Discovery of Bivalent Small Molecule Degraders of Cyclin-dependent Kinase 7 (CDK7)

Abstract

Cyclin-dependent kinase 7, along with cyclin H and MAT1, forms the CDK-activating complex (CAK), which directs cell cycle progression via T-loop phosphorylation of cell cycle CDKs. Pharmacological inhibition of CDK7 leads to selective anti-cancer effects in cellular and in vivo models, motivating several ongoing clinical investigations of this target. Current CDK7 inhibitors are either reversible or covalent inhibitors of its catalytic activity. We hypothesized that small molecule targeted protein degradation (TPD) might result in differentiated pharmacology due to loss of scaffolding functions. Here we report the design and characterization of a potent CDK7 degrader that is comprised of an ATP-competitive CDK7 binder linked to a CRL2^VHL recruiter. JWZ-5–13 effectively degrades CDK7 in multiple cancer cells and leads to a potent inhibition of cell proliferation. Additionally, compound JWZ-5–13 displayed bioavailability in a pharmacokinetic study conducted in mice. Therefore, JWZ-5–13 is a useful chemical probe to investigate the pharmacological consequences of CDK7 degradation.

Graphical abstract

A potent and selective CDK7 degrader was developed. JWZ-5–13 induces a rapid and sustained CDK7 degradation and exhibits high proteome-wide selectivity. JWZ-5–13 displays advantages in inhibition of cancer cell proliferation over its parental binder and displays bioavailability in a pharmacokinetic study.

Introduction

Cyclin-dependent kinases (CDKs) are serine/threonine protein kinases that regulate the cell cycle, transcription, and numerous additional cellular pathways.^1,2 There are 21 CDKs and 5 CDK-like genes encoded in the human genome.³ Almost all of the known CDKs are activated upon (i) binding to a specific cyclin and (ii) phosphorylation of their T-loops by a CDK-activating kinase (CAK).³ Dysregulation of the cell cycle or transcription to promote tumorigenesis is frequently a consequence of aberrant expression or mutation of CDKs, cyclins, cyclin-dependent kinase inhibitors (CKIs) or other components of CDK-containing complexes.⁴ The recent success of CDK4/6 inhibitors in the treatment of breast cancer highlights the potential of CDK inhibitors for the treatment of cancer.⁵

Within the CDK family, CDK7 has essential roles in both transcription and cell cycle progression.^{6, 7} In the cytoplasm, CDK7, along with cyclin H and MAT1, comprises the CDK-activating kinase (CAK), which provides the T-loop phosphorylation required for activation of CDKs 1, 2, 4 and 6, driving cell cycle progression.^{8, 9} CDK7 also has a role in the regulation of transcription, exerting as a component of the general transcription factor TFIIH.¹⁰ At active gene promoters, CDK7 phosphorylates the C-terminal domain (CTD) of RNA polymerase II (Pol II), at Serine 5 and Serine 7, to facilitate transcription initiation.¹¹ Additionally, CDK7 phosphorylates CDK9, which in turn phosphorylates the Pol II CTD at Ser2, to drive transcription elongation.¹² Moreover, it was reported that a variety of transcription factors, including p53,¹³ retinoic acid receptor,¹⁴ estrogen receptor,¹⁵ and androgen receptor¹⁶ are regulated by CDK7-mediated phosphorylation.

Aberrant overexpression of CDK7 has been detected in a myriad of cancer types, including hepatocellular carcinoma,¹⁷ ovarian cancer,¹⁸ gastric cancer,¹⁹ and colorectal cancer,²⁰ breast cancer,²¹ and elevated levels of CDK7 are associated with aggressive clinicopathological features and poor prognosis. Accumulating genetic evidence has suggested CDK7 as a promising therapeutic cancer target.²² For examples, knockdown of CDK7 using siRNA reduced gastric cancer cell proliferation and increased the G2/M cell population,²³ and CRISPR/Cas9-mediated knockdown of CDK7 in triple-negative breast cancer (TNBC) cells decreased their proliferation.²⁴ Further, genetic silencing of CDK7 with adenoviral and lentiviral vectors impaired T-loop phosphorylation of other CDKs and reduced the transcription of E2F driven genes, thereby arresting the cell cycle and impeding cell proliferation in mammary epithelial fibroblasts (MEFs).²⁵ CDK7 inhibition has recently been linked to anti-tumor immunity in small cell lung cancer through its induction of genome instability.²⁶ Additionally, inhibition of CDK7 suppress PD-L1 expression by inhibiting MYC activity while boosting antitumor immunity by recruiting infiltrating CD8+ T cells.²⁷ These findings suggest that there could be therapeutic benefit from combined treatment of CDK7 inhibitors and immunotherapy. In addition to cancer, CDK7 has been implicated in the pathogenesis of autosomal dominant polycystic kidney disease (ADPKD).²⁸ Elevated CDK7 immunoreactivity has been reported in susceptible hippocampal neurons of AD patients,²⁹ indicating an involvement of this kinase in the dysregulation of cell cycle proteins in AD neurons.

Therefore, over the past decade, significant efforts from the pharmaceutical industry and academia have propelled the pursuit of small molecule CDK7 inhibitors for cancer therapy.³⁰ These efforts led to the discovery of several CDK7 inhibitors: BS-181,³¹ LDC4297,³² LY3405105,³³ CT7001,³⁴ SY-1365,³⁵ and SY-5609³⁶ (Figure 1). Our group reported the discovery of the first covalent inhibitors including THZ1³⁷ and YKL-5–124³⁸ which react with cysteine (C312) located in the CTD of CDK7. Currently, several CDK7 inhibitors have advanced into clinical trials,^39,40 where they have generally been tolerated but there is limited information regarding tumor responses available in the public domain. One potential problem for CDK7 inhibitors is acquired drug resistance which has been observed for the occupancy driven kinase inhibitor including EGFR, ALK and BTK inhibitor.^{41, 42} Given the compelling preclinical data suggesting CDK7 might be a drug target for cancers, we were motivated to develop bivalent small molecule degraders of CDK7 to investigate the therapeutic differences relative to catalytic inhibitors.

Figure 1.

Chemical structures of representative CDK7 inhibitors

Targeted protein degradation using the proteolysis targeting chimera (PROTAC) technology has emerged as a potentially transformative therapeutic paradigm in drug discovery.^{43, 44} The PROTAC can stabilize a transient ternary complex between the protein of interest (POI) and the ligase, which results in ligase-dependent ubiquitination of the POI and its subsequent destruction by the proteosome. Degraders of disease-causing proteins have a number of potential advantages over inhibitors.⁴⁵ The occupancy-driven pharmacology of inhibitors requires stoichiometric drug binding to the protein target to inhibit its function, which often leads to an inhibitor-induced mutation and then resistance. One notable advantage of PROTACs, supported by evidence from a number of recent studies, is that they can overcome some of the resistance mechanisms to traditional targeted therapies.^{46, 47} To date, PROTACs have been developed for a range of CDKs including CDK2, CDK4, CDK5, CDK6, CDK8, CDK9, CDK12,⁴⁸ but specific CDK7 degraders remain limited.⁴⁹ Here, we report the design and characterization of JWZ-5–13 as a potent CDK7 degrader. Our lead compound JWZ-5–13 significantly degrades CDK7 via the ubiquitin-proteasome system with excellent proteome-wide selectivity and is therefore a useful tool compound for further dissecting the pharmacological consequences of degrading CDK7.

Result and Discussion

Design, synthesis and degradation evaluation of CDK7 degraders

Previously, our group characterized a potent and selective CDK7 inhibitor, YKL-5–124, which covalently labels cysteine (C312) in the C-terminal extension that traverses the mouth of the CDK7 ATP-binding pocket. YKL-5–124 has a Ki value of 2.2 nM and Kinact/ki = 103 nM⁻¹μS⁻¹, its reversible counterpart, YKL-5–167 also displayed inhibition of CDK7/Cyclin H/MAT1 kinase activity with an IC₅₀ of 6.8 nM, highlighting the high binding affinity of this scaffold to CDK7’s ATP-binding pocket.³⁸ To rationally design CDK7 degraders, molecular docking was performed to identify suitable attachment points and exit vectors for linker conjugation (Figure 2B). The acrylamide moiety was identified to be positioned toward the solvent exposed region and may be amenable to derivatization without disrupting binding to CDK7. We then designed and synthesized a set of putative CDK7 degraders, compounds 1 – 9 (Table 1) by conjugating CDK7 binding moiety to the CRBN E3 ligase binder via a diverse set of linkers.

Figure 2.

(A) Chemical structure of covalent CDK7 inhibitor YKL-5–124 and noncovalent inhibitor YKL-5–167 (B) Docking model of YKL-5–124 in the ATP-binding site of CDK7 (PDB : 6XD3).

Table 1.

Chemical Structures and binding affinity of Compounds 1−9

Cp	Linker	IC50 (nM)
1		6.7
2		9.4
3		11.3
4		6.4
5		17.7
6		9.2
7		34.4
8		6.9
9		13.3

The general synthetic route to compounds 1 – 9 is illustrated in Scheme 1. Commercially available heterocycle A1 reacted with ethyl chloroformate, leading to the formation of carbamate protected intermediate A2 which was then condensed with methyl 4-(chlorocarbonyl) benzoate to give intermediate A3. Removal of Boc group under acidic conditions provided A4 which coupled with in situ generated (S)-2-isocyanato-N, N-dimethyl-2-phenylethan-1-amine to afford compound A5. Cleavage of the ethyl carbamate group and methyl ester under basic conditions provided the key intermediate A6. The final products were synthesized using HATU-mediated amide coupling of the intermediate A6 with CRBN binder linker conjugates to provide the corresponding bivalent compounds 1 – 9.

Scheme 1.

Synthesis of Compounds 1–9

^a Reagents and conditions: (a) Et₃N, THF, 0 °C (b) Methyl 4-(chlorocarbonyl)benzoate, DIPEA, DCM, 0 °C (c) TFA, DCM, rt (d) (S)-2-isocyanato-N,N-dimethyl-2-phenylethan-1-amine, DIPEA, DCM, 0 °C (e) 2M NaOH, THF, 60 °C (f) HATU, DIPEA, DMF, rt

We proceeded to evaluate compounds 1 – 9 for the CDK7 binding affinity using a commercial in vitro Adapta Eu kinase assay.³⁸ As shown in the Table 1, all the compounds retained potent inhibition of CDK7 with IC₅₀s ranging from 6.4 nM to 34.4 nM, confirming that the terminal benzene ring of YKL-5–167 could serve as a suitable exit vector. Next, we investigated the degradation of CDK7 induced by this series of compounds in Jurkat cells, a human T lymphocyte cell line previously used to characterize CDK7 inhibitors.^{37, 38} As shown in Figure 3, treatment with either 0.1 μM or 1 μM for 16 hours with each compound did not lead to a complete reduction in CDK7 protein levels. We next sought to employ VHL,⁵⁰ a different E3 ligase substrate adaptor validated for use in PROTACs and synthesized a set of bivalent compounds in which the CDK7 binding moiety was linked with VHL recruiting ligands connected through several exit vectors (Table 2).

Figure 3.

Immunoblot analysis of CDK7 in Jurkat cells treated with CRBN-based compounds for 16 h. Quantified data represents mean ± SEM from three independent biological replicates.

Table 2.

Chemical structures and binding affinity of Compounds 10−22

Cp	Linker	Binder	IC50 (nM)	Cp	Linker	Binder	IC50 (nM)
10		L1	8.7	17		L1	20.1
11		L1	5.6	18		L1	20.3
12		L1	8.1	19		L1	12.6
13		L1	7.8	20		L2	12.1
14		L1	11.1	21		L2	13.4
15		L1	7.6	22		L3	8.6
16		L1	13.7

Reagents and conditions: (a) HATU, DIPEA, DMF, rt (b) 4-Iodobenzoyl chloride, Et₃N, DCM, 0 °C (c) TFA, DCM, rt (d) (S)-2-isocyanato-N,N-dimethyl-2-phenylethan-1-amine, DIPEA, DCM, 0 °C (e) Hex-5-ynoic acid, CuI, Pd(dppf)Cl₂, DMF, 80 °C (f) (S,R,S)-AHPC-Me, HATU, DIPEA, DMF, rt (g) 2M NaOH, THF, 60 °C (h) Pd/C, H₂, MeOH, rt (i) Methyl 5-bromopentanoate, K₂CO₃, DMSO, rt (j) TFA, DCM, rt (k) Oxalyl chloride, DMF, DCM (l) A2, Et₃N, DCM, 0 °C

The synthetic route to the VHL-based compounds is illustrated in Scheme 2. Compounds 10 – 16, 20 – 22 were synthesized using HATU-mediated amide coupling of the intermediate A6 with VHL binder linker conjugates. The synthesis of compound 17 started with condensation of intermediate A2 with 4-iodobenzoyl chloride. Removal of Boc group of A7, followed by coupling with in situ generated (S)-2-isocyanato-N,N-dimethyl-2-phenylethan-1-amine afforded A8. Sonogashira coupling between A8 and 4-ethynylbutyric acid proceeded to give A9 which reacted with VHL ligand (S, R, S)-AHPC-Me {Synonym: (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide} to furnish A10. The cleavage of the ethyl carbamate group under basic conditions provided compound 17 and Pd/C- catalyzed hydrogenation of carbon−carbon triple bond of 17 afforded compound 18. The synthesis of compound 19 commenced from alkylation of commercially available tert-butyl 4-hydroxybenzoate A11. Hydrolysis of tert-butyl ester group in A12 and subsequent conversion of carboxylic acid to acyl chloride afforded intermediate A14, which was coupled with A2 provided A15. Following the same reaction sequence of preparing intermediate of A6, precursor A18 was obtained. Finally, amide coupling reaction with VHL ligand (S, R, S)-AHPC-Me yielded bivalent compound 19.

Scheme 2.

Synthesis of VHL-based Compounds 10 – 22.

The in vitro CDK7 inhibition of these VHL-recruiting bivalent molecules ranged from 5.6 nM to 20.3 nM (Table 2). Notably, the attachment site on the VHL ligands have minimal impact on binding affinity to CDK7. We next evaluated the effect of compounds 10 − 22 on reducing the CDK7 protein level in Jurkat cells at 0.1 μM or 1 μM for 16 h by Western blot (WB) analysis. As shown in Figure 4, the treatment with 1 μM of compound 10 which bears the shortest carbon linker induced a moderate degradation. Increasing the linker length resulted in increased degradation potency as shown by compound 13, which led to more than 80% reduction in CDK7 protein once treated with 0.1 μM or 1 μM. It has been reported that incorporation of the more polar polyethylene glycol (PEG)-based linkers can improve permeability and performance by favoring more folded conformations.⁵¹ However, we found that compounds 14 − 16 bearing PEG linkers had no improved degradation activity compared to compound 13. In contrast, compound 17, which connects the CDK7 binding motif with VHL ligand via an alkyne linker, showed significant improvement and led up to 95% reduction of CDK7 protein at a 16 h timepoint and with 0.1 μM or 1 μM treatment. Its analogue, compound 18 with an alkyl linker showed slightly decreased degradation activity. Compounds 20 and 21, which link to the VHL ligand at its phenolic group and compound 22, connecting a linkage from the thiazole moieties only induced moderate reduction of CDK7 protein levels at 1 μM. Among the analogues we made, compound 17 proved to be the most effective degrader for CDK7. Therefore, compound 17 (JWZ-5–13) was selected for further biological characterization.

Figure 4.

Immunoblot analysis of CDK7 in Jurkat cells treated with VHL-based compounds for 16 h. Quantified data represents mean ± SEM from three independent biological replicates.

Compound 17 induces CDK7 degradation across different cancer cells.

To determine the kinetics of CDK7 degradation, we performed time-course experiments (Figure 5B). Degradation of CDK7 was observable as early as 0.5 hours after treatment of 0.1 μM and continued until 24 hours after treatment. To characterize the potency of compound 17, we treated Jurkat cells with the compound at different concentrations for 6 h. As shown in Figure 5C, compound 17 efficiently induced CDK7 degradation in a dose-dependent manner, no hook effect was observed up to 5 μM. Taken together, these results indicate that compound 17 degrades CDK7 in a time and concentration-dependent manner. Since previous studies have noted cell-specific degradation profiles induced by degrader molecules,^{52, 43} we characterized the degradation profile of compound 17 in different cell lines (Figure 5D). Robust degradation of CDK7 was observed across ovarian adenocarcinoma cell line OVCAR3, B-cell lymphoma cell line SUDHL5, and T lymphoblast cell line Molt4, lung adenocarcinoma cell line A549 upon the treatment with 17 for 6 h. These results demonstrate 17 is a useful pharmacological tool to induce rapid depletion of CDK7 in different cellular contexts.

Figure 5.

(A) Chemical structure of compound 17 (B) Immunoblot analysis of CDK7 in Jurkat cells treated with 0.1 μM of 17 at the indicated time points. (C) Immunoblot analysis of CDK7 in Jurkat cells treated with 17 at the indicated concentration for 6h. Quantified data represents mean ± SEM from three independent biological replicates. (D) Immunoblot analysis of CDK7 in different cells treated with 17 for 6 h.

Compound 17 Induces CDK7 Degradation in a UPS-dependent Manner.

We next investigated the mechanism of action (MOA) for 17-induced CDK7 degradation. In order to investigate whether the observed CDK7 degradation is dependent on the CRL2^VHL E3 ubiquitin ligase, we synthesized a negative control compound 17-Neg, featuring a VHL ligand previously shown to exhibit a substantial reduction in VHL binding affinity⁵³ (Figure 6A). 17-Neg displayed similar in vitro CDK7 inhibition (21.1 nM) to 17. As expected, 17-Neg fails to reduce the level of CDK7 protein in Jurkat cells. To further demonstrate the observed CDK7 degradation is dependent on the ubiquitin-proteasome system, we performed a series of rescue experiments (Figure 6C). Pretreatment with proteasome inhibitors MG132 or a NEDD8 activating E1 enzyme inhibitor (MLN-4924), which inhibits the process of neddylation required for ligase activity rescued CDK7 degradation induced by compound 17. A similar rescue effect was observed with either pretreatment with reversible parental binder YKL-5–167 or VHL ligand (S,R,S)-AHPC-Me. Furthermore, the CDK7 degradation induced by 17 was completely abrogated in cells lacking VHL, compared with wild-type controls (Figure 6D). Taken together, these data demonstrate that 17 degrades CDK7 in a VHL-dependent manner.

Figure 6.

(A) Chemical structure of 17-Neg (B) Immunoblot analysis of CDK7 in Jurkat cells treated with 17 or 17-Neg for 6 h. (C) Immunoblot analysis of CDK7 in Jurkat cells pretreated for 2 h with MG132, MLN-4924, YKL-5–167 and VHL ligand, then treated with 17 for 6 h. (D) Western blotting analysis of CDK7 in Jurkat and VHL-knockout Jurkat cells treated with 17 for 6 h.

Compound 17 is a selective CDK7 degrader across CDKs and proteome.

To assess the selectivity of 17, we evaluated its influence on other CDK proteins (Figure 7A). At concentrations ranging from 0.1 to 0.5 μM, 17 barely affected the protein levels of other CDK family members such as CDK1, 2, 4, 5, 6, 8 and 9. To further assess the intracellular protein degradation selectivity and identify the potential off-targets of 17, we used an unbiased, multiplexed quantitative mass-spectrometry-based proteomics approach. We performed this proteome-wide analysis on OVCAR3 cells 6 h after 0.1 μM compound 17 treatment (Table S1), a time point at which changes in protein abundance would be primarily a result of compound-induced degradation rather than confounding transcriptional or translational changes, secondary to CDK7 degradation. As shown in Figure 7B, in total, out of the 6538 proteins identified among three replicates, only three significantly changed proteins were identified in the 17-treatment group as compared to the DMSO group with 1.75-fold cutoff (p-value < 0.001). As expected, CDK7 was significantly downregulated. Its binding partner in the CAK complex, Cyclin H was also significantly downregulated while the third member of the CAK complex MAT1 was not detected in our experiment. We confirmed dose-dependent downregulation of MAT1 by Western blot (Figure 7C). The concomitant reduction of Cyclin H and MAT1 induced by compound 17 may result from diminished stability of the CAK complex due to loss of the CDK7 subunit after CDK7 degradation or as a result of ‘bystander’ degradation due to direct ubiquitination of Cyclin H or MAT1 as has been reported for other CDKs.^{54, 55} Consistent with our observation, previous studies have documented that genetic knockdown of CDK7 via siRNA resulted in the reduction in protein levels of all three CAK subunits.²¹ Notably, We did not observed LONRF2 degradation by Western blot at concentrations ranging from 0.1 to 5 μM (Figure S1). Taken together, these results demonstrated a high proteome-wide selectivity for compound 17.

Figure 7.

(A) Western blotting analysis of CDKs in Jurkat cells treated with 17 for 6 hours. (B) Quantitative proteomics of OVCAR3 cells following treatment with 0.1 μM 17 for 6 hours (C) Western blotting analysis of CDK7, MAT1, Cyclin H treated with 17 for 6 h in OVCAR3 and Jurkat cells.

CDK7 Degrader 17 induces cell growth inhibition

We next interrogated the impact of CDK7 depletion on cell viability. We treated cells with compound 17 for 72h and used the non-degrading compound 17-Neg and the parental CDK7 inhibitor YKL-5–167 as controls. As illustrated in Figure 8, compound 17 potently inhibited Jurkat cell proliferation in a concentration-dependent manner with an IC₅₀ of 160 nM as measured by CellTiter-Glo. Notably, compound 17 was ~7-fold more potent than the parental compound, YKL-5–167 which exhibited an anti-proliferative IC₅₀ of 1.19 μM. Importantly, the negative control compound 17-Neg, which shared similar CDK7 inhibitory activity as compound 17, but did not induce CDK7 degradation, displayed a much weaker anti-proliferative effect in Jurkat cells. This observation indicates that CDK7 degradation contributes significantly to the cell growth inhibition induced by compound 17. Furthermore, compared with YKL-5–167, we found compound 17 demonstrated enhanced antiproliferative activities against SUDHL5 cells, Molt4 cells and A549 cells, with IC₅₀s of 52 nM, 89 nM and 332 nM, respectively. In OVCAR3 cells compound 17 exhibited efficacies comparable to its parental compound YKL-5–167. Consistently, compound 17-Neg displayed much weaker antiproliferative effect on these cells. Taken together, these results suggest the observed antiproliferative effect of compound 17 arises from its CDK7 degradation activity rather than its CDK7 inhibitory activity in most cell lines that we tested. Moreover, induction of CDK7 degradation by compound 17 provides an effective strategy to suppress the growth of multiple cancer cell types.

Figure 8.

Proliferation assay of 17, 17 −Neg, or YKL-5–167 in different cell lines.

Compound 17 is Bioavailable in Mice.

Lastly, we assessed in vivo pharmacokinetic (PK) properties of compound 17 in mice. Compound concentrations in plasma from mice were determined following a single dose of compound 17 delivered intravenously (IV, 3 mg/kg) or via intraperitoneal injection (IP, 10 mg/kg). Compound 17 exhibited half-lives (T_1/2) of 3.76 h and 2.82 h with low clearance via IV and IP administration, respectively. A single IP injection resulted in the maximum plasma concentration of 3.39 μM 1.0 h post dosing (Figure 9), 6 h post dosing, the concentration of compound 17 was 1.49 μM. In addition, compound 17 was well tolerated by the treated mice, and no clinical signs were observed during the PK study. Taken together, these results suggest that compound 17 could be a valuable chemical tool for investigating the effects of CDK7 degradation in vivo.

Figure 9.

Plasma concentrations of 17 over 24 h, following a single 10 mg/kg IP or 3 mg/kg IV injection. The compound concentration shown at each time point is the mean ± SD from three test mice.

Conclusion

Cyclin-dependent kinase 7 (CDK7) possesses a distinct functional repertoire due to its ability to regulate both transcription and cell cycle progression. While CDK7 inhibitors have been developed as therapeutic agents and have shown promising anticancer activity in numerous preclinical models, none have progressed further. Here, through a medicinal chemistry campaign, we discovered a potent CDK7 degrader compound 17 (JWZ-5–13) which exploits the CRL2^VHL E3 ligase to efficiently reduce CDK7 protein levels in a concentration, time, VHL and UPS-dependent manner. We found that CDK7 degradation could be induced in multiple cell lines and CDK7 degradation resulted in a robust antiproliferative effect. Unbiased global proteomic studies demonstrated the high proteome-wide selectivity for our lead compound. Furthermore, compound 17 was bioavailable in a mouse PK study, which enables the use of compound 17 (JWZ-5–13) as a selective chemical probe to investigate the functions of CDK7 in a variety of biological contexts.

EXPERIMENTAL SECTION

Chemistry General Procedures

Unless otherwise noted, reagents and solvents were obtained from commercial suppliers and were used without further purification. ¹H NMR spectra were recorded on 500 MHz (Bruker A500), and chemical shifts are reported in parts per million (ppm, d) downfield from tetramethylsilane (TMS). Coupling constants (J) are reported in Hz. Spin multiplicities are described as s (singlet), br (broad singlet), d (doublet), t (triplet), q (quartet), and m (multiplet). Mass spectra were obtained on a Waters Micromass ZQ instrument. Preparative HPLC was performed on a Waters Sunfire C18 column (19 × 50 mm, 5μM) using a gradient of 15–95% methanol in water containing 0.05% trifluoroacetic acid over 22 min (28 min run time) at a flow rate of 20 mL/min. All compounds used for biological evaluation have a purity of ⩾95% by UPLC-MS analysis.

Synthesis of (S)-4-((5-((2-(dimethylamino)-1-phenylethyl)carbamoyl)-6,6-dimethyl-1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)carbamoyl)benzoic acid (A6).

Step 1: To a solution of tert-butyl 3-amino-6,6-dimethyl-4,6-dihydropyrrolo[3,4-c]pyrazole-5(1H)-carboxylate A1 (500 mg, 2.0 mmol) and Et₃N (0.42 mL, 3.0 mmol) in THF (5 mL) was added ethyl chloroformate (0.19 mL, 2.0 mmol) dropwise at 0 °C. The mixture was stirred at 0 °C for 1 h. The solvent was removed and the residue was partitioned with ethyl acetate and sat. NaHCO₃. The organic layer was washed with water, brine and dried with Na₂SO₄. The solvent was then removed and purified by flash column chromatography on silica gel to provide A2 as an off white solid (377 mg, 58%). UPLC/MS(ESI): 325.39 [M+H]⁺.

Step 2: To a solution of A2 (324 mg, 1.0 mmol) and DIPEA (387 mg, 3.0 mmol) in DCM (5 mL) was added methyl 4-(chlorocarbonyl)benzoate (237 mg, 1.2 mmol) at 0 °C. The reaction was stirred at room temperature for 4 h. The solvent was removed and purified by flash column chromatography on silica gel to A3 (355 mg, 73%). LC/MS (ESI) m/z = 487.52 (M + H)⁺.

Step 3. A mixture of A3 (355mg, 0.73 mmol) and trifluoroacetic acid (2 mL) in DCM (6 mL) was stirred at rt for 2 hours. The mixture was concentrated to give crude intermediate A4 which was used in the next step without further purification. UPLC/MS(ESI):m/z = 387.40 (M + H)⁺.

Step 4. To a mixture of A4 (250mg, 0.5 mmol) in DCM (5 mL) was added DIPEA (193mg, 1.5mmol) at 0 °C, followed by (S)-2-isocyanato-N,N-dimethyl-2-phenylethan-1-amine (172 mg, 0.75 mmol). The solution was stirred at 0 °C for 4 h. The solvent was then removed and the crude was purified by HPLC to give A5 (153mg, 53%). UPLC/MS(ESI):m/z = 577.60 (M + H)⁺.

Step 5: A mixture of A5 (100 mg, 0.17 mmol), 2N NaOH solution (2 mL) and THF (2 mL) was stirred at 60 °C for 8 h. The mixture was then neutralized by 2N HCl solution (2 mL). The solvent was removed and the crude was purified by HPLC to give A6 (65mg, 78%).

¹H NMR (500 MHz, DMSO- d6) δ 11.09 (s, 1H), 10.23 (s, 1H), 8.15 −7.93 (m, 4H), 7.51 − 7.41 (m, 2H), 7.36 (t, J = 7.7 Hz, 2H), 7.31 − 7.20 (m, 1H), 6.87 (s, 1H), 5.19 − 5.04 (m, 1H), 4.62 (q, J = 12.1 Hz, 2H), 3.13 (d, = 14.8 Hz, 1H), 2.79 (d, J = 12.0 Hz, 1H), 2.49 (s, 6H), 1.65 (s, 3H), 1.58 (s, 3H). UPLC/MS(ESI): m/z = 491.63 (M + H)⁺.

General procedure A for synthesis of final compounds

To a mixture of (S)-4-((5-((2-(dimethylamino)-1-phenylethyl)carbamoyl)-6,6-dimethyl-1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)carbamoyl)benzoic acid A6 (1.0 eq), CRBN or VHL based ligand linkers (1.5 eq.) and DIPEA (3.0 eq) in DMF (1 mL) was added HATU (1.5 eq). The resultant mixture was stirred at room temperature overnight. The solvent was removed under reduced pressure and purified by HPLC to give final compounds.

N1-(5-(((S)-2-(dimethylamino)-1-phenylethyl)carbamoyl)-6,6-dimethyl-1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)-N4-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethyl)terephthalamide (1)

The title compound was prepared according to the general procedure. Compound 1 was obtained as white solid (20.4 mg, 65%).

¹H NMR (500 MHz, DMSO- d6) δ 11.09 (d, J = 6.4 Hz, 2H), 9.05 (s, 1H), 8.86 (t, J =5.3 Hz, 1H), 8.10 (d, J = 8.5 Hz, 2H), 7.96 (d, J = 8.5 Hz, 2H), 7.59 (dd, J = 8.6, 7.0 Hz, 1H), 7.48 − 7.43 (m, 2H), 7.40 (t, J = 7.7 Hz, 2H), 7.34 − 7.28 (m, 1H), 7.26 d, J = 8.7 Hz, 1H), 7.04 d, J =7.0 Hz, 1H), 6.86 (s, 1H), 6.77 (d, J =9.2 Hz, 1H), 5.40 − 5.30 (m, 1H), 5.06 (dd, J = 12.8, 5.5 Hz, 1H), 4.78 d, J = 11.8 Hz, 1H), 4.58 d, J = 11.9 Hz, 1H), 3.62 − 3.47 (m, 5H), 3.40 − 3.30 (m, 1H), 2.89 (d, J = 4.7 Hz, 3H), 2.85 (d, J = 4.8 Hz, 3H), 2.60 (dt, J = 17.6, 3.5 Hz, 1H), 2.09 − 2.01 (m, 1H), 1.69 (s, 3H), 1.60 (s, 3H). ¹³C NMR (126 MHz, DMSO) δ 173.3, 170.6, 169.2, 167.8, 166.6, 164.1, 155.5, 146.8, 140.9, 137.5, 136.7, 136.1, 132.7, 128.9, 128.3, 128.0, 127.8, 127.1, 118.7, 117.7, 116.3, 111.1, 109.8, 106.1, 61.2, 60.4, 53.9, 49.0, 47.6, 44.7, 41.8, 31.5, 26.8, 26.4, 22.7. UPLC/MS(ESI): m/z 789.48 [M+H]⁺.

N1-(5-(((S)-2-(dimethylamino)-1-phenylethyl)carbamoyl)-6,6-dimethyl-1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)-N4-(6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)hexyl)terephthalamide (2).

The title compound was prepared according to the general procedure. Compound 2 was obtained as white solid (11 mg, 38%).

¹H NMR (500 MHz, DMSO- d6) δ 11.07 (d, J = 21.1 Hz, 2H), 8.64 (t, J = 5.6 Hz, 1H), 8.13 − 8.05 (m, 2H), 7.97 d, J = 8.0 Hz, 2H), 7.58 (dd, J = 8.6, 7.1 Hz, 1H), 7.43 (d, J = 7.2 Hz, 2H), 7.37 (t, J = 7.6 Hz, 2H), 7.27 (t, J = 7.3 Hz, 1H), 7.10 (d, J = 8.6 Hz, 1H), 7.03 (d, J = 7.0 Hz, 1H), 6.61 (s, 1H), 6.55 (t, J = 5.9 Hz, 1H), 5.21 (s, 1H), 5.06 (dd, J = 12.7, 5.5 Hz, 1H), 4.74 (d, J = 12.0 Hz, 1H), 4.57 (d, J = 11.8 Hz, 1H), 3.34 (s, 6H), 3.29 (p, J = 6.6 Hz, 4H), 2.95 − 2.85 (m, 1H), 2.72 − 2.55 (m, 6H), 2.09 − 2.01 (m, 1H), 1.68 (s, 3H), 1.60 (s, 3H), 1.55 (m, 2H), 1.39 (dt, J = 7.0, 3.7 Hz, 4H). ¹³C NMR (126 MHz, DMSO) δ 173.3, 170.6, 169.4, 167.8, 165.8, 164.1, 155.5, 146.9, 141.2, 137.9, 136.8, 136.3, 135.9, 132.7, 128.9, 128.2, 127.9, 127.8, 127.1, 117.7, 110.9, 109.5, 61.1, 60.5, 52.1, 51.7, 49.2, 49.0, 42.3, 31.5, 29.5, 29.1, 26.9, 26.7, 26.6, 26.4, 22.6. UPLC/MS(ESI): m/z 845.53 [M+H]⁺.

N1-(5-(((S)-2-(dimethylamino)-1-phenylethyl)carbamoyl)-6,6-dimethyl-1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)-N4-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethyl)terephthalamide (3)

The title compound was prepared according to the general procedure. Compound 3 was obtained as white solid (5.9 mg, 21%).

¹H NMR (500 MHz, DMSO- d6) δ 11.09 (d, J = 20.2 Hz, 2H), 9.12 (s, 1H), 8.69 (t, J = 5.6 Hz, 1H), 8.47 (s, 1H), 8.08 (d, J = 8.3 Hz, 2H), 7.96 (d, J = 8.0 Hz, 2H), 7.57 (dd, J = 8.6, 7.1 Hz, 1H), 7.43 (d, J = 7.5 Hz, 2H), 7.37 (t, J = 7.6 Hz, 2H), 7.29 (d, J = 7.4 Hz, 1H), 7.17 (d, J = 8.6 Hz, 1H), 7.03 (d, J = 7.0 z, 1H), 6.64 (t, J = 5.8 Hz, 1H), 5.24 (s, 1H), 5.05 (dd, J = 12.8, 5.5 Hz, 1H), 4.72 (s, 1H), 4.58 (d, J = 11.8 Hz, 1H), 3.64 (dt, J = 26.2, 5.6 Hz, 4H), 3.49 (dq, J = 12.0, 5.7 Hz, 4H), 3.16 (d, J = 16.7 Hz, 2H), 2.95 − 2.85 (m, 2H), 2.65 − 2.53 (m, 1H), 2.09 − 2.01 (m, 1H), 1.68 (s, 3H), 1.60 (s, 3H). ¹³C NMR (126 MHz, DMSO) δ 173.3, 170.6, 169.4, 167.8, 166.1, 164.1, 155.5, 146.9, 140.9, 137.6, 136.7, 136.0, 132.5, 129.0, 128.2, 128.0, 127.8, 127.1, 119.0, 117.9, 116.6, 111.2, 109.7, 69.2, 69.2, 61.2, 60.5, 53.9, 49.0, 42.2, 42.2, 31.4, 26.8, 26.4, 22.6, 18.5, 17.2, 12.8. UPLC/MS(ESI): m/z 833.53 [M+H]⁺.

N1-(5-(((S)-2-(dimethylamino)-1-phenylethyl)carbamoyl)-6,6-dimethyl-1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)-N4-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethyl)terephthalamide (4)

The title compound was prepared according to the general procedure. Compound 4 was obtained as white solid (14.7 mg, 49%)。 ¹H NMR (500 MHz, DMSO- d6) δ 11.09 (d, J = 9.6 Hz, 2H), 9.04 (s, 1H), 8.66 (t, J = 5.6 Hz, 1H), 8.09 (d, J = 8.5 Hz, 2H), 7.97(d, J = 8.5 Hz, 2H), 7.58 (dd, J = 8.6, 7.0 Hz, 1H), 7.47 − 7.43 (m, 2H), 7.40 (dd, J =8.5, 6.8 Hz, 2H), 7.34 − 7.29 (m, 1H), 7.13 (d, J = 8.6 Hz, 1H), 7.04 (d, J = 7.0 Hz, 1H), 6.77 (d, J = 9.2 Hz, 1H), 6.61 (t, J = 5.9 Hz, 1H), 5.40 − 5.30 (m, 1H), 5.06 (dd, J = 12.9, 5.4 Hz, 1H), 4.78 d, J = 11.9 Hz, 1H), 4.57 (d, J = 11.9 Hz, 1H), 3.66 − 3.51 (m, 10H), 3.48 − 3.38 (m, 4H), 3.36 − 3.30 (m, 1H), 2.89 (d, J =4.6 Hz, 3H), 2.85 (d, J = 4.7 Hz, 3H), 2.63 − 2.53 (m, 2H), 2.09 − 2.01 (m, 1H), 1.69 (s, 3H), 1.60 (s, 3H). ¹³C NMR (126 MHz, DMSO) δ 173.3, 170.6, 169.4, 167.8, 166.0, 164.1, 155.4, 146.9, 140.9, 137.6, 136.7, 136.0, 132.6, 129.0, 128.2, 128.0, 127.8, 127.1, 117.9, 111.2, 109.7, 106.4, 70.2, 70.1, 69.4, 61.2, 60.5, 49.1, 49.0, 47.6, 44.8, 42.2, 41.7, 31.5, 26.8, 26.4, 22.6. UPLC/MS(ESI): m/z 877.54 [M+H]⁺.

N1-(5-(((S)-2-(dimethylamino)-1-phenylethyl)carbamoyl)-6,6-dimethyl-1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)-N4-(2-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxy)ethyl)terephthalamide (5)

The title compound was prepared according to the general procedure. Compound 5was obtained as white solid (6.5mg, 35%). ¹H NMR (500 MHz, DMSO- d6) δ 11.10 (s, 1H), 11.02 (s, 1H), 8.66 (s, 1H), 8.09 (d, J = 8.1 Hz, 2H), 7.95 d, J = 8.1 Hz, 2H), 7.58 (dd, J = 8.6, 7.1 Hz, 1H), 7.38 (d, J = 7.2 Hz, 2H), 7.30 (t, J =7.5 Hz, 2H), 7.20 (t, J =7.3 Hz, 1H), 7.14 (d, J = 8.6 Hz, 1H), 7.04(d, J = 7.0 Hz, 1H), 6.60 (t, J = 5.8 Hz, 1H), 6.27 (s, 1H), 5.06 (dd, J = 12.8, 5.4 Hz, 1H), 4.90 (s, 1H), 4.57 (s, 2H), 3.61 (t, J = 5.4 Hz, 2H), 3.54 (d, J = 3.5 Hz, 9H), 3.45 (dt, J = 11.2, 5.6 Hz, 4H), 3.33 (s, 6H), 2.95 − 2.86 (m, 1H), 2.62 − 2.53 (m, 1H), 2.22 (s, 5H), 2.10 − 1.98 (m, 1H), 1.66 (s, 3H), 1.58 (s, 3H). ¹³C NMR (126 MHz, DMSO) δ 173.3, 170.5, 169.4, 167.8, 164.1, 155.9, 146.9, 136.7, 132.6, 128.5, 128.3, 127.7, 127.2, 126.9, 117.9, 111.2, 109.7, 70.3, 70.2, 70.1, 69.4, 69.3, 60.8, 49.0, 45.4, 42.2, 31.5, 26.7, 26.6, 22.6. UPLC/MS(ESI): m/z 921.55 [M+H]⁺.

N1-(5-(((S)-2-(dimethylamino)-1-phenylethyl)carbamoyl)-6,6-dimethyl-1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)-N4-(5-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)pentyl)terephthalamide (6)

The title compound was prepared according to the general procedure. Compound 6 was obtained as white solid (7.2 mg, 29%). ¹H NMR (500 MHz, DMSO) δ 11.04 (d, = 18.1 Hz, 2H), 8.59 (t, J = 5.7 Hz, 1H), 8.04 (d, J = 8.3 Hz, 2H), 7.91 (d, J = 8.1 Hz, 2H), 7.76 (dd, J = 8.5, 7.2 Hz, 1H), 7.48 (d, J = 8.6 Hz, 1H), 7.38 (dd, J = 14.5, 7.4 Hz, 3H), 7.30 (t, J = 7.6 Hz, 2H), 7.21 (t, J = 12 Hz, 1H), 6.51 (s, 1H), 5.03 (dd, J = 12.8, 5.5 Hz, 1H), 4.64 (s, 1H), 4.52 (d, = 11.9 Hz, 1H), 4.18 (t, J = 6.4 Hz, 1H), 3.55 (s, 1H), 3.31 − 3.26 (m, 6H), 3.07 (s, 1H), 3.01 (q, J = 12 Hz, 3H), 2.87 − 2.78 (m, 2H), 2.59 − 2.49 (m, 3H), 2.01 − 1.95 (m, 1H), 1.77 (p, J = 6.6 Hz, 2H), 1.62 (s, 3H), 1.58 (t, J = 1.4 Hz, 2H), 1.54 (s, 3H), 1.51 − 1.50 (m, 2H). ¹³C NMR (126 MHz, DMSO) δ 173.3, 170.5, 167.3, 165.8, 165.8, 164.1, 156.5, 155.5, 140.9, 137.9, 137.5, 135.9, 133.7, 129.0, 128.2, 128.0, 127.7, 127.1, 120.3, 116.7, 115.6, 69.2, 61.2, 60.4, 53.9, 49.2, 49.1, 47.6, 46.0, 42.1, 31.4, 29.2, 28.6, 26.8, 26.4, 23.3, 22.5. UPLC/MS(ESI): m/z 832.53 [M+H]⁺.

N1-(5-(((S)-2-(dimethylamino)-1-phenylethyl)carbamoyl)-6,6-dimethyl-1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)-N4-(8-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)octyl)terephthalamide (7)

The title compound was prepared according to the general procedure. Compound 7 was obtained as white solid (15 mg, 52%). ¹H NMR (500 MHz, DMSO) δ 11.11 (s, 1H), 11.02 (s, 1H), 8.60 (t, J = 5.7 Hz, 1H), 8.09 (d, J = 8.1 Hz, 2H), 7.95 (d, J = 8.1 Hz, 2H), 7.81 (dd, J = 8.5, 7.3 Hz, 1H), 7.52 (d, J = 8.5 Hz, 1H), 7.44 (d, J = 12 Hz, 1H), 7.32 (t, J = 7.6 Hz, 2H), 7.22 (t, J = 7.3 Hz, 1H), 6.33 (s, 1H), 5.09 (dd, J = 12.8, 5.5 Hz, 1H), 4.95 (s, 1H), 4.59 (s, 2H), 4.21(t, J = 6.4 Hz, 2H), 3.33 (s, 6H), 2.95 − 2.85 (m, 1H), 2.64 − 2.54 (m, 1H), 2.33 − 2.18 (m, 5H), 2.10 − 1.98 (m, 1H), 1.83 − 1.71 (m, 2H), 1.66 (s, 3H), 1.63−1.53 (m, 5H), 1.48 (t, J = 7.8 Hz, 2H), 1.40 − 1.29 (m, 6H). ¹³C NMR (126 MHz, DMSO) δ 173.3, 170.5, 167.3, 165.8, 164.2, 156.5, 155.8, 143.8, 137.9, 137.5, 136.0, 133.7, 129.1, 128.5, 128.2, 127.7, 127.3, 127.2, 127.1, 120.2, 116.7, 115.6, 69.3, 64.2, 60.9, 53.9, 51.5, 49.2, 45.1, 31.4, 29.5, 29.2, 29.1, 28.9, 26.9, 26.7, 26.6, 25.7, 22.5. UPLC/MS(ESI): m/z 874.49 [M+H]⁺.

N1-(5-(((S)-2-(dimethylamino)-1-phenylethyl)carbamoyl)-6,6-dimethyl-1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)-N4-(5-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)pent-4-yn-1-yl)terephthalamide (8)

The title compound was prepared according to the general procedure. Compound 8 was obtained as white solid (12 mg, 42%). ¹H NMR (500 MHz, DMSO) δ 11.14 (s, 1H), 11.09 (s, 1H), 9.18 (s, 1H), 8.75 (t, J = 5.6 Hz, 1H), 8.09 (d, J = 8.2 Hz, 2H), 8.01 − 7.96 (m, 2H), 7.90 − 7.86 (m, 1H), 7.84 (dt, J = 6.2, 1.9 Hz, 2H), 7.49 − 7.36 (m, 4H), 7.32 – 7.26 (m, 1H), 6.78 (d, J = 9.2 Hz, 1H), 5.40 − 5.30 (m, 1H), 5.15 (dd, J = 12.8, 5.4 Hz, 1H), 4.78 (d, J = 11.9 Hz, 1H), 4.58 (d, J = 11.9 Hz, 1H), 3.60 − 3.52 (m, 2H), 3.47 (q, J = 6.0 Hz, 2H), 3.40 − 3.30 (m, 1H), 2.89 (d, J = 4.7 Hz, 3H), 2.85 (d, J = 4.7 Hz, 3H), 2.64 − 2.52 (m, 4H), 2.09 − 2.01 (m, 1H), 1.88 (p, J = 7.0 Hz, 2H), 1.68 (s, 3H), 1.60 (s, 3H). ¹³C NMR (126 MHz, DMSO) δ 173.2, 170.2, 167.0, 166.9, 166.1, 164.1, 155.5, 140.8, 138.6, 137.8, 137.8, 136.0, 132.2, 130.3, 130.1, 129.0, 128.2, 128.0, 127.8, 127.2, 127.1, 126.1, 124.2, 106.1, 96.2, 80.1, 61.2, 60.4, 49.6, 49.1, 47.6, 44.8, 41.7, 39.1, 31.4, 28.3, 26.7, 26.4, 22.4, 17.1. UPLC/MS(ESI): m/z 812.38 [M+H]⁺.

N1-(5-(((S)-2-(dimethylamino)-1-phenylethyl)carbamoyl)-6,6-dimethyl-1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)-N4-(6-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)hex-5-yn-1-yl)terephthalamide (9)

The title compound was prepared according to the general procedure. Compound 9 was obtained as white solid (3.3 mg, 12%). ¹H NMR (500 MHz, DMSO- d6) δ 12.61 (s, 1H), 11.13 (s, 1H), 11.04 (s, 1H), 9.71 (s, 1H), 8.73 (s, 1H), 8.11 (d, J = 8.1 Hz, 2H), 7.98 (d, J = 8.1 Hz, 2H), 7.90 − 7.85 (m, 1H), 7.84 − 7.81 (m, 2H), 7.45 (d, J = 7.7 Hz, 2H), 7.38 (t, J = 7.6 Hz, 2H), 7.29 (t, J = 7.3 Hz, 1H), 6.73 (s, 1H), 5.32 (s, 1H), 5.14 (dd, J = 12.8, 5.5 Hz, 1H), 4.81 (d, J = 12.1 Hz, 1H), 4.57 (d, J = 11.8 Hz, 1H), 2.93 − 2.70 (m, 4H), 2.60 (t, J = 7.0 Hz, 4H), 2.09 − 2.00 (m, 1H), 1.76 (q, J = 7.4, 6.7 Hz, 2H), 1.68 (d, = 6.3 Hz, 4H), 1.60 (s, 3H), 1.21 (d, = 29.7 Hz, 2H). ¹³C NMR (126 MHz, DMSO) δ 173.3, 170.3, 166.8, 166.2, 165.9, 155.4, 140.8, 138.8, 135.1, 132.5, 130.6, 128.9, 128.2, 127.9, 127.8, 127.1, 123.1, 120.5, 99.2, 76.7, 61.1, 49.4, 44.4, 36.7, 31.4, 28.7, 26.4, 25.8, 22.4, 19.2. UPLC/MS(ESI): m/z 826.38 [M+H]⁺.

N1-(5-(((S)-2-(dimethylamino)-1-phenylethyl)carbamoyl)-6,6-dimethyl-1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)-N4-(3-(((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-3-oxopropyl)terephthalamide (10)

The title compound was prepared according to the general procedure. Compound 10 was obtained as white solid (3.6 mg, 25%). ¹H NMR (500 MHz, DMSO- d6) δ 12.61 (s, 1H), 11.05 (s, 1H), 8.99 (s, 1H), 8.68 (s, 1H), 8.42 (d, J =7.8 Hz, 1H), 8.11 (d, J =7.9 Hz, 2H), 7.98 (d, J = 8.7 Hz, 2H), 7.50 − 7.20 (m, 9H), 6.75 (s, 1H), 5.40 − 5.30 (m, 1H), 5.17 (s, 1H), 4.91 (q, J = 7.4 Hz, 1H), 4.82 (d, = 11.8 Hz, 1H), 4.55 (dd, J = 14.4, 10.4 Hz, 2H), 4.45 (t, J = 8.0 Hz, 1H), 4.29 (s, 1H), 3.63 (d, J = 4.4 Hz, 2H), 3.55 − 3.45 (m, 2H), 2.79 (s, 6H), 2.59 (dt, J = 14.7, 7.4 Hz, 1H), 2.46 (s, 3H), 2.09 − 1.98 (m, 1H), 1.85 − 1.75 (m, 1H), 1.69 (s, 3H), 1.61 (s, 3H), 1.38 (d, J = 7.0 Hz, 3H), 0.94 (s, 9H). ¹³C NMR (126 MHz, DMSO) δ 171.1, 170.8, 170.0, 165.8, 164.1, 155.4, 152.0, 148.2, 145.2, 141.1, 137.8, 135.9, 131.6, 130.1, 129.3, 128.9, 128.2, 127.9, 127.8, 127.1, 126.9, 69.2, 61.1, 60.3, 59.0, 57.1, 56.7, 49.1, 48.2, 44.5, 41.6, 38.2, 36.8, 35.7, 35.2, 26.9, 26.4, 22.9, 16.5. UPLC/MS(ESI): m/z 988.56 [M+H]⁺.

N1-(5-(((S)-2-(dimethylamino)-1-phenylethyl)carbamoyl)-6,6-dimethyl-1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)-N4-(4-(((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-4-oxobutyl)terephthalamide (11)

The title compound was prepared according to the general procedure. Compound 11 was obtained as white solid (13.2 mg, 35%) ¹H NMR (500 MHz, DMSO- d6) δ 11.08 (s, 1H), 9.00 (s, 2H), 8.64 (t, J = 5.5 Hz, 1H), 8.38 (d, J = 7.8 Hz, 1H), 8.22 − 8.05 (m, 2H), 8.04 − 7.94 (m, 2H), 7.90 (d, J = 9.3 Hz, 1H), 7.44 (dd, J = 8.5, 2.0 Hz, 4H), 7.40 (td, J = 8.3, 7.9, 2.8 Hz, 4H), 7.36 − 7.27 (m, 1H), 6.77 (d, J = 9.1 Hz, 1H), 5.40 − 5.31 (m, 1H), 4.93 (d, J = 7.2 Hz, 1H), 4.79 (d, J = 11.8 Hz, 1H), 4.56 (dd, J = 17.6, 10.6 Hz, 2H), 4.44 (t, = 8.0 Hz, 1H), 4.30 (s, 1H), 3.67 − 3.51 (m, 4H), 3.39 − 3.26 (m, 4H), 2.89 (d, J =4.8 Hz, 3H), 2.85 (d, J = 4.8 Hz, 3H), 2.46 (s, 3H), 2.34 (dt, J = 14.8, 7.8 Hz, 1H), 2.22 (dt, J = 14.5, 7.3 Hz, 1H), 2.01 (d, J = 14.5 Hz, 1H), 1.85 − 1.75 (m, 3H), 1.69 (s, 3H), 1.60 (s, 3H), 1.38 (d, J = 6.9 Hz, 3H), 0.96 (s, 9H). ¹³C NMR (126 MHz, DMSO) δ 172.3, 171.1, 170.0, 165.9, 164.2, 155.5, 152.0, 148.2, 145.1, 140.8, 137.9, 136.0, 131.6, 130.2, 129.3, 129.0, 128.2, 128.0, 127.8, 127.1, 126.9, 69.3, 61.2, 60.4, 59.0, 57.0, 56.7, 49.1, 48.2, 47.6, 44.8, 41.8, 38.2, 35.7, 33.1, 27.0, 26.9, 26.7, 26.4, 26.1, 22.9, 16.5. UPLC/MS(ESI): m/z 1002.67 [M+H]⁺.

N1-(5-(((S)-2-(dimethylamino)-1-phenylethyl)carbamoyl)-6,6-dimethyl-1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)-N4-(6-(((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-6-oxohexyl)terephthalamide. (12)

The title compound was prepared according to the general procedure. Compound 12 was obtained as off white solid (8.6 mg, 28%). ¹H NMR (500 MHz, DMSO- d6) δ 11.08 (s, 1H), 9.00 (s, 1H), 8.60 (t, J =5.6 Hz, 1H), 8.38 (d, J = 7.8 Hz, 1H), 8.09 (d, J = 8.5 Hz, 2H), 7.96 (d, J = 8.5 Hz, 2H), 7.80 (d, J = 9.3 Hz, 1H), 7.65 (s, 1H), 7.48 − 7.42 (m, 4H), 7.42 − 7.38 (m, 4H), 7.34 − 7.28 (m, 1H), 6.77 (d, J = 9.2 Hz, 1H), 5.40 − 5. 30 (m, 1H), 4.92 (t, J = 7.2 Hz, 1H), 4.78 (d, = 11.8 Hz, 1H), 4.58 (d, J = 11.9 Hz, 2H), 4.53 (d, J = 9.3 Hz, 1H), 4.43 (t, J = 8.0 Hz, 1H), 4.29 (t, J = 3.6 Hz, 1H), 3.66 − 3.50 (m, 5H), 3.35 (td, J = 8.6, 4.2 Hz, 1H), 3.27 (q, J =6.5 Hz, 2H), 2.89 (d, J = 4.8 Hz, 3H), 2.85 (d, J = 4.8 Hz, 3H), 2.46 (s, 3H), 2.27 (dq, J = 13.3, 6.9, 6.1 Hz, 1H), 2.20 − 2.10 (m, 2H), 2.06 − 1.96 (m, 1H), 1.80 (td, . = 8.4, 4.2 Hz, 1H), 1.69 (s, 3H), 1.60 (s, 3H), 1.38 (d, J = 7.0 Hz, 3H), 0.94 (s, 9H). ¹³C NMR (126 MHz, DMSO) δ 172.5, 171.1, 170.1, 165.8, 164.2, 155.5, 151.9, 148.2, 145.1, 140.8, 137.9, 135.9, 131.6, 130.2, 129.5, 129.3, 129.0, 128.2, 128.0, 127.7, 127.1, 126.9, 106.1, 69.2, 61.2, 60.7, 59.0, 56.8, 56.7, 49.1, 48.2, 44.8, 41.7, 38.2, 35.7, 33.9, 30.7, 29.7, 27.0, 26.4, 25.7, 25.3, 22.9, 16.4. UPLC/MS(ESI): m/z 1030.63 [M+H]⁺.

N1-(5-(((S)-2-(dimethylamino)-1-phenylethyl)carbamoyl)-6,6-dimethyl-1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)-N4-(8-(((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-8-oxooctyl)terephthalamide. (13)

The title compound was prepared according to the general procedure. Compound 13 was obtained as white solid (12.3 mg, 36%). ¹H NMR (500 MHz, DMSO- d6) δ 12.53 (s, 1H), 11.09 (s, 1H), 9.20 (s, 1H), 8.99 (s, 1H), 8.62 (t, J = 5.7 Hz, 1H), 8.38 (d, J = 7.8 Hz, 1H), 8.10 (d, J = 8.4 Hz, 2H), 7.96 (d, J = 8.1 Hz, 2H), 7.79 (d, J = 9.3 Hz, 1H), 7.44 (dd, J = 7.8, 5.7 Hz, 4H), 7.42 − 7.36 (m, 4H), 7.33 − 7.27 (m, 1H), 6.77 (d, J = 9.1 Hz, 1H), 5.41 − 5.31 (m, 1H), 5.11 (d, = 3.6 Hz, 1H), 4.98 − 4.88 (m, 1H), 4.80 (d, = 11.8 Hz, 1H), 4.57 (d, J = 11.9 Hz, 1H), 4.52 (d, J = 9.3 Hz, 1H), 4.43 (t, J = 8.0 Hz, 1H), 4.31 − 4.24 (m, 1H), 3.61 (t, J = 3.9 Hz, 2H), 3.55 (t, J = 12.5 Hz, 1H), 3.27 (q, J = 6.7 Hz, 2H), 2.86 (d, J = 19.3 Hz, 6H), 2.46 (s, 3H), 2.27 (dd, J = 14.3, 7.4 Hz, 1H), 2.16 − 2.06 (m, 1H), 2.05 − 1.95 (m, 1H), 1.85 − 1.76 (m, 1H), 1.69 (s, 3H), 1.60 (s, 3H), 1.57 − 1.45 (m, 4H), 1.38 (d, J = 7.0 Hz, 3H), 1.34 − 1.23 (m, 4H), 0.94 (s, 9H). ¹³C NMR (126 MHz, DMSO) δ 172.6, 171.1, 170.1, 165.8, 164.1, 155.5, 151.9, 148.2, 145.2, 140.9, 137.9, 135.9, 131.6, 130.2, 129.3, 129.3, 129.0, 128.2, 128.0, 127.7, 127.1, 126.9, 126.8, 121.3, 118.9, 116.5, 114.1, 69.2, 67.5, 61.2, 60.4, 59.0, 56.8, 56.7, 49.1, 48.2, 47.6, 44.6, 41.7, 38.2, 35.6, 35.4, 29.5, 29.1, 29.0, 27.0, 26.9, 26.8, 26.4, 25.9, 22.9, 22.6, 16.4. UPLC/MS(ESI): m/z 1058.69 [M+H]⁺.

N1-(5-(((S)-2-(dimethylamino)-1-phenylethyl)carbamoyl)-6,6-dimethyl-1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)-N4-(2-(3-(((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-3-oxopropoxy)ethyl)terephthalamide. (14)

The title compound was prepared according to the general procedure. Compound 14 was obtained as white solid (11.7 mg, 33%).

¹H NMR (500 MHz, DMSO- d6) δ 11.09 (s, 1H), 8.99 (s, 2H), 8.66 (t, J = 5.7 Hz, 1H), 8.39 (d, J = 7.8 Hz, 1H), 8.10 (d, J = 8.5 Hz, 2H), 7.99 d, J = 8.5 Hz, 2H), 7.90 d, J = 9.3 Hz, 1H), 7.47 − 7.42 (m, 4H), 7.42 − 7.35 (m, 4H), 7.32 − 7.27 (m, 1H), 6.77 (d, J = 9.1 Hz, 1H), 5.41 − 5.31 (m, 1H), 4.91 (p, J = 7.0 Hz, 1H), 4.78 (d, J = 11.9 Hz, 1H), 4.56 (dd, = 19.3, 10.6 Hz, 2H), 4.44 (t, J = 8.1 Hz, 1H), 4.35 − 4.26 (m, 1H), 3.72 − 3.50 (m, 8H), 3.45 (q, J = 5.5 Hz, 2H), 2.89 (d, J = 4.8 Hz, 3H), 2.84 (d, J = 4.8 Hz, 3H), 2.61 − 2.53 (m, 1H), 2.46 (s, 3H), 2.42 − 2.35 (m, 1H), 2.11 − 1.99 (m, 1H), 1.85 − 1.75 (m, 1H), 1.69 (s, 3H), 1.60 (s, 3H), 1.36 (d, = 7.0 Hz, 3H), 0.93 (s, 9H). ¹³C NMR (126 MHz, DMSO) δ 171.1, 170.5, 169.9, 166.0, 164.2, 155.5, 152.0, 148.2, 145.2, 140.8, 137.6, 136.0, 131.6, 130.2, 129.3, 129.3, 129.0, 128.2, 128.0, 127.8, 127.1, 126.8, 106.1, 69.2, 69.0, 67.1, 61.2, 60.4, 59.1, 56.9, 56.8, 49.1, 48.2, 47.6, 44.8, 41.7, 38.2, 36.1, 35.8, 26.9, 26.7, 26.4, 22.9, 16.4. UPLC/MS(ESI): m/z 1032.73 [M+H]⁺.

N1-(5-(((S)-2-(dimethylamino)-1-phenylethyl)carbamoyl)-6,6-dimethyl-1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)-N4-(2-(2-(3-(((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-3-oxopropoxy)ethoxy)ethyl)terephthalamide. (15).

The title compound was prepared according to the general procedure. Compound 15 was obtained as white solid (6.5 mg, 20%). ¹H NMR (500 MHz, DMSO- d6) δ 11.08 (s, 1H), 9.05 (s, 1H), 8.99 (s, 1H), 8.69 (t, J = 5.6 Hz, 1H), 8.38 (d, J = 7.8 Hz, 1H), 8.10 (d, J = 8.5 Hz, 2H), 7.98 (d, J = 8.5 Hz, 2H), 7.87 (d, J = 9.3 Hz, 1H), 7.46 − 7.42 (m, 4H), 7.42 − 7.35 (m, 4H), 7.33 − 7.29 (m, 1H), 6.77 (d, J = 9.1 Hz, 1H), 5.40 − 5.30 (m, 2H), 4.91 (p, J = 7.3 Hz, 2H), 4.79 (d, J = 11.9 Hz, 1H), 4.55 (dd, J = 19.3, 10.6 Hz, 2H), 4.43 (t, J = 8.1 Hz, 1H), 4.29 (dd, J = 4, 2.4 Hz, 1H), 3.67 − 3.47 (m, 11H), 3.45 (q, J = 6.8, 6.3 Hz, 2H), 3.40 − 3.30 (m, 1H), 2.89 (d, J = 4.8 Hz, 3H), 2.84 (d, J = 4.8 Hz, 3H), 2.46 (s, 3H), 2.36 (dt, J = 14.6, 6.1 Hz, 1H), 2.07 − 1.99 (m, 1H), 1.85 − 1.76 (m, 1H), 1.69 (s, 3H), 1.60 (s, 3H), 1.37 (d, J = 7.0 Hz, 3H), 0.94 (s, 9H). ¹³C NMR (126 MHz, DMSO) δ 171.1, 170.4, 169.9, 166.0, 164.2, 155.5, 152.0, 148.2, 145.1, 140.8, 137.6, 136.0, 131.6, 130.2, 129.3, 129.0, 128.2, 128.0, 127.8, 127.1, 126.8, 118.3, 115.9, 106.1, 70.0, 69.3, 67.4, 61.2, 60.4, 59.0, 56.9, 56.8, 49.1, 48.2, 47.6, 44.8, 41.7, 38.2, 36.2, 35.8, 26.9, 26.7, 26.4, 22.9, 16.4. UPLC/MS(ESI): m/z 1076.70 [M+H]⁺.

N1-(5-(((R)-2-(dimethylamino)-1-phenylethyl)carbamoyl)-6,6-dimethyl-1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)-N4-((S)-14-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidine-1-carbonyl)-15,15-dimethyl-12-oxo-3,6,9-trioxa-13-azahexadecyl)terephthalamide. (16)

The title compound was prepared according to the general procedure. Compound 16 was obtained as white solid (7.2 mg, 19%) ¹H NMR (500 MHz, DMSO- d6) δ 11.08 (s, 1H), 9.13 − 8.92 (m, 2H), 8.69 (t, J =5.6 Hz, 1H), 8.38 (d, J =7.8 Hz, 1H), 8.21 − 8.06 (m, 3H), 8.00 − 7.96 (m, 2H), 7.86 (d, J = 9.3 Hz, 1H), 7.49 − 7.36 (m, 9H), 7.30 (td, J =6.8, 6.4, 1.5 Hz, 1H), 6.77 d, J = 9.2 Hz, 1H), 5.41 − 5.29 (m, 2H), 4.95 − 4.89 (m, 1H), 4.78 d, J = 11.9 Hz, 1H), 4.63 − 4.48 (m, 2H), 4.43 (t, J = 8.0 Hz, 1H), 4.28 (s, 1H), 3.69 − 3.41 (m, 16H), 3.38 − 3.32 (m, 1H), 2.89 (d, J =4.8 Hz, 3H), 2.85 (d, J = 4.8 Hz, 3H), 2.46 (s, 3H), 2.35 (dt, J = 14.5, 6.1 Hz, 1H), 2.05 − 1.95 (m, 1H), 1.80 (td, J = 8.4, 4.3 Hz, 1H), 1.69 (s, 3H), 1.60 (s, 3H), 1.38 (d, J = 7.0 Hz, 3H), 0.94 (s, 9H). ¹³C NMR (126 MHz, DMSO) δ 171.1, 170.4, 169.9, 166.0, 164.2, 155.5, 152.0, 148.2, 145.2, 140.8, 137.6, 136.1, 131.6, 130.2, 129.3, 129.0, 128.5, 128.2, 128.0, 127.8, 127.1, 126.9, 106.1, 70.2, 70.1, 70.0, 69.3, 69.2, 67.4, 61.2, 60.5, 59.0, 56.9, 56.7, 54.0, 49.1, 48.2, 47.6, 44.8, 41.8, 38.2, 36.2, 35.8, 26.9, 26.7, 26.4, 22.9, 16.4. UPLC/MS(ESI): m/z 1120.72 [M+H]⁺.

N-((S)-2-(dimethylamino)-1-phenylethyl)-3-(4-(6-(((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-6-oxohex-1-yn-1-yl)benzamido)-6,6-dimethyl-4,6-dihydropyrrolo[3,4-c]pyrazole-5(1H)-carboxamide (17)

A8 was prepared starting from A2 and 4-iodobenzoyl chloride following a similar procedure with A6. A mixture of A8 (65mg, 0.1 mmol), 5-hexynoicacid (23mg, 0.2 mmol), CuI (10 mg, 0.05mmol), Pd(dppf)Cl₂ (7.4mg, 0.01mmol) under N₂ protection was added Et₃N (31mg, 0.3 mmol) and DMF (2 mL). The mixture was stirred at 80 °C overnight. The solvent was removed and purified by flash column chromatography on silica gel to A9 (41mg, 66%), UPLC/MS(ESI): m/z = 629.31 (M + H)⁺.

To a mixture of A9 (20mg, 0.032 mmol), (S, R, S)-AHPC-Me (14 mg, 0.032 mmol) and DIPEA (12mg, 0.1 mmol) in DMF (1 mL) was added HATU (18 mg, 0.048 mmol). The resultant mixture was stirred at room temperature for 2h. The solvent was removed under reduced pressure and purified by HPLC to give compound A10 (27 mg, 82 %). UPLC/MS(ESI): m/z = 1055.48 (M + H)⁺.

A mixture of A10 (27 mg, 0.025mmol), 2N NaOH solution (1 mL) and THF (1 mL) was stirred at 60 °C for 4 h. The mixture was then neutralized by 2N HCl solution (1 mL). The solvent was removed and the crude was purified by HPLC to give compound 17 (19 mg, 79%)

¹H NMR (500 MHz, DMSO) δ 11.10 (s, 1H), 9.76 (s, 1H), 9.10 (s, 1H), 8.43 (d, J = 7.8 Hz, 1H), 8.02 (d, J = 8.4 Hz, 2H), 7.94 (d, J = 9.2 Hz, 1H), 7.54 (d, J = 8.2 Hz, 2H), 7.45 (dd, J = 8.0, 6.4 Hz, 3H), 7.42 − 7.33 (m, 4H), 7.31 − 7.25 (m, 1H), 6.79 (d, J = 9.0 Hz, 1H), 5.40 − 5.31 (m, 1H), 4.92 (p, J = 7.1 Hz, 1H), 4.83 (d, J = 11.9 Hz, 1H), 4.57 − 4.51 (m, 2H), 4.44 (t, J = 8.0 Hz, 1H), 4.29 (t, J = 3.8 Hz, 1H), 3.72 − 3.64 (m, 2H), 3.64 − 3.53 (m, 2H), 3.52 − 3.44 (m, 2H), 3.38 − 3.29 (m, 1H), 2.86 (d, J = 4.8 Hz, 3H), 2.81 (d, J = 4.8 Hz, 3H), 2.46 (s, 3H), 2.45 − 2.39 (m, 2H), 2.39 − 2.27 (m, 1H), 2.08 − 1.99 (m, 1H), 1.86 − 1.72 (m, 3H), 1.68 (s, 3H), 1.60 (s, 3H), 1.38 (d, J = 7.0 Hz, 3H), 0.95 (s, 9H). ¹³C NMR (126 MHz, DMSO) δ 171.92, 171.12, 170.01, 164.05, 158.91, 158.60, 155.43, 152.39, 147.49, 145.37, 141.04, 132.83, 132.01, 131.81, 129.82, 129.31, 128.95, 128.48, 127.92, 127.21, 127.11, 126.90, 126.79, 106.07, 93.54, 80.85, 72.64, 70.99, 69.23, 61.18, 60.65, 60.31, 59.04, 56.97, 56.75, 49.10, 48.20, 47.53, 44.50, 44.08, 41.63, 40.88, 38.23, 35.68, 34.32, 26.97, 26.93, 26.84, 26.39, 24.98, 22.91, 18.89, 16.13. UPLC/MS(ESI): m/z 983.56 [M+H]⁺.

N-((S)-2-(dimethylamino)-1-phenylethyl)-3-(4-(6-(((S)-1-((2S,4S)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-6-oxohex-1-yn-1-yl)benzamido)-6,6-dimethyl-4,6-dihydropyrrolo[3,4-c]pyrazole-5(1H)-carboxamide (17-Neg)

17-Neg was prepared starting from A9 and (S,S,S)-AHPC-Me following a similar procedure with 17. Compound 17-Neg was obtained as white solid (7.1 mg, 26%)

¹H NMR (500 MHz, DMSO) δ 11.01 (s, 1H), 9.12 (s, 1H), 9.00 (s, 1H), 8.32 (d, J = 7.7 Hz, 1H), 8.03 − 7.97 (m, 2H), 7.94 (d, J = 8.7 Hz, 1H), 7.56 − 7.50 (m, 2H), 7.44 (dd, J = 7.9, 1.9 Hz, 4H), 7.41 − 7.36 (m, 4H), 7.32 − 7.26 (m, 2H), 6.76 (d, J = 9.2 Hz, 1H), 5.39 − 5.31 (m, 1H), 4.96 − 4.88 (m, 1H), 4.76 (d, J = 11.9 Hz, 1H), 4.55 (d, J = 11.9 Hz, 1H), 4.47 (d, J = 8.7 Hz, 1H), 4.35 (dd, J = 8.7, 6.1 Hz, 1H), 4.23 − 4.17 (m, 1H), 3.92 (dd, J = 10.2, 5.6 Hz, 1H), 3.55 (td, J = 12.6, 2.5 Hz, 1H), 3.44 − 3.31 (m, 2H), 2.89 (d, J = 4.7 Hz, 3H), 2.84 (d, J = 4.7 Hz, 3H), 2.46 (s, 3H), 2.44 − 2.38 (m, 3H), 2.35 − 2.27 (m, 2H), 1.83 − 1.71 (m, 2H), 1.68 (s, 3H), 1.59 (s, 3H), 1.38 (d, J = 6.9 Hz, 3H), 0.97 (s, 9H). ¹³C NMR (126 MHz, DMSO) δ 172.23, 171.56, 170.39, 164.14, 159.00, 158.71, 155.47, 152.04, 148.15, 144.79, 140.81, 131.77, 130.21, 129.30, 128.98, 128.45, 127.99, 127.17, 127.05, 126.89, 93.50, 80.82, 69.48, 61.18, 60.44, 58.99, 57.33, 55.98, 49.09, 48.27, 47.57, 44.81, 41.74, 40.85, 37.28, 35.12, 34.21, 26.90, 26.71, 26.42, 24.95, 22.68, 18.86, 16.36. UPLC/MS(ESI): m/z 983.43[M+H]⁺.

N-((S)-2-(dimethylamino)-1-phenylethyl)-3-(4-(6-(((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-6-oxohexyl)benzamido)-6,6-dimethyl-4,6-dihydropyrrolo[3,4-c]pyrazole-5(1H)-carboxamide (18)

The solution of compound 17 (10 mg, 0.01 mmol), Pd/C (10 mg) in MeOH (10 mL) was degassed and subjected to hydrogenation using H₂ gas introduced via a balloon. The mixture was stirred for 6 h and then filtered by Celite. The solvent was removed, and the crude was purified by HPLC to give compound 18 (9 mg, 90%).

¹H NMR (500 MHz, DMSO) δ 10.84 (s, 1H), 8.99 (s, 2H), 8.37 (d, J = 7.8 Hz, 1H), 8.00 − 7.92 (m, 2H), 7.80 (d, J = 9.5 Hz, 1H), 7.46 − 7.36 (m, 5H), 7.36 − 7.28 (m, 4H), 7.22 (d, J = 8.5 Hz, 1H), 7.14 − 7.10 (m, 1H), 6.76 (d, J = 9.2 Hz, 1H), 5.38 − 5. 30 (m, 1H), 4.94 − 4.85 (m, 1H), 4.77 (d, J = 11.9 Hz, 1H), 4.60 − 4.50 (m, 2H), 4.42 (q, J = 7.8 Hz, 1H), 4.29 (dq, J = 6.6, 3.1 Hz, 1H), 3.65 − 3.52 (m, 3H), 3.40 − 3.31 (m, 2H), 2.89 (d, J = 4.8 Hz, 3H), 2.85 (d, J = 4.8 Hz, 3H), 2.64 (t, J = 7.7 Hz, 2H), 2.46 (s, 1H), 2.27 (dt, J = 14.6, 7.7 Hz, 1H), 2.16 − 2.10 (m, 1H), 2.09 (s, 1H), 2.05 − 1.95 (m, 1H), 1.83 − 1.75 (m, 1H), 1.68 (s, 3H), 1.59 (s, 3H), 1.56 − 1.46 (m, 2H), 1.38 (d, J = 7.0 Hz, 2H), 1.36 − 1.22 (m, 3H), 0.93 (s, 9H). ¹³C NMR (126 MHz, DMSO) δ 172.5, 171.1, 170.1, 164.7, 155.5, 152.0, 147.1, 140.8, 131.6, 131.3, 130.2, 129.9, 129.3, 129.0, 128.8, 128.3, 128.0, 127.1, 126.9, 126.2, 69.2, 61.1, 60.4, 59.0, 56.8, 56.7, 49.1, 48.2, 44.8, 41.8, 38.2, 35.7, 35.4, 35.2, 30.8, 28.8, 27.0, 26.9, 26.8, 26.5, 25.7, 22.9, 16.5. UPLC/MS(ESI): m/z 987.56 [M+H]⁺.

N-((S)-2-(dimethylamino)-1-phenylethyl)-3-(4-((5-(((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-5-oxopentyl)oxy)benzamido)-6,6-dimethyl-4,6-dihydropyrrolo[3,4-c]pyrazole-5(1H)-carboxamide (19)

To a mixture of 4-hydroxy-benzoic acid tert-butyl ester (1000 mg, 5.1 mmol), K₂CO₃ (1423 mg, 10.3 mmol) in DMSO (20 mL) was added methyl 5-bromopentanoate (1306 mg, 6.7 mmol) at room temperature. The mixture was stirred for 3 days. The mixture was poured on water (500 mL) and the product was extracted with ether (500 mL). The combined organic extracts concentrated under pressure and purified by flash column chromatography on silica gel to give A12 (1020 mg, 64%).

A solution of A12 (616 mg, 2 mmol) in DCM (10 mL) was added trifluoroacetic acid (3 mL) and then stirred at rt for 2 hours. The mixture was concentrated to give crude intermediate A13 which was used in the next step without further purification.

To a solution of A13 (300 mg, 1.2 mmol) in DCM (5 mL) was added 1 drop DMF. Then oxalyl chloride (151 mg, 1.2 mmol) was added dropwise at 0 °C and the resulting mixture was stirred at rt for 8 h. The solvent was removed to give crude intermediate A14 which was used without further purification.

A18 was prepared starting from A2 and A14 following a similar procedure that used for A6. UPLC/MS(ESI): m/z 563.38 [M+H]⁺.

To a mixture of A18 (20 mg, 0.036 mmol), (S, R, S)-AHPC-Me (17 mg, 0.036 mmol) and DIPEA (14 mg, 0.11 mmol) in DMF (1 mL) was added HATU (20 mg, 0.05 mmol). The resultant mixture was stirred at room temperature for 2h. The solvent was removed under reduced pressure and purified by HPLC to give compound 19 (27 mg, 77 %).

¹H NMR (500 MHz, DMSO) δ 10.79 (s, 1H), 9.11 (s, 1H), 9.00 (s, 1H), 8.38 (d, J = 7.8 Hz, 1H), 8.05 − 7.97 (m, 2H), 7.86 (d, J = 9.3 Hz, 1H), 7.46 − 7.41 (m, 4H), 7.41 − 7.36 (m, 4H), 7.33 − 7.26 (m, 2H), 7.07 − 7.00 (m, 2H), 6.77 (d, J = 9.1 Hz, 1H), 5.40 − 5.30 (m, 1H), 4.93 (p, J = 7.2 Hz, 1H), 4.77 (d, J = 11.8 Hz, 1H), 4.62 − 4.48 55 (dd, J = 10.6, 6.8 Hz, 2H), 4.45 (t, J = 8.1 Hz, 1H), 4.30 (t, J = 3.7 Hz, 1H), 4.06 (t, J = 5.9 Hz, 2H), 3.66 − 3.51 (m, 3H), 3.40 − 3.30 (m, 1H), 2.89 (d, J = 4.6 Hz, 3H), 2.84 (d, J = 4.6 Hz, 3H), 2.45 (s, 3H), 2.35 (dt, J = 13.9, 7.0 Hz, 1H), 2.22 (dt, J = 13.8, 6.8 Hz, 1H), 2.08 − 1.99 (m, 1H), 1.85 − 1.75 (m, 1H), 1.73 (dt, J = 12.3, 5.5 Hz, 2H), 1.68 (s, 3H), 1.66 (s, 1H), 1.60 (d, J = 6.3 Hz, 3H), 1.38 (d, J = 7.0 Hz, 3H), 0.95 (s, 9H). ¹³C NMR (126 MHz, DMSO) δ 172.4, 171.1, 170.1, 164.3, 155.5, 152.1, 148.0, 145.2, 140.8, 138.5, 131.7, 130.2, 130.1, 129.3, 129.0, 128.0, 127.0, 126.9, 125.8, 114.6, 105.9, 69.3, 67.9, 61.2, 60.4, 59.0, 56.9, 56.8, 49.1, 48.2, 47.6, 44.8, 41.7, 38.2, 35.7, 34.9, 28.6, 26.9, 26.7, 26.4, 22.8, 22.5, 16.3.UPLC/MS(ESI): m/z 989.76 [M+H]⁺.

N1-(5-(((S)-2-(dimethylamino)-1-phenylethyl)carbamoyl)-6,6-dimethyl-1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)-N4-(8-(2-((1-(2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)octyl)terephthalamide (20)

The title compound was prepared according to the general procedure. Compound 20 was obtained as white solid (7.3 mg, 46%). ¹H NMR (500 MHz, DMSO) δ 11.07 (d, J = 2.8 Hz, 1H), 9.06 (s, 1H), 8.98 (s, 1H), 8.60 (t, J = 5.6 Hz, 1H), 8.50 (t, J = 6.0 Hz, 1H), 8.11 − 8.04 (m, 2H), 7.95 (dd, J = 8.5, 2.5 Hz, 2H), 7.49 − 7.37 (m, 5H), 7.33 − 7.24 (m, 2H), 6.76 (d, J = 9.2 Hz, 1H), 5.61 (td, J = 9.1, 2.4 Hz, 1H), 5.40 − 5.30 (m, 1H), 4.78 (d, J = 12.0 Hz, 1H), 4.69 − 4.49 (m, 2H), 4.35 (dp, J = 4.1, 2.0 Hz, 1H), 4.33 − 4.17 (m, 2H), 4.04 (t, J = 6.3 Hz, 2H), 3.90 (dd, J = 13.5, 2.4 Hz, 1H), 3.68 − 3.56 (m, 2H), 3.56 − 3.50 (m, 2H), 3.40 − 3.30 (m, 1H), 3.27 (q, J = 6.6 Hz, 2H), 2.88 (d, J = 4.8 Hz, 3H), 2.84 (d, J = 4.8 Hz, 3H), 2.45 (s, 3H), 2.12 − 2.04 (m, 1H), 1.97 − 1.87 (m, 1H), 1.75 (dt, J = 12.7, 6.5 Hz, 2H), 1.67 (d, J = 11.0 Hz, 3H), 1.60 (d, J = 6.5 Hz, 3H), 1.57 − 1.51 (m, 2H), 1.46 (q, J = 7.0 Hz, 2H), 1.39 − 1.30 (m, 7H), 1.25 − 1.19 (m, 2H), 0.95 (s, 9H). ¹³C NMR (126 MHz, DMSO) δ 172.3, 169.4, 168.6, 165.8, 164.2, 156.4, 155.5, 155.2, 151.9, 148.3, 141.2, 140.8, 137.9, 135.9, 131.8, 131.3, 129.1, 129.0, 128.2, 128.1, 128.0, 127.7, 127.4, 127.3, 127.1, 121.2, 112.1, 106.2, 79.5, 77.7, 69.4, 68.2, 61.2, 60.5, 59.3, 57.8, 57.2, 57.0, 55.9, 49.1, 47.6, 44.8, 41.8, 38.4, 37.7, 36.5, 29.5, 29.2, 29.1, 26.9, 26.7, 26.6, 26.5, 26.1, 16.4. UPLC/MS(ESI): m/z 1132.73 [M+H]⁺.

N1-(5-(((S)-2-(dimethylamino)-1-phenylethyl)carbamoyl)-6,6-dimethyl-1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)-N4-(2-(2-(2-(2-((1-(2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)ethoxy)ethoxy)ethyl)terephthalamide (21)

The title compound was prepared according to the general procedure. Compound 21 was obtained as white solid (8.2 mg, 32%) ¹H NMR (500 MHz, DMSO) δ 11.06 (s, 1H), 9.03 (s, 1H), 8.97 (s, 1H), 8.68 (t, J = 5.6 Hz, 1H), 8.50 (t, J = 6.0 Hz, 1H), 8.11 − 8.04 (m, 2H), 7.99 − 7.92 (m, 2H), 7.50 − 7.34 (m, 5H), 7.33 − 7.24 (m, 2H), 7.03 (d, J = 1.7 Hz, 1H), 6.97 (dd, J = 7.8, 1.7 Hz, 1H), 6.76 (d, J = 9.1 Hz, 1H), 5.38 − 5.29 (m, 1H), 4.77 (d, J = 11.9 Hz, 1H), 4.62 − 4.48 (m, 2H), 4.36 − 4.21 (m, 2H), 4.21 − 4.11 (m, 2H), 3.81 – 3.77 (m, 2H), 3.67 − 3.62 (m, 4H), 3.62 − 3.51 (m, 6H), 3.44 (q, J = 5.9 Hz, 2H), 3.38 − 3.30 (m, 1H), 2.88 (d, J = 4.7 Hz, 3H), 2.84 (d, J = 4.8 Hz, 3H), 2.45 (s, 3H), 2.13 − 2.05 (m, 1H), 1.95 − 1.87 (m, 1H), 1.68 (s, 3H), 1.60 (s, 3H), 1.42 − 1.30 (m, 2H), 1.26 − 1.16 (m, 2H), 0.95 (s, 9H). ¹³C NMR (126 MHz, DMSO) δ 172.3, 169.4, 168.5, 166.0, 164.2, 156.3, 155.5, 151.9, 148.3, 140.8, 137.6, 136.0, 131.8, 131.3, 129.0, 128.2, 128.0, 127.8, 127.6, 127.1, 121.5, 112.6, 106.2, 79.5, 70.5, 70.1, 69.5, 69.4, 69.3, 68.4, 61.2, 60.5, 59.3, 57.2, 57.0, 49.1, 47.6, 44.9, 41.8, 26.7, 26.6, 26.5, 16.4. UPLC/MS(ESI): m/z 1136.63 [M+H]⁺.

N1-(5-(5-(4-((S)-1-((2S,4R)-1-((S)-2-acetamido-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)ethyl)phenyl)thiazole-4-carboxamido)pentyl)-N4-(5-(((S)-2-(dimethylamino)-1-phenylethyl)carbamoyl)-6,6-dimethyl-1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)terephthalamide (22)

The title compound was prepared according to the general procedure. Compound 22 was obtained as off white solid (12.1 mg, 55%). ¹H NMR (500 MHz, DMSO) δ 11.05 (s, 1H), 9.08 (s, 1H), 9.00 (s, 1H), 8.61 (t, J = 5.6 Hz, 1H), 8.43 (t, J = 6.0 Hz, 1H), 8.38 (d, J = 7.8 Hz, 1H), 8.10 − 8.04 (m, 2H), 7.97 − 7.91 (m, 2H), 7.89 (d, J = 9.3 Hz, 1H), 7.51 − 7.46 (m, 2H), 7.45 − 7.36 (m, 5H), 7.34 − 7.25 (m, 3H), 6.75 (d, J = 9.1 Hz, 1H), 5.38 − 5.28 (m, 1H), 4.90 (p, J = 7.2 Hz, 1H), 4.77 (d, J = 11.9 Hz, 1H), 4.56 (d, J = 11.9 Hz, 1H), 4.50 (d, J = 9.4 Hz, 2H), 4.42 (t, J = 8.1 Hz, 2H), 4.29 − 4.24 (m, 1H), 3.40 − 3.30 (m, 2H), 3.29 − 3.16 (m, 4H), 2.88 (d, J = 4.6 Hz, 3H), 2.83 (d, J = 4.7 Hz, 3H), 2.08 (s, 3H), 2.04 − 1.97 (m, 2H), 1.87 (s, 3H), 1.82 − 1.75 (m, 2H), 1.67 (s, 3H), 1.59 (s, 3H), 1.57 − 1.47 (m, 5H), 1.39 − 1.28 (m, 6H), 0.93 (s, 9H). ¹³C NMR (126 MHz, DMSO) δ 171.1, 170.1, 169.6, 165.8, 164.2, 162.1, 155.5, 152.8, 145.9, 144.2, 141.1, 140.8, 137.9, 135.9, 130.2, 129.0, 128.9, 128.2, 128.0, 127.7, 127.1, 126.0, 69.2, 61.2, 60.5, 59.0, 56.9, 56.8, 49.1, 48.2, 47.6, 44.9, 41.8, 39.1, 38.2, 35.6, 29.3, 29.2, 26.9, 26.7, 26.5, 24.4, 23.0, 22.8. UPLC/MS(ESI): m/z 1073.69 [M+H]⁺.

Molecular docking

Docking studies were carried out using the Maestro program from Schrödinger. CDK7-THZ1 complex was extracted from the THZ1 bound CDK7-cyclin H-MAT1Δ219 complex (PDB Code: 6XD3). Protein Preparation and LigPrep wizards were applied to prepare the protein and ligand using default parameters. Then covalent docking was conducted using the protocols in Covalent Docking wizard to target the reactive residue Cys312. The docked ligand was confined to a THZ1 centered box with length ≤ 20Å. Hinge hydrogen bonds between the ligand and backbone carbonyl oxygen of Asp92, backbone carbonyl oxygen and NH of Met94 were set as constraints to ensure the proper binding mode. The output docking poses must match at least 2 constraints. Thorough docking mode was selected. Default values were adopted for other parameters.

In Vitro Kinase Assays

Adapta Eu kinase assays were conducted for CDK7/Cyclin H/MNAT1 at ThermoFisher Scientific using Km ATP concentrations.

Cell Culture

The cell lines MOLT4, Jurkat, SUDHL5, OVCAR3, and A549 were procured from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) from Gibco (Grand Island, NY, USA), along with 100 units/mL penicillin and 100 μg/mL streptomycin. Incubation occurred at 37 °C with 5% CO₂ in a humidified atmosphere. Mycoplasma contamination was conducted using the MycoAlert mycoplasma detection kit (Lonza, Basel, Switzerland), with all cell lines consistently testing negative.

Western blotting analysis

Cell Lysis and Protein Extraction: Place the cell culture dish on ice for initial cell stabilization. Rinse cells with ice-cold phosphate-buffered saline (PBS). Remove PBS and add ice-cold Radioimmunoprecipitation Assay (RIPA) buffer, comprising 50mM Tris-HCl, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS at pH 7.4 (1 mL per 10^7 cells). Augment the RIPA buffer with Protease Inhibitor (Roche) and Phosstop Phosphatase Inhibitor (Roche). Incubate the cell lysate on ice for 30 minutes.

Centrifugation and Lysate Processing: Centrifuge the lysate tubes and carefully place them on ice. Aspirate the supernatant into a fresh tube kept on ice. Discard the cellular pellet. Quantify the protein concentration of the lysate using the Bicinchoninic Acid (BCA) protocol (Pierce).

Sample Preparation: Prepare total cell lysates in 4× sample loading buffer (Bio-Rad, 1610747). Boil the lysate samples for 10 minutes at 95 °C.

Electrophoresis: Subject equivalent protein amounts to 4–20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Membrane Transfer: Transfer the separated proteins onto an Immobilon^®-NC Transfer Membrane (Millipore, Bedford, MA, USA).

Blocking and Antibody Incubation: Block the membrane using Intercept^® (TBS) Blocking Buffer (LI-COR Biosciences, Lincoln, NE, USA). Incubate the membrane with primary antibodies （CDK7 (Cell Signaling Technology, 2916, 1:1000)； Cyclin H (Cell Signaling Technology, cat#2927S,1:1000)；MAT1 (Santa Cruz, sc-13142, 1:1000)； VHL (Cell Signaling Technology, 68547, 1:1000)；CDK1 (Abcam, cat# ab131450,1:1000), CDK2 (Cell Signaling Technology, cat# 2546S, 1:1000), CDK4 (Cell Signaling Technology, cat# 12790S, 1:1000), CDK5 (Cell Signaling Technology, cat# 12134S, 1:1000), CDK6 (Cell Signaling Technology, cat# 3136S, 1:1000), CDK8 (Cell Signaling Technology, cat# 4106S, 1:1000), CDK9 (Cell Signaling Technology, cat# 2316S, 1:1000); LONRF2 (Abcam, cat# ab229210, 1:1000) β-actin (Cell Signaling Technology, 3700S, 1:5000) at 4 °C overnight

Secondary Antibody Incubation and Imaging: The membranes were subjected to incubation with IRDye^®800-labeled goat anti-rabbit IgG (LI-COR Biosciences, cat# 926–32211) or IRDye 680RD goat anti-Mouse IgG (LI-COR Biosciences, cat# 926–68070) secondary antibodies at room temperature for a duration of 1 hour. Subsequent to the incubation period, membrane detection was carried out using the Li-COR Odyssey CLx system.

Mass spectrometry

Sample Preparation.

OVCAR3 cells were grown in RPMI supplemented with 10 % FBS and 1 % penicillin/streptomycin and maintained free of mycoplasma at 37 °C and 5% CO₂. Cells were plated in triplicate 10 cm plates per condition and allowed to adhere for 24 hours prior to drug treatment or DMSO control. After 6 hours, cells were washed twice in ice cold PBS and harvested in PBS with Halt^™ dual protease and phosphatase inhibitor (ThermoFisher, cat. No. 78441) by manually scraping the cells from the plates with cell lifters. Cells were centrifuged at 2500 rpm for 5 min. at 4 °C, residual buffer was aspirated, and cell pellets were flash frozen in liquid nitrogen and stored at −80°C until further processing.

Sample preparation was performed as described. Pellets were thawed and cells were lysed in 2% SDS, 150 mM NaCl, 50 mM Tris pH 7.4 supplemented with Halt^™ Protease and Phosphatase Inhibitor Single-Use Cocktail, EDTA Free (ThermoFisher, cat. No. 78443). Lysate was run through Qiashredder filters (Qiagen, cat. No. 79656) by centrifugation and flow-through was collected. Disulfide reduction was performed by adding dithiothreitol (DTT) to a final concentration of 5 mM and heating to 37°C for 1 hour. Cysteines were alkylated with15 mM iodoacetamide (final concentration) for 30 min. in the dark at room temperature (RT). Protein concentration was measured using a Micro BCA^™ Protein Assay Kit (ThermoFisher, cat. No. 23235) according to the manufacturer’s protocol. Detergent was removed by methanol/chloroform protein precipitation. Precipitates were solubilized in freshly prepared 8 M urea in 200 mM EPPS, pH 8.5 for 10 min at 37 °C, the urea concentration was then reduced by dilution with 200 mM EPPS, pH 8.5 to 2 M urea. Lys-C protease (Wako, cat. No. 129–02541) was added at an enzyme-to-substrate ratio of 1:75, and samples were incubated overnight at RT at which time 200 mM EPPS was added to a final urea concentration of 0.5 M, and digestion was completed by addition of trypsin (Promega, cat. No. V5113) at an enzyme-to-substrate ratio of 1:75 for 6h at 37 °C.

A digest check was performed by pooling 1 μg of protein from each sample; only those samples with a missed cleavage rate <10% as measured by LC-MS/MS were processed further. 150 μg of protein per sample was labelled using a TMT18 plex Mass Tag Labelling Kit (ThermoFisher, cat. No. A52045). Hydroxylamine was added to a final concentration of 0.5% (v/v) for 15 min. at RT to quench the TMT labelling reactions. TMT labelling efficiency was measured by LC-MS3 analysis, a ratio check was performed. Formic acid (FA) was added to a final volume of 2% (v/v) to pH < 3.0 and 150 μg per sample was combined and de-salted using a SepPak tC18 Vac RC Cartridge (50 mg, Waters, cat. No. WAT054960). HPLC fractionation was performed over a period of 75 minutes using an Agilent 1200 Series instrument with a flow rate of 600 μl/minute. Peptides were collected in a 96-well plate over a 65 min-gradient of 13–44%B with Buffer A comprising 5% acetonitrile, 10 mM ammonium bicarbonate, pH 8 and Buffer B comprising 90% acetonitrile, 10 mM ammonium bicarbonate, pH8. Fractions were pooled to generate a total of 24 aliquots, followed by sample clean-up using the Stage Tip protocol with C18 Empore^™ Extraction Disks (Fisher Scientific, cat. No. 14-386-2). The matrix was primed with methanol and equilibrated with 70% acetonitrile, 1% FA followed by washing twice with 1% FA. Samples were loaded in 1% FA, followed again by two 1% FA washes, and finally peptides were eluted using 70% acetonitrile, 1% FA. Samples were dried before resuspension in MS Loading Buffer (3% acetonitrile, 5% FA).

MS Analysis

Peptide separation was performed on 75 μm columns packed with 2.6 μm Accucore beads (Thermo Fisher Scientific). Peptides were injected onto 30–40 cm, 100 and 75 μm (internal diameter) columns, respectively, and separated using an EASY-nLC 1200 HPLC (ThermoFisher Scientific). The flow rate was 450 nL/min for the 100 μm columns and 300 nL/min for the 75 μm columns with a gradient of 6–28%B over 170 minutes with Buffer A comprising 3% acetonitrile, 0.4% FA and Buffer B comprising 100% acetonitrile, 0.4% FA for the 100 μm columns and 5–35%B over 240 minutes with Buffer A comprising 0.125% FA and Buffer B comprising 95% acetonitrile, 0.125% FA for the 75 μm columns. The columns were heated to 60 °C using a column heater (constructed in-house). Samples from the HPLC were injected into an Orbitrap Fusion Lumos Tribrid MS (ThermoFisher, cat. No. FSN02–10000) using a multi-notch MS3 method^3,4. MS scans were performed in the Orbitrap over a scan range of 400–1400 m/z with dynamic exclusion. Turbo rate scans were performed in the Ion Trap with a collision energy of 35% and maximum injection times of 200 ms. TMT quantification was performed using SPS-MS3 in the Orbitrap with a scan range of 100–1000 m/z and an HCD collision energy of 55%. Orbitrap resolution was 50,000 (dimensionless units) with a maximum injection time of 450 ms. MS isolation windows were varied depending on the charge state.

MS data analysis.

Mass spectrometric data (Thermo “.RAW” files) were converted to mzXML format, to correct monoisotopic m/z measurements, and to perform a post-search calibration. Peptide spectrum matches were assigned with SEQUEST (v.28 (rev. 12), (c) 1998–2007 Molecular Biotechnology, Univ. of Washington, J.Eng/S.Morgan/J.Yates licensed to Thermo Fisher Scientific Inc.) based software. The quality of peptide identifications by SEQUEST was determined with a target-decoy approach, where each peptide spectra was searched against a composite database of size-sorted forward and reverse protein sequences of the human proteome (Uniprot 02/2014) that also contained common contaminant proteins. For each peptide, identification scores (Xcorr, ΔCn, and precursor mass error) were computed by SEQUEST for target and decoy hits. Linear discriminant analysis that combines the 3 SEQUEST identification score parameters into an optimal discriminant score was performed. For each set, the false discovery rate (FDR) was computed as twice the number of reverse peptides identified divided by the total number of peptide identifications above any given discriminant score threshold⁷. Peptides were filtered to achieve an FDR < 1%. During peptide assignment for all data, oxidized methionine (+15.9949 Da) was searched dynamically. All peptide searches considered TMT modification (+229.1629 Da) on N-termini and lysine residues as static modifications. For each set, the FDR for protein identification was set to <1%, and shared peptides were then collapsed into proteins using rules of parsimony: a peptide that could be mapped to multiple proteins was assigned only to the largest protein. Peptides with a total TMT value of >200 and an isolation specificity of > 0.7 were included in the final dataset.

Proliferation Assays

Cell viability was assessed through the utilization of the CellTiter-Glo assay (Promega, Madison, WI, USA). For cell proliferation assays, cancer cells were seeded at a low density (10^4/ml) in 384-well white plates and subjected to triplicate treatments with the specified compounds. Dimethyl sulfoxide (DMSO) concentrations were standardized across all conditions. The compounds were dispensed from either 10 mM or 1 mM stock solutions employing a D300 drug printing robot (Tecan). Seventy-two hours post-application of the compounds, Cell Titer Glo reagent (Promega) was administered and allowed to incubate for 10–15 minutes at room temperature before luminescence was measured using a PHERAStar plate reader (BMG LABTECH).

Statistical analysis

Analysis was executed through GraphPad Prism software and Microsoft Excel. The presentation of data follows the format of mean ± standard deviation. Statistical significance was ascertained employing the Students t-test. IC₅₀ determination was conducted utilizing a log-transformed, non-linear regression curve fit analysis. Statistical significance was attributed to differences with a p-value less than 0.05.

Mouse pharmacokinetic study

All protocols were reviewed and approved by the Institutional Animal Care and Use. Committee at Shanghai Chempartner, which is AAALAC accredited. The IV and IP dosing solution was prepared in 5%DMSO+5%Tween80+90%Saline at 0.6 and 2 mg/mL, respectively. Three male CD1 mice were administered intravenously at a single 3 mg/kg dose. Blood samples were collected at 0, 0.083, 0.25, 0.5, 1, 2, 4, 8 and 24 h post dose. Three male CD1 mice were administered intraperitoneally at a single 10 mg/kg dose. Blood samples were collected at 0, 0.25, 0.5, 1, 2, 4, 6, 8 and 24 h post dose. At each time point, blood samples were collected from three mice. Blood samples were put on wet ice and centrifuged to obtain plasma samples (2000 g, 5 min under 4 °C) within 15 minutes of blood collection. Plasma samples were stored at approximately −70°C until analysis. All samples were processed for analysis by protein precipitation using acetonitrile and analyzed with LC-MS/MS-39 (Triple Quad 6500+).

Highlights.

• JWZ-5–13 significantly degrades CDK7 in low nanomolar in multiple cell line.

• JWZ-5–13 exhibits high proteome-wide selectivity.

• JWZ-5–13 exhibited advantages in inhibition of cancer cell proliferation over its parental binder.

• JWZ-5–13 displayed bioavailability in a pharmacokinetic study.