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A “Click Chemistry Platform” for the Rapid Synthesis of Bispecific Molecules for Inducing Protein Degradation Ryan P. Wurz,*,† Ken Dellamaggiore,‡ Hannah Dou,∥ Noelle Javier,∥ Mei-Chu Lo,∥ John D. McCarter,§ Dane Mohl,‡ Christine Sastri,‡ J. Russell Lipford,‡ and Victor J. Cee† †

Departments of Therapeutic Discovery−Medicinal Chemistry, ‡Oncology Research and §Discovery Technologies, Amgen Inc., One Amgen Center Drive, Thousand Oaks, California 91320, United States ∥ Department of Therapeutic Discovery−Discovery Technologies, Amgen Inc., 1120 Veterans Boulevard, South San Francisco, California 94080, United States S Supporting Information *

ABSTRACT: Proteolysis targeting chimeras (PROTACs) are bispecific molecules containing a target protein binder and an ubiquitin ligase binder connected by a linker. By recruiting an ubiquitin ligase to a target protein, PROTACs promote ubiquitination and proteasomal degradation of the target protein. The generation of effective PROTACs depends on the nature of the protein/ligase ligand pair, linkage site, linker length, and linker composition, all of which have been difficult to address in a systematic way. Herein, we describe a “click chemistry” approach for the synthesis of PROTACs. We demonstrate the utility of this approach with the bromodomain and extraterminal domain-4 (BRD4) ligand JQ-1 (3) and ligase binders targeting cereblon (CRBN) and Von Hippel−Lindau (VHL) proteins. An AlphaScreen proximity assay was used to determine the ability of PROTACs to form the ternary ligase−PROTAC−target protein complex and a MSD assay to measure cellular degradation of the target protein promoted by PROTACs.

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INTRODUCTION The development of bispecific molecules or proteolysis targeting chimeras (PROTACs)1,2 as potential drugs capable of recruiting target proteins to the cellular machinery for elimination has created alternative options to tackle traditionally “difficult to drug” proteins. This approach “hijacks” the function of a ligase to transfer ubiquitin subunits to the target protein, thereby tagging it for recognition by the proteasome and coding its destruction.3 A variety of protein types have been successfully degraded using this approach, including transcription factors,4,5 kinases5−8 and nuclear epigenetic readers8−11 using a variety of ligases,12 including Von Hippel−Lindau (VHL),4,5,9,13 mouse double minute 2 homologue (MDM2),14 F-box/WD repeatcontaining protein (β-TrCP),15 cereblon,6,8−10 and inhibitor of apoptosis protein (IAP).4,6,7 Arguably one of the more potent examples of PROTACs in the literature to date involves the degradation of the BET bromodomain protein BRD4. Of the PROTACs employing BRD4 ligand JQ-1 (3),16 MZ-1 (1),11 and dBET1 (2)10 have successfully used both VHL and cereblon E3 ligase ligands, respectively, for BRD4 protein degradation and serve as excellent benchmarks for the development of novel PROTACs (Figure 1). A comprehensive survey of the targeted proteolysis literature for commonality of linker composition and length yielded little insight into the ideal linker and suggests a degree of empiricism in PROTAC design. Furthermore, the ligase ligand may have a dramatic influence on activity of the PROTAC6 depending on the cell line because E3 ligase expression levels may vary. Given these complexities to PROTAC design and that a © 2017 American Chemical Society

Figure 1. PROTACs for the degradation of BET bromodomain BRD4 protein derived from ligand 3.

comprehensive study of ligase ligand/linker length activity relationship with a BRD4 targeting PROTAC has not been forthcoming in the literature, we wanted to advance our own understanding of the SAR of these bispecific molecules.17 To facilitate the discovery process for new PROTACs, a reliable linking strategy was sought to couple ligase ligand-containing motifs and target protein ligands in a parallel manner, which led us to the Huisgen 1,3-dipolar cycloaddition reaction. Special Issue: Inducing Protein Degradation as a Therapeutic Strategy Received: December 8, 2016 Published: April 5, 2017 453

DOI: 10.1021/acs.jmedchem.6b01781 J. Med. Chem. 2018, 61, 453−461

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Figure 2. General strategy using “click chemistry” for parallel PROTAC synthesis. POI = protein of interest.

Arguably, this bond forming reaction is one of the most reliable. This copper-catalyzed reaction couples azides and alkynes forming a new triazole ring and is commonly referred to as “click chemistry” due to its ease of use.18,19 This reaction is typically high yielding, requiring stoichiometric quantities of each component and boasts excellent functional group compatibility under mild reaction conditions. This approach would allow for the parallel synthesis of libraries of PROTACs provided the necessary azides and alkynes could be prepared. To test this approach, ligands for VHL and cereblon were modified with terminal alkynes with linkers containing varying ethylene glycol units. A ligand for the target protein would have to be modified with an azide at a suitable position on the molecule so as to minimally influence affinity of the ligand for its target protein and serve as the azide input for the “click reaction” (Figure 2).

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CHEMISTRY For a proof-of-concept for this “click chemistry” platform, we decided to focus on the synthesis of PROTACs targeting the BET bromodomain, BRD4. 3 was used as the targeting ligand and converted into an amide containing a pendant azide necessary for the “click reaction”. This was accomplished by TFA-mediated deprotection of the tert-butyl ester of 3 followed by amide formation with 2-azidoethanamine, furnishing azide 4 in high yield (eq 1).

This involved CuSO4 as a catalyst, sodium ascorbate for catalyst regeneration, and 0.1 M in THF with 3−4 drops of water to aid in solubilizing the copper catalyst. The reactions were typically very clean and often went to completion within 3 h, resulting in 55−90% isolated yields for the 10-membered PROTAC library (Table 1).

For the ligase portion of the “click reaction”, ligands for cereblon and VHL ligases were modified with terminal alkyne motifs containing ethylene glycol units of varying lengths. Thalidomide modified with an ethereal linkage at the 4-position has resulted in potent PROTACs in previous literature reports, such as 2,10 and served as a useful starting point for this investigation. These ligands for cereblon could be synthesized in an expeditious manner from 4-hydroxyphthalic anhydride (5) following treatment with 3-amino-piperidine-2,6-dione (6) to afford 4-hydroxy-thalidomide (7).20 Alkylation of the phenol with either propargyl bromide or propargyl-tosylates containing varying ethylene glycol units afforded the requisite library of cereblon ligase ligands (8a−e, IMiD 0−4) necessary for the “click reaction” (eq 2).21 In a similar manner, a library of VHL ligands modified with terminal alkynes containing varying ethylene glycol units were prepared. To access this library, VHL-amine (9)13 was treated with the corresponding propargyl-glycolic-acid chlorides to generate the library of VHL ligands (10a−e, VHL 0−4) for the “click reaction” (eq 3). With both azide and alkyne components in hand, we then subjected them to typical conditions for a click reaction (Scheme 1).

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RESULTS AND DISCUSSION To evaluate the activity of the 10 new PROTACs, a cell-free proximity assay utilizing AlphaScreen technology (amplified luminescent proximity homogeneous assay, PerkinElmer, Waltham, MA)22 was developed to determine if the bispecific molecules could induce ternary complex formation between the E3 ligase, PROTAC, and BRD4 protein. The proximity assay uses Glutathione Donor beads to bind the glutathione-Stransferase (GST)-tagged BRD4 protein and Nickel Chelate AlphaScreen Acceptor beads to capture the poly-His-tagged E3 ligase and reports the luminescence arising from proximity of E3 ligase-bound acceptor beads and BRD4-bound donor beads through ternary complex formation. To determine cellular degradation of BRD4, we used the Meso Scale Diagnostics (MSD, Rockville, MD) platform and a commercial detection antibody to quantify BRD4 content in cellular lysates. The PROTACs’ activity was assessed in NCI-H661 cells23 at a 4 h time point. The benchmark BRD4 PROTACs 1 and 2 were 454

DOI: 10.1021/acs.jmedchem.6b01781 J. Med. Chem. 2018, 61, 453−461

Journal of Medicinal Chemistry

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Scheme 1

Table 1. PROTAC Synthesis Using the “Click Reaction”a

a

PROTAC

yield (%)

PROTAC

yield (%)

11a 11b 11c 11d 11e

90 67 70 73 69

12a 12b 12c 12d 12e

79 60 72 68 55

Isolated yields following purification by silica gel chromatography.

Table 2. Proximity Assay Results and BRD4 Degradation in a NCI-H661 Cell Line for Cereblon/BRD4 and VHL/BRD4 PROTACs compd

proximity assaya POCmax

3 2 11a 11b 11c 11d 11e 1 12a 12b 12c 12d 12e

0d 100d 116d 36d 118d 113d 148d 100e 46e 22e 11e 15e 3e

protein degradationa H661 cells (4 h) DC50 (μM) cLogPb >5 0.14 0.49 >5 >5 0.45 0.20 0.008 0.059 0.083 0.24 0.46 0.63

4.82 2.49 2.87 2.74 2.60 2.46 2.33 4.90 4.29 4.17 4.04 3.90 3.77

solubility PBS/FaSIF/ 0.01 N HCl (μM)c

Figure 3. Proximity assay data for JQ-1-IMiD PROTACs.

(Figure 3) consistent with the three-body binding equilibria in which excess bridging molecules out-compete ternary complex formation.25 We used the maximal normalized signal (POCmax) to compare the ability of PROTACs to induce ternary complex formation because POCmax is directly related to the maximal amount of ternary complex that can be formed and it has better resolution than other parameters such as the concentration of the half-maximal response. 2 was used as the positive control, and its POCmax was set to be 100. Most of the IMiD-derived PROTACs were able to induce ternary complex formation to a similar extent as the positive control with the exception of 11b (Figure 3). In the MSD assay, the PROTAC with the longest linker studied (11e) was found to be the most active, resulting in a DC50 value of 0.20 μM which was very similar to 2 with a DC50 value of 0.14 μM (Figure 4).26 The poor cellular activity of 11b and 11c relative to shorter (11a) and longer (11d, 11e) linker lengths was an unexpected result, and this highlights the importance of investigating linker length in the optimization of PROTACs. Forming a ternary cereblon/PROTAC/BRD4 complex is a prerequisite for the subsequent ubiquitin transfer and proteasomal degradation of BRD4, but it is not the only factor that determines the level of cellular protein degradation. Other factors such as optimal geometries may play a key role in the kinetics of ubiquitin transfer and therefore cause a loose correlation between POCmax and DC50 from the two assays as observed for these CRBN/BRD4 PROTACs. The cLogP values and solubilities of the IMiD PROTACs (11a−e) prepared using click chemistry were similar to the benchmark IMiD PROTAC 2, and this deviation in physical properties presumably had little impact on their activity versus the benchmark.

146/434/281 60/97/171 31/191/171 111/247/242 76/237/237 130/216/310 158/273/317 97/164/450 0/90/314 38/178/343 71/183/293 168/294/394 206/313/500

a

Data represents an average of at least 2 separate determinations. DC50, concentration of a PROTAC at which the cellular protein content is reduced by half. bLog P values were calculated via a Pipeline Pilot Web Port interface (Biovia, Inc.) to the Daylight software suite (version 4.81, Daylight Chemical Information Systems, Inc.). cn = 1 for pH solubility data, PBS = phosphate-buffered saline, FaSIF = fasted state simulated intestinal fluid. dBenchmark 2 was assigned POCmax = 100 in the proximity assay for IMiD containing PROTACs. e Benchmark 1 was assigned POCmax = 100 in the proximity assay for VHL containing PROTACs.

used as positive controls for these studies and for points of comparison (see Table 2). The data generated from these two assays represent the first systematic study of the effect of linker length on the activity of the PROTACs for the cereblon and VHL ligases in a given series.24 In the cereblon/PROTAC/BRD4 proximity assay, the IMiDderived PROTACs showed a bell-shaped dose−response curve 455

DOI: 10.1021/acs.jmedchem.6b01781 J. Med. Chem. 2018, 61, 453−461

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in this case was successful in identifying VHL/BRD4 as a suitable protein/ligase pair and establishing that shorter linker lengths produce more potent PROTACs, but additional linker composition SAR may still be necessary to achieve maximum potency in a series. There was a modest correlation between the POCmax in the proximity assay and the DC50 in the BRD4 MSD assay. The VHL-derived PROTACs showed much greater differences in ternary complex formation as measured by POCmax than their IMiD-derived counterparts. Experiments involving in vitro reconstitution of ligase complexes and BRD4 will be necessary to fully understand these observations. To confirm that the BRD4 protein disappearance was driven by ternary complex formation between cereblon/PROTAC/ BRD4 protein and was proteasome-mediated, a series of control experiments were performed with the NCI-H661 cell line. Dosing of this cell line alone with the PROTAC 11e at 1 μM resulted in a >90% reduction in BRD4 protein after 4 h versus a DMSO control, whereas cotreatment with 10 μM 3, 10 μM thalidomide, or a proteasome inhibitor (20 μM MG132)27 all blocked the 11e-induced degradation of BRD4 consistent with ternary complex formation and that loss of BRD4 protein is proteasome mediated. We also show that 10 μM of the azide/ alkyne components individually or codosed (4 and 8e) resulted in no appreciable degradation of the BRD4 protein, indicating that the nonlinked components of the PROTAC are not sufficient for degradation (Figure 7A). A similar set of experiments was run to confirm that the BRD4 protein disappearance was driven by ternary complex formation between VHL/ PROTAC/BRD4 protein and was also proteasome-mediated (Figure 7B). In this case, cotreatment with VHL ligand (13)13 resulted in blockage of 12b-induced degradation of BRD4.

Figure 4. MSD assay data for JQ-1-IMiD PROTACs in a NCI-H661 cell line (4 h time point).

Figure 5. Proximity assay data for JQ-1-VHL PROTACs.

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For the VHL-derived PROTACs, 1 was used as the positive control (POCmax = 100) for the proximity assay, and the PROTACs resulting from the “click ligation” showed moderateto-low complex formation relative to 1 (Figure 5). In this case, the presence of the triazole motif in the linker resulted in a small decline in solubility and a small decrease in cLogP when compared to the benchmark VHL PROTAC (1). Nevertheless, the activity of some of the VHL/BRD4 PROTACs were measurable in the BRD4 protein assay, with low double-digit nanomolar activities favoring the PROTACs with the shortest linker lengths (Figure 6). These PROTACs were still ca. 10-fold

CONCLUSION In conclusion, we have described the use of a “click chemistry” platform to prepare a 10-membered library of PROTACs using both cereblon and VHL ligase ligands and evaluated these molecules in their ability to form ternary complexes between the ligase/PROTAC/BRD4 protein as well as their activity in the targeted degradation of BRD4 protein in two lung carcinoma cell lines. Their mechanism of activity is supported by a number of control experiments that suggest the necessity for ternary complex formation and that the process is proteasome mediated. These examples build upon the earlier study by Lebraud and co-workers demonstrating the utility of click chemistry for the synthesis of cell penetrant PROTACs.8 This report represents the first comprehensive study of linker length/activity relationship as well as the relationship between protein degradation and the ability to form strong ternary complexes as measured by a proximity assay. Conceptually, this platform represents a powerful new tool to access libraries of PROTACs as its principles can be easily applied to other ligases and target proteins, the results of which will be reported in due course.

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EXPERIMENTAL SECTION

Biology. BRD4 MSD Assay. NCI-H661 (ATCC, 2.5 × 104 cells/ well) or NCI-H838 (ATCC, 4.0 × 104 cells/well) were seeded in 96-well flat-bottom plates (Corning 3904). Cells were incubated overnight at 37 °C then dosed for 4 h with compound dose responses. Cells were lysed in MSD lysis buffer (R60TX-2) containing protease inhibitors (Roche 04 693 116001) and phosphatase inhibitors (Roche 04 906 837001). BRD4 protein levels were determined by MSD following the standard bind, spot-coating MSD protocol

Figure 6. MSD assay data for JQ-1-VHL (12a−e) PROTACs in a NCI-H661 cell line (4 h time point).

less active than the benchmark 1, suggesting that the triazole moiety negatively impacts the activity because 12a and 12b have roughly the same linker length as 1. This was not observed with the CRBN/BRD4 PROTACs. Therefore, the platform approach 456

DOI: 10.1021/acs.jmedchem.6b01781 J. Med. Chem. 2018, 61, 453−461

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Figure 7. Control experiments: (A) 11e PROTAC in the NCI-H661 cell line (4 h time point); (B) 12b PROTAC in NCI-H661 cell line (4 h time point). (https://www.mesoscale.com/~/media/files/technical%20notes/ assay%20development%20plates%20uncoated.pdf). BRD4 capture antibody (Cell Signaling no. 13440) was diluted 1:50 into 0.03% Triton X-100 in PBS. BRD4 detection antibody (Atlas no. AMAb90841) was diluted 1:500 into 0.6% blocker A (MSD R93BA-4) in 1× wash buffer. CRBN-DDB1 Protein Expression. Two expression vectors were constructed with either full-length human CRBN with a thrombincleavable N-terminal histidine tag or full-length DDB1 cloned behind the polyhedron promotor of pORB1 (Orbigin INC). Virus stocks of each were prepared in Sf9 insect cells using the BestBac 2.0 cotransfection kit according to the manufacturer’s instructions (Expression Systems LLC) and titered by Expression Systems. Recombinant heterodimer was prepared using cotransfection of HisCRBN and DDB1 in 10 L WAVE bioreactor culture of Sf9 cells at 2E6/mL. Cells were infected using HisCRBN and DDB1 virus stocks at an MOI of 1:4, respectively. Zinc acetate was added to the culture to a final concentration of 50 μM. The culture was harvested after 48 h, and cells were pelleted at 4000g. The pelleted cells were resuspended in 50 mM Tris pH 7.4, 500 mM NaCl, 7.5 mM imidazole, 10% glycerol with Roche Complete EDTA-free proteinase cocktail and lysed by microfluidizer. The lysate was clarified by centrifugation at 10000g and filtration through a 1 μm serum filter (Pall). The lysate was bound at 5 mL/min to a HisTrap excel 5 mL column. The column was washed with 80 CVs of resuspension buffer and eluted with 10 CVs of an elution buffer of 50 mM Tris pH 7.4, 500 mM NaCl, 500 mM imidazole, and 10% glycerol. Elution fractions containing heterodimer were concentrated and purified by sizing on a HiLoad 26/600 Superdex 200 pg in a buffer of PBS 1 mM DTT. HisCRBN DDB1 eluted as a single peak and yielded approximately 1 mg per liter of culture. CRBN-DDB1/BRD4 and VHL/BRD4 Proximity Assays. To detect ternary complex formation induced by PROTACs, we used AlphaScreen technology to measure luminescence arising from proximity of CRBN- or VHL-bound acceptor beads and BRD4bound donor beads. The proximity assay was performed in a 384-well white OptiPlate (PerkinElmer) in a total volume of 30 μL. For CRBNDDB1/BRD4 proximity assay, the reaction mixture contained 60 nM His-tagged CRBN-DDB1, 60 nM GST-tagged BRD4 (49−170) (Sigma-Aldrich), 20 μg/mL Nickel Chelate AlphaScreen Acceptor, and Glutathione Donor beads (PerkinElmer) and serially diluted test compounds in the binding buffer of 50 mM HEPES, pH 7.4, 200 mM NaCl, 1 mM TCEP, and 0.1% BSA. 10 μL of 3× test compounds (in 3% DMSO) were preincubated with 10 μL of 3× CRBN-DDB1/ BRD4 mixture at rt for 60 min. Then 10 μL of 3× Nickel Chelate AlphaScreen Acceptor and Glutathione Donor beads were added and assay plates were further incubated at rt for 60 min prior to luminescence detection on an Envision 2102 plate reader (PerkinElmer). VHL/BRD4 proximity assay was performed under the same assay conditions but replacing His-tagged CRBN-DDB1 with His-tagged VHL(1−154) (Abcam). For data analysis, the maximal luminescence

signal was normalized to POCmax (maximal percent of control) using eq 4:

POCmax = 100

(St − Sc −) (Sc + − Sc −)

(4)

where St is the maximal luminescence signal of test compound, Sc+ is the maximal luminescence signal of positive control, and Sc− is the average luminescence signal of DMSO control. Chemistry. Unless otherwise noted, all reagents were commercially available and used as received. All final compounds possessed purity ≥95% as determined by high performance liquid chromatography (HPLC). The HPLC method used the following conditions: HALO-C18 column (3 mm × 50 mm, 2.7 μm) at 40 °C with a 2.0 mL/min flow rate; solvent A of 0.1% TFA in water, solvent B of 0.1% TFA in MeCN; 5−95% gradient A:B over 1.5 min. Flow from the UV detector was split (50:50) to the MS detector, which was configured with APIES as ionizable source. 1H NMR spectra were recorded on a 400 MHz Bruker NMR spectrometer at ambient temperature. Data are reported as follows: chemical shift (ppm, δ units) from an internal standard, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, and br = broad), coupling constant (Hz), and integration. 13C NMR spectra were recorded on a 600 MHz Bruker NMR spectrometer at ambient temperature (32768 scans). Silica gel chromatography was performed using an ISCO Combiflash RF medium pressure liquid chromatography system. Benchmark BRD4 PROTACs 1,11 and 2,10 and VHL-amine (9)13 were synthesized according to previously reported literature protocols. The IMiD containing PROTACs were isolated as a mixture of diastereomers which included racemic mixtures at the IMiD stereocenter with enantio-pure (S)-configuration at the JQ-1 stereocenter. (S)-N-(2-Azidoethyl)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6Hthieno[3,2-f ][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamide (4). Step 1. (S)-2-(4-(4-Chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f ][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetic acid compound with 2,2,2-trifluoroacetic acid (1:1) prepared according to Ciulli and co-workers.11 (S)-tert-Butyl 2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6Hthieno[3,2-f ][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate (Selleckchem.com, catalogue no. S7110, 897 mg, 1.96 mmol) was treated with DCM (6 mL) and TFA (4 mL, 51.9 mmol) and allowed to stir at rt for 2 h. LC-MS analysis of the reaction mixture indicated complete tert-butyl ester deprotection m/z (ESI, +ve) 401.0 (M + H)+. The reaction mixture was concentrated to dryness, affording crude (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f ][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetic acid compound with 2,2,2trifluoroacetic acid (1:1) (1.01 g, 1.96 mmol, 100% yield) as a viscous yellow tar (assuming quantitative yield of the mono(TFA) salt). 1 H NMR (400 MHz, DMSO-d6) δ ppm 3.49 (2 H, dd, J = 16.6, 6.8 Hz), 3.37 (1 H, dd, J = 16.6, 7.4 Hz), 2.65−2.70 (3 H, m), 2.14 (1 H, s). 19F NMR (376 MHz, DMSO-d6) δ ppm −74.94 (1 F, s), assume mono(TFA) salt. m/z (ESI, +ve) 401.1 (M + H)+. 457

DOI: 10.1021/acs.jmedchem.6b01781 J. Med. Chem. 2018, 61, 453−461

Journal of Medicinal Chemistry

Article

Step 2. (S)-2-(4-(4-Chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2f ][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetic acid compound with 2,2,2-trifluoroacetic acid (1:1) (1.01 g, 1.96 mmol) was treated with 2-azidoethanamine hydrochloride (Enamine Chemicals, 289 mg, 2.36 mmol), DMF (10 mL), and Hunig’s base (1.72 mL, 9.82 mmol) followed by HATU (933 mg, 2.45 mmol) in one portion. The reaction mixture was then allowed to stir at rt for 30 min. LC-MS analysis of the reaction mixture indicated ca. 50% conversion to the desired product m/z (ESI, +ve) 469.1 (M + H)+. The reaction mixture was treated with additional Hunig’s base (1.0 mL), and after another 30 min, LC-MS analysis indicated clean conversion to the desired product. The reaction mixture was treated with a saturated solution of NaHCO3 and extracted with EtOAc (2 × 50 mL), washed with brine (3 × 25 mL), and dried over MgSO4, filtered, and concentrated. The crude residue was purified on an ISCO Combiflash RF (40 g Grace Reveleris column, using a gradient of 0−60% [20% MeOH in DCM] in DCM (product eluted with ca. 30−38% [20% MeOH in DCM]/DCM)), affording (S)-N-(2-azidoethyl)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl6H-thieno[3,2-f ][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamide (4, 931 mg, 1.99 mmol, > 99% yield) as a light-yellow foam after drying in a vacuum oven at 45 °C overnight. 1H NMR (400 MHz, DMSO-d6) δ ppm 8.45 (1 H, br t, J = 5.2 Hz), 7.42−7.50 (4 H, m), 4.51 (1 H, dd, J = 8.2, 5.9 Hz), 3.33−3.44 (3 H, m), 3.17−3.29 (3 H, m), 2.59 (3 H, s), 2.41 (3 H, s), 1.62 (3 H, s). 13C NMR (151 MHz, DMSO-d6) δ ppm 170.4, 165.1, 163.5, 162.8, 155.6, 150.3, 137.2, 135.7, 132.7, 131.2, 130.6, 130.3, 130.1, 128.9, 54.3, 50.5, 38.72, 38.65, 38.1, 36.3, 31.2, 28.3, 14.5, 13.2, 11.8. m/z (ESI, +ve) 469.1 (M + H)+. 2-(2,6-Dioxopiperidin-3-yl)-4-(prop-2-yn-1-yloxy)isoindoline-1,3dione (8a). Compound 7 (10 g, 36.5 mmol), propargyl bromide (6.1 g, 51.1 mmol), and Na2CO3 (5.8 g, 54.7 mmol) in DMF (80 mL) was stirred at 60 °C for 24 h. The solvent was evaporated, water (150 mL) was added, and then extracted with EtOAc (3 × 200 mL). The combined organic layers were washed with brine (100 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give crude product which was purified by reverse phase column chromatography (RP-18, H2O/MeOH = 1:1) to furnish the title compound (8a, 6.0 g, 19.2 mmol, 53% yield) as a tan solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 11.06 (1 H, br s), 7.86 (1 H, t, J = 7.9 Hz), 7.57 (1 H, d, J = 8.1 Hz), 7.51 (1 H, d, J = 7.1 Hz), 5.05−5.13 (3 H, m), 3.69 (1 H, t, J = 2.2 Hz), 2.82−2.95 (1 H, m), 2.52−2.63 (2 H, m), 1.99−2.10 (1 H, m). 13C NMR (151 MHz, DMSO-d6) δ ppm 173.3, 170.4, 167.2, 165.6, 154.8, 137.3, 133.8, 120.7, 117.3, 116.5, 79.9, 78.7, 56.9, 49.3, 31.4, 22.4. m/z (ESI, +ve) 313.0 (M + H)+. Representative Procedure for the Synthesis of IMiD Alkynes (8b−e). 2-(2,6-Dioxopiperidin-3-yl)-4-(2-(prop-2-yn-1-yloxy)ethoxy)isoindoline-1,3-dione (8b). A mixture of 2-(prop-2-yn-1yloxy)ethyl 4-methylbenzenesulfonate (8.9 g, 35 mmol), 7 (8 g, 29 mmol) and Na2CO3 (4.6 g, 44 mmol) in DMF (100 mL) was stirred at 80 °C for 42 h. The mixture was added to water (300 mL) and extracted with DCM (3 × 300 mL), and the combined organic layers were washed with brine (200 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give crude product. The residue was purified by reverse phase HPLC (0.1% TFA in CH3CN/ water) to furnish the title compound (8b, 6.7 g, 18.4 mmol, 63% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 11.10 (1 H, s), 7.82 (1 H, t, J = 7.9 Hz), 7.53 (1 H, d, J = 8.7 Hz), 7.46 (1 H, d, J = 7.0 Hz), 5.09 (1 H, dd, J = 12.9, 5.4 Hz), 4.32−4.43 (2 H, m), 4.28 (2 H, d, J = 2.3 Hz), 3.79−3.88 (2 H, m), 3.38−3.51 (1 H, m), 2.82− 2.94 (1 H, m), 2.52−2.63 (2 H, m), 1.99−2.07 (1 H, m). 13C NMR (151 MHz, DMSO-d6) δ ppm 173.3, 170.4, 167.3, 165.8, 156.2, 137.5, 133.7, 120.4, 116.8, 115.9, 80.7, 77.8, 69.1, 67.8, 58.4, 49.2, 31.4, 22.5. m/z (ESI, +ve) 357.2 (M + H)+. 2-(2,6-Dioxopiperidin-3-yl)-4-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)isoindoline-1,3-dione (8c). Prepared according to the representative procedure for the synthesis of IMiD alkynes, affording the title compound (8c, 6.7 g, 16 mmol, 55% yield) as an off-white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 11.09 (1 H, s), 7.81 (1 H, t, J = 7.9 Hz), 7.53 (1 H, d, J = 8.5 Hz), 7.46 (1 H, d, J = 7.0 Hz), 5.08 (1 H, dd, J = 12.9, 5.4 Hz), 4.30−4.40 (2 H, m),

4.09−4.19 (2 H, m), 3.76−3.85 (2 H, m), 3.63−3.73 (2 H, m), 3.53− 3.61 (2 H, m), 3.36−3.45 (1 H, m), 2.80−2.94 (1 H, m), 2.52−2.63 (2 H, m), 1.98−2.08 (1 H, m). 13C NMR (151 MHz, DMSO-d6) δ ppm 173.3, 170.4, 167.3, 165.8, 156.3, 137.5, 133.7, 120.6, 116.8, 115.9, 80.8, 77.6, 70.4, 69.4, 69.2, 69.1, 58.0, 49.2, 31.4, 22.5. m/z (ESI, +ve) 401.2 (M + H)+. 2-(2,6-Dioxopiperidin-3-yl)-4-(2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)ethoxy)isoindoline-1,3-dione (8d). Prepared according to the representative procedure for the synthesis of IMiD alkynes, affording the title compound (8d, 5.4 g, 12 mmol, 41% yield) as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ ppm 11.09 (1 H, s), 7.81 (1 H, t, J = 7.9 Hz), 7.53 (1 H, d, J = 8.5 Hz), 7.46 (1 H, d, J = 7.3 Hz), 5.08 (1 H, dd, J = 12.9, 5.4 Hz), 4.29−4.40 (2 H, m), 4.12 (2 H, d, J = 2.3 Hz), 3.73−3.88 (2 H, m), 3.58−3.71 (2 H, m), 3.52−3.55 (6 H, m), 3.40 (1 H, t, J = 2.3 Hz), 3.32 (2 H, s), 2.82−2.95 (1 H, m), 2.52− 2.63 (2 H, m), 1.98−2.08 (1 H, m). 13C NMR (151 MHz, DMSO-d6) δ ppm 173.3, 170.4, 167.3, 165.8, 156.3, 137.5, 133.7, 120.6, 116.8, 115.9, 80.8, 77.5, 70.6, 70.3, 70.0, 69.4, 69.2, 69.0, 58.0, 49.2, 31.4, 22.5. m/z (ESI, +ve) 445.2 (M + H)+. 4-(3,6,9,12-Tetraoxapentadec-14-yn-1-yloxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (8e). Prepared according to the representative procedure for the synthesis of IMiD alkynes, affording the title compound (8e, 6.8 g, 13.3 mmol, 45% yield) as a pale-yellow solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 11.09 (1 H, s), 7.81 (1 H, t, J = 7.9 Hz), 7.53 (1 H, d, J = 8.5 Hz), 7.46 (1 H, d, J = 7.3 Hz), 5.08 (1 H, dd, J = 12.6, 5.4 Hz), 4.28−4.41 (3 H, m), 4.08− 4.17 (3 H, m), 3.75−3.86 (3 H, m), 3.62−3.67 (3 H, m), 3.54−3.57 (2 H, m), 3.39−3.44 (1 H, m), 2.82−2.94 (1 H, m), 2.52−2.63 (2 H, m), 1.98−2.07 (1 H, m). 13C NMR (151 MHz, DMSO-d6) δ ppm 173.3, 170.4, 167.3, 165.7, 156.3, 137.5, 133.7, 120.6, 116.8, 115.9, 80.8, 77.6, 70.6, 70.3, 70.2, 70.0, 69.4, 69.2, 69.0, 58.0, 49.2, 31.4, 22.5. m/z (ESI, +ve) 489.2 (M + H)+. Representative Procedure for the Synthesis of VHL-alkynes (10a−e). (2S,4R)-1-((S)-3,3-Dimethyl-2-(2-(prop-2-yn-1-yloxy)acetamido)butanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (10a).28 To a solution of 2-(prop2-yn-1-yloxy)acetic acid (684 mg, 6.0 mmol, 1.0 equiv) in DCM (50 mL) was added (COCl)2 (1.90 g, 15.0 mmol, 1.31 mL, 2.5 equiv) followed by DMF (438 mg, 6 mmol, 0.5 mL, 1.0 equiv) dropwise. After addition, the mixture was stirred at 15 °C for 1 h, then the mixture was concentrated under vacuum. The crude acid chloride was diluted with THF (50 mL), 9 (2.8 g, 6.0 mmol, 1.0 equiv) and TEA (3.04 g, 30 mmol, 4.16 mL, 5.0 equiv), and then the mixture was stirred at 40 °C for 12 h. The mixture was quenched with water (80 mL) and extracted with EtOAc (2 × 80 mL), and the combined organic phase was washed with brine (100 mL), dried over anhydrous Na2SO4, filtered, and concentrated under vacuum. The residue was purified by prep-HPLC affording the title compound (10a, 1.40 g, 2.64 mmol, 44% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 9.03 (1 H, s), 8.64 (1 H, t, J = 5.9 Hz), 7.47−7.54 (1 H, m), 7.45 (4 H, s), 5.20 (1 H, br s), 4.60 (1 H, d, J = 9.5 Hz), 4.44−4.52 (1 H, m), 4.37−4.44 (2 H, m), 4.25−4.35 (4 H, m), 3.97−4.12 (2 H, m), 3.61−3.75 (2 H, m), 3.56 (1 H, t, J = 2.3 Hz), 2.10 (1 H, br s), 1.95 (1 H, s), 0.93−1.03 (9 H, m). 13C NMR (151 MHz, DMSO-d6) δ ppm 172.2, 169.6, 168.4, 151.9, 148.2, 139.9, 131.6, 130.2, 129.4, 129.2, 127.9, 80.0, 78.4, 69.4, 68.5, 59.2, 58.4, 57.1, 56.3, 42.2, 38.4, 36.2, 26.8, 26.7, 16.4. m/z (ESI, +ve) 527.2 (M + H)+. (2S,4R)-1-((S)-3,3-Dimethyl-2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)acetamido)butanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (10b). Prepared according to the representative procedure for the synthesis of VHL-alkynes, affording the title compound (10b, 2.0 g, 40% yield) as a light-yellow syrup. 1H NMR (400 MHz, DMSO-d6) δ ppm 8.98 (1 H, s), 8.60 (1 H, t, J = 6.1 Hz), 7.48 (1 H, br d, J = 9.5 Hz), 7.40 (4 H, s), 5.15 (1 H, d, J = 3.5 Hz), 4.57 (1 H, d, J = 9.5 Hz), 4.39−4.47 (2 H, m), 4.37 (1 H, br d, J = 13.7 Hz), 4.12−4.30 (4 H, m), 3.96 (2 H, s), 3.56−3.70 (7 H, m), 3.36−3.44 (1 H, m), 1.90 (1 H, br s), 0.88−1.00 (9 H, m). 13 C NMR (151 MHz, DMSO-d6) δ ppm 172.3, 169.6, 169.1, 151.9, 148.2, 139.9, 131.6, 130.2, 129.4, 129.2, 128.6, 127.9, 80.6, 77.73, 77.70, 70.7, 70.5, 70.0, 69.4, 68.7, 59.2, 58.0, 57.0, 56.2, 42.2, 40.9, 38.4, 36.2, 26.8, 26.7, 16.4, 16.4. m/z (ESI, +ve) 571.2 (M + H)+. 458

DOI: 10.1021/acs.jmedchem.6b01781 J. Med. Chem. 2018, 61, 453−461

Journal of Medicinal Chemistry

Article

(2S,4R)-1-((S)-2-(tert-Butyl)-4-oxo-6,9,12-trioxa-3-azapentadec14-yn-1-oyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (10c). Prepared according to the representative procedure for the synthesis of VHL-alkynes, affording the title compound (10c, 1.0 g, 40% yield) as a light-yellow syrup. 1H NMR (400 MHz, DMSO-d6) δ ppm 9.03 (1 H, s), 8.64 (1 H, br t, J = 6.0 Hz), 7.41−7.51 (6 H, m), 5.19 (1 H, d, J = 3.5 Hz), 4.61 (1 H, d, J = 9.5 Hz), 4.36−4.52 (4 H, m), 4.24−4.36 (2 H, m), 4.12−4.22 (3 H, m), 4.01 (2 H, s), 3.56−3.74 (13 H, m), 3.40−3.52 (3 H, m), 2.65 (1 H, br s), 2.57−2.60 (21 H, m), 2.03−2.25 (1 H, m), 1.95 (1 H, ddd, J = 12.9, 8.7, 4.6 Hz), 0.93−1.04 (9 H, m). 13C NMR (151 MHz, DMSO-d6) δ ppm 172.2, 169.6, 169.1, 151.9, 148.2, 139.9, 131.6, 130.2, 129.4, 129.2, 127.9, 80.8, 77.6, 70.9, 70.1, 70.0, 69.4, 69.01, 68.99, 59.2, 58.0, 57.0, 56.2, 42.2, 38.4, 36.2, 26.8, 26.8, 26.7, 16.4, 16.4. m/z (ESI, +ve) 615.2 (M + H)+. (2S,4R)-1-((S)-2-(tert-Butyl)-4-oxo-6,9,12,15-tetraoxa-3-azaoctadec-17-yn-1-oyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (10d). Prepared according to the representative procedure for the synthesis of VHL-alkynes, affording the title compound (10d, 3.6 g, 32% yield) as a light-yellow syrup. 1 H NMR (400 MHz, DMSO-d6) δ ppm 8.98 (1 H, s), 8.59 (1 H, t, J = 6.1 Hz), 7.40 (5 H, s), 5.15 (1 H, d, J = 3.5 Hz), 4.56 (1 H, d, J = 9.5 Hz), 4.39−4.47 (2 H, m), 4.32−4.39 (2 H, m), 4.19−4.31 (2 H, m), 4.10−4.15 (3 H, m), 3.96 (2 H, s), 3.62−3.69 (3 H, m), 3.52− 3.62 (11 H, m), 3.38−3.42 (2 H, m), 2.53−2.55 (35 H, m), 2.42−2.47 (4 H, m), 2.05 (1 H, br dd, J = 12.5, 7.8 Hz), 1.90 (1 H, ddd, J = 12.8, 8.7, 4.5 Hz), 0.89−0.99 (9 H, m). 13C NMR (151 MHz, DMSO-d6) δ ppm 172.2, 169.6, 169.1, 151.9, 148.2, 139.9, 131.6, 130.2, 129.2, 128.6, 127.9, 80.8, 77.6, 70.9, 70.1, 70.03, 70.00, 69.4, 69.01, 68.99, 59.2, 58.0, 57.0, 56.2, 42.2, 38.4, 36.2, 26.8, 26.7, 16.4. m/z (ESI, +ve) 637.2 (M + H)+. (2S,4R)-1-((S)-2-(tert-Butyl)-4-oxo-6,9,12,15,18-pentaoxa-3-azahenicos-20-yn-1-oyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (10e). Prepared according to the representative procedure for the synthesis of VHL-alkynes, affording the title compound (10e, 6.0 g, 39% yield) as a light-yellow oil. 1H NMR (400 MHz, DMSO-d6) δ ppm 8.98 (1 H, s), 8.59 (1 H, br t, J = 5.9 Hz), 7.34−7.46 (6 H, m), 5.15 (1 H, d, J = 3.3 Hz), 4.56 (1 H, d, J = 9.7 Hz), 4.40−4.47 (2 H, m), 4.31−4.39 (2 H, m), 4.19−4.29 (2 H, m), 4.13 (3 H, d, J = 2.3 Hz), 3.96 (2 H, s), 3.57−3.69 (7 H, m), 3.44−3.57 (18 H, m), 3.38−3.42 (2 H, m), 2.60 (1 H, br s), 2.52−2.55 (21 H, m), 2.42−2.46 (4 H, m), 2.01−2.11 (1 H, m), 1.90 (1 H, ddd, J = 12.9, 8.8, 4.5 Hz), 0.88−0.99 (9 H, m). 13C NMR (151 MHz, DMSO-d6) δ ppm 172.2, 169.6, 169.1, 151.9, 148.2, 139.9, 131.6, 130.2, 129.4, 129.2, 128.6, 127.9, 80.8, 77.6, 70.9, 70.3, 70.2, 70.1, 69.4, 69.0, 59.2, 58.0, 57.0, 56.2, 42.2, 40.9, 38.4, 36.2, 26.8, 26.8, 26.7, 16.4. m/z (ESI, +ve) 703.4 (M + H)+. Representative Procedure for the “Click Ligation”. In a 3 mL vial was weighed (2S,4R)-1-((S)-3,3-dimethyl-2-(2-(prop-2-yn-1-yloxy)acetamido)butanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (10a, 51 mg, 0.097 mmol), (+)-sodium lascorbate (3.8 mg, 0.019 mmol), copper(II) sulfate powder (3.1 mg, 0.019 mmol), and (S)-N-(2-azidoethyl)-2-(4-(4-chlorophenyl)-2,3,9trimethyl-6H-thieno[3,2-f ][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamide (4, 45.4 mg, 0.097 mmol). The reaction mixture was treated with THF (1 mL, ca. 0.1M) and 3−4 drops of water, and then the headspace of the vial was purged briefly with argon and stirred at rt for 16 h. LC-MS analysis of the crude reaction mixture indicated clean conversion to the desired product m/z (ESI, +ve) 995.4 (M + H)+. The reaction mixture was transferred to a 5 g silica gel sample loader using a pipet and purified on an ISCO Combiflash RF (12 g Redisep Gold column, using a gradient of 0−100% [20% MeOH in DCM] in DCM (product eluted with ca. 85−95% [20% MeOH in DCM]/ DCM)), affording the desired (2S,4R)-1-((S)-2-(2-((1-(2-(2-((S)-4(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f ][1,2,4]triazolo[4,3a][1,4]diazepin-6-yl)acetamido)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol5-yl)benzyl)pyrrolidine-2-carboxamide (12a, 76.1 mg, 0.076 mmol, 79% yield) as a white amorphous solid after drying overnight in a vacuum oven at 45 °C.

2-((S)-4-(4-Chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f ][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-N-(2-(4-(((2-(2,6-dioxopiperidin-3yl)-1,3-dioxoisoindolin-4-yl)oxy)methyl)-1H-1,2,3-triazol-1-yl)ethyl)acetamide (11a). Prepared according to the general “click ligation” method, affording the title compound (11a, 79.6 mg, 0.10 mmol, 90% yield) as a white amorphous solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 11.08 (1 H, s), 8.44 (1 H, br t, J = 5.4 Hz), 8.31 (1 H, s), 7.72− 7.84 (2 H, m), 7.40−7.50 (5 H, m), 5.75 (1 H, s), 5.42 (2 H, s), 5.06 (1 H, dd, J = 12.7, 5.5 Hz), 4.44−4.52 (3 H, m), 3.44−3.64 (2 H, m), 3.22 (2 H, br d, J = 7.0 Hz), 2.80−2.93 (1 H, m), 2.58 (3 H, s), 2.40 (3 H, s), 1.96−2.05 (1 H, m), 1.62 (3 H, s). 13C NMR (151 MHz, DMSO-d6) δ ppm 173.2, 170.6, 170.4, 167.2, 165.7, 163.6, 155.8, 155.6, 142.3, 137.4, 137.2, 135.7, 133.8, 132.7, 131.2, 130.7, 130.4, 130.1, 128.9, 125.8, 120.9, 117.0, 116.1, 62.7, 55.4, 54.2, 49.4, 49.2, 38.0, 31.4, 31.2, 22.5, 14.6, 14.5, 13.2, 11.8. m/z (ESI, +ve) 781.2 (M + H)+. 2-((S)-4-(4-Chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f ][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-N-(2-(4-((2-((2-(2,6-dioxopiperidin3-yl)-1,3-dioxoisoindolin-4-yl)oxy)ethoxy)methyl)-1H-1,2,3-triazol1-yl)ethyl)acetamide (11b). Prepared according to the general “click ligation” method, affording the title compound (11b, 89.9 mg, 0.11 mmol, 67% yield) as a light-yellow amorphous solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 11.11 (1 H, s), 8.40−8.46 (1 H, m), 8.20 (1 H, s), 7.79 (1 H, t, J = 7.5 Hz), 7.40−7.52 (5 H, m), 5.09 (1 H, dd, J = 12.8, 5.4 Hz), 4.66 (2 H, s), 4.41−4.50 (3 H, m), 4.29−4.38 (2 H, m), 3.80−3.88 (2 H, m), 3.54−3.62 (1 H, m), 3.22 (2 H, br d, J = 7.2 Hz), 2.81−2.92 (1 H, m), 2.58 (3 H, s), 2.39 (3 H, s), 1.97− 2.05 (1 H, m), 1.61 (3 H, s). 13C NMR (151 MHz, DMSO-d6) δ ppm 173.2, 170.59, 170.57, 170.4, 167.3, 165.8, 163.6, 156.3, 137.5, 137.2, 135.7, 133.71, 133.69, 132.7, 131.22, 131.21, 130.7, 130.4, 130.3, 130.0, 128.9, 124.7, 120.4, 116.8, 115.9, 100.0, 69.2, 68.1, 64.3, 55.4, 54.2, 49.4, 49.3, 49.2, 49.1, 38.0, 31.4, 22.5, 14.5, 13.1, 11.8. m/z (ESI, +ve) 825.2 (M + H)+. 2-((S)-4-(4-Chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f ][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-N-(2-(4-((2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)ethoxy)ethoxy)methyl)-1H1,2,3-triazol-1-yl)ethyl)acetamide (11c). Prepared according to the general “click ligation” method, affording the title compound (11c, 80.0 mg, 0.092 mmol, 70% yield) as a light-yellow amorphous solid. 1 H NMR (400 MHz, DMSO-d6) δ ppm 11.10 (1 H, s), 8.44 (1 H, br t, J = 5.6 Hz), 8.12 (1 H, s), 7.79 (1 H, t, J = 7.8 Hz), 7.39−7.53 (6 H, m), 5.08 (1 H, dd, J = 12.7, 5.3 Hz), 4.40−4.54 (5 H, m), 4.27−4.35 (2 H, m), 3.73−3.84 (2 H, m), 3.61−3.68 (2 H, m), 3.49−3.60 (4 H, m), 3.22 (2 H, br d, J = 7.0 Hz), 2.81−2.93 (1 H, m), 2.59 (3 H, s), 2.40 (3 H, s), 1.96−2.06 (1 H, m), 1.62 (3 H, s). 13C NMR (151 MHz, DMSO-d6) δ ppm 173.3, 170.6, 170.4, 167.3, 165.8, 163.6, 156.3, 155.6, 144.4, 137.4, 137.2, 135.7, 133.7, 132.7, 131.2, 130.7, 130.4, 130.0, 128.9, 124.7, 120.5, 116.8, 115.9, 70.5, 69.5, 69.3, 69.2, 64.0, 55.4, 54.2, 49.26, 49.23, 49.1, 38.0, 31.4, 22.5, 14.5, 13.2, 11.8. m/z (ESI, +ve) 869.4 (M + H)+. 2-((S)-4-(4-Chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f ][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-N-(2-(4-((2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)ethoxy)ethoxy)ethoxy)methyl)-1H-1,2,3-triazol-1-yl)ethyl)acetamide (11d). Prepared according to the general “click ligation” method, affording the title compound (11d, 83.3 mg, 0.091 mmol, 73% yield) as an off-white amorphous solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 11.09 (1 H, s), 8.43 (1 H, br t, J = 5.7 Hz), 8.11 (1 H, s), 7.79 (1 H, t, J = 7.8 Hz), 7.37−7.55 (6 H, m), 5.08 (1 H, dd, J = 12.8, 5.4 Hz), 4.38−4.53 (5 H, m), 4.31−4.37 (2 H, m), 3.72−3.82 (2 H, m), 3.60−3.65 (2 H, m), 3.44−3.58 (8 H, m), 3.16−3.27 (3 H, m), 2.75−2.93 (1 H, m), 2.53− 2.62 (5 H, m), 2.38−2.43 (3 H, m), 1.96−2.06 (1 H, m), 1.62 (3 H, s). 13 C NMR (151 MHz, DMSO-d6) δ ppm 173.3, 170.6, 170.4, 167.3, 165.7, 163.6, 156.3, 155.5, 150.4, 144.3, 137.4, 137.2, 135.7, 133.7, 132.7, 131.2, 130.7, 130.4, 130.0, 128.9, 124.7, 120.5, 116.8, 115.9, 105.9, 91.8, 88.4, 75.3, 73.8, 70.6, 70.3, 70.2, 69.4, 69.3, 69.2, 64.0, 55.4, 54.2, 49.3, 49.2, 38.0, 31.4, 31.2, 22.5, 14.5, 13.2, 11.8. m/z (ESI, +ve) 913.4 (M + H)+. 2-((S)-4-(4-Chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f ][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-N-(2-(4-(13-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)-2,5,8,11-tetraoxatridecyl)1H-1,2,3-triazol-1-yl)ethyl)acetamide (11e). Prepared according to 459

DOI: 10.1021/acs.jmedchem.6b01781 J. Med. Chem. 2018, 61, 453−461

Journal of Medicinal Chemistry

Article

the general “click ligation” method, affording the title compound (11e, 76.2 mg, 0.080 mmol, 69% yield) as an off-white amorphous solid. 1 H NMR (400 MHz, DMSO-d6) δ ppm 11.10 (1 H, s), 8.44 (1 H, br t, J = 5.5 Hz), 8.12 (1 H, s), 7.80 (1 H, t, J = 7.9 Hz), 7.41−7.55 (6 H, m), 5.09 (1 H, dd, J = 12.8, 5.4 Hz), 4.32−4.54 (7 H, m), 4.09 (1 H, br d, J = 5.1 Hz), 3.72−3.84 (2 H, m), 3.57−3.69 (3 H, m), 3.47−3.57 (10 H, m), 3.23 (2 H, br d, J = 7.0 Hz), 3.18 (3 H, d, J = 3.7 Hz), 2.83−2.95 (1 H, m), 2.60 (3 H, s), 2.41 (3 H, s), 1.98−2.08 (1 H, m), 1.63 (3 H, s). 13C NMR (151 MHz, DMSO-d6) δ ppm 173.3, 170.6, 170.4, 167.3, 165.7, 163.6, 156.3, 155.5, 150.3, 144.3, 137.4, 137.2, 135.7, 133.7, 132.7, 131.2, 130.7, 130.4, 130.0, 128.9, 124.7, 120.5, 116.8, 115.9, 91.8, 88.4, 75.3, 73.8, 70.6, 70.2, 69.4, 69.3, 69.2, 64.0, 54.2, 49.3, 49.2, 49.1, 38.0, 31.4, 22.5, 14.5, 13.2, 11.8. m/z (ESI, +ve) 957.4 (M + H)+. (2S,4R)-1-((S)-2-(2-((1-(2-(2-((S)-4-(4-Chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f ][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (12a). Prepared according to the general “click ligation” method, affording (12a, 76.1 mg, 0.076 mmol, 79% yield) as a white amorphous solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 8.97 (1 H, s), 8.58 (1 H, t, J = 5.9 Hz), 8.45 (1 H, t, J = 5.6 Hz), 8.20 (1 H, s), 7.47 (1 H, s), 7.48 (2 H, d, J = 7.9 Hz), 7.35−7.45 (7 H, m), 5.15 (1 H, d, J = 3.5 Hz), 4.63 (2 H, s), 4.56 (1 H, d, J = 9.6 Hz), 4.50 (1 H, t, J = 7.1 Hz), 4.31−4.47 (5 H, m), 4.20−4.31 (1 H, m), 4.00 (2 H, d, J = 4.7 Hz), 3.53−3.69 (4 H, m), 3.21−3.27 (2 H, m), 2.55−2.63 (3 H, m), 2.43−2.46 (3 H, m), 2.41 (3 H, s), 1.98−2.10 (1 H, m), 1.90 (1 H, td, J = 8.7, 4.4 Hz), 1.62 (3 H, s), 0.87−0.97 (9 H, m). 13C NMR (151 MHz, DMSO-d6) δ ppm 172.2, 170.6, 169.6, 168.7, 163.6, 155.5, 151.9, 150.3, 148.2, 143.5, 139.9, 137.2, 135.7, 132.7, 131.6, 131.2, 130.7, 130.4, 130.2, 130.0, 129.4, 129.2, 128.9, 127.9, 125.1, 69.4, 69.0, 64.2, 59.2, 57.1, 56.2, 54.2, 49.3, 42.2, 38.4, 38.0, 36.2, 26.7, 26.7, 16.4, 14.5, 13.2, 11.8. m/z (ESI, +ve) 995.3 (M + H)+. (2S,4R)-1-((S)-2-(2-(2-((1-(2-(2-((S)-4-(4-Chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f ][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)ethoxy)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (12b). Prepared according to the general “click ligation” method, affording (12b, 61.5 mg, 0.059 mmol, 60% yield) as an off-white amorphous solid. 1 H NMR (400 MHz, DMSO-d6) δ ppm 8.95−8.98 (1 H, m), 8.59 (1 H, t, J = 6.0 Hz), 8.43 (1 H, br t, J = 5.7 Hz), 8.14 (1 H, s), 7.45− 7.52 (3 H, m), 7.33−7.45 (6 H, m), 5.15 (1 H, d, J = 3.5 Hz), 4.52− 4.61 (3 H, m), 4.38−4.52 (4 H, m), 4.36 (2 H, br d, J = 6.1 Hz), 4.20−4.32 (1 H, m), 3.95 (2 H, s), 3.47−3.70 (8 H, m), 3.19−3.27 (2 H, m), 3.17 (1 H, d, J = 5.1 Hz), 2.59 (3 H, s), 2.42 (3 H, s), 2.40 (3 H, s), 1.98−2.10 (1 H, m), 1.90 (1 H, ddd, J = 12.9, 8.7, 4.5 Hz), 1.62 (3 H, s), 0.87−0.99 (9 H, m). 13C NMR (151 MHz, DMSO-d6) δ ppm 172.2, 171.8, 170.6, 170.0, 169.7, 169.1, 168.2, 163.6, 155.5, 152.0, 151.9, 150.3, 148.2, 144.2, 139.9, 137.2, 135.7, 132.7, 131.6, 131.2, 130.7, 130.5, 130.4, 130.2, 130.0, 129.4, 129.2, 128.9, 128.6, 127.9, 124.8, 124.7, 109.0, 70.8, 70.7, 70.1, 70.1, 69.9, 69.3, 69.03, 68.97, 67.4, 64.1, 64.0, 59.4, 59.2, 57.0, 56.2, 56.0, 55.4, 54.2, 49.3, 49.1, 38.4, 38.0, 36.6, 36.2, 26.8, 26.7, 26.7, 22.7, 18.4, 16.43, 16.38, 14.5, 13.2, 11.8. m/z (ESI, +ve) 1039.4 (M + H)+. (2S,4R)-1-((S)-12-(tert-Butyl)-1-(1-(2-(2-((S)-4-(4-chlorophenyl)2,3,9-trimethyl-6H-thieno[3,2-f ][1,2,4]triazolo[4,3-a][1,4]diazepin6-yl)acetamido)ethyl)-1H-1,2,3-triazol-4-yl)-10-oxo-2,5,8-trioxa-11azatridecan-13-oyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (12c). Prepared according to the general “click ligation” method, affording (12c, 87.6 mg, 0.081 mmol, 72% yield) as an off-white amorphous solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 8.94−9.00 (1 H, m), 8.59 (1 H, t, J = 6.0 Hz), 8.43 (1 H, t, J = 5.7 Hz), 8.07−8.14 (1 H, m), 7.46−7.51 (2 H, m), 7.30− 7.45 (7 H, m), 5.14 (1 H, d, J = 3.5 Hz), 4.56 (1 H, d, J = 9.8 Hz), 4.47−4.53 (3 H, m), 4.39−4.47 (4 H, m), 4.36 (2 H, br d, J = 6.3 Hz), 4.25 (1 H, br dd, J = 15.7, 5.8 Hz), 4.09 (3 H, q, J = 5.3 Hz), 3.96 (2 H, s), 3.64−3.70 (1 H, m), 3.53−3.63 (11 H, m), 3.23 (2 H, br d, J = 7.0 Hz), 3.17 (9 H, d, J = 5.3 Hz), 2.59 (3 H, s), 2.43 (3 H, s), 2.41 (3 H, s), 2.04 (1 H, br d, J = 8.4 Hz), 1.85−1.98 (1 H, m), 1.62 (3 H, s), 1.25 (2 H, br d, J = 8.2 Hz), 0.93 (9 H, s). 13C NMR (151 MHz,

DMSO-d6) δ ppm 172.2, 170.6, 169.6, 169.1, 163.6, 155.5, 151.9, 150.3, 148.2, 144.3, 139.9, 137.2, 135.7, 132.7, 131.6, 131.2, 130.7, 130.4, 130.2, 130.0, 129.4, 129.2, 128.9, 128.6, 128.0, 124.7, 100.0, 70.9, 70.2, 70.08, 70.07, 69.4, 69.2, 64.0, 59.2, 57.0, 56.2, 54.2, 49.3, 49.1, 46.2, 42.2, 38.4, 38.0, 36.6, 36.2, 26.7, 26.7, 16.44, 16.39, 14.5, 13.2, 11.8. m/z (ESI, +ve) 1083.3 (M + H)+. (2S,4R)-1-((S)-15-(tert-Butyl)-1-(1-(2-(2-((S)-4-(4-chlorophenyl)2,3,9-trimethyl-6H-thieno[3,2-f ][1,2,4]triazolo[4,3-a][1,4]diazepin6-yl)acetamido)ethyl)-1H-1,2,3-triazol-4-yl)-13-oxo-2,5,8,11-tetraoxa-14-azahexadecan-16-oyl)-4-hydroxy-N-(4-(4-methylthiazol5-yl)benzyl)pyrrolidine-2-carboxamide (12d). Prepared according to the general “click ligation” method, affording (12d, 61.8 mg, 0.055 mmol, 68% yield) as a pale-green amorphous solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 8.97 (1 H, s), 8.58 (1 H, t, J = 5.9 Hz), 8.43 (1 H, t, J = 5.6 Hz), 8.11 (1 H, s), 7.45−7.51 (2 H, m), 7.34−7.44 (7 H, m), 5.14 (1 H, d, J = 3.5 Hz), 4.55 (1 H, d, J = 9.6 Hz), 4.48−4.52 (3 H, m), 4.38−4.47 (4 H, m), 4.36 (2 H, br d, J = 6.5 Hz), 4.20−4.31 (1 H, m), 4.08 (2 H, q, J = 5.3 Hz), 3.95 (2 H, s), 3.63−3.69 (1 H, m), 3.57−3.63 (3 H, m), 3.45−3.57 (13 H, m), 3.22 (2 H, d, J = 7.2 Hz), 3.16 (7 H, d, J = 5.3 Hz), 2.59 (3 H, s), 2.43 (3 H, s), 2.40 (3 H, s), 2.03 (1 H, br d, J = 8.2 Hz), 1.84−1.94 (1 H, m), 1.62 (3 H, s), 0.93 (9 H, s). 13C NMR (151 MHz, DMSO-d6) δ ppm 173.3, 172.2, 170.6, 169.6, 169.1, 163.6, 155.6, 151.9, 148.2, 144.3, 139.9, 139.4, 137.2, 135.7, 132.7, 131.6, 131.2, 130.7, 130.3, 130.2, 130.0, 129.4, 129.2, 128.9, 128.6, 127.9, 124.7, 70.9, 70.29, 70.26, 70.14, 70.06, 69.36, 69.35, 64.0, 59.2, 57.0, 56.2, 54.2, 49.3, 49.1, 38.4, 38.0, 36.2, 26.7, 26.6, 16.43, 16.39, 14.5, 13.2, 11.8. m/z (ESI, +ve) 1127.4 (M + H)+. (2S,4R)-1-((S)-18-(tert-Butyl)-1-(1-(2-(2-((S)-4-(4-chlorophenyl)2,3,9-trimethyl-6H-thieno[3,2-f ][1,2,4]triazolo[4,3-a][1,4]diazepin6-yl)acetamido)ethyl)-1H-1,2,3-triazol-4-yl)-16-oxo-2,5,8,11,14-pentaoxa-17-azanonadecan-19-oyl)-4-hydroxy-N-(4-(4-methylthiazol5-yl)benzyl)pyrrolidine-2-carboxamide (12e). Prepared according to the general “click ligation” method and isolated (12e, 61.6 mg, 0.053 mmol, 55% yield) as an off-white amorphous solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 8.97 (1 H, s), 8.59 (1 H, t, J = 6.0 Hz), 8.43 (1 H, t, J = 5.8 Hz), 8.12 (1 H, s), 7.44−7.49 (2 H, m), 7.37−7.44 (7 H, m), 5.14 (1 H, d, J = 3.5 Hz), 4.56 (1 H, d, J = 9.6 Hz), 4.32− 4.53 (6 H, m), 4.20−4.29 (1 H, m), 3.96 (2 H, s), 3.63−3.70 (1 H, m), 3.48−3.63 (4 H, m), 3.23 (2 H, d, J = 7.0 Hz), 2.59 (3 H, s), 2.42− 2.45 (3 H, m), 2.41 (3 H, s), 2.02−2.10 (2 H, m), 1.86−1.95 (1 H, m), 1.62 (3 H, s), 0.91−0.98 (9 H, m). 13C NMR (151 MHz, DMSO-d6) δ ppm 172.2, 170.6, 169.6, 169.1, 163.6, 155.5, 151.9, 150.3, 148.2, 144.3, 139.9, 137.2, 135.7, 132.7, 131.6, 131.2, 130.7, 130.3, 130.2, 130.0, 129.4, 129.2, 128.9, 128.6, 127.9, 124.7, 70.8, 70.3, 70.24, 70.23, 70.21, 70.13, 70.05, 70.01, 69.4, 64.0, 59.2, 57.0, 56.2, 55.4, 54.2, 49.3, 38.4, 38.0, 36.6, 36.2, 31.2, 26.8, 26.6, 16.4, 13.2. m/z (ESI, +ve) 1172.2 (M + H)+.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.6b01781. 1 H NMR and 13C NMR spectra are provided for all of the novel compounds reported (PDF) Molecular formula strings (CSV)

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AUTHOR INFORMATION

Corresponding Author

*Phone: 805-313-5400. E-mail: [email protected]. ORCID

Ryan P. Wurz: 0000-0003-1413-5208 Author Contributions

All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest. 460

DOI: 10.1021/acs.jmedchem.6b01781 J. Med. Chem. 2018, 61, 453−461

Journal of Medicinal Chemistry

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Article

and the hypoxia inducible factor (HIF) alpha subunit with in vitro nanomolar affinities. J. Med. Chem. 2014, 57, 8657−8663. (14) Schneekloth, A. R.; Pucheault, M.; Tae, H. S.; Crews, C. M. Targeted intracellular protein degradation induced by a small molecule: en route to chemical proteomics. Bioorg. Med. Chem. Lett. 2008, 18, 5904−5908. (15) Sakamoto, K. M.; Kim, K. B.; Kumagai, A.; Mercurio, F.; Crews, C. M.; Deshaies, R. J. Protacs: chimeric molecules that target proteins to the Skp1-Cullin-F box complex for ubiquitination and degradation. Proc. Natl. Acad. Sci. U. S. A. 2001, 98, 8554−8559. (16) Filippakopoulos, P.; Qi, J.; Picaud, S.; Shen, Y.; Smith, W. B.; Fedorov, O.; Morse, E. M.; Keates, T.; Hickman, T. T.; Felletar, I.; Philpott, M.; Munro, S.; McKeown, M. R.; Wang, Y.; Christie, A. L.; West, N.; Cameron, M. J.; Schwartz, B.; Heightman, T. D.; La Thangue, N.; French, C. A.; Wiest, O.; Kung, A. L.; Knapp, S.; Bradner, J. E. Selective inhibition of BET bromodomains. Nature 2010, 468, 1067−1073. (17) For a recent report on the synthesis of a phthalimide ligand library, see: Lohbeck, J.; Miller, A. K. Practical synthesis of a phthalimide-based cereblon ligand to enable PROTAC development. Bioorg. Med. Chem. Lett. 2016, 26, 5260−5262. (18) For a recent review on azide-alkyne cycloaddition-click chemistry, see: Singh, M. S.; Chowdhury, S.; Koley, S. Advances of azide-alkyne cycloaddition-click chemistry over the recent decade. Tetrahedron 2016, 72, 5257−5283. (19) For a recent report on the use of click chemistry to prepare analogues of thalidomide, see: Ronnebaum, J. M.; Luzzio, F. A. Synthesis of 1,2,3-triazole “click” analogues of thalidomide. Tetrahedron 2016, 72, 6136−6141. (20) The thalidomide motif was used as a racemate because it is known that its center racemizes in vivo, see for example: Tian, C.; Xiu, P.; Meng, Y.; Zhao, W.; Wang, Z.; Zhou, R. Enantiomerization mechanism of thalidomide and the role of water and hydroxide ions. Chem. - Eur. J. 2012, 18, 14305−14313. (21) See Supporting Information for details of the synthesis of this library. (22) For more information, see: http://www.perkinelmer.com (accessed February 8, 2017). (23) The NCI-H661 cell line is a human large cell lung carcinoma cell line and found to have excellent levels of activity for both VHL and cereblon ligases and is also adherent making this cell line ideal for the MSD assay format. (24) Similar data was also generated for a NCI-H838 cell line and this data is provided in the Supporting Information. (25) Douglass, E. F., Jr.; Miller, C. J.; Sparer, G.; Shapiro, H.; Spiegel, D. A. A comprehensive mathematical model for three-body binding equilibria. J. Am. Chem. Soc. 2013, 135, 6092−6099. (26) These PROTACs also showed similar activities in the NCIH838 cell line (nonsmall cell lung carcinoma) see: Table 1 in the Supporting Information. (27) Meiners, S.; Heyken, D.; Weller, A.; Ludwig, A.; Stangl, K.; Kloetzel, P.-M.; Krüger, E. Inhibition of proteasome activity induces concerted expression of proteasome genes and de novo formation of mammalian proteasomes. J. Biol. Chem. 2003, 278, 21517−21525. (28) See Supporting Information for the comprehensive experimental procedures and for NMR spectra.

ACKNOWLEDGMENTS We thank Chris Wilde for NMR studies, Bill Romanow and Steve Thibault for DDB1/cereblon protein expression and purification and Sasha Kamb, Philip Tagari, Margaret ChuMoyer and Chris Fotsch for their support of this program.

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ABBREVIATIONS USED BRD4, member of the BET bromodomain family; DC50, concentration of a PROTAC at which the cellular protein content is reduced by half; DCM, dichloromethane; IMiD, ligand for cereblon E3 ligase; PROTAC, proteolysis targeting chimeras; TEA, triethylamine; TFA, trifluoroacetic acid; THF, tetrahydrofuran; VHL, von Hippel−Lindau protein

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REFERENCES

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DOI: 10.1021/acs.jmedchem.6b01781 J. Med. Chem. 2018, 61, 453−461