NOVEL METHOD FOR SYNTHESIZING NCA COMPOUNDS

Abstract

A novel method for synthesizing NCA compounds. Also, a new use of a peptide coupling agent. The method makes it possible to obtain NCA compounds from ?-amino-acids, under mild and non-racemic reaction conditions, and in the absence of constraining reagents of use, such as phosgene, which may lead to the formation of undesirable by-products.

Claims

1.-15. (canceled)

16. A method for preparing of a NCA compound, comprising: a step of contacting a N-protected ?-amino-acid compound with propane-phosphonic acid anhydride, in an organic solvent, to obtain said compound NCA.

17. The method of preparation according to claim 16, wherein said compound NCA is of Formula 1-A, prepared from a N-protected ?-amino-acid compound of Formula 2-A, according to the following reaction scheme: ##STR00049## Formula 1-A wherein: R.sup.1 and R.sup.2 independently represent: a hydrogen atom, a group chosen from: a linear or branched C.sub.1 to C.sub.20 alkyl, a linear or branched C.sub.2 to C.sub.20 alkenyl, C.sub.3 to C.sub.10 cycloalkyl, C.sub.3 to C.sub.10 heterocycloalkyl, wherein the heteroatom is selected from N, O, and S, aryle, C.sub.1 to C.sub.20 alkyl-aryl, in particular benzyl, heteroaryl, wherein the heteroatom is selected from N, O, and S, in particular 3-methylindole, C.sub.1 to C.sub.20 alkyl-heteroaryl, wherein the heteroatom is selected from N, O, and S, a halogen, in particular a fluorine atom, said alkyl, alkyl-aryl or alkyl-heteroaryl may be substituted on at least one carbon of the alkyl radical by one or more groups selected from: OR.sup.4, wherein R.sup.4 is selected from H, linear or branched C.sub.1 to C.sub.20 alkyl, linear or branched C.sub.1 to C.sub.20 heteroalkyl, C.sub.3 to C.sub.10 cycloalkyl, C.sub.3 to C.sub.10 heterocycloalkyl, aryl, C.sub.1 to C.sub.20 alkyl-aryl, heteroaryl and C.sub.1 to C.sub.20 alkyl-heteroaryl and a protective group, in particular selected from tBDMS, t-butyl, benzyl, trityl and xanthyl, OC(O)R.sup.5, wherein R.sup.5 is selected from H, linear or branched C.sub.1 to C.sub.20 alkyl, linear or branched C.sub.1 to C.sub.20 heteroalkyl, C.sub.3 to C.sub.10 cycloalkyl, C.sub.3 to C.sub.10 heterocycloalkyl, aryl, C.sub.1 to C.sub.20 alkyl-aryl, heteroaryl and C.sub.1 to C.sub.20 alkyl-heteroaryl, C(O)R.sup.6, wherein R.sup.6 is selected from H, linear or branched C.sub.1 to C.sub.20 alkyl, linear or branched C.sub.1 to C.sub.20 heteroalkyl, C.sub.3 to C.sub.10 cycloalkyl, C.sub.3 to C.sub.10 heterocycloalkyl, aryl, C.sub.1 to C.sub.20 alkyl-aryl, heteroaryl and C.sub.1 to C.sub.20 alkyl-heteroaryl, C(O)OR.sup.7, wherein R.sup.7 is selected from H, linear or branched C.sub.1 to C.sub.20 alkyl, linear or branched C.sub.1 to C.sub.20 heteroalkyl, C.sub.3 to C.sub.10 cycloalkyl, C.sub.3 to C.sub.10 heterocycloalkyl, aryl, C.sub.1 to C.sub.20 alkyl-aryl, heteroaryl and C.sub.1 to C.sub.20 alkyl-heteroaryl, NR.sup.8R.sup.9, wherein R.sup.8 and R.sup.9 are independently selected from H, linear or branched C.sub.1 to C.sub.20 alkyl, linear or branched C.sub.1 to C.sub.20 heteroalkyl, C.sub.3 to C.sub.10 cycloalkyl, C.sub.3 to C.sub.10 heterocycloalkyl, aryl, C.sub.1 to C.sub.20 alkyl-aryl, heteroaryl and C.sub.1 to C.sub.20 alkyl-heteroaryl, (NH)CNHR.sup.10, wherein R10 is selected from H, linear or branched C.sub.1 to C.sub.20 alkyl, linear or branched C.sub.1 to C.sub.20 heteroalkyl, C.sub.3 to C.sub.10 cycloalkyl, C.sub.3 to C.sub.10 heterocycloalkyl, aryl, C.sub.1 to C.sub.20 alkyl-aryl, heteroaryl and C.sub.1 to C.sub.20 alkyl-heteroaryl and a protective group, in particular NO.sub.2, Pbf, Pmc, Mtr or Boc, NR.sup.11C(O)R.sup.12, wherein R.sup.11 and R.sup.12 are independently selected from H, linear or branched C.sub.1 to C.sub.20 alkyl, linear or branched C.sub.1 to C.sub.20 heteroalkyl, C.sub.3 to C.sub.10 cycloalkyl, C.sub.3 to C.sub.10 heterocycloalkyl, aryl, linear or branched C.sub.1 to C.sub.20 alkyl-aryl, heteroaryl, linear or branched C.sub.1 to C.sub.20 alkyl-heteroaryl, linear or branched C.sub.1 to C.sub.20 O-alkyl, linear or branched C.sub.1 to C.sub.20 O-alkyl-aryl, the radical C(O)R.sup.12 being in particular a protective group such as Boc, Cbz, Alloc or Fmoc, C(O)NR.sup.13R.sup.14, wherein R.sup.13 and R.sup.14 are independently selected from H, linear or branched C.sub.1 to C.sub.20 alkyl, linear or branched C.sub.1 to C.sub.20 heteroalkyl, C.sub.3 to C.sub.10 cycloalkyl, C.sub.3 to C.sub.10 heterocycloalkyl, aryl, C.sub.1 to C.sub.20 alkyl-aryl, heteroaryl and C.sub.1 to C.sub.20 alkyl-heteroaryl, SR.sup.15, wherein R.sup.15 is selected from H, linear or branched C.sub.1 to C.sub.20 alkyl, linear or branched C.sub.1 to C.sub.20 heteroalkyl, C.sub.3 to C.sub.10 cycloalkyl, C.sub.3 to C.sub.10 heterocycloalkyl, aryl, C.sub.1 to C.sub.20 alkyl-aryl, heteroaryl and C.sub.1 to C.sub.20 alkyl-heteroaryl and a protective group, in particular trityl or acetamidomethyl (Acm), a halogen, in particular selected from F, Cl, Br and I, said aryl, alkyl-aryl, heteroaryl and alkyl-heteroaryl may be substituted on the aromatic or heteroaromatic ring by one or more groups selected from: a linear or branched C.sub.1 to C.sub.20 alkyl, C.sub.3 to C.sub.10 cycloalkyl, C.sub.3 to C.sub.10 heterocycloalkyl, OR.sup.16, wherein R.sup.16 is selected from H, linear or branched C.sub.1 to C.sub.20 alkyl, linear or branched C.sub.1 to C.sub.20 heteroalkyl, C.sub.3 to C.sub.10 cycloalkyl, C.sub.3 to C.sub.10 heterocycloalkyl, aryl, C.sub.1 to C.sub.20 alkyl-aryl, heteroaryl and C.sub.1 to C.sub.20 alkyl-heteroaryl and a protective group, in particular selected from tBDMS, t-butyl, benzyl, trityl and xanthyl, OC(O)R.sup.17, wherein R.sup.17 is selected from H, linear or branched C.sub.1 to C.sub.20 alkyl, linear or branched C.sub.1 to C.sub.20 heteroalkyl, C.sub.3 to C.sub.10 cycloalkyl, C.sub.3 to C.sub.10 heterocycloalkyl, aryl, C.sub.1 to C.sub.20 alkyl-aryl, heteroaryl and C.sub.1 to C.sub.20 alkyl-heteroaryl, C(O)R.sup.18, wherein R.sup.18 is selected from H, linear or branched C.sub.1 to C.sub.20 alkyl, linear or branched C.sub.1 to C.sub.20 heteroalkyl, C.sub.3 to C.sub.10 cycloalkyl, C.sub.3 to C.sub.10 heterocycloalkyl, aryl, C.sub.1 to C.sub.20 alkyl-aryl, heteroaryl and C.sub.1 to C.sub.20 alkyl-heteroaryl, C(O)OR.sup.19, wherein R.sup.19 is selected from H, linear or branched C.sub.1 to C.sub.20 alkyl, linear or branched C.sub.1 to C.sub.20 heteroalkyl, C.sub.3 to C.sub.10 cycloalkyl, C.sub.3 to C.sub.10 heterocycloalkyl, aryl, C.sub.1 to C.sub.20 alkyl-aryl, heteroaryl and C.sub.1 to C.sub.20 alkyl-heteroaryl, NR.sup.20R.sup.21, wherein R.sup.20 and R.sup.21 are independently selected from H, linear or branched C.sub.1 to C.sub.20 alkyl, linear or branched C.sub.1 to C.sub.20 heteroalkyl, C.sub.3 to C.sub.10 cycloalkyl, C.sub.3 to C.sub.10 heterocycloalkyl, aryl, C.sub.1 to C.sub.20 alkyl-aryl, heteroaryl and C.sub.1 to C.sub.20 alkyl-heteroaryl, (NH)CNHR.sup.22, wherein R.sup.22 is selected from H, linear or branched C.sub.1 to C.sub.20 alkyl, linear or branched C.sub.1 to C.sub.20 heteroalkyl, C.sub.3 to C.sub.10 cycloalkyl, C.sub.3 to C.sub.10 heterocycloalkyl, aryl, C.sub.1 to C.sub.20 alkyl-aryl, heteroaryl and C.sub.1 to C.sub.20 alkyl-heteroaryl and a protective group, in particular NO.sub.2, Pbf, Pmc, Mtr or Boc, NR.sup.23C(O)R.sup.24, wherein R.sup.23 and R.sup.24 are independently selected from H, linear or branched C.sub.1 to C.sub.20 alkyl, linear or branched C.sub.1 to C.sub.20 heteroalkyl, C.sub.3 to C.sub.10 cycloalkyl, C.sub.3 to C.sub.10 heterocycloalkyl, aryl, linear or branched C.sub.1 to C.sub.20 alkyl-aryl, heteroaryl, linear or branched C.sub.1 to C.sub.20 alkyl-heteroaryl, linear or branched C.sub.1 to C.sub.20 O-alkyl, linear or branched C.sub.1 to C.sub.20 O-alkyl-aryl, the radical C(O)R.sup.24 being in particular a protective group such as Boc, Cbz, Alloc or Fmoc, C(O)NR.sup.25R.sup.26, wherein R.sup.25 and R.sup.26 are independently selected from H, linear or branched C.sub.1 to C.sub.20 alkyl, linear or branched C.sub.1 to C.sub.20 heteroalkyl, C.sub.3 to C.sub.10 cycloalkyl, C.sub.3 to C.sub.10 heterocycloalkyl, aryl, C.sub.1 to C.sub.20 alkyl-aryl, heteroaryl and C.sub.1 to C.sub.20 alkyl-heteroaryl, SR.sup.27, wherein R.sup.27 is selected from H, linear or branched C.sub.1 to C.sub.20 alkyl, linear or branched C.sub.1 to C.sub.20 heteroalkyl, C.sub.3 to C.sub.10 cycloalkyl, C.sub.3 to C.sub.10 heterocycloalkyl, aryl, C.sub.1 to C.sub.20 alkyl-aryl, heteroaryl and C.sub.1 to C.sub.20 alkyl-heteroaryl and a protective group, in particular trityl or acetamidomethyl (Acm), a halogen, in particular selected from F, Cl, Br and I, R.sup.1 and R.sup.2 may form a cycle, one of the groups R.sup.1 or R.sup.2 is in particular a hydrogen atom, R.sup.3 represents: a hydrogen atom, a linear or branched C.sub.1 to C.sub.20 alkyl group, R.sup.3 is in particular a hydrogen atom, said group R.sup.3 may form a cycle with R.sup.1 or R.sup.2, said compound of Formula 1 may be in the form of a solvate or a hydrate, said groups NR.sup.8R.sup.9, (NH)CNHR.sup.10, NR.sup.20R.sup.21 and (NH)CNHR.sup.22, heteroaryl, alkyl-heteroaryl and/or heterocycloalkyl may be in a salified form, and Formula 2-A wherein R.sup.1, R.sup.2 and R.sup.3 are as defined above for Formula 1-A, and wherein R.sup.33 represents a linear or branched C.sub.1 to C.sub.20 alkyl group, in particular a tert-butyl, when the compound of Formula 1-A, or the compound of Formula 2-A, comprises a carbon atom, said carbon atom may be .sup.13C, when the compound of Formula 1-A, or the compound of Formula 2-A, comprises a fluorine atom, said fluorine atom may be .sup.18F, when the compound of Formula 1-A, or the compound of Formula 2-A, comprises a hydrogen atom, said hydrogen atom may be deuterium, the asymmetric centers of said compound of Formula 1-A, and said compound of Formula 2-A, are of R or S configuration, or a mixture thereof.

18. The method according to claim 16, wherein the compound of Formula 1-A and the compound of Formula 2-A are such that: R.sup.1 and R.sup.2 independently represent: a hydrogen atom, a group chosen from: a linear or branched C.sub.1 to C.sub.10 alkyl, 3-methylindole, said alkyl may be substituted by one or more groups selected from: OR.sup.4, wherein R.sup.4 is a t-butyl group, (NH)CNHR.sup.10, wherein R.sup.10 is selected from a protective group, in particular NO.sub.2, Pbf, Pmc, Mtr or Boc, NR.sup.11C(O)R.sup.12, wherein R.sup.11 is H, and the radical C(O) R.sup.12 is a protective group such as Boc, Cbz, Alloc or Fmoc, SR.sup.15, wherein R.sup.15 is trityl or acetamidomethyl (Acm), R.sup.3 represents: a hydrogen atom, a linear or branched C.sub.1 to C.sub.20 alkyl group, said group R.sup.3 may form a cycle with R.sup.1 or R.sup.2, R.sup.33 represents a tert-butyl group, said compound of Formula 1-A may be in the form of a solvate or hydrate, said groups NR.sup.8R.sup.9, (NH)CNHR.sup.10, NR.sup.20R.sup.21 and (NH) CNHR.sup.22 and/or 3-methylindole, of the compound of Formula 1-A may be in a salified form, the asymmetric centers of said compound of Formula 1-A, and said compound of Formula 2-A, are of R or S configuration, or a mixture thereof.

19. The method according to claim 16, wherein the ?-amine acid compound is selected from: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic), 2-amino-2-methylpropanoic acid (Aib), and norleucine (Nle), said ?-amino-acid compound being N-protected on the ? amine function by a linear or branched C.sub.1 to C.sub.20C(O)O-alkyl substituent, in particular by a -tert-butyloxycarbonyl group, said amino acidsbeing optionally protected, on the side chain, on the carboxylic acid functions, amine functions, thiol functions, guanidine functions, amide functions and/or alcohol functions, by a protective group, in particular selected from tert-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz) fluorenylmethyloxycarbonyl (Fmoc), alloc, tert-butyloxy (OtBu), formyl (For), 2,2,4,6, 7-pentamethylhydrobenzofuran-5-sulfonyl (Pbf), 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc), 4-methoxy-2,3,6-trimethylbenzenesulfonyl (Mtr), trityl (Trt), trifluoroacetyl, acetamidomethyl (Acm), and xanthyl (Xan).

20. The method according to claim 16, wherein the compound NCA is selected from the following structures: ##STR00050## ##STR00051##

21. The method according to claim 16, wherein the step of contacting a N-protected ?-amino-acid compound with propane-phosphonic acid anhydride is in the presence of an organic base, at room temperature.

22. The method according to claim 21, wherein: said organic base being selected in particular from triethylamine, 1,8-diazabicyclo [5.4.0] undec-7-ene, diisopropylethylamine, N-dimethylaminopyridine, N-methylmorpholine or pyridine.

23. The method according to claim 16, wherein the step of contacting a N-protected ?-amino-acid compound with propane-phosphonic acid anhydride is in the absence of an organic base, at a temperature between 40 and 80? C.

24. The method according to claim 16, further comprising: after obtaining the NCA, a step of purification by at least one aqueous wash.

25. The method according to claim 16, further comprising: a step of purification of the NCA compound, wherein said ?-amino-acid compound is N-protected on the ? amine function by a linear or branched C.sub.1 to C.sub.20 C(O)O-alkyl substituent.

26. The method according to claim 16, wherein: said organic solvent is selected from ethyl acetate or dimethylformamide.

27. The method according to claim 16, wherein: the preparation of the NCA compound is done in the presence of an organic base in an amount of 0.25 to 3 molar equivalents relative to the N-protected ?-amino-acid compound.

28. The method according to claim 16, wherein: propane-phosphonic acid anhydride is used in an amount of 1 to 4 molar equivalents relative to the N-protected ?-amino-acid compound.

29. The method according to claim 16, wherein: the step of purification of the NCA compound, is a recrystallization step.

30. The method according to claim 16, wherein said ?-amino-acid compound is N-protected on the ? amine function by a -tert-butyloxycarbonyl group, said method being optionally implemented in continuous flow.

31. An NCA compound, of the following structure: ##STR00052##

32. A solution comprising an NCA compound according to claim 31, said solution being devoid of phosgene decomposition products, diphosgene decomposition products and triphosgene decomposition products, in particular devoid of hydrochloric acid.

33. A solution comprising an NCA compound prepared according to claim 16, said solution being devoid of phosgene decomposition products, diphosgene decomposition products and triphosgene decomposition products, in particular devoid of hydrochloric acid.

Description

[0387] FIG. 1 show the analytical data obtained for the compound (L) Trp (Boc)-NCA of Example 2.

[0388] FIG. 1A shows the NMR spectrum of the proton,

[0389] FIG. 1B shows the LC chromatogram, and

[0390] FIG. 1C shows the mass spectrum.

[0391] FIG. 2 show an analysis by chiral HPLC on diastereoisomers obtained according to Example 22.

[0392] FIG. 2A shows the LC chromatogram of the racemic mixture,

[0393] FIG. 2B shows the LC chromatogram of the diastereomer S, S, and

[0394] FIG. 2C shows the LC chromatogram of the diastereoisomer R,S.

EXAMPLES

[0395] Materials and Methods

[0396] Reagents and solvents were used as procured from commercial suppliers, and without further purification. The .sup.13C and .sup.1H NMR spectra in DMSO-d.sub.6 and CDCl.sub.3 were performed on a Bruker AVANCE 600 MHz spectrometer, equipped with a BBFO helium cryoprobe at 298 K. Chemical shifts (?) are reported in parts per million using non-deuterated residual solvents as internal references. The spectra were processed and visualized with Topspin 3.2 (Bruker Biospin).

[0397] LC/MS analyses were performed using a 25?4.6 mm C18 Chromolith Flash inverted phase column. A flow rate of 3 ml/min and a gradient of (0 to 100%) of B over 2.5 min were used. Voting agent A: water/0.1% HCO.sub.2H; eluent B: acetonitrile/0.1% HCO.sub.2H. UV detection was performed at 214 nm. The electrospray mass spectra were acquired at a solvent rate of 200 ?L/min. Nitrogen was used for both nebulizing and drying gas. Data were obtained in a scanning mode ranging from 100 to 1000 m/z or from 250 to 1500 m/z at intervals of 0.7 s.

Example 1: General Procedure for the Synthesis of NCA Compoundsin the Presence of Pyridine

[0398] Boc-AA-OH (1 eq) was solubilized in ethyl acetate or dimethylformamide. Pyridine (3 eq) was added, followed by T3P?/AcOEt 50% (2 eq), drip. The reaction mixture was stirred at room temperature for 2 hours, and the progress of the reaction was analyzed by HPLC and LC/MS.

[0399] The reaction mixture was diluted in ethyl acetate and washed twice with cold water and then with an aqueous solution saturated with NaCl. The organic phase was dried on anhydrous magnesium sulphate, filtered and concentrated under vacuum. The expected product was purified by recrystallization.

[0400] The compounds of Examples 2 to 18 and 24 to 34 below were prepared according to this general procedure, using ethyl acetate as reaction solvent and on a scale of 1 gram of starting amino acid.

Example 2: Preparation of (L)Trp(Boc)-NCA

[0401] ##STR00018##

[0402] Yield 81%

[0403] White solid, mp 152-153? C., [?]20.sub.D (c 1.00, CH.sub.2Cl.sub.2)=?49.1?, .sup.1H NMR (600 MHz, CDCl.sub.3) ?=8.04 (brd, J=8 Hz, 1H), 7.43-7.37 (m, 2H), 7.29-7.22 (m, 1H), 7.20-7.13 (m, 1H), 6.36 (brs, 1H), 4.50-4.44 (dd, J=3.64, 8.64 Hz, 1H), 3.33-3.25 (dd, J=3.64, 14.69 Hz, 1H), 3.03-3.93 (dd, J=8.64, 14.69 Hz, 1H), 1.57 (s, 9H), .sup.13C NMR (125 MHz, CDCl.sub.3) ?=168.8, 152.0, 149.4, 135.5, 129.3, 125.0, 124.8, 122.9, 118.4, 115.6, 113.1, 84.3, 57.6, 28.1, 27.7, LC/MS (ESI.sup.+) t.sub.R1.81 min, m/z 331 [M+H]+ m/z 231 [M+H?Boc].sup.+.

Example 3: Preparation of (L)Val-NCA

[0404] ##STR00019##

[0405] Recrystallized in an AcOEt/hexane mixture

[0406] Yield 78%

[0407] White solid, mp 62-63? C., .sup.1H NMR (600 MHz, CDCl.sub.3) ?=6.95 (brs, 1H), 4.28-4.21 (d, J=4.20 Hz, 1H), 2.34-2.20 (m, 1H), 1.14-1.08 (d, J=6.97 Hz, 3H), 1.07 (d, J=6.97 Hz, 3H), .sup.13C NMR (125 MHz, CDCl.sub.3) ?=168.7, 153.4, 63.1, 30.7, 18.2, 16.6, LC/MS (ESI+) t R 1.68 min, m/z 144 [M+H].sup.+.

Example 4: Preparation of (L)Tyr(OtBu)-NCA

[0408] ##STR00020##

[0409] Yield 90%

[0410] White solid, mp 108-109? C., [?]19.sub.D (c 1.00, CH.sub.2Cl.sub.2)=?78.1?, .sup.1H NMR (600 MHz, CDCl.sub.3) ?=7.00-6.96 (d, J=8.44 Hz, 2H), 6.89-6.86 (d, J=8.44 Hz, 2H), 5.67-5.55 (m, 1H), 4.42-4.37 (ddd, J=0.83, 4.06, 8.86 Hz, 1H), 3.18-3.13 (dd, J=4.06, 14.15 Hz, 1H), 2.86-2.80 (dd, J=8.86, 14.15, 1H), 1.24 (s, 9H), .sup.13C NMR (125 MHz, CDCl.sub.3) ?=168.5, 155.3, 151.3, 129.6, 128.5, 124.7, 78.8, 58.8, 37.3, 28.8, LC t.sub.R 1.53 min.

Example 5: Preparation of (L)Phe-NCA

[0411] ##STR00021##

[0412] Recrystallized in an AcOEt/hexane mixture

[0413] Yield 87%

[0414] White solid, mp 88-89? C., [?]18 D (c 1.00, CH.sub.2Cl.sub.2)=?94.6, .sup.1H NMR (600 MHz, CDCl.sub.3) ?=7.35-7.25 (m, 3H), 7.18-7.12 (m, 2H), 6.45-6.09 (m, 1H), 4.53-4.47 (dd, J=4.21, 8.24 Hz, 1H), 3.27-3.19 (m, 1H), 3.02-2.94 (dd, J=8.24, 14.29, 1H). .sup.13C NMR (125 MHz, CDCl.sub.3) ?=168.6, 151.8, 133.8, 129.2, 128.0, 58.8, 37.8, LC t.sub.R 2.25 min.

Example 6: Preparation of (L)Asp(OBzl)-NCA

[0415] ##STR00022##

[0416] Recrystallized in an AcOEt/hexane mixture

[0417] Yield 87%

[0418] White solid, mp 103-104? C., [?]19 D (c 1.00, CH2Cl2)=?42.3?, .sup.1H NMR (600 MHz, DMSO-d.sub.6) ?=8.99 (s, 1H), 7.40-7.30 (m, 5H), 5.13 (s, 2H), 4.70-4.68 (t, J=4.57 Hz, 1H), 3.10-3.04 (dd, J=5.05, 17.86 Hz, 1H), 2.93-2.87 (dd, J=4.35, 17.86 Hz, 1H), .sup.13C NMR (125 MHz, DMSO-d.sub.6) ?=171.4, 169.7, 152.6, 136.0, 128.9, 128.6, 128.5, 66.7, 54.0, 35.1, LC t.sub.R 2.55 min.

Example 7: Preparation of (L)Thr(OBzl)-NCA

[0419] ##STR00023##

[0420] Recrystallized in an AcOEt/hexane mixture

[0421] Yield 81%

[0422] White solid, mp 120-121? C., .sup.1H NMR (600 MHz, CDCl.sub.3) ?=7.27-7.14 (m, 5H), 6.41 (brd, J=9.86 Hz, 1H), 4.53-4.50 (d, J=11.53 Hz, 1H), 4.35-4.32 (d, J=11.53 Hz, 1H), 4.09-4.06 (d, J=4.97 Hz, 1H), 3.84-3.78 (m, 1H), 1.25-1.23 (d, J=6.27 Hz, 3H), .sup.13C NMR (125 MHz, CDCl.sub.3) ?=167.5, 152.5, 152.5, 136.9, 128.5, 128.1, 127.9, 127.8, 73.1, 71.3, 62.7, 15.9, LC t.sub.R 1.40 min.

Example 8: Preparation of (L)Gln(Xan)-NCA

[0423] ##STR00024##

[0424] Yield 80%

[0425] White solid, mp 156-157? C., .sup.1H NMR (600 MHz, DMSO-d.sub.6) ?=9.08 (s, 1H), 8.90-8.85 (d, J=8.80 Hz, 1H), 7.39-7.35 (m, 2H), 7.35-7.31 (m, 2H), 7.17-7.11 (m, 4H), 6.30-6.26 (d, J=8.81 Hz, 1H), 4.55-4.50 (t, J=5.92 Hz, 1H), 2.34-2.22 (m, 2H), 2.11-2.02 (m, 1H), 2.02-1.93 (m, 1H), .sup.13C NMR (125 MHz, DMSO-d.sub.6) ?=171.9, 170.8, 152.3, 150.9, 129.6, 129.5, 129.4, 123.9, 122.2, 122.2, 116.5, 56.9, 42.8, 30.5, 27.3, LC t.sub.R 1.57 min.

Example 9: Preparation of (L)Ala-NCA

[0426] ##STR00025##

[0427] Recrystallized in an AcOEt/hexane mixture

[0428] Yield 76%

[0429] White solid, mp 84-85? C., .sup.1H NMR (600 MHz, CDCl.sub.3) ?=6.41 (m, 1H), 4.33-4.26 (dd, J=7.07, 14.06 Hz, 1H), 1.47-1.45 (d, J=7.07 Hz, 3H), .sup.13C NMR (125 MHz, CDCl.sub.3) ?=170.0, 152.0, 53.3, 17.7.

Example 10: Preparation of (L)Ser(OBzl)-NCA

[0430] ##STR00026##

[0431] Recrystallized in an AcOEt/hexane mixture

[0432] Yield 77%

[0433] White solid, mp 65-66? C., .sup.1H NMR (600 MHz, DMSO-d.sub.6) ?=9.10 (s, 1H), 7.40-7.17 (m, 6H), 4.66 (s, 1H), 4.59-4.48 (m, 2H), 3.81-3.71 (d, J=11.09 Hz, 1H), 3.67-3.60 (d, J=11.09 Hz, 1H), .sup.13C NMR (125 MHz, DMSO-d.sub.6) ?=170.4, 152.7, 138.0, 128.8, 128.1, 127.8, 72.9, 68.1, 58.8, LC t.sub.R 1.36 min.

Example 11: Preparation of (L) Glu(OBzl)-NCA

[0434] ##STR00027##

[0435] Recrystallized in an AcOEt/hexane mixture

[0436] Yield 78%

[0437] White solid, mp 110-111? C., [?]19.sub.D (c 1.00, CH2Cl2)=?13.6?, .sup.1H NMR (600 MHz, DMSO-d.sub.6) ?=9.08 (s, 1H), 7.40-7.29 (m, 5H), 5.10 (s, 2H), 4.49-4.44 (m, 1H), 2.54-2.48 (m, 2H), 2.10-2.00 (m, 1H), 1.99-1.89 (m, 1H), .sup.13C NMR (125 MHz, DMSO-d.sub.6) ?=172.1, 171.7, 152.3, 136.4, 128.9, 128.5, 128.4, 128.4, 66.1, 56.6, 29.5, 26.8, LC t.sub.R 1.48 min.

Example 12: Preparation of (L)Cys(Trt)-NCA

[0438] ##STR00028##

[0439] Yield 82%

[0440] White solid, mp 122-123? C., .sup.1H NMR (600 MHz, DMSO-d.sub.6) ?=9.16 (s, 1H), 7.39-7.23 (m, 18H), 4.42-4.39 (t, J=5.16 Hz, 1H), 2.53-2.50 (dd, J=2.37, 5.16 Hz, 2H), .sup.13C NMR (125 MHz, DMSO-d.sub.6) ?=170.3, 152.0, 144.2, 129.4, 128.6, 127.4, 66.7, 56.8, 32.9, LC t.sub.R 2.00 min.

Example 13: Preparation of (L)Lys (Boc)-NCA

[0441] ##STR00029##

[0442] Recrystallized in an AcOEt/hexane mixture

[0443] Yield 85%

[0444] White solid, mp 142-143? C., [?]19.sub.D (DCM) [c=1]=?37.0?, .sup.1H NMR (600 MHz, DMSO-d.sub.6) ?=9.05 (s, 1H), 6.81-6.72 (m, 1H), 4.46-4.38 (t, J=6.14 Hz, 1H), 2.94-2.85 (dd, J=6.14, 12.63 Hz, 2H), 1.76-1.68 (m, 1H), 1.67-1.59 (m, 1H), 1.42-1.30 (m, 12H), 1.30-1.21 (m, 1H), .sup.13C NMR (125 MHz, DMSO-d.sub.6) ?=172.1, 156.0, 152.4, 77.8, 57.4, 31.1, 29.3, 28.7, 22.0, LC t.sub.R 1.39 min.

Example 14: Preparation of (L)Tic-NCA

[0445] ##STR00030##

[0446] Recrystallized in an AcOEt/hexane mixture

[0447] Yield 76%

[0448] White solid, mp 166-167? C., .sup.1H NMR (600 MHz, DMSO-d.sub.6) ?=7.34-7.18 (m, 4H), 4.81-4.74 (d, J=16.60 Hz, 1H), 4.64-4.60 (dd, J=5.13, 11.62 Hz, 1H), 4.49-4.43 (d, J=16.60 Hz, 1H), 3.25-3.18 (dd, J=11.62, 15.31 Hz, 1H), 3.18-3.12 (dd, J=5.13, 15.31 Hz, 1H), .sup.13C NMR (125 MHz, DMSO-d.sub.6) ?=170.1, 151.0, 131.6, 130.8, 129.8, 127.3, 127.3, 127.0, 54.6, 42.3, 29.2, LC t.sub.R 1.39 min.

Example 15: Preparation of (L)Leu-NCA

[0449] ##STR00031##

[0450] Recrystallized in an AcOEt/hexane mixture

[0451] Yield 86%

[0452] White solid, mp 66-67? C., .sup.1H NMR (600 MHz, DMSO-d.sub.6) ?=9.11 (s, 1H), 4.49-4.41 (dd, J=5.45, 8.77 Hz, 1H), 1.80-1.67 (m, 1H), 1.63-1.51 (m, 2H), 0.94-0.83 (t, J=7.78 Hz, 6H), .sup.13C NMR (125 MHz, DMSO-d.sub.6) ?=172.4, 152.4, 56.0, 40.5, 24.5, 23.1, 21.7, LC t.sub.R 1.18 min.

Example 16: Preparation of (L)Lys (Fmoc)-NCA

[0453] ##STR00032##

[0454] Recrystallized in an AcOEt/hexane mixture

[0455] Yield 88%

[0456] White solid, mp 101-102? C., [?]20.sub.D=?41.6 (c 1.00, CH.sub.2Cl.sub.2), .sup.1H NMR (600 MHz, DMSO-d.sub.6) ?=9.07 (s, 1H), 7.88-7.87 (d, J=7.52 Hz, 2H), 7.68-7.67 (d, J=7?.53, 2H), 7.42-7.39 (dd, J=7.53, 14.85 Hz, 2H), 7.34-7.31 (dd, J=7.53, 14.85 Hz, 2H), 7.27 (m, 1H), 4.43 (m, 1H), 4.30 (m, 2H), 4.22 (m, 1H), 2.98 (m, 2H), 1.73 (m, 1H), 1.66 (m, 1H), 1.49-1.25 (m, 6H), .sup.13C NMR (125 MHz, DMSO-d.sub.6) ?=172.1, 156.5, 152.4, 144.4, 141.2, 128.0, 127.5, 125.5, 120.5, 65.6, 57.4, 47.2, 31.1, 29.2, 22.0, LC t.sub.R 1.57 min.

Example 17: Preparation of (L)Arg(Pbf)-NCA

[0457] ##STR00033##

[0458] Recrystallized in an AcOEt/hexane mixture

[0459] Yield 86%

[0460] White solid, mp 122-123? C., [?].sup.19.sub.D (c 1.00, CH.sub.2Cl.sub.2)=?17.3?, .sup.1H NMR (600 MHz, CDCl.sub.3) ?=7.71 (s, 1H), 6.28 (s, 3H), 4.28 (m, 1H), 3.64 (m, 1H), 2.84 (s, 2H), 2.42 (s, 3H), 2.35 (s, 3H), 1.97 (s, 3H), 1.84 (m, 1H), 1.75 (m, 2H), 1.65-1.45 (m, 3H), 1.35 (s, 9H), .sup.13C NMR (125 MHz, CDCl.sub.3) ?=170.5, 159.0, 156.4, 152.6, 138.2, 132.2, 124.9, 117.7, 86.6, 67.9, 57.2, 43.1, 28.5, LC t.sub.R 1.56 min.

Example 18: Preparation of (D)-Asp(OBzl)-NCA

[0461] Yield 79%

[0462] [?].sup.19.sub.D (c 1.00, CH.sub.2Cl.sub.2)+42.6?.

Example 19: Setting Up an Operating Unit

[0463] (L)Leu-NCA from 10 g Boc-(L)Leu-OH

[0464] Boc-(L)leu-OH (10 g) was solubilized in ethyl acetate (300 ml). T3P? (2 equivalents), dissolved in ethyl acetate was added dropwise followed by pyridine (3 equivalents). The reaction mixture was stirred for 1 hour at room temperature. Water/ice (300 ml) was added, and the organic phase was recovered, washed 2 times with a cooled NaCl saturated aqueous solution (2?300 ml), dried over MgSO.sub.4, filtered and concentrated under vacuum. Hexane (50 ml) was added to the oily residue, resulting in crystallization. The expected product was recrystallized in an AcOEt/hexane mixture, to obtain (L) Leu-NCA (5.9 g), which was stored under argon at ?20? C.

[0465] Yield 86%.

[0466] (L)Phe-NCA from 20 g of Boc-(L)Phe-OH

[0467] Boc-(L)Phe-OH (20 g) was solubilized in ethyl acetate (1500 ml). T3P? (2 equivalents), dissolved in ethyl acetate was added dropwise followed by pyridine (3 equivalents). The reaction mixture was stirred for 1 hour at room temperature. Water/ice (1000 ml) was added, and the organic phase was recovered, washed 2 times with a cooled NaCl-saturated aqueous solution (2?500 ml), dried over MgSO.sub.4, filtered and concentrated under vacuum. Hexane (100 ml) was added to the oily residue, resulting in crystallization. The expected product was recrystallized in an AcOEt/hexane mixture, to obtain (L) Phe-NCA (11.4 g), which was stored under argon at ?20? C. Yield 79%.

[0468] (L)Glu(OBzl)-NCA from 25 g of Boc-(L)Glu(OBzl)-OH

[0469] Boc-(L)Glu(OBzl)-OH (25 g) was solubilized in ethyl acetate (1500 ml). T3P? (2 equivalents), dissolved in ethyl acetate was added dropwise followed by pyridine (3 equivalents). The reaction mixture was stirred for 1 hour at room temperature. Water/ice (1000 ml) was added, and the organic phase was recovered, washed 2 times with a cooled NaCl-saturated aqueous solution (2?500 ml), dried over MgSO.sub.4, filtered and concentrated under vacuum. Hexane (100 ml) was added to the oily residue, resulting in crystallization. The expected product was recrystallized in an AcOEt/hexane mixture, to obtain (L) Glu (OBzl)-NCA (15 g), which was stored under argon at ?20? C. Yield 79%.

[0470] (L)Ala-NCA from 50 g Boc-(L)Ala-OH

[0471] Boc-(L)Ala-OH (50 g) was solubilized in ethyl acetate (1500 ml). T3P? (2 equivalents), dissolved in ethyl acetate was added dropwise followed by pyridine (3 equivalents). The reaction mixture was stirred for 1 hour at room temperature. Water/ice (1000 ml) was added, and the organic phase was recovered, washed 2 times with a cooled NaCl-saturated aqueous solution (2?500 ml), dried over MgSO.sub.4, filtered and concentrated under vacuum. Hexane (100 ml) was added to the oily residue, resulting in crystallization. The expected product was recrystallized in an AcOEt/hexane mixture, to obtain (L) Ala-NCA (22 g), which was stored under argon at ?20? C. Yield 73%.

[0472] (L)Lys-NCA from 10 g Boc-(L)Lys-OH

[0473] Boc-(L)Lys-(Boc)-OH (10 g) was solubilized in ethyl acetate (300 ml). T3P? (2 equivalents), dissolved in ethyl acetate was added dropwise followed by pyridine (3 equivalents). The reaction mixture was stirred for 1 hour at room temperature. Water/ice (300 ml) was added, and the organic phase was recovered, washed 2 times with a cooled NaCl saturated aqueous solution (2?300 ml), dried over MgSO.sub.4, filtered and concentrated under vacuum. Hexane (50 ml) was added to the oily residue, resulting in crystallization. The expected product was recrystallized in an AcOEt/hexane mixture, to obtain (L)Lys-(Boc)-NCA (6.3 g), which was stored under argon at ?20? C. Yield 80%.

Example 20: Preparation of (L)Tyr(OtBu)-NCAUse of Different Bases

[0474] ##STR00034##

[0475] The general procedure of Example 1 was used, replacing pyridine with bases Et.sub.3N, iPr.sub.2EtN or DBU. After 2 hours of stirring at room temperature in ethyl acetate, the expected NCA product was formed in a majority manner, as analyzed by LC.

TABLE-US-00001 Test Base Yield (%) 1 Et.sub.3N 69 2 iPr.sub.2EtN 79 3 DBU 63

Example 21: Preparation of NCA-(L)-Arg(Pbf)in Absence of Base

[0476] ##STR00035##

[0477] Boc-(L)-Arg(Pbf)-OH (1 eq) was solubilized in ethyl acetate or dimethylformamide. T3P?/AcOEt 50% (4 eq) was added drop by drop. The reaction mixture was stirred at 70? C. for 2 hours, and the progress of the reaction was analyzed by HPLC and LC/MS. The reaction mixture was diluted in ethyl acetate and washed twice with cold water and then with an aqueous solution saturated with NaCl. The organic phase was dried on anhydrous magnesium sulphate, filtered and concentrated under vacuum. The expected product was purified by recrystallization. LC t.sub.R 1.58 min.

Example 22: Study of Stereochemistry

[0478] This test aims to confirm that the method according to the present invention is not racemic and isolates NCA compounds with retention of enantiomeric excess. The compounds (L) Asp (OBzl)-NCA and (D) Asp (OBzl)-NCA were prepared according to the procedure of Example 1, from Boc-(L) Asp (OBzl) OH and Boc (D) Asp (OBzl) OH respectively. Dimethylformamide was used as a reaction solvent.

[0479] The NCA compounds thus obtained were then reacted with (S)-1-(4-methoxyphenyl) ethane-1-amine to obtain diastereoisomers S, S and R, S.

[0480] An analysis by HPLC (Colone Zorbax SB-C18 50*2.1 mm 1.8 ?m; flow rate 0.5 mL/min with a gradient (0-100%) B over 25 min; eluent A: water/0.1% HCO.sub.2H; eluent B: acetonitrile/0.1% HCO.sub.2H. UV detection was performed at 214 nm).

##STR00036##

[0481] FIG. 2 shows the chromatograms thus obtained. Only one peak was observed in both cases (FIGS. 2B and 2C), confirming the retention of stereochemistry during the process. This result was also confirmed by .sup.1H NMR analysis of S,S and R,S diastereomers.

Example 23: Comparative ExampleDCC Versus T3P?

[0482] ##STR00037##

TABLE-US-00002 Test Conditions Outcome (assessed by HPLC) 1 DCC/Pyridine, 30 minutes NCA traces * predominant starting amino acid 2 DCC, 30 minutes NCA traces predominant starting amino acid 3 T.sub.3P?/Pyridine, predominant NCA 30 minutes Traces of starting amino acid * traces of NCA means <3% conversion.

[0483] In addition, the coupling agents HATU (2 equivalents) and BOP (2 equivalents) were also tested, in the presence of pyridine, with the same results as those obtained in the case of DCC, i.e. only traces of NCA were observed.

Example 24: Preparation of Gly-NCA

[0484] ##STR00038##

[0485] Recrystallized in an AcOEt/hexane mixture

[0486] Yield 80%

[0487] White solid, mp>200? C.

Example 25: Preparation of (D)Ala-NCA

[0488] ##STR00039##

[0489] Recrystallized in an AcOEt/hexane mixture

[0490] Yield 80%

[0491] White solid, mp 112-113? C., [?].sup.20.sub.D=?4 (c=1.00, CH.sub.2Cl.sub.2).

Example 26: Preparation of (L)Ile-NCA

[0492] ##STR00040##

[0493] Recrystallized in an AcOEt/hexane mixture

[0494] Yield 86%

[0495] White solid, mp 66-68? C., [?]20.sub.D=?29 (c=1.00, CH.sub.2Cl.sub.2).

Example 27: Preparation of (L)Lys (Tfa)-NCA

[0496] ##STR00041##

[0497] Recrystallized in an AcOEt/hexane mixture

[0498] Yield 84%

[0499] White solid, mp 92-93? C., [?].sup.20.sub.D=?31 (c=1.00, CH.sub.2Cl.sub.2).

Example 28: Preparation of (L)Cys (S-Bzl)-NCA

[0500] ##STR00042##

[0501] Recrystallized in an AcOEt/hexane mixture

[0502] Yield 91%

[0503] White solid, mp 100-102? C., [?]20.sub.D=?60 (c=1.00, CH.sub.2Cl.sub.2).

Example 29: Preparation of N-Me(L)Ala-NCA

[0504] ##STR00043##

[0505] Recrystallized in an Ether/hexane mixture

[0506] Yield 87%

[0507] White solid, mp 131-132? C., [?]20.sub.D=+10 (c=1.00, CH2Cl2), .sup.1H NMR (600 MHz, DMSO-d.sub.6) ? 4.42-4.37 (dd, J=7.13, 14.06 Hz, 1H), 2.84 (s, 3H), 1.39 (d, J=7.13 Hz); .sup.13C NMR (100 MHz, DMSO-d.sub.6): ? 171.2, 152.1, 57.2, 28.4, 14.5.

Example 30: Preparation of N-Benzyl-Gly-NCA

[0508] ##STR00044##

[0509] Recrystallized in an Ether/hexane mixture

[0510] Yield 82%

[0511] White solid, mp 152-153? C., .sup.1H NMR (600 MHz, DMSO-d.sub.6) ? 7.37 (m, 5H), 4.50 (s, 2H), 4.17 (s, 2H); .sup.13C NMR (100 MHz, DMSO-d.sub.6): ? 167.5, 153.1, 135.7, 129.1, 128.2, 128.2, 50.0, 47.1.

Example 31: Preparation of (L)Pro-NCA

[0512] ##STR00045##

[0513] Recrystallized in an AcOEt/hexane mixture

[0514] Yield 85%

[0515] White solid, mp 45-47? C., [?].sup.20.sub.D=?99 (c=1.00, CH.sub.2Cl.sub.2).

Example 32: Preparation of (D)Asp(OBzl)-NCA

[0516] ##STR00046##

[0517] Recrystallized in an AcOEt/hexane mixture

[0518] Yield 85%

[0519] White solid, mp 123-124? C., [?]19.sub.D=+42.3 (c=1.00, CH2 Cl2) .sup.1H NMR (600 MHz, DMSO-d.sub.6) ? 8.99 (s, 1H), 7.41-7.34 (m, 5H), 5.13 (s, 2H), 4.71-4.69 (t, J=4.57 Hz, 1H), 3.11-3.05 (dd, J=4.86, 17.87 Hz, 1H), 2.93-2.87 (dd, J=4.86, 17.87 Hz, 1H); .sup.13C NMR (100 MHz, DMSO-d.sub.6): ? 171.4, 169.7, 152.6, 136.0, 128.9, 128.6, 128.5, 66.7, 54.0, 35.1.

Example 33: Preparation of Aib-NCA

[0520] ##STR00047##

[0521] Recrystallized in an AcOEt/hexane mixture

[0522] Yield 82%

[0523] White solid, mp 72-73? C., .sup.1H NMR (400 MHz, DMSO-d.sub.6): ? 9.07 (s, 1H), 1.41 (s, 6H); .sup.13C NMR (100 MHz, DMSO-d.sub.6): 175.0, 150.8, 59.6, 25.0.

Example 34: Preparation of (L)Asn(Xan)-NCA

[0524] ##STR00048##

[0525] Recrystallized in an AcOEt/hexane mixture

[0526] Yield 89%

[0527] White solid, mp 145-146? C., [?]20.sub.D=?55.6 (c=1.00, dioxane), .sup.1H NMR (400 MHz, DMSO-d.sub.6): ? 9.08-9.06 (d, J=8.80 Hz, 1H), 8.97 (s, 1H), 7.39-7.33 (m, 4H), 7.18-7.14 (m, 4H), 6.30-6.28 (d, J=8.80 Hz, 1H), 4.66-4.64 (t, J=4.46 Hz, 1H), 2.86-2.81 (dd, J=4.33, 16.82 Hz, .sup.2H) 2.74-2.69 (dd, J=4.33, 16.82 Hz, 2H); 13C NMR (100 MHz, DMSO-d.sub.6): ? 172.1, 167.9, 152.9, 150.9, 129.6, 124.0, 121.9, 121.8, 116.6, 54.3, 43.1, 36.2.