- 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]

- 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]

- 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]

- 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]

The provided document does not contain detailed pharmacokinetic parameters (e.g., half-life, Cmax, AUC, bioavailability) for KB-0742. However, 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.

Istisociclib is a highly bioavailable, selective serine/threonine cyclin-dependent kinase 9 (CDK9) inhibitor. CDK9 is the catalytic subunit of positive transcription elongation factor b (PTEF-b; PTEFb), an RNA polymerase II (RNA Pol II) elongation factor, and possesses potential antitumor activity. After oral administration, istisociclib targets and blocks the phosphorylation and kinase activity of CDK9, thereby preventing PTEFb-mediated RNA Pol II activation and subsequently inhibiting the transcription of various anti-apoptotic proteins and oncogenic transcription factors, including MYC and androgen receptor (AR). This induces cell cycle arrest and apoptosis, and inhibits tumor cell proliferation. CDK9 regulates transcriptional elongation by phosphorylating serine 2 (p-Ser2-RNAPII) of RNA Pol II and is an important cofactor for various oncogenic transcription factors. It is upregulated in various tumor cell types and plays a key role in the transcriptional regulation of RNA polymerase II-mediated anti-apoptotic proteins. The survival of tumor cells depends on anti-apoptotic proteins.

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- 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]

C16H25N5

287.40

287.21

C, 66.86; H, 8.77; N, 24.37

2416873-83-9

2416874-75-2

146502834

White to off-white solid at room temperature

2.6

2

4

5

21

333

2

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

VYKCLMALANGCDF-STQMWFEESA-N

InChI=1S/C16H25N5/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/t12-,13-/m0/s1

(1S,3S)-3-N-(5-pentan-3-ylpyrazolo[1,5-a]pyrimidin-7-yl)cyclopentane-1,3-diamine

Istisociclib; KB-0742; 2416873-83-9; KB0742; F7J6KSY5I8

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: ~175 mg/mL (608.9 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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