Injectable solution at pH 7 comprising at least one basal insulin wherein the pI is comprised from 5.8 to 8.5 and a co-polyamino acid bearing carboxylate charges and hydrophobic radicals

Abstract

A composition includes co-polyamino acid bearing carboxylate charges and at least one hydrophobic radical -Hy that are chosen among the co-polyamino acids according to formula XXXb: ##STR00001##
wherein, D represents, independently, either a group —CH.sub.2— (aspartic acid) or a group —CH.sub.2—CH.sub.2— (glutamic acid), X represents a cationic entity chosen from the group comprising alkali cations, Rb and R′b, identical or different, are either a hydrophobic radical -Hy, or a radical chosen from the group consisting of an H, a C2 to C10 linear acyl group, a C3 to C10 branched acyl group, a benzyl, a terminal “amino acid” unit and a pyroglutamate, at least one of Rb and R′b is a hydrophobic radical -Hy, n+m represents the degree of polymerization DP of the co-polyamino acid, namely the mean number of monomeric units per co-polyamino acid chain and 5≤n+m≤250.

Claims

1. A composition in the form of an injectable aqueous solution, wherein the pH is comprised from 6.0 to 8.0, comprising at least: a basal insulin which isoelectric point pI is comprised from 5.8 to 8.5; a co-polyamino acid comprising glutamic or aspartic units that is a co-polyamino acid according to formula I:
[Q(PLG).sub.k][Hy].sub.j[Hy].sub.j′  Formula I wherein: j≥1; 0≤j′≤n′1 and j+j′≥1 and k=2, Q is Q[*].sub.k, wherein Q[*].sub.k is a divalent linear or branched radical or spacer ([Q′].sub.q)[*].sub.k, where * represents binding sites of the different represented elements, 1≤q≤5 and k=2, wherein Q′ is a radical selected from the group consisting of formula III and formula IV and forms ([Q′].sub.q)[*].sub.k: ##STR00166## ##STR00167## wherein: 1≤t≤8, at least one of u.sub.1″ or u.sub.2″ is other than 0, if u.sub.1″≠0 then u.sub.1′≠0 and if u.sub.2″≠0 then u.sub.2″≠0, u.sub.1′ and u.sub.2′ are identical or different, 2≤u≤4, 0≤u.sub.1′≤4, 0≤u.sub.1″≤4, 0≤u.sub.2′≤4, 0≤u.sub.2″≤4, and Fa, Fb, Fa′, and Fb′ are identical or different and each represents function —NH— or —CO, the co-polyamino acid according to formula I bearing at least one hydrophobic radical -Hy, carboxylate charges and two chains of glutamic or aspartic units PLG bound together by the divalent linear or branched radical or spacer, the divalent linear or branched radical or spacer being bound to two glutamic or aspartic unit chains PLG by an amide function and, the amide function binding the divalent linear or branched radical or spacer to the two chains of glutamic or aspartic units results from a reaction between an amine function and an acid function respectively borne either by the Q′ that forms the divalent linear or branched radical or spacer or by a glutamic or aspartic unit, and the hydrophobic radical -Hy being bound either to a terminal amino acid unit and then j≥1, or to a carboxyl function borne by one of the chains of the glutamic or aspartic units PLG and then j′=n′1 and n′1 is the mean number of monomeric units bearing a hydrophobic radical -Hy, wherein the hydrophobic radical -Hy is a radical according to formula X: ##STR00168## wherein GpR is selected from the group consisting of radicals according to formulas VII, VII′ and VII″: ##STR00169## GpG and GpH are identical or different and selected from the group consisting of radicals according to formulas XI and XI′: ##STR00170## GpA is a radical according to formula VIII: ##STR00171## wherein A′ is selected from the group consisting of radicals according to formulas VIII′, VIII″ and VIII′″: ##STR00172## GpL is a radical according to formula XII: ##STR00173## GpC is a radical according to formula IX: ##STR00174## the * indicates the binding sites of the different represented elements bound by amide functions; a is an integer equal to 0 or to 1 and a′=1 if a=0 and a′=1, 2 or 3 if a=1: a′ is an integer equal to 1, to 2 or to 3; b is an integer equal to 0 or to 1; c is an integer equal to 0 or to 1, and if c is equal to 0 then d is equal to 1 or to 2; d is an integer equal to 0, to 1 or to 2; e is an integer equal to 0 or to 1: g is an integer equal to 0, to 1, to 2, to 3 to 4 to 5 or to 6: h is an integer equal to 0, to 1, to 2, to 3 to 4 to 5 or to 6: l is an integer equal to 0 or 1 and l′=1 if 1=0 and l′=2 if l=1; r is an integer equal to 0, to 1 or to 2, and s′ is an integer equal to 0 or 1; A, A.sub.1, A.sub.2 and A.sub.3 identical or different are linear or branched alkyl radicals comprising from 1 to 8 carbon atoms and/or substituted by a radical from a saturated, unsaturated or aromatic ring; B is a radical selected from the group consisting of a non-substituted ether comprising from 4 to 14 carbon atoms and from 1 to 5 oxygen atoms, non-substituted polyether radical comprising from 4 to 14 carbon atoms and from 1 to 5 oxygen atoms, a linear alkyl radical comprising from 1 to 9 carbon atoms, a branched alkyl radical comprising from 1 to 9 carbon atoms, a linear alkyl radical comprising an aromatic nucleus and comprising from 1 to 9 carbon atoms, and a branched alkyl radical comprising an aromatic nucleus and comprising from 1 to 9 carbon atoms: C.sub.x is a linear or branched monovalent alkyl radical, and/or comprising a cyclic part, wherein x indicates the number of carbon atoms and: when the hydrophobic radical -Hy bears 1-GpC, then 9≤x≤25, when the hydrophobic radical -Hy bears 2-GpC, then 9≤x≤15, when the hydrophobic radical -Hy bears 3-GpC, then 7≤x≤13, when the hydrophobic radical -Hy bears 4-GpC, then 7≤x≤11, when the hydrophobic radical -Hy bears 5 or more -GpC, then 6≤x≤11, G is a branched alkyl radical of 1 to 8 carbon atoms, the alkyl radical bearing one or a plurality of free carboxylic acid function(s); R is a radical selected from the group consisting of a linear or branched, divalent alkyl radical comprising from 1 to 12 carbon atoms, a linear or branched, divalent alkyl radical comprising from 1 to 12 carbon atoms bearing one or a plurality of functions —CONH.sub.2 and a non-substituted ether or polyether radical comprising from 4 to 14 carbon atoms and from 1 to 5 oxygen atoms; the hydrophobic radical(s) -Hy according to formula X being bound to the PLG: via a covalent bond between a carbonyl of the hydrophobic radical -Hy and a nitrogen atom borne by the PLG thus forming an amide function obtained from the reaction of an amine function borne by the PLG and an acid function borne by the precursor -Hy′ of the hydrophobic radical -Hy, or via a covalent bond between a nitrogen atom of the hydrophobic radical -Hy and a carbonyl borne by the PLG thus forming an amide function obtained from the reaction of an amine function of a precursor -Hy′ of the hydrophobic radical -Hy and an acid function borne by a PLG, a ratio M between the number of hydrophobic radicals and the number of glutamic or aspartic units being between 0<M≤0.5; when a plurality of hydrophobic radicals are borne by a co-polyamino acid then they are identical or different; a degree of polymerization DP in glutamic or aspartic units for the PLG chains is comprised from 5 to 250; and free carboxylic acid functions are in a form of alkali cation salt selected from the group consisting of Na.sup.+ and K.sup.+.

2. The composition according to claim 1, wherein the co-polyamino acid bearing carboxylate charges and at least one hydrophobic radical -Hy is a co-polyamino acid according to formula XXXb: ##STR00175## wherein, D represents, independently, either a group —CH.sub.2— (aspartic acid) or a group —CH.sub.2—CH.sub.2— (glutamic acid), X represents a cationic entity selected from the group consisting of alkali cations, Rb and R′b, which are identical or different, are either a hydrophobic radical -Hy, or a radical selected from the group consisting of an OH, an amino group, a terminal amino acid unit and a pyroglutamate, at least one of Rb and R′b being a hydrophobic radical -Hy, Q and Hy are as defined above, n+m represents the degree of polymerization DP of the co-polyamino acid, namely the mean number of monomeric units per co-polyamino acid chain and 5≤n+m≤250.

3. The composition according to claim 1, wherein the co-polyamino acid bearing carboxylate charges and at least one hydrophobic radical -Hy is a co-polyamino acid according to formula XXXa hereinafter: ##STR00176## wherein, D represents, independently, either a group —CH.sub.2— (aspartic acid) or a group —CH.sub.2—CH.sub.2— (glutamic acid), X represents a cationic entity selected from the group consisting of alkali cations, Ra and R′a, which are identical or different, are either a hydrophobic radical -Hy, or a radical selected from the group consisting of an H, a C2 to C10 linear acyl group, a C3 to C10 branched acyl group, a benzyl, a terminal amino acid unit and a pyroglutamate, at least one of Ra and R′a being a hydrophobic radical -Hy, Q and -Hy are as defined in Formula I, n+m represents the degree of polymerization DP of the co-polyamino acid, namely the mean number of monomeric units per co-polyamino acid chain and 5≤n+m≤250.

4. The composition according to claim 1, wherein the co-polyamino acid bearing carboxylate charges and at least one hydrophobic radical -Hy is a co-polyamino acid according to formula XXXb′: ##STR00177## wherein: D represents, independently, either a group —CH.sub.2— (aspartic acid) or a group —CH.sub.2—CH.sub.2— (glutamic acid), X represents a cationic entity selected from the group consisting of alkali cations, Q and Hy are as defined in Formula I, Rb and R′b, which are identical or different, are either a hydrophobic radical -Hy, or a radical selected from the group consisting of an —OH, an amine group, a terminal amino acid unit and a pyroglutamate, at least one of Rb and R′b is a hydrophobic radical -Hy, n1+m1 represents the number of glutamic units or aspartic units of the PLG chains of the co-polyamino acid bearing a radical -Hy, n2+m2 represents the number of glutamic units or aspartic units of the PLG chains of the co-polyamino acid not bearing a radical -Hy, n1+n2=n′ and m1+m2=m′, n′+m′ represents the degree of polymerization DP of the co-polyamino acid, namely the mean number of monomeric units per co-polyamino acid chain and 5≤n′+m′≤250.

5. The composition according to claim 1, wherein the co-polyamino acid bearing carboxylate charges and at least one hydrophobic radical -Hy is a co-polyamino acid according to formula XXXa′ hereinafter: ##STR00178## wherein: D represents, independently, either a group —CH.sub.2— (aspartic acid) or a group —CH.sub.2—CH.sub.2— (glutamic acid), X represents a cationic entity selected from the group consisting of alkali cations, Ra and R′a, which are identical or different, are either a hydrophobic radical -Hy, or a radical selected from the group consisting of an H, a C2 to C10 linear acyl group, a C3 to C10 branched acyl group, a benzyl, a terminal amino acid unit and a pyroglutamate, and at least one of Ra and R′a is hydrophobic radical -Hy, Q and Hy are as defined in Formula I, n.sub.1+m.sub.1 represents the number of glutamic units or aspartic units of the PLG chains of the co-polyamino acid bearing a radical -Hy, n.sub.2+m.sub.2 represents the number of glutamic units or aspartic units of the PLG chains of the co-poly amino acid not bearing a radical -Hy, n.sub.1+n.sub.2=n′ and m.sub.1+m.sub.2=m′.

6. The composition according to claim 1, wherein the co-polyamino acid bearing carboxylate charges and hydrophobic radicals is a co-polyamino acid selected from the group according to formulas XXXa, XXXa′, XXXb and XXXb′: ##STR00179## wherein the group D is a group —CH.sub.2— (aspartic unit), X represents a cationic entity selected from the group consisting of alkali cations, Ra and R′a, which are identical or different, are either a hydrophobic radical -Hy, or a radical selected from the group consisting of an a C2 to C10 linear acyl group, a C3 to C10 branched acyl group, a benzyl, a terminal amino acid unit and a pyroglutamate, and at least one of Ra and R′a is a hydrophobic radical -Hy, Rb and R′b, which are identical or different, are either a hydrophobic radical -Hy, or a radical selected, from the group consisting of an —OH, an amine group, a terminal amino acid unit and a pyroglutamate, at least one of Rb and R′b is a hydrophobic radical -Hy, Q and Hy are as defined in Formula I, n1+m1 represents the number of glutamic units or aspartic units of the PLG chains of the co-polyamino acid bearing a radical -Hy, n2+m2 represents the number of glutamic units or aspartic units of the PLG chains of the co-polyamino acid not bearing a radical -Hy, n1+n2=n′ and m1+m2=m′, and n′+m′ represents the degree of polymerization DP of the co-polyamino acid, namely the mean number of monomeric units per co-polyamino acid chain and 5≤n′+m′≤250.

7. The composition according to claim 1, wherein the co-polyamino acid bearing carboxylate charges and hydrophobic radicals is a co-polyamino acid selected from the group according to formulas XXXa, XXXa′, XXXb or XXXb′: ##STR00180## wherein the group D is a group —CH.sub.2—CH.sub.2— (glutamic unit), X represents a cationic entity chosen from the group consisting of alkali cations, Ra and R′a, which are identical or different, are either a hydrophobic radical -Hy, or a radical selected from the group consisting of an H, a C2 to C10 linear acyl group, a C3 to C10 brandied acyl group, a benzyl, a terminal amino acid unit and a pyroglutamate, and at least one Ra and R′a is a hydrophobic radical -Hy, Rb and R′b, which are identical or different, are either a hydrophobic radical -Hy, or a radical selected from the group consisting of an —OH, an amine group, a terminal amino acid unit and a pyroglutamate, at least one of Rb and R′b is a hydrophobic radical -Hy, Q and Hy are as defined in Formula n1+m1 represents the number of glutamic units or aspartic units of the PLG chains of the co-polyamino acid bearing a radical -Hy, n2+m2 represents the number of glutamic units or aspartic units of the PLG chains of the co-polyamino acid not bearing a radical -Hy, n1+n2=n′ and m1+m2=m′, and n′+m′ represents the degree of polymerization DP of the co-polyamino acid, namely the mean number of monomeric units per co-polyamino acid chain and 5≤n′m′≤250.

8. The composition according to claim 1, wherein the basal insulin which isoelectric point is comprised from 5.8 to 8.5 is insulin glargine.

9. The composition according to claim 1, wherein it comprises from 40 to 500 U/mL of basal insulin which isoelectric point is comprised from 5.8 to 8.5.

10. The composition according to claim 1, wherein the concentration of co-polyamino acid bearing carboxylate charges and hydrophobic radicals is not more than 60 mg/mL.

11. The composition according to claim 1, wherein the concentration of co-polyamino acid bearing carboxylate charges and hydrophobic radicals is not more than 40 mg/mL.

12. The composition according to claim 1, wherein the concentration of co-polyamino acid bearing carboxylate charges and hydrophobic radicals is not more than 20 mg/mL.

13. The composition according to claim 1, wherein the concentration of co-polyamino acid bearing carboxylate charges and hydrophobic radicals is not more than 10 mg/mL.

14. A co-polyamino acid bearing carboxylate charges and at least one hydrophobic radical according to formula X, according to formula I:
[Q(PLG).sub.k][Hy].sub.j[Hy].sub.j′  Formula I wherein: j≥1; 0≤j′≤n′1 and j+j′≥1 and k=2, Q is Q[*].sub.k is, wherein Q[*].sub.k is a divalent linear or branched radical or spacer ([Q′].sub.q)[*].sub.k, where * represents binding sites of the different represented elements, 1≤q≤5 and k=2, wherein Q′ is of formula III and forms ([Q′].sub.q)[*].sub.k: ##STR00181## wherein 1≤t≤8, and Fa and Fa′ are identical or different and each represents function or —CO, said co-polyamino acid according to formula I bearing at least one hydrophobic radical -Hy, carboxylate charges and two chains of glutamic or aspartic units PLG bound together by the divalent linear or branched radical or spacer, the divalent linear or branched radical or spacer being bound to two glutamic or aspartic unit chains PLG by an amide function and, the amide function binding the divalent linear or branched radical or spacer to the two chains of glutamic or aspartic units results from a reaction between an amine function and an acid function respectively borne either by the Q′ that forms the divalent linear or branched radical or spacer or by a glutamic or aspartic unit, the hydrophobic radical -Hy being bound either to a terminal amino acid unit and then j≥1, or to a carboxyl function borne by one of the chains of the glutamic or aspartic units PLG and then j′=n′1 and n′1 is the mean number of monomeric units bearing a hydrophobic radical -Hy, and wherein the at least one hydrophobic radical -Hy according to formula X is as defined hereinafter: ##STR00182## wherein GpR is selected from the group consisting of radicals according to formulas VII, VII′ and VII″: ##STR00183## GpG and GpH are identical or different and are selected from the group consisting of radicals according to formulas XI and XI′: ##STR00184## GpA is a radical according to formula VIII ##STR00185## wherein A′ is selected from the group consisting of radicals according to VIII′, VIII″ or VIII′″ ##STR00186## GpL is a radical according to formula XII ##STR00187## GpC is a radical according to formula IX: ##STR00188## the * indicates the binding sites of the different groups bound by the amide function; a is an integer equal to 0 or to 1 and a′=1 if a=0 and a′=2 or 3 if a=1; a′ is an integer equal to 1, to 2 or to 3; h is an integer equal to 0 or to 1; c is an integer equal to 0 or to 1, and if c is equal to 0 then d is equal to 1 or to 2; d is an integer equal to 0, to 1 or to 2; e is an integer equal to 0 or to 1; g is an integer equal to 0, to 1, to 2, to 3 to 4 to 5 or to 6; h is an integer equal to 0, to 1, to 2, to 3 to 4 to 5 or to 6; l is an integer equal to 0 or 1 and l′=1 if l=0 and l′=2 if l=1; r is an integer equal to 0, to 1 or to 2, and s′ is an integer equal to 0 or 1; A, A.sub.1, A.sub.2 and A.sub.3 identical or different are linear or branched alkyl radicals comprising from 1 to 8 carbon atoms and/or substituted by a radical from a saturated, unsaturated or aromatic ring; B is a radical selected from the group consisting of a non-substituted ether comprising from 4 to 14 carbon atoms and from 1 to 5 oxygen atoms, a non-substituted polyether radical comprising from 4 to 14 carbon atoms and from 1 to 5 oxygen atoms, a linear alkyl radical comprising from 1 to 9 carbon atoms, a branched alkyl radical comprising from 1 to 9 carbon atoms, a linear alkyl radical comprising an aromatic nucleus and comprising from 1 to 9 carbon atoms, and a branched alkyl radical comprising an aromatic nucleus and comprising from 1 to 9 carbon atoms; C.sub.x is a linear or branched monovalent alkyl radical, optionally comprising a cyclic part, wherein x indicates the number of carbon atoms and: when the hydrophobic radical -Hy bears 1-GpC, then 9≤x≤25, when the hydrophobic radical -Hy bears 2-GpC, then 9≤x≤15, when the hydrophobic radical -Hy bears 3-GpC, then 7≤x≤13, when the hydrophobic radical -Hy bears 4-GpC, then 7≤x≤11, when the hydrophobic radical -Hy bears 5 or more -GpC, then 6≤x≤11, G is a branched alkyl radical of 1 to 8 carbon atoms, said alkyl radical bearing one or a plurality of free carboxylic acid function(s); and R is a radical selected from the group consisting of a linear or branched, divalent alkyl radical comprising from 1 to 12 carbon atoms, a linear or branched, divalent alkyl radical comprising from 1 to 12 carbon atoms bearing one or a plurality of functions CONH.sub.2, and a non-substituted ether or polyether radical comprising from 4 to 14 carbon atoms and from 1 to 5 oxygen atoms.

15. A method for improving the physicochemical stability of the composition according to claim 1, by adding one or more ionic species selected from the group of anions, cations and zwitterions.

Description

EXAMPLES

(1) Part A—Synthesis of Hydrophobic Intermediate Compounds Hyd for Obtaining the Radicals -Hy.

(2) TABLE-US-00002 # HYDROPHOBIC INTERMEDIATE COMPOUNDS A1  A2  A3  A4  A5  A6  A7  A8  A9  00 A10 01 A11 02 A12 03 A14 04 A15 05 A16 06 A17 07 A18 08 A19 09 A21 0 A22 A23 A26 A27

Example A1: Molecule A1

(3) To a solution of L-proline (300.40 g, 2.61 mol) in 2 N aqueous sodium hydroxide (1.63 L) at 0° C. is added slowly over 1 h myristoyl chloride (322 g, 1.30 mol) in solution in dichloromethane (DCM, 1.63 L). At the end of the addition, the reaction medium is returned to 20° C. in 3 h, then stirred for a further 2 h. The mixture is cooled to 0° C. then a 37% HCl aqueous solution (215 mL) is added in 15 min. The reaction medium is stirred for 3 h from 0° C. to 20° C., then cooled to 3° C. 37% HCl (213 mL) is added in 15 min and the mixture is stirred for 1 h from 0° C. to 20° C. The organic phase is separated, washed with a 10% HCl aqueous solution (3×430 mL), an aqueous solution saturated with NaCl (430 mL), dried on Na.sub.2SO.sub.4, filtered on cotton then concentrated under reduced pressure. The residue is solubilized in heptane (1.31 L) at 50° C., then the solution is progressively returned to ambient temperature. After initiating crystallization using a glass rod, the medium is once again heated to 40° C. for 30 min then returned to ambient temperature for 4 h. A white solid of molecule A1 is obtained after filtration on a sintered filter, washing with heptane (2×350 mL) and drying under reduced pressure.

(4) Yield: 410 g (97%)

(5) .sup.1H NMR (CDCl.sub.3, ppm): 0.88 (3H); 1.28 (20H); 1.70 (2H); 1.90-2.10 (3H); 2.36 (2H); 2.51 (1H); 3.47 (1H); 3.56 (1H); 4.61 (1H).

(6) LC/MS (ESI): 326.4; 651.7; (calculated ([M+H].sup.+): 326.3; ([2M+H].sup.+): 651.6).

Example A2: Molecule A2

(7) Molecule 1: Product obtained by the reaction between decanoyl chloride and L-proline.

(8) Using a similar method to the one used for the preparation of molecule A1 and applied to decanoyl chloride (75.0 g, 393.27 mmol) and to L-proline (90.55 g, 786.53 mmol), a colorless oil of molecule 1 is obtained after washing the organic phase with a 10% HCl aqueous solution (3×125 mL), an aqueous solution saturated with NaCl (125 mL), drying on Na2SO4, cotton filtration then concentration under reduced pressure.

(9) Yield: 104.64 g (99%)

(10) .sup.1H NMR (CDCl.sub.3, ppm): 0.86 (3H); 1.10-1.51 (12H); 1.56-1.80 (2H); 1.83-2.46 (6H); 3.42-3.66 (2H); 4.37-4.41 (0.1H); 4.53-4.60 (0.9H); 10.12 (1H). LC/MS (ESI): 270.1; (calculated ([M+H].sup.+): 270.2).

(11) Molecule A2

(12) To a solution of molecule 1 (90.0 g, 334.09 mmol) in THF (600 mL) at 0° C. are added successively N-hydroxysuccinimide (NHS, 40.4 g, 350.80 mmol) followed by dicyclohexylcarbodiimide (DCC, 72.38 g, 350.80 mmol) in solution in THF (60 mL). After 16 h of stirring at ambient temperature, the reaction medium is filtered and introduced onto a solution of L-lysine hydrochloride (30.51 g, 167.05 mmol) and N,N-diisopropylethylamine (DIPEA, 97.16 g, 751.71 mmol) in water (66 mL) and the mixture is stirred for 48 h at 20° C. After concentration under reduced pressure, water (360 mL) is added and the mixture obtained is treated by successive addition of ethyl acetate (AcOEt, 500 mL) followed by a 5% Na.sub.2CO.sub.3 aqueous solution (1 L). The aqueous phase is then washed once again with AcOEt (200 mL), acidified by adding a 6 N HCl aqueous solution and the product is extracted with dichloromethane (DCM, 3×250 mL). The organic phase is dried on Na.sub.2SO.sub.4, filtered and concentrated under vacuum. The white solid obtained after crystallization in AcOEt is solubilized in DCM (400 mL), the organic phase is washed with a 1 N HCl aqueous solution (200 mL) followed by an aqueous solution saturated with NaCl (200 mL), dried on Na.sub.2SO.sub.4, filtered and concentrated under vacuum. A white solid of molecule A2 is obtained after crystallization in AcOEt.

(13) Yield: 75.90 g (70%)

(14) .sup.1H NMR (DMSO-d6, ppm): 0.85 (6H); 1.10-2.04 (42H); 2.07-2.30 (4H); 2.92-3.08 (2H); 3.28-3.57 (4H); 4.07-4.28 (2H); 4.32-4.40 (1H); 7.66-7.73 (0.6H); 7.96-8.09 (1H); 8.27 (0.4H); 12.51 (1H).

(15) LC/MS (ESI): 649.5 (calculated ([M+H].sup.+): 649.5).

Example A3: Molecule A3

(16) Molecule 2: Product obtained by the reaction between lauroyl chloride and L-proline.

(17) Using a similar method to the one used for the preparation of molecule A1 and applied to lauroyl chloride (27.42 g, 685.67 mmol) and to L-proline (60.0 g, 247.27 mmol), a white solid of molecule 2 is obtained.

(18) Yield: 78.35 g (96%)

(19) .sup.1H NMR (CDCl.sub.3, ppm): 0.87 (3H); 1.26 (16H); 1.70 (2H); 1.90-2.10 (3H); 2.35 (2H); 2.49 (1H); 3.48 (1H); 3.56 (1H); 4.60 (1H).

(20) LC/MS (ESI): 298.1 (calculated ([M+H].sup.+): 298.2).

(21) Molecule A3

(22) Using a similar method to the one used for the preparation of molecule A2 applied to molecule 2 (42.49 g, 142.86 mmol) and to L-lysine hydrochloride (13.7 g, 75.0 mmol), a white solid of molecule A3 is obtained after crystallization in acetone.

(23) Yield: 30.17 g (60%)

(24) .sup.1H NMR (DMSO-d6, ppm): 0.86 (6H); 1.07-2.05 (50H); 2.08-2.30 (4H); 2.93-3.09 (2H); 3.28-3.57 (4H); 4.08-4.29 (2H); 4.33-4.41 (1H); 7.70 (0.6H); 7.97-8.07 (1H); 8.28 (0.4H); 12.52 (1H).

(25) LC/MS (ESI): 705.6; (calculated ([M+H].sup.+): 705.6).

Example A4: Molecule A4

(26) Using a similar method to the one used for the preparation of molecule A2 applied to molecule A1 (200.0 g, 614.44 mmol) and to L-lysine hydrochloride (56.11 g, 307.22 mmol), a white solid of molecule A4 is obtained after crystallization in ethyl acetate.

(27) Yield: 176.0 g (95%)

(28) .sup.1H NMR (DMSO-d.sub.6, ppm): 0.85 (6H); 1.08-1.51 (48H); 1.53-2.04 (10H); 2.08-2.30 (4H); 2.93-3.09 (2H); 3.31-3.55 (4H); 4.10-4.40 (3H); 7.68 (0.6H); 7.97 (1H); 8.27 (0.4H); 12.50 (1H).

(29) LC/MS (ESI): 761.8; (calculated ([M+H].sup.+): 761.6).

Example A5: Molecule A5

(30) Molecule 3: Product obtained by the reaction between Fmoc-Lys(Fmoc)-OH and 2-Cl-trityl chloride resin.

(31) To a suspension of Fmoc-Lys(Fmoc)-OH (7.32 g, 12.40 mmol) in DCM (60 mL) at ambient temperature is added DIPEA (4.32 mL, 24.80 mmol). After complete solubilization (10 min), the solution obtained is poured onto 2-Cl-trityl chloride resin (100-200 mesh, 1% DVB, 1.24 mmol/g) (4.00 g, 4.96 mmol) previously washed with DCM, in a reaction vessel suitable for solid substrate peptide synthesis. After 2 h of stirring at ambient temperature, HPLC grade methanol (0.8 mL/g resin, 3.2 mL) is added and the medium is stirred at ambient temperature for 15 min. The resin is filtered, washed successively with DCM (3×60 mL), DMF (2×60 mL), DCM (2×60 mL), isopropanol (1×60 mL) and DCM (3×60 mL).

(32) Molecule 4: product obtained by the reaction between molecule 3 and an 80:20 DMF/piperidine mixture.

(33) Molecule 3, previously washed with DMF, is treated with an 80:20 DMF/piperidine mixture (60 mL). After 30 min of stirring at ambient temperature, the resin is filtered, washed successively with DMF (3×60 mL), isopropanol (1×60 mL) and DCM (3×60 mL).

(34) Molecule 5: Product obtained by the reaction between molecule 4 and 8-(9-Fluorenylmethyloxycarbonyl-amino)-3,6-dioxaoctanoic acid (Fmoc-O2Oc-OH).

(35) To a suspension of Fmoc-O2Oc-OH (9.56 g, 24.80 mmol) and 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU, 9.43 g, 24.80 mmol) in a 1:1 DMF/DCM mixture (60 mL) is added DIPEA (8.64 mL, 49.60 mmol). After complete solubilization, the solution obtained is poured onto molecule 4. After 2 h of stirring at ambient temperature, the resin is filtered, washed successively with DMF (3×60 mL), isopropanol (1×60 mL) and DCM (3×60 mL).

(36) Molecule 6: Product obtained by the reaction between molecule 5 and an 80:20 DMF/piperidine mixture.

(37) Using a similar method to the one used for molecule 4 applied to molecule 5, molecule 6 is obtained.

(38) Molecule 7: Product obtained by the reaction between molecule 6 and lauric acid.

(39) Using a similar method to the one used for molecule 5 applied to molecule 6 and to lauric acid (4.97 g, 24.80 mmol) in DMF (60 mL), molecule 7 is obtained.

(40) Molecule 8: product obtained by the reaction between molecule 7 and an 80:20 dichloromethane/1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) mixture.

(41) Molecule 7 is treated with an 80:20 dichloromethane/1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) mixture (60 mL). After 20 min of stirring at ambient temperature, the resin is filtered and washed successively with dichloromethane (2×60 mL). The solvents are evaporated under reduced pressure. Two co-evaporations are then carried out on the residue with dichloromethane (60 mL) followed by diisopropylether (60 mL). A white solid of molecule 8 is obtained after recrystallization in acetonitrile.

(42) Yield: 2.63 g (66% in 6 stages)

(43) .sup.1H NMR (CDCl.sub.3, ppm): 0.87 (6H); 1.09-1.66 (40H); 1.77-1.98 (2H); 2.13-2.29 (4H); 3.24-3.75 (18H); 3.95-4.07 (4H); 4.65-4.70 (1H); 6.23-6.37 (1H); 6.39-6.62 (1H); 6.74-6.91 (1H); 7.38-7.54 (1H).

(44) LC/MS (ESI): 801.6 (calculated ([M+H].sup.+): 801.6).

(45) Molecule 9: product obtained by the reaction between molecule 8 and N-Boc ethylenediamine.

(46) To a solution of molecule 8 (2.63 g, 3.29 mmol) in chloroform (20 mL) at ambient temperature are added successively N-hydroxybenzotriazole (HOBt, 654 mg, 4.27 mmol) and N-Boc ethylenediamine (BocEDA, 580 mg, 3.62 mmol). The mixture is cooled to 0° C. then (3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC, 819 mg, 4.27 mmol) is added. The reaction medium is stirred for 15 min at 0° C. followed by 18 h at ambient temperature. The organic phase is washed with an aqueous solution saturated with NH.sub.4Cl (2×10 mL), an aqueous solution saturated with NaHCO.sub.3 (2×10 mL), and an aqueous solution saturated with NaCl (2×10 mL). The organic phase is dried on Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. A white solid of molecule 9 is obtained after purification by silica gel chromatography (eluent: dichloromethane, methanol).

(47) Yield: 2.37 g (76%)

(48) .sup.1H NMR (CDCl.sub.3, ppm): 0.87 (6H); 1.08-1.47 (34H); 1.43 (9H); 1.48-1.70 (7H); 1.78-1.87 (1H); 2.14-2.25 (4H); 3.16-3.71 (22H); 3.92-4.04 (4H); 4.47-4.52 (1H); 5.33 (1H); 6.10 (1H); 6.65-7.01 (1H); 7.11-7.30 (2H); 7.47-7.63 (1H).

(49) Molecule A5

(50) To a solution of molecule 9 (2.37 g, 2.51 mmol) in dichloromethane (50 mL) at ambient temperature is added a 4 M HCl solution in dioxane (6.3 mL) then the medium is stirred for 2 h at ambient temperature. After concentration under reduced pressure, the residue is solubilized in dichloromethane (50 mL) then washed with a 1 N NaOH aqueous solution (2×12.5 mL) and an aqueous solution saturated with NaCl (25 mL). The organic phase is dried on Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. A white solid of molecule A5 is obtained after recrystallization in acetonitrile.

(51) Yield: 1.57 g (74%)

(52) .sup.1H NMR (CDCl.sub.3, ppm): 0.87 (6H); 1.08-1.43 (34H); 1.48-1.71 (7H); 1.74-1.93 (3H); 2.14-2.25 (4H); 2.79-2.86 (2H); 3.17-3.71 (20H); 3.93-4.05 (4H); 4.47-4.54 (1H); 6.08-6.29 (1H); 6.84-7.01 (1H); 7.15-7.32 (2H); 7.50-7.64 (1H).

(53) LC/MS (ESI): 843.6 (calculated ([M+H].sup.+): 843.7).

Example A6: Molecule A6

(54) Molecule 10: Product obtained by hydrogenating retinoic acid.

(55) A solution of retinoic acid (19.0 g, 63.24 mmol) in methanol (450 mL) in the presence of 10% palladium on carbon (1.9 g) is placed in a hydrogen atmosphere (1 atm) at ambient temperature. After overnight, the reaction medium is filtered on a sintered filter and the filtrate is then concentrated under reduced pressure. A colorless oil of molecule 10 is obtained.

(56) Yield: 19.50 g (99%)

(57) .sup.1H NMR (CDCl.sub.3, ppm): 0.45-2.01 (35H); 2.10-2.17 (1H); 2.33-2.38 (1H); 11.14 (1H).

(58) LC/MS (ESI): 309.3; (calculated ([M−H].sup.−): 309.3).

(59) Molecule 11: Product obtained by coupling Boc-1-amino-4,7,10-trioxa-13-tridecane amine (BocTOTA) and molecule 10.

(60) Using a similar method to the one used for the preparation of molecule 9 applied to molecule 10 (19.3 g, 62.15 mmol) and to BocTOTA (23.9 g, 74.58 mmol), an orange oil of molecule 11 is obtained.

(61) Yield: 37.05 g (97%)

(62) .sup.1H NMR (CDCl.sub.3, ppm): 0.43-1.71 (49H); 2.13-2.17 (1H); 3.17-3.24 (2H); 3.32-3.39 (2H); 3.51-3.66 (12H); 4.77 (0.1H); 4.94 (0.9H); 6.13 (0.9H); 6.29 (0.1H).

(63) LC/MS (ESI): 613.5; (calculated ([M+H].sup.+): 613.5).

(64) Molecule A6

(65) Using a similar method to the one used for the preparation of molecule A5 applied to molecule 11 (34.9 g, 56.94 mmol), an orange oil of molecule A6 is obtained.

(66) Yield: 28.5 g (97%)

(67) .sup.1H NMR (CDCl.sub.3, ppm): 0.41-1.96 (42H); 2.13 (1H); 2.78 (2H); 3.31-3.36 (2H); 3.53 (4H); 3.55-3.58 (4H); 3.60-3.63 (4H); 6.43 (1H).

(68) LC/MS (ESI): 513.5; (calculated ([M+H].sup.+): 513.5).

Example A7: Molecule A7

(69) Molecule 12: Product obtained by the reaction between molecule 4 and Fmoc-Glu(OtBu)-OH.

(70) To a suspension of Fmoc-Glu(OtBu)-OH (10.55 g, 24.80 mmol) and HATU (9.43 g, 24.80 mmol) in a 1:1 DMF/dichloromethane mixture (60 mL) is added DIPEA (8.64 mL, 49.60 mmol). After complete solubilization, the solution obtained is poured onto molecule 4. After 2 h of stirring at ambient temperature, the resin is filtered, washed successively with DMF (3×60 mL), isopropanol (1×60 mL) and dichloromethane (3×60 mL).

(71) Molecule 13: Product obtained by the reaction between molecule 12 and a 50:50 DMF/morpholine mixture.

(72) Molecule 12, previously washed with DMF, is treated with a 50:50 DMF/morpholine mixture (60 mL). After 1 h 15 of stirring at ambient temperature, the resin is filtered, washed successively with DMF (3×60 mL), isopropanol (1×60 mL) and dichloromethane (3×60 mL).

(73) Molecule 14: Product obtained by the reaction between molecule A1 and molecule 13.

(74) Using a similar method to the one used for molecule 12 applied to molecule 13 and to molecule A1 (8.07 g, 24.80 mmol) in DMF (60 mL), molecule 14 is obtained.

(75) Molecule A7

(76) Using a similar method to the one used for the preparation of molecule 8 and applied to molecule 14, a white solid of molecule A7 is obtained after purification by silica gel chromatography (eluent: DCM, methanol).

(77) Yield: 2.92 g (52% in 6 stages)

(78) .sup.1H NMR (DMSO-d6, ppm): 0.85 (6H); 1.07-2.32 (88H); 2.95-3.09 (2H); 3.28-3.60 (4H); 4.06-4.19 (1.7H); 4.21-4.38 (2.6H); 4.40-4.46 (0.7H); 7.56-7.63 (0.7H); 7.78-8.09 (2.6H); 8.22-8.31 (0.7H); 12.64 (1H).

(79) LC/MS (ESI): 1131.8 (calculated ([M+H].sup.+): 1131.8).

Example A8: Molecule A8

(80) Molecule 15: Product obtained by the reaction between decanoic acid and L-leucine.

(81) Using a similar method to the one used for the preparation of molecule A2 applied to decanoic acid (8.77 g, 50.94 mmol) and to L-leucine (7.00 g, 53.36 mmol), a white solid of molecule 15 is obtained.

(82) Yield: 9.17 g (66%)

(83) .sup.1H NMR (DMSO-d6, ppm): 0.82-0.89 (9H); 1.18-1.65 (17H); 2.04-2.14 (2H); 4.19-4.23 (1H); 7.98 (1H); 12.40 (1H).

(84) LC/MS (ESI): 286.2 (calculated ([M+H].sup.+): 286.2).

(85) Molecule 16: Product obtained by the reaction between molecule 15 and L-lysine methyl ester.

(86) To a solution of molecule 15 (9.16 g, 32.11 mmol) in THF (160 mL) are added successively triethylamine (8.12 g, 80.27 mmol) and 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) and the medium is stirred for 30 min at ambient temperature. L-lysine methyl ester dihydrochloride (3.93 g, 16.86 mmol) is added and the reaction medium is stirred for 3 h then concentrated under reduced pressure. The residue is diluted with AcOEt (200 mL), the organic phase is filtered and washed with a 1 N HCl aqueous solution then with water, dried on Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. A white solid of molecule 16 is obtained after triturating the residue in acetonitrile.

(87) Yield: 7.33 g (66%)

(88) .sup.1H NMR (DMSO-d6, ppm): 0.80-0.91 (18H); 1.06-1.72 (38H); 2.03-2.16 (4H); 2.91-3.07 (2H); 3.60 (1.15H); 3.61 (1.85H); 4.13-4.28 (2H); 4.33-4.44 (1H); 7.79-7.92 (3H); 8.13-8.26 (1H).

(89) LC/MS (ESI) 695.7 (calculated ([M+H].sup.+): 695.6).

(90) Molecule 17: Product obtained by saponifying molecule 16.

(91) To a solution of molecule 16 (7.33 g, 10.55 mmol) in a THF/methanol/water mixture (105 mL) is added LiOH (505.13 mg, 21.09 mmol) at 0° C. then the medium is stirred for 20 h at ambient temperature and concentrated under reduced pressure. The aqueous phase is acidified with a 1 N HCl solution to pH 1 and the solid formed is filtered, washed with water and dried under reduced pressure to get a white solid of molecule 17.

(92) Yield: 7.09 g (99%)

(93) .sup.1H NMR (DMSO-d6, ppm): 0.80-0.89 (18H); 1.18-1.73 (40H); 2.03-2.16 (4H); 2.91-3.05 (2H); 4.03-4.13 (1H); 4.21-4.27 (1H); 4.31-4.40 (1H); 7.79-8.02 (4H).

(94) LC/MS (ESI): 681.7 (calculated ([M+H].sup.+): 681.6).

(95) Molecule 18: Product obtained by the reaction between molecule 17 and N-Boc ethylenediamine.

(96) Using a similar method to that used for the preparation of molecule 16 applied to molecule 17 (7.09 g, 10.41 mmol) and to N-Boc ethylenediamine (1.83 g, 11.45 mmol), a white solid of molecule 18 is obtained after trituration in acetonitrile.

(97) Yield: 6.64 g (77%)

(98) .sup.1H NMR (DMSO-d6, ppm): 0.80-0.91 (18H); 1.15-1.73 (49H); 2.03-2.18 (4H); 2.92-3.13 (6H); 4.05-4.30 (3H); 6.71-6.83 (1H); 7.69-8.23 (5H).

(99) LC/MS (ESI): 824.0 (calculated ([M+H].sup.+): 823.7).

(100) Molecule A8

(101) Using a similar method to the one used for molecule A5 applied to molecule 18 (3.00 g, 3.64 mmol) without basic washing, a beige solid of molecule A8 in hydrochloride salt form is obtained after co-evaporating the residue 4 times in methanol.

(102) Yield: 2.66 g (96%)

(103) .sup.1H NMR (DMSO-d6, ppm): 0.80-0.91 (18H); 1.15-1.76 (40H); 2.03-2.19 (4H); 1.78-2.89 (2H); 2.91-3.07 (2H); 3.22-3.37 (2H); 4.08-4.14 (1H); 4.17-4.28 (2H); 7.81-8.36 (8H).

(104) LC/MS (ESI): 723.7 (calculated ([M+H].sup.+): 723.6).

Example A9: Molecule A9

(105) Molecule 19: 13-Methyltetradecanoic acid.

(106) In a dry triple neck round bottom flask under argon, magnesium (5.50 g, 226.3 mmol) chips are introduced. The magnesium is covered with anhydrous THF (25 mL) and a few drops of 1-bromo-2-methylpropane are added at ambient temperature to initiate the reaction. After observing an exotherm and slight turbidity of the medium, the remaining 1-bromo-2-methylpropane (28.42 g, 207 mmol) diluted in THF (60 mL) is added dropwise in 1 h whereas the temperature of the medium remains stable from 65 to 70° C. The reaction medium is then reflux heated for 2 h.

(107) In a triple neck round bottom flask under argon, to a solution of CuCl (280 mg, 2.83 mmol) dissolved in N-methylpyrrolidone (NMP) previously distilled at 0° C. is added dropwise a solution of 11-bromoundecanoic acid (25 g, 94.27 mmol) dissolved in THF (60 mL). To this solution is then added dropwise the slightly warm organomagnesium solution diluted in THF (50 mL) in order to maintain the temperature of the medium below 25° C. The mixture is then stirred at ambient temperature for 16 h. The medium is cooled to 0° C. and the reaction is stopped by slowly adding a 1 N HCl aqueous solution to pH 1 (300 mL) and the medium is extracted with hexane (100 mL) and with ethyl acetate (2×75 mL). After washing the organic phase with a 1 N HCl aqueous solution (100 mL), water (100 mL) and drying on Na.sub.2SO.sub.4, the solution is filtered and concentrated under vacuum to produce a brown solid. After purification by flash chromatography (cyclohexane, ethyl acetate), a white solid is obtained.

(108) Yield: 18.1 g (79%)

(109) .sup.1H NMR (CDCl.sub.3, ppm): 0.87 (6H); 1.11-1.18 (2H); 1.20-1.38 (16H); 1.51 (1H); 1.63 (2H); 2.35 (2H).

(110) Molecule 20: Product obtained by reacting molecule 19 and L-leucine.

(111) To a solution of molecule 19 (18.05 g, 74.46 mmol) in THF (745 mL) at ambient temperature are added successively DCC (14.63 g, 70.92 mmol) and NHS (8.16 g, 70.92 mmol). After 40 h of stirring at ambient temperature, the medium is cooled to 0° C. for 20 min, filtered on a sintered filter. L-leucine (9.77 g, 74.46 mmol), DIPEA (86 mL) and water (150 mL) are added to the filtrate. After 20 h of stirring at ambient temperature, the medium is diluted with a saturated aqueous solution of NaHCO.sub.3 (200 mL). The aqueous phase is washed with ethyl acetate (2×200 mL) and acidified with a 2 N HCl solution to pH 1. The precipitate is filtered, rinsed thoroughly with water and dried under vacuum at 50° C. Three times, the solid is triturated in pentane, sonicated then filtered to produce a white solid.

(112) Yield: 18.8 g (75%)

(113) .sup.1H NMR (CDCl.sub.3, ppm): 0.86 (6H); 0.96 (6H); 1.12-1.18 (2H); 1.20-1.78 (22H); 2.24 (2H); 4.58-4.63 (1H); 5.89 (1H).

(114) LC/MS (ESI): 356.2; (calculated ([M+H].sup.+): 356.6).

(115) Molecule 21: Product obtained by the reaction between molecule 20 and Boc-tri(ethyleneglycol)diamine.

(116) To a solution of molecule 20 (16.7 g, 46.97 mmol) in THF (235 mL) are added successively DIPEA (20.3 mL) and TBTU at ambient temperature. After 20 min of stirring, Boc-tri(ethyleneglycol)diamine (14 g, 56.36 mmol) is added. After stirring at ambient temperature for 5 h, the mixture is concentrated under vacuum. The residue is taken up with ethyl acetate (500 mL), washed with a saturated aqueous solution of NaHCO3 (3×200 mL), a 1 N HCl aqueous solution (3×200 mL) and an aqueous solution saturated with NaCl (3×200 mL). After drying on Na2SO4, filtration and concentration under vacuum, the residue is purified by flash chromatography (cyclohexane, ethyl acetate, methanol) to produce a colorless oil.

(117) Yield: 23.5 g (85%)

(118) .sup.1H NMR (CDCl.sub.3, ppm): 0.86 (6H); 0.93 (6H); 1.10-1.17 (2H); 1.19-1.08 (31H); 2.18 (2H); 3.23-3.65 (12H); 4.41-4.56 (1H); 5.12-5.47 (1H); 5.99-6.11 (0.75H); 6.48-6.65 (1H); 7.30-7.40 (0.25H).

(119) Molecule A9

(120) Using a similar method to the one used for the preparation of molecule A5 applied to molecule 21 (23.46 g, 40.04 mmol) without basic washing, the residue obtained after concentration under vacuum is triturated in an acetonitrile/acetone mixture. The supernatant is removed and the pasty residue is dried under vacuum. The residue is triturated in acetone (150 mL) and the white solid of molecule A9 in hydrochloride salt form is filtered, rinsed with acetone then dried under vacuum.

(121) Yield: 13.0 g (64%)

(122) .sup.1H NMR (DMSO-d6, ppm): 0.79-0.90 (12H); 1.09-1.61 (24H); 2.03-2.17 (2H); 2.92-2.98 (2H); 3.15-3.23 (2H); 3.40 (2H); 3.50-3.58 (4H); 3.61 (2H); 4.30-4.23 (1H); 7.88-8.14 (5H).

(123) LC/MS (ESI): 486.4; (calculated ([M−Cl].sup.+): 486.8).

Example A10: Molecule A10

(124) Molecule 22: Product obtained by the reaction between octanoyl chloride and L-proline.

(125) Using a similar method to the one used for the preparation of molecule A1 and applied to octanoyl chloride (150.0 g, 0.922 mol) and to L-proline (212.3 g, 1.844 mol), a colorless oil of molecule 22 is obtained after washing the organic phase with a 10% HCl aqueous solution (3×300 mL), an aqueous solution saturated with NaCl (300 mL), drying on Na.sub.2SO.sub.4, filtration on cotton, concentration under reduced pressure, then the residue is purified by flash chromatography (eluent: DCM, MeOH)

(126) Yield: 134 g (60%)

(127) .sup.1H NMR (CDCl.sub.3, ppm): 0.87 (3H); 1.10-1.52 (8H); 1.57-1.74 (2H); 1.79-2.52 (6H); 3.37-3.67 (2H); 4.37-4.42 (0.07H); 4.53-5.63 (0.93H); 9.83 (1H).

(128) LC/MS (ESI): 242.1; (calculated ([M+H].sup.+): 242.2).

(129) Molecule 23: Product obtained by coupling molecule 22 and L-leucine.

(130) To a solution of molecule 22 (132 g, 0.547 mol) in THF (924 mL) cooled to a temperature below 5° C. are added successively NHS (66.1 g, 0.574 mol) and DCC (118.5 g, 0.574 mol). After 21 h of stirring, the precipitate is removed by precipitation and the filtrate is added in 30 min to a solution of L-lysine (41.98 g, 0.287 mol) in a mixture of deionized water (82 mL) and DIPEA (476 mL, 2.735 mol) at 15° C. After 23 h of stirring at ambient temperature, the reaction medium is concentrated under reduced pressure to produce an oily residue which is diluted in water (1.3 L). The aqueous phase is washed twice with AcOEt (2×0.5 L), cooled to a temperature below 10° C., acidified by adding a 6 N HCl solution (120 mL) to pH 1 then extracted three times with DCM (3×0.6 L). The organic phases are combined, washed with a saturated NaCl solution (0.6 L), dried on Na.sub.2SO.sub.4 then concentrated under reduced pressure. The foam obtained is taken up with acetone (240 mL) at reflux for 2 h. After leaving overnight at 10° C., pentane (240 mL) is added dropwise. After 1 h of stirring, the precipitate is recovered by filtering under vacuum, washed with a 1:1 mixture of pentane and acetone (150 mL) then dried under a vacuum.

(131) Yield: 83.9 g (52%)

(132) .sup.1H NMR (CDCl.sub.3, ppm): 0.87 (6H); 1.06-1.78 (25H); 1.80-2.41 (13H); 2.80-3.72 (6H); 4.30-4.39 (0.15H); 4.46-4.70 (2.85H); 7.84 (1H); 7.93 (1H).

(133) LC/MS (ESI): 593.5; (calculated ([M+H].sup.+): 593.4).

(134) Molecule 24: Product obtained by coupling molecule 23 and L-lysine methyl ester (LysOMe).

(135) To molecule 23 (76.26 g, 0.129 mol) are successively added HOPO (3.57 g, 32.1 mmol), LysOMe dihydrochloride (15.0 g, 64.3 mmol) and EDC (34.53 g, 0.18 mol). Then DMF (600 mL) previously cooled to 5° C. is added. After dissolution, triethylamine (43.9 mL, 0.315 mol) is added dropwise while maintaining the temperature below 5° C. for 2 h after addition. After leaving overnight at ambient temperature, the reaction medium is poured onto a mixture of water/ice (2 kg) and DCM (0.5 L). After 15 min of stirring, the phases are separated. The aqueous phase is extracted twice with DCM (2×0.4 L). The organic phases are combined, washed with a 1 N HCl solution (0.5 L) then with a saturated NaCl solution (0.5 L), dried on Na.sub.2SO.sub.4, concentrated under reduced pressure, then the residue is purified by flash chromatography (eluent: DCM, MeOH).

(136) Yield: 56.7 g (67%)

(137) .sup.1H NMR (CDCl.sub.3, ppm): 0.87 (12H); 1.10-2.40 (82H); 2.86-3.72 (17H); 4.16-4.60 (7H); 6.83-8.01 (6H).

(138) Molecule A10

(139) A solution of molecule 24 (4.0 g, 3.05 mmol) in ethylenediamine (30 mL) is heated at 50° C. overnight. The reaction medium is then diluted with methyl-tetrahydrofuran then the organic phase is washed 4 times with a saturated NaCl solution (4×30 mL) then 2 times with water (2×50 mL) before being dried on Na.sub.2SO.sub.4 and concentrated under reduced pressure. The residue is solubilized in acetonitrile at reflux for 30 min then the solution is cooled to ambient temperature under stirring overnight. The white precipitate is then recovered by filtering under vacuum, washed with cold acetonitrile (2×20 mL) then dried under vacuum.

(140) Yield: 3.0 g (74%)

(141) .sup.1H NMR (CDCl.sub.3, ppm): 0.87 (12H); 1.09-2.37 (84H); 2.74-4.56 (25H); 6.85-8.00 (7H).

(142) LC/MS (ESI): 1338.0 (calculated ([M+H].sup.+): 1338.0).

Example A11: Molecule A11

(143) Molecule 25: Product obtained by the reaction between molecule 13 and lauric acid.

(144) Using a similar method to the one used for molecule 5 applied to molecule 13 (28 mmol) and lauric acid (28.04 g, 140 mmol) in DMF (330 mL), molecule 25 is obtained.

(145) Molecule A11

(146) Using a similar method to the one used for molecule 8 applied to molecule 25, a white solid of molecule A11 is obtained after recrystallization in acetonitrile.

(147) Yield: 13.9 g (56% in 6 stages)

(148) .sup.1H NMR (DMSO-d6, ppm): 0.85 (6H); 1.05-1.61 (60H); 1.62-1.75 (2H); 1.78-1.91 (2H); 2.04-2.27 (8H); 2.96-3.06 (2H); 4.08-4.13 (1H); 4.17-4.22 (1H); 4.27-4.34 (1H); 7.82 (1H); 7.86 (1H); 7.90 (1H); 8.03 (1H); 12.54 (1H).

(149) LC/MS (ESI+): 881.7 (calculated ([M+H].sup.+): 881.7).

Example A12: Molecule A12

(150) Molecule 26: Product obtained by the reaction between molecule 13 and Fmoc-Glu(OtBu)-OH.

(151) Using a similar method to the one used for molecule 5 applied to molecule 13 (9.92 mmol) and to Fmoc-Glu(OtBu)-OH (21.10 g, 49.60 mmol) in N-methyl-2-pyrrolidone (NMP, 120 mL), molecule 26 is obtained.

(152) Molecule 27: Product obtained by the reaction between molecule 26 and an 80:20 NMP/piperidine mixture.

(153) Using a similar method to the one used for molecule 4 applied to molecule 26, using NMP instead of DMF, molecule 27 is obtained.

(154) Molecule 28: Product obtained by the reaction between molecule 27 and Fmoc-Glu(OtBu)-OH.

(155) Using a similar method to the one used for molecule 26 applied to molecule 27 and to Fmoc-Glu(OtBu)-OH (21.10 g, 49.60 mmol), molecule 28 is obtained.

(156) Molecule 29: Product obtained by the reaction between molecule 28 and an 80:20 NMP/piperidine mixture.

(157) Using a similar method to the one used for molecule 27 applied to molecule 28, molecule 29 is obtained.

(158) Molecule 30: Product obtained by the reaction between molecule 29 and molecule A1.

(159) Using a similar method to the one used for molecule 26 applied to molecule 29 (4.96 mmol) and to molecule A1 (8.07 g, 24.80 mmol), molecule 30 is obtained.

(160) Molecule A12

(161) Using a similar method to the one used for molecule 8 applied to molecule 30, a white solid of molecule A12 is obtained after purification by flash chromatography (DCM, MeOH).

(162) Yield: 4.6 g (50% in 10 stages)

(163) .sup.1H NMR (CD.sub.3OD, ppm): 0.90 (6H); 1.22-2.53 (140H); 3.12-3.25 (2H); 3.43-3.80 (4H); 4.17-4.54 (9H).

(164) LC/MS (ESI+): 1894.5 (calculated ([M+Na].sup.+): 1894.2).

Example A14: Molecule A14

(165) Molecule 33: product obtained by reacting N-□-Boc-L-Lysine and palmitoyl chloride

(166) Using a similar method to the one used for the preparation of molecule A1 applied to N-□-Boc-L-Lysine (53.76 g, 218.28 mmol) and to palmitoyl chloride (50.00 g, 181.90 mmol), a white solid of molecule 33 is obtained after recrystallizing 2 times in acetonitrile and purification by flash chromatography (eluent: dichloromethane, methanol).

(167) Yield: 49.10 g (70%)

(168) .sup.1H NMR (DMSO-d.sub.6, ppm): 0.85 (3H); 1.09-1.66 (32H); 1.37 (9H); 2.01 (2H); 2.93-3.06 (2H); 3.78-3.85 (1H); 6.61-6.68 (0.2H); 6.96-6.98 (0.8H); 7.66-7.75 (1H); 12.38 (1H).

(169) LC/MS (ESI): 385.1 (calculated ([M-Boc+H].sup.+): 385.3).

(170) Molecule 34: Product obtained by the reaction between molecule 33 and methyl iodide.

(171) To a solution of molecule 33 (23.40 g, 48.28 mmol) in DMF (200 mL) at ambient temperature are added K.sub.2CO.sub.3 (10.01 g, 72.41 mmol) followed by methyl iodide (5.96 mL, 98.55 mmol). The medium is stirred for 48 h. Water (350 mL) is added and the suspension is stirred for 15 min. The mixture is then filtered on a sintered filter and the solid obtained is rinsed with water (2×250 mL) and dried under vacuum. The solid is then solubilized in DCM (300 mL). The solution is washed with water (200 mL) then with an aqueous solution saturated with NaCl (200 mL), dried on Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. A white solid of molecule 34 is obtained after recrystallization in acetonitrile.

(172) Yield: 19.22 g (80%)

(173) .sup.1H NMR (CDCl.sub.3, ppm): 0.87 (3H); 1.06-2.23 (34H); 1.43 (9H); 3.09-3.33 (2H); 3.72 (3H); 3.94-4.35 (1H); 4.69-5.23 (1H); 5.33-5.75 (1H).

(174) LC/MS (ESI): 543.3 (calculated ([M−H+HCOOH].sup.−): 543.4).

(175) Molecule 35: Product obtained by hydrolyzing molecule 34 with hydrochloric acid

(176) Using a similar method to that used for the preparation of molecule A5 applied to molecule 34 in solution in a 1:1 DCM/methanol mixture (385 mL), a white solid of molecule 35 is obtained after concentration under reduced pressure and co-evaporation with DCM followed by methanol.

(177) Yield: 16.73 g (99%)

(178) .sup.1H NMR (DMSO-d.sub.6, ppm): 0.85 (3H); 1.08-1.50 (30H); 1.67-1.84 (2H); 2.03 (2H); 2.94-3.13 (2H); 3.74 (3H); 3.92-4.01 (1H); 7.77-7.87 (1H); 8.25-8.73 (3H).

(179) LC/MS (ESI): 399.2 (calculated ([M+H].sup.+): 399.4).

(180) Molecule A14

(181) To a suspension of molecule 35 (14.70 g, 33.79 mmol) in a mixture of methyl-THF (338 mL) and DMF (30 mL) are added successively DIPEA (17.70 mL, 101.40 mmol) followed by a solution of succinic anhydride (5.07 g, 50.68 mmol) in THF (60 mL). The medium is stirred for 4 h at ambient temperature. Methyl-THF (100 mL) is added and the organic phase is washed with a 5% HCl aqueous solution (300 mL). The aqueous phase is extracted with methyl-DCM (2×150 mL). The combined organic phases are washed with water (2×150 mL) then with an aqueous solution saturated with NaCl (150 mL), dried on Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The crude product is purified by flash chromatography (eluent: DCM, methanol) then solubilized in methyl-THF. The purified product is then suspended in water. The suspension is stirred by sonication for 20 min followed by magnetic stirring for 30 min. A white solid of molecule A14 is obtained after filtration and drying under reduced pressure.

(182) Yield: 12.99 g (77%)

(183) .sup.1H NMR (DMSO-d.sub.6, ppm): 0.85 (3H); 1.08-1.71 (32H); 2.02 (2H); 2.29-2.45 (4H); 2.94-3.04 (2H); 3.61 (3H); 4.14-4.22 (1H); 7.70 (1H); 8.20 (1H); 12.04 (1H).

(184) LC/MS (ESI): 499.3 (calculated ([M+H].sup.+): 499.4).

Example A15: Molecule A15

(185) molecule 36: product obtained by coupling L-proline and palmitoyl chloride

(186) Using a similar method to the one used for the preparation of molecule A1 applied to L-proline (38.05 g, 906.00 mmol) and to palmitoyl chloride (14.01 g, 350.16 mmol), a white solid of molecule 36 is obtained.

(187) Yield: 47.39 g (96%)

(188) .sup.1H NMR (CDCl.sub.3, ppm): 0.88 (3H); 1.19-1.45 (24H); 1.58-1.74 (2H); 1.88-2.14 (3H); 2.15-2.54 (3H); 3.47 (1H); 3.58 (1H); 4.41 (0.1H); 4.61 (0.9H) 6.60-8.60 (1H).

(189) LC/MS (ESI): 354.5 (calculated ([M+H].sup.+): 354.3).

(190) Molecule 37: Product obtained by the reaction between molecule 36 and N-Bocethylenediamine.

(191) Using a similar method to the one used for molecule 9 applied to molecule 36 (75.1 g, 212.4 mmol), a white solid of molecule 37 is obtained after trituration in diisopropylether (3×400 mL) and vacuum-drying at 40° C.

(192) Yield: 90.4 g (86%).

(193) .sup.1H NMR (CDCl.sub.3, ppm): 0.88 (3H); 1.20-1.37 (24H); 1.44 (9H); 1.54-1.70 (2H); 1.79-1.92 (1H); 1.92-2.04 (1H); 2.03-2.17 (1H); 2.17-2.44 (3H); 3.14-3.36 (4H); 3.43 (1H); 3.56 (1H); 4.29 (0.1H); 4.51 (0.9H); 4.82 (0.1H); 5.02 (0.9H); 6.84 (0.1H); 7.22 (0.9H).

(194) Molecule 38: Product obtained by hydrolyzing molecule 37 with hydrochloric acid

(195) Using a similar method to the one used for the preparation of molecule A5 applied to molecule 37 (38.17 g, 76.99 mmol), a white solid of molecule 38 is obtained.

(196) .sup.1H NMR (CDCl.sub.3, ppm): 0.88 (3H); 1.07-1.40 (24H); 1.49-1.63 (2H); 1.77-2.18 (4H); 2.18-2.45 (2H); 3.14-3.32 (2H); 3.42-3.63 (2H); 3.63-3.84 (2H); 4.37 (0.1H); 4.48 (0.9H); 6.81-8.81 (4H).

(197) LC/MS (ESI): 396.5; (calculated ([M+H].sup.+): 396.4).

(198) Molecule A15

(199) Using a similar method to the one used for the preparation of molecule A14 applied to molecule 38 (10.00 g, 253.00 mmol), a white solid of molecule A15 is obtained.

(200) Yield: 10.00 g (80%)

(201) .sup.1H NMR (DMSO, ppm): 0.85 (3H); 1.07-1.51 (26H); 1.69-2.02 (4H); 2.08-2.53 (6H); 3.01-3.18 (4H); 3.39-3.58 (2H); 4.13-4.18 (0.7H); 4.23-4.27 (0.3H); 7.70-7.78 (1.4H); 7.81-7.86 (0.3H); 8.00-8.04 (0.3H); 12.08 (1H).

(202) LC/MS (ESI): 496.3 (calculated ([M+H].sup.+): 496.4).

Example A16: Molecule A16

(203) Molecule 39: Product obtained by the reaction between molecule 36 and Boc-1-amino-4,7,10-trioxa-13-tridecane amine.

(204) Using a similar method to the one used for the preparation of molecule 9 applied to molecule 36 (17.00 g, 48.08 mmol) and to Boc-1-amino-4,7,10-trioxa-13-tridecane amine (18.49 g, 57.70 mmol), a pale yellow oil of molecule 39 is obtained.

(205) Yield: 31.11 g (98%)

(206) .sup.1H NMR (DMSO-d.sub.6, ppm): 0.85 (3H); 1.17-1.31 (24H); 1.37 (9H); 1.41-1.51 (2H); 1.54-1.67 (4H); 1.69-2.02 (4H); 2.08-2.29 (2H); 2.91-3.00 (2H); 3.01-3.17 (2H); 3.31-3.58 (14H); 4.20 (0.65H); 4.26 (0.35H); 6.29-6.82 (1H); 7.68 (0.65H); 8.02 (0.35H).

(207) LC/MS (ESI): 656.4 (calculated ([M+H].sup.+): 656.5).

(208) Molecule 40: Product obtained by hydrolyzing molecule 39 with hydrochloric acid

(209) Using a similar method to the one used for the preparation of molecule A5 applied to molecule 39 (31.11 g, 47.43 mmol), a yellow wax of molecule 40 is obtained.

(210) Yield: 27 g (97%)

(211) .sup.1H NMR (DMSO-d.sub.6, ppm): 0.85 (3H); 1.18-1.31 (24H); 1.40-1.51 (2H); 1.55-1.67 (2H); 1.70-2.04 (6H); 2.09-2.30 (2H); 2.78-2.89 (2H); 2.99-3.18 (2H); 3.33-3.58 (14H); 4.19 (0.65H); 4.27 (0.35H); 7.55-8.14 (4H).

(212) LC/MS (ESI): 556.3 (calculated ([M+H].sup.+): 556.5).

(213) Molecule A16

(214) Molecule 40 (26.40 g, 44.50 mmol) in hydrochloride form is solubilized in a mixture of DCM (350 mL) and an aqueous solution of NaHCO.sub.3 (350 mL). The organic phase is separated and the aqueous phase is extracted with DCM (2×150 mL). The organic phases are combined dried on Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure to produce a colorless oil. Using a similar method to the one used for the preparation of molecule A14, a yellow resin of molecule A16 is obtained after purification by flash chromatography (eluent: DCM, methanol).

(215) Yield: 19.93 g (68%)

(216) .sup.1H NMR (DMSO-d.sub.6, ppm): 0.85 (3H); 1.18-1.30 (24H); 1.40-1.51 (2H); 1.55-1.67 (4H); 1.70-2.02 (4H); 2.07-2.45 (6H); 2.99-3.18 (4H); 3.33-3.57 (14H); 4.19 (0.65H); 4.26 (0.35H); 7.68 (0.65H); 7.78 (1H); 8.02 (0.35H); 12.03 (1H).

(217) LC/MS (ESI): 656.3 (calculated ([M+H].sup.+): 656.5).

Example A17: Molecule A17

(218) Molecule 41: Product obtained by solid phase peptide synthesis (SPPS)

(219) Molecule 41 is obtained by means of the conventional solid phase peptide synthesis (SPPS) method on 2-chlorotrityl resin

(220) To a solution of 4,7,10-trioxa-1,13-tridecanediamine (TOTA, 76.73 mL, 350 mmol) in DCM (350 mL) is added DIPEA (60.96 mL, 350 mmol). This solution is then poured onto the 2-chlorotrityl resin (47.30 g, 0.74 mmol/g) previously washed with DCM in a reaction vessel suitable for SPPS. After 1.5 h of stirring at ambient temperature, methanol (26 mL) is added and the medium is stirred for 15 min. The resin is filtered, washed successively with DCM (3×350 mL), DMF (2×350 mL), DCM (2×350 mL), isopropanol (1×350 mL) and DCM (3×350 mL). The □-methyl ester of N-Fmoc-L-glutamic acid (1,5 eq) followed by molecule 36 (1.5 eq) are coupled using the coupling agent 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU, 1.5 equivalents) and DIPEA (3 equivalents) in a 1:1 DCM/DMF mixture. A 1:1 DMF/morpholine mixture is used for the cleavage step of the Fmoc protecting group. The resin is washed with DCM, DMF and methanol after each coupling and deprotection step. The cleavage of the product from the resin is carried out using a 1:1 TFA/DCM mixture. The solvents are then evaporated under vacuum; the residue is solubilized in DCM (500 mL) and the organic phase is washed with a 5% Na.sub.2CO.sub.3 aqueous solution (500 mL). After drying on Na.sub.2SO.sub.4, the organic phase is filtered, concentrated under vacuum and a yellow oil of molecule 41 is obtained after drying under reduced pressure.

(221) Yield: 15.95 g (65%)

(222) 1H NMR (DMSO-d6, ppm): 0.85 (3H); 1.16-1.31 (24H); 1.38-1.68 (6H); 1.68-2.37 (12H); 2.58 (2H); 3.01-3.17 (2H); 3.31-3.55 (14H); 3.58 (3H); 4.09-4.18 (0.7H); 4.18-4.29 (1H); 4.36-4.43 (0.3H); 7.62 (0.7H); 7.86 (0.7H); 7.98 (0.3H); 8.23 (0.3H).

(223) LC/MS (ESI): 699.4 (calculated ([M+H]+): 699.5).

(224) Molecule A17

(225) Using a similar method to the one used for the preparation of molecule A14 applied to molecule 41 (14.05 g, 20.10 mmol), a yellow resin of molecule A17 is obtained after purification by flash chromatography (eluent: DCM, methanol).

(226) Yield: 7.70 g (48%)

(227) 1H NMR (DMSO-d6, ppm): 0.85 (3H); 1.17-1.31 (24H); 1.38-1.54 (2H); 1.54-1.68 (4H); 1.68-2.21 (7H); 2.21-2.36 (5H); 2.36-2.44 (2H); 3.01-3.16 (4H); 3.34-3.55 (14H); 3.57 (3H); 4.10-4.18 (0.7H); 4.18-4.30 (1H); 4.40 (0.3H); 7.60 (0.7H); 7.78 (1H); 7.85 (0.7H); 7.95 (0.3H); 8.22 (0.3H); 12.06 (1H).

(228) LC/MS (ESI): 799.5 (calculated ([M+H]+): 799.5).

Example A18: Molecule A18

(229) Molecule 42: Product obtained by the reaction between molecule A1 and Boc-1-amino-4,7,10-trioxa-13-tridecane amine.

(230) Using a similar method to the one used for the preparation of molecule 18 applied to molecule A1 (44.80 g, 137.64 mmol) and to Boc-1-amino-4,7,10-trioxa-13-tridecane amine (52.92 g, 165.16 mmol), an orange oil of molecule 42 is obtained.

(231) Yield: 85.63 g (99%)

(232) 1H NMR (CDCl.sub.3, ppm): 0.87 (3H); 1.08-1.56 (20H); 1.43 (9H); 1.58-1.67 (2H); 1.70-2.00 (6H); 2.04-2.41 (4H); 3.16-3.77 (18H); 4.26-4.29 (0.2H); 4.50-4.54 (0.8H); 4.68-5.10 (1H); 6.74 (0.2H); 7.19 (0.8H).

(233) LC/MS (ESI): 628.4; (calculated ([M+H].sup.+): 628.5).

(234) Molecule 43: Product obtained by hydrolyzing molecule 42 with hydrochloric acid

(235) Using a similar method to the one used for the preparation of molecule A5 applied to molecule 42 (43.40 g, 69.12 mmol), a white solid of molecule 43 in hydrochloride salt form is obtained after trituration in diethylether, solubilisation of the residue in water and lyophilization.

(236) Yield: 38.70 g (98%)

(237) 1H NMR (DMSO, ppm): 0.85 (3H); 1.07-1.38 (20H); 1.41-1.52 (2H); 1.55-1.66 (2H); 1.70-2.02 (6H); 2.08-2.30 (2H); 2.78-2.87 (2H); 3.00-3.16 (2H); 3.29-3.66 (14H); 4.16-4.22 (0.65H); 4.25-4.30 (0.35H); 7.74 (0.65H); 7.86 (3H); 8.10 (0.35H).

(238) LC/MS (ESI): 528.4; (calculated ([M+H].sup.+): 528.4).

(239) Molecule A18

(240) Using a similar method to the one used for the preparation of molecule A14 applied to molecule 43 (13.09 g, 24.8 mmol), a yellow resin of molecule A18 is obtained after purification by flash chromatography (eluent: DCM, methanol).

(241) Yield: 8.53 g (55%)

(242) 1H NMR (DMSO-d6, ppm): 0.86 (3H); 1.10-1.39 (20H); 1.42-1.51 (2H); 1.57-1.67 (4H); 1.71-2.03 (4H); 2.09-2.32 (4H); 2.42 (2H); 3.01-3.17 (4H); 3.36-3.57 (14H); 4.18-4.21 (0.65H); 4.24-4.28 (0.35H); 7.69 (0.65H); 7.80 (1H); 8.03 (0.35H); 12.04 (1H).

(243) LC/MS (ESI): 628.5 (calculated ([M+H].sup.+): 628.5).

Example A19: Molecule A19

(244) Molecule 44: Product obtained by SPPS

(245) By means of a similar SPPS method to the one used for the preparation of molecule 41 and applied to TOTA, to N-Fmoc-L-Leucine, N-Fmoc-L-proline and to myristic acid, an orange oil of molecule 44 is obtained.

(246) Yield: 19.87 g (69%)

(247) .sup.1H NMR (CDCl.sub.3, ppm): 0.72-1.06 (9H); 1.09-1.42 (20H); 1.42-2.40 (17H); 2.80 (2H); 3.22-3.81 (16H); 4.25-4.61 (2H); 6.56-7.23 (2H).

(248) LC/MS (ESI): 641.5; (calculated ([M+H].sup.+): 641.5).

(249) Molecule A19

(250) After a similar method to the one used for the preparation of molecule A14 applied to molecule 44 (13.09 g, 204.42 mmol), 4.81 g of the product obtained by purification by flash chromatography (eluent: DCM, methanol) is solubilized in a mixture of DCM (50 mL) and THF (5.5 mL) then washed with an aqueous solution saturated with NaCl (50 mL), a 0.1 N HCl aqueous solution (50 mL) and an aqueous solution saturated with NaCl (50 mL). The organic phase is dried on Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. A yellow oil of molecule A19 is obtained.

(251) Yield: 4.20 g

(252) .sup.1H NMR (DMSO-d.sub.6, ppm): 0.72-1.02 (9H); 1.08-1.34 (20H); 1.34-2.23 (14H); 2.23-2.35 (3H); 2.42 (2H); 3.01-3.17 (4H); 3.17-3.66 (14H); 4.15-4.44 (2H); 7.53-8.23 (3H); 12.06 (1H).

(253) LC/MS (ESI): 741.5; (calculated ([M+H].sup.+): 741.5).

Example A21: Molecule A21

(254) Molecule 46: Product obtained by SPPS

(255) By means of a similar SPPS method to the one used for the preparation of molecule 41 and applied to TOTA, to N-Fmoc-L-phenylalanine and to molecule A1, an orange oil of molecule 46 is obtained and used without purification.

(256) Yield: 15.07 g (72%)

(257) .sup.1H NMR (CDCl.sub.3, ppm): 0.87 (3H); 1.08-1.42 (20H); 1.42-1.62 (2H); 1.62-1.99 (7H); 1.99-2.26 (3H); 2.72 (2H); 2.86 (2H); 2.94-3.72 (18H); 4.20-4.72 (2H); 6.63-7.37 (7H).

(258) LC/MS (ESI): 675.65; (calculated ([M+H].sup.+): 675.5).

(259) Molecule A21

(260) Using a similar method to the one used for the preparation of molecule A19 applied to molecule 46 (13.79 g, 20.43 mmol), a white solid of molecule A21 is obtained.

(261) Yield: 7.56 g (48%)

(262) .sup.1H NMR (DMSO-d.sub.6, ppm): 0.86 (3H); 1.02-1.42 (21H); 1.42-2.20 (10H); 2.23-2.38 (3H); 2.42 (2H); 2.78-3.18 (6H); 3.23-3.59 (14H); 4.12-4.58 (2H); 7.10-7.30 (5H); 7.53-8.33 (3H); 12.08 (1H).

(263) LC/MS (ESI): 775.5; (calculated ([M+H].sup.+): 775.5).

Example A22: Molecule A22

(264) Using a similar method to the one used for the preparation of molecule A14 applied to molecule A6 (22.15 g, 43.19 mmol), a yellow oil of molecule A22 is obtained.

(265) Yield: 25.19 g (95%)

(266) .sup.1H NMR (DMSO-d.sub.6, ppm): 0.42-1.51 (33H); 1.51-2.05 (8H); 2.29 (2H); 2.41 (2H); 3.07 (4H); 3.38 (4H); 3.43-3.54 (8H); 7.72 (1H); 7.79 (1H); 12.03 (1H).

(267) LC/MS (ESI): 613.5 (calculated ([M+H].sup.+): 613.5).

Example A23: Molecule A23

(268) Molecule 47: Product obtained by hydrogenating phytol.

(269) To a solution of phytol (30.00 g, 101.20 mmol) in THF (450 mL) in argon is added platinum dioxide (PtO.sub.2, 1.15 g, 6.61 mmol). The medium is placed under 1 bar of dihydrogen then stirred for 4 h at ambient temperature. After filtration under celite by rinsing with THF, a black oil of molecule 47 is obtained after concentration under reduced pressure.

(270) Yield: 29.00 g (96%)

(271) .sup.1H NMR (CDCl.sub.3, ppm): 0.84 (6H); 0.86 (6H); 0.89 (3H); 1.00-1.46 (22H); 1.46-1.68 (3H); 3.61-3.73 (2H).

(272) Molecule 48: Product obtained by oxidizing molecule 47

(273) To a solution of molecule 47 (29.0 g, 97.13 mmol) in a dichloroethane/water mixture (485 mL/388 mL) are added successively tetrabutylammonium bromide (16.90 g, 52.45 mmol), acetic acid (150 mL, 2.62 mol) followed by KMnO.sub.4 (46.05 g, 291.40 mmol) in small fractions while maintaining the temperature from 16 to 19° C. The reaction medium is then stirred for 4.5 h at reflux, cooled to 10° C. then acidified to pH 1 with a 6 N HCl solution (20 mL). Na.sub.2SO.sub.3 (53.90 g) is added progressively while maintaining the temperature at 10° C. and the medium is stirred until completely discolored. Water (200 mL) is added, the phases are separated and the aqueous phase is extracted with DCM (2×400 mL). The combined organic phases are washed with a 10% HCl aqueous solution (20 mL), water (2×200 mL), an aqueous solution saturated with NaCl (200 mL), dried on Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. A yellow oil of molecule 48 is obtained after purification by flash chromatography (eluent: cyclohexane, AcOEt).

(274) Yield: 28.70 g (94%)

(275) .sup.1H NMR (CDCl.sub.3, ppm): 0.84 (6H); 0.86 (6H); 0.97 (3H); 1.00-1.41 (20H); 1.52 (1H); 1.96 (1H); 2.14 (1H); 2.35 (1H); 11.31 (1H).

(276) LC/MS (ESI): 311.1 (calculated ([M−H].sup.−): 311.3).

(277) Molecule 49: Product obtained by coupling molecule 48 and methyl L-prolinate.

(278) Using a similar method to the one used for the preparation of molecule 9 applied to molecule 48 (18.00 g, 57.59 mmol) and to methyl L-prolinate hydrochloride (14.31 g, 86.39 mmol) in DCM (380 mL), a yellow oil of molecule 49 is obtained after washing the organic phase with an aqueous solution saturated with NaHCO.sub.3 (2×150 mL), a 10% HCL aqueous solution (2×150 mL), an aqueous solution saturated with NaCl (2×150 mL), followed by drying on Na.sub.2SO.sub.4, filtration and concentration under reduced pressure.

(279) Yield: 23.20 g (95%)

(280) .sup.1H NMR (DMSO-d.sub.6, ppm): 0.78-0.89 (15H); 0.97-1.43 (20H); 1.43-1.56 (1H); 1.70-1.96 (4H); 1.96-2.32 (3H); 3.33-3.56 (2H); 3.59 (0.6H); 3.67 (2.4H); 4.27 (0.8H); 4.57 (0.2H).

(281) LC/MS (ESI): 424.4 (calculated ([M+H].sup.+): 424.4).

(282) Molecule 50: Product obtained by saponifying molecule 49.

(283) Using a similar method to the one used for the preparation of molecule 17 applied to molecule 49 (21.05 g, 49.68 mmol), a yellow oil of molecule 50 is obtained.

(284) Yield: 20.40 g (99%)

(285) .sup.1H NMR (DMSO-d.sub.6, ppm): 0.77-0.91 (15H); 0.97-1.43 (20H); 1.43-1.56 (1H); 1.67-1.96 (4H); 1.96-2.29 (3H); 3.26-3.56 (2H); 4.20 (0.8H); 4.41 (0.2H).

(286) LC/MS (ESI): 410.3 (calculated ([M+H].sup.+): 410.4).

(287) Molecule 51: Product obtained by coupling molecule 50 and Boc-1-amino-4,7,10-trioxa-13-tridecane amine.

(288) Using a similar method to the one used for the preparation of molecule 9 applied to molecule 50 (8.95 g, 21.85 mmol) and to TOTA (8.40 g, 26.21 mmol), a colorless oil of molecule 51 is obtained after purification by flash chromatography (eluent: DCM, AcOEt, methanol).

(289) Yield: 10.08 g (65%)

(290) .sup.1H NMR (DMSO-d.sub.6, ppm): 0.78-0.89 (15H); 0.97-1.43 (29H); 1.43-1.55 (1H); 1.55-1.66 (4H); 1.71-2.30 (7H); 2.95 (2H); 3.00-3.19 (2H); 3.34-3.58 (14H); 4.17-4.29 (1H); 6.30-6.79 (1H); 7.67 (0.65H); 8.00 (0.35H).

(291) LC/MS (ESI): 712.6 (calculated ([M+H].sup.+): 712.6).

(292) Molecule 52: Product obtained by hydrolyzing molecule 42 with hydrochloric acid

(293) Using a similar method to the one used for the preparation of molecule A5 applied to molecule 51 (10.08 g, 14.16 mmol), the residue obtained after concentration under reduced pressure is solubilized in DCM (200 mL). The organic phase is washed with a 2 N NaOH aqueous solution (2×100 mL), dried on Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. A colorless oil of molecule 52 in neutral amine form is obtained.

(294) Yield: 8.23 g (95%)

(295) .sup.1H NMR (DMSO-d.sub.6, ppm): 0.78-0.89 (15H); 0.97-1.43 (20H); 1.43-1.69 (6H); 1.69-2.30 (8H); 2.56 (2H); 2.99-3.19 (2H); 3.31-3.58 (14H); 4.15-4.29 (1H); 7.70 (0.65H); 8.04 (0.35H).

(296) LC/MS (ESI): 612.5 (calculated ([M+H]+): 612.5).

(297) Molecule A23

(298) Using a similar method to the one used for the preparation of molecule A14 applied to molecule 52 (15.40 g, 25.17 mmol), a yellow oil of molecule A23 is obtained.

(299) Yield: 15.19 g (85%)

(300) .sup.1H NMR (DMSO-d.sub.6, ppm): 0.76-0.91 (15H); 0.98-2.26 (32H); 2.29 (2H); 2.41 (2H); 2.98-3.18 (4H); 3.32-3.63 (14H); 4.15-4.29 (1H); 7.68 (0.7H); 7.78 (1H); 8.01 (0.3H); 12.02 (1H).

(301) LC/MS (ESI): 712.5 (calculated ([M+H].sup.+): 712.5).

Example A26: Molecule A26

(302) Molecule 55: Product obtained by SPPS

(303) Molecule 55 is obtained by means of the conventional solid phase peptide synthesis (SPPS) method on 2-chlorotrityl chloride (CTC) resin (47.56 g, 0.74 mmol/g).

(304) The grafting of the first amino acid Fmoc-Glu(OtBu)-OH (2.5 equivalents) is performed in DCM (10 V), in the presence of DIPEA (5.0 equivalents). The unreacted sites are capped with methanol (0.8 mL/g resin) at the end of the reaction.

(305) The protected amino acids Fmoc-Glu(OtBu)-OH (1.5 equivalents (×2)) and molecule A1 (1.5 equivalents) are coupled in DMF (10 V), in the presence of HATU (1.0 equivalent with respect to the acid) and DIPEA (2.0 equivalents with respect to the acid).

(306) The protecting groups Fmoc are removed using an 80:20 DMF/piperidine solution (10 V).

(307) The product is cleaved from the resin using an 80:20 DCM/HFIP solution (10 V).

(308) After concentration under reduced pressure, two co-evaporations are performed on the residue with dichloromethane followed by diisopropylether. The product is purified by silica gel chromatography (dichloromethane, methanol). A colorless gum of molecule 55 is obtained.

(309) Yield: 21.4 g (69% in 8 stages)

(310) .sup.1H NMR (DMSO-d6, ppm): 0.85 (3H); 1.16-1.30 (20H); 1.34-1.41 (27H); 1.41-1.53 (2H); 1.67-2.33 (18H); 3.26-3.60 (2H); 4.09-4.44 (4H); 7.73 (0.65H); 7.85 (0.65H); 7.93-8.04 (1H); 8.17 (0.35H); 8.27 (0.35H); 12.64 (1H).

(311) LC/MS (ESI+): 881.7 (calculated ([M+H].sup.+): 881.6).

(312) Molecule 56: Product obtained by the reaction between molecule 55 and 2-phthalimido ethylamine.

(313) Using a similar method to the one used for the preparation of molecule 9 applied to molecule 55 (21.38 g, 24.26 mmol) and to 2-phthalimido ethylamine hydrochloride (HCl.PhthalEDA, 6.60 g, 29.12 mmol) in DCM and in the presence of DIPEA (5.07 mL, 29.12 mmol), a beige foam of molecule 56 is obtained without purification.

(314) Yield: 25.56 g (100%)

(315) .sup.1H NMR (DMSO-d6, ppm): 0.85 (3H); 1.17-1.30 (20H); 1.34-1.41 (27H); 1.41-1.52 (2H); 1.56-2.32 (18H); 3.18-3.69 (6H); 4.01-4.43 (4H); 7.64-8.30 (8H).

(316) LC/MS (ESI): 1053.8; (calculated ([M+H].sup.+): 1053.6).

(317) Molecule A26

(318) Molecule 56 (25.56 g, 24.26 mmol) is solubilized in a solution of 40% methylamine in MeOH (242.5 mL, 2.38 mol) at 4° C. then the mixture is stirred at ambient temperature for 5 h. Silica is added to the reaction medium then the mixture is concentrated under reduced pressure. The residue is purified by silica gel chromatography (solid deposition, dichloromethane, methanol, NH3) to produce molecule A26 in the form a pale yellow gum. This product is solubilized in DCM (250 mL) then the solution is washed with a 10% HCl aqueous solution. The aqueous phase is extracted with DCM (100 mL). The combined organic phases are dried on Na2SO4, filtered then concentrated under reduced pressure to produce the hydrochloride of molecule A26 in the form of a white solid.

(319) Yield: 13.5 g (58%)

(320) .sup.1H NMR (DMSO-d6, ppm): 0.85 (3H); 1.18-1.30 (20H); 1.34-1.42 (27H); 1.42-1.53 (2H); 1.66-2.02 (9H); 2.02-2.39 (9H); 2.79-2.91 (2H); 3.25-3.64 (4H); 4.08-4.46 (4H); 7.68-8.37 (7H).

(321) LC/MS (ESI): 923.8; (calculated ([M+H].sup.+): 923.6).

Example A27: Molecule A27

(322) Molecule A27 is obtained by means of the conventional solid phase peptide synthesis (SPPS) method on 2-chlorotrityl chloride (CTC) resin (24.00 g, 1.37 mmol/g).

(323) The grafting of the first amino acid Fmoc-6-aminohexanoic acid (1.5 equivalents) is performed in DCM (10 V), in the presence of DIPEA (2.5 equivalents). The unreacted sites are capped with methanol (0.8 mL/g resin) at the end of the reaction.

(324) The protected amino acid Fmoc-Glu-OMe (1.5 equivalents) and palmitic acid (1.5 equivalents) are coupled in DMF (10 V), in the presence of HATU (1.0 equivalent with respect to the acid) and DIPEA (1.5 equivalents with respect to the acid).

(325) The protecting groups Fmoc are removed using an 80:20 DMF/piperidine solution (10 V).

(326) The product is cleaved from the resin using an 80:20 DCM/HFIP solution (10 V).

(327) After concentration under reduced pressure, two co-evaporations are performed on the residue with dichloromethane followed by toluene. The product is purified by recrystallization in ethyl acetate. A white solid of molecule A27 is obtained.

(328) Yield: 11.54 g (68% in 6 stages)

(329) .sup.1H NMR (CDCl.sub.3, ppm): 0.88 (3H); 1.19-1.35 (24H); 1.35-1.44 (2H); 1.50-1.70 (6H); 1.91-2.01 (1H); 2.14-2.40 (7H); 3.14-3.34 (2H); 3.75 (3H); 4.51-4.59 (1H); 6.53 (1H); 6.70 (1H).

(330) LC/MS (ESI+): 513.4 (calculated ([M+H].sup.+): 513.4).

(331) Part B—Hydrophobic Co-Polyamino Acid Synthesis

(332) TABLE-US-00003 # CO-POLYAMINO ACIDS BEARING CARBOXYLATE CHARGES AND HYDROPHOBIC RADICALS B1 B2 B3 0 B4 B5 B6 B9 B13 0 B14 B15 B16 B17 B18 B19 0 B20 B21 B23 B24 B25 0 B26 B27 B28 B29 B30 B31

Example B1: Co-Polyamino Acid B1—Sodium Poly-L-glutamate Modified at its Extremities by Molecule A1 and having a Number Average Molar Mass (Mn) of 3600 g/mol

(333) Co-polyamino acid B1-1: poly-L-benzylglutamate obtained from the polymerization of γ-benzyl-L-glutamate N-carboxyanhydride initiated by ethylenediamine and modified at its extremities by molecule A1.

(334) In an oven-dried flask, γ-benzyl-L-glutamate N-carboxyanhydride (34.74 g, 132 mmol) is solubilized in anhydrous DMF (78 mL). The mixture is then stirred until complete dissolution, cooled to 0° C., then ethylene diamine (0.205 g, 3.41 mmol) is introduced rapidly and the medium is stirred at 0° C.

(335) In parallel, molecule A1 (2.26 g, 6.94 mmol) is solubilized in DMF (44 mL), then NHS (0.82 g, 7.12 mmol) and DCC (1.47 g, 7.12 mmol) are added successively. After stirring overnight at ambient temperature, the heterogeneous mixture is filtered on a sintered filter. The filtrate is then added to the polymer solution kept at 0° C. After 24 h, the solution is placed at ambient temperature. After 6 h of stirring, the reaction medium is poured onto diisopropylether (IPE, 1.8 L). The precipitate is filtered on a sintered filter, washed with IPE (3×30 mL) and dried at 30° C. under reduced pressure.

(336) Co-Polyamino Acid B1

(337) Co-polyamino acid B1-1 is diluted in trifluoroacetic acid (TFA, 132 mL), then the solution is cooled to 4° C. A 33% HBr solution in acetic acid (92.5 mL, 0.528 mol) is then added dropwise. The mixture is stirred at ambient temperature for 2 h, then poured dropwise onto a 1:1 (v/v) mixture of diisopropylether and water under stirring (0.8 L). After 2 h of stirring, the heterogeneous mixture is left to stand overnight. The white precipitate is recovered by filtration, washed with IPE (2×66 mL) then with water (2×66 mL). The solid obtained is then solubilized in water (690 mL) by adjusting the pH to 7 by adding an aqueous solution of 1 N sodium hydroxide. After solubilization, the theoretical concentration is adjusted to 20 g/L theoretical by adding water (310 mL), the solution is filtered on a 0.45 μm filter then purified by ultrafiltration against a 0.9% NaCl solution, followed by water until the conductimetry of the permeate is less than 50 μS/cm. The solution obtained is filtered on a 0.2 μm filter and stored at 2-8° C.

(338) Dry extract: 24.3 mg/g

(339) DP (estimated as per .sup.1H NMR): 40

(340) As per .sup.1H NMR: i=0.050

(341) The calculated average molar mass of co-polyamino acid B1 is 6719 g/mol.

(342) HPLC-Organic SEC (PEG calibrator): Mn=3600 g/mol.

Example B2: Co-Polyamino Acid B2—Sodium Poly-L-glutamate Modified at its Extremities by Stearic Acid and having a Number Average Molar Mass (Mn) of 3400 g/mol

(343) Co-polyamino acid B2-1: poly-L-benzylglutamate obtained from the polymerization of γ-benzyl-L-glutamate N-carboxyanhydride initiated by hexamethylenediamine.

(344) In an previously oven-dried flask, γ-benzyl-L-glutamate N-carboxyanhydride (30.0 g, 114 mmol) is solubilized in anhydrous DMF (67 mL). The mixture is then stirred until complete dissolution, cooled to 0° C., then hexamethylenediamine (0.442 g, 3.8 mmol) is introduced rapidly. After 23 h of stirring at 0° C., a 4 M HCl solution in dioxane (4.7 mL, 18.8 mmol) is added then the reaction medium is poured in 5 min onto a mixture of methanol (94 mL) and IPE (375 mL). The precipitate is filtered on a sintered filter, washed with IPE (2×70 mL) and dried at 30° C. under reduced pressure.

(345) Co-polyamino acid B2-2: poly-L-benzylglutamate modified at its extremities by stearic acid.

(346) To a solution of stearic acid (0.851 g, 2.99 mmol) in DMF (20 mL) at 0° C. are added successively HATU (1.484 g, 3.89 mmol) and DIPEA (1.166 g, 9.02 mmol). The solution is then introduced onto a solution of co-polyamino acid B2-1 (10.0 g) and triethylamine (TEA, 0.309 g, 3.04 mmol) in DMF (110 mL) at 0° C., and the medium is stirred for 18 h from 0° C. to ambient temperature. Dichloromethane (390 mL) is added, the organic phase is washed with 0.1 N HCl aqueous solution (3×190 mL), an aqueous solution saturated with NaHCO.sub.3 (2×190 mL), an aqueous solution saturated with NaCl (2×190 mL) followed by water (190 mL). The medium is then poured onto IPE (1.4 L). The precipitate is filtered on a sintered filter, washed with IPE (2×100 mL) and dried at 30° C. under reduced pressure.

(347) Co-Polyamino Acid B2

(348) Using a similar method to the one used for the preparation of co-polyamino acid B1 applied to co-polyamino acid B2-2 (8.80 g, 36.5 mmol), a sodium poly-L-glutamate modified at its extremities with stearic acid is obtained.

(349) Dry extract: 17.9 mg/g

(350) DP (estimated as per .sup.1H NMR): 30

(351) As per .sup.1H NMR: i=0.0657

(352) The calculated average molar mass of co-polyamino acid B2 is 5174 g/mol.

(353) HPLC-Organic SEC (PEG calibrator): Mn=3400 g/mol.

Example B3: Co-polyamino Acid B3—Sodium Poly-L-glutamate Modified at its Extremities by Molecule A2 and having a Number Average Molar Mass (Mn) of 3000 g/mol

(354) Co-polyamino acid B3-1: poly-L-benzylglutamate obtained from the polymerization of γ-benzyl-L-glutamate N-carboxyanhydride initiated by ethylenediamine.

(355) Using a similar method to the one used for the preparation of co-polyamino acid B2-1 applied to ethylene diamine (0.765 g, 12.73 mmol) and to γ-benzyl-L-glutamate N-carboxyanhydride (80.0 g, 304 mmol), co-polyamino acid B3-1 is obtained.

(356) Co-polyamino acid B3-2: poly-L-benzylglutamate modified at its extremities by A2.

(357) Using a similar method to the one used for the preparation of co-polyamino acid B2-2 applied to co-polyamino acid B3-1 (30.0 g, 5.56 mmol) and to molecule A2 (7.94 g, 12.24 mmol), a poly-L-benzylglutamate modified at its extremities with molecule A2 is obtained.

(358) Co-Polyamino Acid B3

(359) To a solution of co-polyamino acid B3-2 (36.6 g, 133.5 mmol) in N,N-dimethylacetamide (DMAc, 146 mL) is added 5% palladium on alumina (7.3 g), then the solution is placed at 60° C. at 10 bar of hydrogen. After leaving overnight, the reaction medium is filtered on a sintered filter then on a 0.2 μm PTFE filter. The filtrate is then placed under stirring before adding water (1.4 L) previously acidified to pH 2 with a 1 N HCl solution (14 mL) dropwise. After overnight, the precipitate is filtered on a sintered filter, washed with water (4×110 mL) and dried at 30° C. under reduced pressure.

(360) The solid obtained is then solubilized in water (1.09 L) by adjusting the pH to 7 by adding an aqueous solution of 1 N sodium hydroxide (121 mL). After solubilization, the solution is basified by adding 1 N sodium hydroxide (26 mL) up to pH 12. After 2 h, the solution is neutralized by adding 1 N HCl solution (28 mL). The theoretical concentration is adjusted to 12 g/L theoretical by adding water (650 mL) and ethanol (1040 mL) then the solution is filtered on an R53SLP carbon filter (3M) at a rate of 12 mL/min, then on a 0.2 μm PES filter. The solution is then purified by ultrafiltration against a 0.9% NaCl solution, followed by water until the conductimetry of the permeate is less than 50 μS/cm. The solution obtained is filtered on a 0.2 μm filter and stored at 2-8° C.

(361) Dry extract: 21.6 mg/g

(362) DP (estimated as per .sup.1H NMR): 24

(363) As per .sup.1H NMR: i=0.0808

(364) The calculated average molar mass of co-polyamino acid B3 is 4948 g/mol.

(365) HPLC-Organic SEC (PEG calibrator): Mn=3000 g/mol.

Example B4: Co-Polyaminoacid B4—Sodium Poly-L-glutamate Modified at its Extremities by Molecule A3 and having a Number Average Molar Mass (Mn) of 2500 g/mol

(366) Co-polyamino Acid B4-1: poly-L-benzylglutamate obtained from the polymerization of γ-benzyl-L-glutamate N-carboxyanhydride initiated by ethylenediamine.

(367) Using a similar method to the one used for the preparation of co-polyamino acid B2-1 applied to ethylene diamine (1.644 g, 27.35 mmol) and to γ-benzyl-L-glutamate N-carboxyanhydride (100.0 g, 380 mmol), co-polyamino acid B4-1 is obtained.

(368) Co-polyamino acid B4-2: poly-L-benzylglutamate modified at its extremities by molecule A3.

(369) Using a similar method to the one used for the preparation of co-polyamino acid B2-2 applied to co-polyamino acid B4-1 (10.0 g, 3.12 mmol) and to molecule A3 (4.412 g, 6.26 mmol), a poly-L-benzylglutamate modified at its extremities with molecule A3 is obtained.

(370) Co-Polyamino Acid B4

(371) Using a similar method to the one used for the preparation of co-polyamino acid B3 applied to co-polyamino acid B4-2 (12.0 g, 37.3 mmol), a sodium poly-L-glutamate modified at its extremities with molecule A3 is obtained.

(372) Dry extract: 21.7 mg/g

(373) DP (estimated as per .sup.1H NMR): 14

(374) As per .sup.1H NMR: i=0.134

(375) The calculated average molar mass of co-polyamino acid B4 is 3464 g/mol.

(376) HPLC-Organic SEC (PEG calibrator): Mn=2500 g/mol.

Example B5: Co-polyamino Acid B5—Sodium Poly-L-glutamate Modified at its Extremities by Molecule A3 and having a Number Average Molar Mass (Mn) of 2800 g/mol

(377) Co-polyamino acid B5-1: poly-L-benzylglutamate obtained from the polymerization of γ-benzyl-L-glutamate N-carboxyanhydride initiated by Ethylenediamine.

(378) Using a similar method to the one used for the preparation of co-polyamino acid B2-1 applied to ethylene diamine (0.95 g, 15.83 mmol) and to γ-benzyl-L-glutamate N-carboxyanhydride (100.0 g, 380 mmol), co-polyamino acid B5-1 is obtained.

(379) Co-polyamino acid B5-2: Poly-L-benzylglutamate modified at its extremities by molecule A3.

(380) Using a similar method to the one used for the preparation of co-polyamino acid B2-2 applied to co-polyamino acid B5-1 (20.0 g, 3.71 mmol) and to molecule A3 (5.233 g, 7.42 mmol), a poly-L-benzylglutamate modified at its extremities with molecule A3 is obtained.

(381) Co-Polyamino Acid B5

(382) Using a similar method to the one used for the preparation of co-polyamino acid B1 applied to co-polyamino acid B5-2 (15.6 g, 55.93 mmol), a sodium poly-L-glutamate modified at its extremities with molecule A3 is obtained.

(383) Dry extract: 27.4 mg/g

(384) DP (estimated as per .sup.1H NMR): 24

(385) As per .sup.1H NMR: i=0.077

(386) The calculated average molar mass of co-polyamino acid B5 is 4956 g/mol.

(387) HPLC-Organic SEC (PEG calibrator): Mn=2800 g/mol.

Example B6: Co-polyamino Acid B6: Sodium poly-L-glutamate Modified at its Extremities by Molecule A4 and having a Number Average Molar Mass (Mn) of 2900 g mol

(388) Co-polyamino Acid B6-1: poly-L-benzylglutamate obtained from the polymerization of γ-benzyl-L-glutamate N-carboxyanhydride initiated by ethylenediamine.

(389) Using a similar method to the one used for the preparation of co-polyamino acid B2-1 applied to ethylene diamine (0.951 g, 15.83 mmol) and to γ-benzyl-L-glutamate N-carboxyanhydride (100.0 g, 380 mmol), co-polyamino acid B6-1 is obtained.

(390) Co-polyamino acid B6-2: poly-L-benzylglutamate modified at its extremities by molecule A4.

(391) Using a similar method to the one used for the preparation of co-polyamino acid B2-2 applied to co-polyamino acid B6-1 (20.0 g, 3.71 mmol) and to molecule A4 (6.649 g, 8.74 mmol), a poly-L-benzylglutamate modified at its extremities with molecule A4 is obtained.

(392) Co-Polyamino Acid B6

(393) Using a similar method to the one used for the preparation of co-polyamino acid B1 applied to co-polyamino acid B6-2 (19.7 g, 69.47 mmol), a sodium poly-L-glutamate modified at its extremities with molecule A4 is obtained.

(394) Dry extract: 28.7 mg/g

(395) DP (estimated as per .sup.1H NMR): 24

(396) As per .sup.1H NMR: i=0.0812

(397) The calculated average molar mass of co-polyamino acid B6 is 5135 g/mol.

(398) HPLC-Organic SEC (PEG calibrator): Mn=2900 g/mol.

Example B9: Co-polyamino acid B9—Sodium Poly-L-glutamate Modified at its Extremities by Molecule A7 wherein the Side Chains are Deprotected and having a Number Average Molar Mass (Mn) of 3200 g/mol

(399) Co-polyamino acid B9-1: poly-L-benzylglutamate obtained from the polymerization of γ-benzyl-L-glutamate N-carboxyanhydride initiated by ethylenediamine.

(400) Using a similar method to the one used for the preparation of co-polyamino acid B2-1 applied to ethylene diamine (0.96 g, 15.94 mmol) and to γ-benzyl-L-glutamate N-carboxyanhydride (100.0 g, 380 mmol), co-polyamino acid B9-1 is obtained.

(401) Co-polyamino acid B9-2: poly-L-benzylglutamate modified at its extremities by molecule A7.

(402) Using a similar method to the one used for the preparation of co-polyamino acid B2-2 applied to co-polyamino acid B9-1 (25.0 g, 4.64 mmol) and to molecule A7 (10.49 g, 9.27 mmol), a poly-L-benzylglutamate modified at its extremities with molecule A7 is obtained.

(403) Co-polyamino acid B9-3: poly-L-benzylglutamate modified at its extremities by molecule A7 wherein the side chains are deprotected.

(404) Co-polyamino acid B9-2 (18.6 g) is solubilized in TFA (100 mL). After 2 h under stirring, the reaction medium is concentrated under reduced pressure.

(405) Co-Polyamino Acid B9

(406) Using a similar method to the one used for the preparation of co-polyamino acid B3 applied to co-polyamino acid B9-3 (18.0 g, 59.0 mmol), a sodium poly-L-glutamate modified at its extremities with molecule A7 is obtained.

(407) Dry extract: 21.8 mg/g

(408) DP (estimated as per .sup.1H NMR): 24

(409) As per .sup.1H NMR: i=0.0833

(410) The calculated average molar mass of co-polyamino acid B9 is 5776 g/mol.

(411) HPLC-Organic SEC (PEG calibrator): Mn=3200 g/mol.

Example B13: Co-polyamino Acid B13—Sodium Poly-L-glutamate Modified at its Extremities by Molecule A11 wherein the Esters are Deprotected and having a Number Average Molar Mass (Mn) of 3200 g/mol

(412) Co-polyamino acid B13-1: poly-L-benzylglutamate obtained from the polymerization of γ-benzyl-L-glutamate N-carboxyanhydride initiated by ethylenediamine.

(413) Using a similar method to the one used for the preparation of co-polyamino acid B2-1 applied to ethylene diamine (4.76 g, 15.94 mmol) and to γ-benzyl-L-glutamate N-carboxyanhydride (500.0 g, 1900 mmol), co-polyamino acid B13-1 is obtained.

(414) Co-polyamino acid B13-2: poly-L-benzylglutamate modified at its extremities by molecule A11.

(415) To a solution of co-polyamino acid B13-1 (12.0 g) in DMF (40 mL) at 0° C. are successively added a solution of molecule A11 (5.88 g, 6.67 mmol) in DMF (20 mL), N-2-hydroxypyridine oxide (HOPO, 0.82 g, 7.34 mmol), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) (1.66 g, 8.68 mmol), followed by DIPEA (0.97 mL, 5.56 mmol). The reaction medium is stirred at 0° C. for 16 h and at 20° C. for 2 h. Dichloromethane (150 mL) is added and the organic phase is washed with a 0.1 N HCl aqueous solution (6×75 mL), dried on Na.sub.2SO.sub.4 then filtered. The organic phase is then poured onto IPE (600 mL), then left to stand for 18 h. The white precipitate is recovered by filtration, washed with IPE (2×150 mL) then dried under reduced pressure at 30° C.

(416) Co-polyamino acid B13-3: poly-L-benzylglutamate modified at its extremities by molecule A11 wherein the esters are deprotected

(417) Co-polyamino acid B13-2 is solubilized in TFA (60 mL), and the solution is stirred for 2 h at ambient temperature then is poured dropwise onto diisopropylether under stirring (600 mL). After 18 h, the white precipitate is recovered by filtration, triturated with IPE and dried under reduced pressure.

(418) Co-Polyamino Acid B13

(419) Using a similar method to the one used for the preparation of co-polyamino acid B3 applied to co-polyamino acid B13-3 (14.5 g), a sodium poly-L-glutamate modified at its extremities with molecule A11 wherein the esters are deprotected is obtained.

(420) Dry extract: 18.0 mg/g

(421) DP (estimated as per .sup.1H NMR): 24

(422) As per .sup.1H NMR: i=0.079

(423) The calculated average molar mass of co-polyamino acid B13 is 5194 g/mol.

(424) HPLC-Organic SEC (PEG calibrator): Mn=3200 g/mol

Example B14: Co-polyamino Acid B14—Sodium poly-L-glutamate Modified at its Extremities by Molecule A12 wherein the Esters are Deprotected and having a Number Average Molar Mass (Mn) of 3700 g/mol

(425) Co-polyamino acid B14-1: poly-L-benzylglutamate obtained from the polymerization of γ-benzyl-L-glutamate N-carboxyanhydride initiated by ethylenediamine.

(426) Using a similar method to the one used for the preparation of co-polyamino acid B2-1 applied to ethylene diamine (4.76 g, 15.94 mmol) and to γ-benzyl-L-glutamate N-carboxyanhydride (500.0 g, 1900 mmol), co-polyamino acid B14-1 is obtained.

(427) Co-polyamino acid B14-2: poly-L-benzylglutamate modified at its extremities by molecule A12

(428) Using a similar method to the one used for the preparation of co-polyamino acid B2-2 applied to molecule A12 (2.67 g, 1.43 mmol) and to co-polyamino acid B14-1 (3.5 g), a poly-L-benzylglutamate modified at its two extremities with molecule A2 is obtained.

(429) Co-polyamino acid B14-3: poly-L-benzylglutamate modified at its extremities by molecule A12 wherein the esters are deprotected

(430) Using a similar method to the one used for the preparation of co-polyamino acid B13-3 applied to co-polyamino acid B14-2, a poly-L-benzylglutamate modified at its two extremities with molecule A12 wherein the esters are deprotected is obtained.

(431) Co-Polyamino Acid B14

(432) Using a similar method to the one used for the preparation of co-polyamino acid B3 applied to co-polyamino acid B14-3 (1.97 g), in a hydrogen atmosphere (1 atm, 48 h, 65° C.), a sodium poly-L-glutamate modified at its two extremities with molecule A12 wherein the esters are deprotected is obtained.

(433) Dry extract: 13.2 mg/g

(434) DP (estimated as per .sup.1H NMR): 24

(435) As per .sup.1H NMR: i=0.072

(436) The calculated average molar mass of co-polyamino acid B14 is 6537 g/mol.

(437) HPLC-Organic SEC (PEG calibrator): Mn=3700 g/mol

Example B15: Co-polyamino Acid B15—Butyltetracarboxylic Acid Substituted with Molecule A13 wherein the Esters are Deprotected and having a Number Average Molar Mass (Mn) of 2700 g/mol

(438) Molecule A13

(439) ##STR00160##

(440) Molecule 31: Product obtained by polymerization of γ-benzyl-L-glutamate N-carboxyanhydride initiated by N-Boc-ethylenediamine.

(441) A solution of BocEDA (12.00 g, 74.9 mmol) in DMF (12 mL) is prepared. In a reaction vessel, γ-benzyl-L-glutamate N-carboxyanhydride (78.87 g, 300.0 mmol) is solubilized in DMF (165 mL) at 25° C. The mixture is then stirred until complete dissolution, cooled to −10° C., then the BocEDA solution is introduced rapidly. The reaction medium is stirred at 0° C. for 4 h then a solution of HCl in 1,4-dioxane (3.33 M, 19.8 mL, 65.34 mmol) is added. The reaction medium is stirred at ambient temperature then the solution is poured onto a solution of MeOH/IPE (245 mL/990 mL) cooled by an ice bath. After 62 h of stirring at ambient temperature, the white precipitate is filtered on a sintered filter, washed with IPE (2×160 mL) and dried at 30° C. under reduced pressure.

(442) .sup.1H NMR (DMSO-d6, ppm): 1.35 (9H); 1.70-2.10 (10H); 2.26-2.65 (10H); 2.85-3.18 (4H); 3.85 (1H); 4.14-4.42 (4H); 4.87-5.24 (10H); 6.34-6.86 (1H); 7.11-7.56 (25H); 7.90-8.44 (7H); 8.69 (1H).

(443) DP (estimated as per .sup.1H NMR): 5.0

(444) The calculated average molar mass of molecule 31 in hydrochloride salt form is 1292.9 g/mol.

(445) Molecule 32: Product obtained by coupling molecule 31 and molecule A1.

(446) Molecule 31 (10.0 g, 7.73 mmol) is solubilized in a mixture of DCM (90 mL) and DIPEA (1.585 g, 9.32 mmol) at 0° C. To this solution are added successively HOPO (1.242 g, 11.18 mmol), molecule A1 (3.335 g, 10.25 mmol) and EDC (2.141 g, 11.17 mmol). After stirring overnight, the reaction medium is washed twice with a 0.1 N HCl solution (2×100 mL), twice with a 5% Na.sub.2CO.sub.3 aqueous solution (2×100 mL) followed by a saturated NaCl solution (100 mL). The organic phase is dried on Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue is solubilized in DCM (30 mL) and the solution is poured onto isopropyl alcohol (600 mL) under stirring at 0° C. The precipitate formed is recovered by vacuum filtration then dried under vacuum at 30° C.

(447) Yield: 7.58 g (62%)

(448) .sup.1H NMR (CDCl.sub.3, ppm): 0.87 (3H); 1.06-2.76 (58.6H); 3.06-4.45 (12.4H); 4.88-5.25 (10.8H); 5.72-8.40 (34.4H).

(449) DP (estimated as per .sup.1H NMR): 5.4

(450) The calculated average molar mass of molecule 32 in hydrochloride salt form is 1651.6 g/mol.

(451) Molecule A13

(452) After solubilizing molecule 32 (5.93 g, 3.59 mmol) in DCM (40 mL), the solution is cooled to 0° C. and TFA (40 mL) is added. The reaction medium is stirred at 0° C. for 3 h then is dry concentrated under reduced pressure at ambient temperature. The residue is taken up in DCM (120 mL) and washed with an aqueous carbonate buffer solution at pH 10.4 (3×240 mL) then by a 0.1 N HCl aqueous solution (2×240 mL). The organic solution is dried on Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. A white solid of molecule A13 in hydrochloride salt form is obtained.

(453) Yield: 5.17 g (91%)

(454) .sup.1H NMR (TFA-d, ppm): 0.87 (3H); 1.06-1.46 (20H); 1.46-1.68 (2H); 1.68-2.81 (28H); 3.13-4.59 (12.5H); 4.83-5.25 (11H); 7.02-9.13 (37H)

(455) DP (estimated as per .sup.1H NMR): 5.5

(456) The calculated average molar mass of molecule A13 in hydrochloride salt form is 1609.8 g/mol.

(457) Co-polyamino acid B15-1: Molecule A13 (3.47 g, 2.16 mmol) is solubilized in DCM (17 mL) then is added successively at 0° C. butyltetracarboxylic acid (BTCA, 115 mg, 0.49 mmol), HOPO (275 mg, 2.48 mmol), DIPEA (377 μL, 2.16 mmol) followed by EDC (473 mg, 2.47 mmol). After stirring overnight at 0° C., the reaction medium is poured onto MeOH (220 mL) under stirring at 0° C. After overnight, the white precipitate is recovered by vacuum filtration, triturated with cold MeOH then dried under vacuum at 30° C.

(458) Co-Polyamino Acid B15

(459) A solution of co-polyamino acid B15-1 (2.33 g, 0.362 mmol) in DMAc (33 mL) is placed in a hydrogen atmosphere (1 atm) in the presence of 5% palladium on alumina (465 mg) then the solution is heated to 60° C. After leaving overnight, the solution is cooled, filtered under Celite® then the filtrate is poured onto a 15% NaCl solution at pH 2 (500 mL). After leaving overnight, the precipitate is filtered on a sintered filter then washed twice with a 15% NaCl solution (2×8 mL). The solid obtained is then solubilized in water (70 mL) by adjusting the pH to 7 by adding an aqueous solution of 1 N sodium hydroxide. After solubilization, the solution is filtered on a 0.45 μm filter then purified by ultrafiltration against a 0.9% NaCl solution, followed by water until the conductimetry of the permeate is less than 50 μS/cm. The solution obtained is filtered on a 0.2 μm filter and stored at 2-8° C.

(460) Dry extract: 25.8 mg/g

(461) .sup.1H NMR (D.sub.2O, ppm): 0.90 (10.2H); 1.18-1.46 (68H); 1.53-1.9 (6.8H); 1.86-3.04 (101.2H); 3.17-3.80 (20.4H); 4.19-4.68 (22.1H)

(462) DP (estimated as per .sup.1H NMR): 5.5

(463) As per .sup.1H NMR: i=3.4

(464) The calculated average molar mass of co-polyamino acid B15 is 4261.3 g/mol.

(465) HPLC-Organic SEC (PEG calibrator): Mn=2700 g/mol.

Example B16: Co-polyamino Acid B16—Sodium Poly-L-glutamate Modified at its Extremities by Molecule A14 wherein the Esters are Deprotected and having a Number Average Molar mass (Mn) of 3200 g/mol

(466) Co-polyamino acid B16-1: poly-L-benzylglutamate obtained from the polymerization of γ-benzyl-L-glutamate N-carboxyanhydride initiated by 1-amino-4,7,10-trioxa-13-tridecane amine (TOTA).

(467) Using a similar method to the one used for the preparation of co-polyamino acid B2-1 applied to TOTA (13.96 g, 63.37 mmol) and to γ-benzyl-L-glutamate N-carboxyanhydride (400.0 g, 1519 mmol), co-polyamino acid B16-1 is obtained.

(468) Co-Polyamino Acid B16

(469) To a solution of molecule A14 (6.74 g, 13.5 mmol) in DMAc (38 mL) are successively added HOPO (1.65 g, 14.8 mmol)m, and EDC (3.36 g, 17.6 mmol).

(470) To a solution of co-polyamino acid B16-1 (30.0 g) in DMAc (113 mL) at ambient temperature are successively added DIPEA (1.90 mL, 13.5 mmol) followed by the solution of molecule A14 previously prepared.

(471) After 24 h of stirring at ambient temperature, DMAc (82 mL) is added and the solution is placed at 60° C. under 10 bar of hydrogen in the presence of 5% palladium on alumina (7.0 g). After 17 h of reaction, the reaction medium is filtered on a sintered filter then on a 0.2 μm PTFE filter.

(472) The filtrate is placed under stirring, then a 300 g/L sodium carbonate solution (46 mL) followed by acetone (275 mL) are then added successively dropwise. After 3 h, the precipitate is filtered on a sintered filter, washed with acetone (3×70 mL) and dried under reduced pressure.

(473) After solubilizing the solid obtained in water (1.3 L) then diluting with ethanol (0.7 L), the solution is basified by adding 10 N sodium hydroxide (13 mL) until a pH of 13 is obtained. After 3 h of stirring at ambient temperature, the solution is neutralized by adding 1 N HCl solution (190 mL) then the solution is filtered on an R53SLP carbon filter (3M), then on a 0.2 μm PES filter. The solution is then purified by ultrafiltration against a 0.9% NaCl solution, followed by water until the conductimetry of the permeate is less than 50 μS/cm. The solution obtained is filtered on a 0.2 μm filter and stored at 2-8° C.

(474) Dry extract: 21.4 mg/g

(475) DP (estimated as per .sup.1H NMR): 24

(476) As per .sup.1H NMR: i=0.078

(477) The calculated average molar mass of co-polyamino acid B16 is 4761 g/mol.

(478) HPLC-Organic SEC (PEG calibrator): Mn=3200 g/mol.

Example B17: Co-Polyaminoacid B17—Sodium Poly-L-glutamate Modified at its Two Extremities by Molecule A15 and having a Number Average Molar Mass (Mn) of 3200 g/mol

(479) Co-polyamino acid B17-1: Poly-L-benzylglutamate obtained from the polymerization of γ-benzyl-L-glutamate N-carboxyanhydride initiated by ethylenediamine.

(480) Using a similar method to the one used for the preparation of co-polyamino acid B2-1 applied to ethylenediamine (4.77 g, 79.37 mmol) and to γ-benzyl-L-glutamate N-carboxyanhydride (500.0 g, 1899 mmol), co-polyamino acid B17-1 is obtained.

(481) Co-polyamino acid B17

(482) Using a similar method to the one used for the preparation of co-polyamino acid B16 applied to co-polyamino acid B17-1 (15.0 g) and to molecule A15 (3.45 g) with a saponification step at pH 12 for 50 min, co-polyamino acid B17 is obtained.

(483) Dry extract: 20.3 mg/g

(484) DP (estimated as per .sup.1H NMR): 24

(485) As per .sup.1H NMR: i=0.048

(486) The calculated average molar mass of co-polyamino acid B17 is 4237 g/mol.

(487) HPLC-Organic SEC (PEG calibrator): Mn=3200 g/mol

Example B18: Co-Polyaminoacid B18—Sodium Poly-L-glutamate Modified at its Two Extremities by Molecule A16 and having a Number Average Molar Mass (Mn) of 3150 g/mol

(488) Co-polyamino acid B18-1: poly-L-benzylglutamate obtained from the polymerization of γ-benzyl-L-glutamate N-carboxyanhydride initiated by ethylenediamine.

(489) Using a similar method to the one used for the preparation of co-polyamino acid B2-1 applied to ethylenediamine (4.74 g, 78.89 mmol) and to γ-benzyl-L-glutamate N-carboxyanhydride (498.4 g, 1893 mmol), co-polyamino acid B18-1 is obtained.

(490) Co-Polyamino Acid B18

(491) Using a similar method to the one used for the preparation of co-polyamino acid B17 applied to co-polyamino acid B18-1 (14.0 g) and to molecule A16 (4.26 g), co-polyamino acid B18 is obtained.

(492) Dry extract: 9.7 mg/g

(493) DP (estimated as per .sup.1H NMR): 24

(494) As per .sup.1H NMR: i=0.075

(495) The calculated average molar mass of co-polyamino acid B18 is 4839 g/mol.

(496) HPLC-Organic SEC (PEG calibrator): Mn=3150 g/mol

Example B19: Co-polyamino Acid B19—Sodium Poly-L-glutamate Modified at its Extremities by Molecule A17 wherein the Esters are Deprotected and having a Number Average Molar Mass (Mn) of 3400 g/mol

(497) Using a similar method to the one used for the preparation of co-polyamino acid B16 applied to co-polyamino acid B18-1 (20.39 g) and to molecule A17 (7.553 g), co-polyamino acid B19 is obtained.

(498) Dry extract: 18.6 mg/g

(499) DP (estimated as per .sup.1H NMR): 24

(500) As per .sup.1H NMR: i=0.066

(501) The calculated average molar mass of co-polyamino acid B19 is 4936 g/mol.

(502) HPLC-Organic SEC (PEG calibrator): Mn=3400 g/mol

Example B20: Co-Polyaminoacid B20—Sodium Poly-L-glutamate Modified at its Two Extremities by Molecule A18 and having a Number Average Molar Mass (Mn) of 3200 g/mol

(503) Using a similar method to the one used for the preparation of co-polyamino acid B17 applied to co-polyamino acid B17-1 (12.45 g) and to molecule A18 (3.56 g), co-polyamino acid B20 is obtained.

(504) Dry extract: 16.8 mg/g

(505) DP (estimated as per .sup.1H NMR): 24

(506) As per .sup.1H NMR: i=0.075

(507) The calculated average molar mass of co-polyamino acid B20 is 4784 g/mol.

(508) HPLC-Organic SEC (PEG calibrator): Mn=3200 g/mol

Example B21: Co-Polyaminoacid B21—Sodium Poly-L-glutamate Modified at its Two Extremities by Molecule A19 and having a Number Average Molar Mass (Mn) of 3600 g/mol

(509) Using a similar method to that used for the preparation of co-polyamino acid B17 applied to co-polyamino acid B17-1 (12.16 g) and to molecule A19 (4.16 g), co-polyamino acid B21 is obtained.

(510) Dry extract: 26.4 mg/g

(511) DP (estimated as per .sup.1H NMR): 24

(512) As per .sup.1H NMR: i=0.077

(513) The calculated average molar mass of co-polyamino acid B21 is 5023 g/mol.

(514) HPLC-Organic SEC (PEG calibrator): Mn=3600 g/mol

Example B23: Co-Polyaminoacid B23—Sodium Poly-L-glutamate Modified at its Two Extremities by Molecule A21 and having a Number Average Molar Mass (Mn) of 3350 g/mol

(515) Using a similar method to the one used for the preparation of co-polyamino acid B17 applied to co-polyamino acid B17-1 (18.68 g) and to molecule A21 (7.03 g), co-polyamino acid B23 is obtained.

(516) Dry extract: 23.2 mg/g

(517) DP (estimated as per .sup.1H NMR): 24

(518) As per .sup.1H NMR: i=0.080

(519) The calculated average molar mass of co-polyamino acid B23 is 5140 g/mol.

(520) HPLC-Organic SEC (PEG calibrator): Mn=3350 g/mol

Example B24: Co-polyamino Acid B24—Sodium Poly-L-glutamate Modified at its Extremities by Molecule A1 and having a Number Average Molar Mass (Mn) of 2300 g/mol

(521) Co-polyamino acid B24-1: poly-L-benzylglutamate obtained from the polymerization of γ-benzyl-L-glutamate N-carboxyanhydride initiated by molecule 4 and modified at its extremities by molecule A1.

(522) To a suspension of molecule 4 (9.92 mmol) in anhydrous DMF (80 mL) cooled to 0° C. is rapidly added a solution of γ-benzyl-L-glutamate N-carboxyanhydride (26.11 g, 99.2 mmol) in anhydrous DMF (20 mL) at 0° C. After 24 h of stirring at 0° C., a freshly prepared solution of molecule A1 (16.1 g, 49.6 mmol), HATU (18.9 g, 49.6 mmol) and DIPEA (8.64 mL, 49.6 mmol) in DMF (80 mL) is added to the medium and the mixture is stirred from 0° C. to 25° C. for 3.5 h. The resin is filtered, washed successively with DMF (3×100 mL), isopropanol (1×100 mL) and DCM (3×100 mL). The resin obtained is then treated with an 80:20 DCM/HFIP mixture (120 mL). After 30 min of stirring at ambient temperature, the resin is filtered and washed successively with DCM (3×100 mL). The solvents are evaporated under reduced pressure to produce co-polyamino acid B24-1

(523) Co-Polyamino Acid B24

(524) Using a similar method to the one used for the hydrogenation step of co-polyamino acid B16 applied to co-polyamino acid B24-1 (27.4 g), with a saponification step at pH 12 for 50 min but without the carbofiltration step, co-polyamino acid B24 is obtained.

(525) Dry extract: 14.1 mg/g

(526) DP (estimated as per .sup.1H NMR): 14

(527) As per .sup.1H NMR: i=0.143

(528) The calculated average molar mass of co-polyamino acid B24 is 2899 g/mol.

(529) HPLC-Organic SEC (PEG calibrator): Mn=2300 g/mol.

Example B25: Co-Polyaminoacid B25—Sodium Poly-L-glutamate Modified at its Extremities by Molecule A22 and having a Number Average Molar Mass (Mn) of 3050 g/mol

(530) Using a similar method to the one used for the preparation of co-polyamino acid B17 applied to co-polyamino acid B18-1 (30.0 g) and molecule A22 (8.56 g) using a four-fold greater quantity of 300 g/l sodium carbonate solution to precipitate the polymer after the hydrogenolysis step, co-polyamino acid B25 is obtained.

(531) Dry extract: 23.7 mg/g

(532) DP (estimated as per .sup.1H NMR): 24

(533) As per .sup.1H NMR: i=0.074

(534) The calculated average molar mass of co-polyamino acid B25 is 4743 g/mol.

(535) HPLC-Organic SEC (PEG calibrator): Mn=3050 g/mol

Example B26: Co-Polyaminoacid B26—Sodium poly-L-glutamate Modified at its Two Extremities by Molecule A23 and having a Number Average Molar Mass (Mn) of 3400 g/mol

(536) Using a similar method to the one used for the preparation of co-polyamino acid B25 applied to co-polyamino acid B17-1 (25.78 g) and to molecule A23 (8.27 g), co-polyamino acid B21 is obtained.

(537) Dry extract: 11.8 mg/g

(538) DP (estimated as per .sup.1H NMR): 24

(539) As per .sup.1H NMR: i=0.073

(540) The calculated average molar mass of co-polyamino acid B21 is 4902 g/mol.

(541) HPLC-Organic SEC (PEG calibrator): Mn=3400 g/mol

Example B27: Co-polyamino Acid B27—Butyltetracarboxylic Acid Substituted with Molecule A24 wherein the Esters are Deprotected and having a Number Average Molar Mass (Mn) of 2500 g/mol

(542) Molecule A24

(543) ##STR00161##

(544) Molecule 53: Product obtained by polymerization of γ-benzyl-L-glutamate N-carboxyanhydride initiated by N-Boc-ethylenediamine then capped with molecule 2.

(545) A solution of BocEDA (12.00 g, 74.9 mmol) in DMF (12 mL) is prepared. In a reaction vessel, γ-benzyl-L-glutamate N-carboxyanhydride (78.87 g, 300.0 mmol) is solubilized in DMF (165 mL) at 25° C. The mixture is then stirred until complete dissolution, cooled to −10° C., then the BocEDA solution is introduced rapidly. The reaction medium is stirred at 0° C. for 3 h then are introduced successively DMF (100 mL), molecule 2 (26.73 g, 89.88 mmol), HOPO (9.99 g, 89.88 mmol) and EDC (17.23 g, 89.88 mmol). The reaction mixture is stirred at 0° C. for 1 h, from 0° C. to 20° C. for 2 h then at 20° C. for 16 h. It is then poured onto a 1:1 2-propanol/H.sub.2O solution (10 V) under stirring. After 3 h, the white precipitate is filtered on a sintered filter, washed with a 1:1 2-propanol/H.sub.2O mixture (2×360 mL) and dried at 30° C. under reduced pressure.

(546) Yield: 70 g (71%)

(547) .sup.1H NMR (TFA-d, ppm): 0.99 (3H); 1.34-1.59 (16H); 1.68-2.85 (36H); 3.52-3.62 (2H); 3.79-3.99 (4H); 4.70-4.92 (5.75H); 5.20-5.38 (9.5H); 7.36-7.52 (23.75H).

(548) DP (estimated as per .sup.1H NMR): 4.75

(549) The calculated average molar mass of molecule 53 is 1481.0 g/mol.

(550) Molecule A24

(551) Using a similar method to the one used for the preparation of molecule A13 applied to molecule 53 (34.00 g, 22.96 mmol), a white solid of molecule A24 in hydrochloride salt form is obtained.

(552) Yield: 29.40 g (90%)

(553) .sup.1H NMR (TFA-d, ppm): 1.00 (3H); 1.35-1.61 (16H); 1.79-1.93 (2H); 2.05-2.90 (25H); 3.53-3.65 (2H); 3.79-4.02 (4H); 4.74-4.94 (5.75H); 5.20-5.43 (9.5H); 7.32-7.58 (23.75H).

(554) DP (estimated as per .sup.1H NMR): 4.75

(555) The calculated average molar mass of molecule A13 in hydrochloride salt form is 1417.2 g/mol.

(556) Co-Polyamino Acid B27-1:

(557) Using a similar method to the one used for the preparation of co-polyamino acid B15-1 applied to molecule A24 (11.9 g, 8.40 mmol) and to BTCA (0.41 g, 1.75 mmol) in solution in DMF, a white solid is obtained after drying at 30° C. under reduced pressure.

(558) Co-Polyamino Acid B27

(559) Using a similar method to the one used for the preparation of co-polyamino acid B15 applied to co-polyamino acid B27-1 (9.31 g, 1.64 mmol), under hydrogen pressure (6 bar) and with a saponification step at pH 12 for 1 h prior to the ultrafiltration step, co-polyamino acid B27 is obtained.

(560) Dry extract: 19.9 mg/g

(561) DP (estimated as per .sup.1H NMR): 4.75

(562) As per .sup.1H NMR: i=3.7

(563) The calculated average molar mass of co-polyamino acid B27 is 4085.8 g/mol.

(564) HPLC-Organic SEC (PEG calibrator): Mn=2500 g/mol.

Example B28: Co-polyamino Acid B28—Tricarballylic Acid Substituted with Molecule A25 wherein the Esters are Deprotected and having a Number Average Molar Mass (Mn) of 2200 g/mol

(565) Molecule A25

(566) ##STR00162##

(567) Molecule 54: Product obtained by polymerization of γ-benzyl-L-glutamate N-carboxyanhydride initiated by N-Boc-ethylenediamine then capped with molecule A1.

(568) Using a similar method to the one used for the preparation of molecule 53 applied to BocEDA (6.00 g, 37.45 mmol), to γ-benzyl-L-glutamate N-carboxyanhydride (39.44 g, 150.00 mmol) and to molecule A1 (14.63 g, 44.94 mmol), a white solid of molecule 54 is obtained.

(569) Yield: 23.71 g (40%)

(570) .sup.1H NMR (CDCl.sub.3, ppm): 0.87 (3H); 1.12-2.76 (57.6H); 3.06-4.50 (12.15H); 4.90-5.25 (10.3H); 5.91-8.49 (32.9H).

(571) DP (estimated as per .sup.1H NMR): 5.15

(572) The calculated average molar mass of molecule 54 is 1596.8 g/mol.

(573) Molecule A25

(574) Using a similar method to the one used for the preparation of molecule A13 applied to molecule 54 (23.29 g, 14.59 mmol), a translucent solid of molecule A25 in hydrochloride salt form is obtained.

(575) Yield: 19.08 g (85%)

(576) .sup.1H NMR (CDCl.sub.3, ppm): 0.87 (3H); 1.17-1.32 (20H); 1.48-1.63 (2H); 1.69-2.78 (29.6H); 3.15-4.40 (12.15H); 4.89-5.18 (10.3H); 7.06-9.13 (31.9H).

(577) DP (estimated as per .sup.1H NMR): 5.15

(578) The calculated average molar mass of molecule A25 in hydrochloride salt form is 1533.1 g/mol.

(579) Co-Polyamino Acid B28-1:

(580) Using a similar method to the one used for the preparation of co-polyamino acid B15-1 applied to molecule A25 (3.93 g, 2.56 mmol) and to tricarballylic acid (TCA, 125.2 mg, 0.71 mmol) in solution in DMF, a white solid is obtained after drying at 30° C. under reduced pressure.

(581) Co-Polyamino Acid B28

(582) Using a similar method to the one used for the preparation of co-polyamino acid B15 applied to co-polyamino acid B28-1 (2.98 g, 0.65 mmol) and with a saponification step at pH 12 for 1 h prior to the ultrafiltration step, co-polyamino acid B28 is obtained.

(583) Dry extract: 25.8 mg/g

(584) DP (estimated as per .sup.1H NMR): 5.15

(585) As per .sup.1H NMR: i=3.0

(586) The calculated average molar mass of co-polyamino acid B28 is 3559.2 g/mol.

(587) HPLC-Organic SEC (PEG calibrator): Mn=2200 g/mol.

Example B29: Co-polyamino Acid B29-4,7,10-trioxa-1,13-tridecanediamine (TOTA) Substituted with Molecule A12 wherein the Esters are Deprotected and having a Number Average Molar Mass (Mn) of 2000 g/mol

(588) Co-polyamino acid B29-1:

(589) To a solution of molecule A12 (3.70 g, 1.98 mmol) in chloroform (31 mL) at ambient temperature are added successively HOBt (304 mg, 1.98 mmol) and 4,7,10-trioxa-1,13-tridecanediamine (TOTA, 208 mg, 0.94 mmol). The mixture is cooled to 0° C. then EDC (380 mg, 1.98 mmol) is added. The reaction medium is stirred for 15 min at 0° C. followed by 18 h at ambient temperature. The organic phase is washed with a 0.1 N HCl aqueous solution (2×28 mL), and the organic phase is dried on Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The solid obtained is solubilized in CHCl.sub.3 (40 mL) and the solution is added dropwise to IPE (400 mL) under stirring. The suspension is placed in an ice bath without stirring for 17 h. The suspension is centrifuged at 3200 rpm for 10 min at 25° C. The colorless supernatant is removed and the solid obtained is concentrated under reduced pressure.

(590) Yield: 4.59 g (quant.)

(591) .sup.1H NMR (CDCl.sub.3, ppm): 0.88 (12H); 1.12-1.58 (192H); 1.58-2.17 (48H); 2.17-2.62 (44H); 3.08 (2H); 3.13-3.38 (6H); 3.48 (4H); 3.53-3.66 (12H); 3.74-3.83 (4H); 3.92 (2H); 4.00-4.12 (4H); 4.12-4.33 (10H); 4.37 (2H); 6.72-6.84 (4H); 7.06 (2H); 7.31 (2H); 7.52 (2H); 7.82 (2H); 7.94 (2H); 8.57-8.69 (4H).

(592) Co-Polyamino Acid B29

(593) Molecule B29-1 (3.67 g, 0.93 mmol) is solubilized in TFA (11.5 mL) and the solution is stirred at ambient temperature for 6 h. The solution is poured dropwise onto IPE (18 mL) at 5° C. then water (18 mL) is added. The suspension is placed in an ice bath under stirring for 15 h. The suspension is filtered and triturated with IPE (10 mL) and water (2×10 mL). The residue is dried under reduced pressure then solubilized in a 1 N NaOH solution (56 mL) with regular addition of 1 N NaOH to maintain the pH at 7. The solution is diluted to 20 g/L theoretical with water then purified by ultrafiltration against a 0.9% NaCl solution, followed by water until the conductimetry of the permeate is less than 50 μS/cm. The solution obtained is filtered on a 0.2 μm filter and stored at 2-8° C.

(594) Dry extract: 8.0 mg/g

(595) The calculated average molar mass of co-polyamino acid B29 is 3520 g/mol.

(596) HPLC-Organic SEC (PEG calibrator): Mn=2000 g/mol.

Example B30: Co-polyamino Acid B30—Tricarballylic Acid Substituted with Molecule A26 wherein the Esters are Deprotected and having a Number Average Molar Mass (Mn) of 2100 g/mol

(597) Co-Polyamino Acid B30-1:

(598) Using a similar method to supernatant used for the preparation of co-polyamino acid B15-1 applied to molecule A26 (10.87 g, 11.33 mmol) and to tricarballylic acid (TCA, 0.605 g, 3.43 mmol) in solution in DMF, a white solid is obtained after 2 consecutive precipitations of the product in solution in DMF in a 50:50 H.sub.2O/MeCN mixture (10V), filtration, trituration with a 50:50 H.sub.2O/MeCN mixture followed by drying under reduced pressure at 30° C.

(599) Co-Polyamino Acid B30

(600) Co-polyamino acid B30-1 (8.53 g, 2.95 mmol) is solubilized in TFA (30 mL), and the solution is stirred for 3 h at ambient temperature then is poured dropwise onto water under stirring (300 mL). After 1 h, the white precipitate is recovered by filtration, triturated with water and dried under reduced pressure. The solid obtained is then solubilized in water (350 mL) by adjusting the pH to 7 adding an aqueous solution of 1 N sodium hydroxide. The solution is filtered on a 0.2 μm filter then purified by ultrafiltration against a 0.9% NaCl solution, followed by water until the conductimetry of the permeate is less than 50 μS/cm. The solution obtained is filtered on a 0.2 μm filter and stored at 2-8° C.

(601) Dry extract: 28.8 mg/g

(602) The molar mass of co-polyamino acid B30 is 2585 g/mol.

(603) HPLC-Organic SEC (PEG calibrator): Mn=2100 g/mol.

Example B31: Co-polyamino Acid B31—Sodium Poly-L-glutamate Modified at its Extremities by Molecule A27 wherein the Esters are Deprotected and having a Number Average Molar Mass (Mn) of 3800 g/mol

(604) Using a similar method to the one used for the preparation of co-polyamino acid B16 applied to co-polyamino acid B18-1 (28.6 g) and to molecule A27 (6.799 g), co-polyamino acid B31 is obtained.

(605) Dry extract: 20.5 mg/g

(606) DP (estimated as per .sup.1H NMR): 24

(607) As per .sup.1H NMR: i=0.075

(608) The calculated average molar mass of co-polyamino acid B31 is 4591 g/mol.

(609) HPLC-Organic SEC (PEG calibrator): Mn=3800 g/mol

(610) Part CE—Co-polyamino Acid Counterexamples

(611) TABLE-US-00004 # CO-POLYAMINO ACID COUNTEREXAMPLES CE1 CE2 i = 0.05, DP (m + n) = 43

(612) Co-polyamino acids CE1 and CE2 are synthesized according to the method described in the application WO2017211916.

(613) Part C: Compositions

Example C1

Rapid-Acting Insulin Analog Solution (Humalog®) at 100 U/mL

(614) This solution is a commercial insulin lispro solution marketed by ELI LILLY under the trade name Humalog®. This product is a rapid-acting insulin analog. The excipients in Humalog® are m-cresol (3.15 mg/mL), glycerol (16 mg/mL), disodium phosphate (1.88 mg/mL), zinc oxide (to obtain 0.0197 mg of zinc ion/mL), sodium hydroxide and hydrochloric acid to adjust the pH (pH 7-7.8) and water.

Example C2

Rapid-Acting Insulin Lispro Analog Solution at 100-600 U/mL

(615) This solution is an insulin solution prepared from insulin lispro powder produced by Gan&Lee. This product is a rapid-acting insulin analog. The excipients used are m-cresol, glycerol, zinc oxide, sodium hydroxide and hydrochloric acid to adjust the pH (pH 7-7.8) and water. The zinc concentration is 300 μM per 100 IU/mL of insulin. The concentration of the excipients varies according to of the one of lispro to obtain the desired concentrations in the final formulations.

Example C3

Slow-Acting Insulin Analog Solution (Lantus®) at 100 U/mL

(616) This solution is a commercial insulin glargine solution marketed by SANOFI under the trade name Lantus®. This product is a slow-acting insulin analog. The excipients in Lantus® are zinc chloride (30 μg/mL), m-cresol (2.7 mg/mL), glycerol (20 mg/mL), polysorbate 20 (16 μM), sodium hydroxide and hydrochloric acid to adjust the pH (pH 4) and water.

Example C4

Insulin Glargine Solution at 100-400 U/mL

(617) This solution is an insulin glargine solution prepared from insulin glargine powder produced by Gan&Lee. This product is a slow-acting insulin analog. The excipients used are zinc chloride, m-cresol, glycerol*, sodium hydroxide and hydrochloric acid to adjust the pH (pH 4) and water. The zinc concentration is 460 μM per 100 IU/mL of insulins. The concentration of the other excipients varies according to the one of glargine to obtain the desired concentrations in the final formulations.

(618) Part CA—Compositions Comprising Insulin Glargine

(619) Preparation method CA1: Preparation of a dilute co-polyamino acid/50 U/mL insulin glargine solution at pH 7.1, according to a method using insulin glargine in liquid form (in solution) and a co-polyamino acid in liquid form (in solution).

(620) To a stock solution of co-polyamino acid at pH 7.1 are added concentrated solutions of m-cresol and glycerin so as obtain a co-polyamino acid solution of concentration C.sub.stock co-polyamino acid/excipients (mg/mL). The quantity of excipients added is adjusted in order to obtain a concentration of m-cresol of 35 mM and glycerin of 184 mM in the co-polyamino acid/50 U/mL insulin glargine composition at pH 7.1.

(621) In a sterile container, a volume V.sub.insulin glargine of an insulin glargine solution at a concentration of 100 U/mL described in C3 or C4 is added to a volume V.sub.stock co-polyamino acid/excipients of a co-polyamino acid solution at the concentration C.sub.stock co-polyamino acid/excipients (mg/mL) in order to obtain a dilute co-polyamino acid composition C.sub.dilute co-polyamino acid (mg/mL)/50 U/mL insulin glargine at pH 7.1. A turbidity appears. The pH is adjusted to pH 7.1 by adding concentrated NaOH and the solution is placed under static conditions at 40° C. for 2 h until complete solubilization. This visually clear solution is placed at 4° C.

(622) Preparation method CA2: Preparation of a concentrated co-polyamino acid/insulin glargine composition at pH 7.1 using a co-polyamino acid, according to a dilute composition concentration method.

(623) A co-polyamino acid/50 U/mL insulin glargine composition at pH 7.1 described in example CA1 is concentrated by ultrafiltration on a 3 kDa membrane made of regenerated cellulose (Amicon® Ultra-15 marketed by Millipore). Following this ultrafiltration step, the retentate is clear and the insulin glargine concentration in the composition is determined by reverse phase chromatography (RP-HPLC). The insulin glargine concentration in the composition is then adjusted to the desired value by dilution in a solution of m-cresol/glycerin excipients in order to obtain a final concentration of m-cresol of 35 mM, Tween 20 of 52 μM and an osmolarity of 300 mOsmol/kg. The pH is measured and adjusted to pH 7.1 by adding NaOH and concentrated HCl. This visually clear solution at pH 7.1 has an insulin glargine concentration C.sub.insulin glargine (U/mL) and a co-polyamino acid concentration C.sub.co-polyamino acid (mg/mL)=C.sub.dilute co-polyamino acid (mg/mL)×C.sub.insulin glargine (U/mL)/50 (U/mL).

(624) Preparation method CA3: Preparation of a concentrated co-polyamino acid/insulin glargine solution at pH 7.1, according to a method using insulin glargine in liquid form (in solution) and a co-polyamino acid in liquid form (in solution).

(625) To a stock solution of co-polyamino acid at pH 7.1 is added a glargine solution of 220-400 IU/mL containing the excipients described in example C4. The concentration of the excipients in the glargine solution is adjusted in order to obtain a concentration of m-cresol of 35 mM, glycerin of 184 mM in the co-polyamino acid/insulin glargine composition at pH 7.1. A turbidity appears. The pH is adjusted to pH 7.1 by adding concentrated NaOH and the solution is placed under static conditions at 40° C. for 2 h until complete solubilization. This visually clear solution is placed at 4° C. after adding a volume of concentrated polysorbate 20 solution to obtain a final concentration of 52 μM.

(626) According to preparation methods CA2 or CA3, co-polyamino acid/insulin glargine compositions were prepared for example with insulin glargine concentrations from 200 U/mL to 300 U/mL.

Example CA4

Preparation of Co-polyamino Acid/200 U/ml Insulin Glargine Compositions at pH 7.1

(627) Co-polyamino acid/200 U/mL insulin glargine compositions are prepared according to the method described in CA2 and CA3 in order to obtain an insulin glargine concentration C.sub.insulin glargine=200 U/mL and a co-polyamino acid concentration C.sub.co-polyamino acid (mg/mL).

(628) These compositions are shown in Table 1.

(629) TABLE-US-00005 TABLE 1 Insulin glargine (200 U/mL) compositions in the presence of co-polyamino acids. Co-polyamino acid Insulin Visual Co-polyamino concentration glargine appearance of Composition acid (in mg/ml) (U/mL) solution CA3-1 B5 5 200 clear CA3-2 B6 7 200 clear

(630) The co-polyamino acids allow the solubilization of insulin glargine at neutral pH and lead to a clear solution.

Example CA5

Determination of Minimum Co-polyamino Acid Concentration to Solubilize 50 IU/mL Insulin Glargine at pH 7.1

(631) To a stock solution of co-polyamino acid at pH 7.2±0.3 are added concentrated solutions of zinc chloride, sodium chloride, m-cresol and glycerin. 0.5 mL of an insulin glargine solution at a concentration of 100 U/mL, prepared according to example C3 or C4, is added to a volume of 0.5 mL of the solution of co-polyamino acid and excipients to obtain a co-polyamino acid/50 U/ml insulin glargine composition with the desired composition. The co-polyamino acid concentration varies from one preparation to another: solutions having co-polyamino acid concentrations varying not more than 1 mg/ml are prepared in this way.

(632) Following the addition of the glargine solution, a turbidity appears. The pH is adjusted to pH 7.1 by adding concentrated NaOH and the solution is placed in an oven at 40° C. overnight. After leaving overnight at 40° C., the samples are visually inspected and subjected to a static light scattering measurement at an angle of 173° using a zetasizer (Malvern). The minimum concentration of co-polyamino acid suitable for solubilizing insulin glargine is defined as the lowest concentration for which the co-polyamino acid/insulin glargine mixture at pH 7.1±0.1 is visually clear, is free from visible particles and has a scattered intensity less than 1500 kcps/s.

(633) Preparation method CA5a: Preparation of a co-polyamino acid/insulin glargine solution at pH 7.2, according to a method using insulin glargine in liquid form (in solution) and a co-polyamino acid in liquid form (in solution).

(634) To a stock solution of co-polyamino acid at pH 7-7.5 are added a sodium chloride solution and a ZnCl.sub.2 solution to obtain the targeted concentrations in the co-polyamino acid/insulin glargine composition. A 100-220 U/mL glargine solution described in example C4 is added. The concentrations of the excipients in the glargine solution are adjusted in order to obtain a concentration of m-cresol of 35 mM and of glycerin of 230 mM in the co-polyamino acid/insulin glargine composition. A turbidity appears. The pH is adjusted to pH 7.5 by adding concentrated NaOH and the solution is placed in an oven at 40° C. for 2 h until complete solubilization. The solution obtained is visually clear.

Example CA6

Determination of Minimum Concentration of Co-polyamino Acid B20 to Solubilize 50 IU/ml Insulin Glargine at pH 7.1 in the Presence of Glycerin (184 mM), m-cresol (35 mM) and different NaCl and Zinc Chloride Concentrations

(635) According to the preparation method CA5, the minimum concentration of co-polyamino acid B20 to solubilize insulin glargine is determined for different zinc chloride and sodium chloride contents. The results are described in Table 1a.

Example CA6a

Determination of Minimum Concentration of different Co-polyamino Acids to Solubilize 50 IU/ml Insulin Glargine at pH 7.1 in the Presence of Glycerin (184 mM) and M-Cresol (35 mM)

(636) Using a similar method to the one described in example CA6, the minimum concentration determined for different co-polyamino acids according to the invention is shown in Table 1a.

(637) TABLE-US-00006 TABLE 1a Minimum concentration of different co-polyamino acids to solubilize insulin glargine Co- polyamino Ratio acid [Hy]/[insulin concentration glargine] Co- at limit of at limit of Com- polyamino [ZnCl.sub.2] [NaCl] solubilization solubilization position acid (mM) (mM) (mg/mL) (mol/mol) CA6-19 B14 0.23 — 0.75 0.76 CA6-21 B26 0.23 — 0.63 0.8 CA6-22 B9 0.23 — 0.75 0.76 CA7-23 CE1 0.23 — 1.0 0.9 CA7-24 CE2 0.23 — 1.0 0.8

(638) Co-polyaminoacids B14, B26 and B9 are suitable for solubilizing 50 U/mL glargine at a co-polyamino acid concentration less than or equal to 1 mg/mL.

Example CA6b

Determination of Improvement Percentage of the Minimum Concentration of Different Co-polyamino Acids to Solubilize 50 IU/ml Insulin Glargine at pH 7.1 in the Presence of Glycerin (184 mM), M-Cresol (35 mM) and different NaCl and Zinc Chloride Concentrations

(639) Using a similar method to the one described in example CA6, the reduction percentage of the minimum concentration determined for different co-polyamino acids according to the invention provided by adding NaCl and zinc chloride is shown in Table 1b.

(640) TABLE-US-00007 TABLE 1b Decrease of the minimum concentration of different co-polyamino acids to solubilize insulin glargine in the presence of NaCl and zinc chloride. Reduction of co-polyamino acid concentration at limit of solubilization with respect to reference Co- concentration polyamino [ZnCl.sub.2] [NaCl] without Composition acid (mM) (mM) salts (%) CA6-2 B20 0.23 10 25% CA6-3 B20 0.76 10 50% CA6-4 B17 0.23 10 14% CA6-5 CA6-6 0.40 10 57% CA6-7 B18 0.23 10 25% CA6-8 CA6-9 0.40 10 37% CA6-10 B19 0.23 10 40% CA6-11 CA6-12 0.40 10 70% CA6-13 B23 0.23 10 17% CA6-14 CA6-15 0.40 10 50% CA6-16 B31 0.23 10 45% CA6-17 CA6-18 0.40 10 64% CA7-23-1 CE1 0.23 5   10%≤ CA7-23-2 CE1 0.23 5   10%≤ CA7-24-1 CE2 0.23 10   10%≤ CA7-24-2 CE2 0.23 5   10%≤

(641) Adding salt and/or zinc chloride helps enhance the efficacy of the co-polyamino acids according to the invention with respect to co-polyamino acid/glargine compositions free from salt. Adding 10 mM NaCl makes it possible to reduce the minimum concentration of co-polyamino acid by at least 14% and the supplementary addition of zinc chloride by at least 37% with respect to the reference compositions free from NaCl.

(642) Part CB—Compositions Comprising Insulin Glargine and Insulin Lispro

(643) Preparation method CB1: Preparation of a dilute co-polyamino acid/43 (U/mL) insulin glargine/13.5 (U/mL) insulin lispro composition

(644) To a volume V.sub.co-polyamino acid/dilute insulin glargine of the dilute co-polyamino acid/50 U/mL insulin glargine composition at pH 7.1 described in CA1 is added a volume V.sub.insulin lispro of a 100 U/mL lispro solution and water in order to obtain a co-polyamino acid/43 (U/mL) insulin glargine/13.5 (U/mL) insulin lispro composition.

(645) Preparation method CB2: Preparation of a concentrated co-polyamino acid/insulin glargine/insulin lispro composition at pH 7.1

(646) A co-polyamino acid/43 (U/mL) insulin glargine/13.5 (U/ml) insulin lispro composition described in example CB1 is concentrated by ultrafiltration on a 3 kDa membrane made of regenerated cellulose (Amicon® Ultra-15 marketed by MILLIPORE). Following this ultrafiltration step, the retentate is clear and the insulin glargine concentration in the composition is determined by reverse phase chromatography (RP-HPLC). The insulin glargine and insulin lispro concentrations in the composition are then adjusted to the desired value by dilution in a solution of m-cresol/glycerin excipients in order to obtain a final concentration of m-cresol of 35 mM and an osmolarity of 300 mOsm/kg. During this dilution step, a volume of concentrated polysorbate 20 solution is also added to obtain a final concentration of 52 μM. The pH is measured and adjusted if required to pH 7.1 by adding NaOH and concentrated HCl. This visually clear solution at pH 7.1 has an insulin glargine concentration C.sub.insulin glargine (U/mL), an insulin lispro concentration C.sub.insulin lispro=C.sub.insulin glargine×0.33 and a co-polyamino acid concentration C.sub.co-polyamino acid (mg/mL)=C.sub.dilute co-polyamino acid (mg/mL)×C.sub.insulin glargine (U/mL)/50 (U/mL).

(647) Preparation method CB3: Preparation of a concentrated co-polyamino acid/insulin glargine/insulin lispro composition at pH 7.1

(648) To a volume V.sub.co-polyamino acid/concentrated insulin glargine of the concentrated co-polyamino acid/insulin glargine composition at pH 7.1 described in example CA3 is added a volume V.sub.insulin lispro of a lispro solution described in example C2. A volume of polysorbate 20 solution is also added to obtain a final concentration of 52 μM. The resulting solution at pH 7.1 has an insulin glargine concentration C.sub.insulin glargine (U/mL), an insulin lispro concentration C.sub.insulin lispro=C.sub.insulin glargine×0.33 and a co-polyamino acid concentration C.sub.co-polyamino acid (mg/mL)=C.sub.dilute co-polyamino acid (mg/mL)×C.sub.insulin glargine (U/mL)/50 (U/mL). The concentration of m-cresol is 35 mM and that of glycerin 230 mM.

(649) Preparation method CB4: Preparation of a co-polyamino acid/75 U/mL insulin glargine/25 U/mL insulin lispro composition at pH 7.2

(650) To a volume of the concentrated co-polyamino acid/insulin glargine composition described in example CA5a is added a volume of an insulin lispro solution described in example C2. The pH is adjusted to 7.2 by adding a concentrated hydrochloric acid solution. The visually clear resulting solution has a concentration of insulin glargine of 75 U/mL and of insulin lispro of 25 U/mL. The concentration of m-cresol is 35 mM and that of glycerin 230 mM.

(651) Preparation method CB5: Preparation of a co-polyamino acid/150 U/mL insulin glargine/50 U/mL insulin lispro composition at pH 7.2

(652) To a volume of the concentrated co-polyamino acid/insulin glargine composition described in example CA5a is added a volume of a lispro solution described in example C2. The pH is adjusted to 7.2 by adding a concentrated hydrochloric acid solution. The visually clear resulting solution has a concentration of insulin glargine of 150 U/mL and of insulin lispro of 50 U/mL. The concentration of m-cresol is 35 mM and the one of glycerin 230 mM.

Example CB2 and CB3: Preparation of Co-polyamino Acid/200 U/mL Insulin Glargine/66 U/mL Insulin Lispro Compositions at pH 7.1

(653) Co-polyamino acid/200 U/mL insulin glargine/66 U/ml insulin lispro compositions are prepared according to one of the methods described in examples CB2 and CB3 in order to obtain an insulin glargine concentration C.sub.insulin glargine=200 U/mL, an insulin lispro concentration C.sub.insulin lispro=66 U/mL and a co-polyamino acid concentration C.sub.co-polyamino acid (mg/mL).

(654) These compositions are shown in Table 2.

(655) TABLE-US-00008 TABLE 2 Insulin glargine (200 U/mL) and insulin lispro (66 U/ml) compositions in the presence of co-polyamino acids. Co-polyamino Insulin Visual Co-polyamino acid concentration glargine appearance Composition acid (in mg/ml) (U/mL) of solution CB2-1 B5 5 200 clear CB3-1 B6 7 200 clear

Example CB4: Preparation of Co-polyamino Acid/Insulin Glargine/Insulin Lispro Compositions at pH 7.2

(656) Co-polyamino acid/75 U/ml insulin glargine/25 U/ml insulin lispro compositions are prepared according to the method described in example CB4.

(657) TABLE-US-00009 TABLE 2a Insulin glargine (75 U/mL) and insulin lispro (25 U/ml) compositions in the presence of co-polyamino acids. Co-polyamino Co- acid Visual Com- polyamino concentration [ZnCl.sub.2] [NaCl] appearance of position acid (in mg/ml) (mM) (mM) solution CB4-1 B23 3.1 0.5 0 Clear CB4-2 B23 2.6 0.5 10 Clear CB4-3 B23 2.0 1 10 Clear CB4-4 B23 1.5 0.7 10 Clear CB4-5 B14 1.5 0.5 — Clear CB4-6 B14 1.4 0.5 5 Clear CB4-7 B26 2.0 0.5 Clear CB4-8 B9 1.5 0.5 — Clear CB4-9 B6 2.6 0.5 — Clear CB4-10 CE1 2.0 0.5 — Clear CB4-11 CE2 2.0 0.5 — Clear

Example CB5: Preparation of a Co-Polyamino Acid B23/Insulin Glargine/Insulin Lispro Composition at pH 7.2

(658) Co-polyamino acid/150 U/ml insulin glargine/50 U/ml insulin lispro compositions are prepared according to the method described in example CB5.

(659) TABLE-US-00010 TABLE 2b Insulin glargine (150 U/mL) and insulin lispro (50 U/ml) compositions in the presence of co-polyamino acid B23. Co-polyamino Co- acid Visual Com- polyamino concentration [ZnCl.sub.2] [NaCl] appearance of position acid (in mg/ml) (mM) (mM) solution CB5-1 B23 3.1 1.4 10 Clear

(660) Co-polyamino acid B23 enables the solubilization of insulin glargine in the presence of insulin lispro at neutral pH and lead to a clear solution.

(661) Part CD—Results

(662) Demonstration of Physical Stability of Compositions

Example CD1: Accelerated Stability at 25° C. under Dynamic Conditions

(663) 3×3 mL vials filled with 1 mL of co-polyamino acid/insulin glargine or co-polyamino acid/insulin glargine/prandial insulin composition are placed vertically on an orbital stirrer. The stirrer is placed in an oven at 25° C. and the vials are placed under stirring at 250 rpm. The vials are inspected visually daily/weekly in order to detect the appearance of visible particles or turbidity. This inspection is carried out according to the recommendations of the European Pharmacopeia (EP 2.9.20): the vials are placed under lighting of at least 2000 Lux and are observed against a white background and a black background. The number of days of stability corresponds to the period from which at least 2 vials exhibit visible particles or are turbid.

(664) The accelerated stability result with co-polyamino acid B5 is shown in Table 3.

(665) TABLE-US-00011 TABLE 3 Stability result of co-polyamino acid B5/insulin glargine (200 U/mL)/insulin lispro (66 U/mL) composition at 25° C. under dynamic conditions. Co-polyamino Co- acid Insulin Insulin Stability polyamino concentration glargine Lispro in Composition acid (in mg/ml) (U/mL) (U/mL) days CB2-1 B5 5 200 66 >10

Example CD2: Accelerated Stability at 30° C. under Static Conditions

(666) At least 5×3 mL cartridges filled with 1 mL of co-polyamino acid/insulin glargine/prandial insulin composition are placed in an oven at 30° C. under static conditions. The cartridges are inspected visually twice monthly in order to detect the appearance of visible particles or turbidity. This inspection is carried out according to the recommendations of the European Pharmacopeia (EP 2.9.20): the vials are placed under lighting of at least 2000 Lux and are observed against a white background and a black background. The number of weeks of stability corresponds to the period from which the majority of the cartridges exhibit visible particles or are turbid.

(667) The accelerated stability results under static conditions are shown in Table 4.

(668) TABLE-US-00012 TABLE 4 Stability results of co-polyamino acid/insulin glargine insulin lispro compositions at 30° C. under static conditions. Co-polyamino Co- acid polyamino concentration [ZnCl.sub.2] [NaCl] Stability Composition acid (mg/ml) (mM) (mM) (week) CB4-1 B23 3.1 0.5 0 >17 CB4-2 B23 2.6 0.5 10 >17 CB4-3 B23 2.0 1 10 >17 CB4-5 B14 1.5 0.5 — >23 CB4-7 B26 2.0 0.5 — >6

(669) Co-polyamino acids B23, B14, B26 enable the solubilization of insulin glargine in the presence of insulin lispro at neutral pH and lead to a composition having good physical stability. Adding salt and zinc to the compositions comprising co-polyamino acid B23 enable maintaining good physical stability while using a reduced co-polyamino acid B23 concentration.

Example CD3: Precipitation of Insulin Glargine after Mixing Co-polyamino Acid/75 U/mL Insulin Glargine/25 U/mL Insulin Lispro Compositions in a Simulated Physiological Medium

(670) This test demonstrates the precipitation of insulin glargine following injection in a simulated physiological medium at physiological pH and ionic strength and containing albumin. These conditions help imitate the behavior of the composition following subcutaneous injection. To 100 μL of co-polyamino acid/75 U/ml insulin glargine/25 U/ml insulin lispro are added 100 μL of a 20 mg/mL bovine albumin solution in phosphate buffer at pH 7.4. The phosphate buffer (PBS or phosphate buffer saline) is concentrated such that the NaCl and phosphate contents are 140 mM and 10 mM respectively following mixing with the composition. The precipitation of glargine in this medium is followed at ambient temperature (20-25° C.) by absorbance measurements at 450 nm of the mixtures for 30 minutes. The absorbance measurements are made using a UV-visible multi-well plate reader.

(671) The absorbance increases until it reaches a plateau. The precipitation time of glargine is defined as the time required for the absorbance measured to be greater than or equal to 80% of the value of the plateau.

(672) TABLE-US-00013 TABLE 5 Precipitation time of insulin glargine after mixing the co-polyamino acid/insulin glargine/insulin lispro compositions to a medium simulating the subcutaneous medium. Co-polyamino acid Co-polyamino concentration (in Precipitation time Composition acid mg/ml) (minute) CB4-1 B23 3.1 2 CB4-2 B23 2.6 3 CB4-5 B14 1.5 2 CB4-8 B9 1.5 2 CB4-9 B6 2.6 3 CB4-10 CE1 2.0 4 CB4-11 CE2 2.0 4 CB4-4 B23 1.5 1

(673) The co-polyamino acid/insulin glargine/insulin lispro compositions according to the invention result in rapid precipitation of glargine after mixing with a medium simulating the subcutaneous medium.

(674) Part D Pharmacokinetics

(675) D1: Pharmacokinetic measurement protocol of insulin glargine and insulin lispro formulations.

(676) Studies on dogs were conducted with a view to evaluating the pharmacokinetics of insulins after administering a co-polyamino acid B23/insulin glargine (150 U/mL)/insulin lispro (50 U/mL) composition.

(677) The pharmacokinetic profiles of insulin glargine (sum of the circulating concentration of insulin glargine and of the main metabolite M1 thereof) and of insulin lispro were obtained for this composition.

(678) Ten animals placed under fasting conditions for about 17.5 hours were injected by the subcutaneous route at a dose of 0.68 U/kg of insulin. Blood samples are taken for the 16 h following administration to describe the pharmacokinetics of the insulins. The levels of glargine, glargine-M1 and lispro are determined by means of a specific bioanalysis method.

(679) The pharmacokinetic parameters determined are as follows:

(680) AUC.sub.0-1 h, AUC.sub.0-2 h, AUC.sub.10-16 h, AUC.sub.13-16 h corresponding to the area under the curve of the concentrations of insulin glargine (and the metabolite M1 thereof) as a function of time from 0 to 1 h, 0 and 2 h, 10 and 16 h and 13 and 16 h post-administration, respectively;

(681) AUC.sub.0-30 mm, AUC.sub.0-1 h, AUC.sub.8-16 h corresponding to the area under the curve of the concentrations of insulin lispro as a function of time from 0 to 0.5 h, 0 and 1 h and 8 and 16 h post-administration, respectively;

(682) AUC.sub.last corresponding to the area under the curve from the time 0 to the last measurement time performed on the subject.

(683) Table 6 hereinafter reports different pharmacokinetic parameters of insulin glargine and insulin lispro.

(684) TABLE-US-00014 TABLE 6 Mean pharmacokinetic parameters (ratio of means) of composition CB5-1 comprising co-polyamino acid B23/150 U/mL insulin glargine/50 U/mL insulin lispro. Insulin glargine (150 U/mL) Insulin Lispro (50 U/mL) AUC.sub.0-1 h/ AUC.sub.0-2 h/ AUC.sub.10-16 h/ AUC.sub.13-16 h/ AUC.sub.0-1 h/ AUC.sub.8-16 h/ AUC.sub.last AUC.sub.last AUC.sub.last AUC.sub.last AUC.sub.0-30 min/ AUC.sub.last AUC.sub.last (%) (%) (%) (%) AUC.sub.last (%) (%) (%) CB5-1 19.6 28.7 19.8 8.2 25.4 48.0 3.5

(685) The results obtained demonstrate that, on one hand, the glargine component of the formulation is absorbed rapidly (AUC.sub.0-1 h and AUC.sub.0-2 h) while retaining the basal properties thereof with a significant coverage at the end part of the observation time (AUC.sub.10-16 h and AUC.sub.13-16 h).

(686) Furthermore, the lispro component is rapidly absorbed (AUC.sub.0-30 min and AUC.sub.0-1 h) and retains the prandial properties thereof. Indeed, lispro is no longer observed after 8 h (AUC.sub.8-16 h).