, and washed with MTBE. After drying, 13.92 g (67% of theory) of a colorless solid is obtained.
Elementary analysis:
Cld.: C 69.15 H 6.47 N3.10 S 7.10

Fnd.: C 68.98 H 6.63 N3.01 S 7.02 b) N-(9H-Fluoren-9-ylmethyloxycarbonyl)-N-memyl-S-(4,4'-dmiethoxy1xi1yl)-L-homocysteine
18.97 g (0.042 mol) of N-methyl-S-4,4'-dimethoxytrityl-L-homocysteine (title compound of Example 5a) is suspended in 90 ml of water and 100 ml of THF, and 12.58 g (0.12 mol) of sodium carbonate is added. At 10°C, 14.88 g (0.044 mol) of fluorenylmethylsuccinimidiyl carbonate, dissolved in 50 ml of THF, is added in drops, and it is stirred for three more hours at 10"C. Then, it is mixed with 80 ml of water and 130 ml of ethyl acetate, stirred for ten minutes at room temperature and brought to pH 4 with citric acid. The organic phase is washed twice with 100 ml each of water and once with 100 ml of common salt solution. The aqueous phases are subsequently re-extracted once with 100 ml of ethyl acetate. The combined organic phases are evaporated to the dry state in a vacuum; the residue is chromatographed on silica gel (mobile solvent: dichloromethane/ acetone 6:4). 22.92 g (81% of theory) of product is obtained as a solid, colorless foam.
Elementary analysis:
Cld.: C 73.08 H 5.83 N 2.08 S 4.76
Fnd.: C 72.95 115.94 N 2.01 S 4.68
Example 6
Fmoc-(N-CH1)Hcy(Ti-t)-Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-ClTrt-solid phase
The peptide Fmoc-Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-CITrt solid phase substrated on solid phases produced as described in WO 01/44177 (Example 1, steps 1-3) was treated with 5% piperkiine in 1:1 NMP/DCM (75 ml) for 10 minutes followed by a reaction with 20% pipericiine in NMP (75 ml) for 15 minutes. The solid phase was washed in succession with NMP (3 x 75 ml x 1 minute) and DCM (3 x 75 ml x 1 minute). A nirihydrin analysis performed on a smali sample of the solid phase showed the end of the reaction, and the solid phase was washed with NMP (75 ml). A small portion of the carrier resin was treated withHFIPA and analyzed by

HPLC (see HPLC Method 1 from Diatide). The peak for Fmoc-Tyr(fBu)-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-OH at 21.7 minutes was not detected, but a peak at 12.7 minutes for H-Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-OH was detected. In a separate container, N-a-Fmoc-N-a-methyl-S-tiityl-homocysteines (9.20 g, 15 mmol), HATUreagent (5.70 g, 15 mmol) andHOAt (2.04 g, 1 5 mmol) in 50 ml of NMP were dissolved. DIEA (6.96 ml, 40 mmol) was added to the solution of the protected N-methylhomocysteine. It was stirred for one minute, and the mixture was added to the solid-phase batch. The reaction was shaken for 4 hours under a light argon stream. The solution was separated, and the solid phases were washed in succession with NMP (3 x 75 ml x 1 minute) and DCM (3 x 75 ml x 1 minute). A ninhydrin analysis performed on a small sample of the solid phase showed the end of the reaction. A smaller portion of the carrier resin was treated with HFIPA and analyzed by HPLC (see HPLC Method 1 with diatides). The peak for I-[-Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-OH at 12.7 minutes was not detected, but a peak at 26.7 minutes for Fmoc-(N-CH3)Hcy(Trt)-Tyr(tBu)-D-Trp(Boc)-Lys-(Boc)-TlTr(tBu) OH was detected.







We Claim:
1. Process for the production of N-methyl-homocysteines compounds of general formula I
(Formula Removed)
in which
X1 stands for benzyl (Bn), 4-methoxybenzyl (Mob), diphenylmethyl, bis(4-methoxyphenyl)methyl, 4,4'-dimethoxytriphenylmethyl (DMT), triphenylmethyl (trityl), methoxymethyl (MOM), 9H-fluoren-9-ylmethyl or tert-butylsulfide, and
X2 stands for 9H-fluoren-9-ylmethyloxycarbonyl (Fmoc), 2,7-dibromo-
9H-fluoren-9-ylmethyloxycarbonyl, tert-butyloxycarbonyl (Boc), benzyl
oxycarbonyl (Cbz or Z), trifluoroacetyl, 2,2,2-trichloroethyloxycarbonyl
(Troc), 2-trimethylsilylethyloxycarbonyl (Teoc) or 2-
trimethylsilylethylsulfonyl
wherein the oxazolidinone of formula II
(Formula Removed)
is opened with a reducing agent of the kind such as herein described in the presence of an acid at a temperature of -20°C to + 100°C to form the N-methylmethionine of formula III
(Formula Removed)
and then a sulfur protective group and a nitrogen protective group are introduced in a way that is known in the art.
2. Process as claimed in claim 1, wherein alkylsilanes are used as reducing
agents.
3. Process as claimed in claim 1, wherein as acids, trifluoroacetic acid,
pentafluoropropionic acid, trifluoromethylsulfonic acid or
methanesulfonic acid is used.
4. N-methyl-homocysteines compounds of general formula (I) prepared by
the process as claimed in claim 1.

Documents:

01196-DELNP-2004-Abstract-(30-08-2010).pdf

01196-delnp-2004-abstract.pdf

01196-delnp-2004-assignment.pdf

01196-DELNP-2004-Claims-(30-08-2010).pdf

01196-delnp-2004-claims.pdf

01196-DELNP-2004-Correspondence-Others-(30-08-2010).pdf

01196-delnp-2004-correspondence-others.pdf

01196-DELNP-2004-Description (Complete)-(30-08-2010).pdf

01196-DELNP-2004-Form-1-(30-08-2010).pdf

01196-delnp-2004-form-1.pdf

01196-delnp-2004-form-18.pdf

01196-DELNP-2004-Form-2-(30-08-2010).pdf

01196-delnp-2004-form-2.pdf

01196-delnp-2004-form-3.pdf

01196-delnp-2004-form-6.pdf

01196-delnp-2004-gpa.pdf

01196-delnp-2004-pct-210.pdf

01196-delnp-2004-pct-301.pdf

01196-delnp-2004-pct-304.pdf

1196-DELNP-2004-Abstract-(15-07-2008).pdf

1196-DELNP-2004-Assignment-(21-07-2008).pdf

1196-DELNP-2004-Claims-(15-07-2008).pdf

1196-DELNP-2004-Correspondence-Others-(15-07-2008).pdf

1196-DELNP-2004-Correspondence-Others-(21-07-2008).pdf

1196-DELNP-2004-Correspondence-Others-(25-07-2008).pdf

1196-DELNP-2004-Description (Complete)-(15-07-2008).pdf

1196-DELNP-2004-Form-1-(15-07-2008).pdf

1196-DELNP-2004-Form-2-(15-07-2008).pdf

1196-DELNP-2004-Form-3-(15-07-2008).pdf

1196-DELNP-2004-GPA-(15-07-2008).pdf

1196-DELNP-2004-Petition-137-(15-07-2008).pdf

1196-DELNP-2004-Petition-137-(21-07-2008).pdf

1196-DELNP-2004-Petition-138-(15-07-2008).pdf


Patent Number 245300
Indian Patent Application Number 01196/DELNP/2004
PG Journal Number 02/2011
Publication Date 14-Jan-2011
Grant Date 12-Jan-2011
Date of Filing 05-May-2004
Name of Patentee CIS BIO INTERNATIONAL
Applicant Address RN 306 SACLAY, 91400 SACLAY, FRANCE.
Inventors:
# Inventor's Name Inventor's Address
1 MARC WILLUHN TORFSTRASSE 16, 13353 BERLIN, GERMANY.
2 JOHANNES PLATZEK GROTTKAUER STRASSE 55, 12621 BERLIN, GERMANY.
3 ECKHARD OTTOW MOLTKESTRASSE 48, 12203 BERLIN, GERMANY.
4 ORLIN PETROV FRIEDRICHSHALLER STRASSE 7b, 14199 BERLIN, GERMANY.
5 CLAUDIA BORM STEPHANSTRASSE 9, 10559 BERLIN, GERMANY.
6 DIRK HINZ SCHLIEPER STRASSE 38, 13507 BERLIN, GERMANY.
7 GREGOR MANN BENNIGSENSTRASSE 18, 12159 BERLIN, GERMANY.
8 JOHN LISTER-JAMES 45 OLD STONE WAY, BEDFORD, NH 03110, USA.
9 DAVID M. WILSON 9, ARROWHEAD ROAD, BOW, NH 03304, USA.
PCT International Classification Number C07C 229/12
PCT International Application Number PCT/EP02/11779
PCT International Filing date 2002-10-22
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 102 15 336.1 2002-03-28 U.S.A.
2 60/331,416 2001-11-15 U.S.A.