Human Thymidylate Synthase (Ki = 11 nM)

3,4-Dihydro-2-amino-6 methyl-4-oxo-5-(4-pyridylthio)-quinazoline dihydrochloride (AG337) is a water-soluble, lipophilic inhibitor of thymidylate synthase (TS) designed using X-ray structure - based methodologies to interact at the folate cofactor binding site of the enzyme. AG337, inhibits purified recombinant human TS with a Ki of 11 nM, and displays non-competitive inhibition kinetics. It was further shown to inhibit cell growth in a panel of cell lines of murine and human origin, displaying an IC50 of between 0.39 microM 6.6 microM. TS was suggested as the locus of action of AG337 by the ability of thymidine to antagonize cell growth inhibition and the direct demonstration of TS inhibition in whole cells using a tritium release assay. The demonstration, by flow cytometry, that AG337-treated L1210 cells were arrested in the S phase of the cell cycle was also consistent with a blockage of TS, as was the pattern of ribonucleotide and deoxyribonucleotide pool modulation in AG337-treated cells, which showed significant reduction in TTP levels. The effects of AG337 were quickly reversed on removal of the drug, suggesting, as would be expected for a lipophilic agent, that there is rapid influx and efflux from cells and no intracellular metabolism to derivatives with enhanced retention.[1]
Effects of lipophilic thymidylate synthase (TS) inhibitor AG337 on human head and neck squamous cell carcinoma (HNSCC) cell lines and CCRF-CEM human leukemia cells and sublines with acquired methotrexate (MTX) resistance were assayed using continuous or intermittent drug exposure. During 120-h continuous exposure, HNSCC cell lines A253 and FaDu are equally MTX sensitive (EC50 equals approximately 15nM); AG337 is less potent (EC50 approximately equals 1 microM). A253 is intrinsically resistant to 24-h intermittent MTX exposure (EC50 equals approximately 17 microM; FaDu, EC50 equals approximately 0.3 microM); both HNSCC cell lines are resistant to 24-h AG337 exposure (EC50 >100 microM). CCRF-CEM shows MTX (EC50 =14 nM) and AG337 (EC50 equals approximately 0.6 microM) sensitivity in continuous exposure similar to HNSCC; however, AG337 retains potency against CCRF-CEM cells in intermittent exposure (24-h, EC50 equals approximately 2 microM; 6-h, EC50 equals approximately 48 microM). The reduced folate leucovorin (LV) at > or = 0.1 microM fully protects from growth inhibition by continuous MTX exposure, but growth inhibition by AG337 is reversed only slightly by < or = 100 microM LV. Thymidine fully protects A253 and FaDu against growth inhibition by AG337, while hypoxanthine alone is without effect, suggesting inhibition is TS-specific. CCRF-CEM sublines with acquired MTX-resistance resulting from DHFR overexpression, defective MTX transport, or defective MTX polyglutamylation retain full sensitivity to AG337 in continuous exposure (all EC50 =0.4 microM). These data indicate that AG337 may be useful in therapy of tumors that have acquired resistance to MTX by most common mechanisms.[2]

In vivo, AG337 was highly active against the thymidine kinase-deficient murine L5178Y/TK-lymphoma implanted either i.p. or i.m. following i.p. or oral delivery. Prolonged dosing periods of 5 or 10 days were required for activity, and efficacy was improved with twice-daily dose administration. Dose levels of 25 mg/kg delivered i.p. twice daily for 10 days, 50 mg/kg once daily for 10 days, or 100 mg/kg once daily for 5 days elicited 100% cures against the i.p. tumor. Doses required for activity against the i.m. tumor were higher (100 mg/kg i.p. twice daily for 5 or 10 days) but demonstrated the ability of AG337 to penetrate solid tissue barriers. Oral delivery required doses of > or = 150 mg/kg twice daily for periods of 5-10 days to produce 100% cure rates against both i.m. and i.p. implanted tumors. These results were consistent with the pharmacokinetics parameters determined in rats, for which oral bioavailability of 30-50% was determined, together with a relatively short elimination half life of 2h. Clinical studies with AG337 are currently in progress [1].

Enzyme kinetics[1]
TS activity was measured using purified recombinant human TS by a modified tritium release method. Inhibition constants were determined by steady state analysis against the cofactor (6R,6S)5,10- methylene tetrahydrofolate which was generated in situ by reaction of tetrahydrofolate with formaldehyde under conditions of essentially saturating [5-3H] dUMP. Assays contained 50 mM TRIS-Cl pH 7.6, 10 mM dithiothreitol, 1 mM ethylenediamine-tetraacetic acid, 25 mM MgCl2 , 15 mM formaldehyde, 1% DMSO, 25 lM [5-3H]dUMP (specific activity 2]108 cpm/lmol), tetrahydrofolate (10—80 lM), AG337 (0—100 nM), TS (4 nM) and 1—5 lg/ml bovine serum albumin to stabilize the enzyme. Reactions were carried out at 25°C for 6 min and quenched by the addition of charcoal then centrifuged to remove unreacted dUMP. Tritium release from the 5 position of dUMP was determined by liquid scintillation of the supernatant. The apparent K* (K*,!11) was determined at each concentration of 5,10-methylene tetrahydrofolate by computer-assisted non-linear least squares fitting of the data to the tight binding inhibition equation of Morrison [1].

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Whole cell TS assays[1]
TS activity was measured by a tritium release assay in which cells are incubated with [5-3H]deoxyuridine which is converted to the TS substrate, dUMP, following cellular uptake. Enzyme activity is quantitated as tritium released from [5-3H]dUMP during the TScatalyzed reaction. Each reaction mixture contained 30 lM dUdR, 5 uCi/ml 5-3HdUdR and 8]105 cells. Reactions were carried out at 37°C and 5% CO2 in the presence and absence of AG337 and were initiated by the addition of cells. They were stopped after varying incubation times by the addition of 0.4 ml ice-cold 1 M perchloric acid and processed as described previously.

Cell lines were maintained without antibiotics in RPMI-1640 medium containing 10% heat-inactivated fetal calf serum (Hyclone Laboratories, Logan, Utah) except for L1210, CCRF-CEM and L1210/R6 cells, which were passaged in 5% serum. The R6 line was further maintained in the presence of 2 lM methotrexate but was passaged in its absence a minimum of twice before use in assays. Growth inhibition studies were performed by continuous exposure to AG337 in 96-well flat bottomed microtiter plates using a modification of the colorimetric method of Mosmann [20]. Determination of viable cell number was based on the reduction of MTT (Sigma Chemical, St. Louis, Mo.) to formazan, which occurs as a result of dehydrogenase enzyme activity in the mitochondria of viable cells. The formazan product was quantitated after dissolution in DMSO by absorbance measurement at 540 nm. In each assay, the effects of eight or nine serial twofold dilutions of the test agent were tested in quadruplicate microcultures. IC50 values, representing the concentration of AG337 which reduced the optical density to 50% of that observed in control cultures, were derived from semi-logarithmic plots of cell growth (expressed as a percent of control) versus AG337 concentration. For protection studies designed to address the intracellular locus of activity of AG337, thymidine, hypoxanthine, and folinic acid (Sigma Chemical, St. Louis, Mo.) were present in assays at concentrations of 10, 35 and 0.5 lM, respectively.[1]

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Ribonucleotide and deoxyribonucleotide pool levels[1]
L1210 cells (approximately 5]107 cells at a density of 106 cells/ml) were incubated for 2 h at 37°C either alone or with 10 lM AG337. Ribonucleotide profiles were determined by anion-exchange HPLC of neutralized perchloric acid extracts as described previously. Briefly, cells were pelleted by centrifugation and resuspended in 0.2 ml 0.7 N perchloric acid. The acid precipitate was removed by centrifugation and the supernatant neutralized with solid KHCO3. Deoxyribonucleotides were recovered from the neutralized extracts by a modification of the procedure of Garrett and Santi. Aliquots (175 ul) of extracts were treated with 5 mM deoxyguanosine (10 ul) and 0.5 M NaIO4 (40 ul), followed after several minutes by 4 M methylamine pH 7.5 (25 ul) and incubation at 37°C for 30 min. Excess NaIO4 was reduced by a 5-min incubation at room temperature with 1 M rhamnose (20 ul). A mixture of standards containing ATP (2 mM) and 10 mM dATP, dCTP, TTP and dGTP were processed similarly to verify recovery. Separations were performed on a Partisil-10 SAX column. Ribonucleotide extracts (50 ll) were eluted with a linear gradient of 5 mM NH4 H2 PO4 (pH 2.5) to 0.5 M NH4 H2 PO4 (pH 4.8) over 40 min with detection at 254 nm. Retention times were verified in control runs with standards. Deoxynucleotides were separated on the same column with isocratic elution using a mobile phase of 5 mM NH4 H2 PO4 pH 2.8: 0.5 M NH4 H2 PO4 pH 4.8 (4:6) and detection at 284 and 270 nm.[1]

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Cell cycle kinetics [1]
Cell cycle distributions were determined by flow cytometric analysis from the DNA histogram of nuclei stained with propidium iodide. Exponentially growing cultures of L1210 cells were treated for 20 h with concentrations of either 0.7 uM or 3.5 lM AG337 and the cell cycle kinetics compared to those of untreated cultures. For analysis, cells were fixed in 95% ethanol precooled to -20°C, washed in phosphate-buffered saline containing 5% heat-inactivated fetal calf serum and treated with 20 ug/ml propidium iodide containing 1 mg/ml RNase.

Antitumor studies [1]
The thymidine kinase deficient L5178Y/TK~ tumor was maintained by passage in DBA/2 mice. For experiments, either 1]106 cells (i.p.) or 5]106 cells (i.m.) were injected into B6D2F1 hybrid mice. For i.m. studies, the tumor was implanted in the gastrocnemius muscle. The day of tumor inoculation was designated day 0, and treatment was initiated on day 1, 3 or 5 for 5—10 days. AG337 was dissolved in 5% dextrose and administered by i.p. or oral dosing. Antitumor effects were evaluated in animals bearing i.p. tumors by increase in life span compared to controls which received vehicle alone and for which the mean survival time was 26.3 days after tumor inoculation. Animals surviving to day 100 were designated cured. For the i.m. implanted tumor, the diameter of the leg at the site of inoculation was measured daily and animals were sacrificed at a fixed end point diameter of 14 mm, which represents an approximately 2.5-fold increase in size. Animals were considered cured if the leg diameter at day 35 was less than or equal to the leg diameter on day 0. Thymidine kinasereplete L1210 and P388 tumors were implanted i.p. at 1]106 cells per mouse (day 0) and treatment was initiated on day 3.

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Pharmacokinetics[1]
Studies were conducted in male Sprague-Dawley rats with jugular vein cannulated and exteriorized. After i.v. or oral administration of AG337, blood samples (200 ul) were withdrawn from the cannula and plasma separated by centrifugation. Plasma samples (100 ul) were extracted with 1 ml acetonitrile for 2 min, centrifuged at 2000 rpm for 5 min and the supernatant evaporated at 40°C under nitrogen. Dried samples were reconstituted with 300 ul mobile phase and AG337 levels quantitated by HPLC. Separation was effected using a C18 column (4.6 x 150 mm,5 lm) and isocratic elution with 0.05 M sodium phosphate buffer, pH 4.0 and acetonitrile (80:20); detection was at a wavelength of 230 nm. Plasma AG337 concentration-time data were fitted to multi-exponential equations using nonlinear regression analysis. Pharmacokinetic parameters, as follows, were calculated from the parameter estimates of the y-axis intercepts and rate constants: absorption, distribution, and elimination half lifes, volumes of distribution, and systemic clearance (Cl4 ). Absolute oral bioavailability was calculated as [AUCoral/AUCiv]]100 using model-independent values for AUC.

[1]. AG337, a novel lipophilic thymidylate synthase inhibitor: in vitro and in vivo preclinical studies. Cancer Chemother Pharmacol. 1996;37(6):509-17.

[2]. Characterization of the effect of AG337, a novel lipophilic thymidylate synthase inhibitor, on human head and neck and human leukemia cell lines. Int J Oncol. 1999 Dec;15(6):1245-50.

See also: Nolatrexed (annotation moved to).

C14H14CL2N4OS

357.25

356.026

C, 47.07; H, 3.95; Cl, 19.85; N, 15.68; O, 4.48; S, 8.97

152946-68-4

152946-68-4 (HCl salt); 147149-76-6;

135409535

Light yellow to yellow solid powder

493.6ºCat 760 mmHg

301-302ºC

252.3ºC

4.545

4

4

2

22

408

0

CC1=C(SC2=CC=NC=C2)C3=C(NC(NC3=O)=N)C=C1.Cl.Cl

PJKVJJYMWOCLIJ-UHFFFAOYSA-N

InChI=1S/C14H12N4OS.2ClH/c1-8-2-3-10-11(13(19)18-14(15)17-10)12(8)20-9-4-6-16-7-5-9;;/h2-7H,1H3,(H3,15,17,18,19);2*1H

2-amino-6-methyl-5-pyridin-4-ylsulfanyl-3H-quinazolin-4-one;dihydrochloride

Nolatrexed HCl; Nolatrexed dihydrochloride; AG337; AG 337; AG-337; brand name: Thymitaq.

2934.99.9001

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.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O: ~50 mg/mL (~140.0 mM)
DMSO: ~41.7 mg/mL (~116.6 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.82 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.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (5.82 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 20.8 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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (5.82 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 25 mg/mL (69.98 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with heating and sonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)