CDK7 (IC50 = 13.9 nM); CDK1 (IC50 = 96.9 nM); CDK2 (IC50 = 222 nM); CDK5 (IC50 = 134 nM); CDK9 (IC50 = 194 nM); CDK8 (IC50 = 6830 nM)

THZ2 potently suppresses triple-negative breast cancer cell growth while specifically targeting CDK7, leaving ER/PR⁺ cells unaffected. THZ2 effectively inhibits the clonogenic growth of TNBC cells at low nanomolar doses, with an IC50 of about 10 nM. In triple-negative breast cancer cells, but not in ER/PR⁺ breast cancer cells or normal human cells, THZ2 causes apoptotic cell death[1].

THZ2 (10 mg/Kg) exhibits anti-tumor activity and significantly slows the growth rate of tumors in mice. Tumor tissues isolated from mice treated with THZ2 exhibited decreased proliferation and increased apoptosis in comparison to vehicle-treated tumors, as demonstrated by immunostaining against Ki67 and cleaved Caspase 3, respectively. Body weight is decreased by THZ2 in NOD-SCID mice, indicating that THZ2 may not be as well tolerated in this specific strain of mice[1].

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Treating mice intraperitoneally with THZ2 twice daily at the dose of 10 mg/kg did not give rise to overt toxicity, such as a loss of body weight or behavioral changes (data not shown). To test whether THZ2 has any therapeutic effect on triple-negative breast tumors in an orthotopic xenograft model, we transplanted triple-negative breast tumor cells (MDA-MB-231) into the mammary fat pads of nude mice. When tumors reached approximately ~200 mm3, mice were treated with vehicle or THZ2 (10 mg/Kg). Continuous treatment of THZ2 for 25 days did not affect body weight (Figure S2D), indicating that THZ2 is well tolerated in nude mice. The growth rate of tumors in mice treated with THZ2 was markedly reduced as compared to that of control tumors (Figure 2G), demonstrating an anti-tumor activity of THZ2. We also harvested tumors following short-term (50 hours) or long-term (25 days) treatment, and found that both acute and chronic exposure to THZ2 significantly reduced CTD phosphorylation of RNAPII at all three phosphorylation sites (S2, S5, and S7; Figure 2H, S2E), indicating that CDK7 was efficiently targeted in the tumor cells. Compared to vehicle-treated tumors, tumor tissues isolated from mice treated with THZ2 had reduced proliferation and increased apoptosis, as indicated by immunostaining against Ki67 and cleaved Caspase 3 respectively (Figure S2F). Together, these findings indicate that the CDK7 inhibitor was able to efficiently reduce tumor cell proliferation and induce cell death in vivo.
Researchers further evaluated the anti-tumor effect of CDK7 inhibition in two independent patient-derived xenograft (PDX) models of triple-negative breast tumors, DFBC11–26, DFBC13-11. Both PDX models were established from patients with metastatic TNBC, who had progressed on multiple lines of chemotherapy. Tumor fragments were transplanted into the mammary fat pads of NOD-SCID mice. Our first experiment with THZ2 in NOD-SCID mice led to reduced body weight, suggesting that THZ2 might be less well-tolerated in this particular mouse strain. We therefore proceeded with using THZ1 in the PDX model of TNBC. When tumors grew to an average size of ~80 mm3, mice were treated with THZ1. Although THZ1 has poor pharmacokinetic properties, treating mice with this drug led to a substantial blockage of tumor growth in both patient-derived tumor models (Figure 2I and 2J). Notably, THZ1 treatment resulted in a loss of tumor cellularity and disease regression (Figure 2J and 2K). Analysis of tumor tissues also demonstrated markedly decreased CTD phosphorylation of RNAPII and induced PARP cleavage, an indicator of apoptotic cell death (Figure 2L). These results indicate that CDK7 inhibition has potent anti-tumor activity in patient-derived TNBC in vivo[1].

In vitro IC50 for THZ2’s potency in binding to indicated CDK. The LanthaScreen Eu Kinase Binding assay (Invitrogen) was performed with indicated CDKs and their associated cyclins, in the presence of different concentration of THZ2. The IC50 values indicate the affinity of THZ2 towards the ATP binding pocket of CDK [1].

In the 96-well plate assay, cells are plated at a density of 2000 cells per well, and the following day, they are treated with different concentrations of THZ1 or THZ2. Cells are fixed and stained with crystal violet following a 48-hour incubation period. The staining is then removed by adding 10% acetic acid to each well, and the absorbance is measured at 590 nm using 750 nm as a reference.
Cell growth curve of indicated TNBC cell lines that were treated with increasing concentrations of THZ2 for 7 days. Upon harvest, cells were fixed, stained with crystal violet followed by extraction of the staining for the quantification of proliferation.

Mice: A single 400 rad dose of γ-irradiation is given to naked mice (CrTac:NCr-Foxn1nu) six hours prior to cell transplantation. The fourth pair of mice's mammary fat pads are injected with 100 μL of breast cancer cells per site after the cells are extracted and resuscitated in 40% Matrigel-Basement Membrane Matrix, LDEV-free. Manual calipers are used to measure tumors in two dimensions. The formula for calculating tumor volume is V=0.5× length× width× width. THZ2 (3 mg/mL, prepared in vehicle solutions) at a dose of 10 mg/kg intraperitoneally twice daily is administered to animals with established tumors (mean tumor volume of approximately 200 mm3), which are randomly divided into two groups and treated with vehicle (10% DMSO in D5W, 5% dextrose in water). Tumor volume is measured every two to three days. After being harvested, tumors are cut in half. One half is immediately snap frozen in liquid nitrogen for immunoblotting, and the other half is fixed in formalin for one night before being examined histopathologically and then in 70% ethanol.

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Stability of THZ1 and THZ2 in vivo. Mice were administered by tail vein injection of a single dose of THZ1 or THZ2, and blood samples were collected at different timepoints. Concentrations of THZ1 and THZ2 in plasma samples were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach.[1]

Although THZ1 exhibits high antiproliferative activity in primary TNBC cells, its poor in vivo stability (half-life of 45 minutes in mouse plasma) limits its application in in vivo studies. Therefore, we modified the structure of THZ1 by altering the acrylamide region, changing it from 4-acrylamide-benzamide to 3-acrylamide-benzamide, thus obtaining the analog THZ2 (Figure 2A). The pharmacokinetic properties of THZ2 were significantly improved, with a 5-fold increase in in vivo half-life (Figure 2B). Similar to THZ1, THZ2 selectively targets CDK7 (Figure 2C, Table S1) and effectively inhibits the growth of triple-negative breast cancer cells, but has no inhibitory effect on ER/PR-positive breast cancer cells (Figures 2D, 2E). Low nanomolar doses of THZ2 also effectively inhibited TNBC cell colony formation (IC50 approximately 10 nM; Figures 2F, S2C). Similar to THZ1, THZ2 can induce apoptosis in triple-negative breast cancer cells, but it has no effect on ER/PR-positive breast cancer cells or normal human cells (Figure S2A) and does not cause significant changes in the cell cycle (Figure S2C). Therefore, we screened out a THZ2 analog with better pharmacokinetic properties and comparable efficacy, and selected it for further research. [1]

[1]. CDK7-Dependent Transcriptional Addiction in Triple-Negative Breast Cancer. Cell. 2015 Sep 24;163(1):174-186.

Triple-negative breast cancer (TNBC) is a highly aggressive breast cancer with extremely high genetic complexity, but a relatively uniform transcriptional program. We hypothesized that TNBC may be highly dependent on the sustained transcription of a key group of genes in this gene expression program and therefore may be abnormally sensitive to transcriptional inhibitors. Using kinase inhibitors and CRISPR/Cas9-mediated gene editing, we found that triple-negative breast cancer cells (rather than hormone receptor-positive breast cancer cells) have an abnormal dependence on CDK7 (a transcriptional cyclin-dependent kinase). TNBC cells have a unique dependence on this transcriptional CDK and undergo apoptosis after CDK7 inhibition. The “Achilles cluster” of TNBC-specific genes is particularly sensitive to CDK7 inhibition and is often associated with super-enhancers. We concluded that CDK7 mediates the transcriptional dependence of a key gene cluster in TNBC and that CDK7 inhibition may be an effective way to treat this intractable cancer. [1]

C31H28CLN7O2

566.0527

565.199

C, 65.78; H, 4.99; Cl, 6.26; N, 17.32; O, 5.65

1604810-84-5

1604810-84-5

78357763

Light yellow to khaki solid powder

1.4±0.1 g/cm3

1.735

4.99

4

6

9

41

904

0

ClC1=C([H])N=C(N([H])C2C([H])=C([H])C([H])=C(C=2[H])N([H])C(C2C([H])=C([H])C([H])=C(C=2[H])N([H])C(/C(/[H])=C(\[H])/C([H])([H])N(C([H])([H])[H])C([H])([H])[H])=O)=O)N=C1C1=C([H])N([H])C2=C([H])C([H])=C([H])C([H])=C12

FONRCZUZCHXWBD-VGOFMYFVSA-N

InChI=1S/C31H28ClN7O2/c1-39(2)15-7-14-28(40)35-21-9-5-8-20(16-21)30(41)36-22-10-6-11-23(17-22)37-31-34-19-26(32)29(38-31)25-18-33-27-13-4-3-12-24(25)27/h3-14,16-19,33H,15H2,1-2H3,(H,35,40)(H,36,41)(H,34,37,38)/b14-7+

N-[3-[[5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl]amino]phenyl]-3-[[(E)-4-(dimethylamino)but-2-enoyl]amino]benzamide

THZ-2; THZ 2; 1604810-84-5; N-[3-[[5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl]amino]phenyl]-3-[[(E)-4-(dimethylamino)but-2-enoyl]amino]benzamide; CHEMBL4303287; (E)-N-(3-((5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl)amino)phenyl)-3-(4-(dimethylamino)but-2-enamido)benzamide; THZ2

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 100 mg/mL (~176.7 mM)
Ethanol: ˂1 mg/mL (NaN mM)
Water: ˂1 mg/mL (NaN mM)

Solubility in Formulation 1: ≥ 2.17 mg/mL (3.83 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 21.7 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.17 mg/mL (3.83 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 21.7 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)