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Purity: ≥98%
TP-3654 is a novel and potent inhibitor of Pim-1 and Pim-3 (proviral integration site for moloney murine leukemia virus) kinase that is potentially useful for the treatment of various cancers such as prostate cancer, acute myeloid leukemia, multiple sclerosis and psoriasis. As a second-generation PIM inhibitor, TP-3654 displays submicromolar activity with Ki values of 5 and 42 nM for Pim-1 and Pim-3, respectively in pharmacodynamic biomarker modulation, cell proliferation studies, and colony formation assays using the UM-UC-3 bladder cancer cell line. TP-3654 demostrates favorable human ether-à-go-go-related gene and cytochrome P450 inhibition profiles compared with the first-generation PIM inhibitor, SGI-1776, and exhibits oral bioavailability. In vivo xenograft studies using a bladder cancer cell line show that PIM kinase inhibition can reduce tumor growth, suggesting that PIM kinase inhibitors may be active in human urothelial carcinomas.
| Targets |
Pim-1 (Ki=5 nM); Pim-2 (Ki=239 nM); Pim-3 (Ki=42 nM)
PIM kinases (PIM-1, PIM-2, PIM-3) (Ki values of TP-3654 for PIM kinases were comparable to IC50 determinations; PIM-1–specific cellular EC50 of TP-3654 was 67 nM on average in HEK-293 cells transfected with BAD and PIM-1 (phospho-BAD (S112) Surefire assay)) [1] |
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| ln Vitro |
In the PIM-1/BAD overexpression system, TP-3654 exhibits strong PIM-1 specific cellular action with an average EC50 of 67 nM. TP-3654 therapy lowers phospho-BAD levels in vitro using UM-UC-3 bladder cancer cell line. T24 and UM-UC3 cell colony growth is inhibited by TP-3654, indicating that PIM-1 is required for the proliferation of these two cell lines[1].
1. TP-3654 exhibited submicromolar activity in pharmacodynamic biomarker modulation, cell proliferation studies, and colony formation assays using the UM-UC-3 bladder cancer cell line [1] 2. In HEK293T cells transfected with empty vector, PIM1, PIM1 kinase dead (PIM1KD, K67M) alone or in combination with BAD, PIM-1 overexpression increased cellular phospho-BAD (pBAD) levels at Ser¹¹², while TP-3654 treatment decreased pBAD levels in a dose-dependent manner [1] 3. UM-UC3 cells treated with TP-3654 (0.03, 0.3, 1, 3 μM) for 12 hours showed reduced levels of S112 phosphorylated BAD and Th37/46 phosphorylated 4EBP1, with no significant changes in total BAD and total 4EBP1 levels [1] 4. In two-dimensional colony formation assays, TP-3654 inhibited the growth of T24 bladder cancer cells with an average EC50 of 1.1 ± 0.4 μM (n = 4) and UM-UC3 bladder cancer cells with an average EC50 of 2.2 ± 0.2 μM (n = 2) [1] 5. TP-3654 displayed favorable human ether-à-go-go-related gene (hERG) and cytochrome P450 inhibition profiles compared with the first-generation PIM inhibitor SGI-1776 [1] |
| ln Vivo |
With no appreciable changes in body weight or gross adverse toxicity, oral dosage of 200 mg/kg TP-3654 significantly reduces both UM-UC-3 and PC-3 tumor growth as measured by volume (caliper) and final tumor weight[1].
1. UM-UC-3 bladder carcinoma cells (5 × 10⁶) were implanted into Nu/Nu mice (12 mice per group); oral administration of TP-3654 at 200 mg/kg once daily (QD) for 3 weeks (5 days on, 2 days off) significantly reduced tumor growth (caliper measurements: P = .0028) and tumor weights at the end of the study (P = .02), with no significant change in body weights [1] 2. PC-3 prostate adenocarcinoma cells (7.5 × 10⁶) were implanted into male Nu/Nu mice (12 mice per group); oral administration of TP-3654 at 200 mg/kg QD for 3 weeks (5 days on, 2 days off) significantly reduced tumor growth (caliper measurements: P = .007) and tumor weights (P = .0002), with no significant change in body weights [1] 3. Parental 22RV1 and 22RV1/PIM-1 cells (5 × 10⁶) were implanted into Nu/Nu mice (10 mice per group); intraperitoneal injection of SGI-9481 (25 mg/kg QD for 3 weeks, 5 days on, 2 days off) reduced tumor growth (P = .023), while no direct data of TP-3654 on 22RV1 xenografts were provided [1] 4. Parental NIH-3 T3 and NIH-3 T3/PIM-2 cells (5 × 10⁶) were implanted into Nu/Nu mice (10 mice per group); intraperitoneal injection of SGI-9481 (25 mg/kg QD for 3 weeks, 5 days on, 2 days off) reduced tumor growth (P = .002) [1] |
| Enzyme Assay |
1. Kinase selectivity assay of TP-3654: The IC50 values for the most potently inhibited kinases were determined, and Ki values for PIM kinases were measured (Ki values were comparable to IC50 determinations); the specific experimental process involved incubating TP-3654 with different kinases and detecting kinase activity to calculate inhibitory concentrations [1]
2. PIM-1–specific cellular EC50 assay: HEK-293 cells were transfected with BAD and PIM-1; after transfection, cells were treated with different concentrations of TP-3654, and phospho-BAD (S112) levels were detected using a Surefire assay; EC50 values were calculated using GraphPad Prism software (average EC50 = 67 nM from four independent experiments) [1] |
| Cell Assay |
TP-3654 is tested against 336 kinases at 10 μM ATP concentration. IC50 determinations of PI3K and all inhibited kinases are performed using 10-dose, three-fold serial dilutions of TP-3654 starting with 10 μM at Km ATP concentrations for each kinase.[1].
1. PIM-1 shRNA transfection assay in UM-UC-3 cells: UM-UC-3 bladder carcinoma cells were transfected with PIM-1 shRNAs or control shRNA (nontarget) using lentiviral particles overnight; the medium was changed, and cells were collected at 48 hours posttransduction for RNA extraction (to detect PIM-1 mRNA levels) and protein extraction (to detect PIM-1 protein levels); a portion of cells were seeded in six-well plates at 500 cells per well, fixed and stained after 10 days of growth for two-dimensional colony formation assay (P = .0028 for PIM-1 mRNA, P = .0002 for colony formation) [1] 2. Western blot assay for pBAD and 4EBP1 in HEK293T cells: HEK293T cells were transfected with empty vector, PIM1, PIM1KD (K67M) alone or in combination with BAD using a transfection reagent; 24 hours posttransfection, cells were serum starved overnight and lysed; soluble proteins were extracted, and Western blot was performed to detect phospho-BAD (Ser¹¹²) levels using a fluorescence scanner [1] 3. Western blot assay for pBAD and 4EBP1 in UM-UC3 cells: UM-UC3 cells were treated with TP-3654 at concentrations of 3, 1, 0.3, and 0.03 μM for 12 hours; cells were lysed, and separate identical blots were probed with antibodies against S112 phosphorylated BAD, total BAD, Th37/46 phosphorylated 4EBP1, and total 4EBP1 to detect protein phosphorylation and total protein levels [1] 4. Colony formation assay in T24 and UM-UC3 cells: T24 bladder cancer cells were seeded at 300 cells per well, and UM-UC3 bladder cancer cells were seeded at 500 cells per well in 12-well plates; the next day, cells were treated with titrated concentrations of TP-3654; cells were grown for 10 days (T24) or 6 days (UM-UC3), stained for imaging, and lysed for quantitation by absorbance at 560 nm to calculate EC50 values [1] |
| Animal Protocol |
The study involved mice with tumors measuring 100-200 mm3 and randomized to receive oral dosing of TP-3654 or vehicle control every day for 5 days, with 2 days off for 18-21 days, and tumor volumes and body weights determined weekly[1].
1. UM-UC-3 bladder carcinoma xenograft model: Nu/Nu mice were implanted with 5 × 10⁶ UM-UC-3 cells per mouse (12 mice per group); TP-3654 was administered orally at 200 mg/kg QD for 3 weeks (5 days of dosing followed by 2 days off per week); tumor measurements were obtained by caliper, and tumor weights were recorded at the end of the study; body weights of mice were monitored throughout the experiment [1] 2. PC-3 prostate adenocarcinoma xenograft model: Male Nu/Nu mice were implanted with 7.5 × 10⁶ PC-3 cells per mouse (12 mice per group); TP-3654 was administered orally at 200 mg/kg QD for 3 weeks (5 days on, 2 days off); tumor measurements (caliper) and tumor weights were assessed, and body weights were monitored [1] 3. 22RV1/PIM-1 xenograft model: Nu/Nu mice were implanted with 5 × 10⁶ parental 22RV1 or 22RV1/PIM-1 cells per mouse (10 mice per group); SGI-9481 (25 mg/kg) was administered by intraperitoneal injection QD for 3 weeks (5 days on, 2 days off); tumor measurements and body weights were recorded (no TP-3654 dosing data) [1] 4. NIH-3 T3/PIM-2 xenograft model: Nu/Nu mice were implanted with 5 × 10⁶ parental NIH-3 T3 or NIH-3 T3/PIM-2 cells per mouse (10 mice per group); SGI-9481 (25 mg/kg) was administered by intraperitoneal injection QD for 3 weeks (5 days on, 2 days off); tumor measurements and body weights were recorded (no TP-3654 dosing data) [1] 5. Oral pharmacokinetic assay in rats: Female Sprague-Dawley (SD) rats were dosed with TP-3654 either by intravenous (IV) injection at 2 mg/kg or orally at 40 mg/kg; the drug was formulated with 10% polysorbate 20 to achieve maximum exposure; plasma levels of TP-3654 were determined using liquid chromatography-mass spectrometry (LC-MS) [1] |
| ADME/Pharmacokinetics |
1. TP-3654 showed good oral bioavailability in female SD rats; when formulated with 10% polysorbate 20, the highest drug exposure (AUC) was obtained at an oral dose of 40 mg/kg compared to an intravenous dose of 2 mg/kg [1].
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| References | |
| Additional Infomation |
Nuvisertib is an orally administered, second-generation selective ATP-competitive inhibitor that inhibits Moloney murine leukemia virus proviral integration site (PIM) kinase, exhibiting potential antitumor activity. After oral administration, nuvisertib selectively binds to and inhibits PIM kinase activation. This prevents PIM-mediated signaling pathway activation and inhibits the proliferation of PIM-overexpressing cells. PIMs are constitutively active proto-oncogene serine/threonine kinases, upregulated in various cancers, and play a crucial role in tumor cell proliferation and survival.
1. TP-3654 is a second-generation small molecule inhibitor of PIM kinases (PIM-1, PIM-2, PIM-3). PIM kinases are serine/threonine kinases involved in cancer cell signaling pathways (apoptosis inhibitors, positive regulators of the G1-S phase cell cycle) [1] 2. PIM kinases are upregulated in various cancers (lymphoma, leukemia, multiple myeloma, prostate cancer, gastric cancer, head and neck cancer, urothelial carcinoma), and overexpression is associated with poor prognosis [1] 3. High expression of PIM-1, PIM-2 and PIM-3 has been observed in both non-invasive and invasive tumors. Invasive urothelial carcinoma (137 surgical biopsy/resection specimens) [1] 4. TP-3654 is a potential treatment for human urothelial carcinoma because PIM kinase inhibitors can reduce tumor growth in bladder cancer xenograft models [1] 5. Compared with the first-generation PIM inhibitor SGI-1776, TP-3654 has advantages in hERG/cytochrome P450 inhibition and oral bioavailability [1] |
| Molecular Formula |
C22H25F3N4O
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|---|---|---|
| Molecular Weight |
418.4553
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| Exact Mass |
418.198
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| CAS # |
1361951-15-6
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| Related CAS # |
1418143-09-5 (HCl);1361951-15-6;
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| PubChem CID |
66598080
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| Appearance |
White to light yellow solid powder
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| LogP |
4.6
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
30
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| Complexity |
579
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| Defined Atom Stereocenter Count |
0
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| SMILES |
FC(C1=C([H])C([H])=C([H])C(=C1[H])C1=C([H])N=C2C([H])=C([H])C(=NN21)N([H])C1([H])C([H])([H])C([H])([H])C([H])(C(C([H])([H])[H])(C([H])([H])[H])O[H])C([H])([H])C1([H])[H])(F)F
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| InChi Key |
XRNVABDYQLHODA-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C22H25F3N4O/c1-21(2,30)15-6-8-17(9-7-15)27-19-10-11-20-26-13-18(29(20)28-19)14-4-3-5-16(12-14)22(23,24)25/h3-5,10-13,15,17,30H,6-9H2,1-2H3,(H,27,28)
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| Chemical Name |
2-[4-[[3-[3-(trifluoromethyl)phenyl]imidazo[1,2-b]pyridazin-6-yl]amino]cyclohexyl]propan-2-ol
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| Synonyms |
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO: ~84 mg/mL ( 200.73 mM）
Water: <6 mg/mL Ethanol: Insoluble |
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| Solubility (In Vivo) |
Solubility in Formulation 1: 2.5 mg/mL (5.97 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 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. Solubility in Formulation 2: ≥ 2.08 mg/mL (4.97 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. View More
Solubility in Formulation 3: ≥ 2.08 mg/mL (4.97 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.3897 mL | 11.9486 mL | 23.8971 mL | |
| 5 mM | 0.4779 mL | 2.3897 mL | 4.7794 mL | |
| 10 mM | 0.2390 mL | 1.1949 mL | 2.3897 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
| NCT Number | Recruitment | interventions | Conditions | Sponsor/Collaborators | Start Date | Phases |
| NCT03715504 | Completed | Drug: TP-3654 | Advanced Solid Tumors | Sumitomo Pharma Oncology, Inc. | April 16, 2019 | Phase 1 |
| NCT04176198 | Myelofibrosis | Drug: TP-3654 | Advanced Solid Tumors | Sumitomo Pharma Oncology, Inc. | December 16, 2019 | Phase 2 |
PIM-1 overexpression increases cellular pBAD levels, whereas TP-3654 decreases pBAD levels and PIM-1–driven xenografts.Neoplasia.2014 May;16(5):403-12. |
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Validation of PIM-1 in solid tumor modelsin vitro.Neoplasia.2014 May;16(5):403-12. |
TP-3654 inhibits the growth of established solid tumor xenografts.Neoplasia.2014 May;16(5):403-12. |
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