Abstract
We report the design, synthesis, and characterization of a series of targeted degraders directed against testis-specific serine/threonine kinases TSSK1 and TSSK2. A stable CHO-K1 cell line expressing HiBiT-tagged TSSK1 enabled quantitative cellular profiling and identified compound 5.1 as a potent TSSK1 degrader with a DC50 of 10 nM and a D max of 68% after 48 h. In contrast, ex vivo incubation of CD1 mouse sperm with degrader 5.4 induced up to an 80% reduction of the TSSK2 isoform within 4 h and translated into profound functional loss, resulting in a 97% reduction in sperm motility and near-complete loss of in vitro fertilization. Both alkyl- and PEG-linked degraders displayed metabolic N-dealkylation and high efflux, and therefore will require optimization. These data provide proof of concept that targeted degradation can occur within sperm cells, leading to a disruption of sperm function and encouraging further optimization toward in vivo evaluation for male contraception.
Introduction
The development of a nonhormonal male contraceptive is urgently needed to expand contraceptive options and reduce the high rate of unintended pregnancies, estimated at 120 million worldwide each year. Currently, the options available to men for nonpermanent contraception are limited, with condoms being the primary method despite a reported failure rate of up to 12%. Additional male-centric contraceptive methods would alleviate the burden on womenmany of whom cannot use hormonal agents due to side effects or contraindicationsand respond to strong public demand. For instance, a 2024 multicountry study quantified this demand and reported that, on average, 61% of men had a strong interest in adopting a new male contraceptive once available.
Unfortunately, hormone-based male contraceptives have had limited success in clinical development. − While some candidates have reached advanced status, none have completed phase 3 clinical trials, largely due to issues with bioavailability, efficacy, or undesirable side effects, including changes in libido, hepatotoxicity, depression, and cardiovascular risks. − These safety concerns have hindered the progress of hormonal contraceptives, despite the promising concept. In contrast, nonhormonal male contraceptive methods present an alternative strategy for safer and more targeted fertility control. However, progress in this area has been slow, with only one nonhormonal candidate, YCT-529, advancing to a phase 1a/2b clinical trial (NCT06542237) as a retinoic acid receptor-α antagonist. , This illustrates the need for further research and development of nonhormonal options, as this may offer the potential to avoid the systemic side effects commonly associated with hormonal methods.
Targeted disruption of spermiogenesis, the final phase of spermatogenesis, represents an attractive approach for nonhormonal male contraceptive development. During spermiogenesis, round spermatids undergo extensive remodeling, including chromatin condensation, acrosome formation, and flagellum developmentprocesses essential for sperm motility and fertilization. Within these pathways, the six testis-specific serine/threonine kinases (TSSK1–6) have essential roles. − In particular, TSSK1, TSSK2, TSSK3, and TSSK6 are indispensable for sperm structure and motility, as evidenced by knockout studies showing that their absence leads to male infertility without any systemic health effects. ,,
Despite this potential, the TSSKs lie within the “dark kinome” and remain only minimally characterized. A dual-inhibitor (compound 19) was previously identified, displaying high affinity for TSSK1 (IC50 = 0.86 nM) and TSSK2 (IC50 = 66 nM), but it lacked specificity in a 369-kinase panel. Nevertheless, compound 19 provides a strong starting point for further optimization. Herein, we describe our efforts to repurpose this potent inhibitor into a selective targeted degrader for TSSK1 and TSSK2, thereby laying the groundwork for a novel nonhormonal male contraceptive approach.
Targeted degraders achieve selectivity through a dual-targeting mechanism that distinguishes them from conventional inhibitors. , These degraders are typically heterobifunctional molecules that simultaneously bind both a protein of interest (POI) and an E3 ubiquitin ligase, connected via a linker. The recruited E3 ligase facilitates ubiquitination of the POI, leading to proteasomal degradation and regeneration of the degrader molecule. This mechanism enables continuous elimination of the target protein from the cell, rather than temporarily blocking its function. Selectivity is improved because the degradation event relies on the formation of a ternary complex between the degrader molecule, the target protein, and the E3 ligase. , This complex can be highly specific because it depends on structural features and binding dynamics unique to the target. Additionally, degraders act catalytically and typically require lower concentrations than inhibitors, further reducing off-target interactions. Furthermore, chromatin condensation during spermiogenesis silences protein transcription and translation in sperm cells, preventing the synthesis of new proteins, such as TSSK1 and TSSK2. We expect that this will allow for gradual degradation of these proteins with no competition from protein resynthesis, potentially enabling lower or even intermittent dosing of degraders for male contraception, thus reducing off-target effects. Herein, we report our efforts to develop potent and selective degraders for TSSK1 and TSSK2. We also report for the first time the ability to actively degrade a protein target in sperm cells, leading to the disruption of sperm motility and in vitro fertilization.
Results and Discussion
Design of TSSK1/2 Targeted Degraders
To develop selective targeted degraders for TSSK1 and TSSK2, we utilized compound 19, a previously reported ATP-competitive inhibitor, as the core scaffold (Figure a). While compound 19 exhibited potent activity against TSSK1 and TSSK2, its broad nonselectivity classified it as a promiscuous kinase inhibitor, making it unsuitable for chronic dosing in contraceptive applications due to potential off-target effects. Nonetheless, the conversion of nonselective kinase inhibitors into heterobifunctional degraders has emerged as a promising strategy to enhance selectivity by recruiting E3 ligases that degrade target proteins. −
1.
Design of TSSK1/2 targeted degraders. (a) Chemical structure of compound 19 (TSSK1/2 ligand) (b) TSSK1 AlphaFold homology model with compound 19 docked within the ATP-binding site; (c) Chemical structures of designed TSSK1/2 targeted degraders.
Prior to degrader development, we performed a structural analysis to identify a solvent-exposed region of compound 19 suitable for linker attachment that would avoid disruption of its binding to the ATP-binding pocket of TSSK1/2. In this analysis, Schrödinger’s induced-fit docking with an AlphaFold-derived homology model of TSSK1 enabled us to identify the piperidine ring of compound 19 as a solvent-exposed moiety, making it an ideal candidate for linker functionalization (Figure b).
Based on these findings, we developed a convergent synthetic route to produce a free piperidine derivative of compound 19, enabling its use as a TSSK1/2-binding ligand for degrader design (Scheme ). For the E3 ligase-recruiting ligand, we selected pomalidomide due to its well-established role in targeted protein degradation, extensive pharmacokinetic characterization, and physicochemical properties favorable for oral bioavailability. , Additionally, pomalidomide’s commercial availability with a diverse range of linkers facilitated rapid structure–activity relationship (SAR) exploration. Accordingly, we generated a series of heterobifunctional degraders by coupling the free-piperidine derivative of compound 19 to pomalidomide using alkyl and polyethylene glycol (PEG)-based linkers of varying lengths (Figure c).
1. Synthesis of Compound 19 Derivative 9 .
a Reagents and reaction conditions: (i) Zinc powder, TMSCl, 1,2-dibromoethane, CuI, Pd(dppf), DMA, 85 °C, 18 h, 89%; (ii) TFA, DCM, 2 h, rt; (iii) Acetyl Chloride, DCM, 1 h, rt, 78% over two steps; (iv) H2, Pd/C, MeOH, 72 h, rt, 92%; (v) NaH, THF, 2 h, rt, 25%; (vi) TFA, MeOH/IPA, 70 °C, 18 h, 88%; (vii) HCl/MeOH, 90 °C, 18 h, 58%.
Synthesis of Targeted Degraders
The synthesis of the TSSK1/2 degraders, outlined in Schemes – , begins with the preparation of key intermediate 9 (Scheme ). Initially, a Negishi coupling was performed between commercially available 4-bromo-2-methoxy-1-nitrobenzene (1) and tert-butyl 4-iodopiperidine-1-carboxylate (2) to afford intermediate 3. Given that the Boc protecting group was unstable under subsequent acid-catalyzed conditions, it was replaced with a more stable acetyl protecting group. This transformation was achieved by removing the labile carbamate with trifluoroacetic acid, followed by acetylation of the piperidine with acetyl chloride, yielding intermediate 4. Next, the nitro group was reduced to the corresponding aniline 5 using catalytic hydrogenation. Nucleophilic substitution of 2,4,5-trichloropyrimidine (7) with 2-amino-N-methylbenzenesulfonamide (6) provided intermediate 8. Finally, compound 8 was coupled with the aniline derivative 5 under acidic conditions, followed by removal of the acetyl group using 4 M HCl to produce the key intermediate 9.
5. Synthesis of Methylated Pomalidomide Derivatives .
a Reagents and reaction conditions: (i) Cs2CO3, methyl iodide, DMF, rt, 16 h, 65% (ii) DIPEA, NMP, 100 °C, 16 h 65–70% (iii) HATU, Et3N, DCM, rt, 16 h, 17–20%.
Schemes and illustrate the synthesis of degraders 5.1–5.4 and 5.9–5.11 using similar chemical strategies. In these cases, a commercially available pomalidomide derivative bearing a carboxylic acid-terminated linker was coupled to intermediate 9 via HATU-mediated amide coupling, affording the corresponding degrader derivatives. In contrast, the synthesis of degrader 5.5 (Scheme ) employed an alternate approach, where nucleophilic substitution occurred between 2-chloro-N-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)acetamide and the piperidine moiety of intermediate 9.
2. Synthesis of Target Compounds 5.1, 5.9–5.11 .
a Reagents and reaction conditions: (i) HATU, DIPEA, DCM, rt, 18 h, 16–60%.
3. Synthesis of Target Compounds 5.2–5.4 .
a Reagents and reaction conditions: (i) HATU, DIPEA, DCM, rt, 18 h, 16–24%.
4. Synthesis of Target Compound 5.5 .
a Reagents and reaction conditions: (i) THF, 50 °C, 24 h, 36%.
Scheme highlights the synthesis of methylated pomalidomide compounds 2.8 and 2.10. First, the commercially available 4-fluoropomalidomide (10) was methylated on its glutarimide nitrogen (11) using methyl iodide in the presence of cesium carbonate. This was followed by nucleophilic aromatic substitution using linkers 12i and 13i, yielding compounds 12 and 13, respectively. These were attached to intermediate 9 utilizing HATU-mediated amide coupling to generate final compounds 2.8 and 2.10.
Establishment of Stable HiBiT-TSSK1 Cell Line
While commercial antibodies for TSSK1 were initially considered for quantitative evaluation of degrader activity, we found that these antibodies bind to multiple off-target proteins, many of which have molecular weights similar to TSSK1, thereby precluding reliable interpretation of whole-cell Western blots. Therefore, to evaluate degradation, we developed a Nano-Glo HiBiT Lytic Assay to quantify the in vitro degradation of HiBiT-tagged TSSK1 protein. In this assay, cells expressing HiBiT-tagged TSSK1 are treated with a detection reagent containing LgBiT, which binds selectively to the HiBiT tag and produces a luminescent signal proportional to the amount of intact HiBiT-tagged TSSK1 protein. Treatment with degrader compounds induces targeted ubiquitination and proteasomal degradation, eliminating TSSK1 along with its HiBiT tag and resulting in a measurable decrease in luminescence. Importantly, this cell-based system enables characterization of degradation over days rather than hours, which is not feasible in sperm due to their limited viability. This is particularly useful for degraders, whose activity is inherently time-dependent, and short incubation periods may not accurately reflect true degradation potential. Using this system, we were able to establish a dose-dependent degradation response and calculate both the half-maximal degradation concentration (DC50) and maximum observed protein degradation (D max) for each compound post-treatment.
To reduce variability, we opted to generate a stable cell line expressing HiBiT-tagged TSSK1 rather than relying on transient expression systems (Figure S1). For this purpose, we engineered a piggyBac transposon system for permanent integration into CHO-K1 cells (vector ID: VB231116-1266hdp). CHO-K1 cells were selected as the host system due to their ability to support post-translational modifications, such as glycosylation, which are essential for proper TSSK1 folding and function. The piggyBac system employed a two-component mechanism consisting of a transposon DNA plasmid carrying the HiBiT-tagged TSSK1 sequence along with a puromycin resistance gene. The second component of this approach included mRNA encoding the piggyBac transposase. The transposase recognizes two terminal repeat sequences flanking the transposon and facilitates the insertion of the transposon into the host genome. Notably, the transient nature of transposase mRNA ensures that once integration occurs, the transposon remains stably incorporated into the genome, resulting in persistent and heritable expression of HiBiT-tagged TSSK1. Following transfection, puromycin pressure was applied to isolate and enrich the stably transfected cells.
Single-colony clones were then isolated using limiting-dilution cloning (Figure S2A). From this process, 18 stably transfected cell lines were established, each exhibiting varying levels of HiBiT expression, as determined by luminescence intensity. The clone with the greatest HiBiT signal, SC B1.1, was selected for further expansion and used in the Nano-Glo HiBiT Lytic Assay.
Anti-HiBiT Western Blot
To validate the integrity of the stably transfected HiBiT-TSSK1 SC B1.1 cell line, a Western blot analysis was performed using an anti-HiBiT monoclonal antibody, given the absence of selective anti-TSSK1 antibodies (Figure S3). Lysates exhibited a single, distinct band corresponding to the molecular weight of a His-tagged TSSK1 control, indicating the absence of aberrant fusion proteins resulting from piggyBac transposition. This specificity validates that any observed decreases in Nano-Glo luminescence signals are attributable to the degradation of the intended HiBiT-TSSK1 fusion protein rather than unintended HiBiT-tagged byproducts. To further corroborate target engagement and degradation, subsequent biochemical evaluations using purified His-tagged TSSK1 protein have been completed.
Subcellular Protein Fractionation
To determine the subcellular distribution of HiBiT-TSSK1 in SC B1.1 cells, we performed subcellular fractionation using the Thermo Scientific kit and quantified each fraction by Nano-Glo luminescence (Figure S2B). Equal aliquots of cytosolic, membrane, nuclear-soluble, chromatin-bound, and cytoskeletal fractions were assayed, revealing that 76.8 ± 1.0% of HiBiT-TSSK1 resides in the cytosol, with smaller proportions in the nucleus (16.2 ± 1.0%), membrane (6.2 ± 1.8%), and chromatin-bound fraction (0.7 ± 0.2%), and no detectable signal in the cytoskeletal fraction or PBS wash.
This distribution has direct implications for degradation efficiency. Because targeted degraders rely on colocalization of both the target protein and the E3 ligase, cytosolic and nuclear HiBiT-TSSK1 are the only fractions available for degradation, whereas membrane and chromatin-bound TSSK1 is inaccessible to the proteosome. Therefore, the observed cytosolic and nuclear fractions effectively define the upper limit for measurable degradation in this cellular context.
TSSK1 Degradation and Target Engagement
The cell line SC B1.1 was used with the Nano-Glo HiBiT Lytic Assay to quantitatively measure TSSK1 degradation induced by our compounds, and all degradation results were normalized to cell viability (Figures S4–S6). This assay enabled the evaluation of degradation kinetics over extended time points, which is not feasible using primary sperm cells. Sperm cells have a limited lifespan ex vivo, with viability rapidly declining after only 4 h, making prolonged time-course experiments impossible. In contrast, our transfected cell line expressing HiBiT-TSSK1 allowed us to assess degradation dynamics over days to evaluate a prolonged cellular response. We initiated these experiments by first determining the optimal incubation period for maximal TSSK1 degradation by measuring luminescence at 24-, 48-, and 72 h post-treatment, a time frame that would be unattainable with sperm cells (Table ).
1. Quantification of TSSK1 Degradation over 24, 48, and 72 h .
HiBiT-TSSK1 degradation was evaluated in Nano-Glo HiBiT Lytic assays. Cells were treated with each compound for 24, 48, or 72 h. DC50 and D max values reported as the mean from three independent experiments ± SD. ND = No observed degradation.
Our results indicate that TSSK1 degradation was occurring at 24 h for all compounds except 5.5. However, the degradation potency of all compounds peaked at 48 h, with compound 5.1 exhibiting exceptional activity, with a DC50 of 10.8 nM and nearly a 70% reduction in TSSK1 protein. This broad improvement in activity at 48 h may be attributed to poor cellular permeability of the compounds, with longer incubation times allowing increased intracellular concentrations. By 72 h, we observed that TSSK1 levels had fully recovered, likely due to degrader-mediated metabolism, enabling the cells to regenerate TSSK1 protein.
In contrast, this observed recovery of TSSK1 levels would not occur in sperm cells, as mature sperm cannot synthesize new nuclear-encoded proteins. Protein synthesis in spermatozoa is limited to mitochondrial translation during specific processes such as capacitation, and nuclear-encoded proteins, such as TSSK1, cannot be regenerated once degraded. Therefore, the degradation of TSSK1 in sperm cells would be irreversible, indicating these degraders would be highly effective for targeting this protein.
To distinguish whether observed changes in degradation were attributable to altered target engagement or disruptions in ternary complex formation, we employed an ADP-Glo kinase activity assay to directly assess engagement to the ATP-binding site of TSSK1. In this assay, recombinant His-tagged TSSK1 was used to validate biochemical interactions. The ADP-Glo assay quantifies kinase activity by measuring the luminescent signal generated from ATP-to-ADP conversion during the kinase reaction. Since our degraders are designed with an ATP-competitive binding moiety, increasing concentrations of the degrader will result in dose-dependent inhibition of TSSK1 kinase activity. This enables the determination of IC50 values, which serve as a quantitative measure of target engagement, thereby confirming that the degraders effectively interact with TSSK1 in vitro and supporting the mechanistic basis for the cellular degradation observed in our HiBiT-TSSK1 system (Table ).
2. TSSK1 Degradation and Target Engagement Results .
HiBiT-TSSK1 degradation was evaluated in Nano-Glo HiBiT Lytic assays. Cells were treated with each compound for 48 h. DC50 and D max values were averaged from three independent experiments ± SD. Target engagement (IC50) was measured in an ADP-Glo assay and reported as the mean of three independent experiments ± SD.
Using the optimized 48-h time point, we expanded our investigation to include degraders with longer methylene linkers, and the complete SAR is summarized in Table . The results revealed that the single-methylene linker in compound 5.5 was too short to induce potent degradation, resulting in the weakest degrader in the series, with poor target engagement as indicated by a weak IC50 value. In contrast, extending the linker length to three (5.1) or four (5.9) methylene groups significantly enhanced degrader potency, achieving DC50 values as low as 10 nM and demonstrating improved target engagement with TSSK1. However, further increasing the linker length led to a decline in activity, suggesting an optimal linker length of 3–4 methylenes. A similar trend was observed with PEG linkers. The single-PEG degrader (5.2) achieved a DC50 of 48 nM, whereas adding a second (5.3) and third (5.4) PEG resulted in a progressive decrease in activity while maintaining relatively consistent target engagement, indicating that overly long linkers disrupt ternary complex formation rather than perturbing binding to TSSK1.
After evaluating these compounds for TSSK1 degradation, we next sought to confirm that the observed degradation is mediated by cereblon (CRBN) recruitment. To do this, we prepared methylated analogs of the top-performing methylene degrader (5.1) and the PEG degrader (5.2) by methylating the glutarimide nitrogen (Figure a), a modification known to disrupt a key hydrogen bond required for CRBN binding. In contrast to their parent degraders, both methylated control compounds (2.8 and 2.10) exhibited near complete loss of degradation activity in the HiBiT-TSSK1 cellular assay, such that no definable sigmoidal degradation curve was obtainable and maximum degradation remained below 25% (Figure b and c). This profound loss of degradation efficacy is consistent with the expected loss of CRBN binding resulting from disruption of the glutarimide hydrogen bond and is characteristic of impaired ternary complex formation. This strongly supports that the degrader mechanism relies on CRBN recruitment to induce proteasome-dependent degradation, as opposed to off-target cellular effects or nonspecific protein turnover.
2.
Activity of N-methylated degraders. (a) Structure and activity of TSSK1/2 targeted degraders. (b) Dose–response using active degraders. (c) Dose–response using inactive degraders. HiBiT-TSSK1 degradation was evaluated in Nano-Glo HiBiT Lytic assays. Cells were treated with each compound for 48 h. DC50 and D max values reported as the mean from three independent experiments ± SD. aNo sigmoidal fit possible.
Selectivity Profile of Targeted Degraders
We then evaluated the selectivity of these degraders using a panel of 24 kinases and compared their profiles to those of the pan-kinase inhibitor staurosporine and the TSSK1/2 recruiter ligand (Figure S7). To better reflect the cellular activity of the degraders, assay concentrations were chosen to be approximately ten times the DC50 of the most potent degrader rather than the IC50. Under these conditions, both staurosporine and the TSSK-recruiting ligand (9) exhibited broad, nonselective kinase inhibition, whereas conversion into targeted degraders substantially improved their selectivity profiles. The degraders exhibited reduced off-target kinase inhibition relative to the parent TSSK1/2 recruiter ligand, indicating a more focused inhibition profile with an overall decrease in kinase inhibition.
Despite this improvement, residual inhibition of TAK1-TAB1, CHK1, and MAPKAPK2 was observed in this panel. We attribute the persistence of these interactions to the binding profile of the parent ATP-competitive recruiting ligand (9). The recruiting ligand retains considerable affinity for these off-target kinases at the concentrations tested, and although many off-target interactions were mitigated upon conversion to degrader molecules, engagement at the inhibitor level for TAK1-TAB1, CHK1, and MAPKAPK2 remains. TAK1-TAB1 and MAPKAPK2 are integral components of inflammatory signaling pathways, and inhibition of these kinases could negatively influence inflammatory or apoptotic responses. , Similarly, inhibition of CHK1, a key regulator of the DNA damage response, may interfere with cell cycle progression and genomic stability. Nonetheless, these findings are encouraging as they provide strong evidence that our strategy to improve selectivity is effective.
It is important to note that the selectivity assay was conducted at a relatively low ATP concentration (10 μM), which reflects typical biochemical assay conditions but does not reflect the millimolar ATP concentrations present in intact cells. In cells, ATP levels generally range from 1–5 mM, which substantially exceed the ATP Km values for most kinases. Under cellular ATP conditions, competition is greater than in biochemical assay conditions. This increased competition has been shown to increase the apparent IC50 of ATP-competitive inhibitors as ATP concentration rises. Because the TSSK1/2 degraders compete with ATP, we anticipate that the off-target inhibition observed will be further reduced under physiological conditions. This effect is expected to result in an even cleaner selectivity profile in future in vivo studies.
We acknowledge that, as a current limitation, whether TAK1-TAB1, CHK1, and MAPKAPK2 are subject to proteasomal degradation has not yet been evaluated. This important question will be addressed in future work using unbiased proteomic approaches to directly assess degradation across the kinome. Nonetheless, the observed reduction in off-target kinase inhibition relative to the parent inhibitor supports the conclusion that targeted degrader design substantially improves selectivity while highlighting areas for continued optimization.
Evaluation of 5.1 and 5.2 Pharmacokinetic Profiles
Encouraged by these findings, we evaluated the preliminary in vitro pharmacokinetic profiles of the top-performing methylene- and PEG-linked degraders, focusing on solubility and metabolic stability in mouse and human liver microsomes (Table ). Compound 5.1, containing the methylene linker, exhibited poor solubility of only 3 μM. In contrast, the PEG-linked degrader (5.2) demonstrated a >50-fold increase in solubility (173 μM), as anticipated given the hydrophilic nature of PEG linkers.
3. Pharmacokinetic Profile of Degraders 5.1 and 5.2 .
| Compound | Solubility (μM) | MLM T 1/2 (min) | MLM CLint (mL/min/kg) | HLM T 1/2 (min) | HLM CLint (mL/min/kg) |
|---|---|---|---|---|---|
| Diclofenac | 281 | 42.4 | 143 | 9.76 | 178 |
| 5.1 | 3.66 | 26.3 | 231 | 14.8 | 117 |
| 5.2 | 173 | 3.93 | 1540 | <2.26 | >770 |
Solubility evaluated using fasted-state simulated intestinal fluid. Mouse liver microsome (MLM) and human liver microsome (MLM) assays evaluated metabolic stability for each degrader. Permeability was evaluated using Caco-2 cells. Data obtained by Pharmaron. Diclofenac was used as a control compound during these assays.
Despite the significant solubility differences, both compounds displayed short half-lives in mouse and human liver microsomes. Notably, compound 5.2 exhibited a half-life of less than 4 min in both species, likely due to N-dealkylation of the PEG linker. Compound 5.1 showed moderately improved clearance compared to 5.2 but would still require additional optimization before entering in vivo models. To further investigate metabolic pathways, we assessed stability in the presence and absence of UDPGA to evaluate glucuronidation (Figures S8 and S9). Both compounds displayed equivalent half-lives under these conditions, confirming that metabolism was primarily driven by phase I pathways rather than by glucuronidation and supporting N-dealkylation as the predominant metabolic pathway.
Given the rapid metabolic turnover observed in microsomes, we hypothesized that degrader instability was preventing sustained target engagement in our HiBiT-TSSK1 cell line. To test this, SC B1.1 cells were dosed with fresh aliquots of compounds 5.1 and 5.2 every 24 h over a 72-h period (Figures S10 and S11). Under these repeated dosing conditions, both DC50 and D max stabilized by 48 h and remained constant through 72 h. This differed from our earlier experiments using a single dose, where HiBiT-TSSK1 protein levels had recovered by 72 h. These data support our interpretation that the recovery of HiBiT-TSSK1 protein levels after a single-dose treatment was primarily driven by metabolic turnover of the degrader molecules rather than adaptive changes in TSSK1 expression.
To overcome this metabolic limitation in future analogs, we plan to pursue multiple optimization strategies. Linker rigidification will be prioritized, as increased rigidity has been shown to reduce metabolic susceptibility, particularly toward N-dealkylation pathways. We are also exploring modifications to the CRBN recruiting ligand. For example, phenyl glutarimide cores or strategic fluorination/methylation near the pomalidomide linker attachment site may mitigate both metabolic turnover and efflux. , These structural refinements are expected not only to improve metabolic stability but also to enhance kinase selectivity by preferentially locking ternary complex formation toward either TSSK1 or TSSK2 without compromising degradation potency.
The cellular uptake potential and oral bioavailability of degraders 5.1 and 5.2 were evaluated using a Caco-2 permeability assay (Figures S12 and S13). Both compounds exhibited limited cellular uptake, with apparent permeability coefficients (P app A-B) below 1 × 10–6 cm/s, indicating poor membrane permeability. Notably, high efflux ratios were observed (5.1: 16.57; 5.2: 133.40), suggesting pronounced susceptibility to efflux transporters. These permeability deficits correlate with the delayed HiBiT-TSSK1 degradation kinetics observed in the SC B1.1 cell line, where maximal degradation required prolonged incubation. Likewise, experiments using sperm cells (discussed below) are limited to short incubation periods and show reduced degradation potency, likely due to insufficient accumulation of intracellular degraders. While efflux activity may be mitigated in vivo by systemic factors, the intrinsically low permeability remains a concerning liability for oral bioavailability.
Ex Vivo Degradation of TSSK2 in Mouse Sperm
Following our characterization of degrader activity in the HiBiT-TSSK1 cell line, we evaluated whether targeted degradation could be observed in mature spermatozoa. Although commercial antibodies for TSSK1 exist, none have provided the selectivity required for quantitative whole-cell immunoblotting. In contrast, a high-quality anti-TSSK2 antibody enabled us to monitor the abundance of endogenous TSSK2 in sperm. Furthermore, TSSK1 and TSSK2 share 82% amino acid sequence homology, and the recruiting ligand (compound 19) inhibits both isoforms with potent affinity (TSSK1 IC50 = 0.86 nM, TSSK2 IC50 = 66 nM). For these assays, CD1 mouse spermatozoa were incubated in capacitation medium containing DMSO vehicle or increasing concentrations of five degrader compounds (5–200 μM), and abundance was assessed by anti-TSSK2 immunoblot (Figure ). Because mature sperm have a limited ex vivo lifespan, degradation was evaluated after a single four-hour incubation. This short exposure contrasts with our HiBiT-TSSK1 cell assay, which permits up to 72 h of compound treatment, and accordingly, the observed potencies in sperm were reduced relative to cell line assays. Although both isoforms can be engaged by the same recruiting ligand, we did not expect degradation outcomes to be directly comparable between TSSK1 and TSSK2. Differences in the structural environments of the two isoforms can alter the preferred geometry for productive ternary complex formation, and compounds that are most potent for TSSK1 degradation may not be the most potent for TSSK2. Thus, these experiments were designed to identify the relative efficacy of degraders specifically against TSSK2 in a native cellular context.
3.
(a–e) TSSK2 degradation in CD1 mouse sperm cells after incubation with 5.1–5.5. Compounds 5.2, 5.3, and 5.4 reduced TSSK2 protein levels. Replicate blots found in Figure S14. Results are reported as the average of three independent experiments ± SEM.
Only the three PEG-linked degraders (5.2–5.4) produced substantive TSSK2 reduction in this assay. Compound 5.2, containing a single PEG unit, produced a dose-dependent response with a D max of approximately 77% at 50 μM (complete results in Figure ). Compound 5.3 reached the greatest single-point D max (87%) at 30 μM, but this effect was observed only at that concentration and was characterized by a steep onset above 10 μM with loss of activity at 50 μM. The longest PEG linker, compound 5.4, delivered the most consistent profile, producing robust degradation across the full concentration range and achieving an 80% reduction in TSSK2 at 10 μM. By contrast, the alkyl-linked degraders (5.1 and 5.5) failed to reduce TSSK2 levels under these conditions.
5.
(a–d) Sperm cells incubated with compound 5.1–5.4 displayed significant disruption of total and progressive motility, which resulted in similar reduction for 2-cell cleavage and blastulation rates. (e) Oocytes incubated with untreated sperm cells were devoid of cumulus cells and were successfully fertilized as identified by formation of 2-cell state. (f) Oocytes incubated with treated sperm cells using 10 μM of 5.4 display intact cumulus cells surround oocyte and reduced 2-cell formation. Data are reported as the mean of three independent experiments ± SD. ND = No observed degradation.
Although compound 5.1 was the most active degrader in the HiBiT-TSSK1 cell assay, it failed to reduce TSSK2 levels in spermatozoa. That outcome was initially attributed to poor membrane permeability; however, physicochemical profiling confirmed that 5.1 and 5.2 both exhibit limited passive permeability, whereas only 5.2 induced TSSK2 degradation in sperm. These observations indicate that linker chemistry, rather than permeability, is the dominant factor governing productive ternary complex formation with TSSK2. Therefore, it appears that short alkyl linkers, exemplified by 5.1 and 5.5, are unable to position the ligands in a geometry compatible with the recruitment of the E3 ligase, whereas longer PEG linkers enable effective recruitment and degradation of TSSK2.
Taken together, these data indicate that linker composition and three-dimensional geometry are critical determinants of ternary complex formation and degradation efficacy for TSSK2 and that the molecular requirements for recruiting TSSK2 differ from those for TSSK1. Importantly, the demonstration that multiple PEG-linked degraders induce a marked reduction of an endogenous, testis-specific kinase in mature spermatozoa provides the first direct evidence that sperm can support targeted protein degradation and validates our approach as a viable strategy for disrupting sperm-restricted targets.
Proteasome-Dependent Degradation in Mature Sperm Cells
To confirm that the reduction of TSSK2 observed in sperm cells resulted from targeted protein degradation rather than indirect effects on protein stability, a series of mechanistic experiments were conducted in capacitated CD1 mouse sperm. These studies were designed to evaluate both the requirement for CRBN engagement and the proteasome in mediating degradation.
We first examined the methylated glutarimide analogs 2.8 and 2.10, which previously served as negative controls in the HiBiT-TSSK1 cellular assay. Methylation of the glutarimide moiety disrupts binding to CRBN and therefore prevents recruitment of the E3 ligase complex. Consistent with this design, treatment of sperm cells with compounds 2.8 or 2.10 across concentrations ranging from 5 to 200 μM produced no detectable reduction in TSSK2 abundance (Figure a and b). In contrast, the corresponding nonmethylated analog of 2.10, compound 5.2, induced strong TSSK2 degradation, reaching levels approaching 80%. The absence of activity for the methylated controls in sperm cells mirrors the results obtained in the HiBiT-TSSK1 cell line and provides strong evidence that degradation requires productive CRBN engagement.
4.
Ex vivo activity with N-methyl analogs and proteasome inhibitor. (a, b) TSSK2 degradation in CD1 mouse sperm cells after incubation with N-methylated degraders 2.8 and 2.10. (c) TSSK2 degradation using 5.4 with and without proteasome inhibitor pretreatment. Replicate blots are found in Figure S15. Results are reported as the mean of three independent experiments ± SEM.
We next evaluated whether TSSK2 degradation depended on proteasome activity. Epoxomicin, a potent and selective irreversible inhibitor of the 20S proteasome, was used to test this hypothesis. Co-treatment of capacitated sperm with degrader 5.4 alone resulted in an approximately 80% reduction in TSSK2 protein levels relative to the DMSO control. However, when sperm were pretreated with epoxomicin, no degradation of TSSK2 was observed (Figure c). The ability of this proteasome inhibitor to fully rescue TSSK2 levels is consistent with a proteasome-dependent degradation mechanism, as inhibition of proteasomal catalytic activity prevents the turnover of ubiquitinated proteins.
Taken together, these experiments provide strong mechanistic support for the conclusion that the observed loss of TSSK2 in sperm occurs via a CRBN- and proteasome-dependent degradation pathway. The inactivity of methylated glutarimide analogs confirms that recruitment of the CRBN E3 ligase is required, while the ability of epoxomicin to completely block degradation establishes that the proteasome is responsible for the clearance of the ubiquitinated kinase. Importantly, these findings provide direct experimental evidence that mature sperm retain the molecular machinery necessary for pharmacologically induced, CRBN-mediated targeted degradation.
Functional Effects of TSSK2 Degradation on Sperm Motility and Fertilization
After confirming TSSK2 degradation in sperm cells, we then evaluated whether this translated into impaired sperm function (Figure a–d) using computer-assisted sperm analysis (CASA). CD1 mouse sperm were incubated for 4 h with compounds 5.2–5.4, dosed at the concentration that induced maximal TSSK2 loss, as identified by anti-TSSK2 immunoblots. Compounds 5.1 and 5.5 were also included for comparison, despite showing no detectable TSSK2 degradation.
Total motility and progressive motility were measured by CASA following compound treatment, and all evaluated degraders, except 5.5, produced significant reductions in both. The results from these motility assays indicate that the PEG-linked degraders are the most efficacious. Compound 5.4, which contains a three-unit PEG linker, produced the strongest effect and reduced both total and progressive motility by nearly 97% despite being dosed at the lowest active concentration in the series. The shorter PEG-linked compounds 5.2 and 5.3 reduced motility by approximately 90 and 80%, respectively, yet both were dosed at higher concentrations than 5.4. Interestingly, compound 5.1 reduced motility by close to 70% even as it did not yield measurable TSSK2 degradation in sperm. This profile is consistent with the potent TSSK1 degradation observed in the HiBiT-TSSK1 cellular assay, and TSSK1 degradation is the most likely explanation for the observed motility defect. However, this conclusion cannot be directly confirmed due to the lack of selective TSSK1 antibodies. By contrast, compound 5.5 produced no detectable motility defects even at concentrations up to 50 μM. Nevertheless, this result was expected, as 5.5 was 50-fold less potent as a TSSK1 degrader than 5.1 and was completely inactive toward degrading TSSK2. This has allowed us to use compound 5.5 as a negative control in these functional assays, as it retains strong target engagement with TSSK1 (IC50 = 207 nM). Therefore, the lack of effect with 5.5 argues against nonspecific ATP-site inhibition or acute toxicity as the primary cause of motility loss for the active degraders.
Compounds 5.1–5.4 were evaluated for their effects on fertilization using in vitro fertilization (IVF) assays with CD1 mouse sperm. Sperm were incubated in capacitation medium either with DMSO, without DMSO, or in the presence of degrader compounds at concentrations previously used in motility experiments. After incubation, sperm were applied to cumulus-intact oocytes, and early embryonic development was assessed by monitoring two-cell cleavage and blastulation. The two-cell stage provides a rapid readout of successful fertilization and zygotic genome activation, while blastulation serves as a measure of later embryonic competence and viability.
Compared to controls, PEG-linked degraders 5.2, 5.3, and 5.4 resulted in a strong reduction of two-cell cleavage rates (approximately 95%), whereas compound 5.1 showed a more moderate reduction (approximately 65%), consistent with its weaker inhibition of sperm motility. Furthermore, blastulation was essentially abolished in samples treated with degraders 5.2, 5.3, and 5.4, indicating a substantial impact on male fertility. Physical inspection revealed that oocytes incubated with untreated sperm were cleared of cumulus cells, while oocytes exposed to degrader-treated sperm retained intact cumulus layers (Figure e and f). This observation further supports that the treated sperm lack the motility necessary to disperse cumulus cells and successfully fertilize oocytes.
Collectively, these findings represent the first proof of concept that targeted degradation of TSSK family kinases may serve as a potent nonhormonal contraceptive strategy. The ability of these degraders to block not only motility but also fertilization and early embryo development demonstrates their potential to comprehensively interrupt male fertility and provide strong justification for advancing optimized degrader candidates into future in vivo efficacy studies.
Conclusions
We report the design, synthesis, and characterization of the first series of targeted degraders directed against the TSSK1/2 family, derived from a previously reported nonselective ATP-site inhibitor. To enable quantitative evaluation of TSSK1 degradation, we established a stable CHO-K1 cell line expressing HiBiT-tagged TSSK1, as commercially available antibodies lacked the selectivity required for whole-cell characterization. This system further enabled degradation profiling over extended time courses of up to 72 h, beyond what is feasible in sperm cells due to limited viability. Using this platform, compound 5.1 was identified as the most potent TSSK1 degrader, achieving a DC50 of approximately 10 nM after 48 h.
We further demonstrate for the first time that mature spermatozoa possess functional cellular machinery capable of supporting targeted protein degradation. In ex vivo experiments using CD1 mouse sperm, a four-hour incubation with degrader 5.4 resulted in approximately 80% depletion of TSSK2. This loss translated into pronounced functional impairment, with sperm motility reduced by up to 97% and in vitro fertilization effectively abolished. Collectively, these findings establish proof-of-concept that pharmacologically induced degradation of TSSK family kinases can produce physiological effects consistent with a nonhormonal male contraceptive mechanism.
Mechanistic studies support a CRBN-dependent mode of action, as methylation of the pomalidomide glutarimide nitrogen substantially diminished degrader activity in both the HiBiT-TSSK1 cellular assay and in ex vivo sperm experiments. The methylated analogs failed to induce detectable degradation and pretreatment with the proteasome inhibitor epoxomicin rescued TSSK2 expression. These findings together support a cereblon- and proteasome-dependent degradation mechanism functioning in mature sperm cells.
However, physicochemical and metabolic evaluation identified several liabilities in the current leads, including rapid metabolic N-dealkylation and pronounced efflux. These properties were reflected in cellular studies, in which sustained degradation over 72 h required repeated dosing, whereas a single administration allowed recovery of TSSK1 expression. Future optimization efforts will therefore focus on linker rigidification, evaluation of alternative CRBN recruiting ligands, and exploration of prodrug approaches to enhance permeability, metabolic stability, and overall bioavailability.
Further analysis of the degrader series revealed distinct linker length requirements for optimal degradation of the two isoforms. Short alkyl linkers favored TSSK1 degradation, exemplified by compound 5.1, whereas longer PEG-based linkers, such as in compound 5.4, were necessary for efficient TSSK2 degradation in sperm. These observations are consistent with isoform-specific structural features and differences in ternary complex formation that influence productive engagement with CRBN, which may be exploited to bias selectivity toward either TSSK1 or TSSK2, as well as overall kinase selectivity.
For progression toward in vivo application, degraders with improved pharmacokinetic profiles will be required. However, we envision future in vivo studies to use oral dosing strategies at substantially lower exposures than those used ex vivo, potentially with intermittent administration every few days to maintain target engagement. The profound functional impairment observed following a single four-hour ex vivo exposure suggests that effective contraceptive efficacy may be achievable in vivo once metabolic stability and selectivity liabilities are addressed. Nonetheless, a comprehensive in vivo evaluation will be essential to characterize reversibility, safety, and effectiveness of the contraceptive effect.
Experimental Section
Materials
All chemical reagents and solvents were purchased from commercial vendors and used without further purification. Compounds were plated onto 384-well ProxiPlate Plus microplates (PerkinElmer; catalog # 600828) using a LabCyte ECHO550 nanoliter dispensing system. Luminescent assays were all measured using a PerkinElmer Enspire plate reader. His6-tagged recombinant TSSK1 was purchased from Invitrogen (catalog # PV3505). CHO-K1 cells and cell media (F12K) were obtained from ATCC (catalog # ATCC 30-2004). HiBiT-TSSK1 DNA plasmid (vector ID: VB231116-1266hdp) and piggyBac mRNA (catalog # R007S) were purchased from VectorBuilder. 96-well half a rea assay plates (Catalog# 3885) were purchased directly from Corning. Nano-Glo HiBiT Lytic Detection System (catalog # N3040), CellTiter-Glo Luminescent Cell Viability Assay (catalog # G7572), ADP-Glo Kinase Assay (catalog # V9101), anti-HiBiT antibodies (catalog # N7200), and Kinase Selectivity Profiling SystemsGeneral Panel (catalog # V6928) were purchased from Promega. Lipofectamine MessengerMax was purchase from Thermo Fisher Scientific (catalog # LMRNA001).
General Chemistry
All chemical reagents and solvents were purchased from commercial vendors and used without further purification. All reactions that required anhydrous conditions were run under a nitrogen atmosphere. Reaction progress was monitored either by thin-layer chromatography (TLC) using silica gel 60 F254 plates and visualized under UV light at 254 nm or an Agilent 1260 Infinity II LC/MSD iQ system. All tested compounds are >95% pure by HPLC analysis. Purity analysis and observation of molecular m/z were obtained using a Waters ACQUITY UPLC/MS system. Mass spectrometric analysis was performed on an Agilent InfinityLab LC/MSD iQ instrument in positive ion mode. The UPLC analyses and mass spectra (UPLC-MS) were obtained on a Waters ACQUITY system (Waters, Milford, CT, USA) with a QDa Mass Detector with ESI inlet and UV PDA detector. The Waters UPLC BEH C18, 1.7 μm (2.1 × 50 mm) column was used at 40 °C. Samples were dissolved in 200–600 μL of DMSO, depending on their solubility. The sample was then diluted to approximately 0.1 mg/mL in MeOH for injection into the UPLC/MS. The UPLC/MS parameters were as follows: mobile Phase A (10 mM ammonium bicarbonate in water), mobile Phase B (ACN), a flow rate of 0.6 mL/min, injection volume of 7.5 μL, and a run time of 6.0 min. Gradient operation as follows: Hold at 95:5 A:B for 0.5 min. Linear gradient to 5:95 A:B for 3 min. Hold at 5:95 A:B for 0.5 min. Linear gradient to 95:5 A:B for 0.5 min. Hold at 95:5 A:B for 1.5 min. The mass detector ran a positive scan from 150–1000 Da. The PDA quantified the peaks at 254 nm. Compounds were purified by normal-phase or reverse-phase flash chromatography and was performed using a Teledyne ISCO Combiflash NextGEN 300+ liquid chromatography system using RediSep Gold silica gel flash columns (20–40 μm). 1H and 13C NMR were performed on a Bruker Avance NEO 600/151-MHz spectrometer or a Bruker Avance 400/100-MHz spectrometer. Chemical shifts are reported in ppm and referenced to residual solvent peaks: CHCl3 in CDCl3 at 7.26 ppm for 1H NMR and 77.16 ppm for 13C NMR; DMSO in DMSO-d6 at 2.50 ppm for 1H NMR and 39.52 ppm for 13C NMR. Coupling constants (J) are given in Hertz (Hz), and splitting patterns are designated as singlet (s), doublet (d), triplet (t), quartet (q).
In Silico Protocols
The AlphaFold-predicted structure of human TSSK1 (UniProt accession Q9BXA7) was prepared using Schrödinger Maestro’s Protein Preparation Wizard, where bond orders were assigned, hydrogens were added, termini were capped, missing side chains were modeled, and the structure was minimized using the OPLS4 force field. Compound 19 was prepared using LigPrep, with ionization states generated at pH 7.4 ± 2.0 and geometry minimized using the OPLS4 force field. Induced-fit docking (IFD) was performed in Schrödinger by centering the receptor grid on ATP-binding site residues 18–26. Docking of compound 19 was followed by refinement of protein side chains within 5 Å of the ligand and redocking with extra precision (XP) to generate 20 binding models.
Establishment of the HiBiT-TSSK1 CHO-K1 Cell Line
CHO-K1 cells were cultured in F-12K medium supplemented with 10% FBS and 1% Pen/Strep at 37 °C in a 5% CO2 incubator. Cells were initially expanded in T-75 flasks until 70–80% confluency. For transfection, cells were detached with 0.25% trypsin, counted, and seeded into 6-well plates at 300,000 cells/well in 2 mL fresh medium. After 24 h, cells reached 70–80% confluency for transfection. The HiBiT-TSSK1 donor vector (containing the HiBiT tag fused to TSSK1) and PiggyBac transposase mRNA were combined at a 5:1 mass ratio (DNA:mRNA) and diluted in Lipofectamine MessengerMax at two transfection reagent-to-genetic material ratios (5:1 and 7:1 v/m). Each mixture was further diluted in 50 μL OptiMEM, vortexed for 30 s, and incubated at rt for 15 min. For each transfection condition (5:1 or 7:1 v/m), three wells were transfected with 1300, 260, or 50 ng total genetic material. The transfection complexes were added dropwise to designated wells while gently swirling the plate. Cells were incubated with the complexes for 24 h. After transfection, the medium was replaced with F-12K containing 50 μg/mL puromycin. Cells were maintained under puromycin selection for 7 days, with fresh medium replenished every 48 h. Surviving colonies were expanded for an additional 7 days in puromycin-free medium. Cells from each well were detached with 0.25% trypsin, resuspended in F-12K, and diluted to 0.5 cells/mL. A 1 mL aliquot of this suspension was plated into each well of four 24-well plates to isolate single-cell clones. Plates were monitored for 14 days, and wells containing single colonies were identified. Clonal lines were assessed for HiBiT-TSSK1 expression using the Nano-Glo HiBiT Lytic Detection System.
Anti-HiBiT Western Blot
SCB1.1 HiBiT-TSSK1 cells were maintained in standard culture conditions and seeded into T-25 flasks. Cells were grown to 60–80% confluence, the medium was aspirated, and the monolayers were rinsed twice with 2 mL of ice-cold phosphate-buffered saline. The residual buffer was removed, and 1 mL of ice-cold RIPA lysis buffer (96.7% Water, 7% Polyethylene glycol octylphenyl ether, 1% Sodium chloride, 0.9% 1,3-Propanediol, 2-amino-2-(hydroxymethyl)-, hydrochloride, 0.8% Cholan-24-oic acid, 3,12-dihydroxy-, monosodium salt, (3.alpha.,5.beta.,12.alpha.), 0.5% Sodium lauryl sulfate) supplemented with protease and phosphatase inhibitor cocktails (ThermoFisher Scientific; catalog #78440) was added directly to each flask. Plates were incubated on ice for 30 min with intermittent gentle agitation to facilitate lysis. Lysates were transferred to chilled microcentrifuge tubes and clarified by centrifugation at 16,000g for 5 min at 4 °C. Supernatants were collected, and total protein concentration was measured using NanoDrop2000 UV–vis Spectrophotometer. Aliquots were flash frozen and stored at −80 °C prior to shipment. Autowestern analysis was performed by RayBiotech using their automated capillary immunoassay platform and validated antibody panel. Samples were submitted to RayBiotech at the recommended protein concentration and volume, as per the company’s sample submission guidelines. Immunodetection was performed with an anti-HiBiT primary antibody for HiBiT-tagged TSSK1 and anti-GAPDH as a loading control. The chemiluminescent signal was recorded and quantified using the provider’s analysis software, enabling comparison of HiBiT-TSSK1 abundance, normalized to GAPDH. RayBiotech’s automated capillary immunoassay method provides quantitative band-intensity output and was used to confirm the expression of the HiBiT-TSSK1 construct in SCB1.1 cells.
Subcellular Fractionation
Subcellular protein extraction was performed using the Thermo Scientific Subcellular Protein Fractionation Kit for Tissues (catalog #78840), following the manufacturer’s protocol with minor modifications. SC B1.1 cells were harvested by trypsinization, pelleted by centrifugation at 500g for 5 min, washed twice with ice-cold PBS, and centrifuged again under the same conditions. The resulting cell pellet was resuspended in Cytoplasmic Extraction Buffer (CEB) and incubated on ice with constant agitation. Subsequent extraction steps were performed sequentially using Membrane Extraction Buffer (MEB), Nuclear Extraction Buffer (NEB), and Pellet Extraction Buffer (PEB), each followed by centrifugation to yield the cytoplasmic, membrane, nuclear-soluble, and cytoskeletal fractions, respectively. To isolate the chromatin-bound fraction, the nuclear pellet was resuspended in NEB supplemented with CaCl2 and micrococcal nuclease and incubated according to the manufacturer’s instructions. All extraction buffers were supplemented with the recommended concentration of protease inhibitor. All fractions were kept on ice throughout the procedure until analysis. The concentration of HiBiT-tagged protein in each fraction was quantified using the Nano-Glo HiBiT Lytic Detection System (Promega).
Nano-Glo HiBiT Lytic Assay and CellTiter-Glo Cell Viability
The HiBiT-TSSK1 cell line SC B1.1 was seeded in white 96-well half a rea plates (Corning #3885) at 2,500 cells/well in 50 μL of F-12K medium (10% FBS, 1% Pen/Strep). Plates were incubated at 37 °C (5% CO2) for 24 h. Eight-point dose–response curves were prepared for each degrader compound via serial dilution in F-12K medium (final DMSO ≤ 0.5%). For treatment, 40 μL of fresh F-12K medium and 10 μL of compound dilutions were added to each well (final volume: 50 μL/well). Plates were incubated for 24–72 h under standard culture conditions. After incubation, the media were aspirated, and 30 μL of 1X PBS was added to each well. This was followed by 30 μL of either Nano-Glo HiBiT Lytic Detection Reagent or 30 μL of CellTiter-Glo Reagent (prepared per manufacturer’s instructions). The cells were then incubated at rt for 10 min with gentle shaking. Luminescence was measured using a plate reader (integration time: 0.5 s/well). HiBiT-TSSK1 signals were normalized to CellTiter-Glo viability values per well to correct for cytotoxicity. Percent degradation was calculated relative to DMSO-treated controls. Dose–response curves were generated, and DC50 values were determined via nonlinear regression analysis (log[inhibitor] vs normalized response – variable slope) in Prism 10.
ADP-Glo Kinase Activity Assay
Evaluation of His6-TSSK1 kinase activity was performed using 384-well ProxiPlate Plus microplates (PerkinElmer). The reaction buffer consisted of 20 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 0.015% Brij-35, 4 mM DTT, and 0.1 mg/mL BSA. His6-TSSK1 was diluted in reaction buffer to a final concentration of 0.8 ng/μL. An eight-point dose–response curve for test compounds was prepared in triplicate using a LabCyte ECHO550 nanoliter dispensing system. Two controls were included on each plate: a high control containing no inhibitors and a low control containing no inhibitor and no TSSK1. After compound addition, 2 ng/well His6-TSSK1 (2.5 μL) was dispensed into each well, and the plate was centrifuged at 1000g for 10 s. The plate was incubated at rt for 120 min, then a 2.5 μL solution containing 10 μM ATP and 0.4 μg/μL AMARA peptide substrate was added to each well. The plate was centrifuged again at 1000g for 10 s, then agitated for an additional 60 s to ensure thorough mixing. The kinase reaction was incubated for 60 min at rt, followed by the manufacturer’s protocol for the Promega ADP-Glo assay kit to terminate the reaction and measure ADP production. Luminescence was detected using a PerkinElmer Enspire plate reader. Dose–response curves were generated, and IC50 values were calculated using nonlinear regression analysis (log[inhibitor] vs normalized response – variable slope) in Prism 10 software.
Selectivity Panel
Kinase selectivity profiling was performed using the Promega Kinase Selectivity Profiling System: General Panel, which includes 24 representative kinases, in 384-well ProxiPlate Plus microplates (PerkinElmer). Test compounds were plated at a final concentration of 100 nM in duplicate using a LabCyte ECHO550 acoustic liquid handler. Kinases and their respective reaction components were prepared according to the manufacturer’s instructions. Kinase solutions were added to the assay plate and preincubated with test compounds for 1 h at rt. Substrate solutions, containing 10 μM ATP and the corresponding peptide substrate for each kinase, were then added in a 1:1 ratio to initiate the reactions. The kinase reactions were allowed to proceed for 1 h at rt. Reactions were terminated using the Promega ADP-Glo Kinase Assay Kit according to the manufacturer’s protocol. Luminescence was measured using a PerkinElmer Enspire plate reader. Percent activity was calculated by normalizing luminescence values to high controls (kinase + substrate, no inhibitor) and low controls (substrate only, no kinase/inhibitor). Data analysis, including percent activity calculations and visualization of selectivity profiles, was performed using Prism 10 software.
Compound Solubility in Fasted State Simulated Intestinal Fluid (FaSSIF)
FaSSIF-V2 medium was prepared by dissolving NaOH (1.392 g), maleic acid (2.220 g), and NaCl (4.010 g) in ultrapure water, adjusting the pH to 6.5 with 1 M NaOH, and diluting to 1,000 mL; FaSSIF-V2 powder (1.790 g) was then added to 500 mL of this buffer, stirred until dissolved, and diluted to 1,000 mL. Stock solutions of test compounds (10 mM) and diclofenac control (30 mM) were prepared in DMSO. For solubility determination, 30 μL of each stock was mixed with 970 μL FaSSIF in a 96-well plate, stirred at 1,100 rpm for 2 h at rt, filtered through a 0.45 μm membrane, and diluted with methanol/DMSO (980 μL/10 μL). Standards (3 μM) were prepared by diluting stock solutions in DMSO, methanol, and FaSSIF. Samples were analyzed by LC-MS/MS using a C18 column with gradient elution (0.1% formic acid in ACN) and quantified by MRM detection. Solubility was calculated by comparing sample peak areas to a standard curve, normalized to diclofenac controls, and expressed as percent recovery.
Microsomal Stability
A master solution was prepared by combining phosphate buffer (114.94 mM; 69.6 μL), ultrapure water (0.4 μL), and liver microsomes (20 mg/mL; 2 μL) to achieve final concentrations of 100 mM phosphate buffer and 0.556 mg/mL microsomal protein. NADPH (10 mM) was prepared in ultrapure water. Test compounds (2 mM; 80 nL) were added to 72 μL of the master solution, followed by 8 μL of NADPH (final concentration: 1 mM). Negative controls replaced NADPH with 8 μL water. Verapamil served as the positive control. Final test compound concentration was 2 μM (0.1% DMSO). After prewarming at 37 °C for 10 min, aliquots (10 μL × 4 replicates) were transferred to a quench plate containing 120 μL cold ACN with internal standards (200 nM labetalol, 100 nM tolbutamide, 100 nM ketoprofen) at 0, 15, 30, 45, and 60 min. Quenched samples were centrifuged (3220g, 45 min), and 40 μL of the supernatant was diluted with 40 μL water for LC-MS/MS analysis. A master solution was prepared using microsomes (20 mg/mL; 2 μL), alamethicin (5 mg/mL; 0.4 μL), and MgCl2 (5.75 mM; 69.6 μL) to achieve final concentrations of 0.556 mg/mL microsomal protein, 27.8 μg/mL alamethicin, and 5.56 mM MgCl2. NADPH (20 mM) and UDPGA (40 mM) were prepared in water. Test compounds (2 mM; 80 nL) were added to 72 μL master solution, followed by 4 μL each of NADPH (final: 1 mM) and UDPGA (final: 2 mM). Negative controls replaced NADPH/UDPGA with 8 μL water. Diclofenac served as the positive control. Final test compound concentration was 2 μM (0.1% DMSO). After prewarming (37 °C, 10 min), aliquots were quenched and processed as described earlier. Peak areas were extracted from LC-MS/MS chromatograms.
CACO-2 Permeability
Caco-2 permeability assays were conducted by Pharmaron using 96-well HTS transwell plates. Caco-2 cells were seeded at 6.86 × 105 cells/mL and cultured for 14–18 days to form tight monolayers, with medium changes every other day. Monolayer integrity was confirmed by transepithelial electrical resistance (TEER), with only wells exceeding 230 Ω·cm2 considered acceptable. Test compounds and controls (digoxin, prazosin, and metoprolol) were prepared as 5 μM working solutions in HBSS (10 mM HEPES, pH 7.4), with 2% BSA added for test compounds. Permeability was assessed bidirectionally (apical-to-basolateral and basolateral-to-apical) by adding compounds to the donor side and sampling both compartments after 2 h at 37 °C. Samples were quenched with cold methanol containing internal standards, centrifuged, and analyzed by LC-MS/MS. Monolayer integrity was further verified postassay via Lucifer Yellow leakage testing, with fluorescence measured at 485/530 nm.
Sperm Sample Preparation
The caudal portion of the epididymis was excised from the dissected mice, cut with 2–3 incisions, and placed in 1 mL of modified Toyoda–Yokoyama–Hosi (mTYH) medium, also referred to as capacitating media. This was equilibrated overnight at 37 °C in 5% CO2 to achieve a pH of 7.2–7.4. The medium contains the following components (concentrations are provided in parentheses): NaCl (119.3 mM), KCl (4.7 mM), CaCl2. 2H2O (1.71 mM), KH2PO4 (1.2 mM), MgSO4 × 7H2O (1.2 mM), glucose (5.56 mM), Na pyruvate (0.51 mM), NaHCO3 (25.1 mM), Bovine serum albumin (BSA) (4 mg/mL), and gentamicin (10 μg/mL). After 10 min, the epididymal tissue was removed, and sperm motility was assessed under the microscope using a 10 μL sample from the suspension. At this stage, sperm concentration was approximately 10 million/mL. Subsequently, 100 μL (1 × 106 cells) of the original suspension in TYH media was added to the respective experimental media conditions containing various concentrations of degrader. The cells were incubated in a CO2 incubator for 4 h.
TSSK2 Western Blot
Cauda sperm obtained as described above were incubated in experimental tubes containing 400 μL of media (300 μL of capacitating media + 100 μL of sperm suspension) with varying concentrations of degrader (final DMSO ≤ 0.5%). After 4 h of incubation, the tubes were centrifuged at 12,000 rpm for 5 min. The sperm pellets were then washed twice with 1000 μL of 1× PBS. Sperm pellets were resuspended in nonreducing sample buffer (2% SDS, Tris–HCl pH 7.0), heated at 100 °C for 5 min, and centrifuged for 5 min at 12,000 rpm. Supernatants were supplemented with β-mercaptoethanol to a final concentration of 5% (v/v), boiled for an additional 5 min, and then centrifuged again at 12,000 rpm. The extracted proteins were loaded onto a 10-well 8% gel for SDS-PAGE using a Bio-Rad system at a constant current of 40 mA per gel. Following electrophoresis, proteins in the gel were transferred to PVDF membranes (cat# IVPH00010, Millipore Sigma, MA, USA). PVDF membranes were blocked for 1 h at rt in 5% milk in TBS-T buffer (100 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.01% Tween-20) before being used for Western blotting. Blots were incubated overnight at 4 °C with TSSK2 antibody (cat# H00023617-M01, Abnova) (1:1000) in 5% Milk in TBS-T buffer. The next day, blots were washed three times for 10 min with 10 mL of TBS-T, then incubated for 1 h with antimouse secondary HRP-conjugated antibody (cat#715-035-150, Jackson ImmunoResearch Laboratories) at a 1:10000 dilution. Secondary antibodies were washed three times in 10 mL of TBS-T buffer (5 min per wash). Blots were developed using chemiluminescence reagents with ECL prime (cat# 45-010-090, Amersham). After developing, blots were stripped. They were then blocked again with 5% milk in TBS-T buffer and treated with antitubulin (cat#2144S, Cell Signaling). This procedure was generally used as a loading control.
Proteasome Inhibition
Cauda sperm obtained as described above were pretreated with 10 μM epoxomicin (cat# HY-13821, MedChemExpress) for 30 min in capacitating media. Following pretreatment, degrader 5.4 (10 μM) was added to the tubes containing 400 μL media (300 μL of capacitating media + 100 μL of sperm suspension) and were incubated for an additional 4 h. After incubation, the tubes were centrifuged at 12,000 rpm for 5 min. The sperm pellets were then washed twice with 1000 μL of 1× PBS. Sperm pellets were resuspended in 10 μL of 5× nonreducing Laemmli sample buffer (Laemmli, 1970), heated at 100 °C for 5 min, and centrifuged for 5 min at 12,000 rpm. Supernatants were supplemented with β-mercaptoethanol to a final concentration of 5% (v/v), boiled for an additional 5 min, and then centrifuged again at 12,000 rpm. The extracted proteins were loaded onto a 10-well 8% gel for SDS-PAGE using a Bio-Rad system at a constant current of 40 mA per gel. Following electrophoresis, proteins were transferred to PVDF membranes (cat# IVPH00010, Millipore Sigma, MA, USA). PVDF membranes were blocked for 1 h at rt in 5% milk in TBS-T buffer (100 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.01% Tween-20) before being used for Western blotting. Blots were incubated overnight at 4 °C with TSSK2 antibody (cat# H00023617-M01, Abnova) (1:1000) in 5% milk in TBS-T buffer. The next day, blots were washed three times for 10 min each with 10 mL of TBS-T, then incubated for 1 h with antimouse secondary HRP-conjugated antibody (cat# 715-035-150, Jackson ImmunoResearch Laboratories) at a 1:10,000 dilution. Secondary antibodies were washed three times in 10 mL of TBS-T buffer (5 min per wash). Blots were developed using chemiluminescence reagents with ECL Prime (cat# 45-010-090, Amersham). After developing, blots were stripped, blocked again with 5% milk in TBS-T buffer, and treated with antitubulin (cat# 2144S, Cell Signaling) as a loading control.
Computer-Assisted Sperm Analysis
Spermatozoa were diluted to approximately 1 × 106 concentration and incubated in TYH media with degrader for 4 h. After incubation, the cells were washed, and then the sperm motility was measured using CASA. Briefly, 30 μL of the sperm suspension was loaded onto a prewarmed chamber slide and placed on a microscope stage at 37 °C. Motility was examined using CASA (Hamilton Thorne) with default settings (frames acquired: 90; frame rate: 60 Hz; minimum cell size: 4 pixels; static head size: 0.13–2.43; static head intensity: 0.10–1.52; and static head elongation: 5–100). A minimum of 200 spermatozoa were analyzed across different fields for each treatment in each experiment.
In Vitro Fertilization Assay
Animal care followed OLAW guidelines and was approved by the IACUC at the University of Massachusetts Amherst (#6499). Mouse cumulus–oocyte complexes (COCs) were collected from 8–10-week-old superovulated CD1 females from Charles River Laboratories. To induce superovulation, females were injected with 7.5–10 IU of pregnant mare serum gonadotropin (PMSG). After 48 h, 7.5–10 IU of hCG were injected. COCs were harvested 13.5 h after the hCG injections. COCs were collected in TL-HEPES 13.5 h post-hCG injection and subsequently washed with IVF-TYH media before being allocated to the insemination droplets. Insemination dishes, consisting of four 90 μL droplets of IVF-TYH media covered in mineral oil (Fisher Scientific, cat #0121, Waltham, MA, USA), were prepared and equilibrated overnight at 37 °C in 5% CO2. The COCs were then added to a single droplet in the insemination dish. Following the collection and preparation of sperm as previously described, including treatment with the respective concentration of degrader used in Western blots, approximately 10 μL of sperm suspension was added to each insemination dish. These dishes were returned to the incubator and cultured for 4 h. At 4 h postinsemination, oocytes were removed from the insemination droplets and washed through the remaining clean droplets of IVF-TYH (m-TYH media). Prior to embryo culture, dishes with five 50 μL droplets of Embryomax KSOM (cat# MR-106-D, Sigma-Aldrich) covered in mineral oil were prepared and equilibrated overnight at 37 °C in 5% CO2. Oocytes from each treatment group were transferred into KSOM dishes after being washed through the remaining clean droplets of KSOM media for embryo culture and maintained until 96 h postinsemination for blastocyst evaluation.
Chemistry
tert-Butyl 4-(3-Methoxy-4-nitrophenyl)piperidine-1-carboxylate (3)
Zinc powder (1.409 g, 2.5 equiv) was combined with dry DMA (12 mL) in a nitrogen-flushed round-bottom flask (RBF). A mixture of TMSCl and 1,2-dibromoethane (0.74 mL, 0.4 equiv each) was then added dropwise via syringe over 5 min under vigorous stirring. The reaction mixture was flushed with nitrogen and heated at 80 °C for 20 min. tert-Butyl 4-iodopiperidine-1-carboxylate (5.364 g, 2.0 equiv) was then added, and the reaction was maintained at 80 °C under nitrogen for 1 h. Formation of the organozinc intermediate was monitored by TLC (2:1 hexanes/EtOAc). In a separate nitrogen-purged 2-neck RBF equipped with a reflux condenser, 4-bromo-2-methoxy-1-nitrobenzene (2.000 g, 1.0 equiv), copper(I) iodide (0.164 g, 0.1 equiv), and Pd(dppf)Cl2 (0.315 g, 0.05 equiv) were dissolved in DMA (12 mL). The mixture was flushed with nitrogen. The preformed zinc reagent was transferred to this flask via cannula, and the zinc reaction vessel was rinsed with additional DMA (6 mL) to ensure complete transfer. The combined mixture was heated at 80 °C under nitrogen for 18 h. After cooling to rt, the reaction mixture was transferred to a separatory funnel and diluted with EtOAc (400 mL). The organic layer was washed with saturated ammonium chloride (3 × 30 mL). The combined organic phases were back-extracted with EtOAc (2 × 100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by flash column chromatography (100% hexanes to 40% EtOAc gradient over 12 min). The title compound was obtained in 89% yield (2.565 g) as a pale-yellow oil (Rf = 0.38 in 2:1 hexanes/EtOAc). Chemical Formula: C17H24N2O5; 1H NMR (400 MHz, CDCl3) δ 7.78 (d, J = 8.4, 1H), 6.88 (d, J = 1.3, 1H), 6.83 (dd, J = 8.4, J = 1.3, 1H), 4.24 (bs, 2H), 3.92 (s, 3H), 2.78 (t, J = 12.2, 2H), 2.69 (tt, J = 12.2, J = 3.3, 1H), 1.80 (d, J = 12.4, 2H), 1.58 (qd, J = 13.2, J = 12.4, J = 4.3, 2H), 1.45 (s, 9H).
1-(4-(3-Methoxy-4-nitrophenyl)piperidin-1-yl)ethan-1-one (4)
tert-Butyl 4-(3-methoxy-4-nitrophenyl)piperidine-1-carboxylate (2.565 g, 1.0 equiv) was dissolved in DCM (20 mL, 0.3 M). Trifluoroacetic acid (5.83 mL, 10 equiv) was added dropwise, resulting in a dark red solution. The reaction was stirred at rt for 1 h. The mixture was quenched by the slow addition of 1 M NaOH (50 mL), which caused a color change to light orange, and stirred for 5 min. The solution was then diluted with 1 M NaOH (100 mL) (pH confirmed ≥ 12 using pH paper) and extracted with DCM (3 × 250 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum for 60 min, affording the crude deprotected intermediate as a red solid. The crude intermediate was dissolved in DCM (12 mL, 0.36 M) in a nitrogen-flushed RB flask. Pyridine (0.71 mL, 1.2 equiv) was added, and the flask was sealed under nitrogen. Acetyl chloride (1 M in DCM; 8.77 mL, 1.15 equiv) was added dropwise via syringe over 10 min. The reaction was stirred at rt for 1 h. The mixture was quenched with deionized (DI) water (20 mL) and diluted with EtOAc (500 mL). The organic layer was washed sequentially with DI water (3 × 50 mL) and brine (50 mL), dried over anhydrous sodium sulfate, and concentrated to ∼10% of its original volume. The residue was passed through a silica/Celite plug using EtOAc. The product (1.647 g, 78% yield over 2 steps) was obtained as an orange solid (Rf = 0.85 in 5% MeOH/DCM). Chemical Formula: C14H18N2O4; 1H NMR (400 MHz, CDCl3) δ 7.66 (d, J = 8.4, 1H), 6.82 (s, 1H), 6.74 (d, J = 8.4, 1H), 4.64 (d, J = 13.8, 1H), 3.86 (d, J = 13.8, 1H), 3.82 (s, 3H), 3.08 (t, J = 12.7, 1H), 2.72 (t, J = 12.7, 1H), 2.51 (t, J = 12.7, 1H), 2.02 (s, 3H), 1.79 (m, 2H), 1.51 (m, 2H).
1-(4-(4-Amino-3-methoxyphenyl)piperidin-1-yl)ethan-1-one (5)
1-(4-(3-Methoxy-4-nitrophenyl)piperidin-1-yl)ethan-1-one (1.647 g, 1.0 equiv) was dissolved in methanol (25 mL, 0.24 M) before adding 10% Pd/C (0.315 g, 0.05 equiv) and capping the reaction vessel. The reaction vessel was flushed with hydrogen gas for 5 min to displace air, then stirred under a hydrogen atmosphere (1 atm) at rt for 72 h. After complete reduction, as determined by TLC, the reaction mixture was filtered through a Celite pad to remove Pd/C, and the pad was rinsed with methanol (3 × 10 mL). The combined filtrates were concentrated under reduced pressure. The crude residue was purified by flash column chromatography (gradient: 100% DCM to 5% MeOH over 10 min). Fractions containing the product (Rf = 0.85 in 5% MeOH/DCM) were combined and concentrated to afford the title compound as a red oil (1.345 g, 92% yield). Chemical Formula: C14H20N2O2; 1H NMR (400 MHz, CDCl3) δ 6.60 (m, 3H), 4.73 (m, 1H), 3.83 (m, 6H), 3.10 (td, J = 13.1, J = 2.1, 1H), 2.58 (m, 2H), 2.09 (s, 3H), 1.83 (t, J = 13.2, 2H), 1.55 (qd, J = 26.6, J = 12.4, J = 4.3, 2H).
2-((2,5-Dichloropyrimidin-4-yl)amino)-N-methylbenzenesulfonamide (8)
2-Amino-N-methylbenzenesulfonamide (1.000 g, 1.0 equiv) was dissolved in anhydrous THF (26 mL, 0.20 M) in a nitrogen-flushed RB flask and chilled to 0 °C in an ice bath. Sodium hydride [60% dispersion in mineral oil (0.247 g, 1.15 equiv)] was added in one portion, and the flask was sealed under nitrogen. The mixture was stirred at 0 °C for 5 min to deprotonate the sulfonamide. 2,4,5-Trichloropyrimidine (0.65 mL, 1.05 equiv) was added dropwise via syringe to the chilled reaction mixture. The ice bath was removed, and the reaction was stirred at rt for 2 h. The reaction was quenched by the addition of saturated ammonium chloride (50 mL), and the mixture was extracted with DCM (3 × 100 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude residue was purified by flash column chromatography (gradient: 100% hexanes to 40% EtOAc over 10 min). The product (0.441 g, 25% yield) was obtained as a white solid. Chemical Formula: C11H10Cl2N4O2S; 1H NMR (400 MHz, CDCl3) δ 9.62 (bs, 1H), 8.53 (d, J = 8.4, 1H), 8.27 (s, 1H), 7.95 (dd, J = 7.9, J = 1.4, 1H), 7.67 (td, J = 7.9, J = 1.4, 1H), 7.29 (td, J = 7.7, J = 1.0, 1H), 4.67 (m, 1H), 2.66 (d, J = 5.4, 3H)
2-((2-((4-(1-Acetylpiperidin-4-yl)-2-methoxyphenyl)amino)-5-chloropyrimidin-4-yl)amino)-N-methylbenzenesulfonamide (9i)
1-(4-(4-Amino-3-methoxyphenyl)piperidin-1-yl)ethan-1-one (0.227 g, 1.0 equiv) and 2-((2,5-dichloropyrimidin-4-yl)amino)-N-methylbenzenesulfonamide (0.426 g, 1.4 equiv) were combined in a nitrogen-flushed RB flask. The mixture was suspended in methanol (2 mL) and diluted with isopropyl alcohol (2 mL) to a final concentration of 0.2 M. The flask was sealed under nitrogen, and trifluoroacetic acid (0.35 mL, 5.0 equiv) was added dropwise over several min. The reaction mixture was heated to 100 °C and stirred for 2 h, then cooled to 80 °C and allowed to react for 18 h. After cooling to rt, the solution was quenched with saturated sodium bicarbonate (5 mL) and diluted with additional saturated sodium bicarbonate (25 mL). The aqueous layer was extracted with DCM (3 × 25 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated. The crude residue was purified by flash column chromatography (gradient: 100% EtOAc to 50% MeOH over 8 min). The product (0.436 g, 88% yield) was obtained as a yellow solid (Rf = 0.63 in 4:1 EtOAc/MeOH). Chemical Formula: C25H29ClN6O4S; 1H NMR (400 MHz, CDCl3) δ 9.22 (s, 1H), 8.43 (d, J = 8.3, 1H), 8.05 (d, J = 8.3, 1H), 7.88 (m, 2H), 7.51 (td, J = 7.9, J = 1.2, 1H), 7.46 (s, 1H), 7.19 (t, J = 7.7, 1H), 6.66 (s, 1H), 6.62 (d, J = 8.6, 1H), 6.57 (d, J = 5.1, 1H), 4.68 (d, J = 12.4, 1H), 3.87 (d, J = 13.6, 1H), 3.81 (s, 3H), 3.10 (t, J = 13.0, 1H), 2.64 (td, J = 12.3, J = 3.4, 1H), 2.55 (m, 4H), 2.06 (s, 3H), 1.82 (m, 2H), 1.55 (qd, J = 27.1, J = 13.8, J = 3.8, 2H)
2-((5-Chloro-2-((2-methoxy-4-(piperidin-4-yl)phenyl)amino)pyrimidin-4-yl)amino)-N-methylbenzenesulfonamide (9)
2-((2-((4-(1-Acetylpiperidin-4-yl)-2-methoxyphenyl)amino)-5-chloropyrimidin-4-yl)amino)-N-methylbenzenesulfonamide (9i, 0.399 g, 1.00 equiv) was dissolved in methanol (1 mL) in a sealed reaction vessel. To this solution, 4 M aqueous HCl (3 mL, 0.2 M final) was added dropwise. The mixture was heated to 90 °C and stirred for 18 h, with reaction progress monitored by LC/MS (target mass: 503.16 [M + H]+). After cooling to rt, the reaction mixture was diluted with DCM (200 mL) and transferred to a separatory funnel. The solution was neutralized by slow addition of a saturated sodium bicarbonate solution (50 mL) until pH 8–9 (pH paper). The aqueous layer was extracted with DCM (3 × 100 mL) and EtOAc (1 × 150 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude residue was purified by reversed-phase flash chromatography (C18 column; gradient: 10% water/90% ACN to 100% ACN). Fractions containing the product (LC/MS confirmation) were combined and lyophilized for 72 h. The final compound (0.213 g, 58% yield) was obtained as a white solid. Chemical Formula: C23H27ClN6O3S; 1H NMR (600 MHz, CDCl3) δ 9.11 (bs, 1H), 8.52 (d, J = 8.1, 1H), 8.16 (d, J = 8.1, 1H), 8.15 (s, 1H), 7.99 (dd, J = 8.0, J = 1.6, 1H), 7.62 (td, J = 7.8, J = 1.5, 1H), 7.53 (s, 1H), 7.27 (m, 1H), 6.77 (d, J = 1.6, 1H), 6.74 (dd, J = 8.5, J = 1.5, 1H), 3.89 (s, 3H), 3.28 (d, J = 12.8, 2H), 2.80 (t, J = 11.9, 2H), 2.64 (s, 3H), 2.60 (dt, J = 11.9, J = 3.6, 1H), 1.88 (d, J = 12.8, 2H), 1.76 (qd, J = 25.9, J = 12.6, J = 3.6, 2H); 13C NMR (151 MHz, CDCl3) δ 157.7, 155.6, 155.4, 148.3, 140.6, 136.6, 133.6, 130.0, 127.3, 127.1, 124.4, 123.5, 119.3, 118.6, 108.8, 105.9, 55.8, 46.8, 42.6, 33.9, 29.4.
4-Fluoro-2-(1-methyl-2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (11)
Cesium carbonate (885 mg, 2.72 mmol) was added to a solution of 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (500 mg, 1.81 mmol) in anhydrous N,N-dimethylformamide (5 mL) and stirred for 10 min at rt. Iodomethane (124 μL, 1.99 mmol) was added, and the resulting mixture was stirred overnight at rt. The reaction was quenched with glacial acetic acid (0.2 mL) before the addition of water and extraction with EtOAc. The organic layer was rinsed with brine (5 × 10 mL), and the solvent was removed under reduced pressure. The crude residue was triturated with water, then dried, yielding an off-white, yellow solid (340 mg, 65% yield). 1H NMR (400 MHz, CDCl3) δ 7.80–7.73 (m, 1H), 7.70 (d, J = 7.3 Hz, 1H), 7.42 (td, J = 8.5, 0.9 Hz, 1H), 5.03–4.93 (m, 1H), 3.20 (s, 3H), 2.99 (dd, J = 12.7, 2.7 Hz, 1H), 2.86–2.70 (m, 2H), 2.17–2.07 (m, 1H).
4-((2-(1-Methyl-2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)butanoic Acid (12)
3-(4-Fluoro-1,3-dioxo-2-isoindolinyl)-1-methyl-2,6-piperidinedione (100 mg, 345 μmol), 4-aminobutyric acid (12i, 35.5 mg, 345 μmol), and DIPEA (60 μL, 345 μmol) were dissolved in NMP (1 mL) before heating to 100 °C for 16h. The resulting solution was cooled to rt, then dissolved in EtOAc and washed with 1 N HCl and brine (5 × 10 mL). The organic layer was then dried and condensed under reduced pressure. The crude product was isolated as a yellow-green solid (90 mg, 70% yield). 1H NMR (400 MHz, CDCl3) δ 7.46 (t, J = 7.8 Hz, 1H), 7.05 (d, J = 7.0 Hz, 1H), 6.89 (d, J = 8.5 Hz, 1H), 6.46 (s, 1H), 4.96–4.86 (m, 1H), 3.29 (s, 3H), 3.18 (s, 3H), 2.75 (t, J = 11.0 Hz, 2H), 2.52 (s, 2H), 2.36 (dt, J = 15.5, 7.9 Hz, 2H), 1.93 (t, J = 6.8 Hz, 2H).
3-(2-((2-(1-Methyl-2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy) propanoic Acid (13)
3-(4-Fluoro-1,3-dioxo-2-isoindolinyl)-1-methyl-2,6-piperidinedione (100 mg, 345 μmol), 3-(2-aminoethoxy)propionic acid (13i, 50.5 mg, 379 μmol), and DIPEA (66 μL, 379 μmol) were dissolved in NMP before heating to 100 °C for 16 h. Solution was allowed to cool to rt before being dissolved in EtOAc, then washed with 1 N HCl and brine (x5). The organic layer was then dried and condensed under reduced pressure. Crude product was isolated as a yellowish-green solid (90 mg, 65% yield). 1H NMR (400 MHz, CDCl3) δ 7.49–7.43 (m, 1H), 7.35 (t, J = 8.4 Hz, 0H), 7.07 (d, J = 7.1 Hz, 1H), 6.90 (d, J = 8.5 Hz, 1H), 6.44 (s, 1H), 4.95–4.85 (m, 1H), 3.70 (dq, J = 20.9, 5.7 Hz, 6H), 3.44 (q, J = 4.7 Hz, 2H), 3.18 (s, 3H), 2.80–2.68 (m, 2H), 2.59 (t, J = 6.1 Hz, 2H).
General Procedure for Degrader Synthesis
Compound 9 (1 equiv) and the respective pomalidomide acid (1–1.2 equiv) were combined in a Biotage reaction vessel and dissolved in DCM (0.02 M). HATU (2 equiv) and N-ethyl-N-isopropylpropan-2-amine (3 equiv) were then added before capping the reaction vessel and allowing the reaction to proceed overnight (18–24 h) at rt. Reaction progress was monitored via LC/MS. The DCM was then removed under reduced pressure, and the product was purified using reverse-phase flash chromatography (95:5 water/ACN to 100% ACN). Fractions containing the product, as determined by LC/MS, were combined and lyophilized for 96 h. Yields ranged from 16 to 60%. All products were obtained as yellow solids. HPLC purity for all degrader compounds was >95%.
2-((5-Chloro-2-((4-(1-(4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)butanoyl)piperidin-4-yl)-2-methoxyphenyl)amino)pyrimidin-4-yl)amino)-N-methylbenzenesulfonamide (5.1)
Chemical Formula: C40H42ClN9O8S; mp 177 °C. 1H NMR (600 MHz, CDCl3) δ 9.20 (bs, 1H), 8.49 (d, J = 7.2, 1H), 8.19 (d, J = 3.2, 1H), 8.14 (m, 2H), 7.98 (d, J = 7.9, 1H), 7.61 (t, J = 7.2, 1H), 7.53 (t, J = 7.4, 1H), 7.30 (m, 1H), 7.11 (d, J = 7.1, 1H), 7.04 (d, J = 8.6, 1H), 6.74 (s, 1H), 6.72 (d, J = 8.6, 1H), 6.37 (t, J = 5.5, 1H), 4.93 (dd, J = 12.2, J = 5.2, 1H), 4.84 (d, J = 13.2, 1H), 4.79 (s, 1H), 3.99 (d, J = 13.9, 1H), 3.91 (s, 3H), 3.44 (m, 2H), 3.15 (t, J = 13.9, 1H), 2.90 (d, J = 16.3, 1H), 2.78 (m, 3H), 2.66 (d, J = 5.1, 3H), 2.52 (t, J = 6.3, 2H), 2.14 (m, 1H), 2.06 (m, 2H), 1.93 (d, J = 12.6, 2H), 1.63 (m, 4H); 13C NMR (151 MHz, CDCl3) δ 171.1, 170.3, 169.6, 168.4, 167.7, 155.7, 148.5, 147.1, 136.3, 133.5, 132.5, 130.0, 127.4, 127.0, 124.3, 123.7, 119.6, 118.4, 117.0, 111.7, 110.1, 108.9, 106.0, 55.8, 48.9, 46.2, 42.7, 42.6, 42.3, 42.3, 34.2, 33.1, 31.5, 30.1, 30.0, 29.4, 24.6, 22.9
2-((5-Chloro-2-((4-(1-(3-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)propanoyl)piperidin-4-yl)-2-methoxyphenyl)amino)pyrimidin-4-yl)amino)-N-methylbenzenesulfonamide (5.2)
Chemical Formula: C41H44ClN9O9S; mp 163 °C. 1H NMR (600 MHz, CDCl3) δ 9.14 (bs, 1H), 8.48 (d, J = 8.4, 1H), 8.18 (d, J = 25.9, 1H), 8.11 (s, 1H), 7.95 (d, J = 7.9, 1H), 7.60 (m, 2H), 7.48 (t, J = 7.9, 1H), 7.24 (m, 1H), 7.07 (d, J = 7.0, 1H), 6.88 (d, J = 8.7, 1H), 8.69 (s, 1H), 6.67 (d, J = 8.7, 1H), 6.46 (t, J = 5.4, 1H), 4.84 (dd, J = 12.5, J = 5.5, 1H), 4.78 (m, 2H), 4.01 (d, J = 13.1, 1H), 3.84 (m, 4H), 3.71 (t, J = 5.7, 2H), 3.44 (m, 2H), 3.10 (t, J = 12.3, 1H), 2.77 (m, 2H), 2.63 (m, 6H), 2.05 (m, 2H), 1.86 (m, 2H), 1.60 (m, 6H); 13C NMR (151 MHz, CDCl3) δ 170.9, 169.3, 169.1, 168.3, 167.5, 157.2, 155.5, 148.3, 146.8, 139.6, 136.4, 136.1, 133.4, 132.5, 129.8, 127.3, 127.0, 124.2, 123.6, 119.3, 118.3, 116.7, 111.7, 110.2, 108.8, 105.9, 69.2, 67.4, 55.7, 48.8, 46.4, 42.5, 42.4, 34.1, 33.5, 32.9, 31.3, 29.3, 22.7.
2-((5-Chloro-2-((4-(1-(3-((2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)methoxy)propanoyl)piperidin-4-yl)-2-methoxyphenyl)amino)pyrimidin-4-yl)amino)-N-methylbenzenesulfonamide (5.3)
Chemical Formula: C43H48ClN9O10S; mp 157 °C. 1H NMR (600 MHz, CDCl3) δ 9.19 (s, 1H), 8.46 (d, J = 7.8, 1H), 8.26 (d, J = 17.4, 1H), 8.10 (s, 2H), 7.96 (dd, J = 8.07, J = 1.19, 1H), 7.72 (bs, 1H), 7.59 (t, J = 7.6, 1H), 7.47 (m, 1H), 7.27 (m, 1H), 7.08 (dd, J = 7.20, J = 3.50, 1H), 6.89 (dd, J = 8.60, J = 3.23, 1H), 6.70 (m, 2H), 6.48 (m, 1H), 4.88 (m, 1H), 4.80 (d, J = 12.9, 1H), 4.73 (d, J = 13.5, 1H), 4.26 (q, J = 35.1, J = 13.4, 1H), 4.01 (d, J = 13.8, 1H), 3.85 (m, 4H), 3.71 (m, 7H), 3.45 (m, 2H), 3.10 (q, J = 26.1, J = 12.2, 1H), 2.85 (m, 1H), 2.70 (m, 9H), 2.10 (m, 1H), 1.88 (m, 2H), 1.63 (m, 2H); 13C NMR (151 MHz, CDCl3) δ 171.0, 170.9, 169.3, 169.2, 168.4, 168.3, 167.5, 167.5, 167.5, 155.6, 148.5, 146.8, 136.2, 136.0, 133.4, 132.4, 129.8, 127.4, 124.2, 123.6, 119.5, 118.2, 116.7, 111.7, 110.2, 108.7, 105.9, 70.7, 70.5, 69.5, 67.6, 55.7, 48.8, 42.6, 42.5, 42.3, 34.1, 33.6, 32.8, 31.3, 22.7.
2-((5-Chloro-2-((4-(1-(3-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxy)propanoyl)piperidin-4-yl)-2-methoxyphenyl)amino)pyrimidin-4-yl)amino)-N-methylbenzenesulfonamide (5.4)
Chemical Formula: C45H52ClN9O11S; mp 152 °C. 1H NMR (600 MHz, CDCl3) δ 9.18 (s, 1H), 8.59 (d, J = 16.7, 1H), 8.49 (d, J = 8.4, 1H), 8.12 (m, 2H), 7.97 (dd, J = 8.0, J = 1.4, 1H), 7.62 (m, 2H), 7.48 (t, J = 7.9, 1H) 7.22 (t, J = 7.9, 1H), 7.05 (d, J = 7.0, 1H), 6.86 (d, J = 8.7, 1H) 6.66 (m, 2H), 6.44 (t, J = 5.1, 1H), 5.06 (d, J = 4.6, 1H), 4.85 (dd, J = 12.6, J = 5.3, 1H), 4.69 (d, J = 12.1, 1H), 4.23 (q, J = 17.1, J = 13.1, 2H), 3.98 (d, J = 12.9, 1H), 3.84 (s, 3H), 3.68 (m, 7H), 3.42 (d, J = 4.9, 2H), 3.06 (t, J = 12.9, 1H), 2.71 (m, 9H), 2.14 (s, 2H), 2.06 (m, 2H), 1.85 (m, 3H), 1.60 (m, 3H); 13C NMR (151 MHz, CDCl3) δ 171.1, 169.2, 168.4, 167.5, 157.3, 155.4, 154.8, 148.2, 146.7, 139.4, 136.4, 136.0, 133.3, 132.4, 132.1, 129.8, 128.8, 127.2, 127.0, 124.0, 123.3, 119.3, 118.2, 116.7, 111.6, 110.2, 108.6, 105.9, 70.6, 70.5, 70.5, 69.4, 55.6, 48.8, 45.6, 42.6, 42.4, 42.3, 34.1, 32.8, 31.3, 30.8, 22.7.
(2-(4-(4-((5-Chloro-4-((2-(N-methylsulfamoyl)phenyl)amino)pyrimidin-2-yl)amino)-3-methoxyphenyl)piperidin-1-yl)-N-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)acetamide) (5.5)
Chemical Formula: C38H38ClN9O8S; mp 169 °C. 1H NMR (600 MHz, DMSO-d6) δ 11.23 (s, 1H), 11.14 (s, 1H), 9.37 (s, 1H), 8.83 (d, J = 8.17, 1H), 8.51 (d, J = 8.35, 1H), 8.28 (s, 1H), 8.22 (s, 1H), 7.87 (t, J = 7.74, 1H), 7.78 (m, 2H), 7.61 (d, J = 7.30, 1H), 7.50 (t, J = 7.85, 1H), 7.23 (t, J = 7.55, 1H), 6.96 (s, 1H), 6.86 (d, J = 8.00), 5.13 (dd, J = 13.3, J = 5.41, 1H), 3.79 (s, 3H), 3.24 (m, 1H), 3.02 (t, J = 11.9, 2H), 2.83 (m, 1H), 2.58 (m, 3H), 2.41 (m, 5H), 2.02 (m, 4H), 1.81 (d, J = 12.1, 2H); 13C NMR (151 MHz, DMSO-d6) δ 173.1, 171.0, 170.2, 168.6, 167.2, 158.8, 155.5, 155.3, 151.7, 143.3, 136.9, 136.8, 133.5, 131.8, 129.3, 126.9, 126.4, 124.5, 123.2, 118.4, 118.2, 116.2, 110.5, 62.0, 55.8, 54.4, 49.4, 41.4, 40.5, 33.6, 33.4, 31.3, 29.0, 22.4.
(2-((5-Chloro-2-((4-(1-(5-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)pentanoyl)piperidin-4-yl)-2-methoxyphenyl)amino)pyrimidin-4-yl)amino)-N-methylbenzenesulfonamide) (5.9)
Chemical Formula: C41H44ClN9O8S; mp 200 °C. 1H NMR (600 MHz, CDCl3) δ 9.28 (bs, 1H), 8.44 (d, J = 8.25, 1H), 8.16 (d, J = 13.1, 1H), 8.08 (s, 2H), 7.96 (dd, J = 7.92, J = 1.63, 1H), 7.57 (t, J = 7.39, 1H), 7.49 (t, J = 8.13, 1H), 7.28 (m, 1H), 7.08 (d, J = 7.83, 1H), 6.90 (d, J = 8.58, 1H), 6.72 (s, 1H), 6.69 (d, J = 8.77, 1H), 6.28 (t, J = 4.66, 1H), 4.89 (m, 1H), 4.81 (d, J = 13.2, 1H), 3.98 (d, J = 13.5, 1H), 3.89 (s, 3H), 3.33 (m, 2H), 3.13 (t, J = 12.7, 1H), 2.86 (d, J = 15.1, 1H), 2.75 (m, 3H), 2.64 (d, J = 6.63, 3H), 2.45 (t, J = 6.63, 2H), 2.10 (m, 1H), 1.90 (t, J = 13.6, 2H), 1.78 (m, 4H), 1.60 (m, 5H); 13C NMR (151 MHz, CDCl3) δ 171.0, 170.7, 169.5, 168.3, 167.6, 155.7, 148.5, 146.9, 136.2, 133.4, 132.5, 129.9, 127.4, 124.2, 123.7, 119.6, 118.4, 116.7, 111.5, 109.9, 108.8, 105.9, 55.8, 48.9, 46.3, 42.7, 42.5, 42.4, 34.3, 32.9, 32.9, 31.4, 29.3, 29.1, 29.1, 22.8, 22.6, 22.6.
(2-((5-Chloro-2-((4-(1-(6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)hexanoyl)piperidin-4-yl)-2-methoxyphenyl)amino)pyrimidin-4-yl)amino)-N-methylbenzenesulfonamide) (5.10)
Chemical Formula: C42H46ClN9O8S; mp 162 °C. 1H NMR (600 MHz, CDCl3) δ 9.22 (bs, 1H), 8.49 (d, J = 8.64, 1H), 8.14 (m, 3H), 7.98 (d, J = 8.56, 1H), 7.60 (t, J = 8.56, 1H), 7.51 (t, J = 7.52, 1H), 7.29 (m, 1H), 7.11 (d, J = 6.22, 1H), 6.91 (d, J = 8.41, 1H), 6.73 (m, 2H), 6.23 (t, J = 5.30, 1H), 4.92 (dd, J = 12.3, J = 5.60, 1H), 4.80 (dd, J = 33.8, J = 13.9, 2H), 4.00 (d, J = 12.3, 1H), 3.90 (s, 3H), 3.32 (q, J = 14.2, J = 6.47, 2H), 3.15 (t, J = 12.9, 1H), 2.90 (d, J = 16.5, 1H), 2.77 (m, 4H), 2.66 (m, 4H), 2.42 (t, J = 7.58, 2H), 2.14 (m, 1H), 1.92 (t, J = 13.7, 2H), 1.75 (m, 4H), 1.60 (m, 8H); 13C NMR (151 MHz, CDCl3) δ 171.1, 170.9, 169.5, 168.4, 167.6, 155.7, 148.5, 147.0, 136.2, 133.44, 132.5, 129.9, 127.5, 124.3, 123.7, 119.6, 118.4, 116.7, 111.5, 109.9, 108.8, 105.9, 55.8, 48.9, 46.3, 42.7, 42.5, 42.4, 34.3, 34.3, 33.2, 33.0, 33.0, 31.4, 29.3, 29.2, 26.8, 25.0, 22.8.
(2-((5-Chloro-2-((4-(1-(7-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)heptanoyl)piperidin-4-yl)-2-methoxyphenyl)amino)pyrimidin-4-yl)amino)-N-methylbenzenesulfonamide) (5.11)
Chemical Formula: C43H48ClN9O8S; mp 199 °C. 1H NMR (600 MHz, CDCl3) δ 9.28 (bs, 1H), 8.48 (d, J = 9.04, 1H), 8.14 (m, 3H), 7.99 (d, J = 7.71, 1H), 7.60 (t, J = 7.71, 1H), 7.51 (t, J = 9.04, 1H), 7.31 (m, 1H), 7.11 (d, J = 7.06, 1H), 6.91 (d, J = 8.78, 1H), 6.75 (s, 1H), 6.72 (d, J = 8.01, 1H), 6.25 (t, J = 4.77, 1H), 4.92 (dd, J = 12.1, J = 4.46, 1H), 4.84 (d, J = 12.3, 1H), 4.77 (s, 1H), 4.01 (d, J = 13.4, 1H), 3.91 (s, 3H), 3.30 (q, J = 14.7, J = 6.56, 2H), 3.16 (t, J = 11.2, 1H), 2.90 (d, J = 16.2, 1H), 2.78 (m, 3H), 2.66 (m, 4H), 2.41 (t, J = 6.56, 2H), 2.14 (bs, 1H), 1.93 (t, J = 16.2, 2H), 1.72 (m, 4H), 1.49 (m, 6H)
2-((5-Chloro-2-((2-methoxy-4-(1-(3-((2-(1-methyl-2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)propanoyl)piperidin-4-yl)phenyl)amino)pyrimidin-4-yl)amino)-N-methylbenzenesulfonamide (2.8)
Chemical Formula: C41H44ClN9O8S; mp 152 °C. 1H NMR (400 MHz, CDCl3) δ 9.11 (s, 1H), 8.49 (d, J = 8.3 Hz, 1H), 8.16 (d, J = 8.1 Hz, 1H), 8.11 (s, 1H), 7.96 (dd, J = 8.0, 1.6 Hz, 1H), 7.63–7.56 (m, 1H), 7.54–7.47 (m, 2H), 7.08 (d, J = 7.1 Hz, 1H), 7.01 (d, J = 8.5 Hz, 1H), 6.70 (d, J = 9.2 Hz, 2H), 6.34 (t, J = 6.0 Hz, 1H), 4.93–4.87 (m, 1H), 4.80 (dd, J = 13.8, 8.8 Hz, 2H), 3.97 (d, J = 13.6 Hz, 1H), 3.89 (s, 3H), 3.40 (dt, J = 9.7, 6.6 Hz, 2H), 3.19 (s, 3H), 3.11 (d, J = 12.8 Hz, 1H), 2.99–2.92 (m, 1H), 2.76–2.71 (m, 2H), 2.64 (d, J = 5.3 Hz, 4H), 2.49 (t, J = 6.9 Hz, 2H), 2.09–1.99 (m, 3H), 1.91 (d, J = 13.3 Hz, 2H), 1.70–1.51 (m, 3H), 0.86 (q, J = 8.1 Hz, 1H). 13C NMR (101 MHz, CDCl3) δ 171.3, 170.3, 169.1, 167.8, 160.3, 157.5, 155.5, 155.1, 155.1, 148.2, 147.0, 139.4, 136.5, 136.2, 133.5, 132.5, 129.9, 127.3, 127.3, 124.2, 123.4, 119.2, 118.4, 116.9, 111.6, 110.1, 108.7, 55.8, 49.6, 46.2, 42.7, 42.5, 42.2, 34.2, 33.0, 31.9, 30.0, 29.3, 27.3, 24.6, 22.1.
2-((5-Chloro-2-((2-methoxy-4-(1-(3-(2-((2-(1-methyl-2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)propanoyl)piperidin-4-yl)phenyl)amino)pyrimidin-4-yl)amino)-N-methylbenzenesulfonamide (2.10)
Chemical Formula: C42H46ClN9O9S; mp 150 °C. 1H NMR (400 MHz, CDCl3) δ 9.22 (s, 1H), 8.46 (d, J = 8.3 Hz, 1H), 8.09 (d, J = 9.1 Hz, 2H), 7.96 (dd, J = 8.1, 1.6 Hz, 1H), 7.78 (s, 1H), 7.61–7.55 (m, 1H), 7.48 (t, J = 7.8 Hz, 1H), 7.28 (d, J = 8.9 Hz, 1H), 7.09 (d, J = 7.1 Hz, 1H), 6.90 (dd, J = 8.6, 1.8 Hz, 1H), 6.72–6.66 (m, 2H), 6.47 (t, J = 5.6 Hz, 1H), 4.91–4.75 (m, 3H), 4.03 (d, J = 13.3 Hz, 1H), 3.87 (d, J = 8.2 Hz, 5H), 3.72 (t, J = 5.3 Hz, 2H), 3.46 (q, J = 5.4 Hz, 2H), 3.18–3.06 (m, 4H), 2.92 (dd, J = 13.0, 2.8 Hz, 1H), 2.73–2.67 (m, 6H), 2.64 (d, J = 5.3 Hz, 3H), 1.88 (d, J = 13.8 Hz, 2H), 1.61 (pd, J = 13.1, 4.2 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 171.2, 171.2, 169.5, 169.1, 169.0, 167.8, 155.7, 148.5, 146.8, 139.9, 136.2, 136.1, 133.4, 132.5, 129.9, 127.5, 124.3, 123.7, 119.5, 118.3, 116.7, 111.7, 110.4, 108.8, 106.0, 69.3, 67.5, 60.4, 55.8, 49.6, 46.4, 42.6, 42.4, 34.2, 33.6, 32.9, 31.9, 29.3, 27.2, 22.1, 21.1, 14.2.
Supplementary Material
Acknowledgments
This work was supported by an NIH predoctoral fellowship to J.A.H. (NIH training grant 5T32GM132029-5), Male Contraceptive Initiative grant # 223-102, Male Contraceptive Initiative Predoctoral Fellowship to J.A.H.
Glossary
Abbreviations Used
- ACN
acetonitrile
- BSA
bovine serum albumin
- CASA
computer-assisted sperm analysis
- CEB
cytoplasmic extraction buffer
- CHO
Chinese hamster ovary
- COCs
mouse cumulus–oocyte complexes
- CRBN
cereblon
- DCM
dichloromethane;
- DC50
concentration at half-maximum degradation
- DI
deionized
- D max
maximum observed degradation
- DMA
dimethylacetamide
- DTT
dithiothreitol
- EtOAc
ethyl acetate
- FaSSIF
fasted state simulated intestinal fluid
- FBS
fetal bovine serum
- HATU
hexafluorophosphate azabenzotriazole tetramethyl uronium
- HLM
human liver microsome
- HPLC
high-performance liquid chromatography
- HRMS
high resolution mass spectroscopy
- IC50
half maximal inhibitory concentration
- IVF
in vitro fertilization
- MEB
membrane extraction buffer
- MLM
mouse liver microsome
- MRM
multiple reaction monitoring
- mTYH
modified Toyoda–Yokoyama–Hosi
- ND
no observed degradation
- NEB
nuclear extraction buffer
- NICHD
Eunice Kennedy Shriver National Institute of Child Health and Human Development
- NIH
national institute of health
- NMP
N-methyl-2 pyrrolidone
- PBS
phosphate buffered saline
- PEB
pellet extraction buffer
- PMSG
pregnant mare serum gonadotropin
- POI
protein of interest
- PEG
polyethylene glycol
- RBF
round-bottom flask
- SAR
structure activity relationship
- STS
Staurosporine
- TEER
transepithelial electrical resistance
- THF
tetrahydrofuran
- TLC
thin layer chromatography
- TMSCl
trimethylsilyl chloride
- TSSK1
testis specific serine/threonine kinase 1
- TSSK2
testis-specific serine/threonine kinase 2
- UDPGA
uridine 5′ diphosphoglucuronic acid
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.5c03689.
The authors declare no competing financial interest.
References
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