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. 2022 Sep 23;13(12):1495–1503. doi: 10.1039/d2md00284a

Structure–activity relationship study of PROTACs against hematopoietic prostaglandin D2 synthase†

Yuki Murakami 1,2,‡, Hinata Osawa 1,3,‡, Takashi Kurohara 1,‡, Yuta Yanase 1,2, Takahito Ito 1,2, Hidetomo Yokoo 1,4, Norihito Shibata 5, Mikihiko Naito 6, Kosuke Aritake 7, Yosuke Demizu 1,2,3,✉
PMCID: PMC9749925  PMID: 36561070

Abstract

Degradation of hematopoietic prostaglandin D2 synthase (H-PGDS) by proteolysis-targeting chimeras (PROTACs) is expected to be important in the treatment of allergic diseases and Duchenne's muscular dystrophy. We recently reported that PROTAC(H-PGDS)-7 (PROTAC1), which is composed of H-PGDS inhibitor (TFC-007) and cereblon (CRBN) E3 ligase ligand (pomalidomide), showed potent H-PGDS degradation activity. Here, we investigated the structure–activity relationships of PROTAC1, focusing on the C4- or C5-conjugation of pomalidomide, in addition, the H-PGDS ligand exchanging from TFC-007 with the biaryl ether to TAS-205 with the pyrrole. Three new PROTACs were evaluated for H-PGDS affinity, H-PGDS degrading activity, and inhibition of prostaglandin D2 production. All compounds showed high H-PGDS degrading activities, but PROTAC(H-PGDS)-4-TAS-205 (PROTAC3) was slightly less active than the other compounds. Molecular dynamics simulations suggested that the decrease in activity of PROTAC3 may be due to the lower stability of the CRBN-PROTAC-H-PGDS ternary complex.

SAR studies of PROTACs that target H-PGDS, focusing on the E3 ligase ligand and the H-PGDS ligand, are described.graphic file with name d2md00284a-ga.jpg

Introduction

Targeted protein degradation using chemical modalities is a powerful tool for the suppression of protein activity in various diseases. These innovative modalities1–3 include proteolysis-targeting chimeras (PROTACs)4 and specific and non-genetic inhibitors of apoptosis (IAP) protein-dependent protein erasers (SNIPERs).5 Both PROTACs and SNIPERs are heterobifunctional molecules that consist of two ligands connected via an appropriate linker; one ligand targets the protein of interest (POI ligand) and the other recruits an E3 ubiquitin ligase (E3 ligand). PROTACs induce targeted protein degradation by hijacking the ubiquitin–proteasome system (UPS). To date, several E3 ligands have been used in PROTAC design. Representative E3 ligase ligands include cereblon (CRBN) ligands, such as thalidomide and pomalidomide, von Hippel–Lindau (VHL) ligands, and IAP ligands.6 Among these, pomalidomide is one of the most widely used CRBN ligands because of its low molecular weight and straightforward synthesis.7

Overexpression of hematopoietic prostaglandin D2 synthase (H-PGDS) leads to increased levels of prostaglandin D2 (PGD2), which is closely associated with the progression of various diseases, including allergic diseases8,9 and Duchenne's muscular dystrophy.10 To date, several H-PGDS inhibitors,9 such as HQL-79,11,12 BSPT,13 Cibacron-blue,14 TFC-007 (Fig. 1a),15 F092 (ref. 16) and TAS-205 (Fig. 1b),17 have been developed. However, none of these inhibitors has passed clinical trials.

Fig. 1. Chemical structures of (a) TFC-007 and PROTAC(H-PGDS)-7 (PROTAC1), (b) TAS-205, and (c) PROTACs2–4.

Fig. 1

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To effectively suppress the function of H-PGDS, our group embarked on the development of PROTACs targeting H-PGDS. We recently developed PROTAC(H-PGDS)-7 (PROTAC(H-PGDS)-4-TFC: PROTAC1 in this study), in which TFC-007 as a POI ligand was directly conjugated to pomalidomide without any linker moiety (Fig. 1a).18,19 In pomalidomide-containing PROTACs targeting H-PGDS, we found that the shortness and rigidity of the linker are important for H-PGDS degradation activity.19,20

Although PROTAC1 has a rigid structure, we did not investigate whether the relative orientation of the two ligands (TFC-007 and pomalidomide) affects the H-PGDS degradation activity. The pomalidomide ligand can be substituted at either the C4 or C5 position of the phthalimide ring. In the case of a pomalidomide-containing Bruton's tyrosine kinase (BTK)-targeting PROTAC,21 the C4-substituted derivative (MT-541) showed lower degradation activity (DC50 = 90.1 nM) than the corresponding C5-substituted derivative (MT-809, DC50 = 11.6 nM), even though the same linker was used. The effect of the pomalidomide-substitution position on the formation of the E3 ligand–PROTAC–target protein ternary complex is likely to be especially significant for linker-less PROTACs. Therefore, in this study, we investigate rigid PROTACs with different pomalidomide substitution patterns. We also investigate the replacement of the TFC-007-type ligand with TAS-205, which has completed phase II clinical trials and has progressed to phase III trials.22,23

Here, we report three newly designed PROTACs: PROTAC(H-PGDS)-5-TFC-007 (PROTAC2), which is a regioisomer of PROTAC1 with a C5-substituted, rather than C4-substituted, pomalidomide ligand; PROTAC(H-PGDS)-4-TAS-205 (PROTAC3), which is a derivative of PROTAC1 with a TAS-205, rather than TFC-007, E3-ligase ligand; and PROTAC(H-PGDS)-5-TAS-205 (PROTAC4), which is the C5-substituted regioisomer of PROTAC3 (Fig. 1c). The PROTACs were evaluated for H-PGDS binding affinity, H-PGDS degradation activity and inhibition of prostaglandin D2 production. The ternary complexes of each PROTAC with H-PGDS and CRBN were analyzed using molecular dynamics simulations.

Result and discussion

Synthesis

The synthetic routes to the PROTACs1–4 are described in Scheme 1 and Table 1. We first synthesized TFC-007 and TAS-205 derivatives in which the oxygen of the morpholine ring was replaced by an NH group. The TFC-007 derivative 7 was synthesized from ethyl piperidine-4-carboxylate (1) according to a reported procedure.18 The TAS-205 derivative 9 was synthesized from compound 5via a urea-forming reaction using 4-nitrophenyl chloroformate. As shown in Table 1, PROTACs2–4 were synthesized via aromatic substitution reactions between C4- or C5-fluorothalidomide and compounds 7 or 9. In addition, N-methylated PROTACs5–7 were synthesized using the same routes as PROTACs1–4. The N-methylated analogues are considered to have low CRBN affinity and are used to investigate the degradation mechanism.24

Scheme 1. Synthesis of TFC-007 and TAS-205 derivatives. Abbreviations: EDC: 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide; HOBt: 1-hydroxybenzotriazole; DMAP: 4-dimethylaminopyridine.

Scheme 1

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Synthesis of PROTACs 1–7.

graphic file with name d2md00284a-u1.jpg
R1 Reactant (R2, F = C4/C5) Equivalent Yield Product
Inline graphic TFC-007-type (7) 10 (R2 = H, F = C4) X = Y = 3.0a 35%a PROTAC1
11 (R2 = H, F = C5) X = Y = 3.0 6% PROTAC2
12 (R2 = Me, F = C5) X = Y = 3.0 21% PROTAC5
Inline graphic TAS-205-type (9) 10 X = 1.1, Y = 4.2 8% PROTAC3
13 (R2 = Me, F = C4) X = Y = 3.0 9% PROTAC6
11 X = Y = 3.0 2% PROTAC4
12 X = 1.1, Y = 3.0 6% PROTAC7
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a

Previously reported conditions.19 Abbreviations: DIPEA: N,N-diisopropylethylamine.

H-PGDS binding affinity

We investigated the binding affinity of PROTACs1–4 for H-PGDS using a competitive fluorescence polarization (FP) assay. The relationship between the compound concentrations and fluorescence intensity is shown in Fig. 2, and the calculated IC50 values are listed in Table 2. The IC50 of the TFC-007 ligand was determined to be 211.2 ± 11.6 nM, and the IC50 values of PROTACs1 and 2 were determined to be 116.5 ± 19.5 nM and 196.3 ± 105.0 nM, respectively. These results suggest that the position of the pomalidomide substitution does not affect the H-PGDS binding affinity. In addition, the TAS-205-based PRTOACs gave similar results; the IC50 of the TAS-205 ligand was 134.0 ± 8.7 nM, and the IC50 values of PROTACs3 and 4 were 78.1 ± 2.2 nM and 123.9 ± 27.1 nM, respectively.

Fig. 2. Competitive fluorescence polarization assays against H-PGDS for (a) TFC-007, PROTAC1, and PROTAC2; and (b) TAS-205, PROTAC3, and PROTAC4.

Fig. 2

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Summary of FP and western blotting assays.

Compound FP assay Western blotting
IC50 ± SD (nM) DC50 ± SD (pM)
TFC-007 211.2 ± 11.6 n.d.
PROTAC1 116.5 ± 19.5 18.7 ± 1.5
PROTAC2 196.3 ± 105.0 27.6 ± 10.5
TAS-205 134.0 ± 8.7 n.d.
PROTAC3 78.1 ± 2.2 71.4 ± 34.8
PROTAC4 123.9 ± 27.1 23.8 ± 18.4
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H-PGDS degradation activity

The effect of the PROTACs on H-PGDS protein levels was evaluated by western blotting using KU812 cells after 24 h of incubation with the compounds (Fig. 3). All four PROTACs reduced the H-PGDS levels in a dose-dependent manner. There was little difference between the activity of PROTACs1 and 2, with DC50 values of 18.7 ± 1.5 pM and 27.6 ± 10.5 pM, respectively (Table 2). In contrast, the DC50 values of PROTACs3 and 4 were significantly different, at 71.4 ± 34.8 pM and 23.8 ± 18.4 pM, respectively. This indicates an isomer effect between these TAS-205 derivatives.

Fig. 3. H-PGDS protein levels in KU182 cells as measured by western blots after 24 h of incubation with (a) PROTACs1 and 2, and (b) PROTACs3 and 4. The H-PGDS/β-actin ratios were normalized to the DMSO control, which was set at 100%. The data in the bar graph are means ± SEM (n = 3). *p < 0.05, **p < 0.01, ***p < 0.005 compared with the DMSO control in a two-tailed Student's t-test.

Fig. 3

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To confirm whether the newly designed PROTACs hijacked the UPS, co-treatment assays were performed with UPS inhibitors. We investigated the effect of proteasome inhibitor MG132 and the ubiquitin-activating enzyme inhibitor MLN7243.25 In addition, we investigated the activity of the N-methylated E3-ligand derivatives PROTACs5–7, which have lower affinity for CRBN, and evaluated the competition of the PROTACs with the TFC-007 or TAS-205 ligands. The mechanistic study of PROTAC1 was reported previously,19 so only PROTACs2–4 were studied here. PROTAC2 (1 nM) significantly reduced the H-PGDS levels in the absence of UPS inhibitors; however, the addition of MG132 and MLN7243 (10 μM) suppressed the activity of the PROTAC (Fig. 4a). N-Methylated PROTAC5 (10 nM) was not effective at reducing the H-PGDS levels, and the addition of TFC-007 (10 μM) suppressed the activity of PROTAC2 (1 nM). Taken together, these results indicate that the levels of H-PGDS protein are being reduced by the expected UPS-dependent mechanism. Similar results were observed for the TAS-type PROTACs3 and 4; the activity of both PROTACs was suppressed by MG132, MLN7243, and TAS-205 (10 μM) (Fig. 4b and c) and the corresponding N-methylated PROTACs6 and 7 (10 nM) were not active. Therefore, all newly synthesized PROTACs appear to engage the UPS to degrade the H-PGDS protein.

Fig. 4. Investigation of the involvement of the ubiquitin–proteasome system in the reduction of H-PGDS protein levels by (a) PROTACs2 and 5, (b) PROTACs3 and 6, and (c) PROTACs4 and 7. KU812 cells were incubated with the compounds for 6 h. The H-PGDS/β-actin ratios were normalized to the DMSO control, which was set at 100%. The data in the bar graph are means ± SEM (n = 3). *p < 0.05, **p < 0.01, ***p < 0.005 compared with the DMSO control in a two-tailed Student's t-test.

Fig. 4

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To further confirm that PROTAC treatment reduces H-PGDS protein levels in KU812 cells, the concentration of PGD2 was measured by an enzyme-linked immunosorbent assay (ELISA). The H-PGDS inhibitors TFC-007 and TAS-205 (10 nM) decreased PGD2 levels and all PROTACs1–4 (10 nM) decreased PGD2 levels compared with DMSO treatment (Fig. 5). Notably, PROTACs1–4 reduced the PGD2 level more than the corresponding TFC-007 or TAS-205 inhibitors. We previously reported this effect for PROTAC1 and TFC-007.18 This is considered to be due to the combination of both H-PGDS degrading activity and inhibitory activity displayed by the PROTACs.

Fig. 5. Effect of PROTACs1–4 on PGD2 production at 10 nM treatment. *p < 0.05, **p < 0.01, ***p < 0.005 (compared with the DMSO control) in a two-tailed Student's t-test.

Fig. 5

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Molecular dynamics simulations of ternary complexes

In the case of PROTACs based on TFC-007, the two regioisomers did not have significantly different H-PGDS degrading activities, whereas the difference in activities between the two PROTAC regioisomers based on TAS-205 was more pronounced. To investigate these differences, we evaluated the stabilities of the ternary complexes of each PROTAC using molecular dynamics (MD) simulations. The initial structure of each ternary complex was prepared using CRBN (PDB: 4CI3) and H-PGDS (PDB: 5YWX), according to a previously reported method.26,27 The MD simulations in aqueous solution were performed for 310 ps using Amber10: EHT force field. The root-mean-square deviation (RMSD) values for each of the PROTAC ligands are shown in Fig. 6a. The ternary complex of PROTAC3 exhibited the highest RMSD value, suggesting that the structural flexibility of PROTAC3 might be responsible for the reduced activity. The calculated binding mode and protein–ligand interaction in the lowest energy ternary complexes of PROTACs1–4 during MD simulation are shown in Fig. 6b and S1–S4.† In these snaps, there was little change in the interaction between PROTAC1 and 2. On the other hand, comparing PROTAC3 and PROTAC4, the hydrogen bond between Met99 of H-PGDS and the urea carbonyl group of the PROTAC3 was lost. Furthermore, a CH–π interaction between Trp104 of H-PGDS and the piperazine ring of PROTAC4 was observed in the PROTAC4 ternary complex, whereas in PROTAC3, Trp104 formed π–π interaction with the terminal pyrrole ring. These results suggest that in the PROTAC3 ternary complex, the TAS ligand might be moved outward from the H-PGDS and be more easily dissociated than other PROTACs.

Fig. 6. (a) Plot of ligand RMSD values during the MD simulation of PROTACs1–4. (b) Calculated binding modes of PROTACs1–4 in the most stable ternary complexes in MD simulations (CRBN is shown in gray and H-PGDS is shown in brown).

Fig. 6

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Biological experiments with PROTACs1–4 are summarized below. First, the in vitro FP assay showed that the H-PGDS binding affinity of each PROTAC is almost equal to that of the corresponding parent inhibitors (TFC-007 or TAS-205). Isomerization to the C4- or C5-positions did not reduce the binding affinity of the compounds. Second, western blot assays using KU812 cells confirmed that the PROTACs reduce the level of H-PGDS protein, even at low concentrations. The dependence of PROTACs2–4 on the UPS was evidenced by their loss of activity upon the addition of UPS inhibitors (MG132, MLN7243) and the lack of activity of corresponding PROTACS with N-methylated pomalidomide moieties (PROTACs5–7). Furthermore, co-addition of H-PGDS inhibitors (TFC-007, TAS-205) suppressed the activity of the PROTACs, indicating that H-PGDS binding is necessary for H-PGDS degradation. Together, these results indicate that binding to both CRBN and H-PGDS is required for the H-PGDS degradation activity of the PROTACs.

To confirm the observed changes in the expression level of H-PGDS, PGD2 concentrations were evaluated using KU812 cells; all PROTACs1–4 inhibited PGD2 production. As described above, both the C4 and C5 PROTAC regioisomers reduce H-PGDS levels via the expected mechanism, even at low concentrations; however, western blot analysis revealed a slight decrease in the activity of PROTAC3. MD simulations were used to investigate this discrepancy. The simulations indicated that PROTAC3 had larger RMSD than the other PROTACs within the ternary complex with CRBN and H-PGDS; this increased flexibility may be one of the reasons for the reduced activity.

Conclusions

We designed and synthesized three new PROTACs (PROTACs2–4) to investigate the effect of C4- versus C5-substitution at the thalidomide moiety. In addition, TAS-205 was used as a new H-PGDS ligand. All PROTACs reduced the level of H-PGDS protein by hijacking the UPS; however, the C4-substituted PROTAC3 had lower activity. MD simulations indicated that the decrease in activity of PROTAC3 could be due to the low stability of its ternary complex. The structure–activity relationships obtained in this study not only indicate designs that should be avoided in the development of potent H-PGDS-targeting PROTACs but also provide insights into promising designs for other structurally rigid PROTACs.

Experimental

Chemistry

General information

All chemicals were purchased from Sigma-Aldrich Co. LLC, Kanto Chemicals Co. Inc., Tokyo Chemical Industry Co. Ltd., Wako Pure Chemical Industries Ltd., and were used without further purification. Reactions were followed by thin-layer chromatography (TLC) (60 F254, Merck), and spots were visualized by UV irradiation with a handheld UV lamp (254/365 nm) (UVP) and iodine vapor or ninhydrin reagent. Silica gel for column chromatography was Kanto Chemical 60 N (spherical, neutral), NH silica gel (Chromatorex NH-DM1020, Fuji Silicia), or packed columns for medium pressure column chromatography (Hi-Flash column/Inject column Yamazen). Preparative HPLC was performed using a WP300 C18 column (5 μm, 20 mm × 250 mm, GL science) at a flow rate of 10 mL min−1 on a JASCO PU-4180 HPLC, and eluents were detected at 220 nm by a JASCO UV-2075. Analytical HPLC was performed using an Inertsil WP300 C18 column (5 μm, 4.6 mm I.D. × 250 mm, GL Science Inc.) at a flow rate of 1.0 mL min−1 (gradient: 10–90% MeCN–H2O containing 0.1% TFA, 30 min) and eluents were detected at 260 nm by an EXTREMA, Jasco (Tokyo, Japan). 1H-NMR and 13C-NMR spectra were measured on an ECZ 600R spectrometer (JEOL) using deuterated solvents. Chemical shift values (ppm) were corrected for residual solvent signals as internal standards [DMSO-d6: 2.50 for 1H-NMR, 39.5 for 13C-NMR; CD3OD: 3.30 for 1H-NMR, 49.0 for 13C-NMR; CDCl3: 7.26 for 1H-NMR, 77.2 for 13C-NMR]. The splitting modes of the signals are as follows [singlet (s), doublet (d), triplet (t), quartet (q), double of doublets (dd), multiplet (m), broad (br)]. High-resolution mass spectrometry (HRMS) was measured by electrospray ionization using Shimadzu IT-TOF MS (Shimadzu).

Synthesis of PROTACs

Synthesis of compound 2

K2CO3 (734 mg, 5.3 mmol) was added to a solution of 4-fluoronitrobenzene (500 mg, 3.5 mmol) and ethyl piperidine-4-carboxylate (1) (545 μL, 3.5 mmol) in N,N-dimethylformamide (9 mL). The mixture was stirred for 17 h at 50 °C and then cooled to room temperature. The reaction mixture was diluted with EtOAc and washed with water and brine. The organic phase was dried with Na2SO4 and concentrated in vacuo to obtain compound 2 (1.03 g, >99%) as a yellow solid; 1H-NMR (600 MHz, CDCl3): δ 8.12 (d, J = 9.3 Hz, 2H), 6.82 (d, J = 9.3 Hz, 2H), 4.17 (q, J = 7.1 Hz, 2H), 3.89 (dt, J = 13.3, 4.0 Hz, 2H), 3.11–3.06 (m, 2H), 2.58 (dt, J = 10.7, 4.1 Hz, 1H), 2.04 (dd, J = 13.6, 3.6 Hz, 2H), 1.83 (ddd, J = 24.7, 10.9, 3.9 Hz, 2H), 1.27 (t, J = 7.1 Hz, 3H); MS (ESI) m/z 279.1 [M + H]+.

Synthesis of compound 3

Compound 2 (974 mg, 3.5 mmol) was added to a solution of 2.0 M aqueous NaOH (5.25 mL, 10.5 mmol) in THF (8 mL) and H2O (27 mL). The mixture was stirred for 5 h at room temperature. The reaction mixture was then treated with 6.0 M aqueous HCl and diluted with CH2Cl2 and washed with water and brine. The organic phase was dried with Na2SO4 and concentrated in vacuo to obtain compound 3 (520 mg, 53%) as a yellow solid; 1H-NMR (600 MHz, CDCl3): δ 8.12 (d, J = 9.3 Hz, 2H), 6.83 (d, J = 9.3 Hz, 2H), 3.89 (dt, J = 13.3, 3.9 Hz, 2H), 3.14–3.09 (m, 2H), 2.66 (dt, J = 10.5, 4.1 Hz, 1H), 2.09 (dd, J = 13.6, 3.6 Hz, 2H), 1.87 (ddd, J = 24.6, 10.7, 3.7 Hz, 2H); MS (ESI) m/z 251.1 [M + H]+.

Synthesis of compound 4

Compound 3 (500 mg, 2.0 mmol), EDCI·HCl (422 mg, 2.2 mmol), and HOBt·H2O (337 mg, 2.2 mmol) were added to a solution of 4-Boc-piperadine (410 mg, 2.2 mmol) in N,N-dimethylformamide (4 mL). The reaction mixture was stirred for 1.5 h at room temperature. Water was poured into the reaction mixture and the resulting precipitate was collected by filtration. The precipitate was washed with water and dried under vacuum to obtain compound 4 (787 mg, 94%) as a yellow solid; 1H-NMR (600 MHz, DMSO-d6): δ 8.03 (d, J = 9.3 Hz, 2H), 7.01 (d, J = 9.6 Hz, 2H), 4.04 (d, J = 13.1 Hz, 2H), 3.53 (s, 2H), 3.40 (s, 2H), 3.34 (s, 2H), 3.27 (s, 2H), 3.09–3.04 (m, 2H), 2.99–2.95 (m, 1H), 1.70 (d, J = 10.7 Hz, 2H), 1.60–1.53 (m, 2H), 1.40 (s, 9H); 13C-NMR (151 MHz, DMSO-d6): δ 172.3, 154.4, 153.8, 136.2, 125.9 (2C), 112.5 (2C), 79.1, 46.2 (2C), 44.5 (2C), 40.9 (2C), 36.8, 28.0 (3C), 27.5 (2C); HRMS (ESI) m/z calcd for C21H31N4O5 [M + H]+, 419.2289; found, 419.2289.

Synthesis of compound 5

10% Pd/C (150 mg) was added to a solution of compound 4 (1.11 g, 2.65 mmol) in THF (26 mL). The solution was vacuumed and substituted to H2 atmosphere and then stirred for 2.5 h at room temperature. The reaction mixture was filtered through a celite pad. The precipitate was washed with MeOH and dried under vacuum to obtain compound 5 (676 mg, 66%) as a light purple solid; 1H-NMR (600 MHz, DMSO-d6): δ 6.68–6.66 (m, 2H), 6.47 (dt, J = 9.5, 2.7 Hz, 2H), 4.55 (s, 2H), 3.49 (s, 2H), 3.43 (s, 2H), 3.35 (d, J = 4.1 Hz, 4H), 3.28 (s, 2H), 2.64 (t, J = 7.1 Hz, 1H), 2.55–2.52 (m, 2H), 1.68–1.65 (m, 4H), 1.40 (s, 9H); MS (ESI) m/z 289.3 [M + H]+.

Synthesis of compound 6

2-Phenoxy-5-pyrimidinecarboxylic acid (385 mg, 1.78 mmol), EDCI·HCl (341 mg, 1.78 mmol), and HOBt·H2O (273 mg, 1.78 mmol) were added to a solution of compound 5 (630 mg, 1.62 mmol) in N,N-dimethylformamide (7.5 mL). The reaction mixture was stirred for 1.5 h at room temperature. Water was poured into the reaction mixture and the resulting precipitate was collected by filtration. The precipitate was washed with water and dried under vacuum to obtain compound 6 (861 mg, 91%) as a white solid; 1H-NMR (600 MHz, DMSO-d6): δ 10.22 (s, 1H), 9.07 (s, 2H), 7.55 (d, J = 9.3 Hz, 2H), 7.48–7.45 (m, 2H), 7.29 (t, J = 7.4 Hz, 1H), 7.25–7.24 (m, 2H), 6.93 (d, J = 9.0 Hz, 2H), 3.51 (s, 2H), 3.42 (s, 2H), 3.34–3.32 (m, 4H), 3.28 (d, J = 11.7 Hz, 2H), 2.77–2.73 (m, 1H), 2.72–2.68 (m, 2H), 1.70–1.63 (m, 4H), 1.40 (s, 9H); MS (ESI) m/z 587.3 [M + H]+.

Synthesis of compound 8

4-Nitrophenyl chloroformate (242 mg, 1.2 eq.) was dissolved in THF (3 mL), and a solution of compound 5 (389 mg, 1.0 mmol) in THF (3 mL) was added dropwise at −30 °C. After stirring for 30 min at the same temperature, (1-methyl-1H-pyrrol-2-yl) (piperazin-1-yl)methanone (213 mg, 1.1 eq.) and DMAP (61 mg, 0.5 eq.) were added and the mixture was stirred at 45 °C for 24 h. Saturated aqueous sodium bicarbonate solution was added to the reaction solution and extracted with ethyl acetate. The organic layer was washed with water and saturated brine, and then dried over anhydrous sodium sulfate. After removal of the drying agent, the residue obtained by solvent removal under reduced pressure was purified by medium pressure silica gel flash column chromatography (NH silica gel, MeOH/CH2Cl2 = 0/1 to 1/9) to afford compound 8 (133 mg, 22%) as a brown solid; 1H-NMR (600 MHz, DMSO-d6): δ 8.16 (dd, J = 5.2, 1.7 Hz, 1H), 7.27 (d, J = 9.0 Hz, 1H), 6.84 (d, J = 9.3 Hz, 1H), 6.72–6.71 (m, 1H), 6.48 (dd, J = 5.2, 1.7 Hz, 2H), 6.31 (q, J = 1.7 Hz, 1H), 6.10–6.08 (m, 1H), 3.77 (s, 4H), 3.71 (t, J = 5.0 Hz, 3H), 3.61 (d, J = 12.7 Hz, 4H), 3.54–3.48 (m, 7H), 3.41 (d, J = 2.8 Hz, 2H), 3.33 (t, J = 5.0 Hz, 1H), 2.69–2.65 (m, 1H), 2.58 (s, 1H), 1.96 (dd, J = 11.9, 2.6 Hz, 2H), 1.78 (d, J = 12.1 Hz, 2H), 1.49 (s, 9H); 13C-NMR (151 MHz, DMSO-d6): δ 173.3, 162.9, 155.9, 154.3, 154.1, 148.8, 147.6, 131.8, 126.5, 126.4, 124.4, 124.3, 122.1, 117.0, 112.9, 106.8, 106.8, 106.4, 80.1, 50.0, 49.8, 46.7, 45.0, 43.9, 41.3, 38.8, 38.1, 28.4, 28.4, 28.1; HRMS (ESI) m/z calcd for C32H46N7O5 608.3555, found 608.3551 [M + H]+.

Synthesis of compounds 10–13

Compounds 10,28 and 12,28 were prepared according to a reported method. A similar procedure to those of compounds 10 and 12 was employed for the preparation of compounds 11 and 13. For the synthesis of them, 4-fluorophthalic anhydride was used instead of 3-fluorophthalic anhydride and the products were identified as the reported compounds.29

PROTAC1 was synthesized according to the reported procedure.19

Synthesis of PROTAC 2

TFA (500 μL) was added to a solution of compound 6 (51.6 mg, 0.09 mmol) in CH2Cl2 (1.5 mL). The reaction mixture was stirred for 4.5 h at room temperature. The reaction mixture was concentrated in vacuo and then purified by silica gel column chromatography (amino silica, CH2Cl2/MeOH = 10/0 to 9/1). The resulting compound 7 (68.2 mg, 0.247 mmol) and DIPEA (42.0 μL, 0.25 mmol) were added to a solution of compound 11 (40.0 mg, 0.082 mmol) in N,N-dimethylformamide (0.5 mL). The reaction mixture was stirred at 80 °C for 2 h under microwave irradiation. The reaction mixture was cooled to room temperature and filtered through a membrane filter, which was purified by preparative HPLC (gradient 30–60% MeCN–H2O in 40 min). PROTAC2 was obtained as a yellow solid (3.6 mg, 6%); 1H-NMR (600 MHz, DMSO-d6): δ 11.08 (s, 1H), 10.28 (s, 1H), 9.08 (s, 2H), 7.71 (d, J = 8.6 Hz, 1H), 7.59 (s, 2H), 7.48–7.45 (m, 2H), 7.36 (d, J = 1.7 Hz, 1H), 7.30–7.24 (m, 4H), 6.89–7.07 (2H), 5.07 (dd, J = 12.7, 5.5 Hz, 1H), 3.72–3.62 (m, 12H), 2.91–2.85 (m, 2H), 2.60–2.53 (m, 2H), 2.03–2.00 (m, 1H), 1.75 (s, 4H); 13C-NMR (151 MHz, DMSO-d6): δ 172.8, 170.1, 167.5, 167.0, 165.7, 159.7, 154.9, 152.6, 133.8, 129.8, 125.6, 124.9, 124.1, 121.6, 121.4, 118.6, 117.9, 108.0, 48.8, 47.2, 46.5, 44.0, 40.6, 40.0, 39.9, 39.8, 39.6, 39.5, 39.4, 39.2, 39.1, 31.0, 27.8, 22.2; HRMS (ESI) m/z calcd for C40H39N8O7 [M + H]+, 743.2936; found, 743.2928. HPLC purity: >99% (tR = 16.1 min).

A similar procedure to that of PROTAC2 was employed for the preparation of PROTACs3–7. For the synthesis of PROTAC3, compound 10 was used instead of compound 11. For the synthesis of PROTAC4, compound 8 was used instead of compound 6. For the synthesis of PROTACs5–7, corresponding compound 8, 12, and/or 13 was used, respectively.

PROTAC 3

1H-NMR (600 MHz, Methanol-d4): δ 7.71 (t, J = 7.7 Hz, 1H), 7.64 (d, J = 8.6 Hz, 2H), 7.53 (d, J = 8.6 Hz, 2H), 7.44 (d, J = 7.2 Hz, 1H), 7.35 (d, J = 8.3 Hz, 1H), 6.86 (s, 1H), 6.44 (d, J = 3.4 Hz, 1H), 6.11 (s, 1H), 5.12 (dd, J = 12.7, 5.2 Hz, 1H), 3.90 (s, 2H), 3.85–3.82 (m, 6H), 3.78–3.75 (m, 4H), 3.68–3.63 (m, 6H), 3.45 (s, 3H), 3.36 (s, 3H), 2.77–2.74 (m, 2H), 2.22–2.12 (m, 6H); 13C-NMR (151 MHz, Methanol-d4): δ 174.6, 173.9, 171.6, 168.8, 168.2, 165.5, 157.2, 151.1, 137.0, 135.5, 128.2, 125.7, 124.8, 122.7, 122.2, 119.4, 117.0, 114.5, 108.3, 56.6, 56.5, 52.9, 51.6, 50.5, 49.6, 48.4, 46.9, 45.2, 43.2, 35.7, 32.2, 27.9, 23.7; HRMS (ESI) m/z calcd for C40H46N9O7 [M + H]+, 764.3515; found, 764.3514. HPLC purity: 98% (tR = 10.5 min).

PROTAC 4

1H-NMR (600 MHz, Methanol-d4): δ 7.73 (d, J = 8.6 Hz, 1H), 7.65–7.63 (m, 2H), 7.55–7.53 (m, 2H), 7.41 (d, J = 2.4 Hz, 1H), 7.28 (dd, J = 8.6, 2.4 Hz, 1H), 6.85 (t, J = 2.1 Hz, 1H), 6.44 (q, J = 1.8 Hz, 1H), 6.11 (q, J = 2.1 Hz, 1H), 5.08 (dd, J = 12.4, 5.5 Hz, 1H), 3.87 (s, 2H), 3.82 (t, J = 5.2 Hz, 6H), 3.79 (s, 1H), 3.75 (s, 3H), 3.68 (td, J = 11.9, 3.3 Hz, 2H), 3.64–3.62 (m, 4H), 3.59 (t, J = 5.0 Hz, 2H), 3.53 (t, J = 5.2 Hz, 2H), 3.24–3.19 (m, 2H), 2.87–2.84 (m, 1H), 2.77–2.71 (m, 2H), 2.23–2.16 (m, 4H), 2.13–2.10 (m, 1H), 1.31 (t, J = 7.4 Hz, 2H); 13C-NMR (151 MHz, Methanol-d4): δ 175.8, 174.6, 173.9, 173.3, 171.7, 169.3, 168.9, 165.5, 157.2, 156.7, 142.9, 137.8, 135.6, 128.2, 126.1, 125.7, 122.7, 122.2, 121.3, 119.5, 114.5, 109.5, 108.3, 85.7, 56.6, 50.5, 50.0, 49.9, 48.6, 48.4, 48.1, 47.9, 46.0, 45.2, 42.7, 35.7, 32.2, 27.9, 23.8, 9.2; HRMS (ESI) m/z calcd for C40H46N9O7 [M + H]+, 764.3515; found, 764.3513. HPLC purity: 97% (tR = 10.3 min).

PROTAC 5

1H-NMR (600 MHz, DMSO-d6): δ 10.35 (s, 1H), 9.10 (s, 2H), 7.72–7.64 (m, 3H), 7.49–7.46 (m, 2H), 7.37 (d, J = 1.7 Hz, 1H), 7.31–7.25 (m, 4H), 7.11 (s, 2H), 5.15 (dd, J = 13.1, 5.5 Hz, 1H), 3.74–3.64 (m, 12H), 3.01 (s, 3H), 2.98–2.89 (m, 3H), 2.76 (dd, J = 12.9, 2.6 Hz, 1H), 2.06–2.03 (m, 1H), 1.79 (s, 4H); 13C-NMR (151 MHz, DMSO-d6): δ 171.8, 169.8, 167.5, 167.0, 165.7, 159.7, 154.9, 152.6, 133.8, 129.8, 125.6, 125.0, 124.1, 121.6, 121.5, 118.6, 117.9, 108.0, 49.4, 47.1, 46.5, 44.0, 40.7, 31.1, 27.6, 26.6, 21.4; HRMS (ESI) m/z calcd for C41H41N8O7 [M + H]+, 757.3093; found, 757.3084. HPLC purity: >99% (tR = 17.4 min).

PROTAC 6

1H-NMR (600 MHz, DMSO-d6): δ 8.48 (s, 1H), 7.56 (s, 1H), 7.36 (t, J = 7.7 Hz, 1H), 7.20 (s, 2H), 7.03 (d, J = 6.9 Hz, 2H), 6.99 (d, J = 8.3 Hz, 1H), 6.53 (d, J = 1.4 Hz, 1H), 5.98 (t, J = 1.9 Hz, 1H), 5.67–5.66 (m, 1H), 4.80 (dd, J = 12.9, 5.3 Hz, 1H), 3.38 (s, 1H), 3.28–3.23 (m, 5H), 3.13 (t, J = 4.5 Hz, 5H), 2.98 (s, 1H), 2.90 (s, 1H), 2.62 (d, J = 14.1 Hz, 3H), 2.60–2.54 (m, 1H), 2.40–2.34 (m, 6H), 2.19–2.15 (m, 1H), 2.11 (d, J = 1.7 Hz, 6H), 1.68–1.58 (m, 5H); 13C-NMR (151 MHz, DMSO-d6): δ 171.8, 169.7, 167.0, 166.4, 162.3, 162.3, 158.3, 158.0, 154.8, 149.4, 136.0, 133.6, 126.5, 124.7, 123.9, 120.4, 117.0, 115.4, 112.4, 106.6, 51.1, 50.2, 49.4, 44.9, 43.9, 41.2, 35.8, 35.1, 34.3, 31.1, 30.8, 26.6, 21.2; HRMS (ESI) m/z calcd for C41H48N9O7 [M + H]+, 778.3671; found, 778.3677. HPLC purity: 95% (tR = 11.8 min).

PROTAC 7

1H-NMR (600 MHz, Methanol-d4): δ 7.73 (d, J = 8.3 Hz, 1H), 7.65 (d, J = 7.1 Hz, 2H), 7.56 (d, J = 9.3 Hz, 2H), 7.41 (d, J = 2.4 Hz, 1H), 7.28 (dd, J = 8.6, 2.4 Hz, 1H), 6.85 (t, J = 2.1 Hz, 1H), 6.44 (q, J = 1.8 Hz, 1H), 6.11 (dd, J = 4.0, 2.6 Hz, 1H), 5.11 (dd, J = 12.9, 5.3 Hz, 1H), 3.88–3.82 (m, 2H), 3.82 (t, J = 5.2 Hz, 6H), 3.76–3.71 (m, 7H), 3.66 (s, 3H), 3.64–3.59 (m, 5H), 3.55–3.53 (m, 1H), 3.35 (s, 2H), 3.14 (s, 3H), 2.89–2.87 (m, 2H), 2.23–2.03 (m, 5H); 13C-NMR (151 MHz, Methanol-d4): δ 173.7, 171.4, 169.3, 168.9, 165.5, 156.8, 135.6, 128.2, 126.1, 125.7, 123.2, 121.3, 119.5, 114.5, 109.5, 108.3, 69.6, 69.5, 51.1, 50.2, 50.1, 50.1, 50.0, 50.0, 49.9, 49.9, 49.8, 49.8, 49.7, 49.6, 49.6, 48.1, 45.9, 45.2, 42.6, 35.7, 32.5, 28.6, 27.3, 23.0; HRMS (ESI) m/z calcd for C41H48N9O7 [M + H]+, 778.3671; found, 778.3667. HPLC purity: 95% (tR = 11.5 min).

Biology

Reagents

Tissue culture plastics were purchased from Greiner Bio-One (Tokyo, Japan). Penicillin–Streptomycin Mixed Solution were from Nacalai (Kyoto, JAPAN). Roswell Park Memorial Institute-1640 (RPMI-1640) medium were from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS) was from Thermo Fisher Scientific (Waltham, MA, USA). MG132 was purchased from Peptide Institute (Osaka, Japan). MLN7243 was purchased from Active Biochem (Maplewood, NJ, USA). Pomalidomide was purchased from Cayman Chemical (Ann Arbor, 855 MI, USA). TFC-007 (ref. 30) and TAS-205 (ref. 31) were synthesized according to reported procedures.

Binding affinity assay

The fluorescence polarization-based (FP-based) binding assay was performed using Prostaglandin D Synthase (hematopoietic-type) FP-Based Inhibitor Screening Assay Kit Green (600007) (Cayman Chemical). In brief, the binding assays were performed in nonbinding black 384-well and used a recombinant human H-PGDS protein, glutathione, and fluorescence probe in assay buffer to produce a final volume of 47.5 μL. Then, 2.5 μL of test compounds made up as stocks in DMSO was added, and the plate was incubated for 1 h at room temperature. Each was tested against H-PGDS in triplicate at final test compound concentrations (2.50, 1.25 μM, 625, 313, 156, 78.1, 39.0, 19.5, 9.77, 4.88, and 2.44 nM). Plates were then read with excitation wavelengths (470 nm) and emission wavelengths (530 nm) on an EnVision Multilabel Plate Reader (PerkinElmer, Waltham, MA, USA). The measurements of fluorescence polarization of a molecule (mP) are taken in the fluorescence polarization mode. The percentage of inhibition of test compounds was calculated according to the following equationgraphic file with name d2md00284a-t1.jpgwhere mPsample is the value of the wells containing test compounds and mP100% is the value of the maximum binding well. The concentration of test compounds that reduces the mP value by 50% (IC50) was estimated from a graph plotted the mP value versus the concentration of the compounds on semi-log axis.

Cell culture

Human chronic myelogenous leukemia KU812 cells were cultured in RPMI-1640 medium supplemented with 10% FBS and 100 μg mL−1 penicillin–streptomycin mix. KU812 cells were obtained from the Japanese Collection of Research Bioresources (Osaka, Japan) Cell Bank (JCRB0104).32

Western blotting

Cells were lysed with SDS lysis buffer (0.1 M Tris–HCl at pH 8.0, 10% glycerol, 1% SDS) and immediately boiled for 5 min to obtain clear lysates. Protein concentrations were measured using the BCA method (Pierce, Washington, USA). Lysates containing equal amounts of proteins were separated by SDS-PAGE and transferred to PVDF membranes (Merck) for western blot analysis using the appropriate antibodies. Immunoreactive proteins were visualized using the Clarity Western ECL substrate (Bio-Rad, California, USA); light emission intensity was quantified using a ChemiDoc MP Imaging System equipped with Image Lab™ Software (Bio RAD, California, USA). The antibodies used in this study were anti-H-PGDS rabbit polyclonal antibody33 and anti-β-actin mouse mAb (A2228) (Sigma-Aldrich). KU812 cells were treated with the indicated concentrations of compounds for 6 h. Whole-cell lysates were analyzed by western blotting with the indicated antibodies. Numbers below the H-PGDS panels represent H-PGDS/actin, normalized by designating the expression from the vehicle control condition as 100%. The DC50 values were determined by assuming a linear fit between the three data points that span the 50% protein level.

Measurement of PGD2 levels

The PGD2 level in the culture medium was measured by using the ELISA Prostaglandin D2-MOX ELISA Kit (512011) (Cayman Chemicals). KU812 cells were treated with the indicated compounds for 24 h and stimulated with 5 μM calcium ionophore (A23187) (Merck KGaA, Darmstadt, Germany) for 30 min. PGD2 levels in the medium were measured according to the manufacturer's instructions.

Computational analysis

The MD simulations were performed using Molecular Operating Environment (MOE) 2022.02. All the simulations and analysis were calculated using the Amber10: EHT force field. Docking poses of the ternary complex were selected with criteria such as rmsd < 2.5 Å, RMSD_RF_PP < 670 Å2, ligand_E < 3600 kcal mol−1, and ligand_E_PP < Å2. A two-dimensional interaction model was produced with criteria such as H-bond < −0.5 kcal mol−1, ionic < −0.5 kcal mol−1, and distance > 4.5 Å. Glutathione was added to the conformation of docking simulation in the same pose as the crystal structure (PDB: 5YWX). Water molecules were added, and the system was neutralized by the addition of NaCl salt of 0.1 mol L−1 concentration. A periodic boundary condition was utilized to carry out the simulation. In all cases, the cell size was larger than the protein by 10 Å. The temperature of the simulation was controlled by the NAMD. The initial energy minimization process of each simulation was performed by standard protocols in MOE 2022.02. The simulation speed was maintained at 2 fs time step during all the simulations. The system was saved in every 0.5 ps during simulations. First, 10 ps time evolution was performed with a temperature 0 K (minimization step). Next, 100 ps time evolution was performed with a temperature 10–300 K and tether 0.5–100 Å (equilibration step). Then, 100 ps time evolution was performed with a temperature of 300 K (production step). After completion of the MD simulation for all systems, the results were analyzed in MOE 2022.02. For each result, the most stable structure in the production step was selected and calculated ligand interactions and potential energy.

Author contributions

Y. M., H. O., and T. K. contributed equally to this work. T. K., H. Y., N. S., Y. D. designed the research. Y. M., H. O., T. K., H. Y., Y. Y., T. I. performed the experiments and analyzed results. Y. M., H. O., T. K., M. N., K. A., Y. D. wrote and edited the paper. All authors discussed the results and commented on the manuscript.

Conflicts of interest

The authors declare no competing financial interest.

Supplementary Material

MD-013-D2MD00284A-s001

Acknowledgments

We would like to express our deepest appreciation to Dr. Kentaro Kamiya (MOLSIS Inc.) for his help in the PROTAC modelling and MD simulation. This study was supported in part by grants from the Japan Agency for Medical Research and Development (22mk0101197j0002, 22fk0210110j0401, and 22fk0310506j0701 to Y. D.), and Japan Society for the Promotion of Science and the Ministry of Education, Culture, Sports, Science and Technology (JSPS/MEXT KAKENHI Grants Number JP18H05502 to Y. D. and M. N.; 21K05320 to Y. D.; JP21K06075 to N. S.). We thank Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript.

†

Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d2md00284a

Notes and references

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Supplementary Materials

MD-013-D2MD00284A-s001
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