Previously, we reported on the synthesis and estrogen receptor (ER) interaction of imidazoles, which had to be 1-alkyl-4, 5-bis(2-halo-4-hydroxyphenyl) substituted for a high relative binding affinity (RBA >1 %). This led to the assumption that a shielding of the polar heterocyclic system is a prerequisite for ER binding. In continuation of this study we synthesized 2, 4, 5-tris(4-hydroxyphenyl)imidazoles with Cl-or F-atoms in the ortho-positions of the aromatic rings and evaluated whether they mediate sufficient hydrophobicity for ER interaction. 2-(2, 6-Dichloro-3/4-hydroxyphenyl)-4, 5-bis(2-halo-4-hydroxyphenyl)imidazoles were synthesized by reaction of the respective methoxy-substituted benzil with either the 2, 6-dichloro-4-methoxy-or the 2, 6-dichloro-3-methoxybenzaldehyde in ammonium acetate solution. The required ether cleavage was performed subsequently with BBr(3). In the competition experiment with [(3)H]estradiol the imidazoles with the a C2-standing (2, 6-dichloro-4-hydroxyphenyl) ring showed an RBA >0.02 %, but did not activate the luciferase gene in estrogen receptor positive MCF-7-2a breast cancer cells stably transfected with the plasmid ERE(wtc)luc. In the test for antagonistic potency only the 2-(2, 6-dichloro-4-hydroxyphenyl)-4, 5-bis(4-hydroxyphenyl)imidazole 3 antagonized the effects of 1 nM estradiol slightly. From these data, it can be concluded that a C2-standing 2, 6-dichloro-4-hydroxyphenyl ring is not appropriate to optimize the ER interaction of 4, 5-(4-hydroxyphenyl)imidazoles.
Basic side chains determine the pharmacology of selective estrogen receptor modulators such as tamoxifen or raloxifene. In this study we tried to turn the hormonal profile of (4R,5S)/(4S,5R)-4,5-bis(4-hydroxyphenyl)-2-imidazolines from agonistic to antagonistic by introduction of a dimethylaminoethane, a piperidin-1-ylethane, or a pyrrolidin-1-ylethane side chain into one of the 4-hydroxyphenyl rings. The compounds were tested for agonistic and antagonistic activity on hormone sensitive, ERalpha-positive MCF7-2a cells, stably transfected with the plasmid ERE(wtc)luc and on U-2 OS cells transiently transfected with plasmids encoding for ERalpha (pSG5-ERalpha) or ERbeta (pSG5-ERbeta FL) as well as the reporter plasmid (ERE)(2)luc(+). Despite the presence of a basic side chain, the majority of the 4,5-diaryl-2-imidazolines showed agonistic effects. The most active compound, (4R,5S)/(4S,5R)-4-(2-chloro-4-(2-piperidin-1-ylethoxy)phenyl)-5-(2,6-dichloro-4-hydroxyphenyl)-2-imidazoline (5a), achieved at ERalpha an EC(50) value of 0.085 microM and at ERbeta an EC(50) = 0.40 microM. High antagonistic properties only possessed the C2 ethyl substituted compounds 2a and 4a. (4R,5S)/(4S,5R)-2-Ethyl-4-(4-hydroxyphenyl)-5-(4-(2-piperidin-1-ylethoxy)phenyl)-2-imidazoline (2a) reduced the effect of estradiol at ERalpha strongly with IC(50) = 0.038 microM, while its antagonistic properties at ERbeta were distinctly lower (IC(50) = 9.00 microM), probably due to the partial agonistic effects (EC(50) = 0.50 microM).
To determine the role of the enzyme CTP synthetase (EC 6.3.4.2) in the synthesis in situ of CTP in normal and in malignant lymphoblastic cells, the metabolism of radiolabeled pyrimidine ribonucleosides was studied in proliferating normal T lymphocytes and was compared with that of proliferating MOLT-3 cell-line cells and differentiated (non-proliferating) MOLT-3 cells. Both the incorporation of ['4C]uridine into UTP and CTP and the incorporation of [14C]cytidine in CTP, as well as the fluxes of these labeled nucleosides through the nucleotide pools into nucleic acids, were elevated in proliferating MOLT-3 cells compared to proliferating T lymphocytes. Furthermore, the conversion of UTP into CTP was enhanced in proliferating MOLT-3 cells compared to proliferating T lymphocytes, indicating a higher activity of CTP synthetase in the leukemic cells. In non-proliferating MOLT-3 cells, the pyrimidine ribonucleotide fluxes were decreased compared to proliferating MOLT-3 cells and proliferating T lymphocytes. However, the decreased ratio of uracilkytosine ribonucleotides that was found in proliferating T lymphocytes and proliferating MOLT-3 cells compared to non-proliferating blood lymphocytes, was preserved in the differentiated MOLT-3 cells. Moreover, although the fluxes had decreased, most CTP was still synthesized by CTP synthetase in the differentiated MOLT-3 cells. Thus, the elevated activity of CTP synthetase in MOLT-3 cells was independent of the cell growth and maturation stage. We conclude that the increased activity of CTP synthetase is associated with the process of malignant transformation in MOLT-3 cells. Therefore, CTP synthetase offers an attractive target for selective therapy in human acute T-lymphoid leukemia.Previously, we have shown in our laboratories that peripheral blood cells from patients with acute lymphoblastic leukemia, chronic lymphoblastic leukemia and non-Hodgkin lymphoma contain increased amounts of uracil and cytosine nucleotides and contain more uridinediphosphate-sugar compounds with a changed composition than do peripheral blood leukocytes from healthy donors [l -31. Typically, the ratio between uracil and cytosine ribonucleotides is decreased in the leukemic cells [l -31. In proliferating normal T lymphocytes too, an increase in the uracil and cytosine ribonucleotide pools has been observed, with a decreased ratio compared to non-proliferating peripheral blood lymphocytes 141.CTP synthetase catalyses the conversion of UTP into CTP and is the rate-limiting step in the synthesis of cytosine nucleotides from both the de novo and uridine salvage synthesis routes (see Fig. 1). In mouse S 49 T-lymphoma cellline cells the ratio between uracil and cytosine ribonucleotide phate-buffered saline; BrdU, 5-bromo-2'-deoxyuridine.(EC 6.3.4.2); uridine-cytidine kinase (EC 2.7.1.48).pools is balanced by allosteric feedback inhibition of CTP synthetase by the CTP pool [5]. Furthermore, it has been shown by in vitro enzyme analyses that an increased activity of CTP synthetase in tissue homogenates of...
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