| Size | Price | Stock | Qty |
|---|---|---|---|
| 100mg |
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| 250mg |
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Purity: ≥98%
| Targets |
GARFT (Ki = 6.5 ± 1.1 nM) [1]
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|---|---|
| ln Vitro |
LY309887 has a high affinity for human folate receptor (FR)α and FRβ (Ki of 1.78 nM and 18.2 nM, respectively), and is a powerful inhibitor of glycinamide ribonucleotide formyltransferase (GARFT), with a Ki of 6.5 nM for human GARFT. LY309887 exhibits notable cytotoxicity, with an IC50 of 9.9 nM, against the human leukemia cell line CCRF-CEM[1].
LY309887 exhibited potent cytotoxicity against CCRF-CEM human leukemia cells with an IC50 of 2.89 nM, approximately three-fold more potent than lometrexol (IC50 = 9.93 nM). [1] The cytotoxic activity of LY309887 was completely inhibited by the addition of hypoxanthine (100 μM) to the culture medium, indicating selective inhibition of de novo purine biosynthesis. Thymidine (5 μM) alone did not alter the cytotoxic activity, and the combination of hypoxanthine and thymidine had the same effect as hypoxanthine alone. [1] Addition of AICA (300 μM) produced incomplete inhibition of cytotoxicity at higher LY309887 concentrations, suggesting that LY309887 also inhibits AICA-ribonucleotide formyltransferase in addition to GARFT. [1] LY309887 inhibited recombinant monofunctional human GARFT with a Ki of 6.5 nM (tight-binding inhibition). [1] LY309887 is a substrate for folylpolyglutamate synthetase (FPGS) with a lower first-order rate constant (k' = 43) compared to lometrexol (k' = 60). In vitro polyglutamation: incubation with FPGS and 14C-glutamate for 24 h yielded 90 pmol of LY309887 polyglutamates, which were present as tri- (58%), tetra- (35%), and penta- (7%) glutamates. [1] The affinity of LY309887 for folate receptor isoforms: FRα Ki = 1.78 ± 0.18 nM, FRβ Ki = 18.2 ± 4.4 nM, with a β/α selectivity ratio of 10.5, which is twice that of lometrexol (β/α = 5.0). [1] The triglutamylated form of LY309887 (as the 6R,S-diastereomer mixture LY254155) inhibited GARFT with a Ki of 0.25 ± 0.2 nM, which is 10-fold more potent than its monoglutamated parent (Ki = 2.1 nM). [1] |
| ln Vivo |
In mice harboring C3H breast cancer cells, intraperitoneal injections of LY309887 (3 mg/kg–100 mg/kg) completely inhibited the formation of tumors [1].
Against C3H mammary murine tumor: LY309887 dosed intraperitoneally (i.p.) every third day for four doses (q3d×4) starting the day after tumor implantation produced 100% tumor growth inhibition at doses ranging from 3 mg/kg to 100 mg/kg, with less than 20% lethality at all doses studied. In contrast, lometrexol required 100 mg/kg to achieve greater than 60% inhibition. [1] Against PANC-1 human pancreatic xenograft: LY309887 (i.p., q3d×4) showed 65% inhibition at 1 mg/kg and 92% inhibition at 30 mg/kg. The highest tolerated dose was 30 mg/kg; 7 out of 9 mice died at 100 mg/kg. [1] Against BxPC3 pancreatic xenograft: LY309887 (6R-diastereomer) at 40 mg/kg (q3d×4) gave 87% tumor growth inhibition, whereas lometrexol at 50 mg/kg (q2d×5) gave 59% inhibition. [1] Against colon xenografts: LY309887 (6R) at 40 mg/kg (q3d×4) inhibited CX-1 by 64%; at 30 mg/kg (q3d×4) inhibited GC3 by 95%, HC1 by 97%, and VRC5 by 86%. [1] Against LX-1 lung xenograft: LY309887 (6R) at 10 mg/kg (q3d×4) gave 98% inhibition. [1] Against MX-1 mammary xenograft: LY309887 (6R) at 10 mg/kg (q3d×4) gave 52% inhibition. [1] LY309887 demonstrated broad-spectrum antitumor activity in a panel of human xenograft tumors and was more potent than lometrexol in every model tested. [1] |
| Enzyme Assay |
Recombinant monofunctional human GARFT was used for enzyme inhibition assays. Compounds were dissolved in DMSO and diluted with appropriate buffers. Tight-binding inhibition was assessed using the Morrison equation as detailed by Habeck et al. [16]. Ki values were calculated. [1]
FPGS activation assay: Hog liver folylpolyglutamate synthetase (FPGS) activity was measured. The reaction mixture contained 100 mM Tris, 10 mM MgCl2, 5 mM ATP, 20 mM KCl, 100 μg/ml bovine serum albumin, 100 mM β-mercaptoethanol, 1 mM [14C]-L-glutamate, 1 μM substrate (LY309887 or lometrexol), and 2 μg of semi-purified hog liver FPGS at pH 8.9 in a final volume of 0.25 ml. Incubation was at 37°C for 24 hours. Reactions were stopped by boiling for 3 minutes and then centrifuged. Polyglutamates were separated by reversed-phase HPLC. Km, Vmax, and the first-order rate constant (k' = Vmax/Km) were calculated. For LY309887, Km = 6.5 ± 1.1 μM, Vmax = 86 ± 11 μmol/hr/mg, and k' = 43. [1] Folate receptor binding assay: Affinity for FRα (from human KB cell membranes) and FRβ (from human liver membranes) was assessed as described by Habeck et al. [16]. The ability of compounds to displace radiolabeled folic acid was measured, and Ki values were determined. For LY309887, FRα Ki = 1.78 ± 0.18 nM, FRβ Ki = 18.2 ± 4.4 nM. [1] |
| Cell Assay |
CCRF-CEM human leukemia cells were maintained as suspension cultures in RPMI-1640 medium supplemented with 10% dialyzed fetal calf serum and 25 mM HEPES buffer. For cytotoxicity assays, dose-response curves were generated to determine the concentration required for 50% inhibition of growth (IC50). Cells were exposed to various concentrations of LY309887, and IC50 values were calculated. [1]
Reversal studies: IC50 values for LY309887 were determined in the presence of either AICA (300 μM), thymidine (5 μM), hypoxanthine (100 μM), or a combination of thymidine plus hypoxanthine. The results showed that hypoxanthine completely reversed the cytotoxicity, thymidine alone had no effect, and AICA only partially reversed the cytotoxicity at higher drug concentrations. [1] |
| Animal Protocol |
Female C3H mice (for syngeneic mammary tumor) and CD1 nu/nu mice (for human xenografts), weighing approximately 20-25 g at study start, were used. Tumors were carried by serial passage of 1-3 mm tumor fragments implanted subcutaneously via trocar in the axillary region. For the C3H mammary tumor, drug treatment began one day after tumor implantation; for xenograft tumors, treatment began 7 days after tumor implant (14 days for HC1 colon tumor). LY309887 was prepared for dosing in either water or 0.9% NaCl, with NaHCO3 added when necessary to enhance solubility. An intermittent dosing regimen was used: intraperitoneal (i.p.) injection every third day for a total of four doses (q3d×4). For comparison, lometrexol was dosed either q2d×5 or q3d×4. Tumor dimensions were measured with electronic calipers, and tumor weight (mg) was calculated as (tumor width in mm)^2 × (tumor length in mm) × 0.5. Percent tumor growth inhibition was calculated as (1 - (treated group wt / control group wt)) × 100. For C3H mammary studies, tumors were measured on the day after the last treatment day; for xenograft tumors, measurements were taken 5-6 days after the last therapy day. Each study used 7-10 mice per group. [1]
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| ADME/Pharmacokinetics |
LY309887 is a substrate for folylpolyglutamate synthetase (FPGS) with Km = 6.5 μM, Vmax = 86 μmol/hr/mg, and first-order rate constant k' = 43, which is lower than that of lometrexol (k' = 60), indicating less efficient polyglutamation at low substrate concentrations. [1]
In vitro polyglutamation: After 24-hour incubation with FPGS and 14C-glutamate, LY309887 formed tri- (58%), tetra- (35%), and penta- (7%) glutamates, with a total of 90 pmol polyglutamates, whereas lometrexol formed more extensive polyglutamates (tri-, tetra-, penta- at 40%, 39%, 21%). [1] In vivo accumulation in mouse liver: Mice dosed intravenously with equimolar radiolabeled LY309887 accumulated 1.8- to 6-fold less total compound in liver compared to lometrexol over 7 days, depending on dietary folate levels. LY309887 was present primarily as penta- and hexa-glutamates, whereas lometrexol formed hepta- and octa-glutamates under low folate diet. [1] The triglutamate of LY309887 has 10-fold higher potency against GARFT (Ki = 0.25 nM) than the monoglutamate (Ki = 2.1 nM). [1] |
| Toxicity/Toxicokinetics |
In the C3H mammary tumor efficacy study, LY309887 at doses from 3 to 100 mg/kg (i.p., q3d×4) caused less than 20% lethality at all doses studied. [1]
In the PANC-1 xenograft study, the highest tolerated dose of LY309887 was 30 mg/kg (i.p., q3d×4); at 100 mg/kg, 7 out of 9 mice died. [1] LY309887 is expected to have less toxicity than lometrexol based on its reduced polyglutamation and lower affinity for folate receptors. Lometrexol caused delayed cumulative toxicity in Phase I clinical trials, prompting the development of LY309887 as a second-generation inhibitor with a more favorable toxicological profile. [1] In mice on low folate diets, lometrexol accumulated more extensively and formed longer-chain polyglutamates (hepta- and octa-) compared to LY309887 (penta- and hexa-), suggesting lower potential for tissue retention and toxicity for LY309887. [1] |
| References | |
| Additional Infomation |
LY309887 (6R-2',5'-thienyl-5,10-dideazatetrahydrofolic acid) is a second-generation antifolate inhibitor of glycinamide ribonucleotide formyltransferase (GARFT), developed following the clinical observation of delayed cumulative toxicity with the first-generation GARFT inhibitor lometrexol. [1]
Mechanism: By inhibiting GARFT, LY309887 blocks the de novo purine biosynthesis pathway, depriving cells of newly synthesized AMP and GMP required for DNA and RNA synthesis, particularly affecting rapidly proliferating tumor cells. [1] LY309887 is more potent than lometrexol in inhibiting GARFT (9-fold) and shows superior in vivo antitumor activity against a broad spectrum of human xenograft tumors including colon, lung, mammary, and pancreatic cancers. [1] The compound has a thienyl ring modification compared to lometrexol, which alters its biochemical properties including reduced FPGS substrate efficiency and lower folate receptor affinity. [1] LY309887 exhibits selectivity for folate receptor α over β (β/α selectivity ratio 10.5), which may have implications for tumor targeting as some tumors overexpress FRα. [1] The clinical development of LY309887 was motivated by its broad-spectrum in vivo antitumor activity and favorable biochemical and pharmacological properties. [1] |
| Molecular Formula |
C19H23N5O6S
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|---|---|
| Molecular Weight |
449.48082
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| Exact Mass |
449.137
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| CAS # |
127228-54-0
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| Related CAS # |
127228-54-0 (free acid);
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| PubChem CID |
135430850
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| Appearance |
White to off-white solid powder
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| LogP |
1.549
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| Hydrogen Bond Donor Count |
6
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| Hydrogen Bond Acceptor Count |
9
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| Rotatable Bond Count |
9
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| Heavy Atom Count |
31
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| Complexity |
829
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| Defined Atom Stereocenter Count |
2
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| SMILES |
C1[C@H](CNC2=C1C(=O)NC(=N2)N)CCC3=CC=C(S3)C(=O)N[C@@H](CCC(=O)O)C(=O)O
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| InChi Key |
GQCXGHHHNACOGE-SKDRFNHKSA-N
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| InChi Code |
InChI=1S/C19H23N5O6S/c20-19-23-15-11(16(27)24-19)7-9(8-21-15)1-2-10-3-5-13(31-10)17(28)22-12(18(29)30)4-6-14(25)26/h3,5,9,12H,1-2,4,6-8H2,(H,22,28)(H,25,26)(H,29,30)(H4,20,21,23,24,27)/t9-,12+/m1/s1
|
| Chemical Name |
L-Glutamic acid, N-((5-(2-((6R)-2-amino-3,4,5,6,7,8-hexahydro-4-oxopyrido(2,3-d)pyrimidin-6-yl)ethyl)-2-thienyl)carbonyl)-
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| Synonyms |
LY-309887; LY-309887; LY-309887
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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 : ~100 mg/mL (~222.48 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 | 2.2248 mL | 11.1240 mL | 22.2479 mL | |
| 5 mM | 0.4450 mL | 2.2248 mL | 4.4496 mL | |
| 10 mM | 0.2225 mL | 1.1124 mL | 2.2248 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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