Lipid compound containing heteroaromatic ring structure and use thereof

Abstract

A lipid compound containing a heteroaromatic ring structure and use thereof. Provided is use of a compound in the preparation of a lipid carrier. The compound is a compound represented by formula I, a pharmaceutically acceptable salt thereof, or a conjugate thereof. A liposome drug prepared using the lipid compound has a good adjuvant effect, and can effectively activate T cell immunity.

Description

Lipid compounds containing heteroaromatic ring structures and their applications

[0001] This application claims priority to Chinese Patent Application No. 2024112119797, filed on August 30, 2024. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This invention relates to a lipid compound containing a heterocyclic aromatic ring structure and its applications. Background Technology

[0003] In recent years, mRNA vaccines have demonstrated great potential in the prevention and treatment of infectious diseases and cancer. mRNA infectious disease vaccines encode pathogen antigens, enabling the body to rapidly recognize and respond to viral infections. mRNA cancer vaccines, on the other hand, encode tumor-associated antigens, activating the body's immune system to attack cancer cells. However, some existing mRNA vaccines still face challenges in evoking a sufficiently strong immune response. Therefore, scientists are exploring ways to enhance the immunogenicity of vaccines, such as by adding adjuvants, thereby improving their protective efficacy.

[0004] Vaccine adjuvants are crucial components for enhancing vaccine immunogenicity. Their primary function is to stimulate the body's immune system, thereby increasing the immune response to antigens. Adjuvants can promote the activation of antigen-presenting cells and the effective presentation of antigens, enhancing the activation of T cells and B cells, thus improving cellular and humoral immune responses. Furthermore, adjuvants can induce durable immune memory, enabling the body to respond rapidly when encountering real pathogens. The application of adjuvants is particularly widespread in infectious disease and cancer vaccines. By introducing different types of adjuvants, such as MPL and aluminum adjuvants, researchers have been able to significantly improve the protective and therapeutic effects of vaccines.

[0005] TLR agonists are molecules that activate Toll-like receptors (TLRs) and are widely used to enhance immune responses in vaccines and immunotherapies. TLRs are key receptors in the innate immune system, recognizing pathogen-associated molecular patterns, initiating innate immune responses, and activating the adaptive immune system. By activating TLRs, TLR agonists can promote the maturation and function of antigen-presenting cells (APCs), enhance T-cell and B-cell immune responses, and thus improve vaccine immunogenicity. TLR agonists have shown great potential in infectious disease vaccines and tumor immunotherapies, especially when combined with lipid nanoparticle (LNP) technology, enabling more efficient delivery and presentation of antigens, significantly enhancing immune responses and protective effects. These technological advances lay a solid foundation for developing more effective vaccines and immunotherapies.

[0006] Existing lipid nanoparticles (LNPs) often fail to elicit sufficient immune responses or effective T-cell immunity when delivering mRNA sequences for tumor treatment, resulting in poor therapeutic effects of tumor mRNA drugs. To enhance T-cell immune responses, major mRNA companies and research institutions have optimized their products through various methods, including mRNA sequence optimization and encoding specific immunogenic elements into the mRNA, aiming to achieve stronger T-cell immune enhancement.

[0007] Introducing adjuvant lipids (with TLR agonist structures in their substructures) into lipid nanoparticles (LNPs) is an important strategy for enhancing the immunogenicity of mRNA vaccines. In the paper "Adjuvant lipidoid-substituted lipid nanoparticles augment the immunogenicity of SARS-CoV-2 mRNA vaccines" (Han, X., Alameh, MG., Butowska, K. et al. Nat. Nanotechnol. 18, 1105–1114 (2023). https: / / doi.org / 10.1038 / s41565-023-01404-4), researchers demonstrated an innovative approach of replacing traditional lipid nanoparticles with adjuvant lipids, which significantly enhanced the immunogenicity of SARS-CoV-2 mRNA vaccines. The introduction of adjuvant lipids not only improved antibody production but also significantly enhanced T-cell responses. By adding adjuvants to LNPs, the efficacy of mRNA vaccines can be significantly improved, protecting mRNA from degradation, promoting its effective delivery in vivo, and triggering a stronger immune response. Summary of the Invention

[0008] The technical problem this invention aims to solve is to overcome the limited variety of lipid carriers for lipid nanoparticles (LNPs) in the prior art that can elicit sufficient immune responses or T-cell immunity. To address this, this invention provides a lipid compound containing a heterocyclic aromatic ring structure and its applications. Lipid nanoparticles prepared using the lipid compound provided by this invention exhibit good adjuvant effects and can effectively activate T-cell immunity.

[0009] The present invention solves the above-mentioned technical problems through the following technical solution:

[0010] This invention provides the use of a compound in the preparation of lipid carriers, said compound being a compound of formula I, a pharmaceutically acceptable salt thereof, or a conjugate thereof.

[0011] Among them, R 0 For single key or

[0012] R 1 It is an H or lipid fragment;

[0013] R 2 lipid fragments, H, C 1-5 Alkyl, C 1-5 Heteroalkyl, with an R t1 Replacement C 1-5 Alkyl or with an R t1 Replacement C 1-5 Heteroalkyl; each R t1 Independently hydroxyl, C 1-5 Alkyl or with an R t2 Replacement C 1-5 Alkyl; the C 1-5 The heteroatoms in the heteroalkyl group are independently selected from one, two, or three of N, O, and S, and the number of heteroatoms is one, two, or three;

[0014] R t2 It is a hydroxyl group;

[0015] R 3 It is amino or halogen;

[0016] R 4 R 5 and R 6 Independently for H and C 3-6 Cycloalkyl, 3-6 membered heterocyclic alkyl or lipid fragments; or R 4 R 5 Together with the atoms bonded to it, they form C 6-10 Aryl or 5-10 heteroaryl groups;

[0017] And R 1 R 2 R 4 R 5 and R 6 At least one of them is a lipid fragment, said lipid fragment being independently and optionally R f Substituted branched or straight-chain saturated or unsaturated carbon chains; and said carbon chains optionally contain one or more "composed of N(R) e ), O, S, -S(O2)-, C 3-10 Cycloalkyl, 3-10 membered heterocyclic alkyl, C 6-10 "A group consisting of one or more segments of aryl and 5-10 heteroaryl groups";

[0018] Each R e Independently for H and C 1-6 Alkyl, C 3-10 Cycloalkyl or 3-10 membered heterocyclic alkyl;

[0019] Furthermore, the lipid fragment contains at least 11 carbon atoms;

[0020] The coupling of the compounds shown in Formula I represents two, three or more compounds shown in Formula I connected by a connecting unit;

[0021] The heteroatoms in each of the 3-6 membered heterocyclic alkyl groups are independently selected from one, two, or three of N, O, and S, and the number of heteroatoms is independently one, two, or three.

[0022] And the compound shown in Formula I is not

[0023] In one embodiment, certain groups in the compound of Formula I or its pharmaceutically acceptable salt have the following definitions, and the definitions of groups not mentioned are as described in any embodiment of the present invention (hereinafter referred to as "in one embodiment").

[0024] In one embodiment, the lipid fragment is independently R. a1 or

[0025] R a1 C 6-30 alkyl, Or by one or more R f Replacement C 6-30 Alkyl groups; each of the C4 groups 6-30 One or more methylene units in the alkyl group are independently and optionally R c1 Substitute;

[0026] R a2 and R a3 Independently for C 1-30 alkyl, Or by one or more R f Replacement C 1-30 Alkyl groups; each of the C4 groups 1- 30 One or more methylene units in the alkyl group are independently and optionally R c3 Substitute;

[0027] L 1 and L 2 Independently for -(CR) b1 R b2 ) n1 -, n1 is an integer from 1 to 20, R b1 and R b2 Independently H, hydroxyl, C 1-6 Alkyl, C 3-10 Cycloalkyl or 3-10 membered heterocycloalkyl, wherein -(CRb1 R b2 ) n1 One or more of -(CR) b1 R b2 - The unit is independently and optionally controlled by R c2 Substitute;

[0028] Z 1 and Z 2 Independently CH or N;

[0029] R d1 and R d2 Independently for C 1-30 Alkyl or with one or more R f Replacement C 1-30 Alkyl groups, each of the C16 groups 1-30 One or more methylene units in the alkyl group are independently and optionally R c4 Substitute;

[0030] Each R c1 R c2 R c3 and R c4 Independently selected from -N(R) e -, -O-, -S-, -S(O2)-, C 3-10 Cycloalkyl, 3-10 membered heterocyclic alkyl, C 6-10 A group consisting of one or more segments of aryl and 5-10 heteroaryl groups;

[0031] Each R e Independently for H and C 1-6 Alkyl, C 3-10 Cycloalkyl or 3-10 membered heterocyclic alkyl;

[0032] Each R f Independently hydroxyl, C 1-6 Alkyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic alkyl, C 6-10 Aryl or 5-10 heteroaryl groups;

[0033] Each of the 3-10 membered heterocyclic alkyl groups has a heteroatom type independently selected from one, two, or three of N, O, S, and S(O2), and the number of heteroatoms is independently 1, 2, or 3; each of the 5-10 membered heteroaryl groups has a heteroatom type independently selected from one, two, or three of N, O, and S, and the number of heteroatoms is independently 1, 2, or 3.

[0034] In one particular scheme, R a1 In the middle, each of the C 6-30 Alkyl group is C 10-25 Alkyl, such as C15-25 Alkyl groups; each of the C4 groups 5-30 Alkyl groups can be straight-chain alkyl groups, for example...

[0035] In one particular scheme, R a2 and R a3 In the middle, each of the C 1-30 Alkyl groups are independently C 2-25 Alkyl, such as C 2-20 Alkyl; the C 1- 30 Alkyl groups can be straight-chain alkyl groups, for example...

[0036] In one particular scheme, R d1 and R d2 In, the C 1-30 Alkyl groups are independently C 1-16 Alkyl, such as C 1-10 Alkyl groups, for example

[0037] In one scheme, the various Cs 1-6 Alkyl and C 1-5 Alkyl groups are independently C 1-4 Alkyl groups, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, or sec-butyl.

[0038] In one scheme, the various Cs 1-5 In heteroalkyl groups, the heteroatom species is N or O, and the number of heteroatoms is 1, for example...

[0039] In one scheme, the various Cs 3-10 cycloalkyl (e.g., C10) 3-6 cycloalkyl) and C 3-6 The cycloalkyl group is independently cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl, for example, cyclopropyl, cyclobutyl, or cyclohexyl; when C 3-10 When the cycloalkyl group is a subunit, C 3-10 cycloalkyl can be

[0040] In one scheme, the various Cs 6-10 The aryl group is independently phenyl or naphthyl, for example, phenyl; C 6-10 When the aryl group is an alkyl group, C 6-10 Aryl is

[0041] In one scheme, the heteroatom in each 3-6 membered heterocyclic alkyl group is N, and the number of heteroatoms is 1 or 2; for example, piperazine group, and another example...

[0042] In one embodiment, each of the 3-10 membered heterocyclic alkyl groups is independently a 3-6 membered heterocyclic alkyl group; for example... For example When each of the 3-10 membered heterocyclic alkyl groups is a subunit, each of the 3-10 membered heterocyclic alkyl groups can be:

[0043] In one embodiment, each of the 3-10 membered heterocyclic alkyl groups is independently... For example

[0044] In one embodiment, the halogen is fluorine, chlorine, bromine, or iodine; for example, chlorine.

[0045] In one scheme, each of the "multiples" is independently 2, 3, 4 or 5.

[0046] In one embodiment, each of the 5-10 heteroaryl groups is independently a 5-6 heteroaryl group.

[0047] In one particular scheme, R 0 It is a single key.

[0048] In one particular scheme, R 2 For H, C 1-5 Alkyl or C 1-5 Heteroalkyl, the C 1-5 The heteroatoms in the heteroalkyl group are independently selected from one, two, or three of N, O, and S, and the number of heteroatoms is one, two, or three; preferably, R 2 For H, C 1-5 Alkyl or C 1-5 Heteroalkyl, the C 1-5 The heteroatom in the heteroalkyl group is of type O, and the number of heteroatoms is 1, 2 or 3.

[0049] In one particular scheme, R 3 For amino or chlorine, in a certain scheme, R 3 For amino groups, in a certain scheme, R 3 It is chlorine.

[0050] In one particular scheme, R 3 It is a halogen, such as bromine or iodine, or bromine for example.

[0051] In one particular scheme, R 4 For H.

[0052] In one particular scheme, R 5 It is H or a 3-6 membered heterocyclic alkyl group.

[0053] In one particular scheme, R 5 For H.

[0054] In one particular scheme, R 6 It is an H, 3-6 membered heterocyclic alkyl or lipid fragment.

[0055] In one particular scheme, R 6 For H.

[0056] In one particular scheme, R 4 R 5 Together with the atoms bonded to it, they form C 6-10 Aryl.

[0057] In one embodiment, the lipid fragment is:

[0058] In one particular scheme, R a1 Independently for C 10-25 alkyl or The C 10-25 One or more methylene units in the alkyl group are independently and optionally R c1 Alternative; preferably, R a1 Independently for C 10-25 Alkyl, the C 10-25 One or more methylene units in the alkyl group are independently and optionally R c1 Alternative; better, R a1 Independently for C 10-25 Straight-chain alkyl, the C 10-25 One or more methylene units in a straight-chain alkyl group are independently and optionally R c1 Replacement.

[0059] In one particular scheme, R c1 Independently selected from -N(R) e -, -O-, -S-, -S(O2)-, A group consisting of one or more fragments; preferably, R c1 Independently selected from -N(R) e -, -O-, -S-, -S(O2)-, A group consisting of one or more fragments; more preferably, R c1 Independently selected from -N(R) e )-、-O- and A group consisting of one or more segments.

[0060] In one of the schemes, L 1 Independently for -(CR) b1 R b2 ) n1 -, n1 is an integer from 1 to 16, the -(CR b1 R b2 ) n1 One or more of -(CR) b1 R b2 - The unit is independently and optionally controlled by R c2 Alternative; preferably, L 1 -(CR) b1 R b2 ) n1 -, n1 is 1, 2, 3, 4, 5 or 6, the -(CR b1 R b2 ) n1 One or more of -(CR) b1 R b2 - The unit is independently and optionally controlled by R c2 Alternative; better, L 1 -(CR) b1 R b2 ) n1 -, n1 is 1, 2, 3, 4, 5 or 6.

[0061] In one particular scheme, Z 1 Let N be the number of elements in the array.

[0062] In one particular scheme, R a2 and R a3 Independently for C 2-25 alkyl, Or by one or more R f Replacement C 2-25 Alkyl; the C 2-25 One or more methylene units in the alkyl group are independently and optionally R c3 Alternative; preferably, R a2 and R a3 Independently for C 2-25 alkyl or The C 2-25 One or more methylene units in the alkyl group are independently and optionally R c3 Alternative; better, R a2 and R a3 Independently

[0063] In one particular scheme, R a2 and R a3 Independently for C 2-25 straight-chain alkyl, Or by one or more Rf Replacement C 2- 25 Straight-chain alkyl groups; each of the C... 2-25 One or more methylene units in a straight-chain alkyl group are independently and optionally R c3 Replacement.

[0064] In one particular scheme, R c3 Independently selected from -O- C 3-10 cycloalkyl and C 6-10 A group consisting of one or more segments of an aryl group; preferably, R c3 Independently selected from -O- C 3-6 cycloalkyl and C 6-10 A group consisting of one or more segments of an aryl group; more preferably, R c3 Independently selected from -O- and A group consisting of one or two segments.

[0065] In one particular scheme, R c3 Independently selected from -N(R) e )-、-O- and A group consisting of one, two, or three segments.

[0066] In a certain scheme, each R f Independently hydroxyl, C 3-10 Cycloalkyl or 3-10 membered heterocycloalkyl; preferably, each R f Independently hydroxyl, C 3-6 Cycloalkyl or 3-6 membered heterocyclic alkyl.

[0067] In a certain scheme, each R f Independently hydroxyl, C 3-6 cycloalkyl or C 6-10 Aryl, for example C 3-6 cycloalkyl or C 6-10 Aryl.

[0068] In one of the schemes, L 2 Independently for -(CR) b1 R b2 ) n1 -, n1 is an integer from 4 to 15, the -(CR b1 R b2 ) n1 One or more of -(CR) b1 R b2 - The unit is independently and optionally controlled by R c2 Replacement.

[0069] In one particular scheme, R b1 and R b2 Independently H or C 1-6 Alkyl, preferably, R b1 and R b2 For H.

[0070] In one particular scheme, R c2 Independently selected from -O- C 3-10 A group consisting of one or more segments of cycloalkyl and 3-10 membered heterocycloalkyl groups; preferably, R c2 Independently selected from O-, C 3-6 A group consisting of one or more segments of cycloalkyl and 3-6 membered heterocycloalkyl groups.

[0071] In one particular scheme, R c2 It is a 3-6 membered heterocyclic alkyl group.

[0072] In one particular scheme, R c2 Independently selected from -N(R) e )-、-O- and A group consisting of one or more fragments; preferably, R c2 Independently selected from -O- and A group consisting of one or two segments.

[0073] In one particular scheme, Z 2 For CH.

[0074] In one particular scheme, R d1 and R d2 Independently for C 1-16 Alkyl; preferably, R d1 and R d2 Independently for C 1-10 alkyl.

[0075] In a certain scheme, each R e For H.

[0076] In a certain scheme, each R e It is a 3-10 membered heterocyclic alkyl group; preferably a 3-6 membered heterocyclic alkyl group.

[0077] In one particular scheme, R 1 For lipid fragments, R 1 For any of the following options:

[0078] Option 1:

[0079] R 0 For a single bond, R 1C 10-25 Alkyl, the C 10-25 One or more methylene units in the alkyl group are independently and optionally R c1 Replacement, R c1 Independently selected from -N(R) e -, -O-, -S-, -S(O2)- and A group consisting of one or more fragments; R e For H;

[0080] Option 2: R 0 For a single bond, R 1 for

[0081] L 1 The definition is as follows: L 1 -(CR) b1 R b2 ) n1 -, n1 is 1, 2, 3, 4, 5 or 6, the -(CR b1 R b2 ) n1 One or more of -(CR) b1 R b2 - The unit is independently and optionally controlled by R c2 Replacement, R b1 and R b2 Independently for H, R c2 It is a 3-6 membered heterocyclic alkyl group;

[0082] Z 1 Let N be the number of people in the group.

[0083] R a2 and R a3 Independently for C 2-25 straight-chain alkyl, Or by one or more R f Replacement C 2-25 Straight-chain alkyl groups; each of the C... 2-25 One or more methylene units in a straight-chain alkyl group are independently and optionally R c3 Substitute;

[0084] R c3 Independently selected from -N(R) e )-、-O- and A group consisting of one, two, or three segments;

[0085] R e For H;

[0086] Each R f Independently hydroxyl, C 3-6 cycloalkyl or C6-10 Aryl;

[0087] L 2 The definition is as follows:

[0088] L 2 Independently for -(CR) b1 R b2 ) n1 -, n1 is an integer from 4 to 15, the -(CR b1 R b2 ) n1 One or more of -(CR) b1 R b2 - The unit is independently and optionally controlled by R c2 Replacement, R b1 and R b2 For H, R c2 Independently selected from -N(R) e )-、-O- and A group consisting of one or more segments;

[0089] Z 2 For CH;

[0090] R d1 and R d2 Independently for C 1-16 alkyl;

[0091] Option 3: R 0 for R 1 C 10-25 alkyl or The C 10-25 One or more methylene units in the alkyl group are independently and optionally R c1 Replacement, R c1 Independently selected from -N(R) e -, -O-, -S-, -S(O2)- and A group consisting of one or more fragments; R e For H;

[0092] L 1 The definition is as follows: L 1 -(CR) b1 R b2 ) n1 -,

[0093] n1 is 1, 2, 3, 4, 5, or 6, and the -(CR) b1 R b2 ) n1 One or more of -(CR) b1 R b2- The unit is independently and optionally controlled by R c2 Replacement, R b1 and R b2 Independently for H, R c2 It is a 3-6 membered heterocyclic alkyl group.

[0094] Z 1 Let N be the number of people in the group.

[0095] R a2 and R a3 Independently for C 2-25 straight-chain alkyl, Or by one or more R f Replacement C 2-25 Straight-chain alkyl; the C 2-25 One or more methylene units in a straight-chain alkyl group are independently and optionally R c3 Substitute;

[0096] R c3 Independently selected from -N(R) e )-、-O- and A group consisting of one, two, or three segments;

[0097] R e For H;

[0098] Each R f Independently hydroxyl, C 3-6 cycloalkyl or C 6-10 Aryl;

[0099] L 2 The definition is as follows:

[0100] L 2 Independently for -(CR) b1 R b2 ) n1 -, n1 is an integer from 4 to 15, the -(CR b1 R b2 ) n1 One or more of -(CR) b1 R b2 - The unit is independently and optionally controlled by R c2 Replacement, R b1 and R b2 For H, R c2 Independently selected from -N(R) e )-、-O- and A group consisting of one or more segments;

[0101] Z 2 For CH;

[0102] Rd1 and R d2 Independently for C 1-16 alkyl.

[0103] In one particular scheme, R c1 Independently for -O-, Ideally, R c1 Independently for -O-, in The connection direction in lipid fragments is either left-to-right, consistent with the writing order, or right-to-left, opposite to the writing order; preferably, R c1 Independently

[0104] In one particular scheme, R c2 Independently in The connection direction in lipid fragments is independently either from left to right, consistent with the writing order, or independently from right to left, opposite to the writing order; preferably, R c2 Independently Or R c2 Independently for -O-, Better yet, R c2 for Or R c2 for

[0105] In one particular scheme, R c3 for in The connection direction in lipid fragments is either left-to-right, consistent with the writing order, or right-to-left, opposite to the writing order; more preferably, R c3 for

[0106] In one particular scheme, R f Independently hydroxyl,

[0107] In one of the schemes, L 1 Independently methyl, Preferably, the "2" position is related to Z. 1 connect.

[0108] In one of the schemes, L 1 Independently Preferably, the "2" position is related to Z. 1 connect.

[0109] In one of the schemes, L 2 Independently Preferably, the "2" position is related to Z. 2 connect.

[0110] In one particular scheme, R d1 and R d2 Independently

[0111] In one of the solutions, Independent as

[0112] In one particular scheme, R a1 Independently

[0113] In one particular scheme, R a1 for

[0114] In one particular scheme, R a2 and R a3 Independently

[0115] In one particular scheme, R a2 and R a3 Independently

[0116] In one of the solutions, Independently

[0117] In one of the solutions, Independently

[0118] In one particular scheme, R 1 For H,

[0119] In one particular scheme, R 1 for

[0120] In one particular scheme, R 2 For H,

[0121] In one particular scheme, R 2 It is an ethyl group.

[0122] In one particular scheme, R 3 It is either amino or chlorine.

[0123] In one particular scheme, R 3 It can be fluorine, bromine, or iodine.

[0124] In one particular scheme, R 4 For H.

[0125] In one particular scheme, R 5 For H or

[0126] In one particular scheme, R 4 R 5 Together with the atoms attached to it, they form

[0127] In one particular scheme, R 6 For H,

[0128] In one embodiment, the compound represented by Formula I is the compound represented by Formula I-1.

[0129] R 0 For single key or

[0130] R 1 It is a lipid fragment.

[0131] R 2 For H, C 1-5 Alkyl, C 1-5 Heteroalkyl, with an R t1 Replacement C 1-5 Alkyl or with an R t1 Replacement C 1-5 Heteroalkyl; each R t1 Independently hydroxyl, C 1-5 Alkyl or with an R t2 Replacement C 1-5 Alkyl; the C 1-5 The heteroatoms in the heteroalkyl group are independently selected from one, two, or three of N, O, and S, and the number of heteroatoms is one, two, or three;

[0132] R t2 It is a hydroxyl group;

[0133] R 3It is amino or halogen;

[0134] R 4 R 5 and R 6 Independently for H and C 3-6 Cycloalkyl or 3-6 membered heterocycloalkyl; or R 4 R 5 Together with the atoms bonded to it, they form C 6-10 Aryl or 5-10 heteroaryl groups;

[0135] The definition of the lipid fragment is as described in any of the previous schemes.

[0136] In one embodiment, in the compound shown in Formula I-1, R 1 C 10-25 Alkyl, the C 10-25 One or more methylene units in the alkyl group are independently and optionally R c1 Replacement, R c1 Independently selected from -N(R) e -, -O-, -S-, -S(O2)- and A group consisting of one or more fragments; R e For H;

[0137] R 2 R 3 R 4 and R 5 The definition is as described in the previous scheme.

[0138] In one embodiment, the compound shown in Formula I-1 is a compound shown in Formula I-1-A:

[0139] L 1 R a2 R a3 R 2 R 3 R 4 and R 5 The definition is as described in the previous scheme.

[0140] In one embodiment, the compound represented by Formula I is the compound represented by Formula I-1.

[0141] R 1 It is a lipid fragment;

[0142] R 2 For H, C 1-5 Alkyl, C 1-5 Heteroalkyl, with an R t1 Replacement C 1-5Alkyl or with an R t1 Replacement C 1-5 Heteroalkyl; each R t1 Independently hydroxyl, C 1-5 Alkyl or with an R t2 Replacement C 1-5 Alkyl; the C 1-5 The heteroatoms in the heteroalkyl group are independently selected from one, two, or three of N, O, and S, and the number of heteroatoms is one, two, or three;

[0143] R t2 It is a hydroxyl group;

[0144] R 3 It is amino or halogen;

[0145] R 4 For H;

[0146] R 5 and R 6 Independently, it is H or a 3-6 membered heterocyclic alkyl group;

[0147] Or R 4 R 5 Together with the atoms bonded to it, they form C 6-10 Aryl;

[0148] Preferably, the compound represented by Formula I is the compound represented by Formula I-1a.

[0149] R 3 It is amino or halogen;

[0150] R 1 For R a1 or

[0151] R a1 C 10-25 Alkyl; the C 10-25 One or more methylene units in the alkyl group are independently and optionally R c1 Substitute;

[0152] R c1 The selection is from -N(R) e )-、-O- and A group consisting of one or more segments;

[0153] L 1 -(CH2) n1 -, n1 is 1, 2, 3, 4, 5 or 6, the -(CH2) n1 One or more -(CH2)- units in R are independently and optionally controlled by R c2 Substitute;

[0154] R c2 For selected from -O- and A group consisting of one or two segments;

[0155] R a2 and R a3 Independently for C 2-25 alkyl or The C 2-25 One or more methylene units in the alkyl group are independently and optionally R c3 Substitute;

[0156] R c3 For selected from -O- and A group consisting of one or two segments;

[0157] L 2 -(CH2) n1 -, n1 is an integer between 4 and 15, the -(CH2) n1 One or more -(CH2)- units in R are independently and optionally controlled by R c2 Substitute;

[0158] R d1 and R d2 Independently for C 1-10 alkyl;

[0159] More preferably, the compound represented by Formula I is the compound represented by Formula I-1a.

[0160] R 3 It is amino or halogen;

[0161] R 1 for

[0162] L 1 -(CH2) n1 -, n1 is 1, 2, 3, 4, 5 or 6;

[0163] R a2 and R a3 Independently

[0164] L 2 -(CH2) n1 -, n1 is an integer between 4 and 15, the -(CH2) n1 One or more -(CH2)- units in R are independently and optionally controlled by R c2 Substitute; R c2 For selected from -O- and A group consisting of one or two segments;

[0165] R d1 and R d2 Independently for C 1-10 alkyl.

[0166] In one embodiment, the compound represented by Formula I is the compound represented by Formula I-2.

[0167] R 1 It is a lipid fragment;

[0168] R 2 For H, C 1-5 Alkyl, C 1-5 Heteroalkyl, with an R t1 Replacement C 1-5 Alkyl or with an R t1 Replacement C 1-5 Heteroalkyl; each R t1 Independently hydroxyl, C 1-5 Alkyl or with an R t2 Replacement C 1-5 Alkyl; the C 1-5 The heteroatoms in the heteroalkyl group are independently selected from one, two, or three of N, O, and S, and the number of heteroatoms is one, two, or three;

[0169] R t2 It is a hydroxyl group;

[0170] R 3 It is amino or halogen;

[0171] Better place,

[0172] R 2 For H or C 1-5 alkyl;

[0173] R 3 It is a halogen;

[0174] R 1 for

[0175] L 1 -(CH2) n1 -, n1 is 1, 2, 3, 4, 5 or 6, the -(CH2) n1 One or more -(CH2)- units in R are independently and optionally controlled by R c2 Substitute;

[0176] R c2 For selected from -O- and A group consisting of one or two segments;

[0177] R a2 and R a3 Independently for C 2-25 alkyl or The C 2-25 One or more methylene units in the alkyl group are independently and optionally R c3 Substitute;

[0178] R c3 For selected from -O- and A group consisting of one or two segments;

[0179] L 2 -(CH2) n1 -, n1 is an integer between 4 and 15, the -(CH2) n1 One or more -(CH2)- units in R are independently and optionally controlled by R c2 Substitute;

[0180] R d1 and R d2 Independently for C 1-10 alkyl.

[0181] In one embodiment, the compound represented by Formula I is the compound represented by Formula I-2.

[0182] R 1 R 2 R 3 Define any of the following schemes:

[0183] Option 1:

[0184] R 3 It is a halogen; R 1 It is a lipid fragment; the lipid fragment and R 2 The definition is as described in the previous scheme;

[0185] Option 2:

[0186] R 3 It is amino or halogen;

[0187] R 1 C 10-25 Alkyl, the C 10-25 One or more methylene units in the alkyl group are independently and optionally R c1 Replacement, R c1 Independently selected from -N(R) e -, -O-, -S-, -S(O2)- and A group consisting of one or more fragments; R e For H;

[0188] Or, R 1 for

[0189] L 1 The definition is as follows: L 1 -(CR) b1 R b2 ) n1 -,

[0190] n1 is 1, 2, 3, 4, 5, or 6, and the -(CR) b1 R b2 ) n1 One or more of -(CR) b1 R b2 - The unit is independently and optionally controlled by R c2 Replacement, R b1 and R b2 Independently for H, R c2 It is a 3-6 membered heterocyclic alkyl group;

[0191] Z 1 Let N be the number of people in the group.

[0192] R a2 and R a3 Independently for C 2-25 straight-chain alkyl, Or by one or more R f Replacement C 2-25 Straight-chain alkyl; the C 2-25 One or more methylene units in a straight-chain alkyl group are independently and optionally R c3 Substitute;

[0193] R c3 Independently selected from -N(R) e )-、-O- and A group consisting of one, two, or three segments;

[0194] R e For H;

[0195] Each R f Independently for C 3-6 cycloalkyl or C 6-10 Aryl;

[0196] L 2 The definition is as follows:

[0197] L 2 Independently for -(CR) b1 R b2 ) n1 -, n1 is an integer from 4 to 15, the -(CRb1 R b2 ) n1 One or more of -(CR) b1 R b2 - The unit is independently and optionally controlled by R c2 Replacement, R b1 and R b2 For H, R c2 Independently selected from -N(R) e )-、-O- and A group consisting of one or more segments;

[0198] Z 2 For CH;

[0199] R d1 and R d2 Independently for C 1-16 alkyl.

[0200] In one embodiment, the compound represented by Formula I is the compound represented by Formula I-3.

[0201] R 2 It is a lipid fragment; R 3 It is an amino group or a halogen.

[0202] In one embodiment, the compound represented by Formula I is a compound represented by Formula I-4.

[0203] R 6 It is a lipid fragment;

[0204] R 2 For H, C 1-5 Alkyl, C 1-5 Heteroalkyl, with an R t1 Replacement C 1-5 Alkyl or with an R t1 Replacement C 1-5 Heteroalkyl; each R t1 Independently hydroxyl, C 1-5 Alkyl or with an R t2 Replacement C 1-5 Alkyl; the C 1-5 The heteroatoms in the heteroalkyl group are independently selected from one, two, or three of N, O, and S, and the number of heteroatoms is one, two, or three;

[0205] Each R t2 For hydroxyl group; R 3 It is an amino group or a halogen.

[0206] In one embodiment, the compound is a conjugate as shown in Formula I-5.

[0207] R 2 Independently for H and C 1-5 Alkyl, C 1-5 Heteroalkyl, with an R t1 Replacement C 1-5 Alkyl or with an R t1 Replacement C 1-5 Heteroalkyl; each R t1 Independently hydroxyl, C 1-5 Alkyl or with an R t2 Replacement C 1-5 Alkyl; the C 1-5 The heteroatoms in the heteroalkyl group are independently selected from one, two, or three of N, O, and S, and the number of heteroatoms is one, two, or three;

[0208] R t2 It is a hydroxyl group;

[0209] R 3 It is amino or halogen;

[0210] R 4 For H;

[0211] R 5 and R 6 Independently, it is H or a 3-6 membered heterocyclic alkyl group;

[0212] Or R 4 R 5 Together with the atoms bonded to it, they form C 6-10 Aryl;

[0213] L 3 -(CR) b1 R b2 ) n1 -, n1 is an integer from 1 to 20, R b1 and R b2 Independently H, hydroxyl, C 1-6 Alkyl, C 3-10 Cycloalkyl or 3-10 membered heterocycloalkyl, wherein -(CR b1 R b2 ) n1 One or more of -(CR) b1 R b2 - The unit is independently and optionally controlled by R c2 Alternative; preferably, L 3 -(CH2) n1 -, n1 is an integer from 1 to 20, for example

[0214] In one embodiment, the compound of formula I is any one of the following compounds: a compound represented by formula HA-1, HA-1A, HA-11, HA-12, HA-13, HA-13-A, HA-14-C16, HA-14-C16-A, HA-15, HA-15A, HA-16, HA-17, HA-18, HA-18-1, HA-19, HA-19-A, HA-21, HA-22, HA-23, HA-30, HA-30-A, HA-31, HA-32, HA-33, HA-34, HA-35, HA-36, HA-37, HA-TLR-1, HA-TLR-1-A, HA-TLR-1-B, HA-TLRa-A, HA-TLR-2, HA-TLR-2-A, SM-102-HA, HA-1-1, HA-2-1, HA-3-1, 2HA-1, HA-1-4, HA-1-2, HA-1-3, HA-TLR-3, HA-TLR-4, HA-1-5, HA-1-6, HA-1-7, HA-1-8, HA-1-9, HA-1-10, HA-1-11, HA-1-12, HA-1-13, HA-1-14, HA-1-15, HA-1-15-1, HA-1-16, HA-1-17, HA-1-18, HA-TLRb, HA-TLRb-A, HA-38, HA-1A-1, HA-1C-1, HA-38-A, HA-1B-1, HA-1D-1, HA-1-9A, HA-1-9, HA-51A, HA-17B, HA-37-2A, HA-37-3A, HA-37-1A, HA-52A, HA-53B, HA-1-13B, HA-1A-1, HA-1A-R, HA-1A-S, HA-1B-1, HA-11-1, HA-17A, HA-1-10-A, HA-1-18-A, HA-31-A or HA-1B-1.

[0215] In one embodiment, the lipid carrier is used for the delivery of a nucleic acid drug selected from one or more of single-stranded deoxyribonucleic acid (DNA), double-stranded DNA, small interfering RNA (siRNA), self-replicating RNA (samRNA), microRNA (miRNA), circular RNA (circRNA), and messenger RNA (mRNA), such as messenger RNA (mRNA), or firefly luciferase (Fluc) mRNA, enhanced green fluorescent protein (eGFP) mRNA, and BNT113 encoding the human papillomavirus E7 antigen. The mRNA sequence may be an mRNA sequence encoding an infectious disease antigen or an mRNA sequence encoding a tumor antigen; preferably, the mRNA sequence encoding an infectious disease antigen may be an mRNA encoding respiratory syncytial virus antigen (RSV-mRNA), an mRNA encoding varicella-zoster virus antigen (VZV-mRNA), an mRNA encoding rabies virus antigen, an mRNA encoding Mycobacterium tuberculosis antigen, an mRNA encoding novel coronavirus antigen, or an mRNA encoding influenza virus antigen; the tumor may be a solid tumor, such as cervical cancer or non-small cell lung cancer; the liposome may also be used for tumor neoantigen therapy.

[0216] In one embodiment, the BNT113 mRNA encoding the human papillomavirus E7 antigen has the sequence shown in SEQ ID NO:1.

[0217] In one embodiment, the mRNA encoding the rabies virus antigen has a sequence as shown in SEQ ID NO:4.

[0218] In one embodiment, the lipid carrier is a lipid nanoparticle.

[0219] In one embodiment, the lipid carrier further includes one, two, three, or four of the following: lipid component Z, phospholipids, PEG lipids, and sterols; the lipid component Z is selected from one, two, or three of the following: ionizable lipids (positively charged at low pH, uncharged at neutral or high pH), anionic lipids, and cationic lipids (charged lipids, either negatively or positively charged); preferably, the lipid component Z is an ionizable lipid, such as ALC0315. Z can also be SM-102

[0220] In one embodiment, the lipid carrier further includes one, two, three, four, or five of the following: lipid component Z, diluent, phospholipid, PEG lipid, and sterol; the lipid component Z is selected from one, two, or three of the following: ionizable lipids (positively charged at low pH, uncharged at neutral or high pH), anionic lipids, and cationic lipids (anionic and cationic lipids are charged lipids, carrying either a negative or positive charge); preferably, the lipid component Z is an ionizable lipid, such as ALC0315. Z can also be SM-102

[0221] The diluent is a conventional diluent in the art. In one embodiment, the diluent is a citrate buffer, a phosphate buffer, or an acetate buffer; preferably an acetate buffer, such as an acetate buffer with a pH of 4.

[0222] The phospholipid is a conventional phospholipid in the art, preferably a neutral phospholipid. In one embodiment, it is an amphoteric accessory molecule that facilitates the fusion of lipid particles and cell membranes. The phospholipid may be a phospholipid molecule with a polar end and a nonpolar end of a fatty chain, such as distearylphosphatidylcholine (DSPC), dimyristoylphosphatidylcholine (DMPC), dioleoylphosphatidylcholine (DOPC), palmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylethanolamine (DOPE), distearylphosphatidylethanolamine (DSPE), distearylphosphatidylglycerol (DSPG), disqualylphosphatidylcholine (DEPC), palmitoylphosphatidylglycerol (DPPG), dipalmitoylphosphatidic acid (DPPA), docosanoylphosphatidylcholine (DUPC), or palmitoylphosphatidylcholine (POPC).

[0223] In one embodiment, the PEG lipid is a lipid molecule modified with a polyethylene glycol hydrophilic end. The PEG lipid is preferably selected from one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol, such as PEG-DSPE, PEG-DMG, or ALC-0159; the PEG-DSPE is preferably PEG-DSPE-2000.

[0224] The sterol is a conventional sterol in the art, and in one embodiment, the sterol is an animal, plant, or fungal sterol. The sterol is selected from one or more of cholesterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, and α-tocopherol, for example, cholesterol.

[0225] In one embodiment, the lipid carrier comprises any of the compounds, diluents, neutral phospholipids, and sterols described in any one of the present invention; preferably, the lipid carrier is composed of any of the compounds, diluents, neutral phospholipids, and sterols described in any one of the present invention.

[0226] In one embodiment, the lipid carrier comprises any of the compounds, diluents, neutral phospholipids, and PEG lipids described in any one of the present invention; preferably, the lipid carrier is composed of any of the compounds, diluents, neutral phospholipids, and PEG lipids described in any one of the present invention.

[0227] In one embodiment, the lipid carrier comprises any of the compounds described in any one of the present invention, a diluent, a neutral phospholipid, a PEG lipid, and a sterol; preferably, the lipid carrier is composed of any of the compounds described in any one of the present invention, a diluent, a neutral phospholipid, a PEG lipid, and a sterol.

[0228] In one embodiment, the lipid carrier comprises any of the compounds described in any one of the present invention, lipid component Z, neutral phospholipids, PEG lipids, and sterols; preferably, the lipid carrier is composed of any of the compounds described in any one of the present invention, lipid component Z, neutral phospholipids, PEG lipids, and sterols; more preferably, lipid component Z is an ionizable lipid.

[0229] In one embodiment, the lipid carrier comprises any of the compounds described in any one of the present invention, lipid component Z, diluent, neutral phospholipid, PEG lipid, and sterol; preferably, the lipid carrier is composed of any of the compounds described in any one of the present invention, lipid component Z, diluent, neutral phospholipid, PEG lipid, and sterol; more preferably, lipid component Z is an ionizable lipid.

[0230] In one embodiment, the ionizable lipid accounts for 30% to 80%, for example 50%, of the total molar amount of the lipid component Z, sterol, neutral phospholipid and PEG lipid in the lipid carrier.

[0231] In one embodiment, the anionic lipid accounts for 30% to 50% of the total molar amount of the lipid carrier, calculated based on the total molar amount of lipid components Z, sterols, neutral phospholipids, and PEG lipids in the lipid carrier, for example, 40%.

[0232] In one embodiment, the cationic lipid accounts for 30% to 50% of the total molar amount of the lipid carrier, calculated based on the total molar amount of lipid components Z, sterols, neutral phospholipids and PEG lipids in the lipid carrier, for example 40%.

[0233] In one embodiment, the phospholipid in the lipid carrier is calculated based on the total molar amount of lipid component Z, sterol, neutral phospholipid and PEG lipid in the lipid carrier, and the molar amount of the phospholipid is 5% to 20% of the total molar amount, for example 10%.

[0234] In one embodiment, the sterol accounts for 20% to 60% of the total molar amount of the lipid component Z, sterol, neutral phospholipid and PEG lipid in the lipid carrier, for example 38.5%.

[0235] In one embodiment, the PEG lipid accounts for 0.2% to 5% of the total molar amount of the lipid component Z, sterol, neutral phospholipid and PEG lipid in the lipid carrier, for example, 1.5%.

[0236] In one embodiment, the mass percentage of the compound described in any one of the present invention to the lipid component Z in the lipid carrier is 0.5%-60%, for example 1%-50%, or for example 1%, 2.5%, 5%, 10%, 20%, 30%, 40% or 50%, or for example 10%.

[0237] In one embodiment, the lipid carrier comprises the compound, ALC0315, distearate phosphatidylcholine, PEG-DMG-2000, and cholesterol, wherein the compound is...

[0238] In one embodiment, the lipid carrier comprises the compound, ALC0315, a diluent, distearate phosphatidylcholine, PEG-DMG-2000, and cholesterol, wherein the compound is...

[0239] Preferably, the percentage of the mass of the compound to the mass of the ALC0315 is 1%, 2.5%, 5%, or 10%.

[0240] The diluent is an acetate buffer solution with a pH of 4;

[0241] Calculated based on the total molar amounts of ALC0315, distearate phosphatidylcholine, PEG-DMG-2000, and cholesterol in the lipid carrier.

[0242] The molar amount of ALC0315 accounts for 50% of the total molar amount;

[0243] The molar amount of the distearate phosphatidylcholine accounts for 10% of the total molar amount;

[0244] The molar amount of cholesterol accounts for 38.5% of the total molar amount;

[0245] The molar amount of PEG-DMG-2000 accounts for 1.5% of the total molar amount.

[0246] In one embodiment, the lipid carrier comprises the compound, ALC0315, distearate phosphatidylcholine, PEG-DMG-2000, and cholesterol, wherein the compound is...

[0247] In one embodiment, the lipid carrier comprises the compound, ALC0315, a diluent, distearate phosphatidylcholine, PEG-DMG-2000, and cholesterol, wherein the compound is...

[0248] The percentage of the mass of the compound to the mass of the ALC0315 is 10%, 20%, 30%, 40%, or 50%.

[0249] The diluent is an acetate buffer solution with a pH of 4;

[0250] Calculated based on the total molar amounts of ALC0315, distearate phosphatidylcholine, PEG-DMG-2000, and cholesterol in the lipid carrier.

[0251] The molar amount of ALC0315 accounts for 50% of the total molar amount;

[0252] The molar amount of the distearate phosphatidylcholine accounts for 10% of the total molar amount;

[0253] The molar amount of cholesterol accounts for 38.5% of the total molar amount;

[0254] The molar amount of PEG-DMG-2000 accounts for 1.5% of the total molar amount.

[0255] In one embodiment, the lipid carrier comprises the compound, ALC0315, distearate phosphatidylcholine, PEG-DMG-2000, and cholesterol, wherein the compound is any of the following compounds: compounds of formula HA-1, HA-1A, HA-12, HA-14-C16, HA-14-C16-A, HA-TLR-1-A, HA-TLRa, HA-13-A, HA-15, HA-15A, HA-TLR-2, HA-TLR-2-A, HA-11, HA-TLRa-A, HA-TLR-1, and HA-TLR-1-B;

[0256] The mass percentage of the compound relative to the mass of ALC0315 is 10%;

[0257] Calculated based on the total molar amounts of ALC0315, distearate phosphatidylcholine, PEG-DMG-2000, and cholesterol in the lipid carrier.

[0258] The molar amount of ALC0315 accounts for 50% of the total molar amount;

[0259] The molar amount of the distearate phosphatidylcholine accounts for 10% of the total molar amount;

[0260] The molar amount of cholesterol accounts for 38.5% of the total molar amount;

[0261] The molar amount of PEG-DMG-2000 accounts for 1.5% of the total molar amount.

[0262] In one embodiment, the lipid carrier comprises the compound, ALC0315, a diluent, distearate phosphatidylcholine, PEG-DMG-2000, and cholesterol, wherein the compound is any of the following compounds: compounds of formula HA-1, HA-1A, HA-12, HA-14-C16, HA-14-C16-A, HA-TLR-1-A, HA-TLRa, HA-13-A, HA-15, HA-15A, HA-TLR-2, HA-TLR-2-A, HA-11, HA-TLRa-A, HA-TLR-1, and HA-TLR-1-B;

[0263] The mass percentage of the compound relative to the mass of ALC0315 is 10%;

[0264] The diluent is an acetate buffer solution with a pH of 4;

[0265] Calculated based on the total molar amounts of ALC0315, distearate phosphatidylcholine, PEG-DMG-2000, and cholesterol in the lipid carrier.

[0266] The molar amount of ALC0315 accounts for 50% of the total molar amount;

[0267] The molar amount of the distearate phosphatidylcholine accounts for 10% of the total molar amount;

[0268] The molar amount of cholesterol accounts for 38.5% of the total molar amount;

[0269] The molar amount of PEG-DMG-2000 accounts for 1.5% of the total molar amount.

[0270] In one embodiment, the lipid carrier comprises the compound, SM102, distearate phosphatidylcholine, PEG-DMG-2000, and cholesterol, wherein the compound is any of the following compounds: compounds of formula HA-1, HA-1A, HA-12, HA-14-C16, HA-14-C16-A, HA-TLR-1-A, HA-TLRa, HA-13-A, HA-15, HA-15A, HA-TLR-2, HA-TLR-2-A, HA-11, HA-TLRa-A, HA-TLR-1, HA-TLR-1-B;

[0271] The mass percentage of the compound relative to the mass of ALC0315 is 10%;

[0272] The diluent is an acetate buffer solution with a pH of 4;

[0273] Calculated based on the total molar amounts of ALC0315, distearate phosphatidylcholine, PEG-DMG-2000, and cholesterol in the lipid carrier.

[0274] The molar amount of ALC0315 accounts for 50% of the total molar amount;

[0275] The molar amount of the distearate phosphatidylcholine accounts for 10% of the total molar amount;

[0276] The molar amount of cholesterol accounts for 38.5% of the total molar amount;

[0277] The molar amount of PEG-DMG-2000 accounts for 1.5% of the total molar amount.

[0278] In one embodiment, the lipid carrier comprises the compound, SM102, distearate phosphatidylcholine, PEG-DMG-2000, and cholesterol, wherein the compound is any of the following compounds: such as those represented by the formula HA-1-10-A, HA-13, HA-17B, HA-30-A, HA-TLR-1, HA-TLR-1-A, HA-TLR-2, HA-TLR-2-A, HA-TLRa-A, HA-TLRb, HA-TLRb-A, HA-1, HA-11-1, HA-1A, HA-1B-1, HA-33, HA-34, HA-1-13B, HA-1-18-A, HA-17A, or HA-19-A;

[0279] The mass percentage of the compound relative to the mass of ALC0315 is 10%;

[0280] Calculated based on the total molar amounts of ALC0315, distearate phosphatidylcholine, PEG-DMG-2000, and cholesterol in the lipid carrier.

[0281] The molar amount of ALC0315 accounts for 50% of the total molar amount;

[0282] The molar amount of the distearate phosphatidylcholine accounts for 10% of the total molar amount;

[0283] The molar amount of cholesterol accounts for 38.5% of the total molar amount;

[0284] The molar amount of PEG-DMG-2000 accounts for 1.5% of the total molar amount.

[0285] In one embodiment, the lipid carrier comprises the compound, SM102, a diluent, distearate phosphatidylcholine, PEG-DMG-2000, and cholesterol, wherein the compound is any of the following compounds: such as those represented by the formula HA-1-10-A, HA-13, HA-17B, HA-30-A, HA-TLR-1, HA-TLR-1-A, HA-TLR-2, HA-TLR-2-A, HA-TLRa-A, HA-TLRb, HA-TLRb-A, HA-1, HA-11-1, HA-1A, HA-1B-1, HA-33, HA-34, HA-1-13B, HA-1-18-A, HA-17A, or HA-19-A;

[0286] The diluent is an acetate buffer solution with a pH of 4;

[0287] The mass percentage of the compound relative to the mass of ALC0315 is 10%;

[0288] Calculated based on the total molar amounts of ALC0315, distearate phosphatidylcholine, PEG-DMG-2000, and cholesterol in the lipid carrier.

[0289] The molar amount of ALC0315 accounts for 50% of the total molar amount.

[0290] The molar amount of the distearate phosphatidylcholine accounts for 10% of the total molar amount.

[0291] The molar amount of cholesterol accounts for 38.5% of the total molar amount.

[0292] The molar amount of PEG-DMG-2000 accounts for 1.5% of the total molar amount.

[0293] This invention provides a compound, said compound being a compound of formula II, a pharmaceutically acceptable salt thereof, or a conjugate thereof.

[0294] Among them, R 0 R 1 R2 R 3 R 4 R 5 and R 6 The definition is as described in any embodiment of this invention;

[0295] And the compound represented by Formula II satisfies any of the following conditions:

[0296] (1) When R 0 For a single bond, R 1 When it is a lipid fragment, the lipid fragment is R. a1 C 6-30 alkyl, Or by one or more R f Replacement C 6-30 Alkyl; and the C 6-30 One or more methylene units in an alkyl group are independently R c1 Substitute; R c1 For selected from --O-, -S-, -S(O2)-, C 3-10 Cycloalkyl, 3-10 membered heterocyclic alkyl, C 6-10 A group composed of one or more segments of aryl and 5-10 heteroaryl groups;

[0297] (2)R 0 for R 1 For lipid fragments, R 2 C 1-5 Alkyl, R 3 For amino group, R 4 R 5 and R 6 When H is independent, each R f Independently for C 1-6 Alkyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic alkyl, C 6-10 Aryl or 5-10 heteroaryl compounds.

[0298] The present invention also provides a lipid carrier comprising, as described in any of the preceding claims, a compound of formula I or II, a pharmaceutically acceptable salt thereof, or a conjugate thereof, and wherein the compound is not a...

[0299] The present invention also provides a liposomal drug comprising the nucleic acid as described above and the lipid carrier as described above.

[0300] In one embodiment, the nucleic acid is an mRNA encoding luciferase (Luc) or a BNT113 sequence.

[0301] In one embodiment, the nitrogen-to-phosphorus ratio in the liposomal drug is (2-30):1. This nitrogen-to-phosphorus ratio refers to the ratio of the number of moles of ionizable nitrogen atoms in one or more lipid compounds to the number of moles of phosphate groups in the RNA. In this application, when the lipid drug contains ionizable lipids (e.g., ALC0315), the nitrogen-to-phosphorus ratio refers to the ratio of the number of moles of ionizable nitrogen atoms in the ionizable lipids to the number of moles of phosphate groups in the mRNA. Preferably, the nitrogen-to-phosphorus ratio is (2-20):1, more preferably (3-20):1, for example (3-16):1, and even more preferably 6:1.

[0302] In one embodiment, the average particle size of the liposome drug is 40-150 nm, preferably 70-120 nm, such as 89 nm, 90 nm, 91 nm, 92 nm, 93 nm, 103 nm, 106 nm, 107 nm, 108 nm, 109 nm or 110 nm.

[0303] In one embodiment, the average particle size of the liposomal drug is 40-200 nm.

[0304] In one embodiment, the polydispersity index of the liposomal drug is 0.1-0.3, for example, 0.1449, 0.1665, 0.1724, 0.1808, 0.1851, 0.1962, 0.2073, 0.2133, 0.2824, 0.2902 or 0.3142.

[0305] In one embodiment, the encapsulation efficiency of the liposomal drug is 85%-100%, for example 89.23%, 89.71%, 90.35%, 90.36%, 90.38%, 92.79%, 93.42%, 96.53%, 97.14%, 97.56%, or 97.59%.

[0306] In one embodiment, the liposomal drug contains a lipocarrier that encapsulates the nucleic acid as described above.

[0307] Unless otherwise specified, the terms used in this invention have the following meanings:

[0308] Those skilled in the art will understand that, according to conventions used in the art, the structural formulas of the groups described in this invention are... This refers to the fact that the corresponding group is connected to other fragments or groups in the compound through this site.

[0309] In this article, the single dash "-" on both sides of the substituent indicates that the substituent is connected to the parent part by a chemical bond.

[0310] The term "multiple" refers to 2, 3, 4 or 5, preferably 2 or 3.

[0311] The term "pharmaceutically acceptable" means that the salts, solvents, excipients, etc., are generally non-toxic, safe, and suitable for patient use. The term "patient" preferably refers to a mammal, and more preferably a human.

[0312] The term "pharmaceutically acceptable salt" refers to a salt prepared from the compound of the present invention with a relatively non-toxic, pharmaceutically acceptable acid or base.

[0313] When any variable (e.g., R) f When a variable appears multiple times in the definition of a compound, the definition at each position is independent of the definitions at the other positions; their meanings are independent and do not affect each other. Therefore, if a group is surrounded by one, two, or three R... f Group substitution, meaning that the group can be replaced by up to 3 R groups. f Replace, the position R f Definition and other positions R f The definitions are independent of each other. Furthermore, combinations of substituents and / or variables are only permitted if the combination produces a stable compound.

[0314] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0315] The term "alkyl" refers to a straight-chain or branched saturated alkyl group having a specified number of carbon atoms. For example, C 1-30 Alkyl, C 5-30 C 1-6 C 1- 10 Alkyl groups, examples of which include methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl and similar alkyl groups.

[0316] "Heteroalkyl" refers to a saturated alkyl group in which the methylene group (-CH2-) is replaced by a heteroatom (such as O, S, N), including straight-chain or branched heteroalkyl groups; C1-C5 heteroalkyl refers to an alkyl group containing 1-5 carbon atoms in which at least one methylene group is replaced by a heteroatom.

[0317] The term "cycloalkyl" refers to a saturated cyclic alkyl group having a specified number of ring carbon atoms, preferably a saturated cyclic alkyl group having 3-10 ring carbon atoms, more preferably 3-6 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl.

[0318] The term "heterocyclic alkyl" refers to a cyclic group having a specified number of ring atoms (e.g., 3-10), a specified number of heteroatoms (e.g., 1, 2 or 3), and a specified type of heteroatom (1, 2 or 3 of N, O, S and S(O2), preferably 1, 2 or 3 of N, O and S), and each ring is saturated.

[0319] "Aryl" refers to a polycyclic group consisting of an all-carbon monocyclic or fused polycyclic (i.e., a ring sharing adjacent carbon atom pairs) and having a conjugated π-electron system. "6-10 aryl" refers to an all-carbon aryl group containing 6-10 carbon atoms, such as phenyl and naphthyl; phenyl is preferred.

[0320] The term "heteroaryl" refers to a monocyclic or bicyclic aromatic group containing a specified number of ring atoms (e.g., 5 to 10, 5 to 6), a specified number of heteroatoms (e.g., 1, 2 or 3), and a specified type of heteroatom (1, 2 or 3 of N, O and S).

[0321] The positive and progressive effects of this invention are as follows:

[0322] This invention relates to a lipid compound containing a TLR7 (Toll-like receptor 7) activator structure and its applications. TLR7 is a key receptor in the innate immune system, capable of recognizing and responding to pathogen-associated molecular patterns and activating the immune system. By integrating a TLR7 activator into an LNP, we aim to further enhance the immunogenicity of LNP-delivered mRNA. This novel lipid compound not only enhances T-cell immune responses but also provides a more potent and durable anti-tumor effect by activating the innate immune system.

[0323] This invention innovatively integrates TLR activators into lipid nanoparticles, enhancing not only the immunogenicity of mRNA tumor vaccines but also the immune response to mRNA infectious disease vaccines. The widespread use of adjuvants in infectious disease vaccines, tumor vaccines, and tumor therapy demonstrates their significant potential in enhancing immune responses and improving therapeutic efficacy. These technological innovations provide a solid foundation for the future development of vaccines and immunotherapies, and are expected to significantly improve clinical treatment outcomes. Detailed Implementation

[0324] The present invention will be further illustrated by way of embodiments below, but the present invention is not limited to the scope of the embodiments described herein.

[0325] Example 1:

[0326] Step 1:

[0327] Compound 1 (3 g, 12.35 mmol, 1.0 eq.) was dissolved in DCM (400 mL), followed by the addition of compound 2 (1.98 g, 12.35 mmol, 1.0 eq.) and TEA (3.74 g, 37.04 mmol, 3.0 eq.). The reaction mixture was stirred at 70 °C for 2 hours under a nitrogen atmosphere. Thin-layer chromatography (PE:EA = 5:1) showed the reaction was complete, with a new major spot observed. The mixture was quenched with H2O (100 mL) and extracted with EA (100 mL × 3). The organic layers were combined, washed with saturated brine (2 × 100 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (0%–30% EA in PE) to give compound 3 (4 g, 88.9%) as a yellow solid.

[0328] LCMS:Rt:2.063min;MS m / z(ELSD):367.2[M+H] + .

[0329] Step 2

[0330] Compound 1 (4 g, 10.93 mmol, 1.0 eq.) was dissolved in EtOH / H₂O (40 mL), followed by the addition of Fe (1.84 g, 32.79 mmol, 3.0 eq.) and DIEA (1.77 g, 32.79 mmol, 3.0 eq.). The reaction mixture was stirred at 70 °C for 2 hours under a nitrogen atmosphere. Thin-layer chromatography (PE:EA = 2:1) showed the reaction was complete, with a new major spot observed. The mixture was filtered, and the filter cake was washed with MeOH (150 mL). The organic layers were combined, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (0%–60% EA in PE) to give compound 4 (2.75 g, 74.9%) as a yellow solid.

[0331] LCMS:Rt:1.523min;MS m / z(ELSD):337.2[M+H] + ;

[0332] 1H NMR(400MHz, CDCl3)δ7.90(d,J=8.2Hz,1H),7.82(d,J=8.2Hz,1H),7.51–7.39 (m,2H),4.92(s,1H),4.29-3.65(m,3H),3.42(d,J=25.6Hz,4H),1.47(s,9H).

[0333] Step 3

[0334] A solution of compound 4 (2.75 g, 8.18 mmol, 1.0 eq.) and CH(OEt)3 (2.4 g, 16.37 mmol, 2.0 eq.) was stirred at 80 °C for 16 h under a nitrogen atmosphere. Thin-layer chromatography (DCM:MeOH = 10 / 1) showed the reaction was complete, and a new major spot was observed. The mixture was quenched with H2O (100 mL) and extracted with EA (50 mL × 3). The organic layers were combined, washed with saturated brine (2 × 50 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluting with 10% MeOH in DCM) to give compound 5 (2.1 g, 74.2%) as a pale yellow solid.

[0335] LCMS:Rt:1.464min; MS m / z(ELSD):347.2[M+H] + ;

[0336] 1H NMR (400MHz, CDCl3) δ8.21-8,16m,2H),7.90(s,1H),7.76–7.63(m,2H),5.05(s,1H),4.83-4.75(m,2H),3.73-3.69(m,2H),1.46(s,9H).

[0337] Step 4:

[0338] A mixture of compound 5 (2.1 g, 6.25 mmol, 1.0 eq.) and NH3 (30 mL, NH3 in MeOH, 7 M.) was stirred at 140 °C for 48 h. The desired product was obtained by thin-layer chromatography (DCM:MeOH = 10 / 1). The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluting with 10% MeOH in DCM) to give compound 6 (900 mg, 45.5%) as a pale yellow solid.

[0339] LCMS:Rt:0.968min;MS m / z(ELSD):328.2[M+H] + ;

[0340] Step 5:

[0341] Compound 6 (900 mg, 2.75 mmol, 1.0 eq.) was dissolved in DCM (10 mL), and TFA (10 mL) was added at 0 °C and the reaction was carried out under a nitrogen atmosphere. The reaction was allowed to warm to room temperature and stirred for 2 hours. The reaction mixture was quenched with TEA and the pH was adjusted to 8. The mixture was then concentrated under vacuum. The residue was purified by silica gel column chromatography (eluting with 10% MeOH in DCM) to give compound 7 (610 mg, 96.4%) as a white solid.

[0342] LCMS:Rt:1.050min;MS m / z(ELSD):228.1[M+H] + ;

[0343] Step 6:

[0344] Compound 7 (210 mg crude, 0.93 mmol, 1.0 eq.) and compound 8 (553 mg, 1.20 mmol, 1.3 eq.) were dissolved in MeCN (10.0 mL), and K₂CO₃ (383 mg, 2.78 mmol, 3.0 eq.) and KI (154 mg, 0.93 mmol, 1.0 eq.) were added. The mixture was stirred at 90 °C for 16 h under an Ar atmosphere. TLC (DCM:MeOH = 10 / 1) showed that the reaction was complete, and a new major spot was observed. The reaction mixture was quenched with H₂O (50 mL) and extracted with EA (50 mL × 3). The combined organic layers were washed with brine (2 × 100 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography and eluted with 10% MeOH in DCM to give compound 9 (230 mg, 40.9%) as a yellow oil.

[0345] LCMS:Rt:1.301min;MS m / z(ELSD):608.5[M+H] + ;

[0346] Step 7:

[0347] Compound 9 (230 mg, 0.38 mmol, 1.0 eq.) and compound 10 (713 mg, 1.89 mmol, 5.0 eq.) were dissolved in MeCN (10.0 mL), and K₂CO₃ (157 mg, 1.14 mmol, 3.0 eq.) and KI (63 mg, 0.38 mmol, 1.0 eq.) were added. The mixture was stirred at 90 °C for 16 hours under an Ar atmosphere. TLC (DCM:MeOH = 10 / 1) showed that the reaction was complete, and a new major spot was observed. The mixture was quenched with H₂O (30 mL) and extracted with EA (30 mL × 3). The combined organic layers were washed with saturated brine (2 × 100 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography, eluted with 10% MeOH in DCM, to give a crude product as a yellow oil. The residue was purified by preparative high performance liquid chromatography (PreP-HPLC) to obtain HA-1 (33.85 mg, 4.9%) as a yellow oil.

[0348] LCMS: MS m / z (ELSD): 904.8 [M+H] + ;

[0349] 1 H NMR (400MHz, CDCl3) δ8.02–7.89(m,3H),7.66–7.58(m,1H),7.49–7.42(m,1H),4.92–4.75(m,2H),4.56(t,J=6.0Hz,2H),2. 93(t,J=6.0Hz,2H),2.46–2.37(m,4H),2.32–2.22(m,4H),1.61–1.41(m,12H),1.33–1.15(m,53H),0.87(t,J=7.2Hz,12H).

[0350] Example 2:

[0351] Step 1:

[0352] Compound 1 (3 g, 12.35 mmol, 1.0 eq.) was dissolved in DCM (400 mL), and compound 2 (1.98 g, 12.35 mmol, 1.0 eq.) and TEA (3.74 g, 37.04 mmol, 3.0 eq.) were added. The reaction mixture was stirred at 70 °C for 2 h under a nitrogen atmosphere. TLC (PE:EA = 5:1) showed that the reaction was complete, and a new major spot was observed. The mixture was quenched with H2O (100 mL) and extracted with EA (100 mL × 3). The combined organic layers were washed with saturated brine (2 × 100 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (0%–30% EA in PE) to give compound 3 (4 g, 88.9%) as a yellow solid.

[0353] LCMS:Rt:2.063min;MS m / z(ELSD):367.2[M+H] + ;

[0354] Step 2:

[0355] Compound 1 (4 g, 10.93 mmol, 1.0 eq.) was dissolved in EtOH / H₂O (40 mL), and Fe (1.84 g, 32.79 mmol, 3.0 eq.) and DIEA (1.77 g, 32.79 mmol, 3.0 eq.) were added. The reaction mixture was stirred at 70 °C for 2 h under a N₂ atmosphere. TLC (PE:EA = 2:1) showed that the reaction was complete, and a new major spot was observed. The mixture was filtered, and the filter cake was washed with MeOH (150 mL). The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (0%–60% EA in PE) to give compound 4 (2.75 g, 74.9%) as a yellow solid.

[0356] LCMS:Rt:1.523min;MS m / z(ELSD):337.2[M+H] + ;

[0357] 1H NMR(400MHz, CDCl3)δ7.90(d,J=8.2Hz,1H),7.82(d,J=8.2Hz,1H),7.51–7.39 (m,2H),4.92(s,1H),4.29-3.65(m,3H),3.42(d,J=25.6Hz,4H),1.47(s,9H).

[0358] Step 3:

[0359] Compound 4 (2.75 g, 8.18 mmol, 1.0 eq.) and CH(OEt)3 (2.4 g, 16.37 mmol, 2.0 eq.) were dissolved and stirred at 80 °C for 16 h under a nitrogen atmosphere. TLC (DCM:MeOH = 10 / 1) showed that the reaction was complete, and a new major spot was observed. The mixture was quenched with H2O (100 mL) and extracted with EA (50 mL × 3). The combined organic layers were washed with saturated brine (2 × 50 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography and eluted with 10% MeOH in DCM to give compound 5 (2.1 g, 74.2%) as a pale yellow solid.

[0360] LCMS:Rt:1.464min; MS m / z(ELSD):347.2[M+H] + ;

[0361] 1H NMR (400MHz, CDCl3) δ8.21-8,16m,2H),7.90(s,1H),7.76–7.63(m,2H),5.05(s,1H),4.83-4.75(m,2H),3.73-3.69(m,2H),1.46(s,9H).

[0362] Step 4:

[0363] Compound 5 (620 mg, 1.79 mmol, 1.0 eq.) was dissolved in DCM (10 mL), and then hydrochloric acid (5 mL) was added at 0 °C. The reaction was allowed to return to room temperature and stirred for 1 hour. The reaction was monitored for completion by LCMS. The reaction mixture was concentrated under reduced pressure to give some solid, which was then ground with EtOAc (10 mL). The mixture was filtered, and the filter cake was washed with EtOAc (5 mL). The solid was collected and dried under reduced pressure to give compound 6 (450 mg crude).

[0364] LCMS:Rt:1.030min; MS m / z:247.1[M+H] + ;

[0365] Step 5:

[0366] Compound 6 (450 mg crude, 1.83 mmol, 1.0 eq.) and compound 7 (842 mg, 1.83 mmol, 1.0 eq.) were dissolved in MeCN (10.0 mL), and K2CO3 (758 mg, 5.49 mmol, 3.0 eq.) and KI (304 mg, 1.83 mmol, 1.0 eq.) were added. The mixture was stirred at 90 °C for 16 hours under an Ar atmosphere. TLC (DCM:MeOH = 10 / 1) showed that the reaction was complete, and a new major spot was observed. The mixture was quenched with H2O (30 mL) and extracted with EA (30 mL × 3). The combined organic layers were washed with saturated brine (3 × 50 mL), dried on anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give compound 8 (230 mg, 20.5%) as a yellow oil.

[0367] LCMS:Rt:1.330min;MS m / z(ELSD):627.6[M+H] + ;

[0368] Step 6:

[0369] Compound 8 (230 mg, 0.37 mmol, 1.0 eq.) and compound 9 (691 mg, 1.84 mmol, 5.0 eq.) were dissolved in MeCN (10.0 mL), and K2CO3 (153 mg, 1.10 mmol, 3.0 eq.) and KI (61 mg, 0.37 mmol, 1.0 eq.) were added. The mixture was stirred at 90 °C for 16 hours under an Ar atmosphere. TLC (DCM:MeOH = 10 / 1) showed that the reaction was complete, and a new major spot was observed. The mixture was quenched with H2O (30 mL) and extracted with EA (30 mL × 3). The combined organic layers were washed with saturated brine (3 × 50 mL), dried on anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give a yellow oily crude product. The residue was purified by preparative high performance liquid chromatography (PreP-HPLC) to give HA-1A (33.85 mg, 4.9%) as a yellow oil.

[0370] LCMS:Rt:2.384min;MS m / z(ELSD):923.7[M+H] + ;

[0371] CAD: 94.30% purity

[0372] 1H NMR (400MHz, CDCl3) δ8.25–8.18(m,2H),8.13(s,1H),7.79–7.76(m,2H),5.22–4.66(m,4H),3.42–3.10(m,2H),3. 02–2.58(m,4H),2.30–2.24(m,4H),1.61–1.54(m,6H),1.53–1.47(m,8H),1.31–1.21(m,51H),0.90–0.85(m,12H).

[0373] Example 3:

[0374] Step 1:

[0375] Compound 1 (3 g, 12.35 mmol, 1.0 eq.) was dissolved in DCM (400 mL), and compound 2 (1.98 g, 12.35 mmol, 1.0 eq.) and TEA (3.74 g, 37.04 mmol, 3.0 eq.) were added. The reaction mixture was stirred at 70 °C for 2 h under a nitrogen atmosphere. TLC (PE:EA = 5:1) showed that the reaction was complete, and a new major spot was observed. The mixture was quenched with H2O (100 mL) and extracted with EA (100 mL × 3). The combined organic layers were washed with saturated brine (2 × 100 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (0%–30% EA in PE) to give compound 3 (4 g, 88.9%) as a yellow solid.

[0376] LCMS:Rt:2.063min;MS m / z(ELSD):367.2[M+H] + ;

[0377] Step 2

[0378] A solution of compound 1 (4 g, 10.93 mmol, 1.0 eq.) was added to EtOH / H₂O (40 mL), followed by Fe (1.84 g, 32.79 mmol, 3.0 eq.) and DIEA (1.77 g, 32.79 mmol, 3.0 eq.). The reaction mixture was stirred at 70 °C for 2 hours under a nitrogen atmosphere. Thin-layer chromatography (PE:EA = 2:1) showed that the reaction was complete and a new major spot was observed. The mixture was filtered, and the filter cake was washed with MeOH (150 mL). The combined organic phases were dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (0%–60% EA in PE) to give compound 4 (2.75 g, 74.9%) as a yellow solid.

[0379] LCMS:Rt:1.523min;MS m / z(ELSD):337.2[M+H] + ;

[0380] 1H NMR(400MHz, CDCl3)δ7.90(d,J=8.2Hz,1H),7.82(d,J=8.2Hz,1H),7.51–7.39 (m,2H),4.92(s,1H),4.29-3.65(m,3H),3.42(d,J=25.6Hz,4H),1.47(s,9H).

[0381] Step 3

[0382] A solution of compound 4 (2.75 g, 8.18 mmol, 1.0 eq.) and CH(OEt)3 (2.4 g, 16.37 mmol, 2.0 eq.) were stirred at 80 °C for 16 h under a nitrogen atmosphere. Thin-layer chromatography (DCM:MeOH = 10 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was terminated with H2O (100 mL) and extracted with EA (50 mL × 3). The combined organic phases were washed with brine (2 × 50 mL), dried, filtered through anhydrous Na2SO4, and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography and eluted with 10% MeOH in DCM to give compound 5 (2.1 g, 74.2%) as a pale yellow solid.

[0383] LCMS:Rt:1.464min; MS m / z(ELSD):347.2[M+H] + ;

[0384] 1H NMR (400MHz, CDCl3) δ8.21-8,16m,2H),7.90(s,1H),7.76–7.63(m,2H),5.05(s,1H),4.83-4.75(m,2H),3.73-3.69(m,2H),1.46(s,9H).

[0385] Step 4

[0386] A mixture of compound 5 (2.1 g, 6.25 mmol, 1.0 eq.) and NH3 (30 mL, NH3 in MeOH, 7 M) was stirred at 140 °C for 48 hours. Thin-layer chromatography (DCM:MeOH = 10 / 1) showed the desired product. The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with 10% MeOH in DCM, to give compound 6 (900 mg, 45.5%) as a pale yellow solid.

[0387] LCMS:Rt:0.968min;MS m / z(ELSD):328.2[M+H] + ;

[0388] Step 5

[0389] A mixture of compound 6 (900 mg, 2.75 mmol, 1.0 eq.) was dissolved in DCM (10 mL), and then TFA (10 mL) was added at 0 °C. The reaction was carried out under a nitrogen atmosphere. The reaction was allowed to warm to room temperature and stirred for 2 hours. The reaction mixture was terminated with TEA and adjusted to pH 8. The mixture was then concentrated under vacuum. The residue was purified by silica gel column chromatography, eluting with 10% MeOH in DCM, to give compound 7 (610 mg, 96.4%) as a white solid.

[0390] LCMS:Rt:1.050min;MS m / z(ELSD):228.1[M+H] + ;

[0391] Step 6:

[0392] A solution of compound 7 (150 mg, 0.66 mmol, 1.0 eq.) was dissolved in MeCN / CPME (3 mL, 1:1 v / v), followed by the addition of compound 8 (450 mg, 0.99 mmol, 1.5 eq.), KI (219 mg, 1.32 mmol, 2.0 eq.), and K₂CO₃ (456 mg, 3.33 mmol, 5.0 eq.). The mixture was stirred at 90 °C for 36 hours under a nitrogen atmosphere. Thin-layer chromatography (DCM:MeOH = 10 / 1) showed that the reaction was complete, and a new major spot was observed. The reaction was terminated with H₂O (50 mL) and extracted with EA (50 mL × 3). The combined organic phases were washed with brine (3 × 50 mL), dried, filtered through anhydrous Na₂SO₄, and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography and eluted with 10% MeOH in DCM to give compound 9 (210 mg, 52.3%) as a yellow oil.

[0393] LCMS:Rt:1.299min; MS m / z(ELSD):608.6[M+H] + ;

[0394] Step 7:

[0395] A solution of compound 9 (210 mg, 0.15 mmol, 1.0 eq.) was dissolved in MeCN / CPME (3 mL, 1:1 v / v), followed by the addition of compound 10 (240 mg, 0.22 mmol, 1.5 eq.), KI (114 mg, 0.22 mmol, 1.5 eq.), and K₂CO₃ (239 mg, 0.65 mmol, 5.0 eq.). The mixture was stirred at 90 °C for 36 hours under a nitrogen atmosphere. Thin-layer chromatography (DCM:MeOH = 10 / 1) showed that the reaction was complete, and a new major spot was observed. The reaction was terminated with H₂O (50 mL) and extracted with EA (50 mL × 3). The combined organic phases were washed with brine (3 × 50 mL), dried, filtered through anhydrous Na₂SO₄, and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography and eluted with 10% MeOH in DCM to give HA-11 (54.45 mg, 17.9%) as a yellow oil.

[0396] LCMS:Rt:1.789min;MS m / z(ELSD):876.8[M+H] + ;

[0397] CAD:91.2% purity at ELSD; RT=21.070min.

[0398] 1 H NMR(400MHz, CDCl3)δ7.98(d,J=8.1Hz,1H),7.88–7.82(m,2H),7.55(t,J=7.6Hz,1H ),7.34(t,J=7.5Hz,1H),5.73(s,2H),4.86(dd,J=12.5,6.2Hz,1H),4.54(t,J=6.1Hz ,2H),4.05(t,J=6.8Hz,2H),2.93(t,J=6.1Hz,2H),2.41(dd,J=12.3,6.6Hz,4H),2. 27(td,J=7.5,3.5Hz,4H),1.63–1.54(m,6H),1.37–1.13(m,56H),0.91–0.85(m,9H).

[0399] Example 4:

[0400] Step 1:

[0401] A solution of compound 1 (i.e., compound 7 in Example 1) (200 mg, 0.88 mmol, 1.0 eq) was dissolved in MeCN / CPME (3 mL, 1:1 = v / v), followed by the addition of compound 2 (460 mg, 1.32 mmol, 1.5 eq.), KI (293 mg, 1.76 mmol, 2.0 eq.), and K₂CO₃ (608 mg, 4.4 mmol, 5.0 eq.). The mixture was stirred at 90 °C for 36 hours under a nitrogen atmosphere. Thin-layer chromatography (DCM:MeOH = 10 / 1) showed that the reaction was complete, and a new major spot was observed. The reaction was terminated with H₂O (30 mL) and extracted with EA (30 mL × 3). The combined organic phases were washed with brine (2 × 50 mL), dried, filtered through anhydrous Na₂SO₄, and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography and eluted with 10% MeOH in DCM to give compound 3 (81 mg, 19%) as a yellow oil.

[0402] LCMS:Rt:0.897min; MS m / z(ELSD):490.4[M+H] + ;

[0403] Step 2:

[0404] A solution of compound 3 (81 mg, 0.16 mmol, 1.0 eq.) was dissolved in MeCN / CPME (2 mL, 1:1 v / v), followed by the addition of compound 4 (86 mg, 0.25 mmol, 1.5 eq.), KI (41 mg, 0.25 mmol, 1.5 eq.), and K₂CO₃ (90 mg, 0.65 mmol, 5.0 eq.). The mixture was stirred at 90 °C for 36 hours under a nitrogen atmosphere. Thin-layer chromatography (DCM:MeOH = 10 / 1) showed that the reaction was complete, and a new major spot was observed. The reaction was terminated with H₂O (30 mL) and extracted with EA (30 mL × 3). The combined organic phases were washed with brine (3 × 50 mL), dried, filtered through anhydrous Na₂SO₄, and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography and eluted with 10% MeOH in DCM to give HA-12 (25.49 mg, 20%) as a yellow oil.

[0405] LCMS:Rt:1.171min;MS m / z(ELSD):764.7[M+H] + ;

[0406] CAD:91.87% purity at ELSD; RT=16.464min.

[0407] 1 H NMR (400MHz, CDCl3) δ7.97(d,J=8.0Hz,1H),7.89–7.81(m,2H),7.55(t,J=7.6Hz,1H),7.34(t,J=7.6Hz,1H),5.77(s,2H),4.54(t,J=6.0Hz,2H),4. 06(t,J=6.8Hz,4H),2.92(t,J=6.0Hz,2H),2.44–2.37(m,4H),2.28(t,J= 7.6Hz, 4H), 1.65–1.54 (m, 8H), 1.33–1.15 (m, 40H), 0.88 (t, J = 6.8Hz, 6H).

[0408] Example 5:

[0409] Step 1:

[0410] A solution of compound 1 (5 g, 16.61 mmol, 1.0 eq.) was dissolved in DCM (50 mL), followed by the addition of compound 2 (3.6 g, 24.92 mmol, 1.5 eq.), EDCI (6.3 g, 33.22 mmol, 2.0 eq.), DMAP (810 mg, 6.84 mmol, 0.4 eq.), and DIEA (8.6 g, 66.44 mmol, 4.0 eq.). The reaction mixture was stirred at 25 °C for 16 hours under a nitrogen atmosphere. Thin-layer chromatography (PE:EA = 10:1) showed that the reaction was complete, and a new major spot was observed. The mixture was terminated with H2O (200 mL) and extracted with EA (100 mL × 3). The combined organic phases were washed with brine (2 × 200 mL), dried, filtered through anhydrous Na2SO4, and concentrated under reduced pressure. The residue was purified by column chromatography (0%–10% EA in PE) to give compound 3 (6.9 g, 97%) as a yellow oil.

[0411] LCMS:Rt:2.017min; MS m / z(ELSD):328.3[M-100+H] + ;

[0412] 1H NMR (400MHz, CDCl3) δ4.49(s,1H),4.05(t,J=6.7Hz,2H),3.10(t,J=6.9Hz,2H),2.29(t, J=7.5Hz,2H),1.66–1.58(m,4H),1.45(s,8H),1.36–1.21(m,28H),0.87(d,J=7.1Hz,3H).

[0413] Step 2:

[0414] A solution of compound 3 (6.9 g, 16.16 mmol, 1.0 eq.) was dissolved in DCM (15 mL), followed by the addition of TFA (15 mL). The reaction mixture was stirred at 25 °C for 2 hours under a nitrogen atmosphere. Thin-layer chromatography (DCM:MeOH = 15:1) showed that the reaction was complete, and a new major spot was observed. The reaction was terminated with hydrated NaHCO3 (100 mL) and extracted with EA (100 mL × 3). The combined organic phases were washed with brine (2 × 100 mL), dried, filtered through anhydrous Na2SO4, and concentrated under reduced pressure to give crude product 4 (3.2 g) as a yellow solid.

[0415] LCMS:Rt:1.988min;MS m / z(ELSD):328.3[M+H] + ;

[0416] Step 3:

[0417] A solution of compound 5 (1.5 g, 6.20 mmol, 1.0 eq.) was dissolved in DCM (15 mL), followed by the addition of compound 4 (2.02 g, 6.20 mmol, 1.0 eq.) and TEA (1.9 g, 18.60 mmol, 3.0 eq.). The reaction mixture was stirred at 70 °C for 2 hours under a nitrogen atmosphere. Thin-layer chromatography (PE:EA = 15:1) showed that the reaction was complete and a new major spot was observed. The mixture was terminated with H2O (100 mL) and extracted with EA (100 mL × 3). The combined organic phases were washed with brine (2 × 100 mL), dried, filtered through anhydrous Na2SO4, and concentrated under reduced pressure. The residue was purified by column chromatography (0%–40% EA in PE) to give compound 6 (2.5 g, 75.6%) as a yellow solid.

[0418] LCMS:Rt:2.11min;MS m / z(ELSD):534.47[M+H] + ;

[0419] 1H NMR (400MHz, CDCl3) δ7.93 (d, J = 8.6Hz, 2H), 7.75 (t, J = 7.7Hz, 1H), 7.54 (t, J = 7.7Hz, 1H), 6.03 (s, 1H), 4.05 (t, J = 6. 7Hz,2H),3.47(t,J=5.0Hz,2H),2.29(t,J=7.5Hz,2H),1.80–1.56(m,8H),1.48–1.19(m,24H),0.88(t,J=6.8Hz,3H).

[0420] Step 4:

[0421] A solution of compound 6 (2.5 g, 4.69 mmol, 1.0 eq.) was dissolved in EtOH / H₂O (20 mL), followed by the addition of Fe (788 mg, 14.07 mmol, 3.0 eq.) and NH₄Cl (760 mg, 14.07 mmol, 3.0 eq.). The reaction mixture was stirred at 70 °C for 2 hours under a nitrogen atmosphere. Thin-layer chromatography (DCM:MeOH = 10:1) showed that the reaction was complete and a new major spot was observed. The mixture was filtered, and the filter cake was washed with MeOH (100 mL). The filtrate was dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (0%–10% MeOH in DCM) to give compound 7 (1.72 g, 73%) as a yellow solid.

[0422] LCMS:Rt:1.307min;MS m / z(ELSD):504.5[M+H] + ;

[0423] 1H NMR (400MHz, CDCl3) δ7.93(d,J=8.1Hz,1H),7.79(d,J=8.1Hz,1H),7.58–7.35(m,2H),4.05(t,J=6.6Hz,4H) ,3.28(t,J=7.0Hz,2H),2.29(t,J=7.4Hz,2H),1.74–1.57(m,6H),1.49–1.16(m,25H),0.88(t,J=6.3Hz,3H).

[0424] Step 5:

[0425] Compound 7 (1.72 g, 3.42 mmol, 1.0 eq.) and compound 8 (5 mL) were mixed and stirred at 80 °C for 16 hours under a nitrogen atmosphere. Thin-layer chromatography (DCM:MeOH = 10 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was terminated with H2O (100 mL) and extracted with EA (50 mL × 3). The combined organic phases were washed with brine (2 × 50 mL), dried, filtered with anhydrous Na2SO4, and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography and eluted with 10% MeOH in DCM to give compound HA-13-A (459 mg, 26%) as a white solid.

[0426] LCMS:Rt:1.918min; MS m / z(ELSD):514.4[M+H] + ;

[0427] CAD:98.7% purity at ELSD; RT=23.804min.

[0428] 1H NMR (400MHz, CDCl3) δ8.25(d,J=8.2Hz,1H),8.15(d,J=8.1Hz,1H),8.04(s,1H),7.78-7.64(m,2H),4.60(t,J=7.2Hz,2H),4.05(t,J=6 .7Hz,2H),2.28(t,J=7.5Hz,2H),2.10–1.94(m,2H),1.67–1.53(m,4H),1.46-1.38(m,2H),1.36-1.22(m,22H),0.87(t,J=6.8Hz,3H).

[0429] Example 6:

[0430] Step 1:

[0431] A solution of compound 1 (1.5 g, 6.20 mmol, 1.0 eq.) was dissolved in DCM (15 mL), followed by the addition of compound 2 (1.5 g, 6.20 mmol, 1.0 eq.) and TEA (1.9 g, 18.60 mmol, 3.0 eq.). The reaction mixture was stirred at 70 °C for 2 hours under a nitrogen atmosphere. Thin-layer chromatography (PE:EA = 15:1) showed that the reaction was complete and a new major spot was observed. The mixture was terminated with H2O (100 mL) and extracted with EA (100 mL × 3). The combined organic phases were washed with brine (2 × 100 mL), dried, filtered through anhydrous Na2SO4, and concentrated under reduced pressure. The residue was purified by column chromatography (0%–10% EA in PE) to give compound 3 (1.5 g, 53.9%) as a yellow solid.

[0432] LCMS:Rt:3.572min; MS m / z(ELSD):448.5[M+H] + ;

[0433] 1H NMR (400MHz, CDCl3) δ7.94(d,J=8.4Hz,2H),7.75(t,J=7.6Hz,1H),7.54(t,J=7.4Hz,1H),6.07( s,1H),3.48(d,J=3.5Hz,2H),1.84-1.65(m,4H),1.28(d,J=17.1Hz,24H),0.88(t,J=6.8Hz,3H).

[0434] Step 2:

[0435] A solution of compound 3 (1.5 g, 3.36 mmol, 1.0 eq.) was dissolved in EtOH / H₂O (20 mL), followed by the addition of Fe (564 mg, 10.07 mmol, 3.0 eq.) and NH₄Cl (543 mg, 10.07 mmol, 3.0 eq.). The reaction mixture was stirred at 70 °C for 2 hours under a nitrogen atmosphere. Thin-layer chromatography (PE:EA = 10:1) showed that the reaction was complete and a new major spot was observed. The mixture was filtered, and the filter cake was washed with MeOH (100 mL). The filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (0%–20% EA in PE) to give compound 4 (1.07 g, 66.9%) as a yellow solid.

[0436] LCMS:Rt:1.585min; MS m / z(ELSD):418.3[M+H] + ;

[0437] 1H NMR(400MHz, CDCl3) δ7.93(d,J=8.1Hz,1H),7.79(d,J=8.2Hz,1H),7.56–7.39(m,2H),4. 75–3.51(m,3H),3.30-3.27(m,2H),1.74–1.60(m,2H),1.25(s,26H),0.89-0.86(m,3H).

[0438] Step 3:

[0439] A solution of compound 4 (1.07 g, 17.4 mmol, 1.0 eq.) was dissolved in ethanol (5.0 mL), and then compound 5 (39 g, 521 mmol, 30.0 eq.) was added. The mixture was stirred at 50 °C for 16 hours under a nitrogen atmosphere. Thin-layer chromatography (DCM:MeOH = 10 / 1) showed that the reaction was complete and a new major spot was observed. The reaction was terminated with H2O (100 mL) and extracted with EA (50 mL × 3). The combined organic phases were washed with brine (2 × 50 mL), dried, filtered through anhydrous Na2SO4, and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography, eluted with 20% MeOH in DCM, to give HA-14-C16-A (6.1 g, 77.1%) as a yellow oil.

[0440] LCMS:Rt:2.378min;MS m / z(ELSD):428.3[M+H] + ;

[0441] CAD:95.52% purity at ELSD; RT=25.205min.

[0442] 1 H NMR (400MHz, CDCl3) δ8.25(d,J=8.3Hz,1H),8.15(d,J=8.3Hz,1H),8.05(s,1H),7.77–7.63(m,2H),4. 60(t,J=7.3Hz,2H),2.06–1.99(m,2H),1.50–1.34(m,4H),1.33–1.15(m,24H),0.88(t,J=6.8Hz,3H).

[0443] Example 7:

[0444] Step 1:

[0445] A mixture of compound HA-14-C16-A (490 mg, 1.15 mmol, 1.0 eq.) and NH3 (7 M in MeOH) was placed in a sealed tube and stirred at 140 °C for 24 hours. Thin-layer chromatography (MeOH / DCM = 10:1) showed the desired product. The mixture was concentrated under reduced pressure. The residue was purified by column chromatography (0%–10% MeOH in DCM) and pre-separation HPLC to give compound HA-14-C16 (53.26 mg, 11.11%) as a white solid.

[0446] LCMS:Rt:1.270min;MS m / z(ELSD):409.3[M+H] + ;

[0447] CAD:93.35% purity at ELSD; RT=19.186min.

[0448] 1 H NMR (400MHz, CDCl3) δ7.96(d,J=8.0Hz,1H),7.84(d,J=8.4Hz,1H),7.80(s,1H),7.54(t,J=7.6Hz,1H),7.35(t,J=7.6Hz, 1H),5.58(s,2H),4.51(t,J=7.6Hz,2H),2.11–1.89(m,2H),1.43–1.34(m,3H),1.31–1.20(m,23H),0.88(t,J=6.8Hz,3H).

[0449] Example 8:

[0450] Step 1:

[0451] A solution of compound 1 (2.9 g, 13.21 mmol, 1.0 eq.) was dissolved in DCM (30 mL), followed by the addition of compound 2 (3 g, 13.21 mmol, 1.0 eq.), EDCI (5.05 g, 26.43 mmol, 2.0 eq.), HOBt (2.7 g, 19.82 mmol, 1.5 eq.), and DIEA (5.1 g, 39.65 mmol, 3.0 eq.). The reaction mixture was stirred at 25 °C for 16 hours under a nitrogen atmosphere. Thin-layer chromatography (PE:EA = 5:1) showed that the reaction was complete, and a new major spot was observed. The mixture was terminated with H2O (200 mL) and extracted with EA (100 mL × 3). The combined organic phases were washed with brine (2 × 200 mL), dried, filtered through anhydrous Na2SO4, and concentrated under reduced pressure. The residue was purified by column chromatography (0%–30% EA in PE) to give compound 3 (3.78 g, 66%) as a yellow oil.

[0452] LCMS:Rt:2.017min; MS m / z(ELSD):328.3[M-100+H] + ;

[0453] 1H NMR (400MHz, CDCl3) δ5.91(s,1H),4.63(s,1H),3.24(dd,J=12.6,6.9Hz,2H),3.13(t,J=6.7Hz,2H),2.25(t,J=7.5Hz,2H) ,1.67(dd,J=15.0,7.6Hz,2H),1.51(dt,J=14.2,7.0Hz,4H),1.44(s,10H),1.27(d,J=10.6Hz,23H),0.88(t,J=6.8Hz,3H).

[0454] Step 2:

[0455] A solution of compound 3 (3.78 g, 8.89 mmol, 1.0 eq.) was dissolved in DCM (15 mL), followed by the addition of TFA (15 mL). The reaction mixture was stirred at 25 °C for 2 hours under a nitrogen atmosphere. Thin-layer chromatography (DCM:MeOH = 15:1) showed that the reaction was complete, and a new major spot was observed. The reaction was terminated with hydrated NaHCO3 (100 mL) and extracted with EA (100 mL × 3). The combined organic phases were washed with brine (2 × 100 mL), dried, filtered through anhydrous Na2SO4, and concentrated under reduced pressure to give a crude product (3.5 g) as a yellow solid.

[0456] LCMS:Rt:1.078min;MS m / z(ELSD):327.3[M+H] + ;

[0457] Step 3:

[0458] A solution of compound 5 (1.5 g, 6.20 mmol, 1.0 eq.) was dissolved in DCM (15 mL), followed by the addition of compound 4 (2.02 g, 6.20 mmol, 1.0 eq.) and TEA (4 g, 30.99 mmol, 3.0 eq.). The reaction mixture was stirred at 70 °C for 2 hours under a nitrogen atmosphere. Thin-layer chromatography (MeOH:DCM = 15:1) showed that the reaction was complete and a new major spot was observed. The mixture was terminated with H₂O (100 mL) and extracted with EA (100 mL × 3). The combined organic phases were washed with brine (2 × 100 mL), dried, filtered through anhydrous Na₂SO₄, and concentrated under reduced pressure. The residue was purified by column chromatography (0%–10% MeOH in DCM) to give compound 6 (3.25 g, 98%) as a yellow solid.

[0459] LCMS:Rt:2.203min;MS m / z(ELSD):533.3[M+H] + ;

[0460] 1H NMR (400MHz, CDCl3) δ8.41(d,J=8.3Hz,1H),7.95(d,J=7.7Hz,1H),7.73(t,J=7.3Hz,1H),7.56(dd,J=11.4,4.1Hz,1H),5.56(s,1H),3.42 –3.26(m,4H),2.30(t,J=5.9Hz,2H),1.80(d,J=2.8Hz,4H),1.52(dd,J=14.2,6.9Hz,2H),1.28(d,J=25.4Hz,25H),0.88(t,J=6.8Hz,3H).

[0461] Step 4:

[0462] A solution of compound 6 (3.25 g, 6.12 mmol, 1.0 eq.) was dissolved in EtOH / H₂O (30 mL), followed by the addition of Fe (1.1 g, 18.35 mmol, 3.0 eq.) and NH₄Cl (1 g, 14.07 mmol, 3.0 eq.). The reaction mixture was stirred at 70 °C for 2 hours under a nitrogen atmosphere. Thin-layer chromatography (DCM:MeOH = 10:1) showed that the reaction was complete and a new major spot was observed. The mixture was filtered, and the filter cake was washed with MeOH (100 mL). The filtrate was dried over anhydrous Na₂SO₄, filtered again, and concentrated under reduced pressure. The residue was purified by column chromatography (0%–10% MeOH in DCM) to give compound 7 (2.91 g, 94%) as a yellow solid.

[0463] LCMS:Rt:1.276min; MS m / z(ELSD):503.4[M+H] + ;

[0464] Step 5:

[0465] Compound 7 (2.91 g, 5.80 mmol, 1.0 eq.) and compound 8 (5 mL) were mixed and stirred at 80 °C for 16 hours under a nitrogen atmosphere. Thin-layer chromatography (DCM:MeOH = 10 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was terminated with H2O (100 mL) and extracted with EA (50 mL × 3). The combined organic phases were washed with brine (2 × 50 mL), dried, filtered with anhydrous Na2SO4, and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography and eluted with 10% MeOH in DCM to give compound HA-15-A (760 mg, 26%) as a white solid.

[0466] LCMS:Rt:1.906min;MS m / z(ELSD):513.5[M+H] + ;

[0467] CAD:95.88% purity at ELSD; RT=12.191min.

[0468] 1H NMR (400MHz, CDCl3) δ8.23(d,J=8.0Hz,1H),8.15(d,J=8.3Hz,1H),8.07(s, 1H),7.79–7.64(m,2H),5.37(s,1H),4.64(t,J=7.2Hz,2H),3.21(dd,J=12. 7,6.7Hz,2H),2.21(t,J=7.0Hz,2H),2.15–2.02(m,2H),1.81(dt,J=14.6,7 .3Hz,2H),1.50–1.38(m,2H),1.24(d,J=3.7Hz,24H),0.88(t,J=6.8Hz,3H).

[0469] Example 9:

[0470] Step 1;

[0471] A solution of compound 1 (i.e., compound 2 in Example 10) (260 mg, 0.69 mmol, 1.0 eq.) was dissolved in MeCN / CPME (2 mL, 1:1 = v / v), followed by the addition of compound 2 (475 mg, 1.03 mmol, 1.5 eq.), KI (228 mg, 1.37 mmol, 2.0 eq.), and K₂CO₃ (475 mg, 3.44 mmol, 5.0 eq.). The mixture was stirred at 90 °C for 16 hours under a nitrogen atmosphere. Thin-layer chromatography (DCM:MeOH = 10 / 1) showed that the reaction was complete, and a new major spot was observed. The reaction was terminated with H₂O (50 mL) and extracted with EA (50 mL × 3). The combined organic phases were washed with brine (3 × 50 mL), dried, filtered through anhydrous Na₂SO₄, and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography and eluted with 10% MeOH in DCM to give compound 3 (172 mg, 32%) as a yellow oil.

[0472] LCMS:Rt:1.258min;MS m / z(ELSD):759.6[M+H] + ;

[0473] 1H NMR (400MHz, CDCl3) δ8.15(d,J=8.3Hz,1H),7.87(d,J=8.5Hz,1H),7.59(t,J=7.7Hz,1H),7.41(t,J= 7.7Hz,1H),7.34(d,J=8.0Hz,2H),7.01(d,J=8.0Hz,2H),5.78(s,2H),4.92–4.80(m,1H),3.80(s,2H ),3.04–2.88(m,2H),2.62(t,J=7.3Hz,2H),2.25(t,J=7.5Hz,2H),1.82(dd,J=15.6,8.0Hz,4H),1.6 1–1.56(m,2H),1.51–1.42(m,6H),1.35-1.21(m,31H),0.92(t,J=7.4Hz,3H),0.87(t,J=6.8Hz,6H).

[0474] Step 2:

[0475] A solution of compound 3 (170 mg, 0.23 mmol, 1.0 eq.) was dissolved in MeCN / CPME (2 mL, 1:1 v / v), followed by the addition of compound 4 (129 mg, 0.33 mmol, 1.5 eq.), KI (76 mg, 0.46 mmol, 2.0 eq.), and K₂CO₃ (158 mg, 1.15 mmol, 5.0 eq.). The mixture was stirred at 90 °C for 16 hours under a nitrogen atmosphere. Thin-layer chromatography (DCM:MeOH = 10 / 1) showed that the reaction was complete, and a new major spot was observed. The reaction was terminated with H₂O (50 mL) and extracted with EA (50 mL × 3). The combined organic phases were washed with brine (3 × 50 mL), dried, filtered through anhydrous Na₂SO₄, and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography and eluted with 10% MeOH in DCM to give HA-TLR-1-A (49.41 mg, 20.7%) as a yellow oil.

[0476] LCMS:Rt:1.865min;MS m / z(ELSD):1055.8[M+H] + ;

[0477] CAD:97.10% purity at ELSD; RT=20.037min.

[0478] 1H NMR (400MHz, CDCl3) δ8.15(d,J=8.4Hz,1H),7.91(d,J=8.3Hz,1H),7.59(t,J=7.6Hz,1H),7. 40(t,J=7.7Hz,1H),7.28(s,2H),6.97(d,J=7.9Hz,2H),5.79(s,2H),4.87-4.79(m,2H),3.47 (s,2H),3.03–2.96(m,2H),2.37–2.29(m,4H),2.28-2.24(m,4H),1.86-1.78(m,2H),1.55-1. 45(m,7H),1.42-1.39(m,4H),1.35-1.19(m,55H),0.91(t,J=7.4Hz,3H),0.89–0.84(m,12H).

[0479] Example 10:

[0480] Step 1:

[0481] A solution (1 g, 2.09 mmol, 1.0 eq.) of compound 1 (i.e., compound 6 in Example 11) was dissolved in DCM (10 mL), and then TFA (10 mL) was added. The mixture was stirred at 25 °C for 2 hours under a nitrogen atmosphere. Thin-layer chromatography (DCM:MeOH = 10 / 1) showed that the reaction was complete and a new major spot was observed. The reaction was terminated by hydration of NaHCO3 and the pH was adjusted to 8. The mixture was then concentrated under reduced pressure. The residue was purified by silica gel column chromatography and eluted with 10% MeOH in DCM to give compound 2 (732 mg, 96%) as a white solid.

[0482] LCMS:Rt:1.311min; MS m / z(ELSD):379.1[M+H] + ;

[0483] Step 2:

[0484] A solution of compound 2 (260 mg, 0.69 mmol, 1.0 eq.) was dissolved in MeCN / CPME (2 mL, 1:1 v / v), followed by the addition of compound 3 (527 mg, 1.37 mmol, 2.0 eq.), KI (228 mg, 1.37 mmol, 2.0 eq.), and K₂CO₃ (475 mg, 3.44 mmol, 5.0 eq.). The mixture was stirred at 90 °C for 16 hours under a nitrogen atmosphere. Thin-layer chromatography (DCM:MeOH = 10 / 1) showed that the reaction was complete, and a new major spot was observed. The reaction was terminated with H₂O (50 mL) and extracted with EA (50 mL × 3). The combined organic phases were washed with brine (3 × 50 mL), dried, filtered through anhydrous Na₂SO₄, and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography and eluted with 10% MeOH in DCM to give HA-TLR-2-A (89 mg, 14%) as a yellow oil.

[0485] LCMS:Rt:1.305min; MS m / z(ELSD):915.6[M+H] + ;

[0486] CAD:97.55% purity at ELSD; RT=21.027min.

[0487] 1 H NMR (400MHz, DMSO) δ8.15-8.03(m,2H),7.64(t,J=7.6Hz,1H),7.52-7.45(m,2H),7.22(d, J=7.8Hz,1H),7.15(d,J=7.7Hz,1H),6.99(d,J=8.0Hz,1H),6.00(d,J=34.2Hz,2H),4.26( s,1H),4.02–3.88(m,4H),3.40(s,1H),2.99(d,J=6.7Hz,2H),2.95-2.85(m,2H),2.33-2. 15(m,6H),1.82-1.75(m,2H),1.59-1.35(m,12H),1.30–1.11(m,38H),0.89-0.75(m,9H).

[0488] Example 11:

[0489] Step 1:

[0490] A solution of compound 1 (4 g, 16.46 mmol, 1.0 eq.) was dissolved in DCM (40 mL), followed by the addition of compound 2 (3.88 g, 16.46 mmol, 1.0 eq.) and TEA (4.98 g, 49.38 mmol, 3.0 eq.). The reaction mixture was stirred at 70 °C for 2 hours under a nitrogen atmosphere. Thin-layer chromatography (PE:EA = 1:1) showed that the reaction was complete and a new major spot was observed. The mixture was terminated with H2O (150 mL) and extracted with EA (100 mL × 3). The combined organic phases were washed with brine (2 × 100 mL), dried, filtered through anhydrous Na2SO4, and concentrated under reduced pressure. The residue was purified by column chromatography (0%–70% EA in PE) to give compound 3 (5.645 g, 76.7%) as a yellow solid.

[0491] LCMS:Rt:1.867min; MS m / z(ELSD):443.2[M+H] + ;

[0492] Step 2:

[0493] A solution of compound 3 (5.645 g, 12.37 mmol, 1.0 eq.) was dissolved in EtOH / H₂O (60 mL, 5:1, v / v), followed by the addition of Fe (2.146 g, 38.31 mmol, 3.0 eq.) and NH₄Cl (2.07 g, 38.31 mmol, 3.0 eq.). The reaction mixture was stirred at 70 °C for 3 hours under a nitrogen atmosphere. Thin-layer chromatography (PE:EA = 1:3) showed that the reaction was complete and a new major spot was observed. The mixture was filtered, and the filter cake was washed with MeOH (150 mL). The combined organic phases were dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (0%–60% EA in PE) to give compound 4 (3.698 g, 70.3%) as a yellow solid.

[0494] LCMS:Rt:1.727min;MS m / z(ELSD):413.2[M+H] + ;

[0495] Step 3:

[0496] A solution of compound 4 (3 g, 7.28 mmol, 1.0 eq.) was dissolved in THF (50 mL), and then pyridine (2.88 g, 36.44 mmol, 5.0 eq.) and valeryl chloride (917 mg, 7.65 mmol, 1.05 eq.) were added at 0 °C under an argon atmosphere. The mixture was stirred at 25 °C for 16 hours under a nitrogen atmosphere. Thin-layer chromatography (DCM:MeOH = 10 / 1) showed that the reaction was complete and a new major spot was observed. The reaction was terminated with H2O (100 mL) and extracted with EA (80 mL × 3). The combined organic phases were washed with brine (2 × 50 mL), dried, filtered through anhydrous Na2SO4, and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography and eluted with 10% MeOH in DCM to give compound 5 (3.6 g, 98%) as a pale yellow solid.

[0497] LCMS:Rt:1.702min;MS m / z(ELSD):497.2[M+H] + ;

[0498] 1H NMR (400MHz, CDCl3) δ7.93 (d, J=8.4Hz, 1H), 7.86 (d, J=8.4Hz, 1H), 7.61 (t, J= 7.6Hz,1H),7.38(t,J=7.6Hz,1H),7.29-7.15(m,4H),5.43(s,1H),4.90(s,1H ),4.73(d,J=4.9Hz,2H),4.32(d,J=5.6Hz,2H),2.41(t,J=7.6Hz,2H),1.75–1 .62(m,2H),1.46(s,9H),1.38(dt,J=14.2,7.2Hz,2H),0.93(t,J=7.3Hz,3H).

[0499] Step 4:

[0500] A solution of compound 5 (2.75 g, 8.18 mmol, 1.0 eq.) was dissolved in EtOH / H2O (20 mL), followed by the addition of K2CO3 (556 mg, 4.03 mmol, 2.0 eq.). The mixture was stirred at 55 °C for 16 hours under a nitrogen atmosphere. Thin-layer chromatography (DCM:MeOH = 10 / 1) showed that the reaction was complete, and a new major spot was observed. The reaction was terminated with H2O (100 mL) and extracted with EA (80 mL × 3). The combined organic phases were washed with brine (2 × 50 mL), dried, filtered through anhydrous Na2SO4, and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography, eluting with 10% MeOH in DCM, to give compound 6 (1.95 g, 73.6%) as a pale yellow solid.

[0501] LCMS:Rt:1.964min;MS m / z(ELSD):479.3[M+H] + ;

[0502] 1H NMR (400MHz, CDCl3) δ8.16(d,J=8.3Hz,1H),7.86(d,J=8.3Hz,1H),7.60(t,J=7.7Hz,1H),7.42(t,J=7.6Hz,1H),7.25(d,J=8.2Hz,2H),6.99( d,J=7.9Hz,2H),5.78(s,2H),4.84(s,1H),4.28(s,2H),3.07–2.88(m,2H),1.82(dd,J=15.4,7.8Hz,4H),1.43(s,9H),0.93(t,J=7.3Hz,3H).

[0503] Step 5:

[0504] A mixture of compound 6 (1.85 g, 3.87 mmol, 1.0 eq.) was placed in a reactor with NH3 (30 mL, NH3 in MeOH, 7 M) and stirred at 140 °C for 48 hours. Thin-layer chromatography (DCM:MeOH = 10 / 1) showed the desired product. The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with 10% MeOH in DCM, to give compound 7 (880 mg, 49.7%) as a white solid.

[0505] LCMS: Rt: 1.237min; MS m / z (ELSD): 460.3M+H] + ;

[0506] 1H NMR (400MHz, CDCl3) δ7.87(d,J=8.1Hz,1H),7.70(d,J=7.9Hz,1H),7.48(t,J= 7.3Hz,1H),7.26(d,J=6.9Hz,2H),7.21(t,J=7.6Hz,1H),7.00(d,J=8.1Hz,2H ),5.73(s,2H),4.87(s,1H),4.29(d,J=5.2Hz,2H),3.25-3.05(m,2H),2.91–2 .83(m,2H),1.81(dt,J=15.4,7.6Hz,2H),1.43(s,9H),0.94(t,J=7.4Hz,3H).

[0507] Step 6:

[0508] A mixture of compound 6 (880 mg, 1.92 mmol, 1.0 eq.) was dissolved in DCM (10 mL), and then TFA (10 mL) was added at 0 °C under a nitrogen atmosphere. The reaction mixture was allowed to warm to room temperature and stirred for 2 hours. The reaction mixture was terminated with hydrated NaHCO3 and the pH was adjusted to 8. The mixture was then concentrated under vacuum. The residue was purified by silica gel column chromatography, eluting with 10% MeOH in DCM, to give compound 8 (609 mg, 88.5%) as a white solid.

[0509] LCMS:Rt:1.050min;MS m / z(ELSD):228.1[M+H] + ;

[0510] 1H NMR (400MHz, CDCl3) δ7.93(d,J=8.3Hz,1H),7.71(d,J=8.2Hz,1H),7.51(t,J=7.3Hz,1H),7.32(d,J=8.2Hz,2H),7.23(d,J=8.2Hz,1 H),7.00(d,J=8.1Hz,2H),5.72(s,2H),3.86(s,2H),2.90–2.84(m,2H),1.86–1.78(m,2H),1.44-1.55(m,2H),0.94(t,J=7.3Hz,3H).

[0511] Step 7:

[0512] A solution of compound 8 (200 mg, 0.56 mmol, 1.0 eq.) was dissolved in EtOH (3 mL, 95%), followed by the addition of compound 9 (225 mg, 1.23 mmol, 2.2 eq.). The mixture was stirred at 80 °C for 48 hours under a nitrogen atmosphere. Thin-layer chromatography (DCM:MeOH = 10 / 1) showed that the reaction was complete, and a new major spot was observed. The reaction was terminated with H2O (50 mL) and extracted with EA (50 mL × 3). The combined organic phases were washed with brine (3 × 50 mL), dried, filtered through anhydrous Na2SO4, and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography, eluting with 10% MeOH in DCM, to give compound HA-TLRa (50.84 mg, 11%) as a white solid.

[0513] LCMS:Rt:1.060min; MS m / z(ELSD):728.7[M+H] + ;

[0514] CAD:97.99% purity at ELSD; RT=31.007min.

[0515] 1H NMR (400MHz, DMSO) δ7.77(d,J=8.2Hz,1H),7.57(d,J=8.2Hz,1H),7.31(t,J=7.6Hz,1H) ,7.26(d,J=7.6Hz,2H),7.05-6.95(m,3H),6.52(s,2H),5.83(s,2H),4.26(d,J=24.2Hz ,2H),3.55(d,J=8.2Hz,1H),3.46(d,J=14.0Hz,3H),2.89(t,J=7.6Hz,2H),2.31(d,J=8 .5Hz,2H),1.75-1.65(m,2H),1.39-1.31(m,2H),1.29-1.05(m,34H),0.87–0.81(m,9H).

[0516] Example 12:

[0517] In a sealed tube, compound HA-15-A (300 mg, 0.59 mmol, 1.0 eq.) was mixed with ammonia-methanol solution (20 mL, NH3 in MeOH, 7 M) and stirred overnight at 140 °C. The reaction was monitored by TLC (DCM:MeOH = 10 / 1), showing that the reactants were consumed and new spots were formed. The reaction mixture was directly evaporated to dryness under reduced pressure, and the residue was purified by silica gel column chromatography (10% MeOH in DCM) to give a white solid, the target compound HA-15 (85.2 mg, 23.6%).

[0518] LCMS:Rt:1.118min; MS m / z(ELSD):494.4[M+H] + ;

[0519] CAD:96.50% purity at ELSD; RT=12.589min.

[0520] 1H NMR (400MHz, DMSO) δ8.19 (s, 1H), 8.02 (d, J = 8.1Hz, 1H), 7.72 (t, J = 5.6Hz, 1H), 7. 62(d,J=8.3Hz,1H),7.44(t,J=7.7Hz,1H),7.26(t,J=7.5Hz,1H),6.59(s,2H),4.5 8(t,J=7.0Hz,2H),2.97(dd,J=12.7,6.7Hz,2H),2.08(t,J=7.3Hz,2H),1.91–1.7 4(m,2H),1.55(dd,J=15.1,7.7Hz,2H),1.36–1.14(m,27H),0.85(t,J=6.8Hz,3H).

[0521] Example 13:

[0522] Step 1:

[0523] In a flask, compound 1 (2 g, 5.78 mmol, 1.0 eq., prepared according to HA-1) was dissolved in dichloromethane (20 mL), and trifluoroacetic acid (20 mL) was added dropwise to a temperature of 0 °C. The system was then stirred at room temperature for 2 hours. The reaction was monitored by spot TLC (DCM:MeOH = 10 / 1), which showed that the starting material was completely consumed and a main spot was formed. The reaction mixture was directly evaporated to dryness under reduced pressure, dissolved in water (30 mL), and the pH was adjusted to approximately 12 by adding sodium hydroxide (1 M) solution. The mixture was then directly evaporated to dryness under reduced pressure, and the residue was purified by silica gel column chromatography (15% MeOH in DCM) to obtain a white solid, target compound 2 (300 mg, 21%).

[0524] LCMS:Rt:1.167min;MS m / z(ELSD):247.0[M+H] + ;

[0525] Step 2:

[0526] In a flask, KI (220 mg, 1.32 mmol, 2.0 eq.), K₂CO₃ (456 mg, 3.30 mmol, 5.0 eq.) and compound 2 (502 mg, 1.65 mmol, 2.5 eq.) were added to a solution of compound 2 (150 mg, 0.66 mmol, 1.0 eq.) in CPME / MeCN (3 mL, v / v). The mixture was then stirred at 90 °C for 24 h under nitrogen protection. The reaction was monitored by TLC (DCM:MeOH = 10 / 1), showing the formation of a major spot. The reaction was quenched with water (50 mL) and extracted with ethyl acetate (50 mL × 3). The combined organic phases were washed with saturated brine (2 × 20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (10% MeOH in DCM) to give a white solid product HA-19-A (70.07 mg, 14.8%).

[0527] LCMS:Rt:2.279min;MS m / z(ELSD):695.7[M+H] + ;

[0528] CAD:95.34% purity at ELSD; RT=25.386min.

[0529] 1H NMR (400MHz, CDCl3) δ8.22(d,J=9.1Hz,2H),8.02(s,1H),7.72(t,J=7.4Hz,1H),7.65(s,1H),4.64-4.55(m ,2H),3.05-2.94(m,2H),2.52-2.35(m,4H),1.30-1.20(m,50H),1.18-1.15(m,6H),0.88(t,J=6.8Hz,6H).

[0530] Example 14:

[0531] KI (439 mg, 2.64 mmol, 2.0 eq.), K₂CO₃ (912 mg, 6.61 mmol, 5.0 eq.), and compound 2 (1004 mg, 3.30 mmol, 2.5 eq.) were added to compound 1 (300 mg, 1.32 mmol, 1.0 eq.) in a CPME / MeCN solution (6 mL, v / v). After purging with nitrogen, the mixture was stirred at 90 °C for 24 h. The reaction was monitored by spotting on a TLC plate (DCM:MeOH = 10 / 1), showing that the starting material was consumed and new spots were formed. The reaction was quenched with water (50 mL) and extracted with ethyl acetate (50 mL × 3). The combined organic phases were washed with saturated brine (2 × 20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to dryness. The residue was purified by silica gel column chromatography (10% MeOH in DCM) to give the target compound HA-19 (51.4 mg, 10.3%) as a white solid.

[0532] LCMS:Rt:1.865min;MS m / z(ELSD):676.7[M+H] + ;

[0533] CAD:95.10% purity at ELSD; RT=20.031min.

[0534] 1H NMR (400MHz, CDCl3) δ7.99(d,J=7.2Hz,1H),7.85(d,J=7.2Hz,2H),7.54(t,J=7.4Hz,1H),7.33(t,J=7.6Hz,1H),5.5 0(s,1H),4.54(t,J=6.4Hz,2H),2.93(t,J=6.4Hz,2H),2.54–2.33(m,4H),1.30–1.18(m,56H),0.88(t,J=6.8Hz,6H).

[0535] Example 15:

[0536] Step 1:

[0537] Compound 2 (1.6 g, 9.95 mmol, 1.0 eq.), EDCI (3.8 g, 19.91 mmol, 2.0 eq.), DIEA (5.1 g, 39.8 mmol, 5.0 eq.), and DMAP (0.485 g, 3.97 mmol, 0.4 eq.) were added sequentially to a solution of compound 1 (3 g, 9.95 mmol, 1.0 eq.) in dichloromethane (30 mL). The reaction was then stirred overnight at room temperature under nitrogen protection. The reaction was monitored by spot TLC (PE:EA = 5:1), indicating complete consumption of the starting materials. The reaction was quenched with water (200 mL) and extracted with ethyl acetate (100 mL × 3). The combined organic phases were washed with saturated brine (2 × 50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (0%–30% EA in PE) to give a white solid product 3 (2.943 g, 69%).

[0538] LCMS:Rt:2.378min; MS m / z(ELSD):444.3[M-100+H] + ;

[0539] 1H NMR (400MHz, CDCl3) δ4.48(s,1H),3.09(t,J=7.1Hz,2H),2.86(t,J=7.3Hz,2H),2.53(t,J=7.5Hz,2 H),1.69–1.61(m,2H),1.59–1.52(m,2H),1.44(s,10H),1.39–1.18(m,25H),0.88(t,J=6.8Hz,3H).

[0540] Step 2:

[0541] Compound 3 (2.943 g, 6.64 mmol, 1.0 eq.) was dissolved in dichloromethane (DCM) (15 mL), and TFA (15 mL) was slowly added dropwise to the solution at 0 °C. The reaction was then stirred at room temperature for two hours. The reaction was monitored by spot TLC (DCM:MeOH = 15:1), showing the disappearance of the starting material and the formation of a new spot. The reaction was quenched with a saturated sodium bicarbonate solution and extracted with ethyl acetate (100 mL × 3). The organic phase was washed with saturated brine (2 × 100 mL), dried over anhydrous sodium sulfate, filtered, and evaporated under pressure to give a white solid product 4 (2.5 g).

[0542] LCMS:Rt:1.095min;MS m / z(ELSD):344.3[M+H] + ;

[0543] 1H NMR (400MHz, CDCl3) δ7.95(s,2H),2.90–2.80(m,2H),2.57–2.47(m,2H),1.72-1.55(m,6H),1.43–1.17(m,26H),0.88(t,J=6.9Hz,3H).

[0544] Step 3:

[0545] Compound 5 (1.5 g, 6.20 mmol, 1.0 eq.) was dissolved in dichloromethane (30 mL), followed by the addition of compound 6 (2.12 g, 6.20 mmol, 1.0 eq.) and TEA (1.878 g, 18.60 mmol, 3.0 eq.). The reaction was carried out at 70 °C for two hours under nitrogen protection. The reaction was monitored by spot TLC (MeOH:DCM = 15:1), showing complete consumption of the starting material and the formation of new spots. The reaction was quenched with water (100 mL) and extracted with ethyl acetate (100 mL × 3). The organic phase was washed with saturated brine (2 × 100 mL), dried, filtered, and evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (0%–40% EA in PE) to give a yellow solid product 6 (2.157 g, 62%).

[0546] LCMS:Rt:1.660min; MS m / z(ELSD):550.4[M+H] + ;

[0547] 1H NMR (400MHz, CDCl3) δ8.04–7.89(m,2H),7.83–7.70(m,1H),7.61–7.47(m,1H),6.12(s,1H),3.48(dd,J=11.8,7.0Hz,2H),2.94–2.75(m,2H), 2.62–2.43(m,2H),1.79–1.70(m,2H),1.64(dd,J=14.6,7.3Hz,2H),1.55(dt,J=15.0,7.3Hz,2H),1.46–1.21(m,24H),0.88(t,J=6.9Hz,3H).

[0548] Step 4:

[0549] Compound 7 (2.157 g, 3.93 mmol, 1.0 eq.) was dissolved in a mixture of ethanol and water (30 mL), and reduced iron powder (660 mg, 11.79 mmol, 3.0 eq.) and NH4Cl (640 mg, 14.07 mmol, 3.0 eq.) were added. The mixture was then reacted at 70 °C for two hours. The reaction was monitored by spot TLC (DCM:MeOH = 10:1), showing complete consumption of the starting material and the formation of a new spot. The reaction mixture was directly filtered, and the filter cake was washed with methanol (100 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (0%–10% MeOH in DCM) to give compound 8 (1.56 g, 78%) as a yellow solid.

[0550] LCMS:Rt:1.480min; MS m / z(ELSD):520.3[M+H] + ;

[0551] 1H NMR(400MHz, CDCl3) δ7.91(d,J=7.8Hz,1H),7.85–7.73(m,1H),7.47(dq,J=13.7,6.7Hz,2H),3.26(t,J=7.2Hz,2H),2.85 (d,J=7.3Hz,2H),2.53(t,J=7.5Hz,2H),1.68–1.61(m,4H),1.60–1.51(m,3H),1.44–1.22(m,28H),0.87(t,J=6.7Hz,3H).

[0552] Step 5:

[0553] Compound 8 (1.56 g, 3.0 mmol, 1.0 eq.) and compound 9 (15 mL) were mixed together and stirred overnight at 80 °C. The reaction was monitored by spot TLC (DCM: MeOH = 10 / 1), showing complete consumption of the starting material and the formation of a new principal spot. The reaction was quenched with water (100 mL) and extracted with ethyl acetate (50 mL × 3). The combined organic phases were washed with saturated brine (2 × 50 mL), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (10% MeOH in DCM) to give a white solid product HA-30-A (1.42 g, 88%). LCMS: Rt: 2.349 min; MS m / z (ELSD): 530.3 [M+H] + ;

[0554] CAD:96.80% purity at ELSD; RT=25.689min.

[0555] 1H NMR(400MHz, CDCl3)δ8.24(d,J=8.2Hz,1H),8.15(d,J=8.0Hz,1H),8.01(s,1H), 7.73(t,J=7.5Hz,1H),7.67(t,J=7.6Hz,1H),4.59(t,J=7.2Hz,2H),2.90–2.79(m ,2H),2.59–2.48(m,2H),2.02(dd,J=14.9,8.0Hz,2H),1.58–1.51(m,2H),1.47–1 .40(m,2H),1.35(d,J=6.3Hz,4H),1.26(d,J=5.8Hz,20H),0.87(t,J=6.9Hz,3H).

[0556] Example 16:

[0557] Compound 1 (100 mg, 0.20 mmol, 1.0 eq. from HA-34) was dissolved in CPME / MeCN (2 mL, v / v) with KI (67 mg, 0.40 mmol, 2.0 eq.), K₂CO₃ (139 mg, 1.01 mmol, 5.0 eq.), and compound 2 (50 mg, 0.30 mmol, 1.5 eq.). The mixture was then stirred overnight at 90 °C under nitrogen protection. The reaction was monitored by spot TLC (DCM:MeOH = 10 / 1), showing that the starting material was consumed and a new spot was formed. The reaction was quenched with water (50 mL) and extracted with ethyl acetate (50 mL × 3). The organic phase was washed with saturated brine, filtered, and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography with 10% MeOH in DCM to give a pale yellow solid product HA-33 (45 mg, 39.8%). LCMS:Rt:0.949min; MS m / z(ELSD):564.7[M+H] + ;

[0558] CAD:99.21% purity at ELSD; RT=14.243min.

[0559] 1H NMR(400MHz, CDCl3)δ7.98(dd,J=8.2,1.0Hz,1H),7.89–7.81(m,2H),7.54(ddd,J=8.4,7.1,1.3Hz,1 H),7.33(ddd,J=8.2,7.1,1.2Hz,1H),5.59(s,1H),4.55(t,J=6.2Hz,2H),4.05(t,J=6.8Hz,2H),2.9 4(t,J=6.2Hz,2H),2.59–2.50(m,2H),2.44–2.36(m,2H),2.27(t,J=7.5Hz,2H),1.68-1.52(m,4H),1 .32–1.14(m,22H),0.88(t,J=6.9Hz,3H),0.65–0.55(m,1H),0.46–0.33(m,2H),-0.01–-0.05(m,2H).

[0560] Example 17:

[0561] Step 1:

[0562] Compound 1 (1.3 g, 5.73 mmol, 1.0 eq., FROM HA-11) was dissolved in CPME / MeCN (30 mL, v / v) with KI (1.9 g, 11.45 mmol, 2.0 eq.), K₂CO₃ (3.95 g, 28.63 mmol, 5.0 eq.) and compound 2 (1.99 g, 5.73 mmol, 1.0 eq.). The reaction was then stirred at 90 °C for 24 h under nitrogen protection. The reaction was monitored by spot TLC (DCM:MeOH = 10 / 1), showing that the starting material was consumed and a new spot was formed. The reaction was quenched with water (100 mL) and extracted with ethyl acetate (80 mL × 3). The organic phase was washed with saturated brine (2 × 40 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography with 10% MeOH in DCM to give compound 3 (458 mg, 19%) as a pale yellow solid.

[0563] LCMS:Rt:0.890min; MS m / z(ELSD):496.5[M+H] + ;

[0564] 1H NMR (400MHz, CDCl3) δ8.05–7.98(m,2H),7.94(d,J=8.2Hz,1H),7.60(t,J=7 .6Hz,1H),7.45(t,J=7.7Hz,1H),4.66(t,J=5.8Hz,2H),4.05(t,J=6.7Hz,2H ),3.23(t,J=6.0Hz,2H),2.62(t,J=7.1Hz,2H),2.28(t,J=7.5Hz,2H),1.60( d,J=5.6Hz,4H),1.44(s,2H),1.27(d,J=4.4Hz,20H),0.88(t,J=6.8Hz,3H).

[0565] Step 2

[0566] Compound 3 (100 mg, 0.20 mmol, 1.0 eq.) was dissolved in CPME / MeCN (2 mL, v / v) with KI (67 mg, 0.40 mmol, 2.0 eq.), K₂CO₃ (139 mg, 1.01 mmol, 5.0 eq.), and compound 4 (56 mg, 0.30 mmol, 1.5 eq.). The reaction was then stirred at 90 °C for 24 h under nitrogen protection. The reaction was monitored by spot TLC (DCM:MeOH = 10 / 1), showing that the starting material was consumed and a new spot was formed. The reaction was quenched with water H₂O (100 mL) and extracted with ethyl acetate (80 mL × 3). The organic phase was washed with saturated brine (2 × 40 mL), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography with 10% MeOH in DCM to give a pale yellow solid compound HA-34 (45 mg, 31%).

[0567] LCMS:Rt:0.978min;MS m / z(ELSD):600.6[M+H] + ;

[0568] CAD:98.10% purity at ELSD; RT=33.049min.

[0569] 1H NMR (400MHz, CDCl3) δ7.93(dd,J=8.2,1.0Hz,1H),7.85(dd,J=8.4,0.9Hz,1H),7.54(ddd,J=8.4,7.1,1.3Hz,1H ),7.40(s,1H),7.35–7.27(m,3H),7.22(ddd,J=7.3,3.8,1.3Hz,1H),7.15–7.09(m,2H),5.72(s,1H),4.43(t,J= 6.0Hz,2H),4.06(t,J=6.8Hz,2H),2.96(t,J=6.0Hz,2H),2.82–2.70(m,2H),2.65(t,J=7.2Hz,2H),2.46(t,J=6. 8Hz,2H),2.27(t,J=7.5Hz,2H),1.58(ddd,J=22.7,13.3,7.4Hz,4H),1.36–1.09(m,22H),0.88(t,J=6.9Hz,3H).

[0570] Example 18:

[0571] In a flask, compound 1 (166 mg, 0.22 mmol, 1.0 eq.) was dissolved in CPME / MeCN (6 mL, v / v), and KI (75 mg, 0.45 mmol, 2.0 eq.), K₂CO₃ (155 mg, 1.12 mmol, 5.0 eq.), and compound 4 (127 mg, 0.34 mmol, 1.5 eq.) were added. The reaction was stirred at 90 °C for 24 h under nitrogen protection. The reaction was monitored by spotting on a TLC plate (DCM:MeOH = 10 / 1), showing that the starting material was consumed and new spots were formed. The reaction was quenched with water (50 mL) and extracted with ethyl acetate (50 mL × 3). The combined organic phases were washed with saturated brine (2 × 20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography with 10% MeOH in DCM to give a white solid product HA-TLR-1 (59.2 mg, 25%).

[0572] LCMS:Rt:1.591min;MS m / z(ELSD):1037.9[M+H] + ;

[0573] CAD:93.51% purity at ELSD; RT=22.387min.

[0574] 1H NMR (400MHz, CDCl3) δ7.81(d,J=8.4Hz,1H),7.74(d,J=8.2Hz,1H),7.44(t,J=7.7Hz,1H),7.29(s,2H),7. 13(t,J=7.7Hz,1H),6.99(d,J=8.0Hz,2H),5.72(s,2H),4.83(dp,J=18.3,6.1Hz,2H),3.49(s,2H),2.94–2 .84(m,2H),2.34(t,J=7.1Hz,4H),2.26(td,J=7.5,3.3Hz,4H),1.79(dt,J=15.4,7.7Hz,4H),1.60–1.55( m,4H),1.50(d,J=3.1Hz,5H),1.45–1.39(m,5H),1.25(s,52H),0.91(t,J=7.4Hz,3H),0.89–0.84(m,12H).

[0575] Example 19:

[0576] Compound 1 (230 mg, 0.64 mmol, 1.0 eq.) was dissolved in CPME / MeCN (6 mL, v / v), and KI (213 mg, 1.28 mmol, 2.0 eq.), K₂CO₃ (442 mg, 3.20 mmol, 5.0 eq.), and compound 2 (354 mg, 0.77 mmol, 1.2 eq.) were added. The reaction was stirred at 90 °C for 24 h under nitrogen protection. The reaction was monitored by spotting on a TLC plate (DCM:MeOH = 10 / 1), showing that the starting material was consumed and new spots were formed. The reaction was quenched with water (50 mL) and extracted with ethyl acetate (50 mL × 3). The combined organic phases were washed with saturated brine (2 × 20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure. The residue was purified by silica gel with 10% MeOH in DCM to give a white solid product 3 (HA-TLR-1-B) (166 mg, 35%).

[0577] LCMS:Rt:1.183min;MS m / z(ELSD):740.6[M+H] + ;

[0578] CAD:98.23% purity at ELSD; RT=14.589min.

[0579] 1H NMR (400MHz, CDCl3) δ7.81(d,J=8.3Hz,1H),7.72(d,J=8.2Hz,1H),7.44(t,J=7.6Hz,1H),7.28(d ,J=8.1Hz,2H),7.13(t,J=7.7Hz,1H),7.01(d,J=8.1Hz,2H),5.72(s,2H),4.86(p,J=6.2Hz,1H),3 .73(s,2H),2.92–2.83(m,2H),2.58(t,J=7.2Hz,2H),2.26(t,J=7.5Hz,2H),1.79(dd,J=15.3,7.8 Hz,3H),1.53–1.40(m,9H),1.27(d,J=18.7Hz,33H),0.92(t,J=7.4Hz,3H),0.87(t,J=6.8Hz,6H).

[0580] Example 20:

[0581] In a flask, compound 1 (230 mg, 0.64 mmol, 1.0 eq. from HA-TLRa) was dissolved in CPME / MeCN (6 mL, v / v), and KI (213 mg, 0.128 mmol, 2.0 eq.), K₂CO₃ (442 mg, 3.20 mmol, 5.0 eq.), and compound 2 (557 mg, 1.60 mmol, 2.5 eq.) were added. The reaction was stirred at 90 °C for 24 h under nitrogen protection. The reaction was monitored by spotting on a TLC plate (DCM:MeOH = 10 / 1), showing that the starting material was consumed and new spots were formed. The reaction was quenched with water (50 mL) and extracted with ethyl acetate (50 mL × 3). The combined organic phases were washed with brine (2 × 20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography with 10% MeOH in DCM to give a white solid product HA-TLR-2 (71.56 mg, 12%).

[0582] LCMS:Rt:1.113min;MS m / z(ELSD):896.8[M+H] + ;

[0583] CAD:90.87% purity at ELSD; RT=16.696min.

[0584] 1H NMR (400MHz, CDCl3) δ7.82(d,J=8.3Hz,1H),7.74(d,J=7.9Hz,1H),7.45(t,J=7.7Hz,1H),7.28(d,J= 7.8Hz,2H),7.14(t,J=7.5Hz,1H),6.99(d,J=8.0Hz,2H),5.72(s,2H),4.05(t,J=6.8Hz,4H),3.49(s ,2H),2.92–2.84(m,2H),2.35(t,J=6.9Hz,4H),2.27(t,J=7.5Hz,4H),1.79(dt,J=15.3,7.6Hz,4H), 1.43(dd,J=14.9,7.4Hz,7H), 1.28(d,J=14.3Hz,42H), 0.92(t,J=7.4Hz,3H), 0.88(t,J=6.8Hz,6H).

[0585] Example 21:

[0586] Step 1:

[0587] Compound 1 (1 g, 2.09 mmol, 1.0 eq., from HA-TLRa) was dissolved in dichloromethane (10 mL), and the mixture was cooled to 0 °C and trifluoroacetic acid (10 mL) was added dropwise. The reaction was then brought to room temperature and stirred for two hours. The reaction mixture was quenched with saturated sodium bicarbonate solution and pH = 8, and then evaporated to dryness. The residue was purified by silica gel column chromatography with 10% MeOH in DCM to give compound 2 (762 mg, 96%) as a white solid.

[0588] LCMS:Rt:1.053min; MS m / z(ELSD):379.2[M+H] + ;

[0589] Step 2:

[0590] Compound 2 (186 mg, 0.49 mmol, 1.0 eq.) was dissolved in ethanol (4 mL, 95%) and compound 3 (199 mg, 1.08 mmol, 2.2 eq.) was added. The mixture was then stirred at 80 °C for 48 hours under nitrogen protection. The reaction was monitored by TLC (DCM:MeOH = 10 / 1), showing that the starting material was consumed and new products were formed. The reaction was quenched with water (50 mL), extracted with ethyl acetate (50 mL × 3), and the combined organic phases were washed with saturated brine (3 × 50 mL), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography with 10% MeOH in DCM to give a yellow oily product HA-TLRa-A (10.68 mg, 3%).

[0591] LCMS:Rt:1.130min;MS m / z(ELSD):747.6[M+H] + ;

[0592] CAD:89.91% purity at ELSD; RT=17.239min.

[0593] 1H NMR (400MHz, CDCl3) δ8.16(d,J=8.5Hz,1H),7.84(s,1H),7.59(t,J=7.6Hz,1H),7.39(dd,J =21.6,13.7Hz,3H),7.02(d,J=15.7Hz,2H),5.80(s,2H),3.66(d,J=11.6Hz,3H),3.50–3.36 (m,1H),3.12–2.83(m,2H),2.43(d,J=61.7Hz,3H),1.89–1.79(m,2H),1.46(dd,J=15.2,7. 6Hz, 4H), 1.31 (s, 4H), 1.25 (d, J = 4.6Hz, 34H), 0.93 (t, J = 7.4Hz, 3H), 0.89 (d, J = 6.5Hz, 6H).

[0594] Example 22:

[0595] Step 1:

[0596] Pyridine (2.6 g, 32.7 mmol, 5.0 eq.) and compound 2 (0.85 g, 6.9 mmol, 1.05 eq.) were slowly added dropwise to a dry THF (30 mL) solution of compound 1 (2.2 g, 6.5 mmol, 1.0 eq.) under N2 protection at 0 °C. The mixture was then stirred at rt. for 16 h. LCMS showed the formation of the target product. EA (30 mL) was added to the mixture, followed by washing with Sat. NH4Cl (20 mL × 3), water (20 mL), and brine (20 mL × 2) sequentially. The mixture was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (EA volume fraction in PE: 0%–18%) to give compound 3 (1.8 g, 65%) as a yellow oil.

[0597] LCMS:Rt:1.535min; MS m / z(ELSD):425.3[M+H+2] + ;

[0598] Step 2:

[0599] Potassium carbonate (1.18 g, 8.53 mmol, 2.0 eq.) was added to an ethanol / water (5 / 1, 20 mL) solution of compound 3 (1.8 g, 4.27 mmol, 1.0 eq.). The mixture was stirred at 55 °C for 16 h. Liquid chromatography-mass spectrometry (LC-MS) showed that the reaction was complete. The mixture was concentrated to remove ethanol, and dichloromethane (50 mL) was added to the residue. The solution was washed with water (20 mL) and saturated brine (20 mL × 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (0%–25% ethyl acetate in petroleum ether) to give compound 4 (2.0 g, 82% yield) as a yellow solid.

[0600] LCMS:Rt:1.760min;MS m / z(ELSD):406.9[M+H+2] + ;

[0601] Step 3:

[0602] Trifluoroacetic acid (4 mL) was added dropwise to a solution of compound 4 (2.0 g, 4.95 mmol, 1.0 eq.) in dichloromethane (20 mL). The mixture was stirred at room temperature for 2 hours under nitrogen protection. Liquid chromatography-mass spectrometry (LC-MS) showed the formation of the target product. The mixture was concentrated under reduced pressure to remove trifluoroacetic acid; the residue was quenched with aqueous sodium bicarbonate solution, and the pH was adjusted to 8, followed by concentration under reduced pressure. The residue was purified by silica gel column chromatography (using a 10% methanol solution in dichloromethane as eluent) to give compound 5 (1.5 g, 79.8% yield) as a white solid.

[0603] LCMS:Rt:1.128min;MS m / z(ELSD):305.1[M+H] + ;

[0604] Step 4:

[0605] To a cyclopentyl methyl ether / acetonitrile (1 / 1 v / v, 16 mL) solution of compound 5 (500 mg, 1.64 mmol, 1.0 eq.) and compound 6 (907.9 mg, 1.97 mmol, 1.2 eq.), potassium carbonate (1.13 g, 8.20 mmol, 5.0 eq.) and potassium iodide (544.5 mg, 3.28 mmol, 2.0 eq.) were added, and the mixture was stirred at 90 °C for 3 days under argon protection. Liquid chromatography-mass spectrometry (LC-MS) showed the formation of the target product. The mixture was combined with the material from pages 462-130 and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (0%–5% methanol in dichloromethane) to give compound 7 (560 mg, 41.5% yield) as a yellow oil.

[0606] LCMS:Rt:1.247min;MS m / z(ELSD):685.9[M+H] + ;

[0607] 1H NMR (400MHz, CDCl3) δ8.26–8.17(m,2H),7.68(dtd,J=15.1,7.1,1.4Hz,2H),4.95(d,J=5.8Hz ,2H),4.89–4.83(m,1H),4.80(dd,J=13.2,6.0Hz,2H),3.64(q,J=7.2Hz,2H),3.21(t,J=6.8H z,2H),2.63(t,J=7.2Hz,2H),2.27(t,J=7.6Hz,2H),1.58(d,J=14.4Hz,6H),1.49(d,J=5.6Hz ,4H),1.44(d,J=6.8Hz,2H),1.28(s,6H),1.25(dd,J=7.6,6.4Hz,24H),0.86(t,J=6.8Hz,6H).

[0608] Step 5:

[0609] To a solution of compound 7 (500 mg, 0.73 mmol, 1.0 eq.) and compound 8 (381 mg, 1.10 mmol, 1.5 eq.) in cyclopentyl methyl ether / acetonitrile (10.0 mL, v / v), potassium carbonate (503.7 mg, 3.65 mmol, 5.0 eq.) and potassium iodide (242.7 mg, 1.46 mmol, 2.0 eq.) were added, and the mixture was stirred at 95 °C for 24 h under argon protection. Thin-layer chromatography (dichloromethane:methanol = 10 / 1) showed that the reaction was complete and a new main spot was observed. The mixture was filtered, and the filtrate was concentrated under reduced pressure to give a yellow oily crude product. The residue was purified by preparative high-performance liquid chromatography (PreP-HPLC) to give HA-1-13B (298.2 mg, yield 42.8%) as a yellow oil.

[0610] LCMS:Rt:2.015min;MS m / z(ELSD):954.0[M+H] + ;

[0611] CAD:97.22% purity at ELSD; RT=10.034min in 25min.

[0612] 1 H NMR (400MHz, CDCl3) δ8.27–8.17(m,2H),7.71(t,J=7.2Hz,1H),7.63(t,J=7.2Hz,1H),4.94 (s,2H),4.89–4.82(m,1H),4.74(t,J=7.2Hz,2H),4.05(t,J=6.8Hz,2H),3.62(q,J=7.2Hz, 2H),2.94(t,J=7.2Hz,2H),2.47(t,J=6.0Hz,4H),2.27(td,J=7.6,4.0Hz,4H),1.60(s,4H) ,1.59–1.55(m,2H),1.50(d,J=5.6Hz,4H),1.35–1.22(m,55H),0.87(td,J=6.8,2.4Hz,9H).

[0613] Example 23:

[0614] Step 1:

[0615] To a mixture of acetonitrile (10.0 mL) and cyclopentyl methyl ether (10 mL) containing compounds 6 (300 mg, 1.219 mmol) and 7 (400 mg, 1.158 mmol), potassium carbonate (842 mg, 6.095 mmol) and potassium iodide (405 mg, 2.438 mmol) were added, and the mixture was stirred at 90 °C for 16 hours under an argon atmosphere. Thin-layer chromatography (dichloromethane:methanol = 10 / 1) showed that the reaction was complete and a new main spot was observed. The reaction was quenched by adding water (30 mL) to the mixture, and extracted with ethyl acetate (30 mL × 3). The organic layers were combined, washed with brine (3 × 50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, using methanol in dichloromethane as an eluent increasing from 0% to 10%, to give compound 8 (270 mg, 43% yield) as a colorless oil.

[0616] LCMS:Rt:1.013min;MS m / z(ELSD):515.4[M+H] + ;

[0617] Step 2:

[0618] Potassium carbonate (296 mg, 2.14 mmol) and potassium iodide (143 mg, 0.856 mmol) were added to an acetonitrile / cyclopentyl methyl ether (5 mL / 5 mL) solution of compound 8 (220 mg, 0.428 mmol) and compound 9 (592 mg, 1.284 mmol). The mixture was stirred at 90 °C for 16 hours under an argon atmosphere. Thin-layer chromatography (dichloromethane:methanol = 10 / 1) showed that the reaction was complete and a new main spot was observed. The reaction was quenched by adding water (30 mL) to the mixture, and extracted with ethyl acetate (30 mL × 3). The organic layers were combined, washed with brine (3 × 50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a yellow oily crude product. The residue was purified by preparative high-performance liquid chromatography to give HA-1A-1 (52.39 mg, yield 13.7%) as a colorless oil.

[0619] LCMS:Rt:1.941min;MS m / z(ELSD):895.5[M+H] + ;

[0620] CAD: 96.42% purity

[0621] 1H NMR (400MHz, DMSO-d6) δ8.38(d,J=8.0Hz,1H),8.34(s,1H),8.08(d,J=7.2Hz,1H),7.80–7.67(m,2H),4.85–4.67(m,3H),3.98(t,J=6.6Hz,2H),2. 86–2.75(m,2H),2.31(s,4H),2.25–2.17(m,4H),1.57–1.50(m,2H),1.48 –1.38(m,8H),1.31–1.14(m,38H),1.07–0.92(m,14H),0.86–0.79(m,9H).

[0622] Example 24:

[0623] Step 1:

[0624] To a solution of compound 8 (1.2 g, 1.92 mmol, 1.0 eq.) and compound 9 (1.08 g, 2.88 mmol, 1.5 eq.) in cyclopentyl methyl ether / acetonitrile (24.0 mL, v / v), potassium carbonate (1.32 g, 9.6 mmol, 5.0 eq.) and potassium iodide (637.4 mg, 3.84 mmol, 2.0 eq.) were added, and the mixture was stirred at 95 °C for 24 h under argon protection. Thin-layer chromatography (dichloromethane:methanol = 10 / 1) showed that the reaction was complete and a new main spot was observed. The mixture was filtered, and the filtrate was concentrated under reduced pressure to give a yellow oily crude product. The residue was purified by preparative high-performance liquid chromatography (PreP-HPLC) to give HA-1A-R (299.86 mg, yield 16.9%) as a yellow oil.

[0625] LCMS:Rt:2.297min;MS m / z(ELSD):923.6[M+H] + ;

[0626] CAD:97.15% purity at ELSD; RT=9.974min in 25min.

[0627] 1H NMR (400MHz, CDCl3) δ8.21 (dd, J=8.3, 0.9Hz, 1H), 8.16 (d, J=8.3Hz, 1H), 8.0 0(s,1H),7.74–7.67(m,1H),7.66–7.60(m,1H),4.89–4.76(m,2H),4.60(t,J =6.1Hz,2H),2.94(t,J=6.1Hz,2H),2.44–2.37(m,4H),2.26(td,J=7.5,3.4H z,4H),1.65–1.46(m,16H),1.26(d,J=14.8Hz,48H),0.86(t,J=6.7Hz,12H).

[0628] Example 25:

[0629] Step 1:

[0630] To a solution of compound 1 (crude product 2.5 g, 10.16 mmol, 1.0 eq.) and compound 2 (5.6 g, 12.19 mmol, 1.2 eq.) in acetonitrile (10.0 mL), potassium carbonate (758 mg, 5.49 mmol, 3.0 eq.) and potassium iodide (304 mg, 1.83 mmol, 1.0 eq.) were added, and the mixture was stirred at 90 °C for 24 hours under argon protection. Thin-layer chromatography (dichloromethane:methanol = 10 / 1) showed that the reaction was complete and a new main spot was observed. The reaction was quenched by adding water (30 mL) to the mixture, and extracted with ethyl acetate (30 mL × 3). The organic phases were combined, washed with saturated brine (3 × 50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give compound 3 (3.5 g, yield 54.7%) as a yellow oil.

[0631] LCMS:Rt:1.283min;MS m / z(ELSD):627.5[M+H] + ;

[0632] 1H NMR (400MHz, CDCl3) δ8.20(t,J=8.1Hz,2H),8.05(s,1H),7.71(t,J=7.6Hz,1H) ,7.65(dd,J=11.1,4.0Hz,1H),4.87–4.83(m,1H),4.69(t,J=6.1Hz,2H),3.22(t ,J=6.2Hz,2H),2.61(t,J=7.1Hz,2H),2.26(s,2H),1.58(dd,J=14.0,6.9Hz,4H) ,1.49(d,J=5.1Hz,6H),1.44–1.40(m,2H),1.24(s,30H),0.86(t,J=5.3Hz,6H).

[0633] Step 2:

[0634] To a solution of compound 3 (1.7 g, 2.72 mmol, 1.0 eq.) and compound 4 (1.53 g, 4.07 mmol, 1.5 eq.) in cyclopentyl methyl ether / acetonitrile (30.0 mL, v / v), potassium carbonate (1.87 g, 13.58 mmol, 5.0 eq.) and potassium iodide (901.6 mg, 5.43 mmol, 2.0 eq.) were added, and the mixture was stirred at 95 °C for 24 hours under argon protection. Thin-layer chromatography (dichloromethane:methanol = 10 / 1) showed that the reaction was complete and a new main spot was observed. The mixture was filtered, and the filtrate was concentrated under reduced pressure to give a yellow oily crude product. The residue was purified by preparative high-performance liquid chromatography (PreP-HPLC) to give HA-1A-S (301.84 mg, yield 12.0%) as a yellow oil.

[0635] LCMS:Rt:2.115min;MS m / z(ELSD):923.5[M+H] + ;

[0636] CAD:95.63% purity at ELSD; RT=9.504min in 25min.

[0637] 1H NMR (400MHz, CDCl3) δ8.24–8.19(m,1H),8.17(d,J=8.3Hz,1H),8.01(s,1H),7.74–7. 68(m,1H),7.67–7.61(m,1H),4.90–4.82(m,1H),4.82–4.75(m,1H),4.60(t,J=5.8Hz ,2H),2.94(t,J=5.9Hz,2H),2.41(t,J=6.7Hz,4H),2.26(td,J=7.5,3.4Hz,4H),1.62 –1.54(m,8H),1.54–1.50(m,4H),1.25(s,44H),1.16(s,8H),0.87(t,J=6.7Hz,12H).

[0638] Example 26:

[0639] Step 1:

[0640] To a solution of compound 1 (5 g, 24.04 mmol) in dichloromethane (200 mL), m-chloroperoxybenzoic acid (8.3 g, 48.07 mmol) was added. The reaction mixture was stirred at room temperature for 16 hours under an argon atmosphere. LC-MS showed that the reaction was complete (DP). The reaction mixture was quenched with an aqueous sodium sulfite solution (100 mL) and extracted with dichloromethane (150 mL × 3). The combined organic phases were washed with brine (250 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using ethyl acetate (0% to 20%) in dichloromethane as the eluent to give compound 2 (1.45 g, 26.9% yield), a pale yellow solid.

[0641] LCMS:Rt:1.454min;MS m / z(ELSD):225.0[M+H] + ;

[0642] Step 2:

[0643] Phosphorus tribromooxyphos (POBr3) (2.78 g, 8.035 mmol) was added to a solution of compound 2 (1.45 g, 5.357 mmol) in dichloromethane (30 mL). The reaction mixture was stirred at room temperature under an argon atmosphere for 16 hours. LC-MS showed that the reaction was complete (DP). The reaction mixture was quenched with water (50 mL) and extracted with dichloromethane (50 mL × 3). The combined organic phases were washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using ethyl acetate (0% to 10%) in dichloromethane as the eluent to give compound 3 (1.1 g, 71.8% yield), a pale yellow solid.

[0644] LCMS:Rt:1.966min;MS m / z(ELSD):287.0[M+H] + ;

[0645] Step 3:

[0646] To a solution of compound 3 (2.1 g, 7.317 mmol, 1.0 equivalent) in dichloromethane (30 mL), tert-butyl (2-aminoethyl) carbamate (1.173 g, 7.317 mmol, 1.0 equivalent) and triethylamine (TEA) (2.217 g, 21.951 mmol, 3.0 equivalent) were added. The reaction mixture was stirred at 70 °C for 2 h under a nitrogen atmosphere. Thin-layer chromatography (TLC) (petroleum ether (PE): ethyl acetate (EA) = 5:1) showed that the reaction was complete and a new major spot was observed. The reaction mixture was quenched with water (100 mL) and extracted with dichloromethane (70 mL × 3). The combined organic phases were washed with brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (methanol (MeOH) in dichloromethane (0% to 5%)) to give compound 4 (2.5 g, 83.3% yield) as a yellow solid.

[0647] LCMS:Rt:1.958min;MS m / z(ELSD):412.9[M+H] + ;

[0648] Step 4:

[0649] Iron (Fe) (1.71 g, 30.414 mmol, 5.0 equivalent) and ammonium chloride (NH4Cl) (1.63 g, 30.414 mmol) were added to a solution of compound 4 (2.5 g, 6.083 mmol, 1.0 equivalent) in ethanol (EtOH) / water (H2O) (30 mL / 5 mL). The reaction mixture was stirred at 70 °C for 16 h under a nitrogen atmosphere. LCMS showed that the reaction was complete. The mixture was filtered, and the filter cake was washed with methanol (MeOH) (150 mL). The filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (methanol in dichloromethane (containing 0.05% ammonia, 0% to 10%)) to give compound 5 (1.6 g, 69.2% yield) as a yellow solid.

[0650] LCMS:Rt:1.451min;MS m / z(ELSD):383.0[M+3] + ;

[0651] Step 5:

[0652] To a solution of compound 5 (1.4 g, 3.675 mmol, 1.0 equivalent) in toluene (Tol) / dichloroethane (DCE) (12 mL / 6 mL), triethyl orthoformate (CH(OEt)3) (0.817 g, 5.511 mmol, 1.5 equivalent) and pyridine hydrochloride (Py-HCl) (22 mg, 0.184 mmol) were added. The mixture was stirred at 100 °C for 16 h under a nitrogen atmosphere. LC-MS showed complete reaction (DP). The reaction mixture was quenched with an aqueous solution of sodium carbonate (Na2CO3) (50 mL) and extracted with dichloromethane (50 mL × 3). The combined organic phases were washed with brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using methanol (0% to 10%) in dichloromethane as the eluent to give compound 6 (1.3 g, 90.7% yield) as a white solid.

[0653] LCMS:Rt:1.585min;MS m / z(ELSD):390.9[M+H] + ;

[0654] Step 6:

[0655] Trifluoroacetic acid (TFA) (4 mL) was added to a solution of compound 6 (1.3 g, 3.325 mmol) in dichloromethane (20 mL), and the mixture was stirred at room temperature for 2 hours. LC-MS showed that the reaction was complete (DP). The mixture was concentrated. The residue was adjusted to pH 8–9 with sodium hydroxide (NaOH) (1.0 M) and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using methanol (containing 0.05% ammonia, 0%–15%) in dichloromethane as the eluent to give compound 7 (0.8 g, 83% yield) as a white solid.

[0656] LCMS:Rt:0.747min; MS m / z:291.0[M+H] + ;

[0657] Step 7:

[0658] To a solution of compound 7 (600 mg, 2.069 mmol) in acetonitrile (ACN) / cyclopentyl methyl ether (CPME) (8 mL / 8 mL), 955 mg, 2.069 mmol of nonadecanoyl 8-bromooctanoate (955 mg, 2.069 mmol), potassium carbonate (K₂CO₃) (1.43 g, 10.345 mmol), and potassium iodide (KI) (687 mg, 4.138 mmol) were added, and the mixture was stirred at 90 °C for 16 hours. LC-MS showed complete reaction (DP). The mixture was filtered and concentrated. The residue was purified by silica gel column chromatography using methanol (0% to 10%) in dichloromethane as eluent to give compound 8 (480 mg, 34.6% yield), which was red and white in color.

[0659] LCMS:Rt:2.174min;MS m / z(ELSD):671.2[M+H] + ;

[0660] Step 8:

[0661] To a solution of compound 8 (480 mg, 0.715 mmol) in acetonitrile (ACN) / cyclopentyl methyl ether (CPME) (8 mL / 8 mL), nonyl 8-bromooctanoate (750 mg, 2.146 mmol), potassium carbonate (K₂CO₃) (494 mg, 3.575 mmol), and potassium iodide (KI) (238 mg, 1.430 mmol) were added, and the mixture was stirred at 90 °C for 16 hours. LC-MS showed that the reaction was complete (DP). The mixture was filtered and concentrated. The residue was purified by silica gel column chromatography (using methanol (0% to 10%) in dichloromethane as eluent) and separated by preparative high-performance liquid chromatography (prep-HPLC) to give a pale yellow oil HA-1B-1 (91.92 mg, 13.7% yield).

[0662] LCMS:Rt:1.993min;MS m / z(ELSD):941.4[M+H] + ;

[0663] CAD: 90.38% purity

[0664] 1H NMR (400MHz, DMSO-d6) δ8.42–8.36(m,1H),8.34(s,1H),8.08(d,J=7.8Hz,1H),7.79–7.69(m,2H),4.76(s,3H),3.98(t,J=6.6Hz,2H) ,2.86–2.78(m,2H),2.31(s,4H),2.25–2.19(m,4H),1.54–1.39(m,10H),1.29–1.16(m,38H),1.08–0.93(m,14H),0.86–0.79(m,9H).

[0665] Example 27:

[0666] Step 1:

[0667] Compound 2 (4.8 g, 18.79 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI) (4.32 g, 22.55 mmol), and 4-dimethylaminopyridine (DMAP) (1.15 g, 9.395 mmol) were added to a solution of compound 1 (4.189 g, 18.79 mmol) in dichloromethane (60 mL). The mixture was stirred at room temperature under an argon atmosphere for 16 hours. Liquid chromatography-mass spectrometry (LC-MS) showed that the reaction was complete (DP). The reaction was quenched with water (100 mL) and extracted with ethyl acetate (70 mL × 3). The organic phases were combined, washed with brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using ethyl acetate (0% to 10%) in petroleum ether as eluent to give compound 3 (6.2 g, 71.8% yield) as a white solid.

[0668] LCMS:Rt:2.110min;MS m / z(ELSD):460.2[M+H] + ;

[0669] Step 2:

[0670] Compound 4 (2 g, 13.99 mmol), N-methylmorpholine (NMM) (1.42 g, 13.99 mmol), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) (7.98 g, 20.99 mmol) were added to a dichloromethane / dimethylformamide (20 mL / 20 mL) solution of compound 1 (3.75 g, 16.78 mmol). The mixture was stirred at room temperature under an argon atmosphere for 16 hours. Liquid chromatography-mass spectrometry (LC-MS) showed that the reaction was complete (DP). The reaction was quenched with water (100 mL) and extracted with ethyl acetate (70 mL × 3). The organic phases were combined, washed with brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using ethyl acetate (0% to 25%) in petroleum ether as the eluent to give compound 5 (2.1 g, yield 43.2%) as a white solid.

[0671] LCMS:Rt:1.305min;MS m / z(ELSD):350.1[M+H] + ;

[0672] Step 3:

[0673] Compound 3 (931 mg, 2.024 mmol), potassium carbonate (1.4 g, 10.12 mmol), and potassium iodide (672 mg, 4.048 mmol) were added to an acetonitrile / cyclopentyl methyl ether (8 mL / 8 mL) solution of compound 6 (500 mg, 2.024 mmol). The mixture was stirred at 90 °C for 16 hours under an argon atmosphere. Liquid chromatography-mass spectrometry (LC-MS) showed that the reaction was complete (DP). The mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography using methanol (0% to 10%) in dichloromethane as the eluent to give compound 7 (450 mg, 35.5% yield) as a white solid.

[0674] LCMS:Rt:1.101min;MS m / z(ELSD):626.8[M+H] + ;

[0675] Step 4:

[0676] Compound 4 (501 mg, 1.44 mmol), potassium carbonate (497 mg, 3.6 mmol), and potassium iodide (240 mg, 1.44 mmol) were added to an acetonitrile / cyclopentyl methyl ether (8 mL / 8 mL) solution of compound 7 (450 mg, 0.72 mmol). The mixture was stirred at 90 °C for 16 hours under an argon atmosphere. Liquid chromatography-mass spectrometry (LC-MS) showed that the reaction was complete (DP). The mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (using methanol (0% to 10%) in dichloromethane as eluent) and separated by preparative high-performance liquid chromatography to give a white solid HA-11-1 (83.27 mg, yield 12.9%).

[0677] LCMS:Rt:1.276min;MS m / z(ELSD):893.6[M+H] + ;

[0678] CAD: 97.39% purity

[0679] 1 H NMR(400MHz, CDCl3)δ8.21(d,J=8.0Hz,1H),8.18–8.13(m,1H),8.00(s,1H),7.7 2(t,J=7.6Hz,1H),7.65(t,J=7.4Hz,1H),5.91(s,1H),5.64–5.42(m,1H),4.60(t ,J=5.2Hz,2H),3.90(s,1H),3.28–3.21(m,2H),2.99–2.88(m,2H),2.37(s,4H),2 .12(t,J=7.6Hz,4H),1.62–1.47(m,10H),1.33–1.10(m,52H),0.89–0.84(m,9H).

[0680] Example 28:

[0681] Step 1:

[0682] To a dry tetrahydrofuran (15 mL) solution of compound 1 (1.0 g, 2.98 mmol, 1.0 eq.), pyridine (Py, 1.2 g, 14.88 mmol, 5.0 eq.) and compound 2 (381 mg, 3.12 mmol, 1.05 eq.) were slowly added dropwise at 0 °C under nitrogen protection. The mixture was then stirred at room temperature (rt.) for 16 h. Thin-layer chromatography (TLC, petroleum ether / ethyl acetate = 1 / 1) showed the formation of the target product. Ethyl acetate (EA, 30 mL) was added to the mixture, followed by washing with saturated ammonium chloride solution (Sat. NH4Cl, 20 mL × 3), water (20 mL), and saturated brine (brine, 20 mL × 2) sequentially. The mixture was dried over anhydrous sodium sulfate (Na2SO4), filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (0%–18% ethyl acetate in petroleum ether by volume) to give compound 3 (1.1 g, 87% yield), which was a yellow oil.

[0683] Step 2:

[0684] Potassium carbonate (K₂CO₃, 0.85 g, 6.16 mmol, 2.0 eq.) was added to an ethanol / water (EtOH / H₂O, 5 / 1, 15 mL) solution of compound 3 (1.3 g, 3.08 mmol, 1.0 eq.). The mixture was stirred at 55 °C for 16 h. Liquid chromatography-mass spectrometry (LC-MS) showed that the reaction was complete. The mixture was concentrated to remove ethanol, and dichloromethane (DCM, 30 mL) was added to the residue. The solution was washed with water (10 mL) and saturated brine (10 mL × 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (ethyl acetate in petroleum ether, volume fraction 0%–25%) to give compound 4 (0.95 g, yield 76.6%) as a yellow solid.

[0685] LCMS:Rt:1.674min;MS m / z(ELSD):405.3[M+H] + ;

[0686] Step 3:

[0687] Trifluoroacetic acid (TFA, 2 mL) was added dropwise to a solution of compound 4 (950 mg, 2.35 mmol, 1.0 eq.) in dichloromethane (10 mL). The mixture was stirred at room temperature for 2 hours under nitrogen protection. Liquid chromatography-mass spectrometry (LC-MS) showed the formation of the target product. The mixture was concentrated under reduced pressure to remove the trifluoroacetic acid. The residue was quenched with an aqueous solution of sodium bicarbonate (aq. NaHCO3) and the pH was adjusted to 8, followed by concentration under reduced pressure. The residue was purified by silica gel column chromatography (using a 10% methanol solution in dichloromethane as eluent) to give compound 5 (700 mg, 97.9% yield) as a white solid.

[0688] LCMS:Rt:1.067min;MS m / z(ELSD):305.3[M+H] + ;

[0689] Step 4:

[0690] To a solution of compound 5 (700 mg, 2.30 mmol, 1.0 eq.) and compound 6 (1.27 g, 2.76 mmol, 1.2 eq.) in cyclopentyl methyl ether / acetonitrile (CPME / ACN, 1 / 1 v / v, 20 mL), potassium carbonate (1.59 g, 11.5 mmol, 5.0 eq.) and potassium iodide (KI, 763.6 mg, 4.6 mmol, 2.0 eq.) were added, and the mixture was stirred at 90 °C for 3 days under argon (Ar) protection. Liquid chromatography-mass spectrometry (LC-MS) showed the formation of the target product. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (0%–5% methanol in dichloromethane) to give compound 7 (1.2 g, 75.9% yield) as a yellow oil.

[0691] LCMS:Rt:1.313min;MS m / z(ELSD):685.8[M+H] + ;

[0692] 1H NMR(400MHz, CDCl3)δ8.19(t,J=8.4Hz,2H),7.65(ddd,J=15.3,14.4,7.0Hz,2H),4.88–4 .82(m,1H),4.70(dt,J=28.8,6.9Hz,2H),3.98–3.86(m,2H),3.35(t,J=3.0Hz,3H),3.34– 3.28(m,1H),3.17(t,J=6.8Hz,1H),2.63(t,J=7.2Hz,1H),2.26(t,J=7.5Hz,2H),1.62–1 .57(m,2H),1.47(dd,J=20.1,6.1Hz,6H),1.26(d,J=16.4Hz,34H),0.86(t,J=6.8Hz,6H).

[0693] Step 5:

[0694] To a solution of compound 7 (400 mg, 0.58 mmol, 1.0 eq.) and compound 8 (305.3 mg, 0.88 mmol, 1.5 eq.) in cyclopentyl methyl ether / acetonitrile (CPME / MeCN, 8.0 mL, v / v), potassium carbonate (403.0 mg, 2.92 mmol, 5.0 eq.) and potassium iodide (194.2 mg, 1.16 mmol, 2.0 eq.) were added, and the mixture was stirred at 95 °C for 24 h under argon protection. Thin-layer chromatography (TLC, dichloromethane:methanol = 10 / 1) showed that the reaction was complete and a new main spot was observed. The mixture was filtered, and the filtrate was concentrated under reduced pressure to give a yellow oily crude product. The residue was purified by preparative high-performance liquid chromatography (PreP-HPLC) to give HA-17A (111.26 mg, yield 32.1%) as a yellow oil.

[0695] LCMS:Rt:2.016min;MS m / z(ELSD):955.5[M+H+2] + ;

[0696] CAD:99.61% purity at ELSD; RT=9.655min in 25min.

[0697] 1H NMR (400MHz, CDCl3) δ8.20(t,J=7.6Hz,2H),7.68(t,J=7.6Hz,1H),7.61(t,J=7.6Hz,1H), 4.85(p,J=6.0Hz,1H),4.66(t,J=6.8Hz,2H),4.04(t,J=6.8Hz,2H),3.92(t,J=6.6Hz,2H) ,3.36(s,3H),3.31(t,J=6.4Hz,2H),2.89(t,J=6.8Hz,2H),2.44(d,J=5.6Hz,4H),2.27(t d,J=7.6,4.0Hz,4H),1.60(s,4H),1.49(d,J=5.6Hz,4H),1.24(s,54H),0.90–0.83(m,9H).

[0698] Example 29:

[0699] Step 1:

[0700] Compound 2 (3.4 g, 26.19 mmol, 1.2 eq.), tetrakis(triphenylphosphine)palladium (1.26 g, 1.09 mmol, 0.05 eq.), and potassium carbonate (9.05 g, 65.48 mmol, 3.0 eq.) were added to a mixture of compound 1 (5 g, 21.83 mmol, 1.0 eq.) in toluene (50 mL) and water (10 mL). The reaction mixture was stirred at 100 °C for 16 h under a nitrogen atmosphere. Thin-layer chromatography (petroleum ether / ethyl acetate = 10 / 1) showed the formation of a new spot. The reaction was quenched with 100 mL of water and extracted with ethyl acetate (50 mL × 3). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography with petroleum ether / ethyl acetate (1 / 0–10 / 1, v / v) as eluent to give compound 3 (2.12 g, 41% yield) as a colorless oil.

[0701] Step 2:

[0702] Under a nitrogen atmosphere, lithium aluminum hydride (8.53 mL, 21.34 mmol, 2.5 eq.) was added to a mixture of compound 3 (2 g, 8.53 mmol, 1.0 eq.) in tetrahydrofuran (20 mL) at 0 °C. The mixture was allowed to return to room temperature and stirred for 2 hours. Thin-layer chromatography (petroleum ether: ethyl acetate = 5 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was quenched with sodium sulfate decahydrate, filtered, and concentrated under vacuum. The residue was purified by silica gel column chromatography to give compound 4 (1.49 g, 84% yield) as a colorless oil.

[0703] Step 3:

[0704] Under a nitrogen atmosphere, triphenylphosphine (2.24 g, 8.55 mmol, 1.2 eq.) and N-bromosuccinimide (1.52 g, 8.55 mmol, 1.2 eq.) were added to a solution of compound 4 (1.47 g, 7.12 mmol, 1.0 eq.) in dichloromethane (20 mL), and the mixture was stirred at 25 °C for 16 hours. Thin-layer chromatography (petroleum ether: ethyl acetate = 10 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was quenched with 30 mL of water and extracted with ethyl acetate (30 mL × 3). The organic phases were combined, washed with 50 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography with petroleum ether / ethyl acetate (1 / 0–10 / 1, v / v) as eluent to give compound 5 (599 mg, 31% yield) as a yellow oil.

[0705] 1 H NMR (400MHz, CDCl3) δ7.22(t,J=7.8Hz,1H),7.07(d,J=7.7Hz,1H),7.02(d,J=5.7Hz,2H),3.56(t,J=7.8Hz, 2H),3.14(t,J=7.8Hz,2H),2.73–2.47(m,2H),1.65–1.57(m,2H),1.40–1.26(m,6H),0.88(t,J=6.7Hz,3H).

[0706] Step 4:

[0707] Compound 5 (179 mg, 0.67 mmol, 1.5 eq.), potassium iodide (147 mg, 0.89 mmol, 2.0 eq.), and potassium carbonate (307 mg, 2.22 mmol, 5.0 eq.) were added to a mixture of compound 8 (220 mg, 0.44 mmol, 1.0 eq.) in acetonitrile (3 mL) and cyclopentyl methyl ether (3 mL). The mixture was stirred at 90 °C for 16 hours under an argon atmosphere. The mixture was quenched with 40 mL of water and extracted with ethyl acetate (40 mL × 3). The combined organic phases were washed with 40 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (dichloromethane containing 0%–10% methanol) and preparative high-performance liquid chromatography to give a yellow oil, HA-37 (79.97 mg, 28% yield).

[0708] LCMS:Rt:1.101min;MS m / z(ELSD):684.6[M+H] + ;

[0709] CAD:91.82%purity.Rt:13.033min

[0710] 1 H NMR (400MHz, CDCl3) δ7.94(d,J=8.0Hz,1H),7.85(d,J=8.0Hz,1H),7.58-7.50(m,1H),7.44(s,1H),7.36- 7.30(m,1H),7.24-7.17(m,1H),7.04(d,J=8.0Hz,1H),6.98-6.92(m,2H),5.63(s,2H),4.47-4.42(m,2H), 4.09-4.02(m,2H),3.00-2.93(m,2H),2.79-2.71(m,2H),2.67-2.60(m,2H),2.60-2.53(m,2H),2.49-2.4 2(m,2H),2.30-2.23(m,2H),1.64-1.52(m,6H),1.34-1.24(m,20H),1.17-1.08(m,6H),0.90-0.85(m,6H).

[0711] Example 30:

[0712] Step 1:

[0713] Under a nitrogen atmosphere, trifluoroacetic acid (3 mL) was added to a mixture of compound 1 (1 g, 2.326 mmol, 1.0 equivalent) in dichloromethane (15 mL) at 0 °C. The reaction mixture was allowed to return to room temperature and stirred for 2 hours. The reaction mixture was quenched with 1.0 M sodium hydroxide solution and the pH was adjusted to 8. The mixture was then concentrated under vacuum. The residue was purified by silica gel column chromatography using a dichloromethane solution containing 0.05% ammonia (increasing from 0% to 15%) as eluent to give compound 2 (750 mg, 97.6% yield) as a white solid.

[0714] LCMS:Rt:0.780min;MS m / z(ELSD):331.2[M+H] + ;

[0715] Step 2:

[0716] Compound 3 (420 mg, 2.273 mmol) was added to a 5 mL ethanol solution of compound 2 (250 mg, 0.758 mmol), and the mixture was stirred at 80 °C for 48 hours. Liquid chromatography-mass spectrometry (LC-MS) showed the formation of the target product (DP). The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (using methanol in dichloromethane as eluent, increasing from 0% to 15%) and preparative high-performance liquid chromatography (pre-HPLC) to give HA-TLRb-A (123.81 mg, yield 23.3%) as a white solid.

[0717] LCMS:Rt:1.107min;MS m / z(ELSD):699.5[M+H] + ;

[0718] CAD:98.21% purity at ELSD; RT=14.383min.

[0719] 1 ¹H NMR (400MHz, DMSO-d6) δ 8.37–8.29 (m, 1H), 8.10–8.02 (m, 1H), 7.75–7.66 (m, 2H), 4.69–4.51 (m, 2H), 4.24 (s, 2H), 3.37 (s, 2H), 2.99 (t, J = 7.6Hz, 2H), 2.35–2.26 (m, 2H), 2.24–2.18 (m, 2H), 1.92–1.78 (m, 4H), 1.61–1.43 (m, 4H), 1.34–1.04 (m, 38H), 1.00–0.94 (m, 3H), 0.87–0.80 (m, 6H). Example 31:

[0720] Step 1:

[0721] Compound 2 (7.74 g, 41.15 mmol, 1.0 equivalent) and triethylamine (12.47 g, 123.45 mmol, 3.0 equivalent) were added to a solution of compound 1 (10 g, 41.15 mmol, 1.0 equivalent) in dichloromethane (100 mL). The reaction mixture was stirred at 70 °C for 2 h under a nitrogen atmosphere. Thin-layer chromatography (petroleum ether: ethyl acetate = 1:1) showed that the reaction was complete and a new main spot was observed. The reaction was quenched with water (200 mL) and extracted with ethyl acetate (150 mL × 3). The organic layers were combined, washed with brine (2 × 100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, using methanol in dichloromethane as an eluent increasing from 0% to 5%, to give compound 3 (15 g, 92.5% yield) as a yellow solid. LCMS:Rt:1.113min; MS m / z(ELSD):395.1[M+H] + ;

[0722] Step 2:

[0723] Iron powder (3.2 g, 57.107 mmol, 3.0 equivalent) and ammonium chloride (3.06 g, 57.107 mmol, 3.0 equivalent) were added to an ethanol / water (80 mL / 8 mL) solution of compound 3 (7.5 g, 19.036 mmol, 1.0 equivalent). The reaction mixture was stirred at 70 °C for 16 h under a nitrogen atmosphere. Thin-layer chromatography showed that the reaction was complete and a new main spot was observed. The mixture was filtered, and the filter cake was washed with methanol (150 mL). The filtrates were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (using methanol containing 0.05% ammonia in dichloromethane as eluent, increasing from 0% to 10%) to give compound 4 (1.3 g, yield 18.7%) as a white solid.

[0724] LCMS:Rt:0.885min; MS m / z(ELSD):365.2[M+H] + ;

[0725] Step 3:

[0726] Under an argon atmosphere, pyridine (2.82 g, 35.715 mmol, 5.0 equivalent) and valeryl chloride (900 mg, 7.5 mmol, 1.05 equivalent) were added to a tetrahydrofuran (30 mL) solution of compound 4 (2.6 g, 7.143 mmol, 1.0 equivalent) at 0 °C. The mixture was stirred at 25 °C for 16 h under a nitrogen atmosphere. Thin-layer chromatography (dichloromethane:methanol = 10 / 1) showed that the reaction was complete and a new main spot was observed. The reaction was quenched by adding water (100 mL) to the mixture, and extracted with ethyl acetate (80 mL × 3). The organic layers were combined, washed with brine (2 × 50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (using a 10% methanol solution in dichloromethane as eluent) to give compound 5 (2.5 g, 78.1% yield) as a pale yellow solid.

[0727] LCMS:Rt:0.949min; MS m / z(ELSD):450.9[M+H] + ;

[0728] Step 4:

[0729] Potassium carbonate (1.91 g, 13.808 mmol, 2.0 equivalent) was added to an ethanol / water (40 mL / 8 mL) solution of compound 5 (3.1 g, 6.904 mmol, 1.0 equivalent). The mixture was stirred at 55 °C for 16 h under a nitrogen atmosphere. Thin-layer chromatography (dichloromethane:methanol = 10 / 1) showed that the reaction was complete and a new main spot was observed. The reaction was quenched by adding water (100 mL) to the mixture, and extracted with ethyl acetate (80 mL × 3). The organic layers were combined, washed with brine (2 × 50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (using a 10% methanol solution in dichloromethane as eluent) to give compound 6 (3 g, 100% yield) as a pale yellow solid.

[0730] LCMS:Rt:1.101min;MS m / z(ELSD):430.9[M+H] + ;

[0731] Step 5:

[0732] A mixture of compound 6 (2 g, 4.651 mmol, 1.0 equivalent) and ammonia (30 mL, 7 M methanol-ammonia solution) was stirred at 140 °C for 3 days. Thin-layer chromatography (dichloromethane:methanol = 10 / 1) showed the formation of the target product. The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (using 10% methanol-dichloromethane solution as eluent) to give compound 7 (700 mg, yield 36.6%) as a white solid.

[0733] LCMS:Rt:0.844min; MS m / z(ELSD):412.7[M+H] + ;

[0734] Step 6:

[0735] Under a nitrogen atmosphere, trifluoroacetic acid (2 mL) was added to a mixture of compound 6 (700 mg, 1.703 mmol, 1.0 equivalent) and dichloromethane (10 mL) at 0 °C. The reaction mixture was allowed to return to room temperature and stirred for 2 hours. The reaction mixture was quenched with an aqueous sodium bicarbonate solution and the pH was adjusted to 8. The mixture was then concentrated under vacuum. The residue was purified by silica gel column chromatography (using methanol containing 0.05% ammonia in dichloromethane as eluent, increasing from 0% to 15%) to give compound 8 (410 mg, 77.3% yield) as a white solid.

[0736] LCMS:Rt:0.751min;MS m / z(ELSD):312.2[M+H] + ;

[0737] Step 7:

[0738] Compound 9 (444 mg, 2.412 mmol, 3 equivalents) was added to an ethanol (5 mL) solution of compound 8 (250 mg, 0.804 mmol, 1.0 equivalents), and the mixture was stirred at 80 °C for 48 hours under a nitrogen atmosphere. Thin-layer chromatography (dichloromethane:methanol = 10 / 1) showed that the reaction was complete and a new main spot was observed. The reaction was quenched by adding water (50 mL) to the mixture, and extracted with ethyl acetate (50 mL × 3). The organic layers were combined, washed with brine (3 × 50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (using a 10% methanol solution in dichloromethane as eluent) and preparative high-performance liquid chromatography to give compound HA-TLRb (110.61 mg, yield 20.2%) as a white solid.

[0739] LCMS:Rt:1.054min;MS m / z(ELSD):680.4[M+H]+ ;

[0740] CAD:95.20% purity at ELSD; RT=10.039min.

[0741] 1 H NMR (400MHz, DMSO) δ8.00(d,J=8.2Hz,1H),7.60(d,J=7.6Hz,1H),7.39(t,J=7.4 Hz,1H),7.23(t,J=7.4Hz,1H),6.42(s,2H),4.48(t,J=7.6Hz,2H),4.24(d,J=17. 4Hz,2H),3.41(s,2H),2.96–2.85(m,2H),2.38–2.20(m,4H),1.86–1.75(m,4H), 1.58–1.40(m,4H),1.37–1.01(m,38H),0.96(t,J=7.4Hz,3H),0.87–0.81(m,6H).

[0742] Example 32: HA-1-10-A

[0743] Step 1:

[0744] A solution of compound 5 (4 g, 16.53 mmol, 1.0 eq.) was added to dichloromethane (50 mL), followed by compound 6 (3.26 g, 17.36 mmol, 1.05 eq.) and triethylamine (5.01 g, 49.59 mmol, 3.0 eq.). The reaction mixture was stirred at 70 °C for 2 hours under a nitrogen atmosphere. Thin-layer chromatography (PE:EA = 5:1) showed that the reaction was complete and a new major spot was observed. The reaction was quenched with water (100 mL) and extracted with dichloromethane (100 mL × 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (petroleum ether containing 0%–30% ethyl acetate) to give compound 7 (5.53 g, 85%) as a yellow solid.

[0745] LCMS: Rt: 1.929min; MS m / z (ELSD): 395.3[M+H]+;

[0746] Step 2:

[0747] A solution of compound 7 (6.7 g, 16.50 mmol, 1.0 eq.) was added to an ethanol / water mixture (50 mL / 10 mL), followed by iron (2.77 g, 49.50 mmol, 3.0 eq.) and ammonium chloride (2.67 g, 49.50 mmol, 3.0 eq.). The reaction mixture was stirred at 70 °C for 2 hours under a nitrogen atmosphere. Thin-layer chromatography (PE:EA = 2:1) showed that the reaction was complete and a new major spot was observed. The mixture was filtered, and the filter cake was washed with methanol (150 mL). The filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (petroleum ether containing 0%–60% ethyl acetate) to give compound 8 (3.2 g, 42%) as a yellow solid.

[0748] LCMS: Rt: 1.515min; MS m / z (ELSD): 365.3[M+H]+;

[0749] Step 3:

[0750] A solution of compound 8 (2.46 g, 6.75 mmol, 1.0 eq.) was added to tetrahydrofuran (30 mL), followed by compound 9 (683 mg, 7.425 mmol, 1.1 eq.) and pyridine (5.33 g, 67.5 mmol, 10 eq.). The mixture was stirred at room temperature for 1 hour under a nitrogen atmosphere. The reaction was quenched by adding water (30 mL) and extracted with ethyl acetate (50 mL × 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give compound 10 (1.9 g, 67%) as a yellow solid.

[0751] Step 4:

[0752] A mixture of compound 10 (1.79 g, 4.25 mmol, 1.0 eq.) was added to ethanol / water (20 mL / 5 mL), followed by potassium carbonate (1.173 g, 8.5 mmol, 2.0 eq.). The mixture was stirred at 55 °C for 16 hours under a nitrogen atmosphere. The mixture was then concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with dichloromethane containing 10% methanol, to give compound 11 (1.3 g, 76%) as a pale yellow solid.

[0753] Step 5:

[0754] The mixture of compound 11 (1.1 g, 2.06 mmol, 1.0 eq.) in hydrochloric acid / dioxane (10 mL, 4 M) was stirred at room temperature for 2 hours. The reaction mixture was concentrated under vacuum to give crude compound 12 (1.2 g, >100%) as a white solid.

[0755] LCMS: Rt: 1.136min; MS m / z (ELSD): 303.2[M+H]+;

[0756] Step 6:

[0757] A solution of compound 12 (741 mg, 2.45 mmol, 1.0 eq.) was added to dichloromethane (10.0 mL), followed by triethylamine (743 mg, 7.36 mmol, 3.0 eq.) and 3-chloropropane-1-sulfonyl chloride (432 mg, 2.45 mmol, 1.0 eq.). The mixture was stirred at room temperature for 3 hours under an argon atmosphere. The reaction was quenched with water (50 mL) and extracted with ethyl acetate (50 mL × 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with dichloromethane containing 10% methanol, to give a yellow oily compound 13 (342 mg, 33%).

[0758] LCMS: Rt: 1.620min; MS m / z (ELSD): 443.2[M+H]+;

[0759] Step 7:

[0760] A solution of compound 13 (300 mg, 0.679 mmol, 1.0 eq.) was added to N,N-dimethylformamide (8 mL), followed by potassium acetate (100 mg, 1.019 mmol, 1.5 eq.). The mixture was stirred at 120 °C for 16 hours under an argon atmosphere. The reaction was quenched with water (30 mL) and extracted with ethyl acetate (30 mL × 3). The combined organic layers were washed with brine (50 mL × 3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give compound 14 (220 mg, 69.6%) as a yellow oil.

[0761] LCMS: Rt: 1.423min; MS m / z (ELSD): 467.1[M+H]+;

[0762] Step 8:

[0763] A solution of compound 14 (200 mg, 0.43 mmol, 1.0 eq.) was added to methanol (10.0 mL) and water (2 mL), followed by lithium hydroxide monohydrate (180 mg, 4.3 mmol, 10 eq.). The mixture was stirred at 80 °C for 3 hours under an argon atmosphere. The reaction was quenched by adding water (30 mL), and the pH was adjusted to 8-9 with an aqueous sodium bicarbonate solution. The aqueous layer was extracted with ethyl acetate (30 mL × 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give compound 15 (128 mg, 69.9%) as a yellow oil.

[0764] LCMS: Rt: 1.473min; MS m / z (ELSD): 425.3[M+H]+;

[0765] Step 9:

[0766] A solution of compound 15 (128 mg, 0.302 mmol, 1.0 eq.) was added to N,N-dimethylformamide (5 mL), followed by compound 16 (83 mg, 0.364 mmol, 1.2 eq.), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (115 mg, 0.604 mmol, 2.0 eq.), N,N-diisopropylethylamine (156 mg, 1.208 mmol, 4.0 eq.), and 4-dimethylaminopyridine (15 mg, 0.12 mmol, 0.4 eq.). The reaction mixture was stirred at 25 °C for 16 hours under a nitrogen atmosphere. The reaction was quenched with water (20 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was separated and purified by silica gel column chromatography and preparative high performance liquid chromatography to obtain yellow oily HA-1-10-A (45.95 mg, 24%).

[0767] LCMS: Rt: 1.451min; MS m / z (ELSD): 635.5[M+H]+;

[0768] CAD:92.75% purity at ELSD; RT=23.200min.

[0769] 1H NMR (400MHz, CDCl3) δ8.23–8.14(m,1H),8.13–8.03(m,1H),7.70–7.60(m,2H),4.72(t,J=6.2Hz ,1H),4.63–4.43(m,2H),4.16(t,J=6.2Hz,2H),3.21(q,J=6.5Hz,2H),3.06(ddd,J=24.0,11.3, 6.7Hz,4H),2.28(t,J=7.6Hz,2H),2.12(dt,J=12.4,6.2Hz,2H),2.07–1.99(m,2H),1.82–1.74( m,2H),1.62–1.55(m,2H),1.50(t,J=7.5Hz,3H),1.25(d,J=3.4Hz,21H),0.87(d,J=6.1Hz,3H).

[0770] Example 33: HA-1-18-A

[0771] Step 1:

[0772] A solution of compound 1 (3 g, 8.26 mmol, 1.0 eq.) was added to dichloromethane (20 mL), followed by compound 2 (1.89 g, 8.26 mmol, 1.0 eq.) and triethylamine (2.5 g, 24.79 mmol, 3.0 eq.). The reaction mixture was stirred at 70 °C for 2 hours under a nitrogen atmosphere. Thin-layer chromatography (PE:EA = 5:1) showed that the reaction was complete and a new major spot was observed. The reaction was quenched by adding water (50 mL) to the mixture and extracted with dichloromethane (50 mL × 3). The combined organic layers were washed with brine (2 × 80 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound 3 (3.05 g, 76%) as a yellow solid.

[0773] LCMS:Rt:1.666min;MS m / z(ELSD):436.3[M+H] + ;

[0774] Step 2:

[0775] A solution of compound 3 (3.05 g, 7 mmol, 1.0 eq.) was added to an ethanol / water mixture (40 mL, 3 / 1), followed by iron (1.2 g, 21.02 mmol, 3.0 eq.) and ammonium chloride (1.1 g, 21.02 mmol, 3.0 eq.). The reaction mixture was stirred at 70 °C for 3 hours under a nitrogen atmosphere. The mixture was filtered, and the filter cake was washed with methanol (150 mL). The filtrate was concentrated under reduced pressure. The residue was purified by column chromatography to give compound 4 (1.758 g, 43%) as a yellow solid.

[0776] LCMS:Rt:1.820min; MS m / z(ELSD):406.3[M+H] + ;

[0777] Step 3:

[0778] A solution of compound 4 (1.758 g, 4.34 mmol, 1.0 eq.) and triethyl orthoformate (10 mL) was stirred at 80 °C for 16 hours under a nitrogen atmosphere. Thin-layer chromatography (DCM: MeOH = 10 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with dichloromethane containing 10% methanol, to give compound 5 (2.17 g, 98%) as a pale yellow solid.

[0779] LCMS:Rt:1.323min;MS m / z(ELSD):416.3[M+H] + ;

[0780] Step 4:

[0781] Compound 5 (2.117 g, 5.1 mmol, 1.0 eq.) was stirred in a mixture of hydrochloric acid / dioxane (20 mL, 4 M) at room temperature for 2 hours. The reaction mixture was concentrated under vacuum to give a yellow oily compound 6 (1.61 g, >100%), which could be used in the next step without further purification.

[0782] LCMS:Rt:1.118min;MS m / z(ELSD):316.2[M+H] + ;

[0783] Step 5:

[0784] A solution of compound 6 (1.6 g, 5.1 mmol, 1.0 eq.) was added to methanol (20 mL), followed by compound 7 (898 mg, 5.6 mmol, 1.1 eq.) and sodium triacetoxyborohydride (964 mg, 15.30 mmol, 3.0 eq.). The reaction mixture was stirred at room temperature under a nitrogen atmosphere for 16 hours. The reaction was quenched with water (80 mL) and extracted with dichloromethane (80 mL × 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound 8 (1.65 g, 84%) as a yellow solid.

[0785] LCMS:Rt:1.062min;MS m / z(ELSD):459.3[M+H] + ;

[0786] Step 6:

[0787] Under a nitrogen atmosphere, a mixture of compound 8 (1.6 g, 3.49 mmol, 1.0 eq.) was added to dichloromethane (10 mL) at 0 °C, followed by the addition of trifluoroacetic acid (10 mL). The reaction mixture was heated to room temperature and stirred for 2 hours. The reaction mixture was adjusted to pH 8 with an aqueous sodium bicarbonate solution and extracted with ethyl acetate (3 × 50 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound 9 (656 mg, 52.5%) as a yellow solid.

[0788] LCMS:Rt:0.739min;MS m / z(ELSD):359.3[M+H] + ;

[0789] Step 7:

[0790] A solution of compound 9 (650 mg, 1.82 mmol, 1.0 eq.) and compound 10 (857 mg, 1.82 mmol, 1.0 eq.) was added to acetonitrile (10.0 mL) and methyl cyclopentyl ether (10 mL), followed by the addition of potassium carbonate (344 mg, 9.08 mmol, 5.0 eq.) and potassium iodide (603 mg, 3.63 mmol, 2.0 eq.). The mixture was stirred at 90 °C for 16 hours under an argon atmosphere. Thin-layer chromatography (DCM: MeOH = 10 / 1) showed that the reaction was complete and a new major spot was observed. The reaction was quenched with water (50 mL) and extracted with ethyl acetate (50 mL × 3). The combined organic layers were washed with brine (2 × 100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography by elution with dichloromethane containing 10% methanol to give a yellow oily compound 11 (653 mg, 42%).

[0791] Step 8:

[0792] A solution of compound 11 (534 mg, 0.72 mmol, 1.0 eq.) and compound 12 (408 mg, 1.08 mmol, 1.5 eq.) was added to acetonitrile (5 mL) and methyl cyclopentyl ether (5 mL), followed by the addition of potassium carbonate (500 mg, 3.61 mmol, 5.0 eq.) and potassium iodide (240 mg, 1.45 mmol, 2.0 eq.). The mixture was stirred at 90 °C for 16 hours under an argon atmosphere. Thin-layer chromatography (DCM: MeOH = 10 / 1) showed the reaction was complete, and a new major spot was observed. The reaction was quenched with water (530 mL) and extracted with ethyl acetate (50 mL × 3). The combined organic layers were washed with brine (2 × 100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with dichloromethane containing 10% methanol, to give a yellow oily crude product. The residue was purified by preparative high performance liquid chromatography to obtain a white solid HA-1-18-A (178 mg, 13%).

[0793] LCMS: Rt=1.626min, MS m / z(ELSD):1035.9[M+H] + ;

[0794] CAD:87.58% purity at ELSD; RT=23.792min.

[0795] 1H NMR(400MHz, CDCl3)δ8.26–8.15(m,2H),8.06(s,1H),7.76–7.69(m,1H),7. 68–7.61(m,1H),4.90–4.77(m,2H),4.69(t,J=6.3Hz,2H),2.93(t,J=6.3Hz, 2H),2.48(d,J=60.3Hz,16H),2.28(td,J=7.5,3.8Hz,4H),1.59(d,J=17.6Hz ,5H),1.51(dd,J=12.3,5.4Hz,7H),1.34–1.21(m,52H),0.90–0.85(m,12H).

[0796] Example 34: HA-13

[0797] Step 1:

[0798] Sodium azide (622 mg, 9.57 mmol, 3 eq.) was added to a solution of HA-13-A (1.636 g, 3.19 mmol, 1.0 eq.) in N,N-dimethylformamide (20 mL). The reaction mixture was stirred at 50 °C for 16 h under nitrogen protection. The reaction was quenched with water (50 mL), followed by extraction with ethyl acetate (50 mL × 3). The combined organic layers were washed with saturated brine (2 × 100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give compound 1 (1.183 g, 71%) as a yellow oil.

[0799] LCMS:Rt:1.608min; MS m / z(ELSD):521.6[M+H] + ;

[0800] Step 2:

[0801] Triphenylphosphine (8.94 g, 34.12 mmol, 15 eq.) was added to a tetrahydrofuran (10 mL) / water (1 mL) solution of compound 1 (1.183 g, 2.275 mmol, 1.0 eq.). The reaction mixture was stirred at 80 °C for 16 h under nitrogen protection. The reaction was quenched with water (30 mL) and then extracted with ethyl acetate (30 mL × 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give compound 2 (820 mg, 47.8%) as a yellow solid.

[0802] LCMS:Rt:1.113min;MS m / z(ELSD):755.7[M+H] + ;

[0803] Step 3:

[0804] A 1M hydrochloric acid solution (5 mL) of compound 2 (820 mg, 1.09 mmol, 1.0 eq.) was stirred at 120 °C for 3 hours under nitrogen protection. The mixture was concentrated under reduced pressure to give crude compound 3 (401 mg, 100%) as a yellow solid, which could be used in the next reaction without further purification.

[0805] LCMS:Rt:0.891min;MS m / z(ELSD):369.3[M+H] + ;

[0806] Step 4:

[0807] To a solution of compound 3 (300 mg, 0.815 mmol, 1.0 eq.) in dichloromethane (10 mL), 1-nonanol (176 mg, 1.223 mmol, 1.5 eq.), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (313 mg, 1.63 mmol, 2.0 eq.), N,N-diisopropylethylamine (420 mg, 3.26 mmol, 4.0 eq.), and 4-dimethylaminopyridine (37 mg, 0.326 mmol, 0.4 eq.) were added. The reaction mixture was stirred at 25 °C for 16 hours under nitrogen protection. The reaction was quenched with water (20 mL), followed by extraction with ethyl acetate (20 mL × 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography and preparative high-performance liquid chromatography to give a white solid HA-13 (72.78 mg, 18%).

[0808] LCMS:Rt:1.136min;MS m / z(ELSD):495.5[M+H] + ;

[0809] CAD:91.63% purity at ELSD; RT=18.845min.

[0810] 1H NMR(400MHz, CDCl3) δ7.96(d,J=7.3Hz,1H),7.85(d,J=7.7Hz,1H),7.81(s,1H),7 .59–7.50(m,1H),7.40–7.31(m,1H),5.57(s,2H),4.51(t,J=7.2Hz,2H),4.05(t, J=6.7Hz,2H),2.28(t,J=7.5Hz,2H),1.99(dd,J=14.7,7.4Hz,2H),1.68–1.52(m, 4H), 1.41 (dd, J=15.6, 6.9Hz, 2H), 1.31 (d, J=30.0Hz, 22H), 0.88 (t, J=6.8Hz, 3H).

[0811] Example 35: HA-17B

[0812] Step 1:

[0813] Compound 2 (3.7 g, 20.6 mmol, 1.0 eq.) and triethylamine (6.2 g, 61.7 mmol, 3.0 eq.) were added to a solution of compound 1 (5.0 g, 20.6 mmol, 1.0 eq.) in dichloromethane (80 mL). The reaction mixture was stirred at 70 °C for 2 h under nitrogen protection. LC-MS showed that the reaction was complete and a new major spot was observed. The reaction was quenched by adding water (100 mL) and then extracted with ethyl acetate (80 mL × 3). The organic layers were combined, washed with brine (2 × 100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (0%–15% ethyl acetate in dichloromethane) to give compound 3 (7.1 g, 88% yield) as a yellow solid.

[0814] LCMS: Rt: 1.918min; MS m / z (ELSD): 395.2[M+H]+;

[0815] Step 2:

[0816] Ammonium chloride (1.64 g, 30.46 mmol) and iron (1.70 g, 30.46 mmol) were added to an ethanol / water (10 / 1, 45 mL) solution of compound 3 (4.0 g, 10.15 mmol). The mixture was stirred at 90 °C for 2 hours. LC-MS showed the formation of the target product. The mixture was filtered, and dichloromethane / methanol (3 / 1, 30 mL) was added to the filter cake, and the mixture was stirred for 5 minutes. The mixture was filtered again, and the filtrate was concentrated. The residue was purified by column chromatography (0%–20% ethyl acetate in petroleum ether) to give compound 4 (1.5 g, 40.5% yield) as a yellow solid.

[0817] LCMS: Rt: 1.499min; MS m / z (ELSD): 387.3[M+Na]+;

[0818] Step 3:

[0819] Under nitrogen protection, pyridine (1.63 g, 20.6 mmol, 5.0 eq.) and compound 5 (532 mg, 4.33 mmol, 1.0 eq.) were added dropwise to a dry tetrahydrofuran (20 mL) solution of compound 4 (1.5 g, 4.12 mmol, 1.0 eq.) at 0 °C. The mixture was then stirred at room temperature for 16 hours. LC-MS showed the formation of the target product. Ethyl acetate (30 mL) was added to the mixture, followed by washing with saturated ammonium chloride solution (20 mL × 3), water (20 mL), and brine (20 mL × 2). The mixture was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (0%–20% ethyl acetate in petroleum ether) to give compound 6 (1.3 g, 81.3% yield) as a yellow solid.

[0820] LCMS: Rt: 1.416min; MS m / z (ELSD): 451.4[M+H]+;

[0821] Step 4:

[0822] Potassium carbonate (797 mg, 5.78 mmol, 2.0 eq.) was added to an ethanol / water (5 / 1, 15 mL) solution of compound 6 (1.3 g, 2.89 mmol, 1.0 eq.). The mixture was stirred at 55 °C for 16 h. LC-MS showed that the reaction was complete. The mixture was concentrated to remove ethanol, and then dichloromethane (20 mL) was added. The residue was washed with water (10 mL) and brine (10 mL × 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (0%–20% ethyl acetate in petroleum ether) to give compound 7 (1.1 g, 88% yield) as a white solid.

[0823] LCMS:Rt:1.734min; MS m / z(ELSD):377.1[M+H-56] + ;

[0824] Step 5:

[0825] Trifluoroacetic acid (3 mL) was added dropwise to a solution of compound 7 (1.2 g, 2.78 mmol, 1.0 eq.) in dichloromethane (15 mL). The mixture was stirred at room temperature for 2 hours under nitrogen protection. Thin-layer chromatography (methanol / dichloromethane = 1 / 10) showed the formation of the target product. The mixture was concentrated under reduced pressure to remove trifluoroacetic acid, the residue was quenched with aqueous sodium bicarbonate solution, and the pH was adjusted to 8. The mixture was then concentrated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane containing 10% methanol) to give compound 8 (900 mg, 97.6% yield) as a white solid.

[0826] LCMS:Rt:1.166min;MS m / z(ELSD):333.3[M+H] + ;

[0827] Step 6:

[0828] Trifluoroacetic acid (1.22 g, 12.05 mmol, 5 eq.) and compound 9 (554 mg, 3.13 mmol, 1.3 eq.) were added dropwise to a tetrahydrofuran (15 mL) solution of compound 8 (800 mg, 2.41 mmol, 1.0 eq.). The mixture was stirred at room temperature for 16 hours under nitrogen protection. LC-MS showed the formation of the target product. The reaction was quenched by adding water (15 mL) and extracted with ethyl acetate (10 mL × 3). The organic layers were combined, washed with brine (3 × 10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (0–5% methanol in dichloromethane) to give compound 10 (900 mg, 70.3% purity) as a yellow oil.

[0829] LCMS:Rt:1.620min; MS m / z(ELSD):473.1[M+H] + ;

[0830] Step 7:

[0831] Potassium acetate (166.1 mg, 1.69 mmol, 2.0 eq.) was added dropwise to a solution of compound 10 (400 mg, 0.85 mmol, 1.0 eq.) in N,N-dimethylformamide (7 mL). The mixture was stirred at 100 °C for 16 h under nitrogen protection. LC-MS showed the formation of the target product. The reaction was quenched by adding water (10 mL) and extracted with ethyl acetate (10 mL × 3). The organic layers were combined, washed with water (10 mL) and brine (10 mL × 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate in dichloromethane was 0–20%) to give compound 11 (250 mg, yield 47.6%) as a yellow solid.

[0832] LCMS:Rt:1.609min;MS m / z(ELSD):497.0[M+H] + ;

[0833] 1H NMR (400MHz, CDCl3) δ8.19 (dd, J=7.3, 2.4Hz, 1H), 8.16–8.10 (m, 1H), 7.69– 7.64(m,2H),4.67–4.62(m,2H),4.16(t,J=6.2Hz,1H),3.91(t,J=6.3Hz,2H ),3.35(s,3H),3.30–3.25(m,2H),3.23–3.04(m,6H),2.33–2.28(m,1H),2. 13(dd,J=15.5,6.2Hz,1H),2.08–1.98(m,4H),1.81(dt,J=21.2,7.1Hz,3H)

[0834] Step 8:

[0835] Potassium carbonate (111.3 mg, 0.8 mmol, 2.0 eq.) was added dropwise to an ethanol / water (10 / 1, 5 mL) solution of compound 11 (200 mg, 0.4 mmol, 1.0 eq.). The mixture was stirred at room temperature for 2 hours under nitrogen protection. LC-MS showed the formation of the target product. The reaction was quenched by adding water (10 mL) and extracted with ethyl acetate (10 mL × 3). The organic layers were combined, washed with water (10 mL) and brine (10 mL × 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate in dichloromethane was 0-50%) to give compound 12 (120 mg, yield 52.4%) as a yellow solid.

[0836] LCMS:Rt:1.474min; MS m / z(ELSD):454.9[M+H] + ;

[0837] Step 9:

[0838] Under nitrogen protection, compound 13 (60 mg, 0.26 mmol, 1.2 eq.), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (63 mg, 0.33 mmol, 1.5 eq.), and 4-dimethylaminopyridine (7 mg, 0.06 mmol, 0.3 eq.) were added to a solution of compound 12 (100 mg, 0.22 mmol, 1.0 eq.) in dichloromethane (4 mL) at 0 °C. The mixture was stirred at room temperature for 16 hours. LC-MS showed the formation of the target product. Dichloromethane (15 mL) was added to the mixture, and the mixture was washed with water (5 mL) and brine (5 mL × 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by preparative high-performance liquid chromatography to give HA-17B (36.71 mg, yield 20.9%) as a white solid.

[0839] LCMS:Rt:1.461min; MS m / z(ELSD):665.3[M+H] + ;

[0840] CAD:97.49% purity at ELSD; RT=6.670min in 25min.

[0841] 1H NMR (400MHz, CDCl3) δ8.23–8.17(m,1H),8.12(dd,J=6.7,2.9Hz,1H),7.72–7.63(m,2H),4.69–4.61(m,2H ),4.50(t,J=6.3Hz,1H),4.17(t,J=6.3Hz,2H),3.92(t,J=6.1Hz,2H),3.35(s,3H),3.29(t,J=6.1Hz,2H) ,3.20(q,J=6.7Hz,2H),3.10–3.05(m,2H),2.28(t,J=7.6Hz,2H),2.13(dd,J=15.5,6.2Hz,2H),2.07–1.9 9(m,2H),1.80(dd,J=15.0,7.0Hz,2H),1.58(dd,J=14.4,7.2Hz,2H),1.24(s,20H),0.87(t,J=6.8Hz,3H).

[0842] Example 36: HA-30

[0843] Step 1:

[0844] Sodium hydroxide (350 mg, 9.45 mmol, 10.0 eq.) was added to a mixed solution of HA-30-A (600 mg, 0.95 mmol, 1.0 eq.) in methanol (10 mL) and water (1 mL). The reaction mixture was stirred at 50 °C for 2 hours under nitrogen protection. The pH of the mixture was adjusted to 5 with 1 M hydrochloric acid, and then extracted with ethyl acetate (20 mL × 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give crude compound 1 (315 mg, 85.6%) as a white solid.

[0845] LCMS:Rt:1.078min; MS m / z(ELSD):388.3[M+H] + ;

[0846] Step 2:

[0847] Compound 1 (300 mg, 0.78 mmol, 1.0 eq.) was dissolved in an ammonia / methanol solution (20 mL, 7 M) and stirred at 140 °C for 48 hours. The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give compound 2 (100 mg, 35%) as a yellow solid.

[0848] LCMS:Rt:1.363min;MS m / z(ELSD):369.3[M+H] + ;

[0849] Step 3:

[0850] Compound 3 (46 mg, 0.285 mmol, 1.5 eq.), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (73 mg, 0.38 mmol, 2.0 eq.), N,N-diisopropylethylamine (98 mg, 0.76 mmol, 4.0 eq.), and 4-dimethylaminopyridine (9 mg, 0.08 mmol, 0.4 eq.) were added to a solution of compound 2 (70 mg, 0.19 mmol, 1.0 eq.) in N,N-dimethylformamide (3 mL). The reaction mixture was stirred at 25 °C for 16 h under nitrogen protection. The reaction was quenched with water (10 mL) and then extracted with ethyl acetate (10 mL × 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography and preparative high-performance liquid chromatography to give a white solid HA-30 (33.08 mg, 23%).

[0851] LCMS:Rt:1.223min;MS m / z(ELSD):511.5[M+H] + ;

[0852] CAD:90.98% purity at ELSD; RT=16.041min.

[0853] 1 H NMR (400MHz, CDCl3) δ7.97(d,J=7.7Hz,1H),7.90(d,J=8.2Hz,1H),7.84(s,1H),7.59(t, J=7.8Hz,1H),7.41(t,J=7.6Hz,1H),6.28(s,2H),4.52(t,J=7.2Hz,2H),2.85(t,J=7.4H z,2H),2.52(t,J=7.5Hz,2H),2.00(dd,J=14.8,7.4Hz,2H),1.67–1.60(m,2H),1.58–1.5 1(m,2H),1.46–1.40(m,2H),1.34(s,2H),1.26(d,J=6.7Hz,20H),0.87(t,J=6.8Hz,3H).

[0854] Example 37: HA-31-A

[0855] Step 1:

[0856] Compound 2 (6.8 g, 26.59 mmol, 1.0 eq.), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (10 g, 53.19 mmol, 2.0 eq.), N,N-diisopropylethylamine (10 g, 79.79 mmol, 5.0 eq.), and 1-hydroxybenzotriazole (7.18 g, 53.19 mmol, 2 eq.) were added to a solution of compound 1 (5 g, 26.59 mmol, 1.0 eq.) in dichloromethane (50 mL). The reaction mixture was stirred at 25 °C for 16 h under nitrogen protection. The reaction was quenched with water (100 mL) and then extracted with ethyl acetate (100 mL × 3). The combined organic layers were washed with saturated brine (2 × 150 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (dichloromethane containing 0%–10% methanol) to give compound 3 (3.7 g, 33%) as a white solid.

[0857] LCMS:Rt:1.748min;MS m / z(ELSD):427.4[M+H] + ;

[0858] Step 2:

[0859] Trifluoroacetic acid (20 mL) was added to a solution of compound 3 (3.7 g, 8.68 mmol, 1.0 eq.) in dichloromethane (20 mL). The reaction mixture was stirred at 25 °C for 2 hours under nitrogen protection. The reaction was quenched by adjusting the pH to 8 with an aqueous sodium bicarbonate solution, followed by extraction with dichloromethane (100 mL × 3). The combined organic layers were washed with saturated brine (2 × 100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give crude compound 4 (2.68 g, 93%) as a white solid.

[0860] LCMS:Rt:1.054min;MS m / z(ELSD):327.3[M+H] + ;

[0861] Step 3:

[0862] Compound 6 (1.9 g, 6.20 mmol, 1.0 eq.) and triethylamine (1.878 g, 18.60 mmol, 3.0 eq.) were added to a solution of compound 5 (1.5 g, 6.20 mmol, 1.0 eq.) in dichloromethane (30 mL). The reaction mixture was stirred at 70 °C for 2 h under nitrogen protection. Thin-layer chromatography (methanol:dichloromethane = 15:1) showed that the reaction was complete, and a new major spot was observed. The reaction was quenched with water (50 mL) and then extracted with ethyl acetate (50 mL × 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by rapid column chromatography to give compound 7 (2.8 g, 84.8%) as a yellow solid.

[0863] LCMS:Rt:2.128min; MS m / z(ELSD):533.4[M+H] + ;

[0864] Step 4:

[0865] Iron (880 mg, 15.79 mmol, 3.0 eq.) and ammonium chloride (850 mg, 15.79 mmol, 3.0 eq.) were added to an ethanol / water (30 mL / 10 mL) solution of compound 7 (2.8 g, 5.26 mmol, 1.0 eq.). The reaction mixture was stirred at 70 °C for 2 hours under nitrogen protection. Thin-layer chromatography (dichloromethane:methanol = 10:1) showed that the reaction was complete, and a new major spot was observed. The mixture was filtered, and the filter cake was washed with methanol (100 mL). The filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (dichloromethane containing 0%–10% methanol) to give compound 8 (1.84 g, 70.7%) as a yellow solid.

[0866] LCMS:Rt:1.241min; MS m / z(ELSD):503.5[M+H] + ;

[0867] Step 5:

[0868] A mixture of compound 8 (1.84 g, 3.67 mmol, 1.0 eq.) and triethyl orthoformate (10 mL) was stirred at 80 °C for 16 h under nitrogen protection. Thin-layer chromatography (dichloromethane:methanol = 10 / 1) showed the reaction was complete, with a new major spot observed. The reaction was quenched with water (80 mL), followed by extraction with ethyl acetate (50 mL × 3). The combined organic layers were washed with saturated brine (2 × 50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane containing 10% methanol) to give a white solid compound HA-31-A (1.785 g, 95%).

[0869] LCMS:Rt:1.766min; MS m / z(ELSD):513.6[M+H] + ;

[0870] CAD:94.46% purity at ELSD; RT=24.021min.

[0871] 1 H NMR (400MHz, CDCl3) δ8.25–8.19(m,1H),8.18–8.10(m,1H),8.05(s,1H),7.76–7.63(m,2H),5.49(s,1H),4.67(t,J=7.1Hz,2H),3.34 (q,J=6.7Hz,2H),2.16–2.10(m,2H),2.04(dt,J=14.9,7.3Hz,2H),1.65–1.53(m,4H),1.24(d,J=2.4Hz,24H),0.88(t,J=6.8Hz,3H).

[0872] Example 38: HA-31

[0873] Step 1:

[0874] HA-31-A (600 mg, 1.17 mmol, 1.0 eq.) was dissolved in ammonia / methanol solution (20 mL, 7 M) and stirred at 140 °C for 48 hours. The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography and preparative high performance liquid chromatography to give a white solid HA-31 (87.32 mg, 15%).

[0875] LCMS:Rt:1.101min; MS m / z(ELSD):494.6[M+H] + ;

[0876] CAD:89.47% purity at ELSD; RT=17.698min.

[0877] 1 H NMR (400MHz, DMSO) δ8.21 (s, 1H), 8.04 (d, J = 7.3Hz, 1H), 7.75 (t, J = 5.7Hz, 1H), 7 .68–7.55(m,1H),7.45(dd,J=11.2,4.1Hz,1H),7.27(dd,J=11.1,4.1Hz,1H),6. 72(s,2H),4.60(t,J=7.0Hz,2H),3.07(q,J=6.7Hz,2H),1.98(t,J=7.4Hz,2H),1 .89–1.77(m,2H),1.48–1.37(m,4H),1.28–1.14(m,25H),0.85(t,J=6.8Hz,3H).

[0878] Example 39: HA-37-1A

[0879] Step 1:

[0880] Under nitrogen protection, compound 2 (3.3 g, 32.9 mmol, 1.5 eq.), potassium carbonate (9 g, 65.7 mmol, 3 eq.), and ferrocene palladium dichloroethylene (500 mg, 0.68 mmol, 0.03 eq.) were added to a dioxane / water (10 / 1, 60 mL) solution of compound 1 (5 g, 21.9 mmol, 1 eq.). The mixture was stirred at 100 °C for 16 hours. Thin-layer chromatography (petroleum ether / ethyl acetate = 10 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was washed with water (40 mL) and extracted with ethyl acetate (50 mL × 3). The organic layers were combined, washed with water (60 mL × 2) and brine (10 mL × 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (0%–10% ethyl acetate in petroleum ether) to give compound 3 (3.3 g, yield 73.3%), which was a colorless oil.

[0881] 1 H NMR (400MHz, CDCl3) δ7.25–7.09(m,4H),3.71–3.64(m,5H),2.73–2.51(m,2H),1.58–

[0882] 1.48(m,2H),1.40(dq,J=14.3,7.2Hz,2H),0.95(t,J=7.3Hz,3H).

[0883] Step 2:

[0884] Under nitrogen protection, lithium aluminum hydride (2.5 M, 7.0 mL, 17.48 mmol, 1.2 eq.) was added dropwise to a tetrahydrofuran (40 mL) solution of compound 3 (3 g, 14.56 mmol, 1.0 eq.) at 0 °C. The mixture was stirred at room temperature for 16 hours. Thin-layer chromatography (petroleum ether / ethyl acetate = 10 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was cooled to 0 °C, and the reaction was quenched by the slow addition of water (0.7 mL) and 1 M sodium hydroxide (0.7 mL). Anhydrous sodium sulfate (10 g) was added to the mixture, and the mixture was stirred for 5 minutes. The mixture was filtered and concentrated under reduced pressure. The residue was purified by column chromatography (0%–5% ethyl acetate in petroleum ether) to give compound 4 (1.4 g, 54.1% yield) as a colorless oil.

[0885] 1H NMR (400MHz, DMSO-d6) δ7.17–7.05(m,4H),4.67(t,J=5.3Hz,1H),3.55(dd,J=12.9,7.3Hz,2H),2.73(dd,J= 12.9,5.6Hz,2H),2.62–2.55(m,2H),1.55–1.45(m,2H),1.35(dq,J=14.3,7.2Hz,2H),0.91(t,J=7.3Hz,3H).

[0886] Step 3:

[0887] Under nitrogen protection, compound 5 (300 mg, 1.34 mmol, 1.2 eq.), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (428 mg, 2.24 mmol, 2.0 eq.), and 4-dimethylaminopyridine (27.1 mg, 0.22 mmol, 0.2 eq.) were added to a solution of compound 4 (200 mg, 1.12 mmol, 1.0 eq.) in dichloromethane (8 mL) at 0 °C. The mixture was stirred at room temperature for 16 hours. Thin-layer chromatography (petroleum ether / ethyl acetate = 10 / 1) showed that the reaction was complete and a new major spot was observed. Dichloromethane (20 mL) was added to the mixture, and the mixture was washed with water (10 mL) and brine (10 mL × 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (0%–5% ethyl acetate in petroleum ether) to give compound 6 (290 mg, yield 67.7%), which was a colorless oil.

[0888] 1 H NMR (400MHz, CDCl3) δ7.19–7.11(m,4H),4.26(t,J=7.4Hz,2H),3.53(t,J=6.7Hz,1H),3.40(t,J=6.8Hz,1H),2.96(t,J=7.4Hz,2H),2.68–2.60(m, 2H),2.30(t,J=7.5Hz,2H),1.87–1.82(m,1H),1.79–1.73(m,1H),1.66–1 .56(m,4H),1.45–1.38(m,4H),1.34–1.28(m,4H),0.95(t,J=7.3Hz,3H).

[0889] Step 4:

[0890] Under nitrogen protection, pyridine (1.5 g, 19.4 mmol, 4.0 eq.) and compound 8 (0.71 g, 5.82 mmol, 1.2 eq.) were added dropwise to a dry tetrahydrofuran (30 mL) solution of compound 7 (2 g, 4.85 mmol, 1.0 eq.) at 0 °C. The mixture was then stirred at room temperature for 16 hours. LC-MS showed the formation of the target product. Ethyl acetate (30 mL) was added to the mixture, followed by washing with saturated ammonium chloride solution (20 mL × 3), water (20 mL), and brine (20 mL × 2). The mixture was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (ethyl acetate in dichloromethane was 0%–25%) to give compound 9 (2.1 g, 87.5% yield) as a yellow solid.

[0891] LCMS:Rt:1.542min; MS m / z(ELSD):499.2[M+H] + ;

[0892] Step 5:

[0893] Potassium carbonate (1.3 g, 9.24 mmol, 2.0 eq.) was added to an ethanol / water (10 / 1, 30 mL) solution of compound 9 (2.3 g, 4.62 mmol, 1.0 eq.). The mixture was stirred at 55 °C for 16 h under nitrogen protection. LC-MS showed that the reaction was complete. The reaction was quenched by adding water (20 mL) and extracted with ethyl acetate (50 mL × 3). The combined organic layers were washed with water (50 mL) and brine (50 mL × 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give compound 10 (2 g, 90.9% yield) as a yellow solid.

[0894] LCMS:Rt:1.795min;MS m / z(ELSD):481.2[M+H] + ;

[0895] Step 6:

[0896] Trifluoroacetic acid (3 mL) was added dropwise to a solution of compound 10 (2.0 g, 4.17 mmol, 1.0 eq.) in dichloromethane (15 mL). The mixture was stirred at room temperature for 2 hours. LC-MS showed that the reaction was complete. The mixture was quenched with an aqueous sodium bicarbonate solution and the pH was adjusted to 8. The mixture was then concentrated under vacuum. The residue was purified by silica gel column chromatography (dichloromethane containing 10% methanol) to give compound 11 (1.4 g, 88.6% yield) as a white solid.

[0897] LCMS:Rt:1.233min;MS m / z(ELSD):381.3[M+H] + ;

[0898] Step 7:

[0899] Potassium carbonate (363 mg, 2.63 mmol, 1.0 eq.) and potassium iodide (175 mg, 1.05 mmol, 2.0 eq.) were added to a cyclopentyl methyl ether / acetonitrile (1 / 1, 5 mL) solution of compound 11 (200 mg, 0.63 mmol, 1.5 eq.) and compound 12 (290 mg, 0.63 mmol, 1.5 eq.). The mixture was stirred at 90 °C for 3 days under argon protection. LC-MS showed the formation of the target product. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (0%–5% methanol in dichloromethane) to give compound 13 (200 mg, 50.0% yield) as a colorless oil.

[0900] LCMS:Rt:1.270min;MS m / z(ELSD):761.6[M+H] + ;

[0901] Step 8:

[0902] Potassium carbonate (181 mg, 1.31 mmol, 5.0 eq.) and potassium iodide (87 mg, 0.53 mmol, 2.0 eq.) were added to a cyclopentyl methyl ether / acetonitrile (1 / 1, 4 mL) solution of compound 13 (200 mg, 0.26 mmol, 1.0 eq.) and compound 6 (152 mg, 0.40 mmol, 1.5 eq.). The mixture was stirred at 95 °C for 3 days under argon protection. LC-MS showed the formation of the target product. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (0%–5% methanol in dichloromethane) followed by preparative high-performance liquid chromatography to give HA-37-1A (33.64 mg, 12.46% yield) as a colorless oil.

[0903] LCMS:Rt:1.353min; MS m / z(ELSD):1063.9[M+H] + ;

[0904] CAD:98.77% purity at ELSD; RT=8.317min in 25min.

[0905] 1H NMR (400MHz, CDCl3) δ8.15(d,J=8.4Hz,1H),7.93(d,J=8.4Hz,1H),7.59(t,J=7.7Hz,1H),7.41(t,J=7.7Hz,1H),7.26–7.24(m,2 H),7.18–7.11(m,4H),6.96(d,J=7.9Hz,2H),5.89(s,2H),4.94–4.79(m,1H),4.24(t,J=7.4Hz,2H),3.87(t,J=6.3Hz,2H),3.47( s,2H),3.29(s,3H),3.26(t,J=6.3Hz,2H),2.95(t,J=7.4Hz,2H),2.66–2.58(m,2H),2.32(s,4H),2.26(td,J=7.5,4.5Hz,4H),1. 55(d,J=7.6Hz,2H),1.52–1.47(m,4H),1.40(dd,J=14.7,7.3Hz,6H),1.24(s,40H),0.94(t,J=7.3Hz,3H),0.86(t,J=6.7Hz,6H).

[0906] Example 40: HA-51A

[0907] Step 1:

[0908] Compound 2 (4.2 g, 20.6 mmol, 1.0 eq.) and triethylamine (6.2 g, 61.7 mmol, 3.0 eq.) were added to a solution of compound 1 (5.0 g, 20.6 mmol, 1.0 eq.) in dichloromethane (100 mL). The reaction mixture was stirred at 70 °C for 2 h under nitrogen protection. Thin-layer chromatography (petroleum ether: ethyl acetate = 3:1) showed that the reaction was complete and a new major spot was observed. This mixture was combined with the mixture on page 041, washed with water (50 mL) and brine (2 × 50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (0%–15% ethyl acetate in petroleum ether) to give compound 3 (8.5 g, 84.2% yield) as a yellow solid.

[0909] LCMS: Rt: 1.106min; MS m / z (ELSD): 409.3[M+H]+;

[0910] Step 2:

[0911] Ammonium chloride (1.6 g, 34.3 mmol, 3.5 eq.) and iron (1.64 g, 34.3 mmol, 3.5 eq.) were added to an ethanol / water (10 / 1, 50 mL) solution of compound 3 (4 g, 34.3 mmol, 1.0 eq.). The mixture was stirred at 75 °C for 2 h. LCMS showed the formation of the target product. The mixture was filtered, and dichloromethane / methanol (3 / 1, 30 mL) was added to the filter cake, and the mixture was stirred for 5 min. The mixture was filtered again, and the filtrate was concentrated. The residue was purified by column chromatography (0%–35% ethyl acetate in petroleum ether) to give compound 4 (3.8 g, 82.6% yield) as a yellow solid.

[0912] LCMS: Rt: 1.524min; MS m / z (ELSD): 379.2[M+H]+;

[0913] Step 3:

[0914] Under nitrogen protection, pyridine (1.36 g, 17.2 mmol, 5.0 eq.) and compound 5 (440 mg, 3.61 mmol, 1.0 eq.) were added dropwise to a dry tetrahydrofuran (20 mL) solution of compound 4 (1.3 g, 3.44 mmol, 1.0 eq.) at 0 °C. The mixture was then stirred at room temperature for 16 hours. LC-MS showed the formation of the target product. Ethyl acetate (40 mL) was added to the mixture, followed by washing with saturated ammonium chloride solution (20 mL × 3), water (20 mL), and brine (20 mL × 2). The mixture was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (0%–20% ethyl acetate in petroleum ether) to give compound 6 (1.4 g, 88.1% yield) as a white solid.

[0915] LCMS: Rt: 1.612min; MS m / z (ELSD): 463.6[M+H]+;

[0916] Step 4:

[0917] Potassium carbonate (0.84 g, 6.06 mmol, 2.0 eq.) was added to an ethanol / water (10 / 1, 20 mL) solution of compound 6 (1.4 g, 3.03 mmol, 1.0 eq.). The mixture was stirred at 55 °C for 16 h under nitrogen protection. LC-MS showed that the reaction was complete. The mixture was quenched with water (20 mL). The mixture was extracted with ethyl acetate (50 mL × 3). The organic layers were combined, washed with water (50 mL) and brine (50 mL × 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give compound 7 (2 g, 90.9% yield) as a yellow solid.

[0918] LCMS:Rt:1.943min; MS m / z(ELSD):445.6[M+H] + ;

[0919] Step 5:

[0920] Trifluoroacetic acid (3 mL) was added dropwise to a solution of compound 7 (1.3 g, 2.92 mmol, 1.0 eq.) in dichloromethane (15 mL). The mixture was stirred at room temperature for 2 hours. LC-MS showed that the reaction was complete. The mixture was quenched with an aqueous sodium bicarbonate solution and the pH was adjusted to 8. The mixture was then concentrated under vacuum. The residue was purified by silica gel column chromatography (dichloromethane containing 10% methanol) to give compound 8 (1.0 g, 100% yield) as a white solid.

[0921] LCMS:Rt:1.272min; MS m / z(ELSD):345.3[M+H] + ;

[0922] Step 6:

[0923] Potassium carbonate (600 mg, 4.35 mmol, 5.0 eq.) and potassium iodide (289 mg, 1.74 mmol, 2.0 eq.) were added to a cyclopentyl methyl ether / acetonitrile (1 / 1, 15 mL) solution of compound 8 (300 mg, 0.87 mmol, 1.0 eq.) and compound 9 (905 mg, 2.6 mmol, 3.0 eq.). The mixture was stirred at 95 °C for 2 days under argon protection. LC-MS showed the formation of the target product. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (0%–5% methanol in dichloromethane) followed by preparative high-performance liquid chromatography to give compound HA-51A (263.77 mg, 34.3% yield) as a colorless oil.

[0924] LCMS:Rt:1.237min; MS m / z(ELSD):881.9[M+H] + ;

[0925] CAD:99.78% purity at ELSD; RT=6.443min in 25min.

[0926] 1H NMR(400MHz, CDCl3)δ8.19(d,J=7.9Hz,1H),8.11(d,J=8.0Hz,1H),7.71–7.59(m,2H) ,4.56–4.44(m,2H),4.04(t,J=6.8Hz,4H),3.05–2.93(m,2H),2.36(s,6H),2.27(t,J =7.5Hz,4H),1.92(td,J=15.4,7.8Hz,4H),1.59(dd,J=13.8,6.7Hz,8H),1.53–1.49( m, 4H), 1.31 (dd, J = 29.0, 22.9Hz, 42H), 1.01 (t, J = 7.4Hz, 3H), 0.87 (t, J = 6.8Hz, 6H).

[0927] Example 41: HA-52A

[0928] Step 1:

[0929] Compound 2 (1.5 g, 10.3 mmol, 1.0 eq.), potassium iodide (3.4 g, 20.6 mmol), and potassium carbonate (7.1 g, 51.7 mmol) were added to a cyclopentyl methyl ether / acetonitrile (1 / 1, 80 mL) solution of compound 1 (5.5 g, 11.9 mmol, 1.2 eq.). The mixture was stirred at 90 °C for 16 h. LC-MS showed the formation of the target product. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (0%–5% methanol in dichloromethane) to give compound 3 (2.8 g, 52% yield) as a brown oil.

[0930] LCMS: Rt: 1.307min; MS m / z (ELSD): 526.9[M+H]+;

[0931] 1 H NMR: (400MHz, CDCl3) δ4.91–4.79(m,1H),2.83(t,J=6.5Hz,2H),2.59(t,J=7.2Hz,2H),2.43(t,J=6.5Hz,2H),2.27(t, J=7.5Hz,2H),1.62(d,J=7.0Hz,2H),1.51–1.47(m,4H),1.45(s,9H),1.31(s,8H),1.25(s,24H),0.87(t,J=6.8Hz,6H).

[0932] Step 2:

[0933] Compound 4 (5.5 g, 16.0 mmol, 3.0 eq.), potassium iodide (1.8 g, 10.7 mmol, 2.0 eq.), and potassium carbonate (3.7 g, 26.7 mmol, 5.0 eq.) were added to a cyclopentyl methyl ether / acetonitrile (1 / 1, 80 mL) solution of compound 3 (2.8 g, 5.33 mmol, 1.0 eq.). The mixture was stirred at 94 °C for 3 days. LC-MS showed the formation of the target product. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (0%–3% methanol in dichloromethane) to give compound 5 (3.5 g, 83.3% yield) as a light brown oil.

[0934] LCMS: Rt: 1.432min; MS m / z (ELSD): 795.0[M+H]+;

[0935] 1 H NMR: (400MHz, CDCl3) δ4.91–4.81(m,1H),4.05(t,J=6.7Hz,2H),2.73(t,J=7.2Hz,2H),2.35(dd,J=14.1,7.0Hz,4H),2.28(td,J=7.5,5.1H z,4H),1.60(dd,J=14.1,7.3Hz,8H),1.50(d,J=5.5Hz,4H),1.44(s,9H),1.41(s,2H),1.27(d,J=15.0Hz,51H),0.88(dd,J=7.6,6.0Hz,9H).

[0936] Step 3:

[0937] Compound 5 (3.5 g, 4.4 mmol) was dissolved in trifluoroacetic acid / dichloromethane (1 / 3, 40 mL) and stirred at room temperature for 1 hour. Thin-layer chromatography (dichloromethane / methanol = 10 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was quenched with aqueous sodium bicarbonate solution and the pH was adjusted to 8. The mixture was then concentrated under vacuum. The residue was purified by silica gel column chromatography (0%–5% methanol in dichloromethane) to give compound 6 (3 g, 92% yield) as a colorless oil.

[0938] LCMS: Rt: 1.438min; MS m / z (ELSD): 739.0[M+H]+;

[0939] 1H NMR: MC25-462-049-P (400MHz, CDCl3) δ10.67 (s, 1H), 4.90–4.81 (m, 1H), 4.0 5(t,J=6.8Hz,2H),3.39(d,J=6.7Hz,2H),3.05(d,J=8.4Hz,4H),2.85(s,2H) ,2.29(td,J=7.4,5.2Hz,4H),1.69(s,4H),1.64–1.57(m,6H),1.50(d,J=5.6 Hz, 4H), 1.35 (s, 12H), 1.28 (d, J = 20.4Hz, 36H), 0.87 (dd, J = 7.6, 6.1Hz, 9H).

[0940] Step 4:

[0941] Compound 8 (527 mg, 8.64 mmol, 1.0 eq.) and triethylamine (2.6 mg, 25.7 mmol, 3.0 eq.) were added to a solution of compound 7 (2 g, 8.23 ​​mmol, 1.0 eq.) in dichloromethane (35 mL). The reaction mixture was stirred at 70 °C for 2 h under nitrogen protection. LC-MS showed that the reaction was complete. Dichloromethane (30 mL) was added to the mixture, and the mixture was washed with water (30 mL) and brine (2 × 30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (0%–35% ethyl acetate in petroleum ether) to give compound 9 (2.0 g, 90.9% yield) as a yellow solid.

[0942] LCMS: Rt: 1.640min; MS m / z (ELSD): 267.8[M+H]+;

[0943] 1 H NMR(400MHz,DMSO-d6)δ8.48(d,J=8.5Hz,1H),7.86–7.80(m,2H),7.75(s,1H),7.65(ddd,J =8.3,5.6,2.7Hz,1H),4.93(t,J=5.6Hz,1H),3.63(q,J=5.6Hz,2H),3.21(q,J=5.5Hz,2H).

[0944] Step 5:

[0945] Ammonium chloride (1.21 g, 22.47 mmol, 3 eq.) and iron (1.26 g, 22.47 mmol, 3 eq.) were added to an ethanol / water (10 / 1, 25 mL) solution of compound 9 (2.0 g, 7.49 mmol, 1.0 eq.). The mixture was stirred at 90 °C for 2 h. LCMS showed the formation of the target product. The mixture was filtered, and dichloromethane / methanol (3 / 1, 30 mL) was added to the filter cake, and the mixture was stirred for 5 min. The mixture was filtered again, and the filtrate was concentrated. The residue was purified by column chromatography (0%–35% ethyl acetate in petroleum ether) to give compound 10 (1.4 g, 78.7% yield) as a white solid.

[0946] LCMS: Rt: 1.146min; MS m / z (ELSD): 240.1[M+H+2]+;

[0947] Step 6:

[0948] Under nitrogen protection, pyridine (2.17 g, 27.5 mmol, 5.0 eq.) and compound 11 (738 mg dissolved in 6.8 mL of dichloromethane, 6.0 mmol, 1.1 eq.) were added dropwise to a dry tetrahydrofuran (15 mL) solution of compound 10 (1.3 g, 5.5 mmol, 1.0 eq.) at 0 °C. The mixture was then stirred at room temperature for 16 hours. LC-MS showed the formation of the target product. Ethyl acetate (30 mL) was added to the mixture, followed by washing with saturated ammonium chloride solution (20 mL × 3), water (20 mL), and brine (20 mL × 2). The mixture was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (0%–30% ethyl acetate in petroleum ether) to give compound 12 (500 mg, 28.2% yield) as a white solid.

[0949] LCMS:Rt:1.052min;MS m / z(ELSD):324.1[M+H] + ;

[0950] Step 7:

[0951] Potassium carbonate (427 mg, 3.10 mmol) was added to an ethanol / water (5 / 1, 8 mL) solution of compound 12 (500 mg, 1.55 mmol). The mixture was stirred at 55 °C for 16 hours. LC-MS showed that the reaction was complete. The mixture was concentrated to remove ethanol, and then dichloromethane (20 mL) was added. The residue was washed with water (10 mL) and brine (10 mL × 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (0%–30% ethyl acetate in petroleum ether) to give compound 13 (450 mg, 95.3% yield) as a white solid.

[0952] LCMS:Rt:1.440min;MS m / z(ELSD):305.9[M+H] + ;

[0953] Step 8:

[0954] Under nitrogen protection, at 0 °C, compound 6 (986 mg, 1.33 mmol, 1.2 eq.), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (323 mg, 1.67 mmol, 1.5 eq.), and 4-dimethylaminopyridine (37 mg, 0.22 mmol, 0.2 eq.) were added to a solution of compound 13 (340 mg, 1.11 mmol, 1 eq.) in dichloromethane (15 mL). The mixture was stirred at room temperature for 16 hours. LCMS showed the formation of the target product. This mixture was combined with the mixture on page 077, and dichloromethane (25 mL) was added. The mixture was washed with water (20 mL) and brine (20 mL × 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a brown oil. The residue was purified by column chromatography (0%–5% methanol in dichloromethane) and then purified by preparative high performance liquid chromatography to obtain compound HA-52A (111.56 mg, yield 7.6%), which was a colorless oil.

[0955] LCMS:Rt:1.494min; MS m / z(ELSD):1026.1[M+H] + ;

[0956] CAD:97.32% purity at ELSD; RT=8.661min in 25min.

[0957] 1H NMR (400MHz, CDCl3) δ8.28–8.23(m,1H),8.19(dd,J=8.2,1.4Hz,1H),7.67(dtd,J=13.5 ,7.0,3.6Hz,2H),4.95(t,J=6.0Hz,2H),4.89–4.81(m,1H),4.55(t,J=6.0Hz,2H),4.04( t,J=6.8Hz,2H),3.91(t,J=6.1Hz,2H),3.37–3.30(m,5H),2.64(s,2H),2.27(td,J=7.5, 3.7Hz,10H),1.62–1.56(m,6H),1.49(d,J=5.7Hz,4H),1.25(s,52H),0.90–0.84(m,9H).

[0958] Example 42: HA-37-2A

[0959] Step 1:

[0960] Under nitrogen protection, compound 2 (5.1 g, 39.5 mmol, 1.5 eq.), potassium carbonate (10.9 g, 78.9 mmol, 3 eq.), and ferrocene palladium dichloroethylene (500 mg, 0.68 mmol, 0.026 eq.) were added to a dioxane / water (10 / 1, 70 mL) solution of compound 1 (6 g, 26.3 mmol, 1 eq.). The mixture was stirred at 100 °C for 16 h. Thin-layer chromatography (petroleum ether / ethyl acetate = 10 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was washed with water (40 mL) and extracted with ethyl acetate (50 mL × 3). The organic layers were combined, washed with water (60 mL × 2) and brine (10 mL × 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (0%–5% ethyl acetate in petroleum ether) to give compound 3 (5 g, yield 81.3%), which was a colorless oil.

[0961] 1 H NMR (400MHz, CDCl3) δ7.25–7.20(m,1H),7.09(d,J=5.7Hz,3H),3.69(s,3H),3.60(s,2 H),2.65–2.54(m,2H),1.61(d,J=6.8Hz,2H),1.36–1.27(m,6H),0.88(t,J=6.6Hz,3H).

[0962] Step 2:

[0963] Under nitrogen protection, lithium aluminum hydride (2.5 M, 5.1 mL, 3.2 mmol, 1.5 eq.) was added dropwise to a tetrahydrofuran (25 mL) solution of compound 3 (2 g, 8.54 mmol, 1.0 eq.) at 0 °C. The mixture was stirred at room temperature for 16 hours. Thin-layer chromatography (petroleum ether / ethyl acetate = 10 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was cooled to 0 °C, and the reaction was quenched by the slow addition of water (0.5 mL) and 1 M sodium hydroxide (0.5 mL). Anhydrous sodium sulfate (10 g) was added to the mixture, and the mixture was stirred for 5 minutes. The mixture was filtered and concentrated under reduced pressure. The residue was purified by column chromatography (0%–5% ethyl acetate in petroleum ether) to give compound 4 (1.5 g, 85.2% yield) as a colorless oil.

[0964] 1 H NMR(400MHz, DMSO-d6)δ7.16(t,J=7.5Hz,1H),7.03–6.96(m,3H),4.60(t,J=5.2Hz,1H),3.58(td,J=7.1,5.3Hz,2H), 2.68(t,J=7.1Hz,2H),2.55–2.51(m,2H),1.53(dd,J=14.8,7.2Hz,2H),1.26(t,J=6.7Hz,6H),0.85(t,J=6.9Hz,3H).

[0965] Step 3:

[0966] Under nitrogen protection, triphenylphosphine (1.27 g, 4.85 mmol, 2.0 eq.) and N-bromosuccinimide (864 mg, 4.85 mmol, 2.0 eq.) were added to a solution of compound 4 (500 mg, 2.43 mmol, 1 eq.) in dichloromethane (7 mL) at 0 °C. The mixture was stirred at room temperature for 3 hours. Thin-layer chromatography (petroleum ether / ethyl acetate = 10 / 1) showed that the reaction was complete. Dichloromethane (15 mL) was added to the mixture, and the mixture was washed with water (10 mL × 2) and brine (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (pure petroleum ether) to give compound 5 (400 mg, yield 61.5%) as a colorless oil.

[0967] 1H NMR (400MHz, CDCl3) δ7.23(dd,J=9.5,6.1Hz,1H),7.08(d,J=7.7Hz,1H),7.02(d,J=5.7Hz,2H),3.57(dd,J=9.9,5.6Hz,2H), 3.14(t,J=7.8Hz,2H),2.65–2.55(m,2H),1.61(dd,J=14.5,7.7Hz,2H),1.33(dd,J=13.2,4.8Hz,6H),0.89(t,J=6.6Hz,3H).

[0968] Step 4:

[0969] Potassium carbonate (360 mg, 2.61 mmol, 5.0 eq.) and potassium iodide (96 mg, 0.58 mmol, 2.0 eq.) were added to a cyclopentyl methyl ether / acetonitrile (1 / 1, 4 mL) solution of compound 6 (220 mg, 0.29 mmol, 1.0 eq.) and compound 5 (155 mg, 0.58 mmol, 2.0 eq.). The mixture was stirred at 95 °C for 2 days under argon protection. LC-MS showed the formation of the target product. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (0%–5% methanol in dichloromethane) to give a yellow oil (4.2 g), which was further purified by preparative high-performance liquid chromatography to give compound HA-37-2A (26.64 mg, 9.7%) as a colorless oil.

[0970] LCMS:Rt:1.381min;MS m / z(ELSD):949.4[M+H] + ;

[0971] CAD:94.98%purity.Rt:8.416min

[0972] 1H NMR (400MHz, CDCl3) δ8.16(d,J=8.3Hz,1H),7.92(d,J=8.8Hz,1H),7.59(t,J=7.7Hz,1H),7.40(t,J=7.7Hz,1H) ,7.24(s,2H),7.11(t,J=7.5Hz,1H),6.94(d,J=19.9Hz,4H),6.87(d,J=7.6Hz,1H),5.89(s,2H),4.91–4.80(m,1 H),3.87(t,J=6.2Hz,2H),3.57(s,2H),3.30–3.22(m,5H),2.66(s,4H),2.55–2.49(m,2H),2.43(s,2H),2.26(t ,J=7.5Hz,2H),1.55(d,J=7.6Hz,2H),1.49(s,4H),1.42(s,2H),1.26(d,J=14.4Hz,38H),0.86(t,J=6.5Hz,9H).

[0973] Example 43: HA-37-3A

[0974] Step 1:

[0975] Potassium carbonate (685 mg, 3.3 mmol, 5.0 eq.) and potassium iodide (329 mg, 1.32 mmol, 2.0 eq.) were added to a cyclopentyl methyl ether / acetonitrile (1 / 1, 8 mL) solution of compound 1 (300 mg, 0.66 mmol, 1.0 eq.) and compound 2 (413 mg, 0.79 mmol, 1.2 eq.). The mixture was stirred at 90 °C for 3 days under argon protection. LC-MS showed the formation of the target product. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (0%–5% methanol in dichloromethane) to give compound 3 (300 mg, 53.2% yield) as a pale yellow oil.

[0976] LCMS:Rt:1.882min; MS m / z(ELSD):574.5[M+H] + ;

[0977] 1H NMR(400MHz, CDCl3)δ8.24–8.15(m,2H),7.65(dtd,J=15.0,7.0,1.4Hz,2H), 4.74(t,J=6.8Hz,2H), 4.04(t,J=6.8Hz,2H), 3.93(t,J=6.3Hz,2H), 3.39–3. 30(m,5H),3.17(t,J=6.8Hz,2H),2.62(t,J=7.2Hz,2H),2.28(t,J=7.5Hz,2H ),1.61(s,2H),1.48–1.40(m,2H),1.34–1.23(m,20H),0.87(t,J=6.9Hz,3H).

[0978] Step 2:

[0979] Potassium carbonate (301 mg, 2.18 mmol, 5.0 eq.) and potassium iodide (145 mg, 0.87 mmol, 2.0 eq.) were added to a cyclopentyl methyl ether / acetonitrile (1 / 1, 4 mL) solution of compound 3 (250 mg, 0.44 mmol, 1.0 eq.) and compound 5 (233 mg, 0.87 mmol, 2.0 eq.). The mixture was stirred at 95 °C for 2 days under argon protection. LC-MS showed the formation of the target product. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (0%–5% methanol in dichloromethane) followed by preparative high-performance liquid chromatography to give compound HA-37-3A (46.55 mg, 13.9%) as a colorless oil.

[0980] LCMS:Rt:1.282min;MS m / z(ELSD):761.8[M+H] + ;

[0981] CAD:87.39%purity.Rt:14.130min

[0982] 1H NMR (400MHz, CDCl3) δ8.20(d,J=8.4Hz,2H),7.68(t,J=7.4Hz,1H),7.60(t,J=7.2Hz,1H),7.18(t,J=7.8Hz,1H),7. 01(d,J=7.7Hz,1H),6.93(d,J=1.6Hz,2H),4.64(t,J=6.9Hz,2H),4.05(t,J=6.8Hz,2H),3.91(t,J=6.3Hz,2H),3.35 (s,3H),3.28(t,J=6.3Hz,2H),2.97(t,J=6.8Hz,2H),2.76(dd,J=9.6,5.3Hz,2H),2.66(dd,J=9.7,5.4Hz,2H),2.59 –2.50(m,4H),2.28(t,J=7.5Hz,2H),1.61(s,2H),1.57(d,J=7.9Hz,2H),1.36–1.20(m,28H),0.87(t,J=6.4Hz,6H).

[0983] Example 44: HA-53B

[0984] Step 1:

[0985] Under nitrogen protection, pyridine (1.57 g, 19.8 mmol, 5.0 eq.) and compound 2 (532 mg dissolved in 4.5 mL dichloromethane, 4.36 mmol, 1.1 eq.) were added dropwise to a dry tetrahydrofuran (20 mL) solution of compound 1 (1.5 g, 3.97 mmol, 1.0 eq.) at 0 °C. The mixture was then stirred at room temperature for 16 hours. LC-MS showed the formation of the target product. Ethyl acetate (40 mL) was added to the mixture, followed by washing with water (20 mL) and brine (20 mL × 2), drying over anhydrous sodium sulfate, filtration, and concentration under reduced pressure. The residue was purified by column chromatography (0%–20% ethyl acetate in petroleum ether) to give compound 3 (1.2 g, 65.2% yield) as a yellow solid.

[0986] LCMS: Rt: 1.480min; MS m / z (ELSD): 465.4[M+H]+;

[0987] Step 2:

[0988] Potassium carbonate (712 mg, 5.2 mmol, 2.0 eq.) was added to an ethanol / water (5 / 1, 15 mL) solution of compound 3 (1.2 g, 1.58 mmol, 1.0 eq.). The mixture was stirred at 55 °C for 16 hours. LC-MS showed that the reaction was complete. The mixture was concentrated to remove ethanol, and then ethyl acetate (30 mL) was added. The residue was washed with water (10 mL) and brine (10 mL × 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (0%–30% ethyl acetate in petroleum ether) to give compound 4 (1.1 g, 94.2% yield) as a white solid.

[0989] LCMS:Rt:1.789min; MS m / z(ELSD):447.4[M+H] + ;

[0990] Step 3:

[0991] Trifluoroacetic acid (3 mL) was added dropwise to a solution of compound 4 (1.1 g, 2.46 mmol, 1.0 eq.) in dichloromethane (15 mL). The mixture was stirred at room temperature for 2 hours. LC-MS showed that the reaction was complete. The mixture was quenched with aqueous sodium bicarbonate solution and the pH was adjusted to 8. The mixture was then concentrated under vacuum. The residue was purified by silica gel column chromatography (dichloromethane containing 10% methanol) to give compound 5 (500 mg, yield 58.5%) as a white solid.

[0992] LCMS:Rt:1.189min; MS m / z(ELSD):347.3[M+H] + ;

[0993] Step 4:

[0994] Potassium carbonate (698 mg, 5.06 mmol, 5.0 eq.) and potassium iodide (335 mg, 2.02 mmol, 2.0 eq.) were added to a cyclopentyl methyl ether / acetonitrile (1 / 1, 10 mL) solution of compound 5 (350 mg, 1.01 mmol, 1.0 eq.) and compound 6 (560 mg, 1.21 mmol, 1.2 eq.). The mixture was stirred at 95 °C for 3 days under argon protection. Thin-layer chromatography (dichloromethane / methanol = 10 / 1) showed the formation of the target product. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (0%–5% methanol in dichloromethane) to give compound 7 (200 mg, 19.4% yield) as a yellow oil.

[0995] LCMS:Rt:1.202min;MS m / z(ELSD):727.7[M+H] + ;

[0996] Step 5:

[0997] Potassium carbonate (190 mg, 1.38 mmol, 1.0 eq.) and potassium iodide (91 mg, 0.55 mmol, 2.0 eq.) were added to a cyclopentyl methyl ether / acetonitrile (1 / 1, 3.5 mL) solution of compound 7 (200 mg, 0.28 mmol, 1.0 eq.) and compound 8 (106 mg, 0.55 mmol, 2.0 eq.). The mixture was stirred at 95 °C for 2 days under argon protection. LC-MS showed the formation of the target product. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (0%–5% methanol in dichloromethane) to give a yellow oil (4.2 g), which was further purified by preparative high-performance liquid chromatography to give compound HA-53B (10.17 mg, 4.3%) as a yellow oil.

[0998] LCMS:Rt:1.159min;MS m / z(ELSD):839.6[M+H] + ;

[0999] CAD:90.4%purity.Rt:11.928min

[1000] 1 H NMR(400MHz, CDCl3)δ8.20(dd,J=8.3,1.3Hz,1H),8.15–8.10(m,1H),7.66(dtd,J=15.1 ,7.0,1.4Hz,2H),4.90–4.80(m,1H),4.65–4.55(m,2H),3.92(t,J=6.3Hz,4H),3.39–3.3 1(m,5H),3.28(t,J=6.4Hz,2H),2.41(s,5H),2.27(t,J=7.5Hz,2H),1.96(s,2H),1.56( d,J=15.0Hz,4H),1.49(d,J=4.9Hz,8H),1.27(d,J=15.8Hz,38H),0.87(t,J=6.9Hz,6H).

[1001] Example 46: m-RNA (BNT113 mRNA)-LNP formulation.

[1002] (1) Formulation preparation:

[1003] ALC0315, DSPC, cholesterol, and PEG-DMG-2000 were all purchased from Xiamen Sinobond Biotechnology Co., Ltd.

[1004] Nanoparticles can be prepared by rapidly mixing two fluid streams, namely an aqueous phase and an alcohol phase, using a rapid mixing method such as a microfluidic device. One of the two fluid streams contains an aqueous solution of mRNA with therapeutic / preventive effects, and the other is an ethanol organic phase containing lipid components.

[1005] The cationic lipid ALC0315 is a current standard in the art. To compare the effects of various lipid compounds in this application on the delivery efficiency and adjuvant effect of the original four-component LNP formulation, this study uses a standard LNP formulation containing 50 mol% ALC0315, 10 mol% DSPC, 38.5 mol% cholesterol, and 1.5 mol% PEG-DMG as the basis for this study. One or more compounds from Examples 1-45 of this application are then added to this formulation to constitute the lipid components.

[1006] The lipid nanoparticle component was dissolved in 100% ethanol at a specified lipid molar ratio as the organic phase. The mRNA was dissolved in citrate buffer (pH 4.0) or acetate buffer (pH 4.0) as the aqueous phase. The mRNA used was an antigen-encoded therapeutic sequence (e.g., the BNT113 sequence), in which each uridine was replaced with N1-methylpseudouridine. After preparation via rapid mixing, the ethanol in the mRNA-LNP was removed and dispersed in the target buffer (pH 4.0 acetate buffer) using an ultrafiltration tube (100 kDa) or tangential flow filtration (TFF) device. The LNP was aseptically filtered using a 0.22 μM filter and stored at 4°C for further use.

[1007] LNP DLS characterization:

[1008] DLS - The hydrodynamic diameter and polydispersity index (PDI) of LNPs were measured using high-throughput dynamic light scattering (DLS) (Zetasizer Lab, Malvern Panalytical). LNPs were diluted to appropriate concentrations with 1×PBS and analyzed.

[1009] Concentration and encapsulation efficacy testing:

[1010] The concentrations of mRNA used for assay were determined using the Qubit-iT RiboGreen kit according to the manufacturer's instructions. Encapsulation efficiency was determined by measuring the mRNA concentrations in uncleaved and cleaved LNPs, as detailed in Tables 1 and 2. The formulations in Tables 1 and 2 encapsulate the BNT113 mRNA sequence (SEQ ID NO:1) encoding the E7 antigen, and the buffer is acetate at pH 4.0.

[1011] Table 1

[1012] Table 2

[1013] (2) Efficacy Test 1: The compound can be used to prepare drugs that activate specific T cells.

[1014] Six- to seven-week-old female C57BL / 6 mice (purchased from Zhuhai Baishitong Biotechnology Co., Ltd.) were used. All animal experiments were conducted in accordance with the "Guidelines for the Management and Use of Laboratory Animals".

[1015] C57BL / 6 mice (n=3) were administered mRNA-LNP formulations containing varying mass percentages of HA-1A lipid molecules via intravenous (iv) at a dose of 0.5 mg / kg. On day 7 post-administration, peripheral blood samples were collected, and E7-specific T cells in CD8+ cells were analyzed using flow cytometry (Beckman CytoFLEX). + The proportion of T cells. The results are shown in Table 3.

[1016] Table 3

[1017] The results of the examples show that all samples in this example were able to induce E7-specific T cell responses, that is, on CD8. + E7-specific T cells were detected in the T cell population.

[1018] (3) Efficacy Test 2: Verification of the tumor-suppressing effect of the compounds of this invention

[1019] Six- to seven-week-old female C57BL / 6 mice (purchased from Zhuhai Baishitong Biotechnology Co., Ltd.) were used. All animal experiments were conducted in accordance with the "Guidelines for the Management and Use of Laboratory Animals".

[1020] A tumor-bearing model was constructed using TC-1 tumor cells, purchased from Xiamen Yimo Biotechnology. 5 x 10^5 cells were subcutaneously implanted into each mouse. The tumor volume was calculated using the formula: 0.5 x length x width^2. When the tumor volume reached approximately 40 mm², the tumor was considered to have a tumor-bearing capacity. 3Mice were randomly divided into four groups of five each. Each experimental group was administered intravenously (iv) with LNP formulations containing different proportions of HA-1 lipid molecules (HA-1-LNP-02, HA-1-LNP-03, and HA-1-LNP-04), at a dose of 0.5 mg / kg. The LNPs encapsulated mRNA encoding the E7 antigen and were administered on days 0, 5, and 12. Control mice were given a Tris-treated control formulation. Tumor volume changes were monitored in each group after administration. Results are shown in Table 4.

[1021] Table 4

[1022] The experimental results showed that tumor growth was inhibited in all experimental groups, and the tumor-inhibiting effect was dose-dependent as the proportion of HA-1 compound in the LNP preparation increased.

[1023] (4) Effect Test 3: Effect of the compound of the present invention combined with the ALC-0315 formulation on E7-specific CD8 T cells in the spleen

[1024] Six- to seven-week-old female C57BL / 6 mice (purchased from Zhuhai Baishitong Biotechnology Co., Ltd.) were used. All animal experiments were conducted in accordance with the "Guidelines for the Management and Use of Laboratory Animals".

[1025] C57BL / 6 mice (n=3) were immunized on days 0 and 7 by intravenous injection of different formulations of E7 mRNA-LNP at a dose of 0.5 mg / kg. Mice were dissected on day 14, and spleens were collected to prepare single-cell suspensions. The proportion of E7-specific T cells in CD8 T cells was detected and analyzed using flow cytometry (Beckman CytoFLEX). The results are listed in Table 5.

[1026] Table 5

[1027] Example 47: Effect of the compounds of the present invention on the expression level of mRNA-encoded proteins

[1028] The LNP formulation was prepared according to Example 46, using Luc-mRNA (SEQ ID NO:5). The particle size and polydispersity index (PDI) of the obtained formulation were measured using a high-throughput dynamic light scattering instrument (Zetasizer Lab, Malvern Panalytical). The sample was diluted to an appropriate concentration with 0.5×PBS before detection. The mRNA concentration was determined using a Qubit-iT RiboGreen kit. The mRNA encapsulation efficiency of the formulation was calculated by comparing the difference in mRNA content before and after LNP cleavage. The relevant results are listed in Table 6.

[1029] Table 6

[1030] LNP formulation containing 5 μg Luc-mRNA was administered via tail vein injection to 6-week-old female BALB / c mice weighing 17-19g at a dose of 0.5 mg / kg. Six hours after administration, the mice were intraperitoneally injected with a fluorescent imaging substrate, and the fluorescent protein signal expressed by the LNP-delivered mRNA in vivo was detected using the IVIS Spectrum small animal in vivo imaging system. The results are expressed as average radiation intensity.

[1031] The experimental results are shown in Table 7. The results indicate that the introduction of HA-1A did not reduce the IVIS signal intensity after the mRNA-encoded protein catalyzed the substrate; in fact, the corresponding chemiluminescent signal intensity increased by approximately 37% compared to the control formulation.

[1032] Table 7

[1033] Example 48: Effects of the compound of the present invention combined with the SM-102 formulation on E7-specific CD8 T cells in peripheral blood and spleen

[1034] SM-102, DSPC, cholesterol, and PEG-DMG-2000 were all purchased from Xiamen Sinobond Biotechnology Co., Ltd. The nanoparticles were prepared using a rapid mixing method, such as rapidly mixing an aqueous phase and an alcohol phase using a microfluidic device. The aqueous phase was a buffer solution containing reporter, therapeutic, and / or prophylactic mRNA, and the alcohol phase was an ethanol solution containing dissolved lipid components.

[1035] In this embodiment, cationic lipid SM-102 was selected as the control system, which is a standard lipid molecule commonly used in the art. To compare the effects of the lipid compounds described in the embodiments of this application on adjuvant activity, a standard LNP organic phase containing 50 mol% SM-102, 10 mol% DSPC, 38.5 mol% cholesterol, and 1.5 mol% PEG-DMG was first prepared. Based on this, one or more compounds described in the embodiments of this application were further added to the lipid organic phase to construct the LNP formulation of the present invention.

[1036] Each lipid component was dissolved in 100% ethanol at a predetermined molar ratio to form the organic phase; the mRNA was dissolved in citrate or acetate buffer at pH 4.0 to form the aqueous phase. The mRNA used was the BNT113 sequence (SEQ ID NO:1) encoding the E7 antigen, in which all uridines were replaced with N1-methylpseudouridines. After rapid mixing to prepare LNPs, the ethanol content in the formulation was reduced to 0.5% or below through an ultrafiltration tube (100 kDa) or tangential flow filtration (TFF) and replaced with the target buffer. The resulting mRNA-LNPs were sterilely filtered through a 0.22 μm filter membrane and stored at 4°C for later use.

[1037] The particle size and polydispersity index (PDI) of the obtained LNPs were determined using a high-throughput dynamic light scattering instrument (Zetasizer Lab, Malvern Panalytical). Samples were diluted to an appropriate concentration with 0.5×PBS before detection. mRNA content was determined using the Qubit-iT RiboGreen kit. The mRNA encapsulation efficiency was calculated by comparing the mRNA content before and after LNP lysis. The relevant detection results are listed in Table 8.

[1038] Table 8

[1039] Effects of mRNA-LNP on E7-specific CD8 T cells in the spleen

[1040] Sixty-three female C57BL / 6 mice aged 6–8 weeks (n=3 per group) were purchased from Guangzhou Yongnuo Biotechnology Co., Ltd. The mice were administered an intramuscular injection (im) of an LNP preparation containing E7 mRNA. The mRNA used was a self-prepared BNT113 mRNA (SEQ ID NO:1), and all uridine was replaced with N1-methylpseudouridine. The immunization regimen consisted of two administrations on days 0 and 7, with each administration at a dose of 0.5 mg / kg.

[1041] Peripheral blood was collected before the second administration on day 7, and spleen tissue was collected by dissection on day 14.

[1042] Flow cytometry detection

[1043] Spleens were mechanically ground and filtered through a 70 μm filter to prepare a single-cell suspension. After centrifugation at 1500 rpm and 4°C for 5 min, the suspension was treated with erythrocyte lysis buffer and lysed at room temperature for 5–10 min. The reaction was then terminated, followed by centrifugation at 1500 rpm and 4°C for 5 min, followed by discarding the supernatant and resuspending the cells to obtain the spleen single-cell suspension. The cells were then stained with commercially available antibodies PE-E7 MHC dextramer, PE-Cy7 CD8, and eFluor780live / dead, and analyzed using a Beckman CytoFLEX flow cytometer to determine the proportion of E7-specific CD8 T cells in the total CD8 T cell population. See Table 9 for details.

[1044] Table 9

[1045] Example 50: Application of the compound of the present invention in the tumor pipeline combined with ALC-0315 formulation: efficacy - effect of mRNA-LNP on E7-specific CD8 T cells in the spleen.

[1046] Nine female C57BL / 6 mice, approximately 6 to 8 weeks old, were purchased from Guangzhou Yongnuo Biotechnology Co., Ltd., divided into groups of three, for the study described in this example. Each mouse was temporarily restrained, and mRNA-LNP was injected intravenously (IV) via the tail vein for each experiment. The mRNAs included BNT113 mRNA expressing the HPV E7 antigen, mRNA encoding the immune-enhancing element CD40L (SEQ ID NO:2), and mRNA encoding the immune-enhancing element caTLR4 (SEQ ID NO:3), all of which were pre-prepared. Specifically, group 1 received an IV injection of 2.5 μg CD40L mRNA-LNP + 2.5 μg caTLR4 mRNA-LNP + 5 μg E7 mRNA-LNP1; group 2 received an IV injection of 2.5 μg CD40L mRNA-LNP + 5 μg E7 mRNA-LNP2; and group 3 received an IV injection of 2.5 μg caTLR4 mRNA-LNP + 5 μg E7 mRNA-LNP2. Spleen tissue was collected for analysis on the 7th day after the first injection.

[1047] The basic information of the formulation is shown in Table 10. The proportions of ALC0315, cholesterol, DSPC, and DMG-PEG-2000 are the same as those in Example 46. In the E7 mRNA-LNP2 formulation, the mass percentage of HA-1 to ALC0315 is 10%.

[1048] Table 10

[1049] Flow cytometry

[1050] The spleen was mechanically ground thoroughly and then passed through a 70 μM filter to produce a single-cell suspension. After centrifugation at 1500 rpm for 5 min at 4 degrees Celsius, the cells were resuspended in erythrocyte lysis buffer. Lysis was stopped at room temperature for 5-10 min, followed by centrifugation at 1500 rpm for 5 min at 4 degrees Celsius. The supernatant was discarded, and the single-cell suspension was resuspended to complete the preparation of the single-cell suspension. The single-cell suspension was stained with commercially available antibodies PE E7 MHC dextranmer, PE-cy7 CD8, Percp cy5.5 CD4, efluo450 CD45, and efluor780 live / dead. The samples were analyzed using Beckman flow cytometry to determine the percentage of E7-specific CD8 T cells in the total CD8 T cell population. The results are shown in Table 11.

[1051] Table 11

[1052] The results showed that adjuvant-encapsulated mRNA elicited a higher CD8 T cell immune response on day 7 post-immunization compared to conventionally encapsulated mRNA.

[1053] Example 51: Application of the compound of the present invention in the ALC-0315 formulation in the infectious disease pipeline: efficacy-effect of mRNA-LNP on serum RABV-G specific binding antibody IgG.

[1054] Eight female BALB / c mice, approximately 6 to 8 weeks old, were divided into two groups of four, numbered #1, #2, #3, and #4 respectively. These mice were purchased from Guangzhou Yongnuo Biotechnology Co., Ltd. and used for the study described in this example. Each mouse was temporarily restrained. For each experiment, the mice were injected intramuscularly (IM) with mRNA-LNP containing 5 μg of the mRNA expressing the rabies glycoprotein RABV-G antigen (SEQ ID NO:4). The basic formulation information is shown in Table 12. The proportions of ALC0315, cholesterol, DSPC, and DMG-PEG-2000 were the same as in Example 22. In the RABV-G HA-1A formulation, the mass percentage of HA-1 to ALC0315 was 10%.

[1055] Six days after administration, peripheral blood was collected by sampling through the mandible. After standing at room temperature for 1 hour, the blood was centrifuged at 10,000 rpm for 20 minutes. The clear supernatant was collected as serum for testing. The level of RABV-G binding antibody IgG was detected by ELISA and expressed as RABV-G specific IgG endpoint titer. The results are shown in Table 13.

[1056] Table 12

[1057] Table 13

[1058] The results showed that in BALB / c mice, intramuscular injection of 5 μg of mRNA vaccine expressing RABV-G antigen, and detection of serum RABV-G specific binding antibody IgG levels on day 6 post-administration, all animals using adjuvants showed high efficacy (above 4000) in the early stage of immunization.

[1059] mRNA sequence 5'-3'

[1060] BNT113 (SEQ ID NO:1):

[1061] CD40L (SEQ ID NO:2)

[1062] caTLR4 (SEQ ID NO:3)

[1063] RABV-G (SEQ ID NO:4)

[1064] Luc-mRNA (SEQ ID NO:5)

Claims

The use of a compound in the preparation of a lipid carrier, said compound being a compound of formula I, a pharmaceutically acceptable salt thereof, or a conjugate thereof. in, R 0 For single key or R 1 It is an H or lipid fragment; R 2 lipid fragments, H, C 1-5 Alkyl, C 1-5 Heteroalkyl, with an R t1 Replacement C 1-5 Alkyl or with an R t1 Replacement C 1-5 Heteroalkyl; each R t1 Independently hydroxyl, C 1-5 Alkyl or with an R t2 Replacement C 1-5 Alkyl; the C 1-5 The heteroatoms in the heteroalkyl group are independently selected from one, two, or three of N, O, and S, and the number of heteroatoms is independently one, two, or three; R t2 It is a hydroxyl group; R 3 It is amino or halogen; R 4 R 5 and R 6 H and C independently 3-6 Cycloalkyl, 3-6 membered heterocyclic alkyl or lipid fragments; or R 4 R 5 Together with the atoms bonded to it, they form C 6-10 Aryl or 5-10 heteroaryl groups; And R 1 R 2 R 4 R 5 and R 6 At least one of them is a lipid fragment, said lipid fragment being independently and optionally R f Substituted branched or straight-chain saturated or unsaturated carbon chains; and said carbon chains optionally contain one or more "composed of N(R) e ), O, S, -S(O2)-, C 3-10 Cycloalkyl, 3-10 membered heterocyclic alkyl, C 6-10 "A group consisting of one or more segments of aryl and 5-10 heteroaryl groups"; Each R f Independently hydroxyl, C 1-6 Alkyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic alkyl, C 6-10 Aryl or 5-10 heteroaryl groups; Furthermore, the lipid fragment contains at least 11 carbon atoms; Each R e H and C independently 1-6 Alkyl, C 3-10 Cycloalkyl or 3-10 membered heterocyclic alkyl; The coupling of the compounds shown in Formula I represents two, three or more compounds shown in Formula I connected by a connecting unit; The heteroatoms in each of the 3-6 membered heterocyclic alkyl groups are independently selected from one, two, or three of N, O, and S, and the number of heteroatoms is independently one, two, or three. And the compound shown in Formula I is not The application as described in claim 1, characterized in that, The lipid fragment is independently R a1 or R a1 C 6-30 alkyl, Or by one or more R f Replacement C 6-30 Alkyl groups; each of the C4 groups 6-30 One or more methylene units in the alkyl group are independently and optionally R c1 Substitute; R a2 and R a3 Independently for C 1-30 alkyl, Or by one or more R f Replacement C 1-30 Alkyl groups; each of the C4 groups 1- 30 One or more methylene units in the alkyl group are independently and optionally R c3 Substitute; L 1 and L 2 Independently for -(CR) b1 R b2 ) n1 -, n1 is an integer from 1 to 20, R b1 and R b2 Independently H, hydroxyl, C 1-6 Alkyl, C 3-10 Cycloalkyl or 3-10 membered heterocycloalkyl, wherein -(CR b1 R b2 ) n1 One or more of -(CR) b1 R b2 - The unit is independently and optionally controlled by R c2 Substitute; Z 1 and Z 2 Independently CH or N; R d1 and R d2 Independently for C 1-30 Alkyl or with one or more R f Replacement C 1-30 Alkyl groups, each of the C16 groups 1-30 One or more methylene units in the alkyl group are independently and optionally R c4 Substitute; Each R c1 R c2 R c3 and R c4 Independently selected from -N(R) e -, -O-, -S-, -S(O2)-, C 3-10 Cycloalkyl, 3-10 membered heterocyclic alkyl, C 6-10 A group consisting of one or more segments of aryl and 5-10 heteroaryl groups; Each R e H and C independently 1-6 Alkyl, C 3-10 Cycloalkyl or 3-10 membered heterocyclic alkyl; Each R f Independently hydroxyl, C 1-6 Alkyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic alkyl, C 6-10 Aryl or 5-10 heteroaryl groups; Each of the 3-10 membered heterocyclic alkyl groups has a heteroatom type independently selected from one, two, or three of N, O, S, and S(O2), and the number of heteroatoms is independently 1, 2, or 3; each of the 5-10 membered heteroaryl groups has a heteroatom type independently selected from one, two, or three of N, O, and S, and the number of heteroatoms is independently 1, 2, or 3. Preferably, the compound satisfies one or more of the following conditions: (1)R a1 In the middle, each of the C 6-30 Alkyl group is C 10-25 Alkyl, such as C 15-25 Alkyl groups; each of the C4 groups 6-30 Alkyl groups can be straight-chain alkyl groups, for example... (2)R a2 and R a3 In the middle, each of the C 1-30 Alkyl groups are independently C 2-25 Alkyl, such as C 2-20 Alkyl; the C 1-30 Alkyl groups can be straight-chain alkyl groups, for example... (3)R d1 and R d2 In, the C 1-30 Alkyl groups are independently C 1-16 Alkyl, such as C 1-10 Alkyl groups, for example (4) Each of the C 1-6 Alkyl and C 1-5 Alkyl groups are independently C 1-4 Alkyl groups, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, or sec-butyl; (5) Each of the C 1-5 In heteroalkyl groups, the heteroatom species is N or O, and the number of heteroatoms is 1, for example... (6) Each of the C 3-10 cycloalkyl and C 3-6 The cycloalkyl group is independently cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl, for example, cyclopropyl, cyclobutyl, or cyclohexyl; when C 3-10 When the cycloalkyl group is a subunit, C 3-10 cycloalkyl can be (7) Each of the C 6-10 The aryl group is independently phenyl or naphthyl, for example, phenyl; C 6-10 When the aryl group is an alkyl group, C 6-10 Aryl is (8) The heteroatom in each 3-6 membered heterocyclic alkyl group is N, and the number of heteroatoms is 1 or 2; for example, piperazine group, and for example... (9) Each of the 3-10 membered heterocyclic alkyl groups is independently a 3-6 membered heterocyclic alkyl group; for example For example Or each of the 3-10 membered heterocyclic alkyl groups is independently... For example When each of the 3-10 membered heterocyclic alkyl groups is a subunit, each of the 3-10 membered heterocyclic alkyl groups can be: (10) The halogen is fluorine, chlorine, bromine or iodine; for example, chlorine; (11) Each of the "multiples" refers independently to 2, 3, 4 or 5; (12) Each of the 5-10 member heteroaryl groups is independently a 5-6 member heteroaryl group; (13)R 0 It is a single bond; (14)R 2 For H, C 1-5 Alkyl or C 1-5 Heteroalkyl, the C 1-5 The heteroatoms in the heteroalkyl group are independently selected from one, two, or three of N, O, and S, and the number of heteroatoms is one, two, or three; preferably, R 2 For H, C 1-5 Alkyl or C 1-5 Heteroalkyl, the C 1-5 The heteroatom in the heteroalkyl group is of type O, and the number of heteroatoms is 1, 2 or 3; (15)R 3 It is an amino or chlorine, such as R 3 For chlorine; or, R 3 Halogens, such as bromine or iodine, or bromine for example; (16)R 4 For H; (17)R 5 It is H or a 3-6 membered heterocyclic alkyl group, such as R 5 For H; (18)R 6 It is an H, a 3-6 membered heterocyclic alkyl group, or a lipid fragment, such as R. 6 For H; (19)R 4 R 5 Together with the atoms bonded to it, they form C 6-10 Aryl. The application as described in claim 2 is characterized in that, The compound satisfies one or more of the following conditions: (1) The lipid fragment is (2)R a1 Independently for C 10-25 alkyl or The C 10-25 One or more methylene units in the alkyl group are independently and optionally R c1 Alternative; preferably, R a1 Independently for C 10-25 Alkyl, the C 10-25 One or more methylene units in the alkyl group are independently and optionally R c1 Instead, better, R a1 Independently for C 10-25 Straight-chain alkyl, the C 10-25 One or more methylene units in a straight-chain alkyl group are independently and optionally R c1 Substitute; (3)R c1 Independently selected from -N(R) e -, -O-, -S-, -S(O2)-, A group consisting of one or more segments; preferably, R c1 Independently selected from -N(R) e -, -O-, -S-, -S(O2)-, A group consisting of one or more fragments; more preferably, R c1 Independently selected from -N(R) e )-、-O- and A group consisting of one or more segments; (4)L 1 Independently for -(CR) b1 R b2 ) n1 -, n1 is an integer from 1 to 16, the -(CR b1 R b2 ) n1 One or more of -(CR) b1 R b2 - The unit is independently and optionally controlled by R c2 Substitute; Ideally, L 1 Independently for -(CR) b1 R b2 ) n1 -, n1 is 1, 2, 3, 4, 5 or 6, the -(CR b1 R b2 ) n1 One or more of -(CR) b1 R b2 - The unit is independently and optionally controlled by R c2 Substitute; Better yet, L 1 Independently for -(CR) b1 R b2 ) n1 -, n1 is 1, 2, 3, 4, 5 or 6; (5)Z 1 Let N be the number of people in the group. (6)R a2 and R a3 Independently for C 2-25 alkyl, Or by one or more R f Replacement C 2-25 Alkyl; the C 2-25 One or more methylene units in the alkyl group are independently and optionally R c3 Alternative; preferably, R a2 and R a3 Independently for C 2-25 straight-chain alkyl, Or by one or more R f Replacement C 2-25 Straight-chain alkyl groups; each of the C... 2-25 One or more methylene units in a straight-chain alkyl group are independently and optionally R c3 Substitute; Better yet, R a2 and R a3 Independently for C 2-25 alkyl or The C 2-25 One or more methylene units in the alkyl group are independently and optionally R c3 Substitute; Furthermore, even better, R a2 and R a3 Independently (7)R c3 Independently selected from -O- C 3-10 cycloalkyl and C 6-10 A group consisting of one or more segments of an aryl group; preferably, R c3 Independently selected from -O- C 3-6 cycloalkyl and C 6-10 A group consisting of one or more segments of an aryl group; more preferably, R c3 Independently selected from -O- and A group consisting of one or two segments; Or, R c3 Independently selected from -N(R) e )-、-O- and A group consisting of one, two, or three segments; (8) Each R f Independently hydroxyl, C 3-10 Cycloalkyl or 3-10 membered heterocycloalkyl; preferably, each R f Independently hydroxyl, C 3-6 Cycloalkyl or 3-6 membered heterocycloalkyl; or, each R f Independently hydroxyl, C 3-6 cycloalkyl or C 6-10 Aryl, for example C 3-6 cycloalkyl or C 6-10 Aryl; (9)L 2 Independently for -(CR) b1 R b2 ) n1 -, n1 is an integer from 4 to 15, the -(CR b1 R b2 ) n1 One or more of -(CR) b1 R b2 - The unit is independently and optionally controlled by R c2 Substitute; (10)R c2 Independently selected from -O- C 3-10 A group consisting of one or more segments of cycloalkyl and 3-10 membered heterocycloalkyl groups; preferably, R c2 Independently selected from O-, C 3-6 A group consisting of one or more segments of cycloalkyl and 3-6 membered heterocycloalkyl groups; more preferably, R c2 It is a 3-6 membered heterocyclic alkyl group; Or, R c2 Independently selected from -N(R) e )-、-O- and A group consisting of one or more fragments; more preferably, R c2 Independently selected from -O- and A group consisting of one or two segments; (11)Z 2 For CH; (12)R d1 and R d2 Independently for C 1-16 Alkyl; preferably, R d1 and R d2 Independently for C 1-10 alkyl; (13) Each R e For H or each R e It is a 3-10 membered heterocyclic alkyl group; preferably a 3-6 membered heterocyclic alkyl group; Preferably, the compound satisfies one or more of the following conditions: (1)R c1 Independently for -O-, Ideally, R c1 Independently for -O-, in The connection direction in lipid fragments is independently either from left to right, consistent with the writing order, or independently from right to left, opposite to the writing order; preferably, R c1 Independently (2)R c2 Independently -O-、 in The connection direction in lipid fragments is either left-to-right, consistent with the writing order, or right-to-left, opposite to the writing order; preferably, R c2 Independently Or R c2 Independently for -O-, Better yet, R c2 for (3)R c3 Independently in The connection direction in lipid fragments is either left-to-right, consistent with the writing order, or right-to-left, opposite to the writing order; more preferably, R c3 for Or R c2 for The application as described in claim 1, characterized in that, R 1 For lipid fragments, R 1 For any of the following options: Option 1: R 0 For a single bond, R 1 C 10-25 Alkyl, the C 10-25 One or more methylene units in the alkyl group are independently and optionally R c1 Replacement, R c1 Independently selected from -N(R) e -, -O-, -S-, -S(O2)- and A group consisting of one or more fragments; R e For H; Option 2: R 0 For a single bond, R 1 for L 1 The definition is as follows: L 1 -(CR) b1 R b2 ) n1 -, n1 is 1, 2, 3, 4, 5 or 6, the -(CR b1 R b2 ) n1 One or more of -(CR) b1 R b2 - The unit is independently and optionally controlled by R c2 Replacement, R b1 and R b2 Independently for H, R c2 It is a 3-6 membered heterocyclic alkyl group; Z 1 Let N be the number of people in the group. R a2 and R a3 Independently for C 2-25 straight-chain alkyl, Or by one or more R f Replacement C 2-25 Straight-chain alkyl; the C 2-25 One or more methylene units in a straight-chain alkyl group are independently and optionally R c3 Substitute; R c3 Independently selected from -N(R) e )-、-O- and A group consisting of one, two, or three segments; R e For H; Each R f Independently hydroxyl, C 3-6 cycloalkyl or C 6-10 Aryl; L 2 The definition is as follows: L 2 Independently for -(CR) b1 R b2 ) n1 -, n1 is an integer from 4 to 15, the -(CR b1 R b2 ) n1 One or more of -(CR) b1 R b2 - The unit is independently and optionally controlled by R c2 Replacement, R b1 and R b2 For H, R c2 Independently selected from -N(R) e )-、-O- and A group consisting of one or more segments; Z 2 For CH; R d1 and R d2 Independently for C 1-16 alkyl; Option 3: R 0 for R 1 C 10-25 alkyl or The C 10-25 One or more methylene units in the alkyl group are independently and optionally R c1 Replacement, R c1 Independently selected from -N(R) e -, -O-, -S-, -S(O2)- and A group consisting of one or more fragments; R e For H; L 1 The definition is as follows: L 1 -(CR) b1 R b2 ) n1 -, n1 is 1, 2, 3, 4, 5, or 6, and the -(CR) b1 R b2 ) n1 One or more of -(CR) b1 R b2 - The unit is independently and optionally controlled by R c2 Replacement, R b1 and R b2 Independently for H, R c2 It is a 3-6 membered heterocyclic alkyl group. Z 1 Let N be the number of people in the group. R a2 and R a3 Independently for C 2-25 straight-chain alkyl, Or by one or more R f Replacement C 2-25 Straight-chain alkyl; the C 2-25 One or more methylene units in a straight-chain alkyl group are independently and optionally R c3 Substitute; R c3 Independently selected from -N(R) e )-、-O- and A group consisting of one, two, or three segments; R e For H; Each R f Independently hydroxyl, C 3-6 cycloalkyl or C 6-10 Aryl; L 2 The definition is as follows: L 2 Independently for -(CR) b1 R b2 ) n1 -, n1 is an integer from 4 to 15, the -(CR b1 R b2 ) n1 One or more of -(CR) b1 R b2 - The unit is independently and optionally controlled by R c2 Replacement, R b1 and R b2 For H, R c2 Independently selected from -N(R) e )-、-O- and A group consisting of one or more segments; Z 2 For CH; R d1 and R d2 Independently for C 1-16 alkyl. The application as described in claim 1, characterized in that, The compound satisfies one or more of the following conditions: (1)R f Independently hydroxyl, (2)L 1 Independently methyl, Preferably, the "2" position is related to Z. 1 connect; (3)L 2 Independently Preferably, the "2" position is related to Z. 2 connect; (4)R d1 and R d2 Independently (5)R a1 Independently (6)R a2 and R a3 Independently More preferably, in the compound, for More preferably, the compound satisfies one or more of the following conditions: (1)R 1 For H, (2)R 2 For H, ethyl, (3)R 3 It can be amino, chlorine, fluorine, bromine, or iodine; (4)R 4 For H; (5)R 5 For H or (6)R 4 R 5 Together with the atoms attached to it, they form (7)R 6 For H, The application as described in claim 1, characterized in that, The compound represented by Formula I is a compound as shown in Formula I-1 or I-2. The compound shown in Formula I-1 is selected from any of the following schemes: Option 1: R 1 C 10-25 Alkyl, the C 10-25 One or more methylene units in the alkyl group are independently and optionally R c1 Replacement, R c1 Independently selected from -N(R) e -, -O-, -S-, -S(O2)- and A group consisting of one or more fragments; R e For H; R 2 R 3 R 4 and R 5 The definition is as described in any one of claims 1-5; Option 2: The compound shown in Formula I-1 is the compound shown in Formula I-1-A: L 1 R a2 R a3 R 2 R 3 R 4 and R 5 The definition is as described in any one of claims 1-5; In the compound shown in Formula I-2, R 1 R 2 R 3 Define any of the following schemes: Option 1: R 3 It is a halogen; R 1 It is a lipid fragment; R 2 The definition is as described in any one of claims 1-5; Option 2: R 3 It is amino or halogen; R 1 C 10-25 Alkyl, the C 10-25 One or more methylene units in the alkyl group are independently and optionally R c1 Replacement, R c1 Independently selected from -N(R) e -, -O-, -S-, -S(O2)- and A group consisting of one or more fragments; R e For H; Or, R 1 for L 1 The definition is as follows: L 1 -(CR) b1 R b2 ) n1 -, n1 is 1, 2, 3, 4, 5, or 6, and the -(CR) b1 R b2 ) n1 One or more of -(CR) b1 R b2 - The unit is independently and optionally controlled by R c2 Replacement, R b1 and R b2 Independently for H, R c2 It is a 3-6 membered heterocyclic alkyl group. Z 1 Let N be the number of people in the group. R a2 and R a3 Independently for C 2-25 straight-chain alkyl, Or by one or more R f Replacement C 2-25 Straight-chain alkyl; the C 2-25 One or more methylene units in a straight-chain alkyl group are independently and optionally R c3 Substitute; R c3 Independently selected from -N(R) e )-、-O- and A group consisting of one, two, or three segments; R e For H; Each R f Independently for C 3-6 cycloalkyl or C 6-10 Aryl; L 2 The definition is as follows: L 2 Independently for -(CR) b1 R b2 ) n1 -, n1 is an integer from 4 to 15, the -(CR b1 R b2 ) n1 One or more of -(CR) b1 R b2 - The unit is independently and optionally controlled by R c2 Replacement, R b1 and R b2 For H, R c2 Independently selected from -N(R) e )-、-O- and A group consisting of one or more segments; Z 2 For CH; R d1 and R d2 Independently for C 1-16 alkyl. The application as described in claim 1, characterized in that, The compound represented by Formula I is the same as that represented by Formula I-1. R 1 It is a lipid fragment; R 2 For H, C 1-5 Alkyl, C 1-5 Heteroalkyl, with an R t1 Replacement C 1-5 Alkyl or with an R t1 Replacement C 1-5 Heteroalkyl; each R t1 Independently hydroxyl, C 1-5 Alkyl or with an R t2 Replacement C 1-5 Alkyl; the C 1-5 The heteroatoms in the heteroalkyl group are independently selected from one, two, or three of N, O, and S, and the number of heteroatoms is one, two, or three; R t2 It is a hydroxyl group; R 3 It is amino or halogen; R 4 For H; R 5 and R 6 Independently, it is H or a 3-6 membered heterocyclic alkyl group; Or R 4 R 5 Together with the atoms bonded to it, they form C 6-10 Aryl; Preferably, the compound represented by Formula I is the compound represented by Formula I-1a. R 3 It is amino or halogen; R 1 For R a1 or R a1 C 10-25 Alkyl; the C 10-25 One or more methylene units in the alkyl group are independently and optionally R c1 Substitute; R c1 The selection is made from -N(R) e )-、-O- and A group consisting of one or more segments; L 1 -(CH2) n1 -, n1 is 1, 2, 3, 4, 5 or 6, the -(CH2) n1 One or more -(CH2)- units in R are independently and optionally controlled by R c2 Substitute; R c2 For selected from -O- and A group consisting of one or two segments; R a2 and R a3 Independently for C 2-25 alkyl or The C 2-25 One or more methylene units in the alkyl group are independently and optionally R c3 Substitute; R c3 For selected from -O- and A group consisting of one or two segments; L 2 -(CH2) n1 -, n1 is an integer between 4 and 15, the -(CH2) n1 One or more -(CH2)- units in R are independently and optionally controlled by R c2 Substitute; R d1 and R d2 Independently for C 1-10 alkyl; More preferably, the compound represented by Formula I is the compound represented by Formula I-1a. R 3 It is amino or halogen; R 1 for L 1 -(CH2) n1 -, n1 is 1, 2, 3, 4, 5 or 6; R a2 and R a3 Independently L 2 -(CH2) n1 -, n1 is an integer between 4 and 15, the -(CH2) n1 One or more -(CH2)- units in R are independently and optionally controlled by R c2 Substitute; R c2 For selected from -O- and A group consisting of one or two segments; R d1 and R d2 Independently for C 1-10 alkyl. The application as described in claim 1, characterized in that, The compound is any one of the following compounds: The application as described in any one of claims 1-8 is characterized in that, It meets one or more of the following conditions: (1) The lipid carrier is used for the delivery of nucleic acid drugs, wherein the nucleic acid drugs are selected from one or more of single-stranded deoxyribonucleic acid (DNA), double-stranded DNA, small interfering RNA (siRNA), self-replicating RNA (samRNA), microRNA (miRNA), circular RNA (circRNA), and messenger RNA (mRNA), such as messenger RNA (mRNA), or for example, firefly luciferase (Fluc) mRNA, enhanced green fluorescent protein (eGFP) mRNA, BNT113 mRNA sequence encoding human papillomavirus E7 antigen, mRNA sequence encoding infectious disease antigen, or mRNA sequence encoding tumor antigen; preferably, the mRNA sequence encoding infectious disease antigen is mRNA encoding respiratory syncytial virus antigen (RSV-mRNA), mRNA encoding varicella-zoster virus antigen (VZV-mRNA), mRNA encoding rabies virus antigen, mRNA encoding Mycobacterium tuberculosis antigen, mRNA encoding novel coronavirus antigen, or mRNA encoding influenza virus antigen; the tumor may be a solid tumor, such as cervical cancer or non-small cell lung cancer; (2) The lipid carrier further includes one, two, three, four or five of the following: lipid component Z, diluent, phospholipid, PEG lipid and sterol; or the lipid carrier further includes one, two, three or four of the following: lipid component Z, phospholipid, PEG lipid and sterol. The lipid component Z is selected from one, two, or three of ionizable lipids, anionic lipids, and cationic lipids; (3) The lipid carrier is a lipid nanoparticle. The application as described in claim 9, characterized in that, The lipid carrier satisfies one or more of the following conditions: (1) The lipid component Z is an ionizable lipid, such as ALC0315. Or SM-102 (2) The diluent is citrate buffer, phosphate buffer or acetate buffer; preferably citrate or acetate buffer, such as acetate buffer with pH 4; (3) The phospholipid is a neutral phospholipid, for example, the phospholipid is a phospholipid molecule with a polar end and a nonpolar end of an adipose chain, such as distearylphosphatidylcholine (DSPC), dimyristoylphosphatidylcholine (DMPC), dioleoylphosphatidylcholine (DOPC), palmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylethanolamine (DOPE), distearylphosphatidylethanolamine (DSPE), distearylphosphatidylglycerol (DSPG), disqualylphosphatidylcholine (DEPC), palmitoylphosphatidylglycerol (DPPG), dipalmitoylphosphatidic acid (DPPA), docosylphosphatidylcholine (DUPC) or palmitoylphosphatidylcholine (POPC); (4) The PEG lipid is a lipid molecule modified with a polyethylene glycol hydrophilic end; the PEG lipid is preferably selected from one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol and PEG-modified dialkylglycerol, such as PEG-DSPE, PEG-DMG or ALC-0159; the PEG-DSPE is preferably PEG-DSPE-2000; (5) The sterol is an animal, plant or fungal sterol; for example, the sterol is selected from one or more of cholesterol, sitosterol, ergosterol, campesterol, stigmasterol, brassosterol, tomatine, ursolic acid and α-tocopherol, such as cholesterol. The application as described in claim 10 is characterized in that, The lipid carrier can be any of the following: Option 1: The lipid carrier comprises the compound, diluent, neutral phospholipid, and sterol; for example, the lipid carrier is composed of the compound, diluent, neutral phospholipid, and sterol. Option 2: The lipid carrier comprises the compound, diluent, neutral phospholipid, and PEG lipid; for example, the lipid carrier is composed of the compound, diluent, neutral phospholipid, and PEG lipid. More preferably, the lipid carrier comprises the compound, diluent, neutral phospholipid, PEG lipid, and sterol; for example, the lipid carrier is composed of the compound, diluent, neutral phospholipid, PEG lipid, and sterol. More preferably, the lipid carrier comprises the compound, lipid component Z, diluent, neutral phospholipid, PEG lipid, and sterol; for example, the lipid carrier is composed of the compound, lipid component Z, diluent, neutral phospholipid, PEG lipid, and sterol; or, for example, lipid component Z is an ionizable lipid; or the lipid carrier comprises the compound, lipid component Z, neutral phospholipid, PEG lipid, and sterol; preferably, the lipid carrier is composed of the compound, lipid component Z, neutral phospholipid, PEG lipid, and sterol; more preferably, lipid component Z is an ionizable lipid. The application as described in claim 11, characterized in that, In the lipid carrier, based on the total molar amount of lipid component Z, sterol, neutral phospholipid and PEG lipid in the lipid carrier, the lipid carrier satisfies one or more of the following conditions: (1) The molar amount of the ionizable lipid accounts for 30% to 80% of the total molar amount, for example 50%; (2) The molar amount of the anionic lipid accounts for 30% to 50% of the total molar amount, for example 40%; (3) The molar amount of the cationic lipid accounts for 30% to 50% of the total molar amount, for example 40%; (4) The molar amount of the neutral phospholipid accounts for 5% to 20% of the total molar amount, for example, 10%; (5) The molar amount of the sterol accounts for 20% to 60% of the total molar amount, for example 38.5%; (6) The molar amount of the PEG lipid accounts for 0.2% to 5% of the total molar amount, for example, 1.5%; (7) The mass percentage of the compound to the lipid component Z is 0.5%-60%, for example 1%-50%, or for example 1%, 2.5%, 5%, 10%, 20%, 30%, 40% or 50%, or for example 10%; Preferably, the lipid carrier is selected from any of the following: Option 1: The lipid carrier is composed of the compound, ALC0315, distearylphosphatidylcholine, PEG-DMG-2000, and cholesterol; or, the lipid carrier is composed of the compound, ALC0315, a diluent, distearylphosphatidylcholine, PEG-DMG-2000, and cholesterol. The compound is any of the following compounds: compounds with the formula HA-1, HA-1A, HA-12, HA-14-C16, HA-14-C16-A, HA-TLR-1-A, HA-TLRa, HA-13-A, HA-15, HA-15A, HA-TLR-2, HA-TLR-2-A, HA-11, HA-TLRa-A, HA-TLR-1, HA-TLR-1-B; The mass percentage of the compound relative to the mass of ALC0315 is 10%; Calculated based on the total molar amounts of ALC0315, distearate phosphatidylcholine, PEG-DMG-2000, and cholesterol in the lipid carrier. The molar amount of ALC0315 accounts for 50% of the total molar amount; The molar amount of the distearate phosphatidylcholine accounts for 10% of the total molar amount; The molar amount of cholesterol accounts for 38.5% of the total molar amount; The molar amount of PEG-DMG-2000 accounts for 1.5% of the total molar amount; The diluent is an acetate buffer solution with a pH of 4; Option 2: The lipid carrier is composed of the compound, SM102, distearylphosphatidylcholine, PEG-DMG-2000, and cholesterol; or, the lipid carrier is composed of the compound, SM102, a diluent, distearylphosphatidylcholine, PEG-DMG-2000, and cholesterol. The compound is any of the following compounds: compounds represented by the formula HA-1-10-A, HA-13, HA-17B, HA-30-A, HA-TLR-1, HA-TLR-1-A, HA-TLR-2, HA-TLR-2-A, HA-TLRa-A, HA-TLRb, HA-TLRb-A, HA-1, HA-11-1, HA-1A, HA-1B-1, HA-33, HA-34, HA-1-13B, HA-1-18-A, HA-17A or HA-19-A; The mass percentage of the compound relative to the mass of ALC0315 is 10%; Calculated based on the total molar amounts of ALC0315, distearate phosphatidylcholine, PEG-DMG-2000, and cholesterol in the lipid carrier. The molar amount of ALC0315 accounts for 50% of the total molar amount; The molar amount of the distearate phosphatidylcholine accounts for 10% of the total molar amount; The molar amount of cholesterol accounts for 38.5% of the total molar amount; The molar amount of PEG-DMG-2000 accounts for 1.5% of the total molar amount; The diluent is an acetate buffer solution with a pH of 4. A compound, said compound being a compound of formula II, a pharmaceutically acceptable salt thereof, or a conjugate thereof. in, R 0 R 1 R 2 R 3 R 4 R 5 and R 6 The definition is as described in any one of claims 1-8; And the compound represented by Formula II satisfies any of the following conditions: (1) When R 0 For a single bond, R 1 When it is a lipid fragment, the lipid fragment is R. a1 C 6-30 alkyl, Or by one or more R f Replacement C 6-30 Alkyl; and the C 6-30 One or more methylene units in an alkyl group are independently R c1 Substitute; R c1 Independently selected from -O-, -S-, -S(O2)-, C 3-10 Cycloalkyl, 3-10 membered heterocyclic alkyl, C 6- 10 A group composed of one or more segments of aryl and 5-10 heteroaryl groups; (2)R 0 for R 1 For lipid fragments, R 2 C 1-5 Alkyl, R 3 For amino group, R 4 R 5 and R 6 When H is independent, each R f Independently for C 1-6 Alkyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic alkyl, C 6-10 Aryl or 5-10 heteroaryl compounds. A lipid carrier comprising a compound of formula I as claimed in any one of claims 1-8, a pharmaceutically acceptable salt thereof, or a conjugate thereof, or, as claimed in claim 9, a compound of formula II, a pharmaceutically acceptable salt thereof, or a conjugate thereof, wherein the compound is not... The definition of the lipid carrier may also be as described in any one of claims 9-12. A liposomal drug, characterized in that, It includes the nucleic acid drug as described in claim 9 and the lipid carrier as described in claim 14; Preferably, the liposomal drug satisfies one or more of the following conditions: (1) The nitrogen-to-phosphorus ratio in the liposomal drug is (2-30):1, preferably (2-20):1, more preferably (3-20):1, for example (3-16):1, and for example 6:1; (2) The average particle size of the liposome drug is 40-300nm, preferably 70-120nm, for example 89nm, 90nm, 91nm, 92nm, 93nm, 103nm, 106nm, 107nm, 108nm, 109nm or 110nm. (3) The polydispersity index of the liposomal drug is 0.1-0.3, for example 0.1449, 0.1665, 0.1724, 0.1808, 0.1851, 0.1962, 0.2073, 0.2133, 0.2824, 0.2902 or 0.3142; (4) The encapsulation rate of the liposomal drug is 85%-100%, for example 89.23%, 89.71%, 90.35%, 90.36%, 90.38%, 92.79%, 93.42%, 96.53%, 97.14%, 97.56% or 97.59%; (5) The lipid carrier encapsulates the nucleic acid drug.

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