阅读说明：本技术 一种细胞内吞释放响应的化合物及其应用 (Compound responding to endocytosis release and application thereof ) 是由 徐宇虹 梁瑶瑶 于 2021-05-19 设计创作，主要内容包括：本发明提供了一种细胞内吞释放响应的化合物及其应用。具体地,本发明提供了如式I所示的由亲脂性头部、亲水性链和pH响应的中间基团构成的内吞响应化合物。经本发明化合物修饰的复合物不仅在体液循环中稳定,而且转染效率高。(The invention provides a compound responding to endocytosis release and application thereof. Specifically, the invention provides an endocytosis responsive compound which is composed of a lipophilic head, a hydrophilic chain and a pH responsive intermediate group, as shown in formula I. The compound modified complex of the invention is not only stable in the circulation of body fluid, but also has high transfection efficiency.)

1. A compound is shown as a formula I,

wherein the content of the first and second substances,

R₁a lipophilic head;

R₈a hydrophilic chain (preferably, PEG);

x is selected from the group consisting of: o, S, NR_a；

Y is selected from the group consisting of: o, S, respectively;

R₂selected from the group consisting of: none, R₇-L-R₉-W; and when R is₂Is R₇-L-R₉When W is, R₇To "C ═ N";

R₇selected from the group consisting of: substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C6-C12 aryl; r₇Wherein said substitution means that one or more hydrogens in the group are substituted with one or more (preferably, 1 to 3) substituents selected from the group consisting of: deuterium, halogen (F, Cl, Br, I), cyano, nitro, N (R)_a)₂C1-C6 alkyl, C3-C12 cycloalkyl, C6-C12 aryl, 5-to 12-membered heteroaryl, 5-to 10-membered heterocyclyl;

l is selected from the group consisting of: none, O, S, substituted or unsubstituted C1-C6 alkylene, substituted or unsubstituted C3-C6 cycloalkylene;

R₉is selected from the following group: none, C1-C6 alkylene, substituted or unsubstituted C6-C12 aryl, substituted or unsubstituted 5-to 12-membered heteroAryl, substituted or unsubstituted C3-C12 cycloalkylene, substituted or unsubstituted 5-to 10-membered heterocyclyl;

w is selected from the group consisting of: none, -O-, -S-, -R ' C (O) R ' -, -R ' C (O) OR ' -, -R ' OC (O) R ' -, R ' S (O) R ' -, -R ' S (O)₂R”-、-R’N(R_a)R”-、-R’C(O)N(R_a)R”-、-R’C(O)ON(R_a)R”-、-R’OC(O)N(R_a)R”-、-R’S(O)N(R_a)R”-、-R’S(O)₂N(R_a)R”-、-R’N(R_a)C(O)R”-、-R’N(R_a)OC(O)R”-、-R’N(R_a)C(O)OR”-、-R’N(R_a)S(O)R”-、-R’N(R_a)S(O)₂R”-；

R' and R "are each independently selected from the group consisting of: none, unsubstituted or substituted by 1-3R_bSubstituted C1-C6 alkylene; r_bIs a substituted or unsubstituted group selected from: deuterium, C1-C6 alkyl, phenyl, benzyl, heteroaryl, C3-C6 cycloalkyl, 4-to 7-membered heterocyclyl;

R₃selected from the group consisting of: substituted or unsubstituted C3-C12 cycloalkyl, substituted or unsubstituted 5 to 12 membered heterocyclyl, substituted or unsubstituted C6-C12 aryl, substituted or unsubstituted 5 to 12 membered heteroaryl;

R₁₀each independently selected from the group consisting of: H. deuterium, C1-C6 alkoxy, hydroxy, N (R)_a)₂C1-C6 acylamino (-NR)_a-CO-C1-C6 alkyl), C1-C6 acyloxy (-O-CO-C1-C6 alkyl), C1-C6 alkyl, substituted or unsubstituted C6-C12 aryl, substituted or unsubstituted 5-to 12-membered heteroaryl, substituted or unsubstituted C6-C12 cycloalkylene, substituted or unsubstituted 5-to 10-membered heterocyclyl; and o is 0, 1,2 or 3;

R_ais a substituted or unsubstituted group selected from: H. deuterium, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C6-C12 aryl (preferably phenyl), 5-to 12-membered heteroaryl (preferably 5-or 6-membered heteroaryl), C3-C12 cycloalkyl (preferably C3-C6 cycloalkyl), 4-to 12-membered heterocyclyl (preferably 4-to 7-membered heterocyclyl); or, adjacent 2R_aAnd taken together therewith to form a substituted or unsubstituted 5-to 12-membered heteroaryl (preferably 5-or 6-membered heteroaryl) or substitutedOr an unsubstituted 4-to 12-membered heterocyclic group (preferably a 4-to 7-membered heterocyclic group);

unless specifically defined, the substitution means that one or more (preferably 1 to 3) hydrogens on the group are substituted with a substituent selected from the group consisting of: deuterium, halogen (preferably F, Cl, Br), cyano, hydroxy, nitro, N (R)_d)₂C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 deuterated alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C6-C12 aryl (preferably phenyl), 5-to 12-membered heteroaryl (preferably 5-OR 6-membered heteroaryl), C3-C12 cycloalkyl (preferably C3-C6 cycloalkyl), 4-to 12-membered heterocyclyl (preferably 4-to 7-membered heteroaryl), C1-C6 alkoxy, -C (O) OR_d、-OC(O)R_d、-SO₂R_d、-C(O)R_d、-NR_dC(O)R_d；

R_dEach independently selected from the group consisting of: H. deuterium, C1-C6 alkyl.

2. The compound of claim 1, wherein the compound is of formula II,

wherein the content of the first and second substances,

R₄、R₅and R₆Is as defined in claim 1 wherein R_eThe definition of (1);

R₁、R₂、R₃、R₈x and Y are as defined in claim 1.

3. The compound of claim 1, wherein the compound is of formula III-B,

wherein the content of the first and second substances,

R_1ais a group derived from neutral lipids, or is C10-C20 saturatedAnd or an unsaturated hydrocarbon group;

W_aselected from the group consisting of: -O-, -S-, C (O) -, -C (O) O-, -OC (O) -, S (O) -, -S (O)₂-、-NH-、-C(O)NH-、-C(O)ONH-、-OC(O)NH-、-S(O)NH-、-S(O)₂NH-、-NHC(O)-、-NHOC(O)-、-NHC(O)O-、-NHS(O)-、-NHS(O)₂-; preferably, W_aSelected from the group consisting of: -O-, -OC (O) NH-, -NHC (O) O-;

n is an integer of 2-20 (preferably, n is 2,3, 4, 5, 6, 7 or 8);

R₂、R₃、R₄、R₅、R₆x and Y are as defined in claim 1.

4. The compound of claim 1, wherein said compound is selected from the group consisting of compounds of table 1

TABLE 1 Compounds responsive to release by endocytosis

5. Use of a compound according to claim 1 for the modification of liposomes and/or lipid-drug complexes.

6. An intermediate I is characterized in that the intermediate I is shown as a formula IV,

wherein R is₁、R₃、R₄、R₅、R₆X and Y are as defined in claim 1.

7. An intermediate II is characterized in that the intermediate II is shown as a formula V,

wherein the content of the first and second substances,

W_bis a reactive linking group;

R₁、R₃、R₄、R₅、R₆、R₉l, X and Y are as defined in claim 1.

8. A process for the preparation of a compound according to claim 1, wherein the compound is prepared by process one or process two;

the method comprises the following steps:

(1.1) reacting intermediate I with NH in a first inert solvent₂-R₈Reacting to obtain a compound shown as a formula I; or

The second method comprises the following steps:

(2.1) reacting intermediate I with NH in a first inert solvent₂-R₂-W_bReacting to obtain an intermediate II; and

(2.2) reacting the intermediate II obtained in the step (1) with W in a second inert solvent_c-R₈Reacting to obtain a compound shown as a formula I;

wherein, W_bAnd W_CEach independently is the same or different reactive group; the intermediate I is an intermediate shown as a formula IV, and the intermediate II is an intermediate shown as a formula V; r₂And R₈Is as defined in claim 1.

9. A lipid-drug complex, wherein the lipid-drug complex is a lipid-drug complex modified with the compound of claim 1.

10. A formulation comprising the lipid-drug complex of claim 9.

Technical Field

The invention belongs to the field of gene therapy, and particularly relates to a cell endocytosis release response compound and application thereof.

Background

Gene therapy (gene therapy) is a method of introducing exogenous normal genes into target cells to correct or compensate diseases caused by defective and abnormal genes, thereby achieving the purpose of treatment. Gene therapy has been shown to have great therapeutic potential in the treatment of diseases such as cancer, X-chromosome associated severe immunodeficiency, and the like, for the effective treatment of congenital and acquired diseases. Common gene drugs include small interfering RNA (siRNA), plasmid DNA (pDNA), small hairpin RNA (shRNA), antisense Oligonucleotide (ODN), messenger RNA (mRNA), micro RNA (miRNA), and the like. Plasmid DNA, pDNA, is a small circular double-stranded DNA that naturally occurs in bacteria, sometimes in archaea and eukaryotic cells. pDNA is often considered a replicon and can achieve autonomous replication in different host cells. In genetic engineering, artificially constructed plasmids are often used as vectors for specific genes.

The method for introducing the gene medicine mainly comprises the following steps: physical methods, viral vector mediated methods, and non-viral vector mediated methods. The physical method is to deliver the gene medicine into the target cell by means of electroporation, high-pressure injection, magnetic transfection, gene gun and the like. The physical method can efficiently realize that gene drugs directly enter cells and realize expression, but the current method is difficult to enter clinic due to voltage stimulation, organ injury and the like and is still limited to the animal stage for experiment. Viral vector-mediated gene delivery mainly includes adenovirus transfection, lentivirus transfection and retrovirus transfection. Although viral transfection has the advantages of high efficiency and accuracy, gene delivery using viruses as vectors still has great limitations, including potential tumorigenicity, immunogenicity, wide tissue tropism and difficult preparation. Non-viral vector-mediated methods refer to the delivery of genes by means of liposomes, polymers, inorganic materials, and the like. Can effectively solve the limitation of the viral vector, is far superior to the viral vector in safety and is easy to prepare. Has the advantages of high transfection efficiency, low immunogenicity, easy chemical modification, easy scale-up production and the like.

However, there are problems with the non-viral vectors currently used. For example, when using cationic liposomes for delivery, cationic lipid/nucleic acid complexes encounter a series of extracellular and intracellular barriers, and since the complexes have a strong positive charge, in the extracellular environment, these complexes will first bind to negatively charged albumin in the blood, forming larger particles, and thus are easily recognized and cleared by reticuloendothelial cells, making it difficult to reach the target site. In addition, the cation/nucleic acid complex has more positive charges on the surface, which is also easy to cause cytotoxicity and harm to normal tissues. To solve the above problems, many researchers have tried to modify the surface of the complex with PEG to shield more positive charges, prevent aggregation, thereby improving the stability of the complex in body fluids and extending the circulation time of the complex in the body. However, although conventional PEG (DSPE-PEG2000 is more commonly used) modification shows a very outstanding advantage in stable gene delivery, there is also a problem in that transfection efficiency of the modified preparation is low.

In view of the above, there is an urgent need in the art to develop a new liposome for introducing gene drugs or a complex of the liposome and gene drugs, which has high stability in the extracellular environment, is not easily cleared, has low cytotoxicity, and has high transfection efficiency when transfected with the compound.

Disclosure of Invention

The object of the present invention is to provide a novel compound for introducing a gene drug (e.g., nucleic acid) into a target cell.

In a first aspect of the invention, there is provided a compound of formula I,

wherein the content of the first and second substances,

R₁a lipophilic head;

R₈a hydrophilic chain (preferably, PEG);

x is selected from the group consisting of: o, S, NR_a；

Y is selected from the group consisting of: o, S, respectively;

R₂selected from the group consisting of: none, R₇-L-R₉-W; and when R is₂Is R₇-L-R₉When W is, R₇To "C ═ N";

R₇selected from the group consisting of: substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C6-C12 aryl (preferably, phenyl); r₇Wherein said substitution means that one or more hydrogens in the group are substituted with one or more (preferably, 1 to 3) substituents selected from the group consisting of: deuterium, halogen (F, Cl, Br, I), cyano, nitro, N (R)_a)₂C1-C6 alkyl, C3-C8 cycloalkyl, C6-C10 aryl, 5-to 12-membered heteroaryl, 5-to 10-membered heterocyclyl;

l is selected from the group consisting of: none, O, S, substituted or unsubstituted C1-C6 alkylene, substituted or unsubstituted C3-C6 cycloalkylene;

R₉is selected from the following group: none, C1-C6 alkylene, substituted or unsubstituted C6-C12 aryl (preferably, C6-C10 aryl; more preferably, phenyl), substituted or unsubstituted 5-to 12-membered heteroaryl (preferably, 5-to 10-membered heteroaryl; more preferably, 5-to 6-membered heteroaryl), substituted or unsubstituted C3-C12 cycloalkylene, substituted or unsubstituted 5-to 10-membered heterocyclic;

w is selected from the group consisting of: none, -O-, -S-, -R ' C (O) R ' -, -R ' C (O) OR ' -, -R ' OC (O) R ' -, R ' S (O) R ' -, -R ' S (O)₂R”-、-R’N(R_a)R”-、-R’C(O)N(R_a)R”-、-R’C(O)ON(R_a)R”-、-R’OC(O)N(R_a)R”-、-R’S(O)N(R_a)R”-、-R’S(O)₂N(R_a)R”-、-R’N(R_a)C(O)R”-、-R’N(R_a)OC(O)R”-、-R’N(R_a)C(O)OR”-、-R’N(R_a)S(O)R”-、-R’N(R_a)S(O)₂R”-；

R' and R "are each independently selected from the group consisting of: none, unsubstituted or substituted by 1-3R_bSubstituted C1-C6 alkylene; r_bIs a substituted or unsubstituted group selected from: deuterium, C1-C6 alkyl, phenyl, benzyl, heteroaryl, C3-C6 cycloalkyl, 4-to 7-membered heterocyclyl;

R₃selected from the group consisting of: substituted or unsubstituted C3-C12 cycloalkyl (preferably, C5-C7 cycloalkyl), substituted or unsubstituted 5-to 12-membered heterocyclyl, substituted or unsubstituted C6-C12 aryl (preferably, C6-C10 aryl; more preferably, phenyl), substituted or unsubstituted 5-to 12-membered heteroaryl (preferably, 5-to 10-membered heteroaryl; more preferably, 5-to 6-membered heteroaryl);

R₁₀each independently selected from the group consisting of: H. deuterium, C1-C6 alkoxy, hydroxy, N (R)_a)₂C1-C6 acylamino (-NR)_a-CO-C1-C6 alkyl), C1-C6 acyloxy (-O-CO-C1-C6 alkyl), C1-C6 alkyl, substituted or unsubstituted C6-C12 aryl, substituted or unsubstituted 5-to 12-membered heteroaryl, substituted or unsubstituted C6-C12 cycloalkylene, substituted or unsubstituted 5-to 10-membered heterocyclyl; and o is 0, 1,2 or 3;

R_ais a substituted or unsubstituted group selected from: H. deuterium, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C6-C12 aryl (preferably phenyl), 5-to 12-membered heteroaryl (preferably 5-or 6-membered heteroaryl), C3-C12 cycloalkyl (preferably C3-C6 cycloalkyl), 4-to 12-membered heterocyclyl (preferably 4-to 7-membered heterocyclyl); or, adjacent 2R_aAnd taken together therewith to form a substituted or unsubstituted 5-to 12-membered heteroaryl (preferably 5-or 6-membered heteroaryl) or a substituted or unsubstituted 4-to 12-membered heterocyclyl (preferably 4-to 7-membered heterocyclyl);

unless specifically defined, the substitution means that one or more (preferably 1 to 3) hydrogens on the group are substituted with a substituent selected from the group consisting of: deuterium, halogen (preferably F, Cl, Br), cyano, hydroxy, nitro, N (R)_d)₂C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 deuterated alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C6-C12 aryl (preferably phenyl), 5-to 12-membered heteroaryl (preferably 5-OR 6-membered heteroaryl), C3-C12 cycloalkyl (preferably C3-C6 cycloalkyl), 4-to 12-membered heterocyclyl (preferably 4-to 7-membered heteroaryl), C1-C6 alkoxy, -C (O) OR_d、-OC(O)R_d、-SO₂ R_d、-C(O)R_d、-NR_dC(O)R_d；

R_dEach independently selected from the group consisting of: H. deuterium, C1-C6 alkyl.

In another preferred embodiment, the lipophilic head comprises a lipophilic end derived from a lipid (preferably, a neutral lipid) or is a C10-C20 saturated or unsaturated hydrocarbon group; and the lipophilic end is linked to X through a linking group, or is directly linked to X.

In another preferred embodiment, the C10-C20 saturated or unsaturated hydrocarbyl group contains 0, 1,2, 3,4, or 5 double or triple bonds.

In another preferred embodiment, the C10-C20 saturated or unsaturated alkyl group is a straight chain or branched chain alkyl group.

In another preferred embodiment, the neutral lipids comprise: a steroid lipid, a phosphatidylcholine lipid, a phosphatidylethanolamine lipid, a sphingomyelin lipid, or a combination thereof.

In another preferred embodiment, the sterol lipid comprises: cholesterol (Chol).

In another preferred embodiment, the Phosphatidylcholine (PC) lipid comprises: DPPC (1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine), DMPC (1, 2-dimyristoylphosphatidylcholine), DDPC (1, 2-didecanoyl-sn-glycero-3-phosphorylcholine), DSPC (distearoylphosphatidylcholine), DOPC (dioleoylphosphatidylcholine), or combinations thereof.

In another preferred embodiment, the Phosphatidylethanolamine (PE) lipidoid comprises: DSPE (distearoyl-phosphatidylethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), DLPE (dilauroylphosphatidylethanolamine), or combinations thereof.

In another preferred embodiment, the sphingomyelin lipids comprise: a ceramide.

In another preferred embodiment, the lipophilic end is a group derived from a lipid selected from the group consisting of: chol, DSPE, ceramide.

In another preferred embodiment, R₁Is a group shown as a formula I-A,

R_1a-W_a-L_a-(I-A)

wherein R is_1aIs a group derived from neutral lipids, or is a C10-C20 saturated or unsaturated hydrocarbon group;

W_aselected from the group consisting of: none, -O-, -S-, -R ' C (O) R ' -, -R ' C (O) OR ' -, -R ' OC (O) R ' -, R ' S (O) R ' -, -R ' S (O)₂R”-、-R’N(R_a)R”-、-R’C(O)N(R_a)R”-、-R’C(O)ON(R_a)R”-、-R’OC(O)N(R_a)R”-、-R’S(O)N(R_a)R”-、-R’S(O)₂N(R_a)R”-、-R’N(R_a)C(O)R”-、-R’N(R_a)OC(O)R”-、-R’N(R_a)C(O)OR”-、-R’N(R_a)S(O)R”-、-R’N(R_a)S(O)₂R”-；

L_aIs- (Z)_n-; wherein each Z is independently selected from the group consisting of: c (R)_c)₂C3-C6 cycloalkyl, O, S, NR_a；

n is an integer of 2 to 20, R_cEach independently selected from the group consisting of: hydrogen, deuterium, C1-C6 alkyl (preferably C1-C4 alkyl; more preferably selected from methyl, ethyl);

R_ar ', and R' are as defined above.

In another preferred embodiment, Z is CH₂。

In another preferred embodiment, R_1aIs a group derived from a lipid selected from the group consisting of: cholesterol, DOPC, DOPE, DDPC, DDPE, DLPC, DLPE, DMPC, DMPE, DPPC, DPPE, DSPC, DSPE, DPhyPE, sphingomyelin, ceramide, diglyceride, sphingosine; or R_1aIs a C10-C20 saturated or unsaturated alkyl.

In another preferred embodiment, R_1aIs a group derived from a lipid selected from the group consisting of: chol, DSPE (1, 2-distearoyl-sn-glycero-3-phosphocholine), ceramide (ceramide), DOPE, DSPC; or R_1aIs a C10-C20 saturated or unsaturated alkyl.

In another preferred example, n is 2,3, 4, 5, 6, 7 or 8; preferably, n is 2,3, 4, 5 or 6.

In another preferred embodiment, L_aIs- (CH)₂)_n-; preferably, L_aIs- (CH)₂)_2-8-; more preferably, L_aIs- (CH)₂)_2-6-。

In another preferred embodiment, R 'and R' are absent.

In another preferred embodiment, W_aSelected from the group consisting of: none, -O-, -C (O) NH-, -OC (O) NH-.

In another preferred embodiment, W_aSelected from the group consisting of: none, -O-, -C (O) NH₂-。

In another preferred embodiment, R_1aSelected from the group consisting of: chol, DSPE, ceramide (ceramide); preferably, R_1aIs Chol.

In another preferred embodiment, R₁Is Chol-OC (O) -NH- (CH)₂)_2-5-、Chol-C(O)-NH-(CH₂)_2-5-or Chol-O- (CH)₂)_5-8-; more preferably, R₁Is Chol-O-C (O) -NH- (CH)₂)₃-or Chol-O- (CH)₂)₆-。

In another preferred embodiment, R₁Is Chol-C (O) -NH- (CH)₂)_2-5-or Chol-O- (CH)₂)_5-8-; more preferably, R₁Is Chol-O-C (O) -NH- (CH)₂)₃-or Chol-O- (CH)₂)₆-。

In another preferred embodiment, R₃Selected from the group consisting of: substituted or unsubstituted C3-C12 cycloalkyl (preferably, C3-C7 cycloalkyl), substituted or unsubstituted C6-C12 aryl (preferably, C6-C8 aryl).

In another preferred embodiment, R₃In the group, the substitution means that 1 or 2 hydrogens in the group are substituted with a group selected from the group consisting of: hydroxyl, C1-C6 alkoxy.

In another preferred embodiment, R₃Is a substituted or unsubstituted C6-C8 aryl, and said substitution means that 1 hydrogen in the group is substituted with a group selected from: hydroxyl, C1-C3 alkoxy.

In another preferred embodiment, R₃Selected from the group consisting of:phenyl, hydroxyphenyl, C1-C3 alkoxyphenyl, cyclohexyl; preferably, R3 is phenyl.

In another preferred embodiment, R₃Is a group derived from a compound selected from the group consisting of: preferably, R3 is

In another preferred embodiment, R₇Is a benzene ring or a C1-C6 alkylene group.

In another preferred embodiment, L is selected from the group consisting of: none, O.

In another preferred embodiment, R₉Is selected from the following group: none, C1-C6 alkylene (preferably (CH)₂)_1-2) C6-C12 aryl (preferably phenyl).

In another preferred embodiment, W is selected from the group consisting of: c (O) NH.

In another preferred embodiment, R₂Selected from the group consisting of: none, -phenyl-C (O) NH-, -phenyl-O- (CH)₂)_1-2-C (O) NH-, -phenyl-O-phenyl-C (O) NH-, -phenyl-C (O) NH-, - (CH)₂)_1-2-O-(CH₂)_1-2-C (O) NH- (-C (O) NH-linked to a hydrophilic chain).

In another preferred embodiment, R₈Is PEG (polyethylene glycol).

In another preferred embodiment, R₈PEG with the average molecular weight of 190-22000 (namely PEG)_200～20000)。

In another preferred embodiment, R₈Is PEG with an average molecular weight of 1800-2200 (i.e., PEG)₂₀₀₀)。

In another preferred embodiment, the compound is represented by formula II,

wherein the content of the first and second substances,

R₄、R₅and R₆Is as defined in claim 1 wherein R_eThe definition of (1);

R₁、R₂、R₃、R₈x and Y are as defined in claim 1.

In another preferred embodiment, R₄、R₅And R₆Each independently selected from the group consisting of: H. deuterium, C1-C6 alkoxy, hydroxy, N (R)_a)₂C1-C6 acylamino, C1-C6 acyloxy, C1-C6 alkyl, substituted or unsubstituted C6-C12 aryl, substituted or unsubstituted 5-to 12-membered heteroaryl, substituted or unsubstituted C6-C12 cycloalkylene, substituted or unsubstituted 5-to 10-membered heterocyclic group.

In another preferred embodiment, R₄、R₅And R₆Each independently selected from the group consisting of: H. deuterium, C1-C6 alkoxy; preferably, R₄、R₅And R₆Each independently is H or deuterium.

In another preferred embodiment, the compound is represented by formula III-A,

wherein the content of the first and second substances,

m＝200-20000；

R₁、R₂、R₃、R₄、R₅、R₆x and Y are as defined in claim 1.

In another preferred example, m is 1000-4000; preferably, m is 2000.

In another preferred embodiment, the compound is represented by formula III-B,

wherein the content of the first and second substances,

R_1ais neutral ofA group of lipids, or a saturated or unsaturated hydrocarbon group of C10-C20;

W_aselected from the group consisting of: -O-, -S-, C (O) -, -C (O) O-, -OC (O) -, S (O) -, -S (O)₂-、-NH-、-C(O)NH-、-C(O)ONH-、-OC(O)NH-、-S(O)NH-、-S(O)₂NH-、-NHC(O)-、-NHOC(O)-、-NHC(O)O-、-NHS(O)-、-NHS(O)₂-; preferably, W_aSelected from the group consisting of: -O-, -OC (O) NH-, -NHC (O) O-;

n is an integer of 2-20 (preferably, n is 2,3, 4, 5, 6, 7 or 8);

R₂、R₃、R₄、R₅、R₆x and Y are as defined in claim 1.

In another preferred embodiment, R_1aAs previously defined.

In another preferred embodiment, when Wa is-O-, n is 5, 6, 7 or 8.

In another preferred embodiment, when Wa is-oc (O) NH-or-nhc (O) O-, n is 2,3, 4 or 5.

In another preferred embodiment, the compound is represented by formula III-C or formula III-D,

wherein R is_1a、R₃、R₄、R₅、R₆、R₉、Y、L、W_aThe definitions of W, m and n are as previously described.

In another preferred embodiment, each group in each formula (formula I, formula II, formula III-A, formula III-B, formula III-C or formula III-D) is defined as corresponding to the compound in Table 1.

In another preferred embodiment, the compound is selected from the compounds in table 1.

In a second aspect of the invention, an intermediate I is provided, wherein the intermediate I is shown as a formula IV,

wherein R is₁、R₃、R₄、R₅、R₆X and Y are as defined in the first aspect.

In another preferred embodiment, the intermediate I is shown as a formula IV-A,

wherein R is_1a、W_a、R₃、R₄、R₅、R₆Y and n are as defined above.

In a third aspect of the invention, there is provided an intermediate II, said intermediate being represented by formula V,

wherein the content of the first and second substances,

W_bis a reactive linking group;

R₁、R₃、R₄、R₅、R₆、R₉l, X and Y are as defined in the first aspect.

In another preferred embodiment, W_bSelected from the group consisting of: -R 'COOH, -R' C (O) H, -R 'OH, -R' N (R)_a)H、-R’SO₃H、-R’SO₂H; wherein R' and R_aAs defined above.

In another preferred embodiment, W_bis-R' COOH; preferably, W_bis-COOH.

In another preferred embodiment, the intermediate II is shown as the formula V-A,

wherein the content of the first and second substances,

R_1a、W_a、R₃、R₄、R₅、R₆、Y、L、R₉、W_band n is as defined above.

In another preferred embodiment, W_bSelected from the group consisting of: -COOH, -OH, -NH₂。

In a fourth aspect of the invention, there is provided a process for the preparation of a compound as described in the first aspect, prepared by process one or process two;

the method comprises the following steps:

(1.1) reacting intermediate I with NH in a first inert solvent₂-R₈Reacting to obtain a compound shown as a formula I; or

The second method comprises the following steps:

(2.1) reacting intermediate I with NH in a first inert solvent₂-R₂-W_bReacting to obtain an intermediate II; and

(2.2) reacting the intermediate II obtained in the step (1) with W in a second inert solvent_c-R₈Reacting to obtain a compound shown as a formula I;

wherein, W_bAnd W_cEach independently is the same or different reactive group; the intermediate I is an intermediate shown as a formula IV, and the intermediate II is an intermediate shown as a formula V; r₂And R₈Is as defined in claim 1.

In another preferred embodiment, the reactive group W_bAnd an active group W_cCapable of reacting to form a group W.

In another preferred embodiment, in the first method, the intermediate I is an intermediate shown as a formula IV-A.

In another preferred example, in the second method, the intermediate I is an intermediate shown as a formula IV-A and the intermediate II is an intermediate shown as a formula V-A.

In another preferred embodiment, W_bSelected from the group consisting of: -R 'COOH, -R' C (O) H, -R 'OH, -R' N (R)_a)H、-R’SO₃H、-R’SO₂H; wherein R' and R_aAs defined above; preferably, W_bis-R' COOH.

In another preferred embodiment, W_cSelected from the group consisting of: -R "COOH, -R" C (O) H, -R "OH, -R" N (R)_a)H、-R”SO₃H、-R”SO₂H; wherein R' and R_aAs defined above; preferably, W_cis-R' NH₂。

In another preferred embodiment, in step (1.1), intermediate I is reacted with NH₂-R₈The molar ratio of (0.8-1.2) to (1).

In another preferred embodiment, in step (2.1), intermediate I is reacted with NH₂-R₂-W_bThe molar ratio of (0.8-1.2) to (1).

In another preferred embodiment, in step (2.2), intermediate II is reacted with W_c-R₈The molar ratio of (0.8-1.2) to (1).

In another preferred example, the reaction temperature of the step (1.1) is 20-150 ℃; preferably 20 to 100 ℃.

In another preferred example, the reaction time of the step (1.1) is 2-48 h; preferably 10 to 48 hours.

In another preferred example, the reaction temperature of the step (2.1) is 20-150 ℃; preferably 20 to 100 ℃.

In another preferred example, the reaction time of the step (2.1) is 2-48 h; preferably 10 to 48 hours.

In another preferred embodiment, the reaction temperature in step (2.2) is 0-100 ℃; preferably, 10 to 50 ℃; more preferably, 15 to 40 ℃.

In another preferred example, the reaction time of the step (2.2) is 10-48 h; preferably, 20 to 30 hours; more preferably, 24 hours.

In another preferred embodiment, the first inert solvent is selected from the group consisting of: methanol, ethanol, toluene, dichloromethane, or combinations thereof.

In another preferred embodiment, the second inert solvent is selected from the group consisting of: dichloromethane, methanol, ethanol, or combinations thereof.

In a fifth aspect of the invention there is provided the use of a compound as described in the first aspect for the modification of liposomes and/or lipid-drug complexes.

In another preferred embodiment, the modified liposome and/or lipid-drug complex has an endocytic release response function.

In another preferred embodiment, the lipid-drug complex is a lipid-drug complex formed by compounding a liposome and a drug.

In another preferred embodiment, the drug is a gene drug.

In another preferred embodiment, the genetic agent is a nucleic acid.

In another preferred embodiment, the gene drug is selected from the group consisting of: rna (sirna), plasmid dna (pdna), hairpin rna (shrna), antisense Oligonucleotide (ODN), messenger rna (mrna), micro rna (mirna).

In another preferred embodiment, the liposome comprises a cationic lipid and optionally a helper lipid.

In another preferred embodiment, the cationic lipid comprises: DOTAP (N- [1- (2, 3-dioleyloxy) propyl ] -N, N, N-trimethylammonium salt), DODAC (N, N-dioleyl-N, N-dimethylammonium chloride), DDAB (N, N-distearyl-N, N-dimethylammonium bromide), DODAP (1, 2-dioleyl-3-dimethylammonium-propane), DOTMA (N- (1- (2, 3-dioleyloxy) propyl) -N, N, N-trimethylammonium chloride), DODAP (1, 2-dioleylcarbamoyl-3-dimethylammonium-propane), DLINDAP (1, 2-dioleyl-3-dimethylammonium-propane), DLTAP (dilauryl (C12:0) trimethylammonium propane), DOGS (dioctadecylaminoglycyl spermine), DC-Chol (3 β - [ N- (N ', N' -dimethylaminoethyl) ] -cholesterol), DOSPA (dioleoyloxy-N- [ 2-sperminocarboxamido) ethyl } -N, N-dimethyl-1-propanetrifluoroacetate), DMRIE (1, 2-dimyristoyloxypropyl-3-dimethyl-hydroxyethylammonium bromide), CLinDMA (3-dimethylamino-2- (cholest-5-en-3- β -oxybutan-4-oxy) -1- (cis, cis-9, 12-octadecadienoyloxy) propane), DODMA (N, N-dimethyl-2, 3-dioleyloxy) propylamine), CpLinDMA (2- [5 ' - (cholest-5-en-3 [ beta ] -oxy) -3 ' -oxapentyloxy) -3-dimethyl-1- (cis, cis-9 ', 12 ' -octadecadienoyloxy) propane) and DMOBA (N, N-dimethyl-3, 4-dioleyloxybenzylamine), and DOcarbDAP (1,2-N, N ' -dioleylcarbamoyl-3-dimethylaminopropane).

In another preferred embodiment, the cationic lipid comprises: DOTAP, DOTAM, DOSPA, DOGS, or combinations thereof.

In another preferred embodiment, the helper lipids comprise: a steroid lipid, a phosphatidylcholine lipid, a phosphatidylethanolamine lipid, a sphingomyelin lipid, or a combination thereof; preferably, the helper lipid is a steroid lipid; more preferably, the lipid is cholesterol (Chol).

In another preferred embodiment, the sterol lipid comprises: cholesterol (Chol).

In another preferred embodiment, the Phosphatidylcholine (PC) lipid comprises: DPPC (1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine), DMPC (1, 2-dimyristoylphosphatidylcholine), DDPC (1, 2-didecanoyl-sn-glycero-3-phosphorylcholine), DSPC (distearoylphosphatidylcholine), DOPC (dioleoylphosphatidylcholine), or combinations thereof.

In another preferred embodiment, the Phosphatidylethanolamine (PE) lipidoid comprises: DSPE (distearoyl-phosphatidylethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), DLPE (dilauroylphosphatidylethanolamine), or combinations thereof.

In another preferred embodiment, the sphingomyelin lipids comprise: a ceramide.

In a sixth aspect of the present invention there is provided a lipid-drug complex with a dilute release response by endocytosis, said lipid-drug complex being modified by a compound according to the first aspect.

In another preferred embodiment, the lipid-drug complex is formed by complexing a liposome and a drug.

In another preferred embodiment, the lipid-drug complex is a lipid-gene drug complex (i.e., a lipid-drug complex formed by the complexation of a liposome and a gene drug).

In a seventh aspect of the present invention, there is provided a lipid-drug complex responsive to endocytosis and release, said lipid-drug complex being complexed by a liposome, a drug (preferably a genetic drug) and a compound as described in the first aspect, and said liposome comprising: a cationic lipid and optionally a helper lipid.

In another preferred embodiment, in the lipid-drug complex, the molar ratio of the cationic lipid to the compound according to the first aspect is 1 (0.1-10); preferably, the ratio is 1 (0.2-5); more preferably, it is 1 (1. + -. 0.2).

In another preferred embodiment, in the lipid-drug complex, the molar ratio of the cationic lipid, the compound according to the first aspect and the helper lipid is 1 (0.1-10) to (0.1-10); preferably, 1 (0.2-5) and 0.2-5); more preferably, the ratio is 1 (1 + -0.2) to (1 + -0.2).

In another preferred embodiment, the cationic lipid is as defined in the fifth aspect.

In another preferred embodiment, the helper lipids are as defined in the fifth aspect.

In an eighth aspect of the present invention, there is provided a formulation characterized in that it comprises the lipid-drug complex according to the sixth or seventh aspect.

In a ninth aspect of the present invention, there is provided a kit comprising: a compound as described in the first aspect, a cationic lipid and optionally a helper lipid.

In another preferred embodiment, the kit further comprises a drug (preferably, a gene drug).

In another preferred embodiment, the cationic lipid, helper lipid and/or drug (preferably, a gene drug) are as defined in the fifth aspect.

In a tenth aspect of the present invention, there is provided a method for introducing a drug into a cell, comprising the steps of: contacting a subject with a drug complex according to the seventh aspect or a drug complex according to the eighth aspect, thereby introducing a drug.

In another preferred embodiment, the drug is a gene drug.

In another preferred embodiment, the cationic lipid, helper lipid and/or drug are as defined in the fifth aspect.

In another preferred embodiment, the method is non-therapeutic in vitro.

In another preferred embodiment, the subject is a cell.

In another preferred embodiment, the method comprises the steps of: culturing a drug complex according to the seventh aspect or a drug complex according to the eighth aspect together with a cell, thereby introducing a drug into the cell.

In an eleventh aspect of the invention, there is provided a method of transfecting a cell comprising the steps of: the transfection is performed by contacting the subject (preferably, a cell) with a lipid-gene drug complex modified with a compound of the first aspect.

In another preferred embodiment, the genetic drug is DNA.

In another preferred embodiment, the method is non-therapeutic in vitro. The lipid-gene drug complex is cultured together with cells, thereby introducing the gene drug into the cells.

In another preferred embodiment, the method comprises the steps of: in a twelfth aspect of the present invention, which is modified by the compound of the first aspect, there is provided a method of gene therapy comprising the steps of: administering to a subject a lipid-drug complex according to the seventh aspect or a lipid-drug complex according to the eighth aspect or a formulation according to the eighth aspect.

In another preferred embodiment, the drug is a gene drug.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIGS. 1A and B show the degradation in vitro conditions, respectively.

FIG. 2 shows the transfection of different compounds.

Detailed Description

The inventors have extensively and intensively studied and unexpectedly found that a class of compounds having a specific parent nucleus structure has an excellent phagocytic response function of cells. Cationic liposomes modified with such compounds or containing such compounds can be used for delivery of compounds for gene drugs and provide excellent cell transfection efficiency. The compound comprises a lipophilic head, a hydrophilic chain (such as a PEG long chain), and an intermediate group with a pH response function of a special parent nucleus structure. The cationic liposome modified by the compound or the compound of the cationic liposome containing the compound and a gene drug (such as nucleic acid) is not easy to aggregate and combine with albumin to form large particles under the extracellular environment, and is not easy to be identified and eliminated by reticuloendothelial cells, so that the target part can be reached smoothly. Meanwhile, the compound modified by the compound or containing the compound can be rapidly degraded in an intracellular solvent environment (namely, the compound is broken due to pH response), so that positive charges of the compound of cationic lipid and gene drugs (such as nucleic acid) are exposed, lysosome escape of the nucleic acid is realized, the nucleic acid is released into cytoplasm and enters a cell nucleus, and efficient transfection of the nucleic acid is realized. Based on this, the inventors have completed the present invention.

Term(s) for

As used herein, "halogen" refers to F, Cl, Br, and I. More preferably, the halogen atom is selected from F, Cl and Br.

Unless otherwise indicated, the term "C10-C20 saturated or unsaturated hydrocarbyl" includes alkyl, unsaturated hydrocarbyl containing one or more (e.g., 1,2, 3,4, or 5) double and/or triple bonds. Preferably, the C10-C20 saturated or unsaturated alkyl group is a straight chain or branched chain alkyl group (e.g., an alkyl group containing 1,2 or 3 straight chains).

Unless otherwise indicated, the term "alkyl" by itself or as part of another substituent refers to a straight or branched chain hydrocarbon group having the indicated number of carbon atoms (i.e., C1-C6 represents 1-6 carbons). Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, and the like.

As used herein, "C1-C6 alkoxy" includes straight or branched chain alkoxy groups of 1-6 carbon atoms. Such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy, or the like.

Unless otherwise specified, the term "cycloalkyl" refers to hydrocarbon rings having the indicated number of ring atoms (e.g., C3-C8 cycloalkyl) and being fully saturated or having no more than one double bond between ring vertices. "cycloalkyl" also refers to bicyclic and polycyclic hydrocarbon rings such as bicyclo [2.2.1] heptane, bicyclo [2.2.2] octane, and the like. The term "heterocycloalkyl" refers to a cycloalkyl group containing one to five heteroatoms selected from N, O and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom is optionally quaternized. The heterocycloalkyl group can be a monocyclic, bicyclic, or polycyclic ring system. Non-limiting examples of heterocycloalkyl groups include pyrrolidine, imidazolidine, pyrazolidine, butyrolactam, valerolactam, imidazolidinone, hydantoin, dioxolane, phthalimide, piperidine, 1, 4-dioxane, morpholine, thiomorpholine-S-oxide, thiomorpholine-S, S-oxide, piperazine, pyran, pyridone, 3-pyrroline, thiopyran, pyrone, tetrahydrofuran, tetrahydrothiophene, quinuclidine, and the like. The heterocycloalkyl group can be attached to the rest of the molecule via a ring carbon or a heteroatom. By terms such as cycloalkylalkyl and heterocycloalkylalkyl, it is meant that the cycloalkyl or heterocycloalkyl group is attached to the rest of the molecule through an alkyl or alkylene linker. For example, cyclobutylmethyl-is a cyclobutyl ring linked to a methylene linkage in the remainder of the molecule.

Unless otherwise specified, the term "aryl" denotes a polyunsaturated (usually aromatic) hydrocarbon group which may be a single ring or multiple rings (up to three rings) which are fused together or linked covalently. The term "heteroaryl" refers to an aryl (or ring) containing 1 to 5 heteroatoms selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized and the nitrogen atom is optionally quaternized. The heteroaryl group may be attached to the rest of the molecule through a heteroatom. Non-limiting examples of aryl groups include phenyl, naphthyl and biphenyl groups, while non-limiting examples of heteroaryl groups include pyridyl, pyridazinyl, pyrazinyl, pyrimidinyl, triazinyl, quinolinyl, quinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl, benzotriazinyl, purinyl, benzimidazolyl, benzopyrazolyl, benzotriazolyl, benzisoxazolyl, isobenzofuranyl, isoindolyl, indolizinyl, benzotriazinyl, thienopyridyl, thienopyrimidyl, pyrazolopyrimidinyl, imidazopyridine, benzothiazolyl, benzofuranyl, benzothienyl, indolyl, quinolinyl, isoquinolinyl, isothiazolyl, pyrazolyl, indazolyl, pteridinyl, imidazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiadiazolyl, pyrrolyl, thiazolyl, furanyl, thienyl, and the like. The substituents for each of the above aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below.

For the sake of brevity, the term "aryl" when used in combination with other terms (e.g., aryloxy, arylthio, aralkyl) includes aryl and heteroaryl rings as defined above. Thus, the term "aralkyl" is meant to include those groups in which the aryl group is attached to an alkyl group attached to the rest of the molecule (e.g., benzyl, phenethyl, pyridylmethyl, and the like).

As used herein, the term "heteroatom" is meant to include oxygen (O), nitrogen (N), and sulfur (S).

As used herein, derived from a compound or lipid refers to the reactive group (e.g., amino, hydroxyl, carboxyl, etc.) in the compound or lipid reacting with another compound or compounds to form the corresponding group.

Liposomes

Liposomes (liposomes) are micro vesicles formed by encapsulating drugs in lipid bilayers, and contain phospholipids and cholesterol as the main components. The lipid membrane has a structure similar to a biological membrane, so that the lipid membrane has good biocompatibility, and enters cells in an endocytosis and fusion mode after being wrapped by the drug, so that the drug is delivered to target cells.

Cationic liposome

Cationic liposomes (Cationic liposomes) are liposomes composed of positively charged Cationic lipids and neutral helper lipids. The cationic liposome has the advantages of strong ability of loading macromolecular gene drugs, high in vitro transfection efficiency and the like, is widely applied to in vivo and in vitro researches, and is one of the most used non-viral vectors at present. Cationic lipids consist of a positively charged hydrophilic head, a hydrophobic tail, and a connecting chain connecting the head and tail. The cationic liposome is generally composed of cationic lipid and auxiliary lipid, wherein the head of the cationic lipid is positively charged and can be combined with nucleic acid substances with negative charges through electrostatic interaction; helper lipids are generally uncharged, and have the effect of stabilizing lipid bilayers or assisting the fusion of lipid membranes with endosomal membranes.

Common cationic lipids comprise DOTAP, DOTAM, DOSPA and the like, and the positive charges of different lipid heads are different, so that the loading amount and the toxicity of nucleic acid in the liposome are determined; common neutral lipids are mainly Chol, DOPE, DPPC and the like.

Gene delivery mechanism

The gene delivery mechanism of the cationic lipid/nucleic acid complex is: the cationic liposome and nucleic acid with negative charge are tightly combined through electrostatic action to form small-particle-size lipid/nucleic acid complexes with uniform sizes. The positive-charge lipid/nucleic acid complex is easy to be adsorbed on the surface of a negative cell membrane, enters a cell through an endocytosis or membrane fusion mechanism, the endocytosed lipid/nucleic acid complex releases nucleic acid after escaping through lysosomes, and DNA drugs are released to cytoplasm after escaping, and then further enter a nuclear membrane to enter the nucleus to play a role.

Abbreviations used herein are defined as follows:

compounds responsive to release by endocytosis

The present invention addresses the foregoing problems by providing a class of compounds having an endocytic release response that are relatively stable under body fluid conditions (i.e., pH 7.35-7.45), but will degrade in the tumor microenvironment (pH < 6) or under the cell lysosome conditions (pH around 5), due to the action of hydrogen ions in an acidic environment, pH-responsive chemical bonds in the compounds (e.g., imine bonds (-C ═ N-)) used in the compounds of the invention are partially or completely broken, thereby releasing hydrophilic chains (such as hydrophilic long-chain PEG) and exposing the positive charge property of the cationic liposome (such as DOTAP), the lipid/nucleic acid complex is exposed with more positive charges, so that the lysosome escape capacity of the complex is effectively improved, more DNA is released into cytoplasm, and the transfection efficiency of the liposome complex is improved.

Specifically, the compound of the present invention comprises a lipophilic head for insertion into a liposome (or complex), a hydrophilic chain, and a responsive group connecting the lipophilic head and the hydrophilic chain, wherein the responsive group is pH-responsive. Preferably, the intermediate chain remains stable at a pH greater than 7 (e.g., 7.35 to 7.45) and breaks at a pH less than 6 (e.g., about 5) to release the hydrophilic group.

The main advantages of the invention include:

(a) the compound-modified complex (e.g., lipid-nucleic acid complex) of the present invention is stable in the extracellular environment, is not easily aggregated, is not easily removed, and can smoothly reach a target site.

(b) The compound modified complex of the invention can be rapidly degraded in an intracellular environment (generally within 2 h), and the complex of cationic lipid and gene drug (such as nucleic acid) is exposed, so that lysosome escape is realized.

(c) The compound modified by the compound has high transfection efficiency, the transfection efficiency under serum-free conditions is slightly higher than that of the non-modified cationic liposome, and the transfection efficiency under serum conditions can generally reach 10-20 times that of the non-modified cationic liposome.

(d) The compound modified compound effectively reduces the toxicity of the cationic liposome to cells.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

Preparation of the Compound of example 1

EXAMPLE 1.1 preparation of Compound 6(Cpd6)

The first process is as follows:

and a second process:

(a) synthesis of Compound 1-1: 49.1g (0.259mol) of p-toluenesulfonyl chloride were dissolved in 200ml of pyridine and slowly added at room temperature to a pyridine solution (300ml) containing 50.0g (0.129mol) of cholesterol; after the addition was complete, the mixture was stirred at room temperature overnight. After the reaction, 3000ml of dichloromethane was added, and the mixture was washed with 1mol/L hydrochloric acid solution 3 to 5 times, once with saturated brine, and the organic phase was dried over anhydrous sodium sulfate, most of dichloromethane was removed by rotation, followed by addition of an appropriate amount of methanol to recrystallize to obtain 59.7g of the product (Compound 1-1). The yield is 85.4 percent

(b) Synthesis of Compounds 1-2: 218.3g (1.850mol) of 1, 6-hexanediol was added to a 1, 4-dioxane solution (1000ml) containing 50.0g (0.093mol) of compound 1-1, and the reaction mixture was heated to 80 ℃ and reacted for 5 hours. After the reaction, 5000ml of ethyl acetate was added with stirring, followed by washing with water 3 to 5 times, further washing with saturated brine once, drying the organic phase with anhydrous sodium sulfate, spin-drying and column-chromatography to obtain 31.0g of the product (Compound 1-2). The yield thereof is 69.7%

(c) Synthesis of Compounds 1-3: 18.1g (0.104mol) of methanesulfonic anhydride was dissolved in 200ml of dichloromethane, and a solution of 25.0g (0.052mol) of Compound 1-2 and 18.0g (0.130mol) of triethylamine in dichloromethane (200ml) was slowly added under an ice-water bath. After the addition was completed, the reaction was carried out at room temperature overnight. After the reaction, the reaction mixture was washed with water 3 to 5 times, once with saturated brine, and the organic phase was dried over anhydrous sodium sulfate, then spin-dried and subjected to column chromatography to obtain 21.9g of a product (compound 1 to 3). The yield is 75.2%

(d) Synthesis of Compounds 1-4: 206.3g (0.787mol) of triphenylphosphine and 100g (0.787mol) of benzyl chloride were dissolved in 3000ml of acetonitrile, and then the reaction solution was heated to reflux for 5 hours. After the reaction, the reaction solution was suction-filtered, and the obtained solid was washed three times with acetonitrile to obtain 184.7g of a product (Compound 1-4). The yield is 69.4%

(e) Synthesis of Compounds 1-5: 100g (0.725mol) of 2, 4-dihydroxybenzaldehyde, 66.9g (0.797mol) of sodium hydrogencarbonate, 12.0g (0.072mol) of potassium iodide and 100.0g (0.725mol) of benzyl chloride were added to 1000ml of acetonitrile, and then the reaction solution was heated to reflux overnight. After the reaction, most of the reaction solution was removed by rotation, 5000ml of ethyl acetate was added for extraction, and the organic phase was washed with water 3 to 5 times, once with saturated brine, dried over anhydrous sodium sulfate, removed most of the ethyl acetate by rotation, and then recrystallized by adding methanol to obtain 108.7g of the product (Compound 1 to 5). The yield is 65.8 percent

(f) Synthesis of Compounds 1-6: 156.0g (0.403mol) of Compound 1 to 4 and 87.1g (0.382mol) of Compound 1 to 5 were dissolved in 2000ml of acetonitrile, and 61.1g (0.403mol) of DBU was slowly added to the above-mentioned mixture at room temperature. The reaction was then heated to reflux overnight. After the reaction, most of the reaction solution was removed by rotation, 8000ml of ethyl acetate was added for extraction, and the organic phase was washed with water 3 to 5 times, once with saturated brine, dried over anhydrous sodium sulfate and then subjected to column chromatography to obtain 36.6g of the product (Compound 1 to 6). The yield is 56.8 percent

(g) Synthesis of Compounds 1-7: 35.3g (0.117mol) of the compounds 1 to 6 and 96.6g (0.700mol) of potassium carbonate were dissolved in 500ml of tetrahydrofuran, stirred at room temperature for 15 minutes, and then 177.8g (0.700mol) of iodine was added to the mixture and reacted at room temperature for half an hour. After the reaction, an excess saturated sodium hydrogen sulfite solution was added to neutralize excess iodine, and then 3000ml of ethyl acetate was added for extraction, and the organic phase was washed with water 3 to 5 times, once with saturated brine, dried over anhydrous sodium sulfate and then passed through a column to obtain 16.6g of the product (compound 1 to 7). The yield is 47.6 percent

(h) Synthesis of Compounds 1-8: 17.8g (0.244mol) of DMF and 37.3g (0.244mol) of phosphorus oxychloride were added to 500ml of 1, 2-dichloroethane in an ice-water bath and stirred at room temperature for one hour. Then, 14.7g (0.049mol) of the compounds 1 to 7 were added to the mixture, and the reaction mixture was heated to reflux for reaction overnight. After the reaction, the reaction mixture was washed with water 3 to 5 times, once with saturated brine, and the washed organic phase was dried over anhydrous sodium sulfate and then passed through a column to obtain 11.5g of the product (Compound 1 to 8). The yield is 71.4 percent

(i) Synthesis of Compounds 1-9: 9.2g (0.048mol) of titanium tetrachloride was slowly added to a methylene chloride solution (100ml) containing 10.6g (0.032mol) of the compounds 1 to 8 under an ice-water bath, and then reacted at room temperature for one hour. After the reaction, the reaction mixture was washed with water 3 to 5 times, once with saturated brine, and the organic phase was dried over anhydrous sodium sulfate and then passed through a column to obtain 5.9g of a product (compound 1 to 9). The yield is 77.5 percent

(j) Synthesis of Compounds 1-10: 11.4g (0.021mol) of the compounds 1 to 3, 4g (0.017mol) of the compounds 1 to 9, 8.2g (0.025mol) of cesium carbonate, 0.3g (0.002mol) of potassium iodide were dissolved in 200ml of DMF, and then the reaction solution was heated to 80 ℃ for reaction for half an hour. After completion of the reaction, 1000ml of ethyl acetate was added to the reaction mixture, which was washed with water 3 to 5 times, once with saturated brine, and the organic phase was dried over anhydrous sodium sulfate and then subjected to column chromatography to obtain 10.1g of the product (compound 1 to 10). The yield thereof is 83.7%

(k) Synthesis of compound Cpd 6: 260.4mg (0.364mmol) of Compound 1 to 10, 800.0mg (0.400mmol) of methoxypolyethyleneglycoamine, 0.5g of activated 3Ao molecular sieve were added to 40ml of anhydrous methanol, and the reaction mixture was heated under reflux for 36 hours. After the reaction is finished, the molecular sieve is removed, part of methanol is removed by spinning, and a large amount of solid is separated out after the methanol is cooled. Then, a solid was obtained by suction filtration, and the solid was washed three times with methanol to obtain 0.5g of a product. The yield was 48.4%.¹H NMR(400MHz,CDCl₃)δ0.64(s,3H),0.82-0.84(m,6H),0.88-0.90(m,3H),0.94-1.58(m,29H),1.79-1.92(m,5H),1.95-1.99(m,1H),3.07-3.14(m,1H),3.35(s,3H),3.42-3.47(m,3H),3.51-3.69(m,200H),3.78-3.81(m,5H),3.98-4.01(m,2H),5.30-5.31(m,1H),6.88-6.90(m,1H),6.99(s,1H),7.41-7.49(m,3H),7.72(d,J＝8.4Hz,2H),8.15(d,J＝8.4Hz,1H),8.63(s,1H)。

EXAMPLE 1.2 preparation of Compound 2(Cpd2)

(a) Synthesis of Compound 2-1: 30g (0.067mol) of cholesterol formyl chloride are dissolved in 200ml of dichloromethane and the mixture is slowly added to a solution of 10.2g (0.101mol) of triethylamine and 6.0g (0.080mol) of n-propanolamine in dichloromethane (200ml) under an ice-water bath. After the addition was completed, the reaction was carried out at room temperature overnight. After the reaction, the reaction solution was washed with water 3-5 times, once with saturated brine, and the organic phase was dried over anhydrous sodium sulfate, most of dichloromethane was removed, and an appropriate amount of methanol was added to recrystallize, thereby obtaining 27.1g of the product (i.e., compound 2-1). The yield thereof was found to be 83.4%.

(b) Synthesis of Compound 2-2: the synthesis was carried out in accordance with the procedure for synthesizing the compounds 1 to 3 in reference example 1.1, to obtain 18.8g of the product (compound 2-2) in a yield of 81.7%.

(c) Synthesis of Compounds 2-3: the synthesis was carried out in accordance with the procedure for synthesizing the compounds 1 to 10 in reference example 1.1, to obtain 5.4g of the product (compounds 2 to 3) in a yield of 85.5%.

(d) Synthesis of compound Cpd 2: the synthesis was carried out according to the procedure for the synthesis of compound Cpd6 in example 1.1 to yield 0.5g of product in 38.4% yield.¹H NMR(400MHz,CDCl₃)δ0.64(s,3H),0.82-0.83(m,6H),0.87-0.88(m,3H),0.97(s,4H),1.09-1.53(m,20H),1.79-1.99(m,6H),3.35(s,3H),3.43-3.81(m,200H),4.46-4.48(m,2H),4.92(s,1H),5.33(s,1H),6.88-6.90(m,1H),7.00(s,1H),7.42-7.48(m,3H),7.70-7.71(m,2H),8.15(d,J＝8.4Hz,2H),8.63(s,1H).

EXAMPLE 1.3 Synthesis of Compound 4(Cpd4)

The method comprises the following steps:

step two:

(a) synthesis of Compound 3-1: 10g (0.072mol) of p-nitrophenol, 19.9g (0.144mol) of potassium carbonate and 18g (0.108mol) of ethyl bromoacetate were dissolved in 200ml of acetonitrile, and then the reaction solution was heated to reflux for overnight. After the reaction is finished, most of acetonitrile is removed by spinning, 1000ml of ethyl acetate is added for extraction, then the mixture is washed by water for 3-5 times, the mixture is washed by saturated saline solution for one time, the organic phase is dried by anhydrous sodium sulfate, most of ethyl acetate is removed by spinning, and then petroleum ether is added for recrystallization to obtain 15.5g of a product (namely a compound 3-1). The yield is 96.0 percent

(b) Synthesis of Compound 3-2: 9g (0.04mol) of Compound 3-1 was dissolved in 100ml of methanol, and 10% palladium on carbon (0.9g) was added to the solution, followed by reaction at room temperature overnight using hydrogen as a reducing agent. After the reaction is finished, the palladium-carbon is removed by suction filtration, and after the filtrate is dried by spinning, petroleum ether is added for washing for 3-5 times to obtain 7.1g of a product (namely the compound 3-2). The yield thereof is 91.7%

(c) Synthesis of Compounds 3-3: 7g (0.036mol) of Compound 3-2 was dissolved in 100ml of methanol, and then 1.6g (0.040mol) of solid sodium hydroxide was added, and the reaction solution was stirred vigorously at room temperature for 30 minutes. After the reaction, a small amount of saturated ammonium chloride solution was added to neutralize sodium hydroxide, a large amount of solid precipitated at this time, and the reaction solution was filtered, and the obtained solid was washed with ethyl acetate to obtain 4.5g of the product (i.e., compound 3-3). The yield is 74.5 percent

(d) Synthesis of Compounds 3-4: 1.0g (1.397mmol) of the compound 3-2, 0.2g (1.270mmol) of the compound 1-6, and 1.0g of the activated 3Ao molecular sieve were added to 40ml of anhydrous methanol, and then the reaction solution was heated to reflux for 36 hours. After the reaction is finished, the molecular sieve is removed, part of methanol is removed by spinning, and a large amount of solid is separated out after the methanol is cooled. Then, a solid was obtained by suction filtration, and the solid was washed three times with methanol to obtain 0.5g of a product (i.e., compound 3-4). The yield thereof was found to be 51.3%.

(e) Synthesis of compound 4(Cpd 4): 475.8mg (0.550mmol) of Compound 3-4, 122.0mg (1.0mmol) of 4-dimethylaminopyridine and 1.0g (0.5mmol) of methoxypolyethyleneglycolamine were dissolved in 20ml of anhydrous dichloromethane, and 412.0mg (2.0mmol) of dicyclohexylcarbodiimide was added to the mixture in an ice-water bath and reacted at room temperature overnight. After the reaction, the reaction solution was filtered to remove a small amount of precipitate formed by the reaction, the filtrate was concentrated and added with a suitable amount of diethyl ether, at which time a large amount of flocculent precipitate was precipitated, the mixture was centrifuged to remove the supernatant, and the precipitate was washed with diethyl ether 3-5 times to obtain 0.8g of the product (i.e., Cpd 4). The yield thereof was found to be 57.4%.¹H NMR(400MHz,CDCl₃)δ0.65(s,3H),0.84-0.86(m,6H),0.89-0.90(m,3H),0.98(s,4H),1.05-1.60(m,20H),1.82-1.85(m,10H),2.17-2.22(m,1H),2.32-2.36(m,1H),3.37(s,3H),3.45-3.63(m,227H),4.01-4.04(m,2H),4.50(s,2H),5.32-5.33(m,1H),6.94-7.21(m,5H),7.48-7.53(m,4H),7.76(d,J＝7.2Hz,3H),8.36(d,J＝8.4Hz,1H),8.79(s,1H).

Example 1.4 Synthesis of Cpd1

The synthesis was carried out according to the methods of reference example 1.1 and example 1.2.¹H NMR(400MHz,CDCl₃)δ0.64(s,3H),0.83-0.85(m,6H),0.88-0.89(m,3H),0.98(s,4H),1.05-1.53(m,22H),1.60-1.75(m,6H),1.80-1.98(m,8H),2.85-2.90(m,1H),3.37(s,3H),3.46-3.81(m,219H),4.49-4.56(m,1H),5.05-5.12(m,2H),6.39(s,1H),7.33(s,1H),7.75(s,3H),7.75(s,1H),8.65(s,1H).

Example 1.5 Synthesis of Cpd3

The synthesis was carried out according to the method of example 1.1.¹H NMR(400MHz,CDCl₃)δ0.65(s,3H),0.83-0.85(m,6H),0.89(d,J＝6.8Hz,3H),0.97(s,4H),1.05-1.53(m,20H),1.77-1.99(m,6H),2.17(s,4H),2.32-2.36(m,1H),3.10-3.14(m,1H),3.36(s,3H),3.45-3.67(m,183H),4.02(t,J＝6.4Hz,2H),5.31-5.32(m,1H),6.96-6.99(m,2H),7.05(d,J＝2.0Hz,1H),7.22-7.26(m,3H),7.47-7.54(m,3H),7.75-7.77(m,2H),7.86(d,J＝8.4Hz,2H),8.35(d,J＝8.8Hz,1H),8.77(s,1H).

Example 1.6 Synthesis of Cpd5

The synthesis was carried out according to the method of example 1.3.¹H NMR(400MHz,CDCl₃)δ0.64(s,3H),0.84(d,J＝6.4Hz,6H),0.88(d,J＝6.4Hz,3H),0.97(s,4H),1.09-1.59(m,21H),1.81-1.99(m,6H),2.17-2.35(m,5H),3.08-3.13(m,1H),3.35(s,3H),3.43-3.79(m,213H),4.02(t,J＝6.4Hz,2H),5.31-5.32(m,1H),6.96-7.07(m,6H),7.25(s,3H),7.46-7.53(m,3H),7.76-7.81(m,3H),8.36(d,J＝8.8Hz,1H),8.80(s,1H).

The compounds of the present invention and their structures are shown in Table 1

TABLE 1 Compounds responsive to release by endocytosis

Comparative example 1Cpd7 Synthesis

The present invention synthesizes compound Cpd7 with pH-responsive groups but which does not allow efficient transfection of intracellular genes.

The synthetic route of Cpd7 is as follows:

cpd7 validation:¹H NMR(400MHz,CDCl₃)δ0.62(s,3H),0.81-0.82(m,6H),0.86-0.87(m,3H),0.95(s,54H),1.07-1.55(m,23H),1.78-1.85(m,5H),2.28(s,3H),2.42(s,3H),3.15(s,2H),3.33(s,3H),3.43-3.66(m,187H),4.43(s,1H),4.75(s,1H),5.26-5.31(m,2H),5.66(s,1H),8.00(s,1H).

comparative example 2

Comparative example 2 synthesized according to the invention (Cpd8)

Synthetic reference example 1.1 of Cpd8¹H NMR(400MHz,CDCl₃)δ0.65(s,3H),0.83-0.85(m,6H),0.89(d,J＝6.4Hz,3H),0.97(s,4H),1.04-1.60(m,25H),1.79-1.85(m,4H),1.92-1.99(m,2H),2.14(s,1H),3.08-3.14(m,1H),3.35(s,3H),3.36-3.62(m,193H),3.99(t,J＝6.8Hz,2H),5.31-5.32(m,1H),6.50(s,1H),6.89-6.91(m,1H),6.99-7.00(m,1H),7.38-7.46(m,3H),7.68(d,J＝8.4Hz,1H),7.87-7.89(dd,1H).

Test example 1 degradation Rate

The degradation rate of the synthesized compound under the in vitro condition is simulated, and the degradation rates of the synthesized compound with different structures under different buffer system conditions and different time points are considered.

Separately weighing a certain amount of each of the compounds (Cpd1-5 and Cpd7-8), and adding a certain amount of buffer (NaH at pH 5/7.4) to each of the compounds₂PO₃/Na₂HPO₃Buffer salt) to a final concentration of 10mg/ml, the mixture was placed in a shaker and shaken at 37 ℃ and a rotation speed r of 350rpm/min for 2h and 8h, respectively. The reaction solution was then transferred to a lyophilization vessel for vacuum lyophilization.

After 24h of freeze-drying, 0.5ml of deuterated chloroform is added into each group, the freeze-dried substance is redissolved and transferred into a 1.5ml pointed-bottom centrifuge tube, the centrifuge tube is centrifuged for 5min at the rotating speed r of 15000r/min, the supernatant liquid is absorbed and transferred into a nuclear magnetic tube, and the degradation rate is quantified by hydrogen spectrum, and the result is shown in figures 1A and 1B.

As seen from fig. 1A and 1B, Cpd1, Cpd2, and Cpd7 compounds all degraded completely within 2h at pH 5, and it is concluded that they can achieve rapid degradation in the acidic environment (around pH 5) in the cytosol, thereby exposing the positive charge of the cationic lipid complex and effectively releasing nucleic acids. Even though complete degradation at pH 5 at Cpd3, Cpd4, Cpd5 was not achieved, it was concluded that it is likely that the positive charge of the cationic lipid complex would not be exposed in time in the internal environment and thus effective release of nucleic acid would not be achieved.

When the pH of the compounds Cpd1 and Cpd7 is 7.4, Cpd1 and Cpd7 are degraded by more than 60% in 2h, while Cpd2 is degraded by less than 10%, considering that the concentration of the modified compound is low when the compound is administered, Cpd1 is likely to be completely degraded when the compound does not enter cells, the effect of shielding cationic positive charges by hydrophilic groups cannot be realized, and efficient transfection of nucleic acid cannot be realized.

Therefore, from the results of in vitro degradation experiments, it can be seen that the compounds of the present invention, especially compound Cpd2, are relatively stable under experimental conditions (pH 7.4) that mimic humoral conditions, but degrade under experimental conditions (pH 5 or so) that mimic conditions such as tumor microenvironment or lysosome of cells, with the greatest potential for achieving efficient gene transfection in cells.

Test example 2 transfection experiment

2.1 preparation of the Complex

(1) Preparing a cationic blank liposome: placing appropriate amount of DOTAP/Chol chloroform stock solution in 25ml eggplant-shaped bottle to make DOTAP/Chol molar ratio be 1:1, total lipid concentration be 2mg/ml, total lipid 4mg, rotary-steaming in rotary evaporator under reduced pressure for 30min, and N₂Blowing to dry the residual chloroform, adding 2ml of 10mM Hepes buffer salt solution, and carrying out ultrasonic treatment for 30min to obtain the cationic blank liposome. The particle diameter of the particle is 153.2nm, the polydispersity is 0.212 and the potential is 50.4mV, which are measured by a Malvern laser particle size analyzer.

(2) Preparation of cationic lipid/nucleic acid complexes: taking 100 microliters of the cationic blank liposome, taking a proper amount of PGL3(Luciferase) plasmid stock solution, diluting to 100 microliters, enabling the N/P ratio to be 4/1, quickly and uniformly mixing, and incubating at the constant temperature of 37 ℃ for 0.5h to obtain the cationic lipid/nucleic acid compound. The particle diameter of the particle is 180.6nm, the polydispersity is 0.159 and the potential is 45.9 mV.

(3) preparation of pH-responsive cationic lipid/nucleic acid complexes or PEG-modified cationic lipid/nucleic acid complexes: taking a proper amount of prepared cationic lipid/nucleic acid complex, carrying out lipid post-insertion according to the molar ratio of DOTAP: Chol: post-insertion lipid (namely Cpd.2 or Cpd.8) to 1:1:1, rapidly and uniformly mixing a post-insertion lipid solution and a cationic lipid/nucleic acid complex solution, and incubating at the constant temperature of 37 ℃ for 0.5 h. The post-insert lipid groups are respectively a negative control group Cpd8 and an experimental group Cpd.2, and the post-insert conditions are 37 ℃ and 0.5 h. The particle sizes are all less than 250nm, the PDI is less than 0.3, the potential negative group is 20.0mV, and the experimental group is 21.2 mV.

2.2 cell transfection assay

Culturing non-small cell lung cancer cell A549 to 85% of the cell length in culture bottle, counting the number of the digested cells at 1 × 10⁴Density of Individual cells/well cells were seeded in 96-well plates in a 37 ℃ incubator with 0.5% CO₂Culturing for 18-24h, administering DNA 375ng per well, diluting with 1640 culture medium and 20% FBS-containing 1640 culture medium, diluting and administering for 4h, discarding the culture medium, washing with 1 × PBS, and culturing for 24h by replacing with 10% FBS-containing 1640 culture medium. The medium was discarded, washed with 1 × PBS, 30 μ l of cell lysate was added to each well, after sufficient lysis, 20 μ l of cell lysate was taken out of each well and added to a flow tube, an equal amount of luciferase substrate was temporarily added before measurement, and the mixture was mixed, and the fluorescence intensity was measured by a luminometer, and the results are shown in fig. 2.

As seen in FIG. 2, transfection of the Cpd 8-modified negative control group (DOTAP + Cpd8) was performed without serum (1640) administration<1000, consistent with the group not administered (untreated group), considered untransfected; the unmodified cationic lipidome (DOTAP set, i.e.the cationic lipid/nucleic acid complex prepared in 2.1) was transfected at 1X 10⁶Transfection results for the Cpd 2-modified lipid Complex group (DOTAP + Cpd2 group) were 4X 10⁶The transfection results of the DOTAP + Cpd2 group were about 4 times that of the DOTAP group.

20% FBS was designed to mimic the internal environment of the humoral circulation in vivo, and when 20% FBS was administered, the negative control group DOTAP + Cpd8 remained untransfected, whereas the unmodified cationic DOTAP group showed a significant decrease in transfection efficiency, which was only 6X 10³However, the transfection results of the group of lipid complexes DOTAP + Cpd2 modified by Cpd2 were 1.2X 10⁵The transfection results of the DOTAP + Cpd2 group were about 20 times higher than those of the DOTAP group.

Therefore, in combination with serum-free 1640 transfection and 20% FBS transfection mimicking an in vivo environment, it can be seen that modification of the cationic lipid complex with the compound of the present invention (Cpd2) achieves a relatively stable in vivo environment (pH 7.4) and a rapid degradation in the intracellular environment (pH 5), thereby achieving an effective transfection in vivo.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.