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Purity: ≥98%
| Targets |
SphK2 (sphingosine kinase-2): IC50 = 4.30 ± 0.06 μM; Ki = 6.4 ± 0.7 μM (substrate competitive inhibitor with sphingosine) [1].
SphK1: no inhibition at concentrations up to 10 μM [1]. Ceramide kinase (CERK): no significant inhibition at up to 10 μM [1]. Other protein kinases (e.g., ABL1, AKT1, CDK2, EGFR, ERK2, etc., 11 kinases tested): at 10 μM, K145 showed relative selectivity, with only modest inhibition (percentage of control activity mostly >80%, exact values not provided) [1]. |
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| ln Vitro |
U937 cells treated with K145 (0-10 µM; 24-72 hours) exhibit considerable, concentration-dependent growth inhibition [1]. U937 cells treated with K145 (10 µM) for 24 hours showed a considerable increase in apoptosis [1]. Treatment with K145 (4-8 µM; 3 hours; U937 cells) decreases phosphorylation of ERK and Akt [1]. Treatment with K145 (10 µM) reduced total cellular S1P but did not significantly alter ceramide levels [1].
K145 is readily taken up by U937 cells in a concentration‑dependent manner (measured at 0–10 μM after 3 h, data shown graphically) [1]. Treatment of U937 cells with K145 (10 μM for 3 h) caused a decrease in total cellular S1P levels without significant effects on ceramide levels (various chain‑length ceramide species unchanged) [1]. K145 (10 μM for 3 h) did not significantly affect ceramide‑1‑phosphate (C1P) levels in U937 cells [1]. K145 inhibited the phosphorylation of FTY720 (a SphK2‑specific substrate) in U937 cells in a concentration‑dependent manner (1, 2.5, 5, 10 μM for 3 h), confirming SphK2 selectivity [1]. K145 inhibited the growth of U937 cells cultured in 10% serum in a concentration‑ and time‑dependent manner (IC50 values not provided; tested at 0–10 μM for 24, 48, 72 h by MTT assay) [1]. K145 (10 μM for 24 h) induced apoptosis in U937 cells, mainly late apoptosis (Annexin V/PI staining; small percentage of necrotic cells) [1]. K145 treatment (1, 2, 4, 6, 8, 10 μM for 3 h) decreased phosphorylation of ERK and Akt in U937 cells in a concentration‑dependent manner; inhibition evident at as low as 4 μM [1]. In JC tumor samples from mice treated with K145 (20 mg/kg i.p. for 15 days), p‑ERK and p‑Akt levels were decreased compared to vehicle control [1]. |
| ln Vivo |
The treatment of BALB/c-nu mice with K145 (50 mg/kg; oral gavage; daily; for 15 days) dramatically slowed the growth of U937 tumors in nude mice [1].
K145 administered intraperitoneally (i.p.) at 15 mg/kg daily for 17 days significantly inhibited the growth of U937 xenografts in nude mice (BALB/c‑nu), with tumor growth inhibition (TGI) of 44.2% (tumor weight reduction) compared to vehicle; no body weight loss observed [1]. K145 administered orally by gavage at 50 mg/kg daily for 15 days significantly inhibited U937 xenograft growth in nude mice, with TGI of 51.25% (tumor weight reduction); slight initial body weight decrease that recovered; no apparent toxicity [1]. In a syngeneic mouse model (BALB/c mice bearing JC mammary adenocarcinoma cells), K145 given i.p. at 20 mg/kg and 35 mg/kg daily for 15 days significantly inhibited tumor growth in a dose‑dependent manner (>50% reduction in tumor volume at both doses). Tumor weights were significantly reduced, and no significant changes in body weights or major organs (heart, lung, liver, kidney) were observed [1]. K145 was detected in JC tumor samples from treated mice (20 mg/kg i.p.), and S1P levels in tumors were suppressed compared to vehicle [1]. |
| Enzyme Assay |
SphK activity assay: Lysates from cells overexpressing SphK1 or SphK2 were used. SphK1 activity was measured with 5 μM sphingosine and [γ‑32P]ATP (10 μCi, 1 mM) containing 10 mM MgCl₂ in 0.25% Triton X‑100 (which inhibits SphK2). SphK2 activity was measured with sphingosine (complexed with 4 mg/mL BSA) and [γ‑32P]ATP in the presence of 1 M KCl (conditions optimal for SphK2 and strongly inhibiting SphK1). Reactions were incubated, and labeled S1P was extracted and quantified [1].
Ceramide kinase (CERK) assay: Recombinant human CERK (40 ng) was incubated with 15 μM ceramide and test compound in 50 μL volume for 5 min at RT, then reaction started by adding ATP (5 μM final). After 25 min at 37 °C, 50 μL Kinase‑Glo luminescent reagent was added and incubated for 10 min at RT; luminescence was recorded [1]. SelectScreen kinase profiling: Effect of K145 (10 μM) on activity of 11 kinases (including ABL1, AKT1, CDK2/cyclin A, EGFR, ERK2, etc.) was tested by a commercial service (Invitrogen). Data expressed as percentage of control activity averaged from 2 experiments [1]. FTY720 phosphorylation assay: U937 cells (4 × 10⁶) were incubated with 1 μM FTY720 and indicated concentrations of K145 for 3 h, then FTY720‑P was measured by LC‑MS/MS [1]. Lipidomics analysis (S1P, ceramide, C1P, K145 levels): U937 cells (4 × 10⁶) treated with K145 for 3 h; lipids extracted and analyzed by ESI‑MS/MS (for S1P and C1P) or LC‑ESI‑MS/MS (for ceramide species). K145 levels in cells and tumor samples were also measured by ESI‑MS/MS [1]. |
| Cell Assay |
Cell viability assay [1]
Cell Types: U937 cells Tested Concentrations: 0 µM, 4 µM, 6 µM, 8 µM, 10 µM Incubation Duration: 24 hrs (hours), 48 hrs (hours), 72 hrs (hours) Experimental Results: Dramatically inhibited the growth of U937 cells at a certain concentration Growth-dependent manner. Apoptosis analysis [1] Cell Types: U937 Cell Tested Concentrations: 10 µM Incubation Duration: 24 hrs (hours) Experimental Results: Dramatically induced apoptosis in U937 cells. Western Blot Analysis[1] Cell Types: U937 Cell Tested Concentrations: 4 µM, 8 µM Incubation Duration: 3 hrs (hours) Experimental Results: diminished phosphorylated ERK and Akt. U937 cells (human leukemia) were cultured in RPMI 1640 with 10% FBS at 37 °C, 5% CO₂. For proliferation (MTT assay): 20,000 cells/well in 96‑well plates, treated with K145 (0–10 μM) for 24–72 h, then 10 μL MTT (5 mg/mL) added for 2 h; after centrifugation, 170 μL medium removed and 100 μL DMSO added; absorbance read at 570 nm [1]. Apoptosis assay (Annexin V/PI): U937 cells (1 × 10⁶) treated with K145 (10 μM) for 24 h, washed with cold PBS, resuspended in binding buffer (10 mM HEPES/NaOH pH 7.4, 140 mM NaCl, 2.5 mM CaCl₂), stained with Annexin V‑FITC and propidium iodide for 15 min at RT, analyzed by flow cytometry [1]. Western blot: U937 cells (2 × 10⁵/mL) treated with K145 (1–10 μM) for 3 h in 10% FBS; cells lysed in sample buffer (62.5 mM Tris pH 6.8, 2% SDS, 50 mM DTT, 10% glycerol, protease/phosphatase inhibitors). Proteins (30 μg) separated by SDS‑PAGE, transferred to PVDF, blocked with 5% nonfat milk, probed with primary antibodies (anti‑p‑ERK, total ERK, p‑Akt, total Akt, GAPDH), then HRP‑conjugated secondary antibodies, developed with chemiluminescence [1]. For tumor samples: tumors homogenized in lysis buffer (50 mM Tris‑HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X‑100, 0.25% sodium deoxycholate, 0.1% SDS, 1 mM PMSF, plus protease/phosphatase inhibitor cocktail), then Western blot as above [1]. |
| Animal Protocol |
Animal/Disease Models: BALB/c-nu (nude) mice injected with U937 cells [1]
Doses: 50 mg/kg Route of Administration: po (oral gavage); daily; 15-day Experimental Results: 50 mg/kg dose can inhibit the growth of U937 tumors, and does not Significant toxicity was observed. U937 xenograft in nude mice: Female BALB/c‑nu mice (n = 7 per group) were implanted subcutaneously with 1 × 10⁶ U937 cells. When tumors palpable, mice received daily i.p. injections of vehicle, K145 (15 mg/kg), or tamibarotene (15 mg/kg) for 17 days. Tumor volume measured every three days (V = ab²/2). At study end, tumors weighed; TGI calculated as (mean vehicle tumor weight – mean treated tumor weight)/mean vehicle tumor weight [1]. Oral administration study: Similar U937 xenograft model, mice received daily oral gavage of vehicle, K145 (50 mg/kg), or tamibarotene (20 mg/kg) for 15 days. Tumor volume and body weight measured every other day [1]. Syngeneic JC model: Immunocompetent BALB/c mice (n = 8 per group, 18–20 g) implanted subcutaneously with 1 × 10⁶ JC mammary adenocarcinoma cells. When tumors palpable, mice received daily i.p. injections of vehicle or K145 (20 mg/kg or 35 mg/kg) for 15 days. Tumor volume and body weight measured every other day. At end, tumors weighed; samples collected for Western blot and lipid analysis [1]. Drug formulation: Not specified (dissolved in vehicle, details not provided). |
| References | |
| Additional Infomation |
K145 was designed as a sphingosine analogue based on a thiazolidine-2,4-dione (TZD) scaffold. Molecular modeling studies using homology models of SphK1 and SphK2 (based on diacylglycerol kinase PDB 3T5P) showed that K145 binds favorably to SphK2 via hydrogen bonds with Asp344, Arg351, Gln346, and π‑stacking with Phe350, while unfavorable interactions with Glu180 in SphK1 explain selectivity. The compound was synthesized via a multi‑step route (Figure 3). In vitro, K145 accumulates in cells and suppresses S1P levels. It inhibits proliferation and induces apoptosis in U937 cells, associated with downregulation of ERK and Akt signaling. In vivo, it shows antitumor activity in both xenograft and syngeneic models via i.p. and oral routes with no apparent toxicity. The study suggests that K145 is a promising lead for developing more potent SphK2 inhibitors [1].
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| Molecular Formula |
C18H25CLN2O3S
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|---|---|
| Molecular Weight |
384.920702695847
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| Exact Mass |
384.127
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| CAS # |
1449240-68-9
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| Related CAS # |
K145;1309444-75-4
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| PubChem CID |
76849910
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| Appearance |
White to off-white solid powder
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| LogP |
4.776
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
9
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| Heavy Atom Count |
25
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| Complexity |
459
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| Defined Atom Stereocenter Count |
0
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| SMILES |
CCCCOC1=CC=C(C=C1)CC/C=C\2/C(=O)N(C(=O)S2)CCN.Cl
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| InChi Key |
HADFDMGQKBGVAV-NKBLJONXSA-N
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| InChi Code |
InChI=1S/C18H24N2O3S.ClH/c1-2-3-13-23-15-9-7-14(8-10-15)5-4-6-16-17(21)20(12-11-19)18(22)24-16;/h6-10H,2-5,11-13,19H2,1H3;1H/b16-6-;
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| Chemical Name |
(Z)-3-(2-aminoethyl)-5-(3-(4-butoxyphenyl)propylidene)thiazolidine-2,4-dione hydrochloride
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| Synonyms |
K-145; K-145 HCl; K145; K 145; K145 hydrochloride.
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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 Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
| 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) |
H2O : ~126.7 mg/mL (~329.16 mM)
DMSO : ~50 mg/mL (~129.90 mM) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 0.83 mg/mL (2.16 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 8.3 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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. Solubility in Formulation 2: ≥ 0.83 mg/mL (2.16 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 8.3 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. View More
Solubility in Formulation 3: ≥ 0.83 mg/mL (2.16 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 | 2.5979 mL | 12.9897 mL | 25.9794 mL | |
| 5 mM | 0.5196 mL | 2.5979 mL | 5.1959 mL | |
| 10 mM | 0.2598 mL | 1.2990 mL | 2.5979 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.
 K145 inhibits SphK2 but not SphK1.
K145 accumulates and suppresses the S1P level.PLoS One.2013;8(2):e56471. |
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K145 exhibits antiproliferative and apoptotic effects in U937 cells.
K145 suppresses the growth of U937 tumors in nude mice by oral administration.PLoS One.2013;8(2):e56471. |
K145 suppresses the growth of U937 xenograft in nude mice.
K145 suppresses the growth of JC xenograft in BALB/c mice.PLoS One.2013;8(2):e56471. |
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