Antibody-clpp agonist conjugate

Abstract

The present invention provides an antibody-ClpP agonist conjugate prepared using a ClpP agonist having a novel structure. The antibody-ClpP agonist conjugate has certain in vitro cellular activity.

Description

Antibody-ClpP agonist conjugates Technical Field

[0001] This invention relates to antibody-drug conjugates, and more specifically to antibody-ClpP agonist conjugates. Background Technology

[0002] Casein-lysing peptidase P (ClpP) is a mitochondrial protease involved in the occurrence and development of cancer (Bonner et al., 2021). Inhibition or overactivation of ClpP can suppress tumor growth (Cole et al., 2015). ClpP agonists can activate ClpP, reduce the level of oxidative respiratory complex proteins, impair mitochondrial function, and thus induce tumor cell death (Ishizawa et al., 2019). ClpP agonists have shown antitumor activity in various cancers, including glioma, pancreatic cancer, and ovarian cancer.

[0003] ClpP agonists, as a novel payload mechanism for activating ClpP, exhibit a different mechanism of apoptosis induction compared to commonly used topoisomerase inhibitors like ixotecan and microtubule inhibitors like MMAE. Currently, most payloads used in ADC synthesis struggle to target non-dividing cells in cancer cells, which play crucial roles in maintaining tumor stability, resisting treatment, remodeling tumors, and generating cancer stem cells. However, because ClpP agonists target cellular metabolism, they can potentially kill not only dividing cancer cells but also those in the non-dividing phase. Therefore, further research on ClpP agonists is desired to overcome cancer cell resistance to existing antibody-drug conjugate (ADC) payloads and develop therapeutically effective antibody-drug conjugates. Summary of the Invention

[0004] The main objective of this invention is to prepare antibody-drug conjugates using a novel ClpP agonist.

[0005] To achieve the above objectives, a first aspect of the present invention provides an antibody-ClpP agonist conjugate selected from compounds having the structure of Formula I, or derivatives thereof, tautomers, stereoisomers, pharmaceutically acceptable salts or esters, and solvates.

[0006] in,

[0007] Ab is an antibody, L is a linker, the linker includes one or more sub-linker units, q = 1 to 20, and D is a compound of formula AC-P before binding with L, or a tautomer, stereoisomer, or pharmaceutically acceptable salt thereof with ClpP agonist activity.

[0008] in,

[0009] R a2 R b2 Each of them is independently selected from hydrogen or deuterium.

[0010] The structure of ring A is as follows: i is an integer from 1 to 4, where R a Each of these is independently selected from hydrogen, halogen, cyano, -R a1 -R c or -R a1 -N(R c1 )-Y,R a1 Each of these is independently selected from non-existent, -CH2-, -C(O)-, or -CH2-C(O)-;

[0011] The ring B structure is j is an integer from 1 to 4, where R b Each of these radicals is independently selected from hydrogen, deuterium, halogen, cyano, -R b1 -R c or -R b1 -N(R c1 )-Y,R b1 Each of these is independently selected from non-existent, -CH2-, -C(O)-, or -CH2-C(O)-;

[0012] The R mentioned 1 Selected from hydrogen, C1-C3 alkyl groups, C1-C3 alkyl groups substituted with 3-6-membered nitrogen-containing heterocyclic alkyl groups, -CH2-C(R) 11 )2-R c -CH2-C(R) 11 )2-N(R c1 -Y, -R 12 -R c or -R 12 -N(R c1 )-Y, where R 11 Each of them is independently selected from H or methyl, R 12 Each of them is independently selected from one of C1-C3 alkyl, phenyl, 5-6 heteroaryl, a combination of C1-C3 alkyl and phenyl, or a combination of C1-C3 alkyl and 5-6 heteroaryl;

[0013] Each of the Y terms is independently selected from -C(O)-C(R) y1 )2-(CH2) 0-1 -OH, -C(O)-R y2 -(CH2) 0-2-OH、 in,

[0014] The R mentioned y1 Selected from item i) or item ii): i) One of R y1 Selected from C1-C3 alkyl groups and 3-6 cycloalkyl groups, wherein the alkyl or cycloalkyl group may optionally be substituted with one or more halogens, and the other R y1 For hydrogen, ii). Two R y1 It forms a 3- to 6-membered cycloalkyl group with the attached carbon.

[0015] The R mentioned y2 Selected from 3- to 6-membered cycloalkyl, 3- to 6-membered cycloalkyl, phenyl, C1- to C3 alkynyl, n4 = 1 to 8,

[0016] k is selected from 1 or 2, and the R... x Selected from item iii) or item iv): iii). Each R x Independently selected from hydrogen, deuterium, halogen, carbonyl, -R x1 -R c or -R x1 -N(R c1 )-Y,R x1 Each of the following is independently selected from non-existent, -CH2-, -C(O)-, or -CH2-C(O)-; iv). Two R x It forms 3- to 6-membered cycloalkyl or heterocycloalkyl groups with the attached carbon;

[0017] The R mentioned c Selected from -OH or -NHR c1 R c1 Each of them is independently selected from hydrogen or methyl.

[0018] The rings A, B, and R mentioned above 1 The compound represented by AC-P contains at least one -OH, -NH2, or -NH-, and the compound binds to the linker through the reaction of the contained -OH, -NH2, or -NH-.

[0019] In some embodiments of the first aspect of the present invention, i = 1 to 2.

[0020] In some embodiments of the first aspect of the invention, the R x Each of them is independently selected from hydrogen, deuterium, fluorine, and methyl.

[0021] In some embodiments of the first aspect of the present invention, the ring A is selected from:

[0022] In some embodiments of the first aspect of the invention, the R a Each of them is independently selected from hydrogen, deuterium, halogen, or cyano.

[0023] In some embodiments of the first aspect of the present invention, the ring A is selected from:

[0024] In some embodiments of the first aspect of the present invention, at least one R exists. a Selected from -R a1 -R c or -R a1 -N(R c1 )-Y.

[0025] In some embodiments of the first aspect of the present invention, at least one R exists. a Selected from -R a1 -R c .

[0026] In some embodiments of the first aspect of the present invention, at least one R exists. a Selected from -CH2-NH2.

[0027] In some embodiments of the first aspect of the present invention, the ring A is selected from:

[0028] In some embodiments of the first aspect of the present invention, j = 1 to 2.

[0029] In some embodiments of the first aspect of the present invention, the ring B is selected from:

[0030] Selected from

[0031] In some embodiments of the first aspect of the invention, the R b Each of them is independently selected from halogens or cyano groups.

[0032] In some embodiments of the first aspect of the present invention, the ring B is selected from:

[0033] In some embodiments of the first aspect of the invention, one of the R b Selected from -R b1 -R c or -R b1 -N(R c1 )-Y.

[0034] In some embodiments of the first aspect of the invention, one of the R b Selected from -R b1 -R c .

[0035] In some embodiments of the first aspect of the invention, one of the R b Selected from -OH or -CH2-OH.

[0036] In some embodiments of the first aspect of the present invention, the ring B is selected from:

[0037] In some embodiments of the first aspect of the invention, R b1 Each of them is independently selected from -CH2-, -C(O)-, or -CH2-C(O)-, R c Selected from -NH2.

[0038] In some embodiments of the first aspect of the present invention, the ring B is selected from:

[0039] In some embodiments of the first aspect of the present invention, at least one R exists. b Selected from -R b1 -N(R c1 )-Y.

[0040] In some embodiments of the first aspect of the present invention, the ring B is selected from:

[0041] In some embodiments of the first aspect of the invention, the R 1 Selected from -CH2-C(R) 11 )2-R c or -CH2-C(R) 11 )2-N(R c1 )-Y.

[0042] In some embodiments of the first aspect of the invention, the R 1 Selected from -CH2-C(R) 11 )2-R c .

[0043] In some embodiments of the first aspect of the invention, the R c It is -OH.

[0044] In some embodiments of the first aspect of the invention, the R 1 Selected from:

[0045] In some embodiments of the first aspect of the invention, the R c -NHR c1 .

[0046] In some embodiments of the first aspect of the invention, the R 1 Selected from:

[0047] In some embodiments of the first aspect of the invention, the R 1 Selected from -R 12 -R c .

[0048] In some embodiments of the first aspect of the invention, the R 1 Selected from:

[0049] In some embodiments of the first aspect of the invention, the R 1 Selected from -CH2-C(R) 11 )2-N(R c1 )-Y.

[0050] In some embodiments of the first aspect of the invention, the R 1 Selected from:

[0051] In some embodiments of the first aspect of the invention, the R 1 Selected from:

[0052] In some embodiments of the first aspect of the present invention, Y is selected from:

[0053] -C(O)-C(R y1 )2-(CH 2)0-1 -OH.

[0054] In some embodiments of the first aspect of the present invention

[0055] R y1 The term Y is selected from item i, and Y is selected from:

[0056] or R y1 The term Y is selected from item ii, and Y is selected from:

[0057] In some embodiments of the first aspect of the present invention, Y is selected from:

[0058] -C(O)-R y2 -(CH 2)0-2 -OH.

[0059] In some embodiments of the first aspect of the invention, the R y2 Selected from

[0060] In some embodiments of the first aspect of the present invention, Y is selected from:

[0061] In some embodiments of the first aspect of the present invention, Y is selected from:

[0062] In some embodiments of the first aspect of the present invention, Y is selected from:

[0063] In some embodiments of the first aspect of the present invention, the AC-P structure is selected from:

[0064] in,

[0065] R a Each of these is independently selected from hydrogen, halogen, cyano, -R a1 -R c or -R a1 -N(R c1 )-Y,R a1 Each of these is independently selected from non-existent, -CH2-, -C(O)-, or -CH2-C(O)-;

[0066] R b Each of these is independently selected from hydrogen, halogen, cyano, -R b1 -R c or -R b1 -N(R c1 )-Y,R b1 Each of these is independently selected from non-existent, -CH2-, -C(O)-, or -CH2-C(O)-;

[0067] The R mentioned 1 Selected from -CH2-C(R) 11 )2-R c or -CH2-C(R) 11 )2-N(R c1 )-Y, where R 11 Each of them is independently selected from H or methyl;

[0068] Each of the Y terms is independently selected from -C(O)-CH(R) y1 )-OH,R y1 Selected from hydrogen or methyl.

[0069] In some embodiments of the first aspect of the present invention, the AC-P structure is selected from:

[0070] in,

[0071] Ram Selected from -F or -CN, R an Selected from -H or -F,

[0072] R bn Selected from F or Cl, R bm Selected from -H, -CH2-OH, -CH2-NH2, -CH2-NH-C(O)-CH2-OH, -CH2-C(O)-NH2.

[0073] R 1 Selected from

[0074] In some embodiments of the first aspect of the present invention, the formula AC-P is selected from the structures shown in the table below:

[0075] In some embodiments of the first aspect of the present invention, the formula AC-P is selected from the structures shown in the table below:

[0076] In some embodiments of the first aspect of the present invention, the connector has the following structure:

[0077] in,

[0078] L1 is selected from L 1 In the formula, # is used to indicate the site of antibody binding, and the wavy line is used to indicate the site of binding with L. 2 The site of connection;

[0079] L2 is selected from L 2 In the formula, n1 = 0 - 5, n2 = 0 - 10, n3 = 0 - 3, and the # symbol indicates that it is related to L. 1 The connection site, based on L 3 L 4 Whether it exists or not, the wavy line represents the adjacent L. 3 L 4 Or the connection site of D;

[0080] L3 does not exist or is selected from In L3, the # symbol indicates a relationship with L. 2 The connection site, based on L 4 Whether it exists or not, the wavy line represents the adjacent L. 4 Or the connection site of D;

[0081] L4 does not exist or is selected from L 4 In the middle, R L Independently selected from hydrogen, C1-C3 straight-chain or branched alkyl, C1-C3 straight-chain or branched alkyl hydroxyl, C1-C3 straight-chain or branched alkylamine, based on L 3 Whether it exists or not, the # symbol indicates that it is adjacent to the L. 2 L 3 The connection points are indicated by wavy lines, which represent the connection points with D.

[0082] In some embodiments of the first aspect of the present invention, the antibody specifically binds to a tumor antigen.

[0083] In some embodiments of the first aspect of the present invention, the antibody is selected from anti-EGFR antibody, anti-DLL-3 antibody, anti-PSMA antibody, anti-CD70 antibody, anti-MUC16 antibody, anti-ENPP3 antibody, anti-TDGF1 antibody, anti-CCK-BR antibody, anti-MSLN antibody, anti-TIM-1 antibody, anti-LRRC15 antibody, anti-LIV-I antibody, anti-CanAg / AFP antibody, and anti-claudin. 18.2 Antibodies, anti-Mesothelin antibody, anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-c-MET antibody, anti-SLITRK6 antibody, anti-KIT / CD117 antibody, anti-STEAP1 antibody, anti-SLAMF7 / CS1 antibody, anti-NaPi2B / SLC34A2 antibody, anti-GPNMB antibody, anti-HER3 (ErbB3) antibody, anti-MUC1 / CD227 antibody, anti-AXL antibody, anti-CD166 antibody, anti-B7-H3 (CD276) antibody, anti-PTK7 / CCK4 antibody, anti-PRL R antibody, anti-EFNA4 antibody, anti-5T4 antibody, anti-NOTCH3 antibody, anti-Nectin4 antibody, anti-TROP-2 antibody, anti-CD142 antibody, anti-CA6 antibody, anti-GPR20 antibody, anti-CD174 antibody, anti-CD71 antibody, anti-EphA2 antibody, anti-LYPD3 antibody, anti-FGFR2 antibody, anti-FGFR3 antibody, anti-FRα antibody, anti-CEACAMs antibody, anti-GCC antibody, anti-IntegrinAv antibody, anti-CAIX antibody, anti-P-cadherin antibody, anti-GD3 antibody, anti-Cadherin 6. Antibodies, including anti-LAMPI antibody, anti-FLT3 antibody, anti-BCMA antibody, anti-CD79b antibody, anti-CD19 antibody, anti-CD33 antibody, anti-CD56 antibody, anti-CD74 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD37 antibody, anti-CD47 antibody, anti-CD138 antibody, anti-CD352 antibody, anti-CD25 antibody, anti-CD147 antibody, or anti-CD123 antibody.

[0084] In some embodiments of the first aspect of the invention, q = 1 to 12.

[0085] In some embodiments of the first aspect of the invention, q = 2, 4 or 8.

[0086] In some embodiments of the first aspect of the present invention, the following structures are selected:

[0087] In some embodiments of the first aspect of the present invention, the following structures are selected:

[0088] Among them, Ab1 is Trastuzumab, Ab2 is Sacituzumab, Ab3 is Patritumab, and Ab5 is DS-1471.

[0089] A second aspect of the present invention relates to a linker-ClpP agonist conjugate for preparing any of the antibody-ClpP agonist conjugates described above.

[0090] In some embodiments of the second aspect of the present invention, the linker-ClpP agonist conjugate is selected from:

[0091] The third aspect of the present invention relates to the use of any of the above-described antibody-ClpP conjugates in the preparation of a medicament for the treatment or prevention of: tumors associated with HER2, TROP2, CD147 or HER3 expression, and diseases associated with mitochondrial dysfunction.

[0092] In some embodiments of the third aspect of the present invention, the tumor is a solid tumor or hematologic tumor such as breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, stomach cancer, endometrial cancer, salivary gland cancer, esophageal cancer, lung cancer, colon cancer, rectal cancer, colorectal cancer, bone cancer, skin cancer, thyroid cancer, prostate cancer, melanoma, glioma, neuroblastoma, glioma multiforme, sarcoma, lymphoma, and leukemia.

[0093] In some embodiments of the third aspect of the present invention, the tumor is gastric cancer, and the antibody-ClpP conjugate is selected from:

[0094] The technical effects achieved by this invention are as follows:

[0095] 1. Antibody-ClpP agonist conjugates were prepared using a novel ClpP agonist molecular structure.

[0096] 2. This antibody-ClpP agonist conjugate exhibits certain in vitro cell activity.

[0097] 3. Compounds with therapeutic effects on gastric cancer can be generated from the obtained antibody-ClpP agonist conjugate. Attached Figure Description

[0098] Figures 1a and 1b show the DAR values ​​of AC-ADC1 and trastuzumab determined by HPLC.

[0099] Figures 2a and 2b show the DAR values ​​of AC-ADC2 and trastuzumab determined by HPLC.

[0100] Figures 3a and 3b show the DAR values ​​of AC-ADC3 and trastuzumab determined by HPLC.

[0101] Figure 4 shows the activity comparison of different loads in different cell lines.

[0102] Figure 5 shows the activity of different ADCs in the NCI-N87 cell line.

[0103] Figure 6 shows the activity of AC-ADC1 in different cell lines.

[0104] Figure 7 shows the cell activity of ADCs with different loads and connectors.

[0105] Figure 8 shows the activity of ADCs with different DAR values ​​at the HER2 target site on different cell lines.

[0106] Figure 9 shows the activity of AC-ADC3 in different cell lines.

[0107] Figure 10 shows the activity of AC-ADC4 in different cell lines.

[0108] Figure 11 shows the activity of AC-ADC5 in different cell lines.

[0109] Figure 12 shows the activity of AC-ADC6 in different cell lines.

[0110] Figure 13 shows the effects of different ADCs targeting HER2 on the NCI-N87 xenograft in mice.

[0111] Figure 14 shows the effects of different concentrations of ADCs targeting TROP2 on the anti-xenograft NCI-N87 tumor in mice. Detailed Implementation

[0112] This invention employs a ClpP agonist with a structure different from existing ones in the antibody-loaded conjugate, and uses this ClpP agonist as the payload to synthesize an antibody-ClpP agonist conjugate. Based on this, a therapeutically effective molecular structure can be generated in the successfully synthesized antibody-ClpP agonist conjugate, thereby significantly increasing the therapeutic window of the ClpP small molecule.

[0113] The antibody-ClpP agonist conjugate has a general formula structure. In this invention, Ab represents an antibody, L represents a linker comprising one or more sub-linker units, q = 1 to 20, and D, before binding with L, is a ClpP agonist with a structure different from existing structures, as mentioned above. The following description will proceed in the order of ClpP agonist to antibody-ClpP agonist conjugates.

[0114] In describing this invention, unless otherwise defined, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art.

[0115] When this invention refers to a group as "optionally" being substituted by another group, it means that the hydrogen in the group described by the former is not substituted, or is substituted by the group described by the latter, and whether or not it is substituted is optional.

[0116] When "pharmaceuticalally acceptable" is mentioned in this invention, it means that it is safe and / or effective when used in mammals and can have the expected biological activity. Non-limiting examples of pharmaceutically acceptable salts include: hydrochloride, hydrobromide, hydroiodide, sulfate, hydrogen sulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, phosphate, hydrogen phosphate, dihydrogen phosphate, salicylate, citrate, tartrate, malate, maleate, fumarate, formate, benzoate, acetate, trifluoroacetate, lactate, oleate, ascorbate, glutamate, camphorsulfonic acid, methanesulfonate, ethanesulfonate, benzenesulfonate, or p-toluenesulfonate. The antibody-antibody drug conjugates of this application can also form salts with bases; non-limiting examples of pharmaceutically acceptable salts include: sodium salts, potassium salts, calcium salts, magnesium salts, and meglumine salts.

[0117] When the term "stereoisomer" is used in this invention, it includes meso-racemates, racemates, enantiomers and / or diastereomers.

[0118] ClpP agonists

[0119] The ClpP agonist loaded in this invention for preparing antibody-ClpP agonists has a novel structure different from that in the prior art, having the structure shown in the formula AC-P, or a tautomer, stereoisomer, deuterated product, or pharmaceutically acceptable salt of formula AC-P with ClpP agonist activity.

[0120] in,

[0121] R a2 R b2 Each of them is independently selected from hydrogen or deuterium.

[0122] The structure of ring A is as follows: i is an integer from 1 to 4, where R a Each of these is independently selected from hydrogen, halogen, cyano, -R a1 -R c or -R a1 -N(R c1 )-Y,R a1 Each of these is independently selected from non-existent, -CH2-, -C(O)-, or -CH2-C(O)-;

[0123] The ring B structure is j is an integer from 1 to 4, where R b Each of these radicals is independently selected from hydrogen, deuterium, halogen, cyano, -R b1 -R c or -R b1 -N(R c1 )-Y,R b1 Each of these is independently selected from non-existent, -CH2-, -C(O)-, or -CH2-C(O)-;

[0124] The R mentioned 1 Selected from hydrogen, C1-C3 alkyl groups, C1-C3 alkyl groups substituted with 3-6-membered nitrogen-containing heterocyclic alkyl groups, -CH2-C(R) 11 )2-R c -CH2-C(R) 11 )2-N(R c1 -Y, -R 12 -R c or -R 12 -N(R c1 )-Y, where R 11 Each of them is independently selected from H or methyl, R 12 Each of them is independently selected from one of C1-C3 alkyl, phenyl, 5-6 heteroaryl, a combination of C1-C3 alkyl and phenyl, or a combination of C1-C3 alkyl and 5-6 heteroaryl;

[0125] Each of the Y terms is independently selected from -C(O)-C(R) y1 )2-(CH2) 0-1 -OH, -C(O)-R y2 -(CH2) 0-2 -OH、 in,

[0126] The R mentioned y1 Selected from item i) or item ii): i) One of R y1Selected from C1-C3 alkyl groups and 3-6 cycloalkyl groups, wherein the alkyl or cycloalkyl group may optionally be substituted with one or more halogens, and the other R y1 For hydrogen, ii). Two R y1 It forms a 3- to 6-membered cycloalkyl group with the attached carbon.

[0127] The R mentioned y2 Selected from 3- to 6-membered cycloalkyl, 3- to 6-membered cycloalkyl, phenyl, C1- to C3 alkynyl, n4 = 1 to 8,

[0128] k is selected from 1 or 2, and the R... x Selected from item iii) or item iv): iii). Each R x Independently selected from hydrogen, deuterium, halogen, carbonyl, -R x1 -R c or -R x1 -N(R c1 )-Y,R x1 Each of the following is independently selected from non-existent, -CH2-, -C(O)-, or -CH2-C(O)-; iv). Two R x It forms 3- to 6-membered cycloalkyl or heterocycloalkyl groups with the attached carbon;

[0129] The R mentioned c Selected from -OH or -NHR c1 R c1 Each of them is independently selected from hydrogen or methyl.

[0130] The rings A, B, and R mentioned above 1 The compound represented by AC-P contains at least one -OH, -NH2, or -NH-, and the compound binds to the linker through the reaction of the contained -OH, -NH2, or -NH-.

[0131] The loading of the above-mentioned structure was observed to be at the nM level (10) in cytotoxicity experiments. -9 M) to pM level (10 -12 The activity of M) and the presence of ClpP agonist activity (see the results of the cell viability experiment of the load in the biological evaluation section below).

[0132] The AC-P structure can be selected from:

[0133] The AC-P structure can be selected from:

[0134] in,

[0135] R aEach of these is independently selected from hydrogen, halogen, cyano, -R a1 -R c or -R a1 -N(R c1 )-Y,R a1 Each of these is independently selected from non-existent, -CH2-, -C(O)-, or -CH2-C(O)-;

[0136] R b Each of these is independently selected from hydrogen, halogen, cyano, -R b1 -R c or -R b1 -N(R c1 )-Y,R b1 Each of these is independently selected from non-existent, -CH2-, -C(O)-, or -CH2-C(O)-;

[0137] The R mentioned 1 Selected from -CH2-C(R) 11 )2-R c or -CH2-C(R) 11 )2-N(R c1 )-Y, where R 11 Each of them is independently selected from H or methyl;

[0138] Each of the Y terms is independently selected from -C(O)-CH(R) y1 )-OH,R y1 Selected from hydrogen or methyl.

[0139] Preferably, the AC-P structure of the present invention is selected from the following general formula:

[0140] in,

[0141] R am Selected from -F or -CN, R an Selected from -H or -F,

[0142] R bn Selected from F or Cl, R bm Selected from -H, -CH2-OH, -CH2-NH2, -CH2-NH-C(O)-CH2-OH, -CH2-C(O)-NH2.

[0143] R1 is selected from

[0144] Synthetic routes of ClpP agonists

[0145] The ClpP agonist molecular structure shown by formula AC-P can be synthesized by at least one of the following synthetic routes.

[0146] Synthetic route 1 of ClpP agonists

[0147] Step 1: The compound (AC-P-1M1) is obtained by substitution reaction of ethyl 4-oxopyridine-3-carboxylate (AC-P-S01) and benzyl bromide and its different substituted derivatives (AC-P-S02) (see, for example, CN109320515).

[0148] Step 2: Compound (AC-P-1M1) was condensed with different substituted or unsubstituted benzylhydrazine compounds (AC-P-SO3) to give the fused-ring compound (AC-P-1M2) (see EP3170823 and WO2020 / 239861).

[0149] Benzylhydrazine (AC-P-SO3) can be obtained by the substitution reaction of substituted or unsubstituted benzyl bromide with hydrazine:

[0150] Step 3: Obtain (AC-P-1M3) by substitution reaction of compound (AC-P-M2) with different active functionalized intermediates (AC-P-SO4), or by CN-bond coupling conditional reaction mediated by metal catalysts (such as copper or palladium and their various salts).

[0151] In AC-P-S02 and AC-P-S03 used in the upper-level route, R a Each of the individual elements can be selected from hydrogen, halogen, or cyano; R b Each of these radicals is independently selected from hydrogen, halogen, or cyano. Preferably, one or two R radicals may be attached to the phenyl group of AC-P-SO2. a To prepare a sample containing 1-2 R... a The ring A structure includes:

[0152] For example, ring A can be the structure shown in the table below:

[0153] In the AC-P-S03 used in the upper route, R b Each of these radicals is independently selected from hydrogen, halogen, or cyano. Preferably, one or two R radicals may be attached to the phenyl group of AC-P-SO3. b To prepare a sample containing 1-2 R... b The ring B structure. The ring B structure includes:

[0154] For example, ring B can be the structure shown in the table below:

[0155] Furthermore, the ClpP agonists with cyclic A and cyclic B structures prepared above can further convert Ra and Rb into -R through a reaction. a1 -R c Substituents in the form of -CH2-NH2, -OH, or -CH2-OH.

[0156] When the prepared ClpP agonist R a R b When one of the groups is a cyano group, it can be further converted to -CH2-NH2 through a reaction. The preparation method can be found in Example 15.

[0157] For example, the resulting ring A can be the structure shown in the table below:

[0158] For example, the resulting ring B can be the structure shown in the table below:

[0159] When the prepared ClpP agonist R a R b Contains -NH(R) c1 When ), it can be further converted into -N(R) through a reaction. c1 The preparation method of -Y can be found in Example 16. This yields a product in the form -R. b1 -N(R c1 )-Y of R a R b Substituents.

[0160] For example, the resulting ring B can be the structure shown in the table below:

[0161] When the prepared ClpP agonist R a R b When it contains halogen, preferably -Br, it can be further substituted to -CH2OH through reaction. The preparation method can be found in Example 14.

[0162] For example, the resulting ring B can be the structure shown in the table below:

[0163] For example, the ring B structure can be the structure shown in the table below:

[0164] This synthetic route can be used to prepare R in the aforementioned AC-P structure. 1 Alkyl groups selected from C1 to C3, alkyl groups substituted with 3 to 6-membered nitrogen-containing heterocyclic alkyl groups from C1 to C3, and -R 12 -R c or -R12 -N(R c1 The structure of )-Y.

[0165] For example, R 1 It can be structured as shown in the table below:

[0166] For example, R X It can be: -H, -D, -F, -CH3, k = 1 or 2.

[0167] Synthetic route 2 of ClpP agonists

[0168] Step 1: (AC-P-2M1) is obtained by substitution reaction of compound (AC-P-1M2) with different epoxides (AC-P-SO5) under heating or alkaline conditions (see, for example, [Synlett, 2008, #7, pp. 1005-1008]).

[0169] Step 2: The OH compound (AC-P-2M1) is converted into an active amine intermediate (AC-P-2M2) by photo-extending reaction (Mitsunobu reaction). The preferred reaction conditions are PPh3 and DIAD.

[0170] Step 3: Hydrolyze compound (AC-P-2M2) to remove the protecting group, yielding amino compound (AC-P-2M3). The preferred reaction conditions are hydrazine hydrate.

[0171] Step 4: Condensation reaction. The amino compound (AC-P-2M3) undergoes a condensation reaction with different carboxylic acid compounds (AC-P-SO7) to obtain the corresponding amide compound (AC-P-2M4). The preferred reaction conditions are HATU and DIPEA.

[0172] In the above reaction route, compound (AC-P-1M2) can be prepared by referring to the method of synthetic route 1. Ring A and its R... a Ring B and its R b R x All groups can be referenced from the groups listed in synthetic route 1. R can be synthesized using synthetic route 2. 1 -CH2-C(R) 11 )2-R c -CH2-C(R) 11 )2-N(R c1 ClpP agonists in the form of )-Y structure.

[0173] First, R can be prepared from step 1 of synthetic route 2. 1 -CH2-C(R) 11 ClpP agonist of 2-OH.

[0174] For example, R 1 It can be structured as shown in the table below:

[0175] R can be prepared by continuing steps 2 and 3. 1 -CH2-C(R) 11 ClpP agonist of )2-NH2.

[0176] For example, R 1 It can be structured as shown in the table below:

[0177] Furthermore, when R in AC-P-2M1 1 -CH2-C(R) 11 When 2-OH is present, it can be converted to R through a reaction. 1 -CH2-C(R) 11 )2-NH-CH3, the synthesis of which can be found in Example 3.

[0178] For example, R 1 It can be structured as shown in the table below:

[0179] From step 4 to prepare R 1 -CH2-C(R) 11 )2-N(R c1 ClpP agonists of )-Y.

[0180] For example, the carboxylic acid compound HO-Y (AC-P-SO7) used in step 4 can have the structure shown in the table below:

[0181] For example, the obtained R 1 It can be structured as shown in the table below:

[0182] Synthetic route 3 for ClpP agonists

[0183] Step 1: The cyclohexane compound (AC-P-3M1) was obtained by condensation of ethyl 1-N-Boc-4-oxo-3-piperidinecarboxylate (AC-P-S08) with different substituted or unsubstituted benzylhydrazine compounds (see EP3170823 and WO2020 / 239861).

[0184] Step 2: The hydroxyl compound (AC-P-3M2) is obtained by reacting the compound (AC-P-3M1) with different epoxides (AC-P-SO5) under heating or alkaline conditions (e.g., see EP3170823).

[0185] Step 3: Hydrolyze compound (AC-P-3M2) to remove the Boc protecting group to obtain amino compound (AC-P-3M3). The preferred reaction conditions are TFA.

[0186] Step 4: (AC-P-3M3) undergoes a substitution reaction with different substituted or unsubstituted benzyl bromide (AC-P-S09) to give compound (AC-P-3M4) (see, for example, CN109320515).

[0187] Among the ClpP agonists obtained directly from synthetic route 3, or those that can be further prepared through other chemical reactions after synthetic route 3, ring A and its R... a Ring B and its R b R x All can refer to the groups listed in synthetic route 1.

[0188] The compound (AC-P-3M4) obtained from the reaction of synthetic route 3 also yields a compound with the same structure as AC-P-2M1 in step 1 of synthetic route 2. Furthermore, by continuing with steps 2 to 4 of synthetic route 2, amino compounds (AC-P-2M3) and amide compounds (AC-P-2M4) can be prepared.

[0189] Synthetic route 4 of ClpP agonists

[0190] Step 1: (AC-P-4M2) is obtained by substitution reaction of compound (AC-P-4M1) with different active functionalized intermediates (AC-P-SO4), or by CN-bond coupling condition mediated by metal catalysts (such as copper or palladium and their various salts).

[0191] Step 2: Hydrolyze compound (AC-P-4M2) to remove the Boc protecting group, yielding the amino compound (AC-P-4M3). The preferred reaction conditions are TFA.

[0192] Step 3: (AC-P-4M3) undergoes a substitution reaction with different substituted or unsubstituted benzyl bromide (AC-P-S09) to give compound (AC-P-4M4) (see, for example, CN109320515).

[0193] Among the ClpP agonists obtained directly from synthetic route 4, or those that can be further prepared through other chemical reactions after synthetic route 4, ring A and its R... a Ring B and its R b R X R 1 All can refer to the groups listed in synthetic route 1.

[0194] Based on at least one of the above synthetic routes 1 to 4, compounds with structures including but not limited to the following can be prepared:

[0195] Antibody-ClpP agonist conjugates

[0196] connector

[0197] All the connectors in this invention are prepared by known methods.

[0198] The linker has the general formula L before reacting with the antibody and ClpP agonist: 1 -L 2 -L 3 -L 4 -L 5 ,in,

[0199] L 1 These are various linker units capable of conjugating with antibodies. They enable the linker-load to conjugate with the antibody via lysine or cysteine ​​residues in the antibody. The conjugation methods are known and can be found in the section "Preparation of Antibody-ClpP Agonist Conjugates" below. Specifically, the structure of the linker unit before conjugation with the antibody includes, but is not limited to:

[0200] After conjugation with an antibody, L 1 It then has a structure including, but not limited to, the following:

[0201] L 1 In the formula, # is used to indicate the site of antibody binding, and the wavy line is used to indicate the site of binding with L. 2 The site of connection.

[0202] L 2 These are various extension units, including but not limited to:

[0203] L 2 In the diagram, n1 = 0-5, n2 = 0-10, n3 = 0-3, and the # symbol indicates a relationship with L. 1 The connection site, based on L 3 L 4 Whether it exists or not, the wavy line represents the adjacent L. 3 L 4 L 5 Or the binding site of the ClpP agonist of this invention.

[0204] L 3 It may be absent or consist of various amino acid units. When present, an amino acid unit may include multiple amino acid subunits, including but not limited to... In L3, the # symbol indicates a relationship with L. 2 The connection site, based on L 4 Whether it exists or not, the wavy line represents the adjacent L. 4 L 5 Or the binding site of the ClpP agonist of this invention.

[0205] L 4 The spacer unit may be absent or may be of various types. When present, the spacer unit allows the coupling compound to release its complete active payload within the cell. In this invention, the payload is a ClpP agonist, L... 4 Including but not limited to:

[0206] L 4 In the middle, R L Independently selected from hydrogen, C1-C3 straight-chain or branched alkyl, C1-C3 straight-chain or branched alkyl hydroxyl, C1-C3 straight-chain or branched alkylamine, based on L 3 Whether it exists or not, the # symbol indicates that it is adjacent to the L. 2 L 3 The connection point, indicated by the wavy line, is related to L. 5 Or the binding site of the ClpP agonist of this invention.

[0207] L 5 This is because it is able to react with the reactive functional group R in ClpP. c Reactive functional groups that can chemically bond, including those capable of reacting with the reactive functional group -NHR. C1 Various reactive functional groups that are chemically bonded, such as those that form amide bonds through addition, elimination, or substitution reactions; those that form amide bonds through condensation reactions; or those that are chemically bonded to the reactive functional group -OH, such as those that form CO bonds through substitution reactions. Further, refer to the functional groups shown in synthetic routes 1-3 below.

[0208] Based on knowledge in this field, various groups that can be present in the linker to improve the performance of antibody-drug conjugates may also exist.

[0209] Synthesis of linker-ClpP agonists

[0210] The linker-ClpP agonist compounds of the present invention can be synthesized via the following linker-loading synthetic routes. In the illustrated reaction routes, using... The present invention refers to the invention containing the reactive functional group -NHR C1 ClpP agonists. D-OH refers to the ClpP agonists of this invention containing the reactive functional group -OH. The reactive functional group can react with linkers to form linker-loads via the following reaction pathway.

[0211] Connector-Load General Synthesis Route 1:

[0212] For those containing the reactive functional group -NHR C1 ClpP agonists, one way to connect the load and linker is through addition elimination or substitution reactions to generate amide bonds.

[0213] For example:

[0214] Connector-Load General Synthesis Route 2:

[0215] Contains the reactive functional group -NHR C1 Another way to connect the load and linker is through a ClpP agonist via a condensation reaction to generate amide bonds.

[0216] For example:

[0217] Connector-Load General Synthesis Route 3:

[0218] ClpP agonists containing the reactive functional group -OH can form linker-loads via the following synthetic route.

[0219] Using the above synthetic route, linker-ClpP agonist structures, including but not limited to those listed in the table below, can be prepared:

[0220] Antibody

[0221] The antibody is selected at the discretion of those skilled in the art. It is typically chosen based on the selected tumor target / antigen, selecting antibodies capable of binding to it. The selected antibody can be a full-length antibody or an antibody fragment with antigen-binding function. Its immunoglobulin molecule types include, but are not limited to, IgG, IgE, IgM, IgD, and IgA, and categories include, but are not limited to, IgG1, IgG2, IgG3, IgG4, IgG1A, and IgA2. The antibody is not limited to selection from: anti-EGFR antibody, anti-DLL-3 antibody, anti-PSMA antibody, anti-CD70 antibody, anti-MUC16 antibody, anti-ENPP3 antibody, anti-TDGF1 antibody, anti-CCK-BR antibody, anti-MSLN antibody, anti-TIM-1 antibody, anti-LRRC15 antibody, anti-LIV-I antibody, anti-CanAg / AFP antibody, and anti-claudin. 18.2 Antibodies, anti-Mesothelin antibody, anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-c-MET antibody, anti-SLITRK6 antibody, anti-KIT / CD117 antibody, anti-STEAP1 antibody, anti-SLAMF7 / CS1 antibody, anti-NaPi2B / SLC34A2 antibody, anti-GPNMB antibody, anti-HER3 (ErbB3) antibody, anti-MUC1 / CD227 antibody, anti-AXL antibody, anti-CD166 antibody, anti-B7-H3 (CD276) antibody, anti-PTK7 / CCK4 antibody, anti-PRL R antibody, anti-EFNA4 antibody, anti-5T4 antibody, anti-NOTCH3 antibody, anti-Nectin4 antibody, anti-TROP-2 antibody, anti-CD142 antibody, anti-CA6 antibody, anti-GPR20 antibody, anti-CD174 antibody, anti-CD71 antibody, anti-EphA2 antibody, anti-LYPD3 antibody, anti-FGFR2 antibody, anti-FGFR3 antibody, anti-FRα antibody, anti-CEACAMs antibody, anti-GCC antibody, anti-IntegrinAv antibody, anti-CAIX antibody, anti-P-cadherin antibody, anti-GD3 antibody, anti-Cadherin 6. Antibodies, including anti-LAMPI antibody, anti-FLT3 antibody, anti-BCMA antibody, anti-CD79b antibody, anti-CD19 antibody, anti-CD33 antibody, anti-CD56 antibody, anti-CD74 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD37 antibody, anti-CD47 antibody, anti-CD138 antibody, anti-CD352 antibody, anti-CD25 antibody, anti-CD147 antibody, or anti-CD123 antibody.

[0222] Synthesis of antibody-ClpP agonist conjugates

[0223] Methods for conjugating antibodies to linker-loading are known, and typically involve targeting the side chains of lysine or cysteine ​​residues in the antibody. For lysine conjugation, the linker usually contains an N-hydroxysuccinimide, which reacts with the ε-amino group of the lysine to form a stable amide bond, thereby achieving conjugation. For cysteine ​​conjugation, the linker usually contains an N-maleimide, which reacts with the thiol group of the cysteine ​​to form a stable carbon-sulfur bond, thereby achieving conjugation.

[0224] The prepared antibody-loaded conjugates include, but are not limited to, the structures shown in the table below. It should be noted that the -s- between the linker L and the antibody Ab in the structures shown comes from the thiol group contained in the cysteine ​​itself during the aforementioned cysteine ​​conjugation, and is not a connection achieved by adding an additional -S- between L and Ab.

[0225] Thus, using the ClpP agonist with the AC-P structure provided by this invention, an antibody-ClpP agonist conjugate has been successfully prepared. Furthermore, the obtained antibody-load conjugate can produce compounds with tumor therapeutic effects (refer to the PD tumor experimental results in the biological evaluation).

[0226] Example

[0227] The present invention will be specifically illustrated below through examples. The descriptions in the embodiments are merely exemplary and not restrictive, and some embodiments can be found in PCT / CN2024 / 104414 and its references.

[0228] The structures of the compounds described in the examples were determined by nuclear magnetic resonance (NMR). 1 To determine this, use either nuclear magnetic resonance (NMR) or mass spectrometry (MS). 1 The ¹H NMR (high-density spectroscopy) measurements were performed using a Bruker 400MHz NMR spectrometer. The solvents used were deuterated methanol (CD₃OD), deuterated chloroform (CDCl₃), or hexadeuterated dimethyl sulfoxide (DMSO-d₆); the internal standard was tetramethylsilane (TMS). The mass spectrometry (MS) measurements were performed using a Shimaszu (ESI) LCMS-2020 mass spectrometer.

[0229] Examples of ClpP agonists

[0230] Example 1

[0231] The synthesis of 2-(4-chlorobenzyl)-5-(3,5-difluorobenzyl)-1-(2-hydroxyethyl)-1,2,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (AC-P1) is described below:

[0232] Step 1: Methyl 4-oxopiperidin-3-carboxylate (2.00 g, 10.3 mmol, hydrochloride), 1-(bromomethyl)-3,5-difluorobenzene (2.14 g, 10.3 mmol, 1.34 mL), potassium carbonate (1.43 g, 10.3 mmol), and acetonitrile (20 mL) were added to a 40 mL sample vial. After stirring at room temperature for 16 hours, the mixture was quenched with water (40 mL), extracted with ethyl acetate (50 mL × 3), and the organic phases were combined. The samples were washed with brine (150 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was column-passed (ethyl acetate / petroleum ether 0-23%) to give a colorless oily methyl 1-[(3,5-difluorophenyl)methyl]-4-oxopiperidin-3-carboxylate (2.50 g, 85% yield).

[0233] LCMS:298.1[M+H]+.

[0234] Step 2: 2-(4-chlorobenzyl)-5-(3,5-difluorobenzyl)-2,3a,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (1.00 g, 3.36 mmol), (4-chlorophenyl)methylhydrazine (526 mg, 3.36 mmol), sodium acetate (827 mg, 10.0 mmol), and ethanol (15 mL) were added to a 40 mL sample vial. After reacting at room temperature for 18 hours, the reaction was quenched with water (30 mL), extracted with ethyl acetate (40 mL × 3), the organic phases were combined, washed with brine (120 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was passed through a column (methanol / dichloromethane = 1 / 10) to give a red solid 2-(4-chlorobenzyl)-5-(3,5-difluorobenzyl)-2,3a,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (690 mg, yield 52%).

[0235] LCMS:390.2[M+H]+.

[0236] Step 3: 2-(4-chlorobenzyl)-5-(3,5-difluorobenzyl)-2,3a,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (500 mg, 11.3 mmol) and N,N-dimethylformamide (10 mL) were added to a 30 mL microwave-safe tube. Cesium carbonate (1.25 g, 3.85 mmol) was added at 0 °C, and after reacting at room temperature for 10 min, ethylene oxide (288 μL, 6.41 mmol) was added. After reacting at room temperature for 16 hours, the reaction was quenched with water (10 mL), extracted with ethyl acetate (100 mL × 3), the organic phases were combined, washed with saturated brine (300 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was column-passed (in methanol / dichloromethane (0% to 10%)) to give the crude product. The crude product was prepared under high pressure (XBridge ShieldRP 18OBD column, 19×250 mm, 5 μm; mobile phase A: water (10 mmol / L ammonium bicarbonate), mobile phase B: acetonitrile; flow rate: 25 mL / min; gradient: 39% B to 69% B over 7 minutes; wavelength: 254 nm) to give a white solid 2-(4-chlorobenzyl)-5-(3,5-difluorobenzyl)-1-(2-hydroxyethyl)-1,2,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (13.5 mg, yield 2%).

[0237] LCMS(ESI,m / z):434.2[M+H] + .

[0238] 1 H NMR(300MHz,CD3OD)δ(ppm)7.33-7.30(m,2H),7.16-7.13(m,2H),7.04-6.98(m,2H),6.88-6.81(m,1H),5.07(s, 2H),3.81-3.78(m,2H),3.73(s,2H),3.54-3.51(m,2H),3.32-3.29(m,2H),2.81-2.78(m,2H),2.72-2.70(m,2H).

[0239] 19 FNMR(282MHz,CD3OD)δ(ppm)-112.222(s,2F).

[0240] Example 2

[0241] The synthesis of 1-(2-aminoethyl)-2-(4-chlorobenzyl)-5-(3,5-difluorobenzyl)-1,2,4,5,6,7-hexahydro-3H-pyrazole[4,3-c]pyridin-3-one (AC-P2) is described below:

[0242] Step 1: 2-(4-chlorobenzyl)-5-(3,5-difluorobenzyl)-1-(2-hydroxyethyl)-1,2,4,5,6,7-hexahydro-3H-pyrazole[4,3-c]pyridin-3-one (500 mg, 1.15 mmol), phthalimide (339 mg, 2.30 mmol), and dichloromethane (10 mL) were added to a 40 mL sample vial, followed by the addition of triphenylphosphine (1.45 g, 1.73 mmol) and diisopropyl azodicarbonate (340 μL, 1.73 mmol). The resulting mixture was reacted at room temperature for 16 hours and then concentrated. The residue was passed through a column (in anhydrous methanol / dichloromethane (0%–10%)) to give a brown solid 2-(2-(-2-(4-chlorobenzyl)-5-(3,5-difluorobenzyl)-3-oxo-2,3,4,5,6,7-hexahydro-1H-pyrazol[4,3-c]pyridin-1-yl)ethyl)isoindoline-1,3-dione (360 mg, yield 54%).

[0243] LCMS(ESI,m / z):563.1[M+H] + .

[0244] Step 2: Add 2-(2-(-2-(4-chlorobenzyl)-5-(3,5-difluorobenzyl)-3-oxo-2,3,4,5,6,7-hexahydro-1H-pyrazol[4,3-c]pyridin-1-yl)ethyl)isoindoline-1,3-dione (700 mg, 1.24 mmol), hydrazine hydrate (340 μL, 1.73 mmol), and ethanol (20 mL) to a 40 mL sample vial and react at 50 °C for 8 hours. Cool to room temperature, filter, and concentrate the filtrate. The residue was passed through a column (in methanol / dichloromethane (0%–15%)) to give a yellow semi-solid 1-(2-aminoethyl)-2-(4-chlorobenzyl)-5-(3,5-difluorobenzyl)-1,2,4,5,6,7-hexahydro-3H-pyrazole[4,3-c]pyridin-3-one (303.3 mg, 55% yield).

[0245] LCMS(ESI,m / z):433.0[M+H] + .

[0246] 1 H NMR (300MHz, CD3OD) δ (ppm) 7.33-7.30 (m, 2H), 7.17-7.14 (m, 2H), 7.03-6.99 (m, 2H), 6.90-6. 82(m,1H),5.06(s,2H),3.74-3.70(m,4H),3.28(s,2H),2.82-2.79(m,2H),2.69-2.61(m,4H).

[0247] 19 FNMR(282MHz,CD3OD)δ(ppm)-112.125(s,2F).

[0248] Example 3

[0249] The synthesis of 2-(4-chlorobenzyl)-5-(3,5-difluorobenzyl)-1-(2-(methylamino)ethyl)-1,2,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (AC-P3) was carried out via the following route:

[0250] Step 1: Add 2-[(4-chlorophenyl)methyl]-5-[(3,5-difluorophenyl)methyl]-1-(2-hydroxyethyl)-6,7-dihydro-4H-pyrazolo[4,3-c]pyridin-3-one (70.0 mg, 161 μmol), dichloromethane (2 mL), and triethylamine (67.0 μL, 0.48 mmol) to an 8 mL reaction flask. Add methanesulfonyl chloride (613 μL, 194 μmol) at 0 °C. After reacting at room temperature for 2 hours, saturated sodium bicarbonate solution (2 mL) was added to quench the reaction, followed by extraction with dichloromethane (20 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a yellow oily substance, ethyl 2-(2-(4-chlorobenzyl)-5-(3,5-difluorobenzyl)-3-oxo-2,3,4,5,6,7-hexahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)methanesulfonate (80 mg, crude product).

[0251] LCMS: 512.1 [M+H] + .

[0252] Step 2: Add ethyl 2-(2-(4-chlorobenzyl)-5-(3,5-difluorobenzyl)-3-oxo-2,3,4,5,6,7-hexahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)methanesulfonate (80.0 mg, 156 μmol), methylamine (30% ethanol solution, 180 μL, 1.56 mmol), and anhydrous ethanol (5 mL) to an 8 mL sample vial. After reacting at 80 °C for 2 hours, cool to room temperature and concentrate. The residue was subjected to high-pressure preparation (XSelect CSH C18 column, 19×150 mm, 5 μm; mobile phase A: water (0.1% formic acid), mobile phase B: acetonitrile; flow rate: 25 mL / min; gradient: 30% B to 60% B over 7 minutes; wavelength: 254 nm / 220 nm) to give a white semi-solid 2-(4-chlorobenzyl)-5-(3,5-difluorobenzyl)-1-(2-(methylamino)ethyl)-1,2,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridine-3-one carbamate (12.2 mg, 15% yield, formate).

[0253] LCMS 447.2 [M+H] + .

[0254] 1 H NMR (300MHz, CD3OD) δ (ppm) 7.34-7.31 (m, 2H), 7.18-7.15 (m, 2H), 7.05-6.98 (m, 2H), 6.90-6.82 (m, 1H), 5. 06(s,2H),3.87-3.82(m,2H),3.74(s,2H),3.28(s,2H),2.83-2.79(m,2H),2.72-2.64(m,4H),2.42(s,3H).

[0255] 19 FNMR(282MHz,CD3OD)δ(ppm)-112.081(s,2F).

[0256] Example 4

[0257] The synthesis of N-(2-(2-(4-chlorobenzyl)-5-(3,5-difluorobenzyl)-3-oxo-2,3,4,5,6,7-hexahydro-1H-pyrazol[4,3-c]pyridin-1-yl)ethyl)-2-hydroxyacetamide (AC-P4) is described below:

[0258] 1-(2-aminoethyl)-2-(4-chlorobenzyl)-5-(3,5-difluorobenzyl)-1,2,4,5,6,7-hexahydro-3H-pyrazol[4,3-c]pyridin-3-one (15.0 mg, 34.6 μmol), 2-hydroxyacetic acid (12.0 mg, 156 μmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (40.0 mg, 104 μmol), N,N-diisopropylethylamine (18.0 μL, 104 μmol), and N,N-dimethylformamide (0.6 mL) were added to an 8 mL sample vial, and the reaction was carried out at room temperature for 18 hours. The reaction was quenched with water (2 mL), extracted with ethyl acetate (5 mL × 3), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was subjected to high-pressure preparation (YMC-Actus Triart C18 ExRS column, 19×250 mm, 5 μm; mobile phase A: water (10 mmol / L sodium bicarbonate), mobile phase B: acetonitrile; flow rate: 25 mL / min; gradient: 34% B to 64% B over 7 minutes; wavelength: 254 nm) to give a white solid N-(2-(2-(4-chlorobenzyl)-5-(3,5-difluorobenzyl)-3-oxo-2,3,4,5,6,7-hexahydro-1H-pyrazol[4,3-c]pyridin-1-yl)ethyl)-2-hydroxyacetamide (6.5 mg, yield 38%).

[0259] LCMS(ESI,m / z):491.2[M+H] + .

[0260] 1 H NMR(400MHz,CD3OD)δ(ppm)7.32-7.29(m,2H),7.17-7.15(m,2H),7.05-7.00(m,2H),6.88-6.82(m,1H),5.09(s,2 H),4.85(s,2H),3.94(s,2H),3.82-3.78(m,2H),3.74(s,2H),3.27(s,2H),2.81-2.78(m,2H),2.66-2.63(m,2H).

[0261] 19 FNMR(376MHz,CD3OD)δ(ppm)-112.178(s,2F).

[0262] Example 5

[0263] The synthesis of 1-(2-aminoethyl)-2-(4-chloro-2-(hydroxymethyl)benzyl)-5-(3,5-difluorobenzyl)-1,2,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (AC-P5) was performed via the following synthetic route:

[0264] Step 1: 2-(2-bromo-4-chlorobenzyl)-5-(3,5-difluorobenzyl)-1-(2-hydroxyethyl)-1,2,4-,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (370 mg, 721 μmol), phthalimide (160 mg, 1.09 mmol), triphenylphosphine (380 mg, 1.45 mmol), and tetrahydrofuran (6 mL) were added to a 50 mL round-bottom flask, followed by the addition of diisopropyl azodicarbonate (300 μL, 1.52 mmol). The resulting mixture was reacted at room temperature for 16 hours and then concentrated. The residue was column-passed (tetrahydrofuran / petroleum ether (50% to 100%)) to give a crude product (containing triphenylphosphine oxide). The crude product was further subjected to reversed-phase chromatography (C18 reversed-phase column, 20-35 μm, size: 90 g; mobile phase A: water (0.05% ammonium bicarbonate) / mobile phase B: acetonitrile; flow rate: 60 mL / min; gradient: 0% B to 66% B over 33 min; detector: 254 nm) to give a pale yellow solid 2-(2-(2-bromo-4-chlorophenyl)-5-(3,5-difluorobenzyl)-3-oxo-2,3,4,5,6,7-hexahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)ethyl)isoindoline-1,3-dione (300 mg, yield 61%).

[0265] LCMS(ESI,m / z):641.1,643.1[M+H,M+H+2] + .

[0266] Step 2: 2-(2-(2-bromo-4-chlorophenyl)-5-(3,5-difluorobenzyl)-3-oxo-2,3,4,5,6,7-hexahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)ethyl)isoindoline-1,3-dione (240 mg, 373 μmmol), tetrakis(triphenylphosphine)palladium (50 mg, 43 μmol), (tributyltin)methanol (266 mg, 828 μmol), and 1,4-dioxane (6 mL) were added to a 30 mL microwave-safe tube. The tube was rinsed with N2 and sealed. The resulting mixture was stirred at 100 °C for 16 hours under a nitrogen atmosphere. After cooling to room temperature, the mixture was quenched with saturated potassium fluoride aqueous solution (20 mL), extracted with ethyl acetate (100 mL × 3), the organic layers were combined, washed with brine (30 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and evaporated to dryness. The residue was subjected to reversed-phase chromatography (column, C18 spherical, 20-35 μm, size: 40 g; mobile phase A: water (0.05% ammonium bicarbonate) / mobile phase B: acetonitrile; flow rate: 45 mL / min; gradient: 0% B to 40% B over 28 minutes; detector: UV 254 nm) to give a pale yellow oily 2-(2-(4-chloro-2-(hydroxymethyl)benzyl)-5-(3,5-difluorobenzyl)-3-oxo-2,3,4,5,6,7-hexahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)ethyl)isoindoline-1,3-dione (150 mg, yield 29%).

[0267] LCMS(ESI,m / z):593.2[M+H] + .

[0268] Step 3: Add 2-(2-(4-chloro-2-(hydroxymethyl)benzyl)-5-(3,5-difluorobenzyl)-3-oxo-2,3,4,5,6,7-hexahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)ethyl)isoindoline-1,3-dione (60.0 mg, 101 μmol), hydrazine hydrate (85% aqueous solution, 70.0 mg, 1.19 mmol), and ethanol (6 mL) to a 50 mL round-bottom flask. After stirring at room temperature for 60 hours, quench with 1 M sodium hydroxide aqueous solution, extract with dichloromethane (50 mL × 3), combine the organic layers, dry with anhydrous sodium sulfate, filter, concentrate, and evaporate to dryness. The residue was subjected to high-pressure preparation (XBridge prep C18 OBD column, 19×250 mm, 5 μm; mobile phase A: water (10 mmol / L ammonium bicarbonate), mobile phase B: methanol; flow rate: 25 mL / min; gradient: 10 min 50% B to 75% B; wavelength: 254 nm; RT1 (min): 9.57) to give a white solid 1-(2-aminoethyl)-2-(4-chloro-2-(hydroxymethyl)benzyl)-5-(3,5-difluorobenzyl)-1,2,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (6.4 mg, yield 13%).

[0269] LCMS(ESI,m / z):463.2[M+H] + .

[0270] 1 H NMR (400MHz, Methanol-d4) δ7.43(d,J=2.0Hz,1H),7.22(dd,J=8.0,2.0Hz,1H),7.09-6.98(m,2H),6.92-6.81(m,1H),6.69(d,J=8.4 Hz,1H),5.19(s,2H),4.87-4.80(m,2H),4.69(s,2H),3.79-3.67(m,4H),2.88-2.76(m,2H),2.76-2.66(m,2H),2.61(t,J=6.8Hz,2H).

[0271] 19 F NMR (376MHz, Methanol-d4) δ-112.146 (s, 2F).

[0272] Example 6

[0273] The synthesis of N-(2-(2-(4-chloro-2-(hydroxymethyl)benzyl)-5-(3,5-difluorobenzyl)-3-oxo-2,3,4,5,6,7-hexahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)ethyl)-2-hydroxyacetamide (AC-P6) is described below:

[0274] 1-(2-aminoethyl)-2-(4-chloro-2-(hydroxymethyl)benzyl)-5-(3,5-difluorobenzyl)-1,2,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (85.0 mg, 62.4 μmol), 2-hydroxyacetic acid (105 mg, 1.38 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (129 mg, 339 μmol), and N,N-dimethylformamide (4 mL) were added to a 25 mL round-bottom flask, followed by the addition of N,N-diisopropylethylamine (18.0 μL, 104 μmol). After reacting at room temperature for 18 hours, the reaction was quenched with water (30 mL), extracted with ethyl acetate (50 mL × 3), the organic layers were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was subjected to high-pressure preparation (YMC-Actus Triart C18 ExRS column, 19×250 mm, 5 μm; mobile phase A: water (10 mmol / L ammonium bicarbonate), mobile phase B: acetonitrile; flow rate: 25 mL / min; gradient: 28% B to 58% B over 7 minutes; wavelength: 254 nm) to give a white solid N-(2-(2-(4-chloro-2-(hydroxymethyl)benzyl)-5-(3,5-difluorobenzyl)-3-oxo-2,3,4,5,6,7-hexahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)ethyl)-2-hydroxyacetamide (3.9 mg, yield 11%).

[0275] LCMS(ESI,m / z):521.3[M+H] + .

[0276] 1 H NMR (400MHz, CD3OD) δ7.43(d,J=2.0Hz,1H),7.21(dd,J=8.4,2.4Hz,1H),7.09-6.99(m,2H),6.91-6.81(m,1H),6.71(d,J=8.4Hz,1H),5 .19(s,2H),4.88(s,2H),4.70(s,2H),3.93(s,2H),3.82-3.73(m,4H),3.30-3.23(m,2H),2.83(t,J=5.6Hz,2H),2.69(t,J=5.6Hz,2H).

[0277] 19 F NMR(376MHz,CD3OD)δ-112.151(s,2F).

[0278] Example 7

[0279] The synthesis of N-(2-(2-(4-chlorobenzyl)-5-(3,5-difluorobenzyl)-3-oxo-2,3,4,5,6,7-hexahydro-1H-pyrazol[4,3-c]pyridin-1-yl)ethyl)-2-hydroxypropionamide (AC-P7) is shown below:

[0280] (R)-N-(2-(2-(4-chlorobenzyl)-5-(3,5-difluorobenzyl)-3-oxo-2,3,4,5,6,7-hexahydro-1H-pyrazol[4,3-c]pyridin-1-yl)ethyl)-2-hydroxypropamide (5.2 mg, 10 μmol) and (S)-N-(2-(2-(4-chlorobenzyl)-5-(3,5-difluorobenzyl)-3-oxo-2,3,4,5,6,7-hexahydro-1H-pyrazol[4,3-c]pyridin-1-yl)ethyl)-2-hydroxypropamide) The mixture of hydrogen-1H-pyrazol[4,3-c]pyridin-1-yl)ethyl)-2-hydroxypropamide (5.2 mg, 10 μmol) yielded a white solid N-(2-(2-(4-chlorobenzyl)-5-(3,5-difluorobenzyl)-3-oxo-2,3,4,5,6,7-hexahydro-1H-pyrazol[4,3-c]pyridin-1-yl)ethyl)-2-hydroxypropamide (10.4 mg, 99% yield).

[0281] LCMS: 505.3 [M+H] + .

[0282] 1 H NMR (400MHz, Methanol-d4) δ7.35-7.27(m,2H),7.20-7.12(m,2H),7.08-6.98(m,2H),6.91-6.81(m,1H),5.09(s,2H),4.06(q,J= 6.8Hz,1H),3.83-3.75(m,2H),3.79-3.68(m,2H),3.30-3.14(m,4H),2.84-2.75(m,2H),2.74-2.58(m,2H),1.32(d,J=6.8Hz,3H).

[0283] 19 F NMR (376MHz, Methanol-d4) δ-112.163 (s, 2F).

[0284] Example 8

[0285] The synthesis of (R)-N-(2-(2-(4-chlorobenzyl)-5-(3,5-difluorobenzyl)-3-oxo-2,3,4,5,6,7-hexahydro-1H-pyrazol[4,3-c]pyridin-1-yl)ethyl)-2-hydroxypropionamide (AC-P8) is described below:

[0286] 1-(2-aminoethyl)-2-(4-chlorobenzyl)-5-(3,5-difluorobenzyl)-1,2,4,5,6,7-hexahydro-3H-pyrazole[4,3-c]pyridin-3-one (60.0 mg, 0.138 mmol), (R)-2-hydroxypropionic acid (27.2 mg, 0.300 mmol), and N,N-dimethylformamide (1 mL) were added to an 8 mL sample vial, followed by the addition of N,N-diisopropylethylamine (0.100 mL, 0.574 mmol). After stirring at room temperature for 16 hours, the mixture was quenched with water (30 mL), extracted with ethyl acetate (50 mL × 3), and the organic phases were combined. The samples were washed with brine (150 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by TLC preparative plate (elution: methanol / dichloromethane (1 / 10)). Further preparation under high pressure (YMC-Actus Triart C18 column, 30×150mm, 5μm; mobile phase A: water (10mmol / L ammonium bicarbonate + 0.05% ammonia), mobile phase B: acetonitrile; flow rate: 60mL / min; gradient: 25%B to 46%B over 10 minutes; wavelength: 254nm / 220nm) yielded a white solid (R)-N-(2-(2-(2-(4-chlorobenzyl)-5-(3,5-difluorobenzyl)-3-oxo-2,3,4,5,6,7-hexahydro-1H-pyrazol[4,3-c]pyridin-1-yl)ethyl)-2-hydroxypropamide (20mg, yield 28%).

[0287] LCMS: 505.3 [M+H] + .

[0288] 1 H NMR (400MHz, Methanol-d4) δ7.35-7.27(m,2H),7.20-7.12(m,2H),7.08-6.98(m,2H),6.91-6.81(m,1H),5.09(s,2H),4.06(q,J= 6.8Hz,1H),3.83-3.75(m,2H),3.79-3.68(m,2H),3.30-3.14(m,4H),2.84-2.75(m,2H),2.74-2.58(m,2H),1.32(d,J=6.8Hz,3H).

[0289] 19F NMR (376MHz, Methanol-d4) δ-112.16 (s, 2F).

[0290] Example 9

[0291] The synthesis of (S)-N-(2-(2-(4-chlorobenzyl)-5-(3,5-difluorobenzyl)-3-oxo-2,3,4,5,6,7-hexahydro-1H-pyrazol[4,3-c]pyridin-1-yl)ethyl)-2-hydroxypropionamide (AC-P9) is described below:

[0292] 1-(2-aminoethyl)-2-(4-chlorobenzyl)-5-(3,5-difluorobenzyl)-1,2,4,5,6,7-hexahydro-3H-pyrazole[4,3-c]pyridin-3-one (60.0 mg, 0.138 mmol), (S)-2-hydroxypropionic acid (23 mg, 0.25 mmol), and N,N-dimethylformamide (1 mL) were added to an 8 mL sample vial, followed by the addition of N,N-diisopropylethylamine (0.100 mL, 0.574 mmol). After stirring at room temperature for 16 hours, the mixture was quenched with water (30 mL), extracted with ethyl acetate (50 mL × 3), and the organic phases were combined. The samples were washed with brine (150 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by TLC preparative plate (elution: methanol / dichloromethane (1 / 10)). Further preparation was carried out under high pressure (XBridge Prep OBD C18 column, 30×150mm, 5μm; mobile phase A: water (10mmol / L ammonium bicarbonate + 0.05% ammonia), mobile phase B: acetonitrile; flow rate: 60mL / min; gradient: 25%B to 45%B over 10 minutes; wavelength: 254nm / 220nm) to obtain a white solid (R)-N-(2-(2-(2-(4-chlorobenzyl)-5-(3,5-difluorobenzyl)-3-oxo-2,3,4,5,6,7-hexahydro-1H-pyrazol[4,3-c]pyridin-1-yl)ethyl)-2-hydroxypropamide (16mg, yield 22%).

[0293] LCMS: 505.3 [M+H] + .

[0294] 1H NMR (400MHz, Methanol-d4) δ7.33-7.25(m,2H),7.18-7.10(m,2H),7.06-6.96(m,2H),6.89-6.79(m,1H),5.07(s,2H),4.06(q,J= 6.8Hz,1H),3.83-3.75(m,2H),3.79-3.68(m,2H),3.30-3.14(m,4H),2.84-2.75(m,2H),2.74-2.58(m,2H),1.32(d,J=6.8Hz,3H).

[0295] 19 F NMR (376MHz, Methanol-d4) δ-112.164 (s, 2F).

[0296] Example 10

[0297] The synthesis of N-(2-(5-(3,5-difluorobenzyl)-2-(4-fluorobenzyl)-3-oxo-2,3,4,5,6,7-hexahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)ethyl)-2-hydroxyacetamide (AC-P10) is described below:

[0298] Step 1: Ethyl 1-(3,5-difluorobenzyl)-4-oxopiperidin-3-carboxylate (6.0 g, 20 mmol), a stir bar, (4-fluorobenzyl)hydrazine (4.6 g, 26 mmol, hydrochloride), sodium acetate (4.96 g, 60.4 mmol), and ethanol (100 mL) were added to a 500 mL round-bottom flask. The reaction mixture was stirred at -10 °C for 8 hours, quenched with ice water (50 mL), and concentrated under vacuum to remove ethanol. The mixture was extracted with ethyl acetate (100 mL × 3), and the combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was passed through a column (0-75% ethyl acetate / petroleum ether) to give a colorless oily 5-(3,5-difluorobenzyl)-2-(4-fluorobenzyl)-2,3a,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (3.34 g, 44% yield).

[0299] LCMS(ESI): 374.1 [M+H] + .

[0300] Step 2: 5-(3,5-difluorobenzyl)-2-(4-fluorobenzyl)-2,3a,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (3.34 g, 8.95 mmol), cesium carbonate (8.8 g, 27 mmol), and N,N-dimethylformamide (40 mL) were added to a 100 mL round-bottom flask, followed by the addition of ethylene oxide (2.2 mL, 44 mmol) at 0 °C. The reaction mixture was stirred at 25 °C for 36 hours, quenched with water (50 mL), extracted with ethyl acetate (80 mL × 3), the combined organic phases were washed with brine (250 mL × 2), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was passed through a column (0-100% tetrahydrofuran / petroleum ether) to give a colorless oily 5-(3,5-difluorobenzyl)-2-(4-fluorobenzyl)-1-(2-hydroxyethyl)-1,2,4-,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (800 mg, 21% yield).

[0301] LCMS(ESI): 418.3 [M+H] + .

[0302] 1 H NMR(400MHz,CD3OD)δ7.25-7.15(m,2H),7.11-6.94(m,4H),6.91-6.80(m,1H),5.07( s,2H),3.81(t,J=5.0Hz,2H),3.75(s,2H),3.53(t,J=4.9Hz,2H),2.88-2.64(m,4H).

[0303] 19 F NMR (376MHz, CD3OD) δ-112.153(s,2F),-116.582(s,1F).

[0304] Example 11

[0305] The synthesis of 1-(2-aminoethyl)-5-(3,5-difluorobenzyl)-2-(4-fluorobenzyl)-1,2,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (AC-P11) is described below:

[0306] Step 1: 5-(3,5-difluorobenzyl)-2-(4-fluorobenzyl)-1-(2-hydroxyethyl)-1,2,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (800 mg, 1.92 mmol), phthalimide (422 mg, 2.87 mmol), triphenylphosphine (1.0 g, 3.8 mmol), and dichloromethane (15 mL) were added to a 100 mL three-necked round-bottom flask, followed by the dropwise addition of diisopropyl azodicarbonate (760 μL, 3.86 mmol). The reaction mixture was stirred at 25 °C for 16 hours and then concentrated. The residue was passed through a column (0-100% tetrahydrofuran / petroleum ether) to give a yellow solid 2-(2-(5-(3,5-difluorobenzyl)-2-(4-fluorobenzyl)-3-oxo-2,3,4,5,6,7-hexahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)ethyl)isoindole-1,3-dione (800 mg, 76% yield).

[0307] LCMS(ESI): 547.1 [M+H] + .

[0308] Step 2: 2-(2-(5-(3,5-difluorobenzyl)-2-(4-fluorobenzyl)-3-oxo-2,3,4,5,6,7-hexahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)ethyl)isoindoline-1,3-dione (200 mg, 365 μmol), hydrazine hydrate (140 mg, 85% purity), and ethanol (4 mL) were added to a 20 mL sample vial. The reaction mixture was stirred at 50 °C for 8 hours. After cooling to room temperature, the mixture was quenched with 2N sodium hydroxide (20 mL), extracted with dichloromethane (150 mL × 3), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by high-pressure preparation (XBridge Prep OBD C18 column, 19×250mm, 5µm; mobile phase A: water (10mmol / L ammonium bicarbonate), mobile phase B: acetonitrile) (flow rate: 25mL / min; gradient: 30%B to 55%B over 7 minutes; wavelength: 254nm / 220nm) to give a colorless oily 1-(2-aminoethyl)-5-(3,5-difluorobenzyl)-2-(4-fluorobenzyl)-1,2,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (33.1mg, yield 21%).

[0309] LCMS(ESI):417.1[M+H] + .

[0310] 1H NMR(400MHz,CD3OD)δ7.28-7.16(m,2H),7.11-6.96(m,4H),6.92-6.79(m,1H),5.06 (s,2H),3.81-3.68(m,4H),3.29-3.26(m,2H),2.87-2.76(m,2H),2.72-2.58(m,4H).

[0311] 19 F NMR (376MHz, CD3OD) δ-112.13(s,1F),-116.45(s,2F).

[0312] Example 12

[0313] The synthesis of N-(2-(5-(3,5-difluorobenzyl)-2-(4-fluorobenzyl)-3-oxo-2,3,4,5,6,7-hexahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)ethyl)-2-hydroxyacetamide (AC-P12) is described below:

[0314] 1-(2-aminoethyl)-5-(3,5-difluorobenzyl)-2-(4-fluorobenzyl)-1,2,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (145 mg, 348 μmol), 2-hydroxyacetic acid (40.0 mg, 525 μmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (397 mg, 1.04 mmol), N,N-diisopropylethylamine (180 μL, 1.03 mmol), and N,N-dimethylformamide (5 mL) were added to a 10 mL sample vial. The reaction mixture was stirred at 25 °C for 16 hours. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (50 mL × 3), the organic phases were combined, washed with brine (150 mL × 2), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was prepared under high pressure (XSelect-CSH-Fluoro-Pheny column, 19×250mm, 5µm; mobile phase A: water (0.1% formic acid), mobile phase B: acetonitrile; flow rate: 25mL / min; gradient: 5% B to 11% B over 8 minutes; wavelength: 254nm) to give a white solid N-(2-(5-(3,5-difluorobenzyl)-2-(4-fluorobenzyl)-3-oxo-2,3,4,5,6,7-hexahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)ethyl)-2-hydroxyacetamide (25.0 mg, yield 14%).

[0315] LCMS(ESI): 475.1 [M+H] + .

[0316] 1 H NMR (400MHz, CD3OD) δ7.27-7.15(m,2H),7.09-6.97(m,4H),6.91-6.80(m,1H),5.09(s,2H),3.94( s,2H),3.84-3.78(m,2H),3.74(s,2H),3.29-3.24(m,2H),2.86-2.76(m,2H),2.2.69-2.58(m,2H).

[0317] 19 F NMR (376MHz, CD3OD) δ-112.146(s,1F),-116.490(s,2F).

[0318] Example 13

[0319] The synthesis of 2-(2-bromo-4-fluorobenzyl)-5-(3,5-difluorobenzyl)-1-(2-hydroxyethyl)-1,2,4,5,6,7-hexahydro-3H-pyrazole[4,3-c]pyridin-3-one (AC-P13) is described below:

[0320] Step 1: 2-(2-bromo-4-fluorobenzyl)-5-(3,5-difluorobenzyl)-1-(2-hydroxyethyl)-1,2,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (930 mg, 1.87 mmol), phthalimide (570 mg, 3.87 mmol), triphenylphosphine (1.00 g, 3.81 mmol), and tetrahydrofuran (15 mL) were added to a 100 mL round-bottom flask, followed by the addition of diisopropyl azodicarbonate (750 μL, 3.81 mmol). After reacting at room temperature for 16 hours, the mixture was concentrated, and the residue was column-passed (in tetrahydrofuran / petroleum ether (0% to 40%)) to give the crude product (containing triphenylphosphine oxide). The crude product was subjected to reversed-phase chromatography (C18 reversed-phase column, 20-35 μm, size: 80 g; mobile phase A: water (0.05% ammonium bicarbonate) / mobile phase B: acetonitrile; flow rate: 60 mL / min; gradient: 0% B to 50% B over 25 min; detector: 254 nm) to obtain a pale yellow solid 2-(2-(2-bromo-4-fluorobenzyl)-5-(3,5-difluorobenzyl)-3-oxo-2,3,4,5,6,7-hexahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)ethyl)isoindoline-1,3-dione (630 mg, yield 51%).

[0321] LMCS:625.1627.1[M+H,M+H+2] + .

[0322] Step 2: Add 2-(2-(2-bromo-4-fluorobenzyl)-5-(3,5-difluorobenzyl)-3-oxo-2,3,4,5,6,7-hexahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)ethyl)isoindoline-1,3-dione (300 mg, 480 μmol), tributyltin-methanol (308 mg, 959 μmol), tetrakis(triphenylphosphine)palladium (55.0 mg, 48.0 μmol), and 1,4-dioxane (5 mL) to a 20 mL sample vial. React at 80 °C for 16 hours under a nitrogen atmosphere. Cool to room temperature, quench with water (30 mL), extract with ethyl acetate (100 mL × 3), combine the organic phases, dry over anhydrous sodium sulfate, filter, and concentrate. The residue was passed through a column (in tetrahydrofuran / petroleum ether (80%–100%)) to give a brown semi-solid 2-(2-(5-(3,5-difluorobenzyl)-2-(4-fluoro-2-(hydroxymethyl)benzyl)-3-oxo-2,3,4,5,6,7-hexahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)ethyl)isoindoline-1,3-dione (80 mg, 19% yield).

[0323] LCMS 577.2 [M+H] + .

[0324] Step 3: Add 2-(2-(5-(3,5-difluorobenzyl)-2-(4-fluoro-2-(hydroxymethyl)benzyl)-3-oxo-2,3,4,5,6,7-hexahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)ethyl)isoindoline-1,3-dione (80.0 mg, 139 μmol), ethanol (5 mL), and hydrazine hydrate (22.0 μL, 694 μmol) to a 40 mL reaction flask. React at 50 °C for 8 hours, cool to room temperature, and concentrate. The residue was purified by TLC (elution: methanol / dichloromethane (1 / 9)). Further preparation under high pressure (XBridge Prep OBD C18 column, 19×250 mm, 5 μm; mobile phase A: water (10 mmol / L ammonium bicarbonate), mobile phase B: acetonitrile; flow rate: 25 mL / min; gradient: 21% B to 51% B over 7 minutes; wavelength: 254 nm / 220 nm) yielded a white solid 1-(2-aminoethyl)-5-(3,5-difluorobenzyl)-2-(4-fluoro-2-(hydroxymethyl)benzyl)-1,2,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (3.8 mg, yield 6%).

[0325] LCMS:447.1[M+H] + .

[0326] 1H NMR(400MHz,CD3OD)δ(ppm)7.34-7.31(m,1H),7.06-7.01(m,2H),6.97-6.92(m,1H),6.90-6.84(m,1H),6.76-6.73(m,1 H),5.17(m,2H),4.69(s,2H),3.76-3.70(m,4H),3.31(s,2H),2.85-2.82(m,2H),2.73-2.70(m,2H),2.68-2.61(m,2H).

[0327] 19 FNMR (376MHz, CD3OD) δ (ppm) -112.149 (s, 2F), -116.921 (s, 1F).

[0328] Example 14

[0329] The synthesis of N-(2-(5-(3,5-difluorobenzyl)-2-(4-fluoro-2-(hydroxymethyl)benzyl)-3-oxo-2,3,4,5,6,7-hexahydro-1H-pyrazol[4,3-c]pyridin-1-yl)ethyl)-2-hydroxyacetamide and 2-((2-(5-(3,5-difluorobenzyl)-2-(4-fluoro-2-(hydroxymethyl)benzyl)-3-oxo-2,3,4,5,6,7-hexahydro-1H-pyrazol[4,3-c]pyridin-1-yl)ethyl)amino)-2-oxoethyl acetate (AC-P14) is described below:

[0330] Step 1: 1-(2-aminoethyl)-2-(2-bromo-4-fluorobenzyl)-5-(3,5-difluorobenzyl)-1,2,4,5,6,7-hexahydro-3H-pyrazole[4,3-c]pyridin-3-one (510 mg, 823 μmol), 2-hydroxyacetic acid (245 mg, 2.07 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (775 mg, 2.04 mmol) and N,N-dimethylformamide (6 mL) were added to a 50 mL single-necked flask, followed by the addition of N,N-diisopropylethylamine (0.700 mL, 4.02 mmol). The reaction mixture was stirred at room temperature for 16 hours, quenched with water (30 mL), extracted with ethyl acetate (80 mL × 3), washed with brine (250 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was passed through a column (ethyl acetate, petroleum ether (0-90%)) to give a white solid ethyl acetate 2-((2-(2-(-2-bromo-4-fluorobenzyl)-5-(3,5-difluorobenzyl)-3-oxo-2,3,4,5,6,7-hexahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)ethyl)amino)-2-oxoethyl acetate (480 mg, yield 63%).

[0331] LCMS:595.1,597.1[M+H,M+H+2] + .

[0332] Step 2: Add ethyl acetate (438 mg, 735 μmol), tributyltinyl methanol (472 mg, 1.47 mmol), tetraphenylphosphine palladium (85.0 mg, 73.5 μmol), and 1,4-dioxane (10 mL) to a 40 mL sample vial. React at 100 °C for 16 hours under a nitrogen atmosphere. After cooling to room temperature, quench with saturated potassium fluoride solution (80 mL). After stirring for 1 hour, extract with ethyl acetate (100 mL × 3). Combine the organic phases, dry to anhydrous sodium sulfate, filter, and concentrate. The residue was subjected to reversed-phase chromatography (C18 reversed-phase column, 20-35 μm, size: 80 g); mobile phase A: water (0.05% ammonium bicarbonate); mobile phase B: acetonitrile; flow rate: 60 mL / min; gradient: 0% B to 50% B over 40 min; detector: 254 nm, RT1: 22 min, RT2: 23 min) to give an off-white solid N-(2-(5-(3,5-difluorobenzyl)-2-(4-fluoro-2-(hydroxymethyl)benzyl)-3-oxo-2,3,4,5,6,7-hexahydro-1H-pyrazol[4,3-c]pyridin-1-yl)ethyl)-2-hydroxyacetamide (50 mg, yield 12%).

[0333] LCMS:505.25[M+H]+.

[0334] 1 H NMR (400MHz, CD3OD) δ7.18(d,J=9.6Hz,1H),7.07-7.01(m,2H),6.98-6.88(m,1H),6.91-6.81(m,1H),6.81-6.73(m,1H),5.18(s ,2H),4.70(s,2H),3.93(s,2H),3.82-3.73(m,4H),3.32-3.28(m,2H),3.28-3.22(m,2H),2.86-2.79(m,2H),2.72-2.65(m,2H).

[0335] 19 F NMR (376MHz, CD3OD) δ-112.156(s,2F),-116.185(s,1F).

[0336] Example 15

[0337] Synthesis of 2-(2-(aminomethyl)-4-fluorobenzyl)-5-(3,5-difluorobenzyl)-1-(2-hydroxyethyl)-1,2,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (AC-P15), the synthetic route is as follows:

[0338] 2-((5-(3,5-difluorobenzyl)-1-(2-hydroxyethyl)-3-oxo-1,3,4,5,6,7-hexahydro-2H-pyrazolo[4,3-c]pyridin-2-yl)methyl)-5-fluorobenzonitrile (130 mg, 294 μmol), anhydrous methanol (5 mL), and cobalt chloride (76.0 mg, 588 μmol) were added to an 8 mL sample vial. Sodium borohydride (56.0 mg, 1.47 mmol) was added in portions at 0 °C and stirred for 30 minutes. The mixture was quenched with water (20 mL), extracted with ethyl acetate (50 mL × 3), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by TLC preparative plate (elution: methanol / dichloromethane (1 / 9, Rf = 0.4) to give 100 mg of crude product. 50 mg of the crude product was then subjected to high-pressure preparation (Column: XBridge Prep OBD C18 column, 30 × 150 mm, 5 μm; mobile phase A: water (10 mmol / L ammonium bicarbonate), mobile phase B: acetonitrile; flow rate: 60 mL / min; gradient: 17% B to 33% B over 10 min; wavelength: 254 nm / 220 nm) to give a white solid 2-(2-(aminomethyl)-4-fluorobenzyl)-5-(3,5-difluorobenzyl)-1-(2-hydroxyethyl)-1,2,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (9.2 mg, yield 18%).

[0339] LCMS:447.1[M+H] + .

[0340] 1 H NMR(300MHz,CD3OD)δ(ppm)7.20-7.16(m,2H),7.03-6.77(m,5H),5.16(s,2H),4.88(s,2H),3.91 (s,2H),3.83-3.80(m,2H),3.75(s,2H),3.53-3.50(m,2H),2.84-2.81(m,2H),2.77-2.73(m,2H).

[0341] 19 FNMR(282MHz,CD3OD)δ(ppm)-112.202(s,2F),-116.529(s,1F).

[0342] Example 16

[0343] The synthesis of N-(2-((5-(3,5-difluorobenzyl)-1-(2-hydroxyethyl)-3-oxo-1,3,4,5,6,7-hexahydro-2H-pyrazolo[4,3-c]pyridin-2-yl)methyl)-5-fluorobenzyl)-2-hydroxyacetamide (AC-P16) is described below:

[0344] 2-(2-(aminomethyl)-4-fluorobenzyl)-5-(3,5-difluorobenzyl)-1-(2-hydroxyethyl)-1,2,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (40.0 mg, 89.6 μmol), 2-hydroxyacetic acid (13.6 mg, 179 μmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (102 mg, 269 μmol), N,N-dimethylformamide (2 mL), and N,N-diisopropylethylamine (47.0 μL, 269 μmol) were added to a 20 mL sample vial. After reacting at room temperature for 16 hours, the reaction was quenched with water (5 mL), extracted with ethyl acetate (30 mL × 3), the organic phases were combined, washed with brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was subjected to high-pressure preparation (XSelect CSH C18 column, 5 μm, 19 mm × 250 mm; mobile phase A: water (0.1% formic acid), mobile phase B: acetonitrile; flow rate: 25 mL / min; gradient: 3% B to 28% B over 10 min; wavelength: 254 nm / 220 nm) to give a white solid N-(2-((5-(3,5-difluorobenzyl)-1-(2-hydroxyethyl)-3-oxo-1,3,4,5,6,7-hexahydro-2H-pyrazolo[4,3-c]pyridin-2-yl)methyl)-5-fluorobenzyl)-2-hydroxyacetamide (5 mg, yield 10%).

[0345] LCMS: 505.2 [M+H] + .

[0346] 1 H NMR(300MHz,CD3OD)δ(ppm)7.12-7.08(m,1H),7.06-7.02(m,2H),6.98-6.94(m,1H),6.91-6.83(m,1H),6.80-6.75( m,1H),5.20(m,2H),4.80(s,2H),4.03(s,2H),3.80-3.77(m,4H),3.54-3.49(m,2H),3.34(s,2H),2.88-2.75(m,4H).

[0347] 19 FNMR(282MHz,CD3OD)δ(ppm)-112.160--112.163(m,2F),-116.685--117.192(m,1F).

[0348] Example 17

[0349] Example 18

[0350] Example 19

[0351] Synthesis of N-(2-(2-(4-chlorobenzyl)-5-(3-cyanobenzyl)-3-oxo-2,3,4,5,6,7-hexahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)ethyl)-2-hydroxyacetamide (AC-P19). The synthetic route is shown in Example 22.

[0352] Example 20

[0353] Example 21

[0354] Synthesis of 3-((2-(4-chlorobenzyl)-1-(2-hydroxyethyl)-3-oxo-1,2,3,4-6,7-hexahydro-5H-pyrazolo[4,3-c]pyridin-5-yl)methyl)benzonitrile (AC-P21), the synthetic route is shown in Example 22.

[0355] Example 22

[0356] The synthesis of 3-((1-(2-aminoethyl)-2-(4-chlorobenzyl)-3-oxo-1,2,3,4-6,7-hexahydro-5H-pyrazolo[4,3-c]pyridin-5-yl)methyl)benzonitrile (AC-P22) and the synthetic routes of AC-P19, 21, and 22 are as follows:

[0357] Step 1: 2-(4-chlorobenzyl)-1-(2-hydroxyethyl)-1,2,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (582 mg, 1.38 mmol, TFA salt), potassium carbonate (610 mg, 4.41 mmol), and acetonitrile (10 mL) were added to a 50 mL round-bottom flask, followed by the addition of 3-(bromomethyl)benzonitrile (350 mg, 1.79 mmol). After stirring at room temperature for 16 hours, the mixture was quenched with water (30 mL), extracted with ethyl acetate (80 mL × 3), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was passed through a column (methanol / dichloromethane (0-20%)) to give a white solid 3-((2-(4-chlorobenzyl)-1-(2-hydroxyethyl)-3-oxo-1,2,3,4-6,7-hexahydro-5H-pyrazolo[4,3-c]pyridin-5-yl)methyl)benzonitrile (453 mg, 77% yield).

[0358] LCMS(ESI,m / z):423.2[M+H] + .

[0359] Step 2: 3-((2-(4-chlorobenzyl)-1-(2-hydroxyethyl)-3-oxo-1,2,3,4-6,6,7-hexahydro-5H-pyrazolo[4,3-c]pyridin-5-yl)methyl)benzonitrile (453 mg, 1.07 mmol), phthalimide (271 mg, 1.84 mmol), triphenylphosphine (574 mg, 2.19 mmol) and tetrahydrofuran (10 mL) were added to a 50 mL round-bottom flask, followed by the addition of diisopropyl azodicarboxylate (420 μL, 2.13 mmol). The mixture was reacted at room temperature for 2 hours and then concentrated. The residue was subjected to reversed-phase chromatography (C18 reversed-phase column, 20-35 μm, size: 80 g; mobile phase A: water (0.05% ammonium bicarbonate), mobile phase B: acetonitrile; flow rate: 60 mL / min; gradient: from 0% B to 48% B over 28 min; wavelength: 220 nm) to give a yellow solid 3-((2-(4-chlorobenzyl)-1-(2-(1,3-dioxoisoindol-2-yl)ethyl)-3-oxo-1,2,3,4,6,7-hexahydro-5H-pyrazolo[4,3-c]pyridin-5-yl)methyl)benzonitrile (578 mg, yield 83%).

[0360] LCMS(ESI,m / z):552.2[M+H] + .

[0361] Step 3: Add 104 mg (160 μmol) of 3-((2-(4-chlorobenzyl)-1-(2-(1,3-dioxoisoindol-2-yl)ethyl)-3-oxo-1,2,3,4-6,7-hexahydro-5H-pyrazolo[4,3-c]pyridin-5-yl)methyl)benzonitrile and ethanol (3 mL) to an 8 mL sample vial, followed by the addition of hydrazine hydrate (85 μL, 1.4 mmol, 80% purity). React at 50 °C for 3.5 hours, then cool to room temperature and concentrate. The residue was prepared under high pressure (column: XBridge prep OBD C18 column, 30×150 mm, 5 μm; mobile phase A: water (10 mmol / L NH4HCO3), mobile phase B: ACN; flow rate: 60 mL / min; slope: from 18% B to 48% B within 10 min; wavelength: 254 nm / 220 nm) to give a white solid 3-((1-(2-aminoethyl)-2-(4-chlorobenzyl)-3-oxo-1,2,3,4-6,7-hexahydro-5H-pyrazolo[4,3-c]pyridin-5-yl)methyl)benzonitrile (40 mg, yield 57%).

[0362] LCMS(ESI,m / z):422.1[M+H] + .

[0363] 1 H NMR(400MHz,Methanol-d4)δ7.81-7.48(m,4H),7.37-7.30(m,2H),7.21-7.14(m,2H),5.09(s,2H),3.81 (s,2H),3.74(t,J=6.8Hz,2H),3.30(s,2H),2.88-2.80(m,2H),2.74-2.68(m,2H),2.65(t,J=6.8Hz,2H).

[0364] Step 4: Add 3-(1-(2-aminoethyl)-2-(4-chlorobenzyl)-3-oxo-1,2,3,4,6,7-hexahydro-5H-pyrazolo[4,3-c]pyridin-5-yl)methyl)benzonitrile (32.0 mg, 35.6 μmol), 2-hydroxyacetic acid (18.9 mg, 248 μmol), 2-(7-azabenzotriazole)-N,N,N′,N′-tetramethylurea hexafluorophosphate (36.6 mg, 96.0 μmol), N,N-dimethylformamide (1 mL), and N,N-diisopropylethylamine (45 μL, 0.25 mmol) to an 8 mL vial. After reacting at room temperature for 2 hours, the reaction solution was directly passed through reversed-phase chromatography (C18 reversed-phase column, 20-35 μm, size: 20 g; mobile phase A: water (0.05% ammonium bicarbonate), mobile phase B: acetonitrile; flow rate: 30 mL / min; gradient: from 0% B to 34% B over 20 minutes; wavelength: 220 nm) to obtain a grayish-white solid N-(2-(2-(4-chlorobenzyl)-5-(3-cyanobenzyl)-3-oxo-2,3,4-5,6,7-hexahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)ethyl)-2-hydroxyacetamide (13.0 mg, yield 74%).

[0365] LCMS(ESI,m / z):480.2[M+H] + .

[0366] 1 H NMR (400MHz, Methanol-d4) δ7.82-7.77(m,1H),7.77-7.71(m,1H),7.71-7.63(m,1H),7.60-7.51(m,1H),7.36-7.29(m,2H),7.21-7. 14(m,2H),5.11(s,2H),4.85(s,2H),3.96(s,2H),3.86-3.76(m,4H),3.30-3.27(m,2H),2.83(t,J=5.6Hz,2H),2.68(t,J=5.6Hz,2H).

[0367] Example 23

[0368] The synthesis of 2-(2-bromo-4-chlorophenyl)-5-(3,5-difluorobenzyl)-1-(2-hydroxyethyl)-1,2,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one was carried out via the following route:

[0369] Step 1: Ethyl 1-(3,5-difluorobenzyl)-4-oxopiperidin-3-carboxylate (6.00 g, 20.1 mmol), (2-bromo-4-chlorophenyl)hydrazine (7.00 g, 25.7 mmol, hydrochloride), sodium acetate (5.00 g, 60.9 mmol), and EtOH (100 mL) were added to a 250 mL round-bottom flask. The resulting mixture was stirred at -10 °C for 16 hours. The mixture was quenched with ice water (50 mL), extracted with ethyl acetate (150 mL × 3), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was washed with tetrahydrofuran, filtered, and the filtrate was concentrated. The crude product was subjected to column chromatography (tetrahydrofuran / petroleum ether = 3 / 2) to give a pale yellow solid 2-(2-bromo-4-chlorophenyl)-5-(3,5-difluorobenzyl)-2,3a,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (2 g, yield 20%).

[0370] LCMS: 468.1, 470.1 [M+H, M+H+2] + .

[0371] Step 2: 2-(2-bromo-4-chlorobenzyl)-5-(3,5-difluorobenzyl)-2,3a,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (750 mg, 1.46 mmol), cesium carbonate (1.55 g, 4.75 mmol), and N,N-dimethylformamide (15 mL) were added to a 30 mL microwave-safe tube, followed by the addition of ethylene oxide (1.0 mL, 20 mmol). The mixture was stirred at room temperature for 48 hours. Two experiments were performed in parallel, each quenched with water (10 mL). The resulting mixtures were extracted with ethyl acetate (100 mL × 3), the organic phases were combined, washed with brine (300 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was passed through a column (tetrahydrofuran / petroleum ether (0% to 90%)) to give a yellow oil of 2-(2-bromo-4-chlorophenyl)-5-(3,5-difluorobenzyl)-1-(2-hydroxyethyl)-1,2,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (360 mg, 21% yield).

[0372] LCMS(ESI,m / z):512.1,514.1[M+H,M+H+2] + .

[0373] Example 24

[0374] The synthesis of 2-(2-bromo-4-fluorobenzyl)-5-(3,5-difluorobenzyl)-1-(2-hydroxyethyl)-1,2,4,5,6,7-hexahydro-3H-pyrazole[4,3-c]pyridin-3-one was carried out via the following synthetic route:

[0375] Step 1: Ethyl 1-(3,5-difluorobenzyl)-4-oxopiperidin-3-carboxylate (1.00 g, 3.36 mmol), (2-bromo-4-fluorobenzyl)hydrazine (737 mg, 3.36 mmol), sodium acetate (828 mg, 10.1 mmol), and ethanol (10 mL) were added to a 40 mL sample vial. The mixture was stirred at 0 °C for 8 hours, concentrated to remove ethanol, then quenched with ice water (30 mL), extracted with ethyl acetate (100 mL × 3), the combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography (tetrahydrofuran / petroleum ether (50% to 75%)) to give a yellow semi-solid 2-(2-bromo-4-fluorobenzyl)-5-(3,5-difluorobenzyl)-2,3a,4,5,6,7-hexahydro-3H-pyrazole[4,3-c]pyridin-3-one (500 mg, 31% yield).

[0376] LCMS:452.1454.1[M+H,M+H+2] + .

[0377] Step 2: 2-(2-bromo-4-fluorobenzyl)-5-(3,5-difluorobenzyl)-2,3a,4,5,6,7-hexahydro-3H-pyrazole[4,3-c]pyridin-3-one (500 mg, 1.11 mmol), cesium carbonate (1.08 g, 3.32 mmol), and N,N-dimethylformamide (10 mL) were added to a 40 mL sample vial, and 74 μL (5.53 mmol) was added at 0 °C. The resulting mixture was stirred at 25 °C for 36 hours, quenched with water (50 mL), extracted with ethyl acetate (3 × 100 mL), and the combined organic phases were washed with brine (300 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was passed through a column (in tetrahydrofuran / petroleum ether (30%–40%)) to give 2-(2-bromo-4-fluorobenzyl)-5-(3,5-difluorobenzyl)-1-(2-hydroxyethyl)-1,2,4,5,6,7-hexahydro-3H-pyrazole[4,3-c]pyridine-3-one (130 mg). 50 mg was purified by high-pressure preparation (Xbridge Prep Shield RP18 column, 30×150 mm, 5 μm; mobile phase A: water (10 mmol / L ammonium bicarbonate + 0.1% ammonia), mobile phase B: acetonitrile; flow rate: 60 mL / min; gradient: 27% B to 49% B over 9 min; wavelength: 254 nm) to obtain a white solid 2-(2-bromo-4-fluorobenzyl)-5-(3,5-difluorobenzyl)-1-(2-hydroxyethyl)-1,2,4,5,6,7-hexahydro-3H-pyrazole[4,3-c]pyridin-3-one (6.8 mg, yield 1.2%).

[0378] LCMS: 496.1498.1[M+H,M+H+2] + .

[0379] 1 H NMR (400MHz, CD3OD) δ (ppm) 7.45-7.43 (m, 2H), 7.11-7.00 (m, 3H), 6.89-6.78 (m, 1H), 5.16 (s, 2H),3.80-3.75(m,4H),3.55-3.53(s,2H),3.31(s,2H),2.84-2.82(m,2H),2.76-2.74(m,2H).

[0380] 19 FNMR (376MHz, CD3OD) δ (ppm) -112.204 (s, 2F), -114.433 (s, 1F).

[0381] Example 25

[0382] The synthesis of 1-(2-aminoethyl)-2-(2-bromo-4-fluorobenzyl)-5-(3,5-difluorobenzyl)-1,2,4,5,6,7-hexahydro-3H-pyrazole[4,3-c]pyridin-3-one was carried out via the following synthetic route:

[0383] Step 1: 2-(2-bromo-4-fluorobenzyl)-5-(3,5-difluorobenzyl)-1-(2-hydroxyethyl)-1,2,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (930 mg, 1.87 mmol), phthalimide (570 mg, 3.87 mmol), triphenylphosphine (1.00 g, 3.81 mmol), and tetrahydrofuran (15 mL) were added to a 100 mL round-bottom flask, followed by the addition of diisopropyl azodicarbonate (750 μL, 3.81 mmol). After reacting at room temperature for 16 hours, the mixture was concentrated, and the residue was column-passed (in tetrahydrofuran / petroleum ether (0% to 40%)) to give the crude product (containing triphenylphosphine oxide). The crude product was subjected to reversed-phase chromatography (C18 reversed-phase column, 20-35 μm, size: 80 g; mobile phase A: water (0.05% ammonium bicarbonate) / mobile phase B: acetonitrile; flow rate: 60 mL / min; gradient: 0% B to 50% B over 25 min; detector: 254 nm) to obtain a pale yellow solid 2-(2-(2-bromo-4-fluorobenzyl)-5-(3,5-difluorobenzyl)-3-oxo-2,3,4,5,6,7-hexahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)ethyl)isoindoline-1,3-dione (630 mg, yield 51%).

[0384] LMCS:625.1627.1[M+H,M+H+2] + .

[0385] Step 2: 2-(2-(2-bromo-4-fluorobenzyl)-5-(3,5-difluorobenzyl)-3-oxo-2,3,4,5,6,7-hexahydro-1H-pyrazol[4,3-c]pyridin-1-yl)ethyl)isoindoline-1,3-dione (630 mg, 1.01 mmol) and ethanol (10 mL) were added to a 40 mL sample vial, followed by the addition of hydrazine hydrate (360 mg, 6.11 mmol, 85% in water). The resulting mixture was stirred at 50°C for 8 hours, cooled to room temperature, quenched with 1N sodium hydroxide solution (50 mL), extracted with dichloromethane (100 mL × 3), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give a pale yellow solid 1-(2-aminoethyl)-2-(2-bromo-4-fluorobenzyl)-5-(3,5-difluorobenzyl)-1,2,4,5,6,7-hexahydro-3H-pyrazole[4,3-c]pyridin-3-one (510 mg, crude product).

[0386] LC MS: 495.1, 497.1 [M+H, M+H+2] + .

[0387] Example 26

[0388] The synthesis of N-(2-(2-(4-chlorobenzyl)-5-(3,5-difluorobenzyl)-3-oxo-2,3,4-5,6,7-hexahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)ethyl)-14-hydroxy-3,6,9,12-tetraoxatetradecanoamide is described below:

[0389] 1-(2-aminoethyl)-2-(4-chlorobenzyl)-5-(3,5-difluorobenzyl)-1,2,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (74.3 mg, 171 μmol), 14-hydroxy-3,6,9,12-tetraoxatetradecanoic acid (78.0 mg, 309 μmol), 2-(7-azabenzotriazole)-N,N,N′,N′-tetramethylurea hexafluorophosphate (108 mg, 284 μmol), and N,N-dimethylformamide (1 mL) were added to an 8 mL sample vial, followed by the addition of N,N-diisopropylethylamine (150 μL, 851 μmol). After reacting at room temperature for 3 hours, the reaction was quenched with water (10 mL), extracted with ethyl acetate (30 mL × 3), the organic phases were combined, washed with brine (100 mL × 2), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by TLC preparative plate (elution: methanol / dichloromethane (1 / 10)). Further preparation under high pressure (Xselect CSH Prep C18 OBD column, 30×150mm, 5μm; mobile phase A: water (0.1% formic acid), mobile phase B: acetonitrile; flow rate: 60mL / min; gradient: 9%B to 33%B over 8 minutes; detector: UV 220 nm) yielded a colorless oily N-(2-(2-(4-chlorobenzyl)-5-(3,5-difluorobenzyl)-3-oxo-2,3,4-5,6,7-hexahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)ethyl)-14-hydroxy-3,6,9,12-tetraoxatetradecanoamide (45 mg, yield 38%).

[0390] LCMS(ESI,m / z):667.4[M+H] + .

[0391] 1 H NMR(400MHz, Methanol-d4)δ(ppm):7.38-7.30(m,2H),7.22-7.14(m,2H),7.09-7.00(m,2H),6.93-6.83(m,1H),5.12(s,2H),3.99(s,2H),3.84(t ,J=6.0Hz,2H),3.76(s,2H),3.73-3.58(m,14H),3.58-3.44(m,3H),3.38 -3.34(m,1H),3.29(s,2H),2.80(t,J=6.0Hz,2H),2.66(t,J=6.0Hz,2H).

[0392] 19 F NMR (376MHz, CD3OD) δ (ppm): -112.12 (s, 2F).

[0393] Example 27

[0394] The synthesis of 2-(4-chlorobenzyl)-5-(3,5-difluorobenzyl)-1-(2-(methylamino)ethyl)-1,2,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one can be referred to step 1 of Example 28.

[0395] Example 28

[0396] The synthetic route for N-(2-(2-(4-chlorobenzyl)-5-(3,5-difluorobenzyl)-3-oxo-2,3,4,5,6,7-hexahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)ethyl)-2-hydroxy-N-methylacetamide is as follows:

[0397] Step 1: 2-(4-chlorobenzyl)-5-(3,5-difluorobenzyl)-1-(2-(methylamino)ethyl)-1,2,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (formate) (100 mg, crude) was adjusted to pH 8 with saturated sodium bicarbonate solution (20 mL), extracted with ethyl acetate (50 mL × 3), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was subjected to high-pressure preparation (XBridge RP C18 column, 5 μm, 19 mm × 150 mm; mobile phase A: water (10 mmol / L ammonium bicarbonate + 0.1% ammonia), mobile phase B: acetonitrile; flow rate: 25 mL / min; gradient: 25% B to 50% B over 12 minutes; wavelength: 254 nm / 220 nm) to give a white semi-solid 2-(4-chlorobenzyl)-5-(3,5-difluorobenzyl)-1-(2-(methylamino)ethyl)-1,2,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (14.1 mg, yield 15%).

[0398] LCMS: 447.2 [M+H] + .

[0399] 1 H NMR (300MHz, CD3OD) δ (ppm) 7.34-7.31 (m, 2H), 7.16 (d, J = 8.4Hz, 2H), 7.05-6.99 (m, 2H), 6.90-6.82 (m, 1H), 5.0 6(s,2H),3.79-3.74(m,4H),3.28(s,2H),2.83-2.79(m,2H),2.68-2.64(m,2H),2.54-2.50(m,2H),2.29(s,3H).

[0400] 19 FNMR(282MHz,CD3OD)δ(ppm)-112.122(s,2F).

[0401] Step 2: Add 2-(4-chlorobenzyl)-5-(3,5-difluorobenzyl)-1-(2-(methylamino)ethyl)-1,2,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (10.0 mg, 22.4 μmol), 2-hydroxyacetic acid (4.00 mg, 44.8 μmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (26.0 mg, 67.1 μmol), N,N-diisopropylethylamine (12.0 μL, 67.1 μmol), and N,N-dimethylformamide (0.5 mL) to an 8 mL sample vial. The resulting mixture was stirred at room temperature for 16 hours and then subjected to high pressure (YMC Triart C18ExRs column, 20×250mm, 5μm; mobile phase A: water (10mmol / L ammonium bicarbonate + 0.05% ammonia), mobile phase B: acetonitrile; flow rate: 20mL / min; gradient: 30%B to 50%B over 10 minutes, then hold for 12 minutes; wavelength: 254nm / 220nm) to give a white solid N-(2-(2-(4-chlorobenzyl)-5-(3,5-difluorobenzyl)-3-oxo-2,3,4,5,6,7-hexahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)ethyl)-2-hydroxy-N-methylacetamide (7.3mg, yield 64%).

[0402] LCMS:505.0[M+H] + .

[0403] 1 H NMR(300MHz,CD3OD)δ(ppm)7.36-7.31(m,2H),7.20-7.17(m,2H),7.04-6.99(m,2H),6.88-6.82(m,1H),5.09(s,2H),4.16(s, 2H),3.96-3.92(m,1H),3.88-3.84(m,2H),3.74(s,2H),3.36-3.34(m,1H),3.28(s,2H),2.82-2.76(m,5H),2.63-2.59(m,2H).

[0404] 19 FNMR(282MHz,CD3OD)δ(ppm)-112.148(s,2F).

[0405] Example 29

[0406] The synthesis of 5-(3-chloro-5-fluorobenzyl)-2-(4-chlorobenzyl)-1-(2-hydroxyethyl)-1,2,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one was carried out via the following route:

[0407] Step 1: 4-Oxoperidin-3-carboxylic acid ethyl ester hydrochloride (2.00 g, 10.3 mmol), 1-(bromomethyl)-3-chloro-5-fluorobenzene (2.31 g, 10.3 mmol), potassium carbonate (1.43 g, 10.3 mmol), and acetonitrile (20 mL) were added to a 40 mL sample vial. After reacting at room temperature for 15 hours, the mixture was concentrated to remove acetonitrile, quenched with water (20 mL), extracted with ethyl acetate (50 mL × 3), the organic phases were combined, washed with brine (150 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was column-passed (0% to 20% in ethyl acetate / petroleum ether) to give a colorless oily 1-(3-chloro-5-fluorobenzyl)-4-oxoperidin-3-carboxylic acid ethyl ester (2.5 g, 80% yield).

[0408] LCMS:314.1[M+H] + .

[0409] Step 2: Ethyl 1-(3-chloro-5-fluorobenzyl)-4-oxopiperidin-3-carboxylate (1.00 g, 3.19 mmol), (4-chlorobenzyl)hydrazine dihydrochloride (732 mg, 3.19 mmol), sodium acetate (784 mg, 9.56 mmol), and ethanol (15 mL) were added to a 40 mL sample vial. After reacting at room temperature for 18 hours, the mixture was concentrated, quenched with water (50 mL), extracted with ethyl acetate (50 mL × 3), the organic phases were combined, washed with brine (150 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was column-passed (methanol in dichloromethane 1 / 10) to give a yellow oily 5-(3-chloro-5-fluorobenzyl)-2-(4-chlorobenzyl)-2,3a,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (750 mg, yield 58%).

[0410] LCMS:406.2408.2[M+H,M+H+2] + .

[0411] Step 3: 5-(3-chloro-5-fluorobenzyl)-2-(4-chlorobenzyl)-2,3a,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (1.00 g, 2.46 mmol), cesium carbonate (2.41 g, 7.38 mmol), and N,N-dimethylformamide (10 mL) were added to a 30 mL microwave-safe tube, and ethylene oxide (609 mL, 12.3 mmol) was added at 0 °C. After reacting at room temperature for 16 hours, the reaction was quenched with water (30 mL). The mixture was extracted with ethyl acetate (100 mL × 3), the organic phases were combined, washed with brine (300 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was column-passed (30% to 100% in ethyl acetate / petroleum ether, then 0% to 10% in methanol / dichloromethane) to give the crude product. The crude product was then subjected to high-pressure preparation (XBridge Prep OBD C18 column, 30×150mm, 5μm; mobile phase A: water (10mmol / L ammonium bicarbonate + 0.05% ammonia), mobile phase B: acetonitrile; flow rate: 60mL / min; gradient: 42%B to 72%B over 7 minutes; wavelength: 254nm / 220nm) to give a white solid 5-(3-chloro-5-fluorobenzyl)-2-(4-chlorobenzyl)-1-(2-hydroxyethyl)-1,2,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (28.2 mg, yield 2%).

[0412] LCMS:450.0452.0[M+H,M+H+2] + .

[0413] 1 H NMR(400MHz,CD3OD)δ(ppm)7.33-7.30(m,2H),7.26(s,1H),7.16-7.14(m,2H),7.13-7.10(m,2H),5.07(s, 2H),3.80-3.79(m,2H),3.72(s,2H),3.54-3.51(m,2H),3.34(s,2H),2.81-2.78(m,2H),2.73-2.70(m,2H).

[0414] 19 FNMR(376MHz,CD3OD)δ(ppm)-113.230(s,1F).

[0415] Example 30

[0416] The synthesis of 5-(3-chloro-5-fluorobenzyl)-2-(4-chlorobenzyl)-1-(2-(methylamino)ethyl)-1,2,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one was carried out via the following route:

[0417] Step 1: Add 5-(3-chloro-5-fluorobenzyl)-2-(4-chlorobenzyl)-1-(2-hydroxyethyl)-1,2,4,5,6,7-hexahydro-3H-pyrazole[4,3-c]pyridin-3-one (150 mg, 333 μmol), dichloromethane (5 mL), and triethylamine (139 μL, 999 μmol) to a 40 mL sample vial. Add methanesulfonyl chloride (1.27 mL, 400 μmol) at 0 °C. After reacting at room temperature for 2 hours, saturated sodium bicarbonate (20 mL) was added to quench the reaction. The mixture was then extracted with dichloromethane (100 mL × 3), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a yellow oily ethyl 2-(5-(3-chloro-5-fluorobenzyl)-2-(4-chlorobenzyl)-3-oxo-2,3,4,5,6,7-hexahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)methanesulfonate (160 mg, crude product).

[0418] LCMS:528.1530.1[M+H,M+H+2] + .

[0419] Step 2: Ethyl 2-(5-(3-chloro-5-fluorobenzyl)-2-(4-chlorobenzyl)-3-oxo-2,3,4,5,6,7-hexahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)methanesulfonate (160 mg, 303 μmol), ethanol (5 mL), and methylamine (30% ethanol solution, 349 μL, 3.03 mmol) were added to a 40 mL sample vial. After reacting at 80 °C for 3 hours, the mixture was cooled to room temperature and concentrated. The residue was subjected to high-pressure preparation (Xselect CSH C18 OBD column, 30×150 mm, 5 μm; mobile phase A: water (0.1% formic acid), mobile phase B: acetonitrile; flow rate: 60 mL / min; gradient: 3% to 18% B over 10 min; wavelength: 254 nm / 220 nm) to give a white semi-solid 5-(3-chloro-5-fluorobenzyl)-2-(4-chlorobenzyl)-1-(2-(methylamino)ethyl)-1,2,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (11.1 mg, 7% yield, formate).

[0420] LCMS:463.1465.1[M+H,M+H+2] + .

[0421] 1H NMR (300MHz, CD3OD) δ (ppm) 8.52 (brs, 1H), 7.34-7.32 (m, 2H), 7.26 (s, 1H), 7.18-7.11 (m, 4H), 5.05 (s ,2H),3.92-3.87(m,2H),3.74(s,2H),3.26(s,2H),2.84-2.79(m,4H),2.67-2.64(m,2H),2.52(s,3H).

[0422] 19 FNMR(282MHz,CD3OD)δ(ppm)-113.091(s,1F).

[0423] Example 31

[0424] The synthesis of 1-(2-aminoethyl)-5-(3-chloro-5-fluorobenzyl)-2-(4-chlorobenzyl)-1,2,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one can be referred to steps 1 to 2 of Example 32.

[0425] Example 32

[0426] The synthesis of N-(2-(5-(3-chloro-5-fluorobenzyl)-2-(4-chlorobenzyl)-3-oxo-2,3,4,5,6,7-hexahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)ethyl)-2-hydroxyacetamide is described below:

[0427] Step 1: 5-(3-chloro-5-fluorobenzyl)-2-(4-chlorobenzyl)-1-(2-hydroxyethyl)-1,2,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (300 mg, 666 μmol), phthalimide (196 mg, 1.33 mmol), and dichloromethane (5 mL) were added to a 40 mL sample vial, followed by the addition of triphenylphosphine (262 mg, 999 μmol) and diisopropyl azodicarbonate (162 μL, 999 μmol). After reacting at room temperature for 16 hours, the reaction was quenched with water (30 mL), extracted with dichloromethane (100 mL × 3), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was passed through a column (0% to 10% in methanol / dichloromethane) to give a yellow oily 2-(2-(5-(3-chloro-5-fluorobenzyl)-2-(4-chlorobenzyl)-3-oxo-2,3,4,5,6,7-hexahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)ethyl)isoindoline-1,3-dione (300 mg, 59% yield).

[0428] LCMS:579.1581.1[M+H,M+H+2] + .

[0429] Step 2: 2-(2-(5-(3-chloro-5-fluorobenzyl)-2-(4-chlorobenzyl)-3-oxo-2,3,4,5,6,7-hexahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)ethyl)isoindoline-1,3-dione (300 mg, 518 μmol), ethanol (5 mL), and hydrazine hydrate (151 μL, 3.11 mmol) were added to a 40 mL sample vial. After reacting at 50 °C for 8 hours, the mixture was cooled to room temperature, filtered, and the filtrate was concentrated to obtain a yellow oily crude product (150 mg). 50 mg of the crude product was prepared under high pressure (Xselect CSH C18 OBD column, 30×150 mm, 5 μm; mobile phase A: water (0.1% formic acid), mobile phase B: acetonitrile; flow rate: 60 mL / min; gradient: 3% B to 20% B over 10 minutes; wavelength: 254 nm / 220 nm) to obtain a white semi-solid 1-(2-aminoethyl)-5-(3-chloro-5-fluorobenzyl)-2-(4-chlorobenzyl)-1,2,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (10.1 mg formate).

[0430] LCMS:449.0451.0[M+H,M+H+2] + .

[0431] 1 H NMR(300MHz,CD3OD)δ(ppm)8.48(brs,1H),7.34-7.32(m,2H),7.27(s,1H),7.18-7.11(m,4H),5 .05(s,2H),3.95-3.90(m,2H),3.74(s,2H),3.27(s,2H),2.92-2.81(m,4H),2.67-2.64(m,2H).

[0432] 19 FNMR(282MHz,CD3OD)δ(ppm)-113.027(s,1F).

[0433] Step 3: Add 1-(2-aminoethyl)-5-(3-chloro-5-fluorobenzyl)-2-(4-chlorobenzyl)-1,2,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (100 mg, 109 μmol), 2-hydroxyacetic acid (37.0 mg, 491 μmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (124 mg, 327 μmol), N,N-dimethylformamide (5 mL), and N,N-diisopropylethylamine (57 μL, 0.32 μmol) to a 40 mL sample vial. After reacting at room temperature for 18 hours, quench with water (10 mL), extract with ethyl acetate (30 mL × 3), combine the organic phases, wash with brine (100 mL), dry to anhydrous sodium sulfate, filter, and concentrate. The residue was passed through a column (0% to 10% in methanol / dichloromethane) to give a crude product. The crude product was then subjected to high-pressure preparation (XBridge Prep OBD C18 column, 30 × 150 mm, 5 μm; mobile phase A: water (10 mmol / L ammonium bicarbonate + 0.05% ammonia), mobile phase B: acetonitrile; flow rate: 60 mL / min; gradient: 28% B to 44% B over 10 minutes; wavelength: 254 nm / 220 nm) to give a white solid N-(2-(5-(3-chloro-5-fluorobenzyl)-2-(4-chlorobenzyl)-3-oxo-2,3,4,5,6,7-hexahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)ethyl)-2-hydroxyacetamide (18.5 mg, yield 16%).

[0434] LCMS:507.0509.0[M+H,M+H+2] + .

[0435] 1 H NMR (300MHz, CD3OD) δ (ppm) 7.32-7.28 (m, 3H), 7.17-7.10 (m, 4H), 5.09 (s, 2H), 4.86 (s, 2H), 3. 94(s,2H),3.82-3.78(m,2H),3.73(s,2H),3.26(s,2H),2.82-2.78(m,2H),2.66-2.63(m,2H).

[0436] 19 FNMR(282MHz,CD3OD)δ(ppm)-113.196(s,1F).

[0437] Example 33

[0438] The synthesis of N-(2-(5-(3,5-difluorobenzyl)-2-(4-fluorobenzyl)-3-oxo-2,3,4,5,6,7-hexahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)ethyl)-2-hydroxyacetamide is described below:

[0439] Step 1: Ethyl 1-(3,5-difluorobenzyl)-4-oxopiperidin-3-carboxylate (5.0 g, 16 mmol), (4-fluorobenzyl)hydrazine (3.0 g, 21 mmol, hydrochloride), sodium acetate (4.1 g, 49 mmol), and ethanol (50 mL) were added to a 100 mL round-bottom flask and stirred at -10 °C for 8 hours. The mixture was quenched with ice water (50 mL), and the ethanol was removed by concentration under vacuum. The mixture was extracted with ethyl acetate (150 mL × 3), the organic phases were combined, washed with brine (500 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was passed through a column (0-75% ethyl acetate / petroleum ether (0-75%)) to give a colorless oily product 5-(3,5-difluorobenzyl)-2-(4-fluorobenzyl)-2,3a,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (4.0 g, 63% yield).

[0440] LCMS(ESI): 374.1 [M+H] + .

[0441] Step 2: 5-(3,5-difluorobenzyl)-2-(4-fluorobenzyl)-2,3a,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (2.35 g, 6.29 mmol), cesium carbonate (6.2 g, 19 mmol), and N,N-dimethylformamide (20 mL) were added to a 40 mL sample vial, followed by the addition of ethylene oxide (2.5 mL, 50 mmol) at 0 °C. The reaction mixture was stirred at 25 °C for 36 hours, quenched with water (50 mL), extracted with ethyl acetate (80 mL × 3), washed with brine (250 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was column-sected (ethyl acetate / petroleum ether (0-100%)) to give 600 mg of crude product. 50 mg of crude product was subjected to high pressure (Xselect CSH Prep Fluoro-Phenyl column, 19×250, 5 μm; mobile phase A: Water (0.1% formic acid), mobile phase B: acetonitrile; flow rate: 20 mL / min; gradient: 5% B to 25% B over 9 minutes; detector: 254 nm / 220 nm) to obtain a colorless oily 5-(3,5-difluorobenzyl)-2-(4-fluorobenzyl)-1-(2-hydroxyethyl)-1,2,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (2.6 mg, yield 1%).

[0442] LCSM:418.3[M+H] + .

[0443] 1 H NMR(400MHz,CD3OD)δ7.25-7.15(m,2H),7.11-6.94(m,4H),6.91-6.80(m,1H),5.07( s,2H),3.81(t,J=8.0Hz,2H),3.75(s,2H),3.53(t,J=8.0Hz,2H),2.88-2.64(m,4H).

[0444] 19 F NMR (376MHz, CD3OD) δ-112.153(s,2F),-116.582(s,1F).

[0445] LCMS(ESI):418.1[M+H] + .

[0446] Example 34

[0447] The synthesis of 2-(2,4-difluorobenzyl)-5-(3,5-difluorobenzyl)-1-(2-hydroxyethyl)-1,2,4,5,6,7-hexahydro-3H-pyrazole[4,3-c]pyridin-3-one was carried out via the following synthetic route:

[0448] Step 1: Add hydrazine hydrate (9.5 mL, 24 mmol) to a 250 mL three-necked round-bottom flask and cool to 0 °C in an ice-water bath. Then dissolve 1-(bromomethyl)-2,4-difluorobenzene (3.1 mL, 24 mmol) in methanol (20 mL) and add it dropwise to the above solution. Stir the resulting mixture at 0 °C for 16 hours, concentrate to remove methanol, quench with water (50 mL), and extract with diethyl ether (3 × 200 mL). Combine the organic layers, dry with anhydrous sodium sulfate, filter, and concentrate to give a colorless oily (2,4-difluorobenzyl)hydrazine (3.5 g, yield 91%).

[0449] LCMS(ESI): 159.1 [M+H] + .

[0450] Step 2: Ethyl 1-(3,5-difluorobenzyl)-4-oxopiperidin-3-carboxylate (3.9 g, 13 mmol), (2,4-difluorobenzyl)hydrazine (3.5 g, 22 mmol), and ethanol (50 mL) were added to a 250 mL round-bottom flask and cooled to -10 °C. Sodium acetate (5.5 g, 67 mmol) was added. The reaction mixture was stirred at -10 °C for 16 hours and then quenched with water (50 mL). The resulting mixture was extracted with ethyl acetate (3 × 150 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was passed through a column (in tetrahydrofuran / petroleum ether (25%–62%)) to give a yellow oily 2-(2,4-difluorobenzyl)-5-(3,5-difluorobenzyl)-2,3a,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (4.5 g, 87% yield).

[0451] LCMS(ESI):392.2[M+H] + .

[0452] Step 3: 2-(2,4-difluorobenzyl)-5-(3,5-difluorobenzyl)-2,3a,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (1.5 g, 3.8 mmol), cesium carbonate (3.75 g, 11.5 mmol), and N,N-dimethylformamide (20 mL) were added to a 40 mL sample vial and cooled to 0 °C in an ice-water bath. Ethylene oxide (2.0 mL, 40 mmol) was added. The resulting mixture was stirred at 25 °C for 60 hours. Three experiments were performed in parallel. The mixture was quenched with water (50 mL), extracted with ethyl acetate (3 × 100 mL), the combined organic phases were washed with brine (300 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was passed through a column (in ethyl acetate / petroleum ether (0%–90%)) to give a yellow solid 2-(2,4-difluorobenzyl)-5-(3,5-difluorobenzyl)-1-(2-hydroxyethyl)-1,2,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (300 mg, 17% yield). 100 mg of the sample was purified by high-pressure preparation (XBridge Prep OBD C18 column, 30×150 mm, 5 μm; mobile phase A: water (10 mmol / L ammonium bicarbonate + 0.1% ammonia), mobile phase B: acetonitrile; flow rate: 60 mL / min; gradient: 25% B to 45% B over 11 min; wavelength: 254 nm) to obtain 2-(2,4-difluorobenzyl)-5-(3,5-difluorobenzyl)-1-(2-hydroxyethyl)-1,2,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (32.3 mg).

[0453] LCMS(ESI): 436.2 [M+H] + .

[0454] 1 H NMR(300MHz,CD3OD)δ(ppm):7.19-7.67(m,6H),5.11(s,2H),3.88-3.78(m, 2H),3.72(s,2H),3.59-3.50(m,2H),3.29-3.22(m,2H),2.84-2.66(m,4H).

[0455] 19 F NMR (282MHz, CD3OD) δ (ppm): -112.18 (s, 2F), -116.01 (s, 2F).

[0456] Example 35

[0457] Synthesis of 1-(2-aminoethyl)-2-(2,4-difluorobenzyl)-5-(3,5-difluorobenzyl)-1,2,4,5,6,7-hexahydro-3H-pyrazole[4,3-c]pyridin-3-one

[0458] Step 1: Add 2-(2,4-difluorobenzyl)-5-(3,5-difluorobenzyl)-1-(2-hydroxyethyl)-1,2,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (200 mg, 459 μmol), triphenylphosphine (253 mg, 965 μmol), and tetrahydrofuran (5 mL) to a 30 mL microwave tube, and then add diisopropyl azodicarbonate (187 μL, 950 μmol) dropwise. The resulting mixture was stirred at 25°C for 16 hours and then concentrated. The residue was passed through a column (in ethyl acetate / petroleum ether (0%–86%)) to give a yellow solid 2-(-2-(2-(2,4-difluorobenzyl)-5-(3,5-difluorobenzyl)-3-oxo-2,3,4,5,6,7-hexahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)ethyl)isoindoline-1,3-dione (140 mg, yield 53%).

[0459] LCMS(ESI): 565.3 [M+H] + .

[0460] Step 2: Add 2-(-2-(2-(2,4-difluorobenzyl)-5-(3,5-difluorobenzyl)-3-oxo-2,3,4,5,6,7-hexahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)ethyl)isoindoline-1,3-dione (120 mg, 212 μmol), hydrazine hydrate (85% aqueous solution, 44.0 mg, 879 μmol), and ethanol (3 mL) to an 8 mL sample vial. Stir the reaction mixture at 50 °C for 8 hours, cool to room temperature, quench with 5 mL of 2 M sodium hydroxide solution, extract with ethyl acetate (80 mL × 3), combine the organic phases, dry with anhydrous sodium sulfate, filter, and concentrate. The residue was purified by high-pressure preparation (XBridge Prep OBD C18 column, 30×150mm, 5μm; mobile phase A: water (10mmol / L ammonium bicarbonate + 0.1% ammonia), mobile phase B: acetonitrile; flow rate: 60mL / min; gradient: 22%B to 48B over 9min; wavelength: 254nm) to give a white solid 1-(2-aminoethyl)-2-(2,4-difluorobenzyl)-5-(3,5-difluorobenzyl)-1,2,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (27.8mg, yield 29%).

[0461] 1H NMR (400MHz, CD3OD) δ (ppm): 7.19-7.08 (m, 1H), 7.05-6.50 (m, 4H), 5.10 (s, 2H) ),3.79-3.71(m,4H),3.28-3.23(m,2H),2.84-2.76(m,2H),2.72-2.60(m,4H).

[0462] 19 F NMR (376MHz, CD3OD) δ (ppm): -112.04 (s, 1F), -115.74 (s, 2F).

[0463] LCMS(ESI): 435.2 [M+H] + .

[0464] Example 36

[0465] The synthesis of 5-(3,5-difluorobenzyl)-2-(4-hydroxybenzyl)-1-methyl-1,2,4,5,6,7-hexahydro-3H-pyrazole[4,3-c]pyridin-3-one was carried out via the following route:

[0466] Step 1: 2-(4-chlorobenzyl)-5-(3,5-difluorobenzyl)-2,3,4,5,6,7-hexahydro-3H-pyrazole[4,3-c]pyridin-3-one (300 mg, 769 μmol) and tetrahydrofuran (7 mL) were added to a 40 mL sample vial. Sodium hydroxide (69.2 mg, 60% purity) was added at 0 °C, and the mixture was stirred for 10 minutes. Iodomethane (143 μL, 2.31 mmol) was then added, and the reaction mixture was stirred at 0 °C for 30 minutes. The mixture was then allowed to rise naturally to room temperature and stirred for 3 hours. The reaction was quenched with water (30 mL), extracted with ethyl acetate (40 mL × 3), and the organic phases were combined. The mixture was washed with brine (120 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by TLC (elution: methanol / dichloromethane (1 / 15)) to obtain two crude products. The first crude product was prepared under high pressure (column: XBridge prep OBD C18 column, 30×150 mm, 5 μm; mobile phase A: water (10 mmol / L ammonium bicarbonate + 0.05% ammonia), mobile phase B: acetonitrile; flow rate: 60 mL / min; gradient: from 32% B to 62% B over 7 min; wavelength: 254 nm / 220 nm) to give a white solid 2-(4-chlorobenzyl)-5-(3,5-difluorobenzyl)-1-methyl-1,2,4,5,6,7-hexahydro-3H-pyrazole[4,3-c]pyridin-3-one (28.0 mg, yield 8%).

[0467] LCMS(ESI): 404.0 [M+H]+ .

[0468] 1 H NMR(300MHz,CD3OD)δ7.33-7.31(m,2H),7.18-7.16(m,2H),7.03-6.99(m,2H),6.88-6.82(m,1H) ,5.07(s,2H),3.73(s,2H),3.31-3.28(m,2H),3.24(s,3H),2.81-2.78(m,2H),2.62-2.60(m,2H).

[0469] Step 2: Add 2-(4-chlorobenzyl)-5-(3,5-difluorobenzyl)-1-methyl-1,2,4,5,6,7-hexahydro-3H-pyrazole[4,3-c]pyridin-3-one (200 mg, 495 μmol), water (430 μL, 23.87 mmol), 6,6'-diamino-2,2'-bipyridine (5.0 mg, 26 μmol), nickel(II) bromide trihydrate (10 mg, 36 μmol), N-methyldicyclohexylamine (250 mg, 1.28 mmol), phenylsilane (10 mg, 92 mmol), and N,N-dimethylacetamide (2 mL) to a 10 mL microwave tube and stir at 120 °C for 12 hours under a nitrogen atmosphere. Cool to room temperature, quench with water (5 mL), extract with ethyl acetate (30 mL × 3), combine organic phases, wash with brine (100 mL), dry to anhydrous sodium sulfate, filter, and concentrate. The residue was subjected to high pressure (column: Ultimate HS-C18 column, 50 × 250 mm, 8 μm; mobile phase A: water (0.1% ammonium bicarbonate), mobile phase B: acetonitrile; flow rate: 50 mL / min; gradient: 30% B to 70% B over 40 minutes; wavelength: 210 nm) to give a colorless oily 5-(3,5-difluorobenzyl)-2-(4-hydroxybenzyl)-1-methyl-1,2,4,5,6,7-hexahydro-3H-pyrazole[4,3-c]pyridin-3-one (1.1 mg, yield 0.6%).

[0470] LCMS(ESI): 386.2 [M+H] + .

[0471] 1 H NMR(400MHz,CD3OD)δ7.51-6.62(m,7H),3.60-3.44(m,2H),3.37-3.31(m, 3H),2.84-2.56(m,2H),2.18(s,2H),1.40-1.23(m,2H),1.18-1.09(m,2H).

[0472] 19F NMR(376MHz,CD3OD)δ-112.113(m,2F).

[0473] Example 37

[0474] The synthesis of 5-(3-chloro-5-fluorobenzyl)-2-(4-chlorobenzyl)-1-(4-(2-hydroxyethyl)phenyl)-1,2,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one was carried out via the following route:

[0475] Step 1: 5-(3-chloro-5-fluorobenzyl)-2-(4-chlorobenzyl)-2,3a,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (370 mg, 910 μmol), methyl 2-(4-iodophenyl)acetate (505 mg, 1.83 mmol), potassium tert-butoxide (210 mg, 1.87 mmol), DMF (6 mL), CuI (20.0 mg, 105 μmol), and 2-isobutyrylcyclohexanone (20.0 mg, 118 μmol) were added to a 30 mL microwave-safe tube and stirred at 90 °C for 16 hours under a nitrogen atmosphere. After cooling to room temperature, the mixture was quenched with water (10 mL), extracted with ethyl acetate (50 mL × 3), the organic phases were combined, washed with brine (150 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by TLC (elution buffer: ethyl acetate / petroleum ether (4 / 1), Rf = 0.3) to give a black oily product, methyl 2-(4-(5-(3-chloro-5-fluorobenzyl)-2-(4-chlorobenzyl)-3-oxo-2,3,4,5,6,7-hexahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)phenyl)acetate (76 mg, yield 15%).

[0476] LCMS(ESI): 544.0 [M+H] + .

[0477] Step 2: Methyl 2-(4-(5-(3-chloro-5-fluorobenzyl)-2-(4-chlorobenzyl)-3-oxo-2,3,4,5,6,7-hexahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)phenyl)acetate (76.0 mg, 137 μmol) and tetrahydrofuran (6 mL) were added to a 25 mL two-necked round-bottom flask. Then, DIBAL-H (1 mL, 1 M tetrahydrofuran solution) was added under nitrogen atmosphere at 0 °C. The mixture was stirred at room temperature for 12 hours, quenched with water (10 mL), extracted with ethyl acetate (30 mL × 3), the organic phases were combined, washed with brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was subjected to high-pressure preparation (YMC Triart C18 ExRs column, 20 × 250 mm, 5 μm; mobile phase A: water (10 mmol / L ammonium bicarbonate), mobile phase B: acetonitrile; flow rate: 25 mL / min; gradient: 48% B to 78% B over 7 minutes; wavelength: 254 nm / 220 nm) to give a white solid 5-(3-chloro-5-fluorobenzyl)-2-(4-chlorobenzyl)-1-(4-(2-hydroxyethyl)phenyl)-1,2,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (13.6 mg, yield 18%).

[0478] LCMS(ESI): 526.2 [M+H] + .

[0479] 1 H NMR (400MHz, CD3OD) δ (ppm): 7.44-7.36 (m, 2H), 7.34-7.23 (m, 3H), 7.21-7.18 (m, 2H), 7.16-7.00 (m, 2H), 6.98-6.86 (m, 2H), 4.88 -4.84(m,2H),3.89-3.78(m,2H),3.76-3.72(m,2H),3.48-3.31(m,2H),2.87-2.81(m,2H),2.79-2.68(m,2H),2.40-2.37(m,2H).

[0480] 19 F NMR (376MHz, CD3OD) δ (ppm): -113.14 (s, 1F).

[0481] Example 38

[0482] The synthesis of 5-(3-chloro-5-fluorobenzyl)-2-(4-(hydroxymethyl)benzyl)-1-methyl-1,2,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one was carried out via the following synthetic route:

[0483] Step 1: Ethyl 1-(3-chloro-5-fluorobenzyl)-4-oxopiperidin-3-carboxylate (1.40 g, 4.46 mmol), (4-bromobenzyl)hydrazine (1.00 g, 4.21 mmol, hydrochloride), sodium acetate (1.10 g, 13.4 mmol), and ethanol (30 mL) were added to a 100 mL round-bottom flask. The resulting mixture was stirred at 25 °C for 16 hours. The mixture was quenched with water (40 mL), extracted with ethyl acetate (70 mL × 3), the organic phases were combined, washed with brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was passed through a column (in tetrahydrofuran / petroleum ether (70%–80%)) to give a yellow oily 2-(4-bromobenzyl)-5-(3-chloro-5-fluorobenzyl)-2,3a,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (400 mg, yield 21%).

[0484] LCMS(ESI):450.1452.1[M+H,M+H+2] + .

[0485] Step 2: 2-(4-bromobenzyl)-5-(3-chloro-5-fluorobenzyl)-2,3a,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (380 mg, 843 μmol) and tetrahydrofuran (10 mL) were added to a 50 mL three-necked round-bottom flask. Sodium hydride (61.0 mg, 1.52 mmol, 60%) was added in portions at 0 °C. After stirring for half an hour, iodomethane (64.0 μL, 1.02 mmol) was added dropwise. The reaction mixture was stirred at 25 °C for 2 hours. The reaction mixture was quenched with water (20 mL), extracted with ethyl acetate (50 mL × 3), the organic layers were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was passed through a column (in ethyl acetate / petroleum ether (70%–90%)) to give a yellow oily 2-(4-bromobenzyl)-5-(3-chloro-5-fluorobenzyl)-1-methyl-1,2,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (70.0 mg, 17% yield).

[0486] LCMS(ESI):464.1466.1[M+H,M+H+2] + .

[0487] Step 3: 2-(4-bromobenzyl)-5-(3-chloro-5-fluorobenzyl)-1-methyl-1,2,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (70.0 mg, 150 μmol), (tributyltin)methanol (73.0 mg, 227 μmol), tetrakis(triphenylphosphine)palladium (35.0 mg, 30.2 μmol), 1,4-dioxane (5 mL), and a stir bar were added to a 30 mL microwave-safe tube. The reaction mixture was stirred at 80 °C for 16 hours, cooled to room temperature, quenched with water (20 mL), extracted with ethyl acetate (70 mL × 3), the organic layers were combined, washed with brine (30 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and evaporated to dryness. The residue was purified by high-pressure preparation (XBridge Prep OBD C18 column, 30×150mm, 5μm; mobile phase A: water (10mmol / L ammonium bicarbonate + 0.05% ammonia), mobile phase B: acetonitrile; flow rate: 60mL / min; gradient: 25%B to 39%B over 10min; wavelength: 254nm) to give a white solid 5-(3-chloro-5-fluorobenzyl)-2-(4-(hydroxymethyl)benzyl)-1-methyl-1,2,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (5.3mg, yield 8%).

[0488] LCMS(ESI,m / z):416.1[M+H] + .

[0489] 1 H NMR (400MHz, CD3OD) δ (ppm) 7.35-7.31 (m, 1H), 7.28-7.24 (m, 3H), 7.20-7.14 (m, 3H), 5.16-5.13 (m, 1H), 4.87(s,2H),4.45-4.44(m,2H),3.68(s,2H),3.08(s,2H),3.06(s,2H),2.68-2.65(m,2H),2.50(s,2H).

[0490] 19 F NMR(282MHz,CD3OD)δ(ppm)-111.15.

[0491] Example 39

[0492] The synthesis of 5-(3-chloro-5-fluorobenzyl)-2-(4-chlorobenzyl)-1-(5-(hydroxymethyl)thiazolyl-2-yl)-1,2,4,5,6,7-hexahydro-3H-pyrazol[4,3-c]pyridin-3-one was carried out via the following synthetic route:

[0493] Step 1: 5-(3-chloro-5-fluorobenzyl)-2-(4-chlorobenzyl)-2,3a,4,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (333 mg, 819 μmol), methyl 2-bromothiazolium-5-carboxylate (330 mg, 1.49 mmol), cuprous iodide (15.0 mg, 78.7 μmol), potassium tert-butoxide (183 mg, 1.63 mmol), N,N-dimethylformamide (8.5 mL), and 2-isobutyrylcyclohexanone (13.3 mg, 79.0 μmol) were added to a 30 mL microwave tube. The experiment was repeated three times in parallel. The resulting mixture was stirred at 90 °C for 16 hours under a nitrogen atmosphere, cooled to room temperature, quenched with water (50 mL), extracted with ethyl acetate (100 mL × 3), the organic layers were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was subjected to high-pressure preparation (column, C18 spherical, 20-35 μm, size: 120 g; mobile phase A: water (0.05% ammonium bicarbonate) / mobile phase B: acetonitrile; flow rate: 50 mL / min; gradient: 70% B to 80% B over 35 minutes; detection wavelength: 210 nm) to obtain a white solid methyl 2-(5-(3-chloro-5-fluorobenzyl)-2-(4-chlorobenzyl)3-oxo-2,3,4,5,6,7-hexahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)thiazolyl-5-carboxylic acid (20 mg, yield 4%).

[0494] LCMS(ESI):547.1,549.1[M+H,M+H+2] + .

[0495] Step 2: 2-(5-(3-chloro-5-fluorobenzyl)-2-(4-chlorobenzyl)-3-oxo-2,3,4,5,6,7-hexahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)thiazolyl-5-carboxylic acid methyl ester (20 mg, 36.5 μmol) and tetrahydrofuran (5 mL) were added to a 50 mL two-necked flask. Then, under a nitrogen atmosphere at 0 °C, diisobutylaluminum hydride (1.0 M tetrahydrofuran solution, 1.0 mL) was added dropwise. The mixture was slowly brought to room temperature and stirred for 48 hours. The reaction was quenched with water (20 mL), extracted with ethyl acetate (40 mL × 3), and the organic layers were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was subjected to high-pressure preparation (column: XBridge Prep OBD C18 column, 30×150mm, 5µm; mobile phase A: water (10mmol / L ammonium bicarbonate + 0.05% ammonia), mobile phase B: acetonitrile; flow rate: 60mL / min; gradient: 50%B to 66%B over 10 minutes; wavelength: 254nm / 220nm) to give a white solid 5-(3-chloro-5-fluorobenzyl)-2-(4-chlorobenzyl)-1-(5-(hydroxymethyl)thiazolyl-2-yl)-1,2,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (1.7mg, yield 8%).

[0496] LCMS(ESI):519.1,521.1[M+H,M+H+2] + .

[0497] 1 H NMR(300MHz,CD3OD)δ7.33-7.27(m,2H),7.26-7.01(m,6H),5.21(s,2H),4 .82-4.73(m,2H),4.67-4.61(m,2H),3.73-3.63(m,2H),3.91-2.71(m,4H).

[0498] 19 F NMR (282MHz, CD3OD) δ-113.16 (s, 1F).

[0499] Example 40

[0500] The synthesis of (S)-2-(4-chlorobenzyl)-5-(3,5-difluorobenzyl)-1-(2-hydroxypropyl)-1,2,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one was carried out via the following synthetic route:

[0501] 2-(4-chlorobenzyl)-5-(3,5-difluorobenzyl)-2,3a,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (80.0 mg, 205 μmol), cesium carbonate (200 mg, 616 μmol), and N,N-dimethylformamide (2 mL) were added to a 10 mL microwave-safe tube. After stirring at room temperature for 10 minutes, (S)-2-methylethylene oxide (72.0 μL, 1.03 mmol) was added at 0 °C, and the reaction was allowed to proceed for 48 hours at room temperature. The reaction was quenched with water (10 mL), extracted with ethyl acetate (30 mL × 3), the organic phases were combined, washed with brine (100 mL × 2), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was subjected to high-pressure preparation (XBridge Prep OBD C18 column, 30×150mm, 5μm; mobile phase A: water (10mmol / L ammonium bicarbonate + 0.05% ammonia), mobile phase B: acetonitrile; flow rate: 60mL / min; gradient: 30%B to 60%B over 11 minutes; wavelength: 254nm / 220nm) to give a white solid (S)-2-(4-chlorobenzyl)-5-(3,5-difluorobenzyl)-1-(2-hydroxypropyl)-1,2,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (8.2 mg, yield 8%).

[0502] 1 H NMR(300MHz,CD3OD)δ(ppm)7.34-7.30(m,2H),7.16-7.14(m,2H),7.03-7.00(m,2H),6.89-6.82(m,1H),5.20-4.98(m ,2H),3.76-3.68(m,3H),3.64-3.54(m,2H),3.32(s,2H),2.86-2.76(m,3H),2.63-2.58(m,1H),1.07(d,J=6.3Hz,3H).

[0503] 19 FNMR(282MHz, CDCl3)δ(ppm)-112.223(s,2F).

[0504] LCMS: 448.0 [M+H] + .

[0505] Example 41

[0506] The synthesis of (R)-2-(4-chlorobenzyl)-5-(3,5-difluorobenzyl)-1-(2-hydroxypropyl)-1,2,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one was carried out via the following synthetic route:

[0507] 2-(4-chlorobenzyl)-5-(3,5-difluorobenzyl)-2,3a,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (100 mg, 256 μmol), cesium carbonate (251 mg, 770 μmol), and N,N-dimethylformamide (2 mL) were added to a 10 mL microwave-safe tube. After stirring at room temperature for 10 minutes, (R)-2-methylethylene oxide (92.0 mg, 1.28 mmol) was added at 0 °C, and the reaction was allowed to proceed for 48 hours at room temperature. The reaction was quenched with water (10 mL), extracted with ethyl acetate (30 mL × 3), the organic phases were combined, washed with brine (100 mL × 2), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was prepared under high pressure (XBridge Prep OBD C18 column, 30×150mm, 5μm; mobile phase A: water (10mmol / L ammonium bicarbonate + 0.05% ammonia), mobile phase B: acetonitrile; flow rate: 60mL / min; gradient: 30%B to 60%B over 11 minutes; wavelength: 254nm / 220nm) to obtain a colorless oily substance. -2-(4-chlorobenzyl)-5-(3,5-difluorobenzyl)-1-(2-hydroxypropyl)-1,2,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (11.2 mg, 9% yield).

[0508] LCMS: 448.0 [M+H] + .

[0509] 1 H NMR (400MHz, CD3OD) δ (ppm) 7.33-7.30 (m, 2H), 7.16-7.14 (m, 2H), 7.03-7.00 (m, 2H), 6.87-6.83 (m, 1H), 5.19-5.14 (m, 1H), 5.03-4.99 (m,1H),3.76-3.71(m,3H),3.68-3.55(m,2H),3.34(s,1H),3.29(s,1H),2.85-2.77(m,3H),2.62-2.59(m,1H),1.07(d,J=6.4Hz,3H).

[0510] 19 FNMR(376MHz,CD3OD)δ(ppm)-112.215(s,2F).

[0511] Example 42

[0512] Synthesis of 2-(4-chlorobenzyl)-5-(3,5-difluorobenzyl)-1-(2-hydroxy-2-methylpropyl)-1,2,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one

[0513] 2-(4-chlorobenzyl)-5-(3,5-difluorobenzyl)-2,3a,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (100 mg, 256 μmol), cesium carbonate (251 mg, 770 μmol), and N,N-dimethylformamide (2 mL) were added to a 10 mL microwave-safe tube. After stirring at room temperature for 10 minutes, 2-dimethylethylene oxide (92.0 mg, 1.28 mmol) was added at 0 °C, and the reaction was allowed to proceed for 7 days at room temperature. The reaction was quenched with water (5 mL), extracted with ethyl acetate (20 mL × 3), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was subjected to high-pressure preparation (XBridge Prep OBD C18 column, 30×150mm, 5μm; mobile phase A: water (10mmol / L ammonium bicarbonate + 0.05% ammonia), mobile phase B: acetonitrile; flow rate: 60mL / min; gradient: 36%B to 66%B over 10 minutes; wavelength: 254nm / 220nm) to give a white solid 2-(4-chlorobenzyl)-5-(3,5-difluorobenzyl)-1-(2-hydroxy-2-methylpropyl)-1,2,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one (10.0 mg, yield 8%).

[0514] LCMS:462.1[M+H] + .

[0515] 1 H NMR (400MHz, CD3OD) δ (ppm) 7.33-7.29 (m, 2H), 7.08-7.00 (m, 4H), 6.88-6.83 (m, 1H), 5.25(s,2H),3.74(s,2H),3.67(s,2H),3.32(s,2H),2.80-2.74(m,4H),1.15(s,6H).

[0516] 19 FNMR(376MHz,CD3OD)δ(ppm)-112.145(s,2F).

[0517] Examples of linker-ClpP agonists

[0518] Example 43

[0519] The synthesis route for AC-PL-1 is as follows:

[0520] SM1 (49 mg, 0.067 mmol) was dissolved in N,N-dimethylformamide (2 mL), and AC-P2 (30 mg, 0.067 mmol), 1-hydroxybenzotriazole (9 mg, 0.067 mmol), and pyridine (16 mg, 0.201 mmol) were added. The mixture was stirred at room temperature for 16 hours under an argon atmosphere. The product AC-PL-1 (44 mg, 63% yield) was obtained by direct preparation separation (30% MeCN in H2O / FA) purification to a white solid.

[0521] LCMS(ESI)[M+H] + =1045.6.

[0522] 1 H NMR (400MHz, DMSO-d6) δ10.00(s,1H),8.08(d,J=7.5Hz,1H),7.79(d,J=8.6Hz,1H),7.59(d,J=8.5Hz,2H),7.45–7.26(m,4H),7. 19(d,J=8.2Hz,1H),7.13(d,J=7.5Hz,2H),7.06(d,J=19.2Hz,2H),7.00(s,2H),5.97(t,J=5.8Hz,1H),5.41(s,2H),4.90(dd,J=5 0.4,28.5Hz,4H),4.37(d,J=5.3Hz,1H),4.28–4.11(m,1H),3.67(d,J=22.4Hz,4H),3.15–2.86(m,8H),2.66(d,J=11.2Hz,3H),2. 56(s,2H),2.15(ddd,J=22.1,21.7,15.4Hz,4H),1.95(dd,J=13.4,6.6Hz,1H),1.76–1.09(m,12H),0.82(dd,J=11.8,6.8Hz,6H).

[0523] Example 44

[0524] The synthesis route for AC-PL-2 is as follows:

[0525] SM2 (19 mg, 0.03 mmol) was dissolved in N,N-dimethylformamide (1 mL), followed by the sequential addition of N,N-diisopropylethylamine (12 mg, 0.09 mmol) and 2-(7-azobenzotriazole)-N,N,N′,N′-tetramethylurea hexafluorophosphate (15 mg, 0.04 mmol). The reaction mixture was stirred at room temperature for 10 minutes, then AC-P3 (13 mg, 0.03 mmol) was added, and the reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was then purified directly by reversed-phase chromatography (acetonitrile / water containing 0.05% formic acid = 5%-30%) to obtain the target compound AC-PL-2 (14 mg, 45%) as a white solid.

[0526] LCMS(ESI)[M+H]+=1031.5;

[0527] 1H NMR (400MHz, CD3OD) δ7.33–7.26(m,3H),7.26–7.22(m,3H),7.21–7.14(m,3H),7.14–7.12(m,1H),6.99( t,J=9.2Hz,1H),6.78(s,2H),5.10(s,2H),4.74–4.65(m,2H),4.47(dd,J=8.9,6.1Hz,1H),4.10(s,1H),3 .95(s,2H),3.88–3.80(m,6H),3.72(d,J=17.0Hz,1H),3.61(s,1H),3.46(t,J=7.1Hz,2H),3.20–3.14(m ,2H),3.05–2.98(m,1H),2.84–2.71(m,2H),2.24(t,J=7.5Hz,2H),1.66–1.53(m,4H),1.34–1.25(m,3H).

[0528] Examples of antibody-ClpP agonist conjugates

[0529] The antibodies used in the following examples are shown in the table below:

[0530] Example 45

[0531] AC-ADC1 (HER2, DAR3.7) Synthesis:

[0532] The antibody Ab was selected as the anti-HER2 antibody trastuzumab; the q value was selected from 4.

[0533] Column information: SHIMSEMAnkylo HIC Butyl

[0534] Elution conditions: 0%-100% gradient elution—25mM Na3PO4, 25% C3H8O, pH 7

[0535] Sample: Trastuzumab: 2.5 mg / ml

[0536] TCEP: 50 mmol / L

[0537] AC-PL-1AC-ADC: 2 mg / L

[0538] Coupling process:

[0539] 1. Incubate a mixed sample of trastuzumab and TCEP at 37°C;

[0540] 2. After 2 hours, remove the sample and add AC-PL-1 for a new round of incubation;

[0541] 3. After incubation for 1 hour, the coupled ADC was replaced with PBS by replacing the buffer solution using a desalting column.

[0542] 4. The naked antibody and ADC were concentrated using a suitable concentration tube to a final concentration of 10 mg / ml.

[0543] 5. Perform HPLC analysis on the concentrated sample.

[0544] Detailed experimental procedure:

[0545] The antibody was displaced into PBS (pH 7.2-7.4) buffer to obtain the antibody intermediate. 10 mg of the antibody intermediate was taken, and PBS (pH 7.2-7.4) buffer was added to a final volume of 1 mL. 50 mM tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP) stock solution was added, with a TCEP to antibody molar ratio of 2.5:1. The mixture was thoroughly mixed and placed in a metal bath at 37°C for 2 hours. After reduction, 2 mM payload-linker stock solution was added to the reaction system, with a payload to antibody molar ratio of 5:1. The mixture was thoroughly mixed and incubated at room temperature for 1 hour. After coupling, the ADC sample was desalted using a PD10 desalting column to displace the buffer solution into PBS. The sample was then concentrated using ultrafiltration centrifuge tubes to obtain the ADC stock solution, which was aliquoted and stored at -80°C.

[0546] The DAR value was determined by HPLC: DAR = 3.7

[0547] 1. AC-ADC1 (payload 3): The amplified conjugation results of 10mg are as follows: DAR 3.7 (black: trastuzumab; red: conjugated compound). The DAR value was determined by HPLC and the results are shown in Figure 1.

[0548] Example 46

[0549] Synthesis of AC-ADC2 (HER2, DAR4.4)

[0550] Antibody Ab Selection: Anti-HER2 antibody: Trastuzumab (DAR = 4.8)

[0551] Detailed experimental procedure:

[0552] Trastuzumab was displaced into PBS (pH 7.2-7.4) buffer to obtain the antibody intermediate. 10 mg of the antibody intermediate was taken, and PBS (pH 7.2-7.4) buffer was added to a final volume of 1 mL. 50 mM tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP) stock solution was added, with a TCEP to antibody molar ratio of 2.5:1. The mixture was thoroughly mixed and placed in a metal bath at 37°C for 2 hours. After reduction, 2 mM payload-linker stock solution was added to the reaction system, with a payload to antibody molar ratio of 5:1. The mixture was thoroughly mixed and incubated at room temperature for 1 hour. After conjugation, the ADC sample was desalted using a PD10 desalting column to displace the buffer solution into PBS. The sample was then concentrated using ultrafiltration centrifuge tubes to obtain the ADC stock solution, which was aliquoted and stored at -80°C.

[0553] Sample information:

[0554] Antibody: Trastuzumab WM:145531.5d

[0555] payload linker:AC-PL-2WM:1031.51d

[0556] The results of AC-PL-2:10mg conjugation are as follows: DAR = 4.8

[0557] The HPLC determination results of AC-ADC2 are shown in Figure 2.

[0558] Peak value table

[0559] PDACh1280nm

[0560] Example 47

[0561] Synthesis of AC-ADC3 (HER2, DAR8)

[0562] The antibody chosen was the anti-HER2 antibody trastuzumab (DAR = 7.5).

[0563] reduction

[0564] Take 680.7 μL of DPBS buffer solution into a 1.5 mL centrifuge tube, add 249.9 μL of Trastuzumab naked antibody solution (20.01 mg / mL), and add 69.44 μL of 5 mmol / L reducing agent TCEP (tris(2-carboxyethyl)phosphine). The final concentration of naked antibody in the reduction reaction system is 5 mg / mL, and the molar ratio of reducing agent to naked antibody is 10:1. React at 25 °C for 2 hours.

[0565] Couplet

[0566] After the reduction reaction was complete, 68.1 μL of dimethyl sulfoxide (DMSO) was added and mixed well, followed by 43.0 μL of payload4 solution with a concentration of 10 mg / mL. Ultimately, the volume ratio of DMSO in the coupling reaction system was 10%, and the molar ratio of the small molecule to the naked antibody was 12:1. The reaction was continued at 25 °C for 1 hour.

[0567] purification

[0568] After the coupling reaction was completed, centrifugation purification was performed using a desalting column to remove free small molecules. The product was then transferred to a 10 mM NaAc-HAc, pH 5.0 buffer solution. The final product was filtered through a 0.22 μm syringe filter, and its concentration, DAR value, and purity were then measured.

[0569] The DAR value measurement data of AC-ADC3 is shown in Figure 3 below: DAR = 7.5

[0570] Preparation of anti-HER2 antibodies

[0571] The heavy chain (SEQ ID NO:1) and light chain (SEQ ID NO:2) sequences of the target antibody were synthesized and constructed into commercial expression vectors from Haoyang Biotechnology Co., Ltd., named HSP102-GS1 and HSP102-GS3, respectively. The target antibody expression plasmids HSP102-GS1 and HSP102-GS3 were co-transfected into CHO-K1 cells using Freestyle MAX. Cell selection was performed under pressure using the selection markers on the vectors, and cell lines stably expressing the target antibody were obtained through monoclonalization. The cell lines were expanded and fed-batch cultured. The supernatant was collected and purified by affinity using Protein A to obtain the target anti-HER2 antibody, Trastuzumab.

[0572] The preparation methods for AC-ADC4, 5 and 6 are the same as those for AC-ADC3.

[0573] Example 48

[0574] AC-ADC4:

[0575] Example 49

[0576] AC-ADC5:

[0577] Example 50

[0578] AC-ADC6:

[0579] The results for AC-ADC4, 5, and 6 are as follows: (HER3, Trop2, CD147)

[0580] The DAR values ​​of AC-ADC4, 5, and 6 were detected by HPLC, and the detection data are as follows:

[0581] Biological evaluation

[0582] 1. Cell viability assay with load

[0583] The cell lines used in this section are all commercially available, as follows:

[0584] 1.1 Activity assays of different loads on the OCI-AML3 cell line

[0585] use The (CTG) luminescent cell viability assay kit measures the antiproliferative effect of a test substance on cell lines by quantifying ATP. Cells are seeded in 96-well plates and treated with different concentrations of the test substance for 5 days, with each test concentration performed in triplicate.

[0586] First, collect test cells in the logarithmic growth phase, count the cell suspension, and dilute to the desired concentration. Add 50 μL of cell suspension to each well of a 96-well plate. The number of cells seeded per well varies depending on cell size and growth rate; approximately 1000-3000 adherent cells are seeded per well, while approximately 10000 suspension cells are seeded per well. Set up at least three blank control wells containing only culture medium (100 μL / well) and no cells on the same culture plate to obtain background luminescence values. Incubate adherent cells overnight and suspension cells for at least 2 hours.

[0587] Add 20 μl of 0.4 mM analyte to the first V-bottom well, then add 5 μl to 20 μl of DMSO and mix well. Dilute in a 5-fold gradient to obtain 10 concentration gradients including the control.

[0588] Add 1.5 μL of the diluted test compound to 298.5 μL of culture medium, mix well, and then add 50 μL of the culture medium containing the test compound to the cell suspension as shown in the figure. Add 50 μL of control culture medium to the control wells. The final working concentration of the test compound is 1 μM, 5-fold dilution, 3 replicates. After administration, continue incubation in an incubator for 120 hours.

[0589] At the end of the incubation, remove the 96-well plate from the incubator and, after equilibration to room temperature, add 30 μL of [a specific ingredient] to each well. Reagents. Place the 96-well plate on a microplate shaker and mix the contents for 3 minutes to ensure complete cell lysis. Incubate the 96-well plate at room temperature for 10 minutes to allow the fluorescence signal to stabilize. Detect the fluorescence signal using a microplate reader.

[0590] Calculate the percentage of cell viability using the following formula:

[0591] Cell viability percentage (%) = (fluorescence signal value of test wells - fluorescence signal value of blank control wells) / (fluorescence signal value of control wells - fluorescence signal value of blank control wells) × 100%

[0592] GraphPadPrism 10 (GraphPad Software Inc., San Diego, CA) software was used to plot curves and calculate IC50 (pM, 10). -12 M).

[0593] The following known loads were also used for comparison in this experiment:

[0594] The activity test data of different loads on the OCI-AML3 cell line are shown in the table below:

[0595] The above results demonstrate that compound D, used in the preparation of ADCs according to the present invention, exhibits ClpP agonist activity regardless of the substituents used in the general formula, and thus possesses the potential to act as a small molecule toxin in ADCs. Specifically, the IC50 values ​​of AC-P1–5, 8–12, and 19–22 are all less than 0.001 μM, indicating better cytotoxic activity. Furthermore, compared to the reference compound, the cellular activity of the small molecule-loaded compounds is significantly enhanced.

[0596] 1.2. Comparison of the activity of different loads in different stem cell lines

[0597] The following loads were also used for comparison in this section:

[0598] The activity comparison of different loadings in different cell lines is shown in the table below and Figure 4: IC50 loadings (pM, 120hr)

[0599] The above test results show that the drug D of the present invention is active in different cell lines. Furthermore, the activity of the small molecule loading of the present invention is between that of MMAE and PBD, and higher than that of Exatecan.

[0600] 2. Cell viability assay of ADC

[0601] 2.1 Activity assays of different ADCs in the NCI-N87 cell line

[0602] use The (CTG) luminescent cell viability assay kit measures the antiproliferative effect of a test substance on cell lines by quantifying ATP. Cells are seeded in 96-well plates and treated with different concentrations of the test substance for 5 days, with each test concentration performed in triplicate.

[0603] First, collect test cells in the logarithmic growth phase, count the cell suspension, and dilute to the desired concentration. Add 50 μL of cell suspension to each well of a 96-well plate. The number of cells seeded per well varies depending on cell size and growth rate; approximately 1000-3000 adherent cells are seeded per well, while approximately 10000 suspension cells are seeded per well. Set up at least three blank control wells containing only culture medium (100 μL / well) and no cells on the same culture plate to obtain background luminescence values. Incubate adherent cells overnight and suspension cells for at least 2 hours.

[0604] Add the corresponding volume of ADC to the culture medium and mix well to make the final concentration of the first well 100 μg / ml and the final volume 300 μl. Then take 60 μl and add it to 240 μl of culture medium and mix well. Dilute according to a 5-fold gradient to obtain 10 concentration gradients including the control.

[0605] Add 50 μL of diluted ADC drug-containing culture medium to each of the 96-well plates containing cells, and add 50 μL of control culture medium to each control well, so that the initial working concentration of ADC is 50 μg / ml. Dilute 5-fold and repeat 3 times. Incubate for 120 hours.

[0606] At the end of the incubation, remove the 96-well plate from the incubator and, after equilibration to room temperature, add 30 μL of [a specific ingredient] to each well. Reagents. Place the 96-well plate on a microplate shaker and mix the contents for 3 minutes to ensure complete cell lysis. Incubate the 96-well plate at room temperature for 10 minutes to allow the fluorescence signal to stabilize. Detect the fluorescence signal using a microplate reader.

[0607] Calculate the percentage of cell viability using the following formula:

[0608] Cell viability percentage (%) = (fluorescence signal value of test wells - fluorescence signal value of blank control wells) / (fluorescence signal value of control wells - fluorescence signal value of blank control wells) × 100%

[0609] The graphs were plotted using GraphPadPrism 10 (GraphPad Software Inc., San Diego, CA), and the IC50 was calculated.

[0610] The activity tests of different ADCs in the NCI-N87 cell line are shown in the table below and Figure 5:

[0611] The above test results show that the antibody-drug conjugates AC-ADC1, 2 and 3 targeting HER2 disclosed in this paper have significant inhibitory activity against the proliferation of HER2-positive NCI-N87 cells, with AC-ADC3 showing significantly better activity than DS-8201.

[0612] 2.2 Activity assay of AC-ADC1 in different cell lines

[0613] The activity of AC-ADC1 in different cell lines was tested at IC50s (μg / ml, 120hr), and the results are shown in the table below and Figure 6.

[0614] The above test results show that AC-ADC1 has significant inhibitory activity against the proliferation of HER2-positive NCI-N87 cells; at the same time, it has weak inhibitory activity against the proliferation of MDA-MB-231 and HCC44 cells with low HER2 expression, showing a certain selectivity for HER2 expression level; in addition, it also shows a certain selectivity in different cell lines. Although the HER2 expression level of SK-OV-3 cells is comparable to that of NCI-N87, the activity is very different.

[0615] 2.3 Cell viability assays (IC50s, ng / ml, 120hr) for ADCs with different loads and linkers are shown in the table below and Figure 7.

[0616] The above test results indicate that the antibody-drug conjugates targeting HER2 disclosed in this paper have significant inhibitory activity against the proliferation of HER2-positive NCI-N87 cells. Simultaneously, they exhibit weaker inhibitory activity against the proliferation of MDA-MB-231 cells with lower expression levels, demonstrating a certain degree of selectivity. This also suggests that the activity of the ADC is significantly correlated with the activity of the loading, and the linker may also have some influence.

[0617] 2.4. The IC50s (ng / ml, 120hr) of ADCs with different DAR values ​​at the HER2 target site on different cell lines are shown in the table below and Figure 8.

[0618] The above test results indicate that the antibody-drug conjugates targeting HER2 disclosed herein have significant inhibitory activity against the proliferation of HER2-positive NCI-N87 cells. Simultaneously, they exhibit weaker inhibitory activity against the proliferation of MDA-MB-231 cells with lower expression levels, demonstrating a certain degree of selectivity. Furthermore, it is shown that the activity of DAR8 is significantly higher than that of DAR4, and AC-ADC3 shows more than a tenfold increase in activity compared to AC-ADC2.

[0619] 2.5 The activity assays of AC-ADC3 (AC-ADC-HER2) on different cell lines were performed using IC50s (ng / ml, 120hr), as shown in the table below and Figure 9.

[0620] The above test results show that the antibody-drug conjugate AC-ADC3 targeting HER2 has significant inhibitory activity on the proliferation of HER2-positive NCI-N87 cells. At the same time, it has weak inhibitory activity on the proliferation of MDA-MB-231 cells with weaker expression levels, showing a certain degree of selectivity.

[0621] 2.6 Cell viability assays (IC50s, ng / ml, 120hr) for different AC-ADC4 (AC-HER3-ADC) cell lines are shown in the table below and Figure 10.

[0622] The above test results show that the antibody-drug conjugate AC-ADC4 targeting HER3 has significant inhibitory activity against the proliferation of HER3-positive cells MDA-MB-453. At the same time, it has weak inhibitory activity against the proliferation of PANC-1 and NCI-N87 cells with weaker expression levels, showing a certain degree of selectivity.

[0623] 2.7 Cell viability tests of different AC-ADC5 (AC-TROP2-ADC) cell lines are shown in the table below and Figure 11.

[0624] The above test results show that the antibody-drug conjugate AC-ADC5 targeting TROP2 disclosed in this paper has significant inhibitory activity against the proliferation of TROP2-positive cells PANC-1. At the same time, it has weak inhibitory activity against the proliferation of Fadu and NCI-N87 cells with weaker expression levels, showing a certain degree of selectivity.

[0625] 2.8 Cell viability tests of different AC-ADC6 (AC-CD147-ADC) cell lines are shown in the table below and Figure 12.

[0626] The above test results show that the antibody-drug conjugates targeting CD147 disclosed herein have significant inhibitory activity against the proliferation of CD147-positive Fadu cells; at the same time, they also have good inhibitory activity against the proliferation of PANC-1 and MDA-MB-453 cells.

[0627] The test results above demonstrate that, after being conjugated with different antibodies and linkers, the drug D of this invention exhibits selectivity in its inhibitory effect on cell proliferation based on the conjugated antibody. Furthermore, it retains cell proliferation inhibitory activity even when different linkers are used.

[0628] 3. PD Tumor Experiment with ADC

[0629] 3.1 Anti-tumor effect of antibody-drug conjugate targeting HER2 on human gastric cancer cell line NCI-N87 in a Balb / c Nude mouse subcutaneous xenograft model

[0630] 3.1.1 Experimental Objective

[0631] This study investigated the antitumor effects of HER2-targeting antibody-drug conjugates Enhertu and AC-ADC on human gastric cancer cell line NCI-N87 in a Balb / c Nude mouse subcutaneous xenograft model.

[0632] 3.1.2 Experimental Materials

[0633] i. Test drug

[0634] Blank control: PBS

[0635] Enhertu: 6mg / kg

[0636] AC-ADC1 (DAR=4): 6 mg / kg

[0637] AC-ADC2 (DAR=4): 6 mg / kg

[0638] AC-ADC3 (DAR=8): 6 mg / kg

[0639] ii. Laboratory animals

[0640] Balb / c nude mice, female, 5-6 weeks old, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.

[0641] 3.1.3 Experimental Methods

[0642] The harvested fresh NCI-N87 tumor tissue was cut into 30mm pieces. 3 A small piece was implanted subcutaneously below the right scapula of a mouse. When the average tumor volume grew to approximately 120 mm... 3 In this study, tumor-bearing mice were randomly divided into groups of five based on tumor size. The drug was administered via tail vein injection once. Tumor volume and mouse weight were measured twice weekly. Tumor volume (V) was calculated as follows: V = (length × width × width) / 2. The antitumor efficacy of the test drug was assessed using the relative tumor growth inhibitory rate (TGI). The calculation method was as follows: TGI% = (1 - T / C) × 100%. Where T and C are the relative tumor volumes (RTV) of the experimental group and the negative control group at a specific time point, respectively, and T / C% is the relative tumor growth rate, i.e., the percentage of relative tumor volumes (RTV) between the experimental group and the negative control group at a specific time point. The calculation formula is as follows: T / C% = TRTV / CRTV × 100% (TRTV: average RTV of the experimental group; CRTV: average RTV of the negative control group; RTV = Vt / V0, where V0 is the tumor volume of the animal at the time of grouping, and Vt is the tumor volume of the animal after treatment).

[0643] 3.1.4 Experimental Results

[0644] The data of the HER2ADC xenograft NCI-N87 model are shown in the table below and Figure 13.

[0645] The table above and Figure 13 show that, at a single dose of 6 mg / kg, the ADC drug of the present invention has an antitumor effect, and the antibody-drug conjugate ADC3 shows a stronger antitumor effect than Enhertu without showing a decrease in mouse weight.

[0646] 3.2 Antitumor effect of TROP2-targeting antibody-drug conjugate AC-ADC5 on human gastric cancer cell line NCI-N87 in a Balb / c Nude mouse subcutaneous xenograft model

[0647] 3.2.1 Experimental Objective

[0648] This study investigated the antitumor effect of the antibody-drug conjugate AC-ADC5 targeting TROP2 on the human gastric cancer cell line NCI-N87 in a Balb / c Nude mouse subcutaneous xenograft model.

[0649] 3.2.2 Experimental Materials

[0650] i. Test drug

[0651] Blank control: PBS

[0652] AC-ADC5: 3, 1.5, 0.75 mg / kg

[0653] ii. Laboratory animals

[0654] Balb / c nude mice, female, 5-6 weeks old, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.

[0655] 3.2.3 Experimental Methods

[0656] The harvested fresh NCI-N87 tumor tissue was cut into 30mm pieces. 3 A small piece was implanted subcutaneously below the right scapula of a mouse. When the average tumor volume grew to approximately 120 mm... 3 In this study, tumor-bearing mice were randomly divided into groups of five based on tumor size. The drug was administered via tail vein injection once. Tumor volume and mouse weight were measured twice weekly. Tumor volume (V) was calculated as follows: V = (length × width × width) / 2. The antitumor efficacy of the test drug was assessed using the relative tumor growth inhibitory rate (TGI). The calculation method was as follows: TGI% = (1 - T / C) × 100%. Where T and C are the relative tumor volumes (RTV) of the experimental group and the negative control group at a specific time point, respectively, and T / C% is the relative tumor growth rate, i.e., the percentage of relative tumor volumes (RTV) between the experimental group and the negative control group at a specific time point. The calculation formula is as follows: T / C% = TRTV / CRTV × 100% (TRTV: average RTV of the experimental group; CRTV: average RTV of the negative control group; RTV = Vt / V0, where V0 is the tumor volume of the animal at the time of grouping, and Vt is the tumor volume of the animal after treatment).

[0657] 3.2.4 Experimental Results

[0658] The data of the Trop2 ADC xenograft model are shown in the table below and Figure 14.

[0659] The table above and Figure 14 show that when mice were given single intravenous injections of ADC5 at doses of 3, 1.5, and 0.75 mg / kg, respectively, their total glycemic index (TGI) was 85.3%, 53.3%, and 31.4% at 41 days after the end of the experiment, exhibiting a certain dose-related correlation. No weight loss was observed in the mice at any of the three doses.

[0660] The embodiments described in this invention are for illustrative purposes only and do not constitute a limitation on the scope of the claims. Other substantially equivalent substitutions that can be conceived by those skilled in the art are all within the protection scope of this invention.

Claims

1. An antibody-ClpP agonist conjugate, characterized in that... Selected from compounds having the structure of Formula I, or their derivatives, tautomers, stereoisomers, pharmaceutically acceptable salts or esters, and solvates. in, Ab is an antibody, L is a linker, the linker includes one or more sub-linker units, q = 1 to 20, and D is a compound of formula AC-P before binding with L, or a tautomer, stereoisomer, or pharmaceutically acceptable salt thereof with ClpP agonist activity. in, R a2 , each of R b2 is independently selected from hydrogen or deuterium, The structure of ring A is as follows: i is an integer from 1 to 4, where R a Each of these is independently selected from hydrogen, halogen, cyano, -R a1 -R c or -R a1 -N(R c1 )-Y,R a1 Each of these is independently selected from non-existent, -CH2-, -C(O)-, or -CH2-C(O)-; The ring B structure is j is an integer from 1 to 4, where R b Each of these radicals is independently selected from hydrogen, deuterium, halogen, cyano, -R b1 -R c or -R b1 -N(R c1 )-Y,R b1 Each of these is independently selected from non-existent, -CH2-, -C(O)-, or -CH2-C(O)-; R 1 is selected from the group consisting of hydrogen, C1-C3alkyl, C1-C3alkyl substituted with a 3-6 membered nitrogen-containing cycloalkyl, -CH2-C(R 11 )2-R c , -CH2-C(R 11 )2-N(R c1 )-Y, -R 12 -R c , or -R 12 -N(R c1 )-Y, wherein each of R 11 is independently selected from H or methyl, and each of R 12 is independently selected from one of C1-C3alkyl, phenyl, 5-6 membered heteroaryl, a combination of C1-C3alkyl and phenyl, or a combination of C1-C3alkyl and 5-6 membered heteroaryl; Each of the Y terms is independently selected from -C(O)-C(R) y1 )2-(CH2) 0-1 -OH, -C(O)-R y2 -(CH2) 0-2 -OH、 in, The R mentioned y1 Selected from item i) or item ii): i) One of R y1 Selected from C1-C3 alkyl groups and 3-6 cycloalkyl groups, wherein the alkyl or cycloalkyl group may optionally be substituted with one or more halogens, and the other R y1 For hydrogen, ii). Two R y1 It forms a 3- to 6-membered cycloalkyl group with the attached carbon. The R mentioned y2 Selected from 3- to 6-membered cycloalkyl, 3- to 6-membered cycloalkyl, phenyl, C1- to C3 alkynyl, n4＝1～8， k is selected from 1 or 2, said R x is selected from item iii) or item iv): iii). Each R x is independently selected from hydrogen, deuterium, halogen, carbonyl, -R x1 c or -R x1 -N(R c1 )-Y, R x1 Each of R is independently selected from absent, -CH2-, -C(O)-, or -CH2-C(O)-; iv). Two R x form a 3- to 6-membered cycloalkyl or heterocycloalkyl ring with the carbon to which they are attached;​ each of R c is selected from -OH or -NHR c1 , R c1 is independently selected from hydrogen or methyl, The ring A, ring B, R 1 at least one -OH, -NH2or -NH- is present, and the compound represented by AC-P is bound by the -OH, -NH2or -NH- contained reacting with a linker.

2. The antibody-ClpP agonist conjugate as described in claim 1, characterized in that... Formula AC-P is selected from:

3. The antibody-ClpP agonist conjugate as described in claim 1 or 2, characterized in that... The i = 1 to 2.

4. The antibody-ClpP agonist conjugate as described in claim 3, characterized in that... The ring A mentioned above is selected from:

5. The antibody-ClpP agonist conjugate as described in claim 4, characterized in that... The R mentioned a Each of them is independently selected from hydrogen, deuterium, halogen, or cyano.

6. The antibody-ClpP agonist conjugate as described in claim 5, characterized in that... The ring A mentioned above is selected from:

7. The antibody-ClpP agonist conjugate as described in claim 4, characterized in that... at least one R a selected from -R a1 -R c or -R a1 -N(R c1 )-Y.

8. The antibody-ClpP agonist conjugate as described in claim 7, characterized in that... there is at least one R a selected from -R a1 -R c .

9. The antibody-ClpP agonist conjugate as described in claim 8, characterized in that... there is at least one R a is selected from -CH2-NH2.

10. The antibody-ClpP agonist conjugate as described in claim 9, characterized in that... The ring A mentioned above is selected from:

11. The antibody-ClpP agonist conjugate as described in claim 1 or 2, characterized in that... The j = 1 to 2.

12. The antibody-ClpP agonist conjugate as described in claim 11, characterized in that... The ring B mentioned is selected from: Selected from 13. The antibody-ClpP agonist conjugate as described in claim 12, characterized in that... each of said R b is independently selected from halogen or cyano.

14. The antibody-ClpP agonist conjugate as described in claim 13, characterized in that... The ring B mentioned is selected from:

15. The antibody-ClpP agonist conjugate as described in claim 12, characterized in that... one of R b selected from -R b1 -R c or -R b1 -N(R c1 )-Y.

16. The antibody-ClpP agonist conjugate as described in claim 15, characterized in that... one of R b selected from -R b1 -R c .

17. The antibody-ClpP agonist conjugate as described in claim 16, characterized in that... one of R b is selected from -OH or -CH2-OH.

18. The antibody-ClpP agonist conjugate as described in claim 17, characterized in that... The ring B mentioned is selected from:

19. The antibody-ClpP agonist conjugate as described in claim 16, characterized in that... R b1 each of R is independently selected from -CH2-, -C(O)-, or -CH2-C(O)-, R c is selected from -NH2.

20. The antibody-ClpP agonist conjugate as described in claim 19, characterized in that... The ring B mentioned is selected from:

21. The antibody-ClpP agonist conjugate as described in claim 12, characterized in that... there is at least one R b selected from -R b1 -N(R c1 )-Y.

22. The antibody-ClpP agonist conjugate as described in claim 21, characterized in that... The ring B mentioned is selected from:

23. The antibody-ClpP agonist conjugate as described in claim 1 or 2, characterized in that... The R mentioned 1 Selected from -CH2-C(R) 11 )2-R c or -CH2-C(R) 11 )2-N(R c1 )-Y.

24. The antibody-ClpP agonist conjugate as described in claim 23, characterized in that... R is selected from -CH2-C(R 1 R is selected from -CH2-C(R 11 )2-R c .

25. The antibody-ClpP agonist conjugate as described in claim 24, characterized in that... The R mentioned c It is -OH.

26. The antibody-ClpP agonist conjugate as described in claim 25, characterized in that... The R mentioned 1 Selected from:

27. The antibody-ClpP agonist conjugate as described in claim 24, characterized in that... R c -NHR c1 .

28. The antibody-ClpP agonist conjugate as described in claim 27, characterized in that... The R mentioned 1 Selected from:

29. The antibody-ClpP agonist conjugate as described in claim 1 or 2, characterized in that... R 1 selected from -R 12 -R c .

30. The antibody-ClpP agonist conjugate as described in claim 29, characterized in that... The R mentioned 1 Selected from:

31. The antibody-ClpP agonist conjugate as described in claim 1 or 2, characterized in that... The R mentioned 1 Selected from -CH2-C(R) 11 )2-N(R c1 )-Y.

32. The antibody-ClpP agonist conjugate as described in claim 31, characterized in that... The R mentioned 1 Selected from:

33. The antibody-ClpP agonist conjugate as described in claim 1 or 2, characterized in that... The R mentioned 1 Selected from:

34. The antibody-ClpP agonist conjugate as described in claim 1 or 2, characterized in that... The Y mentioned is selected from: -C(O)-C(R y1 )2-(CH 2)0-1 -OH.

35. The antibody-ClpP agonist conjugate as described in claim 34, characterized in that: R y1 The term Y is selected from item i, and Y is selected from: Or R y1 The term Y is selected from item ii, and Y is selected from:

36. The antibody-ClpP agonist conjugate as described in claim 1 or 2, characterized in that... The Y mentioned is selected from: -C(O)-R y2 -(CH 2)0-2 -OH.

37. The antibody-ClpP agonist conjugate as described in claim 2 or 36, characterized in that... The R mentioned y2 Selected from 38. The antibody-ClpP agonist conjugate as described in claim 37, characterized in that... The Y mentioned is selected from:

39. The antibody-ClpP agonist conjugate as described in claim 1 or 2, characterized in that... The Y mentioned is selected from:

40. The antibody-ClpP agonist conjugate as described in claim 1 or 2, characterized in that... The Y mentioned is selected from:

41. The antibody-ClpP agonist conjugate as described in claim 1 or 2, characterized in that... The AC-P structure mentioned above is selected from: in, R a each of R is independently selected from hydrogen, halogen, cyano, -R a1 -R c or -R a1 -N(R c1 )-Y, R a1 each of R is independently selected from absent, -CH2-, -C(O)-, or -CH2-C(O)-; R b each of R is independently selected from hydrogen, halogen, cyano, -R b1 -R c or -R b1 -N(R c1 )-Y, R b1 each of R is independently selected from absent, -CH2-, -C(O)-, or -CH2-C(O)-; R 1 is selected from -CH2-C(R 11 )2-R c or -CH2-C(R 11 )2-N(R c1 )-Y, wherein each of R 11 is independently selected from H or methyl; each of said Y is independently selected from -C(O)-CH(R y1 -OH, R y1 is selected from hydrogen or methyl.

42. The antibody-ClpP agonist conjugate as described in claim 41, characterized in that... The AC-P structure mentioned above is selected from: in, R am selected from -F or -CN, R an selected from -H or -F, R bn selected from F or Cl, R bm selected from -H, -CH2-OH, -CH2-NH2, -CH2-NH-C(O)-CH2-OH, -CH2-C(O)-NH2, R 1 Selected from 43. The antibody-ClpP agonist conjugate as described in claim 1, characterized in that... Formula AC-P is selected from the structures shown in the table below:

44. The antibody-ClpP agonist conjugate as described in claim 1, characterized in that... Formula AC-P is selected from the structures shown in the table below:

45. The antibody-ClpP agonist conjugate as described in claim 1 or 2, characterized in that... The connector has the following structure: in, L1 is selected from L 1 In the formula, # indicates the site of attachment to the antibody, and the wavy line indicates the site of attachment to L 2 . L2 is selected from L 2 In the formula, n1 = 0-5, n2 = 0-10, n3 = 0-3, and the # symbol indicates that it is related to L. 1 The connection site, based on L 3 L 4 Whether it exists or not, the wavy line represents the adjacent L. 3 L 4 Or the connection site of D; L3 does not exist or is selected from In L3, the # symbol indicates a relationship with L. 2 The connection site, based on L 4 Whether it exists or not, the wavy line represents the adjacent L. 4 Or the connection site of D; L4 does not exist or is selected from L 4 In the middle, R L Independently selected from hydrogen, C1-C3 straight-chain or branched alkyl, C1-C3 straight-chain or branched alkyl hydroxyl, C1-C3 straight-chain or branched alkylamine, based on L 3 Whether it exists or not, the # symbol indicates that it is adjacent to the L. 2 L 3 The connection points are indicated by wavy lines, which represent the connection points with D.

46. ​​The antibody-ClpP agonist conjugate as described in claim 1 or 2, characterized in that... The antibody specifically binds to the tumor antigen.

47. The antibody-ClpP agonist conjugate as described in claim 46, characterized in that... The antibodies mentioned are selected from anti-EGFR antibody, anti-DLL-3 antibody, anti-PSMA antibody, anti-CD70 antibody, anti-MUC16 antibody, anti-ENPP3 antibody, anti-TDGF1 antibody, anti-CCK-BR antibody, anti-MSLN antibody, anti-TIM-1 antibody, anti-LRRC15 antibody, anti-LIV-I antibody, anti-CanAg / AFP antibody, anti-claudin 18.2 antibody, anti-Mesothelin antibody, anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-c-MET antibody, anti-SLITRK6 antibody, anti-KIT / CD117 antibody, anti-STEAP1 antibody, anti-SLAMF7 / CS1 antibody, anti-NaPi2B / SLC34A2 antibody, anti-GPNMB antibody, anti-HER3 (ErbB3) antibody, anti-MUC1 / CD227 antibody, anti-AXL antibody, anti-CD166 antibody, anti-B7-H3 (CD276) antibody, anti-PTK7 / CCK4 antibody, and anti-PRL antibody. R antibody, anti-EFNA4 antibody, anti-5T4 antibody, anti-NOTCH3 antibody, anti-Nectin4 antibody, anti-TROP-2 antibody, anti-CD142 antibody, anti-CA6 antibody, anti-GPR20 antibody, anti-CD174 antibody, anti-CD71 antibody, anti-EphA2 antibody, anti-LYPD3 antibody, anti-FGFR2 antibody, anti-FGFR3 antibody, anti-FRα antibody, anti-CEACAMs antibody, anti-GCC antibody, anti-IntegrinAv antibody, anti-CAIX antibody, anti-P-cadherin antibody, anti-GD3 antibody, anti-Cadherin 6. Antibodies, including anti-LAMPI antibody, anti-FLT3 antibody, anti-BCMA antibody, anti-CD79b antibody, anti-CD19 antibody, anti-CD33 antibody, anti-CD56 antibody, anti-CD74 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD37 antibody, anti-CD47 antibody, anti-CD138 antibody, anti-CD352 antibody, anti-CD25 antibody, anti-CD147 antibody, or anti-CD123 antibody.

48. The antibody-ClpP agonist conjugate as described in claim 1 or 2, characterized in that... q＝1～12。 49. The antibody-ClpP agonist conjugate as described in claim 48, characterized in that... q = 2, 4 or 8.

50. The antibody-ClpP agonist conjugate as described in claim 1 or 2, characterized in that... Selected from the following structure:

51. The antibody-ClpP conjugate as described in claim 2, characterized in that... Selected from the following structure: Among them, Ab1 is Trastuzumab, Ab2 is Sacituzumab, Ab3 is Patritumab, and Ab5 is DS-1471.

52. The antibody-ClpP agonist conjugate as described in claim 1, characterized in that... Each of the Rx is independently selected from hydrogen, deuterium, fluorine, and methyl.

53. A linker-ClpP agonist conjugate for preparing the antibody-ClpP agonist conjugate according to any one of claims 1 to 52.

54. The linker-ClpP agonist conjugate as described in claim 53, characterized in that... Selected from:

55. The use of the antibody-ClpP conjugate of any one of claims 1 to 52 in the preparation of a medicament for the treatment or prevention of: tumors associated with HER2, TROP2, CD147 or HER3 expression, aging-related diseases or diseases associated with mitochondrial dysfunction.

56. The application as described in claim 55, characterized in that... The tumors mentioned include solid tumors or hematologic malignancies such as breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, stomach cancer, endometrial cancer, salivary gland cancer, esophageal cancer, lung cancer, colon cancer, rectal cancer, colorectal cancer, bone cancer, skin cancer, thyroid cancer, prostate cancer, melanoma, glioma, neuroblastoma, glioma multiforme, sarcoma, lymphoma, and leukemia.

57. The application as described in claim 56, characterized in that... The antibody-ClpP conjugate is selected from:

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