Discovery of YJZ5118: a Potent and Highly Selective Irreversible CDK12/13 Inhibitor with Synergistic Effects in Combination with Akt Inhibition

Jianzhang Yang; Yu Chang; Kaijie Zhou; Weixue Huang; Jean Ching-Yi Tien; Pujuan Zhang; Wenyan Liu; Licheng Zhou; Yang Zhou; Xiaomei Ren; Rahul Mannan; Somnath Mahapatra; Yuping Zhang; Rudana Hamadeh; Grafton Ervine; Zhen Wang; George Xiaoju Wang; Arul M Chinnaiyan; Ke Ding

doi:10.1021/acs.jmedchem.5c00127

. Author manuscript; available in PMC: 2025 Jun 9.

Published in final edited form as: J Med Chem. 2025 Mar 13;68(6):6718–6734. doi: 10.1021/acs.jmedchem.5c00127

Discovery of YJZ5118: a Potent and Highly Selective Irreversible CDK12/13 Inhibitor with Synergistic Effects in Combination with Akt Inhibition

Jianzhang Yang ^α,^β,^#, Yu Chang ^δ,^#, Kaijie Zhou ^α,^#, Weixue Huang ^α,^#, Jean Ching-Yi Tien ^δ,^Ψ, Pujuan Zhang ^α, Wenyan Liu ^α,^δ, Licheng Zhou ^β, Yang Zhou ^β, Xiaomei Ren ^α, Rahul Mannan ^δ, Somnath Mahapatra ^δ, Yuping Zhang ^δ, Rudana Hamadeh ^δ, Grafton Ervine ^δ, Zhen Wang ^α,^*, George Xiaoju Wang ^δ,^Ψ,^*, Arul M Chinnaiyan ^δ,^Ψ,^Φ,^χ,^ξ,^*, Ke Ding ^α,^*

PMCID: PMC12147761 NIHMSID: NIHMS2082140 PMID: 40080446

Abstract

Cyclin-dependent kinases 12 and 13 (CDK12/13) have emerged as promising therapeutic targets for castration-resistant prostate cancer (CRPC) and other human cancers. Despite the development of several CDK12/13 inhibitors, challenges remain in achieving an optimal balance of potency, selectivity and pharmacokinetic properties. Here, we report the discovery of YJZ5118, a novel, potent and highly selective covalent inhibitor of CDK12/13 with reasonable pharmacokinetic profiles. YJZ5118 effectively inhibited CDK12 and CDK13 with IC₅₀ values of 39.5 and 26.4 nM, respectively, while demonstrating high selectivity over other CDKs. Mass spectrometry analysis, co-crystal structure determination, and pulldown-proteomic experiments confirmed the compound’s covalent binding mode with CDK12/13. Functionally, YJZ5118 efficiently suppressed the transcription of DNA damage response genes, induced DNA damage, and triggered apoptosis. Moreover, the compound significantly inhibited the proliferation of multiple tumor cell lines, particularly prostate cancer cells. Notably, YJZ5118 exhibited synergistic effects with Akt inhibitors both in vitro and in vivo.

Keywords: CDK12, CDK13, covalent inhibitor, castration-resistant prostate cancer, synergistic effect with Akt inhibitors

Graphical Abstract

graphic file with name nihms-2082140-f0001.jpg

INTRODUCTION

Cyclin-dependent kinase 12 (CDK12) and its paralog CDK13 belong to a transcription-associated CDK family,¹ and play indispensable roles in DNA damage response (DDR) and the maintenance of genomic stability.^{2, 3} These kinases cooperatively regulate transcription elongation, splicing, and cleavage and polyadenylation by triggering phosphorylation of serine 2 in the C-terminal domain (CTD) of RNA polymerase II in complex with their essential partner protein, cyclin K (CCNK).⁴ CDK12/13 have emerged as promising therapeutic targets for various human cancers,^{5, 6} as evidenced by the inhibition of cell proliferation following genetic knockdown or pharmacological inhibition/degradation of these targets in both triple-negative breast cancer (TNBC) and castration-resistant prostate cancer (CRPC) cells.^7–11 Furthermore, CDK12 mutations have been identified in 5%–7% of patients with metastatic CRPC (mCRPC).^10–12 Our recent studies have also suggested that CDK12 mutation may serve as synergistic biomarkers for CDK12/13 proteolysis-targeting chimeras (PROTACs) in mCRPC cells.^{13, 14}

Multiple approaches are being explored to selectively suppress CDK12/13, including kinase function inhibition (e.g., 1-5),^{7, 15–19} PROTAC degradation (e.g., 6-8) of CDK12/13,^{14, 20, 21} and molecular glues degrading the partner protein CCNK (e.g., 9-13)^22–26 (Figure 1). Most recently, Carrick Therapeutics initiated a phase 1 clinical trial for CT7439, a novel CCNK degrader.²⁷ Additionally, a derivative of YJ1206, the 1^st reported orally bioavailable CDK12/13 degrader, was nominated as a potential clinical candidate for further development in our team.¹⁴ Although several reversible or irreversible CDK12/13 inhibitors have been reported,^{7, 15–19} none have progressed into clinical trials, likely due to the challenges in balancing antiproliferative activity, target selectivity, pharmacokinetic (PK) profiles and/or safety concerns. Moreover, there is limited in vivo efficacy data for these inhibitors.^{7, 19}

Figure 1. — Representative compounds suppressing/degrading CDK12/13 kinases.

SR-4835 (10) is the 1^st selective CDK12/13 inhibitor to demonstrate in vivo therapeutic efficacy in TNBC models; however, subsequent studies suggest that its activity may be attributed to CCNK degradation.^{23, 24} Interestingly, reversible inhibitors (e.g. 1, 2) generally exhibit significantly weaker antiproliferative activity compared to covalent inhibitors (e.g. 3-5), despite their comparable kinase suppressive potency. This observation highlights the potential advantage of irreversible CDK12/13 inhibitors, such as prolonged target engagement and enhanced ATP-competitive binding within cells.^{28, 29} Additionally, the distinct cysteine residues near the ATP-binding pockets of CDK12 (Cys1039) and CDK13 (Cys1017),¹⁷ present an opportunity to improve target specificity through covalently targeting. Notably, most current covalent CDK12/13 inhibitors are structurally derived from the CDK7 covalent inhibitor THZ1, which targets a similar cysteine residue in the structure (Cys312).^{17, 30} Developing new irreversible inhibitors with alternative chemical scaffolds is highly desirable for further validating selective CDK12/13 inhibition as a viable cancer therapeutic strategy.

Herein, we report the discovery of YJZ5118, a new, potent, and highly selective covalent inhibitor of CDK12/13, by optimization of a structurally distinct reversible inhibitor 2. YJZ5118 effectively inhibited CDK12 and CDK13 with low nanomolar IC₅₀ values, demonstrating improved target specificity. The covalent binding mode of YJZ5118 was extensively validated through mass spectrometry analysis, co-crystal structure determination, and pulldown-proteomic experiments. Furthermore, the compound exhibited reasonable PK parameters and demonstrated promising in vivo efficacy in a VCaP CRPC xenograft mouse model when combined with Akt inhibitors.

RESULTS AND DISCUSSION

Molecule Design.

Compound 2 is a 3-benzyl-1-(trans-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)-1-arylurea-based CDK12/13 inhibitor with strong enzymatic inhibitory activity and excellent kinase selectivity.¹⁶ It effectively inhibited the kinase activities of CDK12 and CDK13 with IC₅₀ values of 28.6 and 17.8 nM, respectively, in the independent ADP-Glo validation assays conducted by ICE Bioscience Inc. (Figure 2). A CDK family panel screening further validated its specificity for CDK12 and CDK13, although its selectivity over CDK7 and CDK9 was moderate, with fold-difference ranging from 18- to 40-fold (Figure 3B). The X-ray co-crystal structure of an analogue of compound 2 with CDK12/CCNK revealed detailed structural interactions (PDB: 6CKX).¹⁶ Combining this structural information with data from the covalent inhibitor THZ531 (3) (PDB: 5ACB), a computational study suggested that the aminopyridine moiety of compound 2 forms two hydrogen bonds with hinge residue Met816, while the central phenyl ring is positioned 6.6 Å from Cys1039 of CDK12. Additionally, the hydrophilic 1-methylpyridin-2(1H)-one group is oriented toward the solvent accessible surface. Although Cys1039 is relatively distant from the reversible inhibitor 2, the conformational flexibility of the corresponding loop suggested the possibility of introducing an acrylamide warhead at the central phenyl ring to covalently target Cys1039 of CDK12, achieving irreversible inhibition against the kinase (14a). We further hypothesized that the solvent exposing 1-methylpyridin-2(1H)-one moiety could be diversified with basic aliphatic amines to facilitate the deprotonation of Cys1039, thereby promoting the covalent bond formation. Incorporating hydrophilic aliphatic amines was also expected to improve aqueous solubility and PK profiles (Figure 2).

Figure 2. — Structure-based design of new irreversible CDK12/13 inhibitors 11. Chemical structures and predicted binding modes of compounds 2 and **14a** with CDK12 (PDB: 5ACB) were shown. Hydrogen bonds were indicated by yellow dashed lines.

Figure 3. — (A) PK profiles of compound **YJZ5118** in mice; (B) Kinase inhibitory IC₅₀ values of compounds 2 and **YJZ5118** against CDK family members.

Chemistry.

Synthesis of compound 14a and the derivatives was illustrated in Scheme 1. Briefly, The commercially available 1-bromo-4-iodo-2-nitrobenzene underwent Buchwald-Hartwig reaction with tert-butyl ((1R,4R)-4-aminocyclohexyl)carbamate to produce intermediate 17, which reacted with benzyl isocyanate 18 to afford urea 19. Removal of Boc group allowed compound 19 to react with 6-fluoronicotinonitrile 20 to yield key intermediate 21. Compound 21 reacted with 1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one under Suzuki coupling conditions to generate compound 22a. Subsequent reduction of the nitro group of compound 22a by Fe powder in acidic conditions, followed by coupling with acryloyl chloride yielded the first designed molecule 14a. Alternatively, hydrogenation of 21 under catalysis of Pd/C yielded the debrominated aniline which reacted with acryloyl chloride to yield analogue 14b. Compound 21 could also be converted to a series of analogs 22c-l by a Buchwald-Hartwig reaction with a series of secondary amine derivatives, which were readily converted to analogues 14c-l under similar procedure to that of 14a and 14b. A biotinylated probe YJZ9149 was also synthesized by coupling compound 14m and 17-oxo-21-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-4,7,10,13-tetraoxa-16-azahenicosanoic acid.

Discovery of YJZ5118 (14k) as a Potent and Highly Selective Irreversible CDK12/13 Inhibitor.

The kinase inhibitory activities of the compounds against CDK12 and CDK13 were preliminarily evaluated using the ADP-Glo kinase assay. Cell growth inhibition was determined in VCaP prostate cancer cells, which were characterized as being sensitive to CDK12/13 inhibition.¹⁴ Previously reported reversible CDK12/13 inhibitor 2 and the irreversible inhibitor 3 were included as the positive reference compounds and exhibited similar kinase inhibitory potencies to their reported data.^{16, 17} Compound 14a, the initially designed molecule, showed strong CDK12/13 kinase inhibitory activities with IC₅₀ values of 37.9 and 14.9 nM, respectively, comparable to the lead molecule 2 (Table 1). However, 14a demonstrated approximately 6-fold higher antiproliferative potency in VCaP cells compared to the lead molecule 2, with an IC₅₀ value of 330.4 nM. These data suggested that 14a may covalently target the kinases, extending target residence time and improving antiproliferative activity.

Table 1.

In Vitro CDK12 and CDK13 Kinase Inhibitory Activity and Cellular Antiproliferative Potency of Compounds 14a–k.



Cpd.	R¹	IC₅₀ (nM)
Cpd.	R¹	CDK12^a	CDK13^a	VCaP

3	-	77.4 (158^b)	63.3 (69^b)	33.5
2	-	28.6 (52^c)	17.8 (10^c)	1950
14a		37.9	14.9	330.4
14b	H	506.0	99.0	96.2
14c		190.6	79.3	44.4
14d		151.3	73.7	133.3
14e		567.5	176.8	57.6
14f		130.3	66.8	39.9
14g		209.1	83.9	76.3
14h		33.1	21.7	4.3
14i		331.3	164	45.5
14j		55.0	12.8	59.9
14k (YJZ5118)		39.5	26.4	23.7

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CDK12/13 inhibition was performed by using an ADP-Glo kinase assay. The data are means from three independent experiments.

The reported IC₅₀ values for CDK12/13 determined by using a radioactive kinase activity assay.¹⁷

The reported IC₅₀ values for CDK12/13 determined by using a LANCE Ultra assay.¹⁶

The methylpyridone group in 2 was originally introduced to improve aqueous solubility;¹⁶ however removal of this moiety in 14a resulted in a 7- to 13-fold reduction in kinase inhibitory potency (14b), with IC₅₀ values of 506 and 99 nM for CDK12 and CDK13, respectively. Interestingly, 14b exhibited 3.4-fold more potency than 14a in the growth inhibition assay against VCaP cells, likely due to its reduced hydrophilicity facilitating better cellular membrane penetration (Supporting Information Table S4).

It has been well documented that introducing an aliphatic amine adjacent to the acrylamide warhead could induce a localized basic environment to benefit the covalent bond formation by facilitating deprotonation of the sulfhydryl moiety of a cysteine.^{31, 32} Hydrophilicity of an aliphatic amine could also improve the PK properties of a lead compound by enhancing aqueous solubility. Thus, a hydrophilic N-methyl-N-(N’,N’-dimethyl)ethylamino group was first introduced and the resulting molecule 14c partially restored CDK12/13 kinase inhibitory activities with IC₅₀ values of 190.6 and 79.3 nM, respectively. Compound 14c also exhibited significant antiproliferative activity, with an IC₅₀ value of 44.4 nM, approximately 44-fold more potent than the lead compound 2.

Replacement of the hydrophilic group in 14c with other cyclic aliphatic amines (14d-g, 14j) generally maintained kinase inhibitory potency and antiproliferative activity. However, compound 14i, in which the aliphatic amine was neutralized by amide formation, exhibited 2- to 3-fold weaker potency against the kinases compared to its counterpart molecule 14f. When a (S)-3,4-dimethylpiperazin-1-yl group was introduced, the resulting compound 14h exhibited the strongest kinase inhibition and cell growth suppression in the series, with IC₅₀ values of 33.1 nM, 21.7 nM, and 4.3 nM for CDK12, CDK13, and VCaP cells, respectively. KinomeScan profiling study further supported the target specificity of 14h (Supporting Information Figure S1 and Table S1).

Despite its potency, compound 14h exhibited unfavorable in vivo PK profile, with a short half-life (T_1/2), poor oral exposure, and a high clearance rate (Supporting Information Table S3). To address these issues, compound 14k (YJZ5118), incorporating a 4-(dimethylamino)piperidin-1-yl group, was developed. YJZ5118 demonstrated similar CDK12/13 kinase inhibitory activities to 14h, with IC₅₀ values of 39.5 and 26.4 nM, respectively. The compound also potently suppressed VCaP cell growth with an IC₅₀ value of 23.7 nM. Although the antiproliferative activity of YJZ5118 was approximate 5.5-fold less potent than 14h, this compound exhibited markedly improved PK properties in mice, with an AUC of 2235.5 h_*ng/mL and T_1/2 of 2.32 h upon 10 mg/kg oral administration (Figure 3A). The oral bioavailability of YJZ5118 was 35.5%, and clearance rate of YJZ5118 was 4-time lower than that of 14h with a value of 28.34 mL/min/kg at 2 mg/kg i.v. dosing. During revising this manuscript, Insilico Medicine reported a new irreversible CDK12/13 inhibitor with oral bioavailability and in vivo efficacy³³. However, YJZ5118 exhibited a better PK profile.

Target specificity of YJZ5118 was assessed against the CDK family, showing over 100-fold selectivity for CDK12/13 over other CDK family members, with the exception of CDK7 (Figure 3B). YJZ5118 exhibited moderate inhibition against CDK7, with an IC₅₀ value of 2263 nM, which was 57- and 86- fold higher than the values against CDK12 and CDK13, respectively. In contrast, the reversible lead molecule 2 exhibited significant inhibition of CDK7 and CDK9, with IC₅₀ values of 678.6 and 514.8 nM, respectively. In conclusion, YJZ5118 represented a new covalent CDK12/13 inhibitor with enhanced target specificity, cytotoxic effect and reasonable PK profiles, making it a promising candidate for further investigation.

Validation of the Covalent Binding Mode of YJZ5118.

To confirm that YJZ5118 binds to its target protein in a covalent manner, we incubated the CDK12/CCNK complex with a 5-fold molar excess of the compound for 2 h at room temperature, followed by a liquid chromatograph-mass spectrometer (LC-MS) analysis. A mass shift consistent with the addition of a single molecule of YJZ5118 to CDK12 was observed (Figure 4A). The covalent binding mode was further characterized by the co-crystal structure of YJZ5118 with CDK12/CCNK complex at 2.5 Å resolution. The structure revealed that, in addition to two hydrogen bonds formed between the 6-aminonicotinonitrile moiety of YJZ5118 with the hinge residue Met816 and one hydrogen bond formed between the urea group and Asp819, the acrylamide group formed a covalent bond with Cys1039 (Figure 4B). To further confirm covalent bond formation in cell lysates, we synthesized a biotinylated analog of YJZ5118 (YJZ9149) to pull down covalently bound targets by streptavidin. As shown in Figure 4C, CDK12 and CDK13 were most enriched proteins in precipitate.

Figure 4. — (A) Mass spectral peaks corresponding to CDK12 and CDK12 covalently labeled with compound **YJZ5118**; (B) Co-crystal structure of CDK12/CCNK and compound **YJZ5118** (PDB: 9JK1). Compound **YJZ5118** is shown in cyan stick structure. The key residues of CDK12 kinase are shown in gray sticks. Hydrogen bonds to key amino acids are indicated by yellow dashed lines; (C) Peptide to spectrum matches (PSM) proteomic analysis of pull-down experiments in whole cell lysate, and the structure of compound **YJZ9149** (biotinylated compound of **YJZ5118**); (D, E) Washout experiments using compound **YJZ5118** and reversible inhibitors 10 and 2 in VCaP cells.

We next investigated the irreversible inhibition of YJZ5118 on cell growth. The previously reported reversible inhibitor 10 was included for comparison. VCaP cells were treated with the compounds for 6 h, after which the compounds were removed, and the cells were cultured in compound-free media for 5 days. As shown in Figure 4D, while cell growth inhibitory effects of compound 10 was nearly completely lost following washout, YJZ5118 maintained potent antiproliferative activity in VCaP cells even after washout (Figure 4D). Furthermore, YJZ5118 inhibited the phosphorylation of RNA polymerase II at Ser2 for an extended period at a low concentration (100 nM), with complete loss of phosphorylation observed at 15 h after washout (Figure 4E). In contrast, the reversible counterpart 2 inhibited the RNA polymerase II Ser2 phosphorylation for a limited time at a higher concentration (10 μM), with the recovery of phosphorylation at 4 h after washout (Figure 4E). Furthermore, YJZ5118 also maintained significant inhibitory effects on the expression of downstream gene RAD51 and markedly increased apoptotic marker c-PARP after pretreatment with YJZ5118 for 2 h and then washout, but similar results were not observed upon the treatment of reversible inhibitor 2 (Supporting Information Figure S3A). These results collectively support that YJZ5118 is an irreversible CDK12/13 kinase inhibitor.

YJZ5118 Suppresses Transcription of DDR genes, and Provokes DNA Damage and Apoptosis in Multiple Cancer Cell Lines.

YJZ5118 was demonstrated to inhibit phosphorylation of RNA polymerase II Ser2 in VCaP cells in a dose- and time-dependent manner (Figure 5A). Previous studies have reported that CDK12 inhibition leads to gene length-dependent elongation defects, causing premature cleavage and polyadenylation (PCPA) and the loss of expression of long genes, a substantial proportion of which are involved in the DDR.¹³ We performed RNA sequencing (RNA-seq) in VCaP cells treated with YJZ5118 for 6 h. The results showed that a significant correlation between gene length and gene expression: longer genes were more likely to be downregulated (Figure 5B).

To assess if inhibition of CDK12/13 alters the expression of DDR genes, we performed quantitative PCR (qPCR) in cells treated with YJZ5118. We found treatment of VCaP cells with YJZ5118 reduced the expression of a cast of DDR genes including Ataxia Telangiectasia Mutated (ATM), Ataxia Telangiectasia and Rad3 Related (ATR), RAD51, and Fanconi Anemia Complementation Group I (FANC1), as early as 4 h post-treatment (Figure 5C). Notably, the expression of other key cancer-related genes, such as NRAS and Enhancer of Zeste Homolog 2 (EZH2), remained unaffected by YJZ5118.

To further investigate the DNA damage upon YJZ5118 treatment, we performed neutral comet assays in VCaP cells and found a significant increase in neutral comet tails compared to DMSO (Figure 5D). Flow cytometry analysis showed that YJZ5118 induced apoptosis in VCaP cell at the concentration of 100 nM, with an apoptosis rate of 28.5% (Figure 5E). Western blot analysis also confirmed that YJZ5118 significantly increased cleaved PARP at a concentration of 100 nM (Figure 5A).

Next, we expanded the cell viability screening to a panel of normal and cancer cell lines from 8 different lineages. The results showed that several prostate cancer cells (i.e., VCaP and DU145), breast cancer cells (i.e., SK-BR-3, MFM223 and MDA-MD-468) and Ewing’s sarcoma cells (i.e., CHLA10 and CB-AGPN), were preferentially sensitive to YJZ5118, with IC₅₀ values of less than 40 nM. In contrast, normal and non-neoplastic cells exhibited much lower sensitive to the compound (Figure 5F).

YJZ5118 Combined with Akt Inhibitors Shows a Synergistic Effect In Vitro and In Vivo.

Recently, our group reported that the CDK12/13 degrader YJ1206 induced Akt phosphorylation and exhibited a synergistic effect with Akt inhibitors.¹⁴ To determine if blocking CDK12/13 kinase activity could also achieve a similar effect, we treated VCaP cells with YJZ5118 for 24 h at different concentrations. As shown in Figure 6A, YJZ5118 treatment increased phosphorylation of Akt and its direct substrate PRAS40 in a dose-dependent manner, while the total Akt levels remained unchanged. Additionally, YJZ5118 treatment triggered DNA damage and apoptosis, as indicated by increased levels of γH2AX and cleaved-PARP (Figure 6A).

Figure 6. — (A) Immunoblots of the indicated proteins at selected concentrations in VCaP cells after treated with compound **YJZ5118** for 24 h. α-Tubulin is used as a loading control; (B) 22RV1 cells were treated with compounds **YJZ5118** and/or **MK2206** at varied concentrations to determine the effect on cell growth and drug synergism, with assessments using the Loewe method. Red peaks in the 3D plots denote synergy with the average synergy scores noted above; (C) Real-time growth curves of 22RV1 cells upon treatment with compound **YJZ5118** and/or Akt inhibitors. Data are presented as mean +/− SD (n = 3) from one of three independent experiments.

To investigate potential synergistic effects between YJZ5118 and Akt inhibitors, we combined YJZ5118 with MK2206, an Akt inhibitor, in 22RV1 cells, which exhibit moderate sensitivity to CDK12/13 inhibition (Figure 5F). The results revealed a significant synergistic effect between YJZ5118 and MK2206, with a synergy score of 10.81 (Figure 6B). IncuCyte assays further demonstrated enhanced efficacy of the combinatorial treatment in 22RV1 cells (Figure 6C). Similar synergistic effects were also observed when YJZ5118 was combined with other Akt inhibitors, including Uprosertib, Ipatasertib, and Afuresertib in 22RV1 cells (Figure 6C).

We further evaluated the in vivo anti-tumor activity of YJZ5118 using a VCaP CRPC mouse model. Compared to the vehicle control, mice treated with YJZ5118 or Uprosertib showed a significant reduction in tumor growth (Figures 7A, B). Encouragingly, the combination treatment of YJZ5118 and Uprosertib markedly suppressed tumor growth, with no significant changes in animal body weights observed (Figure 7C). Endpoint analyses showed that YJZ5118 effectively inhibited the phosphorylation of RNA polymerase II at Serine 2 and significantly increased pAkt (S473) and pPRAS40 levels in tumors compared to the vehicle group (Figure 7E). Additionally, YJZ5118 treatment reduced the expression of DDR genes in tumors, whereas the expression of the CDK12 gene remained unaffected (Figure 7D).

Histological analysis of the tumors further confirmed that inhibition of CDK12/13 kinase activity led to increased pAkt levels (Figure 7F). Moreover, enhanced apoptosis was evident in the H&E-stained tumor sections (Figure 7F). These results demonstrate that YJZ5118 not only provokes Akt phosphorylation but also exhibits a synergistic anti-tumor effect when combined with Akt inhibitors. While both CDK12/13 inhibitor YJZ5118 and CDK12/13 degrader YJ1206 exhibited a similar synergistic anti-tumor effect with Akt inhibition, they bear different mechanisms of action, e.g. prolonged target engagement of covalent CDK12/13 inhibitors and catalytic event-driven pharmacology of CDK12/13 degraders, which could result in distinct profiles with regard to potency, selectivity, PK properties and toxicity.

CONCLUSIONS

In summary, starting from the reversible inhibitor 2, YJZ5118 was developed as a novel covalent inhibitor of CDK12/13 through structure-based optimization. YJZ5118 showed significantly enhanced antiproliferative activity and improved target specificity. The covalent binding mode with CDK12/13 was comprehensively validated through mass spectrometry analysis, co-crystal structure studies, and pulldown-proteomic experiments. YJZ5118 efficiently inhibited RNA polymerase II Ser2 phosphorylation, suppressed transcription of DNA damage response genes, and induced DNA damage and apoptosis. Furthermore, YJZ5118 significantly inhibited the proliferation of multiple tumor cell lines, while normal and non-neoplastic cells were less sensitive. Notably, YJZ5118 was shown to induce Akt phosphorylation via CDK12/13 inhibition. Synergistic anti-tumor effects were observed when YJZ5118 was combined with Akt inhibitors, both in vitro and in vivo, highlighting its therapeutic potential.

EXPERIMENTAL SECTION

General Methods for Chemistry.

All commercially available reagents and solvents were used without further purification. All chemical reactions were monitored by thin-layer chromatography (TLC) plates with visualization under UV light (254 or 365 nm). ¹H NMR spectra were performed with Bruker AV-400/600 spectrometer, ¹³C NMR spectra were recorded on Bruker AV-600 spectrometer at 150 MHz, internal reference was either TMS or deuterated NMR solvent. Low-resolution mass spectra (MS) were recorded on an Agilent 1200 HPLC-MSD mass spectrometer. High resolution mass spectral analysis was recorded on an Applied Biosystems Q-STAR Elite ESI-LC-MS/MS mass spectrometer. Purity of all final compounds was confirmed to be >95% by HPLC analysis with the Agilent 1260 system. The analytical columns were YMC-Triart C18 reversed-phase column, 5 μm, 4.6 mm × 250 mm, and flow rate 1.0 mL/min.

tert-butyl ((1r,4r)-4-((4-bromo-3-nitrophenyl)amino)cyclohexyl)carbamate (14).

To a solution of 1-bromo-4-iodo-2-nitrobenzene 12 (25.0 g, 76.2 mmol) in toluene (400 mL) were added tert-butyl ((1r,4r)-4-aminocyclohexyl)carbamate 13 (16.3 g, 76.2 mmol), Cs₂CO₃ (29.8 g, 91.5 mmol), Pd₂(dba)₃ (1.74 g, 1.9 mmol) and Xantphos (2.2 g, 3.8 mmol). The mixture was evacuated and backfilled with argon (3 cycles). The reaction mixture was then heated at 100 °C for 12 h before being filtered through celite. The reaction solvent was evaporated under reduced pressure and purified by silica gel column chromatography to afford the title compound as yellow solid (26.0 g, yield 82%): ¹H NMR (400 MHz, DMSO-d₆) δ 7.43 (d, J = 8.8 Hz, 1H), 7.09 (d, J = 2.2 Hz, 1H), 6.80 (d, J = 7.7 Hz, 1H), 6.75 (d, J = 8.8 Hz, 1H), 6.33 (d, J = 7.9 Hz, 1H), 3.28–3.09 (m, 2H), 1.93 (d, J = 11.1 Hz, 2H), 1.78 (d, J = 12.3 Hz, 2H), 1.38 (s, 9H), 1.28 (q, J = 12.5, 12.1 Hz, 2H), 1.16 (q, J = 12.0 Hz, 2H). MS (ESI), m/z: 412.0 [M-H]⁻.

tert-butyl ((1r,4r)-4-(3-benzyl-1-(4-bromo-3-nitrophenyl)ureido)cyclohexyl)carbamate (16).

To a solution of tert-butyl ((1r,4r)-4-((4-bromo-3-nitrophenyl)amino)cyclohexyl)carbamate 14 (25 g, 60.3 mmol) and DIPEA (1.59 g, 12.3 mmol) in DMF (10 mL) was added benzyl isocyanate 15 (24 g, 181.0 mmol) at room temperature. The mixture was stirred at 95 °C for 4 h. The solvent was removed under reduced pressure and purified by column chromatography to give yellow solid (26 g, yield 79%): ¹H NMR (400 MHz, DMSO-d₆) δ 7.94 (d, J = 8.5 Hz, 1H), 7.84 (s, 1H), 7.40 (d, J = 8.5, 1H), 7.32–7.22 (m, 3H), 7.21–7.14 (m, 2H), 6.68 (d, J = 8.0 Hz, 1H), 6.43 (t, J = 6.3 Hz, 1H), 4.21–4.09 (m, 3H), 3.00 (s, 1H), 1.81–1.68 (d, J = 11.3 Hz, 4H), 1.35 (s, 9H), 1.28–1.19 (m, 2H), 1.13–1.04 (m, 2H). MS (ESI), m/z: 569.1 [M+Na]⁺.

3-benzyl-1-(4-bromo-3-nitrophenyl)-1-((1r,4r)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)urea (18).

TFA (10 mL) was added to a solution of tert-butyl ((1r,4r)-4-(3-benzyl-1-(4-bromo-3-nitrophenyl)ureido)cyclohexyl)carbamate 16 (22.8 g, 41.6 mmol) in DCM (20 mL) and the mixture was refluxed at 50 °C for 3 h. After that the reaction mixture was concentrated to dryness under reduced pressure, it was then extracted with EtOAc (3x) and washed with H₂O. The combined EtOAc layers were subsequently dried over MgSO₄, and the solvent was removed under reduced pressure. The obtained crude material was used for next step without further purification. To a solution of the above deprotected crude product (14.4 g, 32.2 mmol) in DMF (30 mL) were added 5-cyano-2-fluoropyridine 17 (3.9 g, 32.2 mmol), and Cs₂CO₃ (12.6 g, 38.6 mmol). The mixture was stirred at room temperature for 10 minutes. Then the reaction mixture was heated at 60 °C for another 60 minutes. The reaction mixture was then filtered and the solvent was removed under reduced pressure. The crude material was purified by column chromatography to afford 18 as yellow solid (15.4 g, yield 80%): ¹H NMR (400 MHz, DMSO-d₆) δ 8.30 (d, J = 2.4 Hz, 1H), 7.95 (d, J = 8.4 Hz, 1H), 7.87 (d, J = 2.4 Hz, 1H), 7.61 (d, J = 8.9 Hz, 1H), 7.48 (d, J = 7.6 Hz, 1H), 7.42 (dd, J = 8.4, 2.5 Hz, 1H), 7.33–7.24 (m, 2H), 7.23–7.15 (m, 3H), 6.52–6.40 (m, 2H), 4.27 (t, J = 12.3 Hz, 1H), 4.17 (d, J = 5.9 Hz, 2H), 3.56 (s, 1H), 1.93 (d, J = 12.2 Hz, 2H), 1.83 (d, J = 12.0 Hz, 2H), 1.32 (q, J = 12.4 Hz, 2H), 1.24–1.09 (m, 2H). MS (ESI), m/z: 549.1 [M+H]⁺.

3-benzyl-1-((1r,4r)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)-1-(4-(1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-3-nitrophenyl)urea (19a).

A mixture of compound 18 (300 mg, 0.5 mmol), K₂CO₃ (69 mg, 1.0 mmol), Pd(PPh₃)₄ (57.8 mg, 0.05 mmol), and 1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one (141 mg, 0.6 mmol) in 1,4-dioxane/H₂O (10:1, 60 mL) was evacuated and backfilled with argon. After stirring at 100 °C for 10 h, the solvent was removed under vacuum, and the resultant crude residue redissolved in EtOAc (20 mL), which was washed with H₂O (60 mL). The layers was separated and the aqueous phase was extracted with EtOAc (2 × 20 mL). The combined EtOAc layers were subsequently dried over MgSO₄, and the solvent was removed under reduced pressure. The crude material was purified by silica column chromatography to afford the title compound as white solid (150 mg, yield 52%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.30 (d, J = 2.3 Hz, 1H), 7.95 (d, J = 2.7 Hz, 1H), 7.82 (d, J = 2.0 Hz, 1H), 7.65 – 7.56 (m, 3H), 7.51 (d, J = 7.6 Hz, 1H), 7.39 – 7.35 (m, 1H), 7.32 – 7.26(m, 2H), 7.24 – 7.17 (m, 3H), 6.47 (t, J = 8.8 Hz, 2H), 6.41 (t, J = 6.0 Hz, 1H), 4.29 (t, J = 11.9 Hz, 1H), 4.20 (d, J = 5.9 Hz, 2H), 3.58 – 3.51 (m, 1H) 3.50 (s, 3H), 1.91 (dd, J = 23.5, 12.3 Hz, 4H), 1.35 (q, J = 11.4 Hz, 2H), 1.18 (q, J = 11.4 Hz, 2H). MS (ESI), m/z: 575.6 [M-H]⁻.

3-benzyl-1-((1r,4r)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)-1-(4-((2-(dimethylamino)ethyl)(methyl)amino)-3-nitrophenyl)urea (19c).

A mixture of 3-benzyl-1-(4-bromo-3-nitrophenyl)-1-((1r,4r)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)urea 18 (200 mg, 0.36 mmol), N¹,N¹,N²-trimethylethane-1,2-diamine (44.1 mg, 0.43 mmol), Cs₂CO₃ (237.4 mg·0.72 mmol), Pd₂(dba)₃ (36.6 mg·0.04 mmol) and Xantphos (41.6 mg, 0.07 mmol) in 20 mL toluene : DMF (10 : 1) was evacuated and backfilled with argon (3 cycles) and then heated at 100 °C for 12 h. The mixture was filtered through Celite after cooling, concentrated in vacuo, and purified by column chromatography to afford the title compound as yellow solid (110 mg, yield 50%): ¹H NMR (400 MHz, DMSO-d₆) δ 8.30 (s, 1H), 7.61 (d, J = 8.9 Hz, 1H), 7.54–7.44 (d, J = 11.3 Hz, 2H), 7.32–7.23 (m, 4H), 7.21–7.13 (d, J = 7.4 Hz, 3H), 6.48 (d, J = 8.9 Hz, 1H), 6.19 (t, J = 6.1 Hz, 1H), 4.25 (t, J = 12.6 Hz, 1H), 4.16 (d, J = 5.9 Hz, 2H), 3.51 (s, 1H), 3.28 (t, J = 7.0 Hz, 3H), 3.18 (s, 1H), 2.81 (s, 3H), 2.47 (t, J = 6.8 Hz, 2H), 2.15 (s, 6H), 1.92 (d, J = 11.3 Hz, 2H), 1.80 (d, J = 12.4 Hz, 2H), 1.31 (q, J = 13.2, 12.5 Hz, 2H), 1.12 (q, J = 12.9 Hz, 2H). MS (ESI), m/z: 569.2 [M-H]⁻.

3-benzyl-1-((1r,4r)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)-1-(4-(3-(dimethylamino)azetidin-1-yl)-3-nitrophenyl)urea (19d).

Compound 19d was synthesized from commercially available N,N-dimethylazetidin-3-amine and 18 by following a similar procedure as that of 19c (yield, 40%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.30 (s, 1H), 7.61 (d, J = 9.0 Hz, 1H), 7.56 (s, 1H), 7.48 (d, J = 7.5 Hz, 1H), 7.27 (t, J = 7.8 Hz, 3H), 7.21–7.13 (m, 3H), 6.82 (d, J = 8.9 Hz, 1H), 6.47 (d, J = 8.9 Hz, 1H), 6.18 (t, J = 5.8 Hz, 1H), 4.26 (t, J = 11.9 Hz, 1H), 4.15 (d, J = 5.9 Hz, 2H), 4.00 (t, J = 8.2 Hz, 2H), 3.78–3.66 (m, 2H), 3.51 (s, 1H), 3.12 (p, J = 6.3 Hz, 1H), 2.11 (s, 6H), 1.92 (d, J = 12.3 Hz, 2H), 1.80 (d, J = 12.0 Hz, 2H), 1.31 (q, J =13.9 Hz, 2H), 1.11 (q, J = 12.6 Hz, 2H). MS (ESI), m/z: 569.3 [M+H]⁺.

3-benzyl-1-((1r,4r)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)-1-(4-(3-(dimethylamino)pyrrolidin-1-yl)-3-nitrophenyl)urea (19e).

Compound 19e was synthesized from commercially available N,N-dimethylpyrrolidin-3-amine and 18 by following a similar procedure as that of 19c (yield, 41%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.29 (s, 1H), 7.61 (d, J = 9.0 Hz, 1H), 7.51 (d, J = 2.4 Hz, 1H), 7.48 (d, J = 7.4 Hz, 1H), 7.31–7.22 (m, 3H), 7.21–7.14 (m, 3H), 7.11 (d, J = 8.9 Hz, 1H), 6.47 (d, J = 8.9 Hz, 1H), 6.15 (t, J = 6.0 Hz, 1H), 4.31–4.22 (m, 1H), 4.21–4.08 (m, 2H), 3.51 (s, 1H), 3.43–3.38 (m, 1H), 3.17 (q, J = 8.4, 7.4 Hz, 3H), 2.75 (t, J = 7.8 Hz, 1H), 2.25–2.11 (s, 7H), 1.92 (d, J = 11.9 Hz, 2H), 1.85–1.71 (m, 3H), 1.30 (q, J = 12.4 Hz, 2H), 1.18–1.02 (m, 2H). MS (ESI), m/z: 583.3 [M-H]⁺.

3-benzyl-1-((1r,4r)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)-1-(4-(4-methylpiperazin-1-yl)-3-nitrophenyl)urea (19f).

Compound 19f was synthesized from commercially available 1-methylpiperazine and 18 by following a similar procedure as that of 19c (yield, 58%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.30 (s, 1H), 7.65–7.55 (m, 2H), 7.48 (d, J = 8.4 Hz, 1H), 7.39–7.34 (m, 1H), 7.29 (dd, J = 15.5, 8.0 Hz, 3H), 7.18 (d, J = 7.3 Hz, 3H), 6.48 (d, J = 8.9 Hz, 1H), 6.24 (t, J = 6.2 Hz, 1H), 4.28 (t, J = 10.9 Hz, 1H), 4.17 (d, J = 5.9 Hz, 2H), 3.52 (s, 1H), 3.05 (t, J = 4.5 Hz, 4H), 2.44 (t, J = 4.5 Hz, 4H), 2.23 (s, 3H), 1.93 (d, J = 11.2 Hz, 2H), 1.81 (d, J = 12.1 Hz, 2H), 1.32 (q, J = 12.3, 11.9 Hz, 2H), 1.12 (q, J = 10.1, 8.0 Hz, 2H). MS (ESI), m/z: 567.2 [M-H]⁻.

3-benzyl-1-((1r,4R)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)-1-(4-((1R,4R)-5-methyl-2,5-diazabicyclo[2.2.1]heptan-2-yl)-3-nitrophenyl)urea (19g).

Compound 19g was synthesized from commercially available (1R,4R)-2-methyl-2,5-diazabicyclo[2.2.1]heptane and 18 by following a similar procedure as that of 19c (yield, 40%). MS (ESI), m/z: 581.3 [M+H]⁺.

3-benzyl-1-((1r,4S)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)-1-(4-((S)-3,4-dimethylpiperazin-1-yl)-3-nitrophenyl)urea (19h).

Compound 19h was synthesized from commercially available (S)-1,2-dimethylpiperazine and 18 by following a similar procedure as that of 19h (yield, 50%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.30 (s, 1H), 7.64–7.57 (m, 2H), 7.48 (d, J = 7.6 Hz, 1H), 7.38–7.25 (m, 4H), 7.22–7.14 (m, 3H), 6.48 (d, J = 8.8 Hz, 1H), 6.23 (t, J = 5.8 Hz, 1H), 4.32–4.22 (m, 1H), 4.16 (d, J = 5.9 Hz, 2H), 3.52 (s, 1H), 3.15–3.04 (m, 2H), 2.99 (t, J = 11.1 Hz, 1H), 2.78 (d, J = 12.2 Hz, 1H), 2.65 (t, J = 10.7 Hz, 1H), 2.31–2.13 (m, 5H), 1.92 (d, J = 12.1 Hz, 2H), 1.81 (d, J = 12.4 Hz, 2H), 1.32 (q, J = 12.1 Hz, 2H), 1.11 (q, J = 12.5 Hz, 2H), 1.01 (d, J = 6.3 Hz, 3H). MS (ESI), m/z: 583.3 [M+H]⁺.

1-(4-(4-acetylpiperazin-1-yl)-3-nitrophenyl)-3-benzyl-1-((1r,4r)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)urea (19i).

Compound 19i was synthesized from commercially available 1-(piperazin-1-yl)ethan-1-one and 18 by following a similar procedure as that of 19c (yield, 64%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.30 (s, 1H), 7.63 (s, 1H), 7.61 (d, J = 8.7 Hz, 1H), 7.50 (d, J = 7.7 Hz, 1H), 7.43–7.37 (m, 1H), 7.37–7.32 (m, 1H), 7.28 (t, J = 7.6 Hz, 2H), 7.22–7.15 (m, 3H), 6.48 (d, J = 8.9 Hz, 1H), 6.26 (t, J = 6.0 Hz, 1H), 4.26 (t, J = 12.0 Hz, 1H), 4.16 (d, J = 5.9 Hz, 2H), 3.57 (s, 4H), 3.45 (s, 1H), 3.07 (d, J = 20.0 Hz, 4H), 2.04 (s, 3H), 1.92 (d, J = 12.2 Hz, 2H), 1.82 (d, J = 12.1 Hz, 2H), 1.32 (q, J = 12.3 Hz, 2H), 1.12 (q, J = 11.0, 9.9 Hz, 2H). MS (ESI), m/z: 596.9 [M+H]⁺.

3-benzyl-1-((1r,4r)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)-1-(4-morpholino-3-nitrophenyl)urea (19j).

Compound 19j was synthesized from commercially available morpholine and 18 by following a similar procedure as that of 19c (yield, 51%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.30 (s, 1H), 7.67–7.55 (m, 2H), 7.50 (d, J = 7.6 Hz, 1H), 7.43–7.37 (m, 1H), 7.36–7.31 (m, 1H), 7.28 (t, J = 7.5 Hz, 2H), 7.21–7.15 (d, J = 7.5 Hz, 3H), 6.48 (d, J = 8.9 Hz, 1H), 6.26 (t, J = 6.0 Hz, 1H), 4.27 (t, J = 12.2 Hz, 1H), 4.16 (d, J = 5.9 Hz, 2H), 3.71 (t, J = 4.1 Hz, 4H), 3.52 (s, 1H), 3.05 (t, J = 4.5 Hz, 4H), 1.92 (d, J = 12.2 Hz, 2H), 1.82 (d, J = 12.2 Hz, 2H), 1.32 (q, J = 12.4 Hz, 2H), 1.12 (q, J = 12.6 Hz, 2H). MS (ESI), m/z: 556.1 [M+H]⁺.

3-benzyl-1-((1r,4r)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)-1-(4-(4-(dimethylamino)piperidin-1-yl)-3-nitrophenyl)urea (19k).

Compound 19k was synthesized from commercially available N,N-dimethylpiperidin-4-amine and 18 by following a similar procedure as that of 19c (yield, 40%).¹H NMR (400 MHz, DMSO-d₆) δ 8.30 (d, J = 2.3 Hz, 1H), 7.61 (dd, J = 8.9, 2.3 Hz, 1H), 7.58 (d, J = 2.4 Hz, 1H), 7.49 (d, J = 7.8 Hz, 1H), 7.35 (dd, J = 8.8, 2.5 Hz, 1H), 7.32–7.25 (m, 3H), 7.18 (td, J = 5.5, 3.0 Hz, 3H), 6.48 (d, J = 9.0 Hz, 1H), 6.24 (t, J = 6.1 Hz, 1H), 4.33–4.21 (m, 1H), 4.16 (d, J = 6.1 Hz, 2H), 3.51 (s, 1H), 3.28 (d, J = 12.4 Hz, 2H), 2.86 (td, J = 12.2, 2.3 Hz, 2H), 2.24 (s, 1H), 2.21 (s, 6H), 1.92 (d, J = 12.0 Hz, 2H), 1.82 (t, J = 12.7 Hz, 4H), 1.49 (q, J = 11.9 Hz, 2H), 1.31 (q, J = 14.8, 13.7 Hz, 2H), 1.18–1.04 (m, 2H). MS (ESI), m/z: 597.2 [M+H]⁺.

tert-butyl (1-(4-(3-benzyl-1-((1r,4r)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)-2-nitrophenyl)piperidin-4-yl)(methyl)carbamate (19l).

Compound 19l was synthesized from commercially available tert-butyl methyl(piperidin-4-yl)carbamate and 18 by following a similar procedure as that of 19c (yield, 62%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.30 (d, J = 2.3 Hz, 1H), 7.65 – 7.56 (m, 2H), 7.48 (d, J = 7.7 Hz, 1H), 7.39 – 7.31 (m, 2H), 7.31 – 7.24 (m, 2H), 7.21 – 7.15 (m, 3H), 6.47 (d, J = 8.9 Hz, 1H), 6.24 (t, J = 5.9 Hz, 1H), 4.26 (t, J = 12.0 Hz, 1H), 4.16 (d, J = 5.9 Hz, 2H), 4.07 – 3.75 (m, 1H), 3.59 – 3.45 (m, 1H), 3.30 (t, J = 12.0 Hz, 2H), 2.94 (t, J = 12.0 Hz, 2H), 2.72 (s, 3H), 1.92 (d, J = 11.8 Hz, 2H), 1.85 – 1.72 (m, 4H), 1.64 (d, J = 9.5 Hz, 2H), 1.42 (s, 9H), 1.32 (q, J = 12.9, 12.0 Hz, 2H), 1.11 (q, J = 13.5, 12.8 Hz, 2H). MS (ESI), m/z: 683.4 [M+H]⁺.

N-(5-(3-benzyl-1-((1r,4r)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)-2-(1-methyl-6-oxo-1,6-dihydropyridin-3-yl)phenyl)acrylamide (14a).

To a solution of 19a (810 mg, 1.4 mmol) in H₂O/EtOH (3:7, 30 mL), 0.05 mL of concentrated HCl and Fe powder (602 mg, 7 mmol) were added. The mixture was stirred for 2 h at 70 °C. The mixture was filtered through Celite after cooling, concentrated in vacuo to dryness to afford the crude product as brown solid, which was used in the next step without further purification. To the solution of the crude product and DIPEA (361.2 mg, 2.8 mmol) in anhydrous DCM (30 mL), which was cooled to 0°C, was added acryloyl chloride (216.7 mg, 1.7 mmol) slowly. The reaction mixture was stirred for 10 min at 0 °C to rt for 15 min. The mixture was quenched with H₂O, the pH was adjusted to 7–8 with saturated NaHCO₃ solution, and extracted with EtOAc three times. The combined organic phase was concentrated in vacuo and purified by column chromatography to give the desired compound 14a (352 mg, yield 42%, two steps). ¹H NMR (600 MHz, DMSO-d₆) δ 9.55 (s, 1H), 8.32 (d, J = 2.3 Hz, 1H), 7.85 (d, J = 2.6 Hz, 1H), 7.67–7.59 (m, 2H), 7.56 (s, 1H), 7.44 (dd, J = 9.4, 2.6 Hz, 1H), 7.39 (d, J = 8.1 Hz, 1H), 7.28 (t, J = 7.6 Hz, 2H), 7.19 (dd, J = 17.0, 7.7 Hz, 3H), 7.07 (d, J = 8.1 Hz, 1H), 6.53–6.42 (m, 3H), 6.24 (d, J = 19.0 Hz, 1H), 5.95 (t, J = 6.2 Hz, 1H), 5.74 (d, J = 10.0 Hz, 1H), 4.30 (tt, J = 12.0, 3.7 Hz, 1H), 4.19 (d, J = 5.9 Hz, 2H), 3.51(s, 1H), 3.48 (s, 4H), 1.94 (d, J = 10.6 Hz, 2H), 1.82 (d, J = 10.9 Hz, 2H), 1.34 (q, J = 11.2 Hz, 2H), 1.20 (q, J = 12.3, 11.0 Hz, 2H). ¹³C NMR (151 MHz, DMSO-d₆) δ 164.11, 161.68, 159.51, 156.85, 153.36, 141.63, 141.16, 139.83, 137.77, 136.02, 132.00, 130.90, 130.56, 128.52 (4C), 127.99, 127.55, 127.21 (4C), 126.74, 119.51, 119.24, 116.10, 94.53, 53.79, 49.06, 43.98, 40.51, 37.41, 31.75, 30.66. HRMS (ESI) calcd for C₃₅H₃₅N₇O₃ [M + H]⁺, 602.2874; found, 602.2853. HPLC analysis: MeOH-H₂O (70:30), 5.91 min, 98.7% purity.

N-(3-(3-benzyl-1-((1r,4r)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl)acrylamide (14b).

To a solution of the compound 18 (250 mg, 0.42 mmol) in methanol (20 mL) was added 10% Pd/C (25 mg, 10% w/w) at room temperature. The reaction mixture was stirred under hydrogen balloon for 3 h and then filtered through celite. The filtrate was evaporated to dryness to give the crude material, which was used in the next step without further purification. To the solution of the crude product and DIPEA (81.3 mg, 0.63 mmol) in anhydrous DCM (30 mL), which was cooled to 0°C, was added acryloyl chloride (45.3 mg, 0.5 mmol) slowly. The reaction mixture was stirred for 10 min at 0 °C to rt for 15 min. The mixture was quenched with H₂O, the pH was adjusted to 7–8 with saturated NaHCO₃ solution, and extracted with EtOAc three times. The combined organic phase was concentrated in vacuo and purified by column chromatography to give the desired compound 14b (82 mg, yield 39%, two steps): ¹H NMR (400 MHz, DMSO-d₆) δ 10.28 (s, 1H), 8.30 (d, J = 2.3 Hz, 1H), 7.73 (d, J = 8.2 Hz, 1H), 7.61 (dd, J = 8.8, 2.4 Hz, 1H), 7.56 (d, J = 2.1 Hz, 1H), 7.48 (d, J = 7.6 Hz, 1H), 7.41 (t, J = 8.0 Hz, 1H), 7.30–7.23 (m, 2H), 7.22–7.13 (m, 3H), 6.89 (d, J = 8.5 Hz, 1H), 6.50–6.46 (m, 1H), 6.46–6.39 (m, 1H), 6.28 (dd, J = 16.9, 2.1 Hz, 1H), 5.85 (t, J = 6.0 Hz, 1H), 5.79 (dd, J = 10.0, 2.1 Hz, 1H), 4.34–4.23 (m, 1H), 4.16 (d, J = 6.0 Hz, 2H), 3.50 (s, 1H), 1.93 (d, J = 11.9 Hz, 2H), 1.79 (d, J = 11.6 Hz, 2H), 1.32 (q, J = 12.5 Hz, 2H), 1.17 (q, J = 12.5 Hz, 2H). ¹³C NMR (151 MHz, DMSO-d₆) δ 163.71, 156.84, 153.58, 141.74, 140.40, 138.81, 132.19, 130.07, 128.48 (4C), 127.70, 127.15 (4C), 126.70, 126.59, 122.18, 119.60, 119.09, 94.45, 53.60, 49.07, 43.94, 40.49, 31.76, 30.66. HRMS (ESI) calcd for C₂₉H₃₀N₆O₂ [M + H]⁺, 495.2503; found, 495.2485. HPLC analysis: MeOH-H₂O (70:30), 11.84 min, 95.9% purity.

N-(5-(3-benzyl-1-((1r,4r)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)-2-((2-(dimethylamino)ethyl)(methyl)amino)phenyl)acrylamide (14c).

Compound 14c was synthesized from compound 19c with a similar procedure to that of 14a (yield 38%, two steps). ¹H NMR (400 MHz, DMSO-d₆) δ 10.22 (s, 1H), 8.31 (d, J = 2.3 Hz, 1H), 8.24 (d, J = 2.5 Hz, 1H), 7.61 (dd, J = 8.9, 2.4 Hz, 1H), 7.50 (d, J = 7.6 Hz, 1H), 7.33 (d, J = 8.4 Hz, 1H), 7.30–7.24 (m, 2H), 7.21–7.14 (m, 3H), 6.89 (dd, J = 8.4, 2.5 Hz, 1H), 6.48 (d, J = 8.7 Hz, 1H), 6.45–6.39 (m, 1H), 6.30 (dd, J = 17.0, 2.2 Hz, 1H), 5.82 (dd, J = 9.9, 2.2 Hz, 1H), 5.76 (t, J = 6.0 Hz, 1H), 4.33–4.23 (m, 1H), 4.18 (d, J = 6.0 Hz, 2H), 3.52 (s, 1H), 2.83 (t, J = 5.7 Hz, 2H), 2.72 (s, 3H), 2.40 (t, J = 5.6 Hz, 2H), 2.23 (s, 6H), 1.93 (d, J = 12.1 Hz, 2H), 1.79 (d, J = 11.9 Hz, 2H), 1.32 (q, J = 12.3 Hz, 2H), 1.18 (dd, J = 28.1, 15.7 Hz, 2H). ¹³C NMR (151 MHz, DMSO-d₆) δ 163.64, 159.70, 157.03, 153.58, 142.77, 141.73, 134.93, 134.13, 132.27, 128.49 (4C), 127.67, 127.06 (4C), 126.69, 122.88, 122.48, 119.60, 94.45, 57.38, 56.72, 53.71, 49.0, 46.18 (2C), 43.91, 41.48, 40.52, 31.79, 30.65. HRMS (ESI) calcd for C₃₄H₄₂N₈O₂ [M + H]⁺, 595.3503; found, 595.3508. HPLC analysis: MeOH-H₂O (80:20), 8.92 min, 95.3% purity.

N-(5-(3-benzyl-1-((1r,4r)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)-2-(3-(dimethylamino)azetidin-1-yl)phenyl)acrylamide (14d).

Compound 14d was synthesized from compound 19d with a similar procedure to that of 14a (yield 35%, two steps). ¹H NMR (400 MHz, DMSO-d₆) δ 9.38 (s, 1H), 8.33 (d, J = 2.3 Hz, 1H), 7.61 (dd, J = 8.9, 2.4 Hz, 1H), 7.49 (d, J = 7.6 Hz, 1H), 7.30–7.23 (m, 2H), 7.20–7.14 (m, 3H), 7.11 (d, J = 2.4 Hz, 1H), 6.90 (dd, J = 8.6, 2.4 Hz, 1H), 6.61 (d, J = 8.5 Hz, 1H), 6.55 (dd, J = 17.0, 10.2 Hz, 1H), 6.47 (d, J = 8.9 Hz, 1H), 6.24 (dd, J = 17.0, 2.1 Hz, 1H), 5.74 (dd, J = 10.2, 2.1 Hz, 1H), 5.59 (t, J = 6.1 Hz, 1H), 4.31–4.20 (m, 1H), 4.17 (d, J = 6.0 Hz, 2H), 3.98 (t, J = 7.2 Hz, 2H), 3.60 (t, J = 6.7 Hz, 2H), 3.50 (s, 1H), 3.12 (d, J = 14.5 Hz, H), 2.11 (s, 6H), 1.96–1.85 (m, 2H), 1.76 (d, J = 12.0 Hz,2H), 1.30 ((q, J = 11.9 Hz, 2H)), 1.14 (q, J = 11.7 Hz, 2H). ¹³C NMR (151 MHz, DMSO-d₆) δ 163.95, 159.70, 157.21, 153.63, 145.74, 141.66, 132.15, 129.79, 129.24, 128.52 (4C), 128.29, 127.16 (4C), 126.72, 124.77, 119.62, 114.23, 94.44, 57.44, 56.28, 53.50, 49.06, 46.06, 43.96, 41.93 (2C), 40.51, 31.77, 30.61. HRMS (ESI) calcd for C₃₄H₄₀N₈O₂ [M + H]⁺, 593.3347; found, 593.3329. HPLC analysis: MeOH-H₂O (70:30), 12.20 min, 96.1% purity.

N-(5-(3-benzyl-1-((1r,4r)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)-2-(3-(dimethylamino)pyrrolidin-1-yl)phenyl)acrylamide (14e).

Compound 14e was synthesized from compound 19e with a similar procedure to that of 14a (yield 35%, two steps). ¹H NMR (600 MHz, DMSO-d₆) δ 9.49 (s, 1H), 8.32 (d, J = 2.3 Hz, 1H), 7.61 (dd, J = 9.0, 2.4 Hz, 1H), 7.51 (s, 1H), 7.31–7.23 (m, 2H), 7.21–7.13 (m, 4H), 6.89 (s, 2H), 6.57 (dd, J = 17.0, 10.2 Hz, 1H), 6.48 (d, J = 8.9 Hz, 1H), 6.24 (dd, J = 17.0, 2.0 Hz, 1H), 5.74 (dd, J = 10.1, 2.0 Hz, 1H), 5.58 (s, 1H), 4.26 (td, J = 10.1, 8.3, 6.0 Hz, 1H), 4.17 (d, J = 6.2 Hz, 2H), 3.51 (s, 1H), 3.27–3.15 (m, 3H), 2.66 (p, J = 7.8 Hz, 1H), 2.16 (s, 6H), 2.12–2.05 (m, 1H), 1.92 (s, 2H), 1.80–1.74 (m, 2H), 1.74–1.68 (m, 1H), 1.32 (q, J = 13.0, 12.2 Hz, 2H), 1.24 (d, J = 5.2 Hz, 1H), 1.21–1.11 (m, 2H). ¹³C NMR (151 MHz, DMSO-d₆) δ 164.02, 159.70, 157.19, 153.61, 144.28, 141.64, 132.26, 130.32, 129.22, 128.52 (4C), 127.18 (4C), 126.96, 126.73, 126.08, 119.61, 116.16, 94.44, 65.62, 55.29, 53.49, 49.61, 49.07, 44.42 (2C), 43.97, 40.52, 31.78, 30.63, 30.10. HRMS (ESI) calcd for C₃₅H₄₂N₈O₂ [M + H]⁺, 607.3503; found, 607.3480. HPLC analysis: MeOH-H₂O (70:30), 16.19 min, 99.1% purity.

N-(5-(3-benzyl-1-((1r,4r)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)-2-(4-methylpiperazin-1-yl)phenyl)acrylamide (14f).

Compound 14f was synthesized from compound 19f with a similar procedure to that of 14a (yield 46%, two steps). ¹H NMR (400 MHz, DMSO-d₆) δ 9.08 (s, 1H), 8.31 (d, J = 2.3 Hz, 1H), 8.05 (s, 1H), 7.61 (dd, J = 8.9, 2.4 Hz, 1H), 7.52 (d, J = 7.6 Hz, 1H), 7.31–7.24 (m, 3H), 7.18 (dt, J = 6.4, 1.6 Hz, 3H), 6.95 (dd, J = 8.4, 2.4 Hz, 1H), 6.75 (dd, J = 16.9, 10.2 Hz, 1H), 6.47 (d, J = 8.9 Hz, 1H), 6.32 (dd, J = 17.0, 1.9 Hz, 1H), 5.89–5.79 (m, 2H), 4.34–4.22 (m, 1H), 4.18 (d, J = 6.0 Hz, 2H), 3.69–3.37 (m, 5H), 3.18 (s, 2H) 3.07 (d, J = 19.4 Hz, 2H), 2.88 (s, 3H), 1.92 (d, J = 11.8 Hz, 2H), 1.80 (d, J = 11.5 Hz, 2H), 1.39–1.27 (m, 2H), 1.21–1.09 (m, 2H). ¹³C NMR (151 MHz, DMSO-d₆) δ 163.77, 159.68, 156.95, 153.57, 141.68, 134.73, 133.46, 132.53, 128.50 (4C), 127.80, 127.49, 127.11(4C), 126.72, 124.38, 121.42, 119.60, 94.46, 53.66, 53.30 (2C), 49.01, 48.78 (2C), 43.92, 42.93, 40.52, 31.74, 30.66. HRMS (ESI) calcd for C₃₄H₄₀N₈O₂ [M + H]⁺, 593.3347; found, 593.3365. HPLC analysis: MeOH-H₂O (80:30), 6.87 min, 100% purity.

N-(5-(3-benzyl-1-((1r,4R)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)-2-((1R,4R)-5-methyl-2,5-diazabicyclo[2.2.1]heptan-2-yl)phenyl)acrylamide (14g).

Compound 14g was synthesized from compound 19g with a similar procedure to that of 14a (yield 30%, two steps). ¹H NMR (400 MHz, DMSO-d₆) δ 9.30 (s, 1H), 8.33 (d, J = 2.4 Hz, 1H), 7.61 (dd, J = 8.8, 2.4 Hz, 1H), 7.49 (d, J = 7.6 Hz, 1H), 7.30–7.23 (m, 2H), 7.17 (dt, J = 9.0, 3.0 Hz, 3H), 7.13 (d, J = 2.1 Hz, 1H), 6.90–6.79 (m, 2H), 6.54 (dd, J = 17.0, 10.2 Hz, 1H), 6.47 (d, J = 8.9 Hz, 1H), 6.23 (dd, J = 17.1, 2.1 Hz, 1H), 5.73 (dd, J = 10.1, 2.1 Hz, 1H), 5.59 (d, J = 6.2 Hz, 1H), 4.31–4.20 (m, 1H), 4.17 (d, J = 6.1 Hz, 2H), 4.09 (s, 1H), 3.51 (s, 1H), 3.39 (dd, J = 8.9, 2.4 Hz, 2H), 3.07 (d, J = 9.0 Hz, 1H), 2.81 (d, J = 9.6 Hz, 1H), 2.65 (dd, J = 9.7, 2.3 Hz, 1H), 2.26 (s, 3H), 1.92 (d, J = 11.2 Hz, 2H), 1.85–1.71 (m, 3H), 1.68 (d, J = 9.2 Hz, 1H), 1.31 (dt, J = 15.5, 11.6 Hz, 3H), 1.14 (t, J = 13.5 Hz, 3H). ¹³C NMR (151 MHz, DMSO-d₆) δ 163.80, 159.70, 157.21, 153.62, 143.65, 141.64, 132.28, 130.50, 128.98, 128.52 (4C), 128.12, 127.15 (4C), 126.94, 126.72, 119.61, 116.85, 94.44, 63.28, 60.35, 58.66, 53.53, 49.07, 43.96, 41.82, 40.52, 34.36, 31.78, 29.56. HRMS (ESI) calcd for C₃₅H₄₀N₈O₂ [M + H]⁺, 605.3347; found, 605.3329. HPLC analysis: MeOH-H₂O (70:30), 10.96 min, 100% purity.

N-(5-(3-benzyl-1-((1r,4S)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)-2-((S)-3,4-dimethylpiperazin-1-yl)phenyl)acrylamide (14h).

Compound 14h was synthesized from compound 19h with a similar procedure to that of 14a (yield 40%, two steps). ¹H NMR (400 MHz, DMSO-d₆) δ 9.02 (s, 1H), 8.31 (d, J = 2.3 Hz, 1H), 7.91 (s, 1H), 7.60 (dd, J = 8.9, 2.3 Hz, 1H), 7.49 (d, J = 7.6 Hz, 1H), 7.30–7.24 (m, 2H), 7.21 (d, J = 8.4 Hz, 1H), 7.20–7.15 (m, 3H), 6.92 (dd, J = 8.4, 2.4 Hz, 1H), 6.65 (dd, J = 16.9, 10.2 Hz, 1H), 6.47 (d, J = 8.9 Hz, 1H), 6.27 (dd, J = 17.0, 1.9 Hz, 1H), 5.78 (td, J = 9.6, 3.9 Hz, 2H), 4.33–4.22 (m, 1H), 4.17 (d, J = 6.0 Hz, 2H), 3.51 (s, 1H), 2.95–2.75 (m,4H), 2.45 (d, J = 10.6 Hz, 2H), 2.37 (d, J = 9.4 Hz, 1H), 2.25 (s, 3H), 1.92 (d, J = 12.0 Hz, 2H), 1.78 (d, J = 12.0 Hz, 2H), 1.32 (q, J = 12.5 Hz, 2H), 1.16 (t, J = 12.8 Hz, 2H), 1.01 (d, J = 6.1 Hz, 3H). ¹³C NMR (151 MHz, DMSO-d₆) δ 170.83, 163.61, 159.67, 156.99, 153.59, 142.86, 141.73, 133.64, 132.91, 132.56, 128.49 (4C), 127.52, 127.10 (4C), 126.69, 124.58, 120.84, 119.62, 94.43, 58.86, 57.59, 53.61, 51.79, 49.05, 43.91, 42.64 (2C), 40.49, 31.75, 30.64, 17.22. HRMS (ESI) calcd for C₃₅H₄₂N₈O₂ [M + H]⁺, 607.3503; found, 607.3521. HPLC analysis: MeOH-H₂O (80:20), 7.19 min, 95.1% purity.

N-(2-(4-acetylpiperazin-1-yl)-5-(3-benzyl-1-((1r,4r)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl)acrylamide (14i).

Compound 14i was synthesized from compound 19i with a similar procedure to that of 14a (yield 40%, two steps). ¹H NMR (400 MHz, DMSO-d₆) δ 9.15 (s, 1H), 8.31 (dd, J = 2.4, 0.7 Hz, 1H), 8.02–7.95 (m, 1H), 7.61 (dd, J = 8.8, 2.4 Hz, 1H), 7.50 (d, J = 7.6 Hz, 1H), 7.30–7.24 (m, 2H), 7.22 (d, J = 8.4 Hz, 1H), 7.20–7.15 (m, 3H), 6.92 (dd, J = 8.4, 2.5 Hz, 1H), 6.75 (dd, J = 16.9, 10.2 Hz, 1H), 6.48 (d, J = 8.9 Hz, 1H), 6.30 (dd, J = 17.0, 1.9 Hz, 1H), 5.85–5.78 (m, 2H), 4.26 (d, J = 11.9 Hz, 1H), 4.17 (d, J = 6.0 Hz, 2H), 3.72–3.63 (m, 4H), 3.51 (s, 1H), 2.86 (t, J = 5.0 Hz, 2H), 2.81 (t, J = 5.1 Hz, 2H), 2.06 (s, 3H),1.93 (d, J = 9.7 Hz, 2H), 1.79 (d, J = 11.4 Hz, 2H), 1.32 (q, J = 12.4, 12.0 Hz, 2H), 1.18 (q, J = 12.4, 12.0 Hz, 2H). ¹³C NMR (151 MHz, DMSO-d₆) δ 168.83, 163.71, 159.67, 156.99, 153.59, 141.73, 134.09, 133.26, 132.57, 128.49 (4C), 127.66, 127.49, 127.10 (4C), 126.69, 124.44, 121.28, 119.62, 94.43, 60.24, 53.66, 52.10, 51.61, 49.07, 46.27, 43.91, 41.37, 40.49, 31.75, 30.63, 21.78. HRMS (ESI) calcd for C₃₅H₄₀N₈O₃ [M + H]⁺, 621.3296; found, 621.3295. HPLC analysis: MeOH-H₂O (70:30), 8.39 min, 100% purity.

N-(5-(3-benzyl-1-((1r,4r)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)-2-morpholinophenyl)acrylamide (14j).

Compound 14j was synthesized from compound 19j with a similar procedure to that of 14a (yield 42%, two steps). ¹H NMR (400 MHz, DMSO-d₆) δ 9.13 (s, 1H), 8.31 (dd, J = 2.4, 0.7 Hz, 1H), 7.95 (s, 1H), 7.61 (dd, J = 8.9, 2.4 Hz, 1H), 7.50 (d, J = 7.6 Hz, 1H), 7.30–7.22 (m, 3H), 7.22–7.15 (m, 3H), 6.94 (dd, J = 8.4, 2.5 Hz, 1H), 6.73 (dd, J = 16.9, 10.2 Hz, 1H), 6.48 (d, J = 8.9 Hz, 1H), 6.28 (dd, J = 16.9, 1.9 Hz, 1H), 5.80 (dt, J = 10.2, 3.3 Hz, 2H), 4.35–4.22 (m, 1H), 4.17 (d, J = 6.0 Hz, 2H), 3.83 (t, J = 4.5 Hz, 4H), 3.50 (s, H), 2.86 (t, J = 4.6 Hz, 4H), 1.93 (d, J = 11.9 Hz, 2H), 1.79 (d, J = 11.6 Hz, 2H), 1.32 (q, J = 12.2 Hz, 2H), 1.18 (q, J = 12.2 Hz, 2H). ¹³C NMR (151 MHz, DMSO-d₆) δ 163.69, 159.69, 157.00, 153.59, 142.78, 141.72, 133.92, 133.10, 132.58, 128.49 (4C), 127.59, 127.11 (4C), 126.69, 124.60, 120.99, 119.60, 94.45, 66.64 (2C), 53.69, 52.16 (2C), 49.00, 43.93, 40.52, 31.76, 30.65. HRMS (ESI) calcd for C₃₃H₃₇N₇O₃ [M + H]⁺, 580.3031; found, 5890.3024. HPLC analysis: MeOH-H₂O (70:30), 12.33 min, 99.1% purity.

N-(5-(3-benzyl-1-((1r,4r)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)-2-(4-(dimethylamino)piperidin-1-yl)phenyl)acrylamide (14k, YJZ5118).

Compound YJZ5118 was synthesized from compound 19k with a similar procedure to that of 14a (yield 40%, two steps). ¹H NMR (400 MHz, DMSO-d₆) δ 9.01 (s, 1H), 8.31 (d, J = 2.4 Hz, 1H), 7.92 (s, 1H), 7.60 (dd, J = 9.0, 2.4 Hz, 1H), 7.49 (d, J = 7.5 Hz, 1H), 7.30–7.23 (m, 2H), 7.22–7.13 (m, 4H), 6.90 (dd, J = 8.3, 2.5 Hz, 1H), 6.73 (dd, J = 16.9, 10.2 Hz, 1H), 6.47 (d, J = 8.9 Hz, 1H), 6.28 (dd, J = 17.0, 2.0 Hz, 1H), 5.84–5.72 (m, 2H), 4.32–4.22 (m, 1H), 4.17 (d, J = 6.0 Hz, 2H), 3.50 (s, 1H), 3.07 (d, J = 11.4 Hz, 2H), 2.65 (t, J = 11.2 Hz, 2H), 2.23 (s, 6H), 2.17 (d, J = 11.1 Hz, 1H),1.92 (d, J = 11.9 Hz, 2H), 1.85 (d, J = 11.4 Hz, 2H), 1.78 (d, J = 12.9 Hz, 2H), 1.74–1.68 (m, 2H), 1.32 (q, J = 12.2 Hz, 2H), 1.22–1.09 (m, 2H). ¹³C NMR (151 MHz, DMSO-d₆) δ 163.68, 159.69, 157.06, 153.57, 143.52, 141.69, 133.43, 132.91, 132.60, 128.50 (3C), 127.54, 127.44, 127.10 (4C), 126.71, 124.43, 120.75, 119.60, 94.45, 61.87, 53.69, 51.64 (2C), 49.02, 43.92, 42.07 (2C), 40.47, 31.76, 30.64, 28.79 (2C). HRMS (ESI) calcd for C₃₆H₄₄N₈O₂ [M + H]⁺, 621.3660; found, 621.3666. HPLC analysis: MeOH-H₂O (80:20), 10.27 min, 100% purity.

tert-butyl (1-(2-acrylamido-4-(3-benzyl-1-((1r,4r)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl)piperidin-4-yl)(methyl)carbamate (14l).

Compound 14l was synthesized from compound 19l with a similar procedure to that of 14a (yield 44%, two steps). ¹H NMR (400 MHz, DMSO-d₆) δ 9.04 (s, 1H), 8.30 (d, J = 2.3 Hz, 1H), 7.94 (s, 1H), 7.60 (dd, J = 8.9, 2.3 Hz, 1H), 7.48 (d, J = 7.6 Hz, 1H), 7.30–7.21 (m, 3H), 7.21–7.13 (m, 3H), 6.90 (dd, J = 8.3, 2.4 Hz, 1H), 6.74 (dd, J = 16.9, 10.2 Hz, 1H), 6.47 (d, J = 8.9 Hz, 1H), 6.29 (dd, J = 17.0, 1.8 Hz, 1H), 5.86–5.74 (m, 2H), 4.28 (t, J = 10.9 Hz, 1H), 4.17 (d, J = 6.0 Hz, 2H), 3.92 (s, 1H), 3.06 (d, J = 11.2 Hz, 2H), 2.77 (s, 3H), 2.75–2.68 (m, 2H), 2.06–1.95 (m, 2H), 1.91 (d, J = 12.4 Hz, 2H), 1.78 (d, J = 11.9 Hz, 2H), 1.61 (s, 2H), 1.41 (s, 9H), 1.31 (q, J = 12.4, 10.1 Hz, 3H), 1.15 (q, J = 12.5 Hz, 2H). MS (ESI), m/z: 707.4 [M+H]⁺.

N-(5-(3-benzyl-1-((1r,4r)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)-2-(4-(methylamino)piperidin-1-yl)phenyl)acrylamide (14m).

TFA (2 mL) was added to a solution of tert-butyl (1-(2-acrylamido-4-(3-benzyl-1-((1r,4r)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl)piperidin-4-yl)(methyl)carbamate 14l (185 mg, 0.26 mmol) in DCM (6 mL), and the mixture was stirred at 50 °C for 3 h. The reaction mixture was then concentrated to dryness under reduced pressure. The resultant crude material was dissolved in EtOAc (20 mL) and H₂O (40 mL) was added. Subsequently, the pH was adjusted to 7–8 with saturated NaHCO₃ solution, and extracted with EtOAc three times. The combined organic phase was concentrated in vacuo and purified by column chromatography to give the desired compound 14m (95 mg, yield 60%). ¹H NMR (400 MHz, DMSO-d₆) δ 9.10 (s, 1H), 8.66 (s, 2H), 8.30 (d, J = 2.3 Hz, 1H), 7.90 (s, 1H), 7.60 (dd, J = 8.9, 2.4 Hz, 1H), 7.50 (d, J = 7.6 Hz, 1H), 7.30–7.25 (m, 2H), 7.23 (d, J = 8.4 Hz, 1H), 7.21–7.14 (m, 3H), 6.93 (dd, J = 8.4, 2.4 Hz, 1H), 6.63 (dd, J = 17.0, 10.2 Hz, 1H), 6.47 (d, J = 8.9 Hz, 1H), 6.28 (dd, J = 17.0, 1.8 Hz, 1H), 5.84–5.74 (m, 2H), 4.34–4.23 (m, 1H), 4.18 (d, J = 6.0 Hz, 2H), 3.50 (s, 1H), 3.13 (dd, J = 14.3, 10.4 Hz, 3H), 2.79–2.68 (m, 2H), 2.62 (s, 3H), 2.08 (d, J = 11.9 Hz, 2H), 1.92 (d, J = 11.3 Hz, 2H), 1.87–1.73 (m, 4H), 1.32 (q, J = 12.5 Hz, 2H), 1.14 (q, J = 13.3, 12.0 Hz, 2H). ¹³C NMR (151 MHz, DMSO-d₆) δ 163.62, 159.69, 156.99, 153.57, 142.88, 141.67, 134.01, 133.04, 132.53, 128.51 (4C), 127.50, 127.12 (4C), 126.72, 124.69, 121.25, 119.60, 94.46, 55.42, 53.68, 50.28, 49.03, 43.94, 40.52, 31.75, 30.67, 30.02 (2C), 28.56 (2C). HRMS (ESI) for C₃₅H₄₂N₈O₂ [M+H]⁺, calcd: 607.3503, found: 607.3489. HPLC analysis: MeOH-H₂O (77:23), 14.3 min, 99.9% purity.

N-(1-(2-acrylamido-4-(3-benzyl-1-((1r,4r)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl)piperidin-4-yl)-N-methyl-1-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)-3,6,9,12-tetraoxapentadecan-15-amide (YJZ9149).

To a solution of 14m (65 mg·0.1 mmol), HATU (48.8 mg·0.12 mmol), and DIPEA (20.7 mg, 0.16 mmol) in DMF (8 mL), was added 17-oxo-21-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-4,7,10,13-tetraoxa-16-azahenicosanoic acid (58.3 mg, 0.12 mmol). The mixture was stirred at rt for 30 min. The reaction mixture was concentrated in vacuo and purified by column chromatography to give the desired compound YJZ9149 as white solid (77 mg, yield 71%). ¹H NMR (400 MHz, DMSO-d₆) δ 9.06 (d, J = 7.0 Hz, 1H), 8.30 (d, J = 2.3 Hz, 1H), 7.96 (s, 1H), 7.88–7.78 (m, 1H), 7.60 (dd, J = 8.8, 2.3 Hz, 1H), 7.49 (d, J = 7.6 Hz, 1H), 7.26 (q, J = 9.2, 8.4 Hz, 3H), 7.21–7.15 (m, 3H), 6.91 (dt, J = 8.5, 2.9 Hz, 1H), 6.76 (dd, J = 17.0, 10.4 Hz, 1H), 6.47 (d, J = 8.9 Hz, 1H), 6.41 (s, 1H), 6.35 (s, 1H), 6.30 (dd, J = 16.9, 1.9 Hz, 1H), 5.84–5.76 (d, J = 11.3 Hz, 2H), 4.49–4.39 (m, 0.5H), 4.34–4.22 (m, 2H), 4.17 (d, J = 5.9 Hz, 2H), 4.15–4.10 (m, 1H), 3.90–3.80 (m, 0.5H), 3.65 (t, J = 6.8 Hz, 2H), 3.52–3.48 (m, 14H), 3.21–3.15 (m, 2H), 3.12–3.01 (m, 3H), 2.93 (s, 2H), 2.87–2.71 (m, 4H), 2.66 (t, J = 6.6 Hz, 1H), 2.61–2.55 (m, 2H), 2.06 (td, J = 7.4, 2.6 Hz, 3H), 1.92 (d, J = 11.6 Hz, 2H), 1.78 (d, J = 11.7 Hz, 2H), 1.72–1.57 (m, 2H), 1.57–1.38 (m, 5H), 1.40–1.09 (m, 7H). ¹³C NMR (151 MHz, DMSO-d₆) δ 172.58, 170.41, 170.24, 163.64, 163.17, 159.70, 157.04, 153.58, 143.20, 141.72, 133.70, 133.04, 132.64, 128.49 (3C), 127.66, 127.38, 127.11 (3C), 126.70, 124.21, 121.01, 120.92, 119.60, 94.46, 70.27, 70.20, 70.18, 70.04, 69.64, 67.63, 67.27, 61.50, 59.66, 55.90, 54.23, 53.74, 51.92, 51.69, 50.31, 49.01, 43.93, 40.53, 38.91, 35.56, 34.20, 33.57, 31.78, 30.65, 30.07, 29.86, 29.50, 29.10, 28.67, 28.51, 27.53, 25.73. HRMS (ESI) for C₅₆H₇₇N₁₁O₉S [M+H]⁺, calcd: 1080.5699, found: 1080.5905. HPLC analysis: MeOH-H₂O (70:30), 6.87 min, 99.0% purity.

Cell Culture.

VCaP and 22RV1 cells were purchased from ATCC, and each cell line was cultured according to ATCC guidelines. VCaP cells were cultured in DMEM, GlutaMAX^™, while 22RV1 cells were cultured in RPMI 1640 Medium (ATCC modification). Both media were supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin (P/S). The cells were maintained at 37 °C in 5% CO₂ incubator. Cell lines were tested for mycoplasma using the Lonza MycoAlert Kit following the manufacturer’s protocol.

Western Blotting.

Generally, after incubation with the compounds for the indicated time, the tested cells were washed by cold PBS buffer (Gibco) and lysed with RIPA buffer (ThermoFisher Scientific) supplemented with cOmplete protease inhibitor cocktail tablets (Sigma-Aldrich). The cell lysate was treated with ultrasonic for 20s, following a centrifugation of 14000 rpm at 4°C for 10 min. The protein concentration was measured by BCA protein assay (DC Protein Assay, BIO-RAD). An equal amount of protein was loaded to 4–12% Bis-Tris gels and underwent SDS-PAGE electrophoresis at 120 V for 90 min. The proteins were transferred to a nitrocellulose (NC) membrane (Thermo Scientific) and the membranes were blocked in 5% milk buffer at room temperature for 1h, followed by incubating with primary antibody at the recommended dilution at 4°C for overnight. The membranes were washed by PBST for three times and incubated with anti-rabbit or anti-mouse HRP-conjugated secondary antibody for 1h at room temperature, after which the membranes were imaged by Odyssey CLx Imager (LiCOR Biosciences).

In Vitro Kinase Enzyme Assay.

All the kinase activity were performed using an ADP-Glo kinase assay by ICE Bioscience Inc. (Beijing, China). Kinome selectivity profiles (KINOMEscan profiling) and K_d detection were performed by Eurofins DiscoveryX Corporation (San Diego, CA, USA).

Protein Expression and Purification.

Protein used for crystallization was expressed in Spodoptera frugiperda cells (Sf9) transfected with codon-optimized synthetic genes comprising the kinase domain of human CDK12 (UniProt accession number Q9NYV4, residues 715–1052), the cyclin box domain of human Cyclin K (UniProt accession number O75909, residues 1–267), and full-length CAK1 from Saccharomyces cerevisiae (UniProt accession number P43568, residues 1–368). The expression and purification process were performed as previously described⁸.

Crystallography.

The purified CDK12/CycK protein complex was stored in a buffer of 25 mM HEPES, pH 7.5, 150 mM NaCl and 1 mM TCEP. Prior to setting up crystallization trials, compounds diluted in DMSO was added in a 1:5 ratio to the pooled kinase complexes (10 mg/ml) and incubated for 30 minutes on ice, in order to generate a CDK12/CycK-compound ternary complex. Optimization on pH, buffer pairs, precipitant and ion concentration helped in growing big single crystals from the initial strip or sheet ones using the hanging drop vapor diffusion technique at 291 K by mixing the protein solution in 1:1 with the reservoir solution containing 0.1 M Bis-Tris, pH 5.8, 21.5% PEG 3350, 0.4 M MgCl₂. The crystals were cryo-protected by rapid transfer into a reservoir solution supplemented with 20% glycerol and rapidly flash-frozen in ALS-style pucks that were submerged in liquid nitrogen. The diffraction data were collected at Protein Microcrystal Structure beamline 18U (Shanghai Synchrotron Radiation Facility, Shanghai) and processed using the XDS software package, pointless and aimless. The complex structure of compound YJZ5118 with CDK12/CCNK was determined by molecular replacement using the program PHASER in CCP4 suite. Iterative cycle of refinement was carried out using COOT and PHENIX. Structure figures were created using PyMol.

Real-Time Quantitative PCR Assay.

Total RNA was extracted using RNeasy Mini Kit (Qiagen). RNA concentrations were measured by NanoDrop 2000 spectrophotometer (Thermo Scientific). Reverse transcriptions and qRT-PCR assay were performed as previously described¹⁴.

RNA-seq.

VCaP cells were treated by compound YJZ5118 with the indicated concentrations. After 6 h incubation, RNA was extracted using RNeasy Mini Kit (Qiagen) and quantified by NanoDrop 2000 Spectrophotometers (Thermo Scientific) for RNA-seq as previously described¹⁴.

Comet Assay.

Briefly, VCaP cells were treated with compound YJZ5118 of 100 nM. After 12 h incubation, cells were collected and resuspended in ice-cold PBS. Following the protocol for OxiSelect Comet Assay Kit (Cell Biolabs, STA-351), the comets were observed using a fluorescence microscope and quantified using ImageJ. The data was analyzed using GraphPad Prism software.

Pull-Down Assay.

Briefly, VCaP cells were treated with YJZ9149 for 4 h, and then lysed in RIPA buffer (Thermo Fisher). The cell lysate was incubated with streptavidin beads overnight at 4 °C. The beads were washed with PBST buffer (0.1% Tween) for three times and eluted by 2X SDS loading buffer. The protein was then separated by SDS-PAGE gel electrophoresis.

Cell Proliferation Assay.

Cells were seeded in 96-well plates and incubated at 37 °C and 5% CO₂. After overnight incubation, serial dilutions of tested compounds were added to the plate. The assay was tested with Cell Titer-Glo (Promega) after 5 days. The luminescence signal was detected by the Infinite M1000 Pro plate reader (Tecan), and the IC₅₀ values were calculated by GraphPad Prism 10.

Cell Apoptosis Assay.

Cells were seeded in six-well plates and incubated for 24 h at 37 °C and 5% CO₂. Cells were then treated with the indicated concentrations of the tested compounds for 48 h. The treated cells then were collected, centrifuged and washed twice by cold PBS. Cell apoptosis assay was performed using TUNEL Assay Kit (Cell Signaling Technology) by following the manufacturer’s instruction. Cell samples were analyzed with flow cytometry (SH800S cell sorter, Sony Biotechnology).

Drug Synergism Assessment.

Briefly, 22RV1 cells were exposed to escalating concentrations of each drug for 5 days. Cell viability was determined post-treatment using the CellTiter-Glo Luminescent Cell Viability Assay (Promega) with three biological replicates. Results were calculated as percentage inhibition relative to control. The assessment of synergy was conducted using the Bliss method in SynergyFinder.

IncuCyte Proliferation Assays.

Cell proliferation was quantitatively assessed using the IncuCyte Live-Cell Analysis System (Essen Bioscience). 22RV1 cells were plated in 96-well plates. Following overnight incubation at 37 °C and 5% CO₂, the cells were treated with YJZ5118 at the concentration of 70 nM, with or without AKT inhibitors. Real-time cell proliferation was monitored by capturing phase-contrast images every 4 h using a 10× objective. The IncuCyte software (version 2022A Rev1) was utilized to measure cell confluence continuously as a proxy for proliferation. Data analysis was performed using the software’s built-in analytical tools, focusing on growth curves and confluence metrics (percentage area). Figures were generated using GraphPad Prism software.

Pharmacokinetics Study.

The pharmacokinetic investigation was taken by Shanghai Medicilon Inc. (Project Code: 10017–20023). Specific pathogen free male and female ICR mice provided by Shanghai Xipuer-BK Laboratory Animal Co., Ltd were dosed with tested compounds solution formulation (5% DMSO, 10% Solutol, 85% normal saline, 2.5 or 2 mg/kg for intravenous dose, 10 mg/kg for oral dose). Blood samples were collected at 0.083, 0.25, 0.5, 1, 2, 4, 8 and 24 h in intravenous administration, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h in oral administration. The blood samples were collected from sets of three mice at each time point in labeled microcentrifuge tubes containing heparin sodium as an anticoagulant. Plasma samples were separated by centrifugation (2–8 °C, 6800 g for 6 min) within 1 h and stored below −80 °C until bioanalysis. All samples were processed for analysis by precipitation using acetonitrile and analyzed with a partially validated LC/MS/MS method. Pharmacokinetic parameters were calculated using the noncompartmental analysis tool of WinNonlin Enterprise software.

In Vivo Efficacy Study.

All the animal experiments were performed under an approved animal protocol (Protocol ID: PRO00010006, PI, Arul Chinnaiyan) by the Institutional Animal Care & Use Committee of the University of Michigan. Six- to eight-week-old NSG (Jackson Laboratory) or CB17SCID female (Charles River Laboratory) mice were in a regular SPF housing room prior to cell injection. Briefly, 5 ×10⁶ cells of VCaP CRPC were injected orthotopically into the mammary fat pad of NSG or CB17SCID mice, respectively. After tumor size reached approximately 200–400 mm³, animals were subjected to drug treatment. 0.5 or 1.5 mg/kg of compound YJZ5118 was administered to animals by i.p. injection for 27 days. Vehicle consisted of 20% PEG400, 6% Cremophor EL, and 74% PBS solution. Tumors were collected at the end of the experiment for Western blot analysis.

Histology Analysis.

H&E staining of formalin-fixed paraffin embedded (FFPE) tissue sections and histological assessment was performed as previously described¹⁴.

Computational Modeling Studies.

The structure of CDK12 (PDB: 5ACB) was prepared using Protein Preparation Wizard and the compounds were prepared by LigPrep. The covalent docking was carried out using covalent docking module with default settings (Schrödinger, LLC, New York, NY, 2021).

Supplementary Material

Supporting

NIHMS2082140-supplement-Supporting.pdf^{(5.2MB, pdf)}

ASSOCIATED CONTENT

Supporting Information

The selectivity profiling results of compound 14h; PK profiles of compound 14h and YJZ5118; Caco-2 permeability results of compounds 14a and 14b; Washout results for YJZ5118; Real-time growth curves of VCaP cells upon treatment with YJZ5118; Atomic coordinates and experimental data for the co-crystal structure of compound YJZ5118 with CDK12/CCNK; NMR spectra and HPLC chromatograms for all final compounds;

Molecular formula strings (CSV);

Docking pose of compound 2 in CDK12 (PDB);

Docking pose of compound 14a in CDK12 (PDB);

ACKNOWLEDGMENTS

We acknowledge the financial support from the National Key R&D Program of China (2023YFE0119000, 2023YFF1205104, 2023YFC2506402), the National Natural Science Foundation of China (22037003 and 32071446), Open Project of Shenzhen Bay Laboratory (SZBL2021080601004), Major Program of Guangzhou National Laboratory (GZNL2023A02012), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB1060000), State Key Laboratory of Chemical Biology. This work was funded by a National Cancer Institute (NCI) Prostate Specialized Programs of Research Excellence (SPORE) Grant (P50-CA186786, A.M.C.), an NCI Outstanding Investigator Award (R35-CA231996, A.M.C.), and Prostate Cancer Foundation Challenge Award AWD016479 (A.M.C). A.M.C. is a Howard Hughes Medical Institute Investigator, A. Alfred Taubman Scholar, and American Cancer Society Professor. We also thank the staff at BL10U2/BL02U1/BL17B1/BL18U1/BL19U1 beamlines at Shanghai Synchrotron Radiation Facility (SSRF) of the National Facility for Protein Science in Shanghai (NFPS), Shanghai Advanced Research Institute, Chinese Academy of Sciences, for providing technical support in X-ray diffraction data collection and analysis.

ABBREVIATIONS

CDK12/13: Cyclin-dependent kinases 12 and 13
CPRC: castration-resistant prostate cancer
KD: kinase domain
CCNK: cyclin K
CTD: C-terminal domain
DDR: DNA damage response
PCa: prostate cancer
mCRPC: metastatic castration-resistant prostate cancer
PK: pharmacokinetics
IC₅₀: half inhibition concentration
Xantphos: 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene
tert-BuONa: sodium tert-butoxide
Pd₂(dba)₃: tris(dibenzylideneacetone) dipalladium
DIPEA: N,N-diisopropylethylamine
DMF: N,N-dimethylformamide
TFA: trifluoroacetic acid
DCM: dichloromethane
Cs₂CO₃: cesium carbonate
K₂CO₃: potassium carbonate
K₃PO₄: tripotassium phosphate
Pd(pph₃)₄: Tetrakis(triphenylphosphine)palladium
T_1/2: terminal half-life
C_max: maximum drug plasma concentration
AUC: area under the drug concentration-time curve
CL: plasma clearance rate
F: oral bioavailability
PARP: Poly ADP-ribose polymerase
PCPA: polyadenylation
RNA-seq: RNA sequencing
5-EU: 5-ethynyluridine
i.v.: intravenous administration
i.p.: intraperitoneal injection
HATU: 2-(7-azabenzotriazol-1-yl)-N′,N′,N′-tetramethyluronium hexafluorophosphate
TLC: thin-layer chromatography
TUNEL: terminal dUTP nick end labeling
IHC: immunohistochemistry

Footnotes

The authors declare the following competing financial interest(s): The University of Michigan has filed patent applications on these CDK12/CDK13 covalent inhibitors described in this study in which A.M.C., K.D., X.W., J.Y., Y.C., L.Z., K.Z., W.H., and Z.W. are named as inventors. The work is partially supported by Livzon Pharmaceutical Group Inc., Zhuhai City, China.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supporting

NIHMS2082140-supplement-Supporting.pdf^{(5.2MB, pdf)}

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PERMALINK

Discovery of YJZ5118: a Potent and Highly Selective Irreversible CDK12/13 Inhibitor with Synergistic Effects in Combination with Akt Inhibition

Jianzhang Yang

Yu Chang

Kaijie Zhou

Weixue Huang

Jean Ching-Yi Tien

Pujuan Zhang

Wenyan Liu

Licheng Zhou

Yang Zhou

Xiaomei Ren

Rahul Mannan

Somnath Mahapatra

Yuping Zhang

Rudana Hamadeh

Grafton Ervine

Zhen Wang

George Xiaoju Wang

Arul M Chinnaiyan

Ke Ding

Abstract

Graphical Abstract

INTRODUCTION

Figure 1.

RESULTS AND DISCUSSION

Molecule Design.

Figure 2.

Figure 3.

Chemistry.

Scheme 1.

Discovery of YJZ5118 (14k) as a Potent and Highly Selective Irreversible CDK12/13 Inhibitor.

Table 1.

Validation of the Covalent Binding Mode of YJZ5118.

Figure 4.

YJZ5118 Suppresses Transcription of DDR genes, and Provokes DNA Damage and Apoptosis in Multiple Cancer Cell Lines.

Figure 5.

YJZ5118 Combined with Akt Inhibitors Shows a Synergistic Effect In Vitro and In Vivo.

Figure 6.

Figure 7.

CONCLUSIONS

EXPERIMENTAL SECTION

General Methods for Chemistry.

tert-butyl ((1r,4r)-4-((4-bromo-3-nitrophenyl)amino)cyclohexyl)carbamate (14).

tert-butyl ((1r,4r)-4-(3-benzyl-1-(4-bromo-3-nitrophenyl)ureido)cyclohexyl)carbamate (16).

3-benzyl-1-(4-bromo-3-nitrophenyl)-1-((1r,4r)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)urea (18).

3-benzyl-1-((1r,4r)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)-1-(4-(1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-3-nitrophenyl)urea (19a).

3-benzyl-1-((1r,4r)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)-1-(4-((2-(dimethylamino)ethyl)(methyl)amino)-3-nitrophenyl)urea (19c).

3-benzyl-1-((1r,4r)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)-1-(4-(3-(dimethylamino)azetidin-1-yl)-3-nitrophenyl)urea (19d).

3-benzyl-1-((1r,4r)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)-1-(4-(3-(dimethylamino)pyrrolidin-1-yl)-3-nitrophenyl)urea (19e).

3-benzyl-1-((1r,4r)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)-1-(4-(4-methylpiperazin-1-yl)-3-nitrophenyl)urea (19f).

3-benzyl-1-((1r,4R)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)-1-(4-((1R,4R)-5-methyl-2,5-diazabicyclo[2.2.1]heptan-2-yl)-3-nitrophenyl)urea (19g).

3-benzyl-1-((1r,4S)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)-1-(4-((S)-3,4-dimethylpiperazin-1-yl)-3-nitrophenyl)urea (19h).

1-(4-(4-acetylpiperazin-1-yl)-3-nitrophenyl)-3-benzyl-1-((1r,4r)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)urea (19i).

3-benzyl-1-((1r,4r)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)-1-(4-morpholino-3-nitrophenyl)urea (19j).

3-benzyl-1-((1r,4r)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)-1-(4-(4-(dimethylamino)piperidin-1-yl)-3-nitrophenyl)urea (19k).

tert-butyl (1-(4-(3-benzyl-1-((1r,4r)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)-2-nitrophenyl)piperidin-4-yl)(methyl)carbamate (19l).

N-(5-(3-benzyl-1-((1r,4r)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)-2-(1-methyl-6-oxo-1,6-dihydropyridin-3-yl)phenyl)acrylamide (14a).

N-(3-(3-benzyl-1-((1r,4r)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl)acrylamide (14b).

N-(5-(3-benzyl-1-((1r,4r)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)-2-((2-(dimethylamino)ethyl)(methyl)amino)phenyl)acrylamide (14c).

N-(5-(3-benzyl-1-((1r,4r)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)-2-(3-(dimethylamino)azetidin-1-yl)phenyl)acrylamide (14d).

N-(5-(3-benzyl-1-((1r,4r)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)-2-(3-(dimethylamino)pyrrolidin-1-yl)phenyl)acrylamide (14e).

N-(5-(3-benzyl-1-((1r,4r)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)-2-(4-methylpiperazin-1-yl)phenyl)acrylamide (14f).

N-(5-(3-benzyl-1-((1r,4R)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)-2-((1R,4R)-5-methyl-2,5-diazabicyclo[2.2.1]heptan-2-yl)phenyl)acrylamide (14g).

N-(5-(3-benzyl-1-((1r,4S)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)-2-((S)-3,4-dimethylpiperazin-1-yl)phenyl)acrylamide (14h).

N-(2-(4-acetylpiperazin-1-yl)-5-(3-benzyl-1-((1r,4r)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl)acrylamide (14i).

N-(5-(3-benzyl-1-((1r,4r)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)-2-morpholinophenyl)acrylamide (14j).

N-(5-(3-benzyl-1-((1r,4r)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)-2-(4-(dimethylamino)piperidin-1-yl)phenyl)acrylamide (14k, YJZ5118).

tert-butyl (1-(2-acrylamido-4-(3-benzyl-1-((1r,4r)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl)piperidin-4-yl)(methyl)carbamate (14l).

N-(5-(3-benzyl-1-((1r,4r)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)-2-(4-(methylamino)piperidin-1-yl)phenyl)acrylamide (14m).

N-(1-(2-acrylamido-4-(3-benzyl-1-((1r,4r)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl)piperidin-4-yl)-N-methyl-1-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)-3,6,9,12-tetraoxapentadecan-15-amide (YJZ9149).

Cell Culture.

Western Blotting.

In Vitro Kinase Enzyme Assay.

Protein Expression and Purification.

Crystallography.

Real-Time Quantitative PCR Assay.

RNA-seq.

Comet Assay.

Pull-Down Assay.