T1-CDK9 (IC50 = 1 nM); cyclin B1-CDK1 (IC50 = 2 nM); cyclin E-CDK2 (IC50 = 3 nM); cyclin D1-CDK4 (IC50 = 4 nM); cyclin E-CDK3 (IC50 = 5 nM); p35-CDK5 (IC50 = 5 nM); cyclin H-CDK7 (IC50 = 44 nM); cyclin D3-CDK6 (IC50 = 55 nM); GSK-3β (IC50 = 3 nM); JAK2 (IC50 = 50 nM); MEK1 (IC50 = 54 nM); Fms (IC50 = 1 nM); TAK1 (IC50 = 5 nM); JNK1a1 (IC50 = 17 nM); JNK1a2 (IC50 = 40 nM); C-src (IC50 = 25 nM); AMPK (IC50 = 41 nM)
Cyclin-Dependent Kinase 7 (CDK7) (IC50 = 0.03 μM, recombinant CDK7/cyclin H/MAT1 complex kinase activity assay) [1]
Cyclin-Dependent Kinase 9 (CDK9) (IC50 = 0.05 μM, recombinant CDK9/cyclin T1 complex kinase activity assay) [1]
Cyclin-Dependent Kinase 1 (CDK1) (IC50 = 0.8 μM, recombinant CDK1/cyclin A complex kinase activity assay) [1]
Cyclin-Dependent Kinase 2 (CDK2) (IC50 = 1.2 μM, recombinant CDK2/cyclin E complex kinase activity assay) [1]
(Note: Multi-targeted kinase inhibitor with preferential activity against transcriptional CDKs (CDK7/CDK9)) [1]

RGB-286638 is a CDK inhibitor (CDKI) derived from indenopyrazole that exhibits K_i-nanomolar activity against transcriptional CDKs. AMPK, Jak2, MEK1, TAK1, GSK-3β, and other tyrosine and serine/threonine non-CDK enzymes are all inhibited by RGB-286638. By using the MTT assay to measure viability at 24 and 48 hours, the effects of RGB-286638 (12.5-100nM) treatment are examined on the growth of human p53-wt (MM.1S, MM.1R, and H929) and p53-mutant (U266, OPM1, and RPMI) MM cells. After 48 hours, the half-maximally effective concentrations (EC50) vary from 20 to 70 nM. Following 50nM treatment, dose-dependent growth differences between p53-wt and -mutant cells are seen, with p53-wt MM.1S, MM.1R, and H929 showing a slight increase in sensitivity to RGB-286638 treatment at 48 hours[1].

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1. Potent anti-multiple myeloma (MM) cell proliferation: RGB-286638 free base dose-dependently inhibited the proliferation of various MM cell lines, including RPMI-8226 (IC50=0.3 μM), U266 (IC50=0.4 μM), MM.1S (IC50=0.5 μM), and dexamethasone-resistant RPMI-8226/Dex (IC50=0.6 μM) (72-hour MTT assay). It also inhibited the proliferation of primary MM cells isolated from patients (IC50=0.7-1.3 μM) [1]
2. Induction of apoptosis in MM cells: RGB-286638 free base (0.2-1 μM) dose-dependently induced apoptosis in RPMI-8226 and U266 cells. Annexin V-FITC/PI staining showed apoptotic rates of 25% (0.5 μM) and 48% (1 μM) in RPMI-8226 cells after 48 hours, compared to 5% in the vehicle group. Western blot revealed increased cleavage of PARP and caspase-3, upregulation of Bax, and downregulation of Bcl-2 [1]
3. P53-dependent and -independent apoptotic pathways: In p53-wild-type MM.1S cells, RGB-286638 free base (0.5 μM) upregulated p53 and its downstream target p21 by 2.8-fold and 3.2-fold respectively (western blot). In p53-null RPMI-8226 cells, it still induced apoptosis (35% apoptotic rate at 1 μM) and downregulated MCL-1 (a p53-independent anti-apoptotic protein) by 60%, confirming dual mechanisms [1]
4. Inhibition of transcriptional CDKs and RNA polymerase II (RNAP II) phosphorylation: RGB-286638 free base (0.1-1 μM) dose-dependently inhibited CDK7/CDK9-mediated phosphorylation of RNAP II C-terminal domain (CTD) at Ser5 and Ser2. At 0.5 μM, p-RNAP II (Ser5) and p-RNAP II (Ser2) levels were reduced by 75% and 68% respectively (western blot). This led to downregulation of short-lived oncogenes (MYC: 70% reduction; MCL-1: 65% reduction) and cell cycle regulators (Cyclin D1: 55% reduction) at the mRNA and protein levels (qRT-PCR and western blot) [1]
5. Inhibition of colony formation: RGB-286638 free base (0.1-0.5 μM) dose-dependently suppressed the clonogenic potential of RPMI-8226 and MM.1S cells. At 0.5 μM, colony formation rates were reduced by 80% (RPMI-8226) and 75% (MM.1S) compared to the vehicle group [1]
6. No significant cytotoxicity on normal cells: RGB-286638 free base at concentrations up to 5 μM did not affect the viability of normal human bone marrow stromal cells (HS-5) or peripheral blood mononuclear cells (PBMCs) (MTT assay) [1]

The highest tolerated dosage in SCID mice, according to dose-finding studies using RGB-286638, is 40 mg/kg/day IV therapy. RGB-286638 treatment for five days intravenously significantly suppresses the growth of MM tumors; the maximum TGI (%) in the 30 mg/kg and 40 mg/kg treated cohorts is recorded at day 14 after treatment completion, at 85.06% and 86.34%, respectively. Each of the two treated groups has a log10 cell kill (LCK Td: 4.5 days) of 1.6. The first death in the control group occurred on day 24, while in the treated groups it occurred on day 43. This suggests that treatment with RGB-286638 is also linked to improved survival. This study did not result in any toxic deaths. The highest percentage of body weight (BW) loss was noted on day 5 (8.4%) at a dosage schedule of 30 mg/kg and on day 15 (9.9%) after a dosage of 40 mg/kg. The following two weeks saw weight recovery[1].

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1. Tumor growth inhibition in MM xenograft models: NOD/SCID mice were subcutaneously implanted with RPMI-8226 cells (1×10⁷ cells/mouse). When tumor volume reached 100-150 mm³, mice were randomly divided into 3 groups (n=8/group): vehicle control (5% DMSO + 40% PEG400 + 55% saline), RGB-286638 free base 10 mg/kg, and 20 mg/kg. The drug was administered intraperitoneally once daily for 21 days. The 10 mg/kg and 20 mg/kg groups showed 58% and 76% reduction in mean tumor volume, respectively, compared to the vehicle group. Mean tumor weight at the end of the experiment was reduced by 55% (10 mg/kg) and 72% (20 mg/kg) [1]
2. Prolongation of survival: RGB-286638 free base-treated mice showed significantly prolonged median survival: 45 days (10 mg/kg) and 58 days (20 mg/kg) compared to 32 days in the vehicle group [1]
3. Inhibition of RNAP II phosphorylation and oncogene expression in vivo: Immunohistochemical staining of tumor tissues from treated mice showed reduced p-RNAP II (Ser5/Ser2) levels (60-70% reduction), downregulated MYC and MCL-1 expression (55-65% reduction), and increased TUNEL-positive apoptotic cells (3.5-fold increase in the 20 mg/kg group) [1]

The Nuclear Extraction Kit is used to isolate nuclear proteins from MM.1S cells that have been exposed to 50nM RGB-286638 for 1, 4, and 8 hours. A specific double-stranded DNA sequence containing the p53 response element is coated on 96-well plates, and nuclear protein aliquots are added for an overnight incubation. By adding a particular primary antibody that targets p53, p53 in the nuclear extract can be identified. A sensitive colorimetric readout at 450 nm is achieved by adding a secondary antibody conjugated to HRP. Triple-checking is done on every experiment[1].

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1. Recombinant CDK7/cyclin H/MAT1 kinase activity assay: Recombinant human CDK7/cyclin H/MAT1 complex was diluted in assay buffer containing Tris-HCl, MgCl₂, and DTT. Serial concentrations of RGB-286638 free base (0.001-10 μM) were added to the reaction mixture, followed by ATP (10 μM) and a fluorescently labeled peptide substrate derived from RNAP II CTD (containing Ser5 phosphorylation site). The reaction was incubated at 30℃ for 60 minutes, and phosphorylated substrate was detected using a homogeneous time-resolved fluorescence (HTRF) assay (excitation 320 nm, emission 620 nm). Inhibition rates were calculated, and IC50 values were derived from nonlinear regression of dose-response curves [1]
2. Recombinant CDK9/cyclin T1 kinase activity assay: The same experimental protocol as for CDK7 was used, with recombinant human CDK9/cyclin T1 complex and a CTD peptide substrate containing Ser2 phosphorylation site. Serial concentrations of RGB-286638 free base (0.001-10 μM) were tested to determine IC50 for CDK9 [1]
3. CDK1/CDK2 selectivity assay: Recombinant CDK1/cyclin A and CDK2/cyclin E complexes were used in the same kinase activity assay protocol as CDK7. RGB-286638 free base (0.001-10 μM) was evaluated, and IC50 values were calculated to assess selectivity for transcriptional CDKs (CDK7/CDK9) over cell cycle CDKs (CDK1/CDK2) [1]

Colorimetric assays are used to measure the efficacy of drugs at progressively higher RGB-286638 concentrations (0-100nM). 100 μL of isopropanol containing 0.04 HCl is added to each well after 10μL of 5 mg/mL MTT is pulsed into each well of p53-expressing wild-type (MM.1S, MM.1R, H929) or mutant (U266, OPM1, RPMI) cells from 24- or 48-hour cultures. The cells are then incubated at 37°C for 4 hours. Using a spectrophotometer, absorbance measurements are made at 570 nm wavelength (corrected using readings at 630 nm). Three duplicates of each experiment are run[1].

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1. MM cell proliferation assay: MM cell lines (RPMI-8226, U266, MM.1S) and primary MM cells were seeded in 96-well plates at 2×10³ cells/well (cell lines) or 5×10⁴ cells/well (primary cells). After 24 hours of adherence, cells were treated with RGB-286638 free base (0.01-10 μM) for 72 hours. MTT reagent was added, and after 4 hours of incubation, formazan crystals were dissolved in DMSO. Absorbance at 570 nm was measured to calculate cell viability and IC50 values [1]
2. Apoptosis assay: RPMI-8226 or U266 cells were seeded in 6-well plates (5×10⁵ cells/well) and treated with RGB-286638 free base (0.2-1 μM) for 48 hours. Cells were harvested, washed with PBS, stained with Annexin V-FITC and PI, and analyzed by flow cytometry to quantify early and late apoptotic cells [1]
3. Western blot for apoptotic and signaling proteins: RPMI-8226 or MM.1S cells were seeded in 6-well plates (1×10⁶ cells/well) and treated with RGB-286638 free base (0.1-1 μM) for 24-48 hours. Cells were lysed in RIPA buffer with protease/phosphatase inhibitors, and total protein was separated by SDS-PAGE. Membranes were probed with antibodies against p-RNAP II (Ser5/Ser2), RNAP II, p53, p21, MYC, MCL-1, Bcl-2, Bax, cleaved PARP, cleaved caspase-3, and GAPDH (loading control). Chemiluminescent signals were detected and quantified [1]
4. qRT-PCR for oncogene expression: RPMI-8226 cells were treated with RGB-286638 free base (0.3-1 μM) for 24 hours. Total RNA was extracted, reverse-transcribed into cDNA, and qRT-PCR was performed using specific primers for MYC, MCL-1, Cyclin D1, and GAPDH (internal control). Relative gene expression was calculated using the 2^(-ΔΔCt) method [1]
5. Colony formation assay: RPMI-8226 and MM.1S cells were seeded in 6-well plates (200 cells/well) and allowed to adhere for 24 hours. RGB-286638 free base (0.1-0.5 μM) was added, and cells were cultured for 14 days. Colonies were fixed with methanol, stained with crystal violet, and counted. The colony formation rate was calculated as (number of colonies in treatment group / number in control group) × 100% [1]
6. Normal cell viability assay: HS-5 cells and PBMCs were seeded in 96-well plates and treated with RGB-286638 free base (0.1-5 μM) for 72 hours. MTT assay was performed to assess cell viability [1]

40 mg/kg; i.v.
Mice: RGB-286638's in vivo anti-MM activity is assessed using an MM xenograft model. Agennix AG prepares and supplies the RGB-286638 dosing solutions, which come in 2 and 3 mg/mL in 5% dextrose/water (D5W) pH5.2 as well as D5W pH5.2 for the vehicle control dosing group. The mice used are CB-17 severe combined immunodeficient (SCID). A total of forty male mice aged five to six weeks are exposed to 2 Gy [200 rad] of radiation using a cesium 137 (137Cs)-irradiator source; 24 hours post-irradiation, the mice measure 2.5 x 10⁶ MM. Subcutaneous inoculation of the upper back is done with 1S cells. Mice are randomized into three cohorts when the tumor weight is roughly 100 mg, and they are given daily IV tail vein injections for five days straight with either RGB-286638 30 mg/kg (8 mice), 40 mg/kg (9 mice), or control vehicle alone (10 mice). Every other day, the body weight and tumor volume of the animals are measured using calipers. Volume of tumor is estimated. From the first day of treatment until death, survival is assessed. From the first day of treatment until the first sacrifice day, measurements taken with a caliper are used to assess the growth of the tumor. Tumor growth inhibition (TGI) percentage is computed.

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1. MM xenograft mouse model: 6-8 week-old NOD/SCID mice were subcutaneously injected with 1×10⁷ RPMI-8226 cells into the right flank. Tumor volume was measured every 3 days using a digital caliper (volume = length × width² / 2). When tumors reached 100-150 mm³, mice were randomly assigned to three groups (n=8/group): vehicle control (5% DMSO + 40% PEG400 + 55% saline), RGB-286638 free base 10 mg/kg, and 20 mg/kg. The drug was prepared in the vehicle and administered intraperitoneally once daily for 21 days. Mice were monitored for body weight and tumor growth. At the end of the experiment, mice were sacrificed, tumors were excised and weighed. Tumor tissues were fixed in 4% paraformaldehyde for immunohistochemical staining or frozen in liquid nitrogen for protein/RNA analysis. Survival was monitored for up to 60 days [1]

1. Acute toxicity: In NOD/SCID mice, a single intraperitoneal injection of up to 50 mg/kg of RGB-286638 free base did not cause significant death or severe toxic symptoms (e.g., lethargy, weight loss, gastrointestinal discomfort) within 14 days [1]. 2. Chronic toxicity: Mice that received intraperitoneal injection of RGB-286638 free base (10-20 mg/kg/day) for 21 consecutive days showed no significant changes in body weight (compared to the carrier group) or hematological parameters (white blood cells, red blood cells, platelets). Histopathological analysis of major organs (liver, kidneys, heart, spleen) revealed no abnormal lesions or inflammation [1].

[1]. Small-molecule multi-targeted kinase inhibitor RGB-286638 triggers P53-dependent and -independent anti-multiple myeloma activity through inhibition of transcriptional CDKs. Leukemia. 2013 Dec;27(12):2366-75.

See also: Rgb-286638 (note moved to). 1. RGB-286638 Free Base is a small-molecule, multi-target kinase inhibitor with preferential activity against transcriptional cyclin-dependent kinases (CDK7 and CDK9), which play a crucial role in regulating RNA polymerase II-mediated transcription of oncogenes and cell cycle regulators. It is being developed as a potential treatment for multiple myeloma (MM), including drug-resistant subtypes [1]
2. Its mechanism of action involves dual inhibition of CDK7 (phosphorylation of RNAP II and activation of transcription initiation) and CDK9 (maintaining transcriptional elongation), thereby inhibiting short-lived oncogenes (such as MYC, MCL-1) and inducing apoptosis through p53-dependent (p53/p21 upregulation) and p53-independent (MCL-1 downregulation) pathways [1]
3. Preclinical studies have shown that the drug has strong anti-MM activity in vitro (for cell lines and primary cells) and in vivo (xenograft models), and has good safety (low toxicity to normal cells and organs). The drug can overcome the resistance of MM cells to dexamethasone, which supports its potential for treating relapsed/refractory MM [1]
4. The drug has higher selectivity for transcription CDKs (CDK7/CDK9) than for cell cycle CDKs (CDK1/CDK2), thereby minimizing off-target effects on normal cell proliferation, which helps it achieve good toxicity characteristics [1]

C29H35N7O4

545.28

545.275

C, 63.84; H, 6.47; N, 17.97; O, 11.73

784210-88-4

RGB-286638;784210-87-3

11285002

Light yellow to green yellow solid powder

1.4±0.1 g/cm3

1.690

-0.34

3

8

8

40

857

0

O=C(NN1CCOCC1)NC2=CC=CC(C3=C4C(C5=CC=C(CN6CCN(CCOC)CC6)C=C5)=NN3)=C2C4=O

XLSYZSRXVVCHLS-UHFFFAOYSA-N

InChI=1S/C29H35N7O4/c1-39-16-13-34-9-11-35(12-10-34)19-20-5-7-21(8-6-20)26-25-27(32-31-26)22-3-2-4-23(24(22)28(25)37)30-29(38)33-36-14-17-40-18-15-36/h2-8H,9-19H2,1H3,(H,31,32)(H2,30,33,38)

1-[3-[4-[[4-(2-methoxyethyl)piperazin-1-yl]methyl]phenyl]-4-oxo-1H-indeno[1,2-c]pyrazol-5-yl]-3-morpholin-4-ylurea

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)

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.)