WO2023274265A1 - Benzamide compound and use thereof - Google Patents

Abstract

Provided are a benzamide compound and a use thereof. Specifically, provided is a compound represented by formula I or a salt thereof, wherein R¹ to R⁷, M¹ to M⁶, m1 to m3, and n1 to n6 are as defined herein.

Description

Benzamides and their uses technical field

The disclosure belongs to the field of medicine, and relates to benzamide compounds and applications thereof.

Background technique

Nucleic acid-based drugs, such as messenger RNA (mRNA), antisense oligonucleotides, small interfering RNA (siRNA), plasmids, etc., have broad application prospects. How to safely and effectively deliver them to target organs and target cells in vivo is a major constraint Problems of technological development.

At present, the nucleic acid drug delivery system can be divided into two categories: viral vector system and non-viral system. Liposome-mediated nucleic acid drug delivery is the main method belonging to the non-viral delivery system.

In gene therapy applications, lipid nanoparticles have been demonstrated as delivery vehicles for nucleic acid drugs. Lipid nanoparticles formed from cationic lipids and other co-lipids such as cholesterol, phospholipids, and PEGylated lipids encapsulate nucleic acids, protecting them from degradation and facilitating cellular uptake. In addition, the delivery of liposomal nanoparticles for bioactive components has other advantages, such as good targeting, less side effects, good stability, and high transfection efficiency.

With the rapid development of mRNA-based therapies, such as vaccines, gene therapy, and protein replacement therapy, there is a huge demand for mRNA delivery systems. Therefore, the development of efficient and safe mRNA delivery systems is effective for diseases based on mRNA and other nucleic acid drugs, including The treatment of diseases such as preventive diseases, genetic diseases and tumors is of great significance.

Contents of the invention

The disclosure (The disclosure) provides the compound shown in formula I or its salt

Wherein, M ¹ to M ⁶ are each independently selected from a bond, -C(O)O-a1 and -OC(O)-a1, and a1 is a combination with R ¹ , R ² , R ³ , R ⁴ , R ⁵ or R ⁶ connected bonds, and M ¹ to M ⁶ are not all -C(O)O-a1 or bonds;

R ¹ to R ⁶ are each independently substituted or unsubstituted alkyl or substituted or unsubstituted alkenyl;

R ⁷ are each independently hydrogen or substituted or unsubstituted C _1-6 alkyl;

m1 to m3 are each independently selected from 1, 2, 3, 4, 5, 6, 7 and 8;

n1 to n6 are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10.

In some embodiments, in the compound represented by formula I or a salt thereof, R ¹ to R ⁶ are each independently unsubstituted alkyl or unsubstituted alkenyl.

In some embodiments, in the compound represented by formula I or a salt thereof, R ¹ to R ⁶ are each independently selected from C ₄ -C ₁₄ alkyl or C ₄ -C ₁₄ alkenyl.

In some embodiments, in the compound represented by formula I or its salt, R ¹ is C ₄ -C ₁₄ alkyl (including but not limited to C ₄ alkyl, C ₅ alkyl, C ₆ alkyl, C ₇ alkyl, C ₈ alkyl, C ₉ alkyl, C ₁₀ alkyl, C ₁₁ alkyl, C ₁₂ alkyl, C ₁₃ alkyl, C ₁₄ alkyl). In other embodiments, in the compound represented by formula I or a salt thereof, R ¹ is C ₅ -C ₁₂ alkyl.

In some embodiments, in the compound represented by formula I or its salt, R ² is C ₄ -C ₁₄ alkyl (including but not limited to C ₄ alkyl, C ₅ alkyl, C ₆ alkyl, C ₇ alkyl, C ₈ alkyl, C ₉ alkyl, C ₁₀ alkyl, C ₁₁ alkyl, C ₁₂ alkyl, C ₁₃ alkyl, C ₁₄ alkyl).

In other embodiments, in the compound represented by formula I or a salt thereof, R ² is a C ₅ -C ₁₂ alkyl group.

In some embodiments, in the compound represented by formula I or its salt, R ³ is C ₄ -C ₁₄ alkyl (including but not limited to C ₄ alkyl, C ₅ alkyl, C ₆ alkyl, C ₇ alkyl, C ₈ alkyl, C ₉ alkyl, C ₁₀ alkyl, C ₁₁ alkyl, C ₁₂ alkyl, C ₁₃ alkyl, C ₁₄ alkyl). In other embodiments, in the compound represented by formula I or a salt thereof, R ³ is a C ₅ -C ₁₂ alkyl group.

In some embodiments, in the compound represented by formula I or its salt, R ⁴ is C ₄ -C ₁₄ alkyl (including but not limited to C ₄ alkyl, C ₅ alkyl, C ₆ alkyl, C ₇ alkyl, C ₈ alkyl, C ₉ alkyl, C ₁₀ alkyl, C ₁₁ alkyl, C ₁₂ alkyl, C ₁₃ alkyl, C ₁₄ alkyl).

In other embodiments, in the compound represented by formula I or a salt thereof, R ⁴ is a C ₅ -C ₁₂ alkyl group.

In some embodiments, in the compound represented by formula I or its salt, R ⁵ is C ₄ -C ₁₄ alkyl (including but not limited to C ₄ alkyl, C ₅ alkyl, C ₆ alkyl, C ₇ alkyl, C ₈ alkyl, C ₉ alkyl, C ₁₀ alkyl, C ₁₁ alkyl, C ₁₂ alkyl, C ₁₃ alkyl, C ₁₄ alkyl). In other embodiments, in the compound represented by formula I or its salt, R ⁵ is C ₅ -C ₁₂ alkyl.

In some embodiments, in the compound represented by formula I or its salt, R ⁶ is C ₄ -C ₁₄ alkyl (including but not limited to C ₄ alkyl, C ₅ alkyl, C ₆ alkyl, C ₇ alkyl, C ₈ alkyl, C ₉ alkyl, C ₁₀ alkyl, C ₁₁ alkyl, C ₁₂ alkyl, C ₁₃ alkyl, C ₁₄ alkyl). In other embodiments, in the compound represented by formula I or a salt thereof, R ⁶ is a C ₅ -C ₁₂ alkyl group.

In other embodiments, in the compound represented by formula I or its salt, R ¹ is C ₄ -C ₁₄ alkenyl (including but not limited to C ₄ alkenyl, C ₅ alkenyl, C ₆ alkenyl, C ₇ alkenyl, C ₈ alkenyl, C ₉ alkenyl, C ₁₀ alkenyl, C ₁₁ alkenyl, C ₁₂ alkenyl, C ₁₃ alkenyl, C ₁₄ alkenyl). In other embodiments, in the compound represented by formula I or a salt thereof, R ¹ is C ₅ -C ₁₂ alkenyl.

In some embodiments, in the compound represented by formula I or its salt, R ² is C ₄ -C ₁₄ alkenyl (including but not limited to C ₄ alkenyl, C ₅ alkenyl, C ₆ alkenyl, C ₇ alkenyl, C ₈ alkenyl, C ₉ alkenyl, C ₁₀ alkenyl, C ₁₁ alkenyl, C ₁₂ alkenyl, C ₁₃ alkenyl, C ₁₄ alkenyl). In other embodiments, in the compound represented by formula I or a salt thereof, R ² is C ₅ -C ₁₂ alkenyl.

In some embodiments, in the compound represented by formula I or its salt, R ³ is C ₄ -C ₁₄ alkenyl (including but not limited to C ₄ alkenyl, C ₅ alkenyl, C ₆ alkenyl, C ₇ alkenyl, C ₈ alkenyl, C ₉ alkenyl, C ₁₀ alkenyl, C ₁₁ alkenyl, C ₁₂ alkenyl, C ₁₃ alkenyl, C ₁₄ alkenyl). In other embodiments, in the compound represented by formula I or a salt thereof, R ³ is C ₅ -C ₁₂ alkenyl.

In some embodiments, in the compound represented by formula I or its salt, R ⁴ is C ₄ -C ₁₄ alkenyl (including but not limited to C ₄ alkenyl, C ₅ alkenyl, C ₆ alkenyl, C ₇ alkenyl, C ₈ alkenyl, C ₉ alkenyl, C ₁₀ alkenyl, C ₁₁ alkenyl, C ₁₂ alkenyl, C ₁₃ alkenyl, C ₁₄ alkenyl). In other embodiments, in the compound represented by formula I or a salt thereof, R ⁴ is C ₅ -C ₁₂ alkenyl.

In some embodiments, in the compound represented by formula I or its salt, R ⁵ is C ₄ -C ₁₄ alkenyl (including but not limited to C ₄ alkenyl, C ₅ alkenyl, C ₆ alkenyl, C ₇ alkenyl, C ₈ alkenyl, C ₉ alkenyl, C ₁₀ alkenyl, C ₁₁ alkenyl, C ₁₂ alkenyl, C ₁₃ alkenyl, C ₁₄ alkenyl). In other embodiments, in the compound represented by formula I or a salt thereof, R ⁵ is C ₅ -C ₁₂ alkenyl.

In some embodiments, in the compound represented by formula I or its salt, R ⁶ is C ₄ -C ₁₄ alkenyl (including but not limited to C ₄ alkenyl, C ₅ alkenyl, C ₆ alkenyl, C ₇ alkenyl, C ₈ alkenyl, C ₉ alkenyl, C ₁₀ alkenyl, C ₁₁ alkenyl, C ₁₂ alkenyl, C ₁₃ alkenyl, C ₁₄ alkenyl). In other embodiments, in the compound represented by formula I or a salt thereof, R ⁶ is C ₅ -C ₁₂ alkenyl.

Further, in some embodiments, in the compound represented by formula I or a salt thereof, R ⁵ is C ₄ -C ₁₄ alkenyl, and R ⁶ is C ₄ -C ₁₄ alkenyl.

In some embodiments, in the compound represented by formula I or a salt thereof, R ³ is C ₄ -C ₁₄ alkenyl, and R ⁴ is C ₄ -C ₁₄ alkenyl.

In some embodiments, in the compound represented by formula I or a salt thereof, R ¹ is C ₄ -C ₁₄ alkenyl, and R ² is C ₄ -C ₁₄ alkenyl.

Further, in some embodiments, in the compound represented by formula I or a salt thereof, R ¹ is a C ₄ -C ₁₄ alkyl group, and R ² is a C ₄ -C ₁₄ alkyl group.

In some embodiments, in the compound represented by formula I or a salt thereof, R ³ is C ₄ -C ₁₄ alkyl, and R ⁴ is C ₄ -C ₁₄ alkyl.

In some embodiments, in the compound represented by formula I or a salt thereof, R ⁵ is a C ₄ -C ₁₄ alkyl group, and R ⁶ is a C ₄ -C ₁₄ alkyl group.

On the other hand, some embodiments provide that in the compound represented by formula I or a salt thereof, R ¹ to R ⁶ are each independently C _4-15 alkyl or alkenyl. Other embodiments provide that in the compound represented by formula I or a salt thereof, R ¹ to R ⁶ are each independently C _8-16 alkyl or alkenyl.

In some embodiments, in the compound represented by formula I or a salt thereof, R ¹ is C _4-15 alkyl or C _8-16 alkyl.

In some embodiments, in the compound represented by formula I or a salt thereof, R ² is C _4-15 alkyl or C _8-16 alkyl.

In some embodiments, in the compound represented by formula I or a salt thereof, R ³ is C _4-15 alkyl or C _8-16 alkyl.

In some embodiments, in the compound represented by formula I or its salt, R ⁴ is C _4-15 alkyl or C _8-16 alkyl.

In some embodiments, in the compound represented by formula I or a salt thereof, R ⁵ is C _4-15 alkyl or C _8-16 alkyl.

In some embodiments, in the compound represented by formula I or a salt thereof, R ⁶ is C _4-15 alkyl or C _8-16 alkyl.

In some embodiments, in the compound represented by formula I or a salt thereof, R ¹ to R ⁶ are C _4-15 alkyl or C _8-16 alkyl.

In some embodiments, in the compound represented by formula I or a salt thereof, R ¹ is C _4-15 alkenyl or C _8-16 alkenyl.

In some embodiments, in the compound represented by formula I or a salt thereof, R ² is C _4-15 alkenyl or C _8-16 alkenyl.

In some embodiments, in the compound represented by formula I or a salt thereof, R ³ is C _4-15 alkenyl or C _8-16 alkenyl.

In some embodiments, in the compound represented by formula I or a salt thereof, R ⁴ is C _4-15 alkenyl or C _8-16 alkenyl.

In some embodiments, in the compound represented by formula I or a salt thereof, R ⁵ is C _4-15 alkenyl or C _8-16 alkenyl.

In some embodiments, in the compound represented by formula I or a salt thereof, R ⁶ is C _4-15 alkenyl or C _8-16 alkenyl.

In some embodiments, in the compound represented by formula I or a salt thereof, R ¹ to R ⁶ are C _4-15 alkenyl or C _8-16 alkenyl.

On the other hand, in the compound represented by formula I or a salt thereof provided by some embodiments, R ⁷ is hydrogen or optionally substituted C _1-3 alkyl. In some embodiments, in the compound represented by formula I or a salt thereof, R ⁷ is hydrogen, methyl or ethyl.

In some embodiments, M ¹ to M ⁶ in the compound represented by formula I or its salt are each independently -OC(O)-a1, and a1 is the combination of R ¹ , R ² , R ³ , R ⁴ , R ⁵ or R ⁶ is connected to the bond.

In other embodiments, M ¹ and M ² in the compound represented by formula I or a salt thereof are each independently -C(O)O-a1, and a1 is a bond connecting R ¹ or R ² .

In other embodiments, M ¹ , M ² , M ³ , and M ⁴ in the compound represented by formula I or its salt are each independently -C(O)O-a1, and a1 is the same as R ¹ , R ² , R ³ or R ⁴ bonded.

On the other hand, in some embodiments, in the compound represented by formula I or a salt thereof, M ¹ , M ² , M ³ and M ⁴ are bonds.

In other embodiments, M ⁵ and M ⁶ are bonds in the compound represented by formula I or a salt thereof.

Further, m1 to m3 in the compound represented by formula I or the salt thereof provided by some embodiments are each independently selected from 2, 3 and 4.

In some embodiments, in the compound represented by formula I or a salt thereof, R ¹ to R ⁶ are independently C ₄ -C ₁₄ alkyl or C ₄ -C ₁₄ alkenyl.

In some embodiments, in the compound shown in formula I or its salt, R ¹ or R ² are independently selected from

In some embodiments, n1 to n6 in the compound represented by formula I or a salt thereof are each independently selected from 1, 2, 3, 4 and 5.

In some embodiments, n1 to n6 in the compound represented by formula I or a salt thereof are 3 independently.

In some embodiments, n1 to n6 in the compound represented by formula I or a salt thereof are 4 independently.

In some embodiments, n1 to n6 in the compound represented by formula I or a salt thereof are 5 independently.

In some embodiments, in the compound represented by formula I or a salt thereof, n1 to n6 are each independently selected from 6, 7, 8, 9 and 10.

In some embodiments, n1 to n6 in the compound represented by formula I or a salt thereof are each independently 7.

In some embodiments, n1 to n6 in the compound represented by formula I or a salt thereof are each independently 8.

In some embodiments, n1 to n6 in the compound represented by formula I or a salt thereof are each independently 9.

On the other hand, the compound represented by formula I or its salt in the present disclosure is the compound represented by formula II or its salt,

其中R ¹至R ⁷、m1至m3、n1至n6如式I化合物或其盐中定义。

Wherein R ¹ to R ⁷ , m1 to m3, n1 to n6 are as defined in the compound of formula I or a salt thereof.

In some embodiments, in the compound represented by formula I or formula II or salt thereof, n1 or n2 are each independently selected from 6, 7, 8, 9 and 10.

In some embodiments, in the compound shown in formula II or its salt, R ¹ or R ² are independently selected from

In some embodiments, in the compound represented by formula I or II or a salt thereof, R ⁷ is independently hydrogen.

In some embodiments, in the compound represented by formula II or a salt thereof, m1 to m3 are independently selected from 2, 3 and 4. On the other hand, the compound represented by formula I or its salt in the present disclosure is the compound represented by formula III or its salt

其中R ¹至R ⁷、m1至m3、n1至n6如式I化合物或其盐中定义。

Wherein R ¹ to R ⁷ , m1 to m3, n1 to n6 are as defined in the compound of formula I or a salt thereof.

In some embodiments, in the compound represented by formula III or a salt thereof, R ⁷ is independently hydrogen.

In some embodiments, in the compound represented by formula III or a salt thereof, m1 to m3 are independently selected from 2, 3 and 4.

In some embodiments, n1 to n6 in the compound represented by formula III or a salt thereof are each independently selected from 6, 7, 8, 9 and 10.

In some embodiments, in the compound shown in formula III or its salt, R ¹ to R ⁶ are independently selected from

In some embodiments, in the compound shown in formula III or its salt, R ¹ to R ⁶ are independently

Typical compounds shown in formula I or pharmaceutically acceptable salts thereof include but are not limited to:

The present disclosure also provides an isotopic substitution of the aforementioned compound or a salt thereof, preferably, the isotopic substitution is a deuterium atom substitution.

The present disclosure also provides a lipid particle comprising the aforementioned compound or a salt thereof, or an isotope substitution. Further, in some embodiments, the lipid particle further comprises an active agent, and the active agent is preferably an immunostimulatory oligonucleotide, siRNA, antisense oligonucleotide, mRNA, or a plasmid.

The present disclosure also provides a pharmaceutical composition comprising the aforementioned lipid particles and a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition contains 0.01%-99.99% of pharmaceutically acceptable excipients based on the total weight of the composition. In certain embodiments, the pharmaceutical composition contains 0.1%-99.9% of pharmaceutically acceptable excipients. In certain embodiments, the pharmaceutical composition contains 0.5%-99.5% of pharmaceutically acceptable excipients. In certain embodiments, the pharmaceutical composition contains 1%-99% of pharmaceutically acceptable excipients. In certain embodiments, the pharmaceutical composition contains 2%-98% of pharmaceutically acceptable excipients.

The present disclosure also provides a use of the aforementioned compound or a salt thereof, or an isotope substitution, or the aforementioned lipid particle, or the aforementioned pharmaceutical composition in the preparation of a medicament for inducing an immune response in a subject.

The present disclosure also provides the aforementioned compound or its salt, or isotope substitution, or the aforementioned lipid particle, or the aforementioned pharmaceutical composition in the preparation of medicines for preventing and/or treating diseases or disorders related to polypeptide overexpression the use of.

The present disclosure also provides the aforementioned compound or its salt, or isotope substitution, or the aforementioned lipid particle, or the aforementioned pharmaceutical composition in the preparation of a medicament for preventing and/or treating diseases or conditions related to insufficient expression of polypeptides the use of.

In some embodiments, diseases or conditions described in the present disclosure include, but are not limited to, cancer, infection, autoimmune disease, neurodegenerative disease, and inflammation, such as COVID-19.

The present disclosure also provides a method for inducing an immune response in a subject, comprising administering to the patient the aforementioned compound or a salt thereof, or the aforementioned lipid particle, or the aforementioned pharmaceutical composition.

The present disclosure also provides a method for preventing and/or treating diseases or disorders related to polypeptide overexpression, comprising administering to the patient the aforementioned compound or its salt, or the aforementioned lipid particle, or the aforementioned pharmaceutical composition.

The present disclosure also provides a method for preventing and/or treating a disease or condition related to insufficient expression of a polypeptide, comprising administering to the patient the aforementioned compound or its salt, or the aforementioned lipid particle, or the aforementioned pharmaceutical composition.

In another aspect, the present disclosure also provides the aforementioned compound or salt thereof, or the aforementioned lipid particle, or the aforementioned pharmaceutical composition for inducing an immune response in a subject.

The present disclosure also provides the aforementioned compounds or salts thereof, or the aforementioned lipid particles, or the aforementioned pharmaceutical compositions for preventing and/or treating diseases or disorders related to polypeptide overexpression.

The present disclosure also provides the aforementioned compounds or salts thereof, or the aforementioned lipid particles, or the aforementioned pharmaceutical compositions for preventing and/or treating diseases or conditions associated with insufficient expression of polypeptides.

Salts of compounds described in this disclosure include "acid" addition salts and "base" addition salts. For example, salts formed by acid-base reactions with basic groups (amino groups), including organic or inorganic acids. In addition, the compound salts also include salts formed by quaternization with basic groups (amino groups), and the quaternization reagents include linear or branched chlorinated hydrocarbons.

Compounds of the present disclosure may exist in particular geometric or stereoisomeric forms. This disclosure contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers isomers, (D)-isomers, (L)-isomers, and their racemic and other mixtures, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of this disclosure. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers, as well as mixtures thereof, are included within the scope of this disclosure. Compounds of the present disclosure containing asymmetric carbon atoms can be isolated in optically pure or racemic forms. Optically pure forms can be resolved from racemic mixtures or synthesized by using chiral starting materials or reagents.

Optically active (R)- and (S)-isomers as well as D and L-isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the present disclosure is desired, it can be prepared by asymmetric synthesis or derivatization with chiral auxiliary agents, wherein the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), a diastereoisomeric salt is formed with an appropriate optically active acid or base, and then a diastereomeric salt is formed by a conventional method known in the art. Diastereomeric resolution is performed and the pure enantiomers are recovered. Furthermore, the separation of enantiomers and diastereomers is usually accomplished by the use of chromatography using chiral stationary phases, optionally in combination with chemical derivatization methods (e.g. amines to amino groups formate).

表示未指定构型，即如果化学结构中存在手性异构体，键

可以为

或

或者同时包含

和

两种构型。本公开所述化合物的化学结构中，键

并未指定构型，即可以为Z构型或E构型，或者同时包含两种构型。 In the chemical structures of the compounds described in this disclosure, the bond

Indicates unassigned configuration, i.e. if chiral isomers exist in the chemical structure, the bond

can be

or

or both

with

Two configurations. In the chemical structures of the compounds described in this disclosure, the bond

If the configuration is not specified, it can be the Z configuration or the E configuration, or both configurations.

The compounds and intermediates of the present disclosure may also exist in different tautomeric forms and all such forms are included within the scope of the present disclosure. The term "tautomer" or "tautomeric form" refers to structural isomers of different energies that can interconvert via a low energy barrier. For example, proton tautomers (also known as prototropic tautomers) include interconversions via migration of a proton, such as keto-enol and imine-enamine, lactam-lactim isomerization . An example of a lactam-lactim equilibrium is between A and B as shown below.

All compounds in this disclosure can be drawn as Form A or Form B. All tautomeric forms are within the scope of the present disclosure. The naming of compounds does not exclude any tautomers.

The present disclosure also includes certain isotopically labeled compounds of the disclosure that are identical to those described herein, but wherein one or more atoms are replaced by an atom of an atomic mass or mass number different from that normally found in nature. Examples of isotopes that can be incorporated into compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as ² H, ³ H, ¹¹ C, ¹³ C, ¹⁴ C, ¹³ N, ¹⁵ N, ¹⁵ O, ¹⁷ O, ¹⁸ O, ³¹ P, ³² P, ³⁵ S, ¹⁸ F, ¹²³ I, ¹²⁵ I and ³⁶ Cl, etc.

Unless otherwise stated, when a position is specifically designated as deuterium (D), the position is understood to have an abundance of deuterium (i.e., at least 10 % deuterium incorporation). Exemplary compounds having a natural abundance greater than deuterium can be at least 1000 times more abundant deuterium, at least 2000 times more abundant deuterium, at least 3000 times more abundant deuterium, at least 4000 times more abundant deuterium, at least 5000 times more abundant deuterium, at least 6000 times more abundant deuterium, or more abundant deuterium. The present disclosure also includes various deuterated forms of compounds of formula (I). Each available hydrogen atom attached to a carbon atom can be independently replaced by a deuterium atom. Those skilled in the art can refer to the relevant literature to synthesize the deuterated form of the compound of formula (I). Commercially available deuterated starting materials can be used in the preparation of deuterated forms of compounds of formula (I), or they can be synthesized using conventional techniques using deuterated reagents, including but not limited to deuterated borane, trideuterated Borane tetrahydrofuran solution, deuterated lithium aluminum hydride, deuterated ethyl iodide and deuterated methyl iodide, etc.

"Optionally" or "optionally" means that the subsequently described event or circumstance can but need not occur, and that the description includes instances where the event or circumstance occurs or does not occur. For example, "C _1-6 alkyl optionally substituted by halogen or cyano" means that halogen or cyano may but not necessarily exist, and this description includes the case where the alkyl is substituted by halogen or cyano and the alkyl is not substituted by halogen And the case of cyano substitution.

Explanation of terms:

"Pharmaceutical composition" means a mixture containing one or more compounds described herein, or a physiologically acceptable salt or prodrug thereof, and other chemical components, as well as other components such as physiologically acceptable carriers and excipients. agent. The purpose of the pharmaceutical composition is to promote the administration to the organism, facilitate the absorption of the active ingredient and thus exert biological activity.

"Pharmaceutically acceptable excipients" include, but are not limited to, any adjuvants, carriers, excipients, glidants, sweeteners, diluents, agent, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier.

An "effective amount" or "therapeutically effective amount" as used in the present disclosure includes an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition. An effective amount also means an amount sufficient to allow or facilitate diagnosis. Effective amounts for a particular patient or veterinary subject may vary depending on factors such as the condition being treated, the general health of the patient, the method, route and dosage of administration, and the severity of side effects. An effective amount may be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.

The terms "nucleic acid", "nucleic acid molecule", "oligonucleotide", "polynucleotide" and "nucleotide" are used interchangeably herein. The term refers to polymers of deoxyribonucleotides (DNA), ribonucleotides (RNA) and modified forms thereof, either as individual fragments or as components of larger constructs, linear or branched Stranded, single-stranded, double-stranded, triple-stranded or hybrid forms thereof. The term also includes RNA/DNA hybrids. A polynucleotide may include sense and antisense oligonucleotides or polynucleotide sequences of DNA or RNA. The DNA or RNA molecule can be, for example, but not limited to: complementary DNA (cDNA), genomic DNA, synthetic DNA, recombinant DNA or a hybrid thereof, or an RNA molecule such as, for example, mRNA, shRNA, siRNA, miRNA, anti- Sense RNA and the like. Each possibility represents a separate embodiment of the invention. The term also includes oligonucleotides comprising naturally occurring bases, sugars, and covalent internucleoside linkages, as well as oligonucleotides having non-naturally occurring portions that function like corresponding naturally occurring parts. The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.

The term "leukocyte" relates to white blood cells (WBC) produced and derived from pluripotent hematopoietic stem cells in the bone marrow. White blood cells have a nucleus, and based on their functional or physical properties, the types of white blood cells can be divided into five main types, including neutrophils, eosinophils, basophils, lymphocytes, and monocytes.

"Alkyl" refers to a saturated aliphatic hydrocarbon group including, but not limited to, straight and branched chain alkyl groups. In some embodiments, an alkyl group has 1-4 carbons, also known as C _1-4 alkyl. In some embodiments, an alkyl group has 10-22 carbons, also known as a C _10-22 alkyl. In some embodiments, an alkyl group has 4-22 carbons, also known as a _C4-22 alkyl. In some embodiments, an alkyl group has 4-15 carbons, also known as a C _4-15 alkyl. In some embodiments, the alkyl group has 8-16 carbons, also known as _C8-16 alkyl.

The alkyl group may be unsubstituted or replaced by one or more members selected from halogen, hydroxy, amino, oxo, alkoxycarbonyl, amido, alkylamido, dialkylamido, nitro, Amino, alkylamino, dialkylamino, carboxyl, thio, and thioalkyl groups are substituted. In some embodiments, the alkyl group is a straight chain alkyl. In some embodiments, the alkyl group is a branched chain alkyl.

"Alkenyl" means an unsaturated aliphatic hydrocarbon group and includes straight and branched chain alkenyl groups. In some embodiments, alkenyl groups have 1-4 carbons, also known as C _1-4 alkenyl. In some embodiments, alkenyl groups have 10-22 carbons, also known as C _10-22 alkenyl. In some embodiments, alkenyl groups have 4-22 carbons, also known as _C4-22 alkenyl. Exemplary alkenyl groups include vinyl, propenyl, n-butenyl, isobutenyl, 3-methylbut-2-enyl, n-pentenyl, heptenyl, octenyl, cyclohexyl-butenyl Alkenyl and Decenyl.

Alkenyl can be unsubstituted or replaced by one or more members selected from halogen, hydroxy, amino, oxo, alkoxycarbonyl, amido, alkylamido, dialkylamido, nitro, amino, Group substitution with alkylamino, dialkylamino, carboxy, thio and thioalkyl groups.

The term "hydroxyl" refers to a -OH group.

The term "halogen" refers to fluorine, chlorine, bromine or iodine.

The term "cyano" refers to _-NH2 .

The term "oxo" refers to a =O substituent.

"Substituted" means that one or more hydrogen atoms in a group, preferably up to 5, more preferably 1 to 3 hydrogen atoms are independently substituted by the corresponding number of substituents. It goes without saying that substituents are only in their possible chemical positions and that a person skilled in the art can determine (by experiment or theory) possible or impossible substitutions without undue effort.

Description of drawings

Figure 1: Comparison of intracellular mRNA luciferase expression after delivery of selected lipids (compounds 1-3 and comparative compounds 1 and 2).

Figure 2: Comparison of mRNA luciferase expression levels at injection sites in mice after delivery of selected lipids (compounds 1-3 and comparative compound 1).

Figure 3: Comparison of protein expression levels of growth factors injected intramuscularly in vivo after delivery of selected lipids (compounds 1, 3 and comparative compound 1).

Figure 4: Comparison of mRNA luciferase expression levels in the whole body of mice after delivery of selected lipids (compounds 1, 2 and comparative compound 1).

Figure 5: Comparison of mRNA luciferase expression levels in mouse lungs after delivery of selected lipids (compound 4 and comparative compound 1).

detailed description

The present disclosure is further described below in conjunction with examples, but these examples do not limit the scope of the present disclosure.

The experimental methods in the examples of the present disclosure that do not indicate specific conditions are usually in accordance with conventional conditions, or in accordance with the conditions suggested by the raw material or commodity manufacturers. Reagents without specific sources indicated are conventional reagents purchased in the market.

Compound structures were determined by nuclear magnetic resonance (NMR) or/and mass spectroscopy (MS). NMR shifts (δ) are given in units of 10 ^-6 (ppm). The determination of NMR is to use Bruker AVANCE-400 nuclear magnetic instrument, and measuring solvent is deuterated dimethyl sulfoxide (DMSO-d ₆ ), deuterated chloroform (CDCl ₃ ), deuterated methanol (Methanol-d ₄ ), internal standard is Tetramethylsilane (TMS).

The determination of HPLC uses Agilent1100 high pressure liquid chromatography, GAS15B DAD ultraviolet detector, Water Vbridge C18 150*4.6mm 5um chromatographic column.

The determination of MS uses Agilent6120 triple quadrupole mass spectrometer, G1315D DAD detector, Waters Xbridge C18 4.6*50mm, 5um chromatographic column, scanning in positive/negative ion mode, and the mass scanning range is 80-1200.

The thin-layer chromatography silica gel plate uses Yantai Huanghai HSGF254 silica gel plate, the specification of the thin-layer chromatography (TLC) silica gel plate is 0.2mm±0.03mm, and the specification of the thin-layer chromatography separation and purification product is 0.4mm-0.5mm.

The flash column purification system uses Combiflash Rf150 (TELEDYNE ISCO) or Isolara one (Biotage).

Forward column chromatography generally uses Yantai Huanghai silica gel 200-300 mesh or 300-400 mesh silica gel as the carrier, or Changzhou Santai pre-packed pre-packed ultra-pure normal-phase silica gel column (40-63μm, 60g, 24g, 40g, 120g or other specifications).

The known starting materials in this disclosure can be adopted or synthesized according to methods known in the art, or can be purchased from Shanghai Titan Technology, ABCR GmbH&Co.KG, Acros Organics, Aldrich Chemical Company, Shaoyuan Chemical Technology (Accela ChemBio Inc), Beide Pharmaceutical and other companies.

Unless otherwise specified in the examples, the reactions can all be carried out under a nitrogen atmosphere.

The nitrogen atmosphere means that the reaction bottle is connected to a nitrogen balloon with a volume of about 1 L.

The hydrogen atmosphere means that the reaction bottle is connected to a hydrogen balloon with a capacity of about 1L.

Hydrogen was produced by a QPH-1L hydrogen generator from Shanghai Quanpu Scientific Instrument Company.

Nitrogen atmosphere or hydrogenation atmosphere is usually evacuated and filled with nitrogen or hydrogen, and the operation is repeated 3 times.

Unless otherwise specified in the examples, the solution refers to an aqueous solution.

Unless otherwise specified in the examples, the reaction temperature is room temperature, which is 20°C to 30°C.

The monitoring of the reaction progress in the embodiment adopts thin-layer chromatography (TLC), the developing agent used in reaction, the eluent system of the eluent system of the column chromatography that purification compound adopts and the developing agent system of thin-layer chromatography, the volume of solvent The ratio is adjusted according to the polarity of the compound, and it can also be adjusted by adding a small amount of basic or acidic reagents such as triethylamine and acetic acid.

Example 1

Di(octane-4-yl)9,9'-((3-(3,5-bis((3-(di(dodecylamino))propyl)carbamoyl)benzamido) Propyl)azanyldiyl)dinonanoate

Preparation of Compound E

Under nitrogen protection, compound E1 (13g, 55mmol) and compound E2 (6.5g, 50mmol) were dissolved in DCM (65mL), DMAP (1.8g, 15mmol) and EDCI (14.4g, 75mmol) were added and reacted at room temperature, TLC (EA/PE=1/10) showed that the reaction reached the end point, the reaction solution was mixed with silica gel (100-200 mesh) and concentrated, and 100-200 mesh silica gel column chromatography EA/PE=1/30 gave 14.14 g of oil Compound E3, yield: 81.2%.

¹ H NMR (400MHz, CDCl ₃ ): δ4.92-4.83(m,1H),3.42-3.32(m,2H),2.30-2.23(m,2H),1.87-1.73(m,2H),1.63- 1.18(m,20H),0.96-0.89(m,6H).

Under the protection of nitrogen, compound E3 (9.0g, 25.8mmol) and compound V2 (1.8g, 10.3mmol) were dissolved in DMF (18mL), and K ₂ CO ₃ (3.56g, 25.8mmol) was added to react at 80°C. TLC (MeOH/DCM=1/10) detected that the reaction was complete, diluted with ethyl acetate (100mL), washed 3 times with water, dried over sodium sulfate, concentrated by filtration, purified by silica gel column, MeOH/DCM system, MeOH%: 0%→15 %, to obtain 2.54g oil compound E4, yield 34.6%.

¹ H NMR (400MHz, CDCl ₃ ): δ5.20 (bs, 1H), 4.92-4.83 (m, 2H), 3.47-3.32 (m, 2H), 2.68-2.22 (m, 10H), 1.87-1.39 ( m,46H), 0.93-0.85(m,12H).

Dissolve compound E4 (2.54g, 3.58mmol) in 1,4-dioxane (10mL), add 4M HCl (10mL, 40.0mmol) at room temperature, react at room temperature for 2 hours, LCMS shows that the reaction is complete, and concentrate to After drying, 2.15 g of crude compound E was obtained, with a yield of 88.5%.

MS m/z (ESI): 611.5 [M+1] ⁺ .

Preparation of Compound V

Under the protection of nitrogen, compound V1 (10.7g, 42.9mmol) and compound V3 (3.0g, 17.2mmol) were dissolved in DMF (30mL), potassium carbonate (5.95g, 43.1mmol) was added, and the reaction was heated at 80°C overnight. The reaction solution was diluted with ethyl acetate, washed with water, and the aqueous phase was back-extracted with a small amount of ethyl acetate, the organic phases were combined, washed three times with water, washed once with saline, dried over sodium sulfate, concentrated by filtration, purified by flash column chromatography, and eluted System MeOH/DCM, MeOH%: 0%-5% (5min) → 5%-10% (10min) → 10%-15% (15min) to obtain 3.8 g of oil compound V2, yield 43.2%.

¹ H NMR (400MHz, CDCl ₃ ): δ5.74(s, 1H), 3.25-3.09(m, 2H), 2.55-2.25(m, 6H), 1.69-1.54(m, 2H), 1.50-1.16( m,49H), 0.88(t,6H).

Dissolve compound V2 (2.0g, 3.91mmol) in 1,4-dioxane (18mL), add 4M HCl (6mL, 24.0mmol) at room temperature, react at 50°C for 1.0-3.0 hours, and concentrate to dryness to obtain 1.92g crude compound V, the obtained crude product was directly used in the next step.

MS m/z(ESI):411.5[M+1] ⁺

Step 1: Preparation of compound 1b

Compound V (800mg, 1.66mmol) and compound 1a (185mg, 0.83mmol prepared by the known method "Tetrahedron Letters, 2011, 52(1), 155-158")) were dissolved in DCM (8mL), and added EDCI (478mg, 2.49mmol), DMAP (101mg, 0.83mmol) and triethylamine (340μL, 2.49mmol), the reaction was stirred overnight at room temperature, diluted with ethyl acetate, washed once with water, dried over sodium sulfate, concentrated by filtration, flash column Chromatographic purification, elution system MeOH/DCM, MeOH%: 0%-10% (10min) → 10% (5min) → 10%-20% (10min) → 20%-30% (10min) to obtain 340mg oil Compound 1b, yield 40.8%.

¹ H NMR (400MHz, CDCl ₃ ): δ8.98(s,2H),8.54(s,3H),3.94(s,3H),3.64-3.52(m,4H),2.75-2.60(m,4H) ,2.60-2.37(m,8H),1.93-1.68(m,4H),1.57-1.39(m,8H),1.37-1.03(m,72H),0.88(t,12H).

Step 2: Preparation of compound 1c

Dissolve compound 1b (340mg, 0.337mmol) in THF/MeOH (1/1, 8mL), add LiOH-H ₂ O (33.6mg, 0.80mmol), heat at 50°C for complete reaction, wash with dilute hydrochloric acid and saturated The sodium bicarbonate solution adjusted the pH to 7-8, and left it at room temperature overnight, concentrated, adjusted the pH to about 11 with triethylamine, and then purified it with a large plate (MeOH/DCM=1/5) to obtain 153 g of compound 1c, Yield 45.7%.

¹ H NMR (400MHz, CDCl ₃ ): δ8.90(s,2H),8.57(s,1H),3.72-3.45(m,4H),3.11-2.40(m,12H),2.17-1.85(m, 4H), 1.71-1.46(m, 8H), 1.37-0.96(m, 72H), 0.86(t, 12H).

Step 3: Preparation of compound 1

Under nitrogen protection, compound 1c (200mg, 0.201mmol) was dissolved in THF (5mL), 1 drop of DMF was added, SOCl ₂ (598mg, 5.02mmol) was added, the reaction was heated at room temperature 34°C for 3 hours, and evaporated under reduced pressure The solvent was drained by an oil pump; the crude product was set aside, under nitrogen protection, weighed compound E (177mg, 0.26mmol) in another bottle, dissolved it with anhydrous dichloromethane (3ml), added triethylamine (202mg, 2mmol) , the reaction was placed in an ice-water bath and stirred for 10-30 minutes, the crude product obtained above was added to the reaction solution at one time, and naturally raised to room temperature for overnight reaction, the reaction solution was directly passed through a silica gel column (300-400 mesh, dichloro Methane/methanol=20:1-10:1) separation and purification to obtain 83 mg of oily compound 1 with a yield of 26%.

MS m/z (ESI): 795.3 [M/2+1] ⁺ .

¹ H NMR (400MHz, CDCl ₃ ): δ8.70(s,3H),8.52(bs,3H),4.92-4.83(m,2H),3.62-3.52(m,6H),3.12-2.82(m, 18H), 2.30-2.23(m, 4H), 2.18-2.02(m, 6H), 1.77-1.43(m, 24H), 1.39-1.18(m, 100H), 0.93-0.85(m, 24H).

Example 2

Oct-4-yl 9-((3-(3,5-bis((3-(di(dodecylamino))propyl)carbamoyl)benzamido)propyl)(dodecyl base) amino) nonanoate

Step 1: Preparation of compound 2b

Under the protection of nitrogen, compound E3 (2.5g, 7.15mmol) and 2a (12.4mg, 71.5mmol) were dissolved in EtOH (10mL) and reacted at room temperature (25-30°C) for 24h. The reaction solution was mixed with petroleum ether and water The layers were separated, washed twice with water, dried over sodium sulfate, concentrated by filtration, flash column chromatography, eluent MeOH/DCM, MeOH: 0%→25%, and 1.7g of oily compound 2b was obtained with a yield of 53.8%.

MS m/z (ESI): 443.4 [M+1] ⁺ .

¹ H NMR (400MHz, CDCl ₃ ): δ5.20(bs,1H),4.92-4.83(m,1H),3.27-3.12(m,2H),2.70(t,2H),2.60(t,2H) ,2.27(t,2H),2.20(bs,1H),1.75-1.52(m,4H),1.51-1.42(m,15H),1.41-1.22(m,14H),0.93-0.85(m,6H) .

Step 2: Preparation of Compound 2c

Under the protection of nitrogen, compound V1 (675mg, 2.71mmol) and 2b (1.0g, 2.26mmol) were dissolved in DMF (4mL), potassium carbonate (374mg, 2.71mmol) was added, and the reaction was heated at 80°C overnight. Dilute with ethyl acetate, wash with water, back-extract the aqueous phase with a small amount of ethyl acetate, combine the organic phases, wash 3 times with water, wash once with saline, dry over sodium sulfate, filter and concentrate, purify by flash column chromatography, and the elution system MeOH/ DCM, MeOH%: 0%→25% (30min) to obtain 0.92 g of oily compound 2c, yield 66.7%.

¹ H NMR (400MHz, CDCl ₃ ): δ5.72(bs,1H),4.92-4.83(m,1H),3.27-3.12(m,2H),2.60-2.30(m,4H),2.27(t, 2H), 1.72-1.56(m, 4H), 1.51-1.22(m, 51H), 0.93-0.85(m, 9H).

Step 3: Preparation of compound 2d

Compound 2c (0.92g, 1.5mmol) was dissolved in 1,4-dioxane (2mL), HCl 4M in 1,4-dioxane (2mL, 8mmol) was added at room temperature, room temperature (25- 30° C.), the reaction was almost complete as monitored by LCMS, the reaction solution was directly concentrated and pumped on an oil pump for 2 hours, and the obtained crude compound 2d was directly used in the next step.

MS m/z (ESI): 511.5 [M+1] ⁺ .

Step 4: Preparation of Compound 2

Compound 1c (140mg, 0.14mmol) and 2d (107mg, 0.18mmol) were dissolved in DCM (2mL), HATU (80mg, 0.21mmol) and triethylamine (39μL, 0.28mmol) were added, and the reaction was stirred at room temperature, TLC monitors that the reaction is almost complete, diluted with ethyl acetate, washed once with sodium bicarbonate solution, washed twice with water, concentrated, purified on a preparative plate, developing solvent (MeOH/DCM=1/5)/(EA/PE=1/1) =1.2/0.8, 113mg of oily compound 2 was obtained, yield 54.1%. Purity: 99.4%.

MS m/z (ESI): 1488.9 [M+1] ⁺ .

¹ H NMR (400MHz, CDCl ₃ ): δ8.79-8.21(m, 6H), 4.97-4.82(m, 1H), 3.68-3.50(m, 6H), 2.91-2.41(m, 18H), 2.26( t, J=7.4Hz, 2H), 1.94-1.71(m, 6H), 1.66-1.12(m, 122H), 0.96-0.81(m, 21H).

Example 3

2,2'-((3-(3,5-bis((3-(di(dodecylamino))propyl)carbamoyl)benzamido)propyl)azanyldiyl) Dinonyl diacetate

Step 1: Preparation of compound 3b

Compound 3a (1.0 g, 4.25 mmol, 1.0 eq) and V3 (0.32 g, 1.85 mmol, 2.3 eq) were dissolved in ACN (10 mL), then potassium carbonate (0.59 g, 1.85 mmol, 2.3 eq) and KI were added sequentially ( 0.71g, 1.85mmol, 2.3eq, stirred at 75°C for 4-10h under the protection of nitrogen, the reaction solution was concentrated under reduced pressure to remove the solvent, the residue was dissolved in EA (30ml) and washed with saturated sodium bicarbonate (20ml×2), It was washed once with saturated brine (20 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (100-200 mesh, PE/EA=5:1) to obtain 750 mg of compound 3b with a yield of 37%.

MS m/z (ESI): 543.5 [M+1] ⁺ .

¹ H NMR (400MHz, CDCl ₃ ): δ4.11-4.08(m,4H),3.50(s,4H),2.75(s,2H),1.64-1.61(m,8H),1.43(s,9H) ,1.30-1.26(m,25H),0.89-0.85(m,6H).

Step 2: Preparation of compound 3c

Compound 3b (0.75g, 1.38mmol, 1.0eq) was dissolved in 1,4-dioxane (4mL), and HCl 4M 1,4-dioxane solution (16ml) was added at room temperature, and the reaction was stirred, LCMS detection showed the reaction was complete. Directly concentrated to dryness under reduced pressure to obtain 659 mg of compound 3c, yield: 100%.

MS m/z (ESI): 443.4 [M+1] ⁺ .

Step 3: Preparation of Compound 3

Compound 3c (0.66g, 1.24mmol, 1.3eq) and compound 1c (1.02g, 0.96mmol, 1.0eq) were dissolved in DCM (20ml), then HOBT (0.16g, 1.15mmol, 1.2eq), EDCI (0.37g, 1.91mmol, 2.0eq) and DIEPA (0.62g, 4.78mmol, 5.0eq), stirred under nitrogen protection for 15-20h, the reaction solution was concentrated under reduced pressure to remove the solvent, and the residue was dissolved in EA (30ml) and washed with saturated Washed with sodium bicarbonate (20ml×2), washed once with saturated brine (20mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, purified by reverse phase column chromatography (H ₂ O/ACN, 95% product) to obtain 1.02g of compound 3, yield 77.78%, purity: 96.2%.

MS m/z (ESI): 1420.9 [M+1] ⁺ .

¹ H NMR(400MHz,CD3OD):δ8.49(s,3H),4.12-4.06(m,4H),3.63-3.48(m,9H),3.13-2.81(m,12H),2.01-1.97(m ,6H), 1.80-1.78(m,2H), 1.63-1.58(m,12H), 1.34-1.22(m,98H), 0.91-0.87(m,18H).

Example 4

((((Benzene-1,3,5-tricarbonyl)tri(azanyldiyl))tri(butane-4,1-diyl))tri(azanyltriyl))hexa(hexane -6,1-diyl)hexa(2-hexyldecanoate))

Step 1: Preparation of Compound 4c

Under nitrogen protection, compound 4a (6.4g, 35.3mmol) and 4b (10.0g, 39.0mmol) were dissolved in DCM (64mL), DMAP (1.3g, 10.6mmol) and EDCI (10.2g, 53.2mmol) were added , reacted at room temperature, TLC (EA/PE=1/10) showed that the reaction was complete, concentrated and purified to obtain 8.9 g of oily compound 4c, yield: 57.8%.

¹ H NMR (400MHz, CDCl ₃ ): δ4.07(t, J=6.6Hz, 2H), 3.40(t, J=6.8Hz, 2H), 2.35-2.26(m, 1H), 1.93-1.81(m , 2H), 1.70-1.15 (m, 30H), 0.87 (t, J=6.3Hz, 6H).

Step 2: Preparation of compound 4e

Under nitrogen protection, compound 4c (5.9g, 14.1mmol) and 4d (1.0g, 5.7mmol) were dissolved in DMF (10mL), K ₂ CO ₃ (1.9g, 13.7mmol) was added, reacted at 80°C, TLC (MeOH/DCM=1/10) detected that the basic reaction was complete, diluted with ethyl acetate (100 mL), washed with water, dried the organic phase over sodium sulfate, concentrated by filtration, and purified to obtain 0.96 g of oily compound 4e with a yield of 19.3%.

¹ H NMR (400MHz, CDCl ₃ ): δ5.00(s, 1H), 4.06(t, J=6.7Hz, 4H), 3.18-3.04(m, 2H), 2.06-2.16(m, 8H), 1.72 -1.52 (m, 12H), 1.52-1.10 (m, 65H), 0.87 (t, J=6.1Hz, 12H).

Step 3: Preparation of compound 4f

At room temperature, compound 4e (0.96g, 1.1mmol) was dissolved in 1,4-dioxane (5mL), added HCl (4M 1,4-dioxane, 5mL, 20.0mmol), and reacted at room temperature After 2-6 hours, the reaction solution was directly concentrated to dryness, and the obtained crude product was directly used in the next step.

Step 4: Preparation of Compound 4

Under nitrogen protection, the crude compound 4f (about 1.1mmol) was dissolved in DCM (10mL), compound 4g (84mg, 0.32mmol) was added, triethylamine (354μL, 2.55mmol) was added, and the reaction was carried out at room temperature for 15-18 hours, the reaction solution was mixed with silica gel (100-200 mesh) and concentrated, and 200-300 mesh silica gel column chromatography (MeOH/DCM=1/10)/(EA/PE=1/1)=3/1 obtained the crude product 670 mg, about 320 mg of the part was further purified on a large plate, and the developer was (MeOH/DCM=1/5)/(EA/PE=1/1)=3/1, and 202 mg of oily compound 4 was obtained, and the two-step yield was 26.0% .

¹ H NMR (400MHz, CDCl ₃ ): δ8.40(s, 3H), 7.04(s, 3H), 4.04(t, J=6.6Hz, 12H), 3.55-3.41(m, 6H), 2.62-2.36 (m, 18H), 2.36-2.11 (m, 12H), 1.74-1.50 (m, 36H), 1.50-1.09 (m, 162H), 0.87 (t, J=6.0Hz, 36H).

Example 5

N-(3-(3,5-bis((3-(di(dodecylamino))propyl)carbamoyl)benzamido)propyl)-N-dodecylglycine nonyl ester

Step 1: Preparation of Compound 5c

In a 25mL three-necked flask, sequentially add compound 5a (1.3g, 3.8mmol), DMF (13mL), compound 5b (1.0g, 3.8mmol) and potassium carbonate (524mg, 3.8mmol), replace it three times under nitrogen protection, and start stirring , heating the inner temperature to 110°C for 2h. Sampling LC-MS detection showed the mass spectrum peak of the target product, and a small amount of raw materials remained. Stop the reaction, cool the reaction solution to 25°C, add 150mL of water, then extract with EtOAc (60mL×3), combine the organic phases, wash with water (60mL), saturated brine (60mL), anhydrous sodium sulfate (5g) Dry, filter and evaporate to dryness to obtain crude product. The oily compound 5c (1.860 g, yield: 93.0%) was separated by silica gel column chromatography (PE/EA=20/1-10/1-5/1).

Ms (ESI): m/z 527.5 [M+H] ⁺ .

Step 2: Preparation of compound 5d

In a 25mL single-necked bottle, add compound 5c (1.7g, 3.2mmol), 4N HCl (in dioxane) (10mL) in sequence, replace once under nitrogen protection, start stirring, stir at room temperature 23-26°C for 2h, and sample LC -MS detection showed that the reaction of the raw materials was complete. The reaction was stopped, and concentrated under reduced pressure to obtain white solid compound 5d (1.8 g, yield: 111.6%).

Ms (ESI): m/z 427.4 [M+H] ⁺ .

Step 3: Preparation of Compound 5

In a 100mL three-necked flask, sequentially add compound 1c (2.0g, 2.01mmol), anhydrous DCM (40mL), compound 5d (1.2g, 2.41mmol) and DIEA (2.1mL, 12mmol), and replace three times under the protection of argon, Start stirring, and cool to 5°C in an ice-water bath. Add HATU (917mg, 2.41mmol) at one time and stir for 30 minutes at an internal temperature of less than 10°C, then stir at room temperature for 19h at 25-32°C, sample HPLC detection shows that the reaction of the raw materials is complete, stop the reaction, add 50mL of dichloromethane to dilute, once with saturated carbonic acid Sodium hydrogen solution (30 mL) and saturated brine were washed, dried over anhydrous sodium sulfate (5 g), filtered and evaporated to dryness to obtain a crude product. After 200-300 mesh normal phase silica gel column chromatography (PE/EtOH=0%-60%) to obtain an oily substance, then Pre-TLC (DCM/MeOH=20/1) to obtain an oily compound 5 (526mg, yield: 18.6 %, HPLC purity 94.63%).

MS m/z (ESI): 1404.8 [M+1] ⁺ .

¹ H NMR (400MHz, CDCl ₃ ): δ8.47(s,3H),8.33(s,3H),4.10-4.02(t,2H),3.66-3.52(m,6H),3.32(s,2H) ,2.70-2.48(m,16H),1.74-1.43(m,18H),1.33-1.29(m,12H),1.27-1.14(m,90H),0.89-0.83(m,18H).

Comparative Example 1

Comparative compound 1

Prepared by the method in "Nano Lett.2015, 15, 8099-8107 and WO2016187531A1".

Comparative Example 2

Comparative compound 2

It is prepared by the method in "WO2019099501A1".

Test Example 1: Lipid Particle Composition Delivery Ability

1.1 Preparation method

Compounds 1, 2, 3, 4 and comparative compounds 1, 2 were dissolved in ethanol solution, and mixed with DOPE, cholesterol, DMG-PEG solution dissolved in ethanol at a molar ratio of 20:30:40:0.75 to prepare ethanol lipid substance solution. The mRNA encoding luciferase (GenBank: MN728548.1) was dissolved in citrate buffer to prepare an aqueous mRNA solution. The ethanol lipid solution and mRNA aqueous solution were mixed by microfluidics, and the weight ratio of total lipid to mRNA was about 12-36:1 to prepare liposomes. Ethanol was dialyzed in PBS solution to obtain a liposome nanoparticle (LNP) preparation encapsulating luciferase-encoding mRNA.

1.2 Characterization of lipid nanoparticle composition

The nanometer size and polydispersity index (PDI) of liposomal nanoparticles were detected by dynamic light scattering using Malvern Zetasizer Nano ZS in 173° backscatter detection mode.

Quant-iT RiboGreen RNA Assay Kit RNA Quantitative Detection Kit (purchased from Thermo Fisher Scientific, catalog number R11490) was used to determine the liposome encapsulation efficiency.

The pKa of the cations in the liposomal nanoparticles was determined using a fluorescence assay based on 6-(p-toluidine)-2-naphthalenesulfonic acid sodium salt (TNS). Prepare 150mM NaCl, 10mM sodium phosphate, 10mM sodium citrate, 10mM sodium borate, pH 3-11.5 buffers with different pH. Prepare a 300μM TNS solution and add it to the buffer. The lipid nanoparticles were respectively added into buffer solutions with different pHs, and after being thoroughly mixed, the fluorescence intensity at an excitation wavelength of 325 nm and an emission wavelength of 435 nm was detected at room temperature using a fluorescent microplate reader. For fluorescence data fitting analysis, pKa is the pH value that produces half the maximum fluorescence intensity. See Table 1 for relevant data.

Table 1

1.3 Evaluate the mRNA expression efficiency of the lipid particle composition at the cellular level

Using the liposome nanoparticles corresponding to comparative compounds 1 and 2 as a control, the mRNA expression efficiency at the in vitro cell level of the liposome nanoparticles corresponding to compounds 1-3 was detected.

HEK 293 cells were inoculated into the cell well plate and cultured overnight, and when the cell density reached above 80%, the liposome LNP solution encapsulating luciferase mRNA was added to the culture medium of the cell plate well. After 24 hours, the fluorescence intensity of the expressed luciferase protein was detected using a luciferase reporter gene detection kit (Promega) and a microplate reader. The fluorescence intensity value is the fluorescence value detected by the microplate reader. For each compound, at least three groups of LNPs were used to repeatedly calculate the average fluorescence value intensity and statistical difference, and the data are shown in Table 2 and Figure 1.

In Fig. 1, * means statistically 0.01<P<0.05, indicating statistically significant difference between groups, ** means statistically P<0.01, *** means statistically P<0.001 means extremely significant difference.

Table 2

serial number mean fluorescence intensity value

Compound 1 6.47E+06 Compound 2 4.09E+06 Compound 3 4.58E+06 Comparative compound 1 3.38E+06 Comparative compound 2 2.61E+06

Conclusion: As shown in Table 2 and Figure 1, the lipid nanoparticles corresponding to compounds 1, 2 and 3 can deliver mRNA and express luciferase in cells better than comparative compound 1 and comparative compound 2.

1.4 Evaluate the mRNA expression efficiency of lipid particle composition delivered by intramuscular injection in vivo

In order to evaluate the effective delivery of liposome nanoparticles to mRNA and express the corresponding encoded protein in vivo, a dose of 0.25 mg/kg was injected into the thigh muscle site of 6-8 week-old female BALB/c with luciferase-encapsulated mRNA liposomes. plastid nanoparticles. Six hours later, the luciferase substrate was injected intraperitoneally into each mouse, and the fluorescence pictures of the mice were taken using the IVIS small animal optical in vivo imaging instrument (PerkinElme), and the fluorescence intensity at the injection site was counted. The level of fluorescence intensity represents the level of expression of luciferase protein, which reflects the efficiency of liposome nanoparticle delivery of mRNA in vivo. The fluorescence intensity in Table 3 and Figure 2 is the fluorescence intensity at the injection site of the mouse captured and counted by the IVIS small animal optical live imager. At least 3 groups of LNP corresponding to each compound were repeated to calculate the average fluorescence intensity.

Taking the liposome nanoparticles corresponding to comparative compound 1 as a control, the in vivo intramuscular injection mRNA delivery efficiency of liposome nanoparticles corresponding to compounds 1, 2 and 3 was detected. See Table 3 and Figure 2 for relevant data.

In Fig. 2, * means statistically 0.01<P<0.05, indicating statistically significant difference between groups, ** means statistically P<0.01, *** means statistically P<0.001 means extremely significant difference.

table 3

serial number mean fluorescence intensity value Compound 1 1.80E+08 Compound 2 1.55E+08 Compound 3 4.65E+07 Comparative compound 1 4.19E+07

Conclusion: As the fluorescence intensity shown in Table 3 and Figure 2, the lipid nanoparticles corresponding to compounds 1, 2 and 3 are better than the comparative compound 1 in delivering mRNA in vivo by intramuscular injection.

1.5 Evaluation of protein expression after intramuscular injection of liposomes to deliver mRNA

In order to evaluate the effective delivery of mRNA in vivo by liposome nanoparticles and the expression of the corresponding encoded protein, 6-8 week old female BALB/c thigh muscles were injected with mRNA encapsulated with growth factors at a dose of 0.05 mg/kg (NCBI Reference Sequence: NM_000601.6) Liposome Nanoparticles. After 24 hours, the muscle at the injection site was taken, ground and lysed, and the expression level of growth factor protein (pg/mg) was detected using an ELISA kit, that is, the amount of growth factor protein corresponding to the total protein amount of muscle tissue. The average protein concentration was calculated for at least 3 groups of LNP corresponding to each compound.

With the liposome nanoparticle corresponding to compound 1 as a control, the protein expression level of the liposome nanoparticle corresponding to compound 1 and 3 after intramuscular injection of mRNA was detected, that is, the delivery efficiency of intramuscular injection of mRNA in vivo. See Table 4 and Figure 3 for relevant data.

In Fig. 3, * means statistically 0.01<P<0.05, indicating statistically significant difference between groups, ** means statistically P<0.01, *** means statistically P<0.001 means extremely significant difference.

Table 4

serial number Average protein expression (pg/mg) Compound 1 121.4 Compound 3 94.7 Comparative compound 1 43.6

Conclusion: As shown in Table 4 and Figure 3, the protein expression levels of the LNPs corresponding to compounds 1 and 3 delivered by intramuscular injection were significantly higher than those of the comparison compound 1.

1.6 Evaluation of mRNA expression efficiency of lipid particle composition in vivo tail vein injection

In order to evaluate the effective delivery of mRNA in vivo by liposomal nanoparticles and the expression of the corresponding encoded protein, 6-8 week old female BALB/c tail vein was injected with luciferase-encapsulated mRNA at a dose of 0.5 mg/kg Liposome nanoparticles. Six hours later, luciferase substrate was injected intraperitoneally into each mouse, and IVIS small animal optical in vivo imaging instrument (PerkinElme) was used to take fluorescent pictures of the mice, and the fluorescence intensity of the whole body of the mice was counted. The level of fluorescence intensity represents the level of expression of luciferase protein, which reflects the efficiency of liposome nanoparticle delivery of mRNA in vivo. For each compound, at least three groups of LNPs were used to repeatedly calculate the average fluorescence intensity, and the data are shown in Table 5 and Figure 4.

The fluorescence intensity in Figure 4 is the fluorescence intensity of the whole body of the mouse captured and counted by the IVIS small animal optical live imager.

Using the liposome nanoparticles corresponding to comparative compound 1 as a control, the mRNA delivery efficiency of the in vivo tail vein injection of the liposome nanoparticles corresponding to compounds 1 and 2 was detected.

In Fig. 4, * means statistically 0.01<P<0.05, indicating statistically significant difference between groups, ** means statistically P<0.01, *** means statistically P<0.001 means extremely significant difference.

table 5

serial number mean fluorescence intensity value Compound 1 7.47E+08 Compound 2 9.69E+08 Comparative compound 1 3.98E+08

Conclusion: As the fluorescence intensity shown in Table 5 and Figure 4, the mRNA lipid nanoparticles corresponding to compounds 1 and 2 were injected into the tail vein, and the protein expression was significantly better than that of the comparison compound 1.

1.7 Evaluate the efficiency of mRNA expression in lungs by tail vein injection of lipid particle composition in vivo

In order to evaluate the effective delivery of mRNA in the lung by liposomal nanoparticles and the expression of the corresponding encoded protein, 6-8 week-old female BALB/c tail vein was injected with luciferase-encapsulated mRNA liposome nanoparticles. After 6 hours, after intraperitoneal injection of luciferase substrate to each mouse, the lung tissue of the mice was taken, and fluorescent pictures were taken using the IVIS small animal optical in vivo imaging instrument (PerkinElme), and the fluorescence intensity was counted. The level of fluorescence intensity represents the level of expression of luciferase protein, which reflects the efficiency of liposome nanoparticle delivery of mRNA in vivo. For each compound, at least three groups of LNPs were used to repeatedly calculate the average fluorescence intensity, and the data are shown in Table 6 and Figure 5.

In Fig. 5, * means statistically 0.01<P<0.05, indicating statistically significant difference between groups, ** means statistically P<0.01, *** means statistically P<0.001 means extremely significant difference.

Table 6

serial number mean fluorescence intensity value Compound 4 1.72E+06 Comparative compound 1 4.74E+05

Conclusion: As the fluorescence intensity shown in Table 6 and Figure 5 shows, the mRNA lipid nanoparticles corresponding to compound 4 were injected into the tail vein, and the protein expression in the lung was significantly better than that of the comparison compound 1.

Claims (21)

Compound shown in formula I or its salt

Wherein, M ¹ to M ⁶ are each independently selected from a bond, -C(O)O-a1 and -OC(O)-a1, and a1 is a combination with R ¹ , R ² , R ³ , R ⁴ , R ⁵ or R ⁶ connected bonds, and M ¹ to M ⁶ are not all -C(O)O-a1 or bonds; R ¹ to R ⁶ are each independently substituted or unsubstituted alkyl or substituted or unsubstituted alkenyl; R ⁷ are each independently hydrogen or substituted or unsubstituted C _1-6 alkyl; m1 to m3 are each independently selected from 1, 2, 3, 4, 5, 6, 7 and 8; n1 to n6 are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. The compound or salt thereof according to claim 1, wherein R ¹ to R ⁶ are each independently unsubstituted alkyl or unsubstituted alkenyl, preferably C ₄ -C ₁₄ alkyl or C ₄ -C ₁₄ Alkenyl. The compound or salt thereof according to claim 1 or 2, wherein R ¹ is C ₄ -C ₁₄ alkyl, preferably C ₅ -C ₁₂ alkyl. The compound or salt thereof according to any one of claims 1-3, wherein R ² is C ₄ -C ₁₄ alkyl, preferably C ₅ -C ₁₂ alkyl. The compound or salt thereof according to any one of claims 1-4, wherein R ³ is C ₄ -C ₁₄ alkyl, preferably C ₅ -C ₁₂ alkyl; and/or R ⁴ is C ₄ -C ₁₄ Alkyl, preferably C ₅ -C ₁₂ alkyl; and/or R ⁵ is C ₄ -C ₁₄ alkyl, preferably C ₅ -C ₁₂ alkyl; and/or R ⁶ is C ₄ -C ₁₄ alkyl, preferably C ₅ -C ₁₂ alkyl. The compound or salt thereof according to any one of claims 1-5, wherein R ⁷ is hydrogen or optionally substituted C _1-3 alkyl, preferably hydrogen, methyl or ethyl. The compound or salt thereof according to any one of claims 1-6, wherein M ¹ to M ⁶ are each independently -OC(O)-a1, and a1 is connecting R ¹ , R ² , R ³ , R ⁴ , R ⁵ or R ⁶ bond. The compound or salt thereof according to any one of claims 1-6, wherein M ¹ and M ² are each independently -C(O)O-a1, and a1 is a bond connected to R ¹ or R ² . The compound or salt thereof according to any one of claims 1-6, wherein M ¹ , M ² , M ³ and M ⁴ are each independently -C(O)O-a1, and a1 is the same as R ¹ , R ^2. A bond to which R ³ or R ⁴ is connected. The compound or salt thereof according to any one of claims 1-9, wherein m1 to m3 are each independently selected from 2, 3 and 4. The compound or salt thereof according to any one of claims 1-10, wherein n1 to n6 are each independently selected from 6, 7, 8, 9 and 10. The compound or its salt according to claim 1 or 8, wherein the compound or its salt shown in Formula I is the compound or its salt shown in Formula II,

wherein R ¹ to R ⁷ , m1 to m3, and n1 to n6 are as defined in claim 1 . The compound or its salt according to claim 1 or 7, wherein the compound or its salt shown in Formula I is the compound or its salt shown in Formula III,

wherein R ¹ to R ⁷ , m1 to m3, and n1 to n6 are as defined in claim 1 . ^The compound or salt thereof according to claim ¹ , wherein R and R are each independently selected from

Compound shown in formula I or its salt

An isotope substitution of the compound or salt thereof according to any one of claims 1-15, preferably, the isotope substitution is a deuterium atom substitution. A lipid particle comprising the compound or salt thereof according to any one of claims 1-15, or the isotope substitution according to claim 16, further comprising an active agent, preferably selected from the group consisting of Immunostimulatory oligonucleotides, siRNA, antisense oligonucleotides, mRNA, and plasmids. A pharmaceutical composition comprising the lipid particle according to claim 17 and a pharmaceutically acceptable excipient. The compound or salt thereof according to any one of claims 1-15, or the isotope substitution according to claim 16, or the lipid particle according to claim 17, or the lipid particle according to claim 18 Use of the pharmaceutical composition for the preparation of a medicament for inducing an immune response in a subject. The compound or salt thereof according to any one of claims 1-15, or the isotope substitution according to claim 16, or the lipid particle according to claim 17, or the lipid particle according to claim 18 Use of the pharmaceutical composition in the preparation of a medicament for preventing and/or treating diseases or conditions associated with polypeptide overexpression. The compound or salt thereof according to any one of claims 1-15, or the isotope substitution according to claim 16, or the lipid particle according to claim 17, or the medicine according to claim 18 Use of the composition in the preparation of a medicament for preventing and/or treating a disease or condition associated with underexpression of a polypeptide.

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