Identification of Proteolysis Targeting Chimeras (PROTACs) for Lysine Demethylase 5 and Their Neurite Outgrowth-Promoting Activity

Abstract

Lysine demethylase 5 (KDM5) proteins are involved in various neurological disorders, including Alzheimer’s disease, and KDM5 inhibition is expected to be a therapeutic strategy for these diseases. However, the pharmacological effects of conventional KDM5 inhibitors are insufficient, as they only target the catalytic functionality of KDM5. To identify compounds that exhibit more potent pharmacological activity, we focused on proteolysis targeting chimeras (PROTACs), which degrade target proteins and thus inhibit their entire functionality. We designed and synthesized novel KDM5 PROTAC candidates based on previously identified KDM5 inhibitors. The results of cellular assays revealed that two compounds, 20b and 23b, exhibited significant neurite outgrowth-promoting activity through the degradation of KDM5A in neuroblastoma neuro 2a cells. These results suggest that KDM5 PROTACs are promising drug candidates for the treatment of neurological disorders.

Introduction

Epigenetic modifications, such as methylation and acetylation of histone proteins, control gene expression to regulate various life processes.^1,2) Abnormalities in these modifications can lead to cancer, neurological disorders, and other diseases.^3–5) To counter these abnormalities and the diseases they cause, epigenetic modulation by small molecules is emerging as a promising therapeutic strategy.

Lysine demethylase 5 (KDM5) family proteins (KDM5A–D), which are Fe(II)/α-ketoglutarate-dependent oxidases, oxidatively remove methyl groups from tri- or di-methyl lysine 4 of histone H3 (H3K4me3/2).^6–8) In addition to their enzymatic activity, KDM5s work as scaffolding proteins, recruiting several repressive transcriptional factors including histone deacetylase 1 (HDAC1) and HDAC2.^9–12) In other words, they epigenetically regulate gene expression in cooperation with other proteins rather than independently. Furthermore, KDM5s have been implicated in the development of various forms of cancer, drug resistance, and neurodegenerative disorders such as Alzheimer’s disease.^13–18) Therefore, KDM5 inhibition is regarded as a potential therapeutic strategy for these diseases.

Several KDM5 inhibitors have been identified to date^19–28) (Supplementary Fig. S1). However, these inhibitors have failed to produce any significant pharmacological effects, such as anti-cancer effects, in cells or animal studies. In this context, we decided to explore another strategy for KDM5 inhibition, namely target protein degradation (TPD) utilizing proteolysis targeting chimeras (PROTACs), an approach that has been actively studied for the last decade.^29–35) PROTACs are small molecules composed of two linked ligands: the first ligand binds to a protein of interest (POI), and the second ligand binds to ubiquitin E3 ligase. Essentially, these molecules hijack the ubiquitin-proteasome system to target the POI for degradation. The most unique feature of this approach is that it reduces the cellular levels of the POI itself, unlike conventional drugs, such as enzyme inhibitors, which merely inhibit the catalytic function of the POI. Hence, PROTACs may be uniquely suited to inhibiting proteins containing both enzymatic activity and a scaffolding function, and the resulting degradation would eliminate all functional aspects of the protein at once.^36,37) Notably, we have previously shown that PROTACs targeting such multi-functional proteins inhibited cancer cell growth more strongly than conventional enzyme inhibitors.^38,39) Thus, the TPD approach is an attractive strategy for drug discovery targeting multi-functional proteins. Based on our previous studies of TPD, we embarked on developing KDM5 PROTACs for the treatment of neurodegenerative disorders. Herein, we describe in detail the design and synthesis of KDM5 PROTACs and their neurite outgrowth-promoting activity in neuroblastoma neuro 2a (N2a) cells.

Chemistry

Compounds 14, 20, and 23, which were tested in this study, were prepared using the synthetic routes shown in Charts 1–3. The preparation of 14 is illustrated in Chart 1. tert-Butoxycarbonyl (Boc) protection and the following N-methylation of amine 1 resulted in compound 3. Borylation and Boc deprotection of 3 produced amine 5. Amine 5 reacted with aldehydes 6⁴⁰⁾ in the presence of sodium triacetoxyborohydride to yield compounds 7. Suzuki-Miyaura cross-coupling of 7 with chloride 8²²⁾ provided compounds 9. Compounds 9 were hydrolyzed to generate acids 10. Mitsunobu reaction between compound 11 and 2-[2-(2-tert-butoxyaminoethoxy)ethoxy]ethanol yielded compound 12. Boc deprotection of 12 in the presence of trifluoroacetic acid followed by the salt metathesis reaction resulted in the formation of compound 13 as a hydrochloride salt. The condensation of 13 with 10, followed by treatment with an HCl solution, resulted in the hydrochloride salts of compounds 14.

Chart 1. Synthesis of Compounds 14

Reagents and conditions: (a) (Boc)₂O, Et₃N, CH₂Cl₂, 0 °C to room temperature, 78%; (b) NaH, MeI, dimethylformamide (DMF), 0 °C to room temperature, 99%; (c) bis(pinacolato)diboron, Pd(dppf)Cl₂, KOAc, dimethyl sulfoxide (DMSO), 80 °C, 82%; (d) trifluoroacetic acid (TFA), CH₂Cl₂, 0 °C to room temperature, 84%; (e) NaBH(OAc)₃, ClCH₂CH₂Cl, room temperature, 26% for 7a or 19% for 7b; (f) Pd(dppf)Cl₂, Na₂CO₃, 1,2-dimethoxyethane (DME), H₂O, 110 °C, 15% for 9a or 6% for 9b; (g) NaOH, tetrahydrofuran (THF), MeOH, H₂O, room temperature, 54% for 10a or 91% for 10b; (h) 2-[2-(2-tert-butoxyaminoethoxy)ethoxy]ethanol, di-2-methoxyethyl azodicarboxylate (DMEAD), PPh₃, THF, room temperature, 41%; (i) TFA, CH₂Cl₂, room temperature, then HCl, AcOEt, room temperature; (j) 1-((dimethylamino)(dimethyliminio)methyl)-1H-[1,2,3]triazolo[4,5-b]pyridine 3-oxide hexafluorophosphate (HATU), i-Pr₂NEt, dimethylformamide (DMF), room temperature; (k) HCl, AcOEt, room temperature, 15% of 14a or 16% of 14b.

Chart 2 shows the preparation of 20. Mizoroki–Heck reaction between compounds 15 and hex-5-en-1-ol yielded aldehydes 16 through double-bond rearrangement and isomerization of a generated vinyl alcohol.⁴¹⁾ Reductive amination of compounds 16 with amine 5, followed by Suzuki–Miyaura cross-coupling with compound 8 formed compounds 18. Compounds 18 were converted to acids 19 under basic conditions. Compounds 19 were allowed to react with compound 13 to generate compounds 20. Furthermore, compound 20b was treated with an HCl solution to produce hydrochloride salt.

Chart 2. Synthesis of Compounds 20

Reagents and conditions: (a) hex-5-en-1-ol, Pd(OAc)₂, LiOAc, LiCl, n-Bu₄NCl, DMF 70 °C, 90% for 16a or 90% for 16b; (b) 5, NaBH(OAc)₃, ClCH₂CH₂Cl, room temperature, 48% for 17a or 49% for 17b; (c) Pd(dppf)Cl₂, K₂CO₃, DME, H₂O, 110 °C, 15% for 18a or 34% for 18b; (d) NaOH, THF, MeOH, H₂O, room temperature, quant.; (e) HATU, i-Pr₂NEt, DMF, room temperature 21% for 20a; (f) HATU, i-Pr₂NEt, DMF, room temperature, then HCl, AcOEt, room temperature, 17% for 20b.

Chart 3 shows the preparation of 23. S_N2 reaction between compound 11 and tert-butyl (6-bromohexyl)carbamate yielded compound 21. Removal of the Boc group of 21 yielded compound 22. The target compounds 23 were prepared via condensation of 22 and compounds 19.

Chart 3. Synthesis of Compounds 23

Reagents and conditions: (a) tert-butyl (6-bromohexyl)carbamate, KHCO₃, KI, DMF, 80 °C, 79%; (b) TFA, CH₂Cl₂, room temperature; (c) HCl, AcOEt, room temperature; (d) HATU, i-Pr₂NEt, DMF, room temperature; (e) HCl, AcOEt, room temperature, 52% of 23a or 11% of 23b.

Results and Discussion

Before investigating KDM5 PROTACs, we tested compound 24²²⁾ (Fig. 1A) in a neurite outgrowth assay, which is often used for early screening in the drug discovery of neurodegenerative disorders,⁴²⁾ because compounds that inhibit KDM5A are suggested to be therapeutic candidates for neurodegenerative disorders, including Alzheimer’s disease.¹⁸⁾ As shown in Fig. 1B, treatment of mouse neuroblastoma N2a cells with 0.2 µM of 24 for 24 or 48 h incubation did not strongly induce neurite outgrowth. This indicates that conventional KDM5 inhibitors, which inhibit only the catalytic function of KDM5A, do not exhibit strong neurite outgrowth activity. Based on the assumption that the entire functionality of KDM5A, including the scaffold that interacts with HDAC1 and 2 to control histone deacetylation,⁹⁾ is important for neurite outgrowth, we examined the neurite outgrowth-promoting activity of the KDM5 inhibitor with the HDAC inhibitor vorinostat⁴³⁾ (25) (Fig. 1A). Notably, this combination significantly induced neurite outgrowth compared to 24 or 25 alone (Fig. 1B). This indicates that a two-pronged approach, inducing both histone methylation and acetylation through the inhibition of KDM5A and HDAC, respectively, is an effective means of promoting neurite outgrowth. Based on these results, we envisioned that KDM5A degradation by TPD would also strongly induce neurite outgrowth of N2a cells, as protein degradation would disrupt both its catalytic and scaffolding functions. This idea prompted us to prepare KDM5 PROTAC candidates that could show potent neurite outgrowth activity.

Fig. 1. (A) Structure of KDM5 Inhibitor 24 and Vorinostat (25)

(B) Effect of 24, 25, and cotreatment of 24 with 25 on N2a differentiation after 24 and 48 h treatment. Bars represent the mean values ± standard deviation (S.D.) from three independent experiments. The p-values were determined using Tukey’s multiple comparisons test; * p < 0.05. For representative images showing N2a cells treated with 24 or 25 and cotreated with 24 and 25, see Supplementary Fig. S2.

To design KDM5 PROTACs, we chose compound 24 as a KDM5 ligand.²²⁾ Our previous study on simulation of compound 24 bound to KDM5A suggested that the hexyl chain is directed towards the protein surface.²²⁾ Based on this simulation, we introduced a linker at the terminal of the hexyl group. As the linker structure often affects degradation activities, we tried a number of different linkers, such as amide-conjugated polyethylene glycol linkers (14) and benzene ring-containing linkers (20 and 23) (Fig. 2A). Additionally, we selected thalidomide (26) as an E3 ligand, which is the most widely utilized for PROTAC studies.

Fig. 2. (A) Design of KDM5 PROTACs

(B) Effect of 14, 20, and 23 at 0.2 µM on N2a differentiation after 48 h treatment. (C) Dose-dependent N2a cell-neurite outgrowth activities of 20b, 23b, and 26 after 48 h treatment. (D) Time-dependent N2a cell-neurite outgrowth activities of 20b and 23b at 0.02 µM. Bars represent the mean values ± S.D. from three independent experiments. For representative images showing N2a cells treated with the tested compounds, see Supplementary Figs. S3–S5.

Next, we prepared compounds 14, 20, and 23 as novel KDM5 PROTAC candidates (Fig. 2A) (Charts 1–3) and then screened the synthetic compounds in the neurite outgrowth activity assay. As shown in Fig. 2B, treatment of N2a cells with compounds 14, 20, and 23 promoted neurite outgrowth. Compound 14b was a stronger promoter of neurite outgrowth than compound 14a. This indicates that the introduction of a spacer between the hexyl group of the KDM5 inhibitor and the amide group of the linker is important for neurite outgrowth-promoting activity. Moreover, the replacement of the spacer with a benzene ring, which was expected to contribute to the rigidity of linker orientation, maintained or increased its effectiveness. Importantly, the activities of the meta-substituent compounds 20b and 23b were superior to those of the para-substituent compounds 20a and 23a. Furthermore, the activity of the carbon linker compound 23b was slightly better than that of the polyethylene linker compound 20b. The preliminary structure–activity relationship studies revealed that compounds with a linker containing a meta-substituent benzene ring showed potent neurite outgrowth-promoting activity. Altogether, compounds 20b and 23b exhibited remarkable neurite outgrowth-promoting activity.

We also tested compounds 20b and 23b at different concentrations and incubation times (Figs. 2C, D). Both compounds promoted neurite outgrowth in the range of 0.02–2 µM (Fig. 2C), and both increased the number of neurite cells in a time-dependent manner when applied at 0.02 µM (Fig. 2D). Additionally, we examined the effect of compound 26, an E3 ligand, on neurite outgrowth activity (Fig. 2C). Importantly, 26 did not show strong neurite outgrowth activity relative to compounds 20b and 23b. These findings suggested that KDM5 degradation via the PROTAC compounds resulted in strong neurite outgrowth.

Subsequently, we tested the KDM5A degradation activity of compounds 20b and 23b by means of Western blotting analysis to confirm that they work as KDM5 PROTACs. Treatment of N2a cells with 20b and 23b decreased KDM5A levels in a dose-dependent manner (Fig. 3A). The combination of compounds 24 and 26, which are parent compounds of 20b and 23b, did not reduce KDM5A levels (Fig. 3B). Additionally, the decrease in KDM5A levels by compounds 20b and 23b was disturbed by the proteasome inhibitor MG-132 (Figs. 3C, D). These results suggested that compounds 20b and 23b work as PROTACs and reduce KDM5A levels in N2a cells.

Fig. 3. Western Blot-Detection of KDM5A Levels in N2a Cells

(A) After 24 h treatment with 20b and 23b. (B) After 24 h treatment with 23b and co-treatment with 24 and 26. (C) Co-treatment of 20b with the proteasome inhibitor MG-132. (D) Co-treatment of 23b with the proteasome inhibitor MG-132.

Finally, we investigated the influence of PROTACs 20b and 23b on histone methylation and acetylation. Because H3K4me3 is a substrate of KDM5s, we analyzed the methylation levels of H3K4 in N2a cells treated with the KDM5 PROTACs. As shown in Fig. 4, treatment with 20b and 23b resulted in dose-dependent accumulation of H3K4me3 levels, suggesting that 20b and 23b inhibited the catalytic function of KDM5s in N2a cells. Thereafter, we tested if 20b and 23b affected the levels of acetylated lysine 27 in histone H3 (H3K27Ac), which is both a direct substrate of HDAC1 and 2 and an indirect substrate of KDM5s, through their interaction with HDAC1 and 2. As expected, 20b and 23b also upregulated H3K27Ac levels (Fig. 4). Incidentally, 20b and 23b strongly inhibited KDM5A without affecting HDAC1 in in vitro assays⁴⁴⁾ (Supplementary Table S1). These results indicate that the degradation of KDM5A by 20b and 23b inhibited both the catalytic function of KDM5s to regulate H3K4me3 and the scaffolding function of KDM5s to disturb the deacetylation activity of HDAC1 and 2 in N2a cells.

Fig. 4. Western Blot-Detection of H3K4me3 and H3K27Ac Levels in N2a Cells after 24 h Treatment with Compounds 20b and 23b

Conclusion

In this study, we found that conventional KDM5 inhibitor 24 did not result in strong neurite outgrowth activity of N2a cells, although it has been reported that the inhibition of KDM5s might be effective for neurodegenerative disorders. Meanwhile, the combination of 24 with the HDAC inhibitor vorinostat (25) significantly induced neurite outgrowth of N2a cells, implying that inhibiting both the catalytic activity of KDM5 and its scaffolding connection to HDACs affects neurite outgrowth. In this context, we focused on KDM5 PROTACs that can inhibit both these functions, and we prepared compounds 14, 20, and 23 as KDM5 PROTAC candidates. Among them, compounds 20b and 23b promoted significant neurite outgrowth of N2a cells. Further biological assays suggested that treatment of N2a cells with 20b and 23b degraded KDM5A through PROTAC-mediated proteasomal degradation. Because this degradation affected the entirety of KDM5 functionality, as opposed to traditional KDM5 inhibitors, we believe that KDM5 PROTACs are promising drugs for the treatment of neurological disorders.

Experimental

Chemistry

The chemical reagents and solvents used in this study were commercial products of the highest available purity. Reagents and solvents were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.), FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan), TCI Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan), Nacalai Tesque, Inc. (Kyoto, Japan), and Kanto Chemical Co., Inc. (Tokyo, Japan), and used without purification. All air- and moisture-sensitive reactions were performed under an argon (Ar) atmosphere in dried glassware. NMR spectra were recorded on a JEOL (Tokyo, Japan) ECS400 spectrometer operating at 400 MHz (1H), and Bruker (Billerica, MA, U.S.A.) AVANCE III 700 spectrometer operating at 700 MHz (¹H) or 175 MHz (¹³C). ¹H-NMR and ¹³C-NMR chemical shift values are reported as δ (ppm) relative to the solvent peak or tetramethylsilane (TMS) (DMSO-d₆: ¹H-NMR 2.50, ¹³C-NMR 39.52), and coupling constants are given in Hz. The purity of all tested compounds was >95% as determined via HPLC using a Shimadzu (Kyoto, Japan) UFLC (SPD-M20A UV detector, DGU-20A3R degassing unit, LC-20AD solvent delivery unit, and CBM-20A system) and COSMOSIL packed column (5C18-AR-II, 4.6 ID × 150 mm, Nacalai Tesque, Inc.) at a flow rate of 1 mL/min, with UV detection (λ = 254 nm). HPLC conditions: eluent A: H₂O containing 0.1% TFA; eluent B: acetonitrile containing 0.1% TFA. Gradient: B: 0 to 20 min, 10–90%; 20 to 30 min, 90%; 30 to 40 min, 90–10%. Positive/negative LRMS ion mass spectra were recorded on Bruker HCT-Plus. High-resolution mass spectra (HRMS) were recorded on a LTQ Orbitrap XL (THERMO, Waltham, MA, U.S.A.) or Shimadzu LCMS-IT-TOF mass spectrometer.

Synthesis of 7-{[4-(3-Cyano-6-isopropyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidin-5-yl)phenethyl](methyl)amino}heptanoic Acid (10a)

Step 1: Preparation of tert-butyl (4-bromophenethyl)carbamate (2)

Boc₂O (5.72 g, 26.2 mmol) was added to a solution of 2-(4-bromophenyl)ethylamine (1) (5.00 g, 25.0 mmol) in CH₂Cl₂ (50 mL) and triethylamine (4.20 mL, 30.1 mmol) with cooling in an ice bath. The resulting mixture was stirred at room temperature for 5 h. Thereafter, the reaction was quenched by 10% citric acid and extracted with AcOEt. The organic layer was separated, washed with brine, and dried over MgSO₄. After filtration, concentration under reduced pressure, and purification via flash column chromatography (silica gel, n-hexane/AcOEt = 9/1 to 4/1), 2 was obtained as a colorless solid (5.83 g, 78%); ¹H-NMR (DMSO-d₆, 400 MHz, δ ppm) 7.46 (2H, d, J = 8.3 Hz), 7.15 (2H, d, J = 8.3 Hz), 6.86 (1H, t, J = 5.2 Hz), 3.12 (2H, td, J = 6.8, 6.8 Hz), 2.66 (2H, t, J = 7.0 Hz), 1.35 (9H, s).

Step 2: tert-Butyl (4-bromophenethyl)(methyl)carbamate (3)

A solution of 2 (5.83 g, 19.4 mmol) in DMF (50 mL) was added to a suspension of 60% NaH in oil (0.820 g, 20.5 mmol) at 0 °C. After 15 min, MeI (1.40 mL, 22.5 mmol) was added to the mixture in a dropwise fashion, and the resulting mixture was stirred at 0 °C for 5 h. The reaction mixture was then poured into water and extracted with AcOEt. The organic layer was washed with brine and dried over MgSO₄. After filtration, concentration under reduced pressure, and purification via flash column chromatography (silica gel, n-hexane/AcOEt = 9/1), 3 was obtained as a colorless oil (6.08 g, 99%); ¹H-NMR (DMSO-d₆, 400 MHz, δ ppm) 7.47 (2H, d, J = 8.1 Hz), 7.15 (2H, d, J = 8.1 Hz), 3.36 (2H, t, J = 6.5 Hz), 3.32 (3H, s), 2.72 (2H, t, J = 6.8 Hz), 1.25 (9H, s).

Step 3: Preparation of tert-butyl methyl[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenethyl]carbamate (4)

A solution of 3 (6.08 g, 19.3 mmol), bis(pinacolato)diboron (5.70 g, 22.4 mmol), KOAc (7.30 g, 74.4 mmol), and Pd(dppf)Cl₂ (670 mg, 0.916 mmol) in DMSO (60 mL) was heated at 80 °C for 7 h. After the reaction mixture was filtered, the filtrate was extracted with AcOEt and washed with brine. The organic layer was separated and dried over MgSO₄. After filtration, concentration under reduced pressure, and purification via flash column chromatography (silica gel, n-hexane/AcOEt = 4/1), 4 was obtained as a colorless solid (5.76 g, 82%); ¹H-NMR (DMSO-d₆, 400 MHz, δ ppm), 7.59 (2H, d, J = 7.9 Hz), 7.21 (2H, d, J = 7.4 Hz), 3.36 (2H, t, J = 7.2 Hz), 2.76 (2H, t, J = 7.2 Hz), 2.73 (3H, s), 1.36 (9H, s), 1.28 (12H, s).

Step 4: Preparation of N-methyl-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]ethan-1-amine (5)

Trifluoroacetic acid (TFA) (16 mL) was added to a solution of 4 (5.76 g, 15.9 mmol) in CH₂Cl₂ (35 mL), which was cooled in an ice-bath. The resulting mixture was stirred at 0 °C for 2 h. The solvent was removed under reduced pressure, and saturated NaHCO₃ and AcOEt were added to the resulting residue and separated. The organic layer was washed with brine, dried over MgSO₄, filtered, and the solvent removed under reduced pressure. The residue was purified via flash chromatography (silica gel, n-hexane/AcOEt = 4/1) to give 5 as a colorless solid (3.50 g, 84%); ¹H-NMR (DMSO-d₆, 400 MHz, δ ppm), 8.45 (1H, br s), 7.65 (2H, d, J = 8.1 Hz), 7.28 (2H, d, J = 8.3 Hz), 3.15 (2H, t, J = 9.2 Hz), 2.94–2.89 (2H, m), 2.59 (3H, s), 1.28 (12H, s).

Step 5: Preparation of methyl 7-{methyl[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenethyl]amino}heptanoate (7a)

A solution of methyl 7-oxoheptanoate (6a, 401 mg, 2.53 mmol), 5 (632 mg, 2.42 mmol), and NaBH(OAc)₃ (820 mg, 3.87 mmol) in ClCH₂CH₂Cl (10 mL) was stirred at room temperature overnight. The reaction was quenched with water and extracted with AcOEt. The organic layer was separated, washed with brine, and dried over MgSO₄. After filtration, concentration under reduced pressure, and purification via flash column chromatography (silica gel, CHCl₃/AcOEt = 4/1 to CHCl₃/MeOH = 9/1), 7a was obtained as a colorless oil (253 mg, 26%); ¹H-NMR (DMSO-d₆, 400 MHz, δ ppm), 7.57 (2H, d, J = 8.8 Hz), 7.22 (2H, d, J = 8.8 Hz), 3.57 (3H, s), 2.74–2.64 (2H, m), 2.35–2.23 (4H, m), 2.17 (3H, br s), 1.55–1.43 (2H, m), 1.41–1.32 (2H, m), 1.28 (12H, s), 1.25–1.14 (6H, m).

Step 6: Preparation of methyl 7-{[4-(3-cyano-6-isopropyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidin-5-yl)phenethyl](methyl)amino}heptanoate (9a)

A solution of 7a (147 mg, 0.364 mmol), 8²²⁾ (78.3 mg, 0.331 mmol), Na₂CO₃ (70.2 mg, 0.662 mmol), and Pd(dppf)Cl₂ (24.2 mg, 0.0331 mmol) in DME/H₂O (1.5 mL/0.5 mL) was heated at 110 °C for 3 h. After the reaction mixture was filtered, the filtrate was extracted with AcOEt and washed with brine. The organic layer was separated and dried over MgSO₄. After filtration, concentration under reduced pressure, and purification via flash column chromatography (silica gel, CHCl₃/AcOEt = 4/1 to CHCl₃/MeOH = 9/1), 9a was obtained as a colorless solid (23.5 mg, 15%); ¹H-NMR (DMSO-d₆, 400 MHz, δ ppm), 8.03 (1H, br s), 7.39 (2H, d, J = 7.9 Hz), 7.32 (2H, d, J = 7.9 Hz), 3.57 (3H, s), 2.88–2.76 (4H, m), 2.60–2.53 (3H, m), 2.38 (3H, s), 2.28 (2H, t, J = 7.2 Hz), 1.54–1.42 (4H, m), 1.30–1.23 (4H, m), 1.18 (6H, d, J = 8.2 Hz); MS (ESI) m/z 478 (MH⁺).

Step 7: Preparation of 7-{[4-(3-cyano-6-isopropyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidin-5-yl)phenethyl](methyl)amino}heptanoic acid (10a)

An aqueous solution of NaOH (4N, 0.200 mL, 0.791 mmol) was added to a solution of 9a (126 mg, 0.264 mmol) in MeOH (1.2 mL), and the mixture was stirred at room temperature overnight. 10% aqueous citric acid was added portion-wise, and the resulting mixture was stirred at room temperature. The insoluble material was collected via filtration and washed with water to yield 10a as a colorless solid (66.0 mg, 54%); ¹H-NMR (DMSO-d₆, 400 MHz, δ ppm), 11.9 (1H, br s), 9.24 (1H, br s), 8.02 (1H, s), 7.33 (4H, d, J = 9.6 Hz), 3.11–2.92 (2H, m), 2.90–2.78 (2H, m), 2.75–2.68 (1H, m), 2.52–2.50 (2H, m), 2.51 (3H, s), 2.21 (2H, t, J = 7.3 Hz), 1.68–1.60 (2H, m), 1.54–1.48 (2H, m), 1.36–1.27 (4H, m), 1.23 (6H, d, J = 6.9 Hz); MS (ESI) m/z 464 (MH⁺).

Synthesis of 7-{[4-(3-Cyano-6-isopropyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidin-5-yl)phenethyl](methyl)amino}-N-{2-[2-(2-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl]oxy}ethoxy)ethoxy]ethyl}heptanamide Hydrochloride (14a·HCl)

Step 1: Preparation of {2-[2-(2-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl]oxy}ethoxy)ethoxy]ethyl}carbamate (12)

2-[2-(2-tert-Butoxyaminoethoxy)ethoxy]ethanol (300 mg, 1.20 mmol), DMEAD (383 mg, 1.64 mmol) were added to a solution of 2-(2,6-dioxopiperidin-3-yl)-4-hydroxyisoindoline-1,3-dione (11, 300 mg, 1.09 mmol), and PPh₃ (430 mg, 1.64 mmol) in THF (3 mL) at room temperature. The reaction mixture was stirred at room temperature overnight. The reaction was quenched by water and extracted with AcOEt. The organic layer was separated and dried over MgSO₄. After filtration, concentration under reduced pressure, and purification via flash column chromatography (silica gel, CHCl₃/AcOEt = 4/1 to 1/1), 12 was obtained as a colorless oil (226 mg, 41%); ¹H-NMR (DMSO-d₆, 400 MHz, δ ppm), 11.10 (1H, brs), 7.85–7.78 (1H, m), 7.54 (1H, d, J = 7.8 Hz), 7.46 (1H, d, J = 7.4 Hz), 6.74 (1H, brs), 5.08 (1H, dd, J = 12.8, 5.4 Hz), 4.35 (2H, t, J = 4.5 Hz), 3.80 (2H, t, J = 4.5 Hz), 3.64 (2H, t, J = 4.7 Hz), 3.53–3.47 (2H, m), 3.40–3.36 (2H, m), 3.08–3.01 (2H, m), 2.94–2.84 (1H, m), 2.62–2.51 (2H, m), 2.05–2.01 (1H, m), 1.36 (9H, s).

Step 2: Preparation of 4-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione Hydrochloride (13·HCl)

Trifluoroacetic acid (TFA) (0.34 mL) was added to a solution of 12 (226 mg, 0.448 mmol) in CH₂Cl₂ (2 mL), with cooling in an ice bath. After being stirred at 0 °C for 3 h, the reaction mixture was concentrated under reduced pressure. 4N HCl in AcOEt was added to the residue, and the solvent was removed under reduced pressure to yield 13·HCl (239 mg) as a crude product, which was used in the next reaction without further purification.

Step 3: Preparation of 7-{[4-(3-cyano-6-isopropyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidin-5-yl)phenethyl](methyl)amino}-N-{2-[2-(2-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl]oxy}ethoxy)ethoxy]ethyl}heptanamide (14a)

HATU (20.0 mg, 0.0526 mmol) was added to a solution of 10a (19.9 mg, 0.0429 mmol), i-Pr₂NEt (0.0180 mL, 0.104 mmol), and crude 13·HCl (19.0 mg) in DMF (0.2 mL) at room temperature. The reaction mixture was stirred at room temperature overnight. Water was added to this reaction, followed by extraction with AcOEt twice. The combined organic layer was washed with brine, dried over MgSO₄, filtered, and concentrated. The filtrate was concentrated and purified via reverse phase flash chromatography (MeCN/0.1%TFA) to obtain 14a, which was converted to a hydrochloride salt in the next step.

Step 4: Preparation of 7-{[4-(3-cyano-6-isopropyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidin-5-yl)phenethyl](methyl)amino}-N-{2-[2-(2-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl]oxy}ethoxy)ethoxy]ethyl}heptanamide hydrochloride (14a·HCl)

14a was treated with 4N HCl in AcOEt, and the solvent was removed under reduced pressure to yield 14a·HCl (5.8 mg, 15% from 10a) as a colorless solid; ¹H-NMR (DMSO-d₆, 700 MHz, δ ppm), δ 13.44 (1H, s), 11.13 (1H, s), 8.41 (1H, s), 7.88 (1H, br s), 7.81 (1H, t, J = 8.4 Hz), 7.54 (1H, d, J = 8.6 Hz), 7.50–7.48 (4H, m), 7.47 (1H, d, J = 7.0 Hz), 5.09 (1H, dd, J = 12.9, 5.6 Hz), 4.34 (2H, t, J = 4.3 Hz), 3.81–3.80 (2H, m), 3.66–3.64 (2H, m), 3.53–3.52 (2H, m), 3.41–3.39 (2H, m), 3.32–3.25 (1H, m), 3.19–2.99 (6H, m), 2.91–2.80 (1H, m), 2.82 (3H, s), 2.60–2.58 (2H, m), 2.55–2.49 (2H, m), 2.08–2.06 (2H, m), 2.04–2.00 (1H, m), 1.71–1.63 (2H, m), 1.51–1.46 (2H, m), 1.31–1.25 (4H, m), 1.23 (6H, d, J = 7.0 Hz, 6H); ¹³C-NMR (DMSO-d₆, 175 MHz, δ ppm), 172.22, 171.48, 169.37, 166.20, 164.69, 155.18, 154.40, 147.27, 144.73, 142.85, 138.43, 136.42, 132.62, 130.91, 128.34, 128.25, 119.38, 115.65, 114.81, 113.38, 112.16, 73.37, 69.48, 68.99, 68.56, 68.23, 68.06, 54.83, 54.27, 48.11, 37.80, 34.47, 30.33, 28.60, 27.75, 27.53, 25.20, 24.35, 22.47, 21.37, 20.47, 19.34; HRMS Calcd for C₄₅H₅₅N₈O₉⁺ 851.4092, Found 851.4077; HPLC t_R 12.27 min, 97.84% purity.

Synthesis of 10-{[4-(3-Cyano-6-isopropyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidin-5-yl)phenethyl](methyl)amino}-N-{2-[2-(2-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl]oxy}ethoxy)ethoxy]ethyl}decanamide Hydrochloride (14b·HCl)

14b·HCl was prepared using procedures similar to that described for the synthesis of 10a and 14a·HCl; yellow solid (5.9 mg, 16% from 10b); ¹H-NMR (DMSO-d₆, 700 MHz, δ ppm), 13.43 (1H, s), 11.14 (1H, s), 8.42 (1H, s), 7.84–7.81 (2H, m), 7.54 (1H, d, J = 8.6 Hz), 7.50 (4H, s), 7.47 (1H, d, J = 7.3 Hz), 5.10 (1H, dd, J = 12.9, 5.2 Hz), 4.35 (2H, t, J = 4.3 Hz), 3.81 (2H, t, J = 4.5 Hz), 3.65 (2H, t, J = 4.7 Hz), 3.53 (2H, t, J = 4.7 Hz), 3.50–3.35 (2H, m), 3.32–3.29 (1H, m), 3.22–3.04 (6H, m), 2.94–2.86 (1H, m), 2.85 (3H, s), 2.61–2.57 (2H, m), 2.55–2.48 (2H, m), 2.06–2.02 (1H, m), 2.04 (2H, t, J = 7.0 Hz), 1.72–1.60 (2H, m), 1.48–1.44 (2H, m), 1.29–1.23 (10H, m), 1.23 (6H, d, J = 7.0 Hz); ¹³C-NMR (DMSO-d₆, 175 MHz, δ ppm), 172.21, 171.54, 169.35, 166.19, 164.67, 157.35, 157.16, 155.18, 154.39, 147.23, 144.72, 142.84, 138.27, 136.41, 132.62, 130.94, 128.34, 128.25, 119.36, 115.64, 114.80, 113.36, 112.16, 73.36, 69.47, 68.99, 68.56, 68.22, 68.06, 54.87, 54.42, 52.88, 48.10, 41.15, 37.79, 34.63, 30.32, 28.64, 28.11, 28.07, 28.02, 27.88, 27.75, 25.35, 24.60, 22.67, 21.36, 20.45, 19.34, 17.43, 16.09, 11.84; HRMS Calcd for C₄₈H₆₁N₈O₉⁺ 893.4562, Found 893.4545; HPLC t_R 13.26 min, 95.59% purity.

Synthesis of 4-(6-{[4-(3-Cyano-6-isopropyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidin-5-yl)phenethyl](methyl)amino}hexyl)-N-{2-[2-(2-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl]oxy}ethoxy)ethoxy]ethyl}benzamide (20a)

Step 1: Preparation of ethyl 4-(6-oxohexyl)benzoate (16a)

A solution of 15a (2.42 g, 8.77 mmol), hex-5-en-1-ol (1.05 g, 10.5 mmol), LiOAc (1.44 g, 21.8 mmol), LiCl (371 mg, 8.75 mmol), tetrabutylammonium chloride (1.22 g, 4.39 mmol), and Pd(OAc)₂ (195 mg, 0.869 mmol) in DMF (10 mL) was heated at 70 °C for 6.5 h. After the reaction mixture was filtered, the filtrate was extracted with AcOEt and washed with brine. The organic layer was separated and dried over MgSO₄. After filtration, concentration under reduced pressure, and purification via flash column chromatography (silica gel, n-hexane/AcOEt = 4/1), 16a was obtained as a colorless oil (1.96 g, 90%); ¹H-NMR (DMSO-d₆, 400 MHz, δ ppm), 9.65 (1H, t, J = 1.5 Hz), 7.87 (2H, d, J = 8.4 Hz), 7.34 (2H, d, J = 8.4 Hz), 4.29 (2H, q, J = 7.1 Hz), 2.64 (2H, t, J = 7.6 Hz), 2.41 (2H, td, J = 7.0, 2.0 Hz), 1.63–1.51 (4H, m), 1.33–1.26 (2H, m), 1.31 (3H, t, J = 7.2 Hz).

Steps 2–4: 4-(6-{[4-(3-Cyano-6-isopropyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidin-5-yl)phenethyl](methyl)amino}hexyl)benzoic Acid (19a)

19a was prepared from 8 and 16a using procedures similar to that described for the synthesis of 10a (steps 5–7); yellow solid (25.0 mg, 7% from compound 16a); ¹H-NMR (DMSO-d₆, 400 MHz, δ ppm), 12.8 (1H, brs), 8.01 (1H, brs), 7.86 (2H, d, J = 7.8 Hz), 7.33–7.31 (6H, m), 3.33–3.24 (1H, m), 3.06–2.93 (4H, m), 2.80 (3H, br s), 2.68–2.64 (4H, m), 1.67–1.56 (4H, m), 1.37–1.29 (4H, m), 1.24 (6H, d, J = 9.8 Hz); MS (ESI) m/z 540 (MH⁺).

Step 5: Preparation of 4-(6-{[4-(3-cyano-6-isopropyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidin-5-yl)phenethyl](methyl)amino}hexyl)-N-{2-[2-(2-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl]oxy}ethoxy)ethoxy]ethyl}benzamide (20a)

20a was prepared from 13·HCl and 19a using a procedure similar to that described for the synthesis of 14a·HCl (step 3); yellow solid (9.1 mg, 21%); ¹H-NMR (DMSO-d₆, 700 MHz, δ ppm), 11.14 (1H, s), 8.44 (1H, t, J = 5.6 Hz), 8.04 (1H, s), 7.82–7.76 (3H, m), 7.52 (1H, d, J = 7.0 Hz), 7.47 (1H, d, J = 7.0 Hz), 7.32–7.26 (6H, m), 5.10 (1H, dd, J = 12.9, 5.2 Hz), 4.33–4.23 (2H, m), 3.84–3.80 (2H, m), 3.66 (2H, t, J = 4.7 Hz), 3.57–3.53 (4H, m), 3.43–3.30 (6H, m), 2.92–2.87 (2H, m), 2.74 (1H t, J = 6.5 Hz), 2.65–2.55 (5H, m), 2.54–2.48 (2H, m), 2.04–2.00 (1H, m), 1.63–1.46 (4H, m), 1.35–1.28 (4H, m), 1.30 (6H, d, J = 18.1 Hz); ¹³C-NMR (DMSO-d₆, 175 MHz, δ ppm), 172.70, 169.85, 166.69, 166.00, 165.16, 156.73, 155.67, 145.54, 143.81, 136.87, 133.10, 131.75, 128.00, 127.94, 127.16, 127.07, 126.61, 124.80, 119.83, 116.14, 115.26, 110.56, 73.97, 69.95, 69.55, 68.81, 68.68, 68.54, 59.65, 48.59, 34.72, 34.27, 30.85, 30.82, 30.47, 30.28, 29.11, 28.72, 28.22, 21.96, 21.86, 20.66, 20.44, 13.87; HRMS Calcd for C₅₁H₅₉N₈O₉⁺ 927.4405, Found 927.4390; HPLC t_R 13.73 min, 95.89% purity.

Synthesis of 4-(6-{[4-(3-Cyano-6-isopropyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidin-5-yl)phenethyl](methyl)amino}hexyl)-N-{2-[2-(2-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl]oxy}ethoxy)ethoxy]ethyl}benzamide Hydrochloride (20b·HCl)

20b·HCl was prepared using procedures similar to that described for the synthesis of 20a (steps 1–5) and 14a·HCl (step 4); yellow solid (16.6 mg, 17% from compound 8); ¹H-NMR (DMSO-d₆, 700 MHz, δ ppm), 13.44 (1H, s), 11.15 (1H, d, J = 5.2 Hz, 1H), 8.51 (1H, t, J = 5.6 Hz), 8.43 (1H, s), 7.82 (2H, t, J = 8.0 Hz), 7.74–7.67 (2H, m), 7.53–7.47 (6H, m), 7.39–7.36 (2H, m), 5.10 (1H, dd, J = 12.9, 5.2 Hz), 4.33 (2H, t, J = 4.5 Hz), 3.81 (2H, t, J = 4.5 Hz), 3.67 (2H, t, J = 4.7 Hz), 3.58 (2H, t, J = 4.7 Hz), 3.53 (2H, t, J = 4.7 Hz), 3.52–3.37 (6H, m), 3.35–3.27 (1H, m), 3.22–3.00 (4H, m), 2.92–2.87 (1H, m), 2.86 (3H, s), 2.65–2.59 (4H, m), 2.55–2.48 (2H, m), 2.04–2.01 (1H, m), 1.70–1.66 (2H, m), 1.66–1.63 (2H, m), 1.40–1.32 (4H, m), 1.25 (6H, d, J = 7.0 Hz); ¹³C-NMR (DMSO-d₆, 175 MHz, δ ppm), 172.71, 171.93, 169.86, 166.69, 166.20, 165.17, 155.66, 154.89, 147.74, 145.22, 143.34, 142.15, 138.82, 136.88, 134.24, 133.11, 131.42, 130.98, 128.83, 128.74, 128.04, 127.05, 124.40, 119.84, 116.14, 115.28, 113.86, 112.66, 73.86, 69.96, 69.53, 68.78, 68.69, 68.54, 55.33, 54.83, 48.59, 34.77, 30.82, 30.49, 29.12, 28.25, 28.00, 25.71, 23.07, 22.02, 21.85, 20.96, 19.84; HRMS Calcd for C₅₁H₅₉N₈O₉⁺ 927.4405, Found 927.4388; HPLC t_R 13.80 min, 97.80% purity.

Synthesis of 4-(6-{[4-(3-Cyano-6-isopropyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidin-5-yl)phenethyl](methyl)amino}hexyl)-N-(9-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl]oxy}nonyl)benzamide Hydrochloride (23a·HCl)

Step 1: Preparation of tert-butyl (6-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl]oxy}hexyl)carbamate (21)

A solution of 11 (274 mg, 1.00 mmol), tert-butyl (6-bromohexyl)carbamate (280 mg, 1.00 mmol), KHCO₃ (150 mg, 1.50 mmol), and KI (17.0 mg, 0.102 mmol) in DMF (3 mL) was heated at 80 °C for 14 h. After cooling to room temperature, water and AcOEt were added and separated. The organic layer was washed with brine, dried over MgSO₄. After filtration, concentration under reduced pressure, and purification via flash column chromatography (CHCl₃/AcOEt = 10/1 to 1/1), 21 was obtained as a colorless oil (373 mg, 79%); ¹H-NMR (DMSO-d₆, 400 MHz, δ ppm), 11.08 (1H, brs), 7.82–7.78 (1H, m), 7.51 (1H, d, J = 8.2 Hz), 7.44 (1H, d, J = 7.6 Hz), 6.75 (1H, br s), 5.07 (1H, dd, J = 12.8, 5.4 Hz), 4.20 (2H, t, J = 6.5 Hz), 2.93–2.90 (1H, m), 2.93–2.83 (2H, m), 2.61–2.53 (2H, m), 2.04–2.00 (1H, m), 1.78–1.71 (2H, m), 1.49–1.26 (6H, m), 1.36 (9H, s).

Steps 2: Preparation of 4-[(6-aminohexyl)oxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dionehydrochloride (22·HCl)

22·HCl was prepared as a crude product (404 mg) using a procedure similar to that described for the preparation of 14a·HCl (step 2). This was used directly in the next reaction without further purification.

Step 3: Preparation of 4-(6-{[4-(3-cyano-6-isopropyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidin-5-yl)phenethyl](methyl)amino}hexyl)-N-(6-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl]oxy}hexyl)benzamide hydrochloride (23a·HCl)

23a·HCl was prepared from 19a and 22·HCl using procedures similar to that described for the synthesis of 14a·HCl (steps 3 and 4); yellow solid (26.0 mg, 52%); ¹H-NMR (DMSO-d₆, 700 MHz, δ ppm), 13.43 (1H, s), 11.12 (1H, s), 8.42 (1H, s), 8.41–8.38 (1H, m), 7.81 (1H, dd, J = 7.0, 7.0 Hz), 7.76 (2H, d, J = 8.2 Hz), 7.52 (1H, d, J = 8.6 Hz), 7.50–7.48 (4H, m), 7.44 (1H, d, J = 6.9 Hz), 7.27 (2H, d, J = 8.2 Hz), 5.08 (1H, dd, J = 12.9, 5.6 Hz), 4.21 (2H, t, J = 6.5 Hz), 3.32–3.28 (1H, m), 3.27–3.24 (2H, m), 3.20–2.99 (4H, m), 2.91–2.86 (1H, m), 2.83 (3H, s), 2.65–2.61 (2H, m), 2.60–2.56 (2H, m), 2.53–2.47 (2H, m), 2.03–2.00 (1H, m), 1.79–1.75 (2H, m), 1.73–1.65 (2H, m), 1.65–1.57 (2H, m), 1.57–1.52 (2H, m), 1.52–1.46 (2H, m), 1.41–1.36 (2H, m), 1.36–1.31 (4H, m), 1.24 (6H, d, J = 7.0 Hz); ¹³C-NMR (DMSO-d₆, 175 MHz, δ ppm), 172.20, 169.37, 166.23, 165.28, 164.70, 155.35, 154.38, 147.23, 144.76, 144.71, 142.83, 136.42, 132.60, 131.60, 130.92, 128.32, 128.23, 127.47, 126.55, 126.08, 119.12, 115.54, 114.51, 113.35, 112.14, 73.35, 68.08, 54.82, 54.35, 48.06, 34.14, 30.31, 29.83, 28.62, 28.50, 27.74, 27.48, 25.53, 25.19, 24.45, 22.58, 21.35, 20.45, 19.33; HRMS Calcd for C₅₁H₅₉N₈O₇⁺ 895.4507, Found 895.4490; HPLC t_R 15.16 min, 95.44% purity.

Synthesis of 3-(6-{[4-(3-Cyano-6-isopropyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidin-5-yl)phenethyl](methyl)amino}hexyl)-N-(6-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl]oxy}hexyl)benzamide Hydrochloride (23b·HCl)

23b·HCl was prepared using procedures similar to that described for the synthesis of 23a·HCl (steps 1–3); yellow solid (10.4 mg, 11%); ¹H-NMR (DMSO-d₆, 700 MHz, δ ppm), 13.43 (1H, s), 11.12 (1H, s), 8.47–8.41 (2H, m), 7.80 (1H, dd, J = 8.0, 8.0 Hz), 7.71–7.64 (2H, m), 7.52–7.43 (6H, m), 7.36–7.34 (2H, m), 5.08 (1H, dd, J = 12.9, 5.6 Hz), 4.20 (2H, t, J = 6.5 Hz), 3.33–3.30 (1H, m), 3.28–3.24 (2H, m), 3.17–3.00 (4H, m), 2.90–2.85 (1H, m), 2.82 (3H, s), 2.65–2.61 (2H, m), 2.61–2.56 (2H, m), 2.54–2.47 (2H, m), 2.04–2.00 (1H, m), 1.79–1.75 (2H, m), 1.73–1.66 (2H, m), 1.65–1.59 (2H, m), 1.57–1.52 (2H, m), 1.52–1.47 (2H, m), 1.41–1.37 (2H, m), 1.37–1.31 (4H, m), 1.23 (6H, d, J = 7.0 Hz); ¹³C-NMR (DMSO-d₆, 175 MHz, δ ppm), 172.20, 169.37, 166.22, 165.51, 164.71, 155.35, 141.62, 136.42, 134.06, 132.60, 130.31, 128.30, 128.20, 127.49, 126.50, 123.87, 119.12, 115.54, 114.52, 73.35, 68.08, 54.80, 54.28, 48.06, 34.28, 30.31, 30.00, 28.59, 28.47, 27.74, 27.50, 25.55, 25.21, 24.45, 23.62, 22.52, 21.52, 21.45, 21.35, 20.45, 19.34, 13.36; HRMS Calcd for C₅₁H₅₉N₈O₇⁺ 895.4507, Found 895.4494; HPLC t_R 15.26 min, 95.66% purity.

Neurite Outgrowth Assay

The mouse Neuro-2a (N2a) cell line was obtained from the Japanese Collection of Research Bioresources (JCRB) Cell Bank. The N2a cells were plated at a concentration of 1 × 10⁴ cells/mL in Dulbecco’s modified Eagle’s medium (DMEM) containing high glucose, 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 °C in a humidified atmosphere of 5% CO₂. For the neurite outgrowth assay, the medium was changed to DMEM supplemented with 2% FBS. After incubation with the tested compounds, cell morphology was examined using a microscope (Olympus CKX41). The differentiated cells were defined as those with at least one neurite that was longer than twice the diameter of the cell body. The results were expressed as the percentage of counted cells that were differentiated. These experiments were carried out in triplicate.

Western Blotting

The N2a cells (5 × 10⁵ cells/2 mL/dish) were treated for 24 h with the test compounds at the indicated concentrations in a 10% FBS-supplemented cell culture medium as indicated, and cells were collected and extracted with sodium dodecyl sulfate (SDS) sampling buffer. Protein concentrations of the lysates were determined using a bicinchoninic acid (BCA) protein assay. Equivalent amounts of protein from each lysate were resolved in 5–20% SDS-polyacrylamide gels, and bands were transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, #IPVH00010 for β-actin detection, #ISEQ10100 for H3, H3K4me3, and H3K27Ac detection). After blocking with TBS-T containing 5% skimmed milk, the transblotted membranes were probed with primary antibodies: rabbit monoclonal KDM5A antibody (CST #3876, 1 : 1000 dilution); mouse monoclonal β-actin antibody (Santa Cruz, #sc-47778, 1 : 2000 dilution); rabbit polyclonal H3K4me3 antibody (Abcam, #ab8580, 1 : 5000 dilution); rabbit polyclonal H3K27Ac antibody (Abcam, #ab4729, 1 : 1000 dilution); or rabbit polyclonal histone H3 antibody (Abcam, #ab1791, 1 : 200000 dilution). The probed membranes were washed thrice with TBS-T; incubated with ECL rabbit immunoglobulin G (IgG) horseradish peroxidase (HRP)-linked whole antibody (GE Healthcare Life Science, #NA934, 1 : 2500 dilution), or ECL mouse IgG HRP-linked whole antibody (GE Healthcare Life Science, #NA931, 1 : 2500 dilution); and again, washed thrice with TBS-T. The immunoblots were visualized using enhanced chemiluminescence with chemiluminescent HRP substrate (Millipore, #P90718).

Acknowledgments

The authors would like to thank the members of the Comprehensive Analysis Center, SANKEN, Osaka University for the NMR and HRMS analyses and Mr. Hirokazu Takeshima, Mr. Yuuki Taki, and Ms. Miho Sawada for technical support. This work was supported by Grants for JSPS KAKENHI (19H05295 to Y.I.), “Dynamic Alliance for Open Innovation Bridging Human, Environment and Materials” from MEXT (T.S. and Y.I.), and the Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research; BINDS) from the Japan Agency for Medical Research and Development (AMED) (22ama121041j0001 to T.S. and Y.I.).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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