|
Growth inhibition of CCRF-CEM human leukemic lymphoblasts
|
Homo sapiens
|
4.4
nM
|
|
Journal : J. Med. Chem.
Title : Analogues of the potent nonpolyglutamatable antifolate N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-ornithine (PT523) with modifications in the side chain, p-aminobenzoyl moiety, or 9,10-bridge: synthesis and in vitro antitumor activity.
Year : 2000
Volume : 43
Issue : 8
First Page : 1620
Last Page : 1634
Authors : Rosowsky A, Wright JE, Vaidya CM, Forsch RA, Bader H.
Abstract : Seven N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-o rnithine (2, PT523) analogues were synthesized by modifications of the literature synthesis of the corresponding AMT (1) analogues and were tested as inhibitors of tumor cell growth. In growth assays against cultured CCRF-CEM human leukemic cells exposed to drug for 72 h, the IC(50) values of analogues in which N(10) was replaced by CH(2) and CHMe were found to be 0.55 +/- 0.07 and 0.63 +/- 0.08 nM, and thus these analogues are more potent than 1 (IC(50) = 4.4 +/- 1.0 nM) or 2 (IC(50) = 1.5 +/- 0.39 nM). The 10-ethyl-10-deaza analogue of 2 (IC(50) = 1.2 +/- 0.25 nM) was not statistically different from 2 but was more potent than edatrexate, the 10-ethyl-10-deaza analogue of 1, which had an IC(50) of 3.3 +/- 0.36 nM. In contrast, the analogue of 2 with both an ethyl and a CO(2)Me group at the 10-position had an IC(50) of 54 +/- 4.9 nM, showing this modification to be unfavorable. The 4-amino-1-naphthoic acid analogue of 2 had an IC(50) of 1.2 +/- 0.22 nM, indicating that replacement of the p-aminobenzoic acid (pABA) moiety does not diminish cytotoxicity. The analogues in which the (CH(2))(3) side chain was replaced by slightly longer CH(2)SCH(2) and (CH(2))(2)SCH(2) groups gave IC(50) values of 4.4 +/- 1.1 and 5.0 +/- 0.56 nM and thus were somewhat less potent than the parent molecule. However the analogues in which the aromatic COOH group was at the meta and para positions of the phthaloyl ring had IC(50) values of 7.5 +/- 0.47 and 55 +/- 0.07 nM, confirming the low potency we had previously observed with these compounds against other cell lines. Overall, the results in this study support the conclusion that, while the position of the phthaloyl COOH group and the length of the amino acid side chain in 2 are important determinants of cytotoxic potency, changes in the pABA region and 9, 10-bridge are well-tolerated and can even increase potency.
|
Cell growth inhibition against CEM/MTX cell from human leukemic lymphoblasts
|
Homo sapiens
|
320.0
nM
|
|
Journal : J. Med. Chem.
Title : Methotrexate analogues. 25. Chemical and biological studies on the gamma-tert-butyl esters of methotrexate and aminopterin.
Year : 1985
Volume : 28
Issue : 5
First Page : 660
Last Page : 667
Authors : Rosowsky A, Freisheim JH, Bader H, Forsch RA, Susten SS, Cucchi CA, Frei E.
Abstract : gamma-tert-Butylaminopterin (gamma-tBAMT), the first example of an aminopterin (AMT) gamma-monoester, was synthesized, and new routes to the known N10-methyl analogue gamma-tert-butyl methotrexate (gamma-tBMTX) were developed. The inhibitory effects of gamma-tBAMT on the activity of purified dihydrofolate reductase (DHFR) from L1210 murine leukemia cells, the growth of L1210 cells and CEM human leukemic lymphoblasts in suspension culture, and the growth of several lines of human squamous cell carcinoma of the head and neck in monolayer culture were compared with the effects of gamma-tBMTX and the parent acids AMT and methotrexate (MTX). Patterns of cross-resistance to gamma-tBAMT, gamma-tBMTX, and AMT among several MTX-resistant cell lines were examined. In vivo antitumor activities of gamma-tBAMT and gamma-tBMTX were compared in mice with L1210 leukemia. While the activity of gamma-tBAMT was very close to that of gamma-tBMTX in the DHFR inhibition assay, the AMT ester was more potent than the MTX ester against cells in culture and against L1210 leukemia in vivo. Only partial cross-resistance was shown against gamma-tBMTX and gamma-tBAMT in cultured cells that were resistant to MTX by virtue of a transport defect or a combination of defective transport and elevated DHFR activity.
|
Tested for cell-growth inhibition against human leukemic lymphoblast CEM/MTX cells
|
Homo sapiens
|
320.0
nM
|
|
Journal : J. Med. Chem.
Title : Methotrexate analogues. 34. Replacement of the glutamate moiety in methotrexate and aminopterin by long-chain 2-aminoalkanedioic acids.
Year : 1988
Volume : 31
Issue : 7
First Page : 1338
Last Page : 1344
Authors : Rosowsky A, Bader H, Kohler W, Freisheim JH, Moran RG.
Abstract : Eight previously unreported methotrexate (MTX) and aminopterin (AMT) analogues with the L-glutamate moiety replaced by DL-2-aminoalkanedioic acids containing up to 10 CH2 groups were synthesized from 4-amino-4-deoxy-N10-methylpteroic or 4-amino-4-deoxy-N10-formylpteroic acid. All the compounds were potent inhibitors of purified L1210 mouse leukemia dihydrofolate reductase (DHFR), with IC50's of 0.023-0.034 microM for the MTX analogues and 0.054-0.067 microM for the AMT analogues. The compounds were not substrates for, but were inhibitors of, partially purified mouse liver folylpolyglutamate synthetase (FPGS). Activity was correlated with the number of CH2 groups in the side chain. The IC50's for inhibition of cell growth in culture by the chain-extended MTX analogues were 0.016-0.64 microM against CEM human leukemic lymphoblasts and 0.0012-0.026 microM against L1210 mouse leukemia cells. However, the optimal chain length for growth-inhibitory activity was species-dependent. Our results suggested that CEM cells were inhibited most actively by the analogue with nine CH2 groups, while L1210 cells were most sensitive to the analogue with six CH2 groups. Among the AMT analogues, on the other hand, the most active compound against L1210 cells was the one with nine CH2 groups, which had an IC50 of 0.000 65 microM as compared with 0.0046 microM for MTX and 0.002 microM for AMT. A high degree of cross-resistance was observed between MTX and the chain-extended compounds in two MTX-resistant cell lines, CEM/MTX and L1210/R81. All the MTX analogues were active against L1210 leukemia in mice on a qd X 9 schedule, with optimal increases in lifespan (ILS) of 75-140%. Notwithstanding their high in vitro activity, the AMT analogues were more toxic and less therapeutically effective than MTX analogues of the same chain length even though neither series of compounds possessed FPGS substrate activity. These MTX and AMT analogues are an unusual group of compounds in that they retain the dicarboxylic acid structure of classical antifolates yet are more lipophilic than the parent compounds because they have more CH2 groups and are almost equivalent in vivo to MTX on the same schedule even though they do not form polyglutamates.
|
Cell growth inhibition against CEM cell from human leukemic lymphoblasts
|
Homo sapiens
|
1.0
nM
|
|
Journal : J. Med. Chem.
Title : Methotrexate analogues. 25. Chemical and biological studies on the gamma-tert-butyl esters of methotrexate and aminopterin.
Year : 1985
Volume : 28
Issue : 5
First Page : 660
Last Page : 667
Authors : Rosowsky A, Freisheim JH, Bader H, Forsch RA, Susten SS, Cucchi CA, Frei E.
Abstract : gamma-tert-Butylaminopterin (gamma-tBAMT), the first example of an aminopterin (AMT) gamma-monoester, was synthesized, and new routes to the known N10-methyl analogue gamma-tert-butyl methotrexate (gamma-tBMTX) were developed. The inhibitory effects of gamma-tBAMT on the activity of purified dihydrofolate reductase (DHFR) from L1210 murine leukemia cells, the growth of L1210 cells and CEM human leukemic lymphoblasts in suspension culture, and the growth of several lines of human squamous cell carcinoma of the head and neck in monolayer culture were compared with the effects of gamma-tBMTX and the parent acids AMT and methotrexate (MTX). Patterns of cross-resistance to gamma-tBAMT, gamma-tBMTX, and AMT among several MTX-resistant cell lines were examined. In vivo antitumor activities of gamma-tBAMT and gamma-tBMTX were compared in mice with L1210 leukemia. While the activity of gamma-tBAMT was very close to that of gamma-tBMTX in the DHFR inhibition assay, the AMT ester was more potent than the MTX ester against cells in culture and against L1210 leukemia in vivo. Only partial cross-resistance was shown against gamma-tBMTX and gamma-tBAMT in cultured cells that were resistant to MTX by virtue of a transport defect or a combination of defective transport and elevated DHFR activity.
|
Tested for cell-growth inhibition against human leukemic lymphoblast CEM cells
|
Homo sapiens
|
1.0
nM
|
|
Journal : J. Med. Chem.
Title : Methotrexate analogues. 34. Replacement of the glutamate moiety in methotrexate and aminopterin by long-chain 2-aminoalkanedioic acids.
Year : 1988
Volume : 31
Issue : 7
First Page : 1338
Last Page : 1344
Authors : Rosowsky A, Bader H, Kohler W, Freisheim JH, Moran RG.
Abstract : Eight previously unreported methotrexate (MTX) and aminopterin (AMT) analogues with the L-glutamate moiety replaced by DL-2-aminoalkanedioic acids containing up to 10 CH2 groups were synthesized from 4-amino-4-deoxy-N10-methylpteroic or 4-amino-4-deoxy-N10-formylpteroic acid. All the compounds were potent inhibitors of purified L1210 mouse leukemia dihydrofolate reductase (DHFR), with IC50's of 0.023-0.034 microM for the MTX analogues and 0.054-0.067 microM for the AMT analogues. The compounds were not substrates for, but were inhibitors of, partially purified mouse liver folylpolyglutamate synthetase (FPGS). Activity was correlated with the number of CH2 groups in the side chain. The IC50's for inhibition of cell growth in culture by the chain-extended MTX analogues were 0.016-0.64 microM against CEM human leukemic lymphoblasts and 0.0012-0.026 microM against L1210 mouse leukemia cells. However, the optimal chain length for growth-inhibitory activity was species-dependent. Our results suggested that CEM cells were inhibited most actively by the analogue with nine CH2 groups, while L1210 cells were most sensitive to the analogue with six CH2 groups. Among the AMT analogues, on the other hand, the most active compound against L1210 cells was the one with nine CH2 groups, which had an IC50 of 0.000 65 microM as compared with 0.0046 microM for MTX and 0.002 microM for AMT. A high degree of cross-resistance was observed between MTX and the chain-extended compounds in two MTX-resistant cell lines, CEM/MTX and L1210/R81. All the MTX analogues were active against L1210 leukemia in mice on a qd X 9 schedule, with optimal increases in lifespan (ILS) of 75-140%. Notwithstanding their high in vitro activity, the AMT analogues were more toxic and less therapeutically effective than MTX analogues of the same chain length even though neither series of compounds possessed FPGS substrate activity. These MTX and AMT analogues are an unusual group of compounds in that they retain the dicarboxylic acid structure of classical antifolates yet are more lipophilic than the parent compounds because they have more CH2 groups and are almost equivalent in vivo to MTX on the same schedule even though they do not form polyglutamates.
|
Evaluated in vitro for ability to inhibit the Detroit 98 cell lines
|
Homo sapiens
|
2.0
nM
|
|
Journal : J. Med. Chem.
Title : Synthesis and biological activity of an acyclic analogue of 5,6,7,8-tetrahydrofolic acid, N-[4-[[3-(2,4-diamino-1,6-dihydro-6-oxo-5- pyrimidinyl)propyl]amino]-benzoyl]-L-glutamic acid.
Year : 1990
Volume : 33
Issue : 2
First Page : 561
Last Page : 567
Authors : Kelley JL, McLean EW, Cohn NK, Edelstein MP, Duch DS, Smith GK, Hanlon MH, Ferone R.
Abstract : The synthesis and biological evaluation of N-[4-[[3-(2,4-diamino-1,6-dihydro-6-oxo-5-pyrimidinyl)propyl]amino]- benzoyl]-L-glutamic acid (1) (5-DACTHF, 543U76), an acyclic analogue of 5,6,7,8-tetrahydrofolic acid (THFA), are described. The key intermediate, hemiaminal 8, was prepared in four stages from 3-chloropropionaldehyde diethyl acetal. Reaction of 8 with dimethyl N-(4-aminobenzoyl)-L-glutamate gave the 2,4-bis(acetylamino) derivative 11, which was hydrolyzed with 1 N sodium hydroxide to give 1; the glycine analogue 16 was prepared in a similar manner. The N-methyl analogue 2 and N-formyl analogue 3 were prepared from 11 and 1, respectively. Compounds 1-3 inhibited growth of Detroit 98 and L cells in cell culture, with IC50s ranging from 2 to 0.018 microM. Cell culture toxicity reversal studies and enzyme inhibition tests showed that 1 was cytotoxic but not by the mechanism of the dihydrofolate reductase inhibitor aminopterin. Compound 1 and its polyglutamylated homologues inhibited glycinamide ribonucleotide transformylase (GAR-TFase) and aminoimidazole ribonucleotide transformylase (AICAR-TFase), the folate-dependent enzymes in de novo purine biosynthesis; and 1 was an effective substrate for mammalian folyl-polyglutamate synthetase. The compound inhibited (IC50 = 20 nM) the conversion of [14C]formate to [14C]-formylglycinamide ribonucleotide by MOLT-4 cells in culture. These data suggest that the site of action of 1 is inhibition of purine de novo biosynthesis. Moderate activity was observed against P388 leukemia in vivo.
|
Compound was tested for its inhibitory concentration to inhibit the enzyme Dihydro Folate Reductase (DHFR) from murine L1210 leukemia cells.
|
None
|
20.0
nM
|
|
Journal : J. Med. Chem.
Title : Methotrexate analogues. 28. Synthesis and biological evaluation of new gamma-monoamides of aminopterin and methotrexate.
Year : 1986
Volume : 29
Issue : 9
First Page : 1703
Last Page : 1709
Authors : Rosowsky A, Bader H, Radike-Smith M, Cucchi CA, Wick MM, Freisheim JH.
Abstract : Lipophilic gamma-monoamide derivatives of aminopterin (AMT) were synthesized in high overall yield from 4-amino-4-deoxy-N10-formylpteroic acid and gamma-N-tert-alkyl-, gamma-N-aralkyl-, or gamma-N-arylamides of alpha-benzyl L-glutamate via a modification of the mixed carboxylic-carbonic anhydride coupling method. Coupling was also accomplished with p-nitrophenyl 4-amino-4-deoxy-N10-formylpteroate. Compounds obtained in this manner included the gamma-tert-butylamide, gamma-(1-adamantylamide), gamma-benzylamide, gamma-(3,4-dichlorobenzylamide), gamma-(2,6-dichlorobenzylamide), gamma-anilide, gamma-(3,4-methylenedioxyanilide), and gamma-(3,4-dihydroxanilide) derivatives of AMT. Also prepared, from 4-amino-4-deoxy-N10-methylpteroic acid via diethyl phosphorocyanidate coupling, was the gamma-(3,4-methylenedioxyanilide) of MTX. The methylenedioxyanilides were cleaved smoothly to dihydroxyanilides with boron tris(trifluoroacetate) in trifluoroacetic acid. All the gamma-monoamides were tested as inhibitors of purified dihydrofolate reductase (DHFR) from murine L1210 leukemia cells and as inhibitors of the growth of wild-type L1210 cells and a subline (L1210/R81) with high-level resistance to MTX and AMT based mainly on a defect in drug uptake via active transport. Several compounds were also tested against human leukemic lymphoblasts (CEM cells) and a resistant subline (CEM/MTX) whose resistance is likewise based on uptake. The IC50 of the gamma-monoamides against DHFR was 1.5- to 5-fold higher than that of the parent acids, but the IC50 against cultured cells varied over a much broader range, suggesting that uptake and/or metabolism rather than DHFR binding are principal determinants of in vitro growth inhibitory activity for these compounds. gamma-N-Aryl and gamma-N-aralkyl derivatives appeared to be more potent than gamma-N-tert-alkyl derivatives. Where comparison could be made, AMT gamma-monoamides were more potent than MTX gamma-monoamides. Several of the gamma-monoamides showed potency comparable to that of the parent acid against wild-type L1210 and CEM cells; all of them were more potent than MTX against the L1210/R81 subline; and some of the AMT gamma-monoamides were also more potent than the parent acid against resistant CEM/MTX cells. As a group, however, the gamma-monoamides were considerably more active against the murine cells than against the human cells, suggesting that the former may take up the amides better or may be able to metabolize them more efficiently than the parent acids. All the gamma-monoamides were tested in vivo against L1210 leukemia in mice.(ABSTRACT TRUNCATED AT 400 WORDS)
|
Inhibition of dihydrofolate reductase in HeLa cells
|
None
|
31.0
nM
|
|
Journal : J. Med. Chem.
Title : Novel fluorinated antifolates. Enzyme inhibition and cytotoxicity studies on 2'- and 3'-fluoroaminopterin.
Year : 1983
Volume : 26
Issue : 8
First Page : 1193
Last Page : 1196
Authors : Henkin J, Washtien WL.
Abstract : Two novel analogues of aminopterin with a single fluorine substitution in the 2' (compound 8) or in the 3' (compound 9) position of the p-aminobenzoyl group were synthesized and evaluated as inhibitors of dihydrofolate reductase from two bacterial species and from human HeLa cells. The 2'-fluoro compound was bound essentially the same as aminopterin itself, while the 3'-fluoro derivative bound two- to threefold more tightly in all cases. UV spectral shifts indicated normal binding of the pteridine. Cytotoxicity studies against mouse leukemia L1210 cells and the human stomach cancer line HuTu80 indicated equivalent toxicity of the parent drug with the 2'-fluoro analogue. 3'-Fluoroaminopterin was, however, twice as toxic as aminopterin to both cell lines.
|
Ability to inhibit purified Dihydrofolate reductase from human leukemic lymphoblasts was determined spectrophotometrically at 340 nM.
|
None
|
25.0
nM
|
|
Journal : J. Med. Chem.
Title : Analogues of methotrexate and aminopterin with gamma-methylene and gamma-cyano substitution of the glutamate side chain: synthesis and in vitro biological activity.
Year : 1991
Volume : 34
Issue : 1
First Page : 203
Last Page : 208
Authors : Rosowsky A, Bader H, Freisheim JH.
Abstract : Analogues of methotrexate (MTX) and aminopterin (AMT) modified at the gamma-position of the glutamate side chain were synthesized and evaluated as dihydrofolate reductase (DHFR) inhibitors and tumor cell growth inhibitors. Condesations of 4-amino-4-deoxy-N10-methylpteroic acid (mAPA) with dimethyl DL-4-methyleneglutamate in the presence of diethyl phosphorocyanidate (DEPC) followed by alkaline hydrolysis yielded N-(4-amino-4-deoxy-N10-methylpteroyl)-DL-4-methyleneglutamic acid (gamma-methyleneMTX). Condensation of 4-amino-4-deoxy-N10-formylpteroic acid (fAPA) with dimethyl-DL-4-methyleneglutamate by the mixed carboxylic-carbonic anhydride method yielded N-4-amino-4-deoxypteroyl)-DL-4-methyleneglutamic acid (gamma-methyleneAMT). Also prepared via DEPC coupling was a mixture of the four possible diastereomers of N-(4-amino-4-deoxy-N10-methylpteroyl)-4-cyanoglutamic acid (gamma-cyanoMTX). The requisite intermediate gamma-tert-butyl alpha-methyl 4-cyanoglutamate, as a DL-threo/DL-erythro mixture, was prepared from methyl N alpha-Boc-O-tosyl-L-serinate by reaction with sodium tert-butyl cyanoacetate followed by mild trifluoroacetic treatment to selectively remove the Boc group. The gamma-methylene derivatives of MTX and AMT are attractive because of their potential to act as Michael acceptors within the DHFR active site. gamma-CyanoMTX may be viewed as a congener of the nonpolyglutamated MTX analogue gamma-fluoroMTX. In vitro bioassay data for the gamma-methylene and gamma-cyano compounds support the idea that the active site of DHFR, already known for its ability to tolerate modification of the gamma-carboxyl group of MTX and AMT, can likewise accommodate substitution on the gamma-carbon itself.
|
Inhibition of dihydrofolate reductase (DHFR) from human cells (WI-L2/M4).
|
None
|
25.0
nM
|
|
Journal : J. Med. Chem.
Title : Synthesis and biological activity of methotrexate analogues with two acid groups and a hydrophobic aromatic ring in the side chain.
Year : 1991
Volume : 34
Issue : 2
First Page : 574
Last Page : 579
Authors : Rosowsky A, Bader H, Freisheim JH.
Abstract : The heretofore unknown gamma-(m-carboxyanilide) and gamma-(m-boronoanillide) derivatives of methotrexate (MTX) and the gamma-(m-carboxyanilide) derivatives of aminopterin (AMT) were prepared and tested as inhibitors of dihydrofolate reductase (DHFR) and as inhibitors of cell growth in culture with the aim of comparing their activity with that of N alpha-(4-amino-4-deoxypteroyl)-N delta-hemiphthaloyl-L-ornithine, a potent antifolate whose side chain likewise contains a hydrophobic aromatic ring with an acid group on the ring. All three anilides were potent DHFR inhibitors, with activity comparable to MTX and AMT. The gamma-(m-boronoanilide) displayed growth inhibitory potency similar to that of the hemiphthaloylornithine analogue, with an IC50 of only 0.7 nM. This compound, which is the most potent of the gamma-amides of MTX tested to date, is also the first reported example of an antifolate with a B(OH)2 group in the side chain and is especially novel because of its potential to form a stable tetrahedral boronate complex by reaction with electron rich OH or NH2 groups in the active site of DHFR or other folate enzymes. In antitumor assays against L1210 leukemia in mice, N alpha-(4-amino-4-deoxypteroyl)-N delta-hemiphthaloyl-L-ornithine gave a T/C of greater than 263% at 20 mg/kg (qdx9) and 300% at 16 mg/kg (bidx10), whereas maximally tolerated doses of MTX of 8 mg/kg (qdx9) and 1 mg/kg (bidx10) gave T/C values of 213 and 188%, respectively. MTX gamma-(m-boronoanilide) was also active, with a T/C of 175% at 32 mg/kg (qdx9), the highest dose tested.
|
Tested for inhibitory concentration against human dihydrofolate reductase(DHFR)
|
None
|
11.8
nM
|
|
Journal : J. Med. Chem.
Title : Synthesis and biological activity of N omega-hemiphthaloyl-alpha,omega- diaminoalkanoic acid analogues of aminopterin and 3',5-dichloroaminopterin.
Year : 1994
Volume : 37
Issue : 14
First Page : 2167
Last Page : 2174
Authors : Rosowsky A, Bader H, Wright JE, Keyomarsi K, Matherly LH.
Abstract : Analogues of N alpha-(4-amino-4-deoxypteroyl)-N delta-(hemiphthaloyl)-L-ornithine (PT523) with 3',5'-dichloro substitution in the p-aminobenzoyl moiety or with one less or one more CH2 group in the amino acid moiety were synthesized and tested as inhibitors of dihydrofolate reductase (DHFR) activity and cell growth. Replacement of L-ornithine in PT523 by L-2,4-diaminobutanoic acid or L-lysine did not decrease binding to human recombinant DHFR but resulted in some loss of activity against SCC25 human and SCC VII murine squamous cell carcinoma and against MCF-7 human breast carcinoma in culture. PT523 was several times more potent than methotrexate (MTX), aminopterin (AMT), or trimetrexate (TMQ). 3',5'-Dichloro substitution did not decrease either DHFR binding or cytotoxicity. A new synthetic route to PT523 from 2,4-diamino-6-(hydroxymethyl)pteridine and methyl N alpha-(4-aminobenzoyl)-N delta-phthaloyl-L-ornithinate was investigated but was not found superior to previously described methods. In comparative experiments on the ability of PT523 and MTX to competitively inhibit the influx of (6R)-5,10-dideazatetra-hydrofolate (DDATHF, lometrexol), used here as a surrogate for MTX and reduced folates, the Ki of PT523 was lower than that of MTX in both wild-type CCRF-CEM human leukemic lymphoblasts and the transport- and polyglutamylation-defective subline CEM/MTX. The CCRF-CEM cells were 10-fold more sensitive to PT523 than to MTX, whereas the CEM/MTX cells were 240-fold more sensitive. However, in contrast to other MTX-resistant cells where collateral sensitivity to PT523 has been seen. CEM/MTX cells still showed substantial cross resistance to PT523 which may reflect an unusual heightened ability to utilize exogenous folic acid. The good correlation observed with both cell lines between the cytotoxicity of PT523 and MTX and the ability to inhibit DDATHF influx supported the view that PT523 and MTX share, at least in part, a common protein carrier for membrane transport.
|
Compound was evaluated for Dihydrofolate Reductase (DHFR) inhibition
|
None
|
25.0
nM
|
|
Journal : J. Med. Chem.
Title : Synthesis and biological activity of the 2-desamino and 2-desamino-2-methyl analogues of aminopterin and methotrexate.
Year : 1991
Volume : 34
Issue : 1
First Page : 227
Last Page : 234
Authors : Rosowsky A, Forsch RA, Moran RG, Freisheim JH.
Abstract : The previously undescribed 2-desamino and 2-desamino-2-methyl analogues of aminopterin (AMT) and methotrexate (MTX) were synthesized from 2-amino-5-(chloromethyl)pyrazine-3-carbonitrile. The AMT analogues were obtained via a three-step sequence consisting of condensation with di-tert-butyl N-(4-aminobenzoyl)-L-glutamate, heating with formamidine or acetamidine acetate, and mild acidolysis with trifluoroacetic acid. The MTX analogues were prepared similarly, except that 2-amino-5-(chloromethyl)pyrazine-3-carbonitrile was condensed with 4-(N-methylamino)benzoic acid and the resulting product was annulated with formamidine or acetamidine acetate to obtain the 2-desamino and 2-desamino-2-methyl analogues, respectively, of 4-amino-4-deoxy-N10-methylpteroic acid. Condensation with di-tert-butyl L-glutamate in the presence of diethyl phosphorocyanidate followed by ester cleavage with trifluoroacetic acid was then carried out. Retention of the L configuration in the glutamate moiety during this synthesis was demonstrated by rapid and essentially complete hydrolysis with carboxypeptidase G1 under conditions that likewise cleaved the L enantiomer of MTX but left the D enantiomer unaffected. The 2-desamino and 2-desamino-2-methyl analogues of AMT and MTX inhibited the growth of tumor cells, but were very poor inhibitors of dihydrofolate reductase (DHFR). These unexpected results suggested that activity in intact cells was due to metabolism of the 2-desamino compounds to polyglutamates.
|
Inhibition of dihydrofolate reductase in Escherichia coli
|
Escherichia coli
|
11.0
nM
|
|
Journal : J. Med. Chem.
Title : Novel fluorinated antifolates. Enzyme inhibition and cytotoxicity studies on 2'- and 3'-fluoroaminopterin.
Year : 1983
Volume : 26
Issue : 8
First Page : 1193
Last Page : 1196
Authors : Henkin J, Washtien WL.
Abstract : Two novel analogues of aminopterin with a single fluorine substitution in the 2' (compound 8) or in the 3' (compound 9) position of the p-aminobenzoyl group were synthesized and evaluated as inhibitors of dihydrofolate reductase from two bacterial species and from human HeLa cells. The 2'-fluoro compound was bound essentially the same as aminopterin itself, while the 3'-fluoro derivative bound two- to threefold more tightly in all cases. UV spectral shifts indicated normal binding of the pteridine. Cytotoxicity studies against mouse leukemia L1210 cells and the human stomach cancer line HuTu80 indicated equivalent toxicity of the parent drug with the 2'-fluoro analogue. 3'-Fluoroaminopterin was, however, twice as toxic as aminopterin to both cell lines.
|
Tested in vitro for inhibitory concentration against CCRF-CEM human Leukemic lymphoblast by using DHFR as primary target
|
None
|
3.0
nM
|
|
Journal : J. Med. Chem.
Title : Synthesis and biological activity of N omega-hemiphthaloyl-alpha,omega- diaminoalkanoic acid analogues of aminopterin and 3',5-dichloroaminopterin.
Year : 1994
Volume : 37
Issue : 14
First Page : 2167
Last Page : 2174
Authors : Rosowsky A, Bader H, Wright JE, Keyomarsi K, Matherly LH.
Abstract : Analogues of N alpha-(4-amino-4-deoxypteroyl)-N delta-(hemiphthaloyl)-L-ornithine (PT523) with 3',5'-dichloro substitution in the p-aminobenzoyl moiety or with one less or one more CH2 group in the amino acid moiety were synthesized and tested as inhibitors of dihydrofolate reductase (DHFR) activity and cell growth. Replacement of L-ornithine in PT523 by L-2,4-diaminobutanoic acid or L-lysine did not decrease binding to human recombinant DHFR but resulted in some loss of activity against SCC25 human and SCC VII murine squamous cell carcinoma and against MCF-7 human breast carcinoma in culture. PT523 was several times more potent than methotrexate (MTX), aminopterin (AMT), or trimetrexate (TMQ). 3',5'-Dichloro substitution did not decrease either DHFR binding or cytotoxicity. A new synthetic route to PT523 from 2,4-diamino-6-(hydroxymethyl)pteridine and methyl N alpha-(4-aminobenzoyl)-N delta-phthaloyl-L-ornithinate was investigated but was not found superior to previously described methods. In comparative experiments on the ability of PT523 and MTX to competitively inhibit the influx of (6R)-5,10-dideazatetra-hydrofolate (DDATHF, lometrexol), used here as a surrogate for MTX and reduced folates, the Ki of PT523 was lower than that of MTX in both wild-type CCRF-CEM human leukemic lymphoblasts and the transport- and polyglutamylation-defective subline CEM/MTX. The CCRF-CEM cells were 10-fold more sensitive to PT523 than to MTX, whereas the CEM/MTX cells were 240-fold more sensitive. However, in contrast to other MTX-resistant cells where collateral sensitivity to PT523 has been seen. CEM/MTX cells still showed substantial cross resistance to PT523 which may reflect an unusual heightened ability to utilize exogenous folic acid. The good correlation observed with both cell lines between the cytotoxicity of PT523 and MTX and the ability to inhibit DDATHF influx supported the view that PT523 and MTX share, at least in part, a common protein carrier for membrane transport.
|
Compound was evaluated for the inhibition of Dihydrofolate reductase at concentration ranged from 0.15-0.50 uM
|
None
|
0.0037
nM
|
|
Journal : J. Med. Chem.
Title : Synthesis and potent antifolate activity and cytotoxicity of B-ring deaza analogues of the nonpolyglutamatable dihydrofolate reductase inhibitor Nalpha-(4-amino-4-deoxypteroyl)-Ndelta-hemiphthaloyl- L-ornithine (PT523).
Year : 1998
Volume : 41
Issue : 26
First Page : 5310
Last Page : 5319
Authors : Rosowsky A, Wright JE, Vaidya CM, Bader H, Forsch RA, Mota CE, Pardo J, Chen CS, Chen YN.
Abstract : Six new B-ring analogues of the nonpolyglutamatable antifolate Nalpha-(4-amino-4-deoxypteroyl)-Ndelta-hemiphthaloy l-L-ornithine (PT523, 3) were synthesized with a view to determining the effect of modifications at the 5- and/or 8-position on dihydrofolate reductase (DHFR) binding and tumor cell growth inhibition. The 5- and 8-deaza analogues were prepared from methyl 2-L-amino-5-phthalimidopentanoate and 4-amino-4-deoxy-N10-formyl-5-deaza- and -8-deazapteroic acid, respectively. The 5,8-dideaza analogues were prepared from methyl 2-L-[(4-aminobenzoyl)amino]-5-phthalimidopentanoate and 2, 4-diaminoquinazoline-6-carbonitriles. The Ki for inhibition of human DHFR by the 5-deaza and 5-methyl-5-deaza analogues was about the same as that of 3 (0.35 pM), 11-fold lower than that of aminopterin (AMT, 1), and 15-fold lower than that of methotrexate (MTX, 2). However the Ki of the 8-deaza analogue was 27-fold lower than that of 1, and that of the 5,8-dideaza, 5-methyl-5,8-dideaza, and 5-chloro-5,8-dideaza analogues was approximately 50-fold lower. This trend was consistent with the published literature on the corresponding DHFR inhibitors with a glutamate side chain. In colony formation assays against the human head and neck squamous carcinoma cell line SCC25 after 72 h of treatment, the 5- and 8-deaza analogues were approximately as potent as 3, whereas the 5,8-dideaza analogue was 3 times more potent. 5-Methyl and 5-chloro substitution was also favorable, with the 5-methyl-5-deaza analogue being 2. 5-fold more potent than the 5-deaza analogue. However the effect of 5-methyl substitution was less pronounced in the 5,8-dideaza analogues than in the 5-deaza analogues. The 5-chloro-5,8-dideaza analogue of 3 was the most active member of the series, with an IC50 = 0.33 nM versus 1.8 nM for 3 and 15 nM for MTX. The 5-methyl-5-deaza analogue of 3 was also tested at the National Cancer Institute against a panel of 50 human tumor cell lines in culture and was consistently more potent than 3, with IC50 values in the low-nanomolar to subnanomolar range against most of the tumors. Leukemia and colorectal carcinoma cell lines were generally most sensitive, though good activity was also observed against CNS tumors and carcinomas of the breast and prostate. The results of this study demonstrate that B-ring analogues of 3 inhibit DHFR activity and tumor cell colony formation as well as, or better than, the parent compound. In view of the fact that 3 and its B-ring analogues cannot form polyglutamates, their high cytotoxicity relative to the corresponding B-ring analogues of AMT is noteworthy.
|
Inhibitory activity against Leu22-Phe mutant human Dihydrofolate reductase
|
None
|
0.21
nM
|
|
Journal : J. Med. Chem.
Title : 2,4-Diamino-5-substituted-quinazolines as inhibitors of a human dihydrofolate reductase with a site-directed mutation at position 22 and of the dihydrofolate reductases from Pneumocystis carinii and Toxoplasma gondii.
Year : 1995
Volume : 38
Issue : 5
First Page : 745
Last Page : 752
Authors : Rosowsky A, Mota CE, Queener SF, Waltham M, Ercikan-Abali E, Bertino JR.
Abstract : 2,4-Diaminoquinazoline antifolates with a lipophilic side chain at the 5-position, and in one case with a classical (p-aminobenzoyl)-L-glutamate side chain, were synthesized as potentially selective inhibitors of a site-directed mutant of human dihydrofolate reductase (DHFR) containing phenylalanine instead of leucine at position 22. This mutant enzyme is approximately 100-fold more resistant than native enzyme to the classical antifolate methotrexate (MTX), yet shows minimal cross resistance to the nonclassical antifolates piritrexim (PTX) and trimetrexate (TMQ). Although they were much less potent than trimetrexate and piritrexim, the lipophilic 5-substituted analogues were all found to bind approximately 10 times better to the mutant DHFR than to the wild-type enzyme. The potency of the analogue with a classical (p-aminobenzoyl)-L-glutamate side chain was similarly diminished in comparison with MTX, but the difference in its binding affinity to the two DHFR species was only 5-fold. Thus, by making subtle structural changes in the antifolate molecule, it may be possible to attack resistance due to mutational alterations in the active site of the target enzyme. Also, to test the hypothesis that DHFR from Pneumocystis carinii and Toxoplasma gondii may have a less sterically restrictive active site than the enzyme from mammalian cells, inhibition assays using several of the lipophilic analogues in the series were carried out against the P. carinii and T. gondii reductases in comparison with the enzyme from rat liver. In contrast to their preferential binding to mutant versus wild-type human DHFR, binding of these analogues to the P. carinii and T. gondii enzymes was weaker than binding to rat enzyme. It thus appears that, if the active site of the DHFR from these parasites is less sterically restrictive than the active site of the mammalian enzyme, this difference cannot be successfully exploited by moving the side chain from the 6-position to the 5-position.
|
Compound was tested for its ability to inhibit purified dihydrofolate reductase(DHFR) from L1210/R81 cells
|
None
|
35.0
nM
|
|
Journal : J. Med. Chem.
Title : Methotrexate analogues. 26. Inhibition of dihydrofolate reductase and folylpolyglutamate synthetase activity and in vitro tumor cell growth by methotrexate and aminopterin analogues containing a basic amino acid side chain.
Year : 1986
Volume : 29
Issue : 5
First Page : 655
Last Page : 660
Authors : Rosowsky A, Freisheim JH, Moran RG, Solan VC, Bader H, Wright JE, Radike-Smith M.
Abstract : Analogues of the antitumor antifolate methotrexate (MTX) were synthesized in which the glutamate (Glu) moiety was replaced by ornithine (Orn), 2,4-diaminobutyric acid (Dab), or 2,3-diaminopropionic acid (Dap). An aminopterin (AMT) analogue with Orn in place of Glu was also synthesized. The MTX analogues were obtained by reaction of 4-amino-4-deoxy-N10-methylpteroic acid (mAPA) and N omega-Boc-alpha,omega-diaminoalkanoic acids in the presence of diethyl phosphorocyanidate, followed by deprotection with trifluoroacetic acid (TFA) or by reaction of p-nitrophenyl-mAPA and N omega-Boc-alpha,omega-diaminoalkanoic acids and subsequent treatment with TFA. The AMT analogue (APA-Orn) was synthesized by reaction of p-nitrophenyl 4-amino-4-deoxy-N10-formylpteroate with silylated N delta-Boc-L-ornithine in DMF at 55 degrees C for 3 days (45% yield), saponification (83%), and TFA cleavage (89%). APA-Orn was a potent inhibitor of both dihydrofolate reductase (DHFR) from L1210 mouse leukemia (IC50 = 0.072 microM) and partly purified folylpolyglutamate synthetase (FPGS) from mouse liver (Ki = 0.15 +/- 0.06 microM). The MTX analogue (mAPA-Orn) was likewise active against both enzymes, with an IC50 of 0.160 microM for DHFR and a Ki of 20.4 +/- 7.7 microM for FPGS inhibition. The other MTX analogues and the previously reported lysine derivative (mAPA-Lys) showed DHFR affinity similar to that of mAPA-Orn but lacked activity as FPGS inhibitors. The positively charged amino group appears to be detrimental to cellular uptake, as evidenced by the low cytotoxicity of these compounds (IC50 = 0.40-2.4 microM) in comparison with MTX and AMT (IC50 = 0.002 microM) against wild-type L1210 cells. On the other hand, mAPA-Orn and APA-Orn were both more potent than the corresponding Glu derivatives MTX and AMT against L1210/R81 cells, suggesting that in these MTX-resistant cells there may occur a "self-potentiation" process involving enhanced antifolate activity via interference with the polyglutamylation of reduced folates. APA-Orn is the most potent dual inhibitor of DHFR and FPGS discovered to date, but its effectiveness as a therapeutic agent may require some form of prodrug modification to neutralize the terminal amino group of the side chain.
|
Inhibition of dihydrofolate reductase (DHFR) from murine leukemia cells
|
Mus musculus
|
35.0
nM
|
|
Journal : J. Med. Chem.
Title : Methotrexate analogues. 33. N delta-acyl-N alpha-(4-amino-4-deoxypteroyl)-L-ornithine derivatives: synthesis and in vitro antitumor activity.
Year : 1988
Volume : 31
Issue : 7
First Page : 1332
Last Page : 1337
Authors : Rosowsky A, Bader H, Cucchi CA, Moran RG, Kohler W, Freisheim JH.
Abstract : N delta-Acyl derivatives of the potent folylpolyglutamate synthetase (FPGS) inhibitor N alpha-(4-amino-4-deoxypteroyl)-L-ornithine (APA-L-Orn) were synthesized from N alpha-(4-amino-4-deoxy-N10-formylpteroyl)-L-ornithine by reaction with an N-(acyloxy)succinimide or acyl anhydride, followed by deformylation with base. The N delta-hemiphthaloyl derivative was also prepared from 4-amino-4-deoxy-N10-formylpteroic acid by reaction with persilylated N delta-phthaloyl-L-ornithine, followed by simultaneous deformylation and ring opening of the N delta-phthaloyl moiety with base. The products were potent inhibitors of purified dihydrofolate reductase (DHFR) from L1210 murine leukemia cells, with IC50's ranging from 0.027 and 0.052 microM as compared with 0.072 microM for APA-L-Orn. Several of the N delta-acyl-N10-formyl intermediates also proved to be good DHFR inhibitors. One of them, N alpha-(4-amino-4-deoxy-N10-formylpteroyl)-N delta-(4-chlorobenzoyl)-L- ornithine, had a 2-fold lower IC50 than its deformylated product, confirming that the N10-formyl group is well tolerated for DHFR binding. While N delta-acylation of APA-L-Orn did not significantly alter anti-DHFR activity, inhibition of FPGS was dramatically diminished, supporting the view that the basic NH2 on the end of the APA-L-Orn side chain is essential for the activity of this compound against FPGS. N delta-Acylation of APA-L-Orn markedly enhanced toxicity to cultured tumor cells. However, N delta-acyl derivatives also containing an N10-formyl substituent were less cytotoxic than the corresponding N10-unsubstituted analogues even though their anti-DHFR activity was the same, suggesting that N10-formylation may be unfavorable for transport. Two compounds, the N delta-benzoyl and N delta-hemiphthaloyl derivatives of APA-L-Orn, with IC50's against L1210 cells of 0.89 and 0.75 nM, respectively, were more potent than either methotrexate (MTX) or aminopterin (AMT) in this system. These compounds were also more potent than MTX against CEM human lymphoblasts and two human head and neck squamous cell carcinoma cell lines (SCC15, SCC25) in culture. Moreover, in assays against SCC15/R1 and SCC25/R1 sublines with 10-20-fold MTX resistance, the N delta-hemiphthaloyl derivative of APA-L-Orn showed potency exceeding that of MTX itself against the parental cells.(ABSTRACT TRUNCATED AT 400 WORDS)
|
Inhibition of dihydrofolate reductase (DHFR) from mouse cells (L1210/R71).
|
None
|
35.0
nM
|
|
Journal : J. Med. Chem.
Title : Synthesis and biological activity of methotrexate analogues with two acid groups and a hydrophobic aromatic ring in the side chain.
Year : 1991
Volume : 34
Issue : 2
First Page : 574
Last Page : 579
Authors : Rosowsky A, Bader H, Freisheim JH.
Abstract : The heretofore unknown gamma-(m-carboxyanilide) and gamma-(m-boronoanillide) derivatives of methotrexate (MTX) and the gamma-(m-carboxyanilide) derivatives of aminopterin (AMT) were prepared and tested as inhibitors of dihydrofolate reductase (DHFR) and as inhibitors of cell growth in culture with the aim of comparing their activity with that of N alpha-(4-amino-4-deoxypteroyl)-N delta-hemiphthaloyl-L-ornithine, a potent antifolate whose side chain likewise contains a hydrophobic aromatic ring with an acid group on the ring. All three anilides were potent DHFR inhibitors, with activity comparable to MTX and AMT. The gamma-(m-boronoanilide) displayed growth inhibitory potency similar to that of the hemiphthaloylornithine analogue, with an IC50 of only 0.7 nM. This compound, which is the most potent of the gamma-amides of MTX tested to date, is also the first reported example of an antifolate with a B(OH)2 group in the side chain and is especially novel because of its potential to form a stable tetrahedral boronate complex by reaction with electron rich OH or NH2 groups in the active site of DHFR or other folate enzymes. In antitumor assays against L1210 leukemia in mice, N alpha-(4-amino-4-deoxypteroyl)-N delta-hemiphthaloyl-L-ornithine gave a T/C of greater than 263% at 20 mg/kg (qdx9) and 300% at 16 mg/kg (bidx10), whereas maximally tolerated doses of MTX of 8 mg/kg (qdx9) and 1 mg/kg (bidx10) gave T/C values of 213 and 188%, respectively. MTX gamma-(m-boronoanilide) was also active, with a T/C of 175% at 32 mg/kg (qdx9), the highest dose tested.
|
Inhibitory activity against dihydrofolate reductase(DHFR) isolated from L1210 murine leukemia cells
|
None
|
2.0
nM
|
|
Journal : J. Med. Chem.
Title : Methotrexate analogues. 31. Meta and ortho isomers of aminopterin, compounds with a double bond in the side chain, and a novel analogue modified at the alpha-carbon: chemical and in vitro biological studies.
Year : 1988
Volume : 31
Issue : 4
First Page : 763
Last Page : 768
Authors : Rosowsky A, Bader H, Forsch RA, Moran RG, Freisheim JH.
Abstract : Five heretofore undescribed analogues of methotrexate (MTX) and aminopterin (AMT) were synthesized and tested as dihydrofolate reductase (DHFR) inhibitors and tumor cell growth inhibitors. The meta isomer of AMT was obtained from 2,4-diamino-6-(bromomethyl)pteridine and m-(aminobenzoyl)-L-glutamic acid, while the ortho isomer was obtained via the same route by using alpha-methyl gamma-tert-butyl o-(aminobenzoyl)-L-glutamate instead of the free acid. Analogues of MTX and AMT containing a double bond in the side chain were prepared from dimethyl D,L-2-amino-4-hexenedioate and 4-amino-4-deoxy-N10-methylpteroic acid and 4-amino-4-deoxy-N10-formylpteroic acid, respectively. Finally, a positional isomer of MTX with the CH2CH2COOH moiety moved from the alpha-carbon to the adjacent carboxamide nitrogen was synthesized from 3-[N-(carboxymethyl)amino]propanoic acid diethyl ester and 4-amino-4-deoxy-N10-methylpteroic acid. The positional isomers of AMT were weak DHFR inhibitors and showed very little growth-inhibitory activity against L1210 murine leukemia cells or the MTX-resistant L1210/R81 mutant line in culture. The MTX and AMT analogues with the CH2CH2COOH moiety replaced by a CH2CH = CHCOOH side chain showed anti-DHFR activity similar to that of the previously described saturated compound N-(4-amino-4-deoxy-N10-methylpteroyl)-L-2-aminoadipic acid, but were less potent than the parent drugs. The MTX analogue with the CH2CH2COOH side chain displaced from C to N was weakly bound to DHFR, confirming the importance of an intact CONH moiety, and showed greatly diminished cell growth inhibitory potency relative to MTX. None of the compounds was a substrate for folylpolyglutamate synthetase (FPGS) from mouse liver. Furthermore, inhibition of folic acid polyglutamylation in vitro at equimolar 500 microM concentrations of drug and substrate was negligible. The structural changes embodied in these five novel compounds are therefore too great for binding to the FPGS active site.
|
Tested for inhibition against purified Dihydrofolate reductase from L1210 murine leukemia cells
|
None
|
25.0
nM
|
|
Journal : J. Med. Chem.
Title : Methotrexate analogues. 34. Replacement of the glutamate moiety in methotrexate and aminopterin by long-chain 2-aminoalkanedioic acids.
Year : 1988
Volume : 31
Issue : 7
First Page : 1338
Last Page : 1344
Authors : Rosowsky A, Bader H, Kohler W, Freisheim JH, Moran RG.
Abstract : Eight previously unreported methotrexate (MTX) and aminopterin (AMT) analogues with the L-glutamate moiety replaced by DL-2-aminoalkanedioic acids containing up to 10 CH2 groups were synthesized from 4-amino-4-deoxy-N10-methylpteroic or 4-amino-4-deoxy-N10-formylpteroic acid. All the compounds were potent inhibitors of purified L1210 mouse leukemia dihydrofolate reductase (DHFR), with IC50's of 0.023-0.034 microM for the MTX analogues and 0.054-0.067 microM for the AMT analogues. The compounds were not substrates for, but were inhibitors of, partially purified mouse liver folylpolyglutamate synthetase (FPGS). Activity was correlated with the number of CH2 groups in the side chain. The IC50's for inhibition of cell growth in culture by the chain-extended MTX analogues were 0.016-0.64 microM against CEM human leukemic lymphoblasts and 0.0012-0.026 microM against L1210 mouse leukemia cells. However, the optimal chain length for growth-inhibitory activity was species-dependent. Our results suggested that CEM cells were inhibited most actively by the analogue with nine CH2 groups, while L1210 cells were most sensitive to the analogue with six CH2 groups. Among the AMT analogues, on the other hand, the most active compound against L1210 cells was the one with nine CH2 groups, which had an IC50 of 0.000 65 microM as compared with 0.0046 microM for MTX and 0.002 microM for AMT. A high degree of cross-resistance was observed between MTX and the chain-extended compounds in two MTX-resistant cell lines, CEM/MTX and L1210/R81. All the MTX analogues were active against L1210 leukemia in mice on a qd X 9 schedule, with optimal increases in lifespan (ILS) of 75-140%. Notwithstanding their high in vitro activity, the AMT analogues were more toxic and less therapeutically effective than MTX analogues of the same chain length even though neither series of compounds possessed FPGS substrate activity. These MTX and AMT analogues are an unusual group of compounds in that they retain the dicarboxylic acid structure of classical antifolates yet are more lipophilic than the parent compounds because they have more CH2 groups and are almost equivalent in vivo to MTX on the same schedule even though they do not form polyglutamates.
|
Compound was evaluated for inhibitory effect on dihydrofolate reductase (DHFR) from L1210 cells at Inhibitory constant (n=3)
|
None
|
0.00355
nM
|
|
Journal : J. Med. Chem.
Title : Syntheses and antifolate activity of 5-methyl-5-deaza analogues of aminopterin, methotrexate, folic acid, and N10-methylfolic acid.
Year : 1986
Volume : 29
Issue : 6
First Page : 1080
Last Page : 1087
Authors : Piper JR, McCaleb GS, Montgomery JA, Kisliuk RL, Gaumont Y, Sirotnak FM.
Abstract : Evidence indicating that modifications at the 5- and 10-positions of classical folic acid antimetabolites lead to compounds with favorable differential membrane transport in tumor vs. normal proliferative tissue prompted an investigation of 5-alkyl-5-deaza analogues. 2-Amino-4-methyl-3,5-pyridinedicarbonitrile, prepared by hydrogenolysis of its known 6-chloro precursor, was treated with guanidine to give 2,4-diamino-5-methylpyrido[2,3-d]pyrimidine-6-carbonitrile which was converted via the corresponding aldehyde and hydroxymethyl compound to 6-(bromomethyl)-2,4-diamino-5-methylpyrido[2,3-d]pyrimidine. Reductive condensation of the nitrile 8 with diethyl N-(4-amino-benzoyl)-L-glutamate followed by ester hydrolysis gave 5-methyl-5-deazaaminopterin. Treatment of 12 with formaldehyde and Na(CN)BH3 afforded 5-methyl-5-deazamethotrexate, which was also prepared from 15 and dimethyl N-[(4-methylamino)benzoyl]-L-glutamate followed by ester hydrolysis. 5-Methyl-10-ethyl-5-deazaaminopterin was similarly prepared from 15. Biological evaluation of the 5-methyl-5-deaza analogues together with previously reported 5-deazaaminopterin and 5-deazamethotrexate for inhibition of dihydrofolate reductase (DHFR) isolated from L1210 cells and for their effect on cell growth inhibition, transport characteristics, and net accumulation of polyglutamate forms in L1210 cells revealed the analogues to have essentially the same properties as the appropriate parent compound, aminopterin or methotrexate (MTX), except that 20 and 21 were approximately 10 times more growth inhibitory than MTX. In in vivo tests against P388/0 and P388/MTX leukemia in mice, the analogues showed activity comparable to that of MTX, with the more potent 20 producing the same response in the P388/0 test as MTX but at one-fourth the dose; none showed activity against P388/MTX. Hydrolytic deamination of 12 and 20 produced 5-methyl-5-deazafolic acid and 5,10-dimethyl-5-deazafolic acid, respectively. In bacterial studies on the 2-amino-4-oxo analogues, 5-deazafolic acid proved to be a potent inhibitor of Lactobacillus casei DHFR and also the growth of both L. casei ATCC 7469 and Streptococcus faecium ATCC 8043. Its 5-methyl congener 22 is also inhibitory toward L. casei, but its IC50 for growth inhibition is much lower than its IC50 values for inhibition of DHFR or thymidylate synthase from L. casei, suggesting an alternate site of action.
|
In vitro inhibitory activity against L1210 dihydrofolate reductase in rodent neoplastic cells
|
None
|
0.004
nM
|
|
Journal : J. Med. Chem.
Title : 10-Propargylaminopterin and alkyl homologues of methotrexate as inhibitors of folate metabolism.
Year : 1982
Volume : 25
Issue : 7
First Page : 877
Last Page : 880
Authors : Piper JR, McCaleb GS, Montgomery JA, Kisliuk RL, Gaumont Y, Sirotnak FM.
Abstract : Reported antifolate activity against leukemia L1210 by N-[14-[[(2-amino-4-hydroxy-6-quinazolinyl)methyl]-propargylamino]benzoyl]]-L-glu tamic acid through potent inhibition of thymidylate synthase (EC 2.1.1.45) prompted us to include the propargyl group in a study of the effect on folate metabolism and membrane transport of replacing the 10-methyl group of methotrexate with other groups. Along with the propyl (8a) and octyl (8b) homologues of methotrexate, the propargyl compound 8c was prepared for evaluation. Syntheses of 8a,b were achieved by a standard multistep sequence involving preparation of the side-chain precursors via tosylated intermediates and then their alkylation with 6-(bromomethyl)-2,4-pteridinediamine hydrobromide. The side-chain precursor to 8c was prepared by direct alkylation of diethyl N-(4-aminobenzoyl)-L-glutamate with propargyl bromide and was separated from unchanged amine and dipropargyl coproduct by a combination of methods, including dry-column chromatography and recrystallization. Subsequent steps leading to 8c were like those used to prepare 8a,b. Biological evaluations of the three compounds consisted of studies of their effects on enzyme inhibition [(dihydrofolate reductase (EC 1.5.1.3) and thymidylate synthase)], L1210 cell growth inhibition, cellular membrane transport with various murine cell types (L210, S180, Ehrlich, and epithelial), in vivo (mice) activity vs. L1210 leukemia and S180 ascites, and plasma clearance in mice. The in vivo results vs. S180 ascites offered evidence that 8c might have a better therapeutic index against this tumor than methotrexate, but no other result from either of these compounds suggested significant superiority over methotrexate.
|
Tested for inhibition of dihydrofolate reductase enzyme from mouse
|
None
|
0.0023
nM
|
|
Journal : J. Med. Chem.
Title : Synthesis and antitumor activity of 10-alkyl-10-deazaminopterins. A convenient synthesis of 10-deazaminopterin.
Year : 1982
Volume : 25
Issue : 10
First Page : 1227
Last Page : 1230
Authors : DeGraw JI, Brown VH, Tagawa H, Kisliuk RL, Gaumont Y, Sirotnak FM.
Abstract : Requirements for large-scale synthesis of the potent antitumor drug 10-deazaminopterin have led to development of a facile synthesis of this compound and its 10-alkyl analogues. The lithium diisopropyl amide generated dianions of appropriate p-alkylbenzoic acids were alkylated with 3-methoxyallyl chloride. The resulting 4-(p-carboxyphenyl)-1-methoxy-1-butenes were brominated at pH 7-8 to afford the 2-bromo-4-(p-carboxyphenyl)butyraldehydes. Condensation with 2,4,5,6-tetraminopyrimidine and subsequent in situ oxidation of the resulting dihydropteridines yielded crystalline 10-alkyl-10-deaza-4-amino-4-deoxypteroic acids. The pteroic acids were coupled with diethyl glutamate via the mixed anhydride method, followed by saponification at room temperature, to give the target 10-deazaminopterins. The 10-alkyl compounds were approximately equipotent to 10-deazaminopterin as growth inhibitors of folate-dependent bacteria. Their abilities to inhibit Lactobacillus casei and L1210 derived dihydrofolate reductases were also similar. Transport properties in vitro were suggestive of an improved therapeutic index for the 10-alkyl analogues. Against L1210 in mice, the percent increase in life span at the LD10 dosage was +151% (methotrexate), +178% (10-deazaminopterin), +235% (10-methyl analogue), and +211% (10-ethyl analogue). 10,10-Dimethyl-10-deazaminopterin was less effective at an equimolar dosage, but the ILS at the maximum dose tested (72 mg/kg) was +135%. It was far less toxic than the other analogues possibly because of enhanced clearance.
|
Inhibition of dihydrofolate reductase in Lactobacillus casei
|
Lactobacillus casei
|
5.0
nM
|
|
Journal : J. Med. Chem.
Title : Novel fluorinated antifolates. Enzyme inhibition and cytotoxicity studies on 2'- and 3'-fluoroaminopterin.
Year : 1983
Volume : 26
Issue : 8
First Page : 1193
Last Page : 1196
Authors : Henkin J, Washtien WL.
Abstract : Two novel analogues of aminopterin with a single fluorine substitution in the 2' (compound 8) or in the 3' (compound 9) position of the p-aminobenzoyl group were synthesized and evaluated as inhibitors of dihydrofolate reductase from two bacterial species and from human HeLa cells. The 2'-fluoro compound was bound essentially the same as aminopterin itself, while the 3'-fluoro derivative bound two- to threefold more tightly in all cases. UV spectral shifts indicated normal binding of the pteridine. Cytotoxicity studies against mouse leukemia L1210 cells and the human stomach cancer line HuTu80 indicated equivalent toxicity of the parent drug with the 2'-fluoro analogue. 3'-Fluoroaminopterin was, however, twice as toxic as aminopterin to both cell lines.
|
Inhibition constant (Ki) was determined in Escherichia coli
|
Escherichia coli
|
0.09
nM
|
|
Journal : J. Med. Chem.
Title : Novel fluorinated antifolates. Enzyme inhibition and cytotoxicity studies on 2'- and 3'-fluoroaminopterin.
Year : 1983
Volume : 26
Issue : 8
First Page : 1193
Last Page : 1196
Authors : Henkin J, Washtien WL.
Abstract : Two novel analogues of aminopterin with a single fluorine substitution in the 2' (compound 8) or in the 3' (compound 9) position of the p-aminobenzoyl group were synthesized and evaluated as inhibitors of dihydrofolate reductase from two bacterial species and from human HeLa cells. The 2'-fluoro compound was bound essentially the same as aminopterin itself, while the 3'-fluoro derivative bound two- to threefold more tightly in all cases. UV spectral shifts indicated normal binding of the pteridine. Cytotoxicity studies against mouse leukemia L1210 cells and the human stomach cancer line HuTu80 indicated equivalent toxicity of the parent drug with the 2'-fluoro analogue. 3'-Fluoroaminopterin was, however, twice as toxic as aminopterin to both cell lines.
|
Compound was tested for its inhibitory concentration against human leukemic lymphoblasts (CEM Cells)
|
Homo sapiens
|
1.0
nM
|
|
Journal : J. Med. Chem.
Title : Methotrexate analogues. 28. Synthesis and biological evaluation of new gamma-monoamides of aminopterin and methotrexate.
Year : 1986
Volume : 29
Issue : 9
First Page : 1703
Last Page : 1709
Authors : Rosowsky A, Bader H, Radike-Smith M, Cucchi CA, Wick MM, Freisheim JH.
Abstract : Lipophilic gamma-monoamide derivatives of aminopterin (AMT) were synthesized in high overall yield from 4-amino-4-deoxy-N10-formylpteroic acid and gamma-N-tert-alkyl-, gamma-N-aralkyl-, or gamma-N-arylamides of alpha-benzyl L-glutamate via a modification of the mixed carboxylic-carbonic anhydride coupling method. Coupling was also accomplished with p-nitrophenyl 4-amino-4-deoxy-N10-formylpteroate. Compounds obtained in this manner included the gamma-tert-butylamide, gamma-(1-adamantylamide), gamma-benzylamide, gamma-(3,4-dichlorobenzylamide), gamma-(2,6-dichlorobenzylamide), gamma-anilide, gamma-(3,4-methylenedioxyanilide), and gamma-(3,4-dihydroxanilide) derivatives of AMT. Also prepared, from 4-amino-4-deoxy-N10-methylpteroic acid via diethyl phosphorocyanidate coupling, was the gamma-(3,4-methylenedioxyanilide) of MTX. The methylenedioxyanilides were cleaved smoothly to dihydroxyanilides with boron tris(trifluoroacetate) in trifluoroacetic acid. All the gamma-monoamides were tested as inhibitors of purified dihydrofolate reductase (DHFR) from murine L1210 leukemia cells and as inhibitors of the growth of wild-type L1210 cells and a subline (L1210/R81) with high-level resistance to MTX and AMT based mainly on a defect in drug uptake via active transport. Several compounds were also tested against human leukemic lymphoblasts (CEM cells) and a resistant subline (CEM/MTX) whose resistance is likewise based on uptake. The IC50 of the gamma-monoamides against DHFR was 1.5- to 5-fold higher than that of the parent acids, but the IC50 against cultured cells varied over a much broader range, suggesting that uptake and/or metabolism rather than DHFR binding are principal determinants of in vitro growth inhibitory activity for these compounds. gamma-N-Aryl and gamma-N-aralkyl derivatives appeared to be more potent than gamma-N-tert-alkyl derivatives. Where comparison could be made, AMT gamma-monoamides were more potent than MTX gamma-monoamides. Several of the gamma-monoamides showed potency comparable to that of the parent acid against wild-type L1210 and CEM cells; all of them were more potent than MTX against the L1210/R81 subline; and some of the AMT gamma-monoamides were also more potent than the parent acid against resistant CEM/MTX cells. As a group, however, the gamma-monoamides were considerably more active against the murine cells than against the human cells, suggesting that the former may take up the amides better or may be able to metabolize them more efficiently than the parent acids. All the gamma-monoamides were tested in vivo against L1210 leukemia in mice.(ABSTRACT TRUNCATED AT 400 WORDS)
|
Compound was tested for its inhibitory concentration against human leukemic lymphoblasts (CEM Cells) and a resistant subline (CEM / MTX).
|
Homo sapiens
|
320.0
nM
|
|
Journal : J. Med. Chem.
Title : Methotrexate analogues. 28. Synthesis and biological evaluation of new gamma-monoamides of aminopterin and methotrexate.
Year : 1986
Volume : 29
Issue : 9
First Page : 1703
Last Page : 1709
Authors : Rosowsky A, Bader H, Radike-Smith M, Cucchi CA, Wick MM, Freisheim JH.
Abstract : Lipophilic gamma-monoamide derivatives of aminopterin (AMT) were synthesized in high overall yield from 4-amino-4-deoxy-N10-formylpteroic acid and gamma-N-tert-alkyl-, gamma-N-aralkyl-, or gamma-N-arylamides of alpha-benzyl L-glutamate via a modification of the mixed carboxylic-carbonic anhydride coupling method. Coupling was also accomplished with p-nitrophenyl 4-amino-4-deoxy-N10-formylpteroate. Compounds obtained in this manner included the gamma-tert-butylamide, gamma-(1-adamantylamide), gamma-benzylamide, gamma-(3,4-dichlorobenzylamide), gamma-(2,6-dichlorobenzylamide), gamma-anilide, gamma-(3,4-methylenedioxyanilide), and gamma-(3,4-dihydroxanilide) derivatives of AMT. Also prepared, from 4-amino-4-deoxy-N10-methylpteroic acid via diethyl phosphorocyanidate coupling, was the gamma-(3,4-methylenedioxyanilide) of MTX. The methylenedioxyanilides were cleaved smoothly to dihydroxyanilides with boron tris(trifluoroacetate) in trifluoroacetic acid. All the gamma-monoamides were tested as inhibitors of purified dihydrofolate reductase (DHFR) from murine L1210 leukemia cells and as inhibitors of the growth of wild-type L1210 cells and a subline (L1210/R81) with high-level resistance to MTX and AMT based mainly on a defect in drug uptake via active transport. Several compounds were also tested against human leukemic lymphoblasts (CEM cells) and a resistant subline (CEM/MTX) whose resistance is likewise based on uptake. The IC50 of the gamma-monoamides against DHFR was 1.5- to 5-fold higher than that of the parent acids, but the IC50 against cultured cells varied over a much broader range, suggesting that uptake and/or metabolism rather than DHFR binding are principal determinants of in vitro growth inhibitory activity for these compounds. gamma-N-Aryl and gamma-N-aralkyl derivatives appeared to be more potent than gamma-N-tert-alkyl derivatives. Where comparison could be made, AMT gamma-monoamides were more potent than MTX gamma-monoamides. Several of the gamma-monoamides showed potency comparable to that of the parent acid against wild-type L1210 and CEM cells; all of them were more potent than MTX against the L1210/R81 subline; and some of the AMT gamma-monoamides were also more potent than the parent acid against resistant CEM/MTX cells. As a group, however, the gamma-monoamides were considerably more active against the murine cells than against the human cells, suggesting that the former may take up the amides better or may be able to metabolize them more efficiently than the parent acids. All the gamma-monoamides were tested in vivo against L1210 leukemia in mice.(ABSTRACT TRUNCATED AT 400 WORDS)
|
Inhibition of growth (cytotoxicity) of HuTu 80 cell line in vitro
|
Homo sapiens
|
4.7
nM
|
|
Journal : J. Med. Chem.
Title : Novel fluorinated antifolates. Enzyme inhibition and cytotoxicity studies on 2'- and 3'-fluoroaminopterin.
Year : 1983
Volume : 26
Issue : 8
First Page : 1193
Last Page : 1196
Authors : Henkin J, Washtien WL.
Abstract : Two novel analogues of aminopterin with a single fluorine substitution in the 2' (compound 8) or in the 3' (compound 9) position of the p-aminobenzoyl group were synthesized and evaluated as inhibitors of dihydrofolate reductase from two bacterial species and from human HeLa cells. The 2'-fluoro compound was bound essentially the same as aminopterin itself, while the 3'-fluoro derivative bound two- to threefold more tightly in all cases. UV spectral shifts indicated normal binding of the pteridine. Cytotoxicity studies against mouse leukemia L1210 cells and the human stomach cancer line HuTu80 indicated equivalent toxicity of the parent drug with the 2'-fluoro analogue. 3'-Fluoroaminopterin was, however, twice as toxic as aminopterin to both cell lines.
|
Evaluated in vitro for ability to inhibit the L cell lines.
|
Mus musculus
|
2.3
nM
|
|
Journal : J. Med. Chem.
Title : Synthesis and biological activity of an acyclic analogue of 5,6,7,8-tetrahydrofolic acid, N-[4-[[3-(2,4-diamino-1,6-dihydro-6-oxo-5- pyrimidinyl)propyl]amino]-benzoyl]-L-glutamic acid.
Year : 1990
Volume : 33
Issue : 2
First Page : 561
Last Page : 567
Authors : Kelley JL, McLean EW, Cohn NK, Edelstein MP, Duch DS, Smith GK, Hanlon MH, Ferone R.
Abstract : The synthesis and biological evaluation of N-[4-[[3-(2,4-diamino-1,6-dihydro-6-oxo-5-pyrimidinyl)propyl]amino]- benzoyl]-L-glutamic acid (1) (5-DACTHF, 543U76), an acyclic analogue of 5,6,7,8-tetrahydrofolic acid (THFA), are described. The key intermediate, hemiaminal 8, was prepared in four stages from 3-chloropropionaldehyde diethyl acetal. Reaction of 8 with dimethyl N-(4-aminobenzoyl)-L-glutamate gave the 2,4-bis(acetylamino) derivative 11, which was hydrolyzed with 1 N sodium hydroxide to give 1; the glycine analogue 16 was prepared in a similar manner. The N-methyl analogue 2 and N-formyl analogue 3 were prepared from 11 and 1, respectively. Compounds 1-3 inhibited growth of Detroit 98 and L cells in cell culture, with IC50s ranging from 2 to 0.018 microM. Cell culture toxicity reversal studies and enzyme inhibition tests showed that 1 was cytotoxic but not by the mechanism of the dihydrofolate reductase inhibitor aminopterin. Compound 1 and its polyglutamylated homologues inhibited glycinamide ribonucleotide transformylase (GAR-TFase) and aminoimidazole ribonucleotide transformylase (AICAR-TFase), the folate-dependent enzymes in de novo purine biosynthesis; and 1 was an effective substrate for mammalian folyl-polyglutamate synthetase. The compound inhibited (IC50 = 20 nM) the conversion of [14C]formate to [14C]-formylglycinamide ribonucleotide by MOLT-4 cells in culture. These data suggest that the site of action of 1 is inhibition of purine de novo biosynthesis. Moderate activity was observed against P388 leukemia in vivo.
|
Evaluated for reversal of L cell inhibition, where reversal of toxicity was evaluated by the addition of Hypoxanthine (37 uM)
|
Mus musculus
|
79.0
%
|
|
Journal : J. Med. Chem.
Title : Synthesis and biological activity of an acyclic analogue of 5,6,7,8-tetrahydrofolic acid, N-[4-[[3-(2,4-diamino-1,6-dihydro-6-oxo-5- pyrimidinyl)propyl]amino]-benzoyl]-L-glutamic acid.
Year : 1990
Volume : 33
Issue : 2
First Page : 561
Last Page : 567
Authors : Kelley JL, McLean EW, Cohn NK, Edelstein MP, Duch DS, Smith GK, Hanlon MH, Ferone R.
Abstract : The synthesis and biological evaluation of N-[4-[[3-(2,4-diamino-1,6-dihydro-6-oxo-5-pyrimidinyl)propyl]amino]- benzoyl]-L-glutamic acid (1) (5-DACTHF, 543U76), an acyclic analogue of 5,6,7,8-tetrahydrofolic acid (THFA), are described. The key intermediate, hemiaminal 8, was prepared in four stages from 3-chloropropionaldehyde diethyl acetal. Reaction of 8 with dimethyl N-(4-aminobenzoyl)-L-glutamate gave the 2,4-bis(acetylamino) derivative 11, which was hydrolyzed with 1 N sodium hydroxide to give 1; the glycine analogue 16 was prepared in a similar manner. The N-methyl analogue 2 and N-formyl analogue 3 were prepared from 11 and 1, respectively. Compounds 1-3 inhibited growth of Detroit 98 and L cells in cell culture, with IC50s ranging from 2 to 0.018 microM. Cell culture toxicity reversal studies and enzyme inhibition tests showed that 1 was cytotoxic but not by the mechanism of the dihydrofolate reductase inhibitor aminopterin. Compound 1 and its polyglutamylated homologues inhibited glycinamide ribonucleotide transformylase (GAR-TFase) and aminoimidazole ribonucleotide transformylase (AICAR-TFase), the folate-dependent enzymes in de novo purine biosynthesis; and 1 was an effective substrate for mammalian folyl-polyglutamate synthetase. The compound inhibited (IC50 = 20 nM) the conversion of [14C]formate to [14C]-formylglycinamide ribonucleotide by MOLT-4 cells in culture. These data suggest that the site of action of 1 is inhibition of purine de novo biosynthesis. Moderate activity was observed against P388 leukemia in vivo.
|
Evaluated for reversal of L cell inhibition, where reversal of toxicity was evaluated by the addition of Leucovorin (0.2 uM)
|
Mus musculus
|
6.0
%
|
|
Journal : J. Med. Chem.
Title : Synthesis and biological activity of an acyclic analogue of 5,6,7,8-tetrahydrofolic acid, N-[4-[[3-(2,4-diamino-1,6-dihydro-6-oxo-5- pyrimidinyl)propyl]amino]-benzoyl]-L-glutamic acid.
Year : 1990
Volume : 33
Issue : 2
First Page : 561
Last Page : 567
Authors : Kelley JL, McLean EW, Cohn NK, Edelstein MP, Duch DS, Smith GK, Hanlon MH, Ferone R.
Abstract : The synthesis and biological evaluation of N-[4-[[3-(2,4-diamino-1,6-dihydro-6-oxo-5-pyrimidinyl)propyl]amino]- benzoyl]-L-glutamic acid (1) (5-DACTHF, 543U76), an acyclic analogue of 5,6,7,8-tetrahydrofolic acid (THFA), are described. The key intermediate, hemiaminal 8, was prepared in four stages from 3-chloropropionaldehyde diethyl acetal. Reaction of 8 with dimethyl N-(4-aminobenzoyl)-L-glutamate gave the 2,4-bis(acetylamino) derivative 11, which was hydrolyzed with 1 N sodium hydroxide to give 1; the glycine analogue 16 was prepared in a similar manner. The N-methyl analogue 2 and N-formyl analogue 3 were prepared from 11 and 1, respectively. Compounds 1-3 inhibited growth of Detroit 98 and L cells in cell culture, with IC50s ranging from 2 to 0.018 microM. Cell culture toxicity reversal studies and enzyme inhibition tests showed that 1 was cytotoxic but not by the mechanism of the dihydrofolate reductase inhibitor aminopterin. Compound 1 and its polyglutamylated homologues inhibited glycinamide ribonucleotide transformylase (GAR-TFase) and aminoimidazole ribonucleotide transformylase (AICAR-TFase), the folate-dependent enzymes in de novo purine biosynthesis; and 1 was an effective substrate for mammalian folyl-polyglutamate synthetase. The compound inhibited (IC50 = 20 nM) the conversion of [14C]formate to [14C]-formylglycinamide ribonucleotide by MOLT-4 cells in culture. These data suggest that the site of action of 1 is inhibition of purine de novo biosynthesis. Moderate activity was observed against P388 leukemia in vivo.
|
Evaluated for reversal of L cell inhibition, where reversal of toxicity was evaluated by the addition of Thymidine + Hypoxanthine
|
Mus musculus
|
0.0
%
|
|
Journal : J. Med. Chem.
Title : Synthesis and biological activity of an acyclic analogue of 5,6,7,8-tetrahydrofolic acid, N-[4-[[3-(2,4-diamino-1,6-dihydro-6-oxo-5- pyrimidinyl)propyl]amino]-benzoyl]-L-glutamic acid.
Year : 1990
Volume : 33
Issue : 2
First Page : 561
Last Page : 567
Authors : Kelley JL, McLean EW, Cohn NK, Edelstein MP, Duch DS, Smith GK, Hanlon MH, Ferone R.
Abstract : The synthesis and biological evaluation of N-[4-[[3-(2,4-diamino-1,6-dihydro-6-oxo-5-pyrimidinyl)propyl]amino]- benzoyl]-L-glutamic acid (1) (5-DACTHF, 543U76), an acyclic analogue of 5,6,7,8-tetrahydrofolic acid (THFA), are described. The key intermediate, hemiaminal 8, was prepared in four stages from 3-chloropropionaldehyde diethyl acetal. Reaction of 8 with dimethyl N-(4-aminobenzoyl)-L-glutamate gave the 2,4-bis(acetylamino) derivative 11, which was hydrolyzed with 1 N sodium hydroxide to give 1; the glycine analogue 16 was prepared in a similar manner. The N-methyl analogue 2 and N-formyl analogue 3 were prepared from 11 and 1, respectively. Compounds 1-3 inhibited growth of Detroit 98 and L cells in cell culture, with IC50s ranging from 2 to 0.018 microM. Cell culture toxicity reversal studies and enzyme inhibition tests showed that 1 was cytotoxic but not by the mechanism of the dihydrofolate reductase inhibitor aminopterin. Compound 1 and its polyglutamylated homologues inhibited glycinamide ribonucleotide transformylase (GAR-TFase) and aminoimidazole ribonucleotide transformylase (AICAR-TFase), the folate-dependent enzymes in de novo purine biosynthesis; and 1 was an effective substrate for mammalian folyl-polyglutamate synthetase. The compound inhibited (IC50 = 20 nM) the conversion of [14C]formate to [14C]-formylglycinamide ribonucleotide by MOLT-4 cells in culture. These data suggest that the site of action of 1 is inhibition of purine de novo biosynthesis. Moderate activity was observed against P388 leukemia in vivo.
|
Evaluated for reversal of L cell inhibition, where reversal of toxicity was evaluated by the addition of Thymidine (20 uM)
|
Mus musculus
|
68.0
%
|
|
Journal : J. Med. Chem.
Title : Synthesis and biological activity of an acyclic analogue of 5,6,7,8-tetrahydrofolic acid, N-[4-[[3-(2,4-diamino-1,6-dihydro-6-oxo-5- pyrimidinyl)propyl]amino]-benzoyl]-L-glutamic acid.
Year : 1990
Volume : 33
Issue : 2
First Page : 561
Last Page : 567
Authors : Kelley JL, McLean EW, Cohn NK, Edelstein MP, Duch DS, Smith GK, Hanlon MH, Ferone R.
Abstract : The synthesis and biological evaluation of N-[4-[[3-(2,4-diamino-1,6-dihydro-6-oxo-5-pyrimidinyl)propyl]amino]- benzoyl]-L-glutamic acid (1) (5-DACTHF, 543U76), an acyclic analogue of 5,6,7,8-tetrahydrofolic acid (THFA), are described. The key intermediate, hemiaminal 8, was prepared in four stages from 3-chloropropionaldehyde diethyl acetal. Reaction of 8 with dimethyl N-(4-aminobenzoyl)-L-glutamate gave the 2,4-bis(acetylamino) derivative 11, which was hydrolyzed with 1 N sodium hydroxide to give 1; the glycine analogue 16 was prepared in a similar manner. The N-methyl analogue 2 and N-formyl analogue 3 were prepared from 11 and 1, respectively. Compounds 1-3 inhibited growth of Detroit 98 and L cells in cell culture, with IC50s ranging from 2 to 0.018 microM. Cell culture toxicity reversal studies and enzyme inhibition tests showed that 1 was cytotoxic but not by the mechanism of the dihydrofolate reductase inhibitor aminopterin. Compound 1 and its polyglutamylated homologues inhibited glycinamide ribonucleotide transformylase (GAR-TFase) and aminoimidazole ribonucleotide transformylase (AICAR-TFase), the folate-dependent enzymes in de novo purine biosynthesis; and 1 was an effective substrate for mammalian folyl-polyglutamate synthetase. The compound inhibited (IC50 = 20 nM) the conversion of [14C]formate to [14C]-formylglycinamide ribonucleotide by MOLT-4 cells in culture. These data suggest that the site of action of 1 is inhibition of purine de novo biosynthesis. Moderate activity was observed against P388 leukemia in vivo.
|
Compound was evaluated for the growth inhibition of L1210 cells.
|
Mus musculus
|
2.0
nM
|
|
Journal : J. Med. Chem.
Title : Synthesis and biological activity of methotrexate analogues with two acid groups and a hydrophobic aromatic ring in the side chain.
Year : 1991
Volume : 34
Issue : 2
First Page : 574
Last Page : 579
Authors : Rosowsky A, Bader H, Freisheim JH.
Abstract : The heretofore unknown gamma-(m-carboxyanilide) and gamma-(m-boronoanillide) derivatives of methotrexate (MTX) and the gamma-(m-carboxyanilide) derivatives of aminopterin (AMT) were prepared and tested as inhibitors of dihydrofolate reductase (DHFR) and as inhibitors of cell growth in culture with the aim of comparing their activity with that of N alpha-(4-amino-4-deoxypteroyl)-N delta-hemiphthaloyl-L-ornithine, a potent antifolate whose side chain likewise contains a hydrophobic aromatic ring with an acid group on the ring. All three anilides were potent DHFR inhibitors, with activity comparable to MTX and AMT. The gamma-(m-boronoanilide) displayed growth inhibitory potency similar to that of the hemiphthaloylornithine analogue, with an IC50 of only 0.7 nM. This compound, which is the most potent of the gamma-amides of MTX tested to date, is also the first reported example of an antifolate with a B(OH)2 group in the side chain and is especially novel because of its potential to form a stable tetrahedral boronate complex by reaction with electron rich OH or NH2 groups in the active site of DHFR or other folate enzymes. In antitumor assays against L1210 leukemia in mice, N alpha-(4-amino-4-deoxypteroyl)-N delta-hemiphthaloyl-L-ornithine gave a T/C of greater than 263% at 20 mg/kg (qdx9) and 300% at 16 mg/kg (bidx10), whereas maximally tolerated doses of MTX of 8 mg/kg (qdx9) and 1 mg/kg (bidx10) gave T/C values of 213 and 188%, respectively. MTX gamma-(m-boronoanilide) was also active, with a T/C of 175% at 32 mg/kg (qdx9), the highest dose tested.
|
Ability to inhibit L1210 murine leukemia tumor cell growth in culture after 48 hr of its exposure.
|
Mus musculus
|
2.0
nM
|
|
Journal : J. Med. Chem.
Title : Analogues of methotrexate and aminopterin with gamma-methylene and gamma-cyano substitution of the glutamate side chain: synthesis and in vitro biological activity.
Year : 1991
Volume : 34
Issue : 1
First Page : 203
Last Page : 208
Authors : Rosowsky A, Bader H, Freisheim JH.
Abstract : Analogues of methotrexate (MTX) and aminopterin (AMT) modified at the gamma-position of the glutamate side chain were synthesized and evaluated as dihydrofolate reductase (DHFR) inhibitors and tumor cell growth inhibitors. Condesations of 4-amino-4-deoxy-N10-methylpteroic acid (mAPA) with dimethyl DL-4-methyleneglutamate in the presence of diethyl phosphorocyanidate (DEPC) followed by alkaline hydrolysis yielded N-(4-amino-4-deoxy-N10-methylpteroyl)-DL-4-methyleneglutamic acid (gamma-methyleneMTX). Condensation of 4-amino-4-deoxy-N10-formylpteroic acid (fAPA) with dimethyl-DL-4-methyleneglutamate by the mixed carboxylic-carbonic anhydride method yielded N-4-amino-4-deoxypteroyl)-DL-4-methyleneglutamic acid (gamma-methyleneAMT). Also prepared via DEPC coupling was a mixture of the four possible diastereomers of N-(4-amino-4-deoxy-N10-methylpteroyl)-4-cyanoglutamic acid (gamma-cyanoMTX). The requisite intermediate gamma-tert-butyl alpha-methyl 4-cyanoglutamate, as a DL-threo/DL-erythro mixture, was prepared from methyl N alpha-Boc-O-tosyl-L-serinate by reaction with sodium tert-butyl cyanoacetate followed by mild trifluoroacetic treatment to selectively remove the Boc group. The gamma-methylene derivatives of MTX and AMT are attractive because of their potential to act as Michael acceptors within the DHFR active site. gamma-CyanoMTX may be viewed as a congener of the nonpolyglutamated MTX analogue gamma-fluoroMTX. In vitro bioassay data for the gamma-methylene and gamma-cyano compounds support the idea that the active site of DHFR, already known for its ability to tolerate modification of the gamma-carboxyl group of MTX and AMT, can likewise accommodate substitution on the gamma-carbon itself.
|
Inhibition of growth (cytotoxicity) of L1210 cell line in vitro
|
Mus musculus
|
1.8
nM
|
|
Journal : J. Med. Chem.
Title : Novel fluorinated antifolates. Enzyme inhibition and cytotoxicity studies on 2'- and 3'-fluoroaminopterin.
Year : 1983
Volume : 26
Issue : 8
First Page : 1193
Last Page : 1196
Authors : Henkin J, Washtien WL.
Abstract : Two novel analogues of aminopterin with a single fluorine substitution in the 2' (compound 8) or in the 3' (compound 9) position of the p-aminobenzoyl group were synthesized and evaluated as inhibitors of dihydrofolate reductase from two bacterial species and from human HeLa cells. The 2'-fluoro compound was bound essentially the same as aminopterin itself, while the 3'-fluoro derivative bound two- to threefold more tightly in all cases. UV spectral shifts indicated normal binding of the pteridine. Cytotoxicity studies against mouse leukemia L1210 cells and the human stomach cancer line HuTu80 indicated equivalent toxicity of the parent drug with the 2'-fluoro analogue. 3'-Fluoroaminopterin was, however, twice as toxic as aminopterin to both cell lines.
|
Cell growth inhibition against mouse leukemia L1210 cells
|
Mus musculus
|
2.0
nM
|
|
Journal : J. Med. Chem.
Title : Methotrexate analogues. 25. Chemical and biological studies on the gamma-tert-butyl esters of methotrexate and aminopterin.
Year : 1985
Volume : 28
Issue : 5
First Page : 660
Last Page : 667
Authors : Rosowsky A, Freisheim JH, Bader H, Forsch RA, Susten SS, Cucchi CA, Frei E.
Abstract : gamma-tert-Butylaminopterin (gamma-tBAMT), the first example of an aminopterin (AMT) gamma-monoester, was synthesized, and new routes to the known N10-methyl analogue gamma-tert-butyl methotrexate (gamma-tBMTX) were developed. The inhibitory effects of gamma-tBAMT on the activity of purified dihydrofolate reductase (DHFR) from L1210 murine leukemia cells, the growth of L1210 cells and CEM human leukemic lymphoblasts in suspension culture, and the growth of several lines of human squamous cell carcinoma of the head and neck in monolayer culture were compared with the effects of gamma-tBMTX and the parent acids AMT and methotrexate (MTX). Patterns of cross-resistance to gamma-tBAMT, gamma-tBMTX, and AMT among several MTX-resistant cell lines were examined. In vivo antitumor activities of gamma-tBAMT and gamma-tBMTX were compared in mice with L1210 leukemia. While the activity of gamma-tBAMT was very close to that of gamma-tBMTX in the DHFR inhibition assay, the AMT ester was more potent than the MTX ester against cells in culture and against L1210 leukemia in vivo. Only partial cross-resistance was shown against gamma-tBMTX and gamma-tBAMT in cultured cells that were resistant to MTX by virtue of a transport defect or a combination of defective transport and elevated DHFR activity.
|
Compound was evaluated for cell growth inhibition of murine (L1210) leukemic cells
|
Mus musculus
|
2.0
nM
|
|
Journal : J. Med. Chem.
Title : Synthesis and biological activity of the 2-desamino and 2-desamino-2-methyl analogues of aminopterin and methotrexate.
Year : 1991
Volume : 34
Issue : 1
First Page : 227
Last Page : 234
Authors : Rosowsky A, Forsch RA, Moran RG, Freisheim JH.
Abstract : The previously undescribed 2-desamino and 2-desamino-2-methyl analogues of aminopterin (AMT) and methotrexate (MTX) were synthesized from 2-amino-5-(chloromethyl)pyrazine-3-carbonitrile. The AMT analogues were obtained via a three-step sequence consisting of condensation with di-tert-butyl N-(4-aminobenzoyl)-L-glutamate, heating with formamidine or acetamidine acetate, and mild acidolysis with trifluoroacetic acid. The MTX analogues were prepared similarly, except that 2-amino-5-(chloromethyl)pyrazine-3-carbonitrile was condensed with 4-(N-methylamino)benzoic acid and the resulting product was annulated with formamidine or acetamidine acetate to obtain the 2-desamino and 2-desamino-2-methyl analogues, respectively, of 4-amino-4-deoxy-N10-methylpteroic acid. Condensation with di-tert-butyl L-glutamate in the presence of diethyl phosphorocyanidate followed by ester cleavage with trifluoroacetic acid was then carried out. Retention of the L configuration in the glutamate moiety during this synthesis was demonstrated by rapid and essentially complete hydrolysis with carboxypeptidase G1 under conditions that likewise cleaved the L enantiomer of MTX but left the D enantiomer unaffected. The 2-desamino and 2-desamino-2-methyl analogues of AMT and MTX inhibited the growth of tumor cells, but were very poor inhibitors of dihydrofolate reductase (DHFR). These unexpected results suggested that activity in intact cells was due to metabolism of the 2-desamino compounds to polyglutamates.
|
Compound was evaluated for inhibitory effect on growth of L1210 cells at IC50 (n=4)
|
Mus musculus
|
0.72
nM
|
|
Journal : J. Med. Chem.
Title : Syntheses and antifolate activity of 5-methyl-5-deaza analogues of aminopterin, methotrexate, folic acid, and N10-methylfolic acid.
Year : 1986
Volume : 29
Issue : 6
First Page : 1080
Last Page : 1087
Authors : Piper JR, McCaleb GS, Montgomery JA, Kisliuk RL, Gaumont Y, Sirotnak FM.
Abstract : Evidence indicating that modifications at the 5- and 10-positions of classical folic acid antimetabolites lead to compounds with favorable differential membrane transport in tumor vs. normal proliferative tissue prompted an investigation of 5-alkyl-5-deaza analogues. 2-Amino-4-methyl-3,5-pyridinedicarbonitrile, prepared by hydrogenolysis of its known 6-chloro precursor, was treated with guanidine to give 2,4-diamino-5-methylpyrido[2,3-d]pyrimidine-6-carbonitrile which was converted via the corresponding aldehyde and hydroxymethyl compound to 6-(bromomethyl)-2,4-diamino-5-methylpyrido[2,3-d]pyrimidine. Reductive condensation of the nitrile 8 with diethyl N-(4-amino-benzoyl)-L-glutamate followed by ester hydrolysis gave 5-methyl-5-deazaaminopterin. Treatment of 12 with formaldehyde and Na(CN)BH3 afforded 5-methyl-5-deazamethotrexate, which was also prepared from 15 and dimethyl N-[(4-methylamino)benzoyl]-L-glutamate followed by ester hydrolysis. 5-Methyl-10-ethyl-5-deazaaminopterin was similarly prepared from 15. Biological evaluation of the 5-methyl-5-deaza analogues together with previously reported 5-deazaaminopterin and 5-deazamethotrexate for inhibition of dihydrofolate reductase (DHFR) isolated from L1210 cells and for their effect on cell growth inhibition, transport characteristics, and net accumulation of polyglutamate forms in L1210 cells revealed the analogues to have essentially the same properties as the appropriate parent compound, aminopterin or methotrexate (MTX), except that 20 and 21 were approximately 10 times more growth inhibitory than MTX. In in vivo tests against P388/0 and P388/MTX leukemia in mice, the analogues showed activity comparable to that of MTX, with the more potent 20 producing the same response in the P388/0 test as MTX but at one-fourth the dose; none showed activity against P388/MTX. Hydrolytic deamination of 12 and 20 produced 5-methyl-5-deazafolic acid and 5,10-dimethyl-5-deazafolic acid, respectively. In bacterial studies on the 2-amino-4-oxo analogues, 5-deazafolic acid proved to be a potent inhibitor of Lactobacillus casei DHFR and also the growth of both L. casei ATCC 7469 and Streptococcus faecium ATCC 8043. Its 5-methyl congener 22 is also inhibitory toward L. casei, but its IC50 for growth inhibition is much lower than its IC50 values for inhibition of DHFR or thymidylate synthase from L. casei, suggesting an alternate site of action.
|
Compound was tested for its ability to inhibit growth of L1210 mouse leukemia cells
|
Mus musculus
|
2.0
nM
|
|
Journal : J. Med. Chem.
Title : Methotrexate analogues. 26. Inhibition of dihydrofolate reductase and folylpolyglutamate synthetase activity and in vitro tumor cell growth by methotrexate and aminopterin analogues containing a basic amino acid side chain.
Year : 1986
Volume : 29
Issue : 5
First Page : 655
Last Page : 660
Authors : Rosowsky A, Freisheim JH, Moran RG, Solan VC, Bader H, Wright JE, Radike-Smith M.
Abstract : Analogues of the antitumor antifolate methotrexate (MTX) were synthesized in which the glutamate (Glu) moiety was replaced by ornithine (Orn), 2,4-diaminobutyric acid (Dab), or 2,3-diaminopropionic acid (Dap). An aminopterin (AMT) analogue with Orn in place of Glu was also synthesized. The MTX analogues were obtained by reaction of 4-amino-4-deoxy-N10-methylpteroic acid (mAPA) and N omega-Boc-alpha,omega-diaminoalkanoic acids in the presence of diethyl phosphorocyanidate, followed by deprotection with trifluoroacetic acid (TFA) or by reaction of p-nitrophenyl-mAPA and N omega-Boc-alpha,omega-diaminoalkanoic acids and subsequent treatment with TFA. The AMT analogue (APA-Orn) was synthesized by reaction of p-nitrophenyl 4-amino-4-deoxy-N10-formylpteroate with silylated N delta-Boc-L-ornithine in DMF at 55 degrees C for 3 days (45% yield), saponification (83%), and TFA cleavage (89%). APA-Orn was a potent inhibitor of both dihydrofolate reductase (DHFR) from L1210 mouse leukemia (IC50 = 0.072 microM) and partly purified folylpolyglutamate synthetase (FPGS) from mouse liver (Ki = 0.15 +/- 0.06 microM). The MTX analogue (mAPA-Orn) was likewise active against both enzymes, with an IC50 of 0.160 microM for DHFR and a Ki of 20.4 +/- 7.7 microM for FPGS inhibition. The other MTX analogues and the previously reported lysine derivative (mAPA-Lys) showed DHFR affinity similar to that of mAPA-Orn but lacked activity as FPGS inhibitors. The positively charged amino group appears to be detrimental to cellular uptake, as evidenced by the low cytotoxicity of these compounds (IC50 = 0.40-2.4 microM) in comparison with MTX and AMT (IC50 = 0.002 microM) against wild-type L1210 cells. On the other hand, mAPA-Orn and APA-Orn were both more potent than the corresponding Glu derivatives MTX and AMT against L1210/R81 cells, suggesting that in these MTX-resistant cells there may occur a "self-potentiation" process involving enhanced antifolate activity via interference with the polyglutamylation of reduced folates. APA-Orn is the most potent dual inhibitor of DHFR and FPGS discovered to date, but its effectiveness as a therapeutic agent may require some form of prodrug modification to neutralize the terminal amino group of the side chain.
|
Inhibitory concentration against growth of L1210 cell line
|
Mus musculus
|
2.0
nM
|
|
Journal : J. Med. Chem.
Title : Methotrexate analogues. 33. N delta-acyl-N alpha-(4-amino-4-deoxypteroyl)-L-ornithine derivatives: synthesis and in vitro antitumor activity.
Year : 1988
Volume : 31
Issue : 7
First Page : 1332
Last Page : 1337
Authors : Rosowsky A, Bader H, Cucchi CA, Moran RG, Kohler W, Freisheim JH.
Abstract : N delta-Acyl derivatives of the potent folylpolyglutamate synthetase (FPGS) inhibitor N alpha-(4-amino-4-deoxypteroyl)-L-ornithine (APA-L-Orn) were synthesized from N alpha-(4-amino-4-deoxy-N10-formylpteroyl)-L-ornithine by reaction with an N-(acyloxy)succinimide or acyl anhydride, followed by deformylation with base. The N delta-hemiphthaloyl derivative was also prepared from 4-amino-4-deoxy-N10-formylpteroic acid by reaction with persilylated N delta-phthaloyl-L-ornithine, followed by simultaneous deformylation and ring opening of the N delta-phthaloyl moiety with base. The products were potent inhibitors of purified dihydrofolate reductase (DHFR) from L1210 murine leukemia cells, with IC50's ranging from 0.027 and 0.052 microM as compared with 0.072 microM for APA-L-Orn. Several of the N delta-acyl-N10-formyl intermediates also proved to be good DHFR inhibitors. One of them, N alpha-(4-amino-4-deoxy-N10-formylpteroyl)-N delta-(4-chlorobenzoyl)-L- ornithine, had a 2-fold lower IC50 than its deformylated product, confirming that the N10-formyl group is well tolerated for DHFR binding. While N delta-acylation of APA-L-Orn did not significantly alter anti-DHFR activity, inhibition of FPGS was dramatically diminished, supporting the view that the basic NH2 on the end of the APA-L-Orn side chain is essential for the activity of this compound against FPGS. N delta-Acylation of APA-L-Orn markedly enhanced toxicity to cultured tumor cells. However, N delta-acyl derivatives also containing an N10-formyl substituent were less cytotoxic than the corresponding N10-unsubstituted analogues even though their anti-DHFR activity was the same, suggesting that N10-formylation may be unfavorable for transport. Two compounds, the N delta-benzoyl and N delta-hemiphthaloyl derivatives of APA-L-Orn, with IC50's against L1210 cells of 0.89 and 0.75 nM, respectively, were more potent than either methotrexate (MTX) or aminopterin (AMT) in this system. These compounds were also more potent than MTX against CEM human lymphoblasts and two human head and neck squamous cell carcinoma cell lines (SCC15, SCC25) in culture. Moreover, in assays against SCC15/R1 and SCC25/R1 sublines with 10-20-fold MTX resistance, the N delta-hemiphthaloyl derivative of APA-L-Orn showed potency exceeding that of MTX itself against the parental cells.(ABSTRACT TRUNCATED AT 400 WORDS)
|
Tested for cell-growth inhibition against mouse leukemic L1210 cells
|
Mus musculus
|
2.0
nM
|
|
Journal : J. Med. Chem.
Title : Methotrexate analogues. 34. Replacement of the glutamate moiety in methotrexate and aminopterin by long-chain 2-aminoalkanedioic acids.
Year : 1988
Volume : 31
Issue : 7
First Page : 1338
Last Page : 1344
Authors : Rosowsky A, Bader H, Kohler W, Freisheim JH, Moran RG.
Abstract : Eight previously unreported methotrexate (MTX) and aminopterin (AMT) analogues with the L-glutamate moiety replaced by DL-2-aminoalkanedioic acids containing up to 10 CH2 groups were synthesized from 4-amino-4-deoxy-N10-methylpteroic or 4-amino-4-deoxy-N10-formylpteroic acid. All the compounds were potent inhibitors of purified L1210 mouse leukemia dihydrofolate reductase (DHFR), with IC50's of 0.023-0.034 microM for the MTX analogues and 0.054-0.067 microM for the AMT analogues. The compounds were not substrates for, but were inhibitors of, partially purified mouse liver folylpolyglutamate synthetase (FPGS). Activity was correlated with the number of CH2 groups in the side chain. The IC50's for inhibition of cell growth in culture by the chain-extended MTX analogues were 0.016-0.64 microM against CEM human leukemic lymphoblasts and 0.0012-0.026 microM against L1210 mouse leukemia cells. However, the optimal chain length for growth-inhibitory activity was species-dependent. Our results suggested that CEM cells were inhibited most actively by the analogue with nine CH2 groups, while L1210 cells were most sensitive to the analogue with six CH2 groups. Among the AMT analogues, on the other hand, the most active compound against L1210 cells was the one with nine CH2 groups, which had an IC50 of 0.000 65 microM as compared with 0.0046 microM for MTX and 0.002 microM for AMT. A high degree of cross-resistance was observed between MTX and the chain-extended compounds in two MTX-resistant cell lines, CEM/MTX and L1210/R81. All the MTX analogues were active against L1210 leukemia in mice on a qd X 9 schedule, with optimal increases in lifespan (ILS) of 75-140%. Notwithstanding their high in vitro activity, the AMT analogues were more toxic and less therapeutically effective than MTX analogues of the same chain length even though neither series of compounds possessed FPGS substrate activity. These MTX and AMT analogues are an unusual group of compounds in that they retain the dicarboxylic acid structure of classical antifolates yet are more lipophilic than the parent compounds because they have more CH2 groups and are almost equivalent in vivo to MTX on the same schedule even though they do not form polyglutamates.
|
In vitro inhibition of L1210 cell growth in rodent neoplastic cells
|
Mus musculus
|
1.2
nM
|
|
Journal : J. Med. Chem.
Title : 10-Propargylaminopterin and alkyl homologues of methotrexate as inhibitors of folate metabolism.
Year : 1982
Volume : 25
Issue : 7
First Page : 877
Last Page : 880
Authors : Piper JR, McCaleb GS, Montgomery JA, Kisliuk RL, Gaumont Y, Sirotnak FM.
Abstract : Reported antifolate activity against leukemia L1210 by N-[14-[[(2-amino-4-hydroxy-6-quinazolinyl)methyl]-propargylamino]benzoyl]]-L-glu tamic acid through potent inhibition of thymidylate synthase (EC 2.1.1.45) prompted us to include the propargyl group in a study of the effect on folate metabolism and membrane transport of replacing the 10-methyl group of methotrexate with other groups. Along with the propyl (8a) and octyl (8b) homologues of methotrexate, the propargyl compound 8c was prepared for evaluation. Syntheses of 8a,b were achieved by a standard multistep sequence involving preparation of the side-chain precursors via tosylated intermediates and then their alkylation with 6-(bromomethyl)-2,4-pteridinediamine hydrobromide. The side-chain precursor to 8c was prepared by direct alkylation of diethyl N-(4-aminobenzoyl)-L-glutamate with propargyl bromide and was separated from unchanged amine and dipropargyl coproduct by a combination of methods, including dry-column chromatography and recrystallization. Subsequent steps leading to 8c were like those used to prepare 8a,b. Biological evaluations of the three compounds consisted of studies of their effects on enzyme inhibition [(dihydrofolate reductase (EC 1.5.1.3) and thymidylate synthase)], L1210 cell growth inhibition, cellular membrane transport with various murine cell types (L210, S180, Ehrlich, and epithelial), in vivo (mice) activity vs. L1210 leukemia and S180 ascites, and plasma clearance in mice. The in vivo results vs. S180 ascites offered evidence that 8c might have a better therapeutic index against this tumor than methotrexate, but no other result from either of these compounds suggested significant superiority over methotrexate.
|
Inhibitory concentration of compound in growth of L-1210 cells
|
Mus musculus
|
0.61
nM
|
|
Journal : J. Med. Chem.
Title : Folate analogues altered in the C9-N10 bridge region. 18. Synthesis and antitumor evaluation of 11-oxahomoaminopterin and related compounds.
Year : 1981
Volume : 24
Issue : 9
First Page : 1068
Last Page : 1073
Authors : Nair MG, Bridges TW, Henkel TJ, Kisliuk RL, Gaumont Y, Sirotnak FM.
Abstract : The chemical synthesis of 11-oxahomoaminopterin (1) has been carried out using procedures which were also found to be applicable to the synthesis of 11-oxahomofolic acid (2). Reaction of 1-bromo-4-[p-(caarbomethoxy)phenoxy]-2-butanone (10) with sodium azide gave 1-azido-4-[p-(carbomethoxy)phenoxy]-2-butanone (11). Protection of the carbonyl group of 11 as the ethylene ketal and subsequent base hydrolysis of the product gave 1-azido-4-(p-carboxyphenoxy)-2-butanone ketal (13). The glutamate conjugate 14 was prepared from 13 by the isobutyl chloroformate method and was hydrogenated to diethyl N-[(alpha-amino-2-oxo-4-butanoyl)-p-anisoyl]-L-glutamate ketal (15). Reaction of 15 with 6-chloro-2,4-diamino-5-nitropyrimidine (16) and 2-amino-6-chloro-4-hydroxy-5-nitropyrimidine (17) and deprotection of the corresponding products gave the intermediates 18 and 19, which were elaborated to 1 and 2 using a series of steps involving deprotection, dithionite reduction, cyclization, oxidation, and hydrolysis. Although 11-oxahomoaminopterin showed antifolate activity against two folate-requiring microorganisms and inhibited Lactobacillus casei DHFR, it was inactive against L-1210 leukemia in mice at a maximum dose of 48 mg/kg. Compound Lactobacillus casei DHFR, it was inactive against L-1210 leukemia in mice at a maximum dose of 48 mg/kg. Compound 1 was also tested for its ability to be transported via the methotrexate transport system using the L-1210 and Ehrlich tumor cell lines, and these results are compared with those of related analogues. The growth inhibitory activity of 1 in the L-1210 cell lines in culture was found to be 15 times weaker than that of methotrexate.
|
Inhibition constant (Ki) was determined in Lactobacillus casei
|
Lactobacillus casei
|
0.11
nM
|
|
Journal : J. Med. Chem.
Title : Novel fluorinated antifolates. Enzyme inhibition and cytotoxicity studies on 2'- and 3'-fluoroaminopterin.
Year : 1983
Volume : 26
Issue : 8
First Page : 1193
Last Page : 1196
Authors : Henkin J, Washtien WL.
Abstract : Two novel analogues of aminopterin with a single fluorine substitution in the 2' (compound 8) or in the 3' (compound 9) position of the p-aminobenzoyl group were synthesized and evaluated as inhibitors of dihydrofolate reductase from two bacterial species and from human HeLa cells. The 2'-fluoro compound was bound essentially the same as aminopterin itself, while the 3'-fluoro derivative bound two- to threefold more tightly in all cases. UV spectral shifts indicated normal binding of the pteridine. Cytotoxicity studies against mouse leukemia L1210 cells and the human stomach cancer line HuTu80 indicated equivalent toxicity of the parent drug with the 2'-fluoro analogue. 3'-Fluoroaminopterin was, however, twice as toxic as aminopterin to both cell lines.
|
Compound was tested for its inhibitory concentration to inhibit the growth of wild type L1210 cells.
|
Mus musculus
|
3.0
nM
|
|
Compound was tested for its inhibitory concentration to inhibit the growth of wild type L1210 cells.
|
Mus musculus
|
2.0
nM
|
|
Journal : J. Med. Chem.
Title : Methotrexate analogues. 28. Synthesis and biological evaluation of new gamma-monoamides of aminopterin and methotrexate.
Year : 1986
Volume : 29
Issue : 9
First Page : 1703
Last Page : 1709
Authors : Rosowsky A, Bader H, Radike-Smith M, Cucchi CA, Wick MM, Freisheim JH.
Abstract : Lipophilic gamma-monoamide derivatives of aminopterin (AMT) were synthesized in high overall yield from 4-amino-4-deoxy-N10-formylpteroic acid and gamma-N-tert-alkyl-, gamma-N-aralkyl-, or gamma-N-arylamides of alpha-benzyl L-glutamate via a modification of the mixed carboxylic-carbonic anhydride coupling method. Coupling was also accomplished with p-nitrophenyl 4-amino-4-deoxy-N10-formylpteroate. Compounds obtained in this manner included the gamma-tert-butylamide, gamma-(1-adamantylamide), gamma-benzylamide, gamma-(3,4-dichlorobenzylamide), gamma-(2,6-dichlorobenzylamide), gamma-anilide, gamma-(3,4-methylenedioxyanilide), and gamma-(3,4-dihydroxanilide) derivatives of AMT. Also prepared, from 4-amino-4-deoxy-N10-methylpteroic acid via diethyl phosphorocyanidate coupling, was the gamma-(3,4-methylenedioxyanilide) of MTX. The methylenedioxyanilides were cleaved smoothly to dihydroxyanilides with boron tris(trifluoroacetate) in trifluoroacetic acid. All the gamma-monoamides were tested as inhibitors of purified dihydrofolate reductase (DHFR) from murine L1210 leukemia cells and as inhibitors of the growth of wild-type L1210 cells and a subline (L1210/R81) with high-level resistance to MTX and AMT based mainly on a defect in drug uptake via active transport. Several compounds were also tested against human leukemic lymphoblasts (CEM cells) and a resistant subline (CEM/MTX) whose resistance is likewise based on uptake. The IC50 of the gamma-monoamides against DHFR was 1.5- to 5-fold higher than that of the parent acids, but the IC50 against cultured cells varied over a much broader range, suggesting that uptake and/or metabolism rather than DHFR binding are principal determinants of in vitro growth inhibitory activity for these compounds. gamma-N-Aryl and gamma-N-aralkyl derivatives appeared to be more potent than gamma-N-tert-alkyl derivatives. Where comparison could be made, AMT gamma-monoamides were more potent than MTX gamma-monoamides. Several of the gamma-monoamides showed potency comparable to that of the parent acid against wild-type L1210 and CEM cells; all of them were more potent than MTX against the L1210/R81 subline; and some of the AMT gamma-monoamides were also more potent than the parent acid against resistant CEM/MTX cells. As a group, however, the gamma-monoamides were considerably more active against the murine cells than against the human cells, suggesting that the former may take up the amides better or may be able to metabolize them more efficiently than the parent acids. All the gamma-monoamides were tested in vivo against L1210 leukemia in mice.(ABSTRACT TRUNCATED AT 400 WORDS)
|
Inhibitory concentration against the growth of L1210 murine leukemia cells in tissue culture.
|
Mus musculus
|
2.1
nM
|
|
Journal : J. Med. Chem.
Title : Synthesis of the antileukemic agents 5,10-dideazaaminopterin and 5,10-dideaza-5,6,7,8-tetrahydroaminopterin.
Year : 1985
Volume : 28
Issue : 7
First Page : 914
Last Page : 921
Authors : Taylor EC, Harrington PJ, Fletcher SR, Beardsley GP, Moran RG.
Abstract : Total syntheses from pyridine precursors of 5,10-dideazaaminopterin (1) and 5,10-dideaza-5,6,7,8-tetrahydroaminopterin (2) are described. These compounds exhibit significant in vivo activity against L1210 leukemia that is comparable to that observed with methotrexate.
|
Tested for inhibitory concentration against human SCC25 cell line
|
Homo sapiens
|
6.9
nM
|
|
Journal : J. Med. Chem.
Title : Synthesis and biological activity of N omega-hemiphthaloyl-alpha,omega- diaminoalkanoic acid analogues of aminopterin and 3',5-dichloroaminopterin.
Year : 1994
Volume : 37
Issue : 14
First Page : 2167
Last Page : 2174
Authors : Rosowsky A, Bader H, Wright JE, Keyomarsi K, Matherly LH.
Abstract : Analogues of N alpha-(4-amino-4-deoxypteroyl)-N delta-(hemiphthaloyl)-L-ornithine (PT523) with 3',5'-dichloro substitution in the p-aminobenzoyl moiety or with one less or one more CH2 group in the amino acid moiety were synthesized and tested as inhibitors of dihydrofolate reductase (DHFR) activity and cell growth. Replacement of L-ornithine in PT523 by L-2,4-diaminobutanoic acid or L-lysine did not decrease binding to human recombinant DHFR but resulted in some loss of activity against SCC25 human and SCC VII murine squamous cell carcinoma and against MCF-7 human breast carcinoma in culture. PT523 was several times more potent than methotrexate (MTX), aminopterin (AMT), or trimetrexate (TMQ). 3',5'-Dichloro substitution did not decrease either DHFR binding or cytotoxicity. A new synthetic route to PT523 from 2,4-diamino-6-(hydroxymethyl)pteridine and methyl N alpha-(4-aminobenzoyl)-N delta-phthaloyl-L-ornithinate was investigated but was not found superior to previously described methods. In comparative experiments on the ability of PT523 and MTX to competitively inhibit the influx of (6R)-5,10-dideazatetra-hydrofolate (DDATHF, lometrexol), used here as a surrogate for MTX and reduced folates, the Ki of PT523 was lower than that of MTX in both wild-type CCRF-CEM human leukemic lymphoblasts and the transport- and polyglutamylation-defective subline CEM/MTX. The CCRF-CEM cells were 10-fold more sensitive to PT523 than to MTX, whereas the CEM/MTX cells were 240-fold more sensitive. However, in contrast to other MTX-resistant cells where collateral sensitivity to PT523 has been seen. CEM/MTX cells still showed substantial cross resistance to PT523 which may reflect an unusual heightened ability to utilize exogenous folic acid. The good correlation observed with both cell lines between the cytotoxicity of PT523 and MTX and the ability to inhibit DDATHF influx supported the view that PT523 and MTX share, at least in part, a common protein carrier for membrane transport.
|
Tested for inhibitory concentration against SCC-VII murine squamous carcinoma cell line
|
Mus musculus
|
4.0
nM
|
|
Journal : J. Med. Chem.
Title : Synthesis and biological activity of N omega-hemiphthaloyl-alpha,omega- diaminoalkanoic acid analogues of aminopterin and 3',5-dichloroaminopterin.
Year : 1994
Volume : 37
Issue : 14
First Page : 2167
Last Page : 2174
Authors : Rosowsky A, Bader H, Wright JE, Keyomarsi K, Matherly LH.
Abstract : Analogues of N alpha-(4-amino-4-deoxypteroyl)-N delta-(hemiphthaloyl)-L-ornithine (PT523) with 3',5'-dichloro substitution in the p-aminobenzoyl moiety or with one less or one more CH2 group in the amino acid moiety were synthesized and tested as inhibitors of dihydrofolate reductase (DHFR) activity and cell growth. Replacement of L-ornithine in PT523 by L-2,4-diaminobutanoic acid or L-lysine did not decrease binding to human recombinant DHFR but resulted in some loss of activity against SCC25 human and SCC VII murine squamous cell carcinoma and against MCF-7 human breast carcinoma in culture. PT523 was several times more potent than methotrexate (MTX), aminopterin (AMT), or trimetrexate (TMQ). 3',5'-Dichloro substitution did not decrease either DHFR binding or cytotoxicity. A new synthetic route to PT523 from 2,4-diamino-6-(hydroxymethyl)pteridine and methyl N alpha-(4-aminobenzoyl)-N delta-phthaloyl-L-ornithinate was investigated but was not found superior to previously described methods. In comparative experiments on the ability of PT523 and MTX to competitively inhibit the influx of (6R)-5,10-dideazatetra-hydrofolate (DDATHF, lometrexol), used here as a surrogate for MTX and reduced folates, the Ki of PT523 was lower than that of MTX in both wild-type CCRF-CEM human leukemic lymphoblasts and the transport- and polyglutamylation-defective subline CEM/MTX. The CCRF-CEM cells were 10-fold more sensitive to PT523 than to MTX, whereas the CEM/MTX cells were 240-fold more sensitive. However, in contrast to other MTX-resistant cells where collateral sensitivity to PT523 has been seen. CEM/MTX cells still showed substantial cross resistance to PT523 which may reflect an unusual heightened ability to utilize exogenous folic acid. The good correlation observed with both cell lines between the cytotoxicity of PT523 and MTX and the ability to inhibit DDATHF influx supported the view that PT523 and MTX share, at least in part, a common protein carrier for membrane transport.
|
Cell growth inhibition against human squamous cell carcinoma (SCC25) line
|
Homo sapiens
|
1.6
nM
|
|
Journal : J. Med. Chem.
Title : Methotrexate analogues. 25. Chemical and biological studies on the gamma-tert-butyl esters of methotrexate and aminopterin.
Year : 1985
Volume : 28
Issue : 5
First Page : 660
Last Page : 667
Authors : Rosowsky A, Freisheim JH, Bader H, Forsch RA, Susten SS, Cucchi CA, Frei E.
Abstract : gamma-tert-Butylaminopterin (gamma-tBAMT), the first example of an aminopterin (AMT) gamma-monoester, was synthesized, and new routes to the known N10-methyl analogue gamma-tert-butyl methotrexate (gamma-tBMTX) were developed. The inhibitory effects of gamma-tBAMT on the activity of purified dihydrofolate reductase (DHFR) from L1210 murine leukemia cells, the growth of L1210 cells and CEM human leukemic lymphoblasts in suspension culture, and the growth of several lines of human squamous cell carcinoma of the head and neck in monolayer culture were compared with the effects of gamma-tBMTX and the parent acids AMT and methotrexate (MTX). Patterns of cross-resistance to gamma-tBAMT, gamma-tBMTX, and AMT among several MTX-resistant cell lines were examined. In vivo antitumor activities of gamma-tBAMT and gamma-tBMTX were compared in mice with L1210 leukemia. While the activity of gamma-tBAMT was very close to that of gamma-tBMTX in the DHFR inhibition assay, the AMT ester was more potent than the MTX ester against cells in culture and against L1210 leukemia in vivo. Only partial cross-resistance was shown against gamma-tBMTX and gamma-tBAMT in cultured cells that were resistant to MTX by virtue of a transport defect or a combination of defective transport and elevated DHFR activity.
|
Cell growth inhibition against human squamous cell carcinoma (SCC25/R1) line
|
Homo sapiens
|
4.3
nM
|
|
Journal : J. Med. Chem.
Title : Methotrexate analogues. 25. Chemical and biological studies on the gamma-tert-butyl esters of methotrexate and aminopterin.
Year : 1985
Volume : 28
Issue : 5
First Page : 660
Last Page : 667
Authors : Rosowsky A, Freisheim JH, Bader H, Forsch RA, Susten SS, Cucchi CA, Frei E.
Abstract : gamma-tert-Butylaminopterin (gamma-tBAMT), the first example of an aminopterin (AMT) gamma-monoester, was synthesized, and new routes to the known N10-methyl analogue gamma-tert-butyl methotrexate (gamma-tBMTX) were developed. The inhibitory effects of gamma-tBAMT on the activity of purified dihydrofolate reductase (DHFR) from L1210 murine leukemia cells, the growth of L1210 cells and CEM human leukemic lymphoblasts in suspension culture, and the growth of several lines of human squamous cell carcinoma of the head and neck in monolayer culture were compared with the effects of gamma-tBMTX and the parent acids AMT and methotrexate (MTX). Patterns of cross-resistance to gamma-tBAMT, gamma-tBMTX, and AMT among several MTX-resistant cell lines were examined. In vivo antitumor activities of gamma-tBAMT and gamma-tBMTX were compared in mice with L1210 leukemia. While the activity of gamma-tBAMT was very close to that of gamma-tBMTX in the DHFR inhibition assay, the AMT ester was more potent than the MTX ester against cells in culture and against L1210 leukemia in vivo. Only partial cross-resistance was shown against gamma-tBMTX and gamma-tBAMT in cultured cells that were resistant to MTX by virtue of a transport defect or a combination of defective transport and elevated DHFR activity.
|
Cell growth inhibition against human squamous cell carcinoma (SCC68) line
|
Homo sapiens
|
3.7
nM
|
|
Journal : J. Med. Chem.
Title : Methotrexate analogues. 25. Chemical and biological studies on the gamma-tert-butyl esters of methotrexate and aminopterin.
Year : 1985
Volume : 28
Issue : 5
First Page : 660
Last Page : 667
Authors : Rosowsky A, Freisheim JH, Bader H, Forsch RA, Susten SS, Cucchi CA, Frei E.
Abstract : gamma-tert-Butylaminopterin (gamma-tBAMT), the first example of an aminopterin (AMT) gamma-monoester, was synthesized, and new routes to the known N10-methyl analogue gamma-tert-butyl methotrexate (gamma-tBMTX) were developed. The inhibitory effects of gamma-tBAMT on the activity of purified dihydrofolate reductase (DHFR) from L1210 murine leukemia cells, the growth of L1210 cells and CEM human leukemic lymphoblasts in suspension culture, and the growth of several lines of human squamous cell carcinoma of the head and neck in monolayer culture were compared with the effects of gamma-tBMTX and the parent acids AMT and methotrexate (MTX). Patterns of cross-resistance to gamma-tBAMT, gamma-tBMTX, and AMT among several MTX-resistant cell lines were examined. In vivo antitumor activities of gamma-tBAMT and gamma-tBMTX were compared in mice with L1210 leukemia. While the activity of gamma-tBAMT was very close to that of gamma-tBMTX in the DHFR inhibition assay, the AMT ester was more potent than the MTX ester against cells in culture and against L1210 leukemia in vivo. Only partial cross-resistance was shown against gamma-tBMTX and gamma-tBAMT in cultured cells that were resistant to MTX by virtue of a transport defect or a combination of defective transport and elevated DHFR activity.
|
Cell growth inhibition against human squamous cell carcinoma (SCC68/R1) line
|
Homo sapiens
|
290.0
nM
|
|
Journal : J. Med. Chem.
Title : Methotrexate analogues. 25. Chemical and biological studies on the gamma-tert-butyl esters of methotrexate and aminopterin.
Year : 1985
Volume : 28
Issue : 5
First Page : 660
Last Page : 667
Authors : Rosowsky A, Freisheim JH, Bader H, Forsch RA, Susten SS, Cucchi CA, Frei E.
Abstract : gamma-tert-Butylaminopterin (gamma-tBAMT), the first example of an aminopterin (AMT) gamma-monoester, was synthesized, and new routes to the known N10-methyl analogue gamma-tert-butyl methotrexate (gamma-tBMTX) were developed. The inhibitory effects of gamma-tBAMT on the activity of purified dihydrofolate reductase (DHFR) from L1210 murine leukemia cells, the growth of L1210 cells and CEM human leukemic lymphoblasts in suspension culture, and the growth of several lines of human squamous cell carcinoma of the head and neck in monolayer culture were compared with the effects of gamma-tBMTX and the parent acids AMT and methotrexate (MTX). Patterns of cross-resistance to gamma-tBAMT, gamma-tBMTX, and AMT among several MTX-resistant cell lines were examined. In vivo antitumor activities of gamma-tBAMT and gamma-tBMTX were compared in mice with L1210 leukemia. While the activity of gamma-tBAMT was very close to that of gamma-tBMTX in the DHFR inhibition assay, the AMT ester was more potent than the MTX ester against cells in culture and against L1210 leukemia in vivo. Only partial cross-resistance was shown against gamma-tBMTX and gamma-tBAMT in cultured cells that were resistant to MTX by virtue of a transport defect or a combination of defective transport and elevated DHFR activity.
|
Cell growth inhibition against human squamous cell carcinoma (SCC78/R1) line
|
Homo sapiens
|
14.0
nM
|
|
Journal : J. Med. Chem.
Title : Methotrexate analogues. 25. Chemical and biological studies on the gamma-tert-butyl esters of methotrexate and aminopterin.
Year : 1985
Volume : 28
Issue : 5
First Page : 660
Last Page : 667
Authors : Rosowsky A, Freisheim JH, Bader H, Forsch RA, Susten SS, Cucchi CA, Frei E.
Abstract : gamma-tert-Butylaminopterin (gamma-tBAMT), the first example of an aminopterin (AMT) gamma-monoester, was synthesized, and new routes to the known N10-methyl analogue gamma-tert-butyl methotrexate (gamma-tBMTX) were developed. The inhibitory effects of gamma-tBAMT on the activity of purified dihydrofolate reductase (DHFR) from L1210 murine leukemia cells, the growth of L1210 cells and CEM human leukemic lymphoblasts in suspension culture, and the growth of several lines of human squamous cell carcinoma of the head and neck in monolayer culture were compared with the effects of gamma-tBMTX and the parent acids AMT and methotrexate (MTX). Patterns of cross-resistance to gamma-tBAMT, gamma-tBMTX, and AMT among several MTX-resistant cell lines were examined. In vivo antitumor activities of gamma-tBAMT and gamma-tBMTX were compared in mice with L1210 leukemia. While the activity of gamma-tBAMT was very close to that of gamma-tBMTX in the DHFR inhibition assay, the AMT ester was more potent than the MTX ester against cells in culture and against L1210 leukemia in vivo. Only partial cross-resistance was shown against gamma-tBMTX and gamma-tBAMT in cultured cells that were resistant to MTX by virtue of a transport defect or a combination of defective transport and elevated DHFR activity.
|
Cell growth inhibition against human squamous cell carcinoma (SCC78) line
|
Homo sapiens
|
2.5
nM
|
|
Journal : J. Med. Chem.
Title : Methotrexate analogues. 25. Chemical and biological studies on the gamma-tert-butyl esters of methotrexate and aminopterin.
Year : 1985
Volume : 28
Issue : 5
First Page : 660
Last Page : 667
Authors : Rosowsky A, Freisheim JH, Bader H, Forsch RA, Susten SS, Cucchi CA, Frei E.
Abstract : gamma-tert-Butylaminopterin (gamma-tBAMT), the first example of an aminopterin (AMT) gamma-monoester, was synthesized, and new routes to the known N10-methyl analogue gamma-tert-butyl methotrexate (gamma-tBMTX) were developed. The inhibitory effects of gamma-tBAMT on the activity of purified dihydrofolate reductase (DHFR) from L1210 murine leukemia cells, the growth of L1210 cells and CEM human leukemic lymphoblasts in suspension culture, and the growth of several lines of human squamous cell carcinoma of the head and neck in monolayer culture were compared with the effects of gamma-tBMTX and the parent acids AMT and methotrexate (MTX). Patterns of cross-resistance to gamma-tBAMT, gamma-tBMTX, and AMT among several MTX-resistant cell lines were examined. In vivo antitumor activities of gamma-tBAMT and gamma-tBMTX were compared in mice with L1210 leukemia. While the activity of gamma-tBAMT was very close to that of gamma-tBMTX in the DHFR inhibition assay, the AMT ester was more potent than the MTX ester against cells in culture and against L1210 leukemia in vivo. Only partial cross-resistance was shown against gamma-tBMTX and gamma-tBAMT in cultured cells that were resistant to MTX by virtue of a transport defect or a combination of defective transport and elevated DHFR activity.
|
Ability to inhibit WI-L2 murine leukemia tumor cell growth in culture after 48 hr of its exposure.
|
Homo sapiens
|
7.1
nM
|
|
Journal : J. Med. Chem.
Title : Analogues of methotrexate and aminopterin with gamma-methylene and gamma-cyano substitution of the glutamate side chain: synthesis and in vitro biological activity.
Year : 1991
Volume : 34
Issue : 1
First Page : 203
Last Page : 208
Authors : Rosowsky A, Bader H, Freisheim JH.
Abstract : Analogues of methotrexate (MTX) and aminopterin (AMT) modified at the gamma-position of the glutamate side chain were synthesized and evaluated as dihydrofolate reductase (DHFR) inhibitors and tumor cell growth inhibitors. Condesations of 4-amino-4-deoxy-N10-methylpteroic acid (mAPA) with dimethyl DL-4-methyleneglutamate in the presence of diethyl phosphorocyanidate (DEPC) followed by alkaline hydrolysis yielded N-(4-amino-4-deoxy-N10-methylpteroyl)-DL-4-methyleneglutamic acid (gamma-methyleneMTX). Condensation of 4-amino-4-deoxy-N10-formylpteroic acid (fAPA) with dimethyl-DL-4-methyleneglutamate by the mixed carboxylic-carbonic anhydride method yielded N-4-amino-4-deoxypteroyl)-DL-4-methyleneglutamic acid (gamma-methyleneAMT). Also prepared via DEPC coupling was a mixture of the four possible diastereomers of N-(4-amino-4-deoxy-N10-methylpteroyl)-4-cyanoglutamic acid (gamma-cyanoMTX). The requisite intermediate gamma-tert-butyl alpha-methyl 4-cyanoglutamate, as a DL-threo/DL-erythro mixture, was prepared from methyl N alpha-Boc-O-tosyl-L-serinate by reaction with sodium tert-butyl cyanoacetate followed by mild trifluoroacetic treatment to selectively remove the Boc group. The gamma-methylene derivatives of MTX and AMT are attractive because of their potential to act as Michael acceptors within the DHFR active site. gamma-CyanoMTX may be viewed as a congener of the nonpolyglutamated MTX analogue gamma-fluoroMTX. In vitro bioassay data for the gamma-methylene and gamma-cyano compounds support the idea that the active site of DHFR, already known for its ability to tolerate modification of the gamma-carboxyl group of MTX and AMT, can likewise accommodate substitution on the gamma-carbon itself.
|
Compound was evaluated for cell growth inhibition of human (WI-L2) leukemic cells.
|
Homo sapiens
|
7.0
nM
|
|
Journal : J. Med. Chem.
Title : Synthesis and biological activity of the 2-desamino and 2-desamino-2-methyl analogues of aminopterin and methotrexate.
Year : 1991
Volume : 34
Issue : 1
First Page : 227
Last Page : 234
Authors : Rosowsky A, Forsch RA, Moran RG, Freisheim JH.
Abstract : The previously undescribed 2-desamino and 2-desamino-2-methyl analogues of aminopterin (AMT) and methotrexate (MTX) were synthesized from 2-amino-5-(chloromethyl)pyrazine-3-carbonitrile. The AMT analogues were obtained via a three-step sequence consisting of condensation with di-tert-butyl N-(4-aminobenzoyl)-L-glutamate, heating with formamidine or acetamidine acetate, and mild acidolysis with trifluoroacetic acid. The MTX analogues were prepared similarly, except that 2-amino-5-(chloromethyl)pyrazine-3-carbonitrile was condensed with 4-(N-methylamino)benzoic acid and the resulting product was annulated with formamidine or acetamidine acetate to obtain the 2-desamino and 2-desamino-2-methyl analogues, respectively, of 4-amino-4-deoxy-N10-methylpteroic acid. Condensation with di-tert-butyl L-glutamate in the presence of diethyl phosphorocyanidate followed by ester cleavage with trifluoroacetic acid was then carried out. Retention of the L configuration in the glutamate moiety during this synthesis was demonstrated by rapid and essentially complete hydrolysis with carboxypeptidase G1 under conditions that likewise cleaved the L enantiomer of MTX but left the D enantiomer unaffected. The 2-desamino and 2-desamino-2-methyl analogues of AMT and MTX inhibited the growth of tumor cells, but were very poor inhibitors of dihydrofolate reductase (DHFR). These unexpected results suggested that activity in intact cells was due to metabolism of the 2-desamino compounds to polyglutamates.
|
Compound was evaluated for growth inhibition of WI-L2 cells.
|
Homo sapiens
|
7.1
nM
|
|
Journal : J. Med. Chem.
Title : Synthesis and biological activity of methotrexate analogues with two acid groups and a hydrophobic aromatic ring in the side chain.
Year : 1991
Volume : 34
Issue : 2
First Page : 574
Last Page : 579
Authors : Rosowsky A, Bader H, Freisheim JH.
Abstract : The heretofore unknown gamma-(m-carboxyanilide) and gamma-(m-boronoanillide) derivatives of methotrexate (MTX) and the gamma-(m-carboxyanilide) derivatives of aminopterin (AMT) were prepared and tested as inhibitors of dihydrofolate reductase (DHFR) and as inhibitors of cell growth in culture with the aim of comparing their activity with that of N alpha-(4-amino-4-deoxypteroyl)-N delta-hemiphthaloyl-L-ornithine, a potent antifolate whose side chain likewise contains a hydrophobic aromatic ring with an acid group on the ring. All three anilides were potent DHFR inhibitors, with activity comparable to MTX and AMT. The gamma-(m-boronoanilide) displayed growth inhibitory potency similar to that of the hemiphthaloylornithine analogue, with an IC50 of only 0.7 nM. This compound, which is the most potent of the gamma-amides of MTX tested to date, is also the first reported example of an antifolate with a B(OH)2 group in the side chain and is especially novel because of its potential to form a stable tetrahedral boronate complex by reaction with electron rich OH or NH2 groups in the active site of DHFR or other folate enzymes. In antitumor assays against L1210 leukemia in mice, N alpha-(4-amino-4-deoxypteroyl)-N delta-hemiphthaloyl-L-ornithine gave a T/C of greater than 263% at 20 mg/kg (qdx9) and 300% at 16 mg/kg (bidx10), whereas maximally tolerated doses of MTX of 8 mg/kg (qdx9) and 1 mg/kg (bidx10) gave T/C values of 213 and 188%, respectively. MTX gamma-(m-boronoanilide) was also active, with a T/C of 175% at 32 mg/kg (qdx9), the highest dose tested.
|
Inhibitory activity against Wild-type human DHFR
|
None
|
0.0018
nM
|
|
Journal : J. Med. Chem.
Title : 2,4-Diamino-5-substituted-quinazolines as inhibitors of a human dihydrofolate reductase with a site-directed mutation at position 22 and of the dihydrofolate reductases from Pneumocystis carinii and Toxoplasma gondii.
Year : 1995
Volume : 38
Issue : 5
First Page : 745
Last Page : 752
Authors : Rosowsky A, Mota CE, Queener SF, Waltham M, Ercikan-Abali E, Bertino JR.
Abstract : 2,4-Diaminoquinazoline antifolates with a lipophilic side chain at the 5-position, and in one case with a classical (p-aminobenzoyl)-L-glutamate side chain, were synthesized as potentially selective inhibitors of a site-directed mutant of human dihydrofolate reductase (DHFR) containing phenylalanine instead of leucine at position 22. This mutant enzyme is approximately 100-fold more resistant than native enzyme to the classical antifolate methotrexate (MTX), yet shows minimal cross resistance to the nonclassical antifolates piritrexim (PTX) and trimetrexate (TMQ). Although they were much less potent than trimetrexate and piritrexim, the lipophilic 5-substituted analogues were all found to bind approximately 10 times better to the mutant DHFR than to the wild-type enzyme. The potency of the analogue with a classical (p-aminobenzoyl)-L-glutamate side chain was similarly diminished in comparison with MTX, but the difference in its binding affinity to the two DHFR species was only 5-fold. Thus, by making subtle structural changes in the antifolate molecule, it may be possible to attack resistance due to mutational alterations in the active site of the target enzyme. Also, to test the hypothesis that DHFR from Pneumocystis carinii and Toxoplasma gondii may have a less sterically restrictive active site than the enzyme from mammalian cells, inhibition assays using several of the lipophilic analogues in the series were carried out against the P. carinii and T. gondii reductases in comparison with the enzyme from rat liver. In contrast to their preferential binding to mutant versus wild-type human DHFR, binding of these analogues to the P. carinii and T. gondii enzymes was weaker than binding to rat enzyme. It thus appears that, if the active site of the DHFR from these parasites is less sterically restrictive than the active site of the mammalian enzyme, this difference cannot be successfully exploited by moving the side chain from the 6-position to the 5-position.
|
TP_TRANSPORTER: inhibition of MTX uptake in OAT-K1-expressing MDCK cells
|
None
|
500.0
nM
|
|
Journal : J. Pharmacol. Exp. Ther.
Title : Trans-stimulation effects of folic acid derivatives on methotrexate transport by rat renal organic anion transporter, OAT-K1.
Year : 2000
Volume : 293
Issue : 1
First Page : 1034
Last Page : 1039
Authors : Takeuchi A, Masuda S, Saito H, Hashimoto Y, Inui K.
Abstract : We examined the pharmacological role of the renal organic anion transporter OAT-K1, which localizes predominantly in the brush-border membranes of proximal straight tubules, in the urinary excretion of methotrexate and the possibility of its contribution to "folinic acid rescue." With Madin-Darby canine kidney (MDCK) cells stably transfected with OAT-K1 cDNA, OAT-K1-mediated methotrexate accumulation was inhibited in the presence of various folic acid derivatives. These derivatives included aminopterin, 5-methyltetrahydrofolic acid, unlabeled methotrexate, folinic acid (citrovorum factor, leucovorin), and folic acid with apparent inhibition constant values of 0.5, 1.2, 1.8, 8.2, and 14.1 microM, respectively. In contrast, 10 microM taurocholic acid and sulfobromophthalein did not inhibit OAT-K1-mediated methotrexate accumulation. In addition, methotrexate efflux was stimulated in the presence of inwardly directed gradients of aminopterin, 5-methyltetrahydrofolic acid, unlabeled methotrexate, folinic acid, and folic acid, but not of uric acid, taurocholic acid, and glutathione, indicating that OAT-K1-mediated methotrexate efflux is stimulated by a folic acid derivatives exchange. In conclusion, OAT-K1 was suggested to enhance the apical efflux of highly accumulated methotrexate in tubular epithelial cells and contribute at least in part to folinic acid rescue by exchanging intracellular methotrexate for extracellular folinic acid.
|
Inhibition of Clostridium botulinum recombinant neurotoxin A light chain using SNAPtide as substrate at 10 uM after 1 hr by FRET assay relative to control
|
Clostridium botulinum
|
11.0
%
|
|
Journal : J. Med. Chem.
Title : Paclitaxel is an inhibitor and its boron dipyrromethene derivative is a fluorescent recognition agent for botulinum neurotoxin subtype A.
Year : 2013
Volume : 56
Issue : 7
First Page : 2791
Last Page : 2803
Authors : Dadgar S, Ramjan Z, Floriano WB.
Abstract : We have successfully identified one new inhibitor and one new fluorescent recognition agent for the botulinum neurotoxin subtype A (BoNT/A) using the virtual screening protocol "protein scanning with virtual ligand screening" (PSVLS). Hit selection used an in-house developed holistic binding scoring method. Selected hits were tested experimentally for inhibitory activity using fluorescence resonance energy transfer (FRET) assays against the light chain (catalytic domain) of BoNT/A. Ligand binding was determined against the light and heavy chain BoNT/A complex through either radiolabeled ligand binding assays (nonfluorescent ligands) or fluorescence intensity assays (fluorescent ligands). These experimental assays have confirmed one compound (paclitaxel) to inhibit BoNT/A's proteolytic activity experimentally with an IC50 of 5.2 μM. A fluorescent derivative was also confirmed to bind to the toxin and therefore is a suitable candidate for the rational design of new detection agents and for the development of fluorescence-based multiprobe detection assays.
|
Inhibition of sodium fluorescein uptake in OATP1B1-transfected CHO cells at an equimolar substrate-inhibitor concentration of 10 uM
|
Cricetulus griseus
|
89.56
%
|
|
Journal : Mol. Pharmacol.
Title : Structure-based identification of OATP1B1/3 inhibitors.
Year : 2013
Volume : 83
Issue : 6
First Page : 1257
Last Page : 1267
Authors : De Bruyn T, van Westen GJ, Ijzerman AP, Stieger B, de Witte P, Augustijns PF, Annaert PP.
Abstract : Several recent studies show that inhibition of the hepatic transport proteins organic anion-transporting polypeptide 1B1 (OATP1B1) and 1B3 (OATP1B3) can result in clinically relevant drug-drug interactions (DDI). To avoid late-stage development drug failures due to OATP1B-mediated DDI, predictive in vitro and in silico methods should be implemented at an early stage of the drug candidate evaluation process. In the present study, we first developed a high-throughput in vitro transporter inhibition assay for the OATP1B subfamily. A total of 2000 compounds were tested as potential modulators of the uptake of the OATP1B substrate sodium fluorescein, in OATP1B1- or 1B3-transfected Chinese hamster ovary cells. At an equimolar substrate-inhibitor concentration of 10 µM, 212 and 139 molecules were identified as OATP1B1 and OATP1B3 inhibitors, respectively (minimum 50% inhibition). For 69 compounds, previously not identified as OATP1B inhibitors, concentration-dependent inhibition was also determined, yielding Ki values ranging from 0.06 to 6.5 µM. Based on these in vitro data, we subsequently developed a proteochemometrics-based in silico model, which predicted OATP1B inhibitors in the test group (20% of the dataset) with high specificity (86%) and sensitivity (78%). Moreover, several physicochemical compound properties and substructures related to OATP1B1/1B3 inhibition or inactivity were identified. Finally, model performance was prospectively verified with a set of 54 compounds not included in the original dataset. This validation indicated that 80 and 74% of the compounds were correctly classified for OATP1B1 and OATP1B3 inhibition, respectively.
|
Inhibition of sodium fluorescein uptake in OATP1B3-transfected CHO cells at an equimolar substrate-inhibitor concentration of 10 uM
|
Cricetulus griseus
|
124.5
%
|
|
Journal : Mol. Pharmacol.
Title : Structure-based identification of OATP1B1/3 inhibitors.
Year : 2013
Volume : 83
Issue : 6
First Page : 1257
Last Page : 1267
Authors : De Bruyn T, van Westen GJ, Ijzerman AP, Stieger B, de Witte P, Augustijns PF, Annaert PP.
Abstract : Several recent studies show that inhibition of the hepatic transport proteins organic anion-transporting polypeptide 1B1 (OATP1B1) and 1B3 (OATP1B3) can result in clinically relevant drug-drug interactions (DDI). To avoid late-stage development drug failures due to OATP1B-mediated DDI, predictive in vitro and in silico methods should be implemented at an early stage of the drug candidate evaluation process. In the present study, we first developed a high-throughput in vitro transporter inhibition assay for the OATP1B subfamily. A total of 2000 compounds were tested as potential modulators of the uptake of the OATP1B substrate sodium fluorescein, in OATP1B1- or 1B3-transfected Chinese hamster ovary cells. At an equimolar substrate-inhibitor concentration of 10 µM, 212 and 139 molecules were identified as OATP1B1 and OATP1B3 inhibitors, respectively (minimum 50% inhibition). For 69 compounds, previously not identified as OATP1B inhibitors, concentration-dependent inhibition was also determined, yielding Ki values ranging from 0.06 to 6.5 µM. Based on these in vitro data, we subsequently developed a proteochemometrics-based in silico model, which predicted OATP1B inhibitors in the test group (20% of the dataset) with high specificity (86%) and sensitivity (78%). Moreover, several physicochemical compound properties and substructures related to OATP1B1/1B3 inhibition or inactivity were identified. Finally, model performance was prospectively verified with a set of 54 compounds not included in the original dataset. This validation indicated that 80 and 74% of the compounds were correctly classified for OATP1B1 and OATP1B3 inhibition, respectively.
|
Inhibition of pigeon liver dihydrofolate reductase
|
Columba livia
|
26.0
nM
|
|
Journal : J. Med. Chem.
Title : Analogues of methotrexate.
Year : 1979
Volume : 22
Issue : 7
First Page : 862
Last Page : 868
Authors : Montgomery JA, Piper JR, Elliott RD, Temple C, Roberts EC, Shealy YF.
Abstract : Analogues of methotrexate (MTX) were prepared by alkylation of side-chain precursors with 6-(bromomethyl)-2,4-pteridinediamine followed, where necessary, by saponification of the intermediate esters and, in two cases, by electrophilic substitution reactions in the pyridine ring portion of 3-deazamethotrexate. Effects of the various modifications on their ability to inhibit dihydrofolate reductase, cytotoxicity, and activity against L1210 leukemia in mice were examined in light of recent findings concerning active transport of MTX and related compounds and the binding features of the MTX-dihydrofolate reductase complex.
|
Antiviral activity determined as inhibition of SARS-CoV-2 induced cytotoxicity of Caco-2 cells at 10 uM after 48 hours by high content imaging
|
Homo sapiens
|
8.42
%
|
|
Title : Identification of inhibitors of SARS-CoV-2 in-vitro cellular toxicity in human (Caco-2) cells using a large scale drug repurposing collection
Year : 2020
Authors : Bernhard Ellinger, Denisa Bojkova, Andrea Zaliani, Jindrich Cinatl, Carsten Claussen, Sandra Westhaus, Jeanette Reinshagen, Maria Kuzikov, Markus Wolf, Gerd Geisslinger, Philip Gribbon, Sandra Ciesek
Abstract : To identify possible candidates for progression towards clinical studies against SARS-CoV-2, we screened a well-defined collection of 5632 compounds including 3488 compounds which have undergone clinical investigations (marketed drugs, phases 1 -3, and withdrawn) across 600 indications. Compounds were screened for their inhibition of viral induced cytotoxicity using the human epithelial colorectal adenocarcinoma cell line Caco-2 and a SARS-CoV-2 isolate. The primary screen of 5632 compounds gave 271 hits. A total of 64 compounds with IC50 <20 µM were identified, including 19 compounds with IC50 < 1 µM. Of this confirmed hit population, 90% have not yet been previously reported as active against SARS-CoV-2 in-vitro cell assays. Some 37 of the actives are launched drugs, 19 are in phases 1-3 and 10 pre-clinical. Several inhibitors were associated with modulation of host pathways including kinase signaling P53 activation, ubiquitin pathways and PDE activity modulation, with long chain acyl transferases were effective viral inhibitors.
|
SARS-CoV-2 3CL-Pro protease inhibition percentage at 20µM by FRET kind of response from peptide substrate
|
Severe acute respiratory syndrome coronavirus 2
|
7.055
%
|
|
Title : Identification of inhibitors of SARS-Cov2 M-Pro enzymatic activity using a small molecule repurposing screen
Year : 2020
Authors : Maria Kuzikov, Elisa Costanzi, Jeanette Reinshagen, Francesca Esposito, Laura Vangeel, Markus Wolf, Bernhard Ellinger, Carsten Claussen, Gerd Geisslinger, Angela Corona, Daniela Iaconis, Carmine Talarico, Candida Manelfi, Rolando Cannalire, Giulia Rossetti, Jonas Gossen, Simone Albani, Francesco Musiani, Katja Herzog, Yang Ye, Barbara Giabbai, Nicola Demitri, Dirk Jochmans, Steven De Jonghe, Jasper Rymenants, Vincenzo Summa, Enzo Tramontano, Andrea R. Beccari, Pieter Leyssen, Paola Storici, Johan Neyts, Philip Gribbon, and Andrea Zaliani
Abstract : Compound repurposing is an important strategy being pursued in the identification of effective treatment against the SARS-CoV-2 infection and COVID-19 disease. In this regard, SARS-CoV-2 main protease (M-Pro), also termed 3CL-Pro, is an attractive drug target as it plays a central role in viral replication by processing the viral polyprotein into 11 non-structural proteins. We report the results of a screening campaign involving ca 8.7 K compounds containing marketed drugs, clinical and preclinical candidates, and chemicals regarded as safe in humans. We confirmed previously reported inhibitors of 3CL-Pro, but we have also identified 68 compounds with IC50 lower than 1 uM and 127 compounds with IC50 lower than 5 uM. Profiling showed 67% of confirmed hits were selective (> 5 fold) against other Cys- and Ser- proteases (Chymotrypsin and Cathepsin-L) and MERS 3CL-Pro. Selected compounds were also analysed in their binding characteristics.
|
Antiviral activity determined as inhibition of SARS-CoV-2 induced cytotoxicity of VERO-6 cells at 10 uM after 48 hours exposure to 0.01 MOI SARS CoV-2 virus by high content imaging
|
Chlorocebus sabaeus
|
-0.01
%
|
|
Antiviral activity determined as inhibition of SARS-CoV-2 induced cytotoxicity of VERO-6 cells at 10 uM after 48 hours exposure to 0.01 MOI SARS CoV-2 virus by high content imaging
|
Chlorocebus sabaeus
|
-0.01
%
|
|
Title : Cytopathic SARS-Cov2 screening on VERO-E6 cells in a large repurposing effort
Year : 2020
Authors : Andrea Zaliani, Laura Vangeel, Jeanette Reinshagen, Daniela Iaconis, Maria Kuzikov, Oliver Keminer, Markus Wolf, Bernhard Ellinger, Francesca Esposito, Angela Corona, Enzo Tramontano, Candida Manelfi, Katja Herzog, Dirk Jochmans, Steven De Jonghe, Winston Chiu, Thibault Francken, Joost Schepers, Caroline Collard, Kayvan Abbasi, Carsten Claussen , Vincenzo Summa, Andrea R. Beccari, Johan Neyts, Philip Gribbon and Pieter Leyssen
Abstract : Worldwide, there are intensive efforts to identify repurposed drugs as potential therapies against SARS-CoV-2 infection and the associated COVID-19 disease. To date, the anti-inflammatory drug dexamethasone and (to a lesser extent) the RNA-polymerase inhibitor remdesivir have been shown to be effective in reducing mortality and patient time to recovery, respectively, in patients. Here, we report the results of a phenotypic screening campaign within an EU-funded project (H2020-EXSCALATE4COV) aimed at extending the repertoire of anti-COVID therapeutics through repurposing of available compounds and highlighting compounds with new mechanisms of action against viral infection. We screened 8702 molecules from different repurposing libraries, to reveal 110 compounds with an anti-cytopathic IC50 < 20 µM. From this group, 18 with a safety index greater than 2 are also marketed drugs, making them suitable for further study as potential therapies against COVID-19. Our result supports the idea that a systematic approach to repurposing is a valid strategy to accelerate the necessary drug discovery process.
|
Inhibition of Mycobacterium smegmatis ATCC 607 dihydrofolic reductase
|
Mycobacterium smegmatis
|
0.18
nM
|
|
Journal : Bioorg Med Chem
Title : Insights of synthetic analogues of anti-leprosy agents.
Year : 2019
Volume : 27
Issue : 13
First Page : 2689
Last Page : 2717
Authors : Bera S, Mondal D.
Abstract : Today, the emergence of the phenomenon of drug or multidrug-resistance for community-associated diseases represents a major concern in the world. In these contexts, the chronic infectious disease, leprosy, grounded by a slow-growing bacterium called Mycobacterium leprae or Mycobacterium lepromatosis is a leadingcause of severe disfiguring skin sores and nerve damage in the arms, legs, and skin areas around the body. Even, over 200,000 new leprosy cases are being accounted every year along with the relapsed leprosy cases. Nonetheless, this has been considered a curable disease with a higher dose of multidrug therapy (MDT) for a long period of time. The prolonged action of a high dose of combination drugs administration may cause an adverse reaction that can significantly affect patient compliance, particularly the outbreak of multidrug-resistance in the infected person. To overcome these shortfalls or prevent the resistance-associated problems, researchers are diligently involved in the structural modifications of the clinically used anti-leprosy drugs or the allied compounds for the structure-antimycobacterial activity relationship study. This review article described the detailed synthesis and biological assays of different anti-leprosy compounds reported by several research groups.
|
Cytotoxicity against human HL60 cells assessed reduction in cell viability incubated for 48 hrs in presence of ROS scavenger pyruvate by Alamar blue assay
|
Homo sapiens
|
3.0
nM
|
|
Journal : Bioorg Med Chem
Title : Auxiliary in vitro and in vivo biological evaluation of hydrogen peroxide sensitive prodrugs of methotrexate and aminopterin for the treatment of rheumatoid arthritis.
Year : 2020
Volume : 28
Issue : 2
First Page : 115247
Last Page : 115247
Authors : Previtali V, Petrovic K, Peiró Cadahía J, Troelsen NS, Clausen MH.
Abstract : Rheumatoid arthritis (RA) is a chronic inflammatory disease that causes severe joints damage and other extra-articular alterations. Despite the efficacy of low-dose methotrexate (LD-MTX) in RA treatment, adverse effects are the predominant reasons for discontinuation of therapy. As a therapeutic targeting strategy, the presence of increased concentrations of reactive oxygen species (ROS) in the inflammatory environment can serve as the stimulus for prodrug activation in site-selective drug delivery systems. Our group has previously reported novel ROS sensitive prodrugs (1-3) of MTX and aminopterin (AMT) for site-selective delivery to inflammatory tissue associated with RA, with the aim of reducing side effects in RA therapy. Herein, we investigate the effect and toxicity of the same prodrugs in a rat CIA (collagen-induced arthritis) model of RA. We find that prodrug 1, an arylboronic acid ROS-sensitive MTX-prodrug, displays similar in vivo efficacy as MTX at an equimolar dose, while avoiding adverse effects known to restrict MTX treatment. To further characterize prodrug 1 and its ROS mediated activation, we synthesized compound 4, a negative control lacking the boronic acid moiety. We then investigated the effect of molecules on cell proliferation and cytotoxicity in the presence of the ROS scavenger pyruvate, as well as their stability in buffer and cell media, demonstrating a direct correlation between ROS concentration and the prodrug activity. Moreover, the in vitro ADME properties were investigated, including permeability, rat plasma and microsomal stability.
|
Cytotoxicity against human HL60 cells assessed as reduction in cell viability incubated for 48 hrs in absence of ROS scavenger pyruvate by Alamar blue assay
|
Homo sapiens
|
2.5
nM
|
|
Journal : Bioorg Med Chem
Title : Auxiliary in vitro and in vivo biological evaluation of hydrogen peroxide sensitive prodrugs of methotrexate and aminopterin for the treatment of rheumatoid arthritis.
Year : 2020
Volume : 28
Issue : 2
First Page : 115247
Last Page : 115247
Authors : Previtali V, Petrovic K, Peiró Cadahía J, Troelsen NS, Clausen MH.
Abstract : Rheumatoid arthritis (RA) is a chronic inflammatory disease that causes severe joints damage and other extra-articular alterations. Despite the efficacy of low-dose methotrexate (LD-MTX) in RA treatment, adverse effects are the predominant reasons for discontinuation of therapy. As a therapeutic targeting strategy, the presence of increased concentrations of reactive oxygen species (ROS) in the inflammatory environment can serve as the stimulus for prodrug activation in site-selective drug delivery systems. Our group has previously reported novel ROS sensitive prodrugs (1-3) of MTX and aminopterin (AMT) for site-selective delivery to inflammatory tissue associated with RA, with the aim of reducing side effects in RA therapy. Herein, we investigate the effect and toxicity of the same prodrugs in a rat CIA (collagen-induced arthritis) model of RA. We find that prodrug 1, an arylboronic acid ROS-sensitive MTX-prodrug, displays similar in vivo efficacy as MTX at an equimolar dose, while avoiding adverse effects known to restrict MTX treatment. To further characterize prodrug 1 and its ROS mediated activation, we synthesized compound 4, a negative control lacking the boronic acid moiety. We then investigated the effect of molecules on cell proliferation and cytotoxicity in the presence of the ROS scavenger pyruvate, as well as their stability in buffer and cell media, demonstrating a direct correlation between ROS concentration and the prodrug activity. Moreover, the in vitro ADME properties were investigated, including permeability, rat plasma and microsomal stability.
|
Cytotoxicity against human HeLa cells assessed as reduction in cell viability incubated for 48 hrs in presence of ROS scavenger pyruvate by Alamar blue assay
|
Homo sapiens
|
3.6
nM
|
|
Journal : Bioorg Med Chem
Title : Auxiliary in vitro and in vivo biological evaluation of hydrogen peroxide sensitive prodrugs of methotrexate and aminopterin for the treatment of rheumatoid arthritis.
Year : 2020
Volume : 28
Issue : 2
First Page : 115247
Last Page : 115247
Authors : Previtali V, Petrovic K, Peiró Cadahía J, Troelsen NS, Clausen MH.
Abstract : Rheumatoid arthritis (RA) is a chronic inflammatory disease that causes severe joints damage and other extra-articular alterations. Despite the efficacy of low-dose methotrexate (LD-MTX) in RA treatment, adverse effects are the predominant reasons for discontinuation of therapy. As a therapeutic targeting strategy, the presence of increased concentrations of reactive oxygen species (ROS) in the inflammatory environment can serve as the stimulus for prodrug activation in site-selective drug delivery systems. Our group has previously reported novel ROS sensitive prodrugs (1-3) of MTX and aminopterin (AMT) for site-selective delivery to inflammatory tissue associated with RA, with the aim of reducing side effects in RA therapy. Herein, we investigate the effect and toxicity of the same prodrugs in a rat CIA (collagen-induced arthritis) model of RA. We find that prodrug 1, an arylboronic acid ROS-sensitive MTX-prodrug, displays similar in vivo efficacy as MTX at an equimolar dose, while avoiding adverse effects known to restrict MTX treatment. To further characterize prodrug 1 and its ROS mediated activation, we synthesized compound 4, a negative control lacking the boronic acid moiety. We then investigated the effect of molecules on cell proliferation and cytotoxicity in the presence of the ROS scavenger pyruvate, as well as their stability in buffer and cell media, demonstrating a direct correlation between ROS concentration and the prodrug activity. Moreover, the in vitro ADME properties were investigated, including permeability, rat plasma and microsomal stability.
|
Cytotoxicity against human HeLa cells assessed as reduction in cell viability incubated for 48 hrs in absence of ROS scavenger pyruvate by Alamar blue assay
|
Homo sapiens
|
8.3
nM
|
|
Journal : Bioorg Med Chem
Title : Auxiliary in vitro and in vivo biological evaluation of hydrogen peroxide sensitive prodrugs of methotrexate and aminopterin for the treatment of rheumatoid arthritis.
Year : 2020
Volume : 28
Issue : 2
First Page : 115247
Last Page : 115247
Authors : Previtali V, Petrovic K, Peiró Cadahía J, Troelsen NS, Clausen MH.
Abstract : Rheumatoid arthritis (RA) is a chronic inflammatory disease that causes severe joints damage and other extra-articular alterations. Despite the efficacy of low-dose methotrexate (LD-MTX) in RA treatment, adverse effects are the predominant reasons for discontinuation of therapy. As a therapeutic targeting strategy, the presence of increased concentrations of reactive oxygen species (ROS) in the inflammatory environment can serve as the stimulus for prodrug activation in site-selective drug delivery systems. Our group has previously reported novel ROS sensitive prodrugs (1-3) of MTX and aminopterin (AMT) for site-selective delivery to inflammatory tissue associated with RA, with the aim of reducing side effects in RA therapy. Herein, we investigate the effect and toxicity of the same prodrugs in a rat CIA (collagen-induced arthritis) model of RA. We find that prodrug 1, an arylboronic acid ROS-sensitive MTX-prodrug, displays similar in vivo efficacy as MTX at an equimolar dose, while avoiding adverse effects known to restrict MTX treatment. To further characterize prodrug 1 and its ROS mediated activation, we synthesized compound 4, a negative control lacking the boronic acid moiety. We then investigated the effect of molecules on cell proliferation and cytotoxicity in the presence of the ROS scavenger pyruvate, as well as their stability in buffer and cell media, demonstrating a direct correlation between ROS concentration and the prodrug activity. Moreover, the in vitro ADME properties were investigated, including permeability, rat plasma and microsomal stability.
