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Purity: ≥98%
(R)-FL118 is a novel and potent inhibitor of human survivin expression, activating tumor suppressor p53 as a novel MOA in p53 wild-type cancer cells.
FL118 (10,11‑methylenedioxy‑camptothecin) is a novel camptothecin analogue with a unique methylenedioxy group at positions 10 and 11 of the A‑ring. It was identified through high‑throughput screening using a survivin promoter‑driven luciferase reporter system. Unlike the clinically approved camptothecin derivatives irinotecan and topotecan, FL118 is not a substrate of the efflux pumps ABCG2/BCRP or P‑gp/MDR1, and its antitumor activity is independent of topoisomerase I (Top1) expression. FL118 acts as a “molecular glue degrader” that directly binds to the oncoprotein DDX5 (p68) with high affinity (KD = 34.4 nM), inducing its dephosphorylation and proteasome‑dependent degradation without affecting DDX5 mRNA. Through this mechanism, FL118 downregulates multiple downstream oncogenic targets, including survivin, Mcl‑1, XIAP, cIAP2, c‑Myc, and mutant Kras. In preclinical studies, FL118 exhibits potent antitumor activity against colon, head‑and‑neck, lung, pancreatic, and ovarian cancers, and effectively overcomes irinotecan‑ and topotecan‑resistant human tumor xenografts. With favorable pharmacokinetics (rapid tumor accumulation, long tumor half‑life, and low systemic exposure) and a manageable safety profile, FL118 is a promising anticancer agent currently under clinical development.| Targets |
DDX5 (p68) – a multifunctional DEAD-box RNA helicase and master regulator oncoprotein. FL118 binds directly to DDX5 with high affinity (KD = 34.4 nM) and induces its dephosphorylation and degradation via the ubiquitin-proteasome pathway. This leads to downstream inhibition of survivin, Mcl-1, XIAP, cIAP2, c-Myc, and mutant Kras [3].
Cytoglobin (CYGB) – FL118 upregulates CYGB expression, which mediates its anti-proliferative and anti-migratory effects in ovarian cancer cells [4]. |
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| ln Vitro |
In Vitro: FL118 inhibits the proliferation of A549 (lung), MDA-MB-231 (breast), and RM-1 (mouse prostate) cancer cells with IC50 values ranging from 2.32 nM to 4.53 μM. In A549 cells, IC50 = 8.94 ± 1.54 nM; in MDA-MB-231 cells, IC50 = 24.73 ± 13.82 nM; in RM-1 cells, IC50 = 69.19 ± 8.34 nM. In comparison, irinotecan showed IC50 values of 9.14 μM (A549) and 7.82 μM (MDA-MB-231) [2].
In ovarian cancer ES-2 and SK-OV-3 cells, FL118 inhibits cell proliferation and migration in a dose- and time-dependent manner. At 100 nM, FL118 significantly reduces cell viability and migration distance after 24-72 h. FL118 upregulates cytoglobin (CYGB) mRNA and protein expression in a dose-dependent manner, and CYGB knockdown partially reverses FL118's anti-proliferative and anti-migratory effects. FL118 also inhibits PI3K/AKT/mTOR signaling (increases p-AKT and p-mTOR) and modulates EMT markers (increases E-cadherin, decreases vimentin) [4]. In HCT-8 colon cancer cells, FL118 (1-100 nM) inhibits survivin, Mcl-1, XIAP, cIAP2, and c-Myc expression. It is approximately 25-fold more potent than topotecan in inhibiting cell growth and colony formation. FL118 is not a substrate of P-gp/MDR1 or ABCG2 efflux pumps, and its efficacy is not affected by these transporters. FL118 exhibits high Caco-2 permeability (Papp = 6.76-8.86 × 10⁻⁶ cm/s) with a very low efflux ratio (1.0-1.2) [1]. (R)-FL118, with IC50 values of 392.7, 32.53, 129.3, 138.7, and 92.51 ng/mL in B16, MCF-7, HS578T, DU145, and MPC3 cells, respectively, suppresses the proliferation of melanoma, breast cancer, and prostate cancer cell lines. |
| ln Vivo |
In Vivo: In mice bearing irinotecan-resistant FaDu head-and-neck tumors, FL118 (1.5 mg/kg IP, q2d × 5) effectively eliminated resistant tumors; some regressed tumors that relapsed responded to a second cycle of FL118 treatment [1].
In mice bearing topotecan-resistant FaDu tumors, FL118 (1-1.5 mg/kg IP, q2d × 5) effectively eliminated resistant tumors [1]. In HCT116 colon carcinoma xenografts, FL118 (10 mg/kg IP, once daily for 14 days) inhibited tumor volume by 76%, superior to 5-FU (41% inhibition) and vorinostat (26% inhibition) [1]. In human PDAC patient-derived xenograft (PDX) tumors, FL118 (2.5 mg/kg oral, weekly × 4) regressed PDX19015 and PDX17624 tumors with high DDX5 expression, while PDX12872 with low DDX5 expression showed less sensitivity. Combination with low-dose gemcitabine (40 mg/kg) eliminated PDX12872 tumors [3]. In CRC xenografts, FL118 (5 mg/kg oral, weekly × 4) effectively inhibited tumor growth in high DDX5-expressing SW620 and SW837 tumors, while low DDX5-expressing SW480 and SW948 tumors showed poor sensitivity [3]. In ES-2 ovarian cancer xenografts, FL118 (5 or 10 mg/kg oral, once weekly for 20 days) showed superior antitumor activity compared to topotecan (2 mg/kg, 5×/week). At 10 mg/kg, 60% of tumors were completely abolished. FL118 upregulated CYGB expression in tumor tissues [4]. In a mouse intratracheal pharmacodynamic model, FL118 inhibited IL-6-induced lung pSTAT3 (JAK1/JAK2) with an ED50 of approximately 3 μg/animal [1]. In a mouse ear dermal model, 4% topical FL118 reduced IL-23-induced ear swelling by 48% over 11 days [1]. |
| Enzyme Assay |
Enzyme Assay: For FL118 binding to DDX5, isothermal titration calorimetry (ITC) was performed using purified Flag-DDX5 protein (10 μM) and FL118 (100 μM) in 1× PBS (pH 7.4) with 8% DMSO. The compound was titrated into the protein cell by 20 injections over 60 min (one injection per 3 min) at 25°C. The binding affinity (KD) was calculated as 34.4 nM. For Top1, the KD was 315 nM; no binding to BSA was detected [3].
For FL118 affinity purification, FL118 was coupled to agarose resin beads via a diaminodipropylamine linker through a Mannich reaction. Cancer cell lysates were passed through the FL118 affinity column and a control column in parallel. After stringent washing, bound proteins were eluted with 8 M urea, concentrated, and separated by SDS-PAGE. The ~70 kDa protein band was identified as DDX5 by mass spectrometry [3]. For Caco-2 permeability assay, Caco-2 cell monolayers were grown on collagen-coated polycarbonate membranes. FL118 (1 μM) was added to apical or basolateral side with or without valspodar (1 μM, P-gp inhibitor). Samples were taken at 120 min and analyzed by LC-MS/MS. Apparent permeability (Papp) and efflux ratio were calculated. FL118 showed high permeability (Papp = 6.76-8.86 × 10⁻⁶ cm/s) and low efflux ratio (1.0-1.2) [1]. |
| Cell Assay |
Cell Assay: For cytotoxicity, cells were seeded in 96-well plates (2,000-7,000 cells/well), treated with various concentrations of FL118 for 24-72 h, and viability was measured by MTT or resazurin assays. IC50 values were calculated using Logit approach [1][2][4].
For colony formation, HCT-8 cells (100 cells/well in 12-well plates) were treated with FL118 or topotecan for 2 or 6 h, then washed and cultured for 2 weeks. Colonies were stained with crystal violet and counted [1]. For western blot, cells were lysed in RIPA buffer, proteins separated by SDS-PAGE, transferred to nitrocellulose or PVDF membranes, and probed with antibodies against DDX5, survivin, Mcl-1, XIAP, cIAP2, c-Myc, mutant Kras, p-AKT, p-mTOR, E-cadherin, vimentin, PARP, caspase-3, and loading controls (actin, GAPDH, tubulin). Blots were visualized by ECL [1][3][4]. For siRNA knockdown, cells were transfected with CYGB-specific siRNA or negative control using Lipofectamine RNAiMAX. After 48 h, cells were treated with FL118 (100 nM) and analyzed for proliferation (MTT) and migration (scratch wound assay) [4]. For CRISPR-Cas9 knockout of DDX5, Panc-1 and Mia Paca-2 cells were electroporated with DDX5 sgRNA-Cas9 ribonucleoprotein complexes. Knockout clones were validated by western blot and PCR/T7 endonuclease assay [3]. For scratch wound assay, cells were plated in 6-well plates, grown to 80-90% confluence, scratched with a 200 μL pipette tip, washed with PBS, and treated with FL118 (10-100 nM) in 1% FBS medium for 24 h. Migration distance was measured [4]. |
| Animal Protocol |
Animal Protocol: For pharmacokinetic studies, SCID mice bearing FaDu or SW620 tumors (800 mm³) received a single IV injection of FL118 (1.5 mg/kg) via tail vein. Blood and tumor tissues were collected at 10 min, 1, 4, 12, 24, and 48 h. FL118 was extracted with acidified methanol and analyzed by UPLC with fluorescence detection (Ex 370 nm, Em 510 nm). Irinotecan was used as internal standard [1].
For irinotecan/topotecan resistance models, SCID mice with established SW620 or FaDu tumors were treated with irinotecan (100 mg/kg IP weekly) or topotecan (4 mg/kg IP daily ×5) until tumors acquired resistance. Resistant tumors were then treated with FL118 (0.75-1.5 mg/kg IP, q2d × 5 per cycle) [1]. For xenograft efficacy studies, SCID mice were inoculated subcutaneously with cancer cells (1-3 × 10⁶) or tumor fragments (30-50 mg). When tumors reached 100-200 mm³ (day 0), FL118 was administered orally (2.5-10 mg/kg) weekly ×4, or intraperitoneally (0.75-10 mg/kg) q2d ×5 or daily ×14 days. Tumor volume was measured twice weekly with calipers. Formulation: FL118 (0.1-0.5 mg/mL), DMSO (0-5%), hydroxypropyl-β-cyclodextrin (0.1-0.5%), propylene glycol (2.5%), PEG400 (2.5%) in saline [1][3][4]. For PDX models, human PDAC tumor fragments (30-40 mg) were subcutaneously transplanted into SCID mice. Treatment started when tumors reached 100-200 mm³. FL118 was administered orally at 2.5 mg/kg (1/4 MTD) weekly ×4. Gemcitabine (40 mg/kg IP) was used in combination [3]. For DDX5 KO tumor models, DDX5 KO Panc-1 or Mia Paca-2 cells (2 × 10⁶) were injected subcutaneously into SCID mice, and tumor growth was monitored over time [3]. |
| ADME/Pharmacokinetics |
ADME/Pharmacokinetics: Following a single IV dose of FL118 (1.5 mg/kg) in SCID mice, FL118 rapidly accumulated in tumor tissues with a long half-life (6.85 h in FaDu tumors, 12.75 h in SW620 tumors) but was quickly cleared from circulation (plasma half-life = 1.788 h). Cmax in tumor was 1154 ng/g (FaDu) and 1588 ng/g (SW620); Cmax in plasma was 438 ng/mL. Tumor AUC was 3448-4289 hr·ng/g; plasma AUC was 1042 hr·ng/mL. FL118 concentration in tumor tissue at 10 min post-injection was >2.5-fold higher than in plasma [1].
FL118 has a cLogP of 4.4 and log D of 3.9 (shake flask method). It contains a weakly basic imidazole group (pKa = 4.5) and a weakly acidic fluorophenol group (pKa = 8.7). TPSA = 126 Ų [1]. In rat, IV (0.5 mg/kg): CL = 48 mL/min/kg, CLunbound >48,000 mL/min/kg, Vss = 0.8 L/kg, terminal T1/2 = 2.1 h, oral bioavailability <5%. In dog, IV (0.1 mg/kg infusion): CL = 18 mL/min/kg, CLunbound >18,000 mL/min/kg, Vss = 1.0 L/kg, terminal T1/2 = 2.0 h. Plasma protein binding ≥99.9% [1]. In vitro metabolism in human hepatocytes: major metabolites produced by direct glucuronidation and P450 metabolism; phenol-conjugated metabolites show no JAK inhibition. Consistent across rat and dog [1]. |
| Toxicity/Toxicokinetics |
Toxicity/Toxicokinetics: The maximum tolerated dose (MTD) of FL118 in mice for daily ×5 or q2d ×5 schedules is approximately 1.5 mg/kg. For weekly ×4 schedule, MTD is 10 mg/kg [1][3].
In exploratory 7-day IV toxicology studies in rats and dogs, no adverse test article-related findings were observed. A 36-fold margin was achieved in rat over total plasma AUC based on projected human clinical lung dose (200 μg), and a 55-fold margin in dog plasma AUC. BioLum Ames assay showed no genetic toxicity with or without metabolic activation; CHO in vitro micronucleus test also negative. No hERG inhibition at 1 μM (<1% inhibition). No cough signal in mouse Aδ fiber sensory nerve preparation up to 2 mg [1]. In ovarian cancer xenograft study, FL118 at 10 mg/kg (oral, weekly ×4) caused significant body weight loss compared to control, while 5 mg/kg showed no statistical difference. Topotecan (2 mg/kg, 5×/week) also caused body weight loss but not statistically different from control [4]. In PDX models, FL118 at 2.5 mg/kg (1/4 MTD) was well tolerated with minimal body weight changes. Combination with gemcitabine (40 mg/kg) did not increase toxicity [3]. |
| References |
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| Additional Infomation |
FL118 is a novel camptothecin analogue containing a unique methylenedioxy group at positions 10 and 11 of the A-ring. It was identified through high-throughput screening using a survivin promoter-driven luciferase reporter system. Unlike irinotecan/SN-38 and topotecan, FL118 is not a substrate of ABCG2/BCRP or P-gp/MDR1 efflux pumps, and its antitumor activity is independent of Top1 expression. FL118 selectively inhibits multiple cancer-associated anti-apoptotic proteins (survivin, Mcl-1, XIAP, cIAP2) and functions as a "molecular glue degrader" that binds to, dephosphorylates, and degrades the DDX5 oncoprotein via the proteasome pathway without affecting DDX5 mRNA. DDX5 is a master regulator controlling survivin, Mcl-1, XIAP, cIAP2, c-Myc, and mutant Kras [3].
FL118 shows broad-spectrum preclinical antitumor activity against colon, head-and-neck, lung, pancreatic, ovarian, and other cancers. It overcomes irinotecan and topotecan resistance in human tumor xenograft models. FL118 is currently in clinical development [1][3]. |
| Molecular Formula |
C21H16N2O6
|
|---|---|
| Molecular Weight |
392.361545562744
|
| Exact Mass |
392.101
|
| Elemental Analysis |
C, 64.28; H, 4.11; N, 7.14; O, 24.47
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| CAS # |
151636-76-9
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| Related CAS # |
FL118;135415-73-5
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| PubChem CID |
486154
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| Appearance |
Off-white to yellow solid powder
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| LogP |
1.808
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| Hydrogen Bond Donor Count |
1
|
| Hydrogen Bond Acceptor Count |
7
|
| Rotatable Bond Count |
1
|
| Heavy Atom Count |
29
|
| Complexity |
852
|
| Defined Atom Stereocenter Count |
1
|
| SMILES |
CC[C@]1(C2=C(COC1=O)C(=O)N3CC4=C(C3=C2)N=C5C=C6C(=CC5=C4)OCO6)O
|
| InChi Key |
RPFYDENHBPRCTN-OAQYLSRUSA-N
|
| InChi Code |
InChI=1S/C21H16N2O6/c1-2-21(26)13-5-15-18-11(7-23(15)19(24)12(13)8-27-20(21)25)3-10-4-16-17(29-9-28-16)6-14(10)22-18/h3-6,26H,2,7-9H2,1H3/t21-/m1/s1
|
| Chemical Name |
(4R)-4-Ethyl-4-hydroxy-8,9-methylenedioxy-1H-pyrano[3',4'
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| Synonyms |
(R)-FL118; FL118; (R)-FL-118; FL 118; FL-118
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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 |
| 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 : ~2.7 mg/mL (~6.88 mM)
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|---|---|
| Solubility (In Vivo) |
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.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] Oral Formulations
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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.5487 mL | 12.7434 mL | 25.4868 mL | |
| 5 mM | 0.5097 mL | 2.5487 mL | 5.0974 mL | |
| 10 mM | 0.2549 mL | 1.2743 mL | 2.5487 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.
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