Abstract
Previously, we reported a first-in-class von Hippel–Lindau (VHL)-recruiting mitogen-activated protein kinase kinases 1 and 2 (MEK1/2) degrader, MS432. To date, only two MEK1/2 degrader papers have been published and very limited structure–activity relationships (SAR) have been reported. Here, we describe our extensive SAR studies exploring both von Hippel–Lindau (VHL) and cereblon (CRBN) E3 ligase ligands and a variety of linkers, which resulted in two novel, improved VHL-recruiting MEK1/2 degraders, 24 (MS928) and 27 (MS934), and the first CRBN-recruiting MEK1/2 degrader 50 (MS910). These compounds potently and selectively degraded MEK1/2 by hijacking the ubiquitin-proteasome system, inhibited downstream signaling, and suppressed cancer cell proliferation. Furthermore, concurrent inhibition of BRAF or PI3K significantly potentiated the antitumor activity of degrader 27, suggesting that the combination of MEK1/2 degradation with BRAF or PI3K inhibition may provide potential therapeutic benefits. Finally, besides being more potent, degrader 27 displayed improved plasma exposure levels in mice, representing the best MEK1/2 degrader to date for in vivo studies.
Graphical Abstract
INTRODUCTION
Mitogen-activated protein kinase kinases 1 and 2 (MEK1/2) are critical components of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling transduction pathway, which transmits an extracellular signal into the nucleus through a cascade activation of multiple proteins, including Ras, Raf, MEK, and ERK.1–3 As crucial proteins in the MAPK/ERK signaling, MEK1/2 are phosphorylated and activated by its upstream RAF kinases.4 Activation of MEK1/2 consequently leads to the phosphorylation of ERK at the threonine and tyrosine residues and activation of the ERK signaling.5 The MAPK/ERK signaling pathway is associated with a broad array of cellular processes, including cell proliferation, differentiation, cell survival, and apoptosis.6,7 Aberrant regulation of this pathway through hyperactivation and mutation has been implicated in a variety of human cancers, such as melanoma, nonsmall cell lung cancer (NSCLC), colorectal cancer, primary brain tumors, and hepatocellular carcinoma.1,8–12
Pharmacological inhibition of the MAPK/ERK signaling pathway by targeting the catalytic function of Ras, Raf, MEK, and ERK has resulted in multiple FDA approved drugs and many inhibitors in clinic development.13–20 PD184352 was the first MEK1/2 inhibitor that entered clinical trials.21 However, the development of PD184352 was terminated due to the lack of clinical efficacy.22,23 Optimization of PD184352 led to another clinical MEK1/2 inhibitor PD0325901 (1) (Figure 1) with improved potency, solubility, and metabolic stability.24 PD0325901 is a typical non-ATP competitive MEK1/2 inhibitor, which occupies an allosteric binding pocket adjacent to the ATP binding site.25 Numerous MEK inhibitors have been reported subsequently.26 Although MEK inhibitors can be used as a single agent to treat diseases, the combination of MEK and BRAF inhibitors has shown delayed drug resistance and prolonged progression-free survival.27–29 As a result, combination therapies, such as MEK inhibitor trametinib (2) (Figure 1) with BRAF inhibitor dabrafenib, MEK inhibitor cobimetinib with BRAF inhibitor vemurafenib, and MEK inhibitor binimetinib with BRAF inhibitor encorafenib, have been approved by the FDA for treating BRAF-mutated melanoma.30–33 These combination therapies have shown good efficacies for treating melanoma patients. However, the acquired drug resistance through reactivation and mutation has been reported.34,35 Therefore, new therapeutic strategies to delay or overcome drug resistance are desired.
Figure 1.
Chemical structures of MEK inhibitors 1 and 2, and MEK degraders 3 and 4.
Recently, proteolysis targeting chimeras (PROTACs) have emerged as a novel therapeutic strategy by reducing the protein level of oncological targets.36–39 PROTACs are heterobifunctional small molecules, composing of a ligand that binds the target protein, a ligand of an E3 ligase, and a linker that connects these two ligands. PROTACs bring the target protein and E3 ligase to close proximity, resulting in polyubiquitination of the target protein and its subsequent degradation by hijacking the ubiquitin-proteasome system (UPS).39 The PROTAC technology has been applied to the degradation of the protein targets in the MAPK/ERK signaling pathway, including Ras,40 Raf,41–43 MEK,44,45 and ERK.46 In addition to the important functions of MEK1/2 in the canonical ERK signaling cascade, MEK1/2 possess other biological roles by phosphorylation of MyoD, HSF1, and β-arrestin 2.47–49 Furthermore, MEK1/2 have noncatalytic functions, which have been associated with nuclear export of ERK and PPAR, repression of MyoD transactivation, and regulation of FOXO1 localization.50–53 Therefore, reduction of MEK1/2 protein levels using PROTACs is expected to diminish both catalytic and noncatalytic functions of the proteins and could have more profound pharmacological effects than inhibition of the kinase activity alone.
Recently, we reported a first-in-class PD0325901-derived MEK degrader 3 (MS432) (Figure 1), which recruits the E3 ligase von Hippel–Lindau (VHL).44 Compound 3 potently degraded MEK1/2 proteins and effectively inhibited the proliferation of BRAF-mutated colorectal cancer cell line HT-29 and melanoma cell line SK-MEL-28. Shortly after this first publication, Vollmer et al. reported another VHL-recruiting MEK1/2 degrader 4 (Figure 1), which effectively inhibited the proliferation of melanoma cell line A375.45 To date, these two papers are the only MEK1/2 degrader papers. Furthermore, very limited structure–activity relationships (SAR) were reported in these two studies.
Here, we report extensive SAR studies of the PD0325901-derived MEK1/2 degraders bearing a variety of alkyl or polyethylene glycol (PEG) linkers by recruiting either VHL or cereblon (CRBN) E3 ligase. These SAR studies have led to two improved VHL-recruiting MEK1/2 degraders, 24 (MS928) and 27 (MS934), and the first CRBN-recruiting MEK1/2 degrader 50 (MS910). We also developed a negative control for each of these three MEK1/2 degraders.
RESULTS AND DISCUSSION
Structure–Activity Relationship Studies.
Previously, we evaluated the effect of a very limited number of PD0325901-derived putative MEK1/2 degraders (3, 10, 14, 19, 28, and 35) on degrading MEK1/2 in HT-29 cells by Western blots.44 To directly compare the potency of these previously reported compounds with new compounds we synthesized, we used HT-29 cells as the primary cell line for screening all of the putative degraders in this extensive SAR study.
First, we assessed the antiproliferation potency of VHL-recruiting MEK1/2 compounds bearing alkylene (Table 1) or PEG (Table 2) linkers by treating HT-29 cells for 3 days. As illustrated in Table 1, compounds with shorter alkylene linkers, such as methylene (5), ethylene (6), propylene (7), butylene (8), pentylene (9), and hexylene (10),44 were less potent (GI50 = 1.4–5.0 μM) than the ones with longer linkers (GI50 = 0.4–0.6 μM), such as heptylene (11), octylene (12), nonylene (13), and decylene (14).44 Compounds with one to four PEG units in the linker moiety (15–21) did not display high potency at inhibiting HT-29 cell growth (GI50 = 1.3–4.8 μM, Table 2).
Table 1.
Antiproliferation Potency of Compounds 5–14 in HT-29 Cellsa
 | ||
|---|---|---|
| cpd. | n | HT-29 GI50 (μM) |
| 5 | 1 | 1.8 ± 0.4 |
| 6 | 2 | 5.0 ± 1.1 |
| 7 | 3 | 3.1 ± 0.9 |
| 8 | 4 | 3.0 ± 0.5 |
| 9 | 5 | 2.1 ± 0.1 |
| 10b | 6 | 1.4 ± 0.2 |
| 11 | 7 | 0.5 ± 0.2 |
| 12 | 8 | 0.6 ± 0.1 |
| 13 | 9 | 0.5 ± 0.03 |
| 14b | 10 | 0.4 ± 0.2 |
GI50 values, represented by mean value ± standard deviation (SD), were obtained from at least two independent experiments. In GI50 curves, each concentration point was performed in duplicate or triplicate.
The compounds were previously reported.44
Table 2.
|
GI50 values, represented by mean value ± SD, were obtained from at least two independent experiments. In GI50 curves, each concentration point was performed in duplicate or triplicate.
The compound was previously reported.44
In our previous study, we showed that 14 (GI50 = 0.4 ± 0.2 μM) was able to significantly degrade MEK1/2 protein levels in HT-29 cells at 0.3, 1, and 3 μM concentrations.44 To confirm the MEK1/2 degradation capability of compounds with similar antiproliferation potency, we treated HT-29 cells with 11 (GI50 = 0.5 ± 0.2 μM) or 13 (GI50 = 0.5 ± 0.03 μM) at 0.3, 1, and 3 μM concentrations for 24 h. As illustrated in Figure 2, similar to 14, both 11 and 13 effectively degraded MEK1 and MEK2 proteins.
Figure 2.
Effect of compounds 11, 13, and 14 on MEK1/2 protein levels in HT-29 cells. The cells were treated with dimethyl sulfoxide (DMSO) or serial dilution of indicated compounds for 24 h. MEK1/2 protein levels were determined by Western blots.
We previously showed that the addition of a benzylic methyl group to the VHL binding moiety significantly improved the MEK1/2 degradation potency of the degraders.44 Consistent with this result, compound 3 (GI50 = 130 ± 38 nM, Table 3) showed 3-fold antiproliferation potency improvement over compound 14 (GI50 = 0.4 ± 0.2 μM, Table 1). Shorten the linker length from decylene (3) to nonylene (22) slightly decreased antiproliferation potency (GI50 = 240 ± 55 nM). A longer undecylene linker (23) resulted in slightly better antiproliferation potency (GI50 = 99 ± 8 nM). Interestingly, the introduction of a N-methyl group to the aminopropoxy moiety (24) improved antiproliferation potency (GI50 = 32 ± 8 nM) by about 4-fold compared with 3. In addition, extending the aminopropoxy moiety to an aminobutoxy moiety also led to improved antiproliferation potency. Compared to the aminopropoxy-containing compounds 22, 3, and 23, the corresponding aminobutoxy-bearing analogues 25, 26, and 27 showed approximately 4-, 3-, and 4-fold increased antiproliferation potency in HT-29 cells (GI50 = 55 ± 5 nM for 25; 43 ± 1 nM for 26; and 23 ± 5 nM for 27).
Table 3.
 | ||||
|---|---|---|---|---|
| cpd. | m | n | R | HT-29 GI50 (nM) |
| 22 | 1 | 7 | H | 240 ± 55 |
| 3b | 1 | 8 | H | 130 ± 38 |
| 23 | 1 | 9 | H | 99 ± 8 |
| 24 | 1 | 8 | Me | 32 ± 8 |
| 25 | 2 | 7 | H | 55 ± 5 |
| 26 | 2 | 8 | H | 43 ± 1 |
| 27 | 2 | 9 | H | 23 ± 5 |
GI50 values, represented by mean value ± SD, were obtained from at least two independent experiments. In GI50 curves, each concentration point was performed in duplicate or triplicate.
The compound was previously reported.44
Next, we determined MEK1/2 degradation potencies of the two most potent VHL-recruiting compounds 24 and 27. As shown in Figures 3A, S1A, S1C, and Table 4, both 24 and 27 potently reduced MEK1/2 protein levels (24: DC50 = 18 ± 3 nM for MEK1 and 8 ± 1 nM for MEK2; 27: DC50 = 18 ± 1 nM for MEK1 and 9 ± 3 nM for MEK2) and inhibited phosphorylation of MEK and ERK in a concentration-dependent manner in HT-29 cells. In SK-MEL-28 cells, 24 and 27 also effectively reduced MEK1/2 protein levels (24: DC50 = 16 ± 3 nM for MEK1 and 6 ± 1 nM for MEK2; 27: DC50 = 10 ± 1 nM for MEK1 and 4 ± 1 nM for MEK2) and inhibited phosphorylated MEK and ERK levels (Figures 3B, S1B, S1D, and Table 4). Taken together, these results indicate that compounds 24 and 27 are improved MEK1/2 degraders, which are more potent than the previously reported MEK1/2 degrader, compound 3, at reducing MEK1/2 protein levels, inhibiting MEK and ERK phosphorylation, and suppressing cancer cell growth.
Figure 3.
Compounds 24 and 27 degrade MEK1 and MEK2 and inhibit pMEK and pERK in a concentration-dependent manner. HT-29 (A) and SK-MEL-28 (B) cells were treated with DMSO or serial dilution of indicated compounds for 24 h. The indicated protein levels were determined by Western blots.
Table 4.
MEK1/2 Degradation Potencies of Compounds 24 and 27a
| cell line | target protein | DC50 (compound 24, nM) | DC50 (compound 27, nM) |
|---|---|---|---|
| HT-29 | MEK1 | 18 ± 3 | 18 ± 1 |
| MEK2 | 8 ± 1 | 9 ± 3 | |
| SK-MEL-28 | MEK1 | 16 ± 3 | 10 ± 1 |
| MEK2 | 6 ± 1 | 4 ± 1 |
DC50 values are presented as the mean ± SD from three independent experiments.
In addition to the above VHL-recruiting MEK1/2 degraders, we also investigated SAR of CRBN-recruiting compounds. First, we designed putative MEK1/2 degraders by attaching a variety of linkers to the 4-amino group of pomalidomide and evaluated their antiproliferative activity in HT-29 cells (Table 5). Compared to the previously reported compound 28,44 which possesses the shortest ethylene linker, CRBN-recruiting analogues bearing a longer alkyl linker, such as propylene (29), butylene (30), pentylene (31), hexylene (32), heptylene (33), or octylene (34), did not improve the antiproliferation potency in HT-29 cells. For compounds with PEG linkers, longer linkers with four (38: GI50 = 0.6 ± 0.09 μM) and five (39: GI50 = 0.6 ± 0.1 μM) PEG units resulted in slightly more potent compounds than shorter PEG linkers (35–37). Next, we explored another set of MEK1/2 putative degraders by attaching a variety of alkyl (40–45) and PEG (46–50) linkers to the 5-amino group of pomalidomide (Table 6). In general, derivatization at the 5-position of pomalidomide provided more potent compounds compared to the ones derived from the 4-position. The compound with the longest alkyl (45: GI50 = 0.3 ± 0.1 μM) or PEG (50: GI50 = 0.3 ± 0.06 μM) linker showed the best potency against the growth of HT-29 cells.
Table 5.
|
GI50 values, represented by mean value ± SD, were obtained from at least two independent experiments. In GI50 curves, each concentration point was performed in duplicate or triplicate.
The compound was previously reported.44
Table 6.
Antiproliferation Potency of Compounds 40–50 in HT-29 Cellsa
|
GI50 values, represented by mean value ± SD, were obtained from at least two independent experiments. In GI50 curves, each concentration point was performed in duplicate or triplicate.
Based on the antiproliferation potency of the CRBN-recruiting compounds, we chose seven most potent (GI50 = 0.3–0.5 μM) compounds, 28, 40, 42, 44, 45, 49, and 50, to evaluate their effects on MEK1/2 degradation in HT-29 cells. As illustrated in Figure 4A, compounds 28 and 40 did not show obvious MEK1/2 degradation at 0.3, 1, and 3 μM after 24 h treatment. Interestingly, compound 42 demonstrated significant selectivity at degrading MEK2 over MEK1, which is the first example that significant selectivity for degradation of MEK2 over MEK1 can be achieved. This compound could serve as a good starting point for the development of more selective MEK2 degraders. Compounds 44, 45, 49, and 50 significantly degraded both MEK1 and MEK2 proteins at 0.3, 1, and 3 μM concentrations. These results, however, revealed the significant disconnection between the antiproliferation potency and MEK1/2 degradation activity of these CRBN-recruiting compounds, suggesting that in addition to their MEK1/2 kinase inhibition activity, compounds 28, 40, and 42 may also hit some off-targets, which may include CRBN neo-substrates. A number of studies have reported that some CRBN-recruiting PROTACs indeed degrade CRBN neo-substrates induced by immunomodulatory drugs (IMiDs).54–56 Our results further suggest that antiproliferation potency alone cannot predict the degradation capability of CRBN-recruiting degraders. Because compound 50 was the most effective at both degrading MEK1/2 and inhibiting cell growth among these CRBN-recruiting compounds, we further evaluated the degradation potency of this compound in both HT-29 and SK-MEL-28 cells at a variety of compound concentrations. As shown in Figures 4B, S2, and Table 7, compound 50 potently degraded MEK1 (DC50 = 118 ± 23 nM in HT-29 cells; and 94 ± 3 nM in SK-MEL-28 cells) and MEK2 (DC50 = 55 ± 19 nM in HT-29 cells; and 38 ± 15 nM in SK-MEL-28 cells) and inhibited MEK and ERK phosphorylation in a concentration-dependent manner. To the best of our knowledge, compound 50 is the first potent CRBN-recruiting MEK1/2 degrader.
Figure 4.
(A) Effects of compounds 28, 40, 42, 44, 45, 49, and 50 on MEK1/2 protein levels in HT-29 cells. The cells were treated with DMSO or serial dilution of indicated compounds for 24 h. MEK1/2 protein levels were determined by Western blots and normalized with α-tubulin. (B) Compound 50 reduces MEK1/2 protein levels and inhibits the downstream ERK signaling in a concentration-dependent manner in HT-29 and SK-MEL-28 cells. HT-29 and SK-MEL-28 cells were treated with DMSO or serial dilution of compound 50 for 24 h. The indicated protein levels were determined by Western blots.
Table 7.
MEK1/2 Degradation Potencies of Compound 50a
| cell line | target protein | DC50 (nM) |
|---|---|---|
| HT-29 | MEK1 | 118 ± 23 |
| MEK2 | 55 ± 19 | |
| SK-MEL-28 | MEK1 | 94 ± 3 |
| MEK2 | 38 ± 15 |
DC50 values are presented as the mean ± SD from three independent experiments.
Based on considerations of structural diversity, MEK1/2 degradation potency, and antiproliferation potency, we chose compounds 24 (MS928), 27 (MS934), and 50 (MS910) for further evaluation. In addition, we designed the corresponding control compounds, 51 (MS928N), 52 (MS934N), and 53 (MS910N), for these MEK1/2 degraders. Compounds 51 and 52 are diastereomers of compounds 24 and 27 at the hydroxyproline moiety (Figure 5). It is known that such a diastereoisomer diminishes the binding to the VHL E3 ligase.57 Compound 53 bears a N-methylated glutarimide moiety (Figure 5), which disrupts the binding to the CRBN E3 ligase.58,59
Figure 5.
Chemical structures of MEK degraders and their corresponding controls.
Binding Affinities of Compounds 24, 27, and 50 and Their Control Compounds to MEK1/2.
The binding affinities of PD0325901, 24, 27, 50, and the control compounds (51–53) to MEK1/2 were determined using the KINOMEscan assay from DiscoverX (Table 8 and Figure S3). Compared with MEK1/2 inhibitor PD0325901 (Kd = 0.044 ± 0.004 μM for MEK1; and 0.10 ± 0.03 μM for MEK2), the degraders 24, 27, and 50 showed approximately 9- to 15-fold decreased binding affinities to MEK1 (Kd = 0.40–0.66 μM), and 14- to 24-fold decreased binding affinities to MEK2 (Kd = 1.4–2.4 μM). Considering the significant chemical structure changes at the nonsolvent exposed glycerol moiety of PD0325901, the decreased binding affinities of these degraders were not unexpected. On the other hand, modifications at the linker and the E3 ligand moieties were very well tolerated. In addition, the control compounds (51–53) displayed similar binding affinities to MEK1 and MEK2 as degraders 24, 27, and 50 and all of the three degraders and their control compounds showed comparable binding affinities to MEK1 and MEK2. Interestingly, although the binding affinities of degraders 24, 27, and 50 are stronger for MEK1 over MEK2, these degraders showed slightly better degradation potency for MEK2 over MEK1 (Figures 3 and 4B; Tables 4 and 7), suggesting that MEK1/2 binding affinity is not the most critical factor for determining degradation potency here.
Table 8.
MEK1/2 Binding Affinities of Compounds 24, 27, and 50–53 in KINOMEscan Assaya
| cpd. | MEK1 Kd (μM) | MEK2 Kd (μM) | cpd. | MEK1 Kd (μM) | MEK2 Kd (μM) |
|---|---|---|---|---|---|
| PD0325901 | 0.044 ± 0.004 | 0.10 ± 0.03 | 51 | 0.38 ± 0.07 | 1.7 ± 0.1 |
| 24 | 0.40 ± 0.001 | 1.4 ± 0.54 | 52 | 0.82 ± 0.07 | 2.2 ± 0.03 |
| 27 | 0.40 ± 0.08 | 2.0 ± 0.23 | 53 | 1.2 ± 0.17 | 3.8 ± 0.65 |
| 50 | 0.66 ± 0.1 | 2.4 ± 0.13 |
The experiments were performed in duplicate and the Kd values are presented as the mean ± SD.
Kinetics of MEK1/2 Degradation Induced by Compounds 24, 27, and 50.
We next conducted time-course experiments to understand the kinetics of MEK1/2 degradation and downstream signaling inhibition induced by compounds 24, 27, and 50 in HT-29 cells. As shown in Figure 6, the VHL-recruiting degraders 24 and 27 (at 0.1 μM) induced significant MEK1/2 degradation and ERK phosphorylation inhibition within 2 h, and the maximum degradation (Dmax) and ERK phosphorylation inhibition were reached within 8 h. Compared with the VHL-recruiting degraders, the CRBN-recruiting degrader 50 (at 0.3 μM) showed a slower kinetic effect. Significant MEK1/2 degradation occurred at 4 h, and the maximum degradation (Dmax) was reached within 8–10 h. Consistently, compound 50 inhibited ERK phosphorylation at 8–10 h, albeit less effective compared to compounds 24 and 27.
Figure 6.
Compounds 24, 27, and 50 degrade MEK1 and MEK2 and inhibit pERK signaling in a time-dependent manner. HT-29 cells treated with compound 24 or 27 (0.1 μM) or 50 (0.3 μM) were harvested at indicated time points. The indicated protein levels were determined by Western blots.
Mechanism of Action (MOA) of MEK1/2 Degradation Induced by Compounds 24, 27, and 50.
To demonstrate that the observed MEK1/2 degradation effect of compounds 24, 27, and 50 is VHL- or CRBN-dependent, we first confirmed that control compounds 51, 52, and 53, which possess modified moieties with diminished binding to the VHL or CRBN ligase, did not reduce MEK1/2 protein levels in either HT-29 or SK-MEL-28 cells (Figure 7), indicating that binding to either VHL or CRBN ligase is required for MEK1/2 degradation. In addition, these control compounds were much less potent at inhibiting MEK and ERK phosphorylation compared with degraders 24, 27, and 50 (Figures 3, 4B, and 7). These results suggest that compounds 24, 27, and 50 inhibited the downstream ERK signaling mainly through the reduction of MEK1/2 protein levels instead of inhibition of MEK1/2 kinase activities.
Figure 7.
Control compounds 51–53 do not induce MEK1/2 degradation and are less potent at inhibiting the downstream ERK signaling in HT-29 and SK-MEL-28 cells after 24 h treatment. The indicated protein levels were determined by Western blots.
We next performed a series of rescue experiments to demonstrate that the MEK1/2 degradation induced by compounds 24, 27, and 50 is through hijacking the ubiquitin-proteasome system (Figure 8). Pretreatment of HT-29 cells with MEK1/2 inhibitor PD0325901 (1 μM) effectively blocked the degradation of MEK1/2 induced by 24, 27, or 50, confirming that MEK1/2 binding is required for MEK1/2 degradation. Pretreatment of HT-29 cells with the proteasome inhibitor MG-132 (3 μM) also diminished the MEK1/2 degradation, indicating that the degrader-induced MEK1/2 degradation is mediated by the proteasome. In addition, pretreatment with the neddylation inhibitor MLN4924 (3 μM) rescued MEK1/2 protein levels in HT-29 cells, implying that the degradation requires an active E3 complex. Moreover, the VHL ligand VH 032 (10 μM) was able to diminish the effect of VHL-recruiting degraders 24 and 27, and the CRBN ligand pomalidomide (POMA, 5 μM) also significantly prevented MEK1/2 degradation induced by the CRBN-recruiting degrader 50, further confirming that MEK1/2 degradation by 24 or 27 is VHL-dependent and MEK1/2 degradation by 50 is CRBN-dependent. Interestingly, pretreatment with PD0325901, VH 032, POMA, MG-132, and MLN4924 not only prevented MEK1/2 proteins from degradation but also restored the pMEK level in HT-29 cells (Figure 8). Except for PD0325901, all of these compounds also effectively restored the pERK protein level (Figure 8). Taken together, these studies have demonstrated that MEK1/2 degradation induced by compounds 24, 27, and 50 is through hijacking the ubiquitin-proteasome system.
Figure 8.
Degradation of MEK1/2 induced by compound 24, 27, or 50 is mediated by the ubiquitin-proteasome system. HT-29 cells were pretreated with DMSO, PD0325901 (1 μM), MG-132 (3 μM), MLN4924 (3 μM), VH 032 (10 μM), or pomalidomide (POMA, 5 μM) for 2 h, before 24 or 27 (0.1 μM), or 50 (0.3 μM) was added. The cells were incubated for another 8 h. The indicated protein levels were determined by Western blots.
Selectivity of MEK1/2 Degraders 27 and 50.
To evaluate the selectivity of the VHL-recruiting degrader 27 and CRBN-recruiting degrader 50, we performed unbiased proteomics studies using protein samples from HT-29 cells treated with 27 (0.1 μM), 50 (0.3 μM), 52 (0.1 μM), 53 (0.3 μM), or DMSO for 8 h (Figures 9 and S4). Compared with DMSO control, the global proteome analysis showed that control compounds 52 and 53 did not significantly change the levels of any quantified 5,165 proteins (Figure 9A,B). On the other hand, degraders 27 and 50 only significantly decreased MEK1 and MEK2 protein levels out of the quantified 5165 proteins (Figure 9C,D). It has been reported that CRBN-recruiting PROTAC degraders have the potential to maintain the activities of IMiDs (e.g., degrading neo-substrates of CRBN). Since the global proteomic study was performed using one concentration (0.3 μM) of degrader 50 at a single time point (8 h), it is possible that we could miss some off-targets at elevated degrader concentrations and/or with extended treatment time. Furthermore, the proteomic study did not identify unique peptides for IKZF1/3 proteins. Therefore, we evaluated the protein levels of a few CRBN neo-substrates, including GSPT1, IKZF1/3, and ZFP91, in HT-29 cells treated with degrader 50 at various concentrations for 24 h using Western blotting analysis. As illustrated in Figure S5, after 24 h treatment, degrader 50 did not significantly reduce protein levels of GSPT1, IKZF1, and ZFP91 at concentrations up to 10 μM. However, the IKZF3 protein level was significantly decreased by compound 50 at concentrations above 1 μM. Taken together, these results indicate that: (1) the VHL-recruiting degrader 27 has excellent selectivity for MEK1/2; and (2) the CRBN-recruiting degrader 50 is also selective for MEK1/2 in general. While we did not identify any off-targets in the global proteomic study, Western blotting analysis revealed that compound 50 was able to degrade the CRBN neo-substrate IKZF3, but not other neo-substrates such as GSPT1, IKZF1, and ZFP91. Nevertheless, compound 50 was more potent at degrading MEK1/2 than IKZF3.
Figure 9.
Compounds 27 and 50 selectively degrade MEK1 and MEK2 in HT-29 cells. HT-29 cells were treated with 0.1% DMSO, 27 (0.1 μM), 50 (0.3 μM), 52 (0.1 μM), or 53 (0.3 μM) for 8 h before they were harvested for mass spectrometry analysis. Volcano plots of the −log 10 (p-value) vs the log 2 fold change are displayed. Proteins outside the significance lines are labeled with pink or purple color (FDR = 0.05, S0 = 1). P values were calculated from the data of two technical replicates.
Antiproliferative Activities of Compounds 24, 27, and 50.
To demonstrate the advantages of MEK1/2 protein degradation over kinase activity inhibition, we evaluated the antiproliferative activities of 24, 27, and 50 and their corresponding control compounds 51, 52, and 53 in HT-29 (Figure 10A–C) and SK-MEL-28 (Figure 10D–F) cells. Compared with the control compounds, all three MEK1/2 degraders showed better potency at inhibiting the growth of HT-29 cells. For example, 24 (GI50 = 32 ± 8 nM) was 40-fold more potent than 51 (GI50 = 1300 ± 160 nM); 27 (GI50 = 23 ± 5 nM) was 35-fold more potent than 52 (GI50 = 830 ± 85 nM); and 50 (GI50 = 303 ± 57 nM) was 8-fold more potent than 53 (GI50 = 2600 ± 79 nM). Similar significant differentiation between degraders and control compounds was observed in SK-MEL-28 cells. For example, 24 (GI50 = 56 ± 4 nM) showed 21-fold improved potency over 51 (GI50 = 1200 ± 250 nM); 27 (GI50 = 40 ± 10 nM) showed 32-fold improved potency over 52 (GI50 = 1300 ± 190 nM); and 50 (GI50 = 780 ± 100 nM) showed 3-fold improved potency over 53 (GI50 = 2500 ± 150 nM). Compared with the previously reported compound 3 (GI50 = 130 ± 38 nM for HT-29 cells and 83 ± 15 nM for SK-MEL-28 cells), the new VHL-recruiting degraders 24 and 27 showed improved antiproliferation potency in both HT-29 and SK-MEL-28 cells.
Figure 10.
Compounds 24, 27, and 50 significantly suppress the growth of HT-29 and SK-MEL-28 cells. HT-29 (A–C) and SK-MEL-28 (D–F) cells were treated with serial dilutions of indicated compounds for 3 days, followed by WST-8 assay to get a live cell signal. For each concentration point, in quadruplicate, each mean value ± SD from three independent experiments is shown in the curves. (G–I) Clonogenic assay was conducted in HT-29 cells. HT-29 cells were treated with indicated compounds for 10 days. The Petri dish images are representative of two independent experiments. The cells were fixed and stained with crystal violet.
We also assessed the potency of 24, 27, and 50 and their corresponding controls 51, 52, and 53 at inhibiting colony formation of HT-29 cells (Figure 10G–I). Consistent with the cell antiproliferation assay results, degraders 24, 27, and 50 inhibited colony formation of HT-29 cells much more effectively than the corresponding controls 51, 52, and 53, highlighting potential benefits of MEK1/2 protein degradation over inhibition.
Taken together, these results indicated that MEK1/2 degraders 24, 27, and 50 were more potent at inhibiting cancer cell growth and colony formation than the control compounds that had similar binding affinities to MEK1/2 as the corresponding degraders but cannot degrade MEK1/2. Compared with the MEK1/2 inhibitor PD0325901, 24 and 27 were less potent at inhibiting the cell growth and colony formation, probably due to their decreased binding affinities to both MEK1 and MEK2 and their suboptimal physicochemical properties (e.g., much higher molecular weight) to penetrate the cell membrane.
In addition, compared with compound 4,45 a recently reported VHL-recruiting MEK1/2 degrader, compounds 24 and 27 displayed better potency at inhibiting the growth of HT-29, SK-MEL-28, H3122, and SUDHL1 cells (Figure 11). In H3122 and SUDHL1 cells, western blotting results clearly indicated that compounds 24 and 27, but not the controls 51 and 52, induced MEK1/2 degradation (Figure S6). Compounds 24 and 27 were also more potent than controls 51 and 52 at inhibition of MEK and ERK phosphorylation in both cell lines (Figure S6). Notably, in SUDHL1 cells, compounds 24 (GI50 = 360 ± 50 nM) and 27 (GI50 = 330 ± 100 nM) exhibited similar antiproliferation potency as PD0325901 (GI50 = 350 ± 70 nM) and was much more potent than compound 4 (GI50 = 2400 ± 300 nM) (Figure 11D).
Figure 11.
Antiproliferation effects of compounds 24 and 27 versus compound 4 in four different cancer cell lines. HT-29 (A), SK-MEL-28 (B), H3122 (C), and SUDHL1 (D) cells were treated with serial dilutions of indicated compounds for 3 days, followed by WST-8 assay to get a live cell signal. GraphPad Prism 8 was used to plot column graphs (A–C) or concentration curves (D) and calculate GI50 values (D). Each concentration point was in duplicate (A, B) or triplicate (C, D). The GI50 values (D) are presented as the mean ± SD from three independent experiments. Statistical significance was analyzed by Student’s t-tests for each concentration in the columns comparing to DMSO (A–C), P ≥ 0.05, ns; 0.01 ≤ P < 0.05, *; 0.001 ≤ P < 0.01, **; and P < 0.001, ***.
Combination Treatment.
Concurrent inhibition of MEK and BRAF kinases has shown significant advantages over inhibition of BRAF alone by overcoming the paradoxical MAPK pathway activation.60 Multiple combination therapies with MEK and BRAF inhibitors have been approved by the FDA to treat BRAF-mutated melanoma.30–33 In addition, due to the broad crosstalk between the RAF/MEK/ERK and PI3K/AKT pathways,61 hyperactivation of the PI3K/AKT signaling has been recognized as a resistant mechanism for inhibitors that target the RAF/MEK/ERK pathway.62,63 Therefore, concurrent inhibition of both RAF/MEK/ERK and PI3K/AKT pathways could provide another therapeutic strategy.63
To further explore the therapeutic potentials of MEK degraders, we evaluated the effect of combining our MEK1/2 degrader 27 with either BRAF inhibitor PLX4032 or PI3K inhibitor ZSTK474 on inhibiting the growth of HT-29 and SK-MEL-28 cells. First, we determined that the GI50 values of PLX4032 and ZSTK474 were around 100 and 300 nM, respectively, for HT-29 and SK-MEL-28 cells (Figure S7). We next treated these two types of cells with degrader 27 at a series of concentrations combined with a fixed concentration of either PLX4032 (100 nM) or ZSTK474 (400 nM), which is around their respective GI50 values. As illustrated in Figure 12, both PLX4032 and ZSTK474 significantly potentiated the antiproliferation effect of 27 in HT-29 cells and moderately enhanced the antiproliferation effect of 27 in SK-MEL-28 cells (Figure 12A,B). Therefore, the combination of a MEK degrader with an inhibitor targeting another component of the same RAF/MEK/ERK signaling pathway or a different PI3K/AKT pathway could potentially provide a better therapeutic outcome than the MEK degrader as a single agent.
Figure 12.
Concurrent inhibition of BRAF or PI3K potentiates antiproliferation potency of compound 27. HT-29 (A) and SK-MEL-28 (B) cells were treated with indicated compounds for 3 days, followed by WST-8 assay to get a live cell signal. Each concentration point was in triplicate (A, B). Each mean value ± SD from three independent experiments is shown in the graphs. Statistical significance was analyzed by Student’s t-tests for each concentration in the columns comparing to DMSO. P ≥ 0.05, ns; 0.01 ≤ P < 0.05, *; 0.001 ≤ P < 0.01, **; and P < 0.001, ***.
In Vivo Mouse Pharmacokinetic (PK) Study.
We evaluated the in vivo PK properties of the best two MEK1/2 degraders, 24 and 27, in mice. Following a single intraperitoneal (IP) injection of 24 or 27 at 50 mg/kg dose, compound concentrations in plasma were monitored at 0.5, 2, and 8 h postinjection. As illustrated in Figure 13, the plasma concentrations of both compounds were well above their DC50 and GI50 values in HT-29 or SK-MEL-28 cells. Compound 27 showed better plasma exposure than compound 24 over the 8 h period. In addition, compound 27 was cleared more slowly than compound 24, which could be due to the fact that compound 24 possesses the metabolically labile N-methyl group located in the middle of the linker. Moreover, the plasma exposure level of compound 27 was also higher than that of the previously published MEK degrader 3.44 It is also worth noting that both 24 and 27 were very well tolerated by the studied mice and no clinical signs or adverse effects were observed. In summary, we identified an improved MEK1/2 degrader, 27, which is suitable for in vivo efficacy studies. Compound 27 is not only more potent but also has higher in vivo exposure than the previously reported MEK1/2 degrader 3.
Figure 13.
Plasma concentrations of compounds 24 and 27 following a single intraperitoneal administration at 50 mg/kg for 8 h in male Swiss Albino mice. Plasma concentrations at each time point are presented as the mean ± SD from three mice.
Synthesis of Novel Compounds.
The syntheses of compounds 5–23, 25–50, 52, and 53 are depicted in Scheme 1, using synthetic routes similar to those reported previously.44 Briefly, nucleophilic substitutions between commercially available 1,3-dioxolane 54 or 55 with N-hydroxyphthalimide afforded intermediate 56 or 57, which was converted to alkoxyamine 58 or 59 by hydrazinolysis. Amide condensation of alkoxyamine 58 or 59 with 3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)benzoic acid provided alkoxyamide 60 or 61. Removal of the dioxolane protecting group under acidic conditions unmasked the aldehyde 62 or 63, which was transformed into the putative MEK1/2 degraders through reductive amination reaction with different VHL ligand-based linkers or CRBN ligand-based linkers, which were synthesized according to published procedures.44,64
Scheme 1. Synthesizing Compounds 5–23, 25–50, 52, and 53a.
aReagents and conditions: (a) DBU, N,N-dimethylformamide (DMF), 50 °C, 4 h; (b) N2H4.H2O, MeOH, dichloromethane (DCM), rt, 2 h; (c) 3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)benzoic acid, HOAt, EDCI, NMM, DMSO, rt, overnight; (d) 3 M HCl, tetrahydrofuran (THF), rt, 6 h; (e) different linkers, NaBH3CN, DCM, MeOH, rt, overnight.
The syntheses of compounds 24 and 51 are outlined in Scheme 2. N-Alkylation of known compound 6465 provided the methylated intermediate 65, which was converted to compound 66 or 67 through amide coupling and followed by deprotection. Reductive amination reaction with 62 converted 66 or 67 to compound 24 or 51, respectively.
Scheme 2. Synthesizing Compounds 24 and 51a.
aReagents and conditions: (a) CH3I, NaH, DMF, rt, 18 h; (b) (1) HOAt, EDCI, NMM, DMSO, rt, overnight; (2) trifluoroacetic acid (TFA), DCM, rt, 2 h; (c) 62, NaBH3CN, DCM, MeOH, rt, overnight.
CONCLUSIONS
We conducted extensive SAR studies of the PD0325901-derived MEK1/2 degraders by exploring a large set of linkers and several E3 ligase ligands. From these studies, we identified two novel, improved VHL-recruiting MEK1/2 degraders 24 and 27, both of which potently induced MEK1/2 protein degradation in colorectal cancer cell line HT-29 and melanoma cell line SK-MEL-28 in concentration and time-dependent manners. Our MOA studies confirmed that the MEK1/2 degradation induced by these compounds was through hijacking the ubiquitin-proteasome system. Degrader 27 displayed excellent protein degradation selectivity in a global proteomic study and reduced the protein levels of only MEK1/2 out of over 5000 quantified proteins. Compared with previously reported VHL-recruiting degraders 3 and 4, the new degraders 24 and 27 were more potent at inhibiting the growth of HT-29, SK-MEL-28, H3122, and SUDHL1 cells. In addition, compounds 24 and 27 were more potent than compound 3 at degrading MEK1/2 in HT-29 and SK-MEL-28 cells. Furthermore, degraders 24 and 27 were much more potent at inhibiting cancer cell growth than their corresponding controls that have similar binding affinities to MEK1/2 as degraders 24 and 27, but cannot degrade MEK1/2, indicating potential advantages of pharmacological degradation of MEK1/2 over inhibition of the MEK1/2 kinase activities. Moreover, concurrent pharmacological inhibition of either BRAF or PI3K potentiated the antiproliferation potency of 27 in HT-29 or SK-MEL-28 cells, suggesting that the combination of a MEK1/2 degrader with an inhibitor of BRAF or PI3K may provide potential therapeutic benefits over the MEK1/2 degrader as a single agent. Furthermore, degrader 27 displayed good plasma exposure in mice, which is higher than that of the previously reported degrader 3. Thus, it is suitable for in vivo studies.
In addition to the two improved VHL-recruiting MEK1/2 degraders, we also identified the first CRBN-recruiting MEK1/2 degrader 50, which effectively degraded MEK1/2 and inhibited the growth of HT-29 and SK-MEL-28 cells. Rescue experiments revealed that MEK1/2 degradation induced by 50 was dependent on the ubiquitin-proteasome system and binding to MEK1/2. Global proteomic analysis showed that degrader 50 down-regulated only MEK1/2 proteins. Subsequent Western blotting analysis identified IKZF3, but not other CBRN neo-substrates, including GSPT1, as an off-target of 50. Further optimization is necessary to improve the potency and selectivity of this CRBN-recruiting MEK1/2 degrader.
In summary, our extensive SAR studies led to a novel, potent, and selective VHL-recruiting degrader, 27, which is more potent and has higher plasma exposure in mice than that of previously reported MEK1/2 degraders. Compound 27 is the best MEK1/2 degrader to date for in vivo studies. In addition, the SAR studies resulted in the first CRBN-recruiting MEK1/2 degrader 50, which potently and selectively induced MEK1/2 degradation. Our studies paved the way for further development and optimization of MEK1/2 heterobifunctional small-molecule degraders.
EXPERIMENTAL SECTION
Chemistry General Procedures.
All chemical reagents were purchased from commercial vendors and used in syntheses without further purification. A Teledyne ISCO CombiFlash Rf+ instrument equipped with a variable wavelength UV detector and a fraction collector was used to conduct flash column chromatography. HP C18 RediSep Rf reverse-phase silica columns were also used for the purification of certain polar products. An Agilent 1200 series system with a DAD detector and a 2.1 mm × 150 mm Zorbax 300SB-C18 5 μm column with water containing 0.1% formic acid as solvent A and acetonitrile containing 0.1% formic acid as solvent B at a flow rate of0.4 mL/min for chromatography were used to obtain high-performance liquid chromatography (HPLC) spectra for all final compounds. The gradient program was as follows: 1% B (0–1 min), 1–99% B (1–4 min), and 99% B (4–8 min). A Waters Acquity I-Class ultra-performance liquid chromatography (UPLC) system with a PDA detector was used to generate UPLC spectra for all compounds. Chromatography was performed using a 2.1 mm × 30 mm ACQUITY UPLC BEH C18 1.7 μm column with water containing 3% acetonitrile and 0.1% formic acid as solvent A and acetonitrile containing 0.1% formic acid as solvent B at a flow rate of0.8 mL/min. The gradient program was as follows: 1–99% B (1–1.5 min) and 99–1% B (1.5–2.5 min). High-resolution mass spectra (HRMS) data were obtained in positive ion mode using an Agilent G1969A API-TOF with an electrospray ionization (ESI) source. Nuclear Magnetic Resonance (NMR) spectra were obtained on a Bruker DRX-600 spectrometer with 600 MHz for proton (1H NMR) or a Bruker DXI 800 MHz spectrometer with 800 MHz for proton (1H NMR) or 200 MHz for carbon (13C NMR); chemical shifts are reported in ppm (δ). Preparative HPLC was performed using Agilent Prep 1200 series with a UV detector set to 220 nm. Samples were injected into a Phenomenex Luna 75 mm × 30 mm, 5 μm, C18 column at room temperature. The flow rate was 40 mL/min. A linear gradient was used with 10% (or 50%) of MeOH (A) in H2O (with0.1% TFA) (B) to 100% of MeOH (A). HPLC and UPLC were used to establish the purity of target compounds. All final compounds had >95% purity using the HPLC and UPLC methods described above.
Compounds 5–53 were prepared according to the procedures for the synthesis of compound 3 in our previous work.44
(2S,4R)-1-((S)-11-(tert-Butyl)-1-(3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)phenyl)-1,9-dioxo-3-oxa-2,7,10-triazadodecan-12-oyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (5).
White solid, 40% yield. 1H NMR (600 MHz, methanol-d4) δ 9.30 (d, J = 4.2 Hz, 1H), 7.53–7.47 (m, 3H), 7.47–7.44 (m, 2H), 7.40–7.36 (m, 2H), 7.01 (td, J = 9.1, 6.9 Hz, 1H), 6.68 (td, J = 8.7, 3.8 Hz, 1H), 4.58–4.55 (m, 2H), 4.53 (d, J = 8.4 Hz, 2H), 4.51–4.48 (dt, J = 4.5, 2.5 Hz, 1H), 4.37 (d, J = 15.6 Hz, 1H),4.08 (dt, J = 9.8, 5.0 Hz, 1H), 4.03 (dq, J = 9.9, 5.3 Hz, 1H), 3.98 (d, J = 15.8 Hz, 1H), 3.87 (d, J = 15.8 Hz, 1H), 3.81 (d, J = 11.0 Hz, 1H),3.76 (dd, J = 10.9, 3.8 Hz, 1H), 2.52 (s, 3H), 2.25–2.20 (m, 1H),2.11–2.03 (m, 4H), 1.03 (s, 9H). HPLC > 99%, tR = 4.14 min. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C40H46F3IN7O6S+: 936.2222; found: 936.2047.
(2S,4R)-1-((S)-12-(tert-Butyl)-1-(3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)phenyl)-1,10-dioxo-3-oxa-2,7,11-triazatridecan-13-oyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (6).
White solid, 42% yield. 1H NMR (600 MHz, methanol-d4) δ 8.96 (s, 1H), 7.51–7.45 (m, 3H), 7.44–7.40 (m, 3H), 7.38 (dt, J = 8.3, 1.4 Hz, 1H), 7.05 (td, J = 9.1, 6.9 Hz, 1H), 6.66 (td, J = 8.7, 3.9 Hz, 1H), 4.59–4.53 (m, 3H), 4.50–4.47 (m, 1H), 4.34 (d, J = 15.5 Hz, 1H), 4.03 (t, J = 5.2 Hz, 2H), 3.90 (d, J = 10.9 Hz, 1H), 3.75 (dd, J = 10.9, 3.9 Hz, 1H), 3.35–3.32 (m, 2H),3.27–3.22 (m, 2H), 2.78 (t, J = 6.6 Hz, 2H), 2.47 (s, 3H), 2.25–2.20 (m, 1H), 2.08 (ddd, J = 13.4, 9.3, 4.3 Hz, 1H), 2.05–1.99 (m, 2H),1.02 (s, 9H). HPLC > 99%, tR = 4.18 min. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C41H48F3IN7O6S+: 950.2378; found: 950.2339.
(2S,4R)-1-((S)-13-(tert-Butyl)-1-(3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)phenyl)-1,11-dioxo-3-oxa-2,7,12-triazatetradecan-14-oyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (7).
White solid, 38% yield. 1H NMR (600 MHz, methanol-d4) δ 8.98 (s, 1H), 7.50–7.45 (m, 3H), 7.44–7.38 (m, 3H), 7.37 (dt, J = 8.5, 1.3 Hz, 1H), 7.06 (td, J = 9.2, 7.0 Hz, 1H), 6.64 (td, J = 8.8, 3.9 Hz, 1H), 4.58 (d, J = 6.8 Hz, 1H), 4.56 (dd, J = 3.0, 1.4 Hz, 1H), 4.53 (d, J = 9.7 Hz, 1H), 4.51–4.49 (m, 1H),4.36 (d, J = 15.5 Hz, 1H), 4.03 (dp, J = 11.8, 4.2 Hz, 2H), 3.91 (d, J = 10.9 Hz, 1H), 3.79 (dd, J = 10.9, 3.9 Hz, 1H), 3.22 (t, J = 5.9 Hz, 2H), 3.08 (td, J = 7.5, 1.8 Hz, 2H), 2.48 (s, 3H), 2.48–2.44 (m, 2H),2.22 (ddt, J = 13.2, 7.6, 1.9 Hz, 1H), 2.08 (ddd, J = 13.3, 9.3, 4.4 Hz, 1H), 2.04–1.97 (m, 4H), 1.03 (s, 9H). HPLC > 99%, tR = 4.11 min. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C42H50F3IN7O6S+: 964.2535; found: 964.2422.
(2S,4R)-1-((S)-14-(tert-Butyl)-1-(3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)phenyl)-1,12-dioxo-3-oxa-2,7,13-triazapentadecan-15-oyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (8).
White solid, 36% yield1H NMR (600 MHz, methanol-d4) δ 8.88 (s, 1H), 7.49 (dd, J = 10.7, 1.9 Hz, 1H),7.46 (d, J = 8.2 Hz, 2H), 7.43–7.40 (m, 2H), 7.37 (ddd, J = 8.5, 2.0, 1.0 Hz, 2H), 7.07 (td, J = 9.2, 7.1 Hz, 1H), 6.64 (td, J = 8.7, 3.9 Hz, 1H), 4.61 (s, 1H), 4.59–4.53 (m, 2H), 4.50 (dd, J = 4.2, 2.2 Hz, 1H),4.35 (d, J = 15.5 Hz, 1H), 4.06–4.00 (m, 2H), 3.89 (dt, J = 11.1, 1.7 Hz, 1H), 3.80 (dd, J = 10.9, 3.9 Hz, 1H), 3.22 (t, J = 5.9 Hz, 2H),3.05 (t, J = 7.3 Hz, 2H), 2.47 (s, 3H), 2.34 (q, J = 7.5 Hz, 2H), 2.22 (ddt, J = 13.1, 7.5, 2.0 Hz, 1H), 2.09 (ddd, J = 13.3, 9.2, 4.5 Hz, 1H),2.04–1.97 (m, 2H), 1.81–1.68 (m, 4H), 1.03 (s, 9H). HPLC > 99%, tR = 4.13 min. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C43H52F3IN7O6S+: 978.2691; found: 978.2665.
(2S,4R)-1-((S)-15-(tert-Butyl)-1-(3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)phenyl)-1,13-dioxo-3-oxa-2,7,14-triazahexadecan-16-oyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (9).
White solid, 28% yield. 1H NMR (600 MHz, methanol-d4) δ 8.93 (s, 1H), 7.51–7.45 (m, 3H), 7.42 (d, J = 7.8 Hz, 3H), 7.37 (d, J = 9.1 Hz, 1H), 7.07 (q, J = 8.6 Hz, 1H),6.63 (td, J = 8.7, 4.0 Hz, 1H), 4.64 (s, 1H), 4.61–4.52 (m, 2H), 4.50 (s, 1H), 4.36 (d, J = 15.5 Hz, 1H), 4.04 (t, J = 5.2 Hz, 2H), 3.91 (d, J = 11.0 Hz, 1H), 3.81 (dd, J = 11.0, 3.9 Hz, 1H), 3.23 (t, J = 5.9 Hz, 2H), 3.04 (t, J = 7.9 Hz, 2H), 2.48 (s, 3H), 2.35–2.26 (m, 2H), 2.22 (dd, J = 13.2, 7.8 Hz, 1H), 2.09 (ddd, J = 13.3, 9.1, 4.4 Hz, 1H), 2.02 (dd, J = 11.0, 5.3 Hz, 2H), 1.74 (p, J = 7.8 Hz, 2H), 1.65 (p, J = 7.2 Hz, 2H), 1.42 (p, J = 7.8 Hz, 2H), 1.03 (s, 9H). HPLC > 99%, tR = 4.19 min. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C44H54F3IN7O6S+: 992.2848; found: 992.2816.
(2S,4R)-1-((S)-16-(tert-Butyl)-1-(3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)phenyl)-1,14-dioxo-3-oxa-2,7,15-triazaheptadecan-17-oyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (10).
White solid, 27% yield. 1H NMR (600 MHz, methanol-d4) δ 8.94 (d, J = 6.0 Hz, 1H), 7.51–7.45 (m, 3H), 7.44–7.39 (m, 3H), 7.38 (d, J = 8.8 Hz, 1H), 7.07 (q, J = 8.9 Hz, 1H), 6.63 (td, J = 8.6, 4.0 Hz, 1H), 4.67–4.63 (m, 1H), 4.58–4.55 (m, 1H), 4.53 (d, J = 10.4 Hz, 1H), 4.51–4.49 (m, 1H), 4.36 (d, J = 15.4 Hz, 1H), 4.05 (t, J = 5.1 Hz, 2H), 3.90 (d, J = 11.0 Hz, 1H),3.81 (dd, J = 10.9, 4.0 Hz, 1H), 3.23 (t, J = 5.9 Hz, 2H), 3.10–2.99 (m, 2H), 2.48 (s, 3H), 2.31 (dt, J = 14.9, 7.6 Hz, 1H), 2.28–2.25 (m, 1H), 2.25–2.19 (m, 1H), 2.08 (ddd, J = 13.3, 9.1, 4.4 Hz, 1H), 2.05–1.99 (m, 2H), 1.73 (p, J = 7.8 Hz, 2H), 1.61 (dt, J = 13.7, 6.9 Hz, 2H), 1.42 (p, J = 7.1 Hz, 2H), 1.37 (q, J = 7.2 Hz, 2H), 1.03 (s, 9H). HPLC > 99%, tR = 4.19 min. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C45H56F3IN7O6S+: 1006.3004; found: 1006.2966.
(2S,4R)-1-((S)-17-(tert-Butyl)-1-(3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)phenyl)-1,15-dioxo-3-oxa-2,7,16-triazaoctadecan-18-oyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (11).
White solid, 30% yield. 1H NMR (600 MHz, methanol-d4) δ 8.92 (s, 1H), 7.52–7.45 (m, 3H), 7.44–7.39 (m, 3H), 7.38 (dt, J = 8.6, 1.3 Hz, 1H), 7.07 (td, J = 9.2, 7.0 Hz, 1H), 6.63 (td, J = 8.7, 4.3 Hz, 1H), 4.67–4.62 (m, 1H), 4.60–4.55 (m, 1H), 4.54 (d, J = 12.9 Hz, 1H), 4.51–4.48 (m, 1H), 4.36 (d, J = 15.5 Hz, 1H), 4.06 (t, J = 5.1 Hz, 2H), 3.90 (d, J = 11.0 Hz, 1H), 3.81 (dd, J = 11.0, 3.9 Hz, 1H), 3.23 (t, J = 5.9 Hz, 2H), 3.09–2.97 (m, 2H), 2.48 (s, 3H), 2.32–2.27 (m, 1H), 2.26–2.20 (m, 2H), 2.08 (ddd, J = 13.3, 9.2, 4.5 Hz, 1H), 2.02 (dt, J = 10.1, 5.0 Hz, 2H), 1.72 (p, J = 7.7 Hz, 2H), 1.61 (dp, J = 14.5, 7.1 Hz, 2H), 1.43–1.38 (m, 2H), 1.38–1.28 (m, 4H), 1.03 (s, 9H). HPLC > 99%, tR = 4.17 min. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C46H58F3IN7O6S+: 1020.3161; found: 1020.3086.
(2S,4R)-1-((S)-18-(tert-Butyl)-1-(3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)phenyl)-1,16-dioxo-3-oxa-2,7,17-triazanonadecan-19-oyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (12).
White solid, 31% yield. 1H NMR (600 MHz, methanol-d4) δ 8.97 (s, 1H), 7.51–7.45 (m, 3H), 7.45–7.39 (m, 3H), 7.37 (dt, J = 8.5, 1.3 Hz, 1H), 7.07 (td, J = 9.2, 7.0 Hz, 1H), 6.64 (td, J = 8.7, 4.3 Hz, 1H), 4.64 (s, 1H), 4.60–4.55 (m, 1H),4.54–4.52 (m, 1H), 4.50 (dq, J = 4.1, 2.1 Hz, 1H), 4.36 (d, J = 15.5 Hz, 1H), 4.07 (dd, J = 5.8, 4.5 Hz, 2H), 3.90 (dt, J = 11.2, 1.7 Hz, 1H), 3.81 (dd, J = 10.9, 3.9 Hz, 1H), 3.23 (t, J = 5.8 Hz, 2H), 3.10–2.90 (m, 2H), 2.48 (s, 3H), 2.33–2.26 (m, 1H), 2.25–2.18 (m, 2H),2.08 (ddd, J = 13.3, 9.1, 4.5 Hz, 1H), 2.02 (dt, J = 10.2, 5.1 Hz, 2H),1.72 (p, J = 7.8 Hz, 2H), 1.59 (q, J = 7.2 Hz, 2H), 1.42–1.36 (d, J = 7.9 Hz, 2H), 1.35–1.29 (m, 6H), 1.03 (s, 9H). HPLC > 99%, tR = 4.12 min. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C47H60F3IN7O6S+: 1034.3317; found: 1034.3217.
(2S,4R)-1-((S)-19-(tert-Butyl)-1-(3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)phenyl)-1,17-dioxo-3-oxa-2,7,18-triazaicosan-20-oyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (13).
White solid, 31% yield. 1H NMR (600 MHz, methanol-d4) δ 8.96 (s, 1H), 7.50–7.44 (m, 3H), 7.44–7.39 (m, 3H),7.38 (dt, J = 8.5, 1.3 Hz, 1H), 7.07 (td, J = 9.2, 7.0 Hz, 1H), 6.64 (td, J = 8.7, 4.3 Hz, 1H), 4.63 (s, 1H), 4.59–4.55 (m, 1H), 4.54 (d, J = 12.1 Hz, 1H), 4.50 (dd, J = 4.3, 2.2 Hz, 1H), 4.36 (d, J = 15.5 Hz, 1H), 4.11–4.02 (m, 2H), 3.90 (d, J = 11.0 Hz, 1H), 3.80 (dd, J = 11.0, 3.9 Hz, 1H), 3.24 (t, J = 5.9 Hz, 2H), 3.08–2.99 (m, 2H), 2.48 (s, 3H), 2.29 (ddd, J = 15.2, 8.4, 7.0 Hz, 1H), 2.26–2.19 (m, 2H),2.08 (ddd, J = 13.3, 9.1, 4.5 Hz, 1H), 2.02 (dt, J = 10.3, 5.1 Hz, 2H),1.71 (p, J = 7.7 Hz, 2H), 1.65–1.53 (m, 2H), 1.42–1.36 (m, 2H),1.34–1.25 (m, 8H), 1.03 (s, 9H). HPLC > 99%, tR = 4.22 min. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C48H62F3IN7O6S+: 1048.3474; found: 1078.3372.
(2S,4R)-1-((S)-20-(tert-Butyl)-1-(3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)phenyl)-1,18-dioxo-3-oxa-2,7,19-triazahenicosan-21-oyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (14).
White solid, 24% yield. 1H NMR (600 MHz, methanol-d4) δ 8.94 (s, 1H), 7.50–7.44 (m, 3H), 7.44–7.39 (m, 3H), 7.38 (dt, J = 8.5, 1.3 Hz, 1H), 7.07 (td, J = 9.2, 6.9 Hz, 1H), 6.64 (td, J = 8.7, 4.3 Hz, 1H), 4.64 (s, 1H), 4.59–4.55 (m, 1H),4.54 (d, J = 12.6 Hz, 1H), 4.51–4.49 (m, 1H), 4.36 (d, J = 15.5 Hz, 1H), 4.13–4.03 (m, 2H), 3.90 (d, J = 11.0 Hz, 1H), 3.81 (dd, J = 11.0, 3.9 Hz, 1H), 3.24 (t, J = 5.8 Hz, 2H), 3.10–2.97 (m, 2H), 2.48 (s, 3H), 2.32–2.26 (m, 1H), 2.26–2.19 (m, 2H), 2.08 (ddd, J = 13.4, 9.2, 4.5 Hz, 1H), 2.05–1.99 (m, 2H), 1.71 (p, J = 7.7 Hz, 2H), 1.59 (tq, J = 14.3, 6.9 Hz, 2H), 1.44–1.35 (m, 2H), 1.32–1.29 (m, 6H),1.29–1.26 (m, 4H), 1.03 (s, 9H). HPLC > 99%, tR = 4.26 min. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C49H64F3IN7O6S+: 1062.3630; found: 1062.3556.
(2S,4R)-1-((S)-14-(tert-Butyl)-1-(3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)phenyl)-1,12-dioxo-3,10-dioxa-2,7,13-triazapentadecan-15-oyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (15).
White solid, 33% yield. 1H NMR (600 MHz, methanol-d4) δ 8.98 (s, 1H), 7.50–7.44 (m, 3H), 7.42 (d, J = 8.3 Hz, 2H), 7.40–7.35 (m, 2H), 7.06 (q, J = 8.8 Hz, 1H), 6.62 (td, J = 8.8, 4.0 Hz, 1H), 4.67 (s, 1H), 4.59–4.55 (m, 1H), 4.54 (d, J = 3.3 Hz, 1H), 4.51 (d, J = 10.2 Hz, 1H), 4.35 (d, J = 15.4 Hz, 1H),4.12 (s, 1H), 4.09 (s, 1H), 4.07–4.03 (m, 3H), 3.91–3.84 (m, 3H),3.80 (dd, J = 11.0, 3.7 Hz, 1H), 3.36–3.34 (m, 1H), 3.28 (p, J = 1.7 Hz, 1H), 2.48 (s, 3H), 2.24 (dd, J = 13.1, 7.7 Hz, 1H), 2.12–2.02 (m, 4H), 1.01 (s, 9H). HPLC > 99%, tR = 4.13 min. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C42H50F3IN7O7S+: 980.2484; found: 980.2361.
(2S,4R)-1-((S)-15-(tert-Butyl)-1-(3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)phenyl)-1,13-dioxo-3,10-dioxa-2,7,14-triazahexadecan-16-oyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (16).
White solid, 27% yield. 1H NMR (600 MHz, methanol-d4) δ 9.73 (s, 1H), 7.50 (t, J = 6.7 Hz, 2H), 7.46 (d, J = 8.2 Hz, 2H), 7.43 (dd, J = 10.6, 2.0 Hz, 1H), 7.39 (t, J = 7.2 Hz, 1H), 7.34 (d, J = 8.1 Hz, 1H), 7.01 (d, J = 7.7 Hz, 1H), 6.68–6.54 (m, 1H), 4.63–4.55 (m, 2H), 4.50 (d, J = 14.8 Hz, 2H), 4.43 (d, J = 15.6 Hz, 1H), 4.04 (t, J = 5.4 Hz, 2H), 3.98–3.89 (m, 2H), 3.85–3.68 (m, 6H), 2.57 (s, 6H), 2.26 (t, J = 10.6 Hz, 1H), 2.07 (q, J = 6.3, 5.3 Hz, 4H), 1.01 (s, 9H). HPLC > 99%, tR = 4.15 min. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C43H52F3IN7O7S+: 994.2640; found: 994.2478.
(2S,4R)-1-((S)-17-(tert-Butyl)-1-(3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)phenyl)-1,15-dioxo-3,10,13-trioxa-2,7,16-triazaoctadecan-18-oyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (17).
White solid, 26% yield. 1H NMR (600 MHz, methanol-d4) δ 8.95 (s, 1H), 7.50–7.45 (m, 2H),7.45–7.41 (m, 3H), 7.40–7.35 (m, 2H), 7.16–6.99 (m, 1H), 6.63 (td, J = 8.8, 4.0 Hz, 1H), 4.77–4.70 (m, 1H), 4.59–4.53 (m, 1H),4.50 (d, J = 6.0 Hz, 1H), 4.48–4.38 (m, 2H), 4.08–4.00 (m, 3H),3.96 (d, J = 15.8 Hz, 1H), 3.88 (d, J = 11.2 Hz, 1H), 3.81 (dt, J = 9.8,3.8 Hz, 3H), 3.67–3.61 (m, 4H), 3.30–3.23 (m, 3H), 2.47 (s, 3H),2.33–2.21 (m, 1H), 2.14–2.06 (m, 2H), 2.04 (d, J = 14.1 Hz, 2H),1.03 (s, 9H). HPLC > 99%, tR = 4.19 min. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C44H54F3IN7O8S+: 1024.2746; found: 1024.2692.
(2S,4R)-1-((S)-18-(tert-Butyl)-1-(3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)phenyl)-1,16-dioxo-3,10,13-trioxa-2,7,17-triazanonadecan-19-oyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (18).
White solid, 24% yield. 1H NMR (600 MHz, methanol-d4) δ 9.28 (s, 1H), 7.51–7.47 (m, 3H),7.45 (d, J = 8.1 Hz, 2H), 7.41 (t, J = 6.9 Hz, 1H), 7.38–7.35 (m, 1H),7.05 (q, J = 8.6 Hz, 1H), 6.63 (td, J = 8.6, 4.1 Hz, 1H), 4.64 (s, 1H),4.58–4.53 (m, 1H), 4.52–4.49 (m, 2H), 4.40 (d, J = 15.6 Hz, 1H),4.07 (t, J = 5.2 Hz, 2H), 3.96–3.88 (m, 1H), 3.83–3.76 (m, 3H),3.74–3.63 (m, 3H), 3.58 (t, J = 4.5 Hz, 2H), 3.56–3.52 (m, 2H),3.30–3.23 (m, 3H), 2.57–2.52 (m, 1H), 2.52 (s, 3H), 2.50–2.43 (m, 1H), 2.24 (dd, J = 13.2, 7.6 Hz, 1H), 2.14–2.00 (m, 3H), 1.02 (s, 9H). HPLC > 99%, tR = 4.14 min. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C45H56F3IN7O8S+: 1038.2902; found: 1038.2758.
(2S,4R)-1-((S)-20-(tert-Butyl)-1-(3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)phenyl)-1,18-dioxo-3,10,13,16-tetraoxa-2,7,19-triazahenicosan-21-oyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (19).
White solid, 36% yield. 1H NMR (600 MHz, methanol-d4) δ 8.99 (s, 1H), 7.49–7.45 (m, 3H),7.42 (d, J = 8.2 Hz, 2H), 7.41–7.38 (m, 1H), 7.37 (dt, J = 8.4, 1.3 Hz, 1H), 7.05 (td, J = 9.2, 7.0 Hz, 1H), 6.64 (td, J = 8.8, 4.2 Hz, 1H), 4.68 (s, 1H), 4.56 (dd, J = 7.9, 1.7 Hz, 1H), 4.53 (d, J = 11.4 Hz, 1H),4.51–4.48 (m, 1H), 4.39 (d, J = 15.5 Hz, 1H), 4.14–4.00 (m, 4H),3.88 (d, J = 11.1 Hz, 1H), 3.81 (dd, J = 10.9, 3.8 Hz, 1H), 3.77 (dd, J = 5.7, 4.3 Hz, 2H), 3.71–3.65 (m, 2H), 3.63–3.58 (m, 4H), 3.58–3.55 (m, 2H), 3.30–3.23 (m, 4H), 2.48 (s, 3H), 2.24 (ddt, J = 13.2, 7.5, 1.8 Hz, 1H), 2.08 (ddd, J = 13.4, 9.5, 4.3 Hz, 1H), 2.05–2.00 (m, 2H), 1.04 (s, 9H). HPLC > 99%, tR = 4.23 min. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C46H58F3IN7O9S+: 1068.3008; found: 1068.2944.
(2S,4R)-1-((S)-21-(tert-Butyl)-1-(3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)phenyl)-1,19-dioxo-3,10,13,16-tetraoxa-2,7,20-triazadocosan-22-oyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (20).
White solid, 18% yield. 1H NMR (600 MHz, methanol-d4) δ 9.02 (s, 1H), 7.51–7.44 (m, 3H),7.44–7.39 (m, 3H), 7.38 (dt, J = 8.5, 1.3 Hz, 1H), 7.18–6.99 (m, 1H), 6.64 (td, J = 8.7, 4.2 Hz, 1H), 4.64 (s, 1H), 4.59–4.55 (m, 1H),4.53 (d, J = 15.4 Hz, 1H), 4.49 (d, J = 3.3 Hz, 1H), 4.37 (d, J = 15.5 Hz, 1H), 4.15–4.02 (m, 2H), 3.89 (d, J = 11.0 Hz, 1H), 3.80 (dd, J = 11.0, 3.9 Hz, 1H), 3.78–3.75 (m, 2H), 3.74–3.66 (m, 2H), 3.60–3.57 (dd, J = 5.8, 2.2 Hz, 3H), 3.56–3.53 (m, 5H), 3.30–3.25 (m, 4H), 2.61–2.52 (m, 1H), 2.51–2.39 (m, 4H), 2.27–2.19 (m, 1H),2.09 (dt, J = 8.3, 4.3 Hz, 1H), 2.05 (td, J = 6.7, 4.5 Hz, 2H), 1.03 (s, 9H). HPLC > 99%, tR = 4.10 min. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C47H60F3IN7O9S+: 1082.3165; found: 1082.3084.
(2S,4R)-1-((S)-24-(tert-Butyl)-1-(3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)phenyl)-1,22-dioxo-3,10,13,16,19-pentaoxa-2,7,23-triazapentacosan-25-oyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (21).
White solid, 28% yield. 1H NMR (600 MHz, methanol-d4) δ 8.93 (s, 1H), 7.50–7.44 (m, 3H), 7.43–7.39 (m, 3H), 7.38 (dt, J = 8.5, 1.3 Hz, 1H), 7.06 (td, J = 9.1, 6.8 Hz, 1H), 6.64 (td, J = 8.7, 4.0 Hz, 1H), 4.68–4.63 (m, 1H),4.58–4.54 (m, 1H), 4.54–4.51 (m, 1H), 4.51–4.48 (m, 1H), 4.39–4.33 (m, 1H), 4.11–4.03 (m, 2H), 3.93–3.86 (m, 1H), 3.83–3.76 (m, 3H), 3.75–3.67 (m, 2H), 3.63–3.58 (m, 6H), 3.57 (dq, J = 5.7, 3.2, 2.6 Hz, 6H), 3.30–3.25 (m, 4H), 2.57 (ddd, J = 15.2, 7.2, 5.2 Hz, 1H), 2.50–2.41 (m, 4H), 2.26–2.19 (m, 1H), 2.09 (dt, J = 8.4, 4.2 Hz, 1H), 2.07–2.02 (m, 2H), 1.03 (s, 9H). HPLC > 99%, tR = 4.16 min. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C49H64F3IN7O10S+: 1126.3427; found: 1126.3422.
(2S,4R)-1-((S)-19-(tert-Butyl)-1-(3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)phenyl)-1,17-dioxo-3-oxa-2,7,18-triazaicosan-20-oyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (22).
White solid, 19% yield. 1H NMR (600 MHz, methanol-d4) δ 9.03 (s, 1H), 7.48–7.44 (m, 3H), 7.43 (d, J = 8.2 Hz, 2H), 7.40–7.36 (m, 2H), 7.07 (td, J = 9.2, 6.9 Hz, 1H),6.64 (td, J = 8.7, 4.3 Hz, 1H), 5.03–4.98 (m, 1H), 4.62 (s, 1H),4.59–4.53 (m, 1H), 4.43 (td, J = 4.2, 2.0 Hz, 1H), 4.14–4.04 (m, 2H), 3.87 (d, J = 11.0 Hz, 1H), 3.75 (dd, J = 11.0, 4.0 Hz, 1H), 3.24 (t, J = 5.9 Hz, 2H), 3.12–2.98 (m, 2H), 2.49 (s, 3H), 2.33–2.27 (m, 1H), 2.24 (dd, J = 8.2, 6.3 Hz, 1H), 2.22–2.17 (m, 1H), 2.03 (dt, J = 10.3, 5.0 Hz, 2H), 1.95 (ddd, J = 13.3, 9.0, 4.5 Hz, 1H), 1.72 (p, J = 7.8 Hz, 2H), 1.65–1.55 (m, 2H), 1.51 (d, J = 7.0 Hz, 3H), 1.42–1.37 (m, 2H), 1.35–1.26 (m, 8H), 1.03 (s, 9H). HPLC > 99%, tR = 4.59 min. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C49H64F3IN7O6S+: 1062.3630; found: 1062.3769.
(2S,4R)-1-((S)-21-(tert-Butyl)-1-(3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)phenyl)-1,19-dioxo-3-oxa-2,7,20-triazadocosan-22-oyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (23).
White solid, 17% yield. 1H NMR (600 MHz, methanol-d4) δ 9.12 (d, J = 1.7 Hz, 1H), 7.49–7.42 (m, 5H), 7.39 (dd, J = 16.6, 8.0 Hz, 2H), 7.07 (q, J = 8.5, 8.0 Hz, 1H), 6.70–6.57 (m, 1H), 4.64–4.60 (m, 1H), 4.59–4.54 (m, 1H),4.45–4.41 (m, 1H), 4.15–4.03 (m, 2H), 3.88 (d, J = 10.9 Hz, 1H),3.79–3.72 (m, 1H), 3.24 (t, J = 5.8 Hz, 2H), 3.04 (t, J = 7.8 Hz, 2H),2.55–2.46 (m, 3H), 2.30 (dt, J = 15.6, 8.0 Hz, 1H), 2.26–2.17 (m, 2H), 2.06–2.00 (m, 3H), 1.98–1.90 (m, 1H), 1.75–1.68 (m, 2H),1.65–1.54 (m, 2H), 1.51 (dd, J = 7.1, 1.7 Hz, 3H), 1.42–1.36 (m, 2H), 1.35–1.24 (m, 12H), 1.04 (s, 9H). HPLC > 99%, tR = 4.12 min. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C51H68F3IN7O6S+: 1090.3943; found: 1090.3857.
(2S,4R)-1-((S)-20-(tert-Butyl)-1-(3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)phenyl)-7-methyl-1,18-dioxo-3-oxa-2,7,19-triazahenicosan-21-oyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (24).
White solid, 16% yield. 1H NMR (800 MHz, methanol-d4) δ 8.89 (s, 1H), 7.51 (d, J = 7.9 Hz, 2H), 7.45 (t, J = 10.7 Hz, 3H), 7.39 (s, 1H), 7.36 (d, J = 8.7 Hz, 1H), 7.06 (q, J = 8.3 Hz, 1H), 6.62 (td, J = 8.7, 3.8 Hz, 1H), 5.02 (q, J = 7.2 Hz, 1H), 4.55 (t, J = 7.4 Hz, 1H), 4.49 (s, 1H), 4.47–4.43 (m, 1H), 4.11 (t, J = 7.6 Hz, 1H), 4.06–4.01 (m, 1H), 3.93 (dd, J = 10.8, 5.0 Hz, 1H), 3.68 (dd, J = 11.1, 3.6 Hz, 1H), 3.45 (q, J = 7.7, 6.6 Hz, 1H), 3.04 (td, J = 11.9, 5.0 Hz, 1H), 2.89 (s, 3H), 2.49 (s, 3H),2.27 (dt, J = 14.7, 7.9 Hz, 1H), 2.20 (ddd, J = 13.7, 9.0, 4.8 Hz, 2H),2.10 (dt, J = 12.8, 5.8 Hz, 3H), 1.80–1.67 (m, 2H), 1.59–1.52 (m, 2H), 1.45 (d, J = 7.1 Hz, 3H), 1.36–1.18 (m, 14H), 1.05 (s, 9H). 13C NMR (151 MHz, methanol-d4) δ 175.8, 173.0, 172.1, 168.2, 155.3, 153.6, 153.0, 148.4, 145.6, 144.2, 134.5, 133.6, 132.4, 131.1, 130.3 (2 × C), 127.5 (2 × C), 127.3, 125.6, 125.3, 125.1, 120.8, 120.2, 111.6, 82.3, 77.3, 70.8, 68.8, 60.4, 58.8, 57.8, 57.6, 57.1, 49.9, 40.3, 38.6,36.5, 36.3, 30.3, 30.2, 30.1, 29.9, 27.3, 26.9 (2 × C), 26.8, 25.1, 23.9, 22.2, 15.4. HPLC > 99%, tR = 4.62 min. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C51H68F3IN7O6S+: 1090.3943; found: 1090.3970.
(2S,4R)-1-((S)-20-(tert-Butyl)-1-(3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)phenyl)-1,18-dioxo-3-oxa-2,8,19-triazahenicosan-21-oyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (25).
White solid, 22% yield. 1H NMR (600 MHz, methanol-d4) δ 9.03 (s, 1H), 7.52–7.42 (m, 5H),7.41–7.33 (m, 2H), 7.06 (q, J = 8.7 Hz, 1H), 6.61 (td, J = 8.8, 4.3 Hz, 1H), 5.00 (q, J = 6.8 Hz, 1H), 4.63 (s, 1H), 4.57 (t, J = 8.3 Hz, 1H),4.43 (s, 1H), 3.93 (t, J = 5.5 Hz, 2H), 3.87 (d, J = 11.0 Hz, 1H), 3.75 (dd, J = 11.0, 3.9 Hz, 1H), 3.10 (t, J = 7.4 Hz, 2H), 3.05–2.95 (m, 2H), 2.50 (s, 3H), 2.30 (dt, J = 15.0, 7.7 Hz, 1H), 2.25 (dd, J = 8.1, 6.3 Hz, 1H), 2.22–2.17 (m, 1H), 1.95 (ddd, J = 13.3, 9.0, 4.6 Hz, 1H), 1.88 (p, J = 7.0 Hz, 2H), 1.76 (q, J = 6.1 Hz, 2H), 1.68 (p, J = 7.6 Hz, 2H), 1.63–1.55 (m, 2H), 1.51 (d, J = 7.0 Hz, 3H), 1.42–1.28 (m, 10H), 1.04 (s, 9H). HPLC > 99%, tR = 4.56 min. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C50H66F3IN7O6S+: 1076.3787; found: 1076.3827.
(2S,4R)-1-((S)-21-(tert-Butyl)-1-(3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)phenyl)-1,19-dioxo-3-oxa-2,8,20-triazadocosan-22-oyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (26).
White solid, 23% yield. 1H NMR (600 MHz, methanol-d4) δ 8.93 (s, 1H), 7.50–7.41 (m, 5H),7.37 (dd, J = 8.5, 6.6 Hz, 2H), 7.06 (q, J = 8.8 Hz, 1H), 6.62 (td, J = 8.7, 4.2 Hz, 1H), 5.02–4.98 (m, 1H), 4.65–4.61 (m, 1H), 4.58–4.55 (m, 1H), 4.45–4.42 (m, 1H), 3.93 (t, J = 5.6 Hz, 2H), 3.87 (d, J = 11.1 Hz, 1H), 3.75 (dd, J = 11.0, 4.0 Hz, 1H), 3.10 (t, J = 7.3 Hz, 2H), 3.04–2.95 (m, 2H), 2.48 (s, 3H), 2.30 (dt, J = 15.0, 7.6 Hz, 1H), 2.26–2.23 (m, 1H), 2.22–2.17 (m, 1H), 1.95 (ddd, J = 13.4, 9.1, 4.6 Hz, 1H), 1.88 (t, J = 7.1 Hz, 2H), 1.76 (q, J = 6.1 Hz, 2H),1.68 (p, J = 7.6 Hz, 2H), 1.63–1.56 (m, 2H), 1.51 (d, J = 7.0 Hz, 3H), 1.39–1.35 (m, 2H), 1.34–1.27 (m, 10H), 1.04 (s, 9H). HPLC > 99%, tR = 4.63 min. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C51H68F3IN7O6S+:1090.3943; found, 1090.3991.
(2S,4R)-1-((S)-22-(tert-Butyl)-1-(3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)phenyl)-1,20-dioxo-3-oxa-2,8,21-triazatricosan-23-oyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (27).
White solid, 25% yield. 1H NMR (600 MHz, methanol-d4) δ 9.00 (s, 1H), 7.47–7.41 (m, 5H),7.39 (td, J = 6.4, 3.0 Hz, 1H), 7.37–7.34 (m, 1H), 7.05 (td, J = 9.1, 6.9 Hz, 1H), 6.61 (td, J = 8.7, 4.3 Hz, 1H), 5.00 (q, J = 7.0 Hz, 1H),4.62 (s, 1H), 4.57 (dd, J = 9.1, 7.5 Hz, 1H), 4.44 (dp, J = 4.3, 2.0 Hz, 1H), 3.93 (t, J = 5.6 Hz, 2H), 3.88 (dt, J = 11.3, 1.8 Hz, 1H), 3.75 (dd, J = 11.0, 4.0 Hz, 1H), 3.10 (t, J = 7.3 Hz, 2H), 3.02–2.95 (m, 2H), 2.49 (s, 3H), 2.33–2.27 (m, 1H), 2.26–2.23 (m, 1H), 2.23–2.17 (m, 1H), 1.96 (ddd, J = 13.3, 9.0, 4.5 Hz, 1H), 1.88 (p, J = 7.1 Hz, 2H), 1.75 (h, J = 5.5, 5.0 Hz, 2H), 1.68 (p, J = 7.9 Hz, 2H), 1.63–1.56 (m, 2H), 1.51 (d, J = 7.0 Hz, 3H), 1.41–1.25 (m, 14H), 1.04 (s, 9H); 13C NMR (151 MHz, methanol-d4) δ 176.2, 173.3, 172.0, 167.2, 155.0, 153.4, 152.7, 148.8, 145.2, 134.4, 133.2, 132.4, 131.1, 130.5, 130.2 (2 × C), 128.1, 127.6 (2 × C), 125.4, 125.1, 125.0, 121.4, 120.6, 111.5, 82.1, 77.0, 70.3, 68.9, 60.5, 59.3, 56.7, 49.7, 41.1, 38.8, 36.4, 35.5, 30.4, 30.3 (2 × C), 30.2, 30.1, 30.0, 27.4, 27.0, 26.8 (2 ×C), 26.7, 26.1, 24.4, 22.4, 15.7. HPLC > 99%, tR = 4.61 min. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C52H70F3IN7O6S+: 1104.4100; found: 1104.3876.
N-(3-((2-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)-amino)ethyl)amino)propoxy)-3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)benzamide (28).
White solid, 20% yield. 1H NMR (600 MHz, methanol-d4) δ 7.54 (dd, J = 8.6, 7.1 Hz, 1H),7.37–7.34 (m, 1H), 7.32 (dd, J = 10.7, 1.9 Hz, 1H), 7.29 (dt, J = 8.6,1.2 Hz, 1H), 7.09 (d, J = 8.5 Hz, 1H), 7.06–7.00 (m, 2H), 6.58 (td, J = 8.7, 4.6 Hz, 1H), 4.95–4.91 (m, 1H), 4.19–4.02 (m, 2H), 3.77 (td, J = 5.7, 2.3 Hz, 2H), 3.37 (t, J = 5.8 Hz, 2H), 3.33 (t, J = 5.9 Hz, 2H),2.79 (ddd, J = 17.5, 13.9, 5.4 Hz, 1H), 2.67 (ddd, J = 17.5, 4.6, 2.6 Hz, 1H), 2.52 (qd, J = 13.1, 4.5 Hz, 1H), 2.11–2.00 (m, 2H), 1.94 (dtd, J = 13.1, 5.3, 2.5 Hz, 1H). HPLC > 99%, tR = 4.17 min. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C31H29F3IN6O6+: 765.1140; found: 765.1210.
N-(3-((3-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)-amino)propyl)amino)propoxy)-3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)benzamide (29).
White solid, 21% yield. 1H NMR (600 MHz, methanol-d4) δ 7.50 (dd, J = 8.6, 7.1 Hz, 1H),7.40–7.37 (m, 1H), 7.35 (dd, J = 10.8, 1.9 Hz, 1H), 7.27 (dt, J = 8.5,1.3 Hz, 1H), 7.06 (dd, J = 9.3, 7.0 Hz, 1H), 7.03 (d, J = 2.4 Hz, 1H), 7.02 (d, J = 3.9 Hz, 1H), 6.56 (td, J = 8.7, 4.3 Hz, 1H), 5.01 (dd, J = 12.8, 5.5 Hz, 1H), 4.08 (dd, J = 5.7, 4.4 Hz, 2H), 3.49 (t, J = 6.5 Hz, 2H), 3.27 (t, J = 5.7 Hz, 2H), 3.21 (t, J = 7.2 Hz, 2H), 2.84 (ddd, J = 17.5, 13.9, 5.3 Hz, 1H), 2.73 (ddd, J = 17.4, 4.4, 2.6 Hz, 1H), 2.66 (qd, J = 13.1, 4.5 Hz, 1H), 2.13–2.07 (m, 2H), 2.06–1.99 (m, 3H). HPLC > 99%, tR = 4.16 min. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C32H31F3IN6O6+: 6 6 : 779.1296; found: 779.1603.
N-(3-((4-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)-amino)butyl)amino)propoxy)-3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)benzamide (30).
White solid, 24% yield. 1H NMR (600 MHz, methanol-d4) δ 7.52 (dd, J = 8.6, 7.1 Hz, 1H), 7.40 (dd, J = 10.7, 1.9 Hz, 1H), 7.38–7.35 (m, 1H), 7.27 (dt, J = 8.5, 1.3 Hz, 1H), 7.09–7.03 (m, 1H), 7.02 (d, J = 7.1 Hz, 1H), 6.99 (d, J = 8.5 Hz, 1H), 6.60 (td, J = 8.7, 4.6 Hz, 1H), 5.01–4.93 (m, 1H), 4.17–4.01 (m, 2H), 3.35 (td, J = 6.6, 1.6 Hz, 2H), 3.25 (t, J = 5.8 Hz, 2H),3.13 (t, J = 7.4 Hz, 2H), 2.82 (ddd, J = 17.5, 13.9, 5.3 Hz, 1H), 2.72 (ddd, J = 17.5, 4.5, 2.6 Hz, 1H), 2.63 (qd, J = 13.1, 4.5 Hz, 1H),2.08–1.97 (m, 3H), 1.89 (p, J = 7.3 Hz, 2H), 1.83–1.75 (m, 2H). HPLC > 99%, tR = 4.20 min. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C33H33F3IN6O6+: 793.1453; found: 793.1498.
N-(3-((5-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)-amino)pentyl)amino)propoxy)-3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)benzamide (31).
White solid, 23% yield. 1H NMR (600 MHz, methanol-d4) δ 7.51 (dd, J = 8.6, 7.1 Hz, 1H), 7.42 (dd, J = 10.7, 1.9 Hz, 1H), 7.40–7.37 (m, 1H), 7.31 (dt, J = 8.4, 1.3 Hz, 1H), 7.07–7.00 (m, 2H), 6.98 (d, J = 8.6 Hz, 1H), 6.60 (td, J = 8.8, 4.6 Hz, 1H), 5.00 (dd, J = 12.7, 5.5 Hz, 1H), 4.08 (t, J = 5.3 Hz, 2H), 3.32–3.28 (m, 2H), 3.25 (t, J = 5.7 Hz, 2H), 3.09 (t, J = 7.6 Hz, 2H), 2.84 (ddd, J = 17.4, 13.8, 5.3 Hz, 1H), 2.78–2.71 (m, 1H),2.70–2.62 (m, 1H), 2.09–1.95 (m, 3H), 1.82 (p, J = 7.8 Hz, 2H),1.71 (p, J = 7.0 Hz, 2H), 1.56 (tt, J = 10.0, 6.3 Hz, 2H). HPLC > 99%, tR = 4.19 min. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C34H35F3IN6O6+: 807.1609; found: 807.1524.
N-(3-((6-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)-amino)hexyl)amino)propoxy)-3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)benzamide (32).
White solid, 27% yield. 1H NMR (600 MHz, methanol-d4) δ 7.52 (dd, J = 8.6, 7.0 Hz, 1H), 7.43 (dd, J = 10.6, 1.9 Hz, 1H), 7.39 (ddd, J = 8.9, 5.3, 1.7 Hz, 1H), 7.34 (dt, J = 8.4, 1.3 Hz, 1H), 7.05 (dd, J = 9.3, 7.1 Hz, 1H), 7.03 (d, J = 7.0 Hz, 1H), 7.00 (d, J = 8.5 Hz, 1H), 6.61 (td, J = 8.7, 4.4 Hz, 1H),5.03 (dd, J = 12.7, 5.4 Hz, 1H), 4.08 (t, J = 5.1 Hz, 2H), 3.30–3.28 (m, 2H), 3.24 (t, J = 5.8 Hz, 2H), 3.10–3.02 (m, 2H), 2.88–2.80 (m, 1H), 2.73 (ddd, J = 16.2, 4.0, 2.1 Hz, 1H), 2.67 (td, J = 13.3, 4.4 Hz, 1H), 2.10–2.05 (m, 1H), 2.05–2.00 (m, 2H), 1.76 (q, J = 7.4 Hz, 2H), 1.64 (q, J = 6.9 Hz, 2H), 1.47 (t, J = 3.7 Hz, 4H). HPLC > 99%, tR = 4.22 min. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C35H37F3IN6O6+: 821.1766; found: 821.1712.
N-(3-((7-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)-amino)heptyl)amino)propoxy)-3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)benzamide (33).
White solid, 33% yield. 1H NMR (600 MHz, methanol-d4) δ 7.52 (dd, J = 8.6, 7.0 Hz, 1H), 7.44 (dd, J = 10.7, 1.9 Hz, 1H), 7.39 (ddd, J = 8.7, 5.3, 1.7 Hz, 1H), 7.35 (dt, J = 8.6, 1.4 Hz, 1H), 7.07–7.01 (m, 2H), 7.00 (d, J = 8.5 Hz, 1H), 6.62 (td, J = 8.7, 4.4 Hz, 1H), 5.03 (dd, J = 12.8, 5.4 Hz, 1H),4.20–3.95 (m, 2H), 3.29 (t, J = 7.1 Hz, 2H), 3.23 (t, J = 5.8 Hz, 2H),3.07–3.02 (m, 2H), 2.85 (ddd, J = 17.5, 14.0, 5.4 Hz, 1H), 2.76–2.71 (m, 1H), 2.71–2.66 (m, 1H), 2.12–2.06 (m, 1H), 2.03 (td, J = 6.0,4.4 Hz, 2H), 1.74 (p, J = 7.6 Hz, 2H), 1.65 (p, J = 6.9 Hz, 2H), 1.51–1.35 (m, 6H). HPLC > 99%, tR = 4.27 min. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C36H39F3IN6O6+: 835.1922; found: 835.1902.
N-(3-((8-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)-amino)octyl)amino)propoxy)-3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)benzamide (34).
White solid, 34% yield. 1H NMR (600 MHz, methanol-d4) δ 7.53 (dd, J = 8.6, 7.1 Hz, 1H), 7.45 (dd, J = 10.6, 1.9 Hz, 1H), 7.40 (dd, J = 8.6, 5.5 Hz, 1H), 7.36 (dt, J = 8.4, 1.3 Hz, 1H), 7.05 (dd, J = 9.2, 7.1 Hz, 1H), 7.03 (d, J = 7.1 Hz, 1H), 7.01 (d, J = 8.5 Hz, 1H), 6.63 (td, J = 8.7, 4.4 Hz, 1H), 5.04 (dd, J = 12.8, 5.5 Hz, 1H), 4.08 (dd, J = 5.8, 4.5 Hz, 2H), 3.30–3.28 (m, 2H), 3.23 (t, J = 5.8 Hz, 2H), 3.08–3.00 (m, 2H), 2.85 (ddd, J = 17.5,13.9, 5.3 Hz, 1H), 2.78–2.60 (m, 2H), 2.09 (dtd, J = 10.2, 5.7, 2.5 Hz, 1H), 2.05–1.99 (m, 2H), 1.72 (p, J = 7.6 Hz, 2H), 1.64 (p, J = 7.0 Hz, 2H), 1.41 (d, J = 5.4 Hz, 4H), 1.38–1.33 (m, 4H). HPLC > 99%, tR = 4.31 min. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C37H41F3IN6O6+: 849.2079; found: 849.2108.
N-(3-((2-(2-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethyl)amino) propoxy)-3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)benzamide (35).
White solid, 28% yield. 1H NMR (600 MHz, methanol-d4) δ 7.46 (dd, J = 8.6, 7.1 Hz, 1H), 7.38 (dd, J = 10.7, 2.0 Hz, 1H), 7.35 (dd, J = 7.7, 2.3 Hz, 1H), 7.26 (dt, J = 8.3, 1.3 Hz, 1H), 6.96 (d, J = 7.0 Hz, 1H), 6.95–6.88 (m, 2H), 6.57 (td, J = 8.8, 4.7 Hz, 1H), 4.98–4.93 (m, 1H), 4.15–4.07 (m, 2H),3.93–3.83 (m, 2H), 3.77 (ddd, J = 6.0, 4.5, 1.9 Hz, 2H), 3.45 (dd, J = 5.7, 4.2 Hz, 2H), 3.34 (t, J = 5.2 Hz, 4H), 2.82 (ddd, J = 17.5, 13.8, 5.3 Hz, 1H), 2.71 (ddd, J = 17.5, 4.5, 2.7 Hz, 1H), 2.60 (qd, J = 13.1, 4.5 Hz, 1H), 2.09–2.03 (m, 2H), 2.00 (dtd, J = 13.1, 5.3, 3.0 Hz, 1H). HPLC > 99%, tR = 4.19 min. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C33H33F3IN6O7+: 809.1402; found: 809.1599.
N-(3-((2-(2-(2-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy) ethyl)amino)propoxy)-3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)benzamide (36).
White solid, 27% yield. 1H NMR (600 MHz, methanol-d4) δ 7.54 (dd, J = 8.6, 7.1 Hz, 1H), 7.44 (dd, J = 10.7, 1.9 Hz, 1H), 7.39–7.31 (m, 2H), 7.09–6.97 (m, 3H), 6.63 (td, J = 8.7, 4.1 Hz, 1H), 5.03 (dd, J = 12.8, 5.5 Hz, 1H), 4.04 (t, J = 5.0 Hz, 2H), 3.81 (t, J = 5.0 Hz, 2H), 3.67 (t, J = 5.1 Hz, 2H), 3.66–3.58 (m, 4H), 3.45 (dd, J = 5.8, 4.5 Hz, 2H), 3.27 (t, J = 6.0 Hz, 2H), 3.24 (t, J = 5.1 Hz, 2H), 2.83 (ddd, J = 17.5, 13.9, 5.3 Hz, 1H), 2.76–2.56 (m, 2H), 2.12–2.04 (m, 1H), 2.01 (dt, J = 10.5,5.2 Hz, 2H). HPLC > 99%, tR = 4.18 min. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C35H37F3IN6O8+: 853.1664; found: 853.1614.
N-((1-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)-amino)-3,6,9-trioxa-12-azapenta decan-15-yl)oxy)-3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)benzamide (37).
White solid, 28% yield. 1H NMR (600 MHz, methanol-d4) δ 7.54 (dd, J = 8.5, 7.0 Hz, 1H), 7.46 (dd, J = 10.6, 1.9 Hz, 1H), 7.40–7.31 (m, 2H), 7.04 (dt, J = 15.9, 8.7 Hz, 3H), 6.63 (td, J = 8.7, 4.1 Hz, 1H), 5.04 (dd, J = 12.8, 5.5 Hz, 1H), 4.03 (t, J = 5.1 Hz, 2H), 3.78–3.74 (m, 2H), 3.70 (t, J = 5.2 Hz, 2H), 3.65–3.60 (m, 4H), 3.60 (s, 4H), 3.48 (t, J = 5.1 Hz, 2H), 3.26 (t, J = 6.0 Hz, 2H), 3.24 (t, J = 5.0 Hz, 2H), 2.84 (ddd, J = 17.6, 14.0, 5.3 Hz, 1H), 2.73 (ddd, J = 17.5, 4.3, 2.6 Hz, 1H), 2.71–2.65 (m, 1H), 2.17–2.05 (m, 1H), 2.04–1.97 (m, 2H). HPLC > 99%, tR = 4.18 min. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C37H41F3IN6O9+: 897.1926; found: 897.2312.
N-((1-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)-amino)-3,6,9,12-tetraoxa-15-azaoctadecan-18-yl)oxy)-3,4-di-fluoro-2-((2-fluoro-4-iodophenyl)amino)benzamide (38).
White solid, 27% yield. 1H NMR (600 MHz, methanol-d4) δ 7.54 (dd, J = 8.6, 7.1 Hz, 1H), 7.47 (dd, J = 10.7, 1.9 Hz, 1H), 7.43–7.31 (m, 2H),7.10–6.98 (m, 3H), 6.64 (td, J = 8.7, 4.1 Hz, 1H), 5.04 (dd, J = 12.7,5.6 Hz, 1H), 4.04 (t, J = 5.2 Hz, 2H), 3.80–3.74 (m, 2H), 3.71 (t, J = 5.2 Hz, 2H), 3.67–3.62 (m, 4H), 3.60 (dt, J = 3.8, 2.6 Hz, 2H), 3.59–3.54 (m, 6H), 3.49 (t, J = 5.2 Hz, 2H), 3.28 (t, J = 6.0 Hz, 2H), 3.25 (t, J = 5.0 Hz, 2H), 2.85 (ddd, J = 17.6, 14.0, 5.3 Hz, 1H), 2.76–2.72 (m, 1H), 2.69 (td, J = 13.3, 4.5 Hz, 1H), 2.09 (ddt, J = 13.1, 5.5, 2.8 Hz, 1H), 2.02 (dt, J = 10.5, 5.2 Hz, 2H). HPLC > 99%, tR = 4.21 min. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C39H45F3IN6O10+: 941.2188; found: 941.2169.
N-((1-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)-amino)-3,6,9,12,15-pentaoxa-18-azahenicosan-21-yl)oxy)-3,4-di-fluoro-2-((2-fluoro-4-iodophenyl)amino)benzamide (39).
White solid, 38% yield. 1H NMR (600 MHz, methanol-d4) δ 7.54 (dd, J = 8.5, 7.1 Hz, 1H), 7.47 (dd, J = 10.7, 1.9 Hz, 1H), 7.40 (ddd, J = 9.0,5.3, 1.7 Hz, 1H), 7.37 (ddd, J = 8.5, 2.0, 1.0 Hz, 1H), 7.11–6.99 (m, 3H), 6.64 (td, J = 8.7, 4.2 Hz, 1H), 5.04 (dd, J = 12.8, 5.5 Hz, 1H),4.05 (t, J = 5.2 Hz, 2H), 3.79–3.75 (m, 2H), 3.71 (t, J = 5.2 Hz, 2H),3.65 (s, 4H), 3.64–3.61 (m, 2H), 3.60–3.58 (m, 2H), 3.58–3.56 (m, 4H), 3.54 (dd, J = 6.0, 2.7 Hz, 3H), 3.48 (t, J = 5.2 Hz, 2H), 3.29 (t, J = 6.1 Hz, 2H), 3.26 (t, J = 5.1 Hz, 2H), 2.85 (ddd, J = 17.6, 14.0, 5.3 Hz, 1H), 2.75 (dd, J = 4.4, 2.6 Hz, 1H), 2.74–2.70 (m, 1H), 2.70–2.66 (m, 1H), 2.15–2.07 (m, 1H), 2.06–1.99 (m, 2H). HPLC > 99%, tR = 4.21 min. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C41H49F3IN6O11+: 985.2451; found: 985.2562.
N-(3-((3-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)amino)propyl)amino)propoxy)-3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)benzamide (40).
White solid, 28% yield. 1H NMR (600 MHz, methanol-d4) δ 7.50 (d, J = 8.3 Hz, 1H), 7.36 (dd, J = 10.7, 1.9 Hz, 1H), 7.31 (d, J = 6.6 Hz, 1H), 7.30 (dt, J = 8.5, 1.2 Hz, 1H), 7.04 (td, J = 9.2, 7.0 Hz, 1H), 6.94 (d, J = 2.2 Hz, 1H), 6.81 (dd, J = 8.4, 2.2 Hz, 1H), 6.58 (td, J = 8.7, 4.2 Hz, 1H), 5.02 (dd, J = 12.8, 5.5 Hz, 1H), 4.13–4.02 (m, 2H), 3.36 (t, J = 6.9 Hz, 2H), 3.27 (t, J = 5.7 Hz, 2H), 3.22 (t, J = 7.3 Hz, 2H), 2.85 (ddd, J = 17.4, 13.9, 5.4 Hz, 1H), 2.73 (ddd, J = 17.4, 4.5, 2.6 Hz, 1H), 2.66 (qd, J = 13.1, 4.5 Hz, 1H), 2.13–2.07 (m, 2H), 2.07–2.02 (m, 3H). HPLC > 99%, tR = 4.13 min. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C32H31F3IN6O6+: 779.1296; found: 779.1390.
N-(3-((4-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)amino)butyl)amino)propoxy)-3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)benzamide (41).
White solid, 20% yield. 1H NMR (600 MHz, acetone-d6) δ 9.88 (s, 1H), 9.34 (s, 1H), 8.80 (s, 1H), 7.65–7.57 (m, 1H), 7.54 (d, J = 8.4 Hz, 1H), 7.47 (dd, J = 10.7, 2.0 Hz, 1H), 7.40 (dd, J = 8.5, 1.9 Hz, 1H), 7.08 (td, J = 9.3, 7.1 Hz, 1H), 6.98 (d, J = 2.1 Hz, 1H), 6.90 (dd, J = 8.4, 2.2 Hz, 1H), 6.76 (td, J = 8.8, 5.2 Hz, 1H), 5.04 (dd, J = 12.6, 5.4 Hz, 1H), 4.19 (t, J = 5.2 Hz, 2H), 3.40 (s, 3H), 3.32 (t, J = 6.8 Hz, 3H), 3.00–2.90 (m, 1H),2.85–2.67 (m, 2H), 2.20–2.11 (m, 3H), 2.04–1.98 (m, 2H), 1.84 (p, J = 7.0 Hz, 2H). HPLC > 99%, tR = 4.21 min. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C33H33F3IN6O6+: 793.1453; found: 793.1475.
N-(3-((5-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)amino)pentyl)amino)propoxy)-3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)benzamide (42).
White solid, 17% yield. 1H NMR (600 MHz, methanol-d4) δ 7.53 (d, J = 8.4 Hz, 1H), 7.41 (dd, J = 10.7, 1.9 Hz, 1H), 7.39–7.36 (m, 1H), 7.35–7.31 (m, 1H), 7.05 (q, J = 8.7 Hz, 1H), 6.93 (d, J = 2.1 Hz, 1H), 6.81 (dd, J = 8.4, 2.2 Hz, 1H), 6.61 (td, J = 8.7, 4.2 Hz, 1H), 5.03 (dd, J = 12.6, 5.4 Hz, 1H),4.07 (t, J = 5.1 Hz, 2H), 3.24 (t, J = 5.8 Hz, 2H), 3.20 (t, J = 7.0 Hz, 2H), 3.10–3.03 (m, 2H), 2.91–2.81 (m, 1H), 2.78–2.71 (m, 1H),2.68 (td, J = 13.3, 4.4 Hz, 1H), 2.12–2.06 (m, 1H), 2.05–1.99 (m, 2H), 1.81 (p, J = 7.9 Hz, 2H), 1.70 (p, J = 7.1 Hz, 2H), 1.54 (p, J = 7.7, 7.3 Hz, 2H). HPLC > 99%, tR = 4.29 min. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C34H35F3IN6O6+: 807.1609; found: 807.1607.
N-(3-((6-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)amino)hexyl)amino)propoxy)-3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)benzamide (43).
White solid, 12% yield. 1H NMR (600 MHz, methanol-d4) δ 7.53 (d, J = 8.4 Hz, 1H), 7.43 (dd, J = 10.6, 1.9 Hz, 1H), 7.39 (t, J = 7.1 Hz, 1H), 7.35 (dd, J = 8.5, 1.5 Hz, 1H), 7.05 (q, J = 8.7 Hz, 1H), 6.94 (d, J = 2.3 Hz, 1H), 6.81 (dd, J = 8.4, 2.2 Hz, 1H), 6.62 (td, J = 8.7, 4.3 Hz, 1H), 5.08–4.99 (m, 1H),4.07 (t, J = 5.1 Hz, 2H), 3.24 (t, J = 5.8 Hz, 2H), 3.19 (t, J = 7.1 Hz, 2H), 3.09–3.02 (m, 2H), 2.86 (ddd, J = 22.7, 12.4, 5.4 Hz, 1H),2.78–2.71 (m, 1H), 2.68 (td, J = 13.3, 4.5 Hz, 1H), 2.13–2.05 (m, 1H), 2.06–1.99 (m, 2H), 1.80–1.73 (m, 2H), 1.65 (t, J = 7.0 Hz, 2H), 1.55–1.42 (m, 4H). HPLC > 99%, tR = 4.42 min. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C35H37F3IN6O6+: 821.1766; found: 821.1807.
N-(3-((7-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)amino)heptyl)amino)propoxy)-3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)benzamide (44).
White solid, 15% yield. 1H NMR (600 MHz, methanol-d4) δ 7.53 (d, J = 8.3 Hz, 1H), 7.43 (dd, J = 10.6, 1.9 Hz, 1H), 7.41–7.38 (m, 1H), 7.35 (dt, J = 8.4, 1.3 Hz, 1H), 7.05 (td, J = 9.2, 7.0 Hz, 1H), 6.93 (d, J = 2.2 Hz, 1H), 6.80 (dd, J = 8.4, 2.1 Hz, 1H), 6.62 (td, J = 8.7, 4.3 Hz, 1H), 5.03 (dd, J = 12.8, 5.5 Hz, 1H), 4.07 (t, J = 5.1 Hz, 2H), 3.23 (t, J = 5.8 Hz, 2H), 3.18 (t, J = 7.1 Hz, 2H), 3.08–3.02 (m, 2H), 2.85 (ddd, J = 17.7, 14.0, 5.3 Hz, 1H), 2.77–2.71 (m, 1H), 2.68 (td, J = 13.3, 4.4 Hz, 1H), 2.08 (dtd, J = 14.9, 5.2, 4.3, 2.5 Hz, 1H), 2.03 (dt, J = 6.5, 3.1 Hz, 2H), 1.74 (q, J = 7.7 Hz, 2H), 1.64 (q, J = 7.1 Hz, 2H), 1.47–1.35 (m, 6H). HPLC > 99%, tR = 4.61 min. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C36H39F3IN6O6+: 835.1922; found: 835.1927.
N-(3-((8-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)amino)octyl)amino)propoxy)-3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)benzamide (45).
White solid, 18% yield. 1H NMR (600 MHz, methanol-d4) δ 7.54 (d, J = 8.4 Hz, 1H), 7.45 (dd, J = 10.6, 1.9 Hz, 1H), 7.40 (d, J = 7.4 Hz, 1H), 7.36 (d, J = 8.7 Hz, 1H), 7.09–7.02 (m, 1H), 6.94 (d, J = 2.2 Hz, 1H), 6.81 (dd, J = 8.4,2.2 Hz, 1H), 6.63 (q, J = 4.5 Hz, 1H), 5.03 (dd, J = 12.7, 5.6 Hz, 1H),4.07 (t, J = 5.1 Hz, 2H), 3.23 (t, J = 5.8 Hz, 2H), 3.19 (t, J = 7.1 Hz, 2H), 3.04 (t, J = 7.9 Hz, 2H), 2.90–2.81 (m, 1H), 2.78–2.71 (m, 1H), 2.72–2.65 (m, 1H), 2.12–2.06 (m, 1H), 2.06–2.00 (m, 2H),1.73 (d, J = 7.9 Hz, 2H), 1.68–1.61 (m, 2H), 1.46–1.40 (m, 4H),1.39–1.33 (m, 4H). HPLC > 99%, tR = 4.70 min. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C37H41F3IN6O6+: 849.2079; found: 849.2111.
N-(3-((2-(2-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)amino)ethoxy)ethyl)amino)propoxy)-3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)benzamide (46).
White solid, 38% yield. 1H NMR (600 MHz, methanol-d4) δ 7.50–7.40 (m, 2H), 7.33 (dd, J = 8.5, 1.6 Hz, 1H), 7.29 (t, J = 7.2 Hz, 1H), 7.00 (q, J = 8.6 Hz, 1H),6.86 (d, J = 2.2 Hz, 1H), 6.74 (dd, J = 8.4, 2.2 Hz, 1H), 6.59 (td, J = 8.7, 4.4 Hz, 1H), 5.03 (dd, J = 12.7, 5.5 Hz, 1H), 4.06 (t, J = 5.0 Hz, 2H), 3.87–3.77 (m, 2H), 3.70 (t, J = 5.3 Hz, 2H), 3.33 (t, J = 5.4 Hz, 2H), 3.32–3.30 (m, 4H), 2.85 (ddd, J = 17.2, 13.8, 5.3 Hz, 1H),2.77–2.72 (m, 1H), 2.68 (qd, J = 13.1, 4.4 Hz, 1H), 2.11–2.07 (m, 1H), 2.05 (t, J = 5.8 Hz, 2H). HPLC > 99%, tR = 4.15 min. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C33H33F3IN6O7+: 809.1402; found: 809.1418.
N-(3-((2-(2-(2-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)amino)ethoxy)ethoxy)ethyl)amino)propoxy)-3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)benzamide (47).
White solid, 27% yield. 1H NMR (600 MHz, methanol-d4) δ 7.54 (d, J = 8.4 Hz, 1H),7.46 (dd, J = 10.7, 1.9 Hz, 1H), 7.37 (t, J = 8.8 Hz, 2H), 7.09–7.02 (m, 1H), 7.01 (d, J = 2.2 Hz, 1H), 6.84 (dd, J = 8.4, 2.2 Hz, 1H), 6.63 (td, J = 8.7, 4.1 Hz, 1H), 5.09–5.00 (m, 1H), 4.04 (t, J = 5.2 Hz, 2H),3.77 (t, J = 5.0 Hz, 2H), 3.69–3.56 (m, 6H), 3.38 (t, J = 5.4 Hz, 2H),3.27 (t, J = 6.0 Hz, 2H), 3.23 (t, J = 5.0 Hz, 2H), 2.85 (ddd, J = 18.6, 14.0, 5.4 Hz, 1H), 2.77–2.71 (m, 1H), 2.69 (dd, J = 13.4, 4.3 Hz, 1H), 2.12–2.07 (m, 1H), 2.02 (dd, J = 11.5, 5.9 Hz, 2H). HPLC > 99%, tR = 4.21 min. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C35H37F3IN6O8+: 853.1664; found: 853.1651.
N-((1-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)amino)-3,6,9-trioxa-12-azapenta decan-15-yl)oxy)-3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)benzamide (48).
White solid, 16% yield. 1H NMR (600 MHz, methanol-d4) δ 7.55 (d, J = 8.4 Hz, 1H),7.46 (dd, J = 10.7, 2.0 Hz, 1H), 7.38 (d, J = 8.0 Hz, 1H), 7.35 (dd, J = 8.5, 1.6 Hz, 1H), 7.07–7.02 (m, 1H), 7.01 (d, J = 2.1 Hz, 1H), 6.86 (dd, J = 8.3, 2.2 Hz, 1H), 6.63 (td, J = 8.7, 4.1 Hz, 1H), 5.03 (dd, J = 12.7, 5.5 Hz, 1H), 4.04 (t, J = 5.2 Hz, 2H), 3.81–3.73 (m, 2H), 3.68 (t, J = 5.3 Hz, 2H), 3.62 (qd, J = 3.4, 1.6 Hz, 4H), 3.59 (s, 4H), 3.40 (t, J = 5.3 Hz, 2H), 3.25 (dt, J = 10.1, 5.5 Hz, 4H), 2.84 (ddd, J = 17.1, 13.8, 5.3 Hz, 1H), 2.78–2.71 (m, 1H), 2.67 (td, J = 13.2, 4.3 Hz, 1H), 2.12–2.04 (m, 1H), 2.04–1.99 (m, 2H). HPLC > 99%, tR = 4.27 min. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C37H41F3IN6O9+: 897.1926; found: 897.1900.
N-((1-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)amino)-3,6,9,12-tetraoxa-15-azaoctadecan-18-yl)oxy)-3,4-di-fluoro-2-((2-fluoro-4-iodophenyl)amino)benzamide (49).
White solid, 19% yield. 1H NMR (600 MHz, methanol-d4) δ 7.55 (d, J = 8.3 Hz, 1H), 7.46 (dd, J = 10.6, 1.9 Hz, 1H), 7.39 (d, J = 8.2 Hz, 1H),7.36 (dt, J = 8.5, 1.3 Hz, 1H), 7.09–7.02 (m, 1H), 7.02 (d, J = 2.1 Hz, 1H), 6.86 (dd, J = 8.4, 2.2 Hz, 1H), 6.64 (td, J = 8.7, 4.1 Hz, 1H),5.03 (dd, J = 12.8, 5.4 Hz, 1H), 4.04 (t, J = 5.2 Hz, 2H), 3.81–3.73 (m, 2H), 3.69 (t, J = 5.3 Hz, 2H), 3.66–3.62 (m, 4H), 3.61–3.59 (m, 2H), 3.58–3.55 (m, 6H), 3.40 (t, J = 5.3 Hz, 2H), 3.26 (t, J = 6.1 Hz, 2H), 3.24 (t, J = 5.1 Hz, 2H), 2.84 (ddd, J = 17.7, 14.0, 5.4 Hz, 1H),2.76–2.70 (m, 1H), 2.70–2.64 (m, 1H), 2.11–2.05 (m, 1H), 2.02 (dt, J = 10.5, 5.2 Hz, 2H). HPLC > 99%, tR = 4.35 min. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C39H45F3IN6O10+: 941.2188; found: 941.2445.
N-((1-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)amino)-3,6,9,12,15-pentaoxa-18-azahenicosan-21-yl)oxy)-3,4-di-fluoro-2-((2-fluoro-4-iodophenyl)amino)benzamide (50).
White solid, 17% yield. 1H NMR (600 MHz, methanol-d4) δ 7.44 (d, J = 8.4 Hz, 1H), 7.35 (dd, J = 10.6, 2.0 Hz, 1H), 7.30 (ddd, J = 9.0, 5.3, 1.7 Hz, 1H), 7.27–7.24 (m, 1H), 6.94 (td, J = 9.2, 6.9 Hz, 1H), 6.90 (d, J = 2.2 Hz, 1H), 6.75 (dd, J = 8.4, 2.2 Hz, 1H), 6.53 (td, J = 8.7, 4.2 Hz, 1H), 4.92 (dd, J = 12.7, 5.5 Hz, 1H), 3.95 (t, J = 5.2 Hz, 2H),3.72–3.62 (m, 2H), 3.58 (t, J = 5.3 Hz, 2H), 3.53 (s, 4H), 3.52–3.50 (m, 2H), 3.50–3.48 (m, 2H), 3.47 (td, J = 4.7, 4.0, 1.6 Hz, 3H), 3.44 (dt, J = 4.9, 2.1 Hz, 4H), 3.28 (t, J = 5.3 Hz, 2H), 3.21 (p, J = 1.7 Hz, 1H), 3.18 (t, J = 6.1 Hz, 2H), 3.15 (t, J = 5.0 Hz, 2H), 2.74 (ddd, J = 17.1, 13.7, 5.2 Hz, 1H), 2.67–2.61 (m, 1H), 2.57 (td, J = 13.3, 4.4 Hz, 1H), 1.98 (dtd, J = 10.5, 5.6, 4.8, 2.9 Hz, 1H), 1.93 (dt, J = 10.7, 5.2 Hz, 2H). 13C NMR (151 MHz, methanol-d4) δ 174.5, 171.5, 169.4, 169.0, 168.0, 155.9, 155.2, 153.5, 145.7, 144.0, 135.6, 134.4, 133.6, 132.3, 126.0, 125.6, 125.1, 121.0, 120.1, 118.2, 116.7, 111.4, 106.8, 82.4, 77.2, 71.4, 71.3, 71.2 (2 × C), 71.1 (3 × C), 71.0, 70.2, 66.7, (201 MHz, methanol-d4) δ 175.8, 173.050.1, 48.0, 43.8, 32.0, 25.4, 23.6. HPLC > 99%, tR = 4.37 min. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C41H49F3IN6O11+: 985.2451; found: 985.3103.
(2R,4S)-1-((S)-20-(tert-Butyl)-1-(3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)phenyl)-7-methyl-1,18-dioxo-3-oxa-2,7,19-triazahenicosan-21-oyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (51).
White solid, 20% yield. 1H NMR (800 MHz, methanol-d4) δ 8.89 (s, 1H), 7.48 (d, J = 10.6 Hz, 1H), 7.44 (d, J = 8.0 Hz, 2H), 7.42 (d, J = 7.9 Hz, 2H),7.38 (t, J = 7.3 Hz, 2H), 7.07 (q, J = 8.5 Hz, 1H), 6.64 (td, J = 8.7, 3.8 Hz, 1H), 5.00 (q, J = 7.2 Hz, 1H), 4.63 (s, 1H), 4.57 (t, J = 8.3 Hz, 1H), 4.44 (s, 1H), 4.10 (s, 1H), 4.04 (s, 1H), 3.87 (d, J = 11.0 Hz, 1H), 3.75 (dd, J = 11.2, 4.1 Hz, 1H), 3.45 (d, J = 6.6 Hz, 1H), 3.10–3.00 (m, 1H), 2.90 (s, 3H), 2.48 (s, 3H), 2.29 (dt, J = 15.0, 7.7 Hz, 1H), 2.23 (t, J = 7.2 Hz, 1H), 2.21–2.17 (m, 1H), 2.13–2.08 (m, 2H), 1.96 (ddd, J = 13.3, 8.9, 4.6 Hz, 1H), 1.77 (s, 1H), 1.73 (s, 1H),1.62–1.55 (m, 2H), 1.51 (d, J = 7.1 Hz, 3H), 1.39–1.24 (m, 14H),1.04 (s, 9H); 13C NMR (201 MHz, methanol-d4) δ 175.8, 173.0, 172.1, 168.2, 155.1, 154.9, 153.9, 153.7, 152.7, 148.8, 145.4, 134.5, 133.4, 132.4, 131.3, 130.3 (2 × C), 127.4 (2 × C), 127.3, 125.6, 125.2, 120.8, 120.2, 111.6, 82.3, 77.3, 70.8, 60.4, 58.8, 57.8, 57.6, 57.1, 49.9, 40.3, 38.6, 36.5, 36.3, 30.3 (2 × C), 30.1, 29.9, 27.3, 26.9 (3 ×C), 26.8, 25.1, 23.9, 22.2, 15.6. HPLC > 99%, tR = 4.75 min. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C51H68F3IN7O6S+: 1090.3943; found: 1090.4114.
(2R,4S)-1-((S)-22-(tert-Butyl)-1-(3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)phenyl)-1,20-dioxo-3-oxa-2,8,21-triazatricosan-23-oyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (52).
White solid, 22% yield. 1H NMR (600 MHz, methanol-d4) δ 9.01 (s, 1H), 7.52 (d, J = 8.0 Hz, 2H), 7.47–7.42 (m, 3H), 7.40–7.37 (m, 1H), 7.35 (dd, J = 8.6, 1.7 Hz, 1H), 7.13–6.98 (m, 1H), 6.61 (td, J = 8.7, 4.3 Hz, 1H), 5.02 (q, J = 7.0 Hz, 1H), 4.55 (dd, J = 8.2, 6.5 Hz, 1H), 4.49 (s, 1H), 4.45 (q, J = 4.6 Hz, 1H), 3.93 (q, J = 5.2 Hz, 3H), 3.69 (dd, J = 10.8, 3.5 Hz, 1H), 3.10 (t, J = 7.3 Hz, 2H), 3.02–2.93 (m, 2H), 2.50 (s, 3H),2.33–2.25 (m, 1H), 2.19 (dt, J = 14.4, 7.9 Hz, 2H), 2.10 (dt, J = 12.6,5.8 Hz, 1H), 1.88 (p, J = 7.2 Hz, 2H), 1.75 (p, J = 6.5 Hz, 2H), 1.66 (p, J = 7.7 Hz, 2H), 1.55 (dt, J = 22.0, 7.1 Hz, 2H), 1.45 (d, J = 7.1 Hz, 3H), 1.38–1.31 (m, 2H), 1.31–1.21 (m, 12H), 1.06 (s, 9H); 13C NMR (201 MHz, methanol-d4) δ 175.8, 173.0, 172.1, 167.2, 162.4, 154.8, 153.6, 152.7, 148.8, 145.5, 134.4, 133.2, 132.4, 131.3, 130.3 (2 × C), 127.4 (2 × C), 127.3, 125.4, 125.1, 125.0, 121.4, 120.6, 111.5, 82.1, 77.0, 70.8, 60.4, 58.8, 57.8, 49.9, 38.6, 36.5, 36.3, 30.4 (2 × C),30.3, 30.2, 30.1, 30.0, 27.4, 27.0, 26.9 (4 × C), 26.8, 26.1, 24.4, 22.2, 15.6. HPLC > 99%, tR = 4.84 min. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C52H70F3IN7O6S+: 1104.4100; found: 1104.4016.
3,4-Difluoro-2-((2-fluoro-4-iodophenyl)amino)-N-((1-((2-(1-methyl-2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)amino)-3,6,9,12,15-pentaoxa-18-azahenicosan-21-yl)oxy)benzamide (53).
White solid, 27% yield. 1H NMR (600 MHz, methanol-d4) δ 7.45 (d, J = 8.4 Hz, 1H), 7.36 (dd, J = 10.6, 1.9 Hz, 1H), 7.30 (ddd, J = 9.0, 5.3, 1.8 Hz, 1H), 7.26 (dt, J = 8.5, 1.4 Hz, 1H), 6.94 (td, J = 9.3, 7.0 Hz, 1H), 6.91 (d, J = 2.2 Hz, 1H), 6.76 (dd, J = 8.4, 2.2 Hz, 1H), 6.53 (td, J = 8.7, 4.2 Hz, 1H), 4.95 (dd, J = 12.9, 5.5 Hz, 1H), 3.95 (t, J = 5.2 Hz, 2H), 3.65 (dd, J = 5.7, 4.3 Hz, 2H), 3.59 (t, J = 5.3 Hz, 2H),3.54 (s, 4H), 3.52–3.51 (m, 1H), 3.49 (dd, J = 5.9, 2.5 Hz, 2H),3.48–3.46 (m, 3H), 3.46–3.43 (m, 4H), 3.29 (t, J = 5.3 Hz, 2H),3.21 (p, J = 1.6 Hz, 2H), 3.18 (t, J = 6.1 Hz, 2H), 3.15 (t, J = 5.0 Hz, 2H), 3.03 (s, 3H), 2.79–2.76 (m, 1H), 2.76 (d, J = 4.0 Hz, 1H),2.62–2.48 (m, 1H), 1.97 (ddd, J = 9.9, 5.0, 2.9 Hz, 1H), 1.95–1.91 (m, 2H). 13C NMR (151 MHz, methanol-d4) δ 173.5, 171.3, 169.4, 169.1, 168.0, 155.9, 155.2, 153.6, 145.7, 144.0, 135.7, 134.5, 133.6, 132.3, 126.0, 125.6, 125.2, 121.0, 120.1, 118.2, 116.7, 111.4, 106.8,82.4, 77.2, 71.4, 71.4, 71.3, 71.2 (2 × C), 71.1 (2 × C), 71.1, 70.2, 66.8, 50.8, 48.1, 43.9, 32.3, 27.2, 25.4, 22.9. HPLC > 99%, tR = 4.42 min. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C42H51F3IN6O11+: 999.2607; found: 999.4250.
N-(3-(1,3-Dioxolan-2-yl)propoxy)-3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)benzamide (61).
Compound 61 was prepared according to the previously published procedures for the synthesis of compound 60.44 White solid, 60% yield. 1H NMR (600 MHz, methanol-d4) δ 7.45 (dd, J = 10.7, 1.9 Hz, 1H), 7.35 (ddd, J = 8.5, 2.0, 1.1 Hz, 1H), 7.31 (ddd, J = 8.5, 2.0, 1.0 Hz, 1H), 7.03 (td, J = 9.3, 7.1 Hz, 1H), 6.60 (td, J = 8.7, 4.1 Hz, 1H), 5.74 (s, 1H), 3.95–3.89 (m, 2H), 3.87–3.83 (m, 2H), 3.83–3.80 (m, 2H), 1.77–1.66 (m, 4H). HPLC > 99%, tR = 4.48 min. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C19H19F3IN2O4+: 523.0336; found: 523.0324.
11-((tert-Butoxycarbonyl)(methyl)amino)undecanoic Acid (65).
To a solution of compound 64 (1.0 g, 2.6 mmol) in DMF (4 mL) was added NaH (0.3 g, 7.8 mmol) at 0 °C. The reaction mixture was stirred at rt for 0.5 h before CH3I (0.3 mL, 5.2 mmol) was added at 0 °C. After the reaction mixture was stirred for 18 h, the mixture was poured into ice water and filtered. It was then washed with methanol, and the filtrate was evaporated to obtain a white solid (0.4 g, 46% yield) without purification. 1H NMR (600 MHz, methanol-d4) δ 3.65 (s, 1H), 3.21 (d, J = 7.5 Hz, 1H), 3.01 (t, J = 7.1 Hz, 2H), 2.83 (s, 1H), 2.31 (t, J = 7.4 Hz, 1H), 2.27 (t, J = 7.4 Hz, 2H), 1.60 (t, J = 7.1 Hz, 2H), 1.46–1.42 (m, 10H), 1.36–1.26 (m, 12H).
(2S,4R)-1-((S)-3,3-Dimethyl-2-(11-(methylamino)undecanamido)butanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (66).
To a solution of compound 65 (0.4 g, 1.2 mmol) and (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (0.5 g, 1.2 mmol) in DMSO (5 mL) were added HOAt (0.24 g, 1.8 mmol), EDCI (0.35 g, 1.8 mmol), and N-methylmorpholine (0.4 mL, 3.6 mmol). The reaction mixture was stirred at rt overnight. The reaction mixture was monitored by UPLC. Upon completion, it was purified by reverse-phase column chromatography to provide the title compound 66 (0.5 g, 67% yield) as a white solid. 1H NMR (600 MHz, methanol-d4) δ 8.98 (s, 1H), 7.47–7.41 (m, 4H), 5.00 (q, J = 6.9 Hz, 1H), 4.63 (d, J = 2.6 Hz, 1H), 4.59–4.53 (m, 1H), 4.44 (dp, J = 4.3, 1.9 Hz, 1H),3.88 (dt, J = 11.3, 1.7 Hz, 1H), 3.75 (dd, J = 11.0, 4.0 Hz, 1H), 3.04–2.86 (m, 2H), 2.69 (s, 3H), 2.49 (s, 3H), 2.30 (ddd, J = 14.1, 8.3, 7.1 Hz, 1H), 2.27–2.22 (m, 1H), 2.20 (ddt, J = 13.1, 7.7, 2.0 Hz, 1H),1.96 (ddd, J = 13.3, 9.0, 4.5 Hz, 1H), 1.72–1.63 (m, 2H), 1.59 (dd, J = 15.8, 7.2 Hz, 2H), 1.51 (d, J = 7.0 Hz, 3H), 1.34 (d, J = 13.7 Hz, 12H), 1.04 (s, 9H).
Compound 67 was made by following similar procedures as compound 66.
(2R,4S)-1-((S)-3,3-Dimethyl-2-(11-(methylamino)-undecanamido)butanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (67).
White solid, 60% yield. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C35H56N5O4S+: 642.4048; found: 642.4124.
Cell Culture.
Cells were cultivated in Dulbecco’s modified Eagle’s medium (DMEM), RPMI-1640, or Eagle’s minimum essential medium (EMEM) supplemented with 10% FBS, 100 units/mL of penicillin, and 100 g/mL of streptomycin.
Western Blot.
Cells were lysed on ice for 30 min with the lysis buffer (50 mM Tris pH 7.4, 1% IGEPAL CA-630, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), and 1 mM AESBF), supplemented with protease and phosphatase inhibitor cocktail (A32961, Thermo Fisher Scientific). The samples were centrifuged at 12 000g for 10 min at 4 °C to obtain the supernatant as a cell lysate. Protein concentrations were quantified using the Pierce rapid gold bicinchoninic acid (BCA) protein assay kit. The primary antibodies used were MEK1 (2352, CST), MEK2 (9147, CST), pMEK (9121, CST), ERK (4696, CST), pERK (4370, CST), α-tubulin (T6074, Sigma-Aldrich), IKZF1 (9034, CST), IKZF3 (15103, CST), ZFP91 (PA5–41199, Thermo Fisher Scientific), and GSPT1 (ab126090, Abcam). Fluorescence-labeled IRDye 800CW goat anti-rabbit IgG (926–32211, LI-COR) and IRDye 680CW donkey anti-mouse IgG (926–68072, LI-COR) and the OdysseyCLx imaging system (LICOR) were used to obtain protein signals, which were then analyzed by Image Studio Lite software (LI-COR). DC50 values were obtained with GraphPad Prism 8 from the data of three independent experiments.
Binding Affinity Assays.
For most assays, kinase-tagged T7 phage strains were prepared in an Escherichia coli host derived from the BL21 strain. E. coli were grown to log-phase and infected with T7 phage and incubated with shaking at 32 °C until lysis. The lysates were centrifuged and filtered to remove cell debris. The remaining kinases were produced in HEK-293 cells and subsequently tagged with DNA for qPCR detection. Streptavidin-coated magnetic beads were treated with biotinylated small-molecule ligands for 30 min at rt to generate affinity resins for kinase assays. The liganded beads were blocked with excess biotin and washed with blocking buffer (SeaBlock (Pierce), 1% bovine serum albumin (BSA), 0.05% Tween 20, 1 mM dithiothreitol (DTT)) to remove unbound ligand and to reduce nonspecific binding. Binding reactions were assembled by combining kinases, liganded affinity beads, and test compounds in 1× binding buffer (20% SeaBlock, 0.17 × phosphate-buffered saline (PBS), 0.05% Tween 20, 6 mM DTT). Test compounds were prepared as 111X stocks in 100% DMSO. Kds were determined using an 11-point 3-fold compound dilution series with three DMSO control points. All compounds for Kd measurements are distributed by acoustic transfer (noncontact dispensing) in 100% DMSO. The compounds were then diluted directly into the assays such that the final concentration of DMSO was 0.9%. All reactions were performed in a polypropylene 384-well plate. Each was a final volume of 0.02 mL. The assay plates were incubated at room temperature with shaking for 1 h, and the affinity beads were washed with wash buffer (1 PBS, 0.05% Tween20). The beads were then resuspended in elution buffer (1 PBS,0.05% Tween 20, 0.5 μM nonbiotinylated affinity ligand) and incubated at room temperature with shaking for 30 min. The kinase concentration in the eluates was measured by the quantitative polymerase chain reaction (qPCR).
Proteomics Studies.
Protein Extraction and Digestion.
Cells with indicated treatments were harvested and lysed in lysis buffer (8 M urea, 100 mM tris-HCl pH 8.0). Sonication (5 s on 5 s off, 2 × 30 s) was performed to shear genome DNA. The lysate was centrifugated for 30 min at 3500g at 4 °C and the supernatant was transferred to a clean tube. The protein concentration was determined (BCA assay) and the protein was reduced with 5 mM DTT (dithiothreitol), alkylated with 15 mM IAA (iodoacetamide) in the dark conditions, and then diluted with 3 volume of buffer 25 mM Tris (pH 8.0) and 1 mM CaCl2. The final urea concentration is 2 M. Trypsin was added into protein solution with 1:100 ratio (trypsin:protein) and digested 12–16 h or overnight at rt.
Mass Spectrometry Analysis.
Peptides were cleaned up by C18 stage tips and the concentration was determined (Peptide assay, 23275, Thermo Fisher Scientific). The clean peptides were dissolved in 0.1% formic acid and analyzed on a Q Exactive HF-X coupled with an Easy nanoLC 1200 (Thermo Fisher Scientific, San Jose, CA). Then, 0.5 g of peptides were loaded onto an Acclaim PepMap RSLC C18 Column (150 mm 75 m ID, C18, 2 m, Thermo Fisher). Analytical separation of all peptides was achieved with a 130 min gradient. A linear gradient of 5–30% buffer B over 110 min was executed at a 300 nL/min flow rate followed by a ramp to 100% B in 5 min, and 15 min wash with 100% B, where buffer A was aqueous0.1% formic acid, and buffer B was 80% acetonitrile and 0.1% formic acid.
Liquid chromatography–mass spectrometry (LC–MS) experiments were performed in a data-dependent mode with full MS (externally calibrated to a mass accuracy of <5 ppm and a resolution of 60 000 at m/z 200) followed by high-energy collision-activated dissociation-MS/MS of the top 20 most intense ions with a resolution of 15 000 at m/z 200. High-energy collision-activated dissociation-MS/MS was used to dissociate peptides at a normalized collision energy of 27 eV in the presence of nitrogen bath gas atoms. Dynamic exclusion was 30 s. There were two technical replicates for one treatment, and each sample was subjected to three technical LC–MS replicates.
MS Data Analysis.
Mass spectra processing and peptide identification were performed on the Andromeda search engine in MaxQuant software (Version 1.6.10.43) against a human UniProt database (UP000005640). All searches were conducted with a defined modification of cysteine carbamidomethylation, with methionine oxidation and protein amino-terminal acetylation as dynamic modifications. Peptides were confidently identified using a target-decoy approach with a peptide false discovery rate (FDR) of 1% and a protein FDR of 1%. A minimum peptide length of 7 amino acids was required, maximally two missed cleavages were allowed, the initial mass deviation for precursor ion was up to 7 ppm, and the maximum allowed mass deviation for fragment ions was 0.5 Da. Data processing and statistical analysis were performed on Perseus (Version1.6.10.50). Protein quantitation was performed on technical replicate runs, and a two-sample t-test statistics were used to report statistically significant expression fold-changes.
Cell Viability Assay and Clonogenic Assay.
The assays were conducted according to the protocols reported previously.44
Mouse PK Studies.
The in vivo PK studies were conducted for compounds 24 and 27 using three male Swiss Albino mice per compound. The mice were administered intraperitoneally with solution formulation (5% N-methyl-2-pyrrolidinone (NMP), 5% solutol HS-15, and 90% normal saline) of compound 24 or 27 at a single dose of 50 mg/kg. Sixty microliters of blood samples were collected from each mouse at 0.5, 2, and 8 h. Plasma was harvested by centrifugation of blood and stored at −70 ± 10 °C until analysis. Plasma samples were quantified by the fit-for-purpose LC–MS/MS method (LLOQ: 5.02 ng/mL for plasma). These mouse PK experiments were performed according to the Institutional Animal Care and Use Committee-approved protocol.
Statistic Methods.
For all data, the number of biologically independent experiments and technical replicates, error bars, and P-values are described in figure legends, respectively. At least two independent experiments were conducted for all biological studies. The proteomics study was conducted in duplicate. Two-tailed Student’s t-tests were used for the indicated analyses, respectively, P ≥ 0.05, ns; 0.01 < P < 0.05, *; 0.001 < P < 0.01, **; and P < 0.001, ***.
Supplementary Material
ACKNOWLEDGMENTS
This work was supported in part by the P30CA196521 grant (to J.J.) from the US National Institutes of Health and an endowed professorship from the Icahn School of Medicine at Mount Sinai (to J.J.). This work utilized the AVANCE NEO 600 MHz NMR Spectrometer System that was upgraded with funding from a National Institutes of Health SIG grant 1S10OD025132-01A1. We thank Drs. Poulikos Poulikakos, Ernesto Guccione, E. Premkumar Reddy, and James A. Fagin for providing cell lines.
ABBREVIATIONS USED
- CRBN
cereblon
- ERK
extracellular signal-regulated kinase
- MAPK
mitogen-activated protein kinase
- MEK1 and MEK2
mitogen-activated protein kinase kinases 1 and 2
- MOA
mechanism of action
- NSCLC
nonsmall cell lung cancer
- PROTAC
proteolysis targeting chimera
- SAR
structure–activity relationship
- UPS
ubiquitin-proteasome system
- VHL
von Hippel–Lindau
Footnotes
Supporting Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01609.
MEK1/2 degradation induced by compounds 24 and 27; MEK1/2 degradation induced by compound 50; binding affinity curves of compounds 24, 27, and 50–53; Western blot validation of proteomics studies of compound 50; Neo-substrates of CRBN degradation by compound 50; compounds 24 and 27 degraded MEK1/2 proteins and inhibited downstream ERK signaling in H3122 and SUDHL1 cells; and antiproliferation potencies of PLX4032 and ZSTK474 in HT-29 and SK-MEL-28 cell lines; and 1H, 13C, HPLC, and HRMS spectra of compounds 24, 27, and 50–53 (PDF)
Molecular formula strings and biological data (CSV)
Complete contact information is available at: https://pubs.acs.org/10.1021/acs.jmedchem.0c01609
The authors declare the following competing financial interest(s): J.H., J.W., H.Y., M.K., J.L., and J.J. are inventors of a patent application filed by the Icahn School of Medicine at Mount Sinai.
Contributor Information
Jianping Hu, Mount Sinai Center for Therapeutics Discovery, Departments of Pharmacological Sciences and Oncological Sciences, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, New York 10029, United States.
Jieli Wei, Mount Sinai Center for Therapeutics Discovery, Departments of Pharmacological Sciences and Oncological Sciences, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, New York 10029, United States.
Hyerin Yim, Mount Sinai Center for Therapeutics Discovery, Departments of Pharmacological Sciences and Oncological Sciences, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, New York 10029, United States.
Li Wang, Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States.
Ling Xie, Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States.
Margaret S. Jin, Mount Sinai Center for Therapeutics Discovery, Departments of Pharmacological Sciences and Oncological Sciences, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, New York 10029, United States
Md Kabir, Mount Sinai Center for Therapeutics Discovery, Departments of Pharmacological Sciences and Oncological Sciences, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, New York 10029, United States.
Lihuai Qin, Mount Sinai Center for Therapeutics Discovery, Departments of Pharmacological Sciences and Oncological Sciences, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, New York 10029, United States.
Xian Chen, Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States.
Jing Liu, Mount Sinai Center for Therapeutics Discovery, Departments of Pharmacological Sciences and Oncological Sciences, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, New York 10029, United States.
Jian Jin, Mount Sinai Center for Therapeutics Discovery, Departments of Pharmacological Sciences and Oncological Sciences, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, New York 10029, United States.
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