Amphiphilic derivatives of triazamacrocyclic compounds, products and compositions including same, and synthesis methods and uses thereof
09822083 · 2017-11-21
Assignee
Inventors
Cpc classification
A61K47/186
HUMAN NECESSITIES
A61K9/1272
HUMAN NECESSITIES
A61K47/22
HUMAN NECESSITIES
International classification
A61K47/18
HUMAN NECESSITIES
A61K47/22
HUMAN NECESSITIES
Abstract
The invention relates to amphiphilic derivatives of a triazamacrocyclic compound, as well as to said derivatives as active molecule transporters. The invention also relates to a nanodrug including at least one amphiphilic derivative of a triazamacrocyclic compound and at least one active molecule of a protein such as an antibody, in particular for the treatment of autoimmune diseases or for the treatment of cancer.
Claims
1. A compound of formula (I): ##STR00046## wherein: R.sup.1 has formula (V): ##STR00047## wherein: X represents an alkylene group and selected from C.sub.1-C.sub.4 alkylene groups; Y represents a linear, saturated or unsaturated alkyl chain selected from C.sub.12-C.sub.18 alkyl chains; Z represents a covalent bond and R.sup.4 represents a hydrogen atom, the side chain of an amino acid, or a group of formula (VI): ##STR00048## wherein k is 1 or 2, and Y.sup.1 represents a linear, saturated or unsaturated alkyl chain selected from C.sub.12-C.sub.18 alkyl chains, R.sup.2 represents a hydrogen atom or a linear, branched and/or cyclic, saturated or unsaturated hydrocarbon group selected from groups having 1 to 20 carbon atoms, wherein the hydrocarbon group also includes at least one of a carboxyl group and an amine group covalently bound to a chain of the hydrocarbon group, and wherein the hydrocarbon group has at least one functional cationic group chosen from amino group, guanidino group, imidazole chain-containing group, and quaternary ammonium-containing group; R.sup.3 represents a hydrogen atom or is identical to R′, or is identical to R.sup.2, and m, n and p are equal to 2.
2. The compound according to claim 1, which is selected from the group consisting of: 1,4-didodecyl-N(7′-carboxymethyl-1′,4′,7′-triazacyclononane)-L-aspartate; 1,5-didodecyl-N(7′-carboxymethyl-1′,4′,7′-triazacyclononane)-L-glutamate; 1,4-ditetradecyl-N(7′-carboxymethyl-1′,4′,7′-triazacyclononane)-L-aspartate; 1,5-ditetradecyl-N(7′-carboxymethyl-1′,4′,7′-triazacyclononane)-L-glutamate; 1,4-dihexadecyl-N(7′-carboxymethyl-1′,4′,7′-triazacyclononane)-L-aspartate; 1,5-dihexadecyl-N(7′-carboxymethyl-1′,4′,7′-triazacyclononane)-L-glutamate; 1,4-dioctadecyl-N(7′-carboxymethyl-1′,4′,7′-triazacyclononane)-L-aspartate; 1,5-dioctadecyl-N(7′-carboxymethyl-1′,4′,7′-triazacyclononane)-L-glutamate; 1,4-dioleyl-N(7′-carboxymethyl-1′,4′,7′-triazacyclononane)-L-aspartate; 1,5-dioleyl-N(7′-carboxymethyl-1′,4′,7′-triazacyclononane)-L-glutamate; 1,4-dioleyl-N(1′,4′-dibetainyl-7′-carboxymethyl-1′,4′,7′-triazacyclononane)-L-aspartate; 1,5-dioleyl-N(1′,4′-dibetainyl-7′-carboxymethyl-1′,4′,7′-triazacyclononane)-L-glutamate; 1,4-dioleyl-N(1′,4′-diornithyl-7′-carboxymethyl-1′,4′,7′-triazacyclononane)-L-aspartate; 1,5-dioleyl-N(1′,4′-diornithyl-7′-carboxymethyl-1′,4′,7′-triazacyclononane)-L-glutamate; -1,4-dioleyl-N(1′,4′-diarginyl-7′-carboxymethyl-1′,4′,7′-tri azacyclononane)-L-aspartate; 1,5-dioleyl-N(1′,4′-diarginyl-7′-carboxymethyl-1′,4′,7′-triazacyclononane)-L-glutamate; 1-octadecyl, 4-oleyl-N(7′-carboxymethyl-1′,4′,7′-triazacyclononane)-L-aspartate.
3. A pharmaceutical composition including: an active molecule adapted for administration to a human or animal to establish a medical diagnosis or restore, correct or modify a physiological function by exerting a pharmacological, immunological or metabolic action, wherein the active molecule is selected from the group consisting of proteins, antibodies, nucleic acids and antitumor agents; the compound according to claim 1; and a pharmaceutically acceptable carrier.
4. The compound 1,5-dioleyl-N(7′-carboxymethyl-1′,4′,7′-triazacyclononane)-L-glutamate.
5. The compound according to claim 1, which is selected from the group consisting of: 1,4-didodecyl-N(7′-carboxymethyl-1′,4′,7′-triazacyclononane)-L-aspartate; 1,4-ditetradecyl-N(7′-carboxymethyl-1′,4′,7′-triazacyclononane)-L-aspartate; 1,4-dihexadecyl-N(7′-carboxymethyl-1′,4′,7′-triazacyclononane)-L-aspartate; 1,4-dioctadecyl-N(7′-carboxymethyl-1′,4′,7′-triazacyclononane)-L-aspartate; 1,4-dioleyl-N(7′-carboxymethyl-1′,4′,7′-triazacyclononane)-L-aspartate; 1,4-dioleyl-N(1′,4′-dibetainyl-7′-carboxymethyl-1′,4′,7′-triazacyclononane)-L-aspartate; 1,4-dioleyl-N(1′,4′-diornithyl-7′-carboxymethyl-1′,4′,7′-triazacyclononane)-L-aspartate; 1,4-dioleyl-N(1′,4′-diarginyl-7′-carboxymethyl-1′,4′,7′-triazacyclononane)-L-aspartate; 1-octadecyl, 4-oleyl-N(7′-carboxymethyl-1′,4′,7′-triazacyclononane)-L-aspartate.
6. The compound according to claim 1, which is selected from the group consisting of: 1,5-didodecyl-N(7′-carboxymethyl-1′,4′,7′-triazacyclononane)-L-glutamate; 1,5-ditetradecyl-(7′-carboxymethyl-1′,4′,7′-triazacyclononane)-L-glutamate; 1,5-dihexadecyl-N(7′-carboxymethyl-1′,4′,7′-triazacyclononane)-L-glutamate; 1,5-dioctadecyl-N(7′-carboxymethyl-1′,4′,7′-triazacyclononane)-L-glutamate; 1,5-dioleyl-N(7′-carboxymethyl-1′,4′,7′-triazacyclononane)-L-glutamate; 1,5-dioleyl-N(1′,4′-dibetainyl-7′-carboxymethyl-1′,4′,7′-triazacyclononane)-L-glutamate; 1,5-dioleyl-N(1′,4′-diornithyl-7′-carboxymethyl-1′,4′,7′-triazacyclononane)-L-glutamate; 1,5-dioleyl-N(1′,4′-diarginyl-7′-carboxymethyl-1′,4′,7′-triazacyclononane)-L-glutamate.
7. The pharmaceutical composition according to claim 3, further comprising a colipid, wherein the colipid is dioleoylphosphatidylethanolamine.
Description
(1) The invention will be easier to understand in view of the following non-limiting description, in view of the appended drawings, wherein:
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(5)
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(8)
(9)
(10)
(11)
(12)
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(14)
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(16)
(17) The compounds according to the invention are amphiphilic derivatives of triazamacrocyclic compounds of formula (I):
(18) ##STR00008##
(19) wherein: R.sup.1 responds to formula (II):
(20) ##STR00009##
(21) and wherein: E represents a hydrocarbon, linear or branched, saturated or unsaturated group including 1 to 15 carbon atoms and optionally comprising one or more heteroatoms chosen from nitrogen, oxygen, sulfur, fluorine, chlorine, bromine or iodine; T represents a branched, saturated or unsaturated hydrocarbon group, including 1 to 15 carbon atoms and optionally comprising one or more heteroatoms chosen from nitrogen, oxygen, sulfur, fluorine, chlorine, bromine or iodine; L.sup.1 and L.sup.2, identical or different, represent a linear, branched and/or cyclic, saturated or unsaturated hydrocarbon group, including 6 to 24 carbon atoms and optionally comprising one or more heteroatoms chosen from nitrogen, oxygen, sulfur, fluorine, chlorine, bromine or iodine; q is an integer equal to 0 or 1; r is an integer equal to 0 or 1; s and t, identical or different, are integers equal to 0 or 1, on the condition that at least one of said integers is different from 0; R.sup.2 represents a hydrogen atom or a linear, branched and/or cyclic, saturated or unsaturated hydrocarbon group, including 1 to 20 carbon atoms, comprising one or more heteroatoms chosen from nitrogen, oxygen, sulfur, fluorine, chlorine, bromine or iodine; and having at least one cationic functional group; R.sup.3 represents a hydrogen atom or is identical to R.sup.1, or is identical to R.sup.2. m, n and p, identical or different, are integers equal to 0, 1, 2, 3 or 4.
(22) Above and below, by “heteroatom” we mean an atom chosen from nitrogen, oxygen, sulfur and halogens such as fluorine, chlorine, bromine or iodine. By “azamacrocyle” we mean a cyclic macromolecule containing one or more nitrogen atoms as represented in formula (I).
(23) Preferably, in formula (II):
(24) ##STR00010##
(25) E, which serves as a spacer arm, responds to the following formula (III):
(26) ##STR00011##
(27) wherein: X represents an alkylene group forming a bridge and including 1 to 8 carbon atoms; G1 represents a —CO—, —O—, —S—, —NH— or —NR— group wherein R is an alkyl group, advantageously in C.sub.1 to C.sub.6.
(28) Also preferably, X represents an alkylene group forming a bridge and including 1, 2, 3 or 4 carbon atoms. Still more preferably, X represents an alkylene group forming a bridge and including just 1 carbon atom.
(29) Preferably, in formula (I):
(30) ##STR00012##
(31) T, which serves as a branched spacer arm, represents either the residue of an amino acid or the residue of a glycerol.
(32) By “residue of an amino acid” we mean the atom group that subsists from said amino acid when it is covalently bound: to the spacer arm E in formula (II) or directly to one of the nitrogen atoms of the azamacrocycle in formula (I), and to one and/or the other of groups L.sup.1 and L.sup.2 in formula (II).
(33) By “residue of a glycerol” we mean the atom group that subsists from said glycerol when it is covalently bound: to the spacer arm E in formula (II) or directly to one of the nitrogen atoms of the azamacrocycle in formula (I), and to one and/or the other of groups L.sup.1 and L.sup.2 in formula (II).
(34) Preferably, when T represents the residue of an amino acid in formula (II), T is chosen from the twenty amino acids that are classically involved in the constitution of proteins, namely aspartic acid, glutamic acid, alanine, arginine, asparagine, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine, tryptophan and valine.
(35) Also preferably, it is chosen from aspartic acid, glutamic acid, leucine, isoleucine and lysine, aspartic acid and glutamic acid.
(36) Alternatively, this amino acid may be chosen from rarer amino acids such as, for example, β-alanine, γ-aminobutyric acid, α-aminoadipic acid, hydroxylysine, α,ε-diaminopimelic acid, α,β diaminopropionic acid, α,γ-diaminobutyric acid and ornithine.
(37) Generally, any amino acid may be suitable insofar as the amino acids comprise, by definition, at least two function groups, one carboxylic acid, the other amine, allowing covalent bonding thereof: to the spacer arm E in formula (II) or directly to one of the nitrogen atoms of the azamacrocycle in formula (I), and to one and/or the other of the L.sup.1 and L.sup.2 in formula (II).
(38) Also preferably, T is the residue of an amino acid belonging to series L. It is, however, possible for T to be the residue of an amino acid of series D.
(39) Preferably, in formula (II):
(40) ##STR00013##
(41) L.sup.1 and/or L.sup.2 respond to the following formula (IV):
(42) ##STR00014##
(43) wherein: G.sub.2 represents a —CO—, —O—, —S—, —NH— or —NR— group, in which R is an alkyl group, advantageously in C.sub.1 to C.sub.6, and Y represents a linear saturated or unsaturated alkyl chain in C.sub.8 to C.sub.24. Y may also represent a cyclic or polycyclic group known for being lipophilic, such as a steroid group, for example derived from cholesterol, a polyaromatic group, for example derived from naphthalene, dansyl, anthracene or a group derived from alkaloids.
(44) Also preferably, Y represents a linear saturated or unsaturated alkyl chain in C.sub.12 to C.sub.18.
(45) More preferably still, the derivative of formula (I) according to the invention is such that: R.sup.1 responds to formula (II):
(46) ##STR00015##
(47) wherein: E responds to the following formula (III):
(48) ##STR00016##
(49) wherein: X represents an alkylene group forming a bridge and including 1 to 8 carbon atoms; G1 represents a —CO—, —O—, —S—, —NH— or —NR— group wherein R is an alkyl group in C.sub.1 to C.sub.6; T represents either the residue of an amino acid or the residue of a glycerol; L.sup.1 and/or L.sup.2 respond to the following formula (IV):
(50) ##STR00017##
(51) wherein: G.sub.2 represents a —CO—, —O—, —S—, —NH— or —NR— group, wherein R is an alkyl group in C.sub.1 to C.sub.6, and Y represents a linear, saturated or unsaturated alkyl chain in C.sub.8 to C.sub.24 or a cyclic or polycyclic group.
(52) Among the triazamacrocyclic compounds according to the invention, those in which the spacer arm E is bound by an amide bond or an ester bond to the branched spacer arm T are preferred, T being itself bound by an amide bond or an ester bond to group(s) L.sup.1 and/or L.sup.2. This is for reasons of ease of preparation.
(53) In this case, E responds, preferably, to formulas —X—CO— or —X—NH— in which X has the same meaning as above. L.sup.1 and/or L.sup.2 respond, preferably, to formulas —O—Y, —CO—Y or —NH—Y in which Y has the same meaning as above.
(54) In this case as well it is preferable for R.sup.1 to respond to formula (V):
(55) ##STR00018##
(56) wherein: X and Y have the same meaning as above Z represents: either a covalent bond, in which case R.sup.4 represents a hydrogen atom, a methyl group, the side chain of an amino acid, or a group of formula (VI):
(57) ##STR00019##
(58) wherein k is 1 or 2, and Y has the same meaning as above, or a hydrocarbon group forming a bridge, including 1, 2, 3 or 4 carbon atoms, and capable of comprising one or more heteroatoms chosen from O and N, in which case R.sup.4 represents a primary amine group or a group of formula (VII):
(59) ##STR00020##
(60) wherein Y has the same meaning as above.
(61) Also preferably, the derivative of formula (I) according to the invention is such that: R.sup.1 responds to the formula (V):
(62) ##STR00021##
(63) wherein: X represents an alkylene group forming a bridge and including 1, 2, 3 or 4 carbon atoms; Y represents a linear, saturated or unsaturated alkyl chain in C.sub.12 to C.sub.18; Z represents: either a covalent bond, in which case R.sup.4 represents a hydrogen atom, a methyl group, the side chain of an amino acid, or a group of formula (VI):
(64) ##STR00022##
(65) wherein k is 1 or 2, and Y represents a linear, saturated or unsaturated alkyl chain in C.sub.12 to C.sub.18, or a hydrocarbon group forming a bridge, including 1 to 4 carbon atoms, and capable of comprising one or more heteroatoms chosen from O and N, in which case R.sup.4 represents a primary amine group or a group of formula (VII):
(66) ##STR00023##
(67) wherein Y represents a linear, saturated or unsaturated alkyl chain in C.sub.12 to C.sub.18; R.sup.2 represents a hydrogen atom or a linear, branched and/or cyclic, saturated or unsaturated hydrocarbon group, including 1 to 20 carbon atoms, comprising one or more heteroatoms chosen from nitrogen, oxygen, sulfur, fluorine, chlorine, bromine or iodine; and having at least one cationic functional group; and R.sup.3 represents a hydrogen atom or is identical to R.sup.1, or is identical to R.sup.2.
(68) Advantageously, when T represents, in formula (II):
(69) ##STR00024##
(70) the residue of an amino acid chosen from aspartic acid and glutamic acid, then, in formula (V):
(71) ##STR00025## Z represents a covalent bond; Y represents, preferably, a linear, saturated or unsaturated alkyl chain in C.sub.8 to C.sub.18 and, better yet, in C.sub.12 to C.sub.18; R5 represents a group of formula (VI): —(CH.sub.2).sub.k—CO—O—Y, wherein k is equal to 1 or 2, and Y represents, preferably, a linear, saturated or unsaturated alkyl chain in C.sub.8 to C.sub.18 and, better yet, in C.sub.12 to C.sub.18.
(72) According to yet another preferred provision of the invention, in formula (I):
(73) ##STR00026##
(74) R.sup.2 represents a hydrogen atom or the residue of a cationic amino acid. In the latter case, by “residue of a cationic amino acid” we mean the group of atoms that subsists from an amino acid having at least one cationic functional group when it is covalently bound to one of the nitrogen atoms of the azamacrocycle in formula (I). Preferably, the cationic functional group(s) borne by this cationic amino acid are amino, guanidino, imidazole or quaternary ammonium. In particular, they are chosen from ornithine, lysine, arginine and glycine betaine, glycine betaine being especially preferred.
(75) In formula (I), R.sup.2 represents, preferably, a hydrogen atom.
(76) According to yet another preferred provision of the invention, in formula (I), R.sup.3 more specifically represents a hydrogen atom plus.
(77) Preferably, the compounds according to the invention are amphiphilic derivatives of triazamacrocyclic compounds for which m, n and p are integers equal to 2. Said preferred derivatives are triazacyclononane derivatives, which satisfy formula (VIII):
(78) ##STR00027##
(79) wherein R.sup.1, R.sup.2 and R.sup.3 are as defined above.
(80) Still more specifically, the preferred derivatives responding to formula (VIII) are such that: R.sup.1 responds to formula (V):
(81) ##STR00028##
(82) wherein: X represents an alkylene group forming a bridge and including 1, 2, 3 or 4 carbon atoms; Y represents a linear, saturated or unsaturated alkyl chain in C.sub.12 to C.sub.18; Z represents: either a covalent bond, in which case R.sup.4 represents a hydrogen atom, a methyl group, the side chain of an amino acid, or a group of formula (VI):
(83) ##STR00029##
(84) wherein k is 1 or 2, and Y represents a linear, saturated or unsaturated alkyl chain in C.sub.12 to C.sub.18, or a hydrocarbon group forming a bridge, including 1 to 4 carbon atoms, and capable of comprising one or more heteroatoms chosen from O and N, in which case R.sup.4 represents a primary amine group or a group of formula (VII):
(85) ##STR00030##
(86) wherein Y represents a linear, saturated or unsaturated alkyl chain in C.sub.12 to C.sub.18; R.sup.2 represents a hydrogen atom or a linear, branched and/or cyclic, saturated or unsaturated hydrocarbon group, including 1 to 20 carbon atoms, comprising one or more heteroatoms chosen from nitrogen, oxygen, sulfur, fluorine, chlorine, bromine or iodine; and having at least one functional cationic group; R.sup.3 represents a hydrogen atom or is identical to R.sup.1, or is identical to R.sup.2.
(87) Among the amphiphilic derivatives of triazamacrocyclic compounds according to the invention, the following are especially preferred: 1,4-didodecyl-7-betainyl-1,4,7-triazacyclononane; 1,4-ditetradecyl-7-betainyl-1,4,7-triazacyclononane; 1,4-dihexadecyl-7-betainyl-1,4,7-triazacyclononane; 1,4-dioctadecyl-7-betainyl-1,4,7-triazacyclononane; 1,4-dioleyl-7-betainyl-1,4,7-triazacyclononane; 1,4-didodecyl-N(7′-carboxymethyl-1′,4′,7′-triazacyclononane)-L-aspartate; 1,5-didodecyl-N(7′-carboxymethyl-1′,4′,7′-triazacyclononane)-L-glutamate; 1,4-ditetradecyl-N(7′-carboxymethyl-1′,4′,7′-tri-azacyclononane)-L-aspartate; 1,5-ditetradecyl-N(7′-carboxymethyl-1′,4′,7′-tri-azacyclononane)-L-glutamate; 1,4-dihexadecyl-N(7′-carboxymethyl-1′,4′,7′-tri-azacyclononane)-L-aspartate; 1,5-dihexadecyl-N(7′-carboxymethyl-1′,4′,7′-tri-azacyclononane)-L-glutamate; 1,4-dioctadecyl-N(7′-carboxymethyl-1′,4′,7′-tri-azacyclononane)-L-aspartate; 1,5-dioctadecyl-N(7′-carboxymethyl-1′,4′,7′-tri-azacyclononane)-L-glutamate; 1,4-dioleyl-N(7′-carboxymethyl-1′,4′,7′-triaza-cyclononane)-L-aspartate; 1,5-dioleyl-N(7′-carboxymethyl-1′,4′,7′-triaza-cyclononane)-L-glutamate; 1,4-dioleyl-N(1′,4′-dibetainyl-7′-carboxymethyl-1′,4′,7′-triaza-cyclononane)-L-aspartate; 1,5-dioleyl-N(1′,4′-dibetainyl-7′-carboxymethyl-1′,4′,7′-triaza-cyclononane)-L-glutamate; 1,4-dioctadecyl-7-ornithyl-1,4,7-triazacyclononane; 1,4-dioleyl-7-ornithyl-1,4,7-triazacyclononane; 1,4-dioctadecyl-7-arginyl-1,4,7-triazacyclononane; 1,4-dioleyl-7-arginyl-1,4,7-triazacyclononane; 1,4-dioctadecyl-7-lysyl-1,4,7-triazacyclononane; 1,4-dioleyl-7-lysyl-1,4,7-triazacyclononane; 1,4-dioleyl-N(1′,4′-ornithyl-1′,4′,7′-triaza-cyclononane-7′-carboxymethyl)-L-aspartate; 1,5-dioleyl-N(1′,4′-ornithyl-1′,4′,7′-triaza-cyclononane-7′-carboxymethyl)-L-glutamate; 1,4-dioleyl-N(1′,4′-arginyl-1′,4′,7′-triaza-cyclononane-7′-carboxymethyl)-L-aspartate; and 1,5-dioleyl-N(1′,4′-arginyl-1′,4′,7′-triaza-cyclononane-7′-carboxymethyl)-L-glutamate; 1,4-dioleyl-N(1′,4′-diornithyl-7′-carboxymethyl-1′,4′,7′-triazacyclononane)-L-aspartate; 1,5-dioleyl-N(1′,4′-diornithyl-7′-carboxymethyl-1′,4′,7′-triazacyclononane)-L-glutamate; 1,4-dioleyl-N(1′,4′-diarginyl-7′-carboxymethyl-1′,4′,7′-triazacyclononane)-L-aspartate; 1,5-dioleyl-N(1′,4′-diarginyl-7′-carboxymethyl-1′,4′,7′-triazacyclononane)-L-glutamate; 1-octadecyl-4-oleyl-7-betainyl-1,4,7-triazacyclononane; 1-octadecyl-5-oleyl-N(7′-carboxymethyl-1′,4′,7′-triaza-cyclononane)-L-glutamate; 1-octadecyl, 4-oleyl-N(7′-carboxymethyl-1′,4′,7′-triaza-cyclononane)-L-aspartate; 1-dodecyl-4-oleyl-7-betainyl-1,4,7-triazacyclononane; 1-oleyl-4-dodecyl-7-betainyl-1,4,7-triazacyclononane.
(88) Advantageously, the derivatives according to the invention may include groups enabling their solubility, their cell penetration and their bioavailability to be enhanced.
(89) The invention also relates to the use of a derivative as described above, as a carrier of active molecules.
(90) The active molecules capable of being used include any drug, i.e. any substance capable of being used in humans or animals and capable of being administered to them, in order to establish a medical diagnosis or restore, correct or modify their physiological functions by exerting a pharmacological, immunological or metabolic action.
(91) As non-limiting examples of active molecules capable of being used, it is possible to cite, in particular, proteins such as antibodies, nucleic acids such as genes or siRNA, but also antitumor agents such as bortezomib, cisplatin, carboplatin, ifosfamide, chlorambucil, busulfan, thiotepa, 5-fluorouracil (5FU), fludarabine, methotrexate, vincristine, vinblastine, vinorelbine, paclitaxel, docetaxel, camptothecin derivatives, amsacrine, anthracyclines, epipodophyllotoxin derivatives, doxorubicin, daunorubicin, actinomycin-D, mitomycin-C, plicamycin and bleomycin. It is also possible to cite the ITK's used in different tumor pathologies such as, for example, Imatinib, Dasatinib, Nilotinib and Sunitinib.
(92) Advantageously, the derivatives of the invention are capable of being used as a carrier of nucleic acids or therapeutic molecules such as proteins. More preferably, the derivatives of the invention are used as antibody carriers.
(93) The invention also relates to a pharmaceutical composition including an amphiphilic derivative as described above.
(94) Preferably, said composition includes: a derivative as described above; a colipid; and a pharmaceutically acceptable carrier.
(95) As a non-limiting example of a colipid, it is possible to cite, advantageously, dioleoylphosphatidylethanolamine (DOPE).
(96) The concentration of the colipid in the composition is advantageously between 0 and 50% by weight of the total weight of the composition. More preferably, the concentration of the colipid is between 0 and 30% by weight of the total weight of the composition. Still more preferably, the concentration of the colipid is between 5 and 10% by weight of the total weight of the composition.
(97) The composition is advantageously in the form of an aqueous dispersion of nanoparticles.
(98) The invention also relates to a product including, on the one hand, at least one derivative as described above and, on the other hand, at least one active molecule as described above, as a drug. This type of product may also be called a nanodrug. As indicated above, a nanodrug may be defined as a carrier or a vector of nanometric size capable of carrying an active molecule to a given therapeutic target: a gene, a protein, a cell, a tissue or an organ.
(99) Thus, the invention also relates to a nanodrug including, on the one hand, at least one derivative as described above and, on the other hand, at least one active molecule as described above.
(100) The invention also relates to such a nanodrug for the treatment of autoimmune diseases or for the treatment of cancer.
(101) The invention also relates to a product containing, on the one hand, a nanodrug and, on the other hand, at least one pharmaceutical compound, as a combination product for simultaneously, separate or sequential administration, for the transport of molecules of therapeutic interest, in particular the intracellular delivery of antibodies.
(102) Advantageously, the pharmaceutical compound according to the invention is an anti-inflammatory agent, or an agent reducing the secondary effects associated with the nanodrugs or the active molecules according to the invention.
(103) By simultaneous therapeutic use, in this invention, we mean an administration of both the nanodrug and at least one pharmaceutical compound, by the same route and at the same time or substantially the same time.
(104) By separate therapeutic use, in this invention, we mean in particular an administration of the nanodrug according to the invention and a pharmaceutical compound, at the same time or substantially the same time by different routes.
(105) By sequential therapeutic use, we mean an administration of the nanodrug according to the invention and a pharmaceutical compound at different times, the route of administration being identical or different. More specifically, we mean a mode of administration according to which the entire administration of the nanodrug according to the invention or of a pharmaceutical compound is completed before the administration of the other begins.
(106) It is thus possible to administer the nanodrug according to the invention or a pharmaceutical compound over several months before administering the other. There is no simultaneous treatment in this case. It is also possible to envisage an alternate administration of the nanodrug according to the invention or of the pharmaceutical compound over several weeks.
(107) The route of administration of the composition according to the invention can be oral, parenteral, topical or ocular. Preferably, the pharmaceutical composition is prepared in a form suitable for parenteral application.
(108) Thus, by parenteral route, the composition may be in the form of solutions or suspensions for perfusion or for intramuscular, intravenous or subcutaneous injection.
(109) Alternatively, the composition may be administered orally and may be in the form of tablets, capsules, sugar-coated tablets, syrups, suspensions, solutions, powders, granules, emulsions, suspensions of microspheres or nanospheres or lipid or polymer vesicles allowing controlled release.
(110) Also alternatively, when the composition according to the invention is administered topically, the pharmaceutical composition according to the invention is more specifically intended for the treatment of skin and mucous membranes and may be in liquid, pasty or solid form and more specifically in the form of ointments, aqueous, alcoholic or oily solutions, dispersions of the lotion type, aqueous, anhydrous or lipophilic gels, powders, impregnated pads, detergents, wipes, sprays, foams, sticks, shampoos, compresses, cleansing bases, emulsions of liquid or semi-liquid consistency of the milk type, obtained by dispersion of a fatty phase in an aqueous phase (O/W) or the reverse (W/O), or suspensions or emulsions of soft, semi-liquid or solid consistency of the cream, gel or pomade type. It may also be in the form of suspension of microspheres or nanospheres or lipid or polymer vesicles or polymer or gel patches allowing controlled release.
(111) Finally, the invention also relates to methods for synthesis of the amphiphilic derivatives according to the invention. Such synthesis methods are described in the examples below.
EXAMPLE 1: SYNTHESIS OF COMPOUNDS ACCORDING TO THE Invention
(112) 1. Material:
(113) Most of the reagents and solvents come from Alfa Aesar GmbH (Bischheim, France), Merck KgaA (Darmstadt, Germany), VWR Prolabo (Briare, France), Sigma-Aldrich (Saint Quentin Fallavier, France) and Fluka (division of Sigma-Aldrich, Saint Quentin Fallavier, France). Triazacyclononane (TACN) comes from CheMatech (Dijon, France). All of the anhydrous solvents are purchased at Merck and used as is.
(114) 2. Methods
(115) a) Chromatography
(116) Thin Layer Chromatography (TLC) is performed on aluminum plates 5×7.5 cm coated with silica gel 60 F254 (Merck). The compounds are revealed under UV light (λ=254 nm), then by spraying an aqueous solution of 10% sulfuric acid followed by a step of heating at 250° C. for all of the lipid derivatives, or by aspersion of a 0.2% solution of ninhydrin in ethanol followed by a step of heating at 250° C. for compounds having an amine function.
(117) The flash chromatographic separations are performed on silica gel 60 (230-400 Mesh ASTM) (Merck).
(118) b) Mass Spectrometry
(119) Preparation of the Samples
(120) The products to be analyzed are dissolved (0.01 mg/mL) in a mixture of methanol/water 1/1 (v/v) or acetonitrile/water 1:1 (v/v) and the solutions are directly introduced into the electrospray source (at 5 μL/min) by means of a syringe pump (Harvard Apparatus, Les Ulis, France).
(121) Equipment
(122) The exact mass measurements are performed on a Waters-Micromass device (Manchester, U.K.) LCT, equipped with a pneumatically assisted electrospray (Z-spray) ion source, and provided with an additional nebulizer (Lockspray) for the reference compound (NaI). Nitrogen is used as the desolvation and nebulization gas with a flow rate of 500 and 20 L/h, respectively. The temperatures of the source and the desolvation gas are respectively set at 80 and 120° C. The capillary voltage is ±3.0 kV and the cone voltage is ±100 V (±ESI). The spectra are accumulated at a rate of 3 seconds per scan for a mass range of between 100 and 3500 uma. The resolution used is 5000 FWHM.
(123) The acquisition of data and processing thereof are performed with the MassLynx V3.5 program.
(124) c) NMR .sup.1H
(125) The proton NMR experiments are recorded at a frequency of 400.13 Mhz on a BRUKER Avance DRX400 device equipped with an inverse broadband probe (BBI). The chemical movements are given with respect to an external reference, tetramethylsilane (δ=0 ppm), and the internal calibrations are performed using the residual solvent signal. The solvents deuterated solvents used (CDC13, DMSO-d6) come from Eurisotop (Gif sur Yvette, France). The measurements are performed using a rigorous temperature control at 298 K (±0.1 K).
(126) The spectra are acquired on 16 K points, and converted to 32 K points (zero-filling). An optional treatment with an exponential function (1<LB<5) or a Gaussian function (0.1<GB<0.3; −3<LB<−1) is performed on each of the spectra, as well as a correction of the baseline.
(127) The spectra are processed on a PC using the MestReNova software (Mestrelab Research S.L., Santiago de Compostela, Spain).
(128) 3. Examples of Synthesis Methods
a) Synthesis of 1,4-didodecyl-7-betainyl-1,4,7-triazacyclononane (1) (DL-TACN-Bet)
(129) The synthesis method shown in
(130) ##STR00031##
(131) from commercial 1,4,7-triazacyclononane
i) Step 1: Preparation of 1,4-di(tert-butoxycarbonyl)-1,4,7-triazacyclononane (1a)
(132) ##STR00032##
(133) Triethylamine (460 pL; 3.30 mmol) is added to a solution of 1,4,7-triazacyclononane (TACN) (320.8 mg; 2.48 mmol) in chloroform (20 mL) under agitation and under an inert atmosphere. A solution of di-tert-butyl dicarbonate (1 g; 4.58 mmol) in chloroform (10 mL) is then added very slowly, over a period of 4 h, to the reaction medium. The mixture is then kept under agitation for one night at room temperature and under an inert atmosphere. The solvents are then dry evaporated under a primary vacuum, until a white solid residue is obtained, which is purified by silica gel chromatography (ethyl acetate/methanol 10/1 v/v). The pure compound 1a is thus isolated (521.2 mg; 70%), in the form of a colorless oil.
(134) TLC: Rf=0.5 (CH.sub.2Cl.sub.2/MeOH 9/1 v/v).
(135) HRMS (ESI.sup.+): m/z measured at 330.2394 [M+H].sup.+; calculated at 330.2393 for C.sub.16H.sub.32N.sub.3O.sub.4.
(136) NMR .sup.1H (CDCl.sub.3) δ (ppm): 1.41 (s, 18H, CH.sub.3); 2.85-2.87 (m, 4H, CH.sub.2); 3.15-3.22 (m, 4H, CH.sub.2); 3.35-3.42 (m, 4H, CH.sub.2).
ii) Step 2: Preparation of 3-betainylthiazolidine-2-Thione Chloride (1b)
(137) ##STR00033##
(138) A thionyl chloride solution (1.8 g; 15 mmol) in anhydrous acetonitrile (20 mL) is added drop by drop to the reaction medium containing the glycine betaine (1.17 g; 10 mmol) in suspension in the anhydrous acetonitrile (5 mL), the glycine betaine being first dried at 50° C. for 4 days. The mixture is then kept at 35-40° C. for 1 h under agitation and under an inert atmosphere. The reaction medium is then concentrated under a primary vacuum, then the resulting acyl chloride is dissolved in anhydrous dichloromethane (10 mL). 2-mercaptothiozoline (1.31 g; 11 mml) and triethylamine (1 g; 10 mmol), previously dissolved in 60 mL of anhydrous dichloromethane, are added at 0° C. to the previous solution. The reaction medium is then kept under agitation for 30 minutes at room temperature and under an inert atmosphere.
(139) After concentration of the medium under a primary vacuum, the precipitate formed is washed twice with boiling dichloromethane, then filtered on a frit to yield activated glycine 1b (1.66 g; 65%), in the form of a yellow powder.
(140) TLC: Rf=0.7 (MeOH).
(141) HRMS (ESI.sup.+): m/z measured at 219.0623 [M].sup.+; calculated at 219.0626 for C.sub.8H.sub.15N.sub.2OS.sub.2.
(142) NMR .sup.1H (DMSO-d6) δ (ppm): 3.30 (s, 9H, CH.sub.3); 3.49 (t, 2H, CO—N—CH.sub.2, J=7.6 Hz); 4.57 (t, 2H, CH.sub.2—S, J=7.6 Hz); 5.27 (s, 2H, N.sup.+—CH.sub.2—CO).
iii) Step 3: Preparation of 1,4-di(tert butoxycarbonyl)-7-betainyl-1,4,7-triazacyclononane (1c)
(143) ##STR00034##
(144) Triethylamine (1 mL; 7.2 mmol) is added to a solution of 1,4-di(tert-butoxycarbonyl)-1,4,7-triazacyclononane 1a (150 mg; 0.46 mmol) in N,N-dimethylformamide (DMF) (10 mL) under agitation and under an inert atmosphere. 3-betainylthiazolidine-2-thione chloride 1b (175.8 mg; 0.69 mmol) is then added directly into the reaction medium. The mixture is then kept under agitation for one night at room temperature and under an inert atmosphere. The solvents are then dry evaporated under a primary vacuum. The residue obtained is finally purified by silica gel chromatography (ethyl acetate). The compound 1c is thus isolated in the form of a white solid (chloride salt) (117.3 mg; 55%).
(145) TLC: Rf=0.5 (CH.sub.2Cl.sub.2/MeOH 9/1 v/v).
(146) HRMS (ESI+): m/z measured at 429.3074 [M]+; calculated at 429.3077 for C.sub.21H.sub.41N.sub.4O.sub.5.
iv) Step 4: Preparation of 7-betainyl-1,4,7-triazacyclononane (1d)
(147) ##STR00035##
(148) Trifluoroacetic acid (1 mL) is added to a solution of 1,4-di(tert-butoxycarbonyl)-7-betainyl-1,4,7-triazacyclononane 1c (110 mg; 0.24 mmol) in dichloromethane (1 mL) under agitation. The reaction is kept under agitation for 30 minutes at room temperature. The reaction medium is then concentrated under a primary vacuum, redissolved in dichloromethane and again concentrated under a primary vacuum. The operation is repeated several times in order to remove any traces of trifluoroacetic acid. The residue is dried for one night under a primary vacuum. The pure compound 1d (135 mg; quantitative) is obtained in the form of a pale yellow oil.
(149) TLC: Rf=0 (CH.sub.2Cl.sub.2/MeOH 9/1 v/v).
(150) HRMS (ESI+): m/z measured at 230.2109 [M+H]+; calculated at 230.2107 for C.sub.11H.sub.26N.sub.4O.
v) Step 5: Preparation of Didodecyl Triflate (1e)
(151) ##STR00036##
(152) Trifluoromethanesulfonic anhydride (1.8 g; 1.07 mL; 6.5 mmol) then anhydrous pyridine (0.51 g; 525 μL; 6.5 mmol) are successively added to an anhydrous dichloromethane solution (10 mL) cooled in an ice bath at 0° C., under agitation and under an inert atmosphere. During this addition, a release of smoke is observed and a white precipitate forms in the reaction medium. The ice bath is then removed, then a solution of lauric alcohol (0.93 g; 5 mmol) in anhydrous dichloromethane (4 mL) is added drop by drop into the reaction medium under agitation. The mixture is kept under agitation for 2 hours at room temperature, then the reaction is quenched by adding water. Dichloromethane (20 mL) is added to the reaction medium, and the solution is washed with water (2×10 mL). The aqueous phases are re-extracted with dichloromethane (2 mL) and the organic phases are regrouped, washed with brine (10 mL), dried on sodium sulfate, then filtered on filter paper. The filtrate is evaporated with the Rotavapor to obtain a brown oil. The oil is redissolved in hexane (2 mL) and loaded onto a silica column. The final product is eluted with a mixture of ether/hexane 1/1 v/v, taking care not to produce colored secondary products that are co-eluted with the product. The solvents are dry evaporated with the Rotavapor and the pure product 1e (1.41 g; 89%) is obtained in the form of a colorless oil.
(153) TLC: Rf=0.7 (Hexane/Ether 1/1 v/v).
(154) RMN .sup.1H (CDCl.sub.3) δ (ppm): 4.56 (t, 2H, CF.sub.3SO.sub.3CH.sub.2); 1.91-1.79 (m, 2H, CF.sub.3SO.sub.3CH.sub.2CH.sub.2);
(155) 1.53-1.20 (m, 18H, CH2 (lauryl)); 0.91 (t, 3H, CH.sub.3).
(156) The instability of this product over time requires it to be used very quickly for the next synthesis step.
vi) Step 6: Preparation of 1,4-didodecyl-7-betainyl-1,4,7-triazacyclononane (1)
(157) ##STR00037##
(158) Diisopropylethylamine (78 μL; 0.45 mmol) is added to a solution of 7-betainyl-1,4,7-triazacyclononane 1d (50 mg; 0.09 mmol) in anhydrous chloroform (10 mL) under agitation and under an inert atmosphere. Didodecyltriflate 1e (143.3 mg; 0.45 mmol), freshly prepared from lauric alcohol and triflic anhydride, is added to the reaction medium. The mixture is then kept under agitation for one night at room temperature and under an inert atmosphere. The solvents are then dry evaporated under a primary vacuum. The residue obtained is finally purified by silica gel chromatography (dichloromethane/methanol 1/0 to 9/1 v/v). The pure compound 1 is thus isolated (41.4 mg; 86%) in the form of a white solid.
(159) TLC: Rf=0.5 (CH.sub.2Cl.sub.2/MeOH 9/1 v/v).
(160) HRMS (ESI.sup.+): m/z measured at 566.5866 [M+H].sup.+; calculated at 566.5863 for C.sub.35H.sub.74N.sub.4O.
b) Synthesis of 1,4-dioctadecenyl-7-betainyl-1,4,7-triazacyclononane (2) (DO-TACN-Bet)
(161) The synthesis method shown in
(162) ##STR00038##
(163) This compound 2 is obtained by coupling 7-betainyl-1,4,7-triazacyclononane (1d), the synthesis of which was previously described in point 3.a), with oleyl triflate (2a).
i) Step 1: Synthesis of Oleyl Triflate (2a)
(164) ##STR00039##
(165) Trifluoromethanesulfonic anhydride (1.8 g; 1.07 mL; 6.5 mmol) then anhydrous pyridine (0.51 g; 525 μL; 6.5 mmol) are successively added to an anhydrous dichloromethane solution (10 mL) cooled in an ice bath at 0° C., under agitation and under an inert atmosphere.
(166) During this addition, a release of smoke is observed and a white precipitate forms in the reaction medium. The ice bath is then removed, then a solution of oleic alcohol solution (1.34 g; 5 mmol) in anhydrous dichloromethane (4 mL) is added drop by drop to the reaction medium under agitation. The mixture is kept under agitation for 2 hours at room temperature, then the reaction is quenched by adding water. Dichloromethane (20 mL) is added to the reaction medium, and the solution is washed with water (2×10 mL). The aqueous phases are re-extracted with dichloromethane (2 mL) and the organic phases are regrouped, washed with brine (10 mL), dried on sodium sulfate, then filtered on filter paper. The filtrate is evaporated with the Rotavapor in order to obtain a clear brown liquid. The liquid residue is re-dissolved in hexane (2 mL) and loaded onto a silica column. The final product is eluted with a mixture of ether/hexane 1/1 v/v, taking care not to produce colored secondary products that are co-eluted with the product. The solvents are dry evaporated with the Rotavapor, and a pure product 2a (1.30 g; 65%) is obtained in the form of a colorless liquid.
(167) TLC: Rf=0.8 (Hexane/Ether 1/1 v/v).
(168) NMR .sup.1H (CDCl.sub.3) δ (ppm): 5.42-5.33 (m, 2H, CH═CH); 4.54 (t, 2H, CF3SO3CH.sub.2); 2.13-1.93 (m, 4H, CH.sub.2CH═CHCH.sub.2); 1.90-1.69 (m, 2H, CF3SO3CH.sub.2CH.sub.2); 1.54-1.12 (m, 22H, CH.sub.2 (oleyl)); 0.89 (t, 3H, CH.sub.3).
(169) The instability of this product over time requires it to be used very quickly for the next synthesis step.
ii) Step 2: Synthesis of 1,4-dioctadecenyl-7-betainyl-1,4,7-triazacyclononane (2)
(170) ##STR00040##
(171) Diisopropylethylamine (78 μL; 0.45 mmol) is added to a solution of 7-betainyl-1,4,7-triazacyclononane 1d (50 mg; 0.09 mmol) in anhydrous chloroform (10 mL) under agitation and under an inert atmosphere. Oleyl triflate 2a (180.3 mg; 0.45 mmol), freshly prepared from oleic alcohol and triflic anhydride, is then added directly to the reaction medium. The mixture is then kept under agitation for one night at room temperature and under an inert atmosphere. The solvents are then dry evaporated under a primary vacuum. The residue obtained is finally purified by silica gel chromatography (dichloromethane/methanol 1/0 to 9/1 v/v). The pure compound 2 (53.0 mg; 94%) is thus isolated, in the form of an oil.
(172) TLC: Rf=0.5 (CH.sub.2Cl.sub.2/MeOH 9/1 v/v)
(173) HRMS (ESI.sub.+): m/z measured at 730.7428 [M+H].sub.+; calculated at 730.7428 for C.sub.47H.sub.94N.sub.4O.
c) Synthesis of 1,5-dioleyl-N(7′-carboxymethyl-1′,4′,7′-triazacyclononane)-L-glutamate (3) (DOG-TACN)
(174) The synthesis method shown in
(175) ##STR00041##
(176) This compound 3 is obtained from 1,4-di(tert-butoxycarbonyl)-1,4,7-triazacyclononane (1a), the synthesis of which has previously been described in point 3.a).
i) Step 1: Preparation of 1,4-di(tert-butoxycarbonyl)-7-carboxymethyl-1,4,7-triazacyclononane (3a)
(177) ##STR00042##
(178) Sodium carbonate (67 mg; 0.63 mmol) and ethyl bromoacetate (70 μL; 0.63 mmol) are successively added to a solution of 1,4-di(tert-butoxycarbonyl)-1,4,7-triazacyclononane 1a (173.6 mg; 0.53 mmol) in acetonitrile (10 mL). The mixture is then brought to reflux (bath temperature=80° C.) and kept under agitation for one night at reflux. Then, the reaction is cooled to room temperature and the insoluble products are removed by filtration. The solution is concentrated under a primary vacuum, and the crude product is purified by silica gel chromatography (ethyl acetate) in order to produce the intermediate ester (194 mg; 88%) in the form of a pale yellow oil.
(179) TLC: Rf=0.8 (CH.sub.2Cl.sub.2/MeOH 9/1 v/v).
(180) HRMS (ESI.sub.+): m/z measured at 416.2758 [M+H].sub.+; calculated at 416.2761 for C.sub.20H.sub.38N.sub.3O.sub.6.
(181) Ester (194 mg; 0.47 mmol) is dissolved in methanol (5 mL), then a 1 mol/L (3 mL) soda solution is added to the solution under agitation. The mixture is kept under agitation for 2 h at room temperature. The methanol is then evaporated under vacuum, and the pH of the resulting aqueous solution is adjusted to 5 by adding a 5% citric acid solution (m/v). The aqueous phase is then extracted with dichloromethane (3×20 mL), the organic phases are combined, dried on anhydrous sodium sulfate, and the filtrate is concentrated under a primary vacuum in order to obtain the pure compound 3a (160 mg; 88%), in the form of a colorless oil.
(182) TLC: Rf=0.4 (CH.sub.2Cl.sub.2/MeOH 9/1 v/v).
(183) HRMS (ESI.sub.+): m/z measured at 388.2452 [M+H].sub.+; calculated at 388.2448 for C.sub.18H.sub.34N.sub.3O.sub.6.
(184) NMR .sup.1H (CDCl.sub.3) δ (ppm): 1.30-1.50 (m, 18H, C(CH.sub.3).sub.3(Boc)); 3.10-3.60 (m, 14H, CH.sub.2(TACN), NCH.sub.2CO); 8.20 (br s, 1H, OH).
ii) Step 2: Preparation of 1,5-dioleyl-L-glutamate (3b)
(185) ##STR00043##
(186) L-glutamic acid (0.5 g; 3.4 mmol) and paratoluenesulfonic acid (0.780 g; 4.1 mmol) are dissolved in toluene (100 mL). The mixture is then brought to reflux (bath temperature=120° C.) under agitation for 1 h. The oleic alcohol (2.01 g; 7.5 mmol) is then introduced into the reaction medium, then the reaction is kept under agitation and at reflux for one night. The reaction is then cooled to room temperature, then the toluene is dry evaporated under a primary vacuum. The residue obtained is dissolved in chloroform (50 mL) and washed successively with a saturated aqueous solution of sodium bicarbonate (2×25 mL) with brine (1×25 mL). The organic phase is dried on anhydrous sodium sulfate, and the filtrate is concentrated under a primary vacuum in order to obtain a crude product that is purified by silica gel chromatography (dichloromethane/methanol 4/1 to 1/1 v/v). The pure compound 3b is thus isolated (0.45 g; 20%), in the form of a pale yellow oil.
(187) TLC: Rf=0.4 (CH.sub.2Cl.sub.2/MeOH 98/2 v/v).
(188) HRMS (ESI.sub.+): m/z measured at 648.4931 [M+H].sub.+; calculated at 648.4931 for C.sub.41H.sub.78NO.sub.4.
(189) NMR .sup.1H (CDCl.sub.3) δ (Ppm): 0.90 (t, 6H, CH.sub.2CH.sub.3 (oleyl)); 1.26 (m, 44H, CH.sub.2 (oleyl)); 1.57 (m, 4H, COOCH.sub.2CH.sub.2 (oleyl)); 1.90-2.12 (m, 2H, NH.sub.2CHCH.sub.2 (glutamate)); 2.04 (m, 8H, CH.sub.2CH═CHCH.sub.2 (oleyl)); 2.47 (t, 2H, CH.sub.2CO (glutamate)); 3.59 (t, 1H, NH.sub.2CH (glutamate)); 4.06-4.15 (t, 4H, COOCH.sub.2 (oleyl)); 5.32-5.42 (m, 4H, CH═CH (oleyl)); 7.18, 7.73 (d, 2H, NH.sub.2).
iii) Step 3: Preparation of 1,5-dioleyl-N(1′,4′-di(tert-butoxycarbonyl)-7′-carboxymethyl-1′,4′,7′-triaza-cyclononane)-L-glutamate (3c)
(190) ##STR00044##
(191) PyBOP (72 mg; 0.138 mmol), 1,5-dioctadecenyl-L-glutamate 3b (97.3 mg; 0.150 mmol) and diisopropylethylamine (66 μL; 0.380 mmol) are successively added to a solution of 1,4-di(tert-butoxycarbonyl)-7-carboxymethyl-1,4,7-triazacyclononane 3a (53.3 mg; 0.138 mmol) in anhydrous dichloromethane (2 mL) under agitation and under an inert atmosphere. The reaction is then kept under agitation and under an inert atmosphere for one night at room temperature. The solvents are then dry evaporated under a primary vacuum, and the residue is purified by silica gel chromatography (dichloromethane/methanol 100/1 v/v). The pure compound 3c is thus isolated (124 mg; 88%), in the form of a yellow-orange oil.
(192) TLC: Rf=0.8 (CH.sub.2Cl.sub.2/MeOH 95/5 v/v).
(193) HRMS (ESI.sub.+): m/z measured at 1019.8116 [M+H].sub.+; calculated at 1019.8113 for C.sub.59H.sub.109N.sub.3O.sub.10.
(194) NMR .sup.1H (CDCl.sub.3) δ (ppm): 0.90 (t, 6H, CH.sub.2CH.sub.3 (oleyl)); 1.20-1.60 (m, 66H, C(CH.sub.3).sub.3(Boc), CH.sub.2 (oleyl), COOCH.sub.2CH.sub.2 (oleyl)); 1.90-2.12 (m, 2H, NH.sub.2CHCH.sub.2 (glutamate)); 2.04 (m, 8H, CH.sub.2CH═CHCH.sub.2 (oleyl)); 2.47 (t, 2H, CH.sub.2CO (glutamate)); 3.10-3.60 (m, 15H, CH.sub.2(TACN), NCH.sub.2CO, NH.sub.2CH (glutamate)); 4.06-4.15 (t, 4H, COOCH.sub.2 (oleyl)); 5.32-5.42 (m, 4H, CH═CH (oleyl)); 7.29 (d, 1H, NHCO).
iv) Step 4: Preparation of 1,5-dioleyl-N(7′-carboxymethyl-1′,4′,7′-triazacyclononane)-L-glutamate (3) (DOG-TACN)
(195) Trifluoroacetic acid (1 mL) is added to a solution of 1,5-dioctadecenyl-N(1′,4′-di(tert-butoxycarbonyl)-7′-carboxymethyl-1′,4′,7′-triazacyclononane)-L-glutamate 3c (124 mg; 0.122 mmol) in dichloromethane (1 mL) under agitation. The reaction is kept under agitation for 30 minutes at room temperature. The reaction medium is then concentrated under a primary vacuum, re-dissolved in dichloromethane and again concentrated under a primary vacuum. The operation is repeated several times to remove all traces of trifluoroacetic acid. The residue is dried overnight under a primary vacuum. The pure compound 3 is obtained (126 mg; quantitative) in the form of a pale yellow oil.
(196) TLC: Rf=0 (CH.sub.2Cl.sub.2/MeOH 95/5 v/v).
(197) HRMS (ESI.sub.+): m/z measured at 817.7145 [M+H].sub.+; calculated at 817.7146 for C.sub.49H.sub.93N.sub.4O.sub.5.
d) Synthesis of 1,5-dioleyl-(1′,4′-dibetainyl-1′,4′,7′-triazacyclononane-7′-carboxymethyl)-L-glutamate (DOG-TACN-(Bet)2)
(198) The synthesis method shown in
(199) ##STR00045##
(200) This compound 4 is obtained by coupling 3-betainylthiazolidine-2-thione chloride (1b) with 1,5-dioleyl-N(7′-carboxymethyl-1′,4′,7′-triazacyclononane)-L-glutamate (3), the syntheses of which have previously been described, respectively in points 3.a) and 3.c).
(201) Triethylamine (1 mL; 7.2 mmol) is added to a solution of 1,5-dioctadecenyl-N(7′-carboxymethyl-1′,4′,7′-triazacyclononane)-L-glutamate 3 (100 mg; 0.097 mmol) in N,N dimethylformamide (DMF) (10 mL) under agitation and under an inert atmosphere. 3-betainylthiazolidine-2-thione chloride 1b (74.1 mg; 0.291 mmol) is then added directly to the reaction medium. The mixture is then kept under agitation for one night at room temperature and under an inert atmosphere. The solvents are then dry evaporated under a primary vacuum. The residue obtained is finally purified by silica gel chromatography (dichloromethane/methanol 95/5 to 8/2 v/v). The compound is thus isolated in the form of a white solid (24.2 mg; 23%).
(202) TLC: Rf=0.4 (CH.sub.2Cl.sub.2/MeOH 9/1 v/v)
(203) HRMS (ESI.sub.+): m/z measured at 1017.8672 [M+H].sub.+; calculated at 1017.8671 for C.sub.59H.sub.113N.sub.6O.sub.7.
(204) The example presented above describes the synthesis of triazacyclononane compounds containing a glycine betaine derivative. Similarly, compounds containing derivatives of arginine, lysine and ornithine have been produced. The efficacies of these compounds for the intracellular transport of antibodies differ according to the cell type used, but they are all more or less active. The presence of triacyclononane is essential for obtaining an effective compound.
EXAMPLE 2: BIOLOGICAL APPLICATIONS
(205) 1. Materials and Methods:
(206) The dioleoylphosphatidylethanolamine (DOPE) comes from Sigma-Aldrich (Saint Quentin Fallavier, France). The antibodies to be delivered into the cells are Immunoglobulin G (IgG) from goat serum from Sigma-Aldrich and a p50 human anti-NFkB mouse IgG from BioLegend (San-Diego, Calif., USA). All of the culture media come from Lonza (Basel, Switzerland).
(207) The efficacy of the transport of labeled antibodies into the cells is observed under fluorescence microscope.
(208) 2. Examples of Biological Applications Associated with the Transport of Antibodies into Living Cells
(209) a) Formulations of Amphiphilic Derivatives of Triazamacrocyclic Compounds According to the Invention in the Form of Nanoparticles in Water.
(210) In a pill machine, one of the amphiphilic derivatives of triazamacrocyclic compounds described according to the invention is solubilized in chloroform in the presence of a colipid, dioleoylphosphatidylethanolamine (DOPE), in different molar ratios. The solvent is then evaporated under a light nitrogen current and the lipid film obtained is dried under a primary vacuum for at least 30 minutes. The lipid film is then rehydrated in sterile deionized water for 2 hours at room temperature. The suspension is finally sonicated by means of a sonicator equipped with a microprobe (Sonic Ruptor 250, OMNI International, Kennesaw, USA) in order to obtain an aqueous dispersion of nanoparticles.
(211) The formulations are prepared at a concentration of 1 mmol/L of amphiphilic derivatives of triazamacrocyclic compounds in water. In the following examples, we will refer to formulations prepared “according to example 2.2a”.
(212) b) Influence of DOPE on the Efficacy of the Formulation
(213) Multiple formulations are prepared according to example 2.2a with different molar ratios of DOPE, i.e. respectively with 0%, 5%, 10%, 20% and 50% DOPE. Cos-7 cells are seeded in a 24-well plate (100,000 cells/well) and cultivated in 500 μL of DMEM in the presence of serum in a CO2 incubator for 24 hours. The transport of antibodies is then performed as follows: 1 μg of immunoglobulin G (IgG) from fluorescein-labeled goat serum is diluted in 10 μL of PBS buffer. 2 μL of formulation prepared according to example 2.2a are added and the mixture is incubated for 15 minutes at room temperature. 90 μL of serum-free DMEM are then added and the final mixture is transferred onto the Cos-7 cells in culture. The cells are then incubated in a CO2 incubator. The efficacy of the transport is viewed at 24 h and 48 h under fluorescence microscopy after the cells have been fixed with a formalin solution.
(214) For each formulation tested, the efficacy of the transport is measured at 5 h by flow cytofluorometry. The results presented in
(215) c) Transport of a Fluorescent Antibody into Fibroblasts
(216) NIH3T3 cells are seeded in a 24-well plate (100,000 cells/well) and cultivated in 500 μL of DMEM in the presence of serum in a CO2 incubator for 24 hours.
(217) The transport of antibodies is then performed as follows: 1 μg of IgG from fluorescein-labeled goat serum is diluted in 10 μL of PBS buffer. 2 μL of formulation prepared according to example 2.2a are added and the mixture is incubated for 15 minutes at room temperature. 90 μL of serum-free DMEM are then added and the final mixture is transferred onto the NIH3T3 cells in culture. The cells are then incubated in a CO2 incubator. The efficacy of the transport is viewed at 24 h and 48 h under fluorescence microscopy after the cells have been fixed with a formalin solution.
(218) The results presented in
(219) d) Transport of Fluorescent Antibodies into Other Types of Cells
(220) A549 and RAW264 cells are cultivated on a 24-well plate (100,000 cells/well). Different fluorescent antibodies are then delivered according to the protocol described in example 2.2b. The cells are observed under a fluorescent microscope after 5 h of incubation.
(221) The results presented in
(222) e) Evaluation of the Cell Toxicity of the Compound
(223) HeLa cells are seeded in a 96-well plate (10,000 cells/well) and cultivated in 100 μL of DMEM in the presence of serum in a CO2 incubator for 24 hours. The transport of antibodies is then performed as follows: 0.3 μg of IgG from fluorescein-labeled goat serum is diluted in 2.5 μL of PBS buffer. 0.3 μL of formulation prepared according to example 2.2a is added and the mixture is incubated for 15 minutes at room temperature. 20 μL of serum-free DMEM are then added and the final mixture is transferred onto the HeLa cells in culture. The cells are then incubated in a CO2 incubator. The cell toxicity of the compound is measured at between 5 h and 48 h by an MTT test (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide). The 96-well plate is analyzed on a spectrophotometer at 540 nm.
(224) The results presented in
(225) f) Kinetics of the Transport of an Antibody
(226) NIH3T3 cells are seeded in a 96-well plate (10,000 cells/well) and cultivated in 100 μL of DMEM in the presence of serum in a CO2 incubator for 24 hours. 0.3 μg of IgG from fluorescein-labeled goat serum is transported according to the protocol described in example 2.2e. The cells are then successively washed with PBS and with a trypsin solution, then centrifuged in order to remove as many free antibodies as possible, and are finally fixed with formalin. The intensity of the fluorescence is measured every 2 h, 3 h, 4 h then every 5 hours by a fluorometer at 525 nm.
(227) The results presented in
(228) g) Influence of PBS on the Kinetics of Transport of an Antibody
(229) VERO cells are seed in a 24-well plate (100,000 cells/well) and cultivated in 500 μL of DMEM in the presence of serum in a CO2 incubator for 24 hours.
(230) The transport of antibodies is then performed as follows: 1 μg of IgG from fluorescein-labeled goat serum is diluted in 10 μL of PBS buffer. 1 μL of formulation prepared according to example 2.2a is added and the mixture is incubated for 15 minutes at room temperature. 190 μL of PBS are then added and the final mixture is transferred onto the NIH3T3 cells in culture. The cells are then incubated at room temperature for 20 minutes, then the mixture is replaced with the DMEM culture medium with serum. The efficacy of the transport is viewed at 20 minutes under fluorescence microscopy.
(231) The results presented in
(232) h) Influence of the Ionic Force of the Medium on the Efficacy of the Compound
(233) A549 and BEAS-2B cells are seeded in a 24-well plate (100,000 cells/well) and cultivated in 500 μL of DMEM in the presence of serum in a CO2 incubator for 24 hours.
(234) The transport of antibodies is then performed as follows: 1 μg of IgG from fluorescein-labeled goat serum is diluted in 10 μL of PBS buffer or 10 μL of PBS 0.5× buffer or in 10 μL of PBS 0.1× buffer. 2 μL of formulation prepared according to example 2.2a are added and the mixture is incubated for 15 minutes at room temperature. 90 μL of serum-free DMEM are then added and the final mixture is transferred onto the culture cells. The cells are then incubated in a CO2 incubator. The efficacy of the transport is measured at 5 h by flow cytometry.
(235) The results presented in
(236) The weaker the ionic force of the medium is (PBS 0.5× and PBS 0.1×), the higher the percentage of cells having internalized the antibody is. However, the quantity of antibodies present in each cell is inversely proportional to this.
(237) i) Effect of the Quantity of Antibodies on the Efficacy of the Transport
(238) NIH3T3 cells are seeded in a 24-well plate (100,000 cells/well) and cultivated in 500 μL of DMEM in the presence of serum in a CO2 incubator for 24 hours. The transport of antibodies is performed as described in example 2.2b. The efficacy of the transport is measured at 5 h by flow cytofluorometry. The results presented in
(239) j) Translocation of the NFkb-p50 Protein Viewed, by Means of an Antibody
(240) A549 cells are seeded in a 24-well plate (100,000 cells/well) and cultivated in 500 μL of DMEM in the presence of serum in a CO2 incubator for 24 hours.
(241) The transport of antibodies is then performed as follows: 1 μg of p50 human anti-NFkB mouse IgG (containing 1% bovine serum albumin or BSA) labeled with Alexa Fluor 488 is diluted in 10 μL of PBS buffer. 2 μL of formulation prepared according to example 2.2a are added and the mixture is incubated for 15 minutes at room temperature. 90 μL of serum-free DMEM are then added and the final mixture is transferred onto the NIH3T3 cells in culture.
(242) The cells are then incubated in a CO2 incubator for 4 h. The cells are then treated with phorbol 12-myristate 13-acetate at 75 ng/mL for 3 h. The efficacy of the transport of NFkB p50 is viewed under fluorescence microscopy after the cells have been fixed by a formalin solution.
(243) The results presented in
(244) The labeling is relatively punctiform, which shows the accumulation of the protein in certain areas of the nucleus under the effect of the phorbol ester.
(245) k) Use of the Compound in In Vivo Applications
(246) 50 μg of IgG from DightLight 488-labeled goat serum are diluted in 400 μL of a solution containing 5% glucose.
(247) 6 μL of the formulation prepared according to example 2.2a concentrated ten times are added and the mixture is incubated for 20 minutes at room temperature.
(248) The mixture is then injected into the vein of the tail of an adult mouse. The mouse is sacrificed 2 days later, the tail is removed, ground, homogenized and centrifuged in order to enable the assay of the antibody still present by means of a fluorometer.
(249) The results presented in
(250) l) Biological Activity of an Antibody, Directed Against an Oncoprotein, Associated with One of the Compounds Prepared as Described Above
(251) U87 and 3T6 cells are seeded in a 24-well plate (75,000 cells/well) and cultivated in 500 μL of DMEM containing 10% serum in a CO2 incubator for 16 h. 1 or 2 μg of a monoclonal mouse antibody directed against and intracellular oncoprotein (antitumor antibody) or 1 or 2 μg of a goat serum IgG (control antibody) are transported into the cells according to the protocol described in example 2.2e.
(252) After 48 h of incubation in a CO2 incubator at 37° C., the cells are washed with PBS, then separated in a trypsin solution before being counted.
(253) The results presented in