Catalytic Degraders Effectively Address Kinase Site Mutations in EML4-ALK Oncogenic Fusions

Abstract

Heterobifunctional degraders, known as proteolysis targeting chimeras (PROTACs), theoretically possess a catalytic mode-of-action, yet few studies have either confirmed or exploited this potential advantage of event-driven pharmacology. Degraders of oncogenic EML4-ALK fusions were developed by conjugating ALK inhibitors to cereblon ligands. Simultaneous optimization of pharmacology and compound properties using ternary complex modeling and physicochemical considerations yielded multiple catalytic degraders that were more resilient to clinically relevant ATP-binding site mutations than kinase inhibitor drugs. Our strategy culminated in the design of the orally bioavailable derivative CPD-1224 that avoided hemolysis (a feature of detergent-like PROTACs), degraded the otherwise recalcitrant mutant L1196M/G1202R in vivo, and commensurately slowed tumor growth, while the third generation ALK inhibitor drug lorlatinib had no effect. These results validate our original therapeutic hypothesis by exemplifying opportunities for catalytic degraders to proactively address binding site resistant mutations in cancer.

Graphical Abstract

INTRODUCTION

Targeted protein degradation has emerged as an important therapeutic modality.¹ Molecular glue degraders such as thalidomide and its derivatives EM12, lenalidomide, and pomalidomide (Figure 1) bind cereblon (CRBN), a component of an E3 ubiquitin ligase complex, mediating interactions with neosubstrates, resulting in their polyubiquitination and proteasomal degradation.¹ Additionally, CRBN ligands have been incorporated into heterobifunctional degraders, also called proteolysis targeting chimeras (PROTACs).²⁻⁴ The CRBN binding motif is chemically linked to another ligand of a protein-of-interest (POI) resulting in the hijacking of the ubiquitin ligase machinery, causing degradation of the POI. The potential for an event-driven mechanism of protein modulation provides degraders with an advantage over classical inhibitors because functional pharmacology may still result from fractional binding site occupancy.⁴

Figure 1.

Chemical structures of cereblon molecular glue degraders and ALK kinase inhibitors.

Some studies have provided evidence for the substoichiometric mediated degradation of target proteins by PROTACs, but more work is necessary to illuminate its advantages and substantiate the scope of opportunity for mechanistic differentiation over inhibitors.^5,6

We reasoned that the catalytic degrader mechanism could address a key challenge facing the development of kinase inhibitors for the treatment of cancer. The capacity for target modulation through fractional binding site occupancy may engender PROTACs with greater resilience to ATP-site mutations, but few studies have explored the antiproliferative activity of degraders for clinically relevant mutants. A PROTAC was recently developed to degrade both WT and C481S mutant forms of Bruton’s tyrosine kinase (BTK), the Cys-to-Ser clinical variant emerging following the treatment of cancers with the covalent inhibitor ibrutinib which engages the ATP-site cysteine residue.⁷ In the antiviral field, a PROTAC degrader of the hepatitis C virus (HCV) NS3/4a protease was able to overcome viral variants resistant to enzyme inhibitors.⁸ However, these prior studies did not confirm substoichiometric degradation or specifically use POI in-cell occupancy to drive medicinal chemistry optimization of a catalytic mode of action to enhance mutation resilience.

To test our hypothesis, we chose as a model pathobiological system oncogenic fusions of anaplastic lymphoma kinase (ALK) with the echinoderm microtubule-associated protein-like 4 (EML4) that drive non-small-cell lung cancer (NSCLC) through constitutive activation of the kinase.⁹ Several ALK ATP-competitive inhibitor drugs have been developed for the treatment of EML4-ALK NSCLC, providing a useful benchmark against which to demonstrate differential efficacy. It has been shown that 50% ALK inhibition, and thus 50% occupancy of the ATP-site, is required for clinically relevant tumor growth inhibition (TGI), substantiating a stoichiometric relationship between inhibition of autophosphorylation (measured as p-ALK) and phenotypic efficacy.^10,11 However, mutations in the ATP-binding pocket rapidly emerge in patients treated with inhibitors that reduce drug binding affinity, ameliorating drug efficacy and causing relapse. Common mutations include C1156Y, L1196M, G1202R, and the double mutant L1196M/G1202R.¹² Later generations of ALK inhibitors possess improved mutant profiles over the prototype inhibitor crizotinib,¹³ such as the second generation inhibitors ceritinib,¹⁴ alectinib,¹⁵ and brigatinib¹⁶ and third generation lorlatinib¹⁷ (Figure 1), but resistance continues to emerge for these drugs in the clinic through binding site mutations. A catalytic PROTAC degrader may address this issue by retaining potent downregulation of ALK mutant oncoproteins, even when binding affinity is considerably reduced. Our goal was to develop a degrader of sufficient quality to establish both in vitro and in vivo proof-of-concept.

RESULTS

EML4-ALK exists as several variants that differ in the proportion of EML4 fused to the same cytoplasmic ALK kinase domain.¹⁸ To assess target degradation of a common pathogenic variant (v3), a bioluminescence assay was established in Ba/F3 cells¹⁹ expressing the humanized CRBN protein²⁰ and HiBiT-tagged v3 EML4-ALK to enable quantitative measurement of wild-type (WT) and mutant fusion proteins following compound treatment (see Supporting Information).²¹

To measure ALK kinase site engagement in cells (and the potential for substoichiometric event-driven pharmacology), we developed a NanoBRET occupancy assay that relies on the dose-dependent reduction in BRET signal following displacement of a fluorescent tracer from NanoLuc-tagged ALK.²² In previous work, we found that the BODIPY dye is an efficient BRET acceptor when measured at the 450/520 nm wavelength.²³ To this end we designed a BODIPY-ceritinib analog tracer (structure provided in Figure S4), and to maximize reproducibility, we performed the NanoBRET assay in Ba/F3 cells stably expressing the NanoLuc-ALK kinase domain fusion via lentiviral integration. With degradation and occupancy assays in-hand, the prototype degrader TL13–112 developed by the Gray laboratory (that links ceritinib to a pomalidomide CRBN ligand, Figure 2) was profiled.²⁴ TL13–112 demonstrated proof-of-principle downregulation of EML4-ALK in cells, but it had low degradation activity for the common clinically relevant G1202R mutation that emerges following inhibitor treatment. Importantly, although ceritinib maintains inhibitory potency for the crizotinib resistant gatekeeper mutation L1196M, it does not overcome the G1202R mutation which is proximal to the inhibitors at the entrance to the ATP site and clashes with ligands in this region. Therefore, all PROTACs in this study were screened for their ability to degrade the ceritinib resistant mutation G1202R, as well as WT. Additionally, for TL13–112, there was only a small shift between the cellular occupancy of ALK measured in the NanoBRET assay and the ALK degradation potency. Our initial design strategy was to incorporate flexibility into the ceritinib ligand in the vicinity of mutated residues, which we reasoned may improve mutation resilience. To provide a degrader that would ultimately be suitable for in vivo validation studies, linker optimization focused on reducing hydrogen-bonding capacity and length while increasing rigidity to enhance oral bioavailability,²⁵ although the impact of such changes on ternary complex formation and degradation efficiency was unclear at that time.²⁶

Figure 2.

Design of catalytic EML4-ALK degraders, charting the path from prototype TL13–112 to the advanced in vivo probe CPD-1224. DC₅₀ = concentration required to reduce HiBiT signal of variant 3 EML4-ALK by 50%; D_max (degradation maximum) in parentheses; F = oral bioavailability in mouse. The corresponding dose−response curves for the HiBiT assay are provided in Figure S2.

All PROTACs in this study were prepared using standard reductive amination chemistry that partnered an amine-containing ALK inhibitor with a thalidomide or EM12 derivative possessing the desired aldehyde (see Compound Synthesis section).

Computational models of CRBN/ALK with their respective ligands lenalidomide and a truncated derivative of ceritinib suggested the exit point of ceritinib at the surface of ALK is in close proximity to the CRBN binder lenalidomide as shown in Figure 3, indicating the linker in TL13–112 can be shortened (see Supporting Information). The distance between the ceritinib terminal benzene ring and the lenalidomide amine is ∼11 Å, providing confidence that the objectives of our linker design strategy described above were feasible. Key interacting residues of the ligands are shown and labeled, such as the tritryptophan cage in CRBN for lenalidomide, and the key gatekeeper residue L1196 in ALK. In addition, residues that comprise important polar interactions between ALK and CRBN are also shown such as E1210 in ALK and R373 in CRBN.

Figure 3.

Computational model of CRBN/ALK with lenalidomide and a truncated derivative of ceritinib. The distance between the two ligands (11 Å) is highlighted. Further details are provided in the Supporting Information.

The derivative CPD-198 was rationally designed using this model (Figure 2). The desired flexibility was introduced by replacing the isopropoxy group in ceritinib with the smaller methoxy substituent present also in brigatinib,²⁷ and the piperidine ring was broken to yield a phenethylamine spacer motif, retaining the basic center that is expected to interact with Glu1210 and aid solubility.¹⁴ The polar linker of TL13–112 was replaced with an aliphatic chain attached to the CRBN IMiD via the rigid alkyne, a motif that had been previously shown to retain CRBN binding for a bromodomain and extra-terminal (BET) degrader.²⁸ CPD-198 possessed similar WT degradation potency as TL13–112 but improved activity for the G1202R mutation. Importantly, the shift in potency between ALK occupancy and degradation (7.5-fold) is suggestive of a substoichiometric mechanism per our original goal.

Switching the CRBN ligand binding motif from the phthalimide to isoindolinone scaffold (present in EM12) to provide CPD-985 preserved degradation potency slightly improved metabolic stability relative to CPD-198 (presumably due to lower log D) and possessed good solubility in the biorelevant fed state simulated intestinal fluid (FeSSIF) assay which is a preferred method for assessing PROTAC solubility relevant to predicting oral bioavailability (Figure 2).²⁵ CPD-985 also has higher ALK NanoBRET potency than CPD-198 that may result from higher unbound levels in the cell due to its higher polarity. We decided to benchmark the in vivo pharmacokinetics (PK) of the series and performed oral and intravenous (iv) dosing of CPD-985 in mice. CPD-985 possessed poor oral bioavailability of 0.2%, and hemolysis was also observed, that was confirmed in an in vitro hemolysis assay, using both mouse and human red blood cells (RBCs) (Figure S6).²⁹

We speculated that the detergent-like physicochemistry of CPD-985 consisting of a polar EM12 “head” group attached to a long, flexible lipophilic “tail” may cause significant membrane association and subsequent destabilization, affecting the hemolysis.³⁰ CPD-198 also caused RBC lysis, presumably for these same physicochemical reasons. To address this undesired effect, the focus of the design strategy was to truncate and further rigidify the PROTAC linker in order to ameliorate broad membrane association effects,³¹ and degraders were screened for RBC lysis in vitro before progression to in vivo studies. Furthermore, these physicochemical changes were also expected to improve oral bioavailability.³²

Having established the concept of catalytic degradation of EML4-ALK, we decided to reintroduce the piperidine motif present in ceritinib to increase overall compound rigidity. The resulting degrader CPD-1108 possessed a large shift between ALK occupancy and degradation activity for both WT and G1202R EML4-ALK as expected (Figure 2). However, CPD-1108 was a weak positive in the RBC lysis in vitro assay (see Supporting Information). The linker length was reduced further to produce CPD-1131, which maintained clear substoichiometric degradation activity and good microsomal stability, and RBC lysis was avoided (Figure 2). The oral bioavailability of CPD-1131 was improved considerably to 9% compared to CPD-985, and we were buoyed by this breakthrough to continue employing property-based optimization to enable the delivery of a probe suitable for in vivo efficacy studies.

A challenge in kinase inhibitor anticancer drug discovery is the desire to maintain potent activity often across a plethora of mutants while preserving broad kinome selectivity. Ceritinib itself has a broad off-target kinase profile,³³ and CPD-1131 unfortunately also possessed relatively poor kinome binding selectivity when profiled against a panel of 468 kinases (Figure 4a).³⁴ This contrasts with the degradation selectivity of CPD-1131 determined using a previously reported quantitative mass spectrometry (MS) proteomics platform developed at DFCI.³⁵ Treatment of MOLT4 cells for 6 h with CPD-1131 identified only five proteins that were degraded to any significant extent (Figure 4b). This is likely due to the difficulty in forming productive ternary complexes for many of the binding off-targets because of incompatible protein−protein interfaces or due to the presentation of the bound proteins being incompatible with subsequent ubiquitination.

Figure 4.

(a) CPD-1131 kinase binding selectivity (TREEspot visualization of KINOMEscan data). (b) Quantitative MS proteomics in MOLT4 cells (which do not express ALK) of CPD-1131. (c) Binding selectivity of CPD-1224, highlighting <10-fold selectivity for FAK, FER, FLT3, LTK, and SLK. (d) Degradation proteomics of CPD-1224. Further details are provided in the Supporting Information.

Docking of CPD-1131 into the ALK/CRBN complex suggested attaching the linker to the 5-position of the isoindolinone scaffold may enable even further reductions in linker length (Figure 5). These considerations resulted in the design of CPD-1193 (Figure 2) which exhibited superior degradation potency, albeit with room for further improvements.

Figure 5.

Docking model of CPD-1131 in the ALK/CRBN complex. Glu1210 forms a salt bridge with the basic amine in the linker.

The alkyne was replaced with an alternative rigid connecting element, the azetidine ring, which appears to be used with increasing regularity in PROTAC linker design.³⁶ Both isoindolinone and phthalimide degraders were prepared to explore SAR afforded by subtle changes in CRBN engagement.³⁷ Both degraders retained a catalytic mode of EML4-ALK degradation, though phthalimide CPD-1224 possessed enhanced efficacy (higher D_max values) across the clinically relevant mutations and improved metabolic stability relative to the isoindolinone congener CPD-1793 (Figure 2). Binding modes of CPD-1131 and CPD-1224 were predicted to be very similar (Figure S1). Furthermore, CPD-1224 did not possess RBC lysis and had an impressive oral bioavailability of 28% in mouse (dosed at 10 mg/kg, details provided in the Supporting Information). The formal rotatable bond count of CPD-1224 of 12 does not reflect the rigidity of the molecule, where restricted rotation of the five N−C linkages to benzene and pyrimidine rings is expected to translate to enhanced permeability that helps deliver the observed PK.³² MS proteomics showed that CPD-1224 degraded only a small number of proteins similar to CPD-1131 but has considerably improved kinome binding selectivity (Figure 4c,d). Although CPD-1224 and CPD-1131 both possess excellent degradation selectivity, their off-targets are somewhat different (RSK1 and FER are common), which may reflect the subtle differences in ternary complex conformations between the two degraders effect different ubiquitination profiles, as mentioned above. The gross physicochemical features of CPD-1224 and CPD-1131 could potentially explain the differences in binding selectivity. CPD-1224 is more rigid and less lipophilic than CPD-1131 (cLogP of 6.9 and 8.3, respectively), properties that are known to affect selectivity.

Lipophilic basic compounds are known to accumulate and become trapped in the acidic lysosome where they may induce phospholipidosis. We have observed these effects for other heterobifunctional degraders,³⁸ and ceritinib itself is a reported phospholipogen.³⁹ For these reasons we decided to screen CPD-1224 in a LipidTOX Red imaging assay that detects phospholipid accumulation.⁴⁰ We confirmed the phospholipidosis and cytotoxicity of ceritinib in the liver HepG2 cell line, while CPD-1224 did not possess these effects (Figure S7).

Reinstalling the isopropoxy group present in ceritinib yielded CPD-1451, which was prepared using analogous reductive amination chemistry. CPD-1451 was an inferior degrader to the methoxy containing CPD-1224, vindicating our original strategy and showing that optimal degradation is sensitive to subtle changes in ALK ligand binding motifs (Figure 6). This is likely due to the spatial constraints formed within the ternary complex. Unlike the “free” ALK kinase domain, CRBN in the complex caps the region that was solvent exposed for ceritinib. The isopropoxy group in CPD-1451 would appear to push against side chains that are sterically congested in the complex compared with free ALK (such as R1120). Interestingly, CPD-1451 was equally potent for the L1196M mutant, which may result from increased hydrophobic interactions between the aryl-Cl substitution and the sulfur atom in methionine, thus compensating a binding penalty deriving from the bulkier isopropoxy group. Moreover, switching the ALK ligands for crizotinib (CPD-1402) or alectinib (CPD-1403), maybe unsurprisingly based on the results for CPD-1451, delivered PROTACs with weaker potency and efficacy for the clinical mutations compared to CPD-1224 (Figure 6). CPD-1403 (which has only six rotatable bonds and high microsomal stability) demonstrated 48% oral bioavailability in mouse, suggesting further optimization of potency in this series may deliver degraders with further improved PKPD profiles.

Figure 6.

Modifications to the ALK ligand reveal subtle degradation structure−activity relationships for the EML4-ALK PROTACs. The corresponding dose−response curves for the HiBiT assay are provided in Figure S2.

To further demonstrate the progress of the medicinal chemistry effort toward the stated goal of developing substoichiometric mutation resilient degraders, we plotted degradation capacity as the area-under-the-curve (AUC) of the HiBiT degradation dose−response (for both WT and G1202R) against NanoBRET ALK occupancy for all compounds (Figure 7a,b). Early degraders TL13–112, CPD-198, CPD-985, and another ceritinib-based EML4-ALK PROTAC MS4078,⁴¹ cluster toward higher AUC for both WT and mutant. Optimized degraders such as CPD-1224 clearly differentiate in these AUC-occupancy plots, which we believe are a useful visualization to assess the relationship of the potency and efficacy of degradation with cellular target engagement. To validate the degradation phenotype in a fully human system, we used H3122 cells harboring the EML4-ALK fusion. All tested molecules were active degraders (Figure S3). Further studies could include determination of mutant target engagement using the NanoBRET system to better correlate occupancy versus degradation for the mutants, although we expect the trends shown here for WT will broadly hold.

Figure 7.

EML4-ALK degradation AUC for (a) WT and (b) G1202R versus ALK cellular occupancy.

At this stage we decided CPD-1224 possessed balanced pharmacology and PK suitable for further profiling. Cullin ring ligases (CRLs) require neddylation for activation, and the NEDD8-activating enzyme (NAE) inhibitor MLN4924 is often used as confirmatory evidence that degradation is mediated by CRLs, including the complex CRL4^CRBN. The degradation of WT and G1202R by CPD-1224 was considerably attenuated in the presence of MLN4924 as expected, but downregulation was not completely ablated (Figure 8).

Figure 8.

Degradation of EML4-ALK WT and G1202R by CPD-1224 is inhibited by the neddylation inhibitor MLN4924.

We believe binding of certain ligands to the ATP-site of EML4-ALK results in Hsp90 chaperone deprivation, as seen for certain other kinase inhibitors,^42,43 and further work in our group is currently establishing these mechanistic details, which will be published in due course.

Assessment of the antiproliferative activity of CPD-1224 confirmed excellent translation from the observed mutant degradation to growth inhibitory effects (Figure 9a,b). Ba/F3 cells stably expressing the indicated HiBiT-tagged EML4-ALK v3 were treated with CPD-1224, the N-methyl glutarimide negative control CPD-1224NC (which retains ALK inhibition but does not engage CRBN), and the ALK kinase inhibitors crizotinib, ceritinib, alectinib, brigatinib, and lorlatinib. Unlike the inhibitor drugs and the negative control CPD-1224NC, the catalytic degrader CPD-1224 was able to overcome mutations in the ATP-site, reported as log GI₅₀ (concentration that inhibits cell growth by 50%, Figure 9b). Interestingly, we showed that CPD1224NC retains weak degradation activity (see Supporting Information) possibly because of Hsp90 chaperone deprivation as mentioned above, which may explain its superior antiproliferative activity over ceritinib. Importantly, CPD-1224 was greater than an order of magnitude more potent against the recalcitrant double mutant L1196M/G1202R than CPD-1224NC and the inhibitors.

Figure 9.

(a) CPD-1224 degrades clinically relevant EML4-ALK mutants (4 h treatment in Ba/F3). (b) Degradation translates to potent antiproliferative activity which is superior to the N-methyl glutarimide negative control CPD-1224NC and the approved inhibitor drugs (log GI₅₀ shown in Ba/F3 cells, 72 h treatment).

Based on its excellent in vitro potency and in vivo PK exposure levels that were far in excess of in vitro antiproliferative potency, we decided to perform a more detailed pharmacokinetic−pharmacodynamic (PKPD) and efficacy study of CPD-1224. However, our concern moving forward was that unbound levels in vivo could not be accurately determined due to very high nonspecific binding effects (mouse and human plasma protein binding of >99.9%).³⁸ Our hope was that a 10 mg/kg b.i.d. regimen (the dose used in our original PK study and also used for oral inhibitor drugs such as the third generation lorlatinib previously)⁴⁴ would be sufficient for CPD-1224 to validate our hypothesis in vivo. The compound ALK mutant L1196M/G1202R was chosen as a rigorous test of the catalytic mode of degradation, and the study was performed alongside lorlatinib at the same dose. Ba/F3 cells were implanted in a mouse xenograft model (see Supporting Information). On repeat dosing both compounds achieved the expected exposures in vivo (Figure 10a and Figure S8) and had no significant effect on body weight changes, indicative of being broadly tolerable (Figure S9). CPD-1224 reduced total ALK and p-ALK levels in tumors (Figure 10b,c) in line with in vitro antiproliferation data (Figure 9b). Meanwhile, lorlatinib appeared to increase ALK and p-ALK levels (Figure 10b,c). This was an unexpected result for which we do not have any current explanation. Tumor growth was commensurately inhibited by CPD-1224, while lorlatinib, as expected, had no effect (Figure 10d).

Figure 10.

PKPD and efficacy studies of catalytic degrader CPD-1224. (a) PK of CPD-1224 and lorlatinib in the L1196M/G1202R xenograft mouse model (dotted line represents the GI₅₀ of CPD-1224 for L1196M/G1202R Ba/F3 cells). (b) Western blots showing changes in total ALK and p-ALK levels in tumors following compound treatment. (c) Quantification of WB data. (d) Effects of compound treatment on tumor growth.

DISCUSSION AND CONCLUSIONS

Given the correlation of ALK degradation, p-ALK inhibition, and in vivo efficacy, clearly a higher dose of CPD-1224 will very likely yield increased TGI of the otherwise recalcitrant L1196M/G1202R mutation. Additionally, the development of a PROTAC with even higher potency and/or bioavailability would be expected to further improve TGI. Nevertheless, this work validates our original concept that catalytic degraders possess enhanced resilience to binding site mutations compared to inhibitors. Furthermore, our most advanced degraders exhibit superior pharmacology, pharmacokinetics, and mutant profile than the previously reported ALK PROTACs.⁴⁵⁻⁴⁸

Our hope is that this research has helped establish a useful framework for future catalytic degrader development that will broadly impact anticancer drug discovery. The current therapeutic paradigm employs a strategy whereby inhibitors are developed in a reactive manner to tackle emergent binding site mutations following cancer treatment. We anticipate catalytic degraders may present an opportunity to proactively address the possible emergence of binding site mutants because exceptionally low occupancy is sufficient to effectively downregulate the oncoprotein. Similarly, the strategy may be applied to overcome the emergence of escape mutants in the anti-infectives field.⁸ Our research also highlights a key issue facing PROTAC drug development relating to challenges evaluating unbound compound levels that complicates PKPD understanding, necessitating a certain level of empiricism when advancing such compounds to in vivo validation and efficacy studies.

EXPERIMENTAL SECTION

General Synthetic Methods.

Unless otherwise indicated, reagents and solvents were used as received from commercial suppliers. All reactions were monitored using a Shimadzu LC-20AD high performance liquid chromatography/mass spectrometry (HPLC/MS) system using Kinetex BEH C18 column (2.1 mm × 30 mm, 5 μm particle size). Detection methods used a diode array (DAD). MS mode was positive electrospray ionization. MS range was 100−1000. HPLC method A: the gradient was 5−95% B in 1.50 min, 5% B in 0.01 min, 5−95% B (0.01−0.70 min), 95% B (0.70−1.15 min), 5% B in 1.16 min with a hold at 5% B for 0.34 min; solvent A = 0.04% trifluoroacetic acid in H₂O; solvent B = 0.02% trifluoroacetic acid in acetonitrile; flow rate, 1.5 mL/min. Purification of reaction products was carried out by flash chromatography using Combi-FlashRf with Biotage-Isolera normal-phase silica flash columns or Waters high performance liquid chromatography (HPLC) system using Phenomenex Luna C18 (80 mm × 30 mm × 3 μm), solvent gradient from 0% to 99% acetonitrile in H₂O (0.1% trifluoroacetic acid (TFA) as additive); flow rate, 25 mL/min; or Phenomenex Luna C18 (100 mm × 30 mm × 5 μm), solvent gradient from 0% to 99% acetonitrile in H₂O (0.2% formic acid (FA) as additive); flow rate, 25 mL/min; or Phenomenex Luna C18 (80 mm × 30 mm × 3 μm), solvent gradient from 0% to 99% acetonitrile in H₂O (0.04% hydrochloric acid (HCl) as additive); flow rate, 25 mL/min. All compounds are >90% pure by HPLC. ¹H NMR and ¹³C NMR spectra were obtained using Bruker Avance III spectrometers (400 MHz for ¹H and 100 MHz for ¹³C). Chemical shifts are reported relative to deuterated methanol (δ = 3.31) or dimethyl sulfoxide (DMSO) (δ = 2.50) for ¹H NMR. Spectra are given in ppm (δ) and as br = broad, s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, and coupling constants (J) are reported in hertz.

Hazards.

All cereblon modulators should be treated as teratogens.

Compound Synthesis. General Procedure A: Reductive Amination.

A solution of aldehyde 2-(4-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-methylphenyl)acetaldehyde⁴⁹ (0.1 mmol) and amine hydrochloride (0.1 mmol) in 2.0 mL of MeOH and THF (v/v, 2:1) was stirred at RT under N₂ for 0.5 h. NaBH₃CN (0.3 mmol) was added at 0 °C in portions, and the reaction mixture was stirred at room temperature for 16 h. LCMS showed that most of the starting materials were consumed, and the major peak was the desired target molecule. The mixture was partitioned between ethyl acetate and water. The combined organic phases were washed with water and brine, dried over Na₂SO₄, and concentrated. The crude product was purified by SGC or prep-HPLC to afford the title compound.

Synthesis of 4-(7-((4-((5-Chloro-4-((2-(isopropylsulfonyl)-phenyl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-methylphenethyl)amino)hept-1-yn-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (CPD-198).

Followed general reductive amination procedure A using amine 4-(7-aminohept-1-yn-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione⁴⁹ to obtain CPD-198 (12.5 mg, yield: 6%) as a white solid. ¹H NMR (400 MHz, DMSO-d₆): δ 1.15 (d, J = 6.8 Hz, 6H), 1.46−1.62 (m, 6H), 2.03−2.07 (m, 1H), 2.14 (s, 3H), 2.57−2.62 (m, 2H), 2.76−2.86 (m, 8H), 3.76 (s, 4H), 5.13−5.17 (m, 1H), 6.86 (s, 1H), 7.33 (t, J = 8.0 Hz, 1H), 7.46 (s, 1H), 7.62 (t, J = 8.0 Hz, 1H), 7.81−7.90 (m, 4H), 8.23−8.33 (m, 3H), 8.49−8.51 (m, 1H), 9.48 (brs, 1H). ESI-MS (EI⁺, m/z): 840.3. HRMS (m/z) for C₄₃H₄₇ClN₇O₇S⁺ [M + H]⁺: calcd, 840.2941; found, 840.2941.

Synthesis of 3-(4-(7-((4-((5-Chloro-4-((2-(isopropylsulfonyl)-phenyl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-methylphenethyl)amino)hept-1-yn-1-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (CPD-985).

Followed general procedure A using amine 3-(4-(7-aminohept-1-yn-1-yl)-1-oxoisoindolin-2-yl)-piperidine-2,6-dione⁴⁹ to afford the desired product CPD-985 (130 mg, yield 36%) as a yellow powder. ¹H NMR (400 MHz, DMSO-d₆): δ 1.15 (d, J = 6.8 Hz, 6H), 1.46−1.61 (m, 6 H), 1.88 (s, 2 H), 1.98−2.03 (m, 1 H), 2.13 (s, 3 H), 2.42−2.47 (m, 1 H), 2.56−2.60 (m, 1H), 2.69−2.91 (m, 8 H), 3.42−3.44 (m, 1H), 3.75 (s, 3 H), 4.31 (d, J = 17.7 Hz, 1H), 4.45 (d, J = 17.7 Hz, 1H), 5.11−5.16 (m, 1H), 6.84 (s, 1 H), 7.33 (t, J = 7.6 MHz, 1 H), 7.45 (s, 1 H), 7.51 (t, J = 7.2 MHz, 1 H), 7.59−7.63 (m, 1 H), 7.70 (d, J = 7.2 MHz, 1 H), 7.82 (dd, J = 1.6, 7.6 MHz, 1 H), 8.22 (s, 1 H), 8.25 (s, 1 H), 8.50 (d, J = 8.0 MHz, 1 H), 9.48 (br, 1 H). ESI-MS (EI⁺, m/z): 826.50. HRMS (m/z) for C₄₃H₄₉ClN₇O₆S⁺ [M + H]⁺: calcd, 826.3148; found, 826.3150.

Synthesis o f 3-(4-(7-(4-(4-((5-Chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-methylphenyl)piperidin-1-yl)hept-1-yn-1-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (CPD-1108).

To a solution of amine 5-chloro-N4-(2-(isopropylsulfonyl)phenyl)-N2-(2-methoxy-5-methyl-4-(piperidin-4-yl)phenyl)pyrimidine-2,4-diamine⁴⁹ (50 mg, 0.1 mmol) and 7-(2-(2,6-dioxopiperidin-3-yl)-1- oxoisoindolin-4-yl)hept-6-ynal (35 mg, 0.1 mmol, 1.0 equiv) in 2 mL of THF and 3 mL of MeOH were added 4 Å molecular sieves (50 mg) and HOAc (12 mg, 0.2 mmol, 2.0 equiv). The resulting mixture was stirred at 0 °C for 30 min, and then NaBH(OAc)₃ (63 mg, 0.3 mmol, 3.0 equiv) was added. The resulting mixture was stirred at room temperature under N₂ for 16 h. LCMS showed the reaction was complete. The reaction mixture was purified by prep-HPLC to afford the compound CPD-1108 (7.7 mg, 8.9%) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 11.01 (s, 1H), 9.51 (s, 1H), 8.49 (d, J = 7.8 Hz, 1H), 8.34 (s, 1H), 8.23 (s, 1H), 7.83 (dd, J = 8.0, 1.5 Hz, 1H), 7.72 (d, J = 7.5 Hz, 1H), 7.67−7.63 (m, 1H), 7.59 (t, J = 7.5 Hz, 1H), 7.53 (t, J = 7.6 Hz, 1H), 7.46 (s, 1H), 7.36−7.30 (m, 1H), 6.77 (s, 1H), 5.16 (dd, J = 13.2, 5.1 Hz, 1H), 4.50−4.28 (m, 2H), 3.76 (s, 3H), 3.59 (m, 2H), 3.36 (m, 1H), 3.16−2.86 (m, 6H), 2.65−2.56 (m, 1H), 2.53 (t, J = 6.9 Hz, 2H), 2.45 (m, 1H), 2.18 (s, 3H), 2.07−1.85 (m, 4H), 1.75 (m, 2H), 1.68−1.61 (m, 2H), 1.56−1.44 (m, 2H), 1.16 (d, J = 6.8 Hz, 6H). ESI-MS (EI⁺, m/z): 866.10. HRMS (m/z) for C₄₆H₅₃ClN₇O₆S⁺ [M + H]⁺: calcd, 866.3461; found, 866.3458.

Synthesis of 3 - ( 4 - ( 5 - ( 4 - ( 4 - ( ( 5 - C h l o r o - 4 - ( ( 2 - (isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-methylphenyl)piperidin-1-yl)pent-1-yn-1-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (CPD-1131).

Following the same procedure as that for CPD-1108, using aldehyde 5-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)pent-4-ynal,⁴⁹ the title compound was furnished (5 mg, yield 6%) as a white powder. ¹H NMR (400 MHz, DMSO-d₆) δ 11.01 (s, 1H), 9.49 (s, 1H), 8.54 (s, 1H), 8.24 (d, J = 15.1 Hz, 2H), 7.82 (dd, J = 8.1, 1.3 Hz, 1H), 7.74−7.69 (m, 1H), 7.66 (s, 1H), 7.58 (m, 1H), 7.42 (s, 1H), 7.36−7.30 (m, 1H), 6.86 (s, 1H), 5.12 (dd, J = 13.2, 5.1 Hz, 1H), 4.60−4.25 (m, 2H), 3.77 (s, 3H), 3.08−2.85 (m, 3H), 2.69−2.53 (m, 3H), 2.48−2.29 (m, 3H), 2.15 (s, 3H), 2.11−1.97 (m, 4H), 1.83−1.63 (m, 6H), 1.16 (d, J = 6.8 Hz, 6H). ESI-MS (EI⁺, m/z):838.45. HRMS (m/z) for C₄₄H₄₉ClN₇O₆S⁺ [M + H]⁺: calcd, 838.3148; found, 838.3153.

Synthesis of 3 - ( 5 - ( 3 - ( 4 - ( 4 - ( ( 5 - C h l o r o - 4 - ( ( 2 - (isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-methylphenyl)piperidin-1-yl)prop-1-yn-1-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (CPD-1193).

To a solution of amine (50 mg, 0.1 mmol) and aldehyde 3-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)propiolaldehyde⁴⁹ (30 mg, 0.1 mmol, 1.0 equiv) in THF (2 mL) and MeOH (3 mL) were added 4 Å molecular sieves (50 mg) and HOAc (12 mg, 0.2 mmol, 2.0 equiv). The resulting mixture was stirred at 0 °C for 30 min, and then NaBH(OAc)₃ (63 mg, 0.3 mmol, 3.0 equiv) was added. The resulting mixture was stirred at room temperature under N₂ for 16 h. LCMS showed the reaction was complete. The reaction mixture was purified with prep-HPLC to afford the title compound (20 mg, 25%) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 11.00 (s, 1H), 9.49 (s, 1H), 8.51 (d, J = 8.1 Hz, 1H), 8.28 (s, 1H), 8.22 (s, 1H), 7.81 (dd, J = 8.0, 1.5 Hz, 1H), 7.73 (m, 2H), 7.58 (m, 2H), 7.41 (s, 1H), 7.36−7.29 (m, 1H), 6.89 (s, 1H), 5.12 (dd, J = 13.3, 5.1 Hz, 1H), 4.51−4.29 (m, 2H), 3.77 (s, 3H), 3.61 (s, 2H), 3.45 (m, 1H), 3.03 (d, J = 10.9 Hz, 2H), 2.96−2.86 (m, 1H), 2.69−2.57 (m, 2H), 2.46−2.30 (m, 3H), 2.16 (s, 3H), 2.06−1.96 (m, 1H), 1.84−1.69 (m, 4H), 1.16 (d, J = 6.8 Hz, 6H). ESI-MS (EI⁺, m/z): 810.40. HRMS (m/z) for C₄₂H₄₅ClN₇O₆S⁺ [M + H]⁺: calcd, 810.2835; found, 810.2839.

Synthesis of 5-{3-[(4-{4-[(5-Chloro-4-{[2-(propane-2-sulfonyl)phenyl]amino}pyrimidin-2-yl)amino]-5-methoxy-2-methylphenyl}piperidin-1-yl)methyl]azetidin-1-yl}−2-(2,6-dioxopiperidin-3-yl)-2,3-dihydro-1H-isoindole-1,3-dione (CPD-1224).

Sodium triacetoxyborohydride (792 mg, 3.74 mmol) was added to a mixture of 1-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxoisoindolin-5-yl]azetidine-3-carbaldehyde⁴⁹ (500 mg, 1.25 mmol) and amine (705 mg, 1.25 mmol) in DMF (5 mL) and the reaction mixture stirred at 15 °C for 2 h. After completion of the reaction, the reaction mixture was filtered, and the filtrate was concentrated to give a residue. The residue was purified by prep-HPLC (column, Phenomenex Gemini C18 250 mm × 50 mm × 10 μm; mobile phase, [water (10 mM NH₄HCO₃)−ACN]; B%, 46−80%, 20 min) to give the title compound as a yellow solid (102 mg). ¹H NMR (400 MHz, DMSO-d₆) δ 11.07 (s, 1H), 9.49 (s, 1H), 8.52 (br d, J = 7.6 Hz, 1H), 8.27 (s, 1H), 8.23 (s, 1H), 7.82 (dd, J = 1.3, 7.9 Hz, 1H), 7.66−7.56 (m, 2H), 7.42 (s, 1H), 7.33 (t, J = 7.4 Hz, 1H), 6.87 (s, 1H), 6.79 (d, J = 1.9 Hz, 1H), 6.65 (dd, J = 8.4, 1.9 Hz, 1H), 5.12−4.99 (m, 1H), 4.16 (br t, J = 8.1 Hz, 2H), 3.78 (s, 3H), 3.71 (br dd, J = 8.0, 5.6 Hz, 2H), 3.44 (td, J = 15.5, 6.7 Hz, 1H), 3.14−2.84 (m, 4H), 2.71−2.57 (m, 4H), 2.16 (s, 3H), 2.14−1.92 (m, 4H), 1.78−1.65 (m, 4H), 1.16 (d, J = 6.8 Hz, 6H). HRMS (m/z) for C₄₃H₄₈ClN₈O₇S⁺ [M + H]⁺: calcd, 855.3050; found, 855.3047.

Synthesis of 5-(3-((4-(4-((5-Chloro-4-((2-(isopropylsulfonyl)-phenyl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-methylphenyl)piperidin-1-yl)methyl)azetidin-1-yl)-2-(1-methyl-2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (CPD-1224NC).

A mixture of 5-[3-(dihydroxymethyl)azetidin-1-yl]-2-(1-methyl-2,6 -dioxo-3-piperidyl)isoindoline-1,3-dione (68 mg, 182 μmol), 5-chloro-N4-(2-isopropylsulfonylphenyl)-N2-[2-methoxy-5-methyl-4-(4-piperidyl)phenyl]-2,4-diamine (97 mg, 182 μmol), and NaBH(OAc)₃ (77 mg, 364 μmol) in DMF (1 mL) was stirred at 20 °C for 2 h under a N₂ atmosphere. After completion of the reaction, the mixture was filtered to give a residue which was purified by prep-HPLC (column, Phenomenex Gemini-NX 80 mm × 40 mm × 3 μm; mobile phase, [water (10 mM NH₄HCO₃)−ACN]; B%, 50−80%, 8 min). The title compound was obtained as a yellow solid (20 mg, 23 μmol, 13% yield). ¹H NMR (400 MHz, DMSO-d₆) δ = 9.49 (s, 1H), 8.53 (br s, 1H), 8.29 (s, 1H), 8.23 (s, 1H), 7.82 (d, J = 7.7 Hz, 1H), 7.66−7.56 (m, 2H), 7.41 (s, 1H), 7.33 (t, J = 7.8 Hz, 1H), 6.87 (s, 1H), 6.78 (s, 1H), 6.66 (br d, J = 7.5 Hz, 1H), 5.12 (dd, J = 5.4, 12.8 Hz, 1H), 4.15 (br t, J = 7.8 Hz, 2H), 3.78 (s, 3H), 3.75−3.67 (m, 2H), 3.08−2.96 (m, 6H), 2.76−2.58 (m, 4H), 2.15 (s, 3H), 2.10−1.96 (m, 3H), 1.75−1.66 (m, 4H), 1.16 (d, J = 6.8 Hz, 6H). ESI-MS (M + H⁺, m/z): 869.2.

Synthesis of 3-(5-(3-((4-(4-((5-Chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-methylphenyl)piperidin-1-yl)methyl)azetidin-1-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (CPD-1793).

CPD-1793 was prepared following a similar procedure as CPD-1224 using aldehyde 1-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)azetidine-3-carbaldehyde. ¹H NMR (400 MHz, DMSO-d₆) δ 10.93 (s, 1H), 9.49 (s, 1H), 8.52 (d, J = 8.5 Hz, 1H), 8.26 (s, 1H), 8.23 (s, 1H), 7.82 (dd, J = 8.0, 1.6 Hz, 1H), 7.60 (t, J = 8.0 Hz, 1H), 7.49 (d, J = 8.3 Hz, 1H), 7.42 (s, 1H), 7.33 (t, J = 7.7 Hz, 1H), 6.87 (s, 1H), 6.53−6.45 (m, 2H), 5.03 (dd, J = 13.3, 5.1 Hz, 1H), 4.30 (d, J = 16.9 Hz, 1H), 4.18 (d, J = 16.8 Hz, 1H), 4.04 (t, J = 7.8 Hz, 2H), 3.78 (s, 3H), 3.62−3.55 (m, 2H), 3.45 (p, J = 6.8 Hz, 1H), 3.08−2.83 (m, 3H), 2.69−2.59 (m, 4H), 2.41−2.26 (m, 1H), 2.15 (s, 3H), 2.09 (t, J = 10.4 Hz, 2H), 2.01−1.90 (m, 1H), 1.81−1.58 (m, 4H), 1.16 (d, J = 6.8 Hz, 6H). HRMS (m/z) for C₄₃H₅₀ClN₈O₆S⁺ [M + H]⁺: calcd, 842.3291; found, 842.3278.

Synthesis of 8-[3-(2-{4-[2-(2,6-Dioxopiperidin-3-yl)-1-oxo-2,3-dihydro-1H-isoindol-5-yl]piperidin-1-yl}ethyl)azetidin-1-yl]-9-ethyl-6,6-dimethyl-11-oxo-5H,6H,11H-benzo[b]-carbazole-3-carbonitrile (CPD-1451).

A mixture of 5-chloro-N2-[2-isopropoxy-5-methyl-4-(4-piperidyl)phenyl]-N4-(2-isopropyl-sulfonylphenyl)pyrimidine-2,4-diamine (200 mg, 335 μmol) and 1-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxoisoindolin-5-yl]azetidine-3-carbaldehyde (184 mg, 538 μmol) in MeOH (1.5 mL), DMF (1.5 mL) and HOAc (0.1 mL) was stirred at 15 °C for 1 h under N₂. NaBH(OAc)₃ (143 mg, 673 μmol) was then added. The reaction mixture was stirred at 15 °C for 11 h. The reaction mixture was concentrated under reduced pressure to give a residue which was purified by prep-HPLC (column, Waters Xbridge BEH C18 100 mm × 25 mm × 5 μm; mobile phase, [water (10 mM NH₄HCO₃)−ACN]; B%, 65−85%, 10 min) to give the title compound as a yellow solid (95 mg, 30%). ¹H NMR (400 MHz, DMSO-d₆) δ 11.07 (br s, 1H), 9.46 (br s, 1H), 8.47 (br d, J = 8.2 Hz, 1H), 8.25 (s, 1H), 8.05 (s, 1H), 7.83 (dd, J = 8.0, 1.5 Hz, 1H), 7.66−7.60 (m, 2H), 7.51 (s, 1H), 7.36 (t, J = 7.2 Hz, 1H), 6.85 (s, 1H), 6.79 (d, J = 2.0 Hz, 1H), 6.65 (dd, J = 8.5, 2.0 Hz, 1H), 5.05 (dd, J = 12.8, 5.4 Hz, 1H), 4.59 (td, J = 12.1, 6.0 Hz, 1H), 4.15 (t, J = 8.2 Hz, 2H), 3.70 (dd, J = 8.2, 5.6 Hz, 2H), 3.51−3.38 (m, 1H), 3.09−2.82 (m, 4H), 2.69−2.53 (m, 5H), 2.13 (s, 3H), 2.12−1.96 (m, 3H), 1.71−1.60 (m, 4H), 1.22 (d, J = 6.1 Hz, 6H), 1.67 (d, J = 6.8 Hz, 6H). [M + H⁺] = 883.3.

Synthesis of 5-(3-{[4-(4-{6-Amino-5-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]pyridin-3-yl}−1H-pyrazol-1-yl)piperidin-1-yl]methyl}azetidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)-2,3-dihydro-1H-isoindole-1,3-dione (CPD-1402).

CPD-1402 was prepared following the same procedure as CPD-1451: ¹H NMR (400 MHz, DMSO-d₆) δ 11.06 (s, 1H), 7.97 (s, 1H), 7.76 (s, 1H), 7.63 (d, J = 8.3 Hz, 1H), 7.57 (m, 1H), 7.53 (s, 1H), 7.44 (t, J = 8.7 Hz, 1H), 6.97−6.86 (m, 1H), 6.78 (d, J = 2.1 Hz, 1H), 6.64 (dd, J = 8.3, 2.1 Hz, 1H), 6.08 (q, J = 6.6 Hz, 1H), 5.63 (s, 2H), 5.05 (dd, J = 12.9, 5.4 Hz, 1H), 4.22−4.05 (m, 3H), 3.70 (dd, J = 8.4, 5.3 Hz, 2H), 3.11−2.80 (m, 4H), 2.74−2.54 (m, 3H), 2.24−2.07 (m, 2H), 2.07−1.85 (m, 5H), 1.80 (d, J = 6.6 Hz, 3H). ESI-MS [M + H⁺] = 776.8. HRMS (m/z) [M]⁺: calcd, 775.2321; found, 775.2325.

Synthesis of 8-(4-((1-(2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)azetidin-3-yl)methyl)piperazin-1-yl)-9-ethyl-6,6-dimethyl-11-oxo-6,11-dihydro-5H-benzo[b]carbazole-3-carbonitrile (CPD-1403).

1-[2-(2,6-Dioxo-3-piperidyl)-1,3-dioxoisoindolin-5-yl]azetidine-3-carbaldehyde (8.6 mg, 25.1 μmol) and sodium triacetoxyborohydride (10.6 mg, 50.18 μmol) were added to a solution of 9-ethyl-6,6-dimethyl-11-oxo-8-piperazin-1-yl-5H-benzo-[b]carbazole-3-carbonitrile (20 mg, 25.1 μmol) in DMF (0.5 mL), and the reaction mixture was stirred at 20 °C for 2 h. After completion of the reaction, the reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give a residue which was purified by prep-HPLC (column, Waters Xbridge BEH C18 100 mm × 30 mm × 10 μm; mobile phase, [water (10 mM NH₄HCO₃)−ACN]; B%, 40−70%, 8 min) to give the title compound as a yellow solid (6.0 mg, 31%). ¹H NMR (400 MHz, DMSO-d₆) δ 12.84z (br s, 1H), 11.09 (s, 1H), 8.32 (d, J = 8.2 Hz, 1H), 8.11 (s, 1H), 8.03 (s, 1H), 7.71 (d, J = 8.2 Hz, 1H), 7.63 (dd, J = 8.2, 1.5 Hz, 1H), 7.38 (s, 1H), 6.79 (d, J = 1.8 Hz, 1H), 6.65 (dd, J = 8.4, 1.9 Hz, 1H), 5.06 (dd, J = 12.8, 5.4 Hz, 1H), 4.30 (br t, J = 8.1 Hz, 2H), 3.92 (br dd, J = 7.9, 5.5 Hz, 2H), 3.69−3.52 (m, 4H), 3.31−3.15 (m, 3H), 2.97−2.82 (m, 2H), 2.80−2.58 (m, 5H), 2.33 (m, 1H), 2.05−1.98 (m, 1H), 1.79 (s, 6H), 1.31 (t, J = 7.5 Hz, 3H). [M + H⁺] = 724.2.

Synthesis of N-(4-((5-Chloro-4-((2-(isopropylsulfonyl)-phenyl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-methylphenethyl)-1-(5-(5,5-difluoro-7,9-dimethyl-5H-4l4,5l4-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborinin-3-yl)pentanamido)-N-methyl-3,6,9,12-tetraoxapentadecan-15-amide (BODIPY-Ceritinib tracer).

To a solution of 3-[2-[2-[2-(2-aminoethoxy)-ethoxy]ethoxy]ethoxy]-N-[2-[4-[[5-chloro-4-(2-isopropylsulfonylanilino)pyrimidin-2-yl]amino]-5-methoxy-2-methylphenyl]ethyl]-N-methyl-propanamide⁴⁹ (130 mg, 165 μmol, 1 equiv, HCl) and 5-(2,2-difluoro-10,12-dimethyl-1-aza-3-azonia-2-boranuidatricyclo-[7.3.0.03,7]dodeca-3,5,7,9,11-pentaen-4-yl)pentanoic acid (58 mg, 181.5 μmol, 1.1 equiv) in DMF (2 mL) were added DIPEA (107 mg, 825 μmol, 144 μL, 5 equiv) and HATU (627 mg, 1.65 mmol, 10 equiv). The resulting mixture was stirred at 15 °C for 1 h under N₂. The reaction mixture (combined with another batch of 30 mg scale) was treated with water (5 mL) and extracted with EtOAc (10 mL × 3). The combined organic phases were washed with brine (20 mL), dried with anhydrous Na₂SO₄, filtered, and the filtrate was concentrated in vacuum to give a residue which was purified by prep-HPLC (column, Phenomenex Gemini-NX 80 mm × 40 mm × 3 μm; mobile phase, [water (10 mM NH₄HCO₃)−ACN]; B%, 45−70%, 8 min) to give the title compound (13.4 mg, 97% purity) as a red solid. ¹H NMR (400 MHz, DMSO-d₆) δ = 9.48 (br s, 1H), 8.55−8.52 (m, 1H), 8.25−8.24 (m, 2H), 7.86−7.82 (m, 2H), 7.67−7.63 (m, 2H), 7.50 (d, J = 15.2 Hz, 1H), 7.38−7.30 (m, 1H), 7.10 (d, J = 4.2 Hz, 1H), 6.85 (d, J = 6.4 Hz, 1H), 6.40−6.39 (m, 1H), 6.27 (s, 1H), 3.77 (s, 3H), 3.63 (t, J = 6.7 Hz, 1H), 3.57−3.51 (m, 1H), 3.45−3.35 (m, 12H), 3.32 (s, 2H), 3.22−3.14 (m, 2H), 2.94 (s, 2H), 2.87−2.63 (m, 6H), 2.58−2.52 (m, 1H), 2.50 (s, 3H), 2.45 (t, J = 6.7 Hz, 1H), 2.25 (s, 3H), 2.17 (d, J = 5.7 Hz, 3H), 2.15−2.10 (m, 2H), 1.63−1.56 (m, 4H), 1.16 (d, J = 6.8 Hz, 6H). ¹⁹F NMR (400 MHz, DMSO-d₆) δ = −142.80 (q, 2F). MS: [M + H]⁺ = 1053.4.

ABBREVIATIONS USED

ALK

anaplastic lymphoma kinase

AUC

area-under-the-curve

BRET

bioluminescence resonance energy transfer

CRBN

cereblon

EML4

echinoderm microtubule-associated protein-like 4

FeSSIF

fed state simulated intestinal fluid

HLM

human liver microsome

MS

mass spectrometry

NAE

NEDD8-activating enzyme

NSCLC

non-small-cell lung cancer

PKPD

pharmacokinetic−pharmacodynamic

POI

protein-of-interest

PROTAC

proteolysis targeting chimera

TGI

tumor growth inhibition