The invention relates to a novel method for synthesizing NCA compounds. It also concerns a new use of a peptide coupling agent.
N-carboxyanhydride compounds, or NCAs, are chemical derivatives with high added value industrially used in particular in the production of drugs or polymers. For example, NCAs are intermediates of choice for peptide synthesis under “solvent-free” ecological conditions.
The synthesis of NCA compounds is traditionally carried out with phosgene or a phosgene derivative. However, the use of these reagents is problematic and requires special precautions and facilities, particularly because of their toxicity and dangerousness. In addition, this strategy leads to the concomitant formation of HCl, which is difficult to remove from the NCA product, especially during a large-scale process. However, the presence of residual HCl in the NCA product may give rise to parasitic reactions during its use, for example in a polymerization process.
Some alternative syntheses exist, making it possible to avoid phosgene. Among these methods is the use of carbonyldiimidazole, or diphenyl carbonate. These methods are not industrially applicable, as the presence of secondary products, imidazole and phenol respectively, complicates the purification of the synthesized NCA product. Another method, involving a nitrosation reaction, leads to the concomitant formation of nitric oxide, a toxic compound.
There is therefore a real need for alternatives for the preparation of NCAs compounds, in particular environmentally friendly processes, and to obtain products with a good yield and excellent chemical purity.
One of the objects of the invention is to use the propylphosphonic anhydride reagent in the preparation of NCA compounds.
One of the objects of the invention is to provide a new method for synthesizing NCA compounds, without the concomitant formation of HCl.
Another object of the invention is to provide a method for synthesizing NCA compounds, in the absence of phosgene, or one of its derivatives.
One of the objects of the invention is to provide NCA compounds with good chemical and stereochemical purity.
Another object of the invention is to provide new NCA compounds.
Another object of the invention is to be able to use NCA compounds thanks to their improved chemical purity, in particular as synthetic intermediates in the preparation of a polymer or a UNCA compound.
A first object of the present invention is the use of propane-phosphonic acid anhydride for the preparation of a NCA compound, from a α-amino acid compound N-protected on the α amine function by a linear or branched C1 to C20—C(O)—O-alkyl substituent, in particular by a tert-butyloxycarbonyl group.
The inventors surprisingly found that α-amino acids can be transformed into NCA compounds, by the action of propylphosphonic anhydride. This transformation thus makes it possible to obtain said NCA compounds with good yields, and excellent chemical and stereoisomeric purities.
The transformation is all the more surprising since other agents conventionally used in peptide chemistry, such as DCC, BOP or HATU, do not lead, or very little, to the formation of NCAs.
By “propane-phosphonic acid anhydride” means a molecule whose structure is shown below. It is an anhydride of propylphosphonic acid, which is in the form of a cyclic trimer on which propyl groups are bonded to phosphorus atoms.
The reagent is commercially available, under the name T3P®, in solution at 50 w/w % in several solvents, such as ethyl acetate, dimethylformamide, toluene, or tetrahydrofuran.
By “linear C1 to C20 alkyl” means an alkyl group comprising from 1 to 20 carbon atoms, selected from: methyl C1, ethyl C2, propyl C3, butyl C4, pentyl C5, hexyl C6, heptyl C7, octyl C8, nonyl C9, decyl C10, decyl C11, dodecyl C12, tridecyl C13, tetradecyl C14, pentadecyl C15, hexadecyl C16, heptadencyle C17, octadecyl C18, nonadecyl C19, eicosyl C20, in particular a linear C1 to C10 alkyl group, in particular a linear C1 to C5 alkyl group.
By “branched alkyl” is meant a linear alkyl group as defined above comprising substituents selected from the linear alkyl groups defined above, said linear alkyl groups being also capable of branching. Among the branched alkyl groups include an iso-propyl, sec-butyl, iso-butyl, tert-butyl, sec-pentyl, iso-pentyl, iso-hexyl, iso-heptyl, iso-octyl, iso-nonyl and iso-decyl.
The compounds “NCAs”, or N-CarboxyAnhydrides of α-amino acids, have compounds comprising the unit shown below. These compounds may carry one or more substituents on the carbon α the carbonyl function, as well as on the nitrogen atom.
In the context of the present invention, these NCA compounds are prepared from a N-protected-α-amino acid compound comprising a unit shown below, and wherein R represents an alkyl group C1 to C20, linear or branched.
The group R is preferably a tert-butyl group, giving rise to a N-protected α-amino-acid the α amine function of which is protected by a Boc group.
In the case where the NCA compound carries a substituent other than a hydrogen atom on the nitrogen, this substituent is also present on the corresponding α-amino-acid molecules.
The carboxylic acid function present in the aforementioned N-protected α-amino-acid compound is optionally in the form of carboxylate, especially in the form of a sodium salt, a potassium salt, or a lithium salt.
A specific class of NCA compounds are UNCAs, or N-Urethane-CarboxyAnhydrides of α-amino acids. These compounds comprise, on the nitrogen atom of the NCA ring, a urethane group. UNCA compounds, like other NCAs compounds, may include additional substituents on the carbon of the NCA ring.
The present invention also makes it possible to obtain this specific class of NCA compounds. In this case, it is advisable to use a double-N-protected α-amino acid on the amine function in α, in the form of dicarbamate. Thus, a N-(Boc)2-α-amino-acid, N-(Boc)(Cbz) or N-(Boc)(Fmoc) can for example be used.
According to a particular embodiment, the present invention relates to a use as defined above, wherein the preparation of the NCA compound is made:
The preparation of a compound NCA, from a N-protected α-amino-acid compound, according to the present invention is preferably carried out in the presence of an organic base, said base being able to promote the reaction. However, the reaction also takes place in the absence of an organic base, but it is slower. In this case, it would be necessary to heat the reaction medium, in order to increase the kinetics of the reaction, and to obtain reaction times compatible with an industrial process.
In the case of the presence of an organic base, the reaction takes place at “room temperature”, i.e. at a temperature between 20 and 30° C., in particular about 25° C.
According to another particular embodiment, the present invention relates to a use as defined above, wherein the organic base is a nitrogenous base, in particular selected from triethylamine, 1,8-diazabicyclo [5.4.0] undec-7-ene, diisopropylethylamine, N-dimethylaminopyridine, N-methylmorpholine or pyridine, preferably pyridine.
According to another particular embodiment, the present invention relates to a use as defined above, wherein the preparation of the compound NCA is done:
According to another particular embodiment, the present invention relates to a use as defined above, wherein the preparation of the NCA compound is done in the presence of an organic solvent.
Among the organic solvents used in the preparation of a compound NCA, according to the present invention, mention may be made, but not limited to, ethyl acetate, butyl acetate, tetrahydrofuran, 2-methyltetrahydrofuran, toluene, N, N-dimethylformamide, chlorobenzene, methylene chloride or acetonitrile.
The organic solvent is in particular selected according to the commercial availability of the propane-phosphonic acid anhydride reagent, which is in particular, marketed in 50% solution in ethyl acetate, or dimethylformamide.
According to another particular embodiment, the present invention therefore relates to a use as defined above, wherein the organic solvent is selected from ethyl acetate or dimethylformamide, in particular ethyl acetate.
According to another particular embodiment, the present invention relates to a use as defined above, wherein the propane-phosphonic acid anhydride is used in an amount of 1 to 4 molar equivalents relative to the N-protected α-amino-acid compound.
The amount of propane-phosphonic acid anhydride is in particular 1, 2, or 3 equivalents, in the presence of an organic base, and in particular 2, 3 or 4 equivalents in the absence of an organic base.
According to another particular embodiment, the present invention relates to a use as defined above, wherein the preparation of the NCA compound is made in the presence of an organic base in an amount of 0.25 to 3 molar equivalents relative to the N-protected α-amino-acid compound, in particular 1 to 3 molar equivalents, preferably 3 molar equivalents.
The term “from 0.25 to 3 molar equivalents” also means the following ranges: from 0.25 to 2, from 0.25 to 1, from 0.25 to 0.5, from 0.5 to 3, from 1 to 2, or from 0.5 to 2.
According to another particular embodiment, the present invention relates to a use as defined above, wherein:
An amount of less than 1 base equivalent, compared to the N-protected α-amino-acid compound, may be used. However, a stoichiometric quantity, or greater than the stoichiometric value, of organic base makes it possible to increase the kinetics of the reaction, in order to be able to reach reaction times compatible with an industrial process.
According to another particular embodiment, the present invention relates to a use as defined above, wherein the NCA compound is of Formula 1:
By group “linear C2 to C20 alkenyl” means: a linear alkyl chain comprising from two to 20 carbon atoms, comprise one or more double (s) carbon bond (s). In particular, a linear alkyl chain of 3 to 15 carbon atoms, 3 to 10 carbon atoms, 5 to 20 carbon atoms, 10 to 20 carbon atoms, or 5 to 15 carbon atoms.
By group “branched alkenyl” is meant an alkenyl group as defined above, comprising substituents selected from the list of linear alkyl groups defined above, said linear alkyl groups being also likely to be branched.
By group “C3 to C10 cycloalkyl” means a cycloalkyl group comprising from 3 to 10 carbon atoms, selected from: cyclopropyl C3, cyclobutyl C4, cyclopentyl C5, cyclohexyl C6, cycloheptyl C7, cyclooctyl C8, cyclononyl C9, or cyclodecyl C10.
By group “C3 to C10 hetero cycloalkyl” means a cycloalkyl group comprising from 3 to 10 carbon atoms, and further comprising one or more heteroatoms in the ring, in particular 1 or 2 heteroatom (s).
The term “aryl” refers to an aromatic group comprising 5 to 16 carbon atoms within the aromatic ring, in particular from 6 to 12 carbon atoms, in particular comprising 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 carbon atoms. The aryl groups according to the present invention may also be substituted, in particular by one or more substituents selected from: a linear or branched C1 to C10 alkyl group, a linear or branched C1 to C10 O-alkyl group.
Phenyl, anisyl and naphthyl, o-tolyl, m-tolyl, p-tolyl, o-xylyl, m-xylyl, p-xylyl, are examples of aryl groups according to the present invention.
The term “heteroaryl” refers to an aryl group as defined above, comprising atoms other than carbon atoms, in particular N, O or S within the aromatic ring.
Pyridyl, imidazoyl, indolyl, or furanyl are examples of heteroaryl groups according to the present invention.
Within the meaning of the present invention, the expression “R1 and R2 may form a ring” refers to spirocyclic compounds, preferably from 3 to 10 carbon atoms, as follows, for the general formula 3, wherein R3 is as defined above.
The NCA compounds of Formulas 4, 5 and 6 are specific examples of spirocyclic compounds according to the present invention, comprising respectively a cyclopropyl, cyclopentyl, or cyclohexyl ring.
The expression “said group R3 may form a ring with R1 or R2” refers to the formation of a polycyclic compound, illustrated, non-limitingly, by the following NCA compounds 7 to 9:
When the NCA compound comprises an isotope 13C, or several isotopes 13C, said isotope is either on one of the carbonyl functions of the NCA ring, or on the side chain, represented by R1 and/or R2 in Formula 1. The isotope 13C is preferably present as 13C═O.
When the NCA compound comprises a deuterium isotope, D, or several D isotopes, said isotope (s) are preferably on a non-enolisable position of the NCA cycle, or on a non-exchangeable position of the side chain, represented by R1 and/or R2 in Formula 1.
The expression “the asymmetric centers of said compound of Formula 1 are of R or S configuration, or a mixture of these configurations” refers to the asymmetric centers formed by the configuration of the groups R1 and R2 (Formulas 10 and 11 below), but also to those possibly present on said groups R1 and R2. This situation is illustrated by Formula 12 below, wherein the group R1 is a hydrogen atom, and the group R2 has a racemic asymmetric center. In this example, the carbon bearing the groups R1 and R2 is of S configuration.
The stereochemistry of NCA molecules is determined by the stereochemistry of the N-protected α-amino-acid from which the NCA compound is formed.
The compound of Formula 1 is preferably diastereoisomerically pure, the excess diastereoisomeric being greater than 80%.
The term “greater than 80%”, greater than 90%, greater than 95%, greater than 98% and in particular greater than 99%.
The compound of Formula 1 is preferably enantiomerically pure, the enantiomeric excess being greater than 80%, the N-protected a amino-acid being in particular of L configuration.
The term “greater than 80%”, greater than 90%, greater than 95%, greater than 98% and in particular greater than 99%.
According to another particular embodiment, the present invention relates to a use as defined above, wherein the compound of Formula 1 is such that:
According to another particular embodiment, the present invention relates to a use as defined above, wherein the compound of Formula 1 is such that:
According to another particular embodiment, the present invention relates to a use as defined above, wherein the compound of Formula 1 is such that:
According to another particular embodiment, the present invention relates to a use as defined above, wherein the compound of Formula 1 is such that R3 is a hydrogen atom.
According to this particular embodiment, the compound of Formula 1 has the structure of Formula 13:
wherein R1 and R2 are as defined above.
According to another particular embodiment, the present invention relates to a use as defined above, wherein the compound of Formula 1 is such that R1 and R2 are identical, and are in particular a methyl.
According to another particular embodiment, the present invention relates to a use as defined above, wherein the compound of Formula 1 is such that R1 and R2 form a cycle.
This embodiment has been illustrated above by the NCA compounds of Formulas 3 to 6.
According to another particular embodiment, the present invention relates to a use as defined above, in which the compound of Formula 1 is such that one of R1 or R2 is a hydrogen atom.
According to this particular embodiment, the compound of Formula 1 has the structure of Formula 14, or Formula 14′:
wherein R1, R2 and R3 are as defined above.
According to another particular embodiment, the present invention relates to a use as defined above, wherein at least one of the groups R1 or R2, comprises at least one protective group of carboxylic acid functions, amine functions, thiol functions, guanidine functions, amide functions and/or alcohol functions, 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 (Pme), 4-methoxy-2,3,6-trimethylbenzenesulfonyl (Mtr), trityl (Trt), trifluoroacetyl, acetamidomethyl (Acm), and xanthyl (Xan).
The protective groups used according to the present invention are any protective group available to those skilled in the art (Greene's Protective Groups in Organic Synthesis, 4th edition, Wiley).
According to another particular embodiment, the present invention relates to a use as defined above, wherein the NCA compound is selected from:
The present invention also relates to a use as defined above, wherein the NCA compound is a UNCA compound, in particular a UNCA compound of the following structure:
wherein R′ is selected from linear or branched C1 to C20 alkyl group, in particular a tert-butyl, or an aryl-methyl group, in particular a benzyl group or a fluorenylmethyl group,
wherein R1 and R2 are as defined above.
According to another particular embodiment, the present invention relates to a use as defined above, wherein the NCA compound is selected from the following structures:
A second object of the present invention is a method for preparing an NCA compound, comprising:
According to a particular embodiment, the present invention relates to a method as defined above, wherein the contact step comprises:
According to another particular embodiment, the present invention relates to a method as defined above, wherein the contact step comprises:
Alternatively, the method of the present invention can be implemented in a flow chemistry device.
According to another particular embodiment, the present invention therefore relates to a method as defined above, said method being implemented in continuous flow.
According to another particular embodiment, the present invention relates to a method as defined above, further comprising, after obtaining the NCA, a purification step by at least one aqueous wash.
Aqueous washing makes it possible to remove the propylphosphonic acid formed during the reaction.
According to another particular embodiment, the present invention relates to a method as defined above, further comprising a step of purifying the NCA compound, in particular by recrystallization.
According to another particular embodiment, the present invention relates to a method as defined above, wherein the contact step is carried out for a period of 1 to 24 hours, in particular about 2 hours.
The method according to the present invention generally allows to obtain a total conversion after 2 hours of reaction. However, the reaction time may be longer if the analysis of an aliquot demonstrates the presence of N-protected-α-amino-acid.
According to another particular embodiment, the present invention relates to a method as defined above, in the presence of an organic base, said organic base being a nitrogenous base, in particular selected from triethylamine, 1,8-diazabicyclo [5.4.0] undec-7-ene, diisopropylethylamine, N-dimethylaminopyridine, diisopropylethylamine or pyridine, preferably pyridine.
According to another particular embodiment, the present invention relates to a method as defined above, wherein the organic solvent is selected from ethyl acetate or dimethylformamide, in particular ethyl acetate.
According to another particular embodiment, the present invention relates to a method as defined above, wherein the propane-phosphonic acid anhydride is used in an amount of 1 to 4 molar equivalents relative to the N-protected α-amino-acid compound, in particular 2 molar equivalents.
According to another particular embodiment, the present invention relates to a method as defined above, in the presence of an organic base in an amount of 0.25 to 3 molar equivalents relative to the N-protected α-amino-acid compound, in particular 1 to 3 molar equivalents, preferably 3 molar equivalents.
According to another particular embodiment, the present invention relates to a method as defined above, in the presence of an organic base, wherein the base is pyridine at a rate of 3 molar equivalents relative to the N-protected α-amino-acid compound, wherein the propane-phosphonic acid anhydride is used in an amount of 2 molar equivalents relative to the N-protected α-amino-acid compound, and wherein the stirring step is carried out at room temperature.
According to another particular embodiment, the present invention relates to a method as defined above, wherein the propane-phosphonic acid anhydride is used in an amount of 4 molar equivalents relative to the N-protected α-amino-acid compound, and wherein the stirring step is carried out at a temperature of 50 to 80° C., in the absence of an organic base.
According to another particular embodiment, the present invention relates to a method for preparing a compound NCA, as defined above, comprising:
According to another particular embodiment, the present invention relates to a method for preparing a NCA compound, as defined above, wherein the contact step comprises:
According to another particular embodiment, the present invention relates to a method as defined above, wherein said NCA compound is of Formula 1-A, prepared from a N-protected α-amino-acid compound of Formula 2-A, according to the following reaction scheme:
When the compound of Formula 1-A, or the compound of Formula 2-A, comprises a carbon atom, said carbon atom may be 13C,
when the compound of Formula 1-A, or the compound of Formula 2-A, comprises a fluorine atom, said fluorine atom may be 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.
The carboxylic acid function present in the compound of Formula 2-A is optionally in the form of carboxylate, especially in the form of a sodium salt, a potassium salt, or a lithium salt.
According to another particular embodiment, the present invention relates to a method as defined above, wherein the compound of Formula 1-A and the compound of Formula 2-A are such that:
According to another particular embodiment, the present invention relates to a method as defined above, wherein the compound of Formula 1-A and the compound of Formula 2-A are such that:
According to another particular embodiment, the present invention relates to a method as defined above, wherein the compound of Formula 1-A and the compound of Formula 2-A are such that:
According to another particular embodiment, the present invention relates to a method as defined above, wherein R3 is a hydrogen atom.
According to another particular embodiment, the present invention relates to a method as defined above, wherein R1 and R2 are identical, and represent in particular a methyl.
According to another particular embodiment, the present invention relates to a method as defined above, wherein one of R1 or R2 is a hydrogen atom.
According to another particular embodiment, the present invention relates to a method as defined above, wherein R1 or R2 forms a cycle.
According to another particular embodiment, the present invention relates to a method as defined above, wherein at least one of the groups R1 or R2 comprises at least one protective group, carboxylic acid functions, amine functions, thiol functions, guanidine functions, amide functions and/or alcohol functions, 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).
According to another particular embodiment, the present invention relates to a method as defined above, comprising:
According to another particular embodiment, the present invention relates to a method as defined above, wherein the α-amino-acid compound is selected from:
The present invention also relates to a method as defined above, wherein the NCA compound is a UNCA compound, in particular a UNCA compound of the following structure:
wherein R′ is selected from a linear or branched C1 to C20 alkyl group, in particular a tert-butyl, or an aryl-methyl group, in particular a benzyl group or a fluorenylmethyl group, wherein R1 and R2 are as defined above.
A third object of the present invention is an NCA compound, as obtained by the method as defined above.
The method for preparing a NCA compound according to the invention provides NCA compounds of excellent purities, and devoid of phosgene decomposition products, diphosgene decomposition products and triphosgene decomposition products, in particular devoid of hydrochloric acid. The method also avoids contamination observed when the NCA compound is prepared under nitrosation conditions.
These breakdown products can harm certain uses of NCA compounds, especially in the synthesis of a peptide, where the presence of hydrochloric acid residues for example is troublesome.
However, such compounds according to the present invention can be salified, after their isolation, during a salification step wherein the NCA compound is put in the presence of an acid for example such as hydrochloric acid or triflic acid, non-nucleophilic. However, this is the salification of a salivable function on the side chain, and not of the nitrogen of the NCA cycle whose salified forms are unstable.
A fourth object of the present invention is a novel NCA compound, selected from the following structures:
The invention also relates to a novel NCA compound of the following structure:
According to another particular embodiment, the present invention relates to a novel NCA compound as defined below, selected from:
A fifth object of the present invention is a solution comprising a NCA compound prepared according to the method as defined above, or a NCA compound as defined above, said solution being devoid of phosgene decomposition products, diphosgene decomposition products and triphosgene decomposition products, in particular devoid of hydrochloric acid.
A sixth object of the present invention is the use of an NCA compound prepared according to the method as defined above, or a NCA compound as defined below, in the preparation of a polypeptide, oligopeptide or dendrimer.
A seventh object of the present invention is a method for preparing a polypeptide, comprising a step of contacting a NCA compound prepared according to the method as defined above, or an NCA compound as defined above, with a polymerization initiator, in particular selected from amines, in particular arginine.
An eighth object of the present invention is a polypeptide, as obtained by the method for preparing a polypeptide as defined above.
A ninth object of the present invention is a method for preparing a UNCA compound, comprising a contact step of an NCA compound prepared according to the method as defined above, or an NCA compound as defined above, with:
According to this embodiment, the starting NCA compound comprises an NCA ring in which the nitrogen atom is in the form —NH, allowing to introduce a substituent.
The following examples and figures illustrate the invention, without limiting its scope.
Materials and Methods
Reagents and solvents were used as procured from commercial suppliers, and without further purification. The 13C and 1H NMR spectra in DMSO-d6 and CDCl3 were performed on a Bruker AVANCE 600 MHz spectrometer, equipped with a BBFO helium cryoprobe at 298 K. Chemical shifts (δ) 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).
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% HCO2H; eluent B: acetonitrile/0.1% HCO2H. UV detection was performed at 214 nm. The electrospray mass spectra were acquired at a solvent rate of 200 μL/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.
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.
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.
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.
Yield 81%
White solid, mp 152-153° C., [α]20D (c 1.00, CH2Cl2)=−49.1°, 1H NMR (600 MHz, CDCl3) δ=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), 13C NMR (125 MHz, CDCl3) δ=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+) tR1.81 min, m/z 331 [M+H]+ m/z 231 [M+H−Boc]+.
Recrystallized in an AcOEt/hexane mixture
Yield 78%
White solid, mp 62-63° C., 1H NMR (600 MHz, CDCl3) δ=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), 13C NMR (125 MHz, CDCl3) δ=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]+.
Yield 90%
White solid, mp 108-109° C., [α]19D (c 1.00, CH2Cl2)=−78.1°, 1H NMR (600 MHz, CDCl3) δ=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), 13C NMR (125 MHz, CDCl3) δ=168.5, 155.3, 151.3, 129.6, 128.5, 124.7, 78.8, 58.8, 37.3, 28.8, LC tR 1.53 min.
Recrystallized in an AcOEt/hexane mixture
Yield 87%
White solid, mp 88-89° C., [α]18 D (c 1.00, CH2Cl2)=−94.6, 1H NMR (600 MHz, CDCl3) δ=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). 13C NMR (125 MHz, CDCl3) δ=168.6, 151.8, 133.8, 129.2, 128.0, 58.8, 37.8, LC tR 2.25 min.
Recrystallized in an AcOEt/hexane mixture
Yield 87%
White solid, mp 103-104° C., [α]19 D (c 1.00, CH2Cl2)=−42.3°, 1H NMR (600 MHz, DMSO-d6) δ=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), 13C NMR (125 MHz, DMSO-d6) δ=171.4, 169.7, 152.6, 136.0, 128.9, 128.6, 128.5, 66.7, 54.0, 35.1, LC tR 2.55 min.
Recrystallized in an AcOEt/hexane mixture
Yield 81%
White solid, mp 120-121° C., 1H NMR (600 MHz, CDCl3) δ=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), 13C NMR (125 MHz, CDCl3) δ=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 tR 1.40 min.
Yield 80%
White solid, mp 156-157° C., 1H NMR (600 MHz, DMSO-d6) δ=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), 13C NMR (125 MHz, DMSO-d6) δ=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 tR 1.57 min.
Recrystallized in an AcOEt/hexane mixture
Yield 76%
White solid, mp 84-85° C., 1H NMR (600 MHz, CDCl3) δ=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), 13C NMR (125 MHz, CDCl3) δ=170.0, 152.0, 53.3, 17.7.
Recrystallized in an AcOEt/hexane mixture
Yield 77%
White solid, mp 65-66° C., 1H NMR (600 MHz, DMSO-d6) δ=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), 13C NMR (125 MHz, DMSO-d6) δ=170.4, 152.7, 138.0, 128.8, 128.1, 127.8, 72.9, 68.1, 58.8, LC tR 1.36 min.
Recrystallized in an AcOEt/hexane mixture
Yield 78%
White solid, mp 110-111° C., [α]19D (c 1.00, CH2Cl2)=−13.6°, 1H NMR (600 MHz, DMSO-d6) δ=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), 13C NMR (125 MHz, DMSO-d6) δ=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 tR 1.48 min.
Yield 82%
White solid, mp 122-123° C., 1H NMR (600 MHz, DMSO-d6) δ=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), 13C NMR (125 MHz, DMSO-d6) δ=170.3, 152.0, 144.2, 129.4, 128.6, 127.4, 66.7, 56.8, 32.9, LC tR 2.00 min.
Recrystallized in an AcOEt/hexane mixture
Yield 85%
White solid, mp 142-143° C., [α]19D (DCM) [c=1]=−37.0°, 1H NMR (600 MHz, DMSO-d6) δ=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), 13C NMR (125 MHz, DMSO-d6) δ=172.1, 156.0, 152.4, 77.8, 57.4, 31.1, 29.3, 28.7, 22.0, LC tR 1.39 min.
Recrystallized in an AcOEt/hexane mixture
Yield 76%
White solid, mp 166-167° C., 1H NMR (600 MHz, DMSO-d6) δ=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), 13C NMR (125 MHz, DMSO-d6) δ=170.1, 151.0, 131.6, 130.8, 129.8, 127.3, 127.3, 127.0, 54.6, 42.3, 29.2, LC tR 1.39 min.
Recrystallized in an AcOEt/hexane mixture
Yield 86%
White solid, mp 66-67° C., 1H NMR (600 MHz, DMSO-d6) δ=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), 13C NMR (125 MHz, DMSO-d6) δ=172.4, 152.4, 56.0, 40.5, 24.5, 23.1, 21.7, LC tR 1.18 min.
Recrystallized in an AcOEt/hexane mixture
Yield 88%
White solid, mp 101-102° C., [α]20D=−41.6 (c 1.00, CH2Cl2), 1H NMR (600 MHz, DMSO-d6) δ=9.07 (s, 1H), 7.88-7.87 (d, J=7.52 Hz, 2H), 7.68-7.67 (d, J=7α.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), 13C NMR (125 MHz, DMSO-d6) δ=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 tR 1.57 min.
Recrystallized in an AcOEt/hexane mixture
Yield 86%
White solid, mp 122-123° C., [α]19D (c 1.00, CH2Cl2)=−17.3°, 1H NMR (600 MHz, CDCl3) δ=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), 13C NMR (125 MHz, CDCl3) δ=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 tR 1.56 min.
Yield 79%
[α]19D (c 1.00, CH2Cl2)+42.6°.
(L)Leu-NCA from 10 g Boc-(L)Leu-OH
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 MgSO4, 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.
Yield 86%.
(L)Phe-NCA from 20 g of Boc-(L)Phe-OH
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 MgSO4, 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%.
(L)Glu(OBzl)-NCA from 25 g of Boc-(L)Glu(OBzl)-OH
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 MgSO4, 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%.
(L)Ala-NCA from 50 g Boc-(L)Ala-OH
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 MgSO4, 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%.
(L)Lys-NCA from 10 g Boc-(L)Lys-OH
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 MgSO4, 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%.
The general procedure of Example 1 was used, replacing pyridine with bases Et3N, iPr2EtN 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.
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 tR 1.58 min.
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.
The NCA compounds thus obtained were then reacted with (S)-1-(4-methoxyphenyl) ethane-1-amine to obtain diastereoisomers S, S and R, S.
An analysis by HPLC (Colone Zorbax SB-C18 50*2.1 mm 1.8 μm; flow rate 0.5 mL/min with a gradient (0-100%) B over 25 min; eluent A: water/0.1% HCO2H; eluent B: acetonitrile/0.1% HCO2H. UV detection was performed at 214 nm).
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.
Recrystallized in an AcOEt/hexane mixture
Yield 80%
White solid, mp>200° C.
Recrystallized in an AcOEt/hexane mixture
Yield 80%
White solid, mp 112-113° C., [α]20D=−4 (c=1.00, CH2Cl2).
Recrystallized in an AcOEt/hexane mixture
Yield 86%
White solid, mp 66-68° C., [α]20D=−29 (c=1.00, CH2Cl2).
Recrystallized in an AcOEt/hexane mixture
Yield 84%
White solid, mp 92-93° C., [α]20D=−31 (c=1.00, CH2Cl2).
Recrystallized in an AcOEt/hexane mixture
Yield 91%
White solid, mp 100-102° C., [α]20D=−60 (c=1.00, CH2Cl2).
Recrystallized in an Ether/hexane mixture
Yield 87%
White solid, mp 131-132° C., [α]20D=+10 (c=1.00, CH2Cl2), 1H NMR (600 MHz, DMSO-d6) δ 4.42-4.37 (dd, J=7.13, 14.06 Hz, 1H), 2.84 (s, 3H), 1.39 (d, J=7.13 Hz); 13C NMR (100 MHz, DMSO-d6): δ 171.2, 152.1, 57.2, 28.4, 14.5.
Recrystallized in an Ether/hexane mixture
Yield 82%
White solid, mp 152-153° C., 1H NMR (600 MHz, DMSO-d6) δ 7.37 (m, 5H), 4.50 (s, 2H), 4.17 (s, 2H); 13C NMR (100 MHz, DMSO-d6): δ 167.5, 153.1, 135.7, 129.1, 128.2, 128.2, 50.0, 47.1.
Recrystallized in an AcOEt/hexane mixture
Yield 85%
White solid, mp 45-47° C., [α]20D=−99 (c=1.00, CH2Cl2).
Recrystallized in an AcOEt/hexane mixture
Yield 85%
White solid, mp 123-124° C., [α]19D=+42.3 (c=1.00, CH2 Cl2) 1H NMR (600 MHz, DMSO-d6) δ 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); 13C NMR (100 MHz, DMSO-d6): δ 171.4, 169.7, 152.6, 136.0, 128.9, 128.6, 128.5, 66.7, 54.0, 35.1.
Recrystallized in an AcOEt/hexane mixture
Yield 82%
White solid, mp 72-73° C., 1H NMR (400 MHz, DMSO-d6): δ 9.07 (s, 1H), 1.41 (s, 6H); 13C NMR (100 MHz, DMSO-d6): 175.0, 150.8, 59.6, 25.0.
Recrystallized in an AcOEt/hexane mixture
Yield 89%
White solid, mp 145-146° C., [α]20D=−55.6 (c=1.00, dioxane), 1H NMR (400 MHz, DMSO-d6): δ 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, 2H) 2.74-2.69 (dd, J=4.33, 16.82 Hz, 2H); 13C NMR (100 MHz, DMSO-d6): δ 172.1, 167.9, 152.9, 150.9, 129.6, 124.0, 121.9, 121.8, 116.6, 54.3, 43.1, 36.2.
Number | Date | Country | Kind |
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FR2014040 | Dec 2020 | FR | national |
Filing Document | Filing Date | Country | Kind |
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PCT/EP2021/086981 | 12/21/2021 | WO |