Protein Degradation via CRL4CRBN Ubiquitin Ligase: Discovery and

Article pubs.acs.org/jmc

Protein Degradation via CRL4CRBN Ubiquitin Ligase: Discovery and Structure−Activity Relationships of Novel Glutarimide Analogs That Promote Degradation of Aiolos and/or GSPT1 Joshua D. Hansen,* Kevin Condroski, Matthew Correa, George Muller, Hon-Wah Man, Alexander Ruchelman, Weihong Zhang, Fan Vocanson, Tim Crea, Wei Liu, Gang Lu, Frans Baculi, Laurie LeBrun, Afshin Mahmoudi, Gilles Carmel, Matt Hickman, and Chin-Chun Lu Celgene Corporation, 10300 Campus Point Drive, Suite 100, San Diego, California 92121, United States S Supporting Information *

ABSTRACT: We previously disclosed the identification of cereblon modulator 3 (CC-885), with potent antitumor activity mediated through the degradation of GSPT1. We describe herein the structure−activity relationships for analogs of 3 with exploration of the structurally related dioxoisoindoline class. The observed activity of protein degradation could in part be rationalized through docking into the previously disclosed 3−CRBN−GSPT1 cocrystal ternary complex. For SAR that could not be rationalized through the cocrystal complex, we sought to predict SAR through a QSAR model developed in house. Through these analyses, selective protein degradation could be achieved between the two proteins of interest, GSPT1 and Aiolos.

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INTRODUCTION

and ubiquitination of substrate proteins to the cullin-damaged DNA-binding-RING box-domain protein (CUL4-DDB1RBX1-CRBN) or simply (CRL4CRBN) E3 ubiquitin ligase, with the resulting ubiquitin tagged proteins directed to and subsequently degraded by the 26S proteasome.1−3 Recently, we described the identification of 3 (CC-885), a novel cereblon (CRBN) modulator which was demonstrated to mediate antitumor effects through the recruitment and degradation of G1 to S phase transition 1 protein (GSPT1).4 GSPT1 (also named eRF3a) is a translation termination factor that binds eukaryotic translation termination factor 1 (eFR1) to mediate stop codon recognition and nascent protein release from the ribosome.5−7 Unlike 3, the currently marketed class of immunomodulatory drugs, which include 1 (lenalidomide) and 2 (pomalidomide), do not promote the degradation of GSPT1, demonstrating selectivity favoring zinc finger transcription factors such as Aiolos (IKZF3) and Ikaros (IKZF1). Selectivity in protein degradation has also been reported between lenalidomide and pomalidomide in the comparison of the protein kinase casein kinase 1α (CK1α) where recruitment and degradation are key features to the use of lenalidomide in 5q-deletion-associated myelodysplastic syndrome.8 To investigate the structure−activity requirements and constraints

Induction of protein degradation as a therapeutic strategy has been clinically validated by the class of immunomodulatory drugs which include lenalidomide and pomalidomide (Figure 1). The therapeutic benefits of immunomodulatory drug treatment are connected to their ability to promote recruitment

Special Issue: Inducing Protein Degradation as a Therapeutic Strategy Figure 1. Aiolos and GSPT1 protein degradation by lenalidomide, pomalidomide, and 3. © 2017 American Chemical Society

Received: January 4, 2017 Published: March 30, 2017 492

DOI: 10.1021/acs.jmedchem.6b01911 J. Med. Chem. 2018, 61, 492−503

Journal of Medicinal Chemistry

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governing the selectivity of protein recruitment and degradation for GSPT1/Aiolos, we examined protein degradation using a traditional measurement (EC50) as well as the measure of maximal protein degradation (Ymax) that is induced by compound over a concentration range up to 10 μM (Figure 2) using an enhanced-ProLable (ePL) degradation assay (described in Experimental Section). For analog comparison, the observed level of protein degradation induced by 2 for Aiolos and by 3 for GSPT1 was used as our reference and was set to Ymax = 0, respectively. Since the data were not normalized, a negative Ymax is possible and indicates a greater extent of protein degradation than the control compound reference. The ePL degradation assay was the general method used to generate and interpret SAR described herein. Additionally, protein degradation was examined via Western blot analysis for select compounds (Figure 3). All compounds tested demonstrated comparable efficiency and potency in the degradation of ePL-tagged exogenous GSPT1 and/or Aiolos proteins versus degradation of endogenous GSPT1 and Aiolos proteins measured by Western blot analysis.

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CHEMISTRY The synthesis of urea analogs 7−23 in Scheme 1 began with the Fischer esterification of 4-bromo-2-methylbenzoic acid (4) followed by radical bromination of the tolyl methyl.9 Formation of the bicyclic lactam was accomplished using 3-aminopiperidine-2,6-dione as the nucleophile for bromide displacement followed by further condensation onto the ester to form lactam 5. The requisite aminomethyl functionality was installed via palladium-mediated cyanide insertion to the aryl bromide with subsequent reduction to yield 6. Oxoisoindoline 6 provided a convenient scaffold from which to explore SAR through further elaborations such as arylurea formation shown in Scheme 1.

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RESULTS AND DISCUSSION Concurrent with our oxoisoindoline SAR efforts, we explored alternative scaffolds such as the dioxoisoindoline. In comparison of benchmark urea analogs, shown in Table 1, a clear trend could be observed between the two scaffolds. For example, the unsubstituted phenylurea 7 was able to degrade GSPT1 with Ymax of 9 (Ymax = 9 corresponds to 91% of protein degraded compared to control compound 3 [Figure 2]) while the unsubstituted phenyl urea in the dioxoisoindoline series (8) was less efficient at GSPT1 protein degradation with Ymax = 34. This same trend held for Aiolos, where 7 could promote ∼7% more degradation than control compound pomalidomide (2) giving it a negative numerical value (−7), while 8 could only degrade 56% Aiolos protein (Ymax = 44) as compared to control. Further analysis of the 2-, 3-, or 4-substituted chlorophenyls of the oxoisoindolines (9, 11, and 13) compared to the corresponding dioxoisoindolines (10, 12, and 14) suggests that dioxoisoindolines were in general less efficient protein degraders of both GSPT1 and Aiolos. Comparison between the degree of GSPT1 and Aiolos degradation in analogs 9, 11, and 13 revealed an interesting selectivity pattern of maximal degradation between the two proteins. For example, while the 2-chloro substitution in 9 showed comparable GSPT1 and Aiolos degradation, selectivity was observed in the 3-chloro analog 11 which displayed increased GSPT1 degradation with 18% more GSPT1 degraded than Aiolos (standard error of measure (SEM) for 11 Ymax; GSPT1 = −6 ± 1.0, Aiolos =12 ±

Figure 2. (A) GSPT1 protein degradation curves of 2 (pomalidomide) and 3. (B) Aiolos protein degradation curves of 2 and 3. (C) GSPT1 protein degradation curves of three example compounds to illustrate low levels of protein degradation (high Ymax, 15) and high level of protein degradation (low or negative Ymax, compounds 3 and 10).

1.8; see Supporting Information for all SEM data). A reversal in selectively favoring Aiolos degradation was observed with 4-Cl493



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Figure 3. Comparison protein degradation for selected compounds between the ePL degradation assay and Western blot. (A) OPM2 cells were treated with DMSO, 3, 8, 26, 13, 19, and 22 at the indicated concentration for 8 h, and whole cell extracts were subjected to immunoblot analysis. (B) Table of Ymax values at 10 μM of the selected compounds for comparison to the Western blot analysis.

Scheme 1. Synthesis of 5-yl Substituted Oxoisoindolinesa

Reagents and conditions: (a) MeOH−H2SO4 (50:1), 65 °C, 18 h, 95%; (b) NBS, azo-isobutyronitrile, 85 °C, 18 h, ACN, 66%; (c) 3aminopiperidine-2,6-dione·HCl, triethylamine, rt for 25 h, 44%; (d) 1,1′-bis(diphenylphosphino)ferrocene, ZnCN, ZnOAc, Pd2(dba)3, 120 °C for 20 h, 57%; (e) MsOH, Pd/C, DMA, 50 psi, 40 °C for 20 h, 40%; (f) substituted anilines, CDI, DMF, 80 °C for 16 h, 8−80%. a

Compound 17 was synthesized to confirm the expected loss in potency as predicted by the observed binding mode of the glutarimide moiety in the tritryptophan pocket (Figure 4),10 Methylation of the glutarimide would be expected to disrupt the hydrogen bond network in the glutarimide moiety from H378 to W380, and indeed compound 17 showed a loss of binding to CRBN (data not shown). This disruption of CRBN binding was manifest in reduced levels of protein degradation, demonstrating >1000-fold loss of activity for both GSPT1 and Aiolos EC50 (16 versus 17). The SARs of disubstituted aryl chlorides (18, 19, 22, and 23) were also examined. In comparison to the 2-chloro 9, the 2,4-dichloro 18 showed improved potency and efficiency of degradation for both Aiolos and GSPT1, similar to that observed in the 4-chloro analog 13. The di-2,6-chloro 19 showed comparable GSPT1 potency to 18 but with a marked reduction in maximal degradation. While the potency between 18 and 19 on GSPT1 were comparable, it was interesting to note that 19 demonstrated a 100-fold loss of

derivative 13. To further explore the impact of substitution at the para position, nitrile analog 15 was synthesized and demonstrated an unexpected reversal in selectivity compared to 13, with significant loss in GSPT1 degradation. Although the apparent alterations in selectivity caused by these subtle structural changes could not be readily rationalized through analysis of the X-ray structure, we were encouraged by the apparent potential to discover selective protein degraders suggested by the differentiated selectivity patterns revealed through this series of compounds. With these encouraging results, we sought to further explore substitution on the aryl ring in the oxoisoindoline series, probing potency and selectivity. Highlights of these efforts are outlined in Table 2. When the 4-substituent was changed from chloro (13) to fluoro (16), we observed that the GSPT1 potency (EC50) was equivalent between 13 and 16, and 16 showed a slight loss in ability to degrade protein (Ymax of −2 versus 5). 494



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Table 1. SAR of GSPT1 and Aiolos Protein Degradation Mediated by Compounds in the Oxoisoindoline and Dioxoisoindoline Scaffoldsa,b,c

a All values are the mean of at least three separate assay determinations. bSEM for data available in Supporting Information. cYmax is the % protein remaining at 10 μM, 4 h (see Experimental Section).

Aiolos potency and a large shift for Aiolos maximal degradation (Ymax = 70). A similar trend for GSPT1 degradation selectivity was observed with 2,4,6-trichloro 20; the Aiolos Ymax is high for both 20 and 19 (61/70), although the 4-chloro functionality in 20 might be responsible for the increased GSPT1 activity since both 13 and 18 are highly efficient GSPT1 degraders. This trend of potent GSPT1 activity with a 4-chloro substituent was also observed in 23. While analog 22 with both meta positions

substituted also showed strong GSPT1 degradation, selectivity favoring GSPT1 over Aiolos degradation was observed and to a greater extent than the 3-Cl analog 11. Comparison of the Aiolos results for 19 and 20, which achieve similar levels of maximal degradation (Ymax= 70 and 61, respectively) but show ∼13-fold potency differential (EC50 = 0.32 and 0.02 μM, respectively), further highlights the SAR subtleties of protein degradation. When a large substituent such as phenyl was 495



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Table 2. SAR of GSPT1 and Aiolos Protein Degradationa,b,c

a

All values are the mean of at least three separate assay determinations. bSEM for data available in Supporting Information. cYmax is the % protein remaining at 10 μM, 4 h (see Experimental Section). dNC (not calculated) due to minimal substrate degradation and lack of a sigmoidal curve.

placed in the ortho position (21), we observed selectivity toward more complete GSPT1 degradation (Ymax = 2.2) compared to Aiolos (Ymax = 61). This trend was also noted in compounds 19 and 20, which both contain di-orthosubstitution at terminal phenyl indicating a potential rationale for the selectivity based on an expected conformational restriction of rotation about the phenyl-NH-urea bond.

Our previously reported 3 bound cocrystal structure with CRBN4 suggested the hydrogen bonding networks of the urea carbonyl to CRBN histidine (H353) and of the urea NH to glutamate (E377) were important binding features (Figure 4). To further explore this observation, we made a series of capped urea derivatives (24−26) and noted that methylation of the distal urea nitrogen (24) led to a 44-fold loss in GSPT1 degradation potency and significant loss in maximal degrada496



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Figure 4. Poses of docked 3 (gray) and 3 from x-ray crystallographic structure (green) correspond well. Hydrogen bonding network between cereblon and 3 (W386 omitted for clarity). (Protein Data Bank accession code 5HXB).

Table 3. SAR of GSPT1 and Aiolos Protein Degradationa,b,c

a All values are the mean of at least three separate assay determinations. bSEM for data available in Supporting Information. cYmax is the % protein remaining at 10 μM, 4 h (see Experimental Section). dNC (not calculated) due to minimal substrate degradation and lack of a sigmoidal curve.

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module in Optibrium’s StarDrop software.11 We began by dividing the data sets into training (70%), validation (15%), and test sets (15%) of compounds, each chosen randomly to eliminate any compound selection bias. A library of 321 SMARTS based descriptors (counts of atom types and functionalities)12 and 9 whole-molecule properties were generated within StarDrop for each compound in these data sets. Category models were then constructed using random forest (RF) ensemble methods with predictions based upon the output of a collection of 100 random trees;13 these were found to provide superior accuracy for predictions of GSPT1 and Aiolos EC50 and Ymax when compared to models built with recursive partitioning approaches using decision trees14 and Gaussian processes.15 QSAR Model Results. Binning cutoff values for the categorical models were chosen in order to maximize the accuracy, sensitivity, and specificity of each model over the activity ranges of our data sets (Table 4). The category matrix

tion levels (Ymax = 50, compared to 3 Ymax = 0). Substitution at the proximal urea nitrogen (25) was less disruptive, showing only a 10-fold shift in GSPT1 potency with the ability to degrade 77% of protein levels compared to control. While both of these derivatives can maintain a portion of the observed Hbond interactions with E377, compound 26, with methylation at both urea nitrogens, cannot make this interaction with the protein and we observed a complete lack of ability to degrade either substrate, even at concentrations up to 10 μM. The lack of activity for bis-methylated urea 26 was consistent with the cyclic urea analog 27 which also showed loss of activity on GSPT1. Interestingly, the Aiolos potency was not significantly impacted in 27, providing further evidence for the potential for Aiolos/GSPT1 selectivity. To further explore this result, we synthesized the five-membered cyclic urea 28 which, surprisingly, displayed a selectivity toward GSPT1 degradation. SAR studies in the cyclic ureas led to the identification of 29, which displayed enhanced GSPT1 potency and substantially increased protein degradation (∼88% compared to 28, 48%). Since the activity of 29 was difficult to explain via the crystal structure binding mode, we next explored computational methods as a means to rationalize anomalous GSPT1 activity. Docking Studies. To further understand the underlying structure−activity relationships for GSPT1 recruitment by small molecule CRBN modulators, we considered potential binding modes for analogs of 3. The X-ray crystallographic structure4 of the complex of human CRBN, DNA damage binding protein 1 (DDB1), GSPT1, and 3 (Protein Data Bank accession code 5HXB) was used for docking studies using the Glide software package from Schrödinger, LLC. The docked pose of 3 in the interfacial pocket between CRBN and GSPT1 was observed to reproduce the crystal structure binding mode accurately (Figure 4). The compounds in Tables 1 and 2 were found to dock in a similar fashion to 3, each forming a trio of hydrogen bonds that anchor the glutarimide moieties in the tri-Trp binding site. The one notable exception as discussed previously was the Nmethylation of the glutarimide in 17, which disrupts the hydrogen bonding network, significantly reducing its ability to recruit and degrade GSPT1. Compounds in Table 3 investigate the effects of N-alkylation of the urea, which are all deleterious to GSPT1 degradation activity to varying extents. Docking studies demonstrated either significantly altered binding modes due to disruption of one or more hydrogen bonds between the urea moiety and residues E377 and H353. As previously noted, compound 29 recovered significant GSPT1 degradation ability with the incorporation of the m-phenoxyphenyl ether, and docking studies did not adequately explain this observation. Quantitative Structure−Activity Relationship (QSAR) Model Construction. While docking studies utilizing the existing GSPT1 complex cocrystal structure provide insights into the SAR revealed by this congeneric series of molecules, there is currently no analogous structure of the complex of Aiolos and CRBN with a small molecule bound. Since we desired to construct models that could serve our efforts in a predictive capacity for both GSPT1 and Aiolos degradation, we turned to the wealth of GSPT1 and Aiolos assay data collected for our in-house compound library and used it to develop statistical models. An initial collection of more than 1500 GSPT1 and Aiolos EC50 and Ymax pairs of assay measurements were used to construct a categorical QSAR model using the Auto-Modeler

Table 4. QSAR Models Constructed for Aiolos and GSPT1 EC50 and Ymax model

binning cutoff

units

overall accuracy (test set) (%)

Aiolos EC50 Aiolos Ymax GSPT1 EC50 GSPT1 Ymax

21 10 71 29

nM % control at 4 h nM % control at 4 h

82 80 79 79

for the RF model for Aiolos EC50 with a binning cutoff of 21 nM is shown in Table 5, with predicted categories on the y-axis Table 5. RF Model for Aiolos EC50 with a Binning Cutoff of 21 nM (Test Set)

compared with experimentally observed categories on the xaxis. The overall accuracy is 82% when the model is applied to the test set. A similar plot for Aiolos Ymax data demonstrates an overall accuracy of 80% with a binning cutoff of 10% protein remaining (Table 6). In the case of GSPT1 (Tables 7 and 8), the overall accuracies for EC50 and Ymax were 79%, slightly lower in accuracy than the corresponding Aiolos models. The successful application of these models was demonstrated, as they were applied to more than 1000 additional analogs prepared subsequent to model construction. Aiolos Table 6. RF Model for Aiolos Ymax with Binning Cutoff of 10% (Test Set)

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activity continuum. The subtleties of substitution pattern SAR offer the opportunity to discover compounds across a varied spectrum of maximal degradation, potency, and selectivity, which we hope can lead to profiles of tuned degradation profiles to derive maximal clinical benefit.

Table 7. RF Model for GSPT1 EC50 with a Binning Cutoff of 71 nM (Test Set)

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EXPERIMENTAL SECTION

General. Compounds were named using ChemDraw Ultra. All materials were obtained from commercial sources and used without further purification unless otherwise noted. Chromatography solvents were HPLC grade and used as purchased. All air-sensitive reactions were carried out under a positive pressure of an inert nitrogen atmosphere. Chemical shifts (δ) are reported in ppm downfield of TMS, and coupling constants (J) are given in Hz. Thin layer chromatography (TLC) analysis was performed on Whatman thin layer plates. The purity of final tested compounds was typically determined to be ≥95% by HPLC. Compounds were analyzed for purity using the following method: gradient (5−95% acetonitrile + 0.075% formic acid in water + 0.1% formic acid over 8 min, followed by 95% acetonitrile + 0.075% formic acid for 2 min); flow rate 1 mL/ min, column Phenomenex Luna 5 μm PFP(2) 100A (150 mm × 4.60 mm). Elemental analysis was performed at Robertson Microlit Laboratories, Ledgewood, NJ. Synthesis. Compounds 4−16, 18−20, and 22−23 have been previously described in the patent literature.24,25 1-(4-Fluorophenyl)-3-((2-(1-methyl-2,6-dioxopiperidin-3-yl)1-oxoisoindolin-5-yl)methyl)urea (17). To a 0 °C stirred solution of 16 (295 mg, 0.71 mmol) in DMF (15 mL) was added cesium carbonate (468 mg, 1.43 mmol) followed by dropwise addition of methyl iodide (306 mg, 2.15 mmol). The reaction mixture was stirred for 16 h at room temperature. The reaction mixture was poured into ice cold water (100 mL) and stirred for 30 min. The precipitate was filtered and dried under vacuum. The crude product was purified by reverse phase HPLC to give the title compound (110 mg, 0.26 mmol, 36% yield, HPLC purity >99%). 1H NMR (400 MHz, DMSO-d6) δ 8.66 (s, 1H), 7.70 (d, J = 7.8 Hz, 1H), 7.52 (s, 1H), 7.47−7.38 (m, 3H), 7.06 (t, J = 8.9 Hz, 2H), 6.73 (t, J = 6.1 Hz, 1H), 5.17 (dd, J = 13.4, 5.0 Hz, 1H), 4.55−4.16 (m, 4H), 3.02−2.92 (m, 4H), 2.62−2.56 (m, 1H), 2.48−2.26 (m, 1H), 2.03−1.91 (m, 1H); MS (ESI) m/z 425.19 [M + 1]+. 1-([1,1′-Biphenyl]-2-yl)-3-((2-(2,6-Dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)urea (21). Step A. To a stirred solution of [1,1′-biphenyl]-2-amine (0.5 g, 2.95 mmol) in dichloromethane (30 mL) were added pyridine (0.6 mL, 8.85 mmol) and phenyl carbonochloridate (0.4 mL, 3.54 mmol) at 0 °C. The reaction was stirred at room temperature for 30 min. The reaction was diluted with water and extracted with dichloromethane. The combined organic layers were dried over anhydrous sodium sulfate, concentrated under reduced pressure and the residue obtained was purified by silica gel chromatography to give phenyl [1,1′-biphenyl]-2-ylcarbamate (450 mg, 1.33 mmol, 60% yield). MS (ESI) m/z 290.30 [M + 1]+. Step B. To a stirred solution of phenyl [1,1′-biphenyl]-2ylcarbamate (0.3 g, 1.00 mmol) in DMF (10 mL) was added 5 (309 mg, 0.84 mmol) at 0 °C. The reaction was stirred at 100 °C for 1 h. The reaction mixture was diluted with water, and the resulting precipitate was collected by vacuum filtration. The obtained crude compound was purified by reverse phase HPLC to give 1-([1,1′biphenyl]-2-yl)-3-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)urea (21) (80 mg, 0.17 mmol, 20% yield, HPLC purity >99%). 1 H NMR (300 MHz, DMSO-d6) δ 10.98 (s, 1H), 7.87 (d, J = 8.2 Hz, 1H), 7.68 (d, J = 7.8 Hz, 1H), 7.55−7.33 (m, 7H), 7.32−7.23 (m, 1H), 7.21−7.03 (m, 3H), 5.11 (dd, J = 13.2, 5.1 Hz, 1H), 4.64−4.11 (m, 4H), 2.89−2.87 (m, 1H), 2.60−2.58 (m, 1H), 2.41−2.39 (m, 1H), 2.01−1.99 (m, 1H); MS (ESI) m/z 469.041 [M + 1]+. 1-(3-Chloro-4-methylphenyl)-3-((2-(2,6-dioxopiperidin-3-yl)1-oxoisoindolin-5-yl)methyl)-1-methylurea (24). To stirred solution of 6 (0.4 g, 1.08 mmol) in DMF (10 mL) at room temperature was added 1,1′-carbonyldiimidazole (210 mg, 1.30 mmol), and the mixture was stirred for 1 h. To the reaction mixture

Table 8. RF Model for GSPT1 Ymax with Binning Cutoff of 29% (Test Set)

EC50 and Ymax predictions demonstrated similar performance to our earlier test set assessments, as did those for GSPT1 Ymax, while GSPT1 EC50 predictions demonstrated modest accuracy (see Supporting Information). The predictive capacity of these models has allowed us to thoughtfully and efficiently prioritize the synthesis of selected analogs from large enumerated libraries of proposed compounds.

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CONCLUSIONS The ability to affect protein homeostasis and its association with disease state through targeted protein degradation has exciting implications for drug discovery. In contrast to chimerically linked protein degraders16−18 as a method to utilize the CRBN ligase system, urea-based analogs of 3 can be used to create an interaction hotspot on the surface of CRBN for direct protein−protein interactions.4 We employed a dual strategy of structure-based design and QSAR modeling to more completely rationalize SAR investigations in two series of urea analogs that both demonstrated the ability to recruit and induce degradation of Aiolos and/or GSPT1 protein. During the course of these SAR explorations we observed several categories of activity. This included compounds where Aiolos and GSPT1 were equally degraded but with either low or high potency, as well as profiles that displayed comparable potency but differentiated levels of protein degradation (Aiolos degradation of 7 versus 9). The apparent disconnect between potency and efficiency of protein degradation was not an uncommon observation across the series and may reveal insights into the inherent complexity in the SAR of protein degradation. For example, we measure protein degradation as the sum end point of a process that includes multiple variable steps including (1) compound binding to CRBN, forming the “protein hotspot” or basis for “molecular glue”,19−21 (2) formation of the ternary complex between the CRBN bound compound and substrate such as GSPT1,4 (3) transfer of ubiquitin from the E2, (4) disassociation of ubiquitinated substrate from the ternary complex, (5) potential reverse competition with deubiquitinating enzymes (DUBs), and (6) trafficking to the 26S proteasome for degradation. These steps underpin the currently understood mechanism of action for UPS-dependent protein degradation,3,22,23 and 3 was previously shown to induce protein degradation in a CRBN as well as UPS-dependent manner.4 Compounds with selectivity toward either Aiolos or GSPT1 degradation were noted, also across an 499



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(d, J = 7.8 Hz, 1H), 5.02 (s, 2H), 4.42 (s, 2H), 4.32 (t, J = 7.1 Hz, 2H), 2.81 (s, 3H), 1.46−1.28 (m, 12H). Step F. To a stirred solution of ethyl 2-(bromomethyl)-4-((tertbutoxycarbonyl(methyl)amino)methyl)benzoate (0.7 g, 1.88 mmol) in DMF (20 mL) were added 3-aminopiperidine-2,6-dione hydrochloride (0.3 g, 1.88 mmol) and triethylamine (0.7 mL, 4.70 mmol). The reaction was stirred at 80 °C for 12 h. The reaction mixture was diluted with water and extracted with ethyl acetate. The combined organic layers were washed with water, brine, dried over sodium sulfate, and concentrated under reduced pressure. The resultant residue was purified by silica gel chromatography to give tert-butyl (2(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl(methyl)carbamate (0.4 g, 1.23 mmol, 60% yield). MS (ESI) m/z 388.35 [M + 1]+. Step G. A solution of ethyl tert-butyl (2-(2,6-dioxopiperidin-3-yl)-1oxoisoindolin-5-yl)methyl(methyl)carbamate (0.2 g, 0.61 mmol) in 4 M hydrochloric acid in 1,4-dioxane (5 mL) was stirred at 0 °C for 2 h. The reaction mixture was concentrated under reduced pressure to give 3-(5-((methylamino)methyl)-1-oxoisoindolin-2-yl)piperidine-2,6dione hydrochloride (130 mg, 0.58 mmol, 90% yield). 1H NMR (300 MHz, DMSO-d6) δ 11.00 (s, 1H), 9.11 (s, 2H), 7.81 (d, J = 7.9 Hz, 1H), 7.74 (s, 1H), 7.65 (dd, J = 7.7, 1.5 Hz, 1H), 5.14 (dd, J = 13.2, 5.1 Hz, 1H), 4.56−4.30 (m, 2H), 4.25 (s, 2H), 2.93 (ddd, J = 17.9, 13.4, 5.3 Hz, 1H), 2.56 (s, 4H), 2.40 (dd, J = 13.4, 9.1 Hz, 1H), 2.08− 1.91 (m, 1H); MS (ESI) m/z 288.4 [M + 1]+. Step H. To a 0 °C solution of 3-chloro-4-methylaniline (2.0 g, 14.12 mmol) and phenyl carbonochloridate (2.1 mL, 42.37 mmol) in dichloromethane (30 mL) was added pyridine (3 mL, 16.24 mmol), and the mixture was stirred at 0 °C for 2 h. The reaction mixture was diluted with water and extracted with dichloromethane. The combined organic layers were washed with water, brine, dried over sodium sulfate, and concentrated. The resultant residue was purified by silica gel chromatography to give phenyl 3-chloro-4-methylphenylcarbamate. (1.2 g, 4.59 mmol, 37% yield). MS (ESI) m/z 262.18 [M + 1]+. Step I. To a stirred solution of 3-(5-((methylamino)methyl)-1oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride (0.1 g, 0.30 mmol) in DMF (5 mL) were added the phenyl 3-chloro-4methylphenylcarbamate (50 mg, 0.36 mmol) and triethylamine (0.1 mL, 0.90 mmol). The reaction was stirred at 80 °C for 2 h. The reaction mixture was poured in ice cold water, and solid precipitate was collected by vacuum filtration. The collected precipitate was purified by reverse phase HPLC to give 3-(3-chloro-4-methylphenyl)1-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)-1-methylurea (25) (20 mg, 0.04 mmol, 14% yield, HPLC purity >99%). 1H NMR (400 MHz, DMSO-d6) δ 10.98 (s, 1H), 8.53 (s, 1H), 7.89−7.63 (m, 2H), 7.48 (s, 1H), 7.39 (dd, J = 14.5, 8.1 Hz, 2H), 7.20 (d, J = 8.4 Hz, 1H), 5.11 (dd, J = 13.7, 5.1 Hz, 1H), 4.66 (s, 2H), 4.46 (d, J = 17.4 Hz, 1H), 4.32 (d, J = 17.2 Hz, 1H), 2.95 (s, 4H), 2.60 (d, J = 18.5 Hz, 1H), 2.44−2.30 (m, 1H), 2.25 (s, 3H), 2.00 (d, J = 12.4 Hz, 1H); MS (ESI) m/z 455.16 [M + 1]+. 1-(3-Chloro-4-methylphenyl)-3-((2-(2,6-dioxopiperidin-3-yl)1-oxoisoindolin-5-yl)methyl)-1,3-dimethylurea (26). To a stirred solution of 3-chloro-N,4-dimethylaniline (120 mg, 0.37 mmol) in tetrahydrofuran (10 mL) were added triethylamine (0.2 mL, 1.11 mmol) and triphosgene (43 mg, 0.14 mmol) at 0 °C, and the mixture was stirred at room temperature for 2 h. To the reaction mixture was added 3-(5-((methylamino)methyl)-1-oxoisoindolin-2-yl)piperidine2,6-dione hydrochloride (43 mg, 0.14 mmol), and the mixture was stirred at room temperature for 5 h. The reaction mixture was poured into ice cold water and the desired product precipitated as a solid. The resulting precipitate was dried and purified by reverse phase HPLC to give the title compound (20 mg, 0.04 mmol, 12% yield, HPLC purity >96%). 1H NMR (300 MHz, DMSO-d6) δ 10.98 (s, 1H), 7.69 (d, J = 7.8 Hz, 1H), 7.44 (s, 1H), 7.39−7.33 (m, 1H), 7.28 (d, J = 8.3 Hz, 1H), 7.12 (d, J = 2.3 Hz, 1H), 6.99 (dd, J = 8.2, 2.3 Hz, 1H), 5.11 (dd, J = 13.2, 5.1 Hz, 1H), 4.69−4.18 (m, 4H), 3.32 (s, 3H), 3.10 (s, 3H), 2.92 (m, 1H), 2.60 (d, J = 18.1 Hz, 1H), 2.40 (dd, J = 13.2, 4.4 Hz, 1H), 2.25 (s, 3H), 2.07−1.91 (m, 1H); MS (ESI) m/z 468.97 [M + 1]+.

was added 3-chloro-N,4-dimethylaniline (201 mg, 1.30 mmol), and the mixture was heated at 80 °C for 12 h. The reaction mixture was concentrated under reduced pressure and the resultant residue was purified by reverse phase HPLC to give the title compound (90 mg, 0.19 mmol, 18% yield, HPLC purity >99%). 1H NMR (400 MHz, DMSO-d6) δ 10.98 (s, 1H), 7.66 (d, J = 7.8 Hz, 1H), 7.46 (s, 1H), 7.42−7.30 (m, 3H), 7.17 (dd, J = 8.1, 2.2 Hz, 1H), 6.86 (t, J = 5.9 Hz, 1H), 5.10 (dd, J = 13.3, 5.1 Hz, 1H), 4.57−4.08 (m, 4H), 3.16 (s, 3H), 2.91 (ddd, J = 17.7, 13.5, 5.3 Hz, 1H), 2.65−2.54 (m, 1H), 2.45−2.34 (m, 1H), 2.31 (s, 3H), 2.05−1.95 (m, 1H); MS (ESI) m/z 454.03 [M + 1]+. 3-(3-Chloro-4-methylphenyl)-1-((2-(2,6-dioxopiperidin-3-yl)1-oxoisoindolin-5-yl)methyl)-1-methylurea (25). Step A. To a stirred solution of 1-oxo-1,3-dihydroisobenzofuran-5-carbonitrile (10.0 g, 62.85 mmol) in N-methyl-2-pyrrolidone (100 mL) were added ditert-butyl dicarbonate (27.0 g, 125.70 mmol), sodium borohydride (7.1 g, 188.55 mmol), and nickel chloride hexahydrate (60.0 g, 251.40 mmol) at 0 °C, and the mixture was stirred at room temperature for 12 h. The reaction mixture was diluted with water and extracted with ethyl acetate. The combined organic layers were washed with water, brine, dried over sodium sulfate, and concentrated under reduced pressure. The resultant residue was purified by silica gel chromatography to give tert-butyl ((1-oxo-1,3-dihydroisobenzofuran-5-yl)methyl)carbamate (9.0 g, 34.22 mmol, 56% yield). MS (ESI) m/z 264.22 [M + 1]+. Step B. To a stirred solution of tert-butyl (1-oxo-1,3dihydroisobenzofuran-5-yl)methylcarbamate (5.0 g, 19.04 mmol) in DMF (50 mL) was added sodium hydride (685 mg, 28.56 mmol) at 0 °C, and the mixture was stirred for 30 min. Methyl iodide (3.5 mL, 57.0 mmol) in DMF was added, and the mixture was stirred at room temperature for 2 h. The reaction mixture was diluted with water and extracted with ethyl acetate. The combined organic layers were washed with water, brine, dried over sodium sulfate, and concentrated under reduced pressure. The resultant residue was purified by silica gel chromatography to give tert-butyl methyl((1-oxo-1,3-dihydroisobenzofuran-5-yl)methyl)carbamate (3.0 g, 10.80 mmol, 57% yield). MS (ESI) m/z 278.26 [M + 1]+. Step C. To a stirred solution of tert-butyl methyl((1-oxo-1,3dihydroisobenzofuran-5-yl)methyl)carbamate (1.0 g, 3.61 mmol) in THF (10 mL) was added 2 M solution of sodium hydroxide (10 mL) at 0 °C, and the mixture was stirred at room temperature for 5 h. The reaction mixture was concentrated under reduce pressure to give sodium 4-((tert-butoxycarbonyl(methyl)amino)methyl)-2(hydroxymethyl)benzoate (1.0 g, 3.15 mmol, 80% yield). MS (ESI) m/z 318.34 [M + 1]+. Step D. To a stirred solution of sodium 4-((tert-butoxycarbonyl(methyl)amino)methyl)-2-(hydroxymethyl)benzoate (500 mg, 1.57 mmol) in DMF (50 mL) was added ethyl iodide (0.1 mL, 1.73 mmol) at 0 °C, and the mixture was stirred at room temperature for 12 h. The reaction mixture was diluted with water and extracted with ethyl acetate. The combined organic layers were washed with water, brine, dried over sodium sulfate, and concentrated to give ethyl 4-((tertbutoxycarbonyl(methyl)amino)methyl)-2-(hydroxymethyl)benzoate (0.3 g, 0.92 mmol, 60% yield). 1H NMR (300 MHz, DMSO-d6) δ 7.83 (dd, J = 7.9, 3.5 Hz, 1H), 7.48 (s, 1H), 7.18 (d, J = 8.0 Hz, 1H), 5.23 (s, 1H), 4.81 (d, J = 4.2 Hz, 2H), 4.43 (s, 2H), 4.34−4.18 (m, 2H), 2.79 (d, J = 13.3 Hz, 3H), 1.41 (d, J = 11.1 Hz, 9H), 1.33−1.26 (m, 3H). Step E. To a stirred solution of ethyl 4-((tert-butoxycarbonyl(methyl)amino)methyl)-2-(hydroxymethyl)benzoate (0.3 g, 0.92 mmol) in dichloromethane (20 mL) were added carbon tetrabromide (460 mg, 1.39 mmol) and triphenylphosphine (364 mg, 1.39 mmol) at 0 °C, and the mixture was stirred for 2 h. The reaction mixture was diluted with water and extracted with dichloromethane. The combined organic layers were washed with water, brine, dried over sodium sulfate, and concentrated under reduced pressure. The resultant residue was purified by silica gel chromatography to give ethyl 2(bromomethyl)-4-((tert-butoxycarbonyl(methyl)amino)methyl)benzoate (150 mg, 0.40 mmol, 50% yield). 1H NMR (300 MHz, DMSO-d6) δ 7.87 (d, J = 8.0 Hz, 1H), 7.41 (d, J = 1.8 Hz, 1H), 7.28 500



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3-(5-((3-(3-Chloro-4-methylphenyl)-2-oxotetrahydropyrimidin-1(2H)-yl)methyl)-1-oxoisoindolin-2-yl)piperidine2,6-dione (27). Step A. To a solution of 3-chloro-4-methylaniline (2.0 g, 11.23 mmol) in acetonitrile (20 mL) was added bromopropanol (1.7 g, 12.23 mmol) followed by sodium bicarbonate (1.8 g, 21.42 mmol). The reaction was heated in a microwave reactor for 1 h at 90 °C. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel chromatography to give 3-(3-chloro-4-methylphenylamino)propan-1ol (0.9 g, 4.52 mmol, 40% yield). 1H NMR (300 MHz, CDCl3) δ 6.99 (d, J = 8.2 Hz, 1H), 6.64 (d, J = 2.4 Hz, 1H), 6.45 (dd, J = 8.2, 2.5 Hz, 1H), 3.81 (t, J = 5.9 Hz, 2H), 3.24 (t, J = 6.5 Hz, 2H), 2.24 (s, 3H), 1.87 (p, J = 6.3 Hz, 2H). Step B. To a stirred solution of 3-(3-chloro-4-methylphenylamino)propan-1-ol (0.9 g, 4.52 mmol) in dichloromethane (25 mL) was added tetrabromomethane (2.4 g, 7.23 mmol) followed by triphenylphosphine (1.9 g, 7.23 mmol) at 0 °C, and the mixture was stirred at room temperature for 12 h. The reaction mixture was concentrated under reduced pressure and the obtained residue was purified by silica gel chromatography to give N-(3-bromopropyl)-3chloro-4-methylaniline (0.9 g, 3.43 mmol, 77% yield). 1H NMR (300 MHz, CDCl3) δ 7.03−6.93 (m, 1H), 6.63 (d, J = 2.5 Hz, 1H), 6.44 (dd, J = 8.2, 2.5 Hz, 1H), 3.50 (t, J = 6.4 Hz, 2H), 3.28 (d, J = 6.5 Hz, 2H), 2.24 (s, 3H), 2.17−2.07 (m, 2H). Step C. To a stirred solution of N-(3-bromopropyl)-3-chloro-4methylaniline (50 mg, 0.13 mmol) in acetonitrile (5 mL) was added 6 (40 mg, 0.16 mmol) followed by sodium carbonate (28 mg, 0.27 mmol) and potassium iodide (44 mg, 0.02 mmol). The reaction was stirred at 80 °C for 12 h. The reaction mixture was diluted with water and extracted with ethyl acetate. The organic layer was dried over sodium sulfate, filtered, and the organic phase was concentrated under reduced pressure. The resulting residue was purified by silica gel chromatography to give 3-(5-((3-(3-chloro-4-methylphenylamino)propylamino)methyl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (10 mg, 0.02 mmol, 17% yield). MS (ESI) m/z 455.08 [M + 1]+. Step D. To a stirred solution of 3-(5-((3-(3-chloro-4-methylphenylamino)propylamino)methyl-1-oxoisoindolin-2-yl)piperidine2,6-dione (20 mg, 0.04 mmol) in DMF (1 mL) were added triethylamine (13 mg, 0.13 mmol) and 1,1′-carbonyldiimidazole (8 mg, 0.05 mmol). The reaction was stirred at 80 °C for 12 h. The reaction mixture was diluted with ice−water, extracted with ethyl acetate, dried over sodium sulfate, filtered, and concentrated. The obtained residue was purified by silica gel chromatography to give 3(5-((3-(3-chloro-4-methylphenyl)-2-oxotetrahydropyrimidin-1(2H)yl)methyl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (27) (5 mg, 0.01 mmol, 9% yield, HPLC purity >97%). 1H NMR (400 MHz, DMSOd6) δ 10.98 (s, 1H), 7.71 (d, J = 7.8 Hz, 1H), 7.52 (s, 1H), 7.46−7.36 (m, 2H), 7.29 (d, J = 8.3 Hz, 1H), 7.19 (dd, J = 8.3, 2.3 Hz, 1H), 5.11 (dd, J = 13.2, 5.1 Hz, 1H), 4.62 (s, 2H), 4.50−4.26 (m, 2H), 3.67 (t, J = 5.7 Hz, 2H), 2.92−2.88 (m, 1H), 2.67−2.57 (m, 2H), 2.41−2.33 (m, 2H), 2.30 (s, 4H), 2.03−1.99 (m, 3H). 3-(5-((3-(3-Chloro-4-methyl phenyl)-2-oxoimidazolidin-1yl)methyl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (28). Step A. To 3-chloro-4-methylaniline (3.0 g, 21.27 mmol) in acetonitrile (30 mL) was added bromoethanol (2.9 g, 23.40 mmol) followed by sodium bicarbonate (3.6 g, 42.54 mmol). The reaction was heated in a microwave reactor for 1 h at 90 °C. The reaction mixture was filtered and the filtrate was concentrated resulting in a residue which was purified by silica gel chromatography to give 2-(3-chloro-4-methylphenylamino)ethanol (1.5 g, 8.10 mmol, 38% yield). 1H NMR (300 MHz, DMSO-d6) δ 6.99 (d, J = 8.3 Hz, 1H), 6.60 (d, J = 2.4 Hz, 1H), 6.47 (dd, J = 8.3, 2.4 Hz, 1H), 5.60 (t, J = 5.7 Hz, 1H), 4.66 (t, J = 5.4 Hz, 1H), 3.52 (q, J = 5.8 Hz, 2H), 3.04 (q, J = 5.9 Hz, 2H), 2.15 (s, 3H). Step B. To a solution of 2-(3-chloro-4-methylphenylamino)ethanol (1.5 g, 8.10 mmol) in dichloromethane (15 mL) at 0 °C, was added tetrabromomethane (4.3 g, 12.96 mmol) followed by triphenylphosphine (3.4 g, 12.96 mmol), and the mixture was stirred for 12 h at room temperature. The reaction mixture was concentrated under reduced pressure and the residue obtained was purified by silica gel

chromatography to give N-(2-bromoethyl)-3-chloro-4-methylaniline (1.5 g, 6.07 mmol, 75% yield). 1H NMR (300 MHz, DMSO-d6) δ 7.02 (d, J = 8.3 Hz, 1H), 6.64 (d, J = 2.4 Hz, 1H), 6.50 (dd, J = 8.3, 2.4 Hz, 1H), 5.97 (s, 1H), 3.55 (td, J = 6.2, 1.3 Hz, 2H), 3.44 (t, J = 6.3 Hz, 2H), 2.16 (s, 3H). Step C. To a stirred solution of N-(2-bromoethyl)-3-chloro-4methylaniline (0.9 g, 3.62 mmol) in acetonitrile (10 mL) was added 6 (2.0 g, 5.44 mmol) followed by sodium carbonate (690 mg, 6.51 mmol) and potassium iodide (120 mg, 0.72 mmol). The reaction was stirred for 12 h at 80 °C. The reaction mixture was diluted with water and extracted with ethyl acetate. The organic layer was dried over sodium sulfate and concentrated under reduced pressure. The resulting residue was purified by silica gel chromatography to give 3(5-((2-(3-chloro-4-methylphenylamino)ethylamino)methyl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (0.3 g, 8.10 mmol, 18% yield). 1H NMR (300 MHz, DMSO-d6) δ 10.98 (s, 1H), 7.66 (d, J = 7.7 Hz, 1H), 7.58 (s, 1H), 7.49 (d, J = 7.9 Hz, 1H), 6.99 (d, J = 8.3 Hz, 1H), 6.59 (d, J = 2.3 Hz, 1H), 6.45 (dd, J = 8.3, 2.4 Hz, 1H), 5.63 (s, 1H), 5.11 (dd, J = 13.2, 5.0 Hz, 1H), 4.57−4.09 (m, 2H), 3.09 (d, J = 6.2 Hz, 1H), 2.92 (s, 1H), 2.50 (d, J = 2.1 Hz, 3H), 2.44−2.23 (m, 3H), 2.14 (s, 3H), 2.02 (s, 1H); MS (ESI) m/z 441.20 [M + 1]+. Step D. To a stirred solution of 3-(5-((2-(3-chloro-4-methylphenylamino)ethylamino)methyl)-1-oxoisoindolin-2-yl)piperidine-2,6dione (0.3 g, 0.68 mmol) in DMF (5 mL) was added 1,1′carbonyldiimidazole (331 mg, 2.04 mmol) followed by trimethylamine (0.1 mL, 0.68 mmol). The reaction was stirred at 80 °C for 12 h. The reaction mixture was diluted with water and extracted with ethyl acetate. The organic layer was dried over sodium sulfate and concentrated under reduced pressure. The residue obtained was purified by silica gel chromatography to give 3-(5-((3-(3-chloro-4methylphenyl)-2-oxoimidazolidin-1-yl)methyl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (28) (20 mg, 0.042 mmol, 6% yield, HPLC purity >97%). 1H NMR (300 MHz, DMSO-d6) δ 10.98 (s, 1H), 7.80 (d, J = 2.3 Hz, 1H), 7.73 (d, J = 7.8 Hz, 1H), 7.55 (s, 1H), 7.45 (d, J = 8.0 Hz, 1H), 7.37 (dd, J = 8.4, 2.3 Hz, 1H), 7.29 (d, J = 8.5 Hz, 1H), 5.11 (dd, J = 13.1, 5.0 Hz, 1H), 4.51 (s, 2H), 4.49−4.26 (m, 1H), 3.81 (t, J = 8.1 Hz, 2H), 3.39 (s, 1H), 3.01−2.81 (m, 1H), 2.60 (d, J = 17.2 Hz, 1H), 2.37 (dd, J = 13.2, 4.5 Hz, 1H), 2.28 (s, 3H), 2.01 (s, 1H); MS (ESI) m/z 467.59 [M + 1]+. 3-(1-Oxo-5-((2-oxo-3-(3-phenoxyphenyl)imidazolidin-1-yl)methyl)isoindolin-2-yl)piperidine-2,6-dione (29). Step A. To 3phenoxyaniline (1.5 g, 8.11 mmol) in acetonitrile (20 mL) was added 2-bromoethanol (1.2 g, 9.72 mmol) followed by sodium bicarbonate (2.0 g, 24.32 mmol). The reaction was heated in a microwave reactor for 1 h at 90 °C. The reaction mixture was filtered, and the filtrate was concentrated to give a residue. The crude residue was purified by silica gel chromatography to give 2-(3-phenoxyphenylamino)ethanol (0.6 g, 2.60 mmol, 32% yield). MS (ESI) m/z 230.01 [M + 1]+. Step B. To a stirred solution of 2-(3-phenoxyphenylamino)ethanol (0.8 g, 3.47 mmol) in dichloromethane (15 mL) was added tetrabromomethane (1.8 g, 5.56 mmol) followed by triphenylphosphine (1.4 g, 5.56 mmol) at 0 °C. The reaction was warmed to room temperature and stirred for 12 h. The reaction mixture was concentrated and the obtained residue was purified by silica gel chromatography to give N-(2-bromoethyl)-3-phenoxyaniline (0.8 g, 2.73 mmol, 79% yield). MS (ESI) m/z 292.04 [M + 1]+. Step C. To a stirred solution of N-(2-bromoethyl)-3-phenoxyaniline (150 mg, 0.40 mmol) in acetonitrile (15 mL) was added 6 (178 mg, 0.69 mmol) followed by sodium carbonate (77 mg, 0.73 mmol) and potassium iodide (101 mg, 0.60 mmol). The reaction was stirred for 12 h at 80 °C. The reaction mixture was diluted with water and extracted with ethyl acetate. The organic layer was dried over sodium sulfate, filtered, and concentrated. The obtained residue was purified by silica gel chromatography to give 3-(1-oxo-5-((2-(3-phenoxyphenylamino)ethylamino)methyl)isoindolin-2-yl)piperidine-2,6-dione (50 mg, 103.3 mmol, 25% yield). MS (ESI) m/z 485.2 [M + 1] +. Step D. To a stirred solution of 3-(1-oxo-5-((2-(3-phenoxyphenylamino)ethylamino)isoindolin -2-yl)piperidine-2,6-dione (0.2 g, 0.41 mmol) in DMF (15 mL) were added triethylamine (41 mg, 0.41 mmol) and 1,1′-carbonyldiimidazole (267 mg, 1.65 mmol). The 501



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reaction was stirred at 80 °C for 12 h. The reaction mixture was diluted with water and extracted with ethyl acetate. The organic layer was dried over sodium sulfate, filtered, and concentrated. The obtained residue was purified by silica gel chromatography to give 3-(1-oxo-5((2-oxo-3-(3-phenoxyphenyl)imidazolidin-1-yl)methyl)isoindolin-2yl)piperidine-2,6-dione (29) (70 mg, 0.14 mmol, 33% yield, HPLC purity >99%). 1H NMR (400 MHz, DMSO-d6) δ 10.98 (s, 1H), 7.72 (d, J = 7.8 Hz, 1H), 7.51 (t, J = 2.2 Hz, 2H), 7.48−7.29 (m, 4H), 7.28−7.20 (m, 1H), 7.13 (tt, J = 7.4, 1.2 Hz, 12H), 7.05−6.97 (m, 2H), 6.68−6.60 (m, 1H), 5.11 (dd, J = 13.4, 5.1 Hz, 1H), 4.47 (d, J = 27.0 Hz, 2H), 4.32 (d, J = 17.5 Hz, 2H), 3.86−3.77 (m, 2H), 3.43− 3.34 (m, 2H), 2.90 (d, J = 4.9 Hz, 1H), 2.64−2.55 (m, 1H), 2.43−2.30 (m, 1H), 2.00 (d, J = 12.7 Hz, 1H); MS (ESI) m/z 510.93 [M + 1]+. ePL Degradation Description. To monitor protein degradation, we used the DiscoverX enzyme fragment complementation assay (EFC) technology. This system relies on having two different components of the β-galactosidase enzyme expressed for activity. The large protein fragment of β-galactosidase is included in the InCELL Hunter detection reagent that is added at the end of the assay. The small peptide fragment (the enhanced ProLabel (ePL)) that is required for β-galactosidase activity is expressed on the protein of interest (example, Aiolos and GSPT1). When the ePL tagged protein has been degraded through the E3-ligase mechanism, there is a loss in the β-galactosidase activity. ePL Degradation Assay. DF15 multiple myeloma cells stably expressing ePL-tagged Aiolos (or GSPT1) were generated via lentiviral infection with pLOC-ePL-Aiolos (or GSPT1). Cells were dispensed into a 384-well plate (Corning no. 3712) prespotted with compound. Compounds were dispensed by an acoustic dispenser (ATS acoustic transfer system from EDC Biosystems) into a 384-well in a10 point dose−response curve using 3-fold dilutions starting at 10 μM and going down to 0.0005 μM in DMSO. A DMSO control is added to the assay. 25 μL of medium (RPMI-1640 + 10% heat inactivated FBS + 25 mM Hepes + 1 mM Na pyruvate + 1× NEAA + 0.1% Pluronic F-68 + 1× Pen Strep glutamine) containing 5000 cells was dispensed per well. Assay plates were incubated at 37 °C with 5% CO2 for 4 h. After incubation, 25 μL of the InCELL Hunter detection reagent working solution (DiscoverX, catalog no. 96-0002, Fremont, CA) was added to each well and incubated at room temperature for 30 min protected from light. After 30 min, luminescence was read on a PHERAstar luminometer (Cary, NC). To determine EC50 values for Aiolos or GSPT1 degradation, a fourparameter logistic model (sigmoidal dose−response model),

FIT = A +

926-32210, LICOR), and goat anti-rabbit IRDye-800 antibody (no. 926-32211, LI-COR).

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.6b01911. Molecular formula strings and some data (CSV) Details of preparation of docking studies into crystallographic structure (5HXB), details of preparation of QSAR models, and standard error of measurement for all biological data present (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Phone: 858-795-4910. E-mail: [email protected]. ORCID

Joshua D. Hansen: 0000-0002-4881-5247 Notes

The authors declare no competing financial interest. All authors are currently employees of Celgene except Fan Vocanson, Tim Crea, Gilles Carmel, Matt Hickman, and George Muller who were employees of Celgene at the time of their contributions to this work.

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ACKNOWLEDGMENTS Thanks are extended to K. Blumeyer, B. Lenoir, and G. Khambatta for technical assistance, to Ravi Kumar and his team at GVK Bio for synthetic support, and to L. Hamann and D. Mortensen for discussions and review regarding this manuscript.

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ABBREVIATIONS USED CRBN, cereblon; CUL4, cullin protein; DDB1, damaged DNAbinding protein 1; RBX1, RING box-domain protein; GSPT1, G1 to S phase transition 1; DUB, deubiquitinating enzyme; eRF1, eukaryotic translation termination factor 1; NBS, Nbromosuccinimide; ACN, acetonitrile; HCl, hydrochloric acid; DMA, dimethylacetamide; DMF, diemthylformamide; TLC, thin layer chromatography; HPLC, high performance liquid chromatography

B−A 1+

ASSOCIATED CONTENT

S Supporting Information *

D

( Cx )

where C is the inflection point (EC50), D is the correlation coefficient, and A and B are the low and high limits of the fit, respectively, was used to determine the compound’s EC50 value, which is the halfmaximum effective concentration. The minimum Y is reference to the Y constant. In the Aiolos degradation assay, we use pomalidomide as the control with a Y constant of 0. The maximum limit is the Ymax of DMSO control. All percent of control Aiolos degradation curves were processed and evaluated using Activity Base (IDBS). In the GSPT1 degradation assay, we use 3 as the control with a Y constant of 0. The maximum limit is the Ymax of DMSO control. All percent of control GSPT1 degradation curves were processed and evaluated using Activity Base (IDBS). Immunoblot Analysis. OPM-2 cells were treated with DMSO and test compounds for 8 h as indicated in Figure 3. Cells were then washed with ice-cold 1× PBS twice and lysed in buffer A [50 mM TrisHCl, 150 mM NaCl, 1% Triton X-100, complete protease inhibitor tablet (Roche), phosphatase inhibitor tablet (Roche)]. Whole cell extracts were harvested and subjected to immunoblot analysis with the following antibodies: rabbit anti-GSPT1 polyclonal antibody (no. ab49878, Abcam), rabbit anti-Aiolos monoclonal antibody (no. 15103S, Cell Signaling), rabbit anti-CRBN monoclonal antibody (no. CRBN65, Celgene), mouse anti-actin monoclonal antibody (no. A5316, Sigma-Aldrich), goat anti-mouse IRDye-800 antibody (no.

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REFERENCES

(1) Ito, T.; Ando, H.; Suzuki, T.; Ogura, T.; Hotta, K.; Imamura, Y.; Yamaguchi, Y.; Handa, H. Identification of a primary target of thalidomide teratogenicity. Science 2010, 327, 1345−1350. (2) Kronke, J.; Udeshi, N. D.; Narla, A.; Grauman, P.; Hurst, S. N.; McConkey, M.; Svinkina, T.; Heckl, D.; Comer, C.; Li, X.; Ciarlo, C.; Hartman, E.; Munshi, N.; Schenone, M.; Schreiber, S. L.; Carr, S. A.; Ebert, B. L. Lenalidomide causes selective degradation of IKZF1 and IKZF3 in multiple myeloma cells. Science 2014, 343, 301−305. (3) Lu, G.; Middleton, R. E.; Sun, H.; Naniong, M.; Ott, C. J.; Mitsiades, C. S.; Wong, K. K.; Bradner, J. E.; Kaelin, W. G., Jr. The myeloma drug Lenalidomide promotes the Cereblon-dependent destruction of Ikaros proteins. Science 2014, 343, 305−309. (4) Matyskiela, M.; Lu, G.; Ito, T.; Pagarigan, B.; Lu, C. C.; Miller, K.; Fang, W.; Wang, N. Y.; Nguyen, D.; Houston, J.; Carmel, G.; Tran, T.; Riley, M.; Nosaka, L.; Lander, G. C.; Gaidarova, S.; Xu, S.; Ruchelman, A. L.; Handa, H.; Carmichael, J.; Daniel, T. O.; Cathers, B. E.; Lopez-Girona, A.; Chamberlain, P. P. A novel Cereblon modulator recruits GSPT1 to the CRL4CRBN ubiquitin ligase. Nature 2016, 535, 252−257.

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(5) Cheng, Z.; Saito, K.; Pisarev, A. V.; Wada, M.; Pisareva, V. P.; Pestova, T. V.; Gajda, M.; Round, A.; Kong, C.; Lim, M.; Nakamura, Y.; Svergun, D. I.; Ito, K.; Song, H. Structural insights into eRF3 and stop codon recognition by eRF1. Genes Dev. 2009, 23, 1106−1118. (6) Preis, A.; Heuer, A.; Barrio-Garcia, C.; Hauser, A.; Eyler, D. E.; Berninghausen, O.; Green, R.; Becker, R.; Beckmann, R. Cryoelectron microscopic structures of eukaryotic translation termination complexes containing eRF1-eRF3 or eRF1-ABCE1. Cell Rep. 2014, 8, 59−65. (7) Zhouravleva, G.; Frolova, L.; Le Goff, X.; Le Guellec, R.; IngeVechtomov, S.; Kisselev, L.; Philippe, M. Termination of translation in eukaryotes is governed by two interacting polypeptide chain release factors, eRF1 and eRF3. EMBO J. 1995, 14, 4065−4072. (8) Kronke, J.; Fink, E. C.; Hollenbach, P. W.; MacBeth, K. J.; Hurst, S. N.; Udeshi, N. D.; Chamberlain, P. P.; Mani, D. R.; Man, H. W.; Gandhi, A. K.; Svinkina, T.; Schneider, R. K.; McConkey, M.; Jaras, M.; Griffiths, E.; Wetzler, M.; Bullinger, L.; Cathers, B. E.; Carr, S. A.; Chopra, R.; Ebert, B. L. Lenalidomide induces ubiquitination and degradation of CK1α in del(5q) MDS. Nature 2015, 523, 183−188. (9) Muller, G. W.; Ruchelman, A. L. 5-Substituted isoindoline compounds for the use in cancer. WO 2010053732, 2010. (10) Chamberlain, P. P.; Lopez-Girona, A.; Miller, K.; Carmel, G.; Pagarigan, B.; Chie-Leon, B.; Rychak, E.; Corral, L. G.; Ren, Y. J.; Wang, M.; Riley, M.; Delker, S. L.; Ito, T.; Ando, H.; Mori, T.; Hirano, Y.; Handa, H.; Hakoshima, T.; Daniel, T. O.; Cathers, B. E. Structure of the human Cereblon−DDB1−Lenalidomide complex reveals basis for responsiveness to thalidomide analogs. Nat. Struct. Mol. Biol. 2014, 21, 803−809. (11) StarDrop, version 6.2; Optibrium Ltd., 2016. (12) Obrezanova, O.; Csanyi, G.; Gola, J.; Segall, M. Gaussian processes: a method for automatic QSAR modeling of ADME properties. J. Chem. Inf. Model. 2007, 47 (5), 1847−1857. (13) Breiman, L. Random forests. Machine Learning 2001, 45 (1), 5− 32. (14) Quinlan, R. Induction of decision trees. Machine Learning 1986, 1, 81−106. (15) Quinlan, J. R. C4.5: Programs for Machine Learning; Morgan Kaufmann Publishers, Inc.: San Mateo, CA, 1993. (16) Deshaies, R. J. Protein degradation: prime time for PROTACs. Nat. Chem. Biol. 2015, 11, 634−635. (17) Winter, G. E.; Buckley, D. L.; Paulk, J.; Roberts, J. M.; Souza, A.; Dhe-Paganon, S.; Bradner, J. E. Drug development. Phthalimide conjugation as a strategy for in vivo target protein degradation. Science 2015, 348, 1376−1381. (18) Lu, J.; Qian, Y.; Altieri, M.; Dong, H.; Wang, J.; Raina, K.; Hines, J.; Winkler, J. D.; Crew, A. P.; Coleman, K.; Crews, C. M. Hijacking the E3 ubiquitin ligase cereblon to efficiently target BRD4. Chem. Biol. 2015, 22, 755−763. (19) Chini, A.; Fonseca, S.; Fernandez, G.; Adie, B.; Chico, J. M.; Lorenzo, O.; Garcia-Casado, G.; Lopez-Vidriero, I.; Lozano, F. M.; Ponce, M. R.; Micol, J. L.; Solano, R. The JAZ family of repressors is the missing link in jasmonate signaling. Nature 2007, 448, 666−671. (20) Tan, X.; Calderon-Villalobos, L. I.; Sharon, M.; Zheng, C.; Robinson, C. V.; Estelle, M.; Zheng, N. Mechanism of auxin perception by the TIR1 ubiquitin ligase. Nature 2007, 446, 640−645. (21) Thiel, P.; Kaiser, M.; Ottmann, C. Small-molecule stabilization of protein-protein interactions: an underestimated concept in drug discovery? Angew. Chem., Int. Ed. 2012, 51, 2012−2018. (22) Krönke, J.; Udeshi, N. D.; Narla, A.; Grauman, P.; Hurst, S. N.; McConkey, M.; Svinkina, T.; Heckl, D.; Comer, E.; Li, X.; Ciarlo, C.; Hartman, E.; Munshi, N.; Schenone, M.; Schreiber, S. L.; Carr, S. A.; Ebert, B. L. Lenalidomide causes selective degradation of IKZF1 and IKZF3 in multiple myeloma cells. Science 2014, 343, 301−305. (23) Petzold, G.; Fischer, E. S.; Thomä, N. H. Structural basis of lenalidomide-induced CK1α degradation by the CRL4(CRBN) ubiquitin ligase. Nature 2016, 532, 127−130. (24) Muller, G. W.; Ruchelman, A. L.; Chen, R. 5-Substituted isoindoline compounds. US 2009/0142297, 2009. (25) Muller, G. W.; Ruchelman, A. L.; Chen, R. 5-Substituted isoindoline compounds. WO 2008/027542, 2008.

Article

NOTE ADDED AFTER ASAP PUBLICATION This manuscript published ASAP on 4/13/2017. Figure 2, and Tables 5−8 were corrected, and the revised version was reposted on 4/17/2017.

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Protein Degradation via CRL4CRBN Ubiquitin Ligase: Discovery and

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