Targeted degradation agents for HIV-1 NEF for the treatment of HIV disease

Abstract

Compounds of formula I: TIFF2026521262000196.tif2378 [wherein the formula, the ligand that binds to the Nef protein (NB) is covalently bound to the ligand that binds to the E3 ligase cereblon (CB) via a linker (L)], or its stereoisomers, isotopes, tautomers, or pharmaceutically acceptable salts. The compounds may be used to treat HIV-related conditions of interest, to inhibit the biological function of Nef, or to inhibit the activity of Nef-dependent kinases or any other cellular function affected by Nef.

Description

[Technical Field]

[0001] This application claims the benefit of priority from U.S. Provisional Application No. 63 / 522,932, filed on 23 June 2023, which is incorporated herein by reference.

[0002] Approval of government support This invention was made with government support under grant number AI155054, awarded by the National Institutes of Health. The government has certain rights to this invention. [Background technology]

[0003] background Existing antiretroviral drugs do not eliminate HIV-1 from infected individuals and require lifelong administration to prevent relapse, highlighting the critical need for alternative therapeutic targets. The HIV-1 Nef cofactor is particularly appealing in this regard because it is crucial to the HIV-1 lifecycle in vivo and promotes immune evasion of HIV-infected cells, partly through MHC-I downregulation. However, Nef lacks enzymatic activity and an active site, which complicates conventional mediochemical optimization of existing occupation-based inhibitors due to the lack of correlation between inhibitor analog binding affinity to Nef in vitro and antiretroviral activity in cell assays. Furthermore, Nef functions in both monomeric and dimeric forms. Nef degraders antagonize all undesirable effects of Nef by reducing or completely eliminating levels of the Nef protein. Nef degraders represent an innovative approach to antiretroviral therapy that may provide a pathway to eradicating the viral reservoir. [Overview of the project] [Means for solving the problem]

[0004] overview Compounds of formula I: [ka] [In the formula, the ligand that binds to the Nef protein (NB) is covalently bound to the ligand that binds to the E3 ligase cereblon (CB) via a linker (L)] or stereoisomers, isotopes, tautomers, or pharmaceutically acceptable salts thereof are disclosed herein.

[0005] The compounds may be used to treat the target HIV-related condition, to inhibit the biological function of Nef, or to inhibit the activity of Nef-dependent kinases or any other cellular function affected by Nef.

[0006] The above will become clearer from the following detailed explanation, which will proceed with reference to the attached drawings. [Brief explanation of the drawing]

[0007] [Figure 1] Targeted degradation of HIV-1 Nef by a CRBN-based PROTAC. The cereblon (CRBN) ubiquitin E3 ligase complex (left) is a large multiprotein structure composed of ring box protein 1 (RBX1), cullin 4 (CUL4), DDB1, CRBN, and an E2 subunit conjugated to ubiquitin (Ub). A heterobifunctional Nef PROTAC facilitates the formation of a ternary complex between the HIV-1 Nef protein and the CRBN E3 complex via a CRBN ligand (exemplified by thalidomide, green) using an existing hydroxypyrazole Nef-binding compound (red). Ternary complex formation induces polyubiquitination of Nef and subsequent proteasomal degradation. The Nef PROTAC shown is Example 14, with arrows indicating preferred locations for linker binding on the Nef-binding moiety. [Figure 2a-b]NanoBRET assay for PROTAC-mediated ubiquitination of HIV-1 Nef. A) Assay principle. Nef is fused to nanoluciferase (Nef-nLuc) and co-expressed with a ubiquitin-Halo tag fusion protein (Ub-Halo) in 293T cells. PROTAC promotes the ligation of Ub-Halo to Nef-nLuc, which is detected by bioluminescent energy transfer (BRET) to the Halo tag. B) Evaluation of candidate Nef PROTACs in the NanoBRET assay. Each compound is assayed in a quadruple sequence, and the fluorescence ratio at an average of 618 nm / 460 nm (BRET signal of Ub uptake normalized at the Nef-nLuc level) is shown as a z-score ± SD (error bars smaller than the data point). PROTACs with a z-score ≥ 1.5 (numbered red dots) were further subjected to orthogonal assays for Nef degradation and inhibition of Nef function, along with analog example 31. The z-score is given by (x-μ) / σ [where x = each individual value, μ = mean, and σ = standard deviation]. [Figure 3a] Nef PROTAC treatment restores cell surface CD4 and MHC-I expression in T cells. Human T cell line CEM-T4 was manipulated to express the Nef-GFP fusion protein under the control of a doxycycline (Dox)-inducible promoter. In the absence of Dox, these cells express endogenous CD4 and MHC-I on their surface. Addition of Dox induces Nef-GFP expression and receptor downregulation. A) Representative flow cytometry results from Nef PROTAC Example 29 and cell surface CD4 staining. B) Active Nef PROTAC from NanoBRET ubiquitination assay was screened for cell surface receptor rescue in a triplicate. Bar heights indicate mean ± SE, and individual data points are also shown. [Figure 3B]Nef PROTAC treatment restores cell surface CD4 and MHC-I expression in T cells. Human T cell line CEM-T4 was manipulated to express the Nef-GFP fusion protein under the control of a doxycycline (Dox)-inducible promoter. In the absence of Dox, these cells express endogenous CD4 and MHC-I on their surface. Addition of Dox induces Nef-GFP expression and receptor downregulation. A) Representative flow cytometry results from Nef PROTAC Example 29 and cell surface CD4 staining. B) Active Nef PROTAC from NanoBRET ubiquitination assay was screened for cell surface receptor rescue in a triplicate. Bar heights indicate mean ± SE, and individual data points are also shown. [Figure 4a-c] Evaluation of PROTAC-mediated Nef protein loss. A) Flow cytometry of Nef-GFP protein loss. CEM-T4 cells were treated with doxycycline to induce Nef-GFP expression under conditions that yielded 60-70% positive cells by flow cytometry (see Figure 4). Triple cell cultures were treated with Nef PROTAC analogs at a final concentration of 3 μM. After 24 hours, the percentage of cells showing loss of Nef-GFP protein expression was calculated compared to the DMSO control and presented as mean ± SE, with individual data points also shown. B) Immunoblot analysis. Cells expressing Nef-GFP were treated with eight active PROTACs as in Part A, and after 48 hours, lysates were prepared for immunoblot analysis with Nef and actin antibodies. Representative blots are shown. C) Immunoblot analysis was performed in double series, and band intensity was quantified by LICOR infrared imaging, which was used to calculate the Nef vs. actin protein expression ratio. The bar graph shows the mean values ​​of each ratio along with the individual values. [Figure 5]Representative SPR sensorgrams of Nef PROTAC. The thalidomide-binding domain (CRBN-TBD) of cereblon and full-length Nef (NL4-3 variant) were expressed in E. coli and purified to homogeneity. Each protein was immobilized in one channel of a carboxymethyl dextran biosensor chip, and two Nef PROTAC analogs, Example 2 and Example 14 (structure at the top, Nef-binding moiety in red, and CRBN-binding ligands thalidomide and lenalidomide in green and blue, respectively), were injected over the concentration range shown in the upper left sensorgram. Protein-ligand interactions were tracked for 90 seconds, followed by the dissociation phase for 180 seconds. The obtained data were fitted to a 1:1 Langmuir binding model, and KD values ​​were calculated from the resulting association and dissociation rate constants (KD values ​​are summarized in Table 1). Each concentration was tested in double runs, and individual traces are shown with data in color and the fitted curve superimposed in black. [Figure 6a-b] The active PROTAC stabilizes the Nef-CRBN protein complex in vitro. A) Recombinant purified HIV-1 Nef and CRBN ligand-binding domains were mixed in equimolar ratios and analyzed by size exclusion chromatography (Superdex 75). Since the individual proteins have similar retention volumes, the mixture elutes as a single peak. B) The protein mixture from Part A was incubated with Nef PROTAC Example 2 in the indicated molar ratios at 4°C for 20 minutes prior to SEC. [Figure 7a-b]PROTAC inhibits Nef-dependent enhancement of HIV-1 replication in primary cells. A) Donor PBMCs were infected with wild-type HIV-1NL4-3 (DMSO control), Nef-deficient mutant (ΔNef), or wild-type virus in the presence of each Nef PROTAC at a final concentration of 1 μM. The input virus was 2,500 pg of HIV p24 Gag per well. Replication was assayed by p24 Gag AlphaLISA after 48 hours. Six independent measurements were assayed for each condition, with high and low values ​​of p24 removed. Each bar represents the mean ± SE of the remaining values ​​and indicates the individual data point. The dotted line represents the mean for the ΔNef control. B) Survival rates of uninfected PBMCs were determined using the Cell Titer Blue assay with each 1 μM PROTAC. [Modes for carrying out the invention]

[0008] Detailed explanation term The following explanations of terms and methods are provided to better describe the compound, composition and method, and to guide those skilled in the art in the practice of this disclosure. It should also be understood that the terms used in this disclosure are for illustrative purposes only and are not intended to limit the use of specific embodiments and examples.

[0009] "Acyl" refers to a group having the structure -C(O)R, where R can be, for example, an optionally substituted alkyl, an optionally substituted aryl, or an optionally substituted heteroaryl. A "lower acyl" group contains 1 to 6 carbon atoms.

[0010] "Acyloxy" refers to a group having the structure -OC(O)R-, where R can be, for example, an optionally substituted alkyl, an optionally substituted aryl, or an optionally substituted heteroaryl. A "lower acyloxy" group contains 1 to 6 carbon atoms.

[0011] As used herein, “administration” includes all administration to a subject by another person or by the subject themselves.

[0012] The term "aliphatic" is defined as including alkyl, alkenyl, alkynyl, alkyl halide, and cycloalkyl groups. "Lower aliphatic" groups are branched or unbranched aliphatic groups having 1 to 10 carbon atoms.

[0013] "Alkanediyl," "cycloalkanediyl," "aryldiyl," and "alkanearyldiyl" refer to divalent radicals derived from aliphatic, alicyclic, aryl, and alkanearyl hydrocarbons.

[0014] An "alkenyl" refers to a cyclic, branched, or linear group containing only carbon and hydrogen atoms, and containing one or more double bonds, which may or may not be conjugated. Alkenyl groups may be unsubstituted or substituted. A "lower alkenyl" group contains 1 to 6 carbon atoms.

[0015] The term "alkoxy" refers to linear, branched, or cyclic hydrocarbon structures and combinations thereof that contain 1 to 20 carbon atoms, preferably 1 to 8 carbon atoms (referred to as "lower alkoxys"), and more preferably 1 to 4 carbon atoms, with an oxygen atom at a bond site. An example of an "alkoxy group" is represented by the formula -OR, where R may optionally be an alkyl group substituted with an alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, alkyl halide, alkoxy, or heterocycloalkyl group. Suitable alkoxy groups include methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, i-butoxy, sec-butoxy, tert-butoxycyclopropoxy, and cyclohexyloxy.

[0016] "Alkoxycarbonyl" refers to the alkoxy-substituted carbonyl radical, -C(O)OR, where R represents an optionally substituted alkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, or similar portion.

[0017] The term "alkyl" refers to branched or unbranched saturated hydrocarbon groups with 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl, and tetracosyl. A "lower alkyl" group is a saturated branched or unbranched hydrocarbon having 1 to 6 carbon atoms. Preferred alkyl groups have 1 to 4 carbon atoms. Alkyl groups can be "substituted alkyls" in which one or more hydrogen atoms are substituted with substituents such as halogens, cycloalkyls, alkoxys, aminos, hydroxyls, aryls, alkenyls, or carboxyls. For example, lower alkyls or (C1-C6) alkyls may be methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, pentyl, 3-pentyl, or hexyl. (C3-C6) cycloalkyls may be cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. (C3-C6)cycloalkyl(C1-C6)alkyl may be cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, 2-cyclopropylethyl, 2-cyclobutylethyl, 2-cyclopentylethyl, or 2-cyclohexylethyl. (C1-C6)alkoxy may be methoxy, ethoxy, propoxy, isopropoxy, butoxy, iso-butoxy, sec-butoxy, pentoxy, 3-pentoxy, or hexyloxy. (C2-C6)alkenyl may be vinyl, allyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1,-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, or 5-hexenyl. (C2-C6) Alkinyl may be ethinyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, or 5-hexynyl. (C1-C6) Alkanoyl may be acetyl, propanoyl, or butanoyl.Halo(C1-C6)alkyl can be iodomethyl, bromomethyl, chloromethyl, fluoromethyl, trifluoromethyl, 2-chloroethyl, 2-fluoroethyl, 2,2,2-trifluoroethyl, or pentafluoroethyl. Hydroxy(C1-C6)alkyl can be hydroxymethyl, 1-hydroxyethyl, 2-hydroxyethyl, 1-hydroxypropyl, 2-hydroxypropyl, 3-hydroxypropyl, 1-hydroxybutyl, 4-hydroxybutyl, 1-hydroxypentyl, 5-hydroxypentyl, 1-hydroxyhexyl, or 6-hydroxyhexyl. (C1-C6)alkoxycarbonyl can be methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, pentoxycarbonyl, or hexyloxycarbonyl. (C1-C6)alkylthio can be methylthio, ethylthio, propylthio, isopropylthio, butylthio, isobutylthio, pentylthio, or hexylthio. (C2-C6) Alkanoyloxy may be acetoxy, propanoyloxy, butanoyloxy, isobutanoyloxy, pentanoyloxy, or hexanoyloxy.

[0018] "Alkynyl" refers to a cyclic, branched, or linear group containing only carbon and hydrogen atoms, and unless otherwise specified, typically containing 1 to 12 carbon atoms and one or more triple bonds. Alkynyl groups may be unsubstituted or substituted. "Lower alkynyl" groups contain 1 to 6 carbon atoms.

[0019] The terms "amine" or "amino" refer to a group of formula -NRR', where R and R' can independently be hydrogen or alkyl, alkenyl, alkynyl, acyl, aryl, arylalkyl, cycloalkyl, alkyl halogenated, or heterocycloalkyl. For example, "alkylamino" or "alkylated amino" refers to a -NRR' where at least one of R or R' is alkyl. A suitable amine or amino group is acetamide.

[0020] The term "aminoalkyl" refers to the alkyl group defined above in which at least one hydrogen atom is replaced by an amino group (e.g., -CH2-NH2).

[0021] "Aminocarbonyl" means an amino-substituted carbonyl (carbamoyl) radical, either alone or in combination, and the amino radical may be monosubstituted or disubstituted as needed, for example, alkyl, aryl, acyl, aralkyl, cycloalkyl, cycloalkylalkyl, alkanoyl, alkoxycarbonyl, aralkoxycarbonyl, etc. For example, an aminocarbonyl may be represented by the formula -C(O)NRR', where R and R' are independently, for example, hydrogen, alkyl, alkenyl, alkynyl, acyl, aryl, aralkyl, cycloalkyl, alkyl halogen, or heterocycloalkyl.

[0022] The term "animal" refers to a category that includes living multicellular vertebrate organisms, such as mammals and birds. The term mammal includes both human and non-human mammals. Similarly, the term "subject" includes both human and non-human subjects, including birds and non-human mammals, such as non-human primates, companion animals (dogs and cats, etc.), livestock (pigs, sheep, cattle, etc.), and non-domesticated animals such as large felines. The term "subject" applies regardless of the stage of the organism's life cycle. Therefore, the term "subject" applies to organisms in womb or egg, depending on whether the organism is a mammal or a bird (e.g., poultry or wild birds).

[0023] The term "arylalkyl" refers to an alkyl group in which an aryl group substitutes for a hydrogen atom in an alkyl group. An example of an arylalkyl group is the benzyl group.

[0024] "Aryl" refers to a monovalent unsaturated or aromatic (including pseudoaromatic) carbocyclic group having a monocyclic ring (e.g., phenyl) or multiple fused rings (e.g., naphthyl or anthryl), which may be unsubstituted or substituted as needed. The term "pseudoaromatic" refers to a ring system that is not strictly aromatic but is stabilized by electron delocalization and behaves similarly to an aromatic ring. A "heteroaryl group" is defined as an unsaturated or aromatic (including pseudoaromatic) group having at least one heteroatom incorporated into the ring of an aromatic group. Examples of heteroatoms include, but are not limited to, nitrogen, oxygen, sulfur, and phosphorus. Examples of heteroaryls include, but are not limited to, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isoxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzoxazolyl, and quinoxalinyl. The aryl or heteroaryl group may be substituted with one or more groups, including but not limited to alkyl, alkynyl, alkenyl, aryl, halide, nitro, amino, ester, ketone, aldehyde, hydroxy, carboxylic acid, or alkoxy groups, or the aryl or heteroaryl group may be unsubstituted. The term heteroaryl includes hydroxy-substituted heteroaryls that may exist in tautomeric keto forms, such as 2-hydroxypyridine and pyridine-2-one, and their N-substituted derivatives that always exist in keto forms, such as N-methylpyridine-2-one.

[0025] "Aryloxy" or "heteroaryloxy" refers to a group of the formula -OAr [wherein Ar is either an aryl group or a heteroaryl group].

[0026] The "carbonylamino" group can be -N(R)-C(O)-R [wherein each R is independently a substituent, e.g., alkyl, alkenyl, alkynyl, acyl, aryl, arylalkyl, cycloalkyl, alkyl halide or heterocycloalkyl, or H]. A suitable carbonylamino group is acetamide.

[0027] The terms "carboxylate" or "carboxyl" refer to the group -COO- or -COOH. The carboxyl group can form a carboxylic acid. "Substituting carboxyl" refers to -COOR where R is alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, alkyl halide, or heterocycloalkyl. For example, a substituted carboxyl group can be a carboxylic acid ester or a salt thereof (e.g., carboxylate).

[0028] The terms “co-administration” or “to co-administer” refer to the administration of the compounds disclosed herein with at least one other therapeutic or diagnostic agent within the same general period, and do not require administration at the same precise time (however, co-administration includes all administrations at the same precise time). Therefore, co-administration may occur on the same day or on different days, or in the same week or in different weeks.

[0029] The term "cycloalkyl" refers to a non-aromatic carbon-based ring composed of at least three carbon atoms. Cycloalkyls can be monocyclic or bicyclic rings or ring systems. Examples of cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. The term "heterocycloalkyl" refers to a cycloalkyl group as defined above, in which at least one carbon atom of the ring is substituted with a heteroatom, such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. Heterocycloalkyls can be monocyclic or bicyclic rings or ring systems.

[0030] The term "ester" is, for example, C 1-6Alkyl(carboxyl C) 1-6 This refers to a carboxyl group-containing portion with hydrogen replaced by an alkyl group (or alkyl ester), aryl group (or aralkyl ester), or other similar group. 1-3 Alkyl alkyl groups, such as methyl esters (CO2Me), ethyl esters (CO2Et), and propyl esters (CO2Pr), are preferred, and their reverse esters (e.g., -OCOMe, -OCOEt, and -OCOPr) are also included.

[0031] The term "alkyl halide" or "haloalkyl group" refers to an alkyl group in which one or more hydrogen atoms present on the group are substituted with a halogen (F, Cl, Br, I).

[0032] The term "hydroxyl" is represented by the formula -OH.

[0033] The term "hydroxyalkyl" refers to an alkyl group having at least one hydrogen atom substituted with a hydroxyl group. The term "alkoxyalkyl" is defined as an alkyl group in which at least one hydrogen atom is substituted with the alkoxy group described above.

[0034] "Inhibition" refers to preventing the complete onset of a disease or condition. "Inhibition" also refers to any quantitative or qualitative reduction of biological activity or binding compared to a control.

[0035] A "N-heterocyclic" refers to a monocyclic or bicyclic ring or ring system containing at least one nitrogen heteroatom. The ring or ring system generally contains 1 to 9 carbon atoms in addition to the heteroatom and can be saturated, unsaturated, or aromatic (including pseudoaromatic). The term "pseudoaromatic" refers to a ring system that is not strictly aromatic but is stabilized by electron delocalization and behaves similarly to an aromatic ring. Aromatic rings include pseudoaromatic ring systems such as pyrrolyl rings.

[0036] Examples of 5-membered monocyclic N-heterocycles include pyrrolyl, H-pyrrolyl, pyrrolinyl, pyrrolidinyl, oxazolyl, oxadiazolyl, isoxazolyl (including 1,2,3- and 1,2,4-oxadiazolyl), furazanyl, thiazolyl, isothiazolyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, imidazolyl, imidazolinyl, triazolyl (including 1,2,3- and 1,3,4-triazolyl), tetrazolyl, thiadiazolyl (including 1,2,3- and 1,3,4-thiadiazolyl), and dithiazolyl. Examples of 6-membered monocyclic N-heterocycles include pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, piperidinyl, morpholinyl, thiomorpholinyl, piperazinyl and triazinyl. The heterocycle may be substituted with a wide range of substituents as required, preferably C 1-6 alkyl, C 1-6 alkoxy, C 2-6 alkenyl, C 2-6 alkynyl, halo, hydroxy, mercapto, trifluoromethyl, amino, cyano or mono- or di(C 1-6 alkyl)amino. The N-heterocyclic group may be condensed to a carbocyclic ring such as phenyl, naphthyl, indenyl, azulenyl, fluorenyl, and anthracenyl.

[0037] Examples of 8-, 9- and 10-membered bicyclic heterocycles include 1H thieno[2,3-c]pyrazolyl, indolyl, isoindolyl, benzoxazolyl, benzothiazolyl, benzoisoxazolyl, benzoisothiazolyl, benzimidazolyl, indazolyl, isoquinolinyl, quinolinyl, quinoxalinyl, purinyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, benzotriazinyl and the like. These heterocycles may, if required, be substituted, for example, with C 1-6 alkyl, C 1-6 alkoxy, C 2-6 alkenyl, C 2-6 alkynyl, halo, hydroxy, mercapto, trifluoromethyl, amino, cyano or mono- or di(C 1-6They may be substituted with alkyl)amino groups. Unless otherwise defined, substituted N-heterocycles include pyridinium salts and N-oxide forms of suitable ring nitrogen groups.

[0038] The term "subject" includes both human and non-human subjects, including birds and non-human mammals, such as non-human primates, companion animals (dogs and cats, etc.), livestock (e.g., pigs, sheep, cattle, etc.), and undomesticated animals such as large cats. The term "subject" applies regardless of the stage of the organism's life cycle. Therefore, the term "subject" applies to organisms in womb or egg, depending on whether the organism is a mammal or a bird (e.g., poultry or wild birds).

[0039] "Substituted" or "substituted" means replacing a hydrogen atom of a molecule or R group with one or more additional R groups. Unless otherwise defined herein, the terms "optionally substituted" or "optional substituent" refer to a group that may or may not be further substituted with one, two, three, four or more groups, preferably one, two or three, more preferably one or two groups. A substituent is, for example, C 1-6 Alkyl, C 2-6 Alkenil, C 2-6 Alkinyl, C 3-8 Cycloalkyl, hydroxyl, oxo, C 1-6 Alkoxy, aryloxy, C 1-6 Alkoxyaryl, Halo, C 1-6 Alkyl halos (such as CF3 and CHF2), C 1-6 Alkoxyhalo (OCF3 and OCHF2, etc.), carboxyl, ester, cyano, nitro, amino, substituted amino, disubstituted amino, acyl, ketone, amide, aminoacyl, substituted amide, disubstituted amide, thiol, alkylthio, thioxo, sulfate, sulfonate, sulfinyl, substituted sulfinyl, sulfonyl, substituted sulfonyl, sulfonylamide, substituted sulfonamide, disubstituted sulfonamide, aryl, aryl C1-6 Alkyl, heterocyclyl, and heteroaryl groups can be selected from [wherein each alkyl, alkenyl, alkynyl, cycloalkyl, aryl, and heterocyclyl group, and the groups containing them, may be further substituted as needed]. Optional substituents in the case of N-heterocyclic groups include C 1-6 Alkyl, i.e., NC 1-3 Alkyl, more preferably methyl, and especially N-methyl, are also examples, but are not limited to these.

[0040] "Sulfinyl" refers to the group -S(=O)H.

[0041] The terms "substituted sulfinyl" or "sulfoxide," for example, C 1-6 Alkyl("C 1-6 "Alkyl sulfinyl" or "C 1-6 This refers to a hydrogen-containing sulfinyl group that has been replaced by alkyl sulfoxides, arylsulfinyls, arylalkylsulfinyls, etc. 1-3 Alkyl sulfinyl groups, such as -SO-methyl, -SO-ethyl, and -SO-propyl, are preferred.

[0042] The term "sulfonyl" refers to the group -SO2H. The sulfonyl group can be further substituted with various groups to form, for example, sulfonic acids, sulfonamides, sulfonic acid esters, and sulfones.

[0043] The term "substituted sulfonyl," for example, C 1-6 Alkyl("Sulfonyl C") 1-6 This refers to a sulfonyl group having hydrogen atoms that has been replaced by alkyl, aryl ("arylsulfonyl"), arylalkyl ("arylalkylsulfonyl"), etc. Sulfonyl C 1-3 Alkyl groups, such as -SO2Me, -SO2Et, and -SO2Pr, are preferred.

[0044] The terms "sulfonylamide" or "sulfonamide" refer to the group -SO2NH2.

[0045] The term "tautomer" refers to constituent isomers of an organic compound that readily interconvert through proton transfer, such as the three tautomers of 3-hydroxypyrazole. [ka]

[0046] The "therapeutic dose" refers to the amount of a particular drug sufficient to achieve the desired effect in the subject being treated with that drug. Ideally, a therapeutic dose of a drug is sufficient to inhibit or treat a disease or condition without causing substantial cytotoxic effects in the subject. The therapeutic dose of a drug depends on the subject being treated, the severity of the disease, and the mode of administration of the therapeutic composition.

[0047] "Treatment" refers to a therapeutic intervention involving the administration of a compound or composition to a subject who shows no signs of the disease or only early signs, with the aim of reducing the risk of developing the disease or condition, or reducing the severity of the disease or condition, in order to improve the signs or symptoms of the disease or condition after the onset of the disease or condition. As used herein, the term "improvement" with respect to the disease or condition refers to any observable beneficial effect of the treatment. Beneficial effects can be demonstrated, for example, by delaying the onset of clinical symptoms of the disease in a susceptible subject, by reducing the severity of some or all clinical symptoms of the disease, by slower progression of the disease, by improvement of the subject's overall health or well-being, or by other parameters well known in the art that are specific to a particular disease. The phrase "treating the disease" refers, for example, to inhibiting the complete onset of the disease in a subject at risk of the disease. "Preventing" a disease or condition refers to the prophylactic administration of a composition to a subject who shows no signs of the disease or only early signs, in order to reduce the risk of developing the disease or condition, or to reduce the severity of the disease or condition.

[0048] A “pharmaceutical composition” is a composition comprising one or more disclosed compounds in a certain amount (e.g., a unit dose) together with one or more non-toxic, pharmaceutically acceptable additives, such as carriers, diluents, and / or adjuvants, and optionally other biologically active components. Such pharmaceutical compositions can be prepared by standard pharmaceutical formulation techniques, such as those disclosed in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA (19th Edition).

[0049] The term “pharmaceutically acceptable salt or ester” refers to salts or esters prepared by conventional means, including but not limited to salts of inorganic and organic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, malic acid, acetic acid, oxalic acid, tartaric acid, citric acid, lactic acid, fumaric acid, succinic acid, maleic acid, salicylic acid, benzoic acid, phenylacetic acid, and mandelic acid. The “pharmaceutically acceptable salts” of the compounds in this disclosure also include all salts formed from cations such as sodium, potassium, aluminum, calcium, lithium, magnesium, and zinc, and from bases such as ammonia, ethylenediamine, N-methylglutamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, tris(hydroxymethyl)aminomethane, and tetramethylammonium hydroxide. These salts can be prepared by standard procedures, for example, by reacting a free acid with a suitable organic or inorganic base. Alternatively, any compound listed herein may be administered as a pharmaceutically acceptable salt. "Pharmacologically acceptable salt" includes free acids, bases, and zwitterionic forms. A description of suitable pharmaceutically acceptable salts can be found in Handbook of Pharmaceutical Salts, Properties, Selection and Use, Wiley VCH (2002). Where a compound disclosed herein contains an acidic functional group such as a carboxyl group, suitable pharmaceutically acceptable cation pairs for a carboxyl group are well known to those skilled in the art and include alkalis, alkaline earths, ammonium, quaternary ammonium cations, etc. Such salts are known to those skilled in the art. For further examples of "pharmacologically acceptable salts," see Berge et al., J. Pharm. Sci. 66:1 (1977).

[0050] "Pharmacologically acceptable esters" include those derived from compounds described herein that have been modified to include a carboxyl group. In vivo hydrolyzable esters are esters that are hydrolyzed in the body of humans or animals to produce hydrophilic acids or alcohols. Typical esters include those in which the non-carbonyl portion of the carboxylic acid moiety of the ester group is linear or branched alkyl (e.g., methyl, n-propyl, t-butyl or n-butyl), cycloalkyl, alkoxyalkyl (e.g., methoxymethyl), aralkyl (e.g., benzyl), aryloxyalkyl (e.g., phenoxymethyl), aryl (e.g., halogen, C, if necessary). 1-4 Alkyl, or C 1-4 Examples of carboxylic acid esters include those selected from alkoxy-substituted sulfonate esters, e.g., phenyl or amino sulfonates; alkyl- or aralkylsulfonyl esters, e.g., methanesulfonyl; or amino acid esters, e.g., L-valyl or L-isoleucyl. "Pharmacologically acceptable esters" also include inorganic esters such as mono-, di- or tri-phosphate esters. In such esters, unless otherwise specified, any alkyl moiety present preferably contains 1 to 18 carbon atoms, particularly 1 to 6 carbon atoms, and more specifically 1 to 4 carbon atoms. Any cycloalkyl moiety present in such esters preferably contains 3 to 6 carbon atoms. Any aryl moiety present in such esters preferably contains a phenyl group as optionally substituted, as shown in the definition of carbocyclyl above. Thus, pharmaceutically acceptable esters include C1-C 22 Examples of fatty acid esters include acetyl, t-butyl, or long-chain linear or branched unsaturated or omega-6 monounsaturated fatty acids, such as palmoyl and stearoyl. Alternative aryl or heteroaryl esters include benzoyl and pyridylmethyloyl, all of which may be substituted as defined for carbocyclyl above. Further pharmaceutically acceptable esters include aliphatic L-amino acid esters, such as leucyl, isoleucyl, and especially valyl.

[0051] For therapeutic use, salts of compounds are those whose counterions are pharmaceutically acceptable. However, salts of pharmaceutically unacceptable acids and bases may find uses, for example, in the preparation or purification of pharmaceutically acceptable compounds.

[0052] As mentioned above in this specification, pharmaceutically acceptable acids and base addition salts mean that the compound may include therapeutically active, non-toxic acid and base addition salt forms. Pharmaceutically acceptable acid addition salts can be conveniently obtained by treating the base form with such a suitable acid. Suitable acids include, for example, inorganic acids such as hydrohalic acids, such as hydrochloric acid or hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, or organic acids such as acetic acid, propanoic acid, hydroxyacetic acid, lactic acid, pyruvic acid, oxalic acid (i.e., ethanedioic acid), malonic acid, succinic acid (i.e., butanedioic acid), maleic acid, fumaric acid, malic acid (i.e., hydroxybutanedioic acid), tartaric acid, citric acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclamic acid, salicylic acid, p-aminosalicylic acid, pamoic acid, and others. Conversely, the salt form can be converted back to the free base form by treatment with an appropriate base.

[0053] Compounds containing acidic protons can also be converted to their non-toxic metal or amine addition salt forms by treatment with suitable organic and inorganic bases. Suitable base salt forms include, for example, ammonium salts, alkali and alkaline earth metal salts such as lithium, sodium, potassium, magnesium, and calcium salts, salts with organic bases such as benzathine, N-methyl-D-glucamine, and hydravamin salts, and salts with amino acids such as arginine and lysine.

[0054] Prodrugs of the disclosed compounds are also contemplated herein. A prodrug is an active or inactive compound that, after administration to a target, is chemically modified into an active compound by in vivo physiological actions such as hydrolysis or metabolism. As used herein, the term “prodrug” means a pharmacoagulably acceptable derivative, such as an ester, amide, or phosphate, such that the resulting in vivo bioconversion product of the derivative is an active drug as defined by the compounds described herein. Prodrugs preferably have excellent water solubility, increased bioavailability, and are readily metabolized in vivo to an activity inhibitor. Prodrugs of the compounds described herein can be prepared by modifying functional groups present in the compound such that the modification is cleaved into the parent compound by either routine procedures or in vivo. The suitability and techniques involved in the preparation and use of prodrugs are well known to those skilled in the art. For a general consideration of prodrugs with F or esters, see Svensson and Tunek, Drug Metabolism Reviews 165 (1988) and Bundgaard, Design of Prodrugs, Elsevier (1985).

[0055] The term “prodrug” is also intended to include any covalent carrier that releases the active parent drug of the present invention in vivo when administered to a subject. Because prodrugs often have enhanced properties compared to pharmaceuticals of the active ingredient, such as solubility and bioavailability, the compounds disclosed herein can be delivered in prodrug form. Accordingly, prodrugs of the compounds of this disclosure, methods for delivering prodrugs, and compositions containing such prodrugs are also intended. Prodrugs of the disclosed compounds are typically prepared by modifying one or more functional groups present in the compound so that the modification is cleaved either in normal operation or in vivo to obtain the parent compound. Prodrugs include compounds having phosphonate and / or amino groups functionalized with any group that is cleaved in vivo to yield the corresponding amino and / or phosphonic acid groups, respectively. Examples of prodrugs, but not limited to, include compounds having acylated amino groups and / or phosphonate esters or phosphonate amide groups. In certain examples, prodrugs are lower alkyl phosphonate esters, such as isopropylphosphonate esters.

[0056] Protected derivatives of the disclosed compounds are also intended. Various suitable protecting groups for use with the disclosed compounds are disclosed in Greene and Wuts, Protective Groups in Organic Synthesis; 3rd Ed.; John Wiley & Sons, New York, 1999.

[0057] Generally, protecting groups are removed under conditions that do not affect the rest of the molecule. These methods are well known in the art and include acid hydrolysis, hydrolysis, and the like. One preferred method involves ester removal, for example, cleavage of phosphonate esters using Lewis acidic conditions such as TMS-Br mediated ester cleavage, to obtain free phosphonates. A second preferred method involves removal of protecting groups, such as removal of benzyl groups by hydrolysis utilizing palladium carbon in a suitable solvent system such as alcohol, acetic acid, or mixtures thereof. t-butoxy groups, including t-butoxycarbonyl protecting groups, can be removed using inorganic or organic acids such as HCl or trifluoroacetic acid in a suitable solvent system such as water, dioxane, and / or methylene chloride. Another exemplary protecting group suitable for protecting amino and hydroxyl functional groups is trityl. Other conventional protecting groups are known, and suitable protecting groups can be selected by those skilled in the art with reference to Greene and Wuts, Protective Groups in Organic Synthesis; 3rd Ed.; John Wiley & Sons, New York, 1999. When an amine is deprotected, the resulting salt can be readily neutralized to obtain the free amine. Similarly, when an acidic moiety, such as a phosphonic acid moiety, is deprotected, the compound can be isolated as an acidic compound or a salt thereof.

[0058] Certain examples of the compounds of this disclosure may contain one or more chiral centers. Therefore, these compounds may exist in different stereoisomerized forms. Accordingly, compounds and compositions may be provided as individual pure enantiomers or as stereoisomerized mixtures including racemic mixtures. In certain embodiments, the compounds disclosed herein are synthesized or purified to be substantially enantiopurine, for example, with a 90% enantiomer excess, a 95% enantiomer excess, a 97% enantiomer excess, or an enantiomer excess of more than 99%, for example, enantiopurine.

[0059] The compounds of this disclosure may have at least one chiral or geometric center, or a cis-trans center (C=C, C=N). Unless otherwise specified, all chiral, diastereomer, racemic, meso, rotational isomers and geometric isomers of the structure are intended. The compounds may be isolated as a single isomer or as a mixture of isomers. All tautomers of the compounds are also considered part of this disclosure. The compounds of this disclosure also include all isotopes of the atoms present in the compound, including deuterium, tritium, 18 This may include, but is not limited to, F, etc.

[0060] The following abbreviations have the meanings indicated: [Table 3-1] [Table 3-2]

[0061] compound A proteolytically targeted chimera (PROTAC) for targeted disruption of Nef is disclosed herein. A Nef-binding compound was coupled to a ligand of a ubiquitin E3 ligase via a flexible linker. The resulting bivalent PROTAC induces the formation of a ternary complex between Nef and Cereblon E3 ubiquitin ligase, resulting in polyubiquitination and proteolytic degradation of Nef. As a result, the Nef-targeted PROTAC potently rescues Nef-mediated MHC-I and CD4 downregulation in T cells and inhibits HIV-1 replication in donor PBMCs. This targeted degradation can reverse all Nef effects in HIV-infected cells and provide a robust antiviral response that could not be achieved with previous occupation-based inhibitors.

[0062] In particular, compounds of formula I: [ka] [In the formula, the ligand that binds to the Nef protein (NB) is covalently bound to the ligand that binds to the E3 ligase cereblon (CB) via a linker (L)] or stereoisomers, isotopes, tautomers, or pharmaceutically acceptable salts thereof are disclosed herein.

[0063] In a particular embodiment, the ligands CB that bind to cereblon are CB1-CB22: [ka] [In the formula, Linker, [ka] It is joined at the position marked with, A is either C (=O) or CH2. R a is H, halo, cyano, lower alkyl, lower haloalkyl, lower alkoxy or lower haloalkoxy, R b [is selected from H or a lower alkyl group.]

[0064] In certain embodiments, the linker L comprises 0 to 30 divalent moieties selected from CH2, C≡C, C(=O), NH, NMe, O, S, S(=O), SO2, piperazinyl, piperidinyl, pyrrolidinyl, and azetidinyl, linked together so as not to form OO or NO bonds.

[0065] In a particular embodiment, the ligand for Nef is given by formula NB: [ka] It is a compound of, During the ceremony, The linker is bonded to any position where there is a hydrogen atom. G is either CH or N. R is phenyl, pyridinyl, piperidinyl, pyrrolidinyl, or azetidinyl, and is optionally substituted with 0 to 3 groups independently selected from Y. X, Y, and Z are each independently a halo, cyano, lower alkyl, lower haloalkyl, lower alkoxy, lower haloalkoxy, lower alkylcarbonyl, lower alkoxycarbonyl, or lower alkylsulfonyl. m and n are independently 0, 1, 2, or 3. o is either 1 or 2.

[0066] In one embodiment of CB1, CB2, CB3 and CB5~CB22, R a It is hydrogen.

[0067] In another configuration of CB1, CB2, CB3 and CB5~CB22, R a It is fluorine.

[0068] In one embodiment of CB5~CB10 and CB17, R b It is hydrogen.

[0069] In another embodiment, in CB5~CB10 and CB17, R b It is methyl.

[0070] In another embodiment of CB, the cerebron binder is selected from CB1 or CB2.

[0071] In another embodiment, A in CB1 and CB2 is C (=O).

[0072] In another embodiment, A in CB1 and CB2 is CH2.

[0073] In one embodiment, the linker contains an amide of the formula -C(=O)NH-.

[0074] In one embodiment, the linker contains the ethylene glycol moiety of the formula -OCH2CH2O-.

[0075] In one embodiment, the linker is of the formula [ka] It contains the piperazine portion.

[0076] In one embodiment, the linker is of the formula [ka] It contains the piperidine portion.

[0077] In one embodiment, the linker is a linker containing the formula [ka] It is bound to CB1-CB22 by the piperazine portion.

[0078] In one embodiment, the linker is a linker containing the formula [ka] It is bound to CB1-CB22 by the piperidine portion.

[0079] In one embodiment, the linker is bonded to CB1 to CB22 by the alkyne portion of formula -C≡C- contained in the linker.

[0080] In one embodiment, the linker is bonded to CB1-CB22 by ether oxygen.

[0081] In one embodiment, the linker is bonded to CB1~CB22 by a -CH2- group.

[0082] In one embodiment, the linker is bonded to CB1~CB22 by an -NH- group.

[0083] In one embodiment, the linker is coupled to CB1 by the -NHCH2CH2NH- portion.

[0084] In one embodiment, the linker is coupled to CB1 by a -NMeCH2CH2NH- moiety.

[0085] In one embodiment, the linker is coupled to CB1 by the -OCH2C(=O)NH- moiety.

[0086] In one embodiment, the linker is bonded to CB2 by a 1,4-piperazinyl moiety.

[0087] In one embodiment, the linker is coupled to the NB by the -CH2- portion.

[0088] In one embodiment, the linker is coupled to the NB by a -C(=O)- portion.

[0089] In one embodiment, the linker is bonded to NB by an -O- atom.

[0090] In another embodiment, linker L is derived from formulas II, III, or IV: [ka] [In the formula, L C It consists of 0 to 10 divalent moieties selected from CH2, C≡C, C(=O), NH, NMe, O, S, S(=O), SO2, piperazinyl, piperidinyl, pyrrolidinyl, and azetidinyl, linked together so as not to form OO or NO bonds. L N It consists of 0 to 10 divalent moieties selected from CH2, C≡C, C(=O), NH, NMe, O, S, S(=O), SO2, piperazinyl, piperidinyl, pyrrolidinyl, and azetidinyl, linked together so as not to form OO or NO bonds. L C It is bonded to the cereblon bond site, L N This is the one that is connected to the Nef connection part.

[0091] In another embodiment, linker L is derived from formulas II, III, or IV: [In the formula, L C It consists of 0 to 10 divalent moieties selected from CH2, NH, NMe, O, S, SO2, piperazinyl, piperidinyl, pyrrolidinyl, and azetidinyl, linked together so as not to form OO or NO bonds. L N It consists of 0 to 10 divalent moieties selected from CH2, NH, NMe, O, S, SO2, piperazinyl, piperidinyl, pyrrolidinyl, and azetidinyl, linked together so as not to form OO or NO bonds. L C It is bonded to the cereblon bond site, L N This is the one that is connected to the Nef connection part.

[0092] In another embodiment of L, the linker is L1~L26: [ka] [In the formula, [Any NH can be substituted with methyl to produce NMe, given that p and p' are independently 0 to 7, p'' is 1 to 7, p+p' is < 10, and p+p'' is < 10].

[0093] In other embodiments, the linker is [ka] Selected from.

[0094] In one embodiment of NB, R is phenyl.

[0095] In one embodiment of NB, R is 4-piperidinyl.

[0096] In one embodiment of NB, X is hydrogen, fluorine, or chlorine.

[0097] In one embodiment of NB, X is chlorine.

[0098] In one embodiment of NB, Z is either F or does not exist.

[0099] In one embodiment of NB, Y is either CF3 or does not exist.

[0100] In one embodiment of NB, the linker is bonded to a phenyl ring having substituent Z.

[0101] In one embodiment of NB, the linker is bonded to the para position of the phenyl ring having substituent Z.

[0102] In one embodiment of NB, the linker is coupled to R.

[0103] In one embodiment of NB, R is phenyl, and the linker is bonded at the para position.

[0104] In one embodiment of NB, R is 4-piperidinyl, and the linker is bonded to the piperidine nitrogen atom.

[0105] In one embodiment, NB is NB1, NB2, NB3, NB4, or NB5. [ka] During the ceremony, Linker, [ka] It is joined at the position marked with, X is H or a halogen, Y is a halo, a lower alkylsulfonyl, or a lower haloalkyl. Y' is a lower alkoxycarbonyl, Z is fluorine.

[0106] In one embodiment, X in NB1 to NB3 and NB5 is chlorine.

[0107] In one embodiment, NB1 is coupled to CB1 via a linker L.

[0108] In one embodiment, NB1 is coupled to CB2 via a linker L.

[0109] In one embodiment, NB2 is coupled to CB1 via a linker L.

[0110] In one embodiment, NB2 is coupled to CB2 via a linker L.

[0111] In one embodiment, NB3 is coupled to CB1 via linker L.

[0112] In one embodiment, NB3 is coupled to CB2 via linker L.

[0113] In another embodiment, in a compound of formula I: CB is CB1, L is from equation II, equation III, or equation IV, L C It comprises 0 to 10 divalent moieties selected from CH2, NH, NMe, O, S, SO2, piperazinyl, piperidinyl, pyrrolidinyl, and azetidinyl, linked so as not to form OO or NO bonds. L N It comprises 0 to 10 divalent moieties selected from CH2, NH, NMe, O, S, SO2, piperazinyl, piperidinyl, pyrrolidinyl, and azetidinyl, linked so as not to form OO or NO bonds. L C It is bonded to the cereblon bond site, L N It is connected to the Nef connection part, NB is NB1, and X is Cl.

[0114] In another embodiment, in a compound of formula I: CB is CB2, L is from equation II, equation III, or equation IV, L C It comprises 0 to 10 divalent moieties selected from CH2, NH, NMe, O, S, SO2, piperazinyl, piperidinyl, pyrrolidinyl, and azetidinyl, linked so as not to form OO or NO bonds. L N It comprises 0 to 10 divalent moieties selected from CH2, NH, NMe, O, S, SO2, piperazinyl, piperidinyl, pyrrolidinyl, and azetidinyl, linked so as not to form OO or NO bonds. L C It is bonded to the cereblon bond site, L N It is connected to the Nef connection part, NB is NB1, and X is Cl.

[0115] In another embodiment, in a compound of formula I: CB is CB1, L is from equation II, equation III, or equation IV, L C It comprises 0 to 10 divalent moieties selected from CH2, NH, NMe, O, S, SO2, piperazinyl, piperidinyl, pyrrolidinyl, and azetidinyl, linked so as not to form OO or NO bonds. L N It comprises 0 to 10 divalent moieties selected from CH2, NH, NMe, O, S, SO2, piperazinyl, piperidinyl, pyrrolidinyl, and azetidinyl, linked so as not to form OO or NO bonds. L C It is bonded to the cereblon bond site, L N It is connected to the Nef connection part, NB is NB2, and X is Cl.

[0116] In another embodiment, in a compound of formula I: CB is CB2, L is from equation II, equation III, or equation IV, L C It comprises 0 to 10 divalent moieties selected from CH2, NH, NMe, O, S, SO2, piperazinyl, piperidinyl, pyrrolidinyl, and azetidinyl, linked so as not to form OO or NO bonds. L N It comprises 0 to 10 divalent moieties selected from CH2, NH, NMe, O, S, SO2, piperazinyl, piperidinyl, pyrrolidinyl, and azetidinyl, linked so as not to form OO or NO bonds. L C It is bonded to the cereblon bond site, L N It is connected to the Nef connection part, NB is NB2, and X is Cl.

[0117] In another embodiment, in a compound of formula I: CB is CB1, L is the one in equation IV, L C It comprises 0 to 10 divalent moieties selected from CH2, NH, NMe, O, S, SO2, piperazinyl, piperidinyl, pyrrolidinyl, and azetidinyl, linked so as not to form OO or NO bonds. L N It comprises 0 to 10 divalent moieties selected from CH2, NH, NMe, O, S, SO2, piperazinyl, piperidinyl, pyrrolidinyl, and azetidinyl, linked so as not to form OO or NO bonds. L C It is bonded to the cereblon bond site, L N It is connected to the Nef connection part, NB is NB3, and X is Cl.

[0118] In another embodiment, in a compound of formula I: CB is CB2, L is the one in equation IV, L Ccontains from 0 to 10 divalent moieties selected from CH2, NH, NMe, O, S, SO2, piperazinyl, piperidinyl, pyrrolidinyl and azetidinyl, which are linked so that no O-O or N-O bond is formed, L N contains from 0 to 10 divalent moieties selected from CH2, NH, NMe, O, S, SO2, piperazinyl, piperidinyl, pyrrolidinyl and azetidinyl, which are linked so that no O-O or N-O bond is formed, L C is attached to the celebron binding moiety, L N is attached to the Nef binding moiety, NB is NB3 and X is Cl.

[0119] In another embodiment, the compound of formula I has the structure Ia:

Chemical formula

[0120] In another embodiment, the compound of formula I has the structure Ib

Chemical formula

[0121] In another embodiment, the compound of formula I has structure Ic [ka] It has, In the formula, A is C (=O) or CH2, and L N It contains 4 to 16 divalent moieties selected from CH2, C≡C, C(=O), NH, NMe, O, S, S(=O), SO2, piperazinyl, piperidinyl, pyrrolidinyl, and azetidinyl, which are linked together so as not to form OO or NO bonds.

[0122] In another embodiment, the compound of formula I has structure Id [ka] It has, In the formula, A is C (=O) or CH2, and L C It contains 0 to 6 divalent moieties selected from CH2, C≡C, C(=O), NH, NMe, O, S, S(=O), SO2, piperazinyl, piperidinyl, pyrrolidinyl, and azetidinyl, which are linked together so as not to form an OO or NO bond.

[0123] Examples of difunctional compounds of formula I include the following: [Table 4-1] [Table 4-2] [Table 4-3]

[0124] General explanation of synthesis In the first process, a compound of formula I in which L contains a -C(=O)NH moiety is used in combination with a peptide coupling reagent such as HATU or EDCI and HOBt in the presence of an amine base such as Et3N or i-Pr2NEt to react a cereblon binder CB functionalized with a partial linker L terminated with a carboxylic acid (V) C with a Nef binder NB functionalized with a partial linker L terminated with an amino group (VI): N prepared by reaction:

Chemical formula

[0125] In the second process, a compound of formula I in which L contains a -NHC(=O)- moiety is used in combination with a peptide coupling reagent such as HATU or EDCI and HOBt in the presence of an amine base such as Et3N or i-Pr2NEt to react a cereblon binder CB functionalized with a partial linker L terminated with an amino group (VII) C with a Nef binder NB functionalized with a partial linker L terminated with a carboxylic acid (VIII): N prepared by reaction:

Chemical formula

[0126] In the third process, a compound of formula I in which L is attached to NB by a C(=O) group forming part of a tertiary amide is used in combination with HOBt and a peptide coupling reagent such as HATU or EDCI in the presence of an amine base such as Et3N or i-Pr2NEt to react a cereblon binder CB functionalized with a partial linker L terminated with a carboxylic acid (V) C with a Nef binder NB3 of formula NB3:

Chemical formula

[0127] In the fourth process, a compound of formula I in which L incorporates an acylpiperazine moiety is used in combination with a peptide coupling reagent such as HATU or EDCI and HOBt in the presence of an amine base such as Et3N or i-Pr2NEt to react with a cereblon binder CB functionalized with a partial linker L terminated with a piperazine ring (IX) and a Nef binder NB functionalized with a partial linker L terminated with a carboxylic acid (VIII): C and a Nef binder NB functionalized with a partial linker L terminated with a carboxylic acid (VIII): N prepared by the reaction of:

Chemical formula

[0128] In the fifth process, a hydride reducing agent such as NaCNBH3 or NaBH(OAc)3 is used for the reductive amination of a cereblon binder CB functionalized with a partial linker L terminated with an aldehyde (X) and a Nef binder NB functionalized with a partial linker L terminated with a piperazine (XI) to prepare a compound of formula I in which L incorporates a piperazine moiety: C and a Nef binder NB functionalized with a partial linker L terminated with a piperazine (XI): N prepared by the reaction of:

Chemical formula

[0129] In the sixth process, a hydride reducing agent such as NaCNBH3 or NaBH(OAc)3 is used for the reductive amination of a cereblon binder CB functionalized with a partial linker L terminated with a piperazine (IX) and a Nef binder NB functionalized with a partial linker L terminated with an aldehyde (XII) to prepare a compound of formula I in which L incorporates a piperazine moiety: C and a Nef binder NB functionalized with a partial linker L terminated with an aldehyde (XII): N prepared by the reaction of:

Chemical formula

[0130] [[ID=Z]]In the seventh process, a cereblon binder (XIII) having a hydroxyl group and a compound of formula XIV where Q1 is a leaving group such as iodide, bromide or methanesulfonate, and R z is reacted with a compound [wherein R is an alkyl group such as tert-butyl] in a polar aprotic solvent such as DMF in the presence of an amine base such as i-Pr2NEt to obtain XV, and then R Z is removed by using an acid such as TFA to prepare a functionalized cereblon binder of formula V in which L C is bonded to CB by an ether bond.

Chemical formula

[0131] In the eighth process, a functionalized cereblon binder of formula V in which L C is bonded to CB by an amino bond is prepared by reacting a cereblon binder functionalized with a leaving group Q such as fluoride or chloride (XVI) with an amino compound of formula XVII [wherein R 1 is an alkyl group such as tert-butyl] in a polar aprotic solvent such as DMF or DMSO in the presence of an amine base such as i-Pr2NEt to obtain XVIII, and then removing R Z by using an acid such as TFA. Z is removed.

Chemical formula

[0132] In the ninth process, a functionalized cereblon binder of formula V in which L C is bonded to CB by an alkynyl bond is prepared by reacting a cereblon binder functionalized with a leaving group Q such as bromide or iodide (XVI) with an alkyne of formula XIX [wherein R 1 is an alkyl group such as tert-butyl] in the presence of a palladium catalyst such as Pd(PPh3)2Cl2 or Pd(PPh3)4, a base such as Et3N and a solvent such as CH2Cl2, THF or DMF to obtain XX, and then using an acid such as TFA to remove R Z is an alkyl group such as tert-butyl] to obtain XX, and then using an acid such as TFA to remove RZ Remove. [ka]

[0133] In the tenth process, L C A functionalized cereblon binder of formula VII, in which is bonded to CB by an alkynyl bond, is subjected to a leaving group Q such as bromide or iodide (XVI). 1 A cereblon binder that has been sensually treated, and formula XXI [wherein R Y The alkyne of [an amine protecting group such as Boc] is reacted with a base such as Et3N and a solvent such as CH2Cl2, THF, or DMF, in the presence of a palladium catalyst such as Pd(PPh3)2Cl2 or Pd(PPh3)4 to obtain XXII, and then R is treated with an acid such as TFA. Y It is prepared by removing [something]. [ka]

[0134] In the 11th process, L C The functionalized cereblon binder of formula VII, in which is bonded to CB by an amino bond, is functionalized in a bipolar aprotic solvent such as DMF or DMSO, in the presence of an amine base such as i-Pr2NEt, and leaves off of a leaving group such as fluoride or chloride (XVI). 1 A cereblon binder functionalized with the amino compound of formula XXIII, R Y The amine protecting group, such as Boc, is reacted to obtain XVIII, and then R is treated with an acid such as TFA. Y It is prepared by removing [something]. [ka]

[0135] In the twelfth process, the functionalized Nef binder of formula VI is prepared by reduction of the azide of formula XXV using trimethylphosphine or Ph3P in an aqueous solvent mixture such as water / THF, or by hydrogenation in the presence of palladium carbon: [ka]

[0136] In the 13th process, the functionalization Nef binder of formula VI is obtained using TFA or TBAF, respectively, to form compound formula XXVI [wherein R Y It is prepared by deprotecting a nitrogen protecting group such as Boc or Teoc. [ka]

[0137] In the 14th process, the functionalizing Nef binder of formula VIII is used with NaOH or TFA, respectively, to form formula XXVII [wherein R Z It is prepared by deprotecting a compound of alkyl groups such as methyl or t-butyl. [ka]

[0138] In the 15th process, L N The Nef binder of formula XXV, which is bonded as shown in XXVa and XXVb and terminated with an azide, is converted at a temperature between room temperature and 150°C in a suitable solvent such as ethanol, using an acid catalyst such as HOAc, HCl, or TsOH, to form formula XXVIII or XXX(R Y It is prepared by the reaction of a β-ketoester of a lower alkyl group with hydrazine of formula XXIX. Heat is applied by means including an oil bath, a heated metal block, or a microwave oven. [ka]

[0139] In the 16th process, L N The Nef binder of formula XXVII, which is bonded as shown in XXVIIa and XXVIIb and terminated with an azide, is converted at a temperature between room temperature and 150°C in a suitable solvent such as ethanol, using an acid catalyst such as HOAc, HCl, or TsOH, R Z It is prepared by the reaction of a β-ketoester of formula XXXI or XXXII, in which is a lower alkyl group, with a hydrazine of formula XXIX. Heat is applied by means including an oil bath, a heated metal block, or a microwave oven. [ka]

[0140] In the 17th process, the β-ketoester of formula XXVIII is replaced with the α-unsubstituted β-ketoester of formula XXXIII with the compound of formula XXXIV [wherein Q 1 It is prepared, for example, by alkylation with bromide, iodide, or methanesulfonate, using K2CO3 in DMF supplemented with KI as needed. [ka]

[0141] In the 18th process, the β-ketoester of formula XXVIII is prepared, for example, by Claisen condensation of esters of formulas XXXV and XXXVI using NaH in THF at reflux temperature. [ka]

[0142] β-ketoesters XXX, XXXX, and XXXII are prepared by the same procedure as in steps 17 and 18.

[0143] LC-MS method Column: Phenomenex Luna 3u C18(2)100 A, 75×4.6mm; Mobile phase: A: 0.1% TFA / water, B: 0.1% TFA / CH3CN; Flow rate: 1.5 mL / min; Gradient: 5%B~95%B.

[0144] Synthesis of CRBN-linked intermediates 2-{[2-(2,6-dioxopiperidine-3-yl)-1-oxo-2,3-dihydro-1H-isoindole-4-yl]oxyacetic acid (CB1a) [ka]

[0145] Step 1 t-butyl bromoacetate (0.5 mL, 3.4 mmol) was added to a stirred solution of 3-(4-hydroxy-1-oxo-2,3-dihydro-1H-isoindole-2-yl)piperidine-2,6-dione (926 mg, 3.6 mmol) and i-Pr2NEt (0.64 mL, 3.6 mmol) in dry DMF (10 mL). The mixture was stirred at 40 °C for 2 days and purified directly by preparative HPLC to obtain 2-{[2-(2,6-dioxopiperidine-3-yl)-1-oxo-2,3-dihydro-1H-isoindole-4-yl]oxy}acetate tert-butyl (380 mg, 28%) as an oil. LC-MS t R = 3.90 min, m / z 375, 319. 1 H NMR (300MHz, DMSO-d6) δ = 7.52 - 7.39 (m, 1H), 7.32 (d, J=7.0 Hz, 1H), 7.21 - 7.08 (m, 1H), 5.10 (dd, J=5.1, 13.0 Hz, 1H), 4.82 (s, 2H), 4.48 - 4.13 (m, 2H), 3.03 - 2.75 (m, 1H), 2.63-2.33 (m, 2H), 2.14 - 1.88 (m, 1H), 1.40 (s, 9H).

[0146] Step 2 A solution of tert-butyl 2-{[2-(2,6-dioxopiperidine-3-yl)-1-oxo-2,3-dihydro-1H-isoindole-4-yl]oxy}acetate (380 mg, 1.0 mmol) in 2:1 CH2Cl2 / TFA (9 mL) was stirred at room temperature for 1 day and concentrated. The residue was freeze-dried from the MeCN aqueous solution to obtain the title compound (289 mg, 90%) as a grayish-white solid. 1 H NMR (300MHz, DMSO-d6) δ = 7.52 - 7.40 (m, 1H), 7.32 (d, J=7.5 Hz, 1H), 7.14 (d, J=7.9 Hz, 1H), 5.10 (d, J=8.3 Hz, 1H), 4.82 (s, 2H), 4.45 - 4.15 (m, 2H), 3.02 - 2.78 (m, 1H), 2.64-2.20 (m, 2H), 2.10 - 1.86 (m, 1H).

[0147] 4-[(2-aminoethyl)amino]-2-(2,6-dioxopiperidine-3-yl)-2,3-dihydro-1H-isoindole-1,3-dione(CB1b) [ka]

[0148] Step 1 A mixture of 2-(2,6-dioxopiperidine-3-yl)-4-fluoro-2,3-dihydro-1H-isoindole-1,3-dione (1.75 g, 6.3 mmol), tert-butyl N-(2-aminoethyl)carbamate (1.06 g, 6.6 mmol), i-Pr2NEt (2.3 mL, 12.7 mmol), and dry DMF (30 mL) was stirred in an oil bath at 90°C for 2 days. The mixture was diluted with ELISA (150 mL) and washed with water (2 × 50 mL) and brine (50 mL). The combined aqueous washes were back-extracted with ELISA (50 mL). The combined ELISA layer was dried over Na2SO4 and concentrated under reduced pressure, leaving a black oily substance (4.11 g). Preparative HPLC yielded tert-butyl N-(2-{[2-(2,6-dioxopiperidine-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindole-4-yl]amino}ethyl)carbamate (850 mg, 32%) as a yellow solid. 1 H NMR (300MHz, DMSO-d6) Shift= 7.64 - 7.42 (m, 1H), 7.10 (d, J=8.8 Hz, 1H), 7.04 - 6.93 (m, 2H), 6.68 (br s, 1H), 5.01 (dd, J=5.5, 12.5 Hz, LC-MS t R 3.98 min, m / z 439, 417, 317.

[0149] Step 2 A solution of N-(2-{[2-(2,6-dioxopiperidine-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindole-4-yl]amino}ethyl) tert-butyl carbamate (850 mg, 2.0 mmol) in 2:1 CH2Cl2 / TFA (30 mL) was stirred at room temperature for 0.5 hours. The mixture was concentrated, and the residue was freeze-dried in an aqueous MeCN / 5% HCl solution to obtain the title compound (745 mg, quantitative yield) as its HCl salt.1 H NMR (300MHz, CD3OD) Shift= 7.62 (t, J=7.7 Hz, 1H), 7.15 (d, J=8.3 Hz, 2H), 5.16 - 5.01 (m, 1H), 3.68 (s, 2H), 3.26 - 3.10 (m, 2H), 2.95 - 2.59 (m, 3H), 2.23 - 2.00 (m, 1H).LC-MS t R 2.25 min, m / z 317.

[0150] 4-[(2-aminoethyl)(methyl)amino]-2-(2,6-dioxopiperidine-3-yl)-2,3-dihydro-1H-isoindole-1,3-dione(CB1c) [ka]

[0151] Step 1 A mixture of 2-(2,6-dioxopiperidine-3-yl)-4-fluoro-2,3-dihydro-1H-isoindole-1,3-dione (750 mg, 2.7 mmol), tert-butyl N-[2-(methylamino)ethyl]carbamate (500 mg, 2.9 mmol), i-Pr2NEt (1 mL, 5.5 mmol), and DMF (10 mL) was stirred at 70°C for 20 hours. The mixture was cooled to room temperature, diluted with ELISA (90 mL), washed with water (2 × 20 mL) and brine (20 mL), and dried over Na2SO4. After removal of the solvent, a yellow oily substance (1.58 g) remained. Preparative HPLC yielded tert-butyl N-(2-{[2-(2,6-dioxopiperidine-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindole-4-yl](methyl)amino}ethyl)carbamate (480 mg, 41%) as a yellow solid. 1H NMR (300MHz, DMSO-d6) δ = 7.82 - 7.67 (m, 1H), 7.46 - 7.30 (m, 2H), 6.94 - 6.79 (m, 1H), 5.32 - 5.17 (m, 1H), 3.81 - 3.57 (m, 2H), 3.35 - 3.22 (m, 2H), 3.12 - 2.93 (m, 1H), 2.81 - 2.60 (m, 2H), 2.24 - 2.02 (m, 1H), 1.42 (s, 9H).

[0152] Step 2 A 2:1 CH2Cl2 / TFA (30 mL) solution of N-(2-{[2-(2,6-dioxopiperidine-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindole-4-yl](methyl)amino}ethyl) tert-butyl carbamate (480 mg, 1.1 mmol) was stirred at room temperature for 0.5 hours and concentrated. The residue was freeze-dried from a mixture of MeCN and 5% aqueous HCl to obtain the HCl salt of the title compound (419 mg, quantitative yield) as a yellow solid. 1 H NMR (300MHz, CD3OD) δ = 7.80 - 7.66 (m, 1H), 7.53 - 7.35 (m, 2H), 5.20-5.10 (m, 1H), 3.65-3.57 (m, 2H), 3.41 - 3.30 (m, 2H), 2.98 (s, 3H), 2.94 - 2.58 (m, 3H), 2.21 - 2.06 (m, 1H).

[0153] 2-(2,6-dioxopiperidine-3-yl)-4-{[2-(methylamino)ethyl]amino}-2,3-dihydro-1H-isoindole-1,3-dione(CB1d) [ka]

[0154] The title compound was prepared using the same procedure as described for CB1c, with tert-butyl N-(2-aminoethyl)-N-methylcarbamate used in step 1. LC-MS t R = 2.33 min, m / z 331.

[0155] 4-({2-[2-(2-aminoethoxy)ethoxy]ethyl}amino)-2-(2,6-dioxopiperidine-3-yl)-2,3-dihydro-1H-isoindole-1,3-dione(CB1e) [ka]

[0156] Step 1 A solution of 2-(2,6-dioxopiperidine-3-yl)-4-fluoro-2,3-dihydro-1H-isoindole-1,3-dione (420 mg, 1.5 mmol), N-{2-[2-(2-aminoethoxy)ethoxy]ethyl}carbamate tert-butyl (396 mg, 1.6 mmol), and i-Pr2NEt (0.55 mL, 3.0 mmol) in DMF (10 mL) was stirred at 70°C for 4 hours. The mixture was diluted with ELISA (90 mL), washed with 5% aqueous HCl (2 × 15 mL) and brine (15 mL), and dried over Na2SO4. The solvent was removed to leave a green oily substance (910 mg), which was purified by preparative HPLC to obtain N-{2-[2-(2-{[2-(2,6-dioxopiperidine-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindole-4-yl]amino}ethoxy)ethoxy]ethyl}carbamate tert-butyl (230 mg, 30%) as a yellow solid. 1H NMR (300MHz, DMSO-d6) δ = 7.62 - 7.49 (m, 1H), 7.13 (d, J=8.8 Hz, 1H), 7.02 (d, J=7.0 Hz, 1H), 6.81 - 6.67 (m, 1H), 6.65 - 6.55 (m, 1H), 5.10 - 4.98 (m, 1H), 3.67 - 3.41 (m, 10H), 3.39-3.32 (m, 2H), 3.11 - 2.97 (m, 2H), 2.95 - 2.74 (m, 1H), 2.65 - 2.36 (m, 2H), 2.08 - 1.92 (m, 1H), 1.34 (s, 9H).LC-MS t R = 4.13 min, m / z 505, 405.

[0157] Step 2 A 3:2 CH2Cl2 / TFA (5 mL) solution of N-{2-[2-(2-{[2-(2,6-dioxopiperidine-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindole-4-yl]amino}ethoxy)ethoxy]ethyl}carbamate tert-butyl (230 mg, 0.46 mmol) was stirred at room temperature for 0.5 hours and concentrated. The residue was freeze-dried from a mixture of MeCN and 5% aqueous HCl to obtain the HCl salt of the title compound (114 mg, 57%) as a yellowish-green solid. 1 H NMR (300MHz, DMSO-d6) δ = 8.06 (br s, 2H), 7.65 - 7.50 (m, 1H), 7.14 (d, J=8.8 Hz, 1H), 7.03 (d, J=7.0 Hz, 1H), 6.00 - 5.21 (m, 2H), 5.12-5.00 (m, 1H), 3.70 - 3.52 (m, 8H), 3.50-3.40 (m, 2H), 2.99-2.80 (m, 2H), 2.66 - 2.37 (m, 1H), 2.10 - 1.90 (m, 1H)

[0158] 4-{[2-(2-aminoethoxy)ethyl]amino}-2-(2,6-dioxopiperidine-3-yl)-2,3-dihydro-1H-isoindole-1,3-dione(CB1f) [ka]

[0159] The title compound was prepared using the same procedure as described for CB1e, with N-[2-(2-aminoethoxy)ethyl]carbamate tert-butyl in step 1. LC-MS t R = 2.63 min, m / z 361.

[0160] 2-(2,6-dioxopiperidine-3-yl)-4-{[2-(piperazine-1-yl)ethyl]amino}-2,3-dihydro-1H-isoindole-1,3-dione (CB1g) [ka]

[0161] The title compound was prepared using the same procedure as described for CB1e, with 4-(2-aminoethyl)piperazine-1-carboxylate tert-butyl in step 1. LC-MS t R = 2.25 min, m / z 386.

[0162] 3-{4-[(2-aminoethyl)amino]-1-oxo-2,3-dihydro-1H-isoindole-2-yl}piperidine-2,6-dione(CB1h) [ka]

[0163] Step 1 A mixture of 3-(4-amino-1-oxo-2,3-dihydro-1H-isoindole-2-yl)piperidine-2,6-dione (127 mg, 0.49 mmol), tert-butyl N-(2-oxoethyl)carbamate (67 mg, 0.42 mmol), HOAc (28 μL, 0.49 mmol), and dry MeOH (5 mL) was stirred at room temperature for 2 hours, and NaCNBH3 (62 mg, 0.98 mmol) was added. The mixture was stirred at room temperature overnight and concentrated. The residue was diluted with saturated NaHCO3 aqueous solution (20 mL) and extracted with CH2Cl2 (4 × 25 mL). The combined organic layer was washed with brine (10 mL), dried over Na2SO4, and concentrated, leaving a sticky solid (160 mg). Purification by preparative HPLC yielded N-(2-{[2-(2,6-dioxopiperidine-3-yl)-1-oxo-2,3-dihydro-1H-isoindole-4-yl]amino}ethyl)carbamate tert-butyl (69 mg, 35%). LC-MS t R = 3.55 min, m / z 425, 403, 347, 303.

[0164] Step 2 A 3:1 CH2Cl2 / TFA (4 mL) solution of N-(2-{[2-(2,6-dioxopiperidine-3-yl)-1-oxo-2,3-dihydro-1H-isoindole-4-yl]amino}ethyl) tert-butyl carbamate (69 mg, 0.17 mmol) was stirred at room temperature for 1.5 hours and concentrated. The residue was freeze-dried in an aqueous MeCN / 5% HCl solution to obtain the HCl salt of the title compound (56 mg, 96%) as a grayish-white solid. LC-MS t R = 2.00 min, m / z 303.

[0165] 3-[4-(2-aminoethoxy)-1-oxo-2,3-dihydro-1H-isoindole-2-yl]piperidine-2,6-dione(CB1i) [ka]

[0166] Step 1 t-BuMe2SiCl (16.70 g, 87.2 mmol) was added to a stirred, ice-cold solution of methyl 3-hydroxy-2-methylbenzoate (13.17 g, 79.3 mmol) and imidazole (13.48 g, 198 mmol) in dry DMF (50 mL). The mixture was stirred at room temperature for 20 hours and diluted with ether (300 mL). The mixture was washed with water (2 × 100 mL) and brine (50 mL) and dried over Na2SO4. The solvent was removed to leave a colorless oily substance (27.20 g), which was purified by chromatography in a 120 g silica cartridge and eluted in hexane under a 0-20% siRNA gradient to obtain methyl 3-[(tert-butyldimethylsilyl)oxy]-2-methylbenzoate (20.15 g, 90%). LC-MS t R = 6.58 min, m / z 281.

[0167] Step 2 3-[(tert-butyldimethylsilyl)oxy]-2-methylmethylbenzoate (3.34 g, 11.9 mmol), NBS (2.23 g, 12.6 mmol), AIBN (0.29 g, 1.8 mmol), and CCl4 (30 mL) were placed in a flask. The flask was sealed with a septum and replenished with vacuum evacuation / N2 (3 times). The mixture was stirred in an oil bath at 80°C for 7 hours and concentrated. The residue was dissolved in SiO2 (100 mL), washed with saturated NaHCO3 aqueous solution (2 × 10 mL) and brine (10 mL), and dried over Na2SO4. After removing the solvent, crude 2-(bromomethyl)-3-[(tert-butyldimethylsilyl)oxy]methylbenzoate (4.57 g, quantitative yield) remained as a concentrated oil. LC-MS t R = 6.60 min, m / z 279.

[0168] Step 3 A stirred mixture of crude 2-(bromomethyl)-3-[(tert-butyldimethylsilyl)oxy]methyl benzoate (4.57 g, ≤11.9 mmol), 4-amino-4-carbamoylbutanoate (2.75 g, 14.0 mmol), and MeCN (30 mL) was mixed with i-Pr2NEt (5 mL, 28.0 mmol). The mixture was stirred at room temperature for 1 hour and at 30°C for 6 hours. The mixture was concentrated. The residue was dissolved in RINKAN (90 mL), washed with 5% aqueous HCl (20 mL), saturated aqueous NaHCO3 (20 mL), and 20:1 brine / 5% aqueous HCl (21 mL), and dried over Na2SO4. After removing the solvent, a viscous oily substance (4.81 g) remained, which was purified by chromatography using a 40 g silica cartridge. Elution was performed in hexane under a 20-100% siRNA gradient to obtain methyl 4-{4-[(tert-butyldimethylsilyl)oxy]-1-oxo-2,3-dihydro-1H-isoindole-2-yl}-4-carbamoylbutanoate (3.10 g, 55%) as a foamy substance. LC-MS t R = 4.87 min, m / z 429, 407, 362.

[0169] Step 4 To a stirred, ice-cold solution of methyl 4-{4-[(tert-butyldimethylsilyl)oxy]-1-oxo-2,3-dihydro-1H-isoindole-2-yl}-4-carbamoylbutanoate (3.10 g, 7.6 mmol) in 9:1 DMF / H2O (40 mL), solid K2CO3 (0.53 g, 3.8 mmol) was added. The mixture was stirred at room temperature for 2 hours, cooled in an ice bath, and treated with concentrated HCl (0.65 mL, 7.7 mmol). The mixture was concentrated under high vacuum to leave a viscous oily substance, which was dissolved in MeCN (100 mL). The mixture was filtered through Celite and washed with MeCN (25 mL). The filtrate was concentrated to retain the oily substance, which was freeze-dried from an aqueous MeCN solution to obtain crude methyl 4-carbamoyl-4-(4-hydroxy-1-oxo-2,3-dihydro-1H-isoindole-2-yl)butanoate (2.86 g, quantitative yield) as a sticky solid. LC-MS t R = 2.52 min, m / z 293, 248.

[0170] Step 5 DIAD (0.85 mL, 4.3 mmol) was added dropwise to a stirred, ice-cold solution of methyl 4-carbamoyl-4-(4-hydroxy-1-oxo-2,3-dihydro-1H-isoindole-2-yl)butanoate (506 mg, 1.7 mmol), tert-butyl N-(2-hydroxyethyl)carbamate (420 mg, 2.6 mmol), and Ph3P (1.14 g, 4.3 mmol) in dry THF (10 mL). The mixture was removed from the ice bath and stirred at room temperature for 20 hours. When the mixture was concentrated, a yellow oily substance remained, which was purified by silica gel chromatography (20-100% siRNA gradient in hexane, followed by pure siRNA) and then by preparative HPLC to obtain methyl 4-[4-(2-{[(tert-butoxy)carbonyl]amino}ethoxy)-1-oxo-2,3-dihydro-1H-isoindole-2-yl]-4-carbamoylbutanoate (65 mg, 8%) as a white solid. 1H NMR (300MHz, CD3OD) Shift = 7.51 - 7.42 (m, 1H), 7.41 - 7.33 (m, 1H), 7.24 - 7.13 (m, 1H), 4.99-4.92 (m, 1H), 4.69 - 4.40 (m, 2H), 4.20 - 4.09 (m, 2H), 3.57 (s, 3H), 3.52 - 3.42 (m, 2H), 2.44 - 2.29 (m, 4H), 1.43 (s, 9H).

[0171] Step 6 Solid KOt-Bu (35 mg, 0.31 mmol) was added to a stirred, ice-cold solution of methyl 4-[4-(2-{[(tert-butoxy)carbonyl]amino}ethoxy)-1-oxo-2,3-dihydro-1H-isoindole-2-yl]-4-carbamoylbutanoate (65 mg, 0.15 mmol) in dried THF (2 mL). The mixture was stirred in an ice bath for 3 hours, and HOAc (0.5 mL) was added. The mixture was purified by preparative HPLC to obtain N-(2-{[2-(2,6-dioxopiperidine-3-yl)-1-oxo-2,3-dihydro-1H-isoindole-4-yl]oxy}ethyl)carbamate tert-butyl (20 mg, 33%). LC-MS t R = 3.59 min, m / z 426, 348.

[0172] Step 7 A solution of N-(2-{[2-(2,6-dioxopiperidine-3-yl)-1-oxo-2,3-dihydro-1H-isoindole-4-yl]oxy}ethyl) tert-butyl carbamate (20 mg, 0.05 mmol) in 1:1 CH2Cl2 / TFA was stirred at room temperature for 0.5 hours and concentrated. The residue was freeze-dried using an aqueous MeCN / 5% HCl solution to obtain the HCl salt of the title compound (18 mg, quantitative yield) as a grayish-white solid. LC-MS t R = 0.87 min, m / z 304.

[0173] 3-{4-[2-(2-aminoethoxy)ethoxy]-1-oxo-2,3-dihydro-1H-isoindole-2-yl}piperidine-2,6-dione(CB1j) [ka]

[0174] The title compound was prepared using the same procedure as described for CB1i, with tert-butyl N-[2-(2-hydroxyethoxy)ethyl]carbamate used in step 5. LC-MS t R = 2.30 min, m / z 348.

[0175] 2-(2,6-dioxopiperidine-3-yl)-4-[(2-hydroxyethyl)amino]-2,3-dihydro-1H-isoindole-1,3-dione(CB1k) [ka] The title compound was prepared using aminoethanol, following the same procedure as in Step 1 of CB1b. LC-MS t R = 2.92 min, m / z 318.

[0176] 2-(2-{[2-(2,6-dioxopiperidine-3-yl)-1-oxo-2,3-dihydro-1H-isoindole-4-yl]oxy}ethoxy)acetic acid (CB1l) [ka] The title compound was prepared using the same procedure as described for CB1i, with tert-butyl 2-(2-hydroxyethoxy)acetate in step 5. LC-MS t R = 2.65 min, m / z 363.

[0177] 4-[(3-aminopropyl)amino]-2-(2,6-dioxopiperidine-3-yl)-2,3-dihydro-1H-isoindole-1,3-dione(CB1m) [ka]

[0178] Step 1 A mixture of 2-(2,6-dioxopiperidine-3-yl)-4-fluoro-2,3-dihydro-1H-isoindole-1,3-dione (710 mg, 2.6 mmol), tert-butyl N-(3-aminopropyl)carbamate (470 mg, 2.7 mmol), i-Pr2NEt (1.0 mL, 5.6 mmol), and dry DMF (10 mL) was stirred at 90°C for 1 day. The mixture was diluted with ELISA (90 mL) and washed with water (2 × 20 mL) and brine (10 mL). The combined aqueous washes were back-extracted with ELISA (20 mL). The combined ELISA layer was dried over Na2SO4 and concentrated under reduced pressure, leaving a dark green oily substance (2.03 g). Preparative HPLC yielded N-(3-{[2-(2,6-dioxopiperidine-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindole-4-yl]amino}propyl)carbamate tert-butyl (100 mg, 9%) as a pale green solid. LC-MS t R min, m / z 453, 375, 331.

[0179] Step 2 A solution of N-(3-{[2-(2,6-dioxopiperidine-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindole-4-yl]amino}propyl) tert-butyl carbamate (100 mg, 0.24 mmol) in 3:1 CH2Cl2 / TFA (6 mL) was stirred at room temperature for 0.5 hours. The mixture was concentrated, and the residue was freeze-dried in an aqueous MeCN / 5% HCl solution to obtain the title compound (80 mg, 94%) as its HCl salt. LC-MS t R 2.78 min, m / z 331.

[0180] 2-(2,6-dioxopiperidine-3-yl)-5-{4-[(piperidine-4-yl)methyl]piperazine-1-yl}-2,3-dihydro-1H-isoindole-1,3-dione(CB2a) [ka]

[0181] Step 1 i-Pr2NEt (1.1 mL, 6.1 mmol) was added to a stirred solution of 2-(2,6-dioxopiperidine-3-yl)-5-fluoro-2,3-dihydro-1H-isoindole-1,3-dione (800 mg, 2.9 mmol) and tert-butylpiperazine-1-carboxylate (560 mg, 3.5 mmol) in DMSO (10 mL). The mixture was stirred at 90 °C for 3 hours, cooled to room temperature, and purified by preparative HPLC to obtain 4-[2-(2,6-dioxopiperidine-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindole-5-yl]piperazine-1-carboxylate tert-butyl (410 mg, 32%) as a yellow solid. 1H NMR (300MHz, DMSO-d6) Shift= 7.74 - 7.64 (m, 1H), 7.33 (d, J=2.2 Hz, 1H), 7.23 (d, J=8.8 Hz, 1H), 5.06 (dd, J=5.5, 12.5 Hz, 1H), 3.45 (s, 8H), 2.99 - 2.75 (m, 1H), 2.63 - 2.51 (m, 2H), 2.08 - 1.89 (m, 1H), 1.41 (s, 9H).LC-MS t R = 4.28 min, m / z 386, 342.

[0182] Step 2 To a stirred solution of 4-[2-(2,6-dioxopiperidine-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindole-5-yl]piperazine-1-carboxylate tert-butyl (410 mg, 0.93 mmol) in CH2Cl2 (6 mL), TFA (2 mL) was added. The mixture was stirred at room temperature for 1 hour and concentrated. The residue was freeze-dried using an aqueous solution of MeCN / 5% HCl to obtain the bisHCl salt of 2-(2,6-dioxopiperidine-3-yl)-5-(piperazine-1-yl)-2,3-dihydro-1H-isoindole-1,3-dione (450 mg, quantitative yield) as a yellow solid. LC-MS t R = 2.23 min, m / z 342.

[0183] Step 3 2-(2,6-dioxopiperidine-3-yl)-5-(piperazine-1-yl)-2,3-dihydro-1H-isoindole-1,3-dionebisHCl (450 mg, 1.1 mmol), 4-formylpiperidine-1-carboxylate tert-butyl (460 mg, 2.1 mmol), NaOAc (270 mg, 3.3 mmol), and dried DCE (10 mL) were stirred together, to which MgSO4 (150 mg) was added. The mixture was stirred under N2 for 0.5 hours, and NaBH(OAc)3 (689 mg, 3.3 mmol) was added. The mixture was stirred overnight at room temperature, diluted with water (10 mL), and concentrated under reduced pressure to remove DCE. The residue was diluted with saturated NaHCO3 aqueous solution (20 mL) and extracted with ELISA (2 × 80 mL). The combined siRNA layers were concentrated to leave a yellow solid (680 mg), which was purified by preparative HPLC to obtain the TFA salt (320 mg, 45%) of 4-({4-[2-(2,6-dioxopiperidine-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindole-5-yl]piperazine-1-yl}methyl)piperidine-1-carboxylate tert-butyl. 1 H NMR (300MHz, DMSO-d6) δ = 7.81 - 7.71 (m, 1H), 7.54 - 7.45 (m, 1H), 7.41 - 7.24 (m, 1H), 5.17 - 4.98 (m, 1H), 4.31 - 4.09 (m, 2H), 4.03 - 3.80 (m, 2H), 3.65-3.05 (m, 10H), 2.95-2.40 (m, 4H), 2.10 - 1.89 (m, 2H), 1.82 - 1.62 (m, 2H), 1.38 (s, 9H), 1.17 - 0.93 (m, 1H).LC-MS t R = 3.25 min, m / z 540, 484, 440.

[0184] Step 4 A 3:1 CH2Cl2 / TFA (6 mL) solution of the TFA salt of 4-({4-[2-(2,6-dioxopiperidine-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindole-5-yl]piperazine-1-yl}methyl)piperidine-1-carboxylate tert-butyl (320 mg, 0.49 mmol) was stirred at room temperature for 1 hour and concentrated. The residue was lyophilized using an aqueous MeCN / 5% HCl solution to obtain the bisHCl salt of the title compound (306 mg, quantitative yield) as a solid. LC-MS tR = 2.16 min, m / z 440.

[0185] 3-[1-oxo-5-(piperazine-1-yl)-2,3-dihydro-1H-isoindole-2-yl]piperidine-2,6-dione (CB2b) [ka]

[0186] Step 1 5-bromo-1,3-dihydro-2-benzofuran-1-one (5.45 g, 25.6 mmol), mono-Boc piperazine (4.76 g, 25.6 mmol), xanthophos (1.48 g, 2.6 mmol), Pd2(dba)3 (2.34 g, 2.6 mmol), and powdered K3PO4 (10.86 g, 51.2 mmol) were placed in a flask. The flask was evacuated and replenished with N2 (3 times), and 100 mL of dry dioxane was introduced using a syringe. The flask was evacuated and replenished with N2 (3 times), and the mixture was stirred at 100°C for 1 day. The mixture was cooled to room temperature and filtered through Celite. The filtrate was concentrated. The residue was dissolved in toluene (175 mL), washed with water (50 mL), 5% N-acetylcysteine ​​aqueous solution (50 mL), saturated NaHCO3 aqueous solution (50 mL), and brine (50 mL), and dried over Na2SO4. The solvent was removed to leave a red solid (7.84 g), which was purified by chromatography using an 80 g silica cartridge. Elution was performed in hexane under a 0-70% toluene gradient to obtain 4-(1-oxo-1,3-dihydro-2-benzofuran-5-yl)piperazine-1-carboxylate tert-butyl (3.62 g, 44%). LC-MS t R = 4.31 min, m / z 319.

[0187] Step 2 A stirred mixture of 4-(1-oxo-1,3-dihydro-2-benzofuran-5-yl)piperazine-1-carboxylate tert-butyl (3.62 g, 11.4 mmol), MeOH (30 mL), and THF (15 mL) was mixed with a solution of NaOH (1.82 g, 45.5 mmol) in water (15 mL). The mixture was stirred overnight at room temperature and concentrated to remove the organic solvent. The aqueous residue was diluted with 5% aqueous HCl (100 mL) and extracted with butyl (2 × 150 mL). The combined butyl layers were washed with brine (50 mL), dried over Na₂SO₄, and concentrated, leaving crude 4-{4-[(tert-butoxy)carbonyl]piperazine-1-yl}-2-(hydroxymethyl)benzoic acid (3.71 g, 97%) as an orange solid. 1H NMR (300MHz, CDCl3) Shift = 8.10 - 7.98 (m, 1H), 6.88 (d, J=2.6 Hz, 1H), 6.82 - 6.73 (m, 1H), 4.78 (s, 2H), 3.65 - 3.51 (m, 4H), 3.45 - 3.32 (m, 4H), 1.49 (s, 9H).

[0188] Step 3 To a 1:1 ethyl acetate / MeOH (100 mL) ice-cold solution of stirred crude 4-{4-[(tert-butoxy)carbonyl]piperazin-1-yl}-2-(hydroxymethyl)benzoic acid (2.69 g, 8.0 mmol) of 0.6 M Me3SiCHN2 (50 mL, 30 mmol) in hexane was added dropwise over 15 minutes. The mixture was stirred in an ice bath for 1 hour, and HOAc (5 mL) was added dropwise over 5 minutes. The mixture was stirred in an ice bath for 15 minutes and concentrated. The residue was dissolved in ethyl acetate (200 mL), washed with water (3 × 25 mL), brine (25 mL), saturated NaHCO3 aqueous solution (25 mL), and brine (25 mL), and dried over Na2SO4. After removing the solvent, black tar (2.72 g) remained and was subjected to chromatography in a 40 g silica cartridge. Elution was obtained in hexane under a 0-50% HCl gradient to obtain 4-[3-(hydroxymethyl)-4-(methoxycarbonyl)phenyl]piperazine-1-carboxylate tert-butyl (1.24 g, 44%). LC-MS t R = 4.38 min, m / z 351.

[0189] Step 4 CBr4 (1.76 g, 5.3 mmol) was added to a stirred solution of 4-[3-(hydroxymethyl)-4-(methoxycarbonyl)phenyl]piperazine-1-carboxylate tert-butyl (1.24 g, 3.5 mmol) and PPh3 (1.39 g, 5.3 mmol) in dry THF (20 mL). After stirring at room temperature for 18 hours, further PPh3 (1.39 g, 5.3 mmol) and CBr4 (1.76 g, 5.3 mmol) were added. The mixture was stirred for 2 hours and concentrated. The residue was purified by chromatography in an 80 g silica cartridge and eluted in hexane under a 0-60% siRNA gradient to obtain 4-[3-(bromomethyl)-4-(methoxycarbonyl)phenyl]piperazine-1-carboxylate tert-butyl (550 mg, 37%) as a colorless oil. 1 H NMR (300MHz, cdcl3) shift = 7.92 (d, J=8.8 Hz, 1H), 6.87 (d, J=2.6 Hz, 1H), 6.81 - 6.73 (m, 1H), 4.95 (s, 2H), 3.87 (s, 3H), 3.61-3.54 (m, 4H), 3.37 - 3.23 (m, 4H), 1.48 (s, 9H).

[0190] Step 5 A mixture of 4-[3-(bromomethyl)-4-(methoxycarbonyl)phenyl]piperazine-1-carboxylate tert-butyl (550 mg, 1.3 mmol), 3-aminopiperidine-2,6-dione (256 mg, 2.0 mmol), i-Pr2NEt (0.5 mL, 2.9 mmol), and MeCN (20 mL) was stirred at 80°C for 7 days and concentrated. The residue was purified by preparative HPLC to obtain 4-[2-(2,6-dioxopiperidine-3-yl)-1-oxo-2,3-dihydro-1H-isoindole-5-yl]piperazine-1-carboxylate tert-butyl (180 mg, 31%) as a gray solid. LC-MS t R = 3.83 min, m / z 429.

[0191] Step 6 A solution containing 4-[2-(2,6-dioxopiperidine-3-yl)-1-oxo-2,3-dihydro-1H-isoindole-5-yl]piperazine-1-carboxylate tert-butyl (180 mg, 0.42 mmol) in CH2Cl2 (10 mL) and dioxane (10 mL, 40 mmol) in 4M HCl was stirred at room temperature for 1 day and concentrated, leaving the HCl salt of the title compound (150 mg, quantitative yield) as a white solid. LC-MS t R = 2.05 min, m / z 329.

[0192] 3-(1-oxo-5-{4-[(piperidine-4-yl)methyl]piperazine-1-yl}-2,3-dihydro-1H-isoindole-2-yl)piperidine-2,6-dione(CB2c) [ka]

[0193] The title compound was prepared from 3-[1-oxo-5-(piperazin-1-yl)-2,3-dihydro-1H-isoindole-2-yl]piperidine-2,6-dione (CB2b) following a procedure similar to that of steps 3 and 4 in the preparation of CB2a. LC-MS t R = 1.97 min, m / z 426.

[0194] 2-{[2-(2,6-dioxopiperidine-3-yl)-1-oxo-2,3-dihydro-1H-isoindole-5-yl]oxyacetic acid (CB2d) [ka] The title compound was prepared from 3-(5-hydroxy-1-oxo-2,3-dihydro-1H-isoindole-2-yl)piperidine-2,6-dione following a procedure similar to that used for the preparation of CB1a. LC-MS t R = 2.38 min, m / z 319.

[0195] 5-[(2-aminoethyl)amino]-2-(2,6-dioxopiperidine-3-yl)-2,3-dihydro-1H-isoindole-1,3-dione(CB2e) [ka] Step 1 A stirred mixture of 2-(2,6-dioxopiperidine-3-yl)-5-fluoro-2,3-dihydro-1H-isoindole-1,3-dione (550 mg, 2.0 mmol), N-(2-aminoethyl)carbamate tert-butyl (380 mg, 2.4 mmol), and DMSO (7 mL) was treated with i-Pr2NEt (0.75 mL, 4.2 mmol). The mixture was stirred at 90°C for 20 hours. Preparative HPLC yielded N-(2-{[2-(2,6-dioxopiperidine-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindole-5-yl]amino}ethyl)carbamate tert-butyl (100 mg, 12%) as a greenish solid. LC-MS t R = 3.80 min, m / z 439, 361, 317.

[0196] Step 2 A solution of tert-butyl N-(2-{[2-(2,6-dioxopiperidine-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindole-5-yl]amino}ethyl)carbamate (100 mg, mmol) in CH2Cl2 (3 mL) and TFA (2 mL) was stirred at room temperature for 1 hour and concentrated. The residue was freeze-dried from an aqueous MeCN / 5% HCl solution to obtain the HCl salt of the title compound (76 mg, 89%) as a dark green solid. LC-MS t R = 2.20 min, m / z 317.

[0197] 3-{5-[3-(2-aminoethoxy)propane-1-in-1-yl]-1-oxo-2,3-dihydro-1H-isoindole-2-yl}piperidine-2,6-dione (CB2f) [ka]

[0198] Step 1 3-(5-bromo-1-oxo-2,3-dihydro-1H-isoindole-2-yl)piperidine-2,6-dione (351 mg, 1.08 mmol), N-[2-(propa-2-in-1-yloxy)ethyl]carbamate tert-butyl (214 mg, 1.07 mmol), CuI (41 mg, 0.22 mmol), and PdCl2(PPh3)2 (74 mg, 0.11 mmol) were added to a vial equipped with a stirring rod. The vial was closed with a septum and replenished with vacuum evacuation / N2 (3 times). Dry DMF (2 mL) and Et3N (2 mL) were introduced by syringe. The vial was replenished with vacuum evacuation / N2 (3 times) and stirred at 80°C for 6 hours. The mixture was cooled to room temperature, diluted with siRNA (90 mL), washed with 5% aqueous HCl solution (2 × 25 mL) and brine (25 mL), and dried over Na₂SO₄. After removing the solvent, a dark oily substance (790 mg) remained, which was purified by preparative HPLC to obtain N-[2-({3-[2-(2,6-dioxopiperidine-3-yl)-1-oxo-2,3-dihydro-1H-isoindole-5-yl]propa-2-in-1-yl}oxy)ethyl]carbamate tert-butyl (170 mg, 36%) as a yellow solid. 1 H NMR (300MHz, DMSO-d6) Shift= 7.77 - 7.68 (m, 2H), 7.64 - 7.48 (m, 1H), 6.86 (t, J=5.6 Hz, 1H), 5.10 (dd, J=5.0, 13.2 Hz, 1H), 4.52 - 4.26 (m, 2H), 3.57 - 3.41 (m, 2H), 3.57 - 3.41 (m, 2H), 3.11 (q, J=5.9 Hz, 2H), 2.99 - 2.77 (m, 1H), 2.66 - 2.50 (m, 1H), 2.42 - 2.28 (m, 1H), 2.10 - 1.90 (m, 1st hour), 1.35 (s, 9th hour).

[0199] Step 2 A solution of N-[2-({3-[2-(2,6-dioxopiperidine-3-yl)-1-oxo-2,3-dihydro-1H-isoindole-5-yl]propa-2-in-1-yl}oxy)ethyl]tert-butyl carbamate (170 mg, 0.39 mmol) in 3:1 CH2Cl2 / TFA (6 mL) was stirred at room temperature for 1 hour. The mixture was concentrated, and the residue was freeze-dried in a MecN / 5% HCl aqueous solution to obtain the HCl salt of the title compound (105 mg, 72%) as a yellow solid. LC-MS t R = 2.32 min, m / z 342.

[0200] 2-{[2-(2,6-dioxopiperidine-3-yl)-1-oxo-2,3-dihydro-1H-isoindole-5-yl]oxy}acetaldehyde (CB 2g) [ka] 3-(5-hydroxy-1-oxo-2,3-dihydro-1H-isoindole-2-yl)piperidine-2,6-dione (338 mg, 1.30 mmol), Ph3P (852 mg, 3.25 mmol), 2,2-(diethoxy)ethanol (262 mg, 1.95 mmol), and dry THF (10 mL) were stirred together, to which DIAD (0.64 mL, 3.25 mmol) was added. The mixture was stirred at room temperature for 18 hours, and then 5% aqueous HCl (2 mL) was added. The mixture was stirred over the weekend and filtered. The filtrate was purified by preparative HPLC to obtain the title compound (250 mg, 63%) as a grayish-white solid. LC-MS t R = 2.35 min, m / z 303.

[0201] Synthesis of Nef-bonded intermediates 4-[2-(4-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}phenyl)ethyl]-1-(5-chloro-1H-1,3-benzodiazole-2-yl)-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-5-ol (NB1a) [ka]

[0202] Step 1 A stirred solution of triphenylphosphine (1.40 g, 5.4 mmol) in dry THF was cooled to -40°C, and DIAD (1.05 mL, 5.4 mmol) was added dropwise over 2 minutes. The mixture was stirred in a cooling bath for 10 minutes, and a solution of methyl 4-(4-hydroxyphenyl)butanoate (965 mg, 5.4 mmol) and 2-[2-(2-azidoethoxy)ethoxy]ethane-1-ol (1.05 g, 5.9 mmol) in dry THF (6 mL) was added dropwise over 2 minutes. The mixture was stirred in a cooling bath for 1 hour and at room temperature for 4 hours. The mixture was concentrated, and the residue was subjected to chromatography in an 80 g silica cartridge, eluted in hexane with a 0-70% SiO gradient to obtain methyl 4-(4-{2-[2-(2-azidoethoxy)ethoxy]ethoxy}phenyl)butanoate (1.68 g, 89%). LC-MS t R = 4.86, m / z 374.

[0203] Step 2 A solution of methyl 4-(4-{2-[2-(2-azidoethoxy)ethoxy]ethoxy}phenyl)butanoate (1.68 g, 4.8 mmol) and methyl 4-(trifluoromethyl)benzoate (3.90 g, 19.1 mmol) in toluene (50 mL) was concentrated under reduced pressure to retain the oil, to which 60% NaH (0.96 g, 23.9 mmol) and dry THF (40 mL) were added. The mixture was stirred under N2 at 65°C for 5 hours, cooled to room temperature, diluted with SiO (100 mL), washed with 5% aqueous HCl (20 mL) and brine (20 mL), and dried over Na2SO4. After removing the solvent, a pale yellow solid (6.44 g) was obtained by chromatography on silica gel and elution with a hexane gradient of ethyl acetate to obtain methyl 4-(4-{2-[2-(2-azidoethoxy)ethoxy]ethoxy}phenyl)-2-[4-(trifluoromethyl)benzoyl]butanoate (750 mg, 30%). LC-MS t R = 5.58 min, m / z 546.

[0204] Step 3 A mixture of 4-(4-{2-[2-(2-azidoethoxy)ethoxy]ethoxy}phenyl)-2-[4-(trifluoromethyl)benzoyl]butanoate (160 mg, 0.31 mmol), Boc2O (133 mg, 0.61 mmol), 10% palladium-carbon (Pd on C) (15 mg), and THF (10 mL) was stirred for 2 hours under H2 (1 atm, balloon). The mixture was filtered, and the filtrate was concentrated, leaving a waxy solid (245 mg). Chromatography using a 24g silica cartridge eluted in hexane under a 0-100% siRNA gradient yielded 4-(4-{2-[2-(2-{[(tert-butoxy)carbonyl]amino}ethoxy)ethoxy]ethoxy}phenyl)-2-[4-(trifluoromethyl)benzoyl]butanoate methyl (102 mg, 55%) as a colorless oil. LC-MS t R = 5.60 min, m / z 620, 498.

[0205] Step 4 A solution of methyl 4-(4-{2-[2-(2-{[(tert-butoxy)carbonyl]amino}ethoxy)ethoxy]ethoxy}phenyl)-2-[4-(trifluoromethyl)benzoyl]butanoate (102 mg, 0.17 mmol) and 5-chloro-2-hydrazinyl-1H-1,3-benzodiazole (33 mg, 0.18 mmol) in 4:1 MeOH / HOAc (2.5 mL) was stirred at 50°C for 1 day and at 70°C for 6 days. The mixture was diluted with ELISA (100 mL), washed with 5% aqueous HCl (15 mL) and 4:1 brine / saturated NaHCO3 aqueous solution (15 mL), and dried over Na2SO4. The solvent was removed to leave an oily substance (120 mg), which was purified by preparative HPLC to obtain N-(2-{2-[2-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenoxy)ethoxy]ethoxy}ethyl) tert-butyl carbamate (27 mg, 21%). LC-MS t R= 5.96 min, m / z 730, 630.

[0206] Step 5 To a stirred solution of N-(2-{2-[2-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenoxy)ethoxy]ethoxy}ethyl) tert-butyl carbamate (22 mg, 30 mol) in CH2Cl2 (2 mL), 4 M HCl (2 mL) in dioxane was added. The mixture was stirred at room temperature for 3 hours and then concentrated, leaving behind the HCl salt of 4-[2-(4-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}phenyl)ethyl]-1-(5-chloro-1H-1,3-benzodiazole-2-yl)-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-5-ol (28 mg, quantitative yield) as a yellowish-brown solid. LC-MS t R = 4.45 min, m / z 630.

[0207] 4-(2-{4-[2-(2-aminoethoxy)ethoxy]phenyl}ethyl)-1-(5-chloro-1H-1,3-benzodiazole-2-yl)-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-5-ol (NB1b) [ka]

[0208] Step 1 A stirred mixture of methyl 4-(4-hydroxyphenyl)butanoate (1.04 g, 5.3 mmol), methyl 2-(2-azidoethoxy)ethylmethanesulfonate (1.23 g, 5.9 mmol), KI (978 mg, 5.9 mmol), and DMF (10 mL) was mixed with Cs2CO3 (1.92 g, 5.9 mmol). The mixture was stirred at room temperature for 3 days, diluted with SiO2 (90 mL), washed with water (2 × 15 mL) and brine (15 mL), and dried over Na2SO4. After removing the solvent, a red oily substance remained. This was subjected to chromatography on silica gel, and eluted on an SiO2 / hexane gradient, yielding methyl 4-{4-[2-(2-azidoethoxy)ethoxy]phenyl}butanoate (420 mg, 25%) as the oily substance. LC-MS t R = 4.82 min, m / z 330.

[0209] Steps 2-5 The title compound was obtained by a procedure similar to that described in steps 2-5 of the synthesis of NB1a. LC-MS t R = 4.52 min, m / z 586.

[0210] 4-{2-[4-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethoxy)phenyl]ethyl}-1-(5-chloro-1H-1,3-benzodiazole-2-yl)-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-5-ol(NB1c) [ka]

[0211] Step 1 A mixture of methyl 4-(4-hydroxyphenyl)butanoate (2.50 g, 12.9 mmol), methyl 2-{2-[2-(2-azidoethoxy)ethoxy]ethoxy}ethylmethanesulfonate (4.20 g, 14.0 mmol), KI (2.35 g, 14.2 mmol), Cs2CO3 (4.61 g, 14.2 mmol), and DMF (40 mL) was stirred at 40°C for 4 days. The mixture was diluted with SiO (60 mL) and washed with water (3 × 20 mL). The combined aqueous layer was back-extracted with SiO (40 mL). The combined SiO layer was washed with brine (15 mL), dried over Na2SO4, and concentrated, leaving mobile oil (8.03 g). Chromatography using an 80g silica cartridge, eluting under a 0-40% siRNA gradient in hexane, yielded methyl 4-[4-(2-{2-[2-(2-azidoethoxy)ethoxy]ethoxy}ethoxy)phenyl]butanoate (2.19g, 43%).

[0212] Step 2 A mixture of methyl 4-[4-(2-{2-[2-(2-azidoethoxy)ethoxy]ethoxy}ethoxy)phenyl]butanoate (675 mg, 1.7 mmol) and methyl 4-(trifluoromethyl)butanoate (1.40 g, 6.8 mmol) was mixed with 60% NaH in oil (350 mg, 8.6 mmol), followed by the addition of dry THF (8 mL). The mixture was heated under N2 reflux for 6 hours and cooled to room temperature. The mixture was diluted with SiO2 (90 mL), washed with 5% aqueous HCl (20 mL) and brine (20 mL), and dried over Na2SO4. The solvent was removed to retain the oily substance (2.36 g), which was purified by chromatography using an 80 g silica cartridge. Elution was performed in hexane under a 0-80% siRNA gradient to obtain 4-(4-{2-[2-(2-azidoethoxy)ethoxy]ethoxy}phenyl)-2-[4-(trifluoromethyl)benzoyl]butanoate methyl (660 mg, 68%) as a colorless oil. LC-MS t R = 4.81 min, m / z 590.

[0213] Step 3 A mixture of 4-(4-{2-[2-(2-azidoethoxy)ethoxy]ethoxy}phenyl)-2-[4-(trifluoromethyl)benzoyl]butanoate methyl (305 mg, 0.54 mmol), 5-chloro-2-hydrazinyl-1H-1,3-benzodiazole (105 mg, 0.56 mmol), TsOH.H2O (20 mg, 0.11 mmol), and MeOH (3 mL) was heated in a microwave at 130°C for 3 hours. The mixture was diluted with MeOH (3 mL), filtered, and the filtrate was separated and purified by HPLC to obtain 4-[2-(4-{2-[2-(2-azidoethoxy)ethoxy]ethoxy}phenyl)ethyl]-1-(5-chloro-1H-1,3-benzodiazole-2-yl)-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-5-ol (150 mg, 40%). LC-MS t R = 5.15 min, m / z 700.

[0214] Step 4 A stirred solution of 4-[2-(4-{2-[2-(2-azidoethoxy)ethoxy]ethoxy}phenyl)ethyl]-1-(5-chloro-1H-1,3-benzodiazole-2-yl)-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-5-ol (150 mg, 0.21 mmol) in dry THF (5 mL) was mixed with 1 M Me3P (0.65 mL, 0.65 mmol) in THF. The mixture was stirred at room temperature for 1.5 hours, and water (0.5 mL) was added. After stirring overnight at room temperature, 1 M NaOH aqueous solution (0.5 mL, 0.5 mmol) was added, and stirring was continued for 2 hours. HOAc (1 mL) was added, and the mixture was purified by preparative HPLC to obtain the TFA salt (97 mg, 57%) of 4-{2-[4-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethoxy)phenyl]ethyl}-1-(5-chloro-1H-1,3-benzodiazole-2-yl)-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-5-ol. LC-MS t R = 4.62 min, m / z 674.

[0215] 4-(2-{4-[3-(3-aminopropoxy)propoxy]phenyl}ethyl)-1-(5-chloro-1H-1,3-benzodiazole-2-yl)-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-5-ol(NB1d) [ka]

[0216] Step 1 A mixture of methyl 4-(4-hydroxyphenyl)butanoate (500 mg, 2.6 mmol), Cs2CO3 (925 mg, 2.8 mmol), and DMF (5 mL) was stirred at room temperature for 10 minutes, and 1-chloro-3-(3-chloropropoxy)propane (885 mg, 5.2 mmol) was added. The mixture was stirred at room temperature for 4 days, diluted with toluene (95 mL), washed with 5% aqueous HCl (20 mL) and brine (20 mL), and dried over Na2SO4. The solvent was removed to leave a colorless oily substance (1.62 g), which was subjected to chromatography in a 40 g silica cartridge and eluted in hexane under a 0-40% toluene gradient to obtain methyl 4-{4-[3-(3-chloropropoxy)propoxy]phenyl}butanoate (540 mg, 64%). 1 H NMR (300MHz, CDCl3) Shift= 7.14 - 7.02 (m, 2H), 6.87 - 6.76 (m, 2H), 4.03 (t, J=6.2 Hz, 2H), 3.70 - 3.49 (m, 9H), 2.62-2.55 (m, 2H), 2.40 - 2.25 (m, 2H), 2.09 - 1.83 (m, 6H).

[0217] Step 2 A mixture of methyl 4-{4-[3-(3-chloropropoxy)propoxy]phenyl}butanoate (540 mg, 1.6 mmol), NaN3 (270 mg, 4.1 mmol), water (10 mL), and DMF (10 mL) was stirred at 80°C for 6 days. The mixture was cooled to room temperature, diluted with water (20 mL), and extracted with HCl (2 × 50 mL) / . The combined HCl layers were washed with brine (10 mL), dried over Na2SO4, and concentrated to obtain a yellow oil (830 mg). This was purified by chromatography using a 40 g silica cartridge, and eluted in hexane under a 0-40% HCl gradient to obtain methyl 4-{4-[3-(3-azidopropoxy)propoxy]phenyl}butanoate (470 mg, 85%) as a colorless oil. LC-MS t R = 5.27 min, m / z 358.

[0218] Steps 3-5 The title compound was obtained by performing steps 3-5 using the same procedure as described in steps 2-4 of the synthesis of NB1c. LC-MS t R = 4.65 min, m / z 614.

[0219] 4-(2-{4-[(14-amino-3,6,9,12-tetraoxatetradecane-1-yl)oxy]phenyl}ethyl)-1-(5-chloro-1H-1,3-benzodiazole-2-yl)-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-5-ol (NB1e) [ka]

[0220] The title compound was prepared using a procedure similar to that described in steps 1-4 of the synthesis of NB1c, using 14-azido-3,6,9,12-tetraoxatetradecane-1-ylmethanesulfonate in step 1. LC-MS t R = 4.62 min, m / z 718.

[0221] 1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-{2-[2-(piperazine-1-yl)ethoxy]ethoxy}phenyl)ethyl]-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-5-ol(NB1f) [ka]

[0222] Step 1 A mixture of methyl 4-(4-hydroxyphenyl)butanoate (1.98 g, 10.2 mmol), 2-(2-chloroethoxy)ethane-1-ol (1.27 g, 10.2 mmol), Cs2CO3 (3.65 g, 11.2 mmol), KI (1.86 g, 11.2 mmol), and DMF (20 mL) was stirred at 45°C for 6 days. The mixture was diluted with water (30 mL) and extracted with SiO (3 × 70 mL). The combined SiO layers were washed with brine (20 mL), dried over Na2SO4, and concentrated, leaving a liquid oil (5.67 g). Chromatography on silica gel eluted with an SiO hexane gradient yielded methyl 4-{4-[2-(2-hydroxyethoxy)ethoxy]phenyl}butanoate (640 mg, 22%). LC-MS t R = 3.78 min, m / z 305.

[0223] Step 2 MeSO2Cl (0.26 mL, 3.4 mmol) was added to a stirred, ice-cold solution of methyl 4-{4-[2-(2-hydroxyethoxy)ethoxy]phenyl}butanoate (640 mg, 2.3 mmol) and i-Pr2NEt (0.81 mL, 4.5 mmol) in CH2Cl2 (15 mL). The ice bath was melted, and the mixture was stirred overnight at room temperature. The mixture was concentrated. The residue was dissolved in  (100 mL), washed with 5% aqueous HCl (20 mL) and 9:1 brine / saturated NaHCO3 aqueous solution (10 mL), and dried over Na2SO4. After removing the solvent, methyl 4-(4-{2-[2-(methanesulfonyloxy)ethoxy]ethoxy}phenyl)butanoate (720 mg, 88%) remained as an oily substance. LC-MS tR = 4.30 min, m / z 383.

[0224] Step 3 A mixture of methyl 4-(4-{2-[2-(methanesulfonyloxy)ethoxy]ethoxy}phenyl)butanoate (720 mg, 2.0 mmol), mono-Boc piperazine (460 mg, 4.0 mmol), i-Pr2NEt (0.74 mL, 4.1 mmol), and DMF (20 mL) was stirred at 40°C for 1 day. The mixture was diluted with SiO (100 mL), washed with water (10 mL) and brine (10 mL), and dried over Na2SO4. After removing the solvent, a brown oily substance (1.00 g) remained. This was purified by chromatography using a 40 g silica cartridge and eluted under a 0-10% MeOH gradient in CH2Cl2 to obtain 4-(2-{2-[4-(4-methoxy-4-oxobutyl)phenoxy]ethoxy}ethyl)piperazine-1-carboxylate tert-butyl (780 mg, 86%) as an oily substance. LC-MS t R = 3.85 min, m / z 451.

[0225] Step 3 Step 3 was carried out using the same procedure as described in Step 2 of the synthesis of NB1a.

[0226] Steps 4 and 5 Steps 4 and 5 were carried out using the same procedure as described in steps 4 and 5 of the synthesis of NB1a.

[0227] 1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-{2-[4-(2-{2-[2-(piperazine-1-yl)ethoxy]ethoxy}ethoxy)phenyl]ethyl}3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-5-ol (NB 1g) [ka]

[0228] The title compound was prepared using the same procedure as described in steps 1-4 of the synthesis of NB1f using 2-[2-(2-chloroethoxy)ethoxy]ethane-1-ol in step 1. LC-MS t R =4.08 min, m / z 699.

[0229] 2-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenoxy)acetic acid (NB1h) [ka]

[0230] Step 1 4-(2-hydroxyethyl)phenol (4.97 g, 36.0 mmol), K2CO3 (5.47 g, 39.6 mmol), and DMF (50 mL) were stirred together, and t-butyl bromoacetate (5.3 mL, 36.0 mmol) was added. The mixture was stirred at room temperature for 1 day, diluted with water (150 mL), and extracted with SiO2 (4 × 50 mL). The combined SiO2 layers were washed with water (25 mL) and brine (25 mL) and dried over Na2SO4. The solvent was removed, leaving a viscous oily substance (13.20 g), which was purified by chromatography using an 80 g silica cartridge. Elution in hexane under a 0-70% SiO2 gradient yielded tert-butyl 2-[4-(2-hydroxyethyl)phenoxy]acetate (7.28 g, 80%) as a colorless oily substance. 1 H NMR (300MHz, CDCl3) δ = 7.13 (d, J=8.8 Hz, 2H), 6.84 (d, J=8.4 Hz, 2H), 4.49 (s, 2H), 3.89 - 3.69 (m, 2H), 2.87 - 2.65 (m, 2H), 1.45 (s, 9H).

[0231] Step 2 Methanesulfonyl chloride (5.6 mL, 72.2 mmol) was added to a stirred, ice-cold solution of 2-[4-(2-hydroxyethyl)phenoxy]tert-butyl acetate (7.28 g, 28.9 mmol) and i-Pr2NEt (15.5 mL, 86.6 mmol) in CH2Cl2 (150 mL). The ice bath was melted, and the mixture was stirred overnight at room temperature. The mixture was concentrated, the residue was dissolved in SiO2 (150 mL), and washed with 5% HCl aqueous solution (2 × 50 mL) and brine (50 mL). The SiO2 layer was filtered through Celite. The filtrate was dried over Na2SO4 and concentrated, leaving 2-{4-[2-(methanesulfonyloxy)ethyl]phenoxy}tert-butyl acetate (9.72 g, quantitative yield) as a dark red oily substance. 1 H NMR (300MHz, CDCl3) δ = 7.11 (m, 2H), 6.88 - 6.76 (m, 2H), 4.47 (s, 2H), 4.40 - 4.28 (m, 2H), 3.02 - 2.91 (m, 2H), 2.81 (s, 3H), 1.46 (s, 9H).

[0232] Step 3 A mixture of methyl 3-oxo-3-[4-(trifluoromethyl)phenyl]propanoate (5.00 g, 20.3 mmol), tert-butyl 2-{4-[2-(methanesulfonyloxy)ethyl]phenoxy}acetate (6.84 g, 20.7 mmol), K2CO3 (2.81 g, 20.3 mmol), KI (3.37 g, 20.3 mmol), and DMF (50 mL) was stirred at 70°C for 6 hours. The mixture was cooled, diluted with RINKAN (350 mL), washed with 5% aqueous HCl (50 mL), water (50 mL), and brine (50 mL), and dried over Na2SO4. The solvent was removed, leaving a dark brown oily substance (12.06 g). This was subjected to chromatography using a 120 g silica cartridge, and eluted with 0-25% ELISA in a hexane gradient to obtain a pale yellow oily mixture of the desired C-alkylation product 4-{4-[2-(tert-butoxy)-2-oxoethoxy]phenyl}-2-[4-(trifluoromethyl)benzoyl]methyl butanoate and the O-alkylation product (2)-3-(2-{4-[2-(tert-butoxy)-2-oxoethoxy]phenyl}ethoxy)-3-[4-(trifluoromethyl)phenyl]prop-2-enoate methyl (4.89 g, 50%) in an approximately 3:1 ratio. 1 ¹H NMR (300MHz, CDCl3) δ = 7.99 - 7.88 (m, 2H), 7.78 - 7.65 (m, 2H), 7.11 - 7.02 (m, 2H), 6.88 - 6.74 (m, 2H), 4.54 - 4.44 (s, 2H), 3.64 (s, 3H), 3.12 - 2.94 (m, 1H), 2.70 - 2.54 (m, 2H), 2.45 - 2.16 (m, 2H), 1.48 (s, 9H). Resonances assigned to O-alkylation byproducts have not been reported.

[0233] Step 4 A mixture of methyl 4-{4-[2-(tert-butoxy)-2-oxoethoxy]phenyl}-2-[4-(trifluoromethyl)benzoyl]butanoate and (2)-3-(2-{4-[2-(tert-butoxy)-2-oxoethoxy]phenyl}ethoxy)-3-[4-(trifluoromethyl)phenyl]prop-2-enoate methyl (1.50 g, 3.1 mmol), 5-chloro-2-hydrazinyl-1H-1,3-benzodiazole (685 mg, 3.8 mmol), HOAc (3 mL), and EtOH (12 mL) in an approximately 3:1 ratio was heated in a microwave at 130°C for 3 hours. Preparative HPLC yielded 2-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenoxy) tert-butyl acetate (260 mg, 13%). 1 H NMR (300MHz, CD3OD) δ = 7.86-7.72 (s, 2H), 7.68 - 7.55 (m, 4H), 7.36 - 7.30 (m, 1H), 6.99 - 6.93 (m, 2H), 6.72 - 6.65 (m, 2H), 4.49 (s, 2H), 2.92 - 2.65 (m, 4H), 1.46 (s, 9H)

[0234] Step 5 A solution of 2-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenoxy) tert-butyl acetate (260 mg, 0.42 mmol) in 1:1 CH2Cl2 / TFA (6 mL) was stirred at room temperature for 1 hour and concentrated. The residue was freeze-dried in an aqueous solution of MeCN / 5% HCl to obtain the title compound (220 mg, %). LC-MS t R =5.18 min, m / z 559,557.

[0235] 2-[2-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenoxy)ethoxy]acetic acid (NB1i) [ka]

[0236] Step 1 2-(benzyloxy)ethanol (3.19 g, 21.0 mmol) in 20 mL of dry DMF and 40 mL of dry THF was stirred in an ice-cold solution, to which 60% NaH in oil (1.26 g, 31.5 mmol) was added in several portions. The mixture was stirred in an ice bath for 15 minutes, and t-butyl bromoacetate (6.2 mL, 41.9 mmol) was added over 2 minutes. The ice bath was melted, and the mixture was stirred at room temperature overnight. The mixture was recooled in an ice bath, and brine (40 mL) was added dropwise over 5 minutes. The mixture was concentrated under reduced pressure to remove the bulk THF, and the aqueous residue was extracted with SiO2 (2 × 50 mL). The combined SiO2 layers were washed with water (10 mL) and brine (10 mL) and dried over Na2SO4. After removing the solvent, the fluid oil (8.81 g) remained and was purified by chromatography using an 80 g silica cartridge. Elution in hexane under a 0-50% toluene gradient yielded 2-[2-(benzyloxy)ethoxy] tert-butyl acetate (3.00 g, 54%). 1 H NMR (300MHz, CDCl3) Shift = 7.42-7.30 (m, 5H), 4.60 (s, 2H), 4.12 (s, 2H), 3.85 - 3.64 (m, 4H), 1.60 - 1.32 (m, 9H).

[0237] Step 2 A solution of 2-[2-(benzyloxy)ethoxy]tert-butyl acetate (3.00 g, 11.3 mmol) in MeOH (100 mL) was shaken for 4 hours under H2 (60 psi, Parr) in the presence of 10% wetted palladium-carbon (1.00 g). The mixture was filtered through Celite, and the filtrate was concentrated to leave 2-(2-hydroxyethoxy)tert-butyl acetate (2.06 g, quantitative yield) as an oily substance. 1 ¹H NMR (300MHz, CDCl3) shifts: 4.01 (s, 2H), 3.78 - 3.60 (m, 4H), 3.11 - 2.97 (m, 1H), 1.47 (s, 9H).

[0238] Step 3 To a solution of tert-butyl 2-(2-hydroxyethoxy)acetate (2.06 g, 11.7 mmol) and i-Pr2NEt (4.2 mL, 23.4 mmol) in CH2Cl2 (40 mL), methanesulfonyl chloride (1.4 mL, 17.3 mmol) was added. The ice bath was melted, and the mixture was stirred at room temperature for 6 hours. Further i-Pr2NEt (4.2 mL, 23.4 mmol) and methanesulfonyl chloride (1.4 mL, 17.3 mmol) were added, and the mixture was stirred at room temperature for 18 hours. The mixture was concentrated. The residue was dissolved in HCl (80 mL) and 5% aqueous HCl (30 mL). The layers were separated, and the HCl layer was washed with 5% aqueous HCl (30 mL) and brine (20 mL). The combined aqueous layer was back-extracted with HCl (20 mL). The combined toluene layers were washed with brine (10 mL), dried over Na2SO4, and concentrated, leaving 2-[2-(methanesulfonyloxy)ethoxy] tert-butyl acetate (3.33 g, quantitative yield) as a brown oily substance. 1 H NMR (300MHz, CDCl3) Shift = 4.39 - 4.23 (m, 2H), 3.93 (s, 2H), 3.80 - 3.63 (m, 2H), 3.00 (s, 3H), 1.44 (s, 9H).

[0239] Step 4 A mixture of 4-(2-hydroxyethyl)phenol (1.80 g, 13.0 mmol), Cs2CO3 (4.67 g, 14.3 mmol), and DMF (30 mL) was stirred at room temperature for 15 minutes, and a solution of 2-[2-(methanesulfonyloxy)ethoxy] tert-butyl acetate (3.33 g, 13.0 mmol) in DMF (5 mL) was added. The mixture was stirred at room temperature for 1 day. Potassium iodide (2.17 g, 13.1 mmol) was added, and the mixture was stirred at 40°C for 1 day. The mixture was diluted with HCl (175 mL) and washed with water (2 × 30 mL), 1 M NaOH aqueous solution (30 mL), and brine (30 mL). The water and NaOH aqueous solution were combined and back-extracted with HCl. The combined HCl layer was dried over Na2SO4 and concentrated under reduced pressure, leaving oil (4.61 g). 2-{2-[4-(2-hydroxyethyl)phenoxy]ethoxy}tert-butyl acetate (950 mg, 25%) was obtained as an oily substance by chromatography using an 80 g silica cartridge with elution at a 0-70% HCl gradient in hexane. ¹H NMR (300 MHz, CDCl₃) shifts were 7.20 - 7.04 (m, 2H), 6.93 - 6.77 (m, 2H), 4.18 - 4.11 (m, 2H), 4.09 (s, 2H), 3.95 - 3.86 (m, 2H), 3.85 - 3.76 (m, 2H), 2.83 - 2.74 (m, 2H), and 1.47 (s, 9H).

[0240] Step 5 Methanesulfonyl chloride (0.60 mL, 7.7 mmol) was added to a solution of CH2Cl2 (30 mL) containing 2-{2-[4-(2-hydroxyethyl)phenoxy]ethoxy}tert-butyl acetate (950 mg, 3.2 mmol) and i-Pr2NEt (1.8 mL, 9.7 mmol). The ice bath was melted, and the mixture was stirred at room temperature for 6 hours and concentrated. The residue was dissolved in Depositphotos (90 mL), washed with 5% aqueous HCl (2 × 20 mL) and brine (20 mL), and dried over Na2SO4. After removal of the solvent, 2-(2-{4-[2-(methanesulfonyloxy)ethyl]phenoxy}ethoxy)tert-butyl acetate (1.14 g, 95%) remained as a brown oily substance. 1 H NMR (300MHz, CDCl3) Shift= 7.18 - 7.08 (m, 2H), 6.92 - 6.81 (m, 2H), 4.37 (t, J=6.7 Hz, 2H), 4.20 - 4.10 (m, 2H), 4.05 (s, 2H), 3.97 - 3.85 (m, 2H), 2.98 (t, J=7.0 Hz, 2H), 2.83 (s, 3H), 1.48 (s, 9H).

[0241] Step 6 A mixture of methyl 3-oxo-3-[4-(trifluoromethyl)phenyl]propanoate (750 mg, 3.0 mmol), tert-butyl 2-(2-{4-[2-(methanesulfonyloxy)ethyl]phenoxy}ethoxy) acetate (1.14 g, 3.0 mmol), powdered K2CO3 (420 mg, 3.0 mmol), KI (500 mg, 3.0 mmol), and dried DMF (12 mL) was stirred in an oil bath at 70°C under N2 for 8 hours. The mixture was diluted with ELISA (90 mL), washed with 5% aqueous HCl solution (2 × 20 mL) and brine (20 mL), and dried over Na2SO4. The solvent was removed to leave a fluid oil (1.93 g), which was purified by chromatography using an 80 g silica cartridge. Elution was performed in hexane under a 0-30% SiO gradient to obtain a 2:1 mixture of methyl 4-(4-{2-[2-(tert-butoxy)-2-oxoethoxy]ethoxy}phenyl)-2-[4-(trifluoromethyl)benzoyl]butanoate and the O-alkylation product methyl(2Z)-3-[2-(4-{2-[2-(tert-butoxy)-2-oxoethoxy]ethoxy}phenyl)ethoxy]-3-[4-(trifluoromethyl)phenyl]prop-2-enoate methyl (730 mg, 45%) as an oily substance. 1 ¹H NMR (300MHz, CDCl3) shifts = 8.00 - 7.88 (m, 2H), 7.79 - 7.64 (m, 2H), 7.04 (d, J=8.8 Hz, 2H), 6.84 (d, J=8.3 Hz, 2H), 4.22 - 4.11 (m, 2H), 4.08 (s, 2H), 3.94 - 3.87 (m, 2H), 3.68 (s, 3H), 3.07 - 2.95 (m, 1H), 2.67 - 2.54 (m, 2H), 2.39 - 2.18 (m, 2H), 1.46 (s, 9H). Resonances assigned to O-alkylation byproducts have not been reported.

[0242] Step 7 A mixture of methyl 4-(4-{2-[2-(tert-butoxy)-2-oxoethoxy]ethoxy}phenyl)-2-[4-(trifluoromethyl)benzoyl]butanoate (730 mg, 1.4 mmol), 5-chloro-2-hydrazinyl-1H-1,3-benzodiazole (178 mg, 0.97 mmol), HOAc (1 mL), and MeOH (3 mL) was heated in a microwave at 130°C for 3 hours. Preparative HPLC yielded tert-butyl 2-[2-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenoxy)ethoxy]acetate (132 mg, 14%) as a solid. 1 H NMR (300MHz, METHANOL-d4) Shift = 7.82 - 7.55 (m, 6H), 7.45 - 7.34 (m, 1H), 6.98 - 6.88 (m, 2H), 6.75 - 6.65 (m, 2H), 4.08 (s, 2H), 4.07 - 3.99 (m, 2H), 3.87 - 3.81 (m, 2H), 2.92 - 2.83 (m, 2H), 2.78 - 2.68 (m, 2H), 1.46 (s, 9H).LC-MS t R 6.33 min, m / z 657, 601.

[0243] Step 8 2-[2-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenoxy)ethoxy]acetic acid tert-butyl (128 mg, 0.19 mmol) in CH2Cl2 (3 mL) was mixed with CF3CO2H (3 mL). The mixture was stirred at room temperature for 0.5 hours and then concentrated. The residue was freeze-dried from an aqueous MeCN / 5% HCl solution to obtain the HCl salt (114 mg, 92%) of 2-[2-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenoxy)ethoxy]acetic acid as a yellowish-brown solid. 1 H NMR (300MHz, DMSO-d6) Shift= 7.91 - 7.73 (m, 4H), 7.63 - 7.52 (m, 2H), 7.26 (dd, J=2.2, 8.8 Hz, 1H), 7.03 (d, J=8.8 Hz, 2H), 6.77 (d, J=8.3 Hz, 2H), 4.07 (s, 2H), 4.04 - 3.98 (m, 2H), 3.79 - 3.70 (m, 2H), 2.84 - 2.64 (m, 4H).LC-MS t R 5.28 min, m / z 623, 601.

[0244] 2-[2-(4-{2-[1-(1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenoxy)ethoxy]acetic acid (NB1j) [ka] The title compound was prepared in step 7 using 2-hydrazinyl-1H-1,3-benzodiazole, following the same procedure as described for NB1i. LC-MS t R = 4.48 min, m / z 567.

[0245] 2-(4-{2-[1-(1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-(4-methanesulfonylphenyl)-1H-pyrazole-4-yl]ethyl}phenoxy)acetic acid (NB1k) [ka]

[0246] Step 1 A stirred solution of 1-[4-(methylsulfanyl)phenyl]ethane-1-one (3.01 g, 18.1 mmol) and diethyl carbonate (4.4 mL, 36.2 mmol) in dry THF (60 mL) was to be gradually mixed with 60% NaH in oil (870 mg, 36.2 mmol). The mixture was stirred in an oil bath under N2 at 70°C for 4 hours. After stirring overnight at room temperature, the mixture was poured into an ice-cold 5% HCl aqueous solution (120 mL) and extracted with SiO2 (2 × 60 mL). The combined organic layer was washed with brine (40 mL), dried over Na2SO4, and concentrated, leaving a dark brown oily substance (4.89 g). Chromatography using an 80g silica cartridge, eluting under a 0-70% HCl gradient in hexane, yielded ethyl 3-[4-(methylsulfanyl)phenyl]-3-oxopropanoate (3.00g, 69%) as a yellow oil. 1 H NMR (CDCl3, major tautomer) δ: 7.79-7.88 (m, 2H), 7.19-7.31 (m, 2H), 4.16-4.23 (m, 2H), 3.93 (s, 2H), 2.51 (s, 3H), 1.24 (t, J=7.1 Hz, 3H).

[0247] Step 2 A mixture of ethyl 3-[4-(methylsulfanyl)phenyl]-3-oxopropanoate (1.66 g, 7.0 mmol), tert-butyl 2-{4-[2-(methanesulfonyloxy)ethyl]phenoxy}acetate (2.31 g, 7.0 mmol), K2CO3 (1.06 g, 7.7 mmol), KI (1.27 g, 7.7 mmol), and DMF (15 mL) was stirred under N2 at 70°C for 8 hours. The mixture was diluted with SiO2 (80 mL) and washed with 5% aqueous HCl solution (2 × 20 mL) and brine (20 mL). The combined aqueous washes were back-extracted with SiO2 (20 mL). The combined SiO2 layer was dried over Na2SO4 and concentrated to obtain a brown oily substance (3.86 g). Chromatography using an 80g silica cartridge, eluting under a 0-30% siRNA gradient in hexane, yielded 4-{4-[2-(tert-butoxy)-2-oxoethoxy]phenyl}-2-[4-(methylsulfanyl)benzoyl]ethyl butanoate (1.31g, 40%). 1 H NMR (300MHz, cdcl3) shift= 7.74 - 7.60 (m, 2H), 7.12 (d, J=8.4 Hz, 2H), 6.96 (d, J=8.3 Hz, 2H), 6.70 (d, J=8.3 Hz, 2), 4.42 - 4.31 (m, 2H), 4.16 - 3.90 (m, 3H), 2.58 - 2.45 (m, 2H), 2.40 (s, 3H), 2.25 - 2.04 (m, 2H), 1.37 (s, 9H), 1.13 - 0.95 (m, 3H).LC-MS t R = 5.93 min, m / z 495.

[0248] Step 3 77% m-CPBA (1.27 g, 5.7 mmol) was added to a stirred, ice-cold solution of 4-{4-[2-(tert-butoxy)-2-oxoethoxy]phenyl}-2-[4-(methylsulfanyl)benzoyl]ethyl butanoate (1.31 g, 2.8 mmol) in CH2Cl2 (30 mL). The cooling bath was melted, and the mixture was stirred for 3 hours. The mixture was concentrated, the residue was dissolved in ELISA (90 mL), washed with saturated NaHCO3 aqueous solution (2 × 20 mL), water (10 mL), and brine (10 mL), and dried over Na2SO4. The solvent was removed to retain an oily substance (1.72 g), which was purified by chromatography using a 40 g silica cartridge. Elution was performed in hexane under a 0-100% siRNA gradient to obtain ethyl 4-{4-[2-(tert-butoxy)-2-oxoethoxy]phenyl}-2-(4-methanesulfonylbenzoyl)butanoate (1.04 g, 74%) as a colorless oil. LC-MS t R =5.18 min, m / z 527.

[0249] Steps 4 and 5 The title compound was prepared following the same procedure as in steps 4 and 5 of the preparation of NB1h using 2-hydrazinyl-1H-1,3-benzodiazole in step 4. LC-MS t R =3.78 min, m / z 533.

[0250] 2-[2-(4-{2-[1-(1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-(4-methanesulfonylphenyl)-1H-pyrazole-4-yl]ethyl}phenoxy)ethoxy]acetic acid (NB1l) [ka]

[0251] The title compound was prepared in step 2 using tert-butyl 2-[2-(methanesulfonyloxy)ethoxy]acetate, following the same procedure as described for NB1i. LC-MS t R = 3.82 min, m / z 577.

[0252] 3-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenyl)propanoic acid (NB1m) [ka]

[0253] Step 1 500 mL of RBF was mixed with methyl 4-(4-iodophenyl)butanoate (11.24 g, 37.0 mmol), methyl 4-(trifluoromethyl)benzoate (11.30 g, 55.4 mmol), and 60% NaH in oil (3.70 g, 92.4 mmol). The flask was sealed with a septum, and 100 mL of dry THF was added by syringe. 5 drops of MeOH were added by syringe. The mixture was stirred under N2 at 70°C for 7 hours. The mixture was cooled to room temperature and poured into 150 mL of ice-cold brine. The mixture was extracted with SiO2 (2 × 100 mL). The combined organic layer was washed with brine (50 mL), dried over Na2SO4, and concentrated, leaving a semi-solid (32.59 g). Chromatography using a 120g silica cartridge, eluting under a 0-15% siRNA gradient in hexane, yielded methyl 4-(4-iodophenyl)-2-[4-(trifluoromethyl)benzoyl]butanoate (6.45g, 36%). Furthermore, the product with a purity of approximately 65% ​​was isolated (10.00g). LC-MS t R = 6.25 min, m / z 499.

[0254] Step 2 In a 100 mL RBF flask equipped with a stirring bar, methyl 4-(4-iodophenyl)-2-[4-(trifluoromethyl)benzoyl]butanoate (1.10 g, 2.3 mmol), propargyl alcohol (0.2 mL, 3.5 mmol), PdCl2(PPh3)2 (162 mg, 0.23 mmol), and CuI (22 mg, 0.12 mmol) were added and the flask was sealed with a septum. The flask was evacuated / N2 (3 times) and dried THF (9 mL) and Et3N (3 mL) were added. The flask was evacuated / N2 (3 times) and stirred at room temperature for 16 hours. The mixture was concentrated. The residue was dissolved in ELISA (90 mL), washed with 5% HCl aqueous solution (2 × 30 mL) and brine (30 mL), and dried over Na2SO4. The solvent was removed to leave a brown oily substance (1.43 g), which was purified by chromatography using a 40 g silica cartridge. Elution was performed in hexane under a 0-80% HCl gradient to obtain methyl 4-[4-(3-hydroxypropane-1-in-1-yl)phenyl]-2-[4-(trifluoromethyl)benzoyl]butanoate (850 mg, 91%) as a light brown oily substance. 1 H NMR (300MHz, CDCl3) Shift= 8.05 - 7.91 (m, 2H), 7.76 - 7.65 (m, 2H), 7.35 (d, J=7.9 Hz, 2H), 7.10 (d, J=7.9 Hz, 2H), 4.49 (s, 2H), 4.34 - 4.19 (m, 1H), 3.68 (s, 3H), 2.73-2.62 (m, 2H), 2.42 - 2.21 (m, 2H).

[0255] Step 3 A mixture of 4-[4-(3-hydroxypropane-1-in-1-yl)phenyl]-2-[4-(trifluoromethyl)benzoyl]butanoate methyl (850 mg, 2.1 mmol), wet 10% palladium carbon (500 mg), and MeOH (40 mL) was stirred at room temperature for 3 hours under H2 (1 atm, balloon). The mixture was filtered through Celite. The filtrate was concentrated to retain an oily substance (750 mg), which was purified by chromatography using a 40 g silica cartridge. Elution was performed in hexane under a 0-70% siRNA gradient to obtain 4-[4-(3-hydroxypropyl)phenyl]-2-[4-(trifluoromethyl)benzoyl]butanoate methyl (280 mg, 32%) as a colorless oil. LC-MS t R = 5.20 min, m / z 431.

[0256] Step 4 A stirred solution of methyl 4-[4-(3-hydroxypropyl)phenyl]-2-[4-(trifluoromethyl)benzoyl]butanoate (280 mg, 0.69 mmol) in 9:1 MeCN / H2O (4 mL) was mixed with Bovitt salt (309 mg, 1.0 mmol). The mixture was stirred at room temperature for 1 day, and a further amount of Bovitt salt (103 mg, 0.33 mmol) was added. After stirring for a further 4 hours, the mixture was concentrated. The residue was dissolved in RINKAN (100 mL), washed with 5% aqueous HCl (15 mL) and brine (15 mL), and dried over Na2SO4. After removing the solvent, methyl 4-[4-(3-hydroxypropyl)phenyl]-2-[4-(trifluoromethyl)benzoyl]butanoate (330 mg, quantitative yield) remained as an oily substance. LC-MS t R = 5.03 min, m / z 445.

[0257] Step 5 A mixture of methyl 4-[4-(3-hydroxypropyl)phenyl]-2-[4-(trifluoromethyl)benzoyl]butanoate (330 mg, 0.78 mmol), 5-chloro-2-hydrazinyl-1H-1,3-benzodiazole (157 mg, 0.86 mmol), HOAc (1 mL), and MeOH (3 mL) was heated in a microwave at 130°C for 3 hours. The title compound (55 mg, 12%) was obtained by preparative HPLC. LC-MS t R = 5.45 min, m / z 555.

[0258] 2-[3-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenyl)propoxy]acetic acid (NB1n) [ka]

[0259] Step 1 100 mL of RBF was mixed with methyl 4-(4-iodophenyl)-2-[4-(trifluoromethyl)benzoyl]butanoate (1.96 g, 4.1 mmol), tert-butyl 2-(propa-2-in-1-yloxy)acetate (1.05 g, 6.2 mmol), CuI (79 mg, 0.41 mmol), and Pd(PPh3)2Cl2 (290 mg, 0.41 mmol). The flask was sealed with a septum and replenished with vacuum evacuation / N2 (3 times). Dry CH2Cl2 (16 mL) and Et3N (4 mL) were added by syringe. The flask was replenished with vacuum evacuation / N2 (3 times) and stirred at room temperature for 3 days. The mixture was concentrated. The residue was dissolved in ELISA (90 mL), washed with 5% HCl aqueous solution (2 × 20 mL) and brine (20 mL), and dried over Na2SO4. The solvent was removed, leaving a brown oily substance. This was subjected to chromatography using a 40g silica cartridge, and eluted under a gradient of 0-50% siRNA in hexane, yielding 4-(4-{3-[2-(tert-butoxy)-2-oxoethoxy]propa-1-in-1-yl}phenyl)-2-[4-(trifluoromethyl)benzoyl]butanoate methyl (1.76g, 82%) as a brown oily substance. LC-MS t R = 6.22 min, m / z 541.

[0260] Step 2 A mixture of 4-(4-{3-[2-(tert-butoxy)-2-oxoethoxy]propa-1-in-1-yl}phenyl)-2-[4-(trifluoromethyl)benzoyl]methyl butanoate (1.76 g, 3.4 mmol), moistened 10% palladium carbon (130 mg), and MeOH (35 mL) was stirred at room temperature under H2 (1 atm, balloon) for 5 hours. The flask was flushed with N2, and moistened 10% palladium carbon (300 mg) was added. The mixture was stirred at room temperature under H2 (1 atm, balloon) for 3 hours. The flask was flushed with N2, and the mixture was filtered through Celite. The filtrate was concentrated to retain an oily substance (1.63 g), which was subjected to chromatography in a 40 g silica cartridge. Elution was obtained in hexane under a 0-30% SiO gradient, yielding 4-(4-{3-[2-(tert-butoxy)-2-oxoethoxy]propyl}phenyl)-2-[4-(trifluoromethyl)benzoyl]butanoate methyl (1.21 g, 68%) as a pale oily substance. LC-MS t R = 6.35 min, m / z 545.

[0261] Step 3 A mixture of 4-(4-{3-[2-(tert-butoxy)-2-oxoethoxy]propyl}phenyl)-2-[4-(trifluoromethyl)benzoyl]butanoate (1.21 g, 2.3 mmol), 5-chloro-2-hydrazinyl-1H-1,3-benzodiazole (470 mg, 2.6 mmol), HOAc (1 mL), and MeOH (3 mL) was heated in a microwave at 130°C for 3 hours. Preparative HPLC yielded 2-[3-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenyl)propoxy]tert-butyl acetate (550 mg, 36%) as a white solid. LC-MS t R = 6.67 min, m / z 655, 599.

[0262] Step 4 A solution of 2-[3-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenyl)propoxy]tert-butyl acetate (550 mg, 0.84 mmol) in 1:1 CH2Cl2 / TFA (6 mL) was stirred overnight at room temperature and concentrated. The residue was lyophilized in 5% aqueous HCl / MeCN to obtain the title compound (474 ​​mg, quantitative yield) as a yellowish-brown solid. LC-MS t R = 5.60 min, m / z 599.

[0263] 2-{2-[3-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenyl)propoxy]ethoxy}acetic acid (NB1o) [ka]

[0264] The title compound was prepared using 2-[2-(propa-2-in-1-yloxy)ethoxy]tert-butyl acetate in step 1, following the same procedure as described for NB1n. LC-MS t R = 5.65 min, m / z 643.

[0265] 2-(2-{2-[3-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenyl)propoxy]ethoxy}ethoxy)acetic acid (NB1p) [ka]

[0266] The title compound was prepared using 2-{2-[2-(propa-2-in-1-yloxy)ethoxy]ethoxy}tert-butyl acetate in step 1, following the same procedure as described for NB1n. LC-MS t R = 5.75 min, m / z 687.

[0267] 15-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenyl)-3,6,9,12-tetraoxapentadecanoate (NB1q) [ka]

[0268] The title compound was prepared using tert-butyl 3,6,9,12-tetraoxapentadeca-14-inoate in step 1, following the same procedure as described for NB1n.

[0269] 3-[4-({2-[2-(2-aminoethoxy)ethoxy]ethoxy}methyl)phenyl]-1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-1H-pyrazole-5-ol (NB2a) [ka]

[0270] Step 1 A mixture of 2-[2-(2-chloroethoxy)ethoxy]ethane-1-ol (3.75 g, 22.2 mmol), NaN3 (3.60 g, 55.5 mmol), and water (15 mL) was stirred at 80°C for 1 day, cooled to room temperature, diluted with 1 M NaOH aqueous solution (5 mL, 5.0 mmol), and extracted with ether (3 × 35 mL). The combined ether layer was dried over Na2SO4 and concentrated, leaving 2-{2-[2-azidoethoxy]ethoxy}ethane-1-ol (2.50 g, 64%) as a colorless oily substance.1 1H NMR (300MHz, CDCl) 33 ) δ = 3.75 - 3.63 (m, 8H), 3.62-3.56 (m, 2H), 3.42-3.35 (m, 2H).

[0271] Step 2 A stirred solution of methyl 4-(bromomethyl)benzoate (1.57 g, 6.9 mmol) and 2-{2-[2-azidoethoxy]ethoxy}ethane-1-ol (1.26 g, 7.2 mmol) in dry THF (10 mL) was mixed with 60% NaH in oil (330 mg, 8.3 mmol). The mixture was stirred at room temperature for 5 days, diluted with SiO (90 mL), washed with 5% aqueous HCl (20 mL), saturated aqueous NaHCO3 (20 mL), and brine (20 mL), and dried over Na2SO4. The solvent was removed to leave an oily substance (2.40 g), which was subjected to chromatography using a 40 g silica cartridge. Elution was obtained in hexane under a 0-80% SiO gradient to obtain 4-({2-[2-(2-azidoethoxy)ethoxy]ethoxy}methyl)methyl benzoate (810 mg, 36%) as a colorless oil. 1 1H NMR (300MHz, CDCl) 33 ) δ = 8.01 (d, J=7.9 Hz, 2H), 7.41 (d, J=7.9 Hz, 2H), 4.62 (s, 2H), 3.72-3.60 (m, 10H), 3.43 - 3.31 (m, 2H).

[0272] Step 3 Methyl 4-(4-fluorophenyl)butanoate (280 mg, 1.4 mmol) and 4-(6,9,12-trioxa-1,2λ) 4A mixture of methyl ,3-triazatrideca-1,2-dien-13-yl)benzoate (810 mg, 2.5 mmol) was mixed with 60% NaH in oil (285 mg, 7.1 mmol), followed by the addition of dried THF (5 mL) and MeOH (1 drop). The mixture was heated under N2 reflux for 5 hours, diluted with SiO2 (90 mL), washed with 5% aqueous HCl (15 mL) and brine (15 mL), and dried over Na2SO4. The solvent was removed to leave an oily substance (1.08 g), which was purified by chromatography using a 40 g silica cartridge and eluted in hexane under a 0-100% SiO2 gradient to obtain methyl 2-[4-({2-[2-(2-azidoethoxy)ethoxy]ethoxy}methyl)benzoyl]-4-(4-fluorophenyl)butanoate (170 mg, 24%). 1 H NMR (300MHz, CDCl3) δ = 7.92 - 7.80 (m, 2H), 7.48 - 7.35 (m, 2H), 7.11 (dd, J=5.5, 8.6 Hz, 2H), 7.03 - 6.85 (m, 2H), 4.63 (s, 2H), 4.34 - 4.21 (m, 1H), 3.78 - 3.61 (m, 13H), 3.47 - 3.29 (m, 2H), 2.68-2.60 (m, 2H), 2.40 - 2.19 (m, 2H).

[0273] Step 4 A mixture of 2-[4-({2-[2-(2-azidoethoxy)ethoxy]ethoxy}methyl)benzoyl]-4-(4-fluorophenyl)butanoate methyl (170 mg, 0.35 mmol), 5-chloro-2-hydrazinyl-1H-1,3-benzodiazole (67 mg, 0.37 mmol), TsOH.H2O (14 mg, 0.07 mmol), and MeOH (4 mL) was stirred under N2 at 70°C for 3 days. By preparative HPLC, 3-[4-({2-[2-(2-azidoethoxy)ethoxy]ethoxy}methyl)phenyl]-1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-1H-pyrazole-5-ol (94 mg, 43%) was obtained as a yellowish-brown solid. 1H NMR (300MHz, CD3OD) δ = 7.64 - 7.35 (m, 6H), 7.32 - 7.19 (m, 1H), 7.05 (dd, J=5.5, 8.6 Hz, 2H), 6.94 - 6.79 (m, 2H), 4.58 (s, 2H), 3.77 - 3.56 (m, 10H), 3.43 - 3.20 (m, 2H), 2.78 (s, 4H).LC-MS t R 5.43 min, m / z 622, 620.

[0274] Step 5 3-[4-({2-[2-(2-azidoethoxy)ethoxy]ethoxy}methyl)phenyl]-1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-1H-pyrazole-5-ol (86 mg, 0.14 mmol) was stirred in dry THF (3 mL) to which 1 M Me3P (0.42 mL, 0.42 mmol) in THF was added. The mixture was stirred under N2 at room temperature for 2 hours, and water (0.3 mL) was added. The mixture was stirred at room temperature for 2 days, and 1 M NaOH aqueous solution (0.5, 0.5 mmol) was added. The mixture was stirred at room temperature for 3 hours, diluted with HOAc (1 mL), and purified by preparative HPLC to obtain the TFA salt (76 mg, 77%) of the title compound as a white solid. 1 H NMR (300MHz, CD3OD) δ = 7.65 - 7.38 (m, 6H), 7.35 - 7.23 (m, 1H), 7.14 - 7.00 (m, 2H), 6.95 - 6.77 (m, 2H), 4.61 (s, 2H), 3.80 - 3.59 (m, 12H), 3.18 - 3.06 (m, 2H), 2.81 (s, 4H).LC-MS t R = 4.23 min, m / z 594.

[0275] 3-(4-{[2-(2-aminoethoxy)ethoxy]methyl}phenyl)-1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-1H-pyrazole-5-ol(NB2b) [ka]

[0276] The title compound was prepared using 2-(2-chloroethoxy)ethane-1-ol in step 1, following the same procedure as described for NB2a. LC-MS t R = 4.22 min, m / z 550.

[0277] 3-[4-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethanesulfonyl)phenyl]-1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-1H-pyrazole-5-ol(NB2c) [ka]

[0278] Step 1 250 mL of RBF was mixed with methyl 4-(4-fluorophenyl)-2-(4-iodobenzoyl)butanoate (2.13 g, 5.0 mmol), potassium thioacetate (0.86 g, 7.5 mmol), CuI (95 mg, 0.5 mmol), and 1,10-phenatroline (180 mg, 1.0 mmol). The flask was sealed with a septum and replenished with vacuum evacuation / N2 (3 times). Dry toluene (40 mL) was introduced by syringe, and the flask was replenished with vacuum evacuation / N2 (3 times). The mixture was stirred in an oil bath at 100 °C for 16 hours, cooled to room temperature, diluted with ELISA (50 mL), washed with 5% HCl aqueous solution (2 × 20 mL) and brine (20 mL), and dried over Na2SO4. The solvent was removed to retain the oily substance (2.30 g), which was purified by chromatography using a 40 g silica cartridge. Elution was performed in hexane under a 0-35% siRNA gradient to obtain methyl 2-[4-(acetylsulfanyl)benzoyl]-4-(4-fluorophenyl)butanoate (1.58 g, 84%) as an amber-colored oily substance. LC-MS t R= 5.22 min, m / z 397.

[0279] Step 2 A 1N NaOH aqueous solution (2.2 mL, 2.2 mmol) was added to a stirred solution of methyl 2-[4-(acetylsulfanyl)benzoyl]-4-(4-fluorophenyl)butanoate (820 mg, 2.2 mmol) in THF (8 mL) under N2 conditions. The mixture was stirred under N2 conditions at room temperature for 6 hours. A solution of 2-{2-[2-(2-azidoethoxy)ethoxy]ethoxy}ethyl methanesulfonate (720 mg, 2.4 mmol) in MeOH (4 mL) was added, followed by the addition of a 1N NaOH aqueous solution (2.2 mL, 2.2 mmol). The mixture was stirred at room temperature for 1 day and concentrated to leave an aqueous residue, which was extracted with RINKAN (2 × 50 mL). The combined RINKAN layers were washed with brine (15 mL), dried over Na2SO4, and concentrated to leave an oily substance (1.26 g). Chromatography using a 40g silica cartridge eluted in hexane under a 0-100% siRNA gradient yielded 2-{4-[(2-{2-[2-(2-azidoethoxy)ethoxy]ethoxy}ethyl)sulfanyl]benzoyl}-4-(4-fluorophenyl)butanoate methyl (710 mg, 60%) as an oil. LC-MS t R = 5.28 min, m / z 556.

[0280] Step 3 The same procedure as described in step 3 of the NB1k synthesis was used.

[0281] Steps 4 and 5 The title compound was obtained using a procedure similar to that described in steps 4 and 5 of the synthesis of NB2a. LC-MS t R = 4.30 min, m / z 672.

[0282] 3-{4-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-5-hydroxy-1H-pyrazole-3-yl]phenyl}propanoic acid (NB2d) [ka]

[0283] Step 1 Methyl 4-(4-fluorophenyl)-2-(4-iodobenzoyl)butanoate (2.30 g, 5.4 mmol), CuI (103 mg, 0.54 mmol), and PdCl2(PPh3)2 (379 mg, 0.54 mmol) were added to a flask. The flask was sealed with a septum and replenished with vacuum / N2 (3 times). Dry CH2Cl2 (16 mL), Et3N (4 mL), and t-butylpropiolate (1.1 mL, 8.1 mmol) were introduced by syringe. The flask was replenished with vacuum / N2 (3 times) and stirred at room temperature for 3 days. The mixture was concentrated. The residue was dissolved in 5% aqueous HCl (40 mL) and extracted with ELISA (2 × 50 mL). The combined organic layers were washed with brine and dried over Na2SO4. After removing the solvent, a dark oily substance (3.93 g) remained. This was purified by chromatography using an 80 g silica cartridge, and eluted in hexane under a 0-20% siRNA gradient to obtain 2-{4-[3-(tert-butoxy)-3-oxopropa-1-in-1-yl]benzoyl}-4-(4-fluorophenyl)butanoate methyl (700 mg, 30%). LC-MS t R = 6.22 min, m / z 447.

[0284] Step 2 A mixture of 2-{4-[3-(tert-butoxy)-3-oxopropa-1-in-1-yl]benzoyl}-4-(4-fluorophenyl)butanoate methyl (700 mg, 1.6 mmol), 10% palladium carbon (catalytic amount), and MeOH (40 mL) was stirred at room temperature for 1 day under H2 (1 atm, balloon). The mixture was filtered through Celite, and the filtrate was concentrated to yield oil (700 mg). Chromatography using a 40 g silica cartridge, eluting under a 0-20% HCl gradient in hexane, yielded 2-{4-[3-(tert-butoxy)-3-oxopropyl]benzoyl}-4-(4-fluorophenyl)butanoate methyl (380 mg, 54%). LC-MS t R = 6.05 min, m / z 451.

[0285] Step 3 A mixture of 2-{4-[3-(tert-butoxy)-3-oxopropyl]benzoyl}-4-(4-fluorophenyl)butanoate methyl (380 mg, 0.89 mmol), 5-chloro-2-hydrazinyl-1H-1,3-benzodiazole (205 mg, 1.12 mmol), HOAc (0.75 mL), and MeOH (2.75 mL) was heated in a microwave at 130°C for 3 hours. Preparative HPLC yielded 3-{4-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-5-hydroxy-1H-pyrazole-3-yl]phenyl}propanoate tert-butyl (140 mg, 28%). LC-MS t R = 6.26 min, m / z 561.

[0286] Step 4 A solution of tert-butyl 3-{4-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-5-hydroxy-1H-pyrazole-3-yl]phenyl}propanoate (140 mg, 0.25 mmol) in 1:1 CH2Cl2 / TFA (6 mL) was stirred at room temperature for 16 hours and concentrated. The residue was freeze-dried using an aqueous solution of MeCN / 5% HCl to obtain the title compound (94 mg, 74%) as a grayish-white solid. LC-MS t R = 5.02 min, m / z 505.

[0287] 2-(3-{4-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-5-hydroxy-1H-pyrazole-3-yl]phenyl}propoxy)acetic acid (NB2e) [ka]

[0288] The title compound was obtained using tert-butyl 2-(propa-2-in-1-yloxy)acetate in step 1, following the same procedure as described for 2g of NB. LC-MS t R = 5.12 min, m / z 549.

[0289] 2-[2-(3-{4-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-5-hydroxy-1H-pyrazole-3-yl]phenyl}propoxy)ethoxy]acetic acid (NB2f) [ka]

[0290] The title compound was prepared using 2-[2-(propa-2-in-1-yloxy)ethoxy]tert-butyl acetate in step 1, following the same procedure as described for NB2g. LC-MS t R= 5.17 min, m / z 593.

[0291] 2-{2-[2-(3-{4-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-5-hydroxy-1H-pyrazole-3-yl]phenyl}propoxy)ethoxy]ethoxy}acetic acid (NB 2g) [ka]

[0292] Step 1 To a stirred, ice-cold suspension of 60% NaH (700 mg, 17.3 mmol) in oil in 20 mL of dry THF, a solution of 2-[2-(propa-2-in-1-yloxy)ethoxy]ethane-1-ol (1.78 g, 12.3 mmol) in 10 mL of dry THF was added dropwise over 5 minutes. The mixture was stirred at room temperature for 1 hour, recooled in an ice bath, and treated dropwise with t-butyl bromoacetate (3.65 mL, 24.7 mmol) over 3 minutes. The mixture was warmed to room temperature and stirred for 18 hours. The mixture was poured into 50 mL of ice-cold 5% HCl aqueous solution and extracted with SiO2 (2 × 40 mL). The combined organic layers were washed with brine (15 mL), dried over Na2SO4, and concentrated, leaving a yellow oily substance (5.30 g). Chromatography on silica gel eluted the compound on a hexane gradient of ethyl acetate, yielding 2-{2-[2-(propa-2-in-1-yloxy)ethoxy]ethoxy}tert-butyl acetate (1.08 g, 86%) as a colorless oil. 1 H NMR (300MHz, CDCl3) δ = 4.18 (s, 2H), 3.96 (d, J=0.9 Hz, 1H), 3.65 (br. s., 8H), 2.44 - 2.32 (m, 1H), 1.42 (s, 9H).

[0293] Step 2 In an oven-drying flask equipped with a stirring rod, methyl 4-iodobenzoate (4.77 g, 18.2 mmol), methyl 4-(4-fluorophenyl)butanoate (2.98 g, 15.2 mmol), and 60% NaH in oil (1.30 g, 31.9 mmol) were added. The flask was sealed with a septum, and dry THF (30 mL) was introduced using a syringe, followed by the introduction of MeOH (2 drops). The mixture was stirred under N2 at 70°C for 8 hours, cooled to room temperature, and poured into an ice-cold 2.5% HCl aqueous solution. The mixture was extracted with SiO2 (3 × 35 mL). The combined SiO2 layers were washed with brine (15 mL), dried over Na2SO4, and concentrated, leaving a wet solid (7.85 g). Chromatography using an 80g silica cartridge, eluting under a 0-25% siRNA gradient in hexane, yielded methyl 4-(4-fluorophenyl)-2-(4-iodobenzoyl)butanoate (3.77g, 58%) as a colorless oil. 1 H NMR (300MHz, CDCl3) δ = 7.88 - 7.75 (m, 2H), 7.63 - 7.54 (m, 2H), 7.18 - 7.07 (m, 2H), 6.96 (s, 2H), 4.21 (t, J=7.0 Hz, 1H), 3.68 (s, 3H), 2.70 - 2.54 (m, 2H), 2.41 - 2.16 (m, 2H)

[0294] Step 3 900 mg, 2.1 mmol, 900 mg, 2.1 mmol, 2-{2-[2-(propa-2-in-1-yloxy)ethoxy]ethoxy} tert-butyl acetate, 41 mg, 0.21 mmol, and 150 mg, 0.21 mmol, Pd(PPh3)2Cl2 were added to a flask, and the flask was sealed with a septum. The flask was evacuated and replenished with N2 (3 times), and 10 mL of dry CH2Cl2 and 2.5 mL of Et3N were added by syringe. The flask was evacuated and replenished with N2 (3 times), and the mixture was stirred at room temperature for 2 days to concentrate. The residue was dissolved in 100 mL of HCl, washed with 2 × 10 mL of 5% aqueous HCl solution and brine (10 mL), and dried over Na₂SO₄. The solvent was removed to leave a brown oily substance (1.84 g), which was subjected to chromatography in a 40 g silica cartridge. Elution was obtained in hexane under a gradient of 0 to 60% HCl to obtain 2-{4-[3-(2-{2-[2-(tert-butoxy)-2-oxoethoxy]ethoxy}ethoxy)propa-1-in-1-yl]benzoyl}-4-(4-fluorophenyl)butanoate methyl (760 mg, 64%) as an oily substance. 1 H NMR (300MHz, CDCl3) δ = 7.86-7.78 (m, 2H), 7.55 - 7.46 (m, 2H), 7.15 - 7.05 (m, 2H), 7.03 - 6.87 (m, 2H), 4.45 (s, 2H), 4.31 - 4.20 (m, 1H), 4.03 (s, 2H), 3.80-3.64 (m, 11H), 2.74 - 2.57 (m, 2H), 2.42 - 2.17 (m, 2H), 1.42 (s, 9H).

[0295] Step 4 A solution of 2-{4-[3-(2-{2-[2-(tert-butoxy)-2-oxoethoxy]ethoxy}ethoxy)propa-1-in-1-yl]benzoyl}-4-(4-fluorophenyl)butanoate methyl (760 mg, 1.4 mmol) in MeOH (20 mL) was stirred with 10% palladium carbon (catalyst) under H2 (1 atm, balloon) at room temperature for 4 hours. The flask was flushed with N2 and the mixture was filtered through Celite. The filtrate was concentrated to retain a brown oily substance (730 mg), which was purified by chromatography using a 24 g silica cartridge. Elution was performed in hexane under a 0-70% siRNA gradient to obtain 2-{4-[3-(2-{2-[2-(tert-butoxy)-2-oxoethoxy]ethoxy}ethoxy)propyl]benzoyl}4-(4-fluorophenyl)butanoate methyl (460 mg, 60%) as a brown oily substance. 1 H NMR (300MHz, CDCl3) δ = 7.86 - 7.76 (m, 2H), 7.35-7.24 (m, 2H), 7.18 - 7.07 (m, 2H), 7.01 - 6.89 (m, 2H), 4.33 - 4.20 (m, 1H), 4.02 (s, 2H), 3.77-3.62 (m, 9H), 3.62 - 3.55 (m, 2H), 3.51 - 3.38 (m, 2H), 2.80 - 2.69 (m, 2H), 2.67 - 2.58 (m, 2H), 2.37 - 2.21 (m, 2H), 1.98 - 1.83 (m, 2H), 1.46 (s, 9H)

[0296] Step 5 A mixture of 2-{4-[3-(2-{2-[2-(tert-butoxy)-2-oxoethoxy]ethoxy}ethoxy)propyl]benzoyl}-4-(4-fluorophenyl)butanoate methyl (460 mg, 0.82 mmol), 5-chloro-2-hydrazinyl-1H-1,3-benzodiazole (188 mg, 1.03 mmol), HOAc (0.5 mL), and MeOH (1.5 mL) was heated in a microwave at 130°C for 3 hours. Preparative HPLC yielded 2-{2-[2-(3-{4-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-5-hydroxy-1H-pyrazole-3-yl]phenyl}propoxy)ethoxy]ethoxy}tert-butyl acetate (190 mg, 33%) as a solid. 1 H NMR (300MHz, CD3OD) δ = 7.62 - 7.47 (m, 2H), 7.47 - 7.39 (m, 2H), 7.34 - 7.22 (m, 3H), 7.10 - 6.98 (m, 2H), 6.91 - 6.78 (m, 2H), 4.0 (s, 2H), 3.71 - 3.61 (m, 6H), 3.60 - 3.53 (m, 2H), 3.49 - 3.41 (m, 2H), 2.81 - 2.64 (m, 6H), 1.95 - 1.77 (m, 2H), 1.44 (s, 9H).LC-MS t R = 6.15 min, m / z 693, 637.

[0297] Step 6 A solution of 2-{2-[2-(3-{4-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-5-hydroxy-1H-pyrazole-3-yl]phenyl}propoxy)ethoxy]ethoxy}tert-butyl acetate (165 mg, 0.24 mmol) in 1:1 CH2Cl2 / TFA (6 mL) was stirred at room temperature for 3 hours and concentrated. The residue was freeze-dried from an aqueous MeCN / 5% HCl solution to obtain 2-{2-[2-(3-{4-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-5-hydroxy-1H-pyrazole-3-yl]phenyl}propoxy)ethoxy]ethoxy}acetic acid (136 mg, 90%) as a yellowish-brown solid. 1 H NMR (300MHz, CD3OD) δ = 7.64 - 7.53 (m, 3H), 7.50 - 7.43 (m, 1H), 7.37 - 7.29 (m, 3H), 7.13 - 7.03 (m, 2H), 6.94 - 6.83 (m, 2H), 4.14 (s, 2H), 3.78 - 3.56 (m, 8H), 3.55 - 3.45 (m, 2H), 2.93 - 2.67 (m, 6H), 2.02 - 1.82 (m, 2H).

[0298] 15-{4-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-5-hydroxy-1H-pyrazole-3-yl]phenyl}-3,6,9,12-tetraoxapentadecanoic acid (NB2h) [ka]

[0299] The title compound was prepared using tert-butyl 3,6,9,12-tetraoxapentadeca-14-inoate in Step 1, following the same procedure as described for NB2g.

[0300] 1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-3-(piperidine-4-yl)-1H-pyrazole-5-ol (NB3a) [ka]

[0301] Step 1 A mixture of 4-(3-ethoxy-3-oxopropanoyl)piperidine-1-carboxylate tert-butyl (3.72 g, 12.4 mmol), 1-(2-bromoethyl)-4-fluorobenzene (2.91 g, 14.3 mmol), K2CO3 (1.89 g, 13.7 mmol), KI (2.27 g, 13.7 mmol), and DMF (30 mL) was stirred at 70°C for 8 hours. The mixture was diluted with HCl (175 mL), washed with 5% aqueous HCl (30 mL), water (2 × 30 mL), and brine (30 mL), and dried over Na2SO4. After removal of the solvent, a brown oily substance (6.85 g) remained. Chromatography using an 80g silica cartridge eluted under a 0-40% HCl gradient in hexane yielded 4-{3-ethoxy-2-[2-(4-fluorophenyl)ethyl]-3-oxopropanoyl}piperidine-1-carboxylate tert-butyl (3.59g, 68%) as a pale yellow oil. 1 H NMR (CDCl3) 7.20-7.03 (m, 2H), 7.01-6.85 (m, 2H), 4.25-3.93 (m, 6H), 3.60-3.52 (m, 1H), 2.80-2.45 (m, 6H), 2.20-2.05 (m, 1H), 1.88-1.45 (m, 2H), 1.40 (s, 9H), 1.18-1.27 (m, 3H).LC-MS t R = 5.73 min, m / z 322 [M + Na+].

[0302] Step 2 A mixture of 4-{3-ethoxy-2-[2-(4-fluorophenyl)ethyl]-3-oxopropanoyl}piperidine-1-carboxylate tert-butyl (1.70 g, 4.0 mmol), 5-chloro-2-hydrazinyl-1H-1,3-benzodiazole (920 mg, 5.0 mmol), HOAc (3 mL), and EtOH (12 mL) was heated in a microwave at 130°C for 3 hours. Preparative HPLC yielded 4-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-5-hydroxy-1H-pyrazole-3-yl]piperidine-1-carboxylate tert-butyl (1.02 g, 58%) as a solid. 1 H NMR (CD3OD) δ: 7.52-7.63 (m, 2H), 7.31 (dd, J=8.6, 2.0 Hz, 1H), 7.14-7.23 (m, 2H), 6.93-7.05 (m, 2H), 4.10 (br d, J=13.3 Hz, 2H), 2.62-2.90 (m, 6H), 2.42-2.58 (m, 1H), 1.70-1.48 (br dd, J=17.5, 4.3 Hz, 4H), 1.46 (s, 9H). 19 F NMR (METHANOL-d4) δ: -77.45, -119.47.LC-MS t R 5.72 min, m / z 542, 540, 442, 440.HRMS calc'd for C 28 H 32 ClFN5O3540.2172, found 540.217.

[0303] Step 3 A solution containing 4-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-5-hydroxy-1H-pyrazole-3-yl]piperidine-1-carboxylate tert-butyl (1.02 g, 1.90 mmol) in 3:1 CH2Cl2 / TFA (8 mL) was stirred at room temperature for 1.5 hours and concentrated. The residue was lyophilized from an aqueous MeCN / 5% HCl solution to obtain the HCl salt of the title compound (829 mg, quantitative yield) as a gray solid. LC-MS t R = 3.90 min, m / z 442, 440.

[0304] 2-(2-{4-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-5-hydroxy-1H-pyrazole-3-yl]piperidine-1-yl}-2-oxoethoxy)acetic acid (NB3b) [ka] Diglycolic acid anhydride (66 mg, 0.57 mmol) was added to a stirred solution of HCl salt of 1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-3-(piperidine-4-yl)-1H-pyrazole-5-ol (290 mg, 0.57 mmol) and i-Pr2NEt (0.41 mL, 2.3 mmol) in CH2Cl2 (10 mL). The mixture was stirred at room temperature for 1 day. Further diglycolic acid anhydride (112 mg, 1.14 mmol) was added, and the mixture was stirred at room temperature for 3 hours. The mixture was concentrated, and the residue was partitioned into RINKAN (90 mL) and 5% HCl aqueous solution (20 mL). The RINKAN layer and the solid suspended at the interface were separated and concentrated. The residue was freeze-dried from the MeCN / 5% HCl aqueous solution to obtain the title compound (362 mg, quantitative yield) as a gray solid. LC-MS t R = 4.42 min, m / z 556.

[0305] 4-{2-[4-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethoxy)phenyl]ethyl}-1-{1H-imidazo[4,5-b]pyridine-2-yl}-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-5-ol(NB4a) [ka]

[0306] Step 1 A mixture of 4-[4-(2-{2-[2-(2-azidoethoxy)ethoxy]ethoxy}ethoxy)phenyl]-2-[4-(trifluoromethyl)benzoyl]butanoate methyl (394 mg, 0.69 mmol), {1H-imidazo[4,5-b]pyridine-2-yl}hydrazine (109 mg, 0.73 mmol), TsOH.H2O (25 mg, 0.13 mmol), and MeOH (2 mL) was heated in a microwave at 130°C for 2 hours. Preparative HPLC yielded 4-{2-[4-(2-{2-[2-(2-azidoethoxy)ethoxy]ethoxy}ethoxy)phenyl]ethyl}-1-{1H-imidazo[4,5-b]pyridine-2-yl}-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-5-ol (70 mg, 15%) as a yellowish-brown solid. LC-MS t R = 4.37 min, m / z 667.

[0307] Step 2 4-{2-[4-(2-{2-[2-(2-azidoethoxy)ethoxy]ethoxy}ethoxy)phenyl]ethyl}-1-{1H-imidazo[4,5-b]pyridine-2-yl}-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-5-ol (65 mg, 98 μmol) was added to a stirred, ice-cold suspension of 4-{2-[4-(2-{2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy}ethoxy)phenyl]ethyl}-1-{1H-imidazo[4,5-b]pyridine-2-yl}-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-5-ol (65 mg, 98 μmol) in dry THF (3 mL) as a stirred, ice-cold suspension. 1 M Me3P (0.3 mL, 0.3 mmol) was added to THF. The mixture was stirred in an ice bath for 15 minutes and at room temperature for 2 hours. Further 1 M Me3P (0.2 mL, 0.2 mmol) was added to THF, and stirring at room temperature was continued for 2 hours. The mixture was treated with 1 M NaOH aqueous solution (0.1 mL), stirred for 15 minutes, diluted with HOAc (1 mL), and purified by preparative HPLC to obtain the TFA salt (53 mg, 72%) of the title compound as a pale yellow solid. LC-MS t R = 3.47 min, m / z 641.

[0308] 4-(4-{2-[4-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethoxy)phenyl]ethyl}-1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-1H-pyrazole-3-yl)piperidine-1-carboxylate tert-butyl (NB5a) [ka]

[0309] Step 1 A mixture of 4-(2-hydroxyethyl)phenol (750 mg, 5.4 mmol), 2-{2-[2-(2-azidoethoxy)ethoxy]ethoxy}ethylmethanesulfonate (1.60 g, 5.4 mmol), Cs2CO3 (1.95 g, 6.0 mmol), KI (990 mg, 6.0 mmol), and DMF (10 mL) was stirred at 45°C for 3 days. The mixture was diluted with water (40 mL) and extracted with ELISA (2 × 50 mL). The combined ELISA layers were washed with brine (20 mL), dried over Na2SO4, and concentrated, leaving an oily substance (2.08 g). Chromatography using an 80g silica cartridge, eluting under a 0-100% siRNA gradient in hexane, yielded 2-[4-(2-{2-[2-(2-azidoethoxy)ethoxy]ethoxy}ethoxy)phenyl]ethane-1-ol (930mg, 50%) as a colorless oil. LC-MS t R = 3.72 min, m / z 362.

[0310] Step 2 930 mg, 2.7 mmol of 2-[4-(2-{2-[2-(2-azidoethoxy)ethoxy]ethoxy}ethoxy)phenyl]ethane-1-ol (930 mg, 2.7 mmol) and i-Pr2NEt (1.0 mL, 5.6 mmol) were stirred in CH2Cl2 (20 mL) and ice-cold to which MeSO2Cl (0.32 mL, 4.1 mmol) was added. The mixture was stirred for 4 hours and concentrated. The residue was dissolved in SiO2 (90 mL), washed with 5% aqueous HCl (20 mL) and brine (20 mL), and dried over Na2SO4. After removal of the solvent, 1.18 g, quantitative yield of 2-[4-(2-{2-[2-(2-azidoethoxy)ethoxy]ethoxy}ethoxy)phenyl]ethylmethanesulfonate remained as a colorless oil. LC-MS t R = 4.21 min, m / z 440.

[0311] Step 3 A mixture of 4-(3-ethoxy-3-oxopropanoyl)piperidine-1-carboxylate tert-butyl (730 mg, 2.4 mmol), 2-[4-(2-{2-[2-(2-azidoethoxy)ethoxy]ethoxy}ethoxy)phenyl]ethylmethanesulfonate (1.18 g, 2.8 mmol), K2CO3 (370 mg, 2.6 mmol), KI (445 mg, 2.7 mmol), and DMF (15 mL) was stirred at 70°C for 6 hours. The mixture was diluted with water (50 mL) and extracted with ELISA (2 × 50 mL). The combined siRNA layers were washed with brine (10 mL), dried over Na2SO4, and concentrated to retain an oily substance (2.40 g). This oily substance was purified by chromatography using an 80 g silica cartridge and eluted in hexane under a 0-60% siRNA gradient to obtain 4-(2-{2-[4-(2-{2-[2-(2-azidoethoxy)ethoxy]ethoxy}ethoxy)phenyl]ethyl}3-ethoxy-3-oxopropanoyl)piperidine-1-carboxylate tert-butyl (650 mg, 43%). LC-MS t R = 5.42 min, m / z 643, 521.

[0312] Step 4 A mixture of 4-(2-{2-[4-(2-{2-[2-(2-azidoethoxy)ethoxy]ethoxy}ethoxy)phenyl]ethyl}3-ethoxy-3-oxopropanoyl)piperidine-1-carboxylate tert-butyl (650 mg, 1.05 mmol), 5-chloro-2-hydrazinyl-1H-1,3-benzodiazole (210 mg, 1.15 mmol), HOAc (1 mL), and EtOH (3 mL) was stirred at 80°C for 20 hours. The mixture was cooled to room temperature and purified by preparative HPLC to obtain 4-(4-{2-[4-(2-{2-[2-(2-azidoethoxy)ethoxy]ethoxy}ethoxy)phenyl]ethyl}-1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-1H-pyrazole-3-yl)piperidine-1-carboxylate tert-butyl (510 mg, 66%) as a viscous oil. LC-MS t R = 5.42 min, m / z 739.

[0313] Step 5 A stirred solution of 4-(4-{2-[4-(2-{2-[2-(2-azidoethoxy)ethoxy]ethoxy}ethoxy)phenyl]ethyl}-1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-1H-pyrazole-3-yl)piperidine-1-carboxylate tert-butyl (230 mg, 0.31 mmol) in dry THF (10 mL) was mixed with 1 M Me3P (1 mL, 1.0 mmol) in THF. The mixture was stirred at room temperature for 2 hours, and 1 M NaOH aqueous solution (1 mL, 1.0 mmol) was added. The mixture was stirred overnight and purified by preparative HPLC to obtain the TFA salt (170 mg, 66%) of the title compound as a white solid. LC-MS t R = 4.33 min, m / z 713.

[0314] 2-[3-(4-{2-[1-(1H-1,3-benzodiazole-2-yl)-3-{1-[(tert-butoxy)carbonyl]piperidine-4-yl}-5-hydroxy-1H-pyrazole-4-yl]ethyl}phenyl)propoxy]acetic acid (NB5b) [ka]

[0315] Step 1 Using 2-(4-iodophenyl)ethylmethanesulfonate, the same procedure as in step 1 of the preparation of NB5a was used.

[0316] Step 2 Using 2-(propa-2-in-1-yloxy) tert-butyl acetate, the same procedure as in step 3 of the preparation of 2g of NB was used.

[0317] Step 3 The same procedure as in step 4 of the preparation of NB2g was used.

[0318] Step 4 The same procedure as in step 4 of the preparation of NB5a was used.

[0319] Step 5 To a stirred solution of 4-[1-(1H-1,3-benzodiazole-2-yl)-4-[2-(4-{3-[2-(tert-butoxy)-2-oxoethoxy]propyl}phenyl)ethyl]-5-hydroxy-1H-pyrazole-3-yl]piperidine-1-carboxylate tert-butyl (129 mg, 0.20 mmol) in MeOH (3 mL), 4 M HCl (1 mL) in dioxane was added. The mixture was stirred at room temperature for 10 days and concentrated to obtain the HCl salt of 2-[3-(4-{2-[1-(1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-(piperidine-4-yl)-1H-pyrazole-4-yl]ethyl}phenyl)propoxy]methyl acetate (125 mg, quantitative yield) as an oil.

[0320] Step 6 125 mg, 0.24 mmol) of HCl salt of 2-[3-(4-{2-[1-(1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-(piperidine-4-yl)-1H-pyrazole-4-yl]ethyl}phenyl)propoxy]methyl acetate, 2.5 mL of aqueous 105 K2CO3, and 2.5 mL of dioxane were mixed with Boc2O (80 mg, 0.37 mmol). The mixture was stirred at room temperature for 18 hours. 4 mL of MeOH and 2 mL of 1 M NaOH aqueous solution (2.0 mmol) were added, and stirring was continued for 1 day. The mixture was concentrated, and the residue was partitioned into 100 mL of ELISA and 20 mL of 2.5% aqueous HCl solution. The organic layer was washed with brine (10 mL), dried over Na2SO4, and concentrated. The residue was freeze-dried from an aqueous MeCN solution to obtain the title compound (128 mg, 88%) as a gray solid.

[0321] Compound Synthesis Example 1 3-[4-(2-{4-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-5-hydroxy-1H-pyrazole-3-yl]piperidine-1-yl}-2-oxoethoxy)-1-oxo-2,3-dihydro-1H-isoindole-2-yl]piperidine-2,6-dione [ka]

[0322] EDC.HCl (22 mg, 0.11 mmol) was added to a stirred solution of HCl salt of 1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-3-(piperidine-4-yl)-1H-pyrazole-5-ol (29 mg, 57 μmol), 2-{[2-2,6-dioxopiperidine-3-yl-1-oxo-2,3-dihydro-1H-isoindole-4-yl]oxy}acetic acid (19 mg, 60 μmol), HOBt.H2O (9 mg, 60 μmol), and i-Pr2NEt (60 μL, 0.34 mmol) in dry DMF (1.5 mL). The mixture was stirred at room temperature for 18 hours and purified by preparative HPLC to obtain the TFA salt of the title compound (12 mg, 25%) as a white solid. 1 H NMR (300MHz, DMSO-d6) Shift = 10.99 (s, 1H), 7.56 - 7.46 (m, 2H), 7.46 - 7.41 (m, 1H), 7.34 - 7.29 (m, 1H), 7.26 - 7.02 (m, 6H), 5.19 - 4.89 (m, 3H), 4.49 - 4.15 (m, 4H), 3.97 - 3.77 (m, 1H), 3.14 - 2.97 (m, 1H), 2.78 (d, J=7.0 Hz, 3H), 2.66 - 2.33 (m, 4H), 2.08 - 1.90 (m, 1H), 1.88 - 1.55 (m, 2H), 1.53 - 1.32 (m, 2H).LC-MS t R = 4.63 min, m / z 740.

[0323] Example 2 N-(2-{2-[2-({4-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-5-hydroxy-1H-pyrazole-3-yl]phenyl}methoxy)ethoxy]ethoxy}ethyl)-2-{[2-(2,6-dioxopiperidine-3-yl)-1-oxo-2,3-dihydro-1H-isoindole-4-yl]oxy}acetamide [ka]

[0324] Solid HATU (63 mg, 0.17 mmol) was added to a stirred mixture of 3-[4-({2-[2-(2-aminoethoxy)ethoxy]ethoxy}methyl)phenyl]-1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-1H-pyrazole-5-ol HCl salt (55 mg, 82 μmol), 2-{[2-(2,6-dioxopiperidine-3-yl)-1-oxo-2,3-dihydro-1H-isoindole-4-yl]oxy}acetic acid (53 mg, 0.17 mmol), i-Pr2NEt (80 μL, 0.45 mmol), dried CH2Cl2 (1 mL), and dried DMF (1 mL). The mixture was stirred under N2 at room temperature for 2 hours and concentrated under reduced pressure to remove CH2Cl2. The residue was purified by preparative HPLC, and subsequently freeze-dried in an aqueous solution of MeCN / 5% HCl to obtain the HCl salt (33 mg, 43%) of the title compound as a grayish-white solid. 1H NMR (300MHz, CD3OD) Shift= 7.80 - 7.66 (m, 2H), 7.63 - 7.30 (m, 7H), 7.15 - 6.99 (m, 3H), 6.89 (s, 2H), 5.12 (dd, J=5.1, 13.4 Hz, 1H), 4.63 - 4.61 (m, 2H), 4.59 - 4.57 (m, 2H), 4.47 - 4.43 (m, 2H), 3.69 - 3.66 (m, 4H), 3.63 - 3.56 (m, 6H), 3.50 - 3.43 (m, 2H), 2.96 - 2.82 (m, 3H), 2.80 - 2.65 (m, 3H), 2.51 - 2.29 (m, 1H), 2.19 - 1.99 (m, 1H).LC-MS t R = 4.33 min, m / z 894.

[0325] Example 3 N-(2-{2-[2-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenoxy)ethoxy]ethoxy}ethyl)-2-{[2-(2,6-dioxopiperidine-3-yl)-1-oxo-2,3-dihydro-1H-isoindole-4-yl]oxy}acetamide [ka]

[0326] The title compound was prepared from 4-[2-(4-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}phenyl)ethyl]-1-(5-chloro-1H-1,3-benzodiazole-2-yl)-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-5-ol (NB1a) and 2-{[2-(2,6-dioxopiperidine-3-yl)-1-oxo-2,3-dihydro-1H-isoindole-4-yl]oxy}acetic acid (CB1a) according to the procedure described in Example 2. LC-MS t R = 4.98 min, m / z 931.

[0327] Example 4 N-{2-[2-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenoxy)ethoxy]ethyl}-2-{[2-(2,6-dioxopiperidine-3-yl)-1-oxo-2,3-dihydro-1H-isoindole-4-yl]oxy}acetamide [ka]

[0328] The title compound was prepared from 4-(2-{4-[2-(2-aminoethoxy)ethoxy]phenyl}ethyl)-1-(5-chloro-1H-1,3-benzodiazole-2-yl)-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-5-ol (NB1b) and 2-{[2-(2,6-dioxopiperidine-3-yl)-1-oxo-2,3-dihydro-1H-isoindole-4-yl]oxy}acetic acid (CB1a) according to the procedure described in Example 2. LC-MS t R = 4.43 min, m / z 886.

[0329] Example 5 N-{2-[2-({4-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-5-hydroxy-1H-pyrazole-3-yl]phenyl}methoxy)ethoxy]ethyl}-2-{[2-(2,6-dioxopiperidine-3-yl)-1-oxo-2,3-dihydro-1H-isoindole-4-yl]oxy}acetamide [ka]

[0330] The title compound was prepared from 3-(4-{[2-(2-aminoethoxy)ethoxy]methyl}phenyl)-1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-1H-pyrazole-5-ol and 2-{[2-(2,6-dioxopiperidine-3-yl)-1-oxo-2,3-dihydro-1H-isoindole-4-yl]oxy}acetic acid (CB1a) according to the procedure described in Example 2. LC-MS t R = 4.77 min, m / z 850.

[0331] Example 6 N-[2-(2-{2-[2-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenoxy)ethoxy]ethoxy}ethoxy)ethyl]-2-{[2-(2,6-dioxopiperidine-3-yl)-1-oxo-2,3-dihydro-1H-isoindole-4-yl]oxy}acetamide [ka]

[0332] The title compound was prepared from 4-{2-[4-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethoxy)phenyl]ethyl}-1-(5-chloro-1H-1,3-benzodiazole-2-yl)-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-5-ol (NB1c) and 2-{[2-(2,6-dioxopiperidine-3-yl)-1-oxo-2,3-dihydro-1H-isoindole-4-yl]oxy}acetic acid (CB1a) according to the procedure described in Example 2. LC-MS t R = 5.07 min, m / z 974.

[0333] Example 7 N-{3-[3-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenoxy)propoxy]propyl}-2-{[2-(2,6-dioxopiperidine-3-yl)-1-oxo-2,3-dihydro-1H-isoindole-4-yl]oxy}acetamide [ka]

[0334] The title compound was prepared from 4-(2-{4-[3-(3-aminopropoxy)propoxy]phenyl}ethyl)-1-(5-chloro-1H-1,3-benzodiazole-2-yl)-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-5-ol (NB1d) and 2-{[2-(2,6-dioxopiperidine-3-yl)-1-oxo-2,3-dihydro-1H-isoindole-4-yl]oxy}acetic acid (CB1a) according to the procedure described in Example 1. LC-MS t R = 5.23 min, m / z 914.

[0335] Example 8 3-(4-{2-[4-(2-{2-[2-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenoxy)ethoxy]ethoxy}ethyl)piperazine-1-yl]-2-oxoethoxy}-1-oxo-2,3-dihydro-1H-isoindole-2-yl)piperidine-2,6-dione [ka]

[0336] The title compound was prepared from 1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-{2-[4-(2-{2-[2-(piperazine-1-yl)ethoxy]ethoxy}ethoxy)phenyl]ethyl}-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-5-ol (NB1g) and 2-{[2-(2,6-dioxopiperidine-3-yl)-1-oxo-2,3-dihydro-1H-isoindole-4-yl]oxy}acetic acid (CB1a) according to the procedure described in Example 1. LC-MS t R = 4.45 min, m / z 999.

[0337] Example 9 2-{[2-(2,6-dioxopiperidine-3-yl)-1-oxo-2,3-dihydro-1H-isoindole-4-yl]oxy}-N-(2-{2-[2-(2-{4-[2-(5-hydroxy-1-{1H-imidazo[4,5-b]pyridine-2-yl}-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl)ethyl]phenoxy}ethoxy)ethoxy]ethoxy}ethyl)acetamide [ka]

[0338] The title compound was prepared from 4-{2-[4-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethoxy)phenyl]ethyl}-1-{1H-imidazo[4,5-b]pyridine-2-yl}-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-5-ol (NB4a) and 2-{[2-(2,6-dioxopiperidine-3-yl)-1-oxo-2,3-dihydro-1H-isoindole-4-yl]oxy}acetic acid (CB1a) according to the procedure described in Example 1. LC-MS t R = 3.9 min, m / z 941.

[0339] Example 10 4-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-(2-{4-[2-(2-{2-[2-(2-{[2-(2,6-dioxopiperidine-3-yl)-1-oxo-2,3-dihydro-1H-isoindole-4-yl]oxy}acetamide)ethoxy]ethoxy}ethoxy)ethoxy]phenyl}ethyl)-5-hydroxy-1H-pyrazole-3-yl]piperidine-1-carboxylate tert-butyl [ka]

[0340] The title compound was prepared from 4-(4-{2-[4-(2-{2-[2-(2-(2-aminoethoxy)ethoxy]ethoxy}ethoxy)phenyl]ethyl}-1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-1H-pyrazole-3-yl)piperidine-1-carboxylate tert-butyl (NB5a) and 2-{[2-(2,6-dioxopiperidine-3-yl)-1-oxo-2,3-dihydro-1H-isoindole-4-yl]oxy}acetic acid (CB1a) according to the procedure described in Example 1. LC-MS t R = 4.73 min, m / z 913.

[0341] Example 11 N-[14-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenoxy)-3,6,9,12-tetraoxatetradecane-1-yl]-2-{[2-(2,6-dioxopiperidine-3-yl)-1-oxo-2,3-dihydro-1H-isoindole-4-yl]oxy}acetamide [ka]

[0342] The title compound was prepared from 4-(2-{4-[(14-amino-3,6,9,12-tetraoxatetradecane-1-yl)oxy]phenyl}ethyl)-1-(5-chloro-1H-1,3-benzodiazole-2-yl)-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-5-ol (NB1e) and 2-{[2-(2,6-dioxopiperidine-3-yl)-1-oxo-2,3-dihydro-1H-isoindole-4-yl]oxy}acetic acid (CB1a) according to the procedure described in Example 1. LC-MS t R = 5.02 min, m / z 10¹⁸.

[0343] Example 12 N-(2-{2-[2-(2-{4-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-5-hydroxy-1H-pyrazole-3-yl]benzenesulfonyl}ethoxy)ethoxy]ethoxy}ethyl)-2-{[2-(2,6-dioxopiperidine-3-yl)-1-oxo-2,3-dihydro-1H-isoindole-4-yl]oxy}acetamide [ka]

[0344] The title compound was prepared from 3-[4-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethanesulfonyl)phenyl]-1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-1H-pyrazole-5-ol (NB2c) and 2-{[2-(2,6-dioxopiperidine-3-yl)-1-oxo-2,3-dihydro-1H-isoindole-4-yl]oxy}acetic acid (CB1a) according to the procedure described in Example 1. LC-MS t R = 4.8 min, m / z 972.

[0345] Example 13 N-[2-(2-{2-[2-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenoxy)ethoxy]ethoxy}ethoxy)ethyl]-2-(2-{[2-(2,6-dioxopiperidine-3-yl)-1-oxo-2,3-dihydro-1H-isoindole-4-yl]oxy}ethoxy)acetamide [ka] The title compound was prepared from 4-{2-[4-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethoxy)phenyl]ethyl}-1-(5-chloro-1H-1,3-benzodiazole-2-yl)-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-5-ol (NB1c) and 2-(2-{[2-(2,6-dioxopiperidine-3-yl)-1-oxo-2,3-dihydro-1H-isoindole-4-yl]oxy}ethoxy)acetic acid (CB1l) according to the procedure described in Example 1. LC-MS t R = 5.13 min, m / z 10¹⁸.

[0346] Example 14 2-[2-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenoxy)ethoxy]-N-(2-{[2-(2,6-dioxopiperidine-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindole-4-yl]amino}ethyl)acetamide [ka]

[0347] EDC.HCl (16 mg, 84 μmol) was added to a stirred solution of 2-[2-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenoxy)ethoxy]acetic acid HCl salt (26 mg, 41 μmol), 4-[(2-aminoethyl)amino]-2-(2,6-dioxopiperidine-3-yl)-2,3-dihydro-1H-isoindole-1,3-dione (14.5 mg, 41 μmol), HOBt.H2O (7 mg, 45 μmol), and i-Pr2NEt (40 μL, 0.22 mmol) in dry DMF (1 mL). The mixture was stirred overnight at room temperature and purified by preparative HPLC, yielding the TFA salt (23 mg, 50%) of the title compound as a yellow solid. 1 H NMR (300MHz, DMSO-d6) shift= 8.03 - 7.95 (m, 1H), 7.89 - 7.76 (m, 4H), 7.62 - 7.48 (m, 3H), 7.25 (d, J=2.2 Hz, 1H), 7.16 (d, J=8.8 Hz, 1H), 7.06 - 6.96 (m, 3H), 6.82-6.63 (m, 3H), 5.07 - 4.96 (m, 1H), 4.04 (br s, 2H), 3.92 (s, 2H), 3.78 - 3.69 (m, 2H), 3.42 - 3.22 (m, 4H), 2.95 - 2.77 (m, 1H), 2.76-2.63 (m, 4H), 2.61 - 2.54 (m, 1H), 2.44 - 2.39 (m, 1H), 2.04 - 1.91 (m, 1H).LC-MS t R 5.38 min, m / z 901, 899. HRMS calculation value: C 44 H 39 ClF3N8O8899.2526; Measured value: 899.2529.

[0348] Example 15 2-[3-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenyl)propoxy]-N-(2-{[2-(2,6-dioxopiperidine-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindole-4-yl]amino}ethyl)acetamide [ka]

[0349] The title compound was prepared from 2-[3-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenyl)propoxy]acetic acid (NB1n) and 4-[(2-aminoethyl)amino]-2-(2,6-dioxopiperidine-3-yl)-2,3-dihydro-1H-isoindole-1,3-dione (CB1b) according to the procedure of Example 14. LC-MS t R = 5.52 min, m / z 897.

[0350] Example 16 2-{2-[3-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenyl)propoxy]ethoxy}-N-(2-{[2-(2,6-dioxopiperidine-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindole-4-yl]amino}ethyl)acetamide [ka]

[0351] The title compound was prepared from 2-{2-[3-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenyl)propoxy]ethoxy}acetic acid (NB1o) and 4-[(2-aminoethyl)amino]-2-(2,6-dioxopiperidine-3-yl)-2,3-dihydro-1H-isoindole-1,3-dione (CB1b) according to the procedure of Example 14. LC-MS t R = 5.68 min, m / z 941.

[0352] Example 17 15-{4-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-5-hydroxy-1H-pyrazole-3-yl]phenyl}-N-(2-{[2-(2,6-dioxopiperidine-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindole-4-yl]amino}ethyl)-3,6,9,12-tetraoxapentadecanamide [ka]

[0353] The title compound was prepared from 15-{4-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-5-hydroxy-1H-pyrazole-3-yl]phenyl}-3,6,9,12-tetraoxapentadecanoic acid (NB2h) and 4-[(2-aminoethyl)amino]-2-(2,6-dioxopiperidine-3-yl)-2,3-dihydro-1H-isoindole-1,3-dione (CB1b) according to the procedure of Example 14. LC-MS t R = 5.27 min, m / z 979.

[0354] Example 18 2-[2-(3-{4-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-5-hydroxy-1H-pyrazole-3-yl]phenyl}propoxy)ethoxy]-N-(2-{[2-(2,6-dioxopiperidine-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindole-4-yl]amino}ethyl)acetamide [ka]

[0355] The title compound was prepared from 2-[2-(3-{4-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-5-hydroxy-1H-pyrazole-3-yl]phenyl}propoxy)ethoxy]acetic acid (NB2f) and 4-[(2-aminoethyl)amino]-2-(2,6-dioxopiperidine-3-yl)-2,3-dihydro-1H-isoindole-1,3-dione (CB1b) according to the procedure of Example 14. LC-MS t R = 5.28 min, m / z 891.

[0356] Example 19 2-(3-{4-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-5-hydroxy-1H-pyrazole-3-yl]phenyl}propoxy)-N-(2-{[2-(2,6-dioxopiperidine-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindole-4-yl]amino}ethyl)acetamide [ka]

[0357] The title compound was prepared from 2-(3-{4-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-5-hydroxy-1H-pyrazole-3-yl]phenyl}propoxy)acetic acid (NB2e) and 4-[(2-aminoethyl)amino]-2-(2,6-dioxopiperidine-3-yl)-2,3-dihydro-1H-isoindole-1,3-dione (CB1b) according to the procedure of Example 14. LC-MS t R = 5.25 min, m / z 847.

[0358] Example 20 2-{2-[2-(3-{4-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-5-hydroxy-1H-pyrazole-3-yl]phenyl}propoxy)ethoxy]ethoxy}-N-(2-{[2-(2,6-dioxopiperidine-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindole-4-yl]amino}ethyl)acetamide [ka] EDC.HCl (18 mg, 94 mol) was added to a stirred solution of 2-{2-[2-(3-{4-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-5-hydroxy-1H-pyrazole-3-yl]phenyl}propoxy)ethoxy]ethoxy}acetic acid TFA salt (30 mg, 47 μmol), 4-[(2-aminoethyl)amino]-2-(2,6-dioxopiperidine-3-yl)-2,3-dihydro-1H-isoindole-1,3-dione HCl salt (16.5 mg, 46 μmol), HOBt.H2O (7.5 mg, 49 mol), and i-Pr2NEt (45 μL, 0.25 mmol) in dry DMF (1 mL). The mixture was stirred overnight at room temperature, and the bis-TFA salt of the title compound (16 mg, 29%) was obtained as a yellow solid by preparative HPLC of the directly purified mixture. 1H NMR (300MHz, DMSO-d6) shift= 7.96 (t, J=5.5 Hz, 1H), 7.59 - 7.51 (m, 3H), 7.49 - 7.43 (m, 2H), 7.34 - 7.28 (m, 2H), 7.23 - 7.10 (m, 4H), 7.07 - 6.98 (m, 2H), 6.78 - 6.68 (m, 1H), 5.03 (dd, J=5.5, 13.0 Hz, 1H), 3.87 (s, 2H), 3.61 - 3.22 (m, 14H), 2.96 - 2.34 (m, 9H), 2.07 - 1.93 (m, 1H), 1.89 - 1.69 (m, 2H).LC-MS t R = 5.30 min, m / z 935.

[0359] Example 21 3-{4-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-5-hydroxy-1H-pyrazole-3-yl]phenyl}-N-(2-{[2-(2,6-dioxopiperidine-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindole-4-yl]amino}ethyl)propanamide [ka]

[0360] The title compound was prepared from 3-{4-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-5-hydroxy-1H-pyrazole-3-yl]phenyl}propanoic acid (NB2d) and 4-[(2-aminoethyl)amino]-2-(2,6-dioxopiperidine-3-yl)-2,3-dihydro-1H-isoindole-1,3-dione (CB1b) according to the procedure of Example 14. LC-MS t R = 4.92 min, m / z 803.

[0361] Example 22 3-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenyl)-N-(2-{[2-(2,6-dioxopiperidine-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindole-4-yl]amino}ethyl)propanamide [ka]

[0362] The title compound was prepared from 3-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenyl)propanoic acid (NB1m) and 4-[(2-aminoethyl)amino]-2-(2,6-dioxopiperidine-3-yl)-2,3-dihydro-1H-isoindole-1,3-dione (CB1b) according to the procedure of Example 14. LC-MS t R = 5.35 min, m / z 853.

[0363] Example 23 2-(2-{2-[3-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenyl)propoxy]ethoxy}ethoxy)-N-(2-{[2-(2,6-dioxopiperidine-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindole-4-yl]amino}ethyl)acetamide [ka]

[0364] The title compound was prepared from 2-(2-{2-[3-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenyl)propoxy]ethoxy}ethoxy)acetic acid (NB1p) and 4-[(2-aminoethyl)amino]-2-(2,6-dioxopiperidine-3-yl)-2,3-dihydro-1H-isoindole-1,3-dione (CB1b) according to the procedure of Example 14. LC-MS tR = 5.77 min, m / z 985.

[0365] Example 24 2-[2-(4-{2-[1-(1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-(4-methanesulfonylphenyl)-1H-pyrazole-4-yl]ethyl}phenoxy)ethoxy]-N-(2-{[2-(2,6-dioxopiperidine-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindole-4-yl]amino}ethyl)acetamide [ka]

[0366] The title compound was prepared from 2-[2-(4-{2-[1-(1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-(4-methanesulfonylphenyl)-1H-pyrazole-4-yl]ethyl}phenoxy)ethoxy]acetic acid (NB1k) and 4-[(2-aminoethyl)amino]-2-(2,6-dioxopiperidine-3-yl)-2,3-dihydro-1H-isoindole-1,3-dione (CB1b) according to the procedure of Example 14. LC-MS t R = 4.03 min, m / z 875.

[0367] Example 25 15-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenyl)-N-(2-{[2-(2,6-dioxopiperidine-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindole-4-yl]amino}ethyl)-3,6,9,12-tetraoxapentadecanamide [ka]

[0368] The title compound was prepared from 15-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenyl)-3,6,9,12-tetraoxapentadecanoic acid (NB1q) and 4-[(2-aminoethyl)amino]-2-(2,6-dioxopiperidine-3-yl)-2,3-dihydro-1H-isoindole-1,3-dione (CB1b) according to the procedure of Example 14. LC-MS t R = 5.72 min, m / z 1029.

[0369] Example 26 4-[1-(1H-1,3-benzodiazole-2-yl)-4-{2-[4-(3-{[(2-{[2-(2,6-dioxopiperidine-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindole-4-yl]amino}ethyl)carbamoyl]methoxy}propyl)phenyl]ethyl}-5-hydroxy-1H-pyrazole-3-yl]piperidine-1-carboxylate tert-butyl [ka]

[0370] The title compound was prepared from 2-[3-(4-{2-[1-(1H-1,3-benzodiazole-2-yl)-3-{1-[(tert-butoxy)carbonyl]piperidine-4-yl}-5-hydroxy-1H-pyrazole-4-yl]ethyl}phenyl)propoxy]acetic acid (NB5b) and 4-[(2-aminoethyl)amino]-2-(2,6-dioxopiperidine-3-yl)-2,3-dihydro-1H-isoindole-1,3-dione (CB1b) according to the procedure of Example 14. LC-MS t R = 4.57 min, m / z 902.

[0371] Example 27 2-[2-(4-{2-[1-(1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenoxy)ethoxy]-N-(2-{[2-(2,6-dioxopiperidine-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindole-4-yl]amino}ethyl)acetamide [ka]

[0372] The title compound was prepared from 2-[2-(4-{2-[1-(1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenoxy)ethoxy]acetic acid (NB1j) and 4-[(2-aminoethyl)amino]-2-(2,6-dioxopiperidine-3-yl)-2,3-dihydro-1H-isoindole-1,3-dione (CB1b) according to the procedure of Example 14. LC-MS t R = 4.75 min, m / z 865.

[0373] Example 28 2-(4-{2-[1-(1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-(4-methanesulfonylphenyl)-1H-pyrazole-4-yl]ethyl}phenoxy)-N-(2-{[2-(2,6-dioxopiperidine-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindole-4-yl]amino}ethyl)acetamide [ka]

[0374] The title compound was prepared from 2-(4-{2-[1-(1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-(4-methanesulfonylphenyl)-1H-pyrazole-4-yl]ethyl}phenoxy)acetic acid (NB1k) and 4-[(2-aminoethyl)amino]-2-(2,6-dioxopiperidine-3-yl)-2,3-dihydro-1H-isoindole-1,3-dione (CB1b) according to the procedure of Example 14. LC-MS t R = 4.07 min, m / z 831.

[0375] Example 29 2-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenoxy)-N-(2-{[2-(2,6-dioxopiperidine-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindole-4-yl]amino}ethyl)acetamide [ka]

[0376] EDC.HCl (22 mg, 0.11 mmol) was added to a stirred solution of 2-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenoxy)acetic acid HCl salt (33 mg, 56 μmol), 4-[(2-aminoethyl)amino]-2-(2,6-dioxopiperidine-3-yl)-2,3-dihydro-1H-isoindole-1,3-dione HCl salt (21 mg, 60 μmol), HOBt.H2O (8 mg, 53 μmol), and i-Pr2NEt (60 μL, 0.34 mmol) in dry DMF (1.5 mL). The mixture was stirred at room temperature for 18 hours and then purified directly by preparative HPLC to obtain the TFA salt of the title compound (29 mg, 54%) as a yellow solid. 1 H NMR (300MHz, DMSO-d6) shift = 8.35 - 8.21 (m, 1H), 7.89 - 7.81 (m, 2H), 7.80-7.77 (m, 2H), 7.61 - 7.50 (m, 2H), 7.28 - 7.21 (m, 1H), 7.20 - 7.15 (m, 1H), 7.08 - 6.96 (m, 2H), 6.84 - 6.69 (m, 3H), 5.04 (dd, J=5.5, 12.5 Hz, 1H), 4.39 (s, 2H), 3.47 - 3.22 (m, 4H), 2.99 - 2.78 (m, 1H), 2.73 (s, 4H), 2.61 - 2.35 (m, 2H), 2.09 - 1.92 (m, 1H).LC-MS t R = 5.28 min, m / z 855.

[0377] Example 30 2-(2-{4-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-5-hydroxy-1H-pyrazole-3-yl]piperidine-1-yl}-2-oxoethoxy)-N-(2-{[2-(2,6-dioxopiperidine-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindole-4-yl]amino}ethyl)acetamide [ka]

[0378] The title compound was prepared from 2-(2-{4-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-5-hydroxy-1H-pyrazole-3-yl]piperidine-1-yl}-2-oxoethoxy)acetic acid (NB3b) and 4-[(2-aminoethyl)amino]-2-(2,6-dioxopiperidine-3-yl)-2,3-dihydro-1H-isoindole-1,3-dione (CB1b) according to the procedure of Example 14. LC-MS t R = 4.6 min, m / z 854.

[0379] Example 31 2-[2-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenoxy)ethoxy]-N-(2-{[2-(2,6-dioxopiperidine-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindole-4-yl](methyl)amino}ethyl)acetamide [ka]

[0380] EDC.HCl (11 mg, 57 mol) was added to a stirred solution of 2-[2-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenoxy)ethoxy]acetic acid HCl salt (18 mg, 28 μmol), 4-[(2-aminoethyl)amino]-2-(2,6-dioxopiperidine-3-yl)-2,3-dihydro-1H-isoindole-1,3-dione HCl salt (11 mg, 30 μmol), HOBt.H2O (5 mg, 32 μmol), and i-Pr2NEt (30 μL, 0.17 mmol) in dry DMF (1 mL). The mixture was stirred at room temperature for 1 day and purified by preparative HPLC, yielding the bis-TFA salt of the title compound (16 mg, 49%) as a yellow solid. 1 H NMR (300MHz, DMSO-d6) Shift = 7.91-7.79 (m, 4H), 7.72-7.64 (m, 1H), 7.61 - 7.52 (m, 2H), 7.27 - 7.21 (m, 2H), 7.20 - 7.15 (m, 1H), 7.06 - 6.99 (m, 2H), 6.77-6.70 (m, 2H), 5.13 - 5.00 (m, 1H), 3.95 (br. s., 2H), 3.73 (s, 2H), 3.63 - 3.47 (m, 4H), 3.43 - 3.25 (m, 2H), 2.99 (s, 3H), 2.97-2.65 (m, 5H), 2.58-2.38 (m, 2H), 2.01 - 1.80 (m, 1H).LC-MS t R = 5.23 min, m / z 913.

[0381] Example 32 2-[2-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenoxy)ethoxy]-N-(2-{[2-(2,6-dioxopiperidine-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindole-4-yl]amino}ethyl)-N-methylacetamide [ka]

[0382] The title compound was prepared from 2-[2-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenoxy)ethoxy]acetic acid (NB1i) and 2-(2,6-dioxopiperidine-3-yl)-4-{[2-(methylamino)ethyl]amino}-2,3-dihydro-1H-isoindole-1,3-dione (CB1d) according to the procedure of Example 14. LC-MS t R = 5.38 min, m / z 913.

[0383] Example 33 4-[(2-{4-[2-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenoxy)acetyl]piperazine-1-yl}ethyl)amino]-2-(2,6-dioxopiperidine-3-yl)-2,3-dihydro-1H-isoindole-1,3-dione [ka]

[0384] The title compound was prepared from 2-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenoxy)acetic acid (NB1h) and 2-(2,6-dioxopiperidine-3-yl)-4-{[2-(piperazine-1-yl)ethyl]amino}-2,3-dihydro-1H-isoindole-1,3-dione (CB1g) according to the procedure of Example 14. LC-MS t R = 4.55 min, m / z 924.

[0385] Example 34 4-[(2-{4-[2-(4-{2-[1-(1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-(4-methanesulfonylphenyl)-1H-pyrazole-4-yl]ethyl}phenoxy)acetyl]piperazine-1-yl}ethyl)amino]-2-(2,6-dioxopiperidine-3-yl)-2,3-dihydro-1H-isoindole-1,3-dione [ka]

[0386] The title compound was prepared from 2-(4-{2-[1-(1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-(4-methanesulfonylphenyl)-1H-pyrazole-4-yl]ethyl}phenoxy)acetic acid (NB1k) and 2-(2,6-dioxopiperidine-3-yl)-4-{[2-(piperazine-1-yl)ethyl]amino}-2,3-dihydro-1H-isoindole-1,3-dione (CB1g) according to the procedure of Example 14. LC-MS t R = 3.57 min, m / z 900.

[0387] Example 35 4-[1-(1H-1,3-benzodiazole-2-yl)-4-{2-[4-(3-{2-[4-(2-{[2-(2,6-dioxopiperidine-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindole-4-yl]amino}ethyl)piperazine-1-yl]-2-oxoethoxy}propyl)phenyl]ethyl}-5-hydroxy-1H-pyrazole-3-yl]piperidine-1-carboxylate tert-butyl [ka]

[0388] The title compound was prepared from 2-[3-(4-{2-[1-(1H-1,3-benzodiazole-2-yl)-3-{1-[(tert-butoxy)carbonyl]piperidine-4-yl}-5-hydroxy-1H-pyrazole-4-yl]ethyl}phenyl)propoxy]acetic acid (NB5b) and 2-(2,6-dioxopiperidine-3-yl)-4-{[2-(piperazine-1-yl)ethyl]amino}-2,3-dihydro-1H-isoindole-1,3-dione (CB1g) according to the procedure of Example 14. LC-MS t R = 3.98 min, m / z 971.

[0389] Example 36 4-{[2-(4-{2-[2-(4-{2-[1-(1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-(4-methanesulfonylphenyl)-1H-pyrazole-4-yl]ethyl}phenoxy)ethoxy]acetyl}piperazine-1-yl)ethyl]amino}-2-(2,6-dioxopiperidine-3-yl)-2,3-dihydro-1H-isoindole-1,3-dione [ka]

[0390] The title compound was prepared from 2-[2-(4-{2-[1-(1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-(4-methanesulfonylphenyl)-1H-pyrazole-4-yl]ethyl}phenoxy)ethoxy]acetic acid (NB1l) and 2-(2,6-dioxopiperidine-3-yl)-4-{[2-(piperazine-1-yl)ethyl]amino}-2,3-dihydro-1H-isoindole-1,3-dione (CB1g) according to the procedure of Example 14. LC-MS t R = 3.53 min, m / z 944.

[0391] Example 37 4-{[2-(4-{2-[2-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenoxy)ethoxy]acetyl}piperazine-1-yl)ethyl]amino}-2-(2,6-dioxopiperidine-3-yl)-2,3-dihydro-1H-isoindole-1,3-dione [ka]

[0392] The title compound was prepared from 2-[2-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenoxy)ethoxy]acetic acid (NB1i) and 2-(2,6-dioxopiperidine-3-yl)-4-{[2-(piperazine-1-yl)ethyl]amino}-2,3-dihydro-1H-isoindole-1,3-dione (CB1g) according to the procedure of Example 14. LC-MS t R = 4.52 min, m / z 968.

[0393] Example 38 2-(2-{2-[3-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenyl)propoxy]ethoxy}ethoxy)-N-{2-[2-(2-{[2-(2,6-dioxopiperidine-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindole-4-yl]amino}ethoxy)ethoxy]ethyl}acetamide [ka]

[0394] The title compound was prepared from 2-(2-{2-[3-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenyl)propoxy]ethoxy}ethoxy)acetic acid (NB1p) and 4-({2-[2-(2-aminoethoxy)ethoxy]ethyl}amino)-2-(2,6-dioxopiperidine-3-yl)-2,3-dihydro-1H-isoindole-1,3-dione (CB1e) according to the procedure of Example 14. LC-MS t R = 5.78 min, m / z 1073.

[0395] Example 39 2-(2-{2-[3-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenyl)propoxy]ethoxy}ethoxy)-N-[2-(2-{[2-(2,6-dioxopiperidine-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindole-4-yl]amino}ethoxy)ethyl]acetamide [ka]

[0396] The title compound was prepared from 2-(2-{2-[3-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenyl)propoxy]ethoxy}ethoxy)acetic acid (NB1p) and 4-{[2-(2-aminoethoxy)ethyl]amino}-2-(2,6-dioxopiperidine-3-yl)-2,3-dihydro-1H-isoindole-1,3-dione (CB1f) according to the procedure of Example 14. LC-MS t R = 5.77 min, m / z 1029.

[0397] Example 40 2-{2-[2-(3-{4-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-5-hydroxy-1H-pyrazole-3-yl]phenyl}propoxy)ethoxy]ethoxy}-N-[2-(2-{[2-(2,6-dioxopiperidine-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindole-4-yl]amino}ethoxy)ethyl]acetamide [ka]

[0398] The title compound was prepared from 2-{2-[2-(3-{4-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-5-hydroxy-1H-pyrazole-3-yl]phenyl}propoxy)ethoxy]ethoxy}acetic acid (NB2g) and 4-{[2-(2-aminoethoxy)ethyl]amino}-2-(2,6-dioxopiperidine-3-yl)-2,3-dihydro-1H-isoindole-1,3-dione (CB1f) according to the procedure of Example 14. LC-MS t R = 5.37 min, m / z 979.

[0399] Example 41 4-[1-(1H-1,3-benzodiazole-2-yl)-4-[2-(4-{3-[({2-[2-(2-{[2-(2,6-dioxopiperidine-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindole-4-yl]amino}ethoxy)ethoxy]ethyl}carbamoyl)methoxy]propyl}phenyl)ethyl]-5-hydroxy-1H-pyrazole-3-yl]piperidine-1-carboxylate tert-butyl [ka]

[0400] The title compound was prepared from 2-[3-(4-{2-[1-(1H-1,3-benzodiazole-2-yl)-3-{1-[(tert-butoxy)carbonyl]piperidine-4-yl}-5-hydroxy-1H-pyrazole-4-yl]ethyl}phenyl)propoxy]acetic acid (NB5b) and 4-({2-[2-(2-aminoethoxy)ethoxy]ethyl}amino)-2-(2,6-dioxopiperidine-3-yl)-2,3-dihydro-1H-isoindole-1,3-dione (CB1e) according to the procedure of Example 14. LC-MS t R = 4.62 min, m / z 990.

[0401] Example 42 2-[2-(4-{2-[1-(1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-(4-methanesulfonylphenyl)-1H-pyrazole-4-yl]ethyl}phenoxy)ethoxy]-N-{2-[2-(2-{[2-(2,6-dioxopiperidine-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindole-4-yl]amino}ethoxy)ethoxy]ethyl}acetamide [ka]

[0402] The title compound was prepared from 2-[2-(4-{2-[1-(1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-(4-methanesulfonylphenyl)-1H-pyrazole-4-yl]ethyl}phenoxy)ethoxy]acetic acid (NB1l) and 4-({2-[2-(2-aminoethoxy)ethoxy]ethyl}amino)-2-(2,6-dioxopiperidine-3-yl)-2,3-dihydro-1H-isoindole-1,3-dione (CB1e) according to the procedure of Example 14. LC-MS t R = 4.1 min, m / z 963.

[0403] Example 43 2-[2-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenoxy)ethoxy]-N-(2-{[2-(2,6-dioxopiperidine-3-yl)-1-oxo-2,3-dihydro-1H-isoindole-4-yl]amino}ethyl)acetamide [ka]

[0404] The title compound was prepared from 2-[2-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenoxy)ethoxy]acetic acid (NB1i) and 3-{4-[(2-aminoethyl)amino]-1-oxo-2,3-dihydro-1H-isoindole-2-yl}piperidine-2,6-dione (CB1h) according to the procedure of Example 14. LC-MS t R = 5.0 min, m / z 885.

[0405] Example 44 2-{2-[2-(3-{4-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-5-hydroxy-1H-pyrazole-3-yl]phenyl}propoxy)ethoxy]ethoxy}-N-{2-[2-(2-{[2-(2,6-dioxopiperidine-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindole-4-yl]amino}ethoxy)ethoxy]ethyl}acetamide [ka]

[0406] A stirred solution of 2-{2-[2-(3-{4-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-5-hydroxy-1H-pyrazole-3-yl]phenyl}propoxy)ethoxy]ethoxy}acetic acid HCl salt (23 mg, 36 μmol), 4-({2-[2-(2-aminoethoxy)ethoxy]ethyl}amino)-2-(2,6-dioxopiperidine-3-yl)-2,3-dihydro-1H-isoindole-1,3-dione HCl salt (16 mg, 36 μmol), HOBt.H2O (6 mg, 40 μmol), and i-Pr2NEt (35 μL, 0.19 mmol) in dry DMF (1 mL) was stirred at room temperature for 1 day. The mixture was purified by preparative HPLC to obtain the bis-TFA salt (7 mg, 15%) of the title compound as a yellow solid. 1 H NMR (300MHz, DMSO-d6) shift = 7.67 - 7.60 (m, 1H), 7.59 - 7.50 (m, 3H), 7.50 - 7.43 (m, 1H), 7.35 - 7.28 (m, 1H), 7.24 - 6.97 (m, 7H), 6.65 - 6.53 (m, 1H), 5.13 - 4.95 (m, 1H), 3.86 (s, 2H), 3.60-3.30 (m, 20H), 3.30 - 3.15 (m, 2H), 2.95 - 2.35 (m, 9H), 2.09 - 1.91 (m, 1H), 1.89 - 1.69 (m, 2H).LC-MS t R = 5.33 min, m / z 1023.

[0407] Example 45 2-[2-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenoxy)ethoxy]-N-(2-{[2-(2,6-dioxopiperidine-3-yl)-1-oxo-2,3-dihydro-1H-isoindole-4-yl]oxy}ethyl)acetamide [ka]

[0408] The title compound was prepared from 2-[2-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenoxy)ethoxy]acetic acid (NB1i) and 3-[4-(2-aminoethoxy)-1-oxo-2,3-dihydro-1H-isoindole-2-yl]piperidine-2,6-dione (CB1i) according to the procedure of Example 14. LC-MS t R = 5.1 min, m / z 886.

[0409] Example 46 2-(2-{2-[2-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenoxy)ethoxy]ethoxy}ethoxy)-N-[2-(2-{[2-(2,6-dioxopiperidine-3-yl)-1-oxo-2,3-dihydro-1H-isoindole-4-yl]oxy}ethoxy)ethyl]acetamide [ka]

[0410] The title compound was prepared from 4-{2-[4-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethoxy)phenyl]ethyl}-1-(5-chloro-1H-1,3-benzodiazole-2-yl)-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-5-ol (NB1c) and 3-{4-[2-(2-aminoethoxy)ethoxy]-1-oxo-2,3-dihydro-1H-isoindole-2-yl}piperidine-2,6-dione (CB1j) according to the procedure of Example 14. LC-MS t R = 5.08 min, m / z 10¹⁸.

[0411] Example 47 N-(2-{2-[2-(2-{4-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-5-hydroxy-1H-pyrazole-3-yl]benzenesulfonyl}ethoxy)ethoxy]ethoxy}ethyl)-2-{[2-(2,6-dioxopiperidine-3-yl)-1-oxo-2,3-dihydro-1H-isoindole-5-yl]oxy}acetamide [ka]

[0412] The title compound was prepared from 3-[4-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethanesulfonyl)phenyl]-1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-1H-pyrazole-5-ol (NB2c) and 2-{[2-(2,6-dioxopiperidine-3-yl)-1-oxo-2,3-dihydro-1H-isoindole-5-yl]oxy}acetic acid (CB2d) according to the procedure of Example 1. LC-MS t R = 4.87 min, m / z 972.

[0413] Example 48 3-{5-[2-(4-{2-[2-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenoxy)ethoxy]ethyl}piperazine-1-yl)-2-oxoethoxy]-1-oxo-2,3-dihydro-1H-isoindole-2-yl}piperidine-2,6-dione [ka]

[0414] The title compound was prepared from 1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-{2-[2-(piperazine-1-yl)ethoxy]ethoxy}phenyl)ethyl]-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-5-ol (NB1f) and 2-{[2-(2,6-dioxopiperidine-3-yl)-1-oxo-2,3-dihydro-1H-isoindole-5-yl]oxy}acetic acid (CB2d) according to the procedure of Example 1. LC-MS t R = 4.47 min, m / z 955.

[0415] Example 49 N-[14-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenoxy)-3,6,9,12-tetraoxatetradecane-1-yl]-2-{[2-(2,6-dioxopiperidine-3-yl)-1-oxo-2,3-dihydro-1H-isoindole-5-yl]oxy}acetamide [ka]

[0416] The title compound was prepared from 4-(2-{4-[(14-amino-3,6,9,12-tetraoxatetradecane-1-yl)oxy]phenyl}ethyl)-1-(5-chloro-1H-1,3-benzodiazole-2-yl)-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-5-ol (NB1e) and 2-{[2-(2,6-dioxopiperidine-3-yl)-1-oxo-2,3-dihydro-1H-isoindole-5-yl]oxy}acetic acid (CB2d) according to the procedure of Example 1. LC-MS t R = 5.0 min, m / z 10¹⁸.

[0417] Example 50 N-[2-(2-{2-[2-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenoxy)ethoxy]ethoxy}ethoxy)ethyl]-2-{[2-(2,6-dioxopiperidine-3-yl)-1-oxo-2,3-dihydro-1H-isoindole-5-yl]oxy}acetamide [ka]

[0418] The title compound was prepared from 4-{2-[4-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethoxy)phenyl]ethyl}-1-(5-chloro-1H-1,3-benzodiazole-2-yl)-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-5-ol (NB1c) and 2-{[2-(2,6-dioxopiperidine-3-yl)-1-oxo-2,3-dihydro-1H-isoindole-5-yl]oxy}acetic acid (CB2d) according to the procedure of Example 1. LC-MS t R = 4.97 min, m / z 974.

[0419] Example 51 2-[2-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenoxy)ethoxy]-N-(2-{[2-(2,6-dioxopiperidine-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindole-5-yl]amino}ethyl)acetamide [ka]

[0420] The title compound was prepared from 2-[2-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenoxy)ethoxy]acetic acid (NB1i) and 5-[(2-aminoethyl)amino]-2-(2,6-dioxopiperidine-3-yl)-2,3-dihydro-1H-isoindole-1,3-dione (CB2e) according to the procedure of Example 14. LC-MS t R = 5.15 min, m / z 899.

[0421] Example 52 5-(4-{[1-(2-{2-[2-(3-{4-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-5-hydroxy-1H-pyrazole-3-yl]phenyl}propoxy)ethoxy]ethoxy}acetyl)piperidine-4-yl]methyl}piperazine-1-yl)-2-(2,6-dioxopiperidine-3-yl)-2,3-dihydro-1H-isoindole-1,3-dione [ka]

[0422] EDC.HCl (7 mg, 37 mol) was added to a stirred solution of 2-{2-[2-(3-{4-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-5-hydroxy-1H-pyrazole-3-yl]phenyl}propoxy)ethoxy]ethoxy}acetic acid (12 mg, 18 μmol), 2-(2,6-dioxopiperidine-3-yl)-5-{4-[(piperidine-4-yl)methyl]piperazine-1-yl}-2,3-dihydro-1H-isoindole-1,3-dione bisHCl salt (11.5 mg, 22 μmol), HOBt.H2O (3 mg, 20 μmol), and i-Pr2NEt (25 μL, 0.14 mmol) in dry DMF (1 mL). The mixture was stirred at room temperature for 1 day and purified by preparative HPLC to obtain the tris-HCl salt (3 mg, 12%) of the title compound as a solid. 1 H NMR (300MHz, DMSO-d6) shift= 7.75 (d, J=8.3 Hz, 1H), 7.59 - 7.51 (m, 2H), 7.50 - 7.44 (m, 3H), 7.36 - 7.30 (m, 3H), 7.23 - 7.12 (m, 3H), 7.08 - 6.98 (m, 2H), 5.08 (dd, J=5.3, 12.7 Hz, 1H), 4.41 - 4.05 (m, 10H), 3.85-3.20 (s, 16H), 3.17 - 2.34 (m, 10H), 2.16 - 1.95 (m, 2H), 1.93 - 1.62 (m, 3H), 1.28 - 0.89 (m, 2H).LC-MS t R = 4.52 min, m / z 1058.

[0423] Example 53 5-[4-({1-[2-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenoxy)acetyl]piperidine-4-yl}methyl)piperazine-1-yl]-2-(2,6-dioxopiperidine-3-yl)-2,3-dihydro-1H-isoindole-1,3-dione [ka]

[0424] The title compound was prepared from 2-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenoxy)acetic acid (NB1h) and 2-(2,6-dioxopiperidine-3-yl)-5-{4-[(piperidine-4-yl)methyl]piperazine-1-yl}-2,3-dihydro-1H-isoindole-1,3-dione (CB2a) according to the procedure of Example 14. LC-MS t R = 4.52 min, m / z 973.

[0425] Example 54 5-[4-({1-[2-(4-{2-[1-(1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-(4-methanesulfonylphenyl)-1H-pyrazole-4-yl]ethyl}phenoxy)acetyl]piperidine-4-yl}methyl)piperazine-1-yl]-2-(2,6-dioxopiperidine-3-yl)-2,3-dihydro-1H-isoindole-1,3-dione [ka]

[0426] The title compound was prepared from 2-(4-{2-[1-(1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-(4-methanesulfonylphenyl)-1H-pyrazole-4-yl]ethyl}phenoxy)acetic acid (NB1k) and 2-(2,6-dioxopiperidine-3-yl)-5-{4-[(piperidine-4-yl)methyl]piperazine-1-yl}-2,3-dihydro-1H-isoindole-1,3-dione (CB2a) according to the procedure of Example 14. LC-MS t R = 3.53 min, m / z 954.

[0427] Example 55 5-[4-({1-[2-(2-{4-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-5-hydroxy-1H-pyrazole-3-yl]piperidine-1-yl}-2-oxoethoxy)acetyl]piperidine-4-yl}methyl)piperazine-1-yl]-2-(2,6-dioxopiperidine-3-yl)-2,3-dihydro-1H-isoindole-1,3-dione [ka]

[0428] The title compound was prepared from 2-(2-{4-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-5-hydroxy-1H-pyrazole-3-yl]piperidine-1-yl}-2-oxoethoxy)acetic acid (NB3b) and 2-(2,6-dioxopiperidine-3-yl)-5-{4-[(piperidine-4-yl)methyl]piperazine-1-yl}-2,3-dihydro-1H-isoindole-1,3-dione (CB2a) according to the procedure of Example 14. LC-MS t R = 4.03 min, m / z 977.

[0429] Example 56 5-{4-[(1-{2-[2-(3-{4-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-5-hydroxy-1H-pyrazole-3-yl]phenyl}propoxy)ethoxy]acetyl}piperidine-4-yl)methyl]piperazine-1-yl}-2-(2,6-dioxopiperidine-3-yl)-2,3-dihydro-1H-isoindole-1,3-dione [ka]

[0430] The title compound was prepared from 2-[2-(3-{4-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-5-hydroxy-1H-pyrazole-3-yl]phenyl}propoxy)ethoxy]acetic acid (NB2f) and 2-(2,6-dioxopiperidine-3-yl)-5-{4-[(piperidine-4-yl)methyl]piperazine-1-yl}-2,3-dihydro-1H-isoindole-1,3-dione (CB2a) according to the procedure of Example 14. LC-MS t R = 4.45 min, m / z 10¹⁴.

[0431] Example 57 5-(4-{[1-(2-{2-[3-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenyl)propoxy]ethoxy}acetyl)piperidine-4-yl]methyl}piperazine-1-yl)-2-(2,6-dioxopiperidine-3-yl)-2,3-dihydro-1H-isoindole-1,3-dione [ka]

[0432] The title compound was prepared from 2-{2-[3-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenyl)propoxy]ethoxy}acetic acid (NB1o) and 2-(2,6-dioxopiperidine-3-yl)-5-{4-[(piperidine-4-yl)methyl]piperazine-1-yl}-2,3-dihydro-1H-isoindole-1,3-dione (CB2a) according to the procedure of Example 14. LC-MS t R = 4.73 min, m / z 1064.

[0433] Example 58 5-(4-{[1-(15-{4-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-5-hydroxy-1H-pyrazole-3-yl]phenyl}-3,6,9,12-tetraoxapentadecanoyl)piperidine-4-yl]methyl}piperazine-1-yl)-2-(2,6-dioxopiperidine-3-yl)-2,3-dihydro-1H-isoindole-1,3-dione [ka]

[0434] The title compound was prepared from 15-{4-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-5-hydroxy-1H-pyrazole-3-yl]phenyl}-3,6,9,12-tetraoxapentadecanoic acid (NB2h) and 2-(2,6-dioxopiperidine-3-yl)-5-{4-[(piperidine-4-yl)methyl]piperazine-1-yl}-2,3-dihydro-1H-isoindole-1,3-dione (CB2a) according to the procedure of Example 14. R = 4.5 min, m / z 1103.

[0435] Example 59 5-[4-({1-[15-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenyl)-3,6,9,12-tetraoxapentadecanoyl]piperidine-4-yl}methyl)piperazine-1-yl]-2-(2,6-dioxopiperidine-3-yl)-2,3-dihydro-1H-isoindole-1,3-dione [ka]

[0436] The title compound was prepared from 15-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenyl)-3,6,9,12-tetraoxapentadecanoic acid (NB1q) and 2-(2,6-dioxopiperidine-3-yl)-5-{4-[(piperidine-4-yl)methyl]piperazine-1-yl}-2,3-dihydro-1H-isoindole-1,3-dione (CB2a) according to the procedure of Example 14. R = 4.73 min, m / z 1153.

[0437] Example 60 3-(5-{4-[(1-{2-[3-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenyl)propoxy]acetyl}piperidine-4-yl)methyl]piperazine-1-yl}-1-oxo-2,3-dihydro-1H-isoindole-2-yl)piperidine-2,6-dione [ka]

[0438] The title compound was prepared from 2-[3-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenyl)propoxy]acetic acid (NB1n) and 3-(1-oxo-5-{4-[(piperidine-4-yl)methyl]piperazine-1-yl}-2,3-dihydro-1H-isoindole-2-yl)piperidine-2,6-dione (CB2c) according to the procedure of Example 14. LC-MS t R = 4.45 min, m / z 1007.

[0439] Example 61 3-[5-(4-{[1-(3-{4-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-5-hydroxy-1H-pyrazole-3-yl]phenyl}propanoyl)piperidine-4-yl]methyl}piperazine-1-yl)-1-oxo-2,3-dihydro-1H-isoindole-2-yl]piperidine-2,6-dione [ka]

[0440] The title compound was prepared from 3-{4-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-5-hydroxy-1H-pyrazole-3-yl]phenyl}propanoic acid (NB2d) and 3-(1-oxo-5-{4-[(piperidine-4-yl)methyl]piperazine-1-yl}-2,3-dihydro-1H-isoindole-2-yl)piperidine-2,6-dione (CB2c) according to the procedure of Example 14. LC-MS t R = 4.18 min, m / z 912.

[0441] Example 62 3-{5-[4-({1-[2-(2-{2-[2-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenoxy)ethoxy]ethoxy}ethoxy)acetyl]piperidine-4-yl}methyl)piperazine-1-yl]-1-oxo-2,3-dihydro-1H-isoindole-2-yl}piperidine-2,6-dione [ka]

[0442] The title compound was prepared from 4-{2-[4-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethoxy)phenyl]ethyl}-1-(5-chloro-1H-1,3-benzodiazole-2-yl)-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-5-ol (NB1c) and 3-(1-oxo-5-{4-[(piperidine-4-yl)methyl]piperazine-1-yl}-2,3-dihydro-1H-isoindole-2-yl)piperidine-2,6-dione (CB2c) according to the procedure of Example 14. R = 4.5 min, m / z 1096.

[0443] Example 63 15-{4-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-5-hydroxy-1H-pyrazole-3-yl]phenyl}-N-[2-({3-[2-(2,6-dioxopiperidine-3-yl)-1-oxo-2,3-dihydro-1H-isoindole-5-yl]propa-2-in-1-yl}oxy)ethyl]-3,6,9,12-tetraoxapentadecanamide [ka]

[0444] The title compound was prepared from 15-{4-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-5-hydroxy-1H-pyrazole-3-yl]phenyl}-3,6,9,12-tetraoxapentadecanoic acid (NB2h) and 3-{5-[3-(2-aminoethoxy)propa-1-in-1-yl]-1-oxo-2,3-dihydro-1H-isoindole-2-yl}piperidine-2,6-dione (CB2f) according to the procedure of Example 14. R = 5.1 min, m / z 1004.

[0445] Example 64 2-(3-{4-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-5-hydroxy-1H-pyrazole-3-yl]phenyl}propoxy)-N-[2-({3-[2-(2,6-dioxopiperidine-3-yl)-1-oxo-2,3-dihydro-1H-isoindole-5-yl]propa-2-in-1-yl}oxy)ethyl]acetamide [ka]

[0446] The title compound was prepared from 2-(3-{4-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-5-hydroxy-1H-pyrazole-3-yl]phenyl}propoxy)acetic acid (NB2e) and 3-{5-[3-(2-aminoethoxy)propa-1-in-1-yl]-1-oxo-2,3-dihydro-1H-isoindole-2-yl}piperidine-2,6-dione (CB2f) according to the procedure of Example 14. LC-MS t R = 5.02 min, m / z 872.

[0447] Example 65 15-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenyl)-N-[2-({3-[2-(2,6-dioxopiperidine-3-yl)-1-oxo-2,3-dihydro-1H-isoindole-5-yl]propa-2-in-1-yl}oxy)ethyl]-3,6,9,12-tetraoxapentadecanamide [ka]

[0448] The title compound was prepared from 15-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenyl)-3,6,9,12-tetraoxapentadecanoic acid (NB1q) and 3-{5-[3-(2-aminoethoxy)propa-1-in-1-yl]-1-oxo-2,3-dihydro-1H-isoindole-2-yl}piperidine-2,6-dione (CB2f) according to the procedure of Example 14. LC-MS t R = 5.48 min, m / z 1054.

[0449] Example 66 2-[3-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenyl)propoxy]-N-[2-({3-[2-(2,6-dioxopiperidine-3-yl)-1-oxo-2,3-dihydro-1H-isoindole-5-yl]propa-2-in-1-yl}oxy)ethyl]acetamide [ka]

[0450] The title compound was prepared from 2-[3-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenyl)propoxy]acetic acid (NB1n) and 3-{5-[3-(2-aminoethoxy)propa-1-in-1-yl]-1-oxo-2,3-dihydro-1H-isoindole-2-yl}piperidine-2,6-dione (CB2f) according to the procedure of Example 14. LC-MS t R = 5.47 min, m / z 922.

[0451] Example 67 3-[5-(4-{2-[3-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenyl)propoxy]acetyl}piperazine-1-yl)-1-oxo-2,3-dihydro-1H-isoindole-2-yl]piperidine-2,6-dione [ka]

[0452] The title compound was prepared from 2-[3-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenyl)propoxy]acetic acid (NB1n) and 3-[1-oxo-5-(piperazine-1-yl)-2,3-dihydro-1H-isoindole-2-yl]piperidine-2,6-dione (CB2b) according to the procedure of Example 14. LC-MS t R = 5.33 min, m / z 909.

[0453] Example 68 3-[5-(4-{2-[2-(3-{4-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-5-hydroxy-1H-pyrazole-3-yl]phenyl}propoxy)ethoxy]acetyl}piperazine-1-yl)-1-oxo-2,3-dihydro-1H-isoindole-2-yl]piperidine-2,6-dione [ka]

[0454] The title compound was prepared from 2-[2-(3-{4-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-5-hydroxy-1H-pyrazole-3-yl]phenyl}propoxy)ethoxy]acetic acid (NB2f) and 3-[1-oxo-5-(piperazine-1-yl)-2,3-dihydro-1H-isoindole-2-yl]piperidine-2,6-dione (CB2b) according to the procedure of Example 14. LC-MS t R = 4.93 min, m / z 903.

[0455] Example 69 3-{5-[4-(2-{2-[3-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenyl)propoxy]ethoxy}acetyl)piperazine-1-yl]-1-oxo-2,3-dihydro-1H-isoindole-2-yl}piperidine-2,6-dione [ka]

[0456] The title compound was prepared from 2-{2-[3-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenyl)propoxy]ethoxy}acetic acid (NB1o) and 3-[1-oxo-5-(piperazine-1-yl)-2,3-dihydro-1H-isoindole-2-yl]piperidine-2,6-dione (CB2b) according to the procedure of Example 14. LC-MS t R = 5.3 min, m / z 953.

[0457] Example 70 3-(5-{4-[2-(3-{4-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-5-hydroxy-1H-pyrazole-3-yl]phenyl}propoxy)acetyl]piperazine-1-yl}-1-oxo-2,3-dihydro-1H-isoindole-2-yl)piperidine-2,6-dione [ka]

[0458] The title compound was prepared from 2-(3-{4-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-5-hydroxy-1H-pyrazole-3-yl]phenyl}propoxy)acetic acid (NB2e) and 3-[1-oxo-5-(piperazine-1-yl)-2,3-dihydro-1H-isoindole-2-yl]piperidine-2,6-dione (CB2b) according to the procedure of Example 14. LC-MS t R = 4.87 min, m / z 859.

[0459] Example 71 3-{5-[4-(2-{2-[2-(3-{4-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-5-hydroxy-1H-pyrazole-3-yl]phenyl}propoxy)ethoxy]ethoxy}acetyl)piperazine-1-yl]-1-oxo-2,3-dihydro-1H-isoindole-2-yl}piperidine-2,6-dione [ka]

[0460] The title compound was prepared from 2-{2-[2-(3-{4-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-fluorophenyl)ethyl]-5-hydroxy-1H-pyrazole-3-yl]phenyl}propoxy)ethoxy]ethoxy}acetic acid (NB2g) and 3-[1-oxo-5-(piperazine-1-yl)-2,3-dihydro-1H-isoindole-2-yl]piperidine-2,6-dione (CB2b). LC-MS t R = 4.9 min, m / z 947.

[0461] Example 72 3-[5-(4-{2-[2-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenoxy)ethoxy]acetyl}piperazine-1-yl)-1-oxo-2,3-dihydro-1H-isoindole-2-yl]piperidine-2,6-dione [ka]

[0462] The title compound was prepared from 2-[2-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenoxy)ethoxy]acetic acid (NB1i) and 3-[1-oxo-5-(piperazine-1-yl)-2,3-dihydro-1H-isoindole-2-yl]piperidine-2,6-dione (CB2b) according to the procedure of Example 14. LC-MS t R = 5.02 min, m / z 911.

[0463] Example 73 2-{[2-(2,6-dioxopiperidine-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindole-4-yl]amino}ethyl 2-[2-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenoxy)ethoxy]acetate [ka]

[0464] EDC.HCl (11 mg, 60 μmol) was added to a stirred solution of m2-[2-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenoxy)ethoxy]acetic acid (NB1i, 17 mg, 28 μmol), 2-(2,6-dioxopiperidine-3-yl)-4-[(2-hydroxyethyl)amino]-2,3-dihydro-1H-isoindole-1,3-dione (CB1k, 9 mg, 28 μmol), and DMAP (7 g, 58 μmol) in dry CH2Cl2 (1 mL). The mixture was stirred at room temperature for 16 hours and concentrated. The residue was purified by preparative HPLC to obtain the title compound (6 mg, %) as a yellow solid. LC-MS t R = 5.55 min, m / z 900.

[0465] Example 74 3-{5-[2-(4-{2-[2-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenoxy)ethoxy]ethyl}piperazine-1-yl)ethoxy]-1-oxo-2,3-dihydro-1H-isoindole-2-yl}piperidine-2,6-dione [ka]

[0466] A mixture of 1-(5-chloro-1H-1,3-benzodiazole-2-yl)-4-[2-(4-{2-[2-(piperazine-1-yl)ethoxy]ethoxy}phenyl)ethyl]-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-5-ol (NB1f, 17 mg, 21 μmol), 2-{[2-(2,6-dioxopiperidine-3-yl)-1-oxo-2,3-dihydro-1H-isoindole-5-yl]oxy}acetaldehyde (CB 2 g, 13 mg, 43 μmol), Et3N (12 μL, 85 μmol), MgSO4 (80 mg), and dried CH2Cl2 (2 mL) was stirred under N2 at room temperature for 0.5 hours. The mixture was cooled in an ice bath and solid NaBH(OAc)3 (14 mg, 65 μmol) was added. The mixture was stirred at room temperature for 2 hours, diluted with MeOH (8 mL), and filtered. The filtrate was concentrated, and the residue was purified by preparative HPLC to obtain the bis-TFA salt of the title compound (3.6 mg, 14%) as a white solid. LC-MS t R = 4.77 min, m / z 941.

[0467] Example 75 3-(5-{2-[4-(2-{2-[2-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenoxy)ethoxy]ethoxy}ethyl)piperazine-1-yl]ethoxy}-1-oxo-2,3-dihydro-1H-isoindole-2-yl)piperidine-2,6-dione [ka]

[0468] The title compound was prepared from 4-(2-{4-[(14-amino-3,6,9,12-tetraoxatetradecane-1-yl)oxy]phenyl}ethyl)-1-(5-chloro-1H-1,3-benzodiazole-2-yl)-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-5-ol (NB1e) and 2-{[2-(2,6-dioxopiperidine-3-yl)-1-oxo-2,3-dihydro-1H-isoindole-5-yl]oxy}acetaldehyde (CB2g) according to the procedure of Example 74. LC-MS t R = 4.38 min, m / z 985.

[0469] Example 76 2-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1Hpyrazole-4-yl]ethyl}phenoxy)-N-(3-{[2-(2,6-dioxopiperidine-3-yl)-1,3-dioxo-2,3-dihydro-1Hisoindole-4-yl]aminopropyl)acetamide [ka]

[0470] EDC.HCl (13 mg, 68 μmol) was added to a stirred solution of 2-(4-{2-[1-(5-chloro-1H-1,3-benzodiazole-2-yl)-5-hydroxy-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-4-yl]ethyl}phenoxy)acetic acid HCl salt (NB1h, 20 mg, 34 μmol), 4-[(3-aminopropyl)amino]-2-(2,6-dioxopiperidine-3-yl)-2,3-dihydro-1H-isoindole-1,3-dione HCl salt (CB1m, 14 mg, 38 μmol), HOBt.H2O (5 mg, 37 μmol), and i-Pr2NEt (40 μL, 0.22 mmol) in dry DMF (2.5 mL). The mixture was stirred at room temperature for 18 hours and directly purified by preparative HPLC to obtain the TFA salt of the title compound (12 mg, 36%) as a yellow solid. LC-MS t R= 6.18 min, m / z 869.

[0471] Pharmaceutical composition and method of use Pharmaceutical formulations comprising compounds disclosed herein or pharmaceutically acceptable salts thereof, and at least one pharmaceutically acceptable carrier, excipient, or combination thereof are also disclosed herein. Pharmaceutical formulations may further include additional pharmacologically active agents other than compounds. In certain disclosed embodiments, the pharmacologically active agent is an antiretroviral agent. The antiretroviral agent may be selected from entry inhibitors, CCR5 receptor antagonists, nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, protease inhibitors, integrase inhibitors, maturation inhibitors, capsid inhibitors, or combinations thereof. In certain disclosed embodiments, the antiretroviral agent is selected from maraviroc, enfuvirtide, apraviroc, bicriviroc, zidovudine, didanosine, zalcitabine, stabudine, lamivudine, abacavir, emtricitabine, entecavir, apricitabine, tenofovir, adefovir, efavirenz, nevirapine, delavirdine, etravirine, rilpivirine, saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, lopinavir, atazanavir, fosamprenavir, tipranavir, darunavir, MK-2048, elvitegravir, bevirimat, MPC-9055, renacapavir, cabotegravir, fostemsavir, or a combination thereof.

[0472] Methods for inhibiting the biological function of Nef are also disclosed herein, including contacting Nef with an effective amount of a compound disclosed herein. The biological function of Nef may be selected from HIV infectivity, HIV replication, HIV-1 incubation period and its reversal, Nef-mediated downregulation of the MHC-1 / HIV antigen complex on the surface of infected cells, Nef-mediated effects on other cellular receptors and proteins associated with HIV-1 disease development (e.g., CD4, SERINC5, PD-1, Src and Tec family kinases), and AIDS progression.

[0473] A method for treating Nef-borne diseases is also disclosed, which includes administering an effective amount of a compound disclosed herein to a target. Further embodiments relate to a method for treating HIV, which includes administering an effective amount of a compound disclosed herein to a target.

[0474] Certain disclosed embodiments relate to methods for treating HIV-related conditions, including administering an effective amount of the compound disclosed herein to a subject. HIV-related conditions may be selected from HIV replication, HIV-related CD4+ T cell loss and immunodeficiency, HIV-induced infection, Kaposi's sarcoma, HIV-related nephropathy, AIDS-dementia complex, and combinations thereof. The subject may be suffering from an HIV-related condition. The compound may also be administered to the subject prophylactically. In other embodiments, the subject may be administered the compound prophylactically after exposure.

[0475] The compound may also be administered as a formulation. The formulation may comprise the compound and a pharmaceutically acceptable carrier. The formulation may also further comprise at least one antiretroviral agent, as disclosed herein. The subjects may be animals or humans, and any one of the disclosed embodiments of this method may be carried out in vitro or in vivo.

[0476] Embodiments of the disclosed method may be used when the subject has an HIV-related condition, or the method may be performed prophylactically or post-exposure prophylaxis. HIV-related conditions may be selected from HIV replication, HIV-related CD4+ T cell loss and immunodeficiency, HIV-induced infection, Kaposi's sarcoma, HIV-related nephropathy, AIDS-dementia complex, and combinations thereof.

[0477] The effective dose used in the disclosed method may be the amount best suited to the treatment of the subject. The effective dose may range from greater than 0 to about 1000 mg / kg / day. In certain disclosed embodiments, the effective dose is in the range of 1 mg / kg / day to about 100 mg / kg / day. The subject of the disclosed method may be human or animal, and the method may be performed in vitro or in vivo.

[0478] The compounds disclosed herein may be used to treat Nef-dependent disorders. As disclosed herein, the compounds may be used to treat and / or inhibit biological pathways activated by Nef. Such pathways include, but are not limited to, those involving Src family kinases (e.g., Hck) and Tec family kinases (e.g., Itk and Btk). In certain disclosed embodiments, the compounds may be used to treat or inhibit Nef-dependent HIV-1 replication both in vitro and in vivo. The disclosed compounds may also be used to treat or inhibit Nef-dependent HIV-1 infectivity.

[0479] In other disclosed embodiments, the compounds may be used to treat or inhibit SIV infectivity or replication.

[0480] Certain disclosed embodiments of the compounds disclosed herein are potent and selective inhibitors of Nef-dependent Hck activity and can therefore be used in in vitro, in vivo, and ex vivo settings to modulate or inhibit this activity, prevent Nef-dependent HIV-1 replication, and downregulate MHC-1 and the biological responses arising from such activity. In certain disclosed embodiments, the compounds may be used to inhibit the infectivity and replication of HIV-1 in cell types selected from primary host cells isolated from donors, including, but not limited to, U87MG astroglioma cells, CEM-T4 lymphoblasts, TZM-bl reporter cell lines and CEM-174, as well as peripheral blood mononuclear cells (PBMCs) and CD4+ T lymphocytes derived therefrom. Certain disclosed embodiments of the compounds disclosed herein may be used to inhibit Nef-dependent HIV replication in the range of submicromolar concentrations. Embodiments of the disclosed compounds exhibit in vitro Nef-inducible Hck activation, as well as Nef-inducible Itk activation, at ICs of less than approximately 3.0 μM, more typically less than approximately 2.5 μM, and even more typically less than approximately 2.0 μM. 50 It can show a value.

[0481] In particular, in the embodiments disclosed, the compound can prevent and / or inhibit the Nef-dependent enhancement of HIV-1 infectivity and replication. The compound is not limited to being active against any specific Nef allele. For example, embodiments of the disclosed compound are active against various Nef alleles, particularly those comprising the HIV-1M group clade. Exemplary embodiments of the compound enhance HIV-1 replication in donor PBMCs from 10 nM to about 100 nM, more typically about 50 nM to about 200 nM, and even more typically about 100 nM to about 300 nM IC 50 It can be inhibited by a value.

[0482] In certain disclosed embodiments, the compound may be used to inhibit Nef-dependent HIV replication and infectivity.

[0483] Nef is well known to prevent the cell surface display of MHC-I in complex with HIV-1 antigenic peptide on infected cells, thereby promoting evasion from detection by cytotoxic T lymphocytes. As a result, this effect of Nef may prevent viral clearance from the infected host and contribute to the establishment and maintenance of a persistent viral reservoir. However, the compounds disclosed herein restore MHC-I to the surface of HIV-infected CD4+ T cells (see Figures 5A and 5B). Furthermore, when inhibitor-treated cells were co-cultured with autologous CD8 T cells expanded in the presence of HIV-1 antigenic peptide, the CD8 T cells were activated and exhibited a CTL response against infected target cells despite the presence of Nef. These results suggest that Nef inhibitors, including those described herein, have the potential to enhance the CTL-mediated response against HIV+ cells in vivo as part of a strategy to eliminate latent viral reservoirs.

[0484] The compounds may be administered orally, parenterally (including subcutaneous injection (SC or depot-SC), intravenously (IV), intramuscularly (IM or depot-IM), intrasternal injection or infusion techniques), sublingually, intranasally (inhalation), intrathecally, topically, or rectally. The pharmaceutical compositions may be administered in dosing unit formulations containing conventional non-toxic, pharmaceutically acceptable carriers, adjuvants, and / or vehicles. The compounds are preferably formulated into suitable pharmaceutical formulations such as tablets, capsules, or elixirs for oral administration, or into sterile solutions or suspensions for parenteral administration. Typically, the above compounds are formulated into pharmaceutical compositions using techniques and procedures well known in the art.

[0485] In some embodiments, a pharmaceutical composition is prepared by mixing or combining one or more of the disclosed compounds (including compounds linked to detectable labels or cargo portions) with a suitable pharmaceutically acceptable carrier. Pharmaceutical carriers or vehicles suitable for administering the compounds provided herein include any such carrier known to be suitable for a particular mode of administration. Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, PA, 21 st Edition (2005) describes exemplary compositions and formulations suitable for the pharmaceutical delivery of the compounds disclosed herein. Furthermore, the compounds may be formulated as the sole pharmaceutically active ingredient in a composition, or in combination with other active ingredients.

[0486] When a compound(s) is mixed with or added to a pharmaceutically acceptable carrier, the resulting mixture may be a solution, suspension, emulsion, or the like. Liposome suspensions may also be suitable as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art. The form of the resulting mixture depends on many factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle. If the compound exhibits insufficient solubility, solubilization methods may be used. Such methods are known and include, but are not limited to, the use of cosolvents such as dimethyl sulfoxide (DMSO), surfactants such as Tween®, and dissolution in aqueous sodium bicarbonate. Derivatives of the compound, such as salts or prodrugs, can also be used to formulate effective pharmaceutical compositions. The disclosed compounds can also be prepared with carriers that protect them from rapid elimination from the body, such as sustained-release formulations or coatings. Such carriers include, but are not limited to, controlled-release formulations such as microencapsulated delivery systems.

[0487] The disclosed compounds and / or compositions may be enclosed in multiple or single-dose containers. The compounds and / or compositions may also be provided in a kit, for example, containing components that can be assembled for use. For example, one or more of the disclosed compounds may be provided in lyophilized form, and a suitable diluent may be provided as a separate component for combination before use. In some examples, a kit may contain a disclosed compound and a second therapeutic agent (such as an antiretroviral agent) for co-administration. The compound and the second therapeutic agent may be provided as separate components. The kit may contain multiple containers, each holding one or more unit doses of the compound. The containers are preferably adapted to the desired mode of administration, including but not limited to tablets, gel capsules, sustained-release capsules, etc. for oral administration; depot preparations, pre-filled syringes, ampoules, vials, etc. for parenteral administration; and patches, medipads, creams, etc. for topical administration.

[0488] The active compound is contained in a pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect without undesirable side effects in the subject being treated. The therapeutically effective concentration can be empirically determined by testing the compound in known in vitro and in vivo model systems for the disorder being treated. In some examples, a therapeutically effective amount of the compound is the amount that alleviates or improves at least one symptom of the disorder to which the compound is administered. Typically, the composition is formulated for single-dose administration. The concentration of the active compound in the drug composition depends on the absorption, inactivation, and excretion rates of the active compound, the administration schedule, and the amount administered, as well as other factors known to those skilled in the art.

[0489] In some examples, approximately 0.1 mg to 1000 mg of a disclosed compound, a mixture of such compounds, or a physiologically acceptable salt or ester thereof is formulated in a unit dosage form with a physiologically acceptable vehicle, carrier, excipient, binder, preservative, stabilizer, flavoring agent, etc. The amount of active substance in these compositions or preparations is such that an appropriate dose within the indicated range is obtained. The term “unit dosage form” refers to a physically distinct unit suitable as a unit dose for human subjects and other mammals, each unit containing a predetermined amount of active material calculated to produce a desired therapeutic effect in combination with appropriate pharmaceutical excipients. In some examples, the compositions are formulated in unit dosage forms, with each dose containing one or more of a compound in approximately 1 mg to approximately 1000 mg (e.g., approximately 2 mg to approximately 500 mg, approximately 5 mg to 50 mg, approximately 10 mg to 100 mg, or approximately 25 mg to 75 mg). In other examples, the unit dosage form contains approximately 0.1 mg, approximately 1 mg, approximately 5 mg, approximately 10 mg, approximately 20 mg, approximately 30 mg, approximately 40 mg, approximately 50 mg, approximately 60 mg, approximately 70 mg, approximately 80 mg, approximately 90 mg, approximately 100 mg, approximately 150 mg, approximately 200 mg, approximately 250 mg, approximately 300 mg, approximately 400 mg, approximately 500 mg, approximately 600 mg, approximately 700 mg, approximately 800 mg, approximately 900 mg, approximately 1000 mg or more of the compound(s) of the Disclosure.

[0490] The disclosed compounds or compositions may be administered as a single dose or divided into several smaller doses administered at time intervals. Therapeutic compositions may be administered by repeated-dose protocols (e.g., multiple times daily, daily, weekly, or monthly), by single-dose delivery, or by continuous delivery over a long period. It is understood that the exact dosage, timing, and duration of treatment are functions of the disease being treated and may be determined empirically using known test protocols or by extrapolation from in vivo or in vitro test data (e.g., tests in animal models of HIV infection). It should be noted that concentration and dosage values ​​may also vary depending on the severity of the condition being alleviated. Furthermore, for specific subjects, the treatment plan may be gradually adjusted according to individual needs and the professional judgment of the person administering or managing the composition, and it is understood that the concentration ranges described herein are illustrative only.

[0491] When administered orally as a suspension, these compositions may contain microcrystalline cellulose for bulk, alginic acid or sodium alginate as a suspending agent, methylcellulose as a thickener, and sweeteners / flavoring agents, prepared according to well-known techniques in the field of pharmaceutical formulation. As immediate-release tablets, these compositions may contain microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate, and lactose, as well as / or other excipients, binders, bulking agents, disintegrants, diluents, and lubricants. When oral administration is desired, the compounds are typically provided in compositions that protect the compound from the acidic environment of the stomach. For example, a composition can be formulated in an enteric coating that maintains its integrity in the stomach and releases the active compound in the intestines. The composition can also be formulated in combination with antacids or other such components.

[0492] Oral compositions generally contain an inert diluent or food carrier and may be compressed into tablets or encapsulated in gelatin capsules. For the purpose of oral therapeutic administration, one or more active compounds may be incorporated together with excipients and used in the form of tablets, capsules or lozenges. Pharmaceutically compatible binders and adjuvant materials may be included as part of the composition. Tablets, pills, capsules, lozenges, etc., may contain binders including, but not limited to, tragacanth gum, acacia, corn starch, or gelatin; excipients such as microcrystalline cellulose, starch, or lactose; disintegrants including, but not limited to, alginic acid and corn starch; lubricants such as magnesium stearate; glidants including, but not limited to, colloidal silicon dioxide; sweeteners such as sucrose or saccharin; and flavorings, such as peppermint, methyl salicylate, or fruit flavoring components or compounds of similar properties.

[0493] When the drug unit form is a capsule, it may contain a liquid carrier such as fatty oil in addition to the materials of the types described above. Furthermore, the drug unit form may include coatings of various other materials that modify the physical form of the drug unit, such as sugars and other enteric coatings. The compound can also be administered as an ingredient in elixirs, suspensions, syrups, wafers, chewing gums, etc. In addition to the active compound, syrups may contain sucrose as a sweetener, as well as certain preservatives, dyes and colorants, and flavorings.

[0494] When administered orally, compounds may be administered in conventional dosage forms for oral administration. These dosage forms include conventional solid unit dosage forms such as tablets and capsules, as well as liquid dosage forms such as solutions, suspensions, and elixirs. When using solid dosage forms, they are preferably sustained-release so that the compound only needs to be administered once or twice daily. In some examples, oral dosage forms are administered to subjects one, two, three, four times or more daily. In further examples, compounds may be administered orally to humans in single or divided doses within a dosage range of 1 to 1000 mg / kg body weight. An exemplary dosage range is 0.1 to 200 mg / kg body weight orally (e.g., 0.5 to 100 mg / kg body weight orally) in single or divided doses. For oral administration, compositions may be provided in the form of tablets containing approximately 1 to 1000 milligrams of the active ingredient, particularly 1, 5, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 750, 800, 900, or 1000 milligrams of the active ingredient. However, it will be understood that specific dose levels and dosing frequencies for any particular patient may vary and depend on a variety of factors, including the activity of the particular compound used, its metabolic stability and duration of action, age, weight, general health, sex, diet, mode and timing of administration, excretion rate, drug combinations, severity of the particular condition, and the host being treated.

[0495] Injectable solutions or suspensions may also be formulated using suitable non-toxic, parenterally tolerable diluents or solvents, such as mannitol, 1,3-butanediol, water, Ringer's solution, or isotonic sodium chloride solution, or suitable dispersants or wetting and suspending agents, such as sterile, non-irritating, synthetic monoglycerides or diglycerides containing fixing oils, and fatty acids containing oleic acid. Liquids or suspensions used for parenteral, intradermal, subcutaneous, or topical application may contain any of the following components: sterile diluents, e.g., water for injection, saline solution; fixative oils, natural vegetable oils, e.g., sesame oil, coconut oil, peanut oil, cottonseed oil, etc.; or synthetic fatty vehicles, e.g., ethyl oleate, etc.; polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antimicrobial agents, such as benzyl alcohol and methylparaben; antioxidants, such as ascorbic acid and sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetates, citrates, and phosphates; and agents for adjusting isotonicity, e.g., sodium chloride and dextrose. Parenteral formulations may be enclosed in ampoules, disposable syringes, or multi-dose vials made of glass, plastic, or other suitable materials. Buffers, preservatives, antioxidants, etc., may be incorporated as needed.

[0496] For intravenous administration, suitable carriers include physiological saline, phosphate-buffered saline (PBS), and solutions containing thickeners and solubilizers such as glucose, polyethylene glycol, polypropylene glycol, and mixtures thereof. Liposome suspensions containing tissue-targeted liposomes may also be suitable as pharmaceutically acceptable carriers.

[0497] The compound can be administered parenterally, for example, by IV, IM, depot-IM, SC, or depot-SC. When administered parenterally, therapeutically effective doses of approximately 0.1 to approximately 500 mg / day (e.g., approximately 1 mg / day to approximately 100 mg / day, or approximately 5 mg / day to approximately 50 mg / day) may be delivered. When the depot formulation is used for injection once a month or once every two weeks, the dose may be approximately 0.1 mg / day to approximately 100 mg / day, or approximately 3 mg to approximately 3000 mg per month.

[0498] The compound can also be administered sublingually. When administered sublingually, the compound should be given 1 to 4 times daily at the above dose for IM administration.

[0499] The compound can also be administered intranasally. When administered by this route, suitable dosage forms are nasal spray or dry powder. The dosage of the compound for intranasal administration is the same as the amount for IM administration. When administered by nasal aerosol or inhalation, these compositions may be prepared according to well-known techniques in the field of pharmaceutical formulation, and may be prepared as a solution in physiological saline using benzyl alcohol or other suitable preservatives, absorption enhancers to increase bioavailability, fluorocarbons, and / or other solubilizers or dispersants.

[0500] The compound can be administered intrathecally. When administered via this route, the appropriate dosage form may be parenteral. The dosage of the compound for intrathecal administration is the same as the amount for IM administration.

[0501] The compound can be administered topically. When administered this way, suitable dosage forms are creams, ointments, or patches. When administered topically, exemplary doses range from approximately 0.5 mg / day to approximately 200 mg / day. Since the amount that can be delivered with one patch is limited, two or more patches may be used.

[0502] The compounds can be administered rectally by suppositories. When administered by suppository, exemplary therapeutic doses may range from about 0.5 mg to about 500 mg. When administered rectally in suppository form, these compositions can be prepared by mixing the drug with suitable non-irritating excipients such as cocoa butter or synthetic glyceride esters of polyethylene glycol, which are solid at room temperature but liquefy and / or dissolve in the rectal lumen to release the drug.

[0503] It should be obvious to those skilled in the art that the precise dosage and frequency of administration depend on the specific compound being administered, the specific condition being treated, the severity of the condition being treated, the age, weight, overall physical condition of the particular subject, and any other medications the individual may be taking, as this is well known to the administering physician or other clinician who is proficient in treating retroviral infections, diseases, and related disorders. [Examples]

[0504] result Screening of specific compounds disclosed herein for Nef binding via surface plasmon resonance (SPR) demonstrated that the para positions of the two phenyl rings (indicated by arrows in Figure 1) allow for linker binding without substantially losing binding affinity. Data presented in the following sections demonstrate that a subset of CRBN-based Nef PROTACs induce Nef proteolysis via E3-mediated polyubiquitination and proteasome targeting (Figure 1). Targeted Nef degradation resulted in robust recovery of cell surface MHC-I and CD4 while suppressing HIV-1 replication in primary cells.

[0505] Evaluation of PROTAC induction of Nef ubiquitination using NanoBRET assay Candidate Nef-targeted PROTACs were first screened for induction of Nef ubiquitination in a cell-based NanoBRET assay (Promega; Figure 2A). This assay consists of two key components: 1) HIV-1 Nef (Nef-nLuc) fused to a small luciferase protein, nano-Luc, via its C-terminus; and 2) ubiquitin (Ub-Halo) fused to a self-labeled fluorescent protein known as Halo-Tag16. Halo-Tag is a modified bacterial haloalkane dehalogenase that forms a covalent adduct in the presence of a chloroalkane bound to a fluorescence-generating acceptor (in this case, NanoBRET 618). Nef-nLuc and Ub-Halo were co-expressed in 293T cells, followed by the addition of each PROTAC analog (final 10 μM or 0.1% DMSO as a plate control) and the Ub-Halo ligand. After 24 hours, nanoLuc substrates were added, and donor (nLuc; 480 nm) and acceptor (Halo tag; 618 nm) signals were recorded. The proximity of Nef-nLuc and Ub-Halo due to successful ubiquitination of Nef yielded a BRET signal. Each PROTAC was assayed in a quad-runcopied configuration, and the obtained data were corrected for background and expressed as the fluorescence ratio from 618 nm to 480 nm (BRET signal for Ub uptake normalized to Nef-nLuc levels). The BRET ratio was then normalized to a DMSO control well, and this was used to calculate the z-score based on the normalized ratio for all analogs tested (Figure 2B).

[0506] All PROTACs were assayed in quadruple sequences in a control assay using only Halo-Tag. These controls produced less than 10% of the BRET ratio observed in the presence of Ub-Halo, demonstrating the readout dependency on Nef ubiquitination. Overall, this analysis identified 11 PROTACs that increased Nef ubiquitination by at least 1.5 standard deviations above the mean (z-score > 1.5). These analogs were further subjected to orthogonal assays for induction of Nef degradation and inhibition of Nef function. Example 31 was also subjected to assays for its efficacy in the receptor rescue assay described below.

[0507] T cell-based assay for PROTAC-induced Nef degradation and cell surface receptor downregulation For these experiments, T cell line CEM-T4 was stably transduced with a doxycycline (Dox)-inducible expression vector of the Nef-GFP fusion protein. In the presence of Dox, Nef-GFP was expressed, resulting in downregulation of CD4 and MHC-I from the cell surface, which was quantified by flow cytometry (Figure 3A). CEM / Nef-GFP cells were incubated with each of the active protacs from the Nef-Ub NanoBRET screening at a final concentration of 3 μM, or with DMSO as a control, and Nef expression was induced by the addition of Dox. After 24 hours, treated cells were analyzed for the reversal of cell surface CD4 and MHC-I downregulation. Six of the 12 Nef protacs tested restored cell surface CD4 expression by more than 50%, while seven protacs similarly restored cell surface MHC-I by 50% or more (Figure 3B). No evidence of cytotoxicity was observed at the 3 μM screening concentration. These findings represent a substantial improvement over previous occupation-based Nef inhibitors that reversed Nef-dependent MHC-I downregulation in the range of 5–15% under similar conditions.

[0508] To determine whether the PROTAC-mediated rescue of cell surface receptor expression was due to the degradation of the Nef protein, we also calculated the levels of Nef-GFP in each treated cell population by flow cytometry. All seven PROTACs that restored cell surface receptor expression also induced a significant loss of Nef-GFP signaling, with percentage loss values ​​ranging from 15% to over 50% (Figure 4A). As an independent measure of PROTAC-mediated loss of Nef expression, we also performed quantitative immunoblotting analysis of PROTAC-treated CEM cell lysates using Nef-specific antibodies and actin as a normalization control (Figure 4B). This experiment confirmed PROTAC-dependent loss of Nef protein expression, with values ​​decreasing from 65% to over 95% compared to the DMSO-treated control (Figure 4C). These results provide independent evidence that PROTAC induces Nef degradation, thereby restoring cell surface expression of the receptor essential for immune system recognition in HIV-infected cells.

[0509] Evaluation of direct PROTAC binding to Nef and CRBN by SPR To confirm the interaction between each divalent PROTAC analog and both Nef and CRBN, the inventors used an SPR assay, an established method for small molecule-protein interactions. Eight recombinant Nef proteins representing multiple M-group HIV-1 variants, SIV Nef mac239, and the thalidomide-binding domain of CRBN were expressed in bacteria and purified to homogeneity. The recombinant proteins were immobilized on a carboxymethyl dextran hydrogel biosensor chip, and solubilized PROTACs were injected in triplicates over a concentration range. Following the dissociation phase, the resulting sensorgrams were fitted to a 1:1 Langmuir model, and the rate constants and relationships, and the KD value from KD = kd / ka, were calculated. Representative SPR sensorgrams for active Nef PROTAC Examples 2 and 14 are shown in Figure 6, illustrating the differences in binding kinetics. Both PROTACs bound to the CRBN thalidomide-binding domain to a similar degree, but Example 2 associated more slowly than Example 14 and exhibited a relatively slower dissociation phase. The opposite was true for HIV-1 Nef (NL4-3 variant), with Example 2 showing more rapid association and release compared to Example 14. These kinetic differences may partially reflect alternative linker binding points on the Nef-binding moiety. The shape of the PROTAC sensorgrams containing HIV-1 Nef closely resembles those previously reported for only the structurally relevant Nef-binding component. However, the degree of binding was higher at each PROTAC concentration, which may reflect the higher molecular weight of the PROTAC. Nevertheless, both compounds were highly effective in inducing Nef degradation and restoring CD4 and MHC-I expression to the T cell surface.

[0510] The eight active protacs bound to all Nef variants tested, as well as to the CBRN thalidomide-binding domain with affinities ranging from nM to low μM in almost all cases (Table 1 below), suggesting that Nef protacs based on substituted hydroxypyrazole targeting moieties are broadly active against pandemic-causing M-group HIV-1 variants. Control experiments showed that only Nef-targeting ligands did not bind to CRBN, and neither did thalidomide analogs directly bind to Nef.

[0511] [Table 1] Table 1. Direct binding of active Nef PROTACs to the thalidomide-binding domains of multiple HIV-1 Nef variants, SIV Nef, and cereblon (CRBN) by SPR. Nef proteins include well-characterized B subtype variants SF2 and NL4-3, as well as the C clade signaling molecule / founder (T / F) variant, C / z3618m, and SIV Nef mac239. D The value (M) is K D =k d / k a The relationship was used to calculate the association and dissociation rate constants from the given formula.

[0512] Activated Nef PROTAC stabilizes the ternary Nef-CRBN complex in vitro. Targeted proteolysis requires PROTAC-mediated formation of a ternary complex between a protein target, a divalent PROTAC ligand, and an E3 ligase. To model this mechanism in vitro, we incubated recombinant Nef (NL4-3 variant) and the CRBN thalidomide-binding domain in the presence and absence of an active Nef PROTAC analog (Example 2). In the absence of PROTAC, Nef and CRBN co-eluted from a size exclusion column (Figure 6A). Note that since the retention volumes of the individual proteins are the same, the mixture elutes as a single peak. When pre-incubated in the presence of PROTAC, a new peak with a smaller retention volume (higher molecular weight) was still observed, and the height of this peak increased proportionally to the concentration of PROTAC added to the mixture (Figure 6B). SDS-PAGE of the new peak revealed the presence of both Nef and CRBN. These results provide evidence that the active Nef PROTAC induces a ternary complex between Nef and CRBN, and are consistent with results from NanoBRET and CEM-T4 cells.

[0513] PROTAC inhibits Nef-mediated enhancement of HIV-1 replication in donor PBMCs. HIV-1 Nef is well known to enhance viral replication in donor PBMCs in vitro and is essential for high viral load in vivo. To determine whether antiretroviral activity is retained by Nef PROTAC, we tested the most active analog in HIV-infected PBMCs derived from normal donors. Consistent with previous studies, under conditions where the presence of Nef enhanced replication 3.3-fold, we compared PBMCs with wild-type and Nef-deficient HIV-1. NL4-3 It was infected (Figure 8A).

[0514] Cultures infected with wild-type HIV-1 were incubated with each PROTAC at a final concentration of 1 μM, and the degree of replication was assessed after 48 hours using the p24 Gag AlphaLISA assay. All eight PROTACs showed antiviral activity, and six of the eight analogs completely suppressed the Nef-dependent enhancement of viral replication (Figure 7A). To assess cytotoxicity, uninfected PBMCs were incubated with each Nef PROTAC at 1 μM for 48 hours, followed by viability assessment using the CellTiter-Blue assay. PBMCs treated with seven of the eight PROTACs showed viability of 90% or more compared to the DMSO control (Figure 7B). One exception was Example 20, which showed a 20% decrease in viability. This observation may explain why this analog reduced HIV-1 replication below the ΔNef level. Along with receptor rescue data, these results suggest that targeted degradation of Nef via selective PROTACs has the potential to prevent all Nef functions in HIV-infected cells.

[0515] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0516] Table 2. Binding of HIV-1 Nef PROTAC to cereblon (CRBN), the thalidomide-binding domain of Nef (B clade variants NL4-3 and SF2) and E3 ligase components. D Value. All K D The values ​​are derived from SPRs with Nef and CRBN proteins and their respective analogs immobilized on the biosensor as subjects. The rate constant is obtained from 1:1 Langmuir curve fitting, and K D=k d / k a This was used to generate the dissociation constant from the relationship. For analogs based on VHL E3 ligand, no values ​​are found in the CRBN column. Not all analogs were tested against Nef-SF2.

[0517] Considering the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are merely preferred examples of the invention and should not be construed as limiting the scope of the invention.

Claims

1. Compound of formula I: 【Chemistry 164】 [In the formula, the ligand that binds to the Nef protein (NB) is covalently bonded to the ligand that binds to the E3 ligase cereblon (CB) via a linker (L)], or its stereoisomers, isotopes, tautomers, or pharmaceutically acceptable salts.

2. The ligand CB is CB1 to CB22: 【Chemistry 165】 [In the formula, The aforementioned linker is 【Chemistry 166】 It is joined at the position marked with, A is C (=O) or CH ２ And, R ａ is H, halo, cyano, lower alkyl, lower haloalkyl, lower alkoxy or lower haloalkoxy, R ｂ The compound according to claim 1, wherein is selected from H or a lower alkyl group.

3. The linker L is connected such that no O-O or N-O bonds are formed, CH ２ , C≡C, C(=O), NH, NMe, O, S, S(=O), SO ２ The compound according to claim 1 or 2, comprising 0 to 30 divalent moieties selected from piperazinyl, piperidinyl, pyrrolidinyl, and azetidinyl.

4. The ligand for Nef is formula NB: 【Chemistry 167】 compounds [In the formula, The linker is bonded to any position having a hydrogen atom, G is CH or N, R is phenyl, pyridinyl, piperidinyl, pyrrolidinyl, or azetidinyl, and is optionally substituted with 0 to 3 groups independently selected from Y. X, Y, and Z are each independently a halo, cyano, lower alkyl, lower haloalkyl, lower alkoxy, lower haloalkoxy, lower alkylcarbonyl, lower alkoxycarbonyl, or lower alkylsulfonyl. m and n are independently 0, 1, 2, or 3. The compound according to any one of claims 1 to 3, wherein o is 1 or 2.

5. In CB1, CB2, CB3 and CB5-CB22, R ａ The compound according to any one of claims 2 to 4, wherein is the hydrogen atom of fluorine.

6. In CB5 to CB10 and CB17, R ｂ The compound according to any one of claims 2 to 4, wherein is hydrogen or methyl.

7. The compound according to any one of claims 2 to 4, wherein the ligand CB is CB1 or CB2.

8. In CB1 or CB2, A is C (=O) or CH ２ The compound according to claim 7.

9. where the linker is an amide of the formula -C(=O)NH-, an ethylene glycol moiety of the formula -OCH ２ CH ２ O-, the formula 【Chemical 168】 The piperazine portion of, or formula 【Chemistry 169】 A compound according to any one of claims 1 to 8, comprising the piperidine portion.

10. The linker is a formula included in the linker 【Chemistry 170】 It is bound to CB1-CB22 by the piperazine portion, or The linker is a formula included in the linker 【Chemistry 171】 It is bound to CB1-CB22 by the piperidine moiety, or The linker is bonded to CB1 to CB22 by the alkyne portion of formula -C≡C- contained in the linker, or The linker is bonded to CB1 to CB22 by ether oxygen, or The linker is -CH ２ -Bonded to CB1-CB22 by a group, or The linker is bonded to CB1 to CB22 by an -NH- group, or The linker is -NHCH ２ CH ２ It is either bound to CB1 by the NH- moiety, or The linker is -NMeCH ２ CH ２ It is either bound to CB1 by the NH- moiety, or The linker is bonded to CB1 by the -OCH2C(=O)NH- portion, or The compound according to any one of claims 2 to 8, wherein the linker is bonded to CB2 by a 1,4-piperazinyl moiety.

11. The linker is -CH ２ The compound according to any one of claims 2 to 8 or 10, wherein the linker is bonded to NB by a -C(=O)- moiety, or the linker is bonded to NB by an -O- atom.

12. The linker L is defined by formula II, formula III, or formula IV: 【Chemistry 172】 [In the formula, L Ｃ CH is linked in such a way that no O-O or N-O bonds are formed. ２ , C≡C, C(=O), NH, NMe, O, S, S(=O), SO ２ , comprising 0 to 10 divalent moieties selected from piperazinil, piperidinil, pyrrolidinil and azetidinil, L Ｎ CH is linked in such a way that no O-O or N-O bonds are formed. ２ , C≡C, C(=O), NH, NMe, O, S, S(=O), SO ２ , comprising 0 to 10 divalent moieties selected from piperazinil, piperidinil, pyrrolidinil and azetidinil, L Ｃ It is bonded to the cereblon bond site, L Ｎ It is connected to the Nef junction, or L Ｃ CH is linked in such a way that no O-O or N-O bonds are formed. ２ , NH, NMe, O, S, SO ２ , comprising 0 to 10 divalent moieties selected from piperazinil, piperidinil, pyrrolidinil and azetidinil, L Ｎ CH is linked in such a way that no O-O or N-O bonds are formed. ２ , NH, NMe, O, S, SO ２ , comprising 0 to 10 divalent moieties selected from piperazinil, piperidinil, pyrrolidinil and azetidinil, L Ｃ It is bonded to the cereblon bond site, L Ｎ The compound according to claim 1 or 2, wherein [is bonded to the Nef bond portion].

13. The aforementioned linkers are L1 to L26: 【Chemistry 173】 [In the formula, Any NH can be substituted with methyl to produce NMe, provided that p and p' are independently between 0 and 7, p'' is between 1 and 7, p+p' is < 10, and p+p'' is < 10. A compound selected from any one of claims 1 to 8.

14. The aforementioned linker, 【Chemistry 174】 A compound selected from any one of claims 1 to 8.

15. The compound according to any one of claims 4 to 14, wherein R is phenyl or 4-piperidinyl.

16. The compound according to any one of claims 4 to 15, wherein X is hydrogen, fluorine, or chlorine.

17. The compound according to any one of claims 4 to 16, wherein Z is F or absent.

18. Y is CF ３ The compound according to any one of claims 4 to 17, which is either present or absent.

19. The compound according to any one of claims 4 to 18, wherein the linker is bonded to a phenyl ring having substituent Z.

20. The compound according to any one of claims 4 to 18, wherein the linker is bonded to the para position of a phenyl ring having substituent Z.

21. The compound according to any one of claims 4 to 18, wherein the linker is bonded to the R.

22. The compound according to any one of claims 4 to 18, wherein R is phenyl and the linker is bonded at the para position.

23. The compound according to any one of claims 4 to 18, wherein R is 4-piperidinyl and the linker is bonded to a piperidine nitrogen atom.

24. NB is NB1, NB2, or NB3: 【Chemistry 175】 [In the formula, The aforementioned linker is 【Chemistry 176】 It is joined at the position marked with, X is selected from H or halogen, Y is selected from halo, lower alkylsulfonyl, and lower haloalkyl. Y' is a lower alkoxycarbonyl, The compound according to any one of claims 4 to 14, wherein Z is fluorine.

25. The compound according to claim 24, wherein X in NB1 to NB3 and NB5 is chlorine.

26. The compound according to claim 24 or 25, wherein NB1 is bonded to CB1 via linker L, or NB1 is bonded to CB2 via linker L.

27. The compound according to claim 24 or 25, wherein NB2 is bonded to CB1 via linker L, or NB2 is bonded to CB2 via linker L.

28. The compound according to claim 24 or 25, wherein NB3 is bonded to CB1 via linker L, or NB3 is bonded to CB2 via linker L.

29. The compound of formula I has structure Ia: 【Chemistry 177】 [In the formula, A is C (= O) or CH ２ And R ｃ is H or Me, and R ｄ is H or Me, r is 1, 2 or 3, L Ｎ CH is linked in such a way that no O-O or N-O bonds are formed. ２ , C≡C, C(=O), NH, NMe, O, S, S(=O), SO ２ The compound according to claim 1, having [0 to 6 divalent moieties selected from piperazinyl, piperidinyl, pyrrolidinyl, and azetidinyl].

30. The compound of formula I has structure Ib: 【Chemistry 178】 [In the formula, is C (= O) or CH] ２ And R ｃ is H or Me, and R ｄ is H or Me, r is 1, 2 or 3, L Ｎ CH is linked in such a way that no O-O or N-O bonds are formed. ２ , C≡C, C(=O), NH, NMe, O, S, S(=O), SO ２ The compound according to claim 1, comprising 4 to 16 divalent moieties selected from piperazinyl, piperidinyl, pyrrolidinyl, and azetidinyl.

31. The compound of formula I has structure Ic: 【Chemistry 179】 [In the formula, is C (= O) or CH] ２ And L Ｎ CH is linked in such a way that no O-O or N-O bonds are formed. ２ , C≡C, C(=O), NH, NMe, O, S, S(=O), SO ２ The compound according to claim 1, comprising 4 to 16 divalent moieties selected from piperazinyl, piperidinyl, pyrrolidinyl, and azetidinyl.

32. The compound of formula I has structure Id: 【Chemistry 180】 [In the formula, A is C (= O) or CH ２ And L Ｃ CH is linked in such a way that no O-O or N-O bonds are formed. ２ , C≡C, C(=O), NH, NMe, O, S, S(=O), SO ２ The compound according to claim 1, having [0 to 6 divalent moieties selected from piperazinyl, piperidinyl, pyrrolidinyl, and azetidinyl].

33. The aforementioned compound, Table 5-1 Table 5-2 Table 5-3 Table 5-4 A compound according to claim 1, selected from the following.

34. A pharmaceutical composition comprising at least one compound according to any one of claims 1 to 33 and at least one pharmaceutically acceptable excipient.

35. A pharmaceutical composition comprising at least one compound according to any one of claims 1 to 33 and at least one antiretroviral agent.

36. The pharmaceutical composition according to claim 35, wherein the at least one antiretroviral agent is selected from an entry inhibitor, a CCR5 receptor antagonist, a nucleoside reverse transcriptase inhibitor, a non-nucleoside reverse transcriptase inhibitor, a protease inhibitor, an integrase inhibitor, a maturation inhibitor, or a combination thereof.

37. The pharmaceutical composition according to claim 35, wherein the at least one antiretroviral agent is selected from maraviroc, enfuvirtide, apraviroc, bicriviroc, zidovudine, didanosine, zalcitabine, stabudine, lamivudine, abacavir, emtricitabine, entecavir, apricitabine, tenofovir, adefovir, efavirenz, nevirapine, delavirdin, etravirine, rilpivirine, saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, lopinavir, atazanavir, fosamprenavir, tipranavir, darunavir, MK-2048, elvitegravir, bevirimat, MPC-9055, renacapavir, cabotegravir, fostemsavir, or a combination thereof.

38. A method comprising administering an effective amount of at least one compound selected from any one of claims 1 to 33 to a subject who has, is suspected of having, or is at risk of developing HIV.

39. A method comprising administering an effective amount of the pharmaceutical composition according to any one of claims 34 to 37 to a subject who has, is suspected of having, or is at risk of developing HIV.

40. A method for treating an HIV-related condition in a subject, comprising administering an effective amount of at least one compound selected from any one of claims 1 to 33 to the subject in need of treatment for an HIV-related condition.

41. The method according to claim 40, wherein the HIV-related condition is selected from HIV replication, HIV-related CD4+ T cell loss and immunodeficiency, HIV-induced infection, Kaposi's sarcoma, HIV-related nephropathy, AIDS-dementia complex, or a combination thereof.

42. The method according to claim 40 or 41, wherein the subject is administered the compound prophylactically after exposure.

43. The method according to any one of claims 40 to 42, further comprising co-administering at least one antiretroviral agent to the subject.

44. A method for inhibiting the biological function of Nef, comprising contacting Nef with an effective amount of at least one compound described in any one of claims 1 to 33.

45. The method according to claim 44, wherein the biological function of the Nef is selected from HIV infectivity, HIV replication, MHC-I downregulation, or AIDS progression.

46. A method for inhibiting the activity of a Nef-dependent kinase, comprising contacting the Nef-dependent kinase with an effective amount of at least one compound described in any one of claims 1 to 33.

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