Regulated Induced Proximity Targeting Chimeras (RIPTACs): a Heterobifunctional Small Molecule Strategy for Cancer Selective Therapies

Summary:

We describe a protein proximity inducing therapeutic modality called Regulated Induced Proximity Targeting Chimeras or RIPTACs: heterobifunctional small molecules that elicit a stable ternary complex between a target protein selectively expressed in tumor cells and a pan-expressed protein essential for cell survival. The resulting co-operative protein:protein interaction abrogates the function of the essential protein, thus leading to death selectively in cells expressing the target protein. This approach leverages differentially expressed intracellular proteins as novel cancer targets, with the advantage of not requiring the target to be a disease driver. In this chemical biology study, we design RIPTACs that incorporate a ligand against a model target protein connected via a linker to effector ligands such as JQ1 (BRD4), BI2536 (PLK1), or CDK inhibitors such as TMX3013 or dinaciclib. RIPTACs accumulate selectively in cells expressing the HaloTag-FKBP target, form co-operative intracellular ternary complexes, and induce an anti-proliferative response in target-expressing cells.

Graphical Abstract

eTOC blurb

Raina et al. present a proof-of-concept approach to modulate PPI with heterobifunctional RIPTACs that induce an enduring interaction between target proteins and effector protein. The RIPTACs arrest cancer cell growth by selectively inhibiting a pan-essential effector protein in ternary complex with target proteins expressed at elevated levels in cancer cells.

Introduction:

A classical targeted therapy is typically designed to inhibit a cancer driver protein (e.g., AR, mEGFR, KRAS^G12C) or a pathway on which the cancer cell is selectively dependent (e.g., MEK, PARP). While it has been estimated that there are roughly 300 cancer driver proteins, only about 50 have been successfully targeted with an FDA-approved drug^1,2. Since the advent of the genomics revolution, many targeted therapies have advanced into Phase I clinical trials in oncology. However, the overall likelihood of success for cancer drugs entering the clinic remains low^3,4. Targeted therapies fail for two principal and often related reasons: lack of efficacy and toxicity⁵. For a variety of reasons, the drug discovery process has erroneously identified proteins essential for cell survival (such as mitotic kinases like PLK1 and AURKA/B, and epigenetic regulators like BRD4 and the HDACs) as selective anti-cancer targets⁶. As a result, many drugs against these proteins that have been tested in patients have failed due to the lack of a Therapeutic Index (TI), while a few have received FDA approval albeit with a narrow TI and on-target side effects.

Alternatively, novel methods to selectively trigger cancer cell death have emerged in the past few decades. Antibody-drug conjugates (ADCs), T-cell engagers, and radiopharmaceuticals all deploy a multifunctional agent to engage a cancer cell and selectively cause its death. In the case of an ADC, a toxic payload is delivered to the cancer cell. T-cell recruiters or CAR-T cells activate T cells to secrete perforin and granzyme in the vicinity of a cancer cell, thus killing it. Radiopharmaceuticals also selectively bind cancer cells and their ability to trigger cell death relies on emitting radiation over a short distance. The target protein/antigen for these approaches does not need to be a driver of the disease, thus greatly expanding the number of cancer targets in oncology. For example, lineage-specific antigens, such as CD19, have been successfully employed as targets for ADCs and CAR-T therapy in hematological malignancies^7,8.

Unfortunately, the above modalities rely on engaging cancer cells extracellularly, and as a result are unable to take advantage of most cancer-selective proteins. In addition, their utility is limited by their complexity and cost. More recently, heterobifunctional protein degrading small molecules like Proteolysis Targeting Chimera (PROTACs) have paved the way for “beyond rule of five (bRo5) drugs” as novel therapeutics^9–11. Initial concerns about the potential drawbacks of the physicochemical properties of such heterobifunctional molecules have been allayed by recent positive clinical trial data with oral PROTACs reported by companies like Arvinas, Kymera Therapeutics, and Nurix Therapeutics. PROTACs rely on the induction of novel protein-protein interactions (PPIs) in a ternary complex comprising the drug, its target protein and an E3 ubiquitin ligase. The PPIs impart co-operativity to this protein complex, resulting in high binding affinity, and selective degradation of the target protein^12–14.

We hypothesized that heterobifunctional small molecules could be adapted to selectively induce death in cells that express a distinctive intracellular Target Protein (TP) via induction of proximity between the TP and a pan-essential Effector Protein (EP) required for cell survival. Such a ternary complex would abrogate EP function selectively in TP-expressing cells, causing cell death. The heterobifunctional molecule capable of this would be a Regulated Induced Proximity Targeting Chimera, or RIPTAC. Herein we describe how positive co-operativity, a well-known feature of ternary complexes formed by biologically active heterobifunctional small molecules, allows for selective accumulation inside the cell via TP binding and/or concurrent engagement of the pan-essential protein. This mechanism is leveraged into a treatment modality that delivers a therapeutic window relative to cells lacking the TP. We designed a RIPTAC proof-of-concept study using chemical biology systems with well-characterized target proteins and ligands. Employing a HaloTag-FKBP12^F36V fusion protein as a TP, we demonstrate ternary complex formation with various RIPTACs and their cognate EPs, ultimately resulting in the selective killing of cells expressing the HaloTag-FKBP12^F36V fusion providing proof-of-concept for the RIPTAC modality.

Results:

The HaloTag-FKBP model system:

To generate a model system, we engineered a HEK293-derived cell line with overexpression of FLAG-tagged HaloTag7-FKBP^F36V (hereafter HaloTag-FKBP) with a C-terminal P2A-EGFP sequence as our target protein introduced using lentivirus – “293_HFL” (HaloTag-FKBP-Lentivirus) cells. This allowed access to both covalent (via HaloTag) and non-covalent (via FKBP) means of recruiting the same TP. A cell line overexpressing only EGFP was generated as a control – “293_GFPL” (GFP-Lentivirus) cells. GFP expression-based sorting by flow cytometry allowed selection of a high expressing clone, in which HaloTag-FKBP expression was confirmed by western blotting (Figure S1A). Confirmation that the fusion protein expression was not restricted to any one compartment of the cell was determined with confocal microscopy using the HaloTag binding TAMRA-CA fluorescent reagent (Figure S1A). To characterize the cell line more thoroughly, the concentration of the HaloTag-FKBP protein inside the cell was determined. Whole cell lysates from a fixed number of 293_HFL cells (e.g. from 100,000 cells = “100k”) were probed using a HaloTag antibody alongside a purified recombinant HaloTag-GST protein standard curve (Figure S1B). Assuming a HEK293 cell radius of 6.5 μm, this yielded an intracellular target protein concentration of 1–3 μM. Suitable essential effector proteins were chosen based on published CRISPR dropout screening data and the potency, selectivity, and extent of validation of the chemical matter available to bind them. The final Effector Ligands (ELs) chosen were JQ1 (BET inhibitor), BI2536 (PLK1 inhibitor), TMX3013 (multi-CDK inhibitor), and dinaciclib (multi-CDK inhibitor). The chemical structures of these molecules as well as the FKBP binding ligand are shown in Figure S1C.

RIPTACs show enhanced anti-proliferative activity in HaloTag-FKBP expressing cells:

We synthesized RIPTACs with the above effector ligands at one end, and either a HaloTag-binding chloroalkane (CA), or an FKBP-binding ligand (FKBP) at the other (Figure 1A). Dinaciclib was an exception: we synthesized only chloroalkane-containing RIPTACs for this effector ligand, while choosing to test two distinct linker attachment points on this CDK inhibitor. Binding of RIPTACs to their respective EPs was confirmed in biochemical assays (Table S1). Next, we treated 293_HFL and 293_GFPL cells with these RIPTACs in CellTiter-Glo viability assays in two different formats. First, we tested these compounds in a 7-day assay with continuous RIPTAC exposure. A clear pattern of enhanced anti-proliferative RIPTAC activity in 293_HFL cells was observed for every RIPTAC, shown in Figure 1B (covalent) & Figure 1C (non-covalent). The GI₅₀ values along with the fold-selectivity over the non-target expressing 293_GFPL cell line are summarized in Table 1. Alternatively, RIPTACs were washed out from cells following a 4h treatment and their viability determined after 7 days. Under washout conditions, the potency in 293_HFL cells for RIPTACs incorporating BI-2536 or dinaciclib showed even further increase over potency in 293_GFPL cells (Figure S2A) compared to the differential observed with continuous treatment (Table 1). Neither the ELs themselves nor the FKBP target ligand showed a significant difference in potency between the two cell lines (Figure S2B).

Figure 1. RIPTAC differential biology in 293_HFL model system.

(A) Structures of HaloTag-FKBP fusion protein targeting RIPTACs with JQ1, TMX3013, BI-2536, or dinaciclib as effector ligands. (B) Differential anti-proliferative activity of select covalent RIPTACs in target protein expressing 293_HFL cells in a 7-day Cell TiterGlo assay. (C) Differential anti-proliferative activity of select non-covalent RIPTACs in a 7-day Cell TiterGlo assay. All data representative of 3 independent experiments (N=3)

Table 1. GI₅₀ values and fold-shift of potency in 293_HFL cells over 293_GFPL cells.

Data was obtained under continuous 7-day treatment with the optimal linker length from each RIPTAC series. All data representative of 3 independent experiments (n=3)

effector ligand	JQ1	JQ1	TMX-3013	TMX-3013	BI-2536	BI-2536	dinaciclib
target protein	HaloTag	FKBP	HaloTag	FKBP	HaloTag	FKBP	HaloTag
RIPTAC	JQ1-4PEG-CA	JQ1-4PEG-FKBP	TMX-6PEG-CA	TMX-6PEG-FKBP	BI-2PEG-CA	BI-2PEG-FKBP	DNE-11PEG-CA
293_GFPL GI50 (μM)	> 10	5.442	2.224	> 10	0.377	0.868	0.347
293_HFL GI50 (μM)	0.191	0.506	0.050	0.011	0.002	0.015	0.013
fold-shift	> 50	11	44	> 900	189	58	27

To demonstrate how target protein expression level affects RIPTAC activity, we engineered a variant of the 293_HFL cells wherein expression level of the target protein was doxycycline-inducible, and therefore adjustable. Additionally, the ability to titrate down target protein levels permits testing of whether the increased sensitivity to RIPTACs in 293_HFL cells may have been a consequence of a greater steady-state cellular stress (e.g. from accumulation of misfolded aggregates of FLAG-tagged HaloTag7-FKBP^F36V) making them generally more susceptible to small molecule anti-proliferatives. In the doxycycline-inducible 293_HFL cells, the activity of several RIPTACs was evaluated in CellTiter-Glo viability assay. Unsurprisingly, each of the tested RIPTACs more potently reduced cell viability as the concentration of co-administered doxycycline increased (Figure 2A). Notably, the effects seen with JQ-1 containing RIPTACs, while reproducible, were modest, and required the highest levels of doxycycline induction. This suggests that not all RIPTACs should be expected to be equally robust across cell lines. Given the heterobifunctional nature of the RIPTACs, their ability to recruit the target protein to the effector protein and thereby inhibit the latter is in part dependent on the abundance of the former. Thus, higher concentrations of doxycycline that result in higher levels of target protein will increase RIPTAC activity, as evidenced by their increased potency in cell viability assays. Note that a similar improvement in antiproliferative activity with increased target protein expression was not observed (Figure 2B) for the unmodified inhibitors (BI-2536, JQ1, TMX-3013 and dinaciclib). That finding is not consistent with the overexpressed FLAG-tagged HaloTag-FKBP simply making the cells pan-hypersensitive to test molecules. That the maximum levels of target protein expression did not form cellular aggregates as determined by immunofluorescence in 293_HFL cells (Figure S1A and Figure 5), reinforces their relatively benign nature in absence of RIPTAC. In most cases, our heterobifunctiional RIPTACs were not able to surpass the potency of the ELs themselves in this system, very likely owing to their larger size and therefore more limited permeability. Curiously, the TMX 3013-based RIPTACs (Figure 2A) are an exception to this trend and are actually more potent than the unconjugated inhibitor itself (Figure 2B).

Figure 2. RIPTAC activity depends on target protein abundance.

(A) Activity of RIPTACs on cell viability as expression level of HaloTag-FKBP is titrated using doxycycline. (B) Activity of effector protein inhibitors on cell viability as expression level of HaloTag-FKBP is titrated using doxycycline.

Figure 5. Target protein expression and localization in absence and presence of RIPTAC.

293_HFL cells treated with 1 μM JQ1-6PEG-CA (top) or vehicle (bottom) were immunostained against BRD4 (panels a and b) and the FLAG epitope on HaloTag-FKBP (panels c and d). Merged BRD4 and FLAG channels are shown in panels e and f. Nuclei were counterstained with Hoescht 33342 (panels g and h).

Next, we confirmed that the differential anti-proliferative activity seen with RIPTACs in presence of the target protein was not an artifact of the cell line background. We generated a HT29 derived cell line stably expressing the HaloTag-FKBP protein and tested the RIPTAC “JQ1-4PEG-CA” (JQ1 linked to chloroalkane by 4 PEG linker) in a three-day CellTiter-Glo assay. We observed a clear, albeit modest, potency increase in the TP-expressing HT29_HF cell line (Figure S2C). The difference in RIPTAC potency boost between the HT29 cells (13-fold) and 293 cells (≥ 50 fold) could be based in differences in stoichemetric abundance of the TP or the JQ1-4PEG-CA effector protein BRD4 as differences in either will likely impact RIPTAC-induced complex formation. We confirmed that the observed increased RIPTAC activity in TP-expressing lines were not an artifact of the FKBP immunophilin domain of the fusion protein. We generated 293 cells expressing either Akt-HaloTag (293_AktH), or HaloTag-BTK (293_HBTKL), and in both instances observed RIPTAC potency increases in CellTiter-Glo assays, although not necessarily with all essential protein ligands tested (Figure 2D).

Mechanisms underlying RIPTAC differential biology:

We next sought to determine at a molecular level whether our hypothesis regarding target dependent RIPTAC accumulation could explain the observed potency increases in RIPTAC activity in TP-containing cell lines. We first tested whether the FKBP ligand HLDA-001 showed any target-dependent accumulation in cells. We treated 293_GFPL and 293_HFL cells with 10 nM or 100 nM HLDA-001 for 4h, trypsinized the cells, and analyzed the cell pellet by LCMS (see Methods). We observed >1000x enrichment of the ligand in 293_HFL cells compared to the control cell line lacking the HaloTag-FKBP fusion protein (Figure 3A). Next, we tested select non-covalent RIPTACs -- JQ1-4PEG-FKBP, TMX-6PEG-FKBP and BI-2PEG-FKBP-- using the same method and observed significant accumulation of each in the 293_HFL cell line, although the values were smaller than those observed for HLDA-001 (Figure 3B). RIPTAC accumulation was shown to be target-dependent as evidenced by the loss of TMX-6PEG-FKBP accumulation in 293_HFL cells upon their pre-treatment with an excess of the FKBP ligand HLDA-001 (Figure S3A).

Figure 3. Mechanisms underlying RIPTAC differential biology.

(A) The FKBP ligand HLDA-001 accumulates selectively in 293_HFL cells. (B) Non-covalent RIPTACs also accumulate selectively in 293_HFL cells. (C & D) RIPTAC activity in 7-day viability assays can be competed off by pre-treatment with 10 μM HLDA-001 in the case of non-covalent RIPTACs or 300 nM TAMRA-CA in the case of covalent RIPTACs. (E) RIPTAC DNE-11PEG-CA is 19-fold more potent a CDK9 inhibitor in 293_HFL cells than in control 293_GFPL cells. (F) RIPTAC BI-2PEG-FKBP is a 18-fold more potent PLK1 inhibitor in 293_HFL cells than in control 293_GFPL cells. (G) RIPTAC JQ1-4PEG-CA is a 60-fold more potent BRD4 inhibitor in 293_HFL cells than in control 293_GFPL cells.

Similarly, the differential biology observed in viability assays was shown to be target-dependent as shown by abrogation of increased RIPTAC potency against 293_HFL viability upon competition with HLDA-001 (Figure 3C) or TAMRA-CA (Figure 3D). This data also confirmed that the observed differential biology was not a result of the combined cytotoxicity of the target and effector ligands. The effector ligands by themselves showed no potency changes under competition conditions (Figure S3B). As an additional control, we confirmed the role of the TP in the observed viability effects by treating with inactive RIPTAC analogs, JQ1-4PEG-deschloro and TMX-6PEG-deschloro that cannot bind HaloTag (Figure S3C and Figure S4), as well as the role of the EP with a JQ1 epimer-based RIPTAC, epi-JQ1-6PEG-CA, that cannot bind BRD2/3/4 (Figure S3D and Figure S4). As expected, none of these controls showed an enhanced antiproliferative potency in the 293_HFL cell line relative to the 293_GFPL line.

We next asked whether the increased potency to reduce cell viability seen with RIPTACs is recapitulated by a comparable shift in the cellular inactivation of their respective EPs. We treated 293_GFPL and 293_HFL cells for 4h with increasing concentrations of either the dinaciclib-based RIPTAC, DNE-11PEG-CA, or dinaciclib itself and measured the levels of phosphorylation at Ser2 of RPB1-CTD. The putative kinase for this site is CDK9, which is a known cellular target of dinaciclib. The IC₅₀ of DNE-11PEG-CA for reducing RPB1 phosphorylation in 293_HFL cells was 19 nM compared to 365 nM in 293_GFPL cells, a potency shift of 19.2-fold in favor of the cells expressing the target protein (Figure 3E). These values are highly congruous with the GI₅₀ values of 13 nM and 347 nM observed for the same RIPTAC in viability assays in the two cell lines. In comparison, dinaciclib itself showed no significant difference in IC₅₀ between the cell lines (Figure S3E).

Similarly, both 293_HFL cells and 293_GFPL cells were treated with the PLK1-inhibiting RIPTAC, BI-2PEG-FKBP, or BI-2536 itself to measure how potently they inhibit PLK1. Cyclin B1 is the downstream effector of PLK1, which phosphorylates the former on Ser133 to promote M phase entry. The IC50 of BI-2PEG-FKBP to inhibit Ser133 phosphorylation of cyclin B1 was 14 nM in 293_HFL cells compared to 257 nM in 293_GFPL cells, a potency increase of over 18-fold in favor of the target protein-expressing line (Figure 3F). BI-2536, however, was equipotent in both cell lines (Figure S3F). Finally, the activity of the BRD4-inhibiting RIPTAC, JQ1-4PEG-CA, to block downstream signaling of its effector protein was tested in both cell lines. BRD4 inhibition upregulates the transcription of HEXIM1, a tumor suppressor and key modulator of gene expression. Treatment of both cell lines with increasing amounts of either JQ1-4PEG-CA or JQ1 caused a dose-dependent increase in HEXIM1 transcripts. The EC50 of JQ1-4PEG-CA was 192 nM in 293_HFL cells and 11.4 μM in 293_GFPL cells, which constitutes a nearly 60-fold increase in potency that favors the presence of the target protein in the cell (Figure 3G). Alternatively, JQ1 itself was equally active in promoting HEXIM1 transcription (Figure S3G). Taken altogether, the data strongly suggest that the enhanced antiproliferative activity of the RIPTACs in the target protein-expressing 293_HFL cells is derived from their more potent inactivation effects on their respective effector proteins.

Ternary Complex Formation with RIPTACs:

We next confirmed that the observed differential biology with RIPTACs was due to the formation of TP:RIPTAC:EP ternary complexes. We treated 293_HFL cells with JQ1-based non-covalent RIPTAC JQ1-4PEG-FKBP and covalent RIPTAC JQ1-6PEG-CA for 3h, after which we immunoprecipitated the HaloTag protein with Halo-Trap agarose beads. We observed coimmunoprecipitation of BRD4 with both molecules, but not with the negative control epi-JQ1-6PEG-CA (Figure 4A). We next showed that the BRD4 coimmunoprecipitation seen with JQ1-6PEG-CA and JQ1-4PEG-FKBP could be prevented by pre-treating the 293_HFL cells for 30 min with TAMRA-CA in the case of JQ1-6PEG-CA, and the FKBP ligand HLDA-001 in the case of JQ1-4PEG-FKBP (Figure 4B). In the same experiment, pre-treatment with JQ1 also prevented complex formation with both RIPTACs, but only partially. Using the same Halo-Trap based pulldown protocol, we showed that cellular ternary complexes could also be obtained with treatment of 293_HFL cells with 100 nM of the PLK1-binding non-covalent RIPTAC BI-2PEG-FKBP, 100 nM of the CDK9-binding covalent RIPTAC DNN-9PEG-CA, and 100 nM of the CDK1/2/5-binding covalent RIPTAC TMX-6PEG-CA, but not with negative control ELs, HLDA-001 or the TAMRA-CA HaloTag ligand (Figure S5A). We also established that the cellular ternary complexes formed by RIPTACs were profoundly stable by conducting a washout experiment. We treated 293_HFL cells with 1 μM JQ1-6PEG-CA or 100 nM JQ1-4PEG-FKBP for 3 h, followed by washout, and tracked ternary complex persistence over a period of 72 h. Incredibly, the cellular ternary complexes, even with the non-covalent JQ1-4PEG-FKBP, were readily detectable even 72 h following washout (Figure 4C). The phenomenon of long-lived stable ternary complexes was not restricted to the BRD4-inhibiting class of RIPTACs, in that analogous pulldown experiments with the CDK-binding RIPTAC, DNE-11PEG-CA, and the PLK1-binding RIPTAC, BI-2PEG-FKBP, also showed that even 72 hours after RIPTAC washout, target protein was still readily detected in complex with effector protein (Figure S5B).

Figure 4. Ternary Complex Formation with RIPTACs.

(A) 3h treatment of 293_HFL cells with indicated compounds followed by HaloTag immunoprecipitation using HaloTrap beads demonstrates cellular ternary complex formation with both the non-covalent RIPTAC JQ1-4PEG-FKBP and the covalent RIPTAC JQ1-6PEG-CA, but not with the control molecule epi-JQ1-6PEG-CA that does not bind BRD4. (B) Ternary complex formation with JQ1-6PEG-CA and JQ1-4PEG-FKBP can be competed away with 30 min pre-treatment with TAMRA-CA and HLDA-001, respectively. JQ1 pre-treatment for 30 min partially competes away complex formation with both RIPTACs. (C) 293_HFL cells were treated with the indicated compounds for 3h (T₀), following which the compound treated medium was washout out and replaced with normal growth medium for up to 72h. The HaloTag-FKBP fusion protein was immunoprecipitated at the timepoints shown and the BRD4 protein levels in the complex were detected by immunoblotting.

To determine whether the protein complexes formed by the RIPTACs were true ternary complexes and not haphazard larger order aggregates, 293_HFL cells were treated with CDK-inhibiting RIPTACs for 3h, lysed and subjected to successive rounds of ultrafiltration to separate the lysate proteins/protein complexes based on size (see STAR Methods). The target protein (MW = 46 kDa) was most abundant in the final eluate (proteins < 50 kDa) in the absence of RIPTAC, indicating its uncomplexed, monomeric form (Figure S6). Alternatively, following treatment with RIPTACs, the target protein shifted into the secondary retentate, suggesting that it complexed with one or more other proteins for a combined molecular weight of > 50 kDa, but still not > 100 kDa. This shift is consistent with RIPTAC-dependent formation of ternary complexes with CDKs (e.g. CDK9), but not incorporation into higher order protein aggregates (> 100 kDa). Confirming the absence of large protein aggregates and to demonstrate RIPTAC-induced co-localization of FLAG-HaloTag-FKBP target protein and the corresponding effector protein in cellulo, we performed an immunofluorescence BRD4 co-localization experiment using JQ1-6PEG-CA as a representative RIPTAC (Figure 5). Treating 293_HFL cells with 1 μM JQ1-6PEG-CA for 4 h causes an increase in nuclear FLAG signal (panel c) relative to vehicle-treated control (panel d), reflecting nuclear import of the target protein by our RIPTAC. This heterobifunctional-directed recruitment to BRD4 has been previously observed¹⁵. Note that while the RIPTAC induced the co-localization of the TP with BRD4 in the nucleus, we observed no punctate immunofluorescence indicative of aggregate formation.

Cooperative Binding Promotes Ternary Complex Stability:

Given that ternary complex formation is an underlying mechanistic feature of all RIPTACs, we next tested whether positive cooperativity of binding could in part account for the complex’s stability and the enhanced potency seen with RIPTACs in 293_HFL cells. For this purpose, we first developed an AlphaLISA-based assay, in which we measured the binding of non-covalent RIPTAC JQ1-2PEG-FKBP to BRD4-BD1 in the presence or absence of purified recombinant FKBP. We observed a dramatic (~35-fold, range 25–50-fold) increase in BRD4-BD1 binding of JQ1-2PEG-FKBP upon pre-incubation with FKBP (Figure 6A). No such potency shift was observed for the effector ligand JQ1 or the target ligand HLDA-001 (Figure S7A). This suggests that, as with other heterobifunctional molecules and molecular glues, protein-protein interactions (PPI) play an important role in the pharmacology of RIPTACs, and likely partially explain the selective antiproliferative activity seen in 293_HFL cells. To confirm and extend this finding, we used SPR to measure the dissociation constant (Kd) of JQ1-based RIPTAC for immobilized BRD4 BD1 domain (Figure S7B) showed that the additional presence of HaloTag-FKBP target protein increased the affinity of RIPTAC JQ1-2PEG-FKBP for the effector protein to 85 nM, compared to 470 nM in the absence of the target protein.

Figure 6. Cooperative Binding in Ternary Complex Formation by RIPTACs.

(A) AlphaLISA assay measuring BRD4-BD1 inhibition demonstrates positive co-operativity in biochemical RIPTAC ternary complex formation in presence of the non-covalent RIPTAC JQ1-2PEG-FKBP and FKBP. (B) NanoBRET in cellulo assay measuring cooperativity of effector protein engagement by JQ1-based RIPTACs; (C) by BI2536-based RIPTACs; d) by TMX-3013- and dinaciclib-based RIPTACs; e) by effector protein ligands.

To generate additional evidence in support of co-operativity in RIPTAC-directed ternary complex formation, we utilized the NanoBRET system. This afforded us the additional benefit of performing the evaluation in the more meaningful context of the intracellular environment. Specifically, we transiently transfected NanoLuc fusions of BRD4 or CDK9 or PLK1 into 293_GFPL and 293_HFL cells. We used these cells to study RIPTAC occupancy of the relevant EP ligand binding site using appropriate BRET tracers. We observed enhanced EP occupancy by the RIPTAC in the 293_HFL cells (Figure 6B–E). This observation is consistent with the presence of co-operativity within the RIPTAC ternary complex, while also possibly being a result instead of target-dependent RIPTAC accumulation in 293_HFL cells.

Subcellular Localization of Target Protein Affects RIPTAC Potency:

Having established that RIPTAC differential biology likely benefits from the ability of the TP and EP to form ternary complexes, we postulated that the relative subcellular localization of the two proteins in question likely contributes significantly to the RIPTAC potency differential observed in viability assays. To test this hypothesis, cell lines expressing either a nuclear-localized (293_NLS2HF) or a plasma membrane-localized (293_MYRHF) version of the HaloTag-FKBP fusion protein (Figure S7C) were tested with select RIPTACs in parallel with 293_HFL cells (Figure 7). The covalent RIPTACs incorporating JQ1, TMX3013, and BI2536 all showed significantly reduced potency in the 293_MYRHF cell line compared to 293_HFL cells, yet exhibited equal or better potency in the 293_NLS2HF line. Given the often nuclear-localized role of the effector proteins in cell division, this is perhaps to be expected. Conversely, the non-covalent JQ1-4PEG-FKBP was the only non-covalent RIPTAC that showed a clear preference for the nuclear-localized target protein compared to the plasma membrane-localized one. Non-covalent RIPTACs may therefore be more tolerant towards disparate TP-EP localization than covalent RIPTACs. The activity of control compounds, including all three ELs and the FKBP ligand HLDA-001, did not differ significantly in all four cell lines tested (Figure S7D).

Figure 7. Target Protein Subcellular Localization Affects RIPTAC Activity.

(A) 7 day CellTiter Glo viability assay with indicated covalent RIPTACs in cell lines expressing the HaloTag-FKBP fusion protein selectively in the nucleus (293_NLS2HF), plasma membrane (293_MYRHF), or both (293_HFL). (B) 7 day CellTiter Glo viability assay in the same cell lines using non-covalent RIPTACs.

Discussion

We have described herein a chemical biology proof-of-concept study for a heterobifunctional small molecule modality called RIPTACs, which has potential as an anticancer therapeutic strategy. Our hypothesis stemmed from two previously reported observations.

Firstly, a large number of Fluorine-18 and Gallium-68 Positron Emission Tomography (PET) radiotracers have been used in preclinical in vivo studies to monitor tissue expression of their cognate protein receptors, and many have made their way into clinical use^16–18. Examples of FDA-approved PET ligands include piflufolastat F-18 (PYLARIFY), used to detect PSMA+ prostate cancer and Ga-68 dotatate (NETSPOT) for detection of somatostatin receptor positive neuroendocrine tumors, among others. This suggests that some subset of high-affinity, high selectivity ligands may be prone to accumulation in cells that express their protein target.

Secondly, we know from the field of heterobifunctional small molecules and molecular glues that the apparent K_d of a ligand:protein interaction can be dramatically enhanced by the presence of beneficial PPIs in a ternary complex^19–23. Our work using the HaloTag model system suggests that the ability to form a stable trimer is not restricted to proteins with a chaperone function, like immunophilins, or enzymes with a relatively promiscuous substrate recognition domain, like some E3 ligases.

Our hypothesis relies on the expression of a target protein that we use to block the function of a pan-essential effector protein in a target protein-expression (i.e. cell-selective) manner. There are several additional factors to consider in developing the RIPTAC technology as a platform. For example, the relative abundance of the target protein and the pan-essential effector protein will likely play a role in RIPTAC activity. The HaloTag-FKBP target protein in our work is highly expressed by a CMV promoter and attains micromolar cellular concentrations at steady state. High concentrations in tumor cells have been reported for certain highly amplified oncogenes such as HER2²⁴, and when this work is extended to cancer targets, the relationship between the level of target expression and the abundance of effector protein will be interrogated. As pertains to the former, our doxycycline-inducible HaloTag-FKBP-expressing cells enabled us to test the activity of our RIPTACs over a range of target protein expression levels and thereby more elegantly demonstrate the dependency of their toxicity on target protein abundance. Although even at maximum target protein induction, the efficacy of many of our RIPTACs was 20–30% reduced in the dox-inducible cells compared to their performance in the 293_HFL cells (50% reduced for JQ1-4PEG-CA), the target protein-dependency of their observed toxicity is unambiguous. That the efficacy of each of the RIPTACs tested in both Figure 1 (293_HFL) and Figure 2 (dox-inducible TREx-293) is invariably greater in the former suggests that the maximum concentration of TP in the dox-inducible 293 cells is stoichiometrically less than that in the 293_HFL cells. While either cell system (the doxycycline-inducible TP cell line; and the constitutively-TP expressing line vs. its non-expressing isogenic match) has its relative pros and cons vis-à-vis demonstrating the on-mechanism toxicity of our RIPTACs, we found they served as complementary approaches in our initial investigation of this new modality.

While high expression of TP is helpful in maximizing RIPTAC activity, our previous experience developing PROTACs and their observable “hook effect” (diminished effectiveness at high concentrations) reminded us that more is not necessarily better; and recent studies concerning non-degrader heterobifunctional molecules also have observed a hook effect^15,25. Fortunately, we observed no biological hook effect with our RIPTACs up to and including treatment concentrations at the solubility limit. We reasoned this is due to RIPTACs being still capable of inhibiting their effector protein in either ternary complexes (TP:RIPTAC:EP) or the binary complexes (RIPTAC:TP and RIPTAC:EP) that can form at high RIPTAC concentrations -- PROTACs are not effective in the latter scenario. An alternative scenario is that the neo-PPI formed in the ternary complex is sufficiently stable that increasing concentrations of RIPTAC cannot disrupt the stable complex, thereby avoiding a hook effect.

Thirdly, as we have shown with chemical biology tools, the relative localization of the target and effector proteins can have a significant impact on the activity of the RIPTAC. In the case of BRD4 as an effector protein, for example, nuclear target proteins are clearly the most effective for the RIPTAC approach; while other EP-RIPTAC pairings are more amenable to harnessing non-nuclear TPs, with effectiveness approaching that of nuclear TP in some instances.

Fourthly, we have not examined here whether the RIPTAC mechanism of action involves more than mere inhibition of the active site of the effector protein. Nevertheless, it is reasonable to speculate that in some instances RIPTAC activity may derive from partial or complete mislocalization of the effector protein, or indeed of the target protein, as a result of its incorporation into a stable ternary complex with the target protein bound RIPTAC. Heterobifunctional compounds that affect protein localization in this manner have recently been reported^15,25,26. In fact, the greatest therapeutic index may be achieved with a RIPTAC that shows only weak binding to the effector protein (or non-functional binding) in healthy cells lacking the target protein, but, owing to novel PPIs and the formation of a profoundly stable ternary complex, inactivating the effector protein in tumor cells. Such phenomena are well-documented in the field of molecular glues^27,28.

It is also possible that novel gain-of-function phenotypes may be observed in specific RIPTAC cases. Specifically, examples of heterobifunctional compounds that recruit BRD4 or CDK or the BAF complex to activate transcription in a therapeutically relevant fashion have recently been reported^29–31. Because of the pharmacology of RIPTACs, which bring together a tumor or lineage specific protein with a more ubiquitously expressed essential protein involved in a key cellular process, such cell type specific effects of RIPTACs can easily be envisaged. These may even allow RIPTACs to be successfully leveraged for a therapeutic option in disease areas outside of cancer, where cell killing is not the desired outcome.

Finally, an important feature of the RIPTAC modality is that it is agnostic to the identity of the oncogenic driver of the disease. Conventional targeted therapy relies on the inhibition of the cellular signaling pathway responsible for tumor growth. By availing itself of any distinctive tumor protein – including intracellular ones -- to serve as the target for delivery of its pan-essential inhibitor, RIPTACs serve to combine the desirable attributes of newer anti-cancer modalities (e.g ADCs and T-cell recruiters) with the attractive predictability of small molecule-based approaches. Taken together, our data support further study of RIPTACs as a heterobifunctional small molecule modality for the treatment of cancer. Future work will address several outstanding questions described in this chemical biology proof-of-concept study and focus on advancing bona fide drug-like RIPTACs towards the clinic.

Limitations of the study:

This report presents the RIPTAC concept and demonstrates its therapeutic relevance using several model systems. One limitation of our work herein is that it is a proof-of-concept chemical biology effort and does not demonstrate the applicability of this paradigm to naturally occurring cancer-relevant tumor specific proteins. Another avenue we do not investigate in this manuscript is tissue or lineage specific inhibition of EPs that does not result in cell death but has some other therapeutically relevant outcome. Finally, a limitation of this approach is that bringing two functional proteins together in the cell can occasionally result in novel biology that is not entirely predictable from a priori knowledge of their cellular roles and needs to be investigated on a case-by-case basis. We will expand on these limitations and describe additional use cases of our technology in future work.

STAR Methods:

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Craig M. Crews (craig.crews@yale.edu).

Materials availability

Heterobifunctional molecules, plasmids and cell lines generated in this study are available from the lead contact upon request and completion of an MTA.

Data and code availability

• Original immunoblot images have been deposited at Mendeley and are publicly available as of the date of this publication. The accession number for this data is DOI:10.17632/tnk48294nr.1 . Microscopy data reported in this paper will be shared by the lead contact upon request.

• This paper does not report original code.

• Any additional information required to analyze the data reported in this paper is available from the lead contact upon request.

Experimental model and study participant details

Cell lines

HEK293 cells were purchased from ATCC, and TREx293 cells were obtained from Thermo Fisher Scientific. Both were grown in DMEM (Thermo Fisher Scientific) supplemented with 10% heat inactivated FBS (Thermo Fisher Scientific), 1% penicillin-streptomycin (Thermo Fisher Scientific), and 1 μg/mL puromycin (Invivogen) as needed. HT29 cells were purchased from ATCC and grown in McCoy’s 5A medium (Thermo Fisher Scientific) supplemented with 10% heat inactivated FBS (Thermo Fisher Scientific), 1% penicillin-streptomycin (Thermo Fisher Scientific), and 1 μg/mL puromycin (Invivogen) as needed. Cells were cultured in a humidified 37°C incubator with 5% CO₂. To generate the 293_HFL cell line, the HaloTag7-FKBP sequence was fused to a P2A-EGFP and introduced into the HEK293 cell using polybrene (8 μg/mL)-mediated lentiviral transduction, followed by selection using 1 μg/mL puromycin. To generate the 293_GFPL cell line, a GFP-infected HEK293 line was used as a negative control. In order to induce membrane localization of the fusion protein, an MGSSKSKPK sequence was added to the N-terminus, and an N-terminal PKKKRKV sequence used for nuclear localization. The HT29_HF, 293_AktH and 293_HBTKL cell lines were generated similarly. The doxycycline-inducible HaloTag7-FKBP-expressing cell line was generated by expressing the model fusion protein in TREx293 cells (Thermo Fisher Scientific). HEK293, TREx293 and HT29 cells are all female in origin; because all cell lines were obtained from either a commercial repository with generally established bona fides (ATCC) or from their proprietary source (Thermo Fisher Scientific), no further authentication was performed.

Method details

Chemical syntheses and characterization data:

RIPTACs were synthesized by WuXi AppTec (Shanghai, China). Chemicals used for synthesis were purchased from commercial sources and were used without further purification. Flash chromatography was performed on silica gel columns. 1H NMR spectra were recorded on a Bruker 400 NMR spectrometer (400 MHz for ¹H and 101 MHz for ¹³C). The values of chemical shifts (δ) are reported in p.p.m. Coupling constants (J) are reported in Hz. LCMS were recorded on Agilent 1100 LC and Agilent G1956A. HPLC purifications were performed on a reverse-phase column using a Shimadzu HPLC system or Agilent HPLC system. In many instances, LC-MS characterization was omitted by WuXi AppTec for synthetic intermediates because they lacked a UV (254 nm) -absorbing chromophore, or were poorly UV-absorbing. In such instances, characterization was by ¹H NMR, except in instances where the product of the reaction was carried on to the next step without further purification or characterization. No ¹³C NMR was performed for synthesis intermediates by WuXi AppTec. Full characterization (LC-MS; ¹H NMR; ¹³C NMR; and ¹⁹F NMR, where applicable) was performed for all final synthesis products. Chloroalkane RIPTACs are Series 1–6, and FKBP RIPTACs are Series 7–10. Effector ligands for selected based on a combination of criteria, including prevalance of usage in the field; the existence of one or more solvent-exposed linker attachment points; nanomolar to subnanomolar affinity for their cognate effector protein; and selectivity.

SERIES 1

The synthetic route for JQ1-4PEG-CA

Preparation of Compound 1

Known compound from WO2020/146470 A1 2020-07-16

Preparation of Compound 2

A mixture of tert-butyl N-[2-[2-[2-[2-[2-[2-[2-(6-chlorohexoxy)ethoxy]ethylamino]-2-oxo-ethoxy] ethoxy]ethoxy]ethoxy]ethyl]carbamate (230 mg, 413 μmol, 1.0 equiv) in CH₂Cl₂ (2 ml) and 4 M HCl/dioxane (2.3 ml) was stirred at 25 °C for 1 h. The reaction mixture was concentrated under reduced pressure to afford 2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]-N-[2-[2-(6-chlorohexoxy)ethoxy]ethyl]acetamide (200 mg, 405 μmol, 98% yield, HCl salt) as a yellow solid and carried on to the next step without further purification or characterization.

Preparation of Compound 3

Known compound from J. Med. Chem. 2022, 656573–6592

Preparation of JQ1-4PEG-CA

To a solution of 2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]-N-[2-[2-(6-chlorohexoxy) ethoxy]ethyl]acetamide (200 mg, 0.41 mmol, 1.0 equiv, HCl salt) in DMF (2 ml) were added DIPEA (262 mg, 2.03 mmol, 0.35 ml, 5.0 equiv), HOBt (82.2 mg, 0.61 mmol, 1.5 equiv), EDCI (117 mg, 0.61 mmol, 1.5 equiv) and (9R)-7-(4-chlorophenyl)-4, 5,13-trimethyl-3-thia-1,8,11,12-tetrazatricyclo[8.3.0.02,6]trideca-2(6),4,7,10,12-pentaene-9-carboxylic acid (157 mg, 0.41 mmol, 1.0 equiv). The mixture was stirred at 25 °C for 12 h. The reaction mixture was diluted with water (20 mL) and the mixture was extracted with ethyl acetate (3 × 15 ml). The combined organic phase was washed with brine (10 ml), dried with anhydrous Na₂SO₄ and concentrated under reduced pressure. The residue was purified by prep-HPLC (column: Phenomenex Gemini-NX C18 75*30 mm*3um; mobile phase: [water(0.225%FA)–MeCN]; B%: 48%–78%, 7 min) to afford N-[2-[2-[2-[2-[2-[2-[2-(6-chlorohexoxy)ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethoxy]ethoxy]ethyl]-2-[(9S)-7-(4-chlorophenyl)-4,5,13-trimethyl-3-thia-1,8,11,12-tetrazatricyclo[8.3.0.02,6]trideca-2(6),4,7,10,12-pentaen-9-yl]acetamide (150 mg, 0.17 mmol, 42% yield) as a yellow oil.

¹H NMR (400 MHz, CDCl₃-d): δ 8.31–8.25 (m, 1H), 7.68–7.59 (m, 1H), 7.52–7.39 (m, 4H), 4.54–4.47 (m, 1H), 3.86 (s, 2H), 3.64–3.58 (m, 3H), 3.56 (s, 4H), 3.51–3.30 (m, 17H), 3.30–3.15 (m, 6H), 2.59 (s, 3H), 2.41 (s, 3H), 1.73–1.65 (m, 2H), 1.62 (s, 3H), 1.52–1.43 (m, 2H), 1.41–1.26 (m, 4H).

¹³C NMR (151 MHz, CDCl₃): δ 170.68, 170.17, 163.97, 155.82, 149.99, 136.88, 136.83, 132.33, 131.06, 130.89, 130.61, 130.00, 128.84, 71.40, 71.06, 70.78, 70.76, 70.74, 70.72, 70.53, 70.49, 70.44, 70.18, 69.98, 69.95, 54.51, 45.21, 39.55, 39.28, 38.74, 32.67, 29.61, 26.84, 25.57, 14.56, 13.24, 12.00

LC-MS:

MS (ES⁺): RT = 3.260 min, m/z = 839.3 [M + H⁺].

Spectra:

HRMS [C₃₉H₅₆Cl₂N₆O₈S] Cal: 839.333; Obs: 839.3295

The synthetic route for JQ1-6PEG-CA

Preparation of compound 1

Known compound from WO2023/59581, 2023, A1

Preparation of compound 2

To a solution of tert-butyl N-tert-butoxycarbonyl-N-[2-[2-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]carbamate (1.00 g, 1.90 mmol, 1.0 equiv) in EtOAc (5 ml) and MeCN (5 ml) and H₂O (5 ml) were added RuCl₃ (39.5 mg, 190 μmol, 0.1 equiv) and NaIO₄ (1.63 g, 7.61 mmol, 4.0 equiv) under N₂ atmosphere. The mixture was stirred at 25 °C for 12 h. To the reaction mixture was added water (20 ml). The mixture was filtered and the filtrate was extracted with ethyl acetate (3 × 15 ml). The combined organic phase was washed with brine (18 ml), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (CH₂Cl₂/MeOH = 100/1 to 30/1) to afford 2-[2-[2-[2-[2-[2-[2-[bis(tert-butoxycarbonyl)amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]acetic acid (625 mg, crude) as a yellow oil. This compound was not uv-absorbing, so no LC-MS was performed.

¹H NMR (400 MHz, CDCl3) δ 4.19 (s, 2H), 3.83–3.77 (m, 4H), 3.76–3.59 (m, 20H), 1.52 (s, 18H).

Preparation of compound 4

A mixture of 2-[2-[2-[2-[2-[2-[2-[bis(tert-butoxycarbonyl)amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]acetic acid (1.00 g, 1.85 mmol, 1.0 equiv), 2-[2-(6-chlorohexoxy)ethoxy]ethanamine (482 mg, 1.85 mmol, 1.0 equiv, HCl), HOBt (376 mg, 2.78 mmol, 1.5 equiv), EDCI (710 mg, 3.71 mmol, 2 equiv) and DIEA (1.20 g, 9.27 mmol, 5.0 equiv) in DMF (20 ml) was stirred at 20 °C for 12 h under N₂. The reaction mixture was diluted with brine (35 ml) and the mixture was extracted with EtOAc (3 × 25 ml). The combined organic phase was washed with brine (30 ml), dried with anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (CH₂Cl₂/MeOH= 100/1 to 30/1) and then prep-HPLC (column: Phenomenex luna C18 150*40 mm* 15 μm; mobile phase: [water(0.225%FA)–MeCN]; B%: 50%–80%, 10 min) to afford tert-butyl N-tert-butoxycarbonyl-N-[2-[2-[2-[2-[2-[2-[2-[2-[2-(6-chlorohexoxy)ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]carbamate (790 mg, 1.06 mmol, 57% yield) as a colorless oil.

¹H NMR (400 MHz, CDCl₃) δ 7.20–7.12 (m, 1H), 4.02 (s, 2H), 3.83–3.78 (m, 2H), 3.75–3.45 (m, 36H), 1.87–1.74 (m, 2H), 1.65–1.60 (m, 2H), 1.52 (s, 18H), 1.44–1.36 (m, 2H).

LC-MS:

MS (ES⁺): RT = 1.045 min, m/z = 645.3 [M - Boc + H⁺]; m/z = 545.3 [M - 2Boc + H⁺].

Spectra:

Preparation of compound 5

A mixture of tert-butyl N-tert-butoxycarbonyl-N-[2-[2-[2-[2-[2-[2-[2-[2-[2-(6-chlorohexoxy)ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]carbamate (90.0 mg, 121 μmol, 1.0 equiv) in CH₂Cl₂ (1 ml) and TFA (0.5 ml) was stirred at 20 °C for 1 h. The reaction mixture was concentrated under reduced pressure to afford 2-[2-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-N-[2-[2-(6-chlorohexoxy)ethoxy]ethyl]acetamide (78.0 mg, 118 μmol, 98% yield, TFA salt) as a yellow oil. This compound was not uv absorbing, so no LC-MS was performed, and it was carried on to the next step without further purification or characterization.

Preparation of Compound 1a

Known compound from J. Med. Chem. 2022, 65, 6573–6592

Preparation of JQ1-6PEG-CA

A mixture of 2-[(9S)-7-(4-chlorophenyl)-4,5,13-trimethyl-3-thia-1,8,11,12-tetrazatricyclo[8.3.0.0^2,6]trideca-2(6),4,7,10,12-pentaen-9-yl]acetic acid (100 mg, 249 umol, 1.0 equiv), 2-[2-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-N-[2-[2-(6-chlorohexoxy)ethoxy]ethyl]acetamide (164 mg, 249 umol, 1.0 equiv, TFA salt), HOBt (67.4 mg, 499 μmol, 2.0 equiv), EDCI (143 mg, 748 umol, 3.0 equiv) and DIEA (161 mg, 1.25 mmol, 5.0 equiv) in DMF (8 ml) was stirred at 20 °C for 12 h under N₂. The reaction mixture was diluted with water (20 ml) and the mixture was extracted with ethyl acetate (3 × 15 ml). The combined organic phase was washed with brine (10 m), dried with anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. The residue was purified by prep-HPLC (column: Phenomenex Synergi C18 150*25 mm* 10 μm; mobile phase: [water(0.225%FA)–MeCN]; B%: 48%–78%,10 min), and then prep-HPLC ( column: Phenomenex Synergi C18 150*25 mm* 10 μm; mobile phase: [water(0.225%FA)–MeCN]; B%: 48%–78%,10 min) to afford N-[2-[2-[2-[2-[2-[2-[2-[2-[2-(6-chlorohexoxy)ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]-2-[(9S)-7-(4-chlorophenyl)-4,5,13-trimethyl-3-thia-1,8,11,12-tetrazatricyclo[8.3.0.0^2,6]trideca-2(6),4,7,10,12-pentaen-9-yl]acetamide (159 mg, 170 μmol, 68% yield) as a colorless oil.

¹H NMR (400 MHz, DMSO-d₆) δ 8.28 (t, 1H, J = 5.2 Hz), 7.63 (t, 1H, J = 5.6 Hz), 7.52–7.47 (m, 2H), 7.46–7.40 (m, 2H), 4.54–4.48 (m, 1H), 3.87 (s, 2H), 3.62 (t, 2H, J = 6.8 Hz), 3.58–3.41 (m, 28H), 3.39–3.35 (m, 2H), 3.30–3.21 (m, 6H), 2.60 (s, 3H), 2.42 (s, 3H), 1.76–1.66 (m, 2H), 1.63 (s, 3H), 1.53–1.43 (m, 2H), 1.43–1.26 (m, 4H).

¹³C NMR (101 MHz, CD₃OD): δ 172.88, 172.77, 166.05, 157.03, 152.11, 138.15, 137.91, 133.50, 133.17, 132.01, 131.98, 131.37, 129.78, 72.17, 71.98, 71.60, 71.57, 71.56, 71.54, 71.51, 71.38, 71.35, 71.30, 71.23, 71.19, 70.64, 70.47, 55.19, 45.74, 40.56, 39.77, 38.77, 33.73, 30.57, 27.73, 26.49, 14.44, 12.94, 11.62

LC–MS:

MS (ES⁺): RT = 2.897 min, m/z = 464.4 [M/2 + H⁺].

Spectra:

HRMS [C₄₃H₆₄Cl₂N₆O₁₀S] Cal: 927.3854; Obs: 927.3826

The synthetic route for epi-JQ1-6PEG-CA

Preparation of Compound 3

A mixture of 2-(2-((tert-butoxycarbonyl)amino)ethoxy)acetic acid (160 mg, 0.36 mmol, 1 equiv), 2-(2-((6-chlorohexyl)oxy)ethoxy)ethan-1-amine (94.7 mg, 0.36 mmol, 1 equiv), DIPEA (235 mg, 1.82 mmol, 0.32 ml, 5 equiv) and T₃P (348 mg, 0.55 mmol, 0.32 ml, 50% purity, 1.5 equiv) in CH₂Cl₂ (5 ml) was stirred at 25 °C for 3 h. The reaction mixture was added water (20 ml) and extracted with EtOAc (2 × 20 ml). The combined organic layer was washed with brine (2 × 20 ml), dried with anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. The residue was purified by prep-TLC (CH₂Cl₂/MeOH = 10/1) to afford tert-butyl (18-chloro-5-oxo-3,9,12-trioxa-6-azaoctadecyl)carbamate (180 mg, 0.28 mmol, 77% yield) as a yellow oil.

¹H NMR (CDCl₃, 400 MHz) δ 7.21 (brs, 1H), 4.01 (s, 2H), 3.42–3.71 (m, 34H), 3.31 (t, 2H, J = 5.2 Hz), 1.74–1.81 (m, 2H), 1.55–1.65 (m, 2H), 1.33–1.50 (m, 13H).

LC–MS:

MS (ES⁺): RT = 0.926 min, m/z = 645.3 [M+H⁺].

Spectra:

Preparation of Compound 4

A mixture of tert-butyl-(18-chloro-5-oxo-3,9,12-trioxa-6-azaoctadecyl)carbamate (170 mg, 0.26 mmol, 1 equiv) in TFA (1.5 ml) and CH₂Cl₂ (3 ml) was stirred at 25 °C for 0.5 h. The reaction mixture was concentrated under reduced pressure to afford 2-(2-aminoethoxy)-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)acetamide (170 mg, 0.26 mmol, 98% yield) as a yellow oil. This compound was not uv absorbing, so no LC-MS was performed.

¹H NMR (CDCl₃, 400 MHz) δ 7.68–7.33 (m, 4H), 4.12 (s, 2H), 3.83–3.78 (m, 2H), 3.75–3.71 (m, 2H), 3.66–3.49 (m, 30H), 3.26–3.02 (m, 2H), 1.82–1.74 (m, 2H), 1.65–1.55 (m, 2H), 1.50–1.42 (m, 2H), 1.40–1.32 (m, 2H).

Preparation of compound 5

Known compound from J. Med. Chem. 2022, 65, 6573–6592

Preparation of epi-JQ1-6PEG-CA

A mixture of I-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetic acid (40.0 mg, 100 μmol, 1 equiv), 2-(2-aminoethoxy)-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)acetamide (65.8 mg, 100 μmol, 1 equiv), T₃P (95.2 mg, 150 μmol, 89.0 μL, 50% purity, 1.5 equiv) and DIPEA (64.5 mg, 499 umol, 86.9 uL, 5 equiv) in CH₂Cl₂ (3 ml) was stirred at 25 °C for 16 h. The reaction mixture was added water (15 ml) and extracted with CH₂Cl₂ (2 × 15 ml). The combined organic layer was washed with brine (15 ml), dried with anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. The residue was purified by prep-HPLC (column: Phenomenex Gemini-NX C18 75*30 mm*3 μm;mobile phase: [water(10mM NH₄HCO₃)–-MeCN];B%: 35%–65%, 8 min) to afford compound (R)-N-(18-chloro-5-oxo-3,9,12-trioxa-6-azaoctadecyl)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamide (16.0 mg, 16.3 umol, 16% yield) as a yellow gum.

¹H NMR (DMSO-d₆, 400 MHz) δ 7.45–7.39 (m, 2H), 7.37–7.31 (m, 2H), 7.24–7.12 (m, 1H), 7.05–6.90 (m, 1H), 4.67 (t, 1H, J = 6.8 Hz), 3.99 (2H, s), 3.65–3.69 (m, 19H), 3.63–3.36 (m, 19H), 2.68 (s, 3H), 2.41 (s, 3H), 1.74–1.81 (m, 2H), 1.68 (s, 3H), 1.65–1.55 (m, 2H), 1.41–1.50 (m, 2H), 1.33–1.41 (m, 2H).

LC–MS:

MS (ES⁺): RT = 2.799 min, m/z = 927.3 [M + H⁺];

Spectra:

HRMS [C₄₃H₆₄Cl₂N₆O₁₀S] Cal: 927.3854; Obs: 927.382

SERIES 2

The synthetic route for TMX-6PEG-CA

Preparation of compound 2

To a solution of tert-butyl N-tert-butoxycarbonyl-N-[2-[2-[2-[2-[2-[2-[2-[2-[2-(6-chlorohexoxy)ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]carbamate (577 mg, 774 μmol, 1.0 equiv) in CH₂Cl₂ (2 ml) was added TFA (1 ml).The mixture was stirred at 20 °C for 0.5 h. Analysis by TLC demonstrated the formation of the desired product. The reaction mixture was concentrated under reduced pressure to give 2-[2-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-N-[2-[2-(6-chlorohexoxy)ethoxy]ethyl]acetamide (505 mg, 766 μmol, crude, TFA salt) as a yellow oil. This compound was not uv absorbing, so no LC-MS was performed, and it was used in the next step without further purification.

Preparation of compound 4

To a solution of 2-[2-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-N-[2-[2-(6-chlorohexoxy)ethoxy]ethyl]acetamide (505 mg, 766 μmol, 1.0 equiv, TFA salt) and 4-nitrobenzenesulfonyl chloride (339 mg, 1.53 mmol, 2.0 equiv) in CH₂Cl₂ (10 ml) was added DIEA (495 mg, 3.83 mmol, 667 μl, 5.0 equiv). The mixture was stirred at 20 °C for 0.5 h after which time water was added (50 ml) and the mixture was extracted with EtOAc (50 ml). The combined organic phase was washed with brine (3 × 50 ml), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (EtOAc : MeOH = 1:0 to 10:1 ) to give N-[2-[2-(6-chlorohexoxy)ethoxy]ethyl]-2-[2-[2-[2-[2-[2-[2-[(4-nitrophenyl)sulfonylamino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]acetamide (430 mg, 588 μmol, 77% yield) as a yellow oil.

LC–MS:

MS (ES⁺): RT = 0.882 min, m/z = 730.1 [M + H⁺];

Spectra:

Preparation of compound 5

To a solution of N-[2-[2-(6-chlorohexoxy)ethoxy]ethyl]-2-[2-[2-[2-[2-[2-[2-[(4-nitrophenyl)sulfonylamino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]acetamide (430 mg, 588 μmol, 1.0 equiv) in THF (5.5 ml) and HOAc (1.1 ml) was added Zn (1.93 g, 29.4 mmol, 50.0 equiv), and then it was stirred at 60 °C for 12 h. The pH of the mixture was adjusted to 7–8 by sat. aq. NaHCO₃ (3 ml.), To the reaction mixture was added water (50 ml) and the mixture was extracted with EtOAc (50 ml). The combined organic phase was washed with brine (3 × 50 ml), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to give 2-[2-[2-[2-[2-[2-[2-[(4-aminophenyl)sulfonylamino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-N-[2-[2-(6-chlorohexoxy)ethoxy]ethyl]acetamide (400 mg, crude) as a yellow oil and it was used by next step without further purification.

LC–MS:

MS (ES⁺): RT = 0.881 min, m/z = 700.2 [M + H⁺];

Spectra:

Preparation of TMX-6PEG-CA

To a solution of 2-[2-[2-[2-[2-[2-[2-[(4-aminophenyl)sulfonylamino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-N-[2-[2-(6-chlorohexoxy)ethoxy]ethyl]acetamide (400 mg, 571 μmol, 1.0 equiv), 2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-6-fluoro-benzamide (236 mg, 685 μmol, 1.2 equiv) in i-PrOH (4 ml) was added HCl (69.4 mg, 571 μmol, 68.0 ul, 30% purity, 1.0 equiv), and it was stirred at 95 °C for 12 h. The pH of the reaction mixture was adjusted to 7 by TEA (0.1 ml), then filtered. It was purified by prep-HPLC (column: Waters Xbridge C18 150*50 mm* 10 μm; mobile phase: [water(10 mM NH₄HCO₃)–MeCN]; B%: 38%–68%, min) to afford 2-[[5-bromo-2-[4-[2-[2-[2-[2-[2-[2-[2-[2-[2-(6-chlorohexoxy)ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethylsulfamoyl]anilino]pyrimidin-4-yl]amino]-6-fluoro-benzamide (198 mg, 194 μmol, 34% yield) as a yellow gum.

¹H NMR (400 MHz, CD₃OD-d₄) δ 8.37 (d, J = 8.2 Hz, 1H), 8.29 (s, 1H), 7.85 (d, J = 8.8 Hz, 2H), 7.72 (d, J = 8.8 Hz, 2H), 7.52 (dt, J = 6.5, 8.2 Hz, 1H), 7.00 (dd, J = 8.6, 10.0 Hz, 1H), 3.97 (s, 2H), 3.69– 3.53 (m, 26H), 3.51–3.40 (m, 8H), 3.05 (t, J = 5.4 Hz, 2H), 1.74 (quin, J = 7.0 Hz, 2H), 1.62–1.52 (m, 2H), 1.49–1.33 (m, 4H)

¹³C NMR (151 MHz, CDCl₃): δ 170.20, 167.20 (d, J_CF = 1 Hz), 161.31 (d, J_CF = 246.9 Hz), 157.92, 157.46, 156.74, 143.55, 142.09 (d, J_CF = 4.6 Hz), 132.80, 132.76 (d, J_CF = 12.7 Hz), 128.38, 118.72, 118.65 (d, J_CF = 2.9 Hz), 110.14 (d, J_CF = 25.4 Hz), 109.01 (d, J_CF = 14.5 Hz), 97.36, 71.39, 71.04, 70.69, 70.57, 70.65, 70.62, 70.60, 70.52, 70.42, 70.35, 70.16, 69.92, 69.39, 45.21, 43.13, 38.73, 32.65, 29.59, 26.83, 25.55

¹⁹F NMR (376 MHz, CD₃OD): δ –112.53

LC-MS:

MS (ES⁺): RT = 3.193 min, m/z = 1010.3 [M + H⁺];

Spectra:

HRMS [C₄₁H₆₀BrClFN₇O₁₂S] Cal: 1008.2949; Obs: 1008.2916

SERIES 3

The synthetic route for BI-2PEG-CA

Preparation of compound 1

Known compound from Angew. Chem. Int. Ed. 2006, 45, 4936–4940.

Preparation of compound 2

Known compound from A.C.S. Med. Chem. Lett. 2019, 10, 1443–-1449

Preparation of BI-2PEG-CA

A mixture of 4-[(8-cyclopentyl-7-ethyl-5-methyl-6-oxo-7H-pteridin-2-yl)amino]-3-methoxy-benzoic acid (30.0 mg, 70.5 μmol, 1.0 equiv), 2-[2-(2-aminoethoxy)ethoxy]-N-[2-[2-(6-chlorohexoxy)ethoxy]ethyl]acetamide (34.3 mg, 84.6 μmol, 1.2 equiv, HCl salt), HOBt (14.3 mg, 106 μmol, 1.5 equiv), EDCI (20.3 mg, 106 μmol, 1.5 equiv) and DIPEA (45.6 mg, 353 μmol, 5.0 equiv) in DMF (1 ml) was stirred at 20 °C for 16 h. The reaction mixture was diluted with water (20 ml) and the mixture was extracted with EtOAc (2 × 20 ml). The combined organic layer was washed with brine (30 ml), dried with anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 150*25 mm*10 μm; mobile phase: [water(0.225%FA)–ACN]; B%: 25%–55%,10 min) and prep-HPLC (column: Waters Xbridge 150*25 mm* 5 –m;mobile phase: [water(10 mM NH₄HCO₃)–MeCN] ;B%: 45%–75%, 9 min) to afford N-[2-[2-[2-[2-[2-(6-chlorohexoxy)ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethyl]-4-[(8-cyclopentyl-7-ethyl-5-methyl-6-oxo-7H-pteridin-2-yl)amino]-3-methoxy-benzamide (21.3 mg, 26.9 μmol, 38% yield) as a yellow gum.

¹H NMR (400 MHz, DMSO-d₆): δ 8.45–8.35 (m, 2H), 7.84 (s, 1H), 7.65–7.57 (m, 2H), 7.52–7.46 (m, 2H), 4.41–4.30 (m, 1H), 4.27–4.21 (m, 1H), 3.93 (s, 3H), 3.87 (s, 2H), 3.62–3.53 (m, 8H), 3.49–3.38 (m, 9H), 3.30–3.22 (m, 6H), 2.06–1.98 (m, 1H), 1.93–1.73 (m, 6H), 1.72–1.58 (m, 5H), 1.50–1.41 (m, 2H), 1.40–1.22 (m, 4H), 0.76 (t, 3H, J = 7.6 Hz).

¹³C NMR (101 MHz, CD₃OD): δ 172.55, 169.51, 165.21, 156.09, 153.45, 148.33, 139.08, 134.17, 127.33, 121.29, 117.30, 117.23, 109.99, 72.09, 71.97, 71.24, 71.23, 71.15, 71.07, 70.74, 70.47, 61.29, 60.19, 56.60, 45.74, 40.86, 39.73, 33.68, 30.49, 30.46, 29.76, 28.54, 28.10, 27.69, 26.42, 24.27, 24.03

LC–MS:

MS (ES⁺): RT = 2.488 min, m/z = 776.3 [M + H⁺]

Spectra:

HRMS [C₃₈H₅₈ClN₇O₈] Cal: 776.4108; Obs: 776.4073

SERIES 4

The synthetic route for JQ1-4PEG-deschloro

Preparation of compound 1

See TMX-6PEG-deschloro

Preparation of compound 2

Known compound from Inorganica Chim. Acta, 2011, 36538– 48

Preparation of compound 3

To a solution of 2-(2-heptoxyethoxy)ethanamine (310 mg, 977 μmol, 1.0 equiv, TFA salt) in DMF (3 ml) was added HATU (446 mg, 1.17 mmol, 1.2 equiv), 2-[2-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]acetic acid (343 mg, 977 μmol, 1.0 equiv) and DIEA (379 mg, 2.93 mmol, 3.0 equiv). The mixture was stirred at 25 °C for 2 h. The mixture was concentrated. The residue was purified by prep-HPLC (column: Waters Xbridge 150*25 mm * 5 μm; mobile phase: [water (10 mM NH₄HCO₃)–MeCN]; B%: 38%68%, 10 min) to give tert-butyl N-[2-[2-[2-[2-[2-[2-(2-heptoxyethoxy)ethylamino]-2-oxo-ethoxy]ethoxy]ethoxy]ethoxy]ethyl]carbamate (420 mg, 80% yield).

¹H NMR (400 MHz, CDCl₃): δ 7.15 (s, 1H), 5.06 (s, 1H), 4.02 (s, 2H), 3.71–3.43 (m, 28H), 3.32 (d, J = 5.2 Hz, 2H), 1.71–1.47 (m, 5H), 1.29 (d, J = 5.2 Hz, 10H), 0.89 (t, J = 6.8 Hz, 3H)

LC-MS:

MS (ES⁺): RT = 1.025 min, m/z = 537.3 [M + H⁺];

Spectra:

Preparation of compound 4

To a solution of tert-butyl N-[2-[2-[2-[2-[2-[2-(2-heptoxyethoxy)ethylamino]-2-oxoethoxy]ethoxy]ethoxy]ethoxy]ethyl]carbamate (100 mg, 186 μmol, 1.0 equiv) in DCM (1.5 ml) was added TFA (770 mg, 6.75 mmol, 0.5 ml, 36.2 equiv). The mixture was stirred at 25 °C for 12 h. The mixture was concentrated to give 2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]-N-[2-(2-heptoxyethoxy)ethyl]acetamide (100 mg, 97% yield) as a colorless oil and was used in the next step without further purification.

LC–MS:

MS (ES⁺): RT = 0.518 min, m/z = 437.1 [M + H⁺];

Preparation of JQ1-4PEG-deschloro

To a solution of 2-[(9S)-7-(4-chlorophenyl)-4,5,13-trimethyl-3-thia-1,8,11,12-tetrazatricyclo[8.3.0.02,6]trideca-2(6),4,7,10,12-pentaen-9-yl]acetic acid (73 mg, 181 μmol, 1.0 equiv) in DMF (1.5 ml) was added HATU (83 mg, 218 μmol, 1.2 equiv), 2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]-N-[2-(2-heptoxyethoxy)ethyl]acetamide (100 mg, 182 μmol, 1.0 equiv, TFA salt) and DIEA (117 mg, 908 μmol, 5.0 equiv). The mixture was stirred at 25 °C for 1 h. The mixture was concentrated to give a residue. The residue was purified by prep-HPLC (column: Unisil 3–100 C₁₈ Ultra 150 * 50 mm * 3 μm; mobile phase: [water (0.225%FA)–MeCN]; B%: 52%–82%, 10 min) to give 2-[(9S)-7-(4-chlorophenyl)-4,5,13-trimethyl-3-thia-1,8,11,12-tetrazatricyclo[8.3.0.02,6]trideca-2(6),4,7,10,12-pentaen-9-yl]-N-[2-[2-[2-[2-[2-[2-(2-heptoxyethoxy)ethylamino]-2-oxo-ethoxy]ethoxy]ethoxy]ethoxy]ethyl]acetamide (87 mg, 58% yield) as a yellow gum.

¹H NMR (400 MHz, MeOD-d₄): δ 7.54–7.36 (m, 4H), 4.63 (m, J = 5.2, 9.2 Hz, 1H), 3.97 (s, 2H), 3.67 (s, 12H), 3.63–3.58 (m, 4H), 3.57–3.53 (m, 4H), 3.49–3.40 (m, 7H), 3.28 (d, J = 5.2 Hz, 1H), 2.70 (s, 3H), 2.45 (s, 3H), 1.71 (s, 3H), 1.60–1.50 (m, 2H), 1.30 (d, J = 5.6 Hz, 8H), 1.00–0.81 (m, 3H)

¹³C NMR (151 MHz, CDCl₃): δ 170.68, 170.15, 163.95, 155.82, 149.97, 136.87, 136.83, 132.34, 131.05, 130.86, 130.59, 130.00, 128.83, 71.71, 71.05, 70.77, 70.76, 70.73, 70.53, 70.50, 70.46, 70.12, 69.98, 69.95, 54.51, 39.55, 39.28, 38.74, 31.96, 29.79, 29.30, 26.19, 22.76, 14.55, 14.24, 13.23, 11.99

LC-MS:

MS (ES⁺): RT = 2.868 min, m/z = 820.8 [M + H⁺];

Spectra:

HRMS [C₄₀H₅₉ClN₆O₈S] Cal: 819.3876; Obs: 819.3846

SERIES 5

The synthetic route for TMX-6PEG-deschloro

Preparation of compound 3

To a solution of tert-butyl (2-(2-hydroxyethoxy)ethyl)carbamate (5.0 g, 24.36 mmol, 1.0 equiv) in THF (50 ml) was added NaH (2.44 g, 60.90 mmol, 60% purity, 2.5 equiv) at 0 °C. The mixture was stirred at 0 °C for 30 min. Then 1-iodoheptane (8.26 g, 36.54 mmol, 1.5 equiv) in THF (10 ml) was added to the reaction mixture. The mixture was stirred at 20 °C for 11.5 h. The reaction mixture was quenched by addition of 40 ml of sat. aq. NH₄Cl at 0°C, and then diluted with H₂O (50 ml), and extracted with EtOAc (2 × 100 ml). The combined organic layers were washed with brine (100 ml), dried over Na₂SO₄, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate = 10/1 to 5/1). tert-butyl (2-(2-(heptyloxy)ethoxy)ethyl)carbamate (2.1 g, 6.92 mmol, 28% yield) was obtained as a yellow oil. This compound was not uv absorbing, so no LC-MS was performed.

¹HNMR (400 MHz, CDCl₃): δ 5.14– 4.91 (m, 1H), 3.65– 3.51 (m, 6H), 3.46 (t, J = 6.8 Hz, 2H), 3.33 (m, 2H), 1.64– 1.58 (m, 2H), 1.48– 1.41 (s, 9H), 1.38– 1.22 (m, 8H), 0.96– 0.82 (m, 3H)

Preparation of compound 4

To a solution of tert-butyl (2-(2-(heptyloxy)ethoxy)ethyl)carbamate (200 mg, 659 umol, 1.0 equiv) in CH₂Cl₂ (3 ml) was added TFA (1 ml). The mixture was stirred at 20 °C for 0.5 h. The reaction mixture was concentrated under reduced pressure to give a residue. 2-(2-(heptyloxy)ethoxy)ethan-1-amine (200 mg, crude, TFA salt) was obtained as a yellow oil. This compound was not uv absorbing, so no LC-MS was performed and it was carried on to the next step without further purification.

Preparation of compound 6

To a solution of 2-(2-(heptyloxy)ethoxy)ethan-1-amine (200 mg, crude, TFA salt) and 5-(tert-butoxycarbonyl)-2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatridecan-13-oic acid (230 mg, 426 μmol, 1.0 equiv) in DMF (2 ml) was added DIEA (296 mg, 2.30 mmol, 0.4 ml, 5.4 equiv) and HATU (324 mg, 852 μmol, 2.0 equiv). The mixture was stirred at 20 °C for 0.5 h. The reaction mixture was quenched by addition H₂O (0.1 ml), and then concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Atlantis T₃ 150*30 mm* 5μm; mobile phase: [water(0.225%FA)–MeCN];B%: 58%–88%, 10 min). tert-butyl-(tert-butoxycarbonyl)(8-oxo-3,6,12,15-tetraoxa-9-azadocosyl)carbamate (260 mg, 358 μmol, 84% yield) was obtained as a yellow oil.

¹H NMR (400 MHz, CDCl₃): δ 8.03 (s, 1H), 7.25– 7.14 (m, 1H), 4.02 (s, 2H), 3.82– 3.77 (m, 2H), 3.68– 3.57 (m, 28H), 3.53– 3.48 (m, 4H), 3.45 (t, J = 6.8 Hz, 2H), 1.62– 1.56 (m, 2H), 1.51 (s, 18H), 1.35– 1.27 (m, 8H), 0.94– 0.84 (m, 3H)

LC-MS:

MS (ES⁺): RT = 0.895 min, m/z = 525.3 [M – 2Boc + H⁺]; m/z = 625.3 [M – Boc + H⁺]

Preparation of compound 7

To a solution of tert-butyl-(tert-butoxycarbonyl)(8-oxo-3,6,12,15-tetraoxa-9-azadocosyl)carbamate (260 mg, 358 μmol, 1.0 equiv) in DCM (3 ml) was added TFA (1 ml). The reaction mixture was concentrated under reduced pressure to give a residue. 2-(2-(2-Aminoethoxy)ethoxy)-N-(2-(2-(heptyloxy)ethoxy)ethyl)acetamide (220 mg, crude, TFA salt) was obtained as a yellow oil. This compound was not uv absorbing, so no LC-MS was performed, and it was used in the next step without further purification.

Preparation of compound 9

To a solution of 2-(2-(2-aminoethoxy)ethoxy)-N-(2-(2-(heptyloxy)ethoxy)ethyl)acetamide (220 mg, crude, TFA salt) in DCM (5 ml) was added DIEA (222 mg, 1.72 mmol, 0.3 ml, 5.0 equiv) and 4-nitrobenzenesulfonyl chloride (114 mg, 516 μmol, 1.5 equiv). The mixture was stirred at 20 °C for 0.5 h. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: 3_Phenomenex Luna C18 75*30 mm*3 μm; mobile phase: [water (0.05%HCl)–MeCN]; B%: 50%–70%,6.5 min). N-(2-(2-(Heptyloxy)ethoxy)ethyl)-2-(2-(2-((4-nitrophenyl)sulfonamido)ethoxy)ethoxy)acetamide (170 mg, 239 μmol, 69% yield) was obtained as a yellow oil.

¹HNMR (400 MHz, CD₃OD): δ 8.36 (d, J = 8.7 Hz, 2H), 8.10 (d, J = 8.7 Hz, 2H), 7.23– 7.13 (m, 1H), 6.33– 6.08 (m, 1H), 4.02 (s, 2H), 3.74– 3.43 (m, 32H), 3.20 (m, 2H), 1.69–1.52 (m, 2H), 1.36– 1.24 (m, 8H), 0.97– 0.78 (m, 3H)

LC-MS :

MS (ES⁺) : RT = 0.975 min, m/z = 710.2 [M – 55]

Spectra:

Preparation of compound 10

A mixture of N-(2-(2-(heptyloxy)ethoxy)ethyl)-2-(2-(2-((4-nitrophenyl)sulfonamido)ethoxy)ethoxy)acetamide (170 mg, 239 μmol, 1.0 equiv), Zn (668 mg, 11.97 mmol, 50 equiv) in THF (5 ml) and AcOH (1 ml) was stirred at 60 °C for 2 h. The reaction mixture was adjusted with aq. sat. NaHCO₃ to pH = 7–8, and then the mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (SiO₂, CH₂Cl₂: MeOH = 10:1). 2-(2-(2-((4-Aminophenyl)sulfonamido)ethoxy)ethoxy)-N-(2-(2-(heptyloxy)ethoxy)ethyl)acetamide (100 mg, 147 μmol, 61% yield) was obtained as a colorless oil.

LC-MS:

MS (ES⁺): RT = 0.933 min, m/z = 680.4 [M + H⁺]

Spectra:

Preparation of compound 11

Known compound from Angew. Chem., Int. Ed. 2020, 59, 13865û13870

Preparation of TMX-6PEG-deschloro

To a solution of 2-(2-(2-((4-aminophenyl)sulfonamido)ethoxy)ethoxy)-N-(2-(2-(heptyloxy)ethoxy)ethyl)acetamide (100 mg, 147 μmol, 1.0 equiv) and 2-((5-bromo-2-chloropyrimidin-4-yl)amino)-6-fluorobenzamide (51 mg, 147 μmol, 1.0 equiv) in NMP (3 ml) was added HCl (12 M, 12 μl, 1.0 equiv). The mixture was stirred at 95 °C for 12 h. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: 3_Phenomenex Luna C18 75*30 mm*3 μm; mobile phase: [water(0.05%HCl)–MeCN]; B%: 53%–73%, 6.5 min) to give desired compound 20-(4-((5-bromo-4-((2-carbamoyl-3-fluorophenyl)amino)pyrimidin-2-yl)amino)phenylsulfonamido)-N-(2-(2-(heptyloxy)ethoxy)ethyl)-3,6,9,12,15,18-hexaoxaicosan-1-amide (38 mg, 36 umol, 24% yield, 96% purity, HCl salt) as a yellow gum.

¹HNMR (400 MHz, CD₃OD): δ 8.35 (s, 1H), 7.95 (d, J = 8.4 Hz, 1H), 7.83 (d, J = 8.6 Hz, 2H), 7.64 (d, J = 8.8 Hz, 2H), 7.57– 7.43 (m, J = 6.3, 8.3, 8.3 Hz, 1H), 7.28– 7.14 (m, 1H), 3.98 (s, 2H), 3.67– 3.54 (m, 24H), 3.51–3.40 (m, 8H), 3.06 (t, J = 5.4 Hz, 2H), 1.62– 1.47 (m, 2H), 1.36– 1.25 (m, 8H), 0.89 (m, J = 6.8 Hz, 3H)

¹³C NMR (151 MHz, CDCl₃): δ 170.24, 166.64, 161.15 (d, J_CF = 249.1 Hz), 157.99, 152.47, 145.00, 140.25, 139.57 (d, J_CF = 4.1 Hz), 136.70, 133.31 (d, J_CF = 11.0 Hz), 128.35, 121.74, 119.81 (d, J_CF = 2.5 Hz), 113.43 (d, J_CF = 26 Hz), 110.47 (d, J_CF = 15 Hz), 95.29, 71.71, 71.06, 70.75, 70.68, 70.66, 70.63, 70.59, 70.55, 70.46, 70.44, 70.32, 70.12, 69.92, 69.34, 43.18, 38.78, 31.95, 29.78, 29.30, 26.19, 22.75, 14.24

¹⁹F NMR (376 MHz, CDCl₃): δ –108.37

LC-MS:

MS (ES⁺): RT = 2.462 min, m/z = 987.7, 989.6 [M + H⁺];

Spectra:

SERIES 6

The synthetic route for DNE-11PEG-CA

Preparation of compound 3

To a solution of 2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] ethoxy]ethoxy]ethoxy]ethoxy]ethanol (2.0 g, 3.98 mmol, 1.0 equiv) in THF (40 ml) was added NaH (239 mg, 5.97 mmol, 60% purity, 1.5 equiv) at 0 °C and stirred at 25 °C for 1 h. Then 1-chloro-6-iodo-hexane (980 mg, 3.98 mmol, 1.0 equiv) was added and the mixture was stirred at 25 °C for 12 h. The reaction mixture was quenched by addition of NH₄Cl (5 ml) and then diluted with H₂O (30 ml) and extracted with EtOAc (3 × 30 ml). The combined organic layers were washed with brine (2 × 30 mL), dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, CH₂Cl₂: MeOH from 1/0 to 10/1) to give 2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(6-chlorohexoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethanol (600 mg, 24% yield) as a colorless oil and used in the next step without further purification.

LC-MS:

MS (ES⁺): RT = 0.589 min, m/z = 621.4 [M + H⁺]

Spectra:

Preparation of compound 4

To a solution of 2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(6-chlorohexoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethanol (600 mg, 966 μmol, 1.0 equiv) and TEA (293 mg, 2.90 mmol, 3.0 equiv) in CH₂Cl₂ (5 ml) was added 4-methylbenzenesulfonyl chloride (368 mg, 1.93 mmol, 2.0 equiv) at 0 °C. The mixture was stirred at 25 °C for 12 h. The reaction mixture was diluted with H₂O (30 ml) and extracted with EtOAc (3 × 20 ml). The combined organic layers were washed with brine (2 × 20 ml), dried over Na₂SO₄, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, DCM: MeOH from 1/0 to 10/1) to give 2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(6-chlorohexoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl 4-methylbenzenesulfonate (600 mg, 80% yield) as a yellow oil.

LC–MS:

MS (ES⁺): RT = 0.973 min, m/z = 775.4[M + H⁺];

Preparation of compound 5

Known compound from WO2023059605 A1 2023-04-13

Preparation of DNE-11PEG-CA

To a solution of 2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(6-chlorohexoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl 4-methylbenzenesulfonate (117 mg, 151 μmol, 1.2 equiv) and5-[[[3-ethyl-5-[(2S)-2-(2-hydroxyethyl)-1-piperidyl]pyrazolo[1,5-a]pyrimidin-7-yl]amino]methyl]pyridin-2-ol (50 mg, 126 μmol, 1.0 equiv) in DMF (2 ml) was added K₂CO₃ (35 mg, 2525 μmol, 2.0 equiv). The mixture was stirred at 50 °C for 12 h. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge 150*25 mm*5 μm; mobile phase: [water(NH₄HCO₃)–MeCN]; B%: 58%–88%, 9 min) to give 2-[(2S)-1-[7-[[6-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(6-chlorohexoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-3-pyridyl]methylamino]-3-ethyl-pyrazolo[1,5-a]pyrimidin-5-yl]-2-piperidyl]ethanol (42 mg, 32% yield) as a brown gum.

¹H NMR (400 MHz, CDCl₃): δ 8.12 (d, 1H, J = 2.2 Hz), 7.61 (s, 1H), 7.59 (d, 1H, J = 8.6 Hz), 6.78 (d, 1H, J = 8.4 Hz), 6.38 (t, 1H, J = 5.4 Hz), 5.27 (s, 1H), 5.10–5.01 (m, 1H), 4.49– 4.39 (m, 4H), 3.86– 3.77 (m, 2H), 3.71– 3.68 (m, 2H), 3.68– 3.59 (m, 38H), 3.58– 3.54 (m, 3H), 3.51 (t, 2H, J = 6.8 Hz), 3.44 (t, 2H, J = 6.7 Hz), 3.31 (t, 1H, J = 11.8 Hz), 3.07– 2.95 (m, 1H), 2.58 (d, 2H, J = 2.9, 7.5 Hz), 1.82– 1.31 (m, 16H), 1.23 (t, 3H, J = 7.6 Hz)

¹³C NMR (101 MHz, MeOD) δ 164.73, 158.86, 148.55, 146.93, 146.46, 143.56, 140.08, 127.99, 112.21, 108.01, 72.28, 72.14, 71.66, 71.56, 71.54, 71.53, 71.51, 71.49, 71.17, 70.63, 66.60, 59.44, 45.73, 43.45, 41.50, 33.75, 33.08, 30.55, 30.16, 27.72, 26.70, 26.49, 20.53, 17.14, 15.22

LC-MS:

MS (ES⁺): RT = 2.992 min, m/z = 999.6 [M + H⁺];

Spectra:

HRMS [C₄₉H₈₃ClN₆O₁₃] Cal: 358.1624; Obs: 358.1614

The synthetic route for DNN-9PEG-CA

Preparation of compound 2

To a solution of 2-[2-[2-[2-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethanol (2 g, 4.83 mmol, 1.0 equiv) in THF (20 ml) was added NaH (289 mg, 7.24 mmol, 60 % purity, 1.5 equiv) at 0 °C, the mixture was stirred at 25 °C for 0.5 h, then 1-chloro-6-iodo-hexane (1.55 g, 6.27 mmol, 1.3 equiv) was added. The mixture was stirred at 25 °C for 16 h and quenched with HCl/dioxane (1 M, 8 ml). The mixture was concentrated to give 2-[2-[2-[2-[2-[2-[2-[2-[2-(6-chlorohexoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethanol (2.57 g,). The crude material was used in the next step without further purification

LC–MS:

MS (ES⁺): RT = 0.848 min, m/z = 550.3 [M + H₂O];

Preparation of compound 3

To a solution of 2-[2-[2-[2-[2-[2-[2-[2-[2-(6-chlorohexoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethanol (2.57 g, 4.82 mmol, 1.0 equiv) in CH₂Cl₂ (20 ml) was added TEA (2.44 g, 24.1 mmol, 5.0 equiv) and 4-methylbenzenesulfonyl chloride (2.76 g, 14.4 mmol, 3.0 equiv). The reaction mixture was stirred at 25 °C for 16 h. The mixture was diluted with water (30 ml) and extracted with CH₂Cl₂ (3 × 30 ml). The combined organic layers were dried over Na₂SO₄, filtered, and the filtrate was concentratedunder reduced pressure. The residue was purified by prep-HPLC (column: Waters Xbridge C18 150*50 mm* 10 μm; mobile phase: [water (10 mM NH₄HCO₃)–MeCN]; B%: 42%–72%, 11 min) to give 2-[2-[2-[2-[2-[2-[2-[2-[2-(6-chlorohexoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl 4-methylbenzenesulfonate (500 mg, 15 % yield).

¹H NMR (400 MHz, CDCl₃): δ 7.80 (d, J = 8.4 Hz, 2H), 7.35 (d, J = 8.4 Hz, 2H), 4.20– 4.12 (m, 2H), 3.71– 3.62 (m, 28H), 3.61– 3.57 (m, 6H), 3.56– 3.51 (m, 2H), 3.48– 3.43 (m, 2H), 2.45 (s, 3H), 1.82– 1.73 (m, 2H), 1.65–1.54 (m, 2H), 1.51– 1.32 (m, 4H).

LC–MS:

MS (ES⁺): RT = 0.933 min, m/z = 704.3 [M + H₂O];

Preparation of compound 4

Known compound from WO2023059605 A1 2023-04-13

Preparation of DNN-9PEG-CA

To a solution of 5-[[[3-ethyl-5-[(2S)-2-(2-hydroxyethyl)-1-piperidyl]pyrazolo[1,5-a]pyrimidin-7-yl]amino]methyl]pyridin-2-ol (50 mg, 126 μmol, 1.0 equiv) and 2-[2-[2-[2-[2-[2-[2-[2-[2-(6-chlorohexoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl 4-methylbenzenesulfonate (130 mg, 189 μmol, 1.5 equiv) in DMF (1 ml) was added K₂CO₃ (34 mg, 252 μmol, 2.0 equiv), the mixture was stirred at 50 °C for 12 h. The mixture was filtered and concentrated to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge 150*25 mm* 5 μm;mobile phase: [water(10 mM NH₄HCO₃)-MeCN]; B%: 55%–85%, 9 min) to give 1-[2-[2-[2-[2-[2-[2-[2-[2-[2-(6-chlorohexoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]-5-[[[3-ethyl-5-[(2S)-2-(2-hydroxyethyl)-1-piperidyl]pyrazolo[1,5-a]pyrimidin-7-yl]amino]methyl]pyridin-2-one (50 mg, 44 % yield).

¹H NMR (400 MHz, MeOD): δ 7.80– 7.69 (m, 2H), 7.66– 7.59 (m, 1H), 6.60– 6.53 (m, 1H), 5.63 (s, 1H), 4.47 (s, 2H), 4.21– 4.14 (m, 2H), 4.10– 4.00 (m, 1H), 3.76 (t, J = 4.8 Hz, 2H), 3.67–3.51 (m, 34H), 3.50– 3.47 (m, 5H), 3.20– 3.08 (m, 1H), 2.63– 2.54 (m, 2H), 2.21– 2.10 (m, 1H), 1.83– 1.67 (m, 8H), 1.63– 1.52 (m, 3H), 1.49– 1.34 (m, 4H), 1.25 (t, J = 7.6 Hz, 3H).

¹³C NMR (101 MHz, MeOD) δ 164.16, 149.34, 144.46, 142.36, 139.74, 129.74, 126.93, 120.69, 117.34, 108.02, 72.11, 71.57, 71.52, 71.50, 71.49, 71.48, 71.46, 71.43, 71.41, 71.14, 69.60, 59.07, 50.79, 45.75, 43.12, 33.73, 32.95, 30.54, 30.13, 27.73, 26.48, 26.44, 20.15, 16.78, 15.05

LC-MS:

MS (ES⁺): RT = 2.906 min, m/z = 911.5 [M + H⁺];

Spectra:

HRMS [C₄₅H₇₅ClN₆O₁₁] Cal: 911.5255; Obs: 911.5215

SERIES 7

The synthetic route for JQ1-2PEG-FKBP

Preparation of compound 1.

Known compound from WO2023/59583, 2023, A1

Preparation of compound 2

To a solution of tert-butyl-N-[2-[2-[2-[benzyl(methyl)amino]ethoxy]ethoxy]ethyl]-N-tert-butoxycarbonyl-carbamate (0.5 g, 1.1 mmol, 1.0 equiv) in TFE (10 ml) was added Pd(OH)₂/C (50 mg, 10% purity) under N₂ atmosphere. The mixture was stirred under H₂ (50 psi) at 30 °C for 12 h. The mixture was filtered and concentrated under reduced pressure. The residue was purified by prep-HPLC (column: Waters Xbridge 150*25 mm* 5 μm; mobile phase: [water (10 mM NH₄HCO₃)–MeCN]; B%: 22%–52%, 10 min) to give tert-butyl N-[2-[2-[2-(methylamino)ethoxy]ethoxy]ethyl]carbamate (85 mg, 29% yield).

¹H NMR (400 MHz, CDCl₃): δ 3.64–3.50 (m, 8H), 3.31 (d, J = 4.8 Hz, 2H), 2.85–2.74 (m, 2H), 2.46 (s, 3H), 1.44 (s, 9H).

LC–MS:

MS (ES⁺): RT = 1.302 min, m/z = 363.1 [M + H⁺];

Preparation of compound 3

To a solution of 2-[3-[(1R)-3-(3,4-dimethoxyphenyl)-1-[(2S)-1-[(2S)-2-(3,4,5-trimethoxyphenyl)butanoyl]piperidine-2-carbonyl]oxy-propyl]phenoxy]acetic acid (225 mg, 324 μmol, 1.0 equiv) in DMF (2 ml) was added tert-butyl N-[2-[2-[2-(methylamino)ethoxy]ethoxy]ethyl]carbamate (85 mg, 324 μmol, 1.0 equiv), HATU (148 mg, 389 μmol, 1.2 equiv) and DIEA (84 mg, 648 μmol, 2.0 equiv). The mixture was stirred at 25 °C for 12 h. The mixture was concentrated. The residue was purified by prep-HPLC (column: Waters Xbridge 150*25 mm* 5 μm; mobile phase: [water (10 mM NH₄HCO₃)–MeCN]; B%: 52%–82%, 9 min) to give [1-[3-[2-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethyl-methyl-amino]-2-oxo-ethoxy]phenyl]-3-(3,4-dimethoxyphenyl)propyl] (2S)-1-[(2S)-2-(3,4,5-trimethoxyphenyl)butanoyl]piperidine-2-carboxylate (160 mg, 53% yield) as a colorless oil.

LC–MS:

MS (ES⁺): RT = 1.067 min, m/z = 955.6 [M + 18⁺];

Spectra:

Preparation of compound 4

To a solution of [1-[3-[2-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethyl-methyl-amino]-2-oxo-ethoxy]phenyl]-3-(3,4-dimethoxyphenyl)propyl] (2S)-1-[(2S)-2-(3,4,5-trimethoxyphenyl)butanoyl]piperidine-2-carboxylate (80 mg, 85 μmol, 1.0 equiv) in CH₂Cl₂ (1 ml) was added TFA (0.5 ml). The mixture was stirred at 25 °C for 1 h. The mixture was concentrated to give [1-[3-[2-[2-[2-(2-aminoethoxy)ethoxy]ethyl-methyl-amino]-2-oxo-ethoxy]phenyl]-3-(3,4-dimethoxyphenyl)propyl] (2S)-1-[(2S)-2-(3,4,5-trimethoxyphenyl)butanoyl]piperidine-2-carboxylate (80 mg, 99% yield, TFA salt) as a brown oil and used in the next step without further purification.

LC–MS:

MS (ES⁺): RT = 1.004 min, m/z = 838.5 [M + H⁺];

Spectra:

Preparation of compound 5

Known compound from J. Med. Chem. 2019, 62, 5191–-5216

Preparation of compound 6

To a solution of [(1R)-1-[3-(2-tert-butoxy-2-oxo-ethoxy)phenyl]-3-(3,4-dimethoxyphenyl)propyl] (2S)-1-[(2S)-2-(3,4,5-trimethoxyphenyl)butanoyl]piperidine-2-carboxylate (250 mg, 333 μmol, 1.0 equiv) in CH₂Cl₂ (2 ml) was added TFA (1 ml). The mixture was stirred at 25 °C for 2 h. The mixture was concentrated to give 2-[3-[(1R)-3-(3,4-dimethoxyphenyl)-1-[(2S)-1-[(2S)-2-(3,4,5-trimethoxyphenyl)butanoyl]piperidine-2-carbonyl]oxypropyl] phenoxy]acetic acid (230 mg, 99% yield). This compound was used in the next step without further purification or characterization.

LC-MS:

MS (ES⁺): RT = 0.742 min, m/z = 694.2 [M + H⁺];

Spectra:

Preparation of compound 7

Known compound from J. Med. Chem. 2022, 65, 6573–6592

Preparation of JQ1-2PEG-FKBP

To a solution of 2-[(9S)-7-(4-chlorophenyl)-4,5,13-trimethyl-3-thia-1,8,11,12-tetrazatricyclo[8.3.0.02,6]trideca-2(6),4,7,10,12-pentaen-9-yl]acetic acid (34 mg, 84 μmol, 1.0 equiv) in DMF (1 ml) was added HATU (38 mg, 101 μmol, 1.2 equiv), [1-[3-[2-[2-[2-(2-aminoethoxy)ethoxy]ethyl-methyl-amino]-2-oxo-ethoxy]phenyl]-3-(3,4-dimethoxyphenyl)propyl] (2S)-1-[(2S)-2-(3,4,5-trimethoxyphenyl)butanoyl]piperidine-2-carboxylate (80 mg, 84 μmol, 1.0 equiv, TFA salt) and DIEA (33 mg, 252 μmol, 3.0 equiv). The mixture was stirred at 25 °C for 1 h. The mixture was concentrated. The residue was purified by prep-HPLC (column: Waters Xbridge 150*25 mm* 5 μm; mobile phase: [water (10 mM NH₄HCO₃)-MeCN]; B%: 53%–83%, 8 min) to give [1-[3-[2-[2-[2-[2-[[2-[(9S)-7-(4-chlorophenyl)-4,5,13-trimethyl-3-thia-1,8,11,12-tetrazatricyclo[8.3.0.02,6]trideca-2(6),4,7,10,12-pentaen-9-yl]acetyl]amino]ethoxy]ethoxy]ethyl-methyl-amino]-2-oxo-ethoxy]phenyl]-3-(3,4-dimethoxyphenyl)propyl](2S)-1-[(2S)-2-(3,4,5-trimethoxyphenyl)butanoyl]piperidine-2-carboxylate (58 mg, 57% yield).

¹H NMR (400 MHz, DMSO-d₆): δ 8.31–8.19 (m, 1H), 7.51–7.39 (m, 4H), 7.31–7.10 (m, 1H), 6.94–6.79 (m, 2H), 6.79–6.65 (m, 3H), 6.65–6.48 (m, 4H), 5.84–5.45 (m, 1H), 5.30–5.02 (m, 1H), 4.89–4.80 (m, 2H), 4.50 (t, J = 6.8 Hz, 1H), 4.00 (d, J = 10.4 Hz, 1H), 3.86 (t, J = 7.2 Hz, 1H), 3.74–3.74 (m, 1H), 3.77–3.67 (m, 8H), 3.64–3.59 (m, 2H), 3.58–3.53 (m, 9H), 3.53–3.47 (m, 4H), 3.46–3.39 (m, 3H), 3.32–3.16 (m, 6H), 3.06–3.04 (m, 1H), 2.85 (s, 1H), 2.69–2.58 (m, 3H), 2.40–2.32 (m, 3H), 2.16 (d, J = 14.4 Hz, 1H), 1.95–1.87 (m, 2H), 1.71–1.49 (m, 7H), 1.46–1.34 (m, 1H), 1.22–1.11 (m, 1H), 0.84–0.74 (m, 3H).

¹³C NMR (151 MHz, CDCl₃; multiple rotamers) δ 172.79, 172.78, 172.71, 172.54, 170.77, 170.70, 170.68, 170.66, 170.57, 170.55, 169.48, 168.82, 168.68, 167.94, 167.74, 163.99, 163.96, 163.94, 158.60, 158.46, 158.33, 158.25, 157.92, 155.81, 155.79, 153.59, 153.58, 153.32, 149.99, 149.96, 149.06, 148.99, 148.97, 147.56, 147.54, 147.43, 147.40, 142.21, 142.08, 141.95, 141.54, 141.49, 137.00, 136.97, 136.90, 136.86, 136.81, 136.79, 136.73, 136.25, 136.22, 135.49, 135.43, 133.67, 133.64, 133.46, 133.40, 132.33, 131.04, 131.03, 130.93, 130.90, 130.87, 130.57, 129.98, 129.92, 129.75, 129.72, 128.84, 128.82, 120.32, 120.24, 119.73, 119.54, 119.27, 119.20, 114.34, 114.12, 114.07, 113.84, 113.75, 113.59, 113.44, 113.16, 112.92, 111.84, 111.76, 111.45, 111.39, 105.08, 105.04, 104.67, 75.96, 75.93, 70.91, 70.57, 70.50, 70.40, 70.07, 69.95, 69.26, 68.74, 67.27, 67.19, 67.08, 60.91, 56.43, 56.06, 55.98, 54.54, 52.19, 51.28, 50.93, 50.86, 49.23, 48.21, 43.58, 39.79, 39.53, 39.50, 39.38, 39.32, 38.36, 38.28, 38.17, 36.26, 36.24, 34.06, 31.56, 31.36, 31.32, 30.45, 28.54, 28.52, 26.98, 26.77, 26.72, 25.50, 25.46, 24.66, 21.11, 21.03, 14.54, 13.23, 12.82, 12.79, 12.74, 12.72, 11.98

LC-MS:

MS (ES⁺): RT = 2.435 min, m/z = 1221.6 [M + H⁺];

Spectra:

HRMS [C₆₄H₇₈ClN₇O₁₃S] Cal: 1220.514; Obs: 1220.5098

The synthetic route for JQ1-4PEG-FKBP

Preparation of compound 1

Known compound from US9096844, 2015, B2

Preparation of 3

To a solution of 2-[3-[3-(3,4-dimethoxyphenyl)-1-[1-[(2S)-2-(3,4,5-trimethoxyphenyl)butanoyl]piperidine-2-carbonyl]oxy-propyl]phenoxy]acetic acid (185 mg, 267 μmol, 1.0 equiv) and tert-butyl N-tert-butoxycarbonyl-N-[2-[2-[2-[2-[2-(methylamino)ethoxy]ethoxy]ethoxy]ethoxy]ethyl]carbamate (120 mg, 267 μmol, 1.0 equiv) in DMF (2.0 ml) was added DIEA (138 mg, 1.07 mmol, 4.0 equiv) and HATU (122 mg, 320 μmol, 1.2 equiv). The mixture was stirred at 25 °C for 2 h. The mixture was purified by prep-HPLC (column: Waters Xbridge 150*25 mm* 5 μm; mobile phase: [water(10 mM NH₄HCO₃)–MeCN]; B%: 62%–92%, 8 min) to give [1-[3-[2-[2-[2-[2-[2-[2-[bis(tert-butoxycarbonyl)amino]ethoxy]ethoxy]ethoxy]ethoxy]ethyl-methyl-amino]-2-oxo-ethoxy]phenyl]-3-(3,4-dimethoxyphenyl)propyl]1-[(2S)-2-(3,4,5-trimethoxyphenyl)butanoyl]piperidine-2-carboxylate (200 mg, 64 % yield).

LC–MS:

MS (ES⁺): RT = 1.153 min, m/z = 926.5 [M – 200];

Spectra:

Preparation of 4

To a solution of [(1R)-1-[3-[2-[2-[2-[2-[2-[2-[bis(tert-butoxycarbonyl)amino]ethoxy]ethoxy]ethoxy]ethoxy]ethyl-methyl-amino]-2-oxo-ethoxy]phenyl]-3-(3,4-dimethoxyphenyl)propyl](2S)-1-[(2S)-2-(3,4,5-trimethoxyphenyl)butanoyl]piperidine-2-carboxylate (100 mg, 88 μmol, 1.0 equiv) in CH₂Cl₂ (2.0 ml) was added TFA (1.54 g, 13.5 mmol, 1.0 ml, 152.1 equiv). The mixture was stirred at 25 °C for 1 h. The mixture was concentrated to give the crude product [(1R)-1-[3-[2-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethyl-methyl-amino]-2-oxo-ethoxy]phenyl]-3-(3,4-dimethoxyphenyl)propyl](2S)-1-[(2S)-2-(3,4,5-trimethoxyphenyl)butanoyl]piperidine-2-carboxylate (92 mg, crude, TFA salt). This compound was used in the next step without further purification or characterization.

LC-MS:

MS (ES⁺): RT = 0.797 min, m/z = 926.4 [M + H⁺];

Spectra:

Preparation of JQ1-4PEG-FKBP

To a solution of [(1R)-1-[3-[2-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethyl-methyl-amino]- 2-oxo-ethoxy]phenyl]-3-(3,4-dimethoxyphenyl)propyl] (2S)-1-[(2S)-2-(3,4,5-trimethoxyphenyl)butanoyl] piperidine-2-carboxylate (92 mg, 88 μmol, 1.0 equiv, TFA salt) and 2-[(9S)-7-(4-chlorophenyl)-4,5,13-trimethyl-3-thia-1,8,11,12-tetrazatricyclo[8.3.0.0^2,6]trideca-2(6),4,7,10,12-pentaen-9-yl]acetic acid (35 mg, 88 μmol, 1.0 equiv) in DMF (2.0 ml) was added DIEA (57 mg, 442 μmol, 77 μl, 5.0 equiv) and HATU (40 mg, 106 μmol, 1.2 equiv). The mixture was stirred at 25 °C for 1 h. The mixture was purified by prep-HPLC (column: Phenomenex Gemini-NX C18 75*30 mm*3 μm;mobile phase: [water(10mM NH₄HCO₃)–MeCN]; B%: 50%–80%, 8 min) to give [(1R)-1-[3-[2-[2-[2-[2-[2-[2-[[2-[(9S)-7-(4-chlorophenyl)-4,5,13-trimethyl-3-thia-1,8,11,12-tetrazatricyclo[8.3.0.0^2,6]trideca-2(6),4,7,10,12-pentaen-9-yl]acetyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethyl-methyl-amino]-2-oxo-ethoxy]phenyl]-3-(3,4-dimethoxyphenyl)propyl](2S)-1-[(2S)-2-(3,4,5-trimethoxyphenyl)butanoyl]piperidine-2-carboxylate (57 mg, 49 % yield).

¹H NMR (400 MHz, MeOD): δ 7.49–7.36 (m, 4 H), 7.22– 7.08 (m, 1 H), 6.96– 6.65 (m, 5 H), 6.64– 6.43 (m, 3 H), 5.85– 5.52 (m, 1 H), 5.46– 5.32 (m, 1 H), 4.92–4.89 (m, 1 H), 4.84–4.77 (m, 1 H), 4.62 (dd, J=9.2, 5.2 Hz, 1 H), 4.54– 4.03 (m, 1 H), 3.89– 3.73 (m, 9 H), 3.71–3.51 (m, 26 H), 3.50– 3.40 (m, 3 H), 3.14– 3.10 (m, 1 H), 3.04– 2.81 (m, 2 H), 2.81– 2.61 (m, 4 H), 2.61–2.35 (m, 5 H), 2.32– 2.21 (m, 1 H), 2.09– 1.80 (m, 3 H), 1.78– 1.41 (m, 8 H), 1.30– 1.16 (m, 1 H), 0.91– 0.80 (m, 3 H).

¹³C NMR (151 MHz, CDCl₃; multiple rotamers) δ 172.79, 172.70, 172.53, 170.76, 170.68, 170.66, 170.56, 168.55, 168.40, 167.82, 167.62, 163.93, 158.63, 158.48, 158.35, 158.27, 155.82, 153.58, 153.32, 149.97, 149.06, 148.99, 148.97, 147.56, 147.55, 147.43, 147.41, 142.07, 141.93, 141.54, 141.49, 137.00, 136.97, 136.85, 136.83, 136.75, 136.73, 136.25, 136.22, 135.48, 135.43, 133.67, 133.63, 133.45, 133.40, 132.33, 131.05, 130.86, 130.59, 130.00, 129.92, 129.72, 128.83, 120.32, 120.24, 119.73, 119.50, 119.27, 119.18, 114.30, 114.09, 113.96, 113.89, 113.67, 113.61, 113.48, 112.94, 111.84, 111.76, 111.45, 111.39, 105.08, 105.04, 104.67, 75.96, 75.92, 70.89, 70.74, 70.71, 70.68, 70.50, 70.48, 70.45, 69.98, 69.96, 69.38, 68.83, 68.68, 67.17, 67.15, 67.02, 66.98, 61.02, 60.90, 56.43, 56.12, 56.09, 56.06, 55.99, 54.51, 52.19, 51.29, 50.94, 50.88, 49.20, 48.20, 43.60, 43.59, 39.79, 39.55, 39.26, 39.23, 38.36, 38.30, 38.17, 36.26, 36.24, 33.99, 33.90, 31.57, 31.36, 31.33, 28.54, 28.52, 26.98, 26.72, 25.51, 25.47, 24.66, 21.12, 21.03, 14.55, 13.23, 12.82, 12.79, 12.74, 12.73, 11.99.

LC-MS:

MS (ES⁺): RT = 2.858 min, m/z = 1308.5 [M + H⁺];

Spectra:

HRMS [C₆₈H₈₆ClN₇O₁₅S] Cal: 1308.5664; Obs: 1308.5623

Preparation of compound 2.

Known from WO2023/59583, 2023, A1

SERIES 8

The synthetic route for BI-2PEG-FKBP

Preparation of compound 1

known compound for J. Med. Chem., 2000, 43, 1135

Preparation of compound 3

A mixture of 2-[3-[(1R)-3-(3,4-dimethoxyphenyl)-1-[(2S)-1-[(2S)-2-(3,4,5-trimethoxyphenyl)butanoyl]piperidine-2-carbonyl]oxy-propyl]phenoxy]acetic acid (189 mg, 253 μmol, 1.0 equiv), tert-butyl N-tert-butoxycarbonyl-N-[2-[2-[2-(methylamino)ethoxy]ethoxy]ethyl]carbamate (110 mg, 304 μmol, 1.0 equiv), HOBt (68.5 mg, 507 μmol, 2.0 equiv), EDCI (194 mg, 1.01 mmol, 4.0 equiv) and DIPEA (196 mg, 1.52 mmol, 6.0 equiv) in DMF (5 ml) was stirred at 20 °C for 12 h. To the reaction mixture was added 5 drops water. The residue was purified by prep-HPLC (column: Phenomenex Gemini-NX C18 75*30 mm*3 μm;mobile phase: [water(0.225%FA)–MeCN]; B%: 65%–95%, 7 min) to afford [(1S)-1-[3-[2-[2-[2-[2-[bis(tert-butoxycarbonyl)amino]ethoxy]ethoxy]ethyl-methyl-amino]-2-oxo-ethoxy]phenyl]-3-(3,4-dimethoxyphenyl)propyl] (2R)-1-[(2R)-2-(3,4,5-trimethoxyphenyl)butanoyl]piperidine-2-carboxylate (193 mg, 186 μmol, 73% yield) as a colorless oil. This compound was used in the next step without further purification or characterization.

LC–MS:

MS (ES⁺): RT = 0.850 min, m/z = 1060.4 [M + Na⁺];

Spectra:

Preparation of compound 4

A mixture of [(1S)-1-[3-[2-[2-[2-[2-[bis(tert-butoxycarbonyl)amino]ethoxy]ethoxy]ethyl-methyl-amino]-2-oxo-ethoxy]phenyl]-3-(3,4-dimethoxyphenyl)propyl] (2R)-1-[(2R)-2-(3,4,5-trimethoxyphenyl)butanoyl]piperidine-2-carboxylate (50.0 mg, 48.2 μmol, 1.0 equiv) in TFA (1 ml) and CH₂Cl₂ (2 ml) was stirred at 20 °C for 1 h. The reaction mixture was concentrated under reduced pressuer to afford [(1S)-1-[3-[2-[2-[2-(2-aminoethoxy)ethoxy]ethyl-methyl-amino]-2-oxo-ethoxy]phenyl]-3-(3,4-dimethoxyphenyl)propyl] (2R)-1-[(2R)-2-(3,4,5-trimethoxyphenyl)butanoyl]piperidine-2-carboxylate (45.0 mg, 45.9 μmol, 95% yield, TFA salt) as a yellow oil. This compound was used in the next step without further purification or characterization.

LC–MS:

MS (ES⁺): RT = 0.630 min, m/z = 838.3 [M + H⁺];

Spectra:

Preparation of compound 5

Known compound from A.CS. Med. Chem. Lett. 2019, 10, 1443–1449

Preparation of BI-2PEG-FKBP

A mixture of 4-[[(7R)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo-7H-pteridin-2-yl]amino]-3-methoxy-benzoic acid (20.0 mg, 47.0 μmol, 1.0 equiv), [(1R)-1-[3-[2-[2-[2-(2-aminoethoxy)ethoxy]ethyl-methyl-amino]-2-oxo-ethoxy]phenyl]-3-(3,4-dimethoxyphenyl)propyl] (2S)-1-[(2S)-2-(3,4,5-trimethoxyphenyl)butanoyl]piperidine-2-carboxylate (44.8 mg, 47.0 μmol, 1.0 equiv, TFA salt), HOBt (12.7 mg, 94.0 μmol, 2.0 equiv), EDCI (27.0 mg, 141 μmol, 3.0 equiv) and DIPEA (36.5 mg, 282 μmol, 6.0 equiv) in DMF (2.5 ml) was stirred at 20 °C for 12 h. To the reaction mixture was added 2 drops of water. The mixture was purified byprep-HPLC (column: Phenomenex Gemini-NX C18 75*30 mm*3 μm;mobile phase: [water(0.225%FA)–MeCN]; B%: 38%–68%, 8 min) to afford [(1R)-1-[3-[2-[2-[2-[2-[[4-[[(7R)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo-7H-pteridin-2-yl]amino]-3-methoxy-benzoyl]amino]ethoxy]ethoxy]ethyl-methyl-amino]-2-oxo-ethoxy]phenyl]-3-(3,4-dimethoxyphenyl)propyl] (2S)-1-[(2S)-2-(3,4,5-trimethoxyphenyl)butanoyl]piperidine-2-carboxylate (36.0 mg, 28.2 μmol, 60% yield) as a white solid.

¹H NMR (400 MHz, DMSO-d₆): δ 8.50–8.41 (m, 1H), 8.26–8.16 (m, 1H), 7.81 (s, 1H), 7.59–7.49 (m, 2H), 7.29–7.08 (m, 1H), 6.96–6.42 (m, 9H), 5.81–5.46 (m, 1H), 5.32–5.02 (m, 1H), 4.91–4.79 (m, 2H), 4.50–4.20 (m, 2H), 4.06–3.85 (m, 4H), 3.78–3.63 (m, 9H), 3.62–3.43 (m, 20H), 3.24 (s, 3H), 3.05–3.02 (m, 1H), 2.84 (s, 1H), 2.05–1.43 (m, 20H), 1.41–1.03 (m, 3H), 0.85–0.73 (m, 6H).

¹³C NMR (151 MHz, CDCl₃) δ 172.71, 172.64, 172.40, 170.65, 170.57, 170.39, 169.13, 167.83, 166.91, 162.95, 158.35, 158.21, 158.10, 158.01, 153.44, 153.15, 152.74, 148.90, 148.89, 148.84, 148.82, 147.42, 147.40, 147.29, 147.27, 142.05, 141.95, 141.47, 141.44, 136.87, 136.85, 136.54, 136.03, 136.02, 135.29, 135.24, 133.44, 133.22, 133.20, 129.79, 129.78, 129.62, 129.60, 120.17, 120.08, 119.62, 119.36, 119.11, 118.99, 118.90, 115.82, 115.73, 114.30, 114.04, 113.65, 113.33, 113.13, 112.94, 112.43, 111.68, 111.60, 111.30, 111.25, 110.00, 109.88, 104.87, 104.84, 104.53, 75.77, 75.74, 71.15, 70.37, 70.22, 70.16, 70.00, 69.94, 68.66, 67.84, 66.96, 66.94, 66.83, 60.86, 60.73, 56.27, 56.02, 55.89, 55.83, 52.03, 51.11, 50.78, 50.72, 49.13, 47.90, 43.42, 39.93, 39.82, 39.65, 38.24, 38.16, 37.99, 35.86, 33.69, 31.41, 31.22, 31.19, 29.16, 28.75, 28.37, 28.25, 27.62, 26.82, 25.30, 24.49, 23.31, 23.06, 20.90, 20.83, 20.77, 12.68, 12.65, 12.58, 8.66

LC–MS:

MS (ES⁺): RT = 2.076 min, m/z = 1245.7 [M + H⁺];

Spectra:

HRMS [C₆₇H₈₈N₈O₁₅] Cal: 1245.6442; Obs: 1245.6391

SERIES 9

The synthetic route for TMX-6PEG-FKBP

Preparation of compound 6

Known compound from Angew. Chem., Int. Ed. 2020, 59, 13865–13870.

Preparation of compound 8

Known compound from J. Med. Chem., 2000, 43, 1135 – 1142

Preparation of compound 1.

Known compound from WO2023/59583, 2023, A1.

Preparation of compound 2

To a solution of tert-butyl (2-(2-(2-azidoethoxy)ethoxy)ethyl)(methyl)carbamate (800 mg, 1.72 mmol, 1.0 equiv) in THF (20 ml) was added Pd/C (0.2 g, 10% purity), and then it was degassed and purged with H₂. The reaction mixture was stirred at 25 °C for 12 h under 15 psi pressure. After filtration, the filtrate was concentrated to afford tert-butyl (2-(2-(2-aminoethoxy)ethoxy)ethyl)(methyl)carbamate (800 mg, crude) as a colorless oil, and used for the next step directly without further purification.

¹H NMR:

(400 MHz, CDCl₃) δ 3.77– 3.52 (m, 26H), 3.44– 3.35 (m, 2H), 2.91 (s, 3H), 1.46 (s, 9H)

LC–MS:

MS (ES⁺): RT = 0.555 min, m/z = 439.0 [M + H⁺];

Preparation of compound 4

To a solution of tert-butyl (2-(2-(2-aminoethoxy)ethoxy)ethyl)(methyl)carbamate (800 mg, 1.82 mmol, 1.0 equiv) in CH₂Cl₂ (10 ml) was added 4-nitrobenzenesulfonyl chloride (606 mg, 2.74 mmol, 1.5 equiv) and DIEA (1.18 g, 9.12 mmol, 1.6 ml, 5.0 equiv).The mixture was stirred at 20 °C for 1 h. The reaction mixture was quenched by 5 ml MeOH, and then it was concentrated under reduced pressure to afford crude product. The residue was purified by prep-HPLC (column: Waters Xbridge C18 150*50 mm*10 μm; mobile phase: [water(10 mM NH₄HCO₃)–MeCN]; B%: 38%–68%, 11 min) to afford tert-butyl methyl(2-(2-(2-((4-nitrophenyl)sulfonamido)ethoxy)ethoxy)ethyl)carbamate (240 mg, 384 μmol, 21% yield) as a yellow oil.

¹H NMR:

(400 MHz, CDCl₃) δ 8.36 (d, J = 8.8 Hz, 2H), 8.10 (d, J = 8.8 Hz, 2H), 6.51– 6.03 (m, 1H), 3.71– 3.51 (m, 24H), 3.45– 3.33 (m, 2H), 3.20 (brt, J = 4.6 Hz, 2H), 2.91 (s, 3H), 1.46 (s, 9H)

LC–MS:

MS (ES⁺): RT = 1.361 min, m/z = 524.4 [M -Boc + H⁺];

Preparation of compound 5

To a solution of tert-butyl methyl(2-(2-(2-((4-nitrophenyl)sulfonamido)ethoxy)ethoxy)ethyl)carbamate (240 mg, 384 μmol, 1.0 equiv) in TFE (10 ml) was added Pd/C (50 mg, 10% purity), and then it was degassed and purged with H₂. The resulting solution was stirred at 20 °C for 12 h under 15 psi pressure. The reaction mixture was filtered and the filtrate was concentrated to afford tert-butyl (2-(2-(2-((4-aminophenyl)sulfonamido)ethoxy)ethoxy)ethyl)(methyl)carbamate (220 mg, 370 μmol) as a colorless oil and used for the next step directly.

LC–MS:

MS (ES⁺): RT = 0.867 min, m/z = 494.3 [M + Na] ⁺

Spectra:

Preparation of compound 7

To a solution of tert-butyl (2-(2-(2-((4-aminophenyl)sulfonamido)ethoxy)ethoxy)ethyl)(methyl)carbamate (210 mg, 353 μmol, 1.0 equiv) and 2-((5-bromo-2-chloropyrimidin-4-yl)amino)-6-fluorobenzamide (147 mg, 425 μmol, 1.2 equiv) in iPrOH (4 ml) was added HCl (12 M, 29.47 μl, 1.0 equiv).The mixture was stirred at 95 °C for 12 h. The reaction mixture was concentrated under reduced pressure to afford crude product. The residue was purified by prep-HPLC (column: Phenomenex luna C18 150*40 mm*15 μm; mobile phase: [water (0.1%TFA)–MeCN]; B%: 15%–45%, 11 min) to afford 2-((5-bromo-2-((4-(N-(2-(2-(2-(methylamino)ethoxy)ethoxy)ethyl)sulfamoyl)phenyl)amino)pyrimidin-4-yl)amino)-6-fluorobenzamide (200 mg, 218 μmol, 61% yield, TFA salt) was obtained as a yellow gum.

¹H NMR:

(400 MHz, CD₃OD) δ 10.14 (s, 1H), 9.96 (s, 1H), 8.52 (brs, 2H), 8.39 (s, 1H), 8.24 (brd, J = 8.3 Hz, 1H), 8.14 (brd, J = 17.9 Hz, 2H), 7.85 (d, J = 8.8 Hz, 2H), 7.64 (d, J = 8.8 Hz, 2H), 7.56– 7.46 (m, 2H), 7.09 (t, J = 9.2 Hz, 1H), 6.88– 5.83 (m, 2H), 3.69– 3.61 (m, 2H), 3.48 (brd, J = 3.5 Hz, 15H), 3.46– 3.41 (m, J = 3.1, 5.1 Hz, 4H), 3.38 (t, J = 5.9 Hz, 2H), 3.14– 3.04 (m, 2H), 2.92– 2.82 (m, J = 5.8 Hz, 2H), 2.57 (t, J = 5.4 Hz, 3H)

LC–MS:

MS (ES⁺): RT = 0.800 min, m/z = 804.1 [M + 2] ⁺

Preparation of TMX-6PEG-FKBP

To a solution of 2-(3-((R)-3-(3,4-dimethoxyphenyl)-1-(((S)-1-((R)-2-(3,4,5-trimethoxyphenyl)butanoyl)piperidine-2-carbonyl)oxy)propyl)phenoxy)acetic acid (80 mg, 87 μmol, 1.0 equiv, TFA salt) and 2-((5-bromo-2-((4-(N-(2-(2-(2-(methylamino)ethoxy)ethoxy)ethyl)sulfamoyl)phenyl)amino)pyrimidin-4-yl)amino)-6-fluorobenzamide (67 mg, 96 μmol, 1.1 equiv) in DMF (1 ml) was added HATU (49 mg, 130 μmol, 1.5 equiv) and DIEA (59 mg, 459 μmol, 80 μl, 5.2 equiv).The mixture was stirred at 20 °C for 1 h. The reaction mixture was quenched by (0.1 ml) water. The residue was purified by prep-HPLC (column: Phenomenex Gemini-NX C18 75*30 mm*3 μm; mobile phase: [water(10mM NH₄HCO₃)–MeCN]; B%: 50%–80%, 8 min) to afford (S)-(R)-1-(3-((23-(4-((5-bromo-4-((2-carbamoyl-3-fluorophenyl)amino)pyrimidin-2-yl)amino)phenylsulfonamido)-3-methyl-2-oxo-6,9,12,15,18,21-hexaoxa-3-azatricosyl)oxy)phenyl)-3-(3,4-dimethoxyphenyl)propyl 1-((R)-2-(3,4,5-trimethoxyphenyl)butanoyl)piperidine-2-carboxylate (42 mg, 28 μmol, 32% yield) as a white solid.

LC–MS:

MS (ES⁺): RT = 3.355 min, m/z = 740.4 [M/2 + H⁺]

¹H NMR:

(400 MHz, CD₃OD) δ 8.35 (d, J = 8.3 Hz, 1H), 8.26 (s, 1H), 7.83 (d, J = 8.7 Hz, 2H), 7.71 (d, J = 8.7 Hz, 2H), 7.55– 7.44 (m, 1H), 7.33– 7.08 (m, 1H), 7.04– 6.62 (m, 6H), 6.61–6.54 (m, 2H), 6.53– 6.44 (m, 1H), 5.59– 5.50 (m, 1H), 5.38 (brs, 1H), 4.96– 4.87 (m, 2H), 4.83– 4.78 (m, 1H), 4.12– 4.01 (m, 1H), 3.89– 3.62 (m, 17H), 3.62– 3.41 (m, 24H), 3.17–2.93 (m, 5H), 2.73 (brt, J = 12.9 Hz, 1H), 2.65– 2.33 (m, 2H), 2.27 (brd, J = 13.3 Hz, 1H), 2.15–1.94 (m, 2H), 1.91– 1.79 (m, 1H), 1.73 (brs, 4H), 1.42 (brs, 2H), 0.95– 0.76 (m, 3H)

¹³C NMR (101 MHz, MeOD) δ 174.96, 174.93, 171.92, 171.85, 171.05, 170.41, 168.62, 162.82, 160.37, 159.64, 159.54, 159.24, 158.74, 157.79, 154.83, 154.55, 154.54, 150.32, 148.75, 145.60, 143.34, 136.93, 135.11, 133.83, 133.12, 133.02, 130.71, 128.96, 121.74, 119.88, 115.41, 115.34, 115.11, 114.28, 113.94, 113.51, 113.07, 111.65, 111.41, 106.42, 96.81, 78.09, 71.69, 71.55, 71.49, 71.44, 71.17, 70.51, 69.73, 69.17, 67.08, 61.07, 56.72, 56.52, 56.49, 56.41, 43.99, 36.23, 33.90, 32.24, 29.42, 27.65, 21.89, 12.73

19F NMR:

(376 Hz, CD₃OD) δ –112.92

HRMS [C₇₀H₉₀BrFN₈O₁₉S] Cal: 1479.5263; Obs: 1479.5227

SERIES 10

The synthetic route for HLDA-001

Preparation of compound 1

known compound for J. Med. Chem. 2019, 62, 5191–5216.

Preparation of HLDA-001

To the solution of 2-[3-[(1R)-3-(3,4-dimethoxyphenyl)-1-[(2S)-1-[(2S)-2-(3,4,5-trimethoxyphenyl)butanoyl]piperidine-2-carbonyl]oxy-propyl]phenoxy]acetic acid (50 mg, 72 μmol, 1.0 equiv) and 2-methoxy-N-methyl-ethanamine (10 mg, 112 μmol, 12 μl, 1.6 equiv) in DMF (1 ml) was added DIEA (30 mg, 230 μmol, 0.04 ml, 3.2 equiv) and HATU (41 mg, 108 μmol, 1.5 equiv), then the solution was stirred at 25 °C for 1 h. The solution was filtered to get the filtrate, which was purified by prep-HPLC (column: Phenomenex Gemini-NX C18 75*30 mm*3 μm;mobile phase: [water(0.225%FA–MeCN];B%: 48%–78%, 7 min) to get [(1R)-3-(3,4-dimethoxyphenyl)-1-[3-[2-[2-methoxyethyl(methyl)amino]-2-oxo-ethoxy]phenyl]propyl] (2S)-1-[(2S)-2-(3,4,5-trimethoxyphenyl)butanoyl]piperidine-2-carboxylate (20 mg, 36% yield) as a yellow solid.

¹H NMR (400 MHz, CDCl₃): δ 7.25–7.09 (m, 1H), 6.99–6.60 (m, 3H), 6.70–6.58 (m, 3H), 6.49–6.34 (m, 2H), 5.88–5.39 (m, 2H), 4.87–4.52 (m, 2H), 3.92–3.49 (m, 21H), 3.38–3.29 (m, 3H), 3.22–2.76 (m, 4H), 2.63–2.42 (m, 2H), 2.38–1.98 (m, 4H), 1.76–1.52 (m, 4H), 1.42–1.17 (m, 2H), 0.93–0.81 (m, 3H)

LC–MS:

MS (ES⁺): RT = 2.542 min, m/z = 765.3 [M + H⁺];

Spectra:

HRMS [C₄₂H₅₆N₂O₁₁] Cal: 765.3957; Obs: 765.3924

Reagents:

TAMRA-CA was purchased from Promega (G8251). JQ-1 (HY-13030), BI-2536 (HY-50698), dinaciclib (HY-10492) were purchased from MedChemExpress. Alexa Fluor 647 phalloidin (A22287) and DAPI (62247) were bought from Thermo Fisher Scientific. Kanamycin (K1377), lysozyme (L6876) and doxycycline hyclate (D5207) were obtained from Millipore Sigma. Polybrene (sc-134220) was purchased from Santa Cruz Biotechnology, and DNase 1 (#10104159001) was purchased from Roche. Purified 6xHis-tagged BRD4-BD1 and GST tagged-FKBP12 were supplied by Selvita S.A.

Recombinant protein production:

Protein expression and purification experiments were performed at Selvita S.A. (Kraków, Poland). BRD4-BD1: A DNA construct encompassing genes encoding for human BRD4 bromodomain 1 (BD1, aa 42–168) was codon-optimized for expression in E.coli, synthesized, and cloned into an appropriate expression vector together with the N-terminal 6xHis-tag and TEV cleavage site. The construct was transformed into BL21(DE3) (Thermo Fisher Sci. #ECO114) using a standard protocol. The preculture was prepared by inoculation of several colonies into 20 mL of LB medium supplemented with 100 μg/mL kanamycin and incubated overnight at 37 °C at 200 rpm. This culture was used to seed 500 mL LB-autoinduction medium (LB-AIM, Formedium) supplemented with 100 μg/mL kanamycin and 8 g/L glycerol in 2-L Erlenmeyer flasks and grown at 30 °C shaking at 200 rpm until OD600 reached 0.6–0.8. Then the incubation temperature was lowered to 18 °C and flasks were incubated shaking at 200 rpm overnight. The cell pellets were harvested by centrifugation at 8,000 × g, for 10 minutes, and stored frozen at −20 °C. Pellets were resuspended in the lysis buffer, 25 mM HEPES, pH 7.5, 500 mM NaCl, 5% (w/v) glycerol, 0.5 mM TCEP, 5 mM imidazole, supplemented with protease inhibitor cocktail (Sigma), 0.1 μg/mL DNAse, and 1 mg/mL lysozyme, and lysed by three snap freeze-thaw cycles. The lysates were cleared by centrifugation at 19,000 × g, 50 minutes, and the supernatant was loaded into cOmplete^™ His-Tag Purification Resin (Roche) pre-equilibrated with wash buffer, 25 mM HEPES, pH 7.5, 500 mM NaCl, 5% (w/v) glycerol, 0.5 mM TCEP, 5 mM imidazole. Non-specifically bound proteins were removed with wash buffer and specifically bound proteins were eluted using 25 mM HEPES, pH 7.5, 500 mM NaCl, 5% (w/v) glycerol, 0.5 mM TCEP, 250 mM imidazole. Elution fractions were analyzed using SDS-PAGE. The fractions containing BRD4 BD1 were pooled together, loaded into HiLoad^® 26/600 Superdex^® 75 pg (Cytiva), and eluted with 25 mM HEPES, pH 7.5, 200 mM NaCl, 0.5 mM TCEP. After SDS-PAGE analysis, fractions containing the protein of interest were pooled together and concentrated using Vivaspin 20 centrifugal concentrators with a 5 kDa cut-off (Sartorius). The concentration of BRD4 BD1 was determined spectrophotometrically at 280 nm using the calculated molar extinction coefficient. The content of residual nucleic acid was evaluated spectrophotometrically by measuring the ratio of absorptions A260/A280. FKBP12: For the production of the recombinant peptidyl-prolyl cis-trans isomerase FKBP12 F36V (aa 2–108), the DNA sequence coding for the target protein was codon optimized for expression in E.coli, synthesized, and cloned into the appropriate expression vector together with the N-terminal GST tag and HRV 3C cleavage site. The construct was transformed into BL21(DE3) using a standard protocol. The preculture was prepared using multiple colonies inoculated to the LB medium supplemented with 100 μg/mL kanamycin and incubated overnight at 37 °C at 200 rpm. Then it was used to seed 500 mL LB-autoinduction medium (LB-AIM, Formedium) supplemented with 100 μg/mL kanamycin and 8 g/L glycerol in 2-L Erlenmeyer flasks and grown at 30 °C shaking at 200 rpm until OD600 reached 0.6–0.8. Then the incubation temperature was lowered to 15 °C and flasks were incubated shaking 200 rpm overnight. The cell pellets were harvested by centrifugation at 8,000 × g, for 10 minutes, and stored frozen at −20 °C. Pellets were resuspended in 50 mM Tris, pH 7.2, 250 mM NaCl, 10% (w/v) glycerol, 0.5 mM TCEP, supplemented with 1 mg/mL lysozyme, 0.1 μg/mL DNase, and protease inhibitor cocktail (Sigma). The lysis was performed by three cycles of snap freeze-thaw. The lysate was cleared by centrifugation at 19,000 × g, 50 minutes, and the supernatant was loaded into Glutathione Sepharose 4B GST-tagged protein purification resin (Cytiva) and non-specifically bound proteins were removed by multiple washes with 50 mM Tris, pH 7.2, 250 mM NaCl, 10% (w/v) glycerol, 0.5 mM TCEP. The protein of interest was eluted using the same buffer supplemented with 15 mM reduced glutathione. The N-terminal GST-tag was removed by cleavage with HRV 3C protease in 1:100 (w/w) ratio overnight at 4 °C and then separated from the protein of interest using reverse affinity chromatography. The flow through containing FKBP12 F36V was loaded into HiLoad^® 26/600 Superdex^® 75 pg (Cytiva), and eluted with 50 mM Tris, pH 7.0, 150 mM NaCl, 1 mM TCEP, 10% (w/v) glycerol. After SDS-PAGE analysis, fractions containing FKBP12 F36V were pooled together and concentrated using Vivaspin 20 centrifugal concentrators with a 5 kDa cut-off (Sartorius). The concentration of FKBP12 F36V was determined spectrophotometrically at 280 nm using the calculated molar extinction coefficient. The content of residual nucleic acid was evaluated spectrophotometrically by measuring the ratio of absorptions A260/A280.

Immunoprecipitation and immunoblotting:

293_HFL cells were plated at 5×10⁶ cells per 75 cm² flask (#229341, cell treat scientific) in 15 ml of complete DMEM media. After 24 hours, cells were treated with indicated compounds for 3 h. After the treatments, cells were harvested and washed three times with 1xPBS. Halotag-FKBP Immunoprecipitation was performed using Halo-Trap agarose beads (# OTA-20 Proteintech) as per manufacturer instructions with some modifications. Briefly, HFL cell pellet was resuspended in 500 μL of 1X lysis buffer (#9803, Cell Signaling) for 30 min at 4°C, vortex every 10 min, and then the suspension was pelleted at 14000 × g for 10 minutes at 4°C. The supernatant was precleared with 25 μL of lysis-buffer-washed binding control agarose beads (#bab-20, Proteintech) rotating at 4°C for 1hr. Non-specific aggregates were removed by centrifugation at 2500 × g for 5 mins and 5% of the supernatant was used as the input. Remaining supernatant was incubated with 25 μL of lysis-buffer-washed Halo-Trap agarose beads rotating at 4°C for 1 hr. Halo-trap beads were washed thrice with 500 μL of lysis buffer and fractionated by SDS-PAGE. For lysate ultrafiltration, each lysate was filtered first through a 100 kDa MWCO membrane; the primary retentate (proteins > 100 kDa) was then set aside and the eluate (proteins < 100 kDa) was subsequently filtered through a 50 kDa MWCO membrane to yield a subordinate, secondary retentate (proteins between 100 and 50 kDa) as well as the final eluate (proteins < 50 kDa). Verification of effective separation by size was performed by immunoblotting of the lysate fractions for readily detectable marker proteins (GAPDH = 35 kDa; Akt = 62 kDa; vinculin = 125 kDa). Immunoblotting was done using mouse anti-FKBP (#sc-136962, Santa Cruz), rabbit anti-BRD4 (#13440, Cell Signaling), mouse anti-PLK1 (#ab17056, Abcam), rabbit anti-CDK9 (#2316, Cell Signaling), rabbit anti-phospho-RPB1-CTD Ser2 (#13499, Cell Signaling), rabbit anti-phospho-cyclin B1 Ser133 (#4133 Cell Signaling), rabbit anti-GAPDH (#2118, Cell Signaling), rabbit anti-vinculin (#13901 Cell Signaling), rabbit anti-Akt (#4691 Cell Signaling), mouse anti-HaloTag (#G9211 Promega), rabbit anti-ß-actin (#4970 Cell Signaling), rabbit anti-CDK1 (#ab265590 Abcam), rabbit anti-CDK2 (#18048 Cell Signaling), mouse anti-CDK5 (#12134 Cell Signaling) and rabbit anti-FLAG (anti-DYKDDDDK tag) (#14793 Cell Signaling). Immunoblots were developed by chemiluminscence and quantitated using Image Lab software (Promega). A four-parameter non-linear regression curve fit was applied to dose-response data in Prism (Graphpad Software) to determine the half maximal inhibitory concentration (IC50) for each compound.

CellTiterGlo viability assay:

Cells were seeded in 25 μl growth media in poly-d lysine coated black clear-bottom 384-well plates at 150 cells/well for continuous treatment or 300 cells/well for washout treatment. Following seeding, plates were spun at 300 × g for 30 seconds, then equilibrated to room temperature for 30 minutes, and moved into the incubator. 24 hours after seeding, compounds were titrated in 100% DMSO and diluted in growth medium. 25 μl of the compound/medium mixture was added to cells, bringing the total volume in each well to 50 μl. DMSO was used as a negative control. After treatment, plates were spun at 300 × g for 30 seconds, then cultured at 37°C with 5% CO₂ for 7 days in a humidified tissue culture incubator. For wash out experiments, treatment continued for 4 hours, following which the compound/media was flicked out and wells washed with 75 μl growth medium. Fresh growth medium was added, the plates spun, and cells cultured for 7 days. For competition experiments, 10 μl competition compound was added 1 hour prior to treating with 15 μl test compound, maintaining a total volume of 50 μl. On Day 7 of treatment, cell viability was quantified with CellTiter-Glo 2.0 reagent (Promega). Plates were equilibrated to room temperature for 30 minutes, then 25 μl of CellTiter-Glo 2.0 reagent was added to cells, bringing the total volume in each well to 75 μl. After reagent was added, plates were spun at 3000 × g for 30 seconds then mixed on a shaker for two minutes at 500 rpm and then incubated at room temperature for 10 minutes. Following incubation, plates were spun at 300 × g for 30 seconds, sealed with an optical adhesive cover, and luminescence readings were measured with an EnVision plate reader. Data was normalized to 0 luminescence for baseline. A four-parameter non-linear regression curve fit was applied to dose-response data in GraphPad Prism data analysis software to determine the half maximal growth inhibitory concentration (GI50) for each compound.

Biophysical in vitro co-operativity assay:

An AlphaLISA-based competition assay was developed to assess compound binding to BRD4 BD1 and cooperativity. All samples were assayed in triplicate. All incubations were performed at room temperature, and in low light condition after the addition of AlphaLISA beads (PerkinElmer). 20 nM of His-tagged BRD4 BD1 and 20 nM of biotinylated histone H4 K5/8/12/16(Ac) peptide (AnaSpec) were mixed in 20 mM HEPES pH 7.5, 100 mM NaCl, 0.5 mM TCEP, 0.1% BSA, 0.02% CHAPS, and were added into a 384-well Alpha plate (PerkinElmer). Compounds were serially diluted in the same buffer containing 1% DMSO, in the absence and presence of 8 μM of FKBP12 F36V. The compound samples were incubated for 30 min and were added to the plate. The plate was sealed and incubated for 60 min. After incubation, 10 μg/mL of streptavidin acceptor beads were added to the plate, followed by a 30 min incubation. 10 μg/mL of nickel chelate donor beads were added subsequently, followed by a final 60 min incubation. AlphaLISA signals were detected by using an EnVision plate reader. Data analysis was performed by converting raw data to percent values normalized to DMSO.

NanoBRET assay of in cellulo binding cooperativity:

All the reagents were purchased from Promega. NanoLuc-BRD4 or NanoLuc-PLK1 vector was transiently transfected into both 293_GFPL and 293_HFL cells with transfection carrier DNA using transfection reagent FuGENE HD. NanoLuc-CDK9 vector was transiently transfected into both 293_GFPL and 293_HFL cells along with CCNK expression vector using transfection reagent FuGENE HD. After 24 hours, cells were plated in 96-well solid white plates. Cells were treated with serially-diluted compounds for 4 hours in 37 degree incubator. Corresponding tracer compound for cognate NanoLuc-effector protein was added for 2 hours in 37 degree incubator. NanoBRET Nano-Glo substrate and extracellular NanoLuc inhibitor were added to cells and BRET signal was measured using Envision luminometer.

Biophysical binding constants:

Binding dissociation constants for the RIPTACs (Table S1) for their effector protein (e.g. BRD4-BD1, BRD4-BD2, CDK1/2/4/5/6/9 or PLK1) were determined at Eurofins DiscoveRx using their BROMOscan and KINOMEscan platforms. Briefly, each purified DNA-tagged effector protein under evaluation (e.g. PLK1) was captured individually on a solid support by binding to an immobilized proprietary ligand particular to the platform. Small molecules (i.e. RIPTACs or parent inhibitors) were applied to competitively displace the DNA-tagged protein from the support, and the extent of displacement at each concentration tested is measured by qPCR to provide a dissociation constant (K_d).

Intracellular accumulation:

Cells were treated with indicated compounds in 6-well plates for 4h and harvested by trypsinization. Next, cell pellets were washed 3x with 1mL PBS in microfuge tubes, the residual PBS was aspirated and pellets were weighed. Pellets weighing >10 mg were sent to Drumetix Laboratories (Greensboro, NC) for downstream analysis. Sample preparation: An appropriate volume of methanol:water 1:1 (v/v), typically with a ratio of 4 μL per mg of cell pellet, was added into each unknown cell pellet sample. The mixture was vortexed to homogenize. 20 μL of the unknown cell pellet homogenate was pipetted out into a well in 96-well plates for analysis. After spiking in 20 μL of DMSO:acetonitrile 1:1 (v/v) and 20 μL of 200 ng/mL propranolol and 500 ng/mL diclofenac in methanol:water 1:1 (v/v) as internal standard, 150 μL of chilled acetonitrile was added to precipitate protein. Samples were vortexed and centrifuged at 3500 rpm for 10 min. The supernatant, typically 2 μL, was injected for LC-MS/MS analysis. For calibration standard sample preparation, 20 μL of unknown sample homogenate was replaced with 20 μL of blank cell pellet homogenate with the same solvent volume/cell pellet weight ratio (μL/mg) as in the unknown cell pellet homogenate preparation. 20 μL of DMSO:acetonitrile 1:1 (v/v) was replaced with 20 μL of a standard working solution in DMSO:acetonitrile 1:1 (v/v). Otherwise, the calibration standard sample preparation was the same as the unknown cell pellet sample preparation. The solvent volume/cell pellet weight ratio (μL/mg) in cell pellet homogenization, the volume of DMSO:acetonitrile 1:1 (v/v) or the volume of standard working solution in DMSO:acetonitrile 1:1 (v/v), the volume of acetonitrile added to precipitate protein, and the injection volume may be adjusted to achieve different quantification limits. The concentrations of standard working solutions were usually at 0.3, 3, 30, 300, and 3000 nM, which may by adjusted for different quantification limits and calibration ranges. LC-MS/MS Analysis: Reverse phase chromatography was carried out on a Shimadzu HPLC system with LC-20AD pumps and a SIL-20AC HT autosampler. Separation was performed on an Agilent Zorbax Extend-C18 column (5 μm, 80 Å, 2.1×50 mm) with 0.1% acetic acid 1 mM ammonium in acetonitrile:water 1:9 (v/v) as mobile phase A and 50 mM acetic acid in acetonitrile as mobile phase B at a total flow rate of 0.75 mL/min. In a typical gradient elution, the initial % B is 0. In one minute, % B was increased linearly to 95%. After holding at 95% B for 0.5 min, % B was dropped to 0% and the column was equilibrated with 0% B for 0.5 min to prepare for the next injection. The eluent from the HPLC column was introduced to the TurboIon Spray source attached to an AB Sciex Triple Quad 5500 mass spectrometer, which operated in positive or negative mode, depending on the nature of a test compound. Multiple reaction monitoring (MRM) was used for the detection of test compounds and internal standards (260.1/116.1 for propranolol in positive ion mode; 293.8/249.9 for diclofenac in negative ion mode). The mass spectrometer was set at unit resolution with curtain gas at 30 psi, IS (ionspray voltage) at 5000 V (−4500 V in negative ion mode), TEMP (source temperature) at 500 °C, GS1 (nebulizer gas) at 50 psi, GS2 (heating gas) at 50 psi, and CAD (collision gas) at 6. The other parameters, DP (declustering potential), EP (entrance potential), CE (collision energy), and CXP (collision cell exit potential), were compound dependent and were optimized for each compound. The DP, EP, CE, and CXP values are 66 V, 8 V, 23 eV, and 6 V, respectively, for propranolol in positive ion mode. In negative ion mode, the DP, EP, CE, and CXP values are −50 V, −10 V, −17 eV, and −13 V, respectively, for diclofenac.

Surface Plasmon Resonance (SPR) binding assay:

SPR multi-cycle affinity experiments were performed using a Biacore 8K instrument (Cytiva) at 25°C to characterize binary/ternary binding of compounds to BRD4 BD1. Streptavidin sensor chips were used and the surface was equilibrated in running buffer containing 1X HBS-P+ buffer, 0.5 mM TCEP, 3% DMSO. The same buffer was used to extensively dialyze FKBP12 F36V (residues 2–108) to remove glycerol, prior to ternary binding experiments. For binary binding experiments, 1.2 μg/mL of biotinylated BRD4 BD1 (residues 46–168) was immobilized to a final density of 1500 RU. For ternary binding experiments, 0.2 μg/mL of protein was immobilized to a final density of 250 RU to limit mass transfer effects. Compounds were first serially diluted in DMSO and then transferred to running buffer at varying concentrations. For ternary binding samples, compounds were transferred to running buffer containing 8 μM of the FKBP F36V protein. All samples were injected for 30–40 s of association and 120 s of dissociation at a flow rate of 30 μL/min. Binding data were analyzed using the Biacore Insight evaluation software.

Immunofluorescence:

293- derived HFL, NLS2HF, and MYRHF cells were seeded at 5×10⁵ cells per well in Nunc^™ Lab-Tek^™ II CC2^™ Chamber (#154941, Thermo Scientific) in 500 μL complete DMEM medium. After 48 hours, cells were treated with 0.1 μM TAMRA (H-789) for 1 hr at 37 °C. Following treatment, cells were washed on the chamber twice with 1xPBS. 4% formaldehyde (#12606P, Cell Signaling) was added to fix cells for 15 min at room temperature, followed with 0.1% TritonX-100 permeabilization for 10min. After washing with 1xPBS three times, 300nM Alexa Fluor^® 647 Phalloidin and DAPI in 1xPBS were added to the cells for 30 min at room temperature. Cells were washed three times with 1xPBS, followed by adding 5uL of ProLong^™ Gold. Slides were removed from the chamber and the cells covered overnight with coverslips (#7816, LabScientific). Cells were imaged on a Zeiss (Oberkochen, Germany) LSM 880 Airyscan confocal microscope.

Quantification and statistical analysis

For all dose-response data (e.g. Cell Titer Glo, Nano-BRET, AlphaLISA, immunoblot and qPCR) a four-parameter non-linear regression curve fit was applied in Prism v9.4 (Graphpad Software) to determine the half maximal inhibitory concentration (IC50) for each compound. Error bars represent the standard deviation from the mean value. All data representative of 3 independent experiments (n = 3). SPR data from Biacore 8K was analyzed and fit using Biacore Insight Evaluation v3.0.12.15655 (General Electric) and western blots were quantitated with Image Lab v6.1.0 build 7.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
anti-FKBP	Santa Cruz Biotech	cat# sc-136962
anti-BRD4	Cell Signaling	cat# 13440
anti-PLKl	Abcam	cat# ab17056
anti-CDK9	Cell Signaling Tech.	cat# 2316
anti-phospho-RPBl-CTD Ser2	Cell Signaling Tech.	cat# 13499
anti-phospho-cyclin B1 Ser133	Cell Signaling Tech.	cat# 4133
anti-GAPDH	Cell Signaling Tech.	cat# 2118
anti-vinculin	Cell Signaling Tech.	cat# 13901
anti-Akt	Cell Signaling Tech.	cat# 4691
anti-FLAG (anti-DYKDDDDK tag)	Cell Signaling Tech.	cat# 14793
anti-HaloTag	Promega	cat# G9211
anti-β-actin	Cell Signaling Tech.	cat# 4970
anti-CDK1	Abcam	cat# ab265590
anti-CDK2	Cell Signaling Tech.	cat# 18048
anti-CDK5	Cell Signaling Tech.	cat# 12134
Bacterial and virus strains
E. coli BL21(DE3)	Thermo Fisher Scientific	cat# ECO114
Chemicals, peptides, and recombinant proteins
JQ-1	Med Chem Express	cat# HY-13030
BI-2536	Med Chem Express	cat# HY-50698
dinaciclib	Med Chem Express	cat# HY-10492
TMX3013	WuXi AppTec	cmpd# WX-EW24502–164
TAMRA-CA	Promega	cat# G8251
kanamycin	Millipore Sigma	cat# K1377
DNase 1	Roche	cat#10104159001
lysozyme	Millipore Sigma	cat# L6876
puromycin	Invivogen	cat# ant-pr-1
polybrene	Santa Cruz Biotech.	cat# sc-134220
doxycycline hyclate	Millipore Sigma	cat# D5207
penicillin/streptomycin	Thermo Fisher Scientific
AlexaFluor 647 Phalloidin	Thermo Fisher Scientific	cat# A22287
DAPI	Thermo Fisher Scientific	cat# 62247
6xHis-BRD4 BD1	Selvita S.A.	cat# 6xHis-BRD4 BD1
GST-FKBP12	Selvita S.A.	cat# GST-FKBP12
JQ1-4PEG-CA	this manuscript	n/a
JQ1-4PEG-deschloro	this manuscript	n/a
JQ1-6PEG-CA	this manuscript	n/a
epi-JQ1-6PEG-CA	this manuscript	n/a
JQ1-2PEG-FKBP	this manuscript	n/a
JQ1-4PEG-FKBP	this manuscript	n/a
BI-2PEG-CA	this manuscript	n/a
BI-2PEG-FKBP	this manuscript	n/a
TMX-6PEG-CA	this manuscript	n/a
TMX-6PEG-deschloro	this manuscript	n/a
TMX-6PEG-FKBP	this manuscript	n/a
DNN-9PEG-CA	this manuscript	n/a
DNE-11PEG-CA	this manuscript	n/a
HLDA-001	this manuscript	n/a
D-MEM culture medium
McCoy’s 5A medium
fetal bovine serum
Critical commercial assays
Nano-BRET	Promega	cat# N1662
Cell Titer Glo	Promega	cat# G7572
Halo-Trap	Proteintech	cat# OTA-20
AlphaLISA	PerkinElmer	cat# AS101D; AL125C
Deposited data
original immunoblot images	this manuscript	Mendeley Data accession no. DOI: 10.17632/tnk48294nr.1
Experimental models: cell lines
HEK293	ATCC	cat# CRL-1573
TREx-293T	Thermo Fisher Scientific	cat# R71007
HT29	ATCC	cat# HTB-38
Recombinant DNA
Nanoluc-BRD4	Promega	cat# N1691
Nanoluc-PLK1	Promega	cat# V2841
Nanoluc-CDK9	Promega	cat# NV2871
CCNK expression vector	Promega	cat# NV2661
Software and algorithms
Prism	GraphPad Software	v9.4 for download: https://www.graphpad.com/features
Image Lab	Promega	v6.1.0 build 7 for download: https://www.bio-rad.com/en-us/product/image-lab-software?ID=KRE6P5E8Z
Biacore Insight Evaluation software	General Electric	v3.0.12.15655 for download: https://www.cytivalifesciences.com/en/us/support/software/biacore-downloads/biacore-insight-evaluation-software

Significance:

While specific cell signaling pathway inhibitors have yielded great success in oncology, precision targeting of cancer cell death is one of the great drug discovery challenges facing biomedical research. Attempts to eradicate cancer cells expressing unique target proteins, such as antibody-drug conjugates (ADCs), T-cell engaging therapies, and radiopharmaceuticals have been successful in the clinic, but only can target the limited repertoire of extracellular proteins. More recently, heterobifunctional small molecules such as Proteolysis Targeting Chimera (PROTACs) paved the way for protein proximity inducing therapeutic modalities. Our data suggests that ternary complex formation by itself, a key aspect of such “proxi-drugs”, could be leveraged to selectively kill cancer cells. Our novel therapeutic platform, called Regulated Induced Proximity Targeting Chimeras or RIPTACs, utilizes heterobifunctional small molecules to induce a co-operative protein-protein interaction between a selectively expressed target protein, and a pan-expressed essential cellular protein. This causes more potent inhibition of the essential protein selectively in cells that have the target protein due to increased occupancy of the former compared to cells that lack the target protein. One can envision the utility of the RIPTAC strategy in combatting cancer, where tumor cells often offer a higher expression of many resident proteins. The unique pharmacology of RIPTACs has a major advantage in that it would not require its target protein to be the oncogenic driver, nor be extracellularly-localized. Moreover, we propose that RIPTACs would retain activity in presence of a myriad of bypass signaling mechanisms that render tumors resistant to conventional inhibitors or protein degraders. Herein we used a model HaloTag-FKBP target protein to prove the mechanism of action of RIPTACs. Future work will go beyond chemical biology systems, and aim to demonstrate the applicability of our RIPTAC platform to targets that are selectively overexpressed in both solid tumors and hematological malignancies.

Highlights.

• Heterobifunctional RIPTACs accumulate in cells that express their Target Protein (TP)

• RIPTACs form enduring ternary complexes with TP and essential Effector Proteins (EP)

• The resultant sustained inhibition of the EPs selectively occurs in TP-expressing cells

• This strategy can be leveraged to craft new therapeutics to combat diseases (e.g. cancer)