CDK9/cyclinT1 6 nM (IC50)
- CDK9/cyclin T1 (Positive transcription elongation factor b, P-TEFb): KB-0742 is a potent and selective inhibitor of CDK9. The biochemical IC50 for CDK9/cyclin T1 is 6 nM (at 10 μM ATP). [1]
- Selectivity Profile: KB-0742 shows >50-fold selectivity over all CDK family members profiled (CDK1-8, 12-19) and >100-fold selectivity against cell-cycle CDKs (CDK1-6). Specific IC50 values (μM) for other CDKs: CDK1/cyclin A (>10), CDK2/cyclin A (>10), CDK4/cyclin D1 (>10), CDK6/cyclin D1 (>10), CDK7/cyclin H (0.736), CDK8/cyclin C (0.721), CDK12/cyclin K (>10), CDK13/cyclin K (>10). [1]

- CDK9 Inhibition & Downstream Effects: In 22Rv1 prostate cancer cells, KB-0742 treatment reduces phosphorylation of RNA Polymerase II at Ser2 and Ser7 at low doses (0.1-0.5 μM, 6h). At the highest dose (2.5 μM), it also diminishes phosphorylation at Ser5. Total RNA Pol II and CDK9 protein levels are unaffected. In a time-course study (1 μM), KB-0742 reduces global AR-FL and AR-V protein levels starting at 6h, with complete depletion by 16h, accompanied by reduced AR phosphorylation at Ser81. [1]
- Growth Inhibition (GR50): In 22Rv1 prostate cancer cells, KB-0742 shows a GR50 of 0.18 μM. In MV-4-11 AML cells, the GR50 is 0.097 μM. [1]
- Apoptosis Induction: KB-0742 induces apoptosis in a dose-dependent manner in 22Rv1 cells, as measured by caspase-3/7 activation. Strong induction is observed at higher doses (≥3 μM), with little induction at sub-micromolar doses (0.3 μM). In MV-4-11 AML cells, apoptosis induction is more pronounced at lower concentrations compared to 22Rv1 cells. [1]
- Nascent Transcription Inhibition (SLAM-seq): In 22Rv1 cells, treatment with KB-0742 (1.2 μM) globally downregulates nascent mRNA transcription. By 8h, 95% of high-confidence actively transcribed genes show downregulated nascent transcription. The transcriptional response is similar to that of KI-ARv-03 (4 μM). The drug preferentially downregulates highly transcribed genes, including lineage transcription factors (e.g., FOXA1, SOX4, KLF6) and AR/PSA locus genes, consistent with targeting of super-enhancer-associated transcription. [1]

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RNA Pol II's downstream phosphorylation at Ser2 and Ser7 was significantly reduced, while phosphorylation at Ser5 was lowered, after 6 hours of treatment with KB-0742 (0.1–10 μM; 22Rv1 cells). Beginning around the 6-hour mark of treatment, there is a considerable reduction in the levels of the androgen receptor (AR)-FL and AR-V proteins worldwide, along with a decrease in phospho-AR (Ser81) levels[1]. Treatment with KB-0742 (48–72 hours) has cytostatic effects on leukemia and prostate cancer cell lines. With GR50s of 0.183 μM and 0.288 μM for 22Rv1 cells and MV-4-11 AML cells, respectively, KB -0742 exhibits antiproliferative activity[1]. KB-0742 quickly suppresses emerging transcription in 22Rv1 cells, specifically reducing short half-live transcripts and AR-driven cancerous programs[1].

- 22Rv1 Prostate Cancer Xenograft Model (Mouse): In male CB17-SCID mice bearing subcutaneous 22Rv1 tumors, oral administration of KB-0742 (3 mg/kg, QD, 21 days) is well-tolerated and shows tumor growth inhibition (TGI) of 46% (p=0.007). At 10 mg/kg QD, TGI is 58% (p<0.0001). At 30 mg/kg QD, TGI is 82% (p=0.000003). This is superior to docetaxel (15 mg/kg, i.p., QW, TGI = 70%). A scheduled regimen (30 mg/kg, 3 days on/4 days off) shows TGI of 58% (p<0.00005) and is better tolerated. [1]
- MV-4-11 AML Xenograft Model (Mouse): In female BALB/c nude mice bearing subcutaneous MV-4-11 tumors, oral KB-0742 at 25 mg/kg QD leads to 74% TGI (p<0.00002). Cyclic dosing at 60 mg/kg (3 days on/4 days off) results in 81% TGI (p<0.00001). Lower cyclic doses (15 and 30 mg/kg) show 24% and 40% TGI, respectively. All treatments are well-tolerated with no significant body weight changes. [1]

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In 22Rv1 human prostate cancer cell line-derived xenograft (CDX) models, KB-0742 (3-30 mg/kg; po; daily; over 21 days) dramatically reduces tumor burden while being well tolerated even at high doses[1].

- In Vitro Kinase Activity Assays (HotSpot Kinase Assay): KB-0742 was tested against a panel of 16 CDKs at Reaction Biology Corp. using their HotSpot Kinase Assay. Compounds were tested in 10-dose duplicates (3-fold serial dilution starting at 10 μM) at Km ATP concentrations for each kinase. The control compound staurosporine was used. Normalized data were plotted and IC50 values were calculated in GraphPad PRISM. For kinase-compound pairs where inhibition at 10 μM was <65% or IC50 was out of range, the value was set to 10 μM for heatmap representation. This panel confirmed the high selectivity of KB-0742 for CDK9 (IC50 = 6 nM). [1]
- Thermal Shift Assay (TSA): CDK9/cyclin T1 (2 μM) was assayed with KB-0742 (10 μM) and SYPRO-Orange dye. The temperature was raised at 3°C/min, and fluorescence was measured to assess thermal stabilization of the protein upon compound binding. [1]

- Cell Viability (CellTiter-Glo): Prostate cancer cells were plated in 384-well plates. After 24h, cells were treated with DMSO or KB-0742 for 72h. ATP content was measured using CellTiter-Glo as a proxy for cell viability. Raw luminescence values were averaged and normalized to DMSO controls. [1]
- Growth Rate Inhibition (GR) and Apoptosis (IncuCyte): Cells were plated in 96-well plates with IncuCyte Caspase-3/7 Green Apoptosis Assay Reagent and treated with DMSO or KB-0742. Plates were imaged every 3h for 72h (22Rv1) or 48h (MV-4-11) using an IncuCyte S3. Images were analyzed for confluence (GR metrics) and apoptotic signal (caspase-3/7 positive cells). GR50 values were calculated using the GRcalculator tool. [1]
- Western Blotting: Cells were lysed in RIPA buffer with protease/phosphatase inhibitors. Proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and probed with specific primary antibodies (e.g., anti-CDK9, anti-p-RNA Pol II Ser2, anti-AR, anti-AR pSer81). HRP- or fluorophore-conjugated secondary antibodies were used for detection on a ChemiDoc or Odyssey CLx system. GAPDH or β-actin served as loading controls. [1]
- SLAM-seq (Nascent Transcription Profiling): 22Rv1 cells were pulsed with 800 μM s4U for 30 min, then treated with DMSO, KB-0742 (1.2 μM), or KI-ARv-03 (4 μM) for 2, 4, or 8h. RNA was extracted, treated with iodoacetamide (IAA) to alkylate s4U-labeled RNA, and libraries were prepared using the QuantSeq 3' mRNA-Seq Library Prep Kit. Sequencing was performed on a NextSeq 500. Reads were aligned and processed using the SlamDunk pipeline to quantify nascent mRNA fractions. [1]
- RT-qPCR: For PSA (KLK3) expression, LNCaP cells were treated with compounds for 24h. Cells were lysed, and mRNA was reverse transcribed. qPCR was performed using PrimePCR probe assays for GAPDH (FAM) and KLK3 (Cy5) on a CFX384 Touch system. Fold change was calculated using the ΔΔCt method. [1]

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Western Blot Analysis[1]
Cell Types: 22Rv1 cells
Tested Concentrations: 0.1 μM, 0.5 μM, 1 μM, 10 μM
Incubation Duration: 6 hrs (hours)
Experimental Results: Significant reduction of downstream phosphorylation of RNA Pol II at Ser2 and Ser7, and diminished phosphorylation at Ser5.

- 22Rv1 Xenograft Model: Male CB17-SCID mice (6-8 weeks old) were implanted subcutaneously with 2x10⁶ 22Rv1 cells. When tumors reached ~170 mm³, mice were randomized (n=10/group). KB-0742 was formulated in 10% EtOH, 20% PEG400, and 70% (20% HPβCD) and administered orally (p.o.) at 3, 10, or 30 mg/kg once daily (QD) for 21 days. A separate group received 30 mg/kg on a 3-day on/4-day off schedule. Docetaxel (15 mg/kg, i.p., QW) was used as a comparator. Body weight was measured twice weekly, and tumor volume was calculated as V = 0.5 x a x b². Tumor growth inhibition (TGI) was calculated relative to vehicle. [1]
- MV-4-11 Xenograft Model: Female BALB/c nude mice (6-8 weeks old) were implanted subcutaneously with 1x10⁶ MV-4-11 cells. When tumors reached ~154 mm³, mice were randomized (n=10/group). KB-0742 was formulated as above and administered p.o. at 25 mg/kg QD for 21 days, or on a 3-day on/4-day off schedule at 15, 30, or 60 mg/kg. Tumor volume and body weight were monitored similarly. [1]

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Animal/Disease Models: Male CB17-SCID (severe combined immunodeficient) mouse injected with 22Rv1 human prostate cancer cells[1]
Doses: 3 mg/kg, 10 mg/kg, and 30 mg /kg
Route of Administration: po; daily; over 21 days
Experimental Results: Dramatically diminished tumor growth in castration-resistant prostate cancer (CRPC).

The compound is described as "orally bioavailable," as demonstrated by its in vivo efficacy following oral administration. The formulation used was 10% EtOH, 20% PEG400, and 70% (20% HPβCD). [1]

- In Vitro Selectivity: KB-0742 shows >50-fold selectivity over other CDKs, suggesting a reduced risk of off-target toxicity associated with pan-CDK inhibition. [1]
- In Vivo Tolerability (Body Weight Loss): In the 22Rv1 xenograft model, daily oral administration of KB-0742 at 3 and 10 mg/kg resulted in minimal body weight loss (<10%). At 30 mg/kg QD, body weight loss reached 15% over the course of treatment but stabilized after the first week. This was managed by using a 3-day on/4-day off schedule, which reduced weight loss to 7.5%. In the MV-4-11 model, no significant body weight changes were observed at any dose tested (up to 60 mg/kg cyclic). [1]
- Compound-Anchored Target Engagement (Pull-down): KB-0742 (as a free competitor) was able to displace CDK9 from KI-ARv-03-functionalized beads, confirming reversible target engagement. [1]

[1]. Modulating Androgen Receptor-Driven Transcription in Prostate Cancer with Selective CDK9 Inhibitors. Cell Chem Biol. 2020 Oct 20;S2451-9456(20)30380-9.

- Mechanism of Action (MOA): KB-0742 is a selective, ATP-competitive inhibitor of CDK9, the kinase subunit of the P-TEFb complex. CDK9 phosphorylates Ser2 of the RNA Pol II C-terminal domain (CTD), which is required for productive transcriptional elongation. Inhibition of CDK9 leads to global RNA Pol II stalling, which disproportionately affects short half-life transcripts and genes with high transcriptional output, including oncogenic drivers like AR, MYC, and lineage-specific transcription factors (e.g., FOXA1). In prostate cancer, this leads to reduced AR and AR-V7 protein expression and stability (via reduced Ser81 phosphorylation), disrupting AR-driven oncogenic programs and inducing cell cycle arrest and apoptosis. [1]
- Discovery Path: KB-0742 is the result of medicinal chemistry optimization of the SMM-derived hit KI-ARv-03. Key structural changes include a branched 3-pentane extension on the pyrazolopyrimidine core and inversion of stereochemistry on the cyclopentane-1,3-diamine, which increased potency while maintaining selectivity. [1]
- Comparison to Parent Compound: KB-0742 is approximately 18-fold more potent in inhibiting 22Rv1 cell proliferation (GR50 0.18 μM vs. KI-ARv-03 GR50 3.26 μM) and shows improved biochemical potency (CDK9 IC50 6 nM vs. KI-ARv-03 IC50 68 nM). [1]

C16H27CL2N5

360.3251

359.164

C, 53.33; H, 7.55; Cl, 19.68; N, 19.44

2416874-75-2

2416874-75-2 (HCl);2416873-83-9;

155819527

Light yellow to yellow solid powder

4

4

5

23

333

2

CCC(CC)C1=NC2=CC=NN2C(=C1)N[C@H]3CC[C@@H](C3)N.Cl.Cl

OTUPXOLLHVLWQF-NJHZPMQHSA-N

InChI=1S/C16H25N5.2ClH/c1-3-11(4-2)14-10-16(19-13-6-5-12(17)9-13)21-15(20-14)7-8-18-21;;/h7-8,10-13,19H,3-6,9,17H2,1-2H3;2*1H/t12-,13-;;/m0../s1

(1S,3S)-N1-(5-(pentan-3-yl)pyrazolo[1,5-a]pyrimidin-7-yl)cyclopentane-1,3-diamine dihydrochloride

KB-0742; KB0742; KB 0742 2HCl; Istisociclib; KB-0742 HCl; 2416873-83-9; KB0742; F7J6KSY5I8; KB 0742 dihydrochloride; KB 0742 hydrochloride;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

H2O : 100 mg/mL (277.52 mM)
DMSO : 62.5 mg/mL (173.45 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.77 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 20.8 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: ≥ 2.08 mg/mL (5.77 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (5.77 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 100 mg/mL (277.52 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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