Patent Application
20240197633
The present invention relates to the field of drug delivery, specifically to a PEGylated lipid as pharmaceutical carrier, in particular to a PEGylated lipid with a nitrogen-atom branching center and preparation method thereof, cationic liposomes containing the PEGylated lipid, liposome pharmaceutical compositions containing the PEGylated lipid, formulations of the compositions and application thereof.
Liposome is a microvesicle formed by encapsulating drugs in the lipoid bilayer. Liposomal nanoparticles contain liposomes and drugs, especially nucleic acid drugs, and their structures are similar to biofilms, which makes liposomal nanoparticle a biocompatible and non-toxic nanomaterial. They can encapsulate water-soluble and lipo-soluble drugs, with advantages such as reduced drug dose, sustained release, targeted drug release, and protection of encapsulated nucleic acids from degradation and clearance in serum. In addition, nanoliposome is also an excellent antigen carrier, not only encapsulating a series of antigens with different physicochemical properties and immune adjuvants, protecting protein polypeptide antigens from degradation, but also promoting the phagocytosis and presentation of antigen-presenting cells to antigen and enhancing the specific immune response of the body as well. Therefore, liposomal nanoparticles are widely used in the field of drug delivery.
Cationic lipids bearing positive charges on their surface form cationic liposome-nucleic acid drug complexes by electrostatic interaction with the negatively charged nucleic acids, while the whole cationic liposome-nucleic acid drug complexes bearing positive charges on their surface are prone to generate non-specific adsorption with serum proteins in plasma to form large-size aggregates, and the large-size aggregates are easily cleared by the reticuloendothelial system (RES) to cause short blood circulation time, poor stability and low transportation efficiency. For these reasons, the surfaces of cationic liposomes need to be modified to prepare long circulating cationic liposomes. Currently, the commonly used long circulating cationic liposomes are modified by PEGylated lipids, such as PEG-DMG PEG interacts with the water molecules in solvent via hydrogen bonding to form a hydrated layer on the surface of the modified cationic liposome, and the hydrated layers conceal the positive charges on the surfaces of cationic liposomes to inhibit the protein adsorption and reduce the recognition by the phagocytic system. Therefore, PEGylated lipids are important for preparing long circulating cationic liposomes and further improving the nucleic acid transport efficiency.
Although PEGylated lipids, such as PEG-DMQ have made great progress in drug delivery, there is still a need for novel PEGylated lipids that are suitable for regular therapeutic uses in this field.
The present invention provides novel PEGylated lipids and preparation methods thereof, cationic liposomes containing the PEGylated lipids, pharmaceutical compositions containing the cationic liposome and formulations thereof, especially the nucleic acid pharmaceutical compositions containing the cationic liposome and formulations thereof. The formulations of cationic liposome-nucleic acid pharmaceutical compositions can deliver nucleic acid drugs into cells, and improve the transportation efficiency of nucleic acid drugs, thereby improving the treatment effect of nucleic acid drugs.
The above-mentioned aim can be achieved as follows.
In one embodiment, provided herein is a PEGylated lipid:
A PEGylated lipid having the structure represented by the general formula (2):
wherein, Rc is, at each occurrence, independently a hydrogen atom or a C1-12 alkyl group;
wherein, Rd is, at each occurrence, independently a C1-12 alkyl group, G1 is a (k+1)-valent terminal branching group, j is 0 or 1, and F contains the functional group R01; when j is 0, G1 is absent, and k is 1; when j is 1, G1 protrudes F with the number of k, and k is an integer from 2 to 8;
Four preparation methods of the aforementioned PEGylated lipids are disclosed herein, specifically as follows:
Method-1: The foregoing PEGylated lipids can be prepared by the following steps:
Step 1:
Step 2:
Step 3:
Step 4:
Method-2: The foregoing PEGylated lipids can be prepared by the following steps:
Step 1:
Step 2:
Method-3: The foregoing PEGylated lipids can be prepared by the following steps:
Step 1:
Step 2:
Method-4: The foregoing PEGylated lipids can be prepared by the following steps:
Step 1:
An embodiment as disclosed herein provides a cationic liposome:
A cationic liposome, containing the PEGylated lipid represented by the formula (2).
An embodiment as disclosed herein provides a cationic liposome pharmaceutical composition:
A liposome pharmaceutical composition, containing cationic liposomes and drugs, wherein said cationic liposomes contain the PEGylated lipid represented by the formula (2).
An embodiment as disclosed herein provides a formulation of liposome pharmaceutical composition:
A formulation of liposome pharmaceutical composition, containing the foregoing liposome pharmaceutical composition and pharmaceutically acceptable diluents or excipients.
Compared with the Prior Art, the Present Invention Brings the Following Beneficial Effects:
The nitrogen atom as the branching center of the novel PEGylated lipid in the present invention is easily protonated under physiological pH conditions to produce partial positive charges, and thus the lipid is capable to bind negatively charged nucleic acids, thereby improving the loading efficiency of nucleic acid drugs.
The surfaces of the cationic liposomes can be modified by the novel PEGylated lipids of the present invention to obtain PEGylated cationic liposomes. The presence of the long-chain PEG overcomes the shortcoming of conventional cationic liposomes which are cleared by the phagocytes because of the serum proteins in plasma, improves the stability of cationic liposomes in serum, prolongs the circulation time in vivo, and further improves the transportation efficiency and treatment effect of drugs.
In the present invention, formulations of cationic liposome pharmaceutical compositions containing PEGylated lipids have stronger gene complexation ability and higher biocompatibility, and contribute to improving the therapeutic effect of drugs.
In the present invention, the terminals of the polyethylene glycol chains of the PEGylated lipids can also contain fluorescent groups or targeting groups, which furtherly improve the targeted therapeutic or diagnostic effect of the modified cationic liposome pharmaceutical compositions.
In the present invention, the polyethylene glycol chains of the PEGylated lipids can be degradable. In certain therapeutic areas, such as tumor therapy, the stable PEG coated on the surface of the liposome can keep the liposome components from leaving the endosome so that it prevents the delivery of the liposome contents into the cytoplasm, while the degradable PEG can be degraded to leave the liposome surface in the unique acidic environment of endosomal vacuoles or tumor tissues so that the therapeutic agent loaded in liposomes can be delivered to the target site efficiently.
In the present invention, unless otherwise defined, all terms are defined as follows.
In the present invention, when a structure has isomers, it may refer to any form of the isomers unless otherwise specified. For example, when cis- and trans-isomers are present, it can refer to either a cis-structure or a trans-structure; when E and Z isomers are present, it can refer to either an (E)-structure or a (Z)-structure; and, when the structure has optical activity, it can be either a laevoisomer or a dextroisomer.
In the present invention, the definition of the numerical interval includes both the numerical interval indicated by a dash (e.g., 0-12) and the numerical interval indicated by a wavy line (e.g., 0˜12). In the present invention, an integer interval denoted as an interval can represent the group consisting of all integers within the range of the interval unless otherwise specified, and said range includes two endpoints as well. For example, the integer interval 0-12 represents the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12. The numerical interval in the present invention includes but is not limited to the numerical intervals represented by integers, non-integers, percentages and fractions, and all of the foregoing numerical intervals include two endpoints unless otherwise specified.
In the present invention, a numerical value described with “about” or “approximately” generally indicates a numerical range of ±10% which, in some cases, can be amplified to +15%, not exceeding ±20%, based on the preset numerical value. For example, provided that the molar percentage of steroid lipids in the total lipids in a solvent-containing solution is about 40%, it is generally considered that the molar percentage of steroid lipids is 30%-50%.
In the present invention, unless otherwise specified, the terms “include”, “involve”, “contain”, and similar expressions in the description and claims shall be interpreted as “include but are not limited to”, openly and inclusively.
In the present invention, when two or more objects are “preferably each independently selected from” something and there are multiple levels of preferred options, the objects are not necessarily selected from the preferred options of the same level but allowed to be any of the following cases: one is selected from a wider range of options while the another is selected from a narrower range of options, one is selected from the maximum range of options while another is selected from any allowable options, or all of the objects are selected from the preferred options of the same level. For example, “R1 and R2 are preferably each independently a linear alkyl group, more preferably a C1-25 linear alkyl group, more preferably a C1-17 linear alkyl group” means R1 is a C1-25 linear alkyl group and R2 is a C1-17 linear alkyl group, or R1 is a C1-17 linear alkyl group and R2 is a C1-25 linear alkyl group, or R1 and R2 are both C1-25 linear alkyl groups, or R1 and R2 are both C1-17 linear alkyl groups.
In the present invention, a divalent linking group, e.g., a hydrocarbylene group, an alkylene group, an arylene group, an amide bond, and the like, can use either one of its two connecting ends to be connected to another group, unless otherwise specified. For example, when an amide bond is used as a divalent linking group between C—CH2CH2— and —CH2-D, both C—CH2CH2—C(═O)NH—CH2-D and C—CH2CH2—NHC(═O)—CH2-D are allowable.
In the present invention, when a terminal group of a linking group in a structural formula is easily confused with a substituent group of said linking group, “” is used to mark the location where the linking group is connected to other groups. For example, m the structural formulas
are used to mark the two locations where the divalent linking group is connected to other groups; the two aforementioned structural formulas represent —CH(CH2CH2CH3)2— and —CH2CH2CH(CH3)2—CH2CH2—, respectively.
In the present invention, the range of the number of carbon atoms in a group is denoted as a subscript of C, representing the number of carbon atoms of the group. For example, C1-12 represents a group “having 1 to 12 carbon atoms”, and C1-30 indicates a group “having 1 to 30 carbon atoms”. A “substituted C1-12 alkyl group” refers to a group obtained from one or more hydrogen atoms of a C1-12 alkyl group being replaced by substituents. A “C1-12 substituted alkyl group” refers to a group having 1 to 12 carbon atoms which is obtained from one or more hydrogen atoms of an alkyl group being replaced by substituents. As another example, when a group can be selected from C1-12 alkylene groups, it can be any alkylene group with the number of carbon atoms in the range indicated by the subscript, that is, the group can be selected from the group consisting of C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11 and C12 alkylene groups. In the present invention, a subscript marked in interval form represents any integer within the range unless otherwise specified, and the numerical range includes two endpoints.
In the present invention, heteroatoms are not particularly limited, including but not limited to O, S, N, P, Si, F, Cl, Br, I, B, etc.
In the present invention, the heteroatom used for substitution is referred to as a “substituent atom”, and the group used for substitution is referred to as a “substituent group”.
In the present invention, the term “substituted” indicates that at least one hydrogen atom of any aforementioned group (e.g., aliphatic hydrocarbon groups, hydrocarbon groups, alkyl groups or alkylene groups) is replaced by a bond connected to a non-hydrogen atom, said non-hydrogen atom including but not limited to a halogen atom (F, Cl, Br, or I), an oxo group (═O), a hydroxyl group (—OH), a hydrocarbyloxy group (—ORd, wherein R is a C1-12 alkyl group), a carboxyl group (—COOH), an amine group (—NRcRc, wherein both Rc are each independently H or a C1-12 alkyl group), a C1-12 alkyl group, and a cycloalkyl group. In some embodiments, said substituent group is a C1-12 alkyl group. In another embodiment, said substituent group is a cycloalkyl group. In another embodiment, said substituent group is a halide group, e.g., fluoride. In another embodiment, said substituent group is an oxo group. In another embodiment, said substituent group is a hydroxyl group. In another embodiment, said substituent group is an alkoxy group. In another embodiment, said substituent group is a carboxyl group. In another embodiment, said substituent group is an amine group.
In the present invention, the term “atomic spacing” or “atomic distance” refers to the number of main chain atoms spaced along the main chain, taking into account no pendant groups or side chains. It is usually the shortest atomic distance, which can be used to represent the length of the linking group. For example, the atomic spacing between A and B in A-CO—NH—B is 2, that in A-p-Ph-CH2—B is 5 (wherein p-Ph is p-phenylene), and that in A-CH(CH2CH2CH2CH3)—B is 1. The “main-chain atoms” accounted for the atomic spacing can only be non-hydrogen atoms. Wherein, concerning the divalent linking group with a ring structure, its atomic spacing refers to the minimum number of atoms calculated along the ring. For example, the atomic spacing of p-phenylene, namely 1,4-phenylene, is 4, the atomic spacing of m-phenylene is 3, and the atomic spacing of o-phenylene is 2. For another example, the atomic spacings of —CH2—, —CH(CH3)—, —C(CH3)2—, —CH(CH2Ph)2- and —C(CH2OX)— are all 1.
In the present invention, a “carbon chain linking group” refers to the linking group whose main chain atoms are all carbon atoms, while the pendant chains are allowed to contain heteroatoms or heteroatom-containing groups that replace the hydrogen atoms connected to the main chain carbon atoms. When a “main chain atom” is a heteroatom, it can also be termed a “main chain heteroatom”. For example, A-S—CH2—B, A-O—CH2—B, and
(wherein the atomic spacing is 4) are considered to contain main chain heteroatoms. Carbon chain linking groups can be divided into hydrocarbylene groups and carbon chain linking groups containing heteroatoms in their pendant groups; said pendant groups containing heteroatoms of carbon chain linking groups include but are not limited to an oxo group (═O), a thioxo group (═S), an imino group (connected to the main chain carbon through a carbon-nitrogen double bond), an oxa-hydrocarbon group in the form of an ether bond, a thia-hydrocarbon group in the form of a thioether bond, an aza-hydrocarbon group in the form of a tertiary amino group, etc. The main chain of the “carbon chain linking group” is entirely composed of carbon atoms, and the pendant groups of the carbon chain are allowed to contain heteroatoms, that is, the main chain is constructed by connecting methylene groups or substituted methylene groups. Said substituted methylene group can be substituted by one monovalent substituent, two monovalent substituents, or one divalent substituent (e.g., a divalent oxygen atom, or one that forms a three-membered ring
with a divalent methylene group). Said substituted methylene group can be a methylene group with one hydrogen atom being substituted (e.g., —CH(CH3)—), two hydrogen atoms being substituted respectively (e.g., —(CH3)C(OCH3)—), or two hydrogen atoms being substituted at the same time (e.g., a carbonyl group, a thiocarbonyl group, —C(═NH)—, —C(═N+H2)—, and a cyclic pendant group (e.g.,
wherein the atomic spacing is 1)).
In the present invention, the secondary amino bond and the hydrazine bond refer to the “—NH—” capped with hydrocarbylene groups at both ends, e.g., —CH2—NH—CH2—; while —C(═O)—NH— is termed an amide bond instead of a secondary amino bond.
In the present invention, a compound, a group, or an atom can be substituted and heterosubstituted at the same time, for example, a hydrogen atom can be replaced by a nitrophenyl group, and —CH2—CH2—CH2— can be replaced by —CH2—S—CH(CH3)—.
In the present invention, a “linking bond” is only for connection and contains no atoms. If the definition of a group includes a linking bond, it means the group can be absent.
In the present invention, the expression “at each occurrence, each independently” not only mean that different groups can be each independently selected from the definitions, but also mean that the same group appearing at each different position can also be independently selected from the definition. For example, Z is, at each occurrence, independently selected from the group consisting of —C(═O)—, —OC(═O)—, —C(═O)O—, —OC(═O)O—, —O—, —S—, —C(═O)S—, —SC(═O)—, —NRcC(═O)—, —C(═O)NRc—, —NRcC(═O)NRc—, —OC(═O)NRc—, —NRcC(═O)O—, —SC(═O)NRc—, —NRcC(═O)S—, and
wherein Rc is, at each occurrence, independently a hydrogen atom or a C1-12 alkyl group; in the group “—NRcC(═O)NRc—”, two Rc groups can be the same or different, which are each independently a hydrogen atom or a C1-12 alkyl group.
In the present invention, the term “group” contains at least 1 atom, referring to the radical formed by a compound losing one or more atoms. Relative to a compound, the group formed by a compound losing part of its groups is also termed a residue. The valence of a group is not particularly limited, and examples include a monovalent group, a divalent group, a trivalent group, a tetravalent group, . . . , a hectovalent group, etc. Wherein, groups whose valence is equal to or greater than 2 are collectively defined as linking groups. A linking group can also contain only one atom, such as an oxy group and a thioxy group.
In the present invention, the term “hydrocarbon” refers to a class of compounds that contains only carbon atoms and hydrogen atoms.
In the present invention, hydrocarbons are classified into aliphatic hydrocarbons and aromatic hydrocarbons by category. Hydrocarbons containing neither phenyl rings nor hydrocarbyl-substituted phenyl rings are defined as aliphatic hydrocarbons. Hydrocarbons containing at least one phenyl ring or hydrocarbyl-substituted phenyl ring are defined as aromatic hydrocarbons. An aromatic hydrocarbon can contain aliphatic hydrocarbon groups, such as toluene, diphenylmethane, 2,3-dihydroindene, etc.
In the present invention, hydrocarbons are classified into saturated hydrocarbons and unsaturated hydrocarbons by the degree of saturation. All aromatic hydrocarbons are unsaturated hydrocarbons. Saturated aliphatic hydrocarbons are also termed alkanes. An unsaturated aliphatic hydrocarbon is not particularly limited with respect to the degree of unsaturation. For example, it includes but is not limited to alkenes (containing carbon-carbon double-bonds), alkynes (containing carbon-carbon triple-bonds), dienes (containing two carbon-carbon double-bonds), and the like. When the aliphatic moieties are saturated in an aromatic hydrocarbon, said aromatic hydrocarbon is also termed arylalkane, such as toluene.
In the present invention, the structures of hydrocarbons are not particularly limited, including linear structures without pendant groups, branched structures bearing pendant groups, ring-containing structures containing at least one ring, dendritic structures, comb-like structures, hyperbranched structures, etc. Unless otherwise specified, preferable structures include linear structures without pendant groups, branched structures bearing pendant groups and ring-containing structures, which correspond to linear hydrocarbons, branched hydrocarbons and cyclic hydrocarbons, respectively. Wherein, hydrocarbons without cyclic structures are termed open-chain hydrocarbons, including but not limited to linear structures without pendant groups and branched structures bearing pendant groups. Open-chain hydrocarbons fall into the scope of aliphatic hydrocarbons, so linear hydrocarbons are also described as linear aliphatic hydrocarbons and branched hydrocarbons are also described as branched aliphatic hydrocarbons.
In the present invention, hydrocarbons with any carbon atom replaced by a heteroatom are collectively referred to as hetero-hydrocarbons.
In the present invention, an aliphatic hetero-hydrocarbon refers to the hetero-hydrocarbon derived from an aliphatic hydrocarbon, including aliphatic heterocyclic hydrocarbons and aliphatic open-chain hetero-hydrocarbons. Saturated aliphatic hetero-hydrocarbons are also termed heteroalkanes.
In the present invention, “hydrocarbon group” refers to the residue of a hydrocarbon molecule with at least one hydrogen atom removed. According to the number of the lost hydrogen atoms, hydrocarbon groups can be classified into monovalent hydrocarbon groups (with one hydrogen atom lost), divalent hydrocarbon groups (with two hydrogen atoms lost, also referred to as hydrocarbylene groups), trivalent hydrocarbon groups (with three hydrogen atoms lost), and the like. Accordingly, when n hydrogen atoms are lost, the valence of the resulting hydrocarbon group is n. In the present invention, hydrocarbon groups refer specifically to monovalent hydrocarbon groups, unless otherwise specified.
In the present invention, the source of hydrocarbon group is not particularly limited, for example, aliphatic hydrocarbons or aromatic hydrocarbons, saturated hydrocarbons or unsaturated hydrocarbons, linear hydrocarbons, branched hydrocarbons or cyclic hydrocarbons, hydrocarbons or hetero-hydrocarbons, etc. According to the degree of saturation, e.g., the source can be alkanes, alkenes, alkynes, or dienes, etc.; with respect to cyclic hydrocarbons, the source can be, e.g., alicyclic hydrocarbons or aromatic hydrocarbons, monocyclic hydrocarbons or polycyclic hydrocarbons; and, with respect to heterocyclic hydrocarbons, the source can be, e.g., aliphatic heterocyclic hydrocarbons or aromatic heterocyclic hydrocarbons.
In the present invention, the “aliphatic hydrocarbon group” refers to the residue of an aliphatic hydrocarbon molecule with at least one hydrogen atom removed. Aliphatic hydrocarbon groups in the present invention refer specifically to monovalent aliphatic hydrocarbon groups, unless otherwise specified. Aliphatic hydrocarbon groups include saturated aliphatic hydrocarbon groups and unsaturated aliphatic hydrocarbon groups.
In the present invention, the “alkyl group” refers to a hydrocarbon group obtained from an alkane losing a hydrogen atom at any location, unless otherwise specified. Said alkyl group can be linear or branched, substituted or unsubstituted. Specific examples include that a propyl group refers to either a 1-propyl group or an isopropyl group, and a propylene group refers to a 1,3-propylene group, a 1,2-propylene group or an isopropylidene group.
In the present invention, the “unsaturated hydrocarbon group” refers to a hydrocarbon group obtained from an unsaturated hydrocarbon losing hydrogen atoms. The hydrocarbon group obtained from an unsaturated hydrocarbon losing hydrogen atoms bonded to unsaturated carbon atoms, can be an alkenyl group, an alkynyl group, or a dienyl group, etc., e.g., a propenyl group or a propynyl group. According to the difference of unsaturated bonds, the hydrocarbon group obtained from an unsaturated hydrocarbon losing hydrogen atoms bonded to saturated carbon atoms, can be termed, e.g., an alkenyl hydrocarbon group, an alkynyl hydrocarbon group, a dienyl hydrocarbon group, or the like, and specifically, e.g., an allyl group or a propargyl group.
In the present invention, the “alkenyl” or “alkenyl group” refers to a substituted or unsubstituted, linear or branched alkenyl group which contains two or more carbon atoms (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen eighteen, nineteen, twenty or more carbon atoms) and at least one carbon-carbon double bond. The term “C2-15 alkenyl group” refers to a substituted or unsubstituted, linear or branched alkenyl group which contains 2-15 carbon atoms and at least one carbon-carbon double bond, that is, an alkenyl group can contain one, two, three, four, or more carbon-carbon double bonds. Alkenyl groups in the present invention include substituted and unsubstituted alkenyl groups, unless otherwise specified.
In the present invention, the “alkynyl” or “alkynyl group” refers to a linear or branched, optionally substituted hydrocarbon which contains two or more carbon atoms (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen eighteen, nineteen, twenty or more carbon atoms) and at least one carbon-carbon triple bond. The term “C2-15 alkynyl group” refers to a substituted or unsubstituted, linear or branched alkynyl group which contains 2-15 carbon atoms and at least one carbon-carbon triple bond, that is, an alkynyl group can contain one, two, three, four, or more carbon-carbon triple bonds. Alkynyl groups in the present invention include substituted and unsubstituted alkynyl groups, unless otherwise specified.
In the present invention, the term “molecular weight” represents the mass of a compound, and the term “average molecular weight” represents the mass of a compound component of a general formula in macroscopic matter. Unless otherwise specified, the “average molecular weight” refers to the “number-average molecular weight” (Mn). The number-average molecular weight refers to the molecular weight of polydisperse blocks or substances, or that of monodisperse blocks or substances. Unless otherwise specified, the unit of measurement of “molecular weight” and “average molecular weight” is dalton, Da. The molecular weight of a polyethylene glycol chain can also be measured by “degree of polymerization” which specifically refers to the number of repeating units (oxyethylene units, EO-units) in a compound molecule. Accordingly, “average degree of polymerization”, “number-average degree of polymerization” or “number of EO units” represents the average value or the number average value of the number of repeating units.
In the present invention, for polydisperse cases, when a term such as “equal”, “same”, “equivalent”, or “approximately equal” (including other forms of equivalent expression) is used to describe the molecular weight or the degree of polymerization of a single compound and the number-average molecular weight or the number-average degree of polymerization of a compound component in macroscopic matter, unless otherwise specified, the term does not impose a strict numerical equality but indicates an approximation or an approximate equality in value; said approximation or approximate equality preferably refers to a deviation within ±10%, more preferably a deviation within ±5%, generally based on the preset value. The terms “about” and “approximately” generally indicate a numerical range of ±10% which, in some cases, can be amplified to ±15%, not exceeding ±20%. For example, the deviations of 10 kDa from 11 kDa and 12 kDa are 10% and 20%, respectively. As another example, when the molecular weight of a certain PEG component in the specified general formula is 5 kDa, it is allowed that the corresponding molecular weight or number-average molecular weight is 5 kDa±10%, that is, variable within the range of 4500˜5500 Da. However, for monodisperse cases, the sameness or equality with respect to the number of oxyethylene units in a single compound molecule or general formula thereof indicates the strict numerical equality; for example, if the number of EO units of a certain PEG component is set to 11, then a value being 12 is beyond the scope; whereas, in order to obtain the compound component with the desired number of EO units, the products in macroscopic matter obtained by certain preparation methods may also contain impurities with other numbers of EO units besides the component with the target number of EO units because of the limitation of preparation methods and purification methods; in this case, if the deviation of the average number of EO units from the preset number of EO units is within ±5% (preset value≥10) or within ±0.5 (preset value<10), it can be considered to have obtained the monodisperse product in macroscopic matter with the target component; moreover, when the content of the component with the number of EO units as desired or within the range of average number reaches certain percentages (preferably ≥90%, more preferably >95%, more preferably >96%, more preferably >98%, more preferably 99% to 100%), the obtained product in macroscopic matter falls within the protected scope of the present invention; even if the above mentioned content percentages are not met, the resulting products of insufficient contents and the components in the form of co-products or by-products, as long as prepared by the preparation methods of the present invention or by similar methods using basically the same preparation ideas, whether separation and purification are performed or not, all fall within the protected scope of the present invention.
In the present invention, when Da, kDa, repeating unit, and number of EO units are used to describe the molecular weight of a compound of a general formula containing polydisperse components, for a single molecule, the value falls within a certain range covering the specified value (including endpoints, preferably within the range of 10%); when the number of oxyethylene units is used to describe the preset molecular weight of a compound of a general formula containing monodisperse components, the value is not variable in any range but a discrete point; however, the average number of the EO units might be variable within a certain range (not exceeding ±10% or ±1, preferably not exceeding ±5% or ±0.5) because of heterogeneity in molecular weights of the prepared product. For example, the molecular weight of mPEG is 5 kDa, it means the molecular weight of a single molecule of the general formula is between 4500˜5500 Da, and the average molecular weight of the corresponding component of the prepared product is 5 kDa, that is, the product with the average molecular weight between 4500˜5500 Da is regarded as the target product, and only the components whose molecular weights fall within the scope contribute to the content of the target component; for another example, if the designed mPEG contains 22 oxyethylene units, then the numbers of EO units of all compound molecules of the general formula should be strictly 22, while the prepared product could be a mixture of compounds with the numbers of EO units being 20, 21, 22, 23, and 24, and in this case, when the average number of EO units is within the range of 22±2.2 (preferably in the range of 22±1.1), it is considered to have obtained the target component, meanwhile the components whose molecular weights fall within the numerical range can be regarded as the target component and used to calculate the purity. In the present invention, the product with PDI<1.005 can be considered to be monodisperse with PDI=1.
In the present invention, concerning the polydispersity index PDI in the present invention, different batches of materials can be regarded as the same material without significant difference, as long as the PDI values do not exceed the preset value while the other parameters are the same or considered to be the same.
In the present invention, concerning the percentage, “about” and “approximately” generally refer to a tolerance of 0.5%.
In the present invention, the terms “stable” and “degradable” regarding groups are a pair of opposing concepts.
In the present invention, the term “degradable” (“be degradable” or “can be degraded”) means that a chemical bond can be cleaved into at least two independent residues. If a linking group remains whole, but with its structure altered after chemical changes, then it still belongs to the scope of “being stable”. The conditions to be “degradable” are not particularly limited, which can be physiological conditions in vivo, simulated physiological environments in vitro, or other conditions, preferably physiological conditions in vivo and simulated physiological environments in vitro. Said physiological condition is not particularly limited, including but not limited to physiological environments of serum, heart, liver, spleen, lung, kidney, bone, muscle, fat, brain, lymph node, small intestine, gonad, etc., which are intracellular or in the extracellular matrix, in normal tissues or in pathologic tissues (e.g., tumor, inflammation, etc.). Said simulated physiological environment in vitro is not particularly limited, including but not limited to physiological saline, buffer, culture medium, and the like. The degradation is not particularly limited with respect to the rate, e.g., rapid degradation via enzymolysis, slow hydrolysis under physiological conditions, etc. Said physiological conditions in vivo include physiological conditions during treatment such as ultraviolet radiation, thermal therapy, etc. The conditions for degradation include but are not limited to light, heat, low temperature, enzymatic condition, oxidation-reduction condition, acidic condition, basic condition, physiological condition, simulated physiological environment in vitro, etc., preferably light, heat, enzymatic condition, oxidation-reduction condition, acidic condition, basic condition, etc. Said “degradable” means that the degradation can occur under any stimulation from the above conditions. Said light condition includes but is not limited to visible light, ultraviolet light, infrared light, near-infrared light, mid-infrared light, etc. Said heat condition refers to the temperature higher than the normal physiological temperature, normally the temperature higher than 37° C. and below 45° C., and preferably below 42° C. Said low temperature refers to the temperature below the human physiological temperature, preferably below 25° C., more preferably ≤10° C., with specific examples such as refrigeration temperature, freezer temperature, temperature for liquid nitrogen treatment, 2˜10° C., 4˜8° C., 4° C., 0° C., −20±5° C., etc. Said enzymatic condition is not particularly limited, and all enzymes that can be physiologically generated are included, e.g., peptidases, proteases, lyases, etc. Said oxidation-reduction condition is not particularly limited, e.g., redox transformation or hydrogenation-reduction transformation between a mercapto group and a disulfide bond. Said acidic condition and basic condition mainly refer to the pH conditions of internal body parts such as normal tissues, pathologic tissues, and organs or tissues in treatment; for example, the stomach is under acidic condition, and a tumor site is usually under acidic condition as well. Said “degradable” means that the degradation can be realized through metabolism in vivo (e.g., physiological effect, enzymatic reaction, oxidation-reduction, etc.), microenvironment stimulation in specific areas inside the body (e.g., acidic condition and basic condition), clinical therapeutic stimulation (e.g., light, heat, and low temperature), etc. What should be noted is that, for bond cleavage, some conditions in organic chemistry that are extreme for organisms, e.g., strong acid, strong base, high temperature (e.g., above 100° C.), etc., are not included in the scope of the conditions for degradation of degradable bonds in the present invention. For example, although an ether bond can be cleaved under strong acid conditions (e.g., hydrobromic acid), it is always classified as a stable linking group in the present invention.
In the present invention, as long as a linking group can keep as a whole linking group (i.e., a linking group which can keep covalently linking with the adjacent groups), it would be defined as “stable” (“be stable” or “can remain stable”), wherein chemical changes without breaking the wholeness of the linking group are allowed. Said chemical changes are not particularly limited, including but not limited to isomerization, oxidation reaction, reduction reaction, ionization, protonation, deprotonation, substitution reaction, etc. The conditions to be “stable” are not particularly limited, including but not limited to light, heat, low temperature, enzymatic condition, oxidation-reduction, neutral condition, acidic condition, basic condition, physiological condition, simulated physiological environment in vitro, etc., preferably light, heat, enzymatic condition, oxidation-reduction, acidic condition, basic condition, etc. A group or compound being “stable” indicates that the components can remain stably connected in the metabolic cycle in vivo and the molecular weight will not be reduced due to chain cleavage as long as the group or compound maintains integrity without particular stimulation (e.g., pH condition in specific areas and light, heat, and low temperature in treatment).
In the present invention, for a specific linking group, “stable” is not an absolute concept. For example, an amide bond is much more stable than an ester bond under acidic or basic condition, and in the present invention, “stable” linking groups include amide bonds. However, the peptide bond, for example, is a special kind of amide bond formed via the dehydration condensation of two amino acids that respectively provide an α-carboxyl group and an α-amino group for the reaction, could also be cleaved when encountering specific enzymatic interactions, and therefore it is also within the scope of “degradable” linking groups. Similarly, a urethane group, a thiourethane group, or the like could be either a “stable” linking group or a “degradable” linking group. More commonly, a urethane group, a thiourethane group, and the like tend to degrade slowly, while an amide bond in the form of a non-peptide bond can remain stable in the systemic circulation in vivo. As another example, common ester bonds will degrade under acidic or basic condition, while an ester bond contained in a special structure could also degrade under UV exposure. As another example, even though some chemical bonds will degrade under specific enzymatic conditions, they can still be regarded as stable if their circulation in clinical use (e.g., point-of-site administration) does not or basically not go through said enzymatic conditions.
In the present invention, in order to define more clearly the degradability of a compound structure, provided herein is a criterion for reference which is, a threshold being a specific percentage (e.g., 90%) of the examined chemical bond maintained within a limited time interval. Taking 90% as an example, it usually takes the pharmacokinetic curve of functionalized PEGylated products as a reference, and is based on the dose percentage that meets the clinical evaluation criteria. For example, for intravenously administered PEGylated drugs, when the plasma concentration (calculated in terms of effective drug ingredients including PEGylated drugs and non-PEGylated components after degradation) is lower than 15% of the initial concentration (or another percentage more consistent with the clinical evaluation of the drug), the remaining 85% is taken as the base value; if the proportion of a linking group with its chemical bonds maintained exceeds 90%, then the group is regarded as stable in the present invention; on the contrary, if said 90% is not met, then the linking group belongs to degradable groups. The hydrolytic stabilization and enzymatic degradation reported in the published literature are also included in the present invention. Taking hydrolytic stabilization as an example, the hydrolysis rate in the process of hydrolytic stabilization reported in the published literature is also included herein, preferably referring to the hydrolysis rate less than 1-2% per day (generally 2%) under physiological conditions, in mass or molar weight. The hydrolysis rate of typical chemical bonds can be found in most standard handbooks of chemistry.
In the present invention, the term “hydroxyl protecting group” includes all the groups which can be used as common hydroxyl protecting groups. A hydroxyl protecting group is preferably selected from the group consisting of an alkanoyl group (e.g., an acetyl group and a butyryl group), an aromatic alkanoyl group (e.g., a benzoyl group), a benzyl group, a triphenylmethyl group, a trimethylsilyl group, a t-butyldimethylsilyl group, an allyl group, an acetal group, and a ketal group. The removal of an acetyl group is generally carried out under basic condition, most commonly by the ammonolysis with NH3/MeOH or by the methanolysis catalyzed by methanol anions. The benzyl group is easy to be removed via palladium-catalyzed hydrogenolysis in a neutral solution at room temperature, or via the reduction reaction with metallic sodium in ethanol or liquid ammonia. The triphenylmethyl group is usually removed by catalytic hydrogenolysis. The trimethylsilyl group is usually removed with reagents containing fluoride ions (e.g., tetrabutylammonium fluoride/anhydrous THF, etc.). The t-butyldimethylsilyl ether is relatively stable, which can withstand the ester hydrolysis with alcoholic potassium hydroxide and mild reduction conditions (e.g., Zn/CH3OH and the like), and can be removed by fluoride ions (e.g., Bu4N+F−) in THE solution or by aqueous acetic acid at room temperature.
In the present invention, the “carboxyl protecting group” refers to the protecting group which can be transformed into a carboxyl group through hydrolysis or the deprotection reaction of the carboxyl protecting group itself. A carboxyl protecting group is preferably selected from the group consisting of an alkyl group (e.g., a methyl group, an ethyl group and a butyl group) and an aralkyl group (e.g., a benzyl group), and more preferably selected from the group consisting of a butyl group (tBu), a methyl group (Me), and an ethyl group (Et). In the present invention, the “protected carboxyl group” refers to the group obtained from a carboxyl group being protected by an appropriate carboxyl protecting group, preferably selected from the group consisting of a methoxycarbonyl group, an ethoxycarbonyl group, a t-butyloxycarbonyl group, and a benzyloxycarbonyl group. Said carboxyl protecting group can be removed through hydrolysis catalyzed by acids or alkalis, or through pyrolysis reaction occasionally; for example, t-butyl groups can be removed under mild acidic condition, and benzyl groups can be removed by hydrogenolysis. The reagent used for removing carboxyl protecting groups is selected from the group consisting of TFA, H2O, LiOH, NaOH, KOH, MeOH, EtOH and combinations thereof, preferably selected from the combination of TFA and H2O, the combination of LiOH and MeOH, and the combination of LiOH and EtOH. The deprotection of a protected carboxyl group can produce the corresponding free acid, which can be carried out in the presence of an alkali that forms pharmaceutically acceptable salts with said free acid generated during said deprotection.
In the present invention, the term “amino protecting group” includes all the groups which can be used as common amino protecting groups, such as an aryl C1-6 alkyl group, a C1-6 alkoxy C1-6 alkyl group, a C1-6 alkoxycarbonyl group, an aryloxycarbonyl group, C1-6 alkylsulfonyl group, an arylsulfonyl group, a silyl group, etc. An amino protecting group is preferably selected from the group consisting of a t-butoxycarbonyl group (Boc), a p-methoxybenzyloxycarbonyl group (Moz) and a 9-fluorenylmethoxycarbonyl group (Fmoc). The reagent used for removing amino protecting groups is selected from the group consisting of TFA, H2O, LiOH, MeOH, EtOH and combinations thereof, preferably selected from the combination of TFA and H2O, the combination of LiOH and MeOH, and the combination of LiOH and EtOH. The reagent used for removing the Boc protecting group is TFA or HCl/EA, preferably TFA. The reagent used for removing the Fmoc protecting group is the N,N-dimethylformamide (DMF) solution containing 20% piperidine.
In the present invention, the “activation of a carboxyl group” refers to the activation of a carboxyl group with carboxyl activating agents. The activated carboxyl group can promote the condensation reactions, for example, by inhibiting the generation of racemic impurities, by accelerating the reaction through catalysis, etc. The “carboxyl activating group” refers to the residue of a carboxyl activating agent. Said carboxyl activating agent is selected from the group consisting of N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), N-hydroxy-5-norbornene-2,3-dicarboximide (HONb), and N,N′-dicyclohexylcarbodiimide (DCC), and combinations thereof, preferably selected from the combination of NHS/EDCI, the combination of NHS/DCC, and the combination of HONb/DCC, and more preferably the combination of NHS/EDCI.
In the present invention, the term “cation” refers to the corresponding structure bearing a positive charge, either permanently, or non-permanently but in response to certain conditions (such as pH). Therefore, the cations include permanent cations and those cationisable compounds, groups, or atoms. Permanent cations refer to the corresponding compounds, groups, or atoms that bear positive charges under conditions of any pH value or hydrogen ion activity of their environment. Typically, a positive charge is generated by the presence of quaternary nitrogen atom. When a compound carries multiple such positive charges, it can be called a polycation. A cationisable substance refers to a compound, group or atom that is positively charged at a lower pH and uncharged at a higher pH of its environment. On the other hand, in non-aqueous environments where the pH cannot be determined, a cationisable compound, group, or atom is positively charged at a high hydrogen ion concentration and uncharged at a low concentration or activity of hydrogen ions. It depends on the individual properties of the cationisable or polycationisable compound, especially the pKa of the respective cationisable group or atom, at which pH or hydrogen ion concentration said compound is charged or uncharged. In the diluted aqueous environment, the so-called Henderson-Hasselbalch equation can be used to estimate the fraction of positively charged cationisable compounds, groups or atoms, which is well-known to those skilled in the art. For example, in some embodiments, if a compound or moiety is cationisable, it is preferred that it is positively charged at a pH value of about 1 to 9, preferably 4 to 9, 5 to 8 or even 6 to 8, more preferably at a pH value of or below 9, of or below 8, of or below 7, and most preferably at physiological pH values (e.g., about 7.3 to 7.4), i.e., under physiological conditions, particularly under physiological salt conditions of the cell in vivo. In other embodiments, it is preferred that the cationisable compound or moiety is predominantly neutral at physiological pH values (e.g., about 7.0-7.4), but becomes positively charged at lower pH values. In some embodiments, the preferred range of pKa for the cationisable compound or moiety is about 5 to about 7.
In the present invention, the “cationic component/compound” typically refers to a charged molecule, which is positively charged (cationic) at a pH value of typically about 1 to 9. In some embodiments, the cationic component/compound is preferably charged at a pH value of or below 9 (e.g., 5 to 9), of or below 8 (e.g., 5 to 8), of or below 7 (e.g., 5 to 7), and most preferably at physiological pH values (e.g., about 7.3 to 7.4). Therefore, a cationic peptide, protein, polysaccharide, lipid or polymer according to one embodiment of the present invention is positively charged under physiological conditions, particularly under physiological salt conditions of the cell in vivo.
In the present invention, a lipid nanoparticle, cationic peptide, protein, polysaccharide, lipid, or polymer is uncharged, having a neutral charge or being respectively electrically neutral under physiological conditions, particularly under the physiological salt conditions of the cell in vivo. A cationic peptide or protein preferably contains a larger amount of cationic amino acids, e.g., a greater number of Arg, His, Lys or Orn than other amino acid residues (especially more cationic amino acids than anionic amino acid residues like Asp or Glu), or contains blocks predominantly formed by cationic amino acid residues. The expression “cation” may also refer to “polycationic” components/compounds. The cationic component/compound may also refer to a cationic lipid capable of being positively charged. For example, cationic lipids contain one or more amine groups bearing positive charges, and the preferred cationic lipids are ionizable so that they can exist in a positively charged or neutral form depending on pH. The ionization of the cationic lipid affects the surface charge of a lipid nanoparticle (LNP) under different pH conditions. This charge state can influence plasma protein absorption, blood clearance and tissue distribution as well as the ability to form non-bilayer structures critical to the intracellular delivery of nucleic acids.
In the present invention, the “PEGylated lipid” refers to a molecule containing lipid and polyethylene glycol moieties. In addition to those represented by the general formula (2) in the present invention, PEGylated lipids also include but are not limited to 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)](PEG-DSPE), PEG-cholesterol, PEG-diacylglycamide (PEG-DAG), PEG-dialkyloxypropyl (PEG-DAA), and specifically PEG500-dipalmitoylphosphatidylcholine, PEG2000-dipalmitoylphosphatidylcholine, PEG500-distearylphosphatidylethanolamine PEG2000-distearylphosphatidylethanolamine, PEG500-1,2-dioleoylphosphatidylethanolamine, PEG2000-1,2-dioleoylphosphatidylethanolamine, PEG2000-2,3-distearoylglycerol (PEG-DMG), and the like.
In the present invention, the “neutral lipid” refers to any lipid substance which is uncharged or exists in the form of neutral zwitterion at the chosen pH, preferably phospholipid. This kind of lipid includes but is not limited to 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanola mine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dioleoylphosphatidylserine (DOPS), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), and combinations thereof. The neutral lipid can be synthetic or natural.
In the present invention, a “steroid lipid” is selected from the group consisting of a cholesterol, a coprostanol, sitosterol, an ergosterol, a campesterol, a stigmasterol, a brassicasterol tomatidine, an ursolic acid, a α-tocopherol, and mixtures thereof.
In the present invention, an “amino acid residue” is an amino acid from which, formally, a hydrogen atom has been removed from an amino group and/or from which, formally, a hydroxy group has been removed from a carboxy group and/or from which, formally, a hydrogen atom has been removed from a sulfhydryl group and/or with a protected amino group and/or with a protected carboxyl group and/or with a protected sulfhydryl group.
Imprecisely, an amino acid residue can be described as an amino acid. The source of the amino acid in the present invention is not particularly limited unless otherwise specified, which can be either natural or unnatural, or a mixture of both. The configuration of the amino acid in the present invention is not particularly limited unless otherwise specified, which can be either L-type or D-type, or a mixture of both. In one embodiment of the invention, the amino acid is a hydrophobic amino acid, selected from the group consisting of tryptophan (Trp), phenylalanine (Phe), valine (Val), isoleucine (Ile), leucine (Leu), and tyrosine (Tyr). In another embodiment of the invention, the amino acid is a hydrophilic amino acid, selected from the group consisting of glutamic acid (Glu), aspartic acid (Asp), histidine (His), glutamine (Gln), asparagine (Asn), serine (Ser), threonine (Thr), proline (Pro), glycine (Gly), lysine (Lys), and arginine (Arg), preferably selected from glycine and lysine, and more preferably lysine.
In the present invention, the term “functional group source” refers to those having reaction activity or potential reaction activity, photosensitivity or potential photosensitivity, targeting properties or potential targeting properties. Said “potential” means that the functional group source can be converted into a reactive group through chemical processes including but not limited to functional modification (e.g., grafting, substitution, etc.), deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, leaving group transformation, etc., and can exhibit luminescence or targeting properties through external stimuli such as light, heat, enzymes, specific binding molecules, microenvironment in vivo, etc. Said luminescence is not particularly limited, including but not limited to visible light, fluorescence, phosphorescence, etc.
In the present invention, a variant form refers to a structural form that can be transformed into the target reactive group after any process of chemical change selected from the group consisting of oxidation, reduction, hydration, dehydration, electronic rearrangement, structural rearrangement, salt complexation and decomplexation, ionization, protonation, deprotonation, substitution, deprotection, leaving group transformation, etc.
In the present invention, a “variant form of a reactive group” refers to a form which still has reactivity after the reactive group undergoes at least one process of chemical change selected from oxidation, reduction, hydration, dehydration, electronic rearrangement, structural rearrangement, salt complexation and decomplexation, ionization, protonation, deprotonation, substitution, deprotection, leaving group transformation, etc., or a non-reactive form that has been protected.
In the present invention, the “micro-modification” refers to a chemical modification process that can be completed through simple chemical reaction processes. Said simple chemical reaction processes mainly refer to deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, leaving group transformation, etc.
The “micro variant form” corresponds to the “micro-modification”, referring to the structural form that can be transformed into the target reactive group after simple chemical reaction processes such as deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, leaving group transformation, etc. Said leaving group transformation includes the transformation from an ester form to an acyl chloride form.
The description “any appropriate” in expressions such as “any appropriate linking group”, “any appropriate reactive group”, etc., indicates that the structure accords with the fundamental principle of chemical structure and can enable the preparation method in the present invention to be implemented successfully. A chemical structure described in this way can be considered to have a clear and determined scope.
When at least two structural types are listed, the “any combination” of the listed structural types refers to a combination of any two or more afore-listed types of relevant structures; the number of a structural unit is not limited, which means, the number of any structural unit can be 0, 1, or greater than 1; and, when the number of structural units of the same type is greater than 1, the structural units could be from the same or different chemical structures, wherein the total number of said structural units is at least 2. For example, an arbitrary combination of alkylene groups, divalent cycloalkyl groups, divalent cycloalkenyl groups, divalent cycloalkynyl groups, divalent cyclodienyl groups, arylene groups, carbon-carbon double bonds, carbon-carbon triple bonds, conjugated carbon-carbon double bonds, divalent aliphatic heterocyclic linking groups, divalent aromatic heterocyclic linking groups, and carbon chain linking groups with heteroatom-containing pendant groups, can be, e.g., -Ph-CH2-Ph-(arylene-alkylene-arylene), —CH2-Ph-CH2CH2-(alkylene-arylene-alkylene; wherein, the alkylene group has a quantity of 2, with different chemical structures), or a structure obtained from the phenyl ring of any aforementioned example being replaced with the ring of a hexane, diazepine, or 1-(2-pyridinyl)hexahydro-1H-1,4-diazepine. As another example, cycloalkenyl hydrocarbon group=cycloalkenyl group+hydrocarbylene group=hydrocarbon group substituted by a cycloalkenyl group, and cyclodienyl hydrocarbon group=hydrocarbon group substituted by a cyclodienyl group. In the present invention, the alkylene group (i.e., divalent alkyl group) includes the open-chain alkylene group and the divalent cycloalkyl group; the open-chain alkylene group refers to the divalent alkyl group containing no ring structures, and the divalent cycloalkyl group refers to the ring-containing divalent alkyl group.
In the present invention, the “adjuvant” or “adjuvant component” is typically a (e.g., pharmacological or immunological) agent or composition that may modify (e.g., enhance) the efficacy of other agents (e.g., drugs or vaccines). Conventionally, the term refers in the context of the invention to a compound or composition that serves as a carrier or auxiliary substance for immunogens and/or other pharmaceutically active compounds. It is to be interpreted in a broad sense and refers to a broad spectrum of substances that are able to increase the immunogenicity of antigens incorporated into or co-administered with an adjuvant in question. In the present invention, an adjuvant will preferably enhance the specific immunogenic effect of the active agents of the present invention. Typically, “adjuvant” or “adjuvant component” has the same meaning and can be used mutually. Adjuvants may be divided, e.g., into immuno potentiators, antigenic delivery systems or even combinations thereof.
In the present invention, the “N/P ratio” refers to molar ratio of nitrogen atoms in cationic lipids to phosphoric acid groups in nucleic acids.
In the present invention, the “nucleic acid” refers to DNA, RNA or modified form thereof, including purines and pyrimidine bases in DNA (adenine “A”, cytosine “C”, guanine “G”, thymine “T”), or purines and pyrimidine bases in RNA (adenine “A”, cytosine “C”, guanine “G”, uracil “U”).
In the present invention, the term “RNA” refers to a ribonucleic acid that may be naturally or non-naturally occurring. For example, an RNA may include modified and/or non-naturally occurring components such as one or more nucleobases, nucleosides, nucleotides, or linkers. An RNA may include a cap structure, a chain terminating nucleoside, a stem loop, a polyA sequence, and/or a polyadenylation signal. An RNA may have a nucleotide sequence encoding a polypeptide of interest. For example, an RNA may be a messenger RNA (mRNA). The translation of an mRNA encoding a particular polypeptide, for example, the in vivo translation of an mRNA inside a mammalian cell, may produce the encoded polypeptide. An RNA may be selected from the non-limiting group consisting of small interfering RNA (siRNA), asymmetrical interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), mRNA, single guide RNA (sgRNA), cas9 mRNA and mixtures thereof.
In the present invention, an antisense oligonucleotide or small interfering RNA (siRNA) can inhibit the expression of the target gene and the target protein in vitro or in vivo.
In the present invention, the Fluc mRNA can express the luciferase protein which emits bioluminescence in the presence of fluorescein substrates, so Fluc is commonly used in mammalian cell culture to measure gene expression and cell activity.
In the present invention, the term “inhibiting the expression of a target gene” refers to the ability of nucleic acids to silence, reduce or inhibit the expression of a target gene. To examine the extent of gene silencing, a test sample (e.g., a sample of cells in culture expressing the target gene) is contacted with nucleic acids that inhibit the expression of the target gene. The expression of the target gene in the test sample or test animal is compared to the expression of the target gene in a control sample (e.g., a sample of cells in culture expressing the target gene) which is not contacted with or administered the nucleic acids. The expression of the target gene in the control sample may be assigned a value of 100%. In particular embodiments, inhibition of expression of a target gene is achieved when the level of target gene expression in the test sample or the test mammal relative to the level of target gene expression in the control sample or the control mammal is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%.
In the present invention, assays for determining the level of target gene expression include but are not limited to dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays.
In the present invention, the term “transfection” refers to the introduction of a species (e.g., RNA) into a cell. Transfection may occur, for example, in vitro, ex vivo, or in vivo.
In the present invention, the term “antigen” typically refers to a substance that can be recognized by the immune system, preferably recognized by the adaptive immune system, and triggers an antigen-specific immune response, for example, forming antibodies and/or antigen-specific T cells as a part of the adaptive immune response. Typically, the antigen may be or may contain a peptide or a protein that can be presented to T cells by MHC. In the present invention, the antigen may be a translation product of the provided nucleic acid molecule (preferably mRNA as defined herein). In this context, fragments, variants, and derivatives of peptides and proteins containing at least one epitope are also recognized as antigens.
In the present invention, the term “delivery” refers to providing an entity to the target, for example, delivering drugs and/or therapeutic agents and/or prophylactic agents to subjects, said subjects being tissues and/or cells of human and/or other animals.
In the present invention, the “pharmaceutically acceptable carrier” refers to a diluent, adjuvant, excipient, or vehicle administered together with the therapeutic agent, which is, in a reasonable medical judgment range, suitable for the contact of the human and/or other animal without excessive toxicity, stimulation, hypersensitive response, other problems or complications corresponding to a reasonable benefit/risk ratio. Pharmaceutically acceptable carriers that can be used in the pharmaceutical composition in the present invention include but are not limited to sterile liquids, e.g., water and oil, including the oil from petroleum, animal, plant, or synthetic origin, e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc. When said pharmaceutical composition is administered intravenously, water is an exemplary carrier. Physiological saline, glucose and aqueous glycerol solution can also be used as liquid carriers, and are especially used for injection. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, maltose, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, defatted milk powder, glycerol, propylene glycol, water, ethanol, etc. Said composition can also contain a small amount of humectant, emulsifier or pH buffer as needed. Oral preparations can contain standard carriers, e.g., pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Specifically, excipients include but are not limited to anti-adherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (pigments), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, sorbents, suspending or dispersing agents, sweeteners, and waters for hydration. More specifically, excipients include but are not limited to butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinized starch, phenyl paraben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E (α-tocopherol), vitamin C, and xylitol.
In the present invention, pharmaceutical compositions can act systemically and/or locally. For this purpose, they can be administered by appropriate routes such as injection (e.g., intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection, including instillation) or transdermal administration, or administered by oral, buccal, transnasal, transmucosal or topical routes or in the form of ophthalmic preparation or by inhalation. Regarding these administration routes, the pharmaceutical compositions of the present invention can be administered in a suitable dosage form. The dosage forms include but are not limited to tablets, capsules, lozenges, hard sugar agents, powders, sprays, creams, ointments, suppositories, gels, pastes, emulsions, ointments, aqueous suspensions, injectable solutions, elixirs, and syrups.
In the present invention, vaccines are preventive or therapeutic materials that provide at least one antigen or antigenic function. An antigen or antigenic function can stimulate the body's adaptive immune system to provide an adaptive immune response.
In the present invention, treatment refers to the management and care of patients to resist diseases, obstacles or symptoms, which is intended to delay the development of diseases, obstacles or symptoms, reduce or alleviate symptoms and complications, and/or cure or eliminate diseases, obstacles or symptoms. The patients to be treated are preferably mammals, especially humans.
1.1. A PEGylated Lipid Having the Structure Represented by the General Formula (2):
wherein, Rc is, at each occurrence, independently a hydrogen atom or a C1-12 alkyl group;
wherein, Rd is, at each occurrence, independently a C1-12 alkyl group, G1 is a (k+1)-valent terminal branching group, j is 0 or 1, and F contains the functional group R01; when j is 0, G1 is absent, and k is 1; when j is 1, G1 protrudes F with the number of k, and k is an integer from 2 to 8;
1.1.1. Divalent Linking Group L3, L4, L5, L7, L8, Z, Z1, Z2
In the present invention, the structures of L3, L4, L5, L7, L8, Z, Z1, and Z2 are not particularly limited, each independently including but not limited to linear structures, branched structures or ring-containing structures.
In the present invention, the number of non-hydrogen atoms of L3, L4, L5, L7, L8, Z, Z1, and Z2 are not particularly limited, each preferably ranging independently from 1 to 50, more preferably from 1 to 20, and more preferably from 1 to 10. Said non-hydrogen atom is a carbon atom or a heteroatom. Said heteroatom includes but is not limited to O, S, N, P, Si, B, etc. When the number of non-hydrogen atoms is 1, the non-hydrogen atom can be a carbon atom or a heteroatom. When the number of non-hydrogen atoms is greater than 1, the species of non-hydrogen atoms are not particularly limited, which can be 1, 2 or more than 2 species; when the number of non-hydrogen atoms is greater than 1, they can be any combination of carbon atoms and carbon atoms, carbon atoms and heteroatoms, or heteroatoms and heteroatoms.
In the present invention, two identical or different reactive groups may form a divalent linking group after reaction. The reaction conditions are related to the types of the resulting divalent linking groups, and the prior art can be introduced herein. For example, amino groups can react with active esters, active formates, sulfonate esters, aldehydes, α,β-unsaturated bonds, carboxylic groups, epoxides, isocyanates, and isothiocyanates, respectively, to obtain divalent linking groups such as amide groups, urethane groups, amino groups, imide groups (which can be further reduced to secondary amino groups), amino groups, amide groups, amino alcohols, urea bonds, thiourea bonds, etc.; mercapto groups can react with active esters, active formates, sulfonate groups, mercapto groups, maleimide groups, aldehyde groups, α,β-unsaturated bonds, carboxyl groups, iodoacetamide groups, and anhydride groups, respectively, to obtain divalent linking groups such as thioester groups, thiocarbonate groups, thioether groups, disulfide groups, thioether groups, thiohemiacetal linking groups, thioether groups, thioester groups, thioether groups, thioester groups, etc.; unsaturated bonds can react with mercapto groups to obtain thioether groups; carboxyl groups or acyl halides can respectively react with mercapto groups and amino groups to obtain groups such as thioester bonds, amide bonds, etc.; hydroxyl groups can react with carboxyl groups, isocyanates, epoxides, and chlorocarbonyloxy groups to obtain divalent linking groups such as ester groups, carbamate groups, ether bonds, carbonate groups, etc.; carbonyl groups or aldehyde groups can react with amino groups, hydrazines and hydrazides to obtain divalent linking groups such as imine groups, hydrazone groups, acylhydrazone groups, etc.; reactive groups such as azido groups, alkynyl groups, alkenyl groups, mercapto groups, azido groups, dienyl groups, maleimide groups, 1,2,4-triazoline-3,5-dione groups, dithioester groups, hydroxylamine groups, hydrazide groups, acrylate groups, allyloxy groups, isocyanate groups, tetrazole groups and the like can undergo click reactions to form various divalent linking groups including but not limited to the structures of triazoles, isoxazoles, thioether bonds, and the like.
The stability of divalent linking groups L3, L4, L5, L7, L8, Z, Z1, and Z2 are not particularly limited, wherein any divalent linking group or a divalent linking group consisting of any aforementioned divalent linking group and adjacent heterosubstituted groups thereof is independently a stable linking group STAG or a degradable linking group DEGG
1.1.1.1. Divalent Linking Group L7, L8
In the present invention, wherein, one of L7 and L8 is —OC(═O)O—, —C(═O)—, —O—, —O(CRcRc)sO—, —S—, —C(═O)S—, —SC(═O)—, —NRcC(═O)—, —C(═O)NRc—, —NRcC(═O)NRc—, —OC(═O)NRc—, —NRcC(═O)O—, —SC(═O)NRc—, or —NRcC(═O)S—, and the other is a linking bond, —OC(═O), —C(═O)O—, —OC(═O)O—, —C(═O)—, —O—, —O(CRcRc)sO—, —S—, —C(═O)S—, —SC(═O)—, —NRcC(═O)—, —C(═O)NRc—, —NRcC(═O)NRc—, —OC(═O)NRc—, —NRcC(═O)O—, —SC(═O)NRc—, or —NRcC(═O)S—, wherein, Rc is independently, at each occurrence, a hydrogen atom or an alkyl group, s is 2, 3, or 4.
In one specific embodiment of the present invention, it is preferred that one of L7 and L8 is —OC(═O)O—, —C(═O)—, —O—, —O(CH2)sO—, —S—, —C(═O)S—, —SC(═O)—, —NHC(═O)—, —C(═O)NH—, —NHC(═O)NH—, —OC(═O)NH—, —NHC(═O)O—, —SC(═O)NH—, or —NHC(═O)S—, and the other is a linking bond, —OC(═O)—, —C(═O)O—, —OC(═O)O—, —C(═O)—, —O—, —O(CH2)sO—, —S—, —C(═O)S—, —SC(═O)—, —NHC(═O)—, —C(═O)NH—, —NHC(═O)NH—, —OC(═O)NH—, —NHC(═O)O—, —SC(═O)NH—, or —NHC(═O)S—.
In one specific embodiment of the present invention, it is more preferred that one of the aforementioned L7 and L8 is —OC(═O)O—, —C(═O)—, —O—, —O(CH2)sO—, —S—, —C(═O)S—, —SC(═O)—, —NHC(═O)—, —C(═O)NH—, —NHC(═O)NH—, —OC(═O)NH—, —NHC(═O)O—, —SC(═O)NH—, or —NHC(═O)S—, and the other is a linking bond, —O—, —OC(═O)—, or —C(═O)O—.
In one specific embodiment of the present invention, said L7 and Ls are preferably selected from the following situations:
1.1.1.2. Divalent Linking Group L3
In the present invention, L3 is a linking bond, -L4-, —Z-L4-, -L4-Z—, —Z-L4-Z—, -L4-Z-L5-, —Z-L4-Z-L5-, -L4-Z-L5-Z—, —Z-L4-Z-L5-Z—, or -L4-Z-L4-Z-L5-Z—; said L4 and L5 are carbon-chain linking groups, which are each independently represented by —(CRaRb)t—(CRaRb)o—(CRaRb)p—, wherein, t, o, and p are each independently an integer from 0 to 12, and t, o, and p are not simultaneously 0; Ra and Rb are, at each occurrence, each independently a hydrogen atom or a C1-12 alkyl group; said Z is, at each occurrence, independently selected from the group consisting of —C(═O)—, —OC(═O)—, —C(═O)O—, —OC(═O)O—, —O—, —S—, —C(═O)S—, —SC(═O)—, —NRcC(═O)—, —C(═O)NRc—, —NRcC(═O)NRc—, —OC(═O)NRc—, —NRcC(═O)O—, —SC(═O)NRc—, —NRcC(═O)S—, and
wherein, Rc is, at each occurrence, independently a hydrogen atom or a C1-12 alkyl group;
In one specific embodiment of the present invention, Rc is preferably a hydrogen atom.
In one specific embodiment of the present invention, L3 contains degradable groups, and said degradable group refers to the group which can degrade under any condition selected from the group consisting of light, heat, low temperature, enzymatic condition, oxidation-reduction condition, acidic condition, basic condition, physiological condition, and simulated physiological environment in vitro; the L4 and L5 contained in said L3 are preferably each independently —(CH2)t—; L3 is —(CH2)t—, —(CH2)tZ—, —Z(CH2)t—, —(CH2)tZ(CH2)t—, —Z(CH2)tZ—, —(CH2)tZ(CH2)tZ—, —Z(CH2)tZ(CH2)t—, or —Z(CH2)tZ(CH2)tZ—; wherein, t is an integer from 1 to 12, and Z is, at each occurrence, independently —C(═O)—, —OC(═O)—, —C(═O)O—, —OC(═O)O—, —O—, —S—, —C(═O)S—, —SC(═O)—, —NHC(═O)—, —C(═O)NH—, —NHC(═O)NH—, —OC(═O)NH—, —NHC(═O)O—, —SC(═O)NH—, or —NHC(═O)S—; L3 is preferably —(CH2)t—, —(CH2)tO—, —(CH2)tC(═O)—, —(CH2)tC(═O)O—, —(CH2)tOC(═O)—, —(CH2)tC(═O)NH—, —(CH2)tNHC(═O)—, —(CH2)tOC(═O)O—, —(CH2)tNHC(═O)O—, —(CH2)tOC(═O)NH—, —(CH2)tNHC(═O)NH—, —O(CH2)t, —C(═O)(CH2)t—, —C(═O)O(CH)t—, —(CH2)tC(═O)CH2)—, —C(═O)NH(CH2)t—, —NHC(═O)(CH2)t—, —OC(═O)O(CH2)t—, —NHC(═O)O(CH2)t—, —OC(═O)NH(CH2)t—, —NHC(═O)NH(CH2)t—, —(CH2)tO(CH2)t—, —(CH2)tC(═O)(CH2)t—, —(CH2)tC(═O)O(CH2)t—, —(CH2)tOC(═O)(CH2)t—, —(CH2)tC(═O)NH(CH2)t—, —(CH2)tNHC(═O)(CH2)t—, —(CH2)tOC(═O)O(CH2)t—, —(CH2)tNHC(═O)O(CH2)t—, —(CH2)tOC(═O)NH—, —O(CH2)tNHC(═O)NH(CH2)t—, —O(CH2)tO—, —C(═O)(CH2)tC(═O)—, —C(═O)O(CH2)tC(═O)O—, —OC(═O)(CH2)tOC(═O)—, —C(═O)O(CH2)tOC(═O)—, —OC(═O)(CH2)tC(═O)O—, —OC(═O)O(CH2)tOC(═O)O—, —C(═O)NH(CH2)tC(═O)NH—, —NHC(═O)(CH2)tNHC(═O)—, —NHC(═O)(CH2)tC(═O)NH—, —C(═O)NH(CH2)tNHC(═O)—, —NHC(═O)O(CH2)tNHC(═O)O—, —OC(═O)NH(CH2)tOC(═O)NH—, —NHC(═O)O(CH2)tOC(═O)NH—, —OC(═O)NH(CH2)tNHC(═O)O—, —NHC(═O)NH(CH2)tNHC(═O)NH—, —C(═O)(CH2)tO—, —C(═O)(CH2)tC(═O)O—, —C(═O)(CH2)tOC(═O)—, —C(═O)(CH2)tOC(═O)O—, —C(═O)(CH2)tNHC(═O)O—, —C(═O)(CH2)tOC(═O)NH—, —C(═O)(CH2)tNHC(═O)NH—, —C(═O)(CH2)tC(═O)O(CH2)t—, —C(═O)(CH2)tOC(═O)(CH2)t—, —C(═O)(CH2)tOC(═O)O(CH2)t—, —C(═O)(CH2)tNHC(═O)O(CH2)t—, —C(═O)(CH2)tOC(═O)NH(CH2)t—, —C(═O)(CH2)tNHC(═O)NH(CH2)t—, or —C(═O)(CH2)tC(═O)(CH2)tNHC(═O)O—, wherein t is an integer from 2 to 12; most preferably, L3 is —C(═O)O—, —(CH2)tO—, —(CH2)tC(═O)O—, —(CH2)tOC(═O)—, —(CH2)tO—, —C(═O)(CH2)tO—, —C(═O)(CH2)tOC(═O)—, —C(═O)(CH2)tC(═O)O—, —C(═O)(CH2)tC(═O)NH—, —C(═O)(CH2)tOC(═O)NH—, —C(═O)(CH2)tNHC(═O)O—, —C(═O)(CH2)tOC(═O)NH(CH2)t—, or —C(═O)(CH2)tC(═O)(CH2)tNHC(═O)O—.
1.1.2 Description of Stable and Degradable Groups
In the present invention, a stable linking group STAG or a degradable linking group DEGG can exist within any of the divalent linking groups L3, L4, L5, L7, L8, Z, Z1, and Z2 mentioned above, or within a divalent linking group consisting of any aforementioned divalent linking group and adjacent heterosubstituted groups thereof.
1.1.2.1. Stable Divalent Linking Groups STAG in the Present Invention
The condition of being stable for a stable divalent linking group STAG is not particularly limited, including but not limited to any condition selected from light, heat, low temperature, enzymatic condition, oxidation-reduction, acidic condition, basic condition, physiological condition, simulated physiological environment in vitro, etc., preferably light, heat, enzymatic condition, oxidation-reduction, acidic condition, basic condition, etc.
The type of stable divalent linking group STAG is not particularly limited, including but not limited to an alkylene group, a divalent heteroalkyl group, a carbon-carbon double bond, a carbon-carbon triple bond, a divalent dienyl group, a divalent cycloalkyl group, a divalent cycloalkenyl group, a divalent cycloalkenyl hydrocarbon group, a divalent cycloalkynyl hydrocarbon group, an aromatic ring, an aliphatic heterocyclic group, a heterophenylene group, an aromatic-fused heterocyclic group, a hetero-fused heterocyclic group, a substituted alkylene group, a substituted heteroalkyl group, a substituted divalent heteroalkyl group, a substituted double bond, a substituted triple bond, a substituted dienyl group, a substituted divalent cycloalkyl group, a substituted divalent cycloalkenyl group, a substituted divalent cycloalkenyl hydrocarbon group, a substituted divalent cycloalkynyl hydrocarbon group, a substituted aromatic ring, a substituted aliphatic heterocyclic group, a substituted heterophenylene group, a substituted aromatic-fused heterocyclic group, a substituted hetero-fused heterocyclic group, an ether bond, a thioether bond, a urea bond, a thiourea bond, a carbamate group, a thiocarbamate group, —P(═O)—, a divalent silyl group without active hydrogen atoms, a divalent boron-containing linking group, a secondary amino group, a tertiary amino group, a carbonyl group, a thiocarbonyl group, an amide group, a thioamide group, a sulfonamide group, an enamino group, a triazole group, a 4,5-dihydroisoxazole group, a skeleton of an amino acid or its derivative, and a stable divalent linking group composed of any two or more groups thereof.
Specifically, a stable divalent linking group STAG includes but is not limited to the structures described or listed in the references CN104530413A, CN104530415A and CN104530417A. Taking CN104530417A as an example, the corresponding paragraphs are from [0627] to [0704]. The way in which a STAG is composed of two or more stable divalent linking groups is not particularly limited, which includes but is not limited to those described in the paragraph [0704] of CN104530417A.
1.1.2.2. Degradable Divalent Linking Group DEGG in the Present Invention
The condition of degradable divalent linking groups DEGG to be degradable is not particularly limited, including but not limited to light, heat, low temperature, enzymatic condition, oxidation-reduction, acidic condition, basic condition, physiological condition, simulated physiological environment in vitro, etc., preferably light, heat, enzymatic condition, oxidation-reduction, acidic condition, basic condition, etc.
The divalent linking group formed by the combination of any degradable divalent linking group DEGG and any stable divalent linking group STAG is still a degradable linking group. As for an aromatic ring-containing degradable divalent linking group, it can also be formed by the combination of aromatic rings and degradable divalent linking groups.
The type of degradable divalent linking group DEGG is not particularly limited, including but not limited to those containing any divalent linking group selected from the group consisting of a disulfide bond, a vinylether bond, an ester group, a thioate group, a thioester group, a dithioester group, a carbonate group, a thiocarbonate group, a dithiocarbonate group, a trithiocarbonate group, a carbamate group, a thiocarbamate group, a dithiocarbamate group, an acetal group, a cycloacetal group, a thioacetal group, an azaacetal group, an azacycloacetal group, an azathiaacetal group, a dithioacetal group, a hemiacetal group, a thiohemiacetal group, an azahemiacetal group, a ketal group, a thioketal group, an azaketal group, an azacycloketal group, an azathiaketal group, an imine bond, a hydrazone bond, an acylhydrazone bond, an oxime bond, a thiooxime ether group, a semicarbazone bond, a thiosemicarbazone bond, a hydrazino group, a hydrazide group, a thiocarbohydrazide group, an azocarbohydrazide group, an azothiocarbohydrazide group, a hydrazino formate group, a hydrazino thioformate group, a carbohydrazide group, a thiocarbohydrazide group, an azo group, an isourea group, an isothiourea group, an allophanate group, a thioallophanate group, a guanidino group, an amidino group, an aminoguanidinyl group, a carbamimidamido group, an imino acid group, a thioimidate group, a sulfonate group, a sulfinate group, a sulfonyl hydrazide group, a sulfonyl ureido group, a maleimide group, an orthoester group, a phosphate group, a phosphirate group, a phosphinate group, a phosphonate group, a phosphosilicate group, a silicate group, an amide group, a thioamide group, a sulfonamide group, a polyamide group, a phosphamide group, a phosphoramidite group, a pyrophosphamide group, a cyclophosphamide group, an ifosfamide group, a thiophosphamide group, an aconityl group, a peptide fragment, a skeleton of a nucleotide or its derivative, and a skeleton of a deoxynucleotide or its derivative, and those containing the combination of any two or more said divalent linking groups.
Herein, said carbamate group, thiocarbamate group, amide group, phosphamide group, and the like, can be regarded as either a stable linking group or a degradable linking group, depending on the environmental characteristics of its use.
Specifically, a degradable divalent linking group DEGG includes but is not limited to the structures described or listed in the references CN104530413A, CN104530415A and CN104530417A. Taking CN104530417A as an example, the corresponding paragraphs are from [0705] to [0725].
1.1.3. Alkylene Group B3 and B4
In the present invention, B3 and B4 are each independently a linking bond or a C1-30 alkylene group, more preferably a linking bond or a C1-20 alkylene group.
In one specific embodiment of the present invention, B3 and B4 are both linking bonds.
In one specific embodiment of the present invention, one of B3 and B4 is a linking bond, and the other is a C1-20 alkylene group.
In one specific embodiment of the present invention, B3 and B4 are each independently a C1-20 alkylene group, and specifically, each independently selected from the group consisting of a methylene group, an ethylene group, a propylene group, a butylene group, a pentylene group, a hexylene group, a heptylene group, an octylene group, a nonylene group, a decylene group, an undecylene group, a dodecylene group, a tridecylene group, a tetradecylene group, a pentadecylene group, a hexadecylene group, a heptadecylene group, an octadecylene group, a nonadecylene group, and an eicosylene group.
1.1.4. Aliphatic Hydrocarbon Groups R1, R2
In the present invention, R1 and R2 are each independently a C1-30 aliphatic hydrocarbon group.
In one specific embodiment of the present invention, R1 and R2 are preferably each independently a C5-30 aliphatic hydrocarbon group, more preferably a C10-30 aliphatic hydrocarbon group, and most preferably a C10-20 aliphatic hydrocarbon group. Said aliphatic hydrocarbon group is an alkyl group, an alkenyl group or an alkynyl group.
In one specific embodiment of the present invention, R1 and R2 are preferably each independently a linear alkyl group, a branched alkyl group, a linear alkenyl group, a branched alkenyl group, a linear alkynyl group, or a branched alkynyl group, preferably a linear aliphatic hydrocarbon group, more preferably a C1-25 linear aliphatic hydrocarbon group, more preferably a C1-20 linear aliphatic hydrocarbon group; most preferably selected from the group consisting of a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, a heptyl group, an octyl group, a nonyl group, a decyl group, an undecyl group, a dodecyl group, a tridecyl group, a tetradecyl group, a pentadecyl group, a hexadecyl group, a heptadecyl group, an octadecyl group, a nonadecyl group, an eicosyl group, a (Z)-tridec-8-enyl group, a (Z)-tetradec-9-enyl group, a (Z)-pentadec-8-enyl group, a (Z)-hexadec-9-enyl group, a (Z)-heptadec-5-enyl group, a (Z)-heptadec-8-enyl group, a (E)-heptadec-8-enyl group, a (Z)-heptadec-10-enyl group, a (8Z, 11Z)-heptadec-8,11-dienyl group, a (Z)-octodec-6-enyl group, a (Z)-octodec-9-enyl group, a (E)-octodec-9-enyl group, a (Z)-octodec-11-enyl group, a (9Z, 12Z)-octodec-9,12-dienyl group, a (9Z, 12Z, 15Z)-octodec-9,12,15-trienyl group, a (8Z, 11Z, 14Z)-octodec-8,11,14-trienyl group, a (Z)-eicos-11-enyl group, a (11Z, 14Z)-eicos-11,14-dienyl group, a (Z)-nonadec-10-enyl group, a (10Z, 13Z)-nonadec-10,13-dienyl group, a 2,6,10-trimethylundec-1,5,9-trienyl group, a 3,7,11-trimethyldodec-2,6,10-trienyl group, and a 3,7,11,15-tetramethylhexadec-2-enyltridecyl group.
In one specific embodiment of the present invention, it is preferred that the aforementioned R1 and R2 are both methyl groups.
In one specific embodiment of the aforementioned PEGylated lipid of the present invention, R1 and R2 are each independently a branched alkyl group, a branched alkenyl group, or a branched alkynyl group, and each independently represented as
wherein, Re and Rf are each independently selected from the group consisting of a C1-15 alkyl group, a C2-15 alkenyl group, and a C2-15 alkynyl group, more preferably the group consisting of a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, a heptyl group, an octyl group, a nonyl group, a decyl group, an undecyl group, a dodecyl group, a tridecyl group, a tetradecyl group, a vinyl group, a propenyl group, an allyl group, a butenyl group, an enyl butyl group, a pentenyl group, an enyl pentyl group, a hexenyl group, an enyl hexyl group, a heptenyl group, an enyl heptyl group, an octenyl group, an enyl octyl group, a nonenyl group, an enyl nonyl group, a decenyl group, an enyl decyl group, an ethynyl group, a propynyl group, a propargyl group, a butynyl group, an ynyl butyl group, a pentynyl group, an ynyl pentyl group, a hexynyl group, an ynyl hexyl group, a heptynyl group, an ynyl heptyl group, an octynyl group, an ynyl octyl group, a nonynyl group, an ynyl nonyl group, a decynyl group, and an ynyl decyl group, and more preferably the group consisting of a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, a heptyl group, an octyl group, a nonyl group, and a decyl group. R1 and R2 are further preferably each independently selected from the group consisting of the following structures:
wherein t is an integer from 0 to 12.
1.1.5. R
In the present invention, R is a hydrogen atom, —Rd, —ORd, —NRdRd, —SRd, —C(═O)Rd, —C(═O)ORd, —OC(═O)Rd, —OC(═O)ORd, or
wherein, Rd is, at each occurrence, independently a C1-12 alkyl group, G1 is a (k+1)-valent end-branching group, j is 0 or 1, and F contains the functional group R01; when j is 0, G1 is absent, and k is 1; when j is 1, G1 protrudes F with the number of k, and k is an integer from 2 to 8.
1.1.5.1. Rd
In the present invention, Rd is, at each occurrence, independently a C1-12 alkyl group.
In one specific embodiment of the present invention, Rd is preferably a C1-8 alkyl group; specifically, Rd is preferably a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, a heptyl group, or an octyl group.
1.1.5.2. G1
In the present invention, j is 0 or 1; when j is 0, G1 is absent, and k is 1; when j is 1, G1 exists as a (k+1)-valent terminal branching group and leads to k F containing functional groups, wherein k is an integer from 2 to 8, preferably 2, 3 or 4.
In one specific embodiment of the present invention, when G1 is preferably a branching group with a valence of three or more, G1 is preferably a trivalent or tetravalent branching group; G1 is preferably a trivalent branching group, more preferably a trivalent branching group as a residue of glycerol or amino acid.
In the present invention, when a terminal bifunctionalization is performed, G1 is preferably derived from an alcohol, thiol, primary amine, secondary amine, sulfonate, or halide compound which contains two free or protected hydroxyl groups (e.g., triethanolamine p-toluenesulfonate, glycerol monomercaptoacetate, 3,4-dihydroxy-2′-chloro-acetophenone, and hydroxyl-protected forms thereof), or form an alcohol, thiol, primary amine, secondary amine, sulfonate, or halide compound which contains two free or protected mercapto groups (e.g., dimercaprol and mercapto-protected forms thereof), or from an alcohol, thiol, primary amine, secondary amine, sulfonate, and halide compound which contains two primary amino groups, two secondary amino groups, two protected primary amino groups, or two protected secondary amino groups, etc. Wherein, an exemplary alcohol containing two primary amino groups is 1,3-diamino-2-propanol. The terminal bifunctionalization can be carried out using an aldehyde containing 1 epoxy group, an alcohol containing 1 epoxy group (e.g.,
a sulfonate containing 1 epoxy group, a halide containing 1 epoxy group, or a compound containing one epoxy group and 1 other type of reactive group. The terminal bifunctionalization can also be achieved by the Michael addition reaction of a primary amine and 2 molecules of acrylic acid ester. It is also possible to reduce the disulfide bond after capping with lipoic acid and then proceed to ring-opening, therefore obtaining two mercapto groups at the ends.
In the present invention, when a terminal trifunctionalization is performed, G1 is preferably derived from a tetrafunctionalized small molecule (htetraSM) which contains three hydroxyl groups and one different kind of reactive group, including but not limited to N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid, 3-[tris(hydroxymethyl)methylamino]-1-propanesulfonic acid, methyl 6-O-tosyl-α-D-glucopyranoside, 2-(bromomethyl)-2-(hydroxymethyl)-1,3-propanediol, tris(hydroxymethyl)aminomethane, 2-amino-1,3,4-octadecanetriol, 3-aminopropylsilanetriol, 4-(2-amino-1-hydroxylethyl)-1,2-benzenediol, 4-[1-hydroxy-2-(isopropylamino)ethyl]-1,2-benzenediol, 3,4-dihydroxy-α-(methylaminomethyl)benzyl alcohol, 2,5-anhydro-1-azido-1-deoxy-D-glucitol, 2,3,4-trihydroxybutanal (L-erythrose, D-erythrose, L-(+)-threose and D-(+)-threose), 2,3,4-trihydroxybenzaldehyde, 3,4,5-trihydroxybenzaldehyde, N-[tris(hydroxymethyl)methyl]glycine, 2,3,4-trihydroxybutyric acid (including but not limited to erythorbic acid and threonic acid), 2,4,6-trihydroxybenzoic acid, shikimic acid, 3,4,5-trihydroxybenzoic acid, 2,3,4-trihydroxybenzoic acid, arjunolic acid, 1,4,7-tris(tert-butoxycarbonyl)-1,4,7,10-tetraazacyclododecane, tri-(t-butoxycarbonyl)spermine, etc., and any hydroxyl-protected form thereof. Also included herein is a group consisting of citric acid, laricic acid, N-(2-hydroxyethyl)ethylenediamine-triacetic acid, pentaerythritol triacrylate, aminomethanetrispropionic acid, tri(tert-butyl) aminomethanetrispropionate, etc. Also included herein is the tetravalent silicon-atom branching center obtained via the terminal branching reaction based on alkenyl, trichlorosilane and allylmagnesium chloride, referring to the literature “Macromolecules, Vol. 33, No. 12, 2000”. Also included herein are trifunctionalized small molecules such as 1,4,7-tris(t-butoxycarbonylmethyl)-1,4,7,10-azacyclotetradecane (NOTA), which require an excess amount in the reaction.
1.1.5.3. F containing functional groups
In one specific embodiment of the present invention, F is preferably —(Z2)q—(Z1)q1-R01, wherein, q and q1 are each independently 0 or 1; Z1 and Z2 are each independently a divalent linking group; R01 is a functional group capable of interreacting with bio-related substances.
In one specific embodiment of the present invention, Z1 and Z2 are more preferably each independently selected from the group consisting of -L4-, -L4-Z—, —Z-L4-, —Z-L4-Z—, -L4-Z-L5-, —Z-L4-Z-L5-, and -L4-Z-L5-Z—, wherein, t is an integer from 1 to 12.
In a more specific embodiment of the present invention, R01 is preferably selected from the group consisting of a functional group, a variant form of a functional group, a therapeutic targeting functional group, and a fluorescent functional group; wherein, said variant form includes precursors of functional groups, active forms with functional groups as precursors, substituted active forms, protected forms, and deprotected forms; wherein, said precursors of functional groups refer to structures which can be transformed into the functional group through at least one process selected from the group consisting of oxidation, reduction, hydration, dehydration, electronic rearrangement, structural rearrangement, salt complexation and decomplexation, ionization, protonation, and deprotonation; wherein, the variant forms of functional groups refer to the reactive forms of functional groups after at least one chemical change process selected from the group consisting of oxidation, reduction, hydration, dehydration, electronic rearrangement, structural rearrangement, salt complexation and decomplexation, ionization, protonation, deprotonation, substitution, and deprotection, or refer to the non-reactive forms after protection; said R01 is more preferably a functional group selected from the group consisting of the functional groups in the following Classes A to H and variant forms thereof, or from the group consisting of the functional groups in the following Classes I to J:
Further, R01 is preferably selected from the group consisting of the functional groups in the following Classes A to H and variant forms thereof, and the functional derivatives in Classes I to J; said variant form is a precursor of functional group, an active form with functional group as precursor, a substituted active form, a protected form, or an unprotected form:
are cyclic structures, of which the skeletons contain a nitrogen atom, a nitrogenium ion, a double bond, an azo bond, a triple bond, a disulfide bond, an anhydride group, an imide group, and a dienylene, respectively; said cyclic structures are selected from the group consisting of a carbocycle, a heterocycle, a benzoheterocycle, a substituted carbocycle, a substituted heterocycle, and a substituted benzoheterocycle;
1.1.5.4. Specific Examples of R
In one specific embodiment of the present invention, R preferably contains any atom or group selected from the group consisting of a hydrogen atom, an alkyl group, an alkoxy group, an alcoholic hydroxyl group, a protected alcoholic hydroxyl group, a thiol group, a protected thiol group, a carboxyl group, a protected carboxyl group, an amino group, a protected amino group, an aldehyde group, a protected aldehyde group, an ester group, a carbonate group, a urethane group, a succinimidyl group, a maleimide group, a protected maleimide group, a dimethylamino group, an alkenyl group, an alkenoate group, an azido group, an alkynyl group, a folate group, a rhodamine group, a biotinyl group, a monosaccharide group, and a polysaccharide group, and more preferably selected from the group consisting of H, —CH3, —CH2CH3, —(CH2)tOH, —(CH2)tSH, —OCH3, —OCH2CH3, —(CH2)tNH2, —(CH2)tC(═O)OH, —C(═O)(CH2)tC(═O)OH, —C(═O)CH3, —(CH2)tN3, —C(═O)CH2CH3, —C(═O)OCH3, —OC(═O)OCH3, —C(═O)OCH2CH3, —OC(═O)OCH2CH3, —(CH2)tN(CH3)1, —(CH2)tN(CH2CH3)2, —(CH2)tCHO,
wherein, t is an integer from 0 to 12.
1.1.6. A
In the present invention, A is —(CRaRb)sO— or —O(CRaRb)s—, wherein, s is 2, 3, or 4, Ra and Rb are each independently a hydrogen atom or a C1-12 alkyl group.
In one specific embodiment of the present invention, Ra and Rb are both hydrogen atoms, and s is 2, specifically corresponding to A being CH2CH2O— or —OCH2CH2—.
In the present invention, the number-average molecular weight of the polyethylene glycol chains of the PEGylated lipids is 900, 1000, 1500, 2000, 2500, 3000, 3350, 3500, 4000, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, or 11000.
In one specific embodiment of the present invention, the polyethylene glycol chains of the PEGylated lipids can be monodisperse or polydisperse.
In one specific embodiment of the present invention, the polyethylene glycol chains are preferably polydisperse, and the number-average degree of polymerization (n1) is preferably an integer from approximately 20 to 100, more preferably an integer from approximately 20 to 60, more preferably an integer from approximately 40 to 60, and more preferably 44, 45, 46, 48, 50, 52, 54, 56, 58, or 60.
In one specific embodiment of the present invention, the polyethylene glycol chains are preferably monodisperse, and the number of EO-unit is preferably from 20 to 70, more preferably from 20 to 60, and more preferably 20, 22, 24, 26, 27, 28, 29, 30, 32, 34, 36, 38, 40, 42, 44, 45, 46, 48, 50, 52, 54, 56, 58, or 60.
1.1.7. Examples of Specific General Formulas
In one specific embodiment of the present invention, the structure of PEGylated lipid of the present invention is preferably selected from the group consisting of the following structural formulas:
1.1.8. Specific Structural Examples of PEGylated Lipids
In some specific embodiments of the present invention, PEGylated lipids with the following structures are finally obtained, which include but are not limited to the following structures:
wherein, n1 is an integer from 20 to 250.
2. Preparation of PEGylated Lipids
In the present invention, any of the above-mentioned PEGylated lipids can be prepared by methods including but not limited to the following:
2.1. Method-1:
Step 1:
Step 2:
Step 3:
Step 4:
Wherein, the descriptions of L3, L7, L8, B3, B4, R, R1, R2, and n1 are in line with those of the general formula (2), which are not repeated here.
In one specific embodiment of the present invention, in the foregoing Method-1, said A-1 is preferably R1′—COOH or R2′—COOH, said A-3 is preferably R1—NH2 or R2—NH2, said carboxyl activating agent is preferably selected from the group consisting of N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), N-hydroxy-5-norbornene-2,3-dicarboximide (HONb), and N,N′-dicyclohexylcarbodiimide (DCC), and said PEGylated lipid is preferably prepared by the following routes:
2.2 Method-2:
Step 1:
Step 2:
Wherein, the descriptions of L3, L7, L8, B3, B4, R, R1, R2, and n1 are in line with those of the general formula (2), and no more repeated here.
In one specific embodiment of the present invention, in the foregoing Method-2, said B-3 or B-3′ is R1—NH2 or R2—NH2, said B-4 or B-4′ is R2-L8-B4—Br or R1-L7-B3—Br, and said F1 contains the -Oms group.
2.3. Method-3:
Wherein, the descriptions of L3, L7, L8, B3, B4, R, R1, R2, and n1 are in line with those of the general formula (2), and no more repeated here.
Step 1:
Step 2:
2.4. Method-4:
Step 1:
Step 2:
Step 3:
Wherein, the descriptions of L3, L7, L8, B3, B4, R, R1, R2, and n1 are in line with those of the general formula (2), and no more repeated here.
2.5. Description of Relevant Starting Materials and/or Steps in the Preparation Process
2.5.1. Carboxyl Activating Agent, Condensing Agent, Oxidizing Agent, and Reducing Agent
In the present invention, the “activation of a carboxyl group” refers to the activation of a carboxyl group with carboxyl activating agents. The activated carboxyl group can promote the condensation reactions, for example, by inhibiting the generation of racemic impurities, by accelerating the reaction through catalysis, etc. The “carboxyl activating group” refers to the residue of a carboxyl activating agent. Said carboxyl activating agent is selected from the group consisting of N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), N-hydroxy-5-norbornene-2,3-dicarboximide (HONb), and N,N′-dicyclohexylcarbodiimide (DCC), and combinations thereof, preferably the combination of NHS/EDCI, NHS/DCC, or HONb/DCC, and more preferably the combination of NHS/EDCI.
In the present invention, the condensing agent used in reactions is not particularly limited, but preferably selected from the group consisting of N,N′-dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC-HCl), 2-(7-azobenzotriazole-N,N,N,N-tetramethyluronium hexafluorophosphate (HATU) and (enzotriazole-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU), and most preferably DDC. Generally, the amount of the condensing agent used is 1 to 20 folds of the molar equivalent of the carboxylic acid, preferably 5-10 folds, and a suitable catalyst (such as 4-dimethylaminopyridine) can be added to the reaction.
In the present invention, the oxidizing agent used in reactions is not particularly limited, as long as it is a compound or a combination of compounds that can increase the valence of the substrate; said oxidizing agent is preferably selected from the group consisting of phenyliodine(III)bis(trifluoroacetate), 1,4-benzoquinone, benzyl trimethyl ammonium tribromide, pyridinium dichromate, potassium dichromate, ozone, oxygen, hydrofluoric acid, sodium hypochlorite, cobaltic acetate, cobalt acetate, manganous acetate, palladium(II) acetate, cupric acetate, monoperoxyphthalic acid, iodine, N-iodosuccinimide, iodylbenzene, 2-iodylbenzoic acid, dimethyldioxirane, dimethyl sulfoxide-oxalyl chloride, DDQ, dichlorotris(triphenylphosphine)ruthenium, manganese dioxide, (diacetoxyiodo)benzene, periodic acid, sodium periodate, sodium periodate-osmium tetraoxide, potassium permanganate, sodium perborate, perbenzoic acid, dibenzoyl peroxide, nickel peroxide, hydrogen peroxide, cumyl hydroperoxide, t-butyl hydroperoxide, peracetic acid, m-chloroperbenzoic acid, N-chlorosuccinimide, pyridinium chlorochromate, palladium chloride-cupric chloride, urea hydrogen peroxide adduct, triphenylcarbenium tetrafluoroborate, tributyltin oxide, cobalt trifluoride, vanadium oxytrifluoride, chromium trioxide, manganese triacetate, TEMPO, diammonium cerium nitrate, bromine, pyridine N-oxide, silver oxide, O-ethylperoxycarbonic acid, manganese acetylacetonate, vanadyl acetylacetonate, aluminium isopropoxide, potassium peroxymonosulfate, dichloroiodobenzene, etc., and combinations thereof, and more preferably selected from the group consisting of oxygen, sodium hypochlorite, hydrogen peroxide, dichloroiodobenzene, potassium peroxymonosulfate, etc., and combinations thereof. The molar equivalent of the oxidizing agent used is 1 to 50 folds of that of the hydroxyl groups of intermediate compounds, preferably 1 to 20 folds, and more preferably 5 to 10 folds.
In the present invention, the reducing agents used in reactions are not particularly limited, as long as the Schiff bases formed via the reaction between amines and aldehydes or ketones can be reduced to amine groups; said reducing agents are preferably selected from the group consisting of sodium borohydride, sodium cyanoborohydride, lithium aluminum hydride, borane, diborane, diisobutylaluminum hydride, diisopinocampheylborane, lithium borohydride, zinc borohydride, borane-pyridine complex, borane-methyl sulfide complex, borane-tetrahydrofuran complex, etc., and combinations thereof, and more preferably sodium cyanoborohydride. The molar equivalent of the reducing agent used is 1 to 50 folds of that of the amino groups to be modified, preferably 1 to 20 folds, and more preferably 5 to 10 folds.
In the present invention, the reaction temperature is 0 to 200° C., preferably 0 to 100° C., and more preferably 0 to 25° C. The reaction time is preferably 10 min to 48 h, and more preferably 30 min to 24 h. The obtained product can be purified by a purification means such as extraction, recrystallization, adsorption treatment, precipitation, reverse precipitation, membrane dialysis, supercritical extraction, etc.
In the present invention, the reaction solvent can be absent or an aprotic solvent; said aprotic solvent includes toluene, benzene, xylene, acetonitrile, ethyl acetate, diethyl ether, tert-butyl methyl ether, tetrahydrofuran, chloroform, dichloromethane, dimethylsulfoxide, dimethylformamide, and dimethylacetamide, and is preferably tetrahydrofuran, dichloromethane, dimethylsulfoxide, or dimethylformamide.
In the present invention, the bases used in reactions can be organic bases (e.g., triethylamine, pyridine, 4-dimethylaminopyridine, imidazole, or N,N-diisopropylethylamine), preferably triethylamine or pyridine. The molar equivalent of the base used is 1 to 50 folds of that of carboxylic acids, preferably 1 to 10 folds, and more preferably 2 to 3 folds.
2.5.2. “Protection” and “Deprotection” of Relevant Groups Involved in the Reaction Process
In the present invention, the reaction process also involves the “protection” and “deprotection” processes of relevant groups. In order to prevent a functional group from affecting the reaction, the functional group is usually protected. In addition, when there are two or more functional groups, selectively only the target functional group undergoes the reaction, so the other functional groups should be protected. The protecting group not only provides stable protection to the target functional group, but is also expected to be removed easily as needed. Therefore, in organic synthesis, it is important to remove only the protecting group bonded to the specified functional group under appropriate conditions.
In the present invention, a “carboxyl protecting group” refers to the protecting group which can be transformed into a carboxyl group via hydrolysis or deprotection. A carboxyl protecting group is preferably an alkyl group (e.g., methyl, ethyl, tert-butyl) or an aralkyl group (e.g., benzyl), and more preferably a tert-butyl group (tBu), a methyl group (Me), or an ethyl group (Et). In the present invention, a “protected carboxyl group” refers to the group formed via the protection of a carboxyl group with an appropriate carboxyl protecting group, and is preferably a methoxycarbonyl group, an ethoxycarbonyl group, a t-butoxycarbonyl group, or a benzyloxycarbonyl group. Said carboxyl protecting group can be removed through hydrolysis catalyzed by acids or alkalis, or through pyrolysis reactions occasionally; for example, t-butyl groups can be removed under mild acidic conditions, and benzyl groups can be removed by hydrogenolysis. The reagents used for the removal of carboxyl protecting groups are selected from the group consisting of TFA, H2O, LiOH, NaOH, KOH, MeOH, EtOH, and combinations thereof, preferably the combination of TFA and H2O, the combination of LiOH and MeOH, or the combination of LiOH and EtOH. A protected carboxyl group can undergo deprotection and then produce the corresponding free acid; said deprotection can be conducted in the presence of an alkali, and said alkali forms pharmaceutically acceptable salts with said free acid obtained from said deprotection.
In the present invention, the “amino protecting group” includes all the groups which can be used as common amino protecting groups, such as an aryl C1-6 alkyl group, a C1-6 alkoxy C1-6 alkyl group, a C1-6 alkoxycarbonyl group, an aryloxycarbonyl group, C1-6 alkylsulfonyl group, an arylsulfonyl group, a silyl group, etc. An amino protecting group is preferably selected from the group consisting of a t-butoxycarbonyl group (Boc), a p-methoxybenzyloxycarbonyl group (Moz) and a 9-fluorenylmethoxycarbonyl group (Fmoc). The reagent used for removing amino protecting groups is selected from the group consisting of TFA, H2O, LiOH, MeOH, EtOH and combinations thereof, preferably selected from the combination of TFA and H2O, the combination of LiOH and MeOH, and the combination of LiOH and EtOH. The reagent used for removing the Boc protecting group is TFA or HCl/EA, preferably TFA. The reagent used for removing the Fmoc protecting group is the N,N-dimethylformamide (DMF) solution containing 20% piperidine.
In the present invention, said hydroxyl group protected by a hydroxyl protecting group is not particularly limited, e.g., an alcoholic hydroxyl group, a phenolic hydroxyl group, and the like. Wherein, said amino group protected by an amino protecting group is not particularly limited, selected from the group consisting of a primary amine, a secondary amine, a hydrazine, an amide, and the like. In the present invention, amino groups are not particularly limited, including but not limited to primary amino groups, secondary amino groups, tertiary amino groups, and quaternary ammonium ions.
In the present invention, the deprotection of protected hydroxyl groups is related to the type of hydroxyl protecting group. Said type of hydroxyl protecting group is not particularly limited; for example, benzyl groups, silyl ethers, acetals, or tert-butyl groups can be used to protect terminal hydroxyl groups, and the corresponding deprotection methods include the follows:
A: Deprotection of Benzyl Groups
The deprotection of benzyl groups can be achieved via hydrogenation using a hydrogenation reduction catalyst and a hydrogen donor. As used herein, the water content should be less than 1% in order to facilitate the reaction. When the water content is more than 1%, the cleavage of polyethylene glycol chains will occur and generate low-molecular-weight polyethylene glycol with hydroxyl groups, which can participate in the subsequent polymerization reaction or functionalization reaction to introduce impurities into the target product and may even react with bio-related substances to change the properties of formulations.
The catalyst for hydrogenation reduction is not particularly limited, preferably palladium or nickel, and its carrier is not particularly limited, preferably alumina or carbon, more preferably carbon. The amount of palladium used is 1 to 100 wt % of that of compounds containing protected hydroxyl groups, preferably 1 to 20 wt %. When the amount of palladium used is less than 1 wt %, both the rate of deprotection and the rate of conversion will decrease. The moieties that have not been deprotected are unable to participate in the subsequent polymerization or functionalization, resulting in a low ratio of functionalization of the final product. However, when the amount of palladium used exceeds 100 wt %, it is easy to cause the polyethylene glycol chains to be cleaved.
The reaction solvent is not particularly limited, as long as both the starting materials and the products can be dissolved, but is preferably methanol, ethanol, ethyl acetate, tetrahydrofuran, or acetic acid, more preferably methanol. The hydrogen donor is not particularly limited, but is preferably hydrogen, cyclohexene, 2-propanol, or ammonium formate, etc. The reaction temperature is preferably in the range of 25 to 40° C. When the temperature is higher than 40° C., the cleavage of polyethylene glycol chains is easy to occur.
The reaction time is not particularly limited, which is negatively correlated with the amount of catalyst used, preferably 1 to 5 hours; when the reaction time is shorter than 1 hour, the conversion rate is relatively low; when the reaction time is longer than 5 hours, the cleavage of polyethylene glycol chains is easy to occur.
B: Deprotection of Acetals and Ketals
The compounds used for protecting hydroxyl groups in the forms of acetals or ketals are preferably ethyl vinyl ether, tetrahydropyran, acetone, 2,2-dimethoxypropane, or benzaldehyde, etc. The deprotection of such acetals or ketals can be realized under an acidic condition wherein the pH of the solution is preferably 0 to 4. When the pH is higher than 4, the acidity is too weak for the protecting group to be completely removed. When the pH is lower than 0, the acidity is too strong so that the polyethylene glycol chain tends to be cleaved. The acid is not particularly limited, but preferably acetic acid, phosphoric acid, sulfuric acid, hydrochloric acid, or nitric acid, more preferably hydrochloric acid. The reaction solvent is not particularly limited as long as it allows the starting materials and the products to be dissolved, preferably water. The reaction temperature is preferably 0 to 30° C. When the temperature is lower than 0° C., the reaction rate is relatively slow, and the protecting group cannot be completely removed; when the temperature is higher than 30° C., the cleavage of polyethylene glycol chains is easy to occur under an acidic condition.
C: Deprotection of Silyl Ethers
The protected hydroxyl groups in the forms of silyl ethers include trimethylsilyl ether, triethylsilyl ether, tert-butyldimethylsilyl ether, tert-butyldiphenylsilyl ether, etc. The deprotection of such silyl ethers uses compounds containing fluoride ions; wherein, said compounds are preferably selected from the group consisting of tetrabutylammonium fluoride, tetraethylammonium fluoride, hydrofluoric acid, and potassium fluoride, and more preferably selected from tetrabutylammonium fluoride and potassium fluoride. The molar equivalent of the fluorine-containing reagent used is 5 to 20 folds of that of the protected hydroxyl group, preferably 8 to 15 folds of that of the initiator. When the molar equivalent of the fluorine-containing reagent used is less than 5 folds of that of the protected hydroxyl group, the deprotonation might be incomplete. When the molar equivalent of the deprotection reagent used exceeds 20 folds of that of the protected hydroxyl group, the excess reagents or compounds will bring difficulty in the purification and possibly cause side reactions in subsequent steps. The reaction solvent is not particularly limited, as long as the reactants and the products can be dissolved, but is preferably an aprotic solvent and more preferably tetrahydrofuran or dichloromethane. The reaction temperature is preferably 0 to 30° C.; when the temperature is lower than 0° C., the reaction rate is relatively slow, and the protecting group cannot be completely removed.
D: Deprotection of tert-butyl Groups
The deprotection of tert-butyl groups is carried out under an acidic condition wherein the pH of the solution is preferably 0 to 4. When the pH is higher than 4, the acidity is too weak for the protecting group to be completely removed; when the pH is lower than 0, the acidity is too strong, and the cleavage of polyethylene glycol chains is easy to occur. The acid is not particularly limited, preferably acetic acid, phosphoric acid, sulfuric acid, hydrochloric acid, or nitric acid, more preferably hydrochloric acid. The reaction solvent is not particularly limited as long as the starting materials and the products can be dissolved, preferably water. The reaction temperature is preferably 0° C. to 30° C. When the temperature is lower than 0° C., the reaction rate is relatively slow, and the protective group cannot be completely removed; when the temperature is higher than 30° C., the cleavage of polyethylene glycol chains is easy to occur.
In the following preparation methods for linear functionalization of terminal hydroxyl groups of polyethylene glycol chains, it is preferred that q=0, q1=1, and Z1 is a 1,2-methylene group. When q is not 0, for example, when a linking group such as an amino acid group, a succinyl group, etc., is present between PEG and R01, the prior art capable of generating Z2 or Z1 (including but not limited to alkylation, condensation, click reactions, etc.) can be used, and the preparation process can be carried out referring to the following linear functionalization methods.
2.5.3. Alkylation Reaction
In the present invention, the alkylation reaction is preferably based on hydroxyl groups, mercapto groups, or amino groups, corresponding to the formation of ether bonds, thioether bonds, secondary amino groups, and tertiary amino groups, respectively. Examples are as follows:
2.5.3.1. Alkylation Reaction Between an Alcohol Substrate and a Sulfonate or Halide
An ether intermediate can be obtained via the nucleophilic substitution with a sulfonate or halide on an alcohol substrate under basic conditions. Wherein, the molar equivalent of the sulfonate or halide is 1 to 50 folds of that of the alcohol substrate, preferably 1 to 5 folds. When the molar equivalent of the sulfonate or halide is less than 1 fold of that of the alcohol substrate, the substitution might be incomplete and cause difficulty in the purification process. When the molar equivalent of the sulfonate or halide exceeds 50 folds of that of the alcohol substrate, the excess reagent tends to bring difficulty in the purification process, and might be brought into the subsequent step and therefore cause increased side reactions which further increase the difficulty of purification.
The resulting product is a mixture of ether intermediate, excess sulfonate, and excess halide, and can be purified by means such as anion exchange resin, osmosis, ultrafiltration, etc. Wherein, the anion exchange resin is not particularly limited, as long as the target product can undergo ion-exchange and adsorption in the resin, and is preferably the ion exchange resin of tertiary amines or quaternary ammonia salts, with the matrix being dextran, agarose, polyacrylate, polystyrene, or poly(diphenylethylene), etc. The solvents used for osmosis or ultrafiltration are not limited, generally water or an organic solvent. Said organic solvent is not particularly limited, as long as the product can be dissolved within, but is preferably dichloromethane, chloroform, or the like.
The reaction solvent is not limited, preferably an aprotic solvent such as toluene, benzene, xylene, acetonitrile, ethyl acetate, tetrahydrofuran, chloroform, dichloromethane, dimethylsulfoxide, dimethylformamide, and dimethylacetamide, and more preferably dimethylformamide, dichloromethane, dimethylsulfoxide, or tetrahydrofuran.
The base used can be an organic base (e.g., triethylamine, pyridine, 4-dimethylaminopyridine, imidazole, diisopropylethylamine) or an inorganic base (e.g., sodium carbonate, sodium hydroxide, sodium bicarbonate, sodium acetate, potassium carbonate, potassium hydroxide), preferably an organic base, and more preferably triethylamine or pyridine. The molar equivalent of the base used is 1 to 50, preferably 1 to 10, and more preferably 3 to 5 folds of that of the sulfonate or halide.
2.5.3.2. Alkylation Reaction Between an Amine Substrate and a Sulfonate or Halide
An amine intermediate can be obtained via the nucleophilic substitution between an amine substrate and a sulfonate or halide under a basic condition. Wherein, the amount of the sulfonate or halide is 1 to 50 folds of that of the amine substrate, preferably 1 to 5 folds. When the amount of the sulfonate or halide is less than 1-fold of that of the amine substrate, the substitution may not sufficiently proceed and the purification tends to be difficult. When the amount of the sulfonate or halide exceeds 50 folds of that of the amine substrate, the excess reagent tends to cause difficulty in the purification process and might be brought to the subsequent steps to result in increased side reactions which further increase the difficulty of purification.
The resulting product Is a mixture of amine intermediate, excess sulfonate, and excess halide, the mixture can be purified by purification means such as anion exchange resin, osmosis treatment, ultrafiltration treatment or the like. Wherein, the anion exchange resin is not particularly limited as long as the target product can undergo ion-exchange and adsorption in the resin, and is preferably the ion exchange resin of tertiary amines or quaternary ammonia salts, with the matrix being dextran, agarose, polyacrylate, polystyrene, or poly(diphenylethylene), etc. The solvents used for osmosis or ultrafiltration are not limited, generally water or an organic solvent. Said organic solvent is not particularly limited, as long as the product can be dissolved within, but is preferably dichloromethane, chloroform, or the like.
The reaction solvent is not limited, preferably an aprotic solvent such as toluene, benzene, xylene, acetonitrile, ethyl acetate, tetrahydrofuran, chloroform, dichloromethane, dimethylsulfoxide, dimethylformamide, and dimethylacetamide, and more preferably dimethylformamide, dichloromethane, dimethylsulfoxide, or tetrahydrofuran.
The base used can be an organic base (e.g., triethylamine, pyridine, 4-dimethylaminopyridine, imidazole, diisopropylethylamine) or an inorganic base (e.g., sodium carbonate, sodium hydroxide, sodium bicarbonate, sodium acetate, potassium carbonate, potassium hydroxide), preferably an organic base, and more preferably triethylamine or pyridine. The molar equivalent of the base used is 1 to 50, preferably 1 to 10, and more preferably 3 to 5 folds of that of the sulfonate or halide.
2.5.3.3. Alkylation Reaction Between an Amine Substrate and an Aldehyde Derivative
The amine substrate reacts with an aldehyde derivative to obtain an imine intermediate, which is followed by obtaining an intermediate using reducing agents. Wherein, the molar equivalent of the aldehyde derivative is 1 to 20, preferably 1 to 2, and more preferably 1 to 1.5 folds of that of the amine substrate. When the molar equivalent of the aldehyde derivative exceeds 20 folds of that of the amine substrate, the excess reagent tends to cause difficulty in the purification process, and might be brought into the subsequent step and therefore increase difficulty in the purification. When the molar equivalent of the aldehyde derivative is less than 1 fold of that of the amine substrate, the reaction might be incomplete, causing further difficulty in the purification. Wherein, the resulting product can be obtained after purification by means such as cation exchange resin, osmosis, ultrafiltration, etc. Said cation exchange resin is not particularly limited, as long as it can undergo ion-exchange with quaternary ammonium cations and realize the isolation. The solvents used for osmosis or ultrafiltration treatment are not limited, generally water or an organic solvent. Said organic solvent is not particularly limited, as long as the product can be dissolved within, but is preferably dichloromethane, chloroform, or the like.
The reaction solvent is not limited, preferably an organic solvent such as methanol, ethanol, water, toluene, benzene, xylene, acetonitrile, ethyl acetate, tetrahydrofuran, chloroform, dichloromethane, dimethylsulfoxide, dimethylformamide, dimethylacetamide, etc., and more preferably water or methanol.
The reducing agent is not particularly limited, as long as the imine can be reduced to an amine, but is preferably sodium borohydride, lithium aluminum hydride, sodium cyanoborohydride, or Zn/AcOH, etc., and more preferably sodium cyanoborohydride. The amount of the reducing agent used is generally 0.5 to 50 folds and preferably 1 to 10 folds of that of the aldehyde derivative.
2.5.4. Linear Dual End-Functionalized PEG Derivative biLPEG
Said biLPEG contains a functional group at one end, which can react with amino groups (primary amino groups or secondary amino groups), and forms the divalent linking group L3 via coupling reaction.
The other end of said biLPEG has a structure identical or different from the target structure R, and the corresponding functional group is the same as or different from the target functional group. The functional group on this end of the biLPEG includes but is not limited to the functional groups in the aforementioned classes A-J, including the precursors of any aforementioned functional groups, variant forms with the functional groups as precursors, substituted forms, protected forms, deprotected forms, and the like.
The functional groups at both ends of said biLPEG may be the same or different, and said biLPEG is preferably a linear heterofunctional PEG derivative (biheteroLPEG) with two different functional groups at both ends. Wherein, in the present invention, a pair of heterofunctional groups which can exist simultaneously includes but is not limited to the group consisting of a hydroxyl group paired with a protected hydroxyl group, a hydroxyl group or a protected hydroxyl group with a non-hydroxyl reactive group belonging to classes A˜H (e.g., an amino group, a protected amino group, an ammonium salt group, an aldehyde group, an active ester group, a maleimide group, a carboxyl group, a protected carboxyl group, an alkynyl group, a protected alkynyl group, an azido group, an alkenyl group, an acrylic acid group, an acrylate group, a methacrylate group, an epoxy group, an isocyanato group, etc.), a hydroxyl group or a protected hydroxyl group paired with a functional group or derivative thereof belonging to the classes I-J (such as a targeting group, a photosensitive group, etc.), an active ester group paired with a maleimide group, an active ester group paired with an aldehyde group, an active ester group paired with an azido group, an active ester group paired with an alkynyl group or a protected alkynyl group, an active ester group paired with an acrylate group, an active ester group paired with a methacrylate group, an active ester group paired with an acrylic acid group, a maleimide group paired with an azido group, a maleimide group paired with an alkynyl group or a protected alkynyl group, a maleimide group paired with an acrylate group, a maleimide group paired with a methacrylate group, a maleimide group paired with an acrylic acid group, a maleimide group paired with a carboxyl group, a maleimide group paired with an amino group or a protected amino group or an ammonium salt group, a maleimide group paired with an isocyanato group, a maleimide group paired with a protected mercapto group, an aldehyde group paired with an azido group, an aldehyde group paired with an acrylate group, an aldehyde group paired with a methacrylate group, an aldehyde group paired with an acrylic acid group, an aldehyde group paired with an epoxy group, an aldehyde group paired with a carboxyl group, an aldehyde group paired with an alkynyl group or a protected alkynyl group, an azido group paired with a mercapto group or a protected mercapto group, an azido group paired with an amino group or a protected amino group or an ammonium salt group, an azido group paired with an acrylate group, an azido group paired with a methacrylate group, an azido group paired with an acrylic acid group, an azido group paired with a carboxyl group, an acrylate group paired with an amino group or a protected amino group or an ammonium salt group, an acrylate group paired with an isocyanato group, an acrylate group paired with an epoxy group, an acrylate group paired with a methacrylate group, an acrylate group paired with a carboxyl group, a methacrylate group paired with a carboxyl group, a methacrylate group paired with an amino group or a protected amino group or an ammonium salt group, a methacrylate group paired with an isocyanato group, a methacrylate group paired with an epoxy group, an alkynyl group or a protected alkynyl group paired with an amino or a protected amino group or an ammonium salt group, an alkynyl group or a protected alkynyl group paired with an isocyanato group, an alkynyl group or a protected alkynyl group paired with an acrylate group, an alkynyl group or a protected alkynyl group paired with a methacrylate group, an alkynyl group or a protected alkynyl group paired with acrylic acid group, an alkynyl group or a protected alkynyl group paired with an epoxy group, an alkynyl group or a protected alkynyl group paired with a carboxyl group, a protected alkynyl group paired with an azido group, an acrylic acid group paired with an isocyanato group, an acrylic acid group paired with an acrylate group, an acrylic acid group paired with an epoxy group, an acrylic acid group paired with a carboxyl group, a carboxyl group paired with a mercapto group or a protected mercapto group, a carboxyl group paired with an amino group or a protected amino group or an ammonium salt group, a carboxyl group paired with an isocyanato group, a carboxyl group paired with an epoxy group, an amino group or a protected amino group or an ammonium salt group paired with a mercapto or a protected mercapto group, a targeting group paired with a non-hydroxyl reactive group, a photosensitive group paired with a non-hydroxyl reactive group, and the like. Wherein, said active ester groups include but are not limited to any succinimidyl active ester groups (e.g., a succinimidyl carbonate group), p-nitrophenyl active ester groups, o-nitrophenyl active ester groups, benzotriazole active ester groups, 1,3,5-trichlorophenyl active ester groups, 1,3,5-trifluorophenyl active ester groups, pentafluorophenyl active ester groups, imidazole active ester groups, 2-thioxo-thiazolidine-3-carboxylate groups, and 2-thione-pyrrolidine-1-carboxylate groups, etc., in the present invention; said amino groups include primary amino groups and secondary amino groups. Said ammonium salt is preferably a hydrochloride form of an amino group, such as NH2HCl.
Said biLPEG can be Polydisperse or Monodisperse.
When a biLPEG is polydisperse, the polydispersity index is not particularly limited, while the polydispersity index of the starting material is preferably below 1.15, more preferably below 1.10, more preferably below 1.08, and more preferably below 1.05. The lower the PDI is, the more uniform the molecular weight and the narrower the molecular weight distribution, which leads to a higher quality of modified products when applied in the modification of molecules such as drugs and thus meets the practical needs to a greater extent.
When a biLPEG is monodisperse, PDI=1 and a PEGylated lipid with a defined molecular structure and molecular weight can be obtained. The products prepared with monodisperse starting materials have a more uniform molecular weight distribution; however, due to the limitation of preparation methods, the molecular weight is mostly limited, and the steps are lengthy. The advantage of using polydisperse starting materials is to provide a simple route and a larger range for molecule-weight adjustment.
The methods for preparing monodisperse polyethylene glycol chains can employ techniques in the prior art, including but not limited to the following literatures “J. Org. Chem. 2006, 71, 9884-9886” and cited references therein, “Angew. Chem. 2009, 121, 1274-1278” and cited references therein, “Expert Rev. Mol. Diagn. 2013, 13(4), 315-319” and cited references therein, “Angew. Chem. Int. Ed. 2014, 53, 6411-6413” and cited references therein, “Bioorganic & Medicinal Chemistry Letters, 2015, 25:38-42” and cited references therein, “Angew. Chem. Int. Ed., 2015, 54:3763-3767” and cited references therein, etc.
2.5.5. Linear End-Functionalization of Polyethylene Glycol Chain
The method for linear functionalization of the terminal of a polyethylene glycol chain is not particularly limited, but is relevant to the type of the final functional group or its protected form. It can be the linear functionalization of the terminal hydroxyl group of a polyethylene glycol chain, or the conversion of a reactive group into the target functional group or its protected form, or the combination of both.
The method for linear functionalization of the terminal hydroxyl group of a polyethylene glycol chain, starting from the terminal hydroxyl group of the polyethylene glycol chain, obtains a functional group of classes A-J or its protected form after functionalization. The specific preparation method is as documented in the paragraphs from [0960] to [1205] of the document CN104530417A. The general formula of the reaction is as follows:
The conversion of reactive groups into the target functional groups or protected forms thereof, can be achieved by any of the following approaches: Approach 1: Direct modification. The target functional group or its protected form can be obtained via direct modification of a reactive group. Examples include, the conversion of a carboxyl group to an acyl halide group, a hydrazide group, an ester group, a thioester group, or a dithioester group, the conversion of a hydroxyl group, a mercapto group, an alkynyl group, an amino group, a carboxyl group, or the like to the corresponding protected form, and so on. Another example is, the modification of a hydroxyl group, an amino group, or the like with an anhydride.
Approach 2: Coupling reaction between two reactive groups. Said coupling reaction uses a heterofunctional reagent as the starting material containing 1 type of reactive group and the target functional group or its protected form, to introduce the target functional group or its protected form via the reaction between said reactive group and the terminal reactive group of the polyethylene glycol chain. The reaction between two reactive groups is not particularly limited with respect to the manner and method of the reaction; wherein, the reaction conditions are related to the type of the divalent linking group formed in the reaction, and the prior art can be employed, for example, alkylation, addition reaction of alkenes, addition reaction of alkynes, Schiff base reaction-reduction reaction, condensation reaction, etc. Wherein, the alkylation reaction is preferably selected from those based on a mercapto group or an amino group, corresponding to the formation of a thioether bond, a secondary amino group or a tertiary amino group, respectively. Wherein, the condensation reaction includes but is not limited to those forming an ester group, a thioester group, an amide group, an imine bond, a hydrazone bond, a carbamate group, and the like. Examples also include, introducing the target functional group or its protected form via click reactions by using heterofunctional reagents as the starting materials containing not only an azido group, an alkynyl group, an alkenyl group, a trithioester group, a mercapto group, a dienyl group, a furyl group, a 1,2,4,5-tetrazinyl group, a cyanate group, or the like, but also the target functional group or its protected form. The reaction between two reactive groups brings the formation of new bonds. The representative newly formed divalent linking groups include an amide bond, a urethane bond, an ester bond, a secondary amino bond, a thioether bond, a triazole group, and the like.
Approach 3: The target functional group or its protected form can be obtained via the combination of direct modifications and coupling reactions.
In the present invention, starting materials used in every preparation method can be obtained by purchase or synthetic means.
The intermediates and end-products prepared in the present invention can all be purified by purification methods including but not limited to extraction, recrystallization, adsorption treatment, precipitation, reverse precipitation, membrane dialysis, supercritical extraction and the like. The characterization of the structure and molecular weight of the end-products can use characterization methods including but not limited to NMR, electrophoresis, UV-visible spectrophotometer, FTIR, AFM, GPC, HPLC, MALDI-TOF, circular dichroism spectroscopy, and the like.
3. Cationic Liposomes
3.1. Cationic Liposomes
In the present invention, provided herein is a cationic liposome containing any foregoing PEGylated lipid whose structure is represented by the general formula (2).
In one specific embodiment of the present invention, the cationic liposome preferably contains not only the PEGylated lipid whose structure is represented by the general formula (2), but also one or more types of lipids selected from the group consisting of neutral lipid, steroid lipid, and cationic lipid; more preferably simultaneously contains three types of lipids as neutral lipid, steroid lipid, and cationic lipid.
In one specific embodiment of the present invention, neutral lipids in the cationic liposomes preferably include but are not limited to 1,2-dilinoleoyl-sn-glycero-3-phosphocholines (DLPC), 1,2-dimyristoleoyl-sn-glycero-3-phosphocholines (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholines (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholines (DPPC), 1,2-distearoyl-sn-glycero-3-phosphatidylcholines (DSPC), 1,2-diundecanoyl-sn-glycero-3-phosphatidylcholines (DUPC), 1-plamitoyl-2-oleoyl-sn-glycero-3-phosphocholines (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphatidylcholines (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinyl-sn-glycero-3-phosphocholines (oChemsPC), 1-O-hexadecyl-sn-glycero-3-phosphatidylcholines (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphatidylcholines, 1,2-diarachidonoyl-sn-glycero-3-phosphatidylcholines, 1,2-didecosahexaenoyl-sn-glycero-3-phosphocholines, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamines (DOPE), 1,2-diphytanyl-sn-glycero-3-phosphoethanolamines (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamines, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamines, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamines, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamines, 1,2-didecosahexaenoyl-sn-glycero-3-phosphoethanolamines, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salts (DOPG), dioleoyl phosphatidylserines (DOPS), dipalmitoylphosphatidylglycerols (DPPG), palmitoyloleoyl phosphatidylethanolamines (POPE), distearoyl phosphatidylethanolamines (DSPE), dipalmitoyl phosphatidylethanolamines (DPPE), dimyristoleoyl phosphoethanolamines (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamines (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholines (SOPC), sphingomyelins, phosphatidylcholines, phosphatidylethnolamines, phosphatidylserines, phosphatidylinositols, phosphatidic acids, palmitoyloleoyl phosphatidylcholines, lysophosphatidylcholines, lysophosphatidylethanolamines (LPE), and combinations thereof.
In one specific embodiment of the present invention, PEGylated lipids in the cationic liposomes are, apart from the PEGylated lipids represented by the general formula (2), preferably selected from the group consisting of PEG500-dipalmitoylphosphatidylcholine, PEG2000-dipalmitoylphosphatidylcholine, PEG500-distearylphosphatidylethanolamine, PEG2000-distearylphosphatidylethanolamine, PEG500-1,2-dioleoylphosphatidylethanolamine, PEG2000-1,2-dioleoylphosphatidylethanolamine and PEG2000-2,3-distearoylglycerol, preferably selected from the group consisting of PEG2000-dipalmitoylphosphatidylcholine, PEG2000-distearylphosphatidylethanolamine, and PEG2000-1,2-dioleoylphosphatidylethanolamine, more preferably PEG2000-distearylphosphatidylethanolamine.
In one specific embodiment of the present invention, steroid lipids in the cationic liposomes are preferably selected from the group consisting of cholesterols, coprostanols, sitosterols, ergosterols, campesterols, stigmasterols, brassicasterols, tomatidines, ursolic acids, α-tocopherols, and mixtures thereof.
In one specific embodiment of the present invention, the structure of a cationic lipid in a cationic liposome is preferably represented by the following general formula (1):
wherein, Rd is a C1-12 alkyl group, G1 is a terminal branching group with the valence of k+1, j is 0 or 1, and F contains functional groups; when j is 0, G1 is absent, and k is 1; when j is 1, G1 protrudes F with the number of k, and k is an integer from 2 to 8;
In one specific embodiment of the present invention, the structure of a cationic lipid in a cationic liposome is represented by the general formula (1), and preferably selected from the group consisting of the following structures:
In one specific embodiment of the present invention, the cationic lipids in the cationic liposomes can be selected from not only the cationic lipids represented by the general formula (1), but also the group consisting of N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10), N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), and mixtures thereof.
In one specific embodiment of the present invention, any foregoing cationic liposome preferably contains 20% to 80% cationic lipids represented by the general formula (1), 5% to 15% neutral lipids, 25% to 55% steroid lipids, and 0.5% to 10% PEGylated lipids, and said percentage is the molar percentage of each lipid relative to the total lipids in a solution containing the solvent.
In one specific embodiment of the present invention, in any foregoing cationic liposome, the molar percentage of cationic lipids relative to the total lipids in a solution containing the solvent is preferably 30% to 65%, more preferably about 35%, 40%, 45%, 46%, 47%, 48%, 49%, 50%, or 55%.
In one specific embodiment of the present invention, in any foregoing cationic liposomes, the molar percentage of neutral lipids relative to the total lipids in a solution containing the solvent is preferably 7.5% to 13%, more preferably about 8%, 9%, 10%, 11%, or 12%.
In one specific embodiment of the present invention, in any foregoing cationic liposomes, the molar percentage of steroid lipids relative to the total lipids in a solution containing the solvent is preferably 35% to 50%, more preferably about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50%.
In one specific embodiment of the present invention, in any foregoing cationic liposomes, the molar percentage of PEGylated lipids relative to the total lipids in a solution containing the solvent is preferably 0.5% to 5%, more preferably 1% to 3%, more preferably about 1.5%, 1.6%, 1.7%, 1.8%, or 1.9%.
3.2. Preparation of Cationic Liposomes
In the present invention, cationic liposomes can be prepared by the following methods, including but not limited to thin-film dispersion method, ultrasonic dispersion method, reverse phase evaporation method, freeze-drying method, freeze-thaw method, double emulsion method and/or injection method, preferably thin-film dispersion method, ultrasonic dispersion method and/or reverse phase evaporation method.
In the preparation methods of the cationic liposomes of the present invention, said thin-film dispersion method could include the following steps:
In the preparation methods of the cationic liposomes of the present invention, the ratio of the cationic liposomes to the phosphate buffer solution containing dissolved cryoprotectants could be 1 mg:(0.1˜100) mL, preferably 1 mg:(0.3˜50) mL, and more preferably 1 mg:(0.5˜5) mL.
4. Cationic Liposome-Nucleic Acid Pharmaceutical Composition
In one embodiment of the present invention, provided herein is a cationic liposome pharmaceutical composition containing any foregoing cationic liposome and a drug, wherein the cationic liposome contains any foregoing PEGylated lipid whose structure is represented by the general formula (2).
In one specific embodiment of the present invention, in a cationic liposome pharmaceutical composition, the drug is preferably a nucleic acid drug or an anti-tumor agent; said nucleic acid drug is selected from the group consisting of DNA, antisense nucleic acid, plasmid, mRNA (messenger RNA), interfering nucleic acid, aptamer, miRNA inhibitor (antagomir), microRNA (miRNA), ribozyme, and small interfering RNA (siRNA), and preferably the group consisting of RNA, miRNA and siRNA.
In one specific embodiment of the present invention, the cationic liposome pharmaceutical composition is preferably used as a drug, which is selected from the group consisting of drugs for treating cancer, drugs for treating malignant tumor, anti-infectious agents, antiviral agents, antifungal agents, vaccines, and drugs for treating esophageal carcinoma, gastric cancer, large intestine cancer, nasopharyngeal cancer, brain tumor, cervical carcinoma, leukemia, bone cancer, AIDS, and virus infection.
In the present invention, said drugs are further preferably selected from those including but not limited to doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, streptozotocin, actinomycin D, vincristine, vinblastine, cytosine arabinoside, anthracycline, nitrogen mustard, tioteppa, chlorambucil, rachelmycin, melphalan, carmustine, romustine, busulfan, dibromannitol, mitomycin C, cis-diammineplatinum(II) dichloride, methotrexate, 6-mercaptopurine, 6-thioguanine, cytosine arabinoside, 5-fluorouracil dacarbazine, dibucaine, chlorpromazine, propranolol, demorol, labetalol, clonidine, hydralazine, imipramine, amitriptyline, doxepin, phenytoin, diphenhydramine, chlorpheniramine, promethazine, gentamicin, ciprofloxacin, cefoxitin, miconazole, terconazole, econazole, isoconazole, butoconazole, clotrimazole, itraconazole, nystatin, naftifine, amphotericin B, antiparasitic agents, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, glaucoma drugs, vitamins, tranquilizers, imaging agents, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, colchicine, daunorubicin, quinizarin, mithramycin, 1-dihydrotestosterone, glucocorticoid, procaine, tetracaine, lidocaine, puromycin, and maytansinoid.
In one specific embodiment of the present invention, the N/P ratio of said cationic liposomes to said nucleic acids is preferably (0.1˜100):1, more preferably (0.2˜30):1, and most preferably (0.5˜20):1.
In one specific embodiment of the present invention, the working solution of said formulation of nucleic acid pharmaceutical composition is preferably deionized water, ultrapure water, phosphate buffer, or physiological saline, more preferably phosphate buffer or physiological saline, and most preferably physiological saline; the ratio of the cationic liposomes to the working solution is preferably (0.05˜20) g:100 mL, more preferably (0.1˜10) g:100 mL, and most preferably (0.2˜5) g:100 mL.
5. Formulation of Cationic Liposome Pharmaceutical Composition
In the present invention, provided herein is a formulation of cationic liposome pharmaceutical composition containing any foregoing cationic liposome pharmaceutical composition and pharmaceutically acceptable diluents or excipients; said diluents or excipients are preferably selected from the group consisting of deionized water, ultrapure water, phosphate buffer, and physiological saline, more preferably selected from phosphate buffer and physiological saline, and most preferably physiological saline.
In the present invention, the preparation method of the formulation of cationic liposome-nucleic acid pharmaceutical composition includes the following steps:
The following is a further description with specific examples of the preparation methods of PEGylated lipids, cationic liposomes, and cationic liposome pharmaceutical compositions, and the biological activity assays for cationic liposome pharmaceutical compositions. The specific examples are for further illustration of the present invention, not limiting the protected scope of present invention. Wherein, in the embodiments of preparing PEGylated lipids, the structures of end-products were characterized by NMR, and the molecular weight was determined by MALDI-TOF.
E1-1 corresponds to the general formula (2), wherein, R1 is a tridecyl group, R2 is a tetradecyl group, B3 and B4 are both linking bonds, L7 is a carbonyl group, L8 is a linking bond, L3 is —CH2CH2—, A is —OCH2CH2—, Ra and Rb are both hydrogen atoms, s is 2, n1≈45, R is a methoxy group, and the total molecular weight is approximately 2460 Da.
The preparation process is as follows:
E1-3 corresponds to the general formula (2), wherein, R1 is a tridecyl group, R2 is a tetradecyl group, B3 and B4 are both linking bonds, L7 is a carbonyl group, L8 is a linking bond, L3 is —CH2CH2—, A is —OCH2CH2—, Ra and Rb are both hydrogen atoms, s is 2, n1≈45, R is a hydroxy group, and the total molecular weight is approximately 2450 Da.
The preparation process is as follows:
E2-1 corresponds to the general formula (2), wherein, R1 is a tetradecyl group, R2 is a decyl group, B3, B4, and L7 are all linking bonds, L8 is a carbonyl group, L3 is —CH2CH2—, A is —OCH2CH2—, Ra and Rb are both hydrogen atoms, s is 2, n1≈60, R is a methoxy group, and the total molecular weight is approximately 3070 Da.
The preparation process is as follows:
E3-1 corresponds to the general formula (2), wherein, R1 is a tetradecyl group, R2 is a heptyl group, B3 is a linking bond, B4 is a pentylene group, L7 is a linking bond, L8 is an amide bond (—NHC(═O)—), L3 is a carbonyl group, A is —OCH2CH2—, Ra and Rb are both hydrogen atoms, s is 2, n1≈45, R is a methoxy group, and the total molecular weight is approximately 2500 Da.
The preparation process is as follows:
E4-2 corresponds to the general formula (2), wherein, R1 is a tetradecyl group, R2 is a heptyl group, B3 is a linking bond, B4 is a heptyl group, L7 is a linking bond, L8 is an ether bond (—O—), L3 is —CH2CH2—, A is —OCH2CH2—, Ra and Rb are both hydrogen atoms, s is 2, n1≈45, R is a hydroxy group, and the total molecular weight is approximately 2450 Da.
The preparation process is as follows:
E5-1 corresponds to the general formula (2), wherein, R1 is a tetradecyl group, R2 is a butyl group, B3 is a linking bond, B4 is a heptylene group, L7 is a linking bond, L8 is —OCH2CH2O—, L3 is —CH2CH2—, A is —OCH2CH2—, Ra and Rb are both hydrogen atoms, s is 2, n1≈45, R is a methoxy group, and the total molecular weight is approximately 2460 Da.
The preparation process is as follows:
E6-1 corresponds to the general formula (2), wherein, R1 and R2 are both methyl groups, B3 and B4 are both tridecylene groups, L7 and L8 are both ether bonds (—O—), L3 is —CH2CH2O—, A is —CH2CH2O—, Ra and Rb are both hydrogen atoms, s is 2, n1≈40, R is a methyl group, and the total molecular weight is approximately 2260 Da.
The preparation process is as follows:
E7-1 corresponds to the general formula (2), wherein, R1 and R2 are both heptyl groups, B3 and B4 are both heptylene groups, L7 and L8 are both ether bonds (—O—), L3 is —CH2CH2—, A is —OCH2CH2—, Ra and Rb are both hydrogen atoms, s is 2, n1=45, R is a methoxy group, and the total molecular weight is approximately 2482 Da.
The preparation process is as follows:
E8-1 corresponds to the general formula (2), wherein, R1 and R2 are both heptyl groups, B3 and B4 are both pentylene groups, L7 and L8 are both amide bonds (—NHC(═O)—), L3 is —CH2C(═O)—, A is —OCH2CH2—, Ra and Rb are both hydrogen atoms, s is 2, n1≈60, R is a methoxy group, and the total molecular weight is approximately 3150 Da.
The preparation process is as follows:
E8-2 corresponds to the general formula (2), wherein, R1 and R2 are both heptyl groups, B3 and B4 are both pentylene groups, L7 and L8 are both amide bonds (—NHC(═O)—), L3 is —CH2CH2C(═O)—, A is —OCH2CH2—, Ra and Rb are both hydrogen atoms, s is 2, n1≈150, R is a methoxy group, and the total molecular weight is approximately 7130 Da.
The preparation process is as follows:
The compound S8-8 (13.60 g, 2.0 mmol, Mw≈6800, n1≈150, PDI=1.02) was added into 80 mL water and stirred at room temperature to dissolve. Potassium carbonate (2.76 g, 20.0 mmol), secondary amine compound S8-6 (4.40 g, 10.0 mmol), and tetra-n-butylammonium bromide (0.06 g, 0.2 mmol) were added, and the reaction was stirred at room temperature for 72 h. After completion of the reaction, the reaction solution was extracted twice with dichloromethane (100 mL*2). The organic phases were combined, backwashed once with saturated sodium chloride aqueous solution (100 mL), and then the organic phase was retained, dried with anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product of E8-2. The crude product was purified by column chromatography, concentrated, and dried with an oil pump to obtain the target compound E8-2 (11.12 g). 1H NMR (500 MHz, CDCl3) δ: δ: 4.07 (t, 2H), 3.83-3.46 (m, 598H), 3.36 (s, 3H), 3.26 (t, 2H), 3.25-3.21 (q, 4H), 3.14 (t, 2H), 3.06 (t, 2H), 2.38 (t, 2H), 2.16 (t, 4H), 1.66-1.41 (m, 12H), 1.32-1.26 (m, 20H), 0.87 (t, 6H). The molecular weight of E8-2 was determined to be 7129 Da by MALDI-TOF, PDI=1.02.
E9-1 corresponds to the general formula (2), wherein, R1 is an undecyl group, R2 is a tetradecyl group, B3 is a propylene group, B4 is a linking bond, L7 is an ether bond, Ls is a linking bond, L3 is —C(═O)CH2—, A is —OCH2CH2—, Ra and Rb are both hydrogen atoms, s is 2, n1≈45, R is —CH2CH2NH2, and the total molecular weight is approximately 2500 Da.
The preparation process is as follows:
E10-1 corresponds to the general formula (2), wherein, R1 is a nonyl group, R2 is a decyl group, L7 is —OC(═O)—, L8 is —C(═O)—, B3 is a pentylene group, B4 is a linking bond, L3 is —CH2CH2O—, A is —CH2CH2O—, Ra and Rb are both hydrogen atoms, s is 2, n1≈100, R is a methyl group, and the total molecular weight is approximately 4880 Da.
The preparation process is as follows:
E11-1 corresponds to the general formula (2), wherein, R1 is a pentadecyl group, R2 is a nonyl group, B3 is a linking bond, B4 is a propylene group, L7 is a linking bond, Ls is an amide bond (—C(═O)NH—), L3 is —CH2CH2—, A is —OCH2CH2—, Ra and Rb are both hydrogen atoms, s is 2, n1≈20, R is an ethoxy group, and the total molecular weight is approximately 1390 Da.
The preparation process is as follows:
E12-1 corresponds to the general formula (2), wherein, R1 is an undecyl group, R2 is a tetradecyl dodecyl group, B3 is a propylene group, B4 is a linking bond, L7 is an ether bond, L8 is a linking bond, L3 is —C(═O)CH2CH2C(═O)—, A is —OCH2CH2—, Ra and Rb are both hydrogen atoms, s is 2, n1≈45, R is a methoxy group, and the total molecular weight is approximately 2520 Da.
The preparation process is as follows:
The compound S12-1 (10.50 g, 5.0 mmol, Mw≈2100, n1≈45, PDI=1.02), compound S9-3 (3.19 g, 7.5 mmol) prepared in Example-9, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 1.92 g, 10.0 mmol), 1-hydroxybenzotriazole (HOBt, 1.01 g, 7.5 mmol), and TEA (1.01 g, 10.0 mmol) were dissolved in dichloromethane (100 mL) in sequence, followed by stirring at room temperature overnight. After completion of the reaction, the reaction solution was backwashed twice with 0.1 mol/L HCl aqueous solution (10% NaCl) (100 mL*2) and backwashed once with saturated sodium chloride aqueous solution (100 mL). The organic phase was dried with anhydrous MgSO4, filtered, and concentrated to obtain the crude product. The crude product was purified by column chromatography, concentrated, and dried with an oil pump to obtain the target compound methoxy-PEGylated lipid E12-1 (12.15 g). H NMR (500 MHz, CDCl3) δ: 4.17 (t, 2H), 3.84-3.45 (m, 182H), 3.39 (s, 3H), 3.20 (t, 2H), 3.14 (t, 2H), 2.62 (t, 2H), 2.36 (t, 2H), 1.62-1.46 (m, 6H), 1.24-1.19 (m, 38H), 0.87 (t, 6H). The molecular weight of E12-1 was determined to be 2519 Da by MALDI-TOF, PDI=1.02.
E13-2 corresponds to the general formula (2), wherein, R1 is a dodecyl group, R2 is a decyl group, B3 and B4 are both linking bonds, L7 is a linking bond, Ls is a carbonyl group, L3 is —CH2CH2—, A is —OCH2CH2—, Ra and Rb are both hydrogen atoms, s is 2, n1≈45, R is —OC(═O)CH2CH2COOH, and the total molecular weight is approximately 2480 Da.
The preparation process is as follows:
E14-1 corresponds to the general formula (2), wherein, R1 is a tetradecyl group, R2 is a heptyl group, B3 is a linking bond, B4 is a heptylene group, L7 is a linking bond, L8 is an ether bond, L3 is —CH2CH2—, A is —OCH2CH2—, Ra and Rb are both hydrogen atoms, s is 2, n1≈45, R is
and the total molecular weight is approximately 2570 Da.
The preparation process is as follows:
The compound S14-1 (8.80 g, 4.0 mmol, Mw≈2200, n1≈45, PDI=1.03) was added into 80 mL water and stirred at room temperature to dissolve. Potassium carbonate (5.52 g, 50.0 mmol), secondary amine compound S4-2 (8.52 g, 20.0 mmol), and tetra-n-butylammonium bromide (0.13 g, 0.4 mmol) were added, and the reaction was stirred at room temperature for 72 h. After completion of the reaction, the reaction solution was extracted twice with dichloromethane (100 mL*2). The organic phases were combined, backwashed once with saturated sodium chloride aqueous solution (100 mL), and then the organic phase was retained, dried with anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product of E14-1. The crude product was purified by column chromatography, concentrated, and dried with an oil pump to obtain the target compound succinimide-PEGylated lipid E14-1 (8.35 g). H NMR (500 MHz, CDCl3) δ: 3.87-3.45 (m, 184H), 2.86 (m, 4H), 2.62 (t, 2H), 2.45 (m, 4H), 2.28 (t, 2H), 1.61-1.46 (m, 8H), 1.35-1.19 (m, 36H), 0.88 (t, 6H). The molecular weight of E14-1 was determined to be 2574 Da by MALDI-TOF, PDI=1.03.
E15-2 corresponds to the general formula (2), wherein, R1 is a tridecyl group, R2 is a tetradecyl group, B3 and B4 are both linking bonds, L7 is a carbonyl group, L8 is a linking bond, L3 is —CH2CH2—, A is —OCH2CH2—, Ra and Rb are both hydrogen atoms, s is 2, n1≈45, R is
and the total molecular weight is approximately 2600 Da.
Method-1:
The preparation process is as follows:
Method-2:
The preparation process is as follows:
Under argon atmosphere, into the round-bottom flask containing E1-3 (12.24 g, 5.0 mmol), S15-4 (1.69 g, 10.0 mmol), and DMAP (0.15 g, 1.25 mmol) dissolved in dichloromethane (20 mL), DCC (2.28 g, 11.0 mmol) was added, and the reaction was conducted for 16 h at room temperature. After completion of the reaction, the precipitate was removed by filtering. The reaction solution was backwashed twice with saturated sodium chloride aqueous solution (100 mL). The organic phase was dried with anhydrous MgSO4, filtered, and concentrated, and the residue was purified by silica gel column chromatography to obtain the end-product maleimide-PEGylated lipid E15-2 (11.00 g, 84.7%). 1H NMR (500 MHz, CDCl3) δ: 6.49 (s, 2H), 4.25-4.10 (m, 4H), 3.85-3.46 (m, 180H), 3.35 (t, 2H), 3.17 (t, 2H), 2.73 (t, 2H), 2.24 (t, 2H), 1.58-1.48 (m, 4H), 1.36-1.17 (m, 42H), 0.88 (t, 6H). The molecular weight of E15-2 was determined to be 2598 Da by MALDI-TOF, PDI=1.02.
E16-1 corresponds to the general formula (2), wherein, R1 is an undecyl group, R2 is a tetradecyl group, B3 is a propylene group, B4 is a linking bond, L7 is an ether bond, L8 is a linking bond, L3 is —CH2CH2O—, A is —CH2CH2O—, Ra and Rb are both hydrogen atoms, s is 2, n1≈45, R is —CH2CH2N3, and the total molecular weight is approximately 2520 Da.
The preparation process is as follows:
The compound S16-1 (8.80 g, 4.0 mmol, Mw≈2200, n1≈45, PDI=1.03) was added into 80 mL water and stirred at room temperature to dissolve. Potassium carbonate (5.52 g, 50.0 mmol), secondary amine compound S9-3 (8.52 g, 20.0 mmol), and tetra-n-butylammonium bromide (0.13 g, 0.4 mmol) were added, and the reaction was stirred at room temperature for 72 h. After completion of the reaction, the reaction solution was extracted twice with dichloromethane (100 mL*2). The organic phases were combined, backwashed once with saturated sodium chloride aqueous solution (100 mL), and then the organic phase was retained, dried with anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product of E16-1. The crude product was purified by column chromatography, concentrated, and dried with an oil pump to obtain the target compound azide-PEGylated lipid E16-1 (8.03 g). 1H NMR (500 MHz, CDCl3) δ: 3.85-3.42 (m, 186H), 3.99 (t, 2H), 2.66 (t, 2H), 2.48 (m, 4H), 1.58-1.48 (m, 8H), 1.38-1.19 (m, 38H), 0.85 (t, 6H). The molecular weight of E16-1 was determined to be 2518 Da by MALDI-TOF, PDI=1.03.
E17-2 corresponds to the general formula (2), wherein, R1 is a tridecyl group, R2 is a tetradecyl group, B3 and B4 are both linking bonds, L7 is a carbonyl group, L8 is a linking bond, L3 is —CH2CH2O—, A is —CH2CH2O—, Ra and Rb are both hydrogen atoms, s is 2, n1≈250, R is
and the total molecular weight is approximately 11540 Da.
The preparation process is as follows:
E18-2 corresponds to the general formula (2), wherein, R1 is a tridecyl group, R2 is a tetradecyl group, B3 and B4 are both linking bonds, L7 is a carbonyl group, Ls is a linking bond, L3 is —CH2CH2O—, A is —CH2CH2O—, Ra and Rb are both hydrogen atoms, s is 2, n1≈45, R is
and the total molecular weight is approximately 2580 Da.
The preparation process is as follows:
E19-2 corresponds to the general formula (2), wherein, R1 is a tridecyl group, R2 is a tetradecyl group, B3 and B4 are both linking bonds, L7 is a carbonyl group, L8 is a linking bond, L3 is —CH2CH2O—, A is —CH2CH2O—, Ra and Rb are both hydrogen atoms, s is 2, n1≈45, R is
and the total molecular weight is approximately 2830 Da.
The preparation process is as follows:
E20-1 corresponds to the general formula (2), wherein, R1 is
R2 is a pentadecyl group, B3 is a hexylene group, B4 is a linking bond, L7 is an amide bond (—C(═O)NH—), L8 is a linking bond, L3 is —CH2CH2—, A is —OCH2CH2—, Ra and Rb are both hydrogen atoms, s is 2, n1≈40, R is an methoxy group, and the total molecular weight is approximately 2380 Da.
Method-1:
The preparation process is as follows:
Method-2:
The preparation process is as follows:
E21-1 corresponds to the general formula (2), wherein, R1 is
R2 is a decyl group, B3 is a hexylene group, B4 is a linking bond, L7 is a carboxyl group (—C(═O)O—), L8 is a carbonyl group, L3 is —CH2CH2—, A is —OCH2CH2—, Ra and Rb are both hydrogen atoms, s is 2, n1≈45, R is an methoxy group, and the total molecular weight is approximately 2560 Da.
The preparation process is as follows:
E22-1 corresponds to the general formula (2), wherein, R1 is a tridecyl group, R2 is a tetradecyl group, B3 and B4 are both linking bonds, L7 is a carbonyl group, Ls is a linking bond, L3 is —CH2CH2O—, A is —CH2CH2O—, Ra and Rb are both hydrogen atoms, s is 2, n1≈45, R is
and the total molecular weight is approximately 2910 Da.
E23-1 corresponds to the general formula (2), wherein, R1 is a tridecyl group, R2 is a tetradecyl group, B3 and B4 are both linking bonds, L7 is a carbonyl group, Ls is a linking bond, L3 is —CH2CH2O—, A is —CH2CH2O—, Ra and Rb are both hydrogen atoms, s is 2, n1≈45, R is
and the total molecular weight is approximately 2720 Da.
The preparation process is as follows:
Into a dry and clean 1000 mL round-bottom flask, biotin succinimidyl ester (S23-1, 1.87 g, 5.5 mmol) dissolved in DMF and ethylamine-PEGylated lipid (S22-6, 12.50 g, 5.0 mmol, Mw≈2500, n1≈45, PDI=1.03) dissolved in DCM were added, followed by stirring well. Then, TEA (1.52 g, 15.0 mmol) was added, and the reaction was continued at room temperature until completion as indicated by TLC. After completion of the reaction, the reaction solution was filtered, concentrated, and purified by column chromatography. The eluents were collected, combined, concentrated, and freeze-dried to obtain the PEGylated lipid biotin derivative (E23-1, 9.82 g). 1H NMR (CDCl3) δ: 4.36-4.25 (m, 2H), 3.83-3.45 (m, 184H), 3.35 (t, 2H), 3.20 (m, 1H), 3.18 (t, 2H), 2.81 (t, 2H), 2.26 (t, 2H), 2.22-2.18 (m, 2H), 1.70-1.40 (m, 8H), 1.36-1.21 (m, 42H), 0.87 (t, 6H). The molecular weight of E23-1 was determined to be 2716 Da by MALDI-TOF, PDI=1.03.
E24-1 corresponds to the general formula (2), wherein, R1 and R2 are both methyl groups, B3 and B4 are both tridecylene groups, L7 and L8 are both ether bonds (—O—), L3 is —C(═O)CH2CH2C(═O)NH—, A is —CH2CH2O—, Ra and Rb are both hydrogen atoms, s is 2, n1≈45, R is a methyl group, and the total molecular weight is approximately 2530 Da.
The preparation process is as follows:
Methoxy polyethylene glycol succinate derivative S24-1 containing an amide bond (10.50 g, 5.0 mmol, Mw≈2100, n1≈45, PDI=1.02), compound S6-4 (3.32 g, 7.5 mmol), EDCI (1.92 g, 10.0 mmol), HOBt (1.01 g, 7.5 mmol), and TEA (1.01 g, 10.0 mmol) were dissolved in dichloromethane (100 mL) in sequence, followed by stirring at room temperature overnight. After completion of the reaction, the reaction solution was backwashed twice with 0.1 mol/L HCl aqueous solution (10% NaCl) (100 mL*2) and backwashed once with saturated sodium chloride aqueous solution (100 mL). The organic phase was dried with anhydrous MgSO4, filtered, and concentrated to obtain the crude product. The crude product was purified by column chromatography, concentrated, and dried with an oil pump to obtain the target compound methoxy-PEGylated lipid E24-1 (10.55 g). H NMR (500 MHz, CDCl3) δ: 3.83-3.45 (m, 184H), 3.38 (s, 3H), 3.32 (s, 6H), 3.26-3.17 (m, 4H), 2.43-2.36 (m, 4H), 1.66-1.42 (m, 8H), 1.35-1.19 (m, 36H). The molecular weight of E24-1 was determined to be 2536 Da by MALDI-TOF, PDI=1.02.
E25-1 corresponds to the general formula (2), wherein, R1 and R2 are both methyl groups, B3 and B4 are both tridecylene groups, L7 and L8 are both ether bonds (—O—), L3 is —C(═O)CH2CH2CH2NHC(═O)O—, A is —CH2CH2O—, Ra and Rb are both hydrogen atoms, s is 2, n1≈45, R is a methyl group, and the total molecular weight is approximately 2560 Da.
The preparation process is as follows:
E26-1 corresponds to the general formula (2), wherein, R1 is a nonyl group, R2 is a tridecyl group, B3 is a pentylene group, B4 is a linking bond, L7 is —OC(═O)—, Ls is —C(═O)—, L3 is —CH2CH2C(═O)O—, A is —CH2CH2O—, Ra and Rb are both hydrogen atoms, s is 2, n1≈45, R is a methyl group, and the total molecular weight is approximately 2530.
Method-1:
The preparation process is as follows:
Method-2:
The preparation process is as follows:
P-9 corresponds to the general formula (1), wherein, L1 and L2 are both —C(═O)O—, L3 is a linking bond, B1 and B2 are both
R1 and R2 are both
R3 is a hydrogen atom, A is —(CRaRb)sO—, Ra and Rb are both hydrogen atoms, s is 2, and n is 2.
In this example, multiple groups of cationic liposomes were prepared for comparison. In the compositions of every group of cationic liposomes, the neutral lipids contained were all DSPC, the sterol lipids contained were all cholesterol, and the differences lied in the cationic lipid and the PEGylated lipid components. Wherein, for the control group (L-0), the cationic lipid was DLin-MC3-DMA (MC3 for short) and the PEGylated lipid was PEG2k-DMG (DMG for short); for the experimental groups (L-1 to L-28, and L-31), the cationic lipid was MC3, and the PEGylated lipids were the PEGylated lipids prepared in the present application; for the experimental groups (L-29 to L-30), the cationic lipid was the cationic lipid P-9 prepared in the present application, and the PEGylated lipids were the PEGylated lipids E1-1 or E22-1 prepared in the present application; specifically as shown in Table 1.
The preparation method of cationic liposomes is as follows:
Following the above Steps a-d, the molar ratio of cationic lipid:DSPC:cholesterol PEGylated lipid was adjusted to 48:9:42:1.5. The cationic lipid CL (16.0 μmol), distearoylphosphatidylcholine (DSPC, 3.0 μmol), cholesterol (14.0 μmol), and the PEGylated lipid PL (0.5 μmol) of group L-31 listed in Table 1 were weighed. The other conditions remained unchanged to prepare the cationic liposome powder of L-31.
(1) Determination of Gene Complexation Ability
Gel electrophoresis experiment was used to evaluate the gene complexation ability of LNP/siRNA in each group. 0.8 g agarose was weighed and dissolved in 40 mL TAE solution. The solution was heated in a microwave until the granules of the agarose were dissolved completely, and then cooled. 5 μL nucleic acid dye GelGreen was added into the cooled agarose gel. The agarose gel was added into the gel slot and dried naturally in air. A mixed solution of LNP/siRNA and 2 μL loading buffer was added into the agarose gel well, and the electrophoresis voltage was set to 90 V for the electrophoresis experiment. The electrophoresis was carried out at room temperature for 10 min. The results showed that free siRNA was essentially absent in both the experimental groups (L-1/siRNA˜L-31/siRNA) and the control group (L-0/siRNA), indicating that the formulations of cationic liposome-nucleic acid pharmaceutical compositions modified by the PEGylated lipids of the present invention had a stronger gene complexation ability of mRNA.
Determination of encapsulation efficiency: The LNP/siRNA was ultracentrifuged (4° C., 60000 rpm, 1 h) with an ultracentrifuge, the concentration of unencapsulated siRNA in the supernatant was determined by a nucleic acid quantifier, and the encapsulation efficiency of the liposome for siRNA was calculated. The results were summarized in Table 1, showing that the cationic liposomes of the present invention (L-1˜L-31) had higher encapsulation efficiency for nucleic acid drugs. The DMG is a PEGylated lipid which has been successfully applied to mRNA vaccines. In contrast, the encapsulation efficiency for siRNA of L-1 (containing the PEGylated lipid E1-1), L-3 (containing the PEGylated lipid E2-1), L-7 (containing the PEGylated lipid E6-1), L-12 (containing the PEGylated lipid E10-1), L-16 (containing the PEGylated lipid E14-1), L-17 (containing the PEGylated lipid E15-2), and L-26 (containing the PEGylated lipid E24-1) using the same cationic lipid MC3 were all higher than the group containing DMG; L-29˜L-30 using the cationic lipid prepared in the present invention also showed higher encapsulation efficiency; L-31 adopted a different ratio of lipids, and the prepared cationic liposome showed higher encapsulation efficiency for nucleic acid as well.
Determination of particle size: In this example, the particle size of LNP/siRNA was determined by dynamic light scattering (DLS). The measured sizes of LNP/siRNA had a relatively high uniformity with PDI values all smaller than 0.3. With the PEGylated lipids of the present invention being used, when n1 was in the range of approximately 40 to 100, the particle sizes of the prepared LNP/siRNA were in the range of 90 to 130 nm.
(2) Study on the Stability in Serum
In this experiment, the stability of the PEGylated lipid-modified formulation of cationic liposome-nucleic acid pharmaceutical composition in serum was determined by measuring the variation of the size of liposome particles. In this example, 0.5 mL LNP/siRNA formulation L-0˜L-31 whose N/P ratio was 10/1 was added into the 0.5 mL culture medium containing 10% fetal bovine serum (FBS), followed by stirring at 37° C. Samples were taken at a regular interval to measure the variation of the particle size of the formulation of cationic liposome-nucleic acid pharmaceutical composition, based on which the stability of the formulation of cationic liposome-nucleic acid pharmaceutical composition in serum was analyzed. In other groups, 0.5 mL PBS was added to replace the culture medium to determine the stability of the formulation of cationic liposome-nucleic acid pharmaceutical composition in serum, using the same method as aforementioned. Results of the experiments showed that, in 48 h, compared with L-0, the variations in the particle size of the formulation of cationic liposome-nucleic acid pharmaceutical composition of L-1˜L-31 in PBS solution and serum were all relatively small, indicating that the PEGylated lipid-modified cationic liposomes of the present invention were relatively stable, which had good stability in serum and met the requirements of long circulating cationic liposomes.
(3) Study on the Cytotoxicity (Biocompatibility)
The cytotoxicity of the formulation of cationic liposome-nucleic acid pharmaceutical composition of the present invention was examined by the MTT assay. The formulations of cationic liposome-nucleic acid pharmaceutical compositions were dissolved in culture medium to the required concentration, and an appropriate amount of solubilizer could be added when necessary. With HeLa cells as a cell model, the cell suspension was inoculated onto a 96 well plate at 100 μL/well and a density of 1×104 cells/well. After the inoculation, the cells were incubated in a cell incubator at 37° C., 4% CO2 for 24 h. Then, the culture medium was aspirated and 100 μL culture medium containing 3.3 μg/mL LNP/siRNA (formulation of cationic liposome-nucleic acid pharmaceutical composition prepared in Example-41) was added to each well, 100 μL fresh culture medium was added to the blank control group, and each concentration of each group corresponded to 6 replicate wells. After the formulations of cationic liposome-nucleic acid pharmaceutical compositions were incubated with the HeLa cells for 24 h, 20 μL PBS buffer solution containing 5 mg/mL MTT was added to each well. After the MTT was incubated with the cancer cells for 4 h, the mixed solution of the culture medium and the buffer solution containing MTT was aspirated, and DMSO was added at 150 μL/well to dissolve the purple formazan crystals in living cells. After shaking thoroughly, the absorbance was measured by a microplate reader. The result was calculated according to the measured absorbance value, and showed that compared with the blank control group, the cell viability in groups containing the formulations of cationic liposome-nucleic acid pharmaceutical compositions prepared in the present invention were all over 93%, indicating that the PEGylated lipid-modified formulations of cationic liposome-nucleic acid pharmaceutical composition of the present invention had good biocompatibility
(4) Study on the Nucleic Acid Transport Efficiency
In order to examine the nucleic acid transport efficiency of each group of formulation of cationic liposome-nucleic acid pharmaceutical composition prepared with the PEGylated lipid of the present invention, the Luc-HeLa cells which could stably express the luciferase were used as the cell model to evaluate the nucleic acid transport efficiency of each group of the formulation of cationic liposome-nucleic acid pharmaceutical compositions (as described in Example-29) whose N/P ratio was 10/1. In a 96-well plate, when the cells were cultured to a cell density of 80%, each group of formulation of cationic liposome-nucleic acid pharmaceutical composition was added to the culture medium, and the cells were incubated in a cell incubator at 37° C., 4% CO2 for 24 h. The culture medium was then aspirated, and the culture medium containing 3.3 μg/mL LNP/siRNA was added for further incubation for 24 h. The cells were treated with lyse buffer, and the fluorescence intensity was measured using a luciferase assay kit and a chemiluminescence detector. On the other hand, the naked siRNA at the same dosage was added into the cultured cells to set a blank/siRNA group. Considering the siRNA which was transported into cells could inhibit the expression of the luciferase gene Fluc mRNA, the untreated cells were used as the negative control group, and the expression (i.e., fluorescence intensity) of the target gene in the blank cell group was designated as 100%. The result showed that the fluorescence intensity of the blank/siRNA group was approximately 78%, the fluorescence intensity of the L-0/siRNA group was approximately 43%, the fluorescence intensity of the L-1/siRNA group was approximately 29%, the fluorescence intensity of the L-7/siRNA group was approximately 31%, the fluorescence intensity of the L-26/siRNA group was approximately 39%, and the fluorescence intensity of the L-27/siRNA group was approximately 36%. Compared with the blank cell group and the siRNA group, the control group and the experimental groups showed better inhibitory effects on the luciferase gene, indicating that cationic liposomes could improve the transport efficiency for nucleic acid drugs; compared with the control group L-0/siRNA, the experimental groups had better inhibitory effects on the luciferase gene (the fluorescence intensity was less than 43%) except the group L-10/siRNA, L-12/siRNA, and L-19/siRNA, indicating that the cationic liposomes prepared with the PEGylated lipids of the present invention could improve the nucleic acid transport efficiency of liposomes.
(5) Study on the Uptake Efficiency of Tumor Cells
In order to investigate whether the PEGylated lipid-modified cationic liposomes containing the targeting group folic acid can increase the uptake of cationic liposomes by tumor cells, in the present invention, the nasopharymneal carcinoma cells (KB) with high expression of folate receptors on the cell surface and the hepatoma cells (HepG2) with low expression of folate receptors on the cell surface were respectively used as models to evaluate the cellular uptake of liposomes. The cells were inoculated onto 6-well plates. The supernatant was discarded when the cell growth density reached approximately 80%. Then, 0.2 mL cationic liposome formulation of L-29/siRNA without the targeting group folic acid or L-30/siRNA with the targeting group folic acid, together with 1.8 mL culture medium, was added to each well, and each sampling point corresponded to 3 replicate wells. After 1, 3 and 6 hours of incubation, respectively, the supernatant was retrieved, and the cells were trypsinized, washed with PBS, and transferred into a flow cytometer tube. 1×104 cells were counted and collected with a flow cytometer to calculate the percentage of positive cells (i.e., cells whose fluorescence intensity exceeded 10 units). The uptake efficiency was represented by the percentage of positive cells. Results of the experiments showed that, for the hepatoma cells with low expression of folate receptors, the difference of cellular uptake efficiency between L-29/siRNA without the targeting group folic acid and L-30/siRNA with the targeting group folic acid was relatively small. Compared with the L-29/siRNA without the targeting group folic acid, the L-30/siRNA with the targeting group folic acid permitted a higher cellular uptake efficiency of HepG2 cells by approximately 4%; however, for the nasopharymneal carcinoma cells with high expression of folate receptors, compared with the L-29/siRNA without the targeting group folic acid, the L-30/siRNA with the targeting group folic acid permitted a higher cellular uptake efficiency of KB cells by approximately 16%. It meant that the targeting effect of folic acid and the enhanced transport efficiency of cationic liposomes for nucleic acid drugs can act synergistically and further improve the effects of gene therapy on tumor.
Those described above are only embodiments of the present invention, and are not for setting a limitation on the patent scope of the present invention. Any modification of equivalent structures or equivalent routes according to the present invention, which may be applied in other related art in a direct or an indirect way, should be included into the scope of the present invention.
For those skilled in the art, without departing from the spirit and scope of the present invention, and without unnecessary experimentation, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. While the present invention has given particular examples, it should be understood that the present invention can be further modified. In conclusion, in accordance with the principles of the present invention, the present application is intended to cover any alterations, uses, or improvements of the present invention, including changes departing from the scope disclosed in this application but made using conventional techniques known in the art.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202110379685.5 | Apr 2021 | CN | national |
| 202110780189.0 | Jul 2021 | CN | national |
| 202110839008.7 | Jul 2021 | CN | national |
This application is a U.S. National Phase Application of International Application No. PCT/CN2022/085528, filed Apr. 7, 2022 which claims priority to Chinese Application Nos. 202110379685.5, filed Apr. 8, 2021, 202110780189.0, filed Jul. 9, 2021, and 202110839008.7, filed Jul. 23, 2021.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2022/085528 | 4/7/2022 | WO |
