Neuraminidase treatment modifies the function of electroplax sodium channels in planar lipid bilayers.

Sodium channels from several sources are covalently modified by unusually large numbers of negatively charged sialic acid residues. In the present studies, purified electroplax sodium channels were treated with neuraminidase to remove sialic acid residues and then examined for functional changes in planar lipid bilayers. Neuraminidase treatment resulted in a large depolarizing shift in the average potential required for channel activation. Additionally, desialidated channels showed a striking increase in the frequency of reversible transitions to subconductance states. Thus it appears that sialic acid residues play a significant role in the function of sodium channels, possibly through their influence on the local electric field and/or conformational stability of the channel molecule.

Metabolic effects and kill of human T-cell leukemia by 5-deazaacyclotetrahydrofolate, a specific inhibitor of glycineamide ribonucleotide transformylase.

Metabolic effects and mode of cytotoxicity of 5-deazaacyclotetrahydrofolate (5-DACTHF, BW543U76), a glycineamide ribonucleotide transformylase inhibitor, were studied in MOLT-4 cells, a human T-cell leukemia line. 5-DACTHF inhibits purine synthesis with 50% inhibitory concentration values of 0.5 microM and 0.08 microM following 6- or 24-h exposure to drug, respectively. At 6 h, adenine nucleotide synthesis is preferentially inhibited over guanine nucleotide synthesis. A similar effect was observed with another glycineamide ribonucleotide transformylase inhibitor, 5,10-dideazatetrahydrofolate. GTP was depleted to 40% of control and ATP to 10% of control by 5 microM 5-DACTHF. After a transitory increase, UTP and CTP were depleted to 30% of control. Deoxynucleotides were also depleted by the drug; dCTP was depleted to the greatest extent, followed by dATP, dTTP, and dGTP, respectively. MOLT-4 cell growth was inhibited by 5-DACTHF with a 50% inhibitory concentration of 0.066 microM. Complete reversal was effected by hypoxanthine, and there was no reversal by thymidine. The drug was cytotoxic to MOLT-4 cells in the range 0.25 to 5.0 microM, but a minimum of 48 h was required for trypan blue-staining dead cells to appear. The rate and extent of kill with the thymidylate synthase inhibitor 2-methyl-10-propargyl-5,8-dideazafolate was greater than with 5-DACTHF, which indicates that kill by inhibition of thymidylate synthase is more effective than that by inhibition of purine synthesis. Electron microscopy of MOLT-4 cells exposed to 5-DACTHF showed electron-dense mitochondria and nuclear changes reminiscent of apoptosis. These morphological changes were accompanied by the appearance of DNA strand breaks at approximately 180-base pair intervals (internucleosomal breaks). Concomitant proteolysis of nuclear proteins poly(ADP-ribose) polymerase and lamin B was observed.

Biochemical and chemotherapeutic studies on 2,4-diamino-6-(2,5-dimethoxybenzyl)-5-methylpyrido[2,3-d]pyrimidine (BW 301U), a novel lipid-soluble inhibitor of dihydrofolate reductase.

The lipophilic diaminopyridopyrimidine BW 301U (2,4-diamino-6-(2,5-dimethoxybenzyl)-5-methylpyrido[2,3-d]pyrimidine) is as active as methotrexate as an inhibitor of dihydrofolate reductase and mammalian cell growth. This compound was selected from among related pyridopyrimidines and other lipid-soluble diaminoheterocyclic compounds as having the most favorable combination of properties as a potent inhibitor of dihydrofolate reductase with minimal effects on histamine metabolism. In contrast to methotrexate, entry of BW 301U into cells is rapid and is not temperature dependent, indicating passage across cell membranes by diffusion. There is no competition between BW 301U and leucovorin (folinic acid) for uptake into Sarcoma 180 cells in culture. When BW 301U is added to culture medium, deoxyuridine incorporation ceases within the first few min, and this inhibition persists when cells are transferred to drug-free medium. Both leucovorin and thymidine are required to protect cells in culture from the cytotoxicity of BW 301U. The effect on thymidine biosynthesis appears to be indirect since BW 301U is inactive as an inhibitor of thymidylate synthetase. Hypoxanthine and thymidine restore growth by only 50% in cultures containing BW 301U, and complete restoration of growth requires the further addition of adenosine and either uridine or cytidine to the medium. In vivo, BW 301U is active against Walker 256, L1210, P388, Sarcoma 180, and Ehrlich ascites tumors.

Dihydrofolate reductase in primary brain tumors, cell cultures of central nervous system origin, and normal brain during fetal and neonatal growth.

Dihydrofolate reductase (DHFR) was measured during the development in rats of brain tumors induced following inoculation with avian sarcoma virus. Increasing activity of this enzyme in brain was correlated with the course of primary brain tumor growth. The specific activities of DHFR in primary human brain tumor tissues were comparable to those found in avian sarcoma virus-induced brain tumors in rats. Specific activities of DHFR in cell cultures derived from human and rat primary intracranial gliomas and sarcomas were up to 6 times those found in adult rat liver. The presence of DHFR in neoplasms of central nervous system origin is relevant to the development of folate antagonists which, unlike methotrexate, can readily cross the blood-brain barrier. In normal developing rat brain, DHFR specific activity was high in embryos at 19 days of gestation and declined thereafter, until at 20 days after birth the activity was very low. The methotrexate titration assay was used to measure enzyme levels in the brains of fetal and newborn rats, and good correlation with the spectrophotometric assay was observed. The pattern was different in liver, showing maximum activity 11 days after birth and retaining high activity in adult liver. Both the cofactor requirement and the sensitivity to methotrexate indicate that the enzyme in the brain is DHFR.

Enhanced antitumor activity for the thymidylate synthase inhibitor 1843U89 through decreased host toxicity with oral folic acid.

The purpose of this investigation was to determine whether antitumor selectivity of the third generation thymidylate synthase inhibitor 1843U89 could be enhanced by a combination of the drug with folic acid. The effects of folic acid on toxicity of 1843U89 to the dog and mouse and on antitumor efficacy of 1843U89 in the mouse were studied. These data were compared to the effect of folic acid on the in vitro cell culture antitumor activity of 1843U89. The sensitivity of eight cancer cell lines (three ovarian, one colon, one ileocecal, one epidermoid, one osteosarcoma, and one breast line) to 1843U89 was tested in vitro in the presence and absence of folic acid. Folic acid concentrations greater than 100 microM were required to decrease 1843U89 activity in seven of the cell lines. Only the activity in HCT-8, the ileocecal line, was reserved at folic acid concentrations below 100 microM. Oral folic acid given 30 min prior to an i.v. dose of 1843U89 increased the maximally tolerated dose and the lethal dose of 1843U89, both in dogs and in thymidine-depleted mice. In mice, oral folic acid produced little or no effect upon the antitumor efficacy of 1843U89 in two of three tumor cell lines in vivo. HCT-8, the line that was sensitive to folate reversal in vitro, was also sensitive in vivo. The results show that an oral dose of folic acid 30 min prior to i.v. 1843U89 can block mouse and dog intestinal toxicity without decreasing efficacy of 1843U89 in two of three human tumor lines in the nude mouse. Thus, the data reported here indicate that the antitumor selectivity of 1843U89 may be enhanced through a combination of 1843U89 with oral folic acid.

Biochemical and cellular pharmacology of 1843U89, a novel benzoquinazoline inhibitor of thymidylate synthase.

Studies on a series of benzoquinazoline folate analogues as inhibitors of human thymidylate synthase led to the selection of 1843U89 for further evaluation. This compound had a Ki of 90 pM versus human thymidylate synthase and was noncompetitive with (6R,S)-5,10-methylenetetrahydrofolate. It was a good substrate for the addition of the second glutamate by hog liver folylpolyglutamate synthetase, having a Vmax/Km value 7.8-fold higher than (6R,S)-tetrahydrofolate. The data indicate that 1843U89 was transported into cells via the reduced folate carrier. The Kt for 1843U89 in MOLT-4 cells was 0.33 microM, which was 3-fold lower than that for methotrexate and 16-fold lower than that for (6S-5-formyltetrahydrofolate. V/K values were 20.3 for 1843U89 versus 1.2 and 1.9 for methotrexate and (6S)-5-formyltetrahydrofolate, respectively. It was a potent inhibitor of the growth of human cells, having 50% inhibitory concentrations below 1 nM for all cell lines tested. Growth inhibition was reversed by thymidine alone, indicating that thymidylate synthase was the only site of action of this compound. Growth inhibition was not affected by (6R,S-5-formyltetrahydrofolate at concentrations below 5 microM. However, the 50% inhibitory concentration increased when the concentration in the medium was increased to 100 microM, presumably due to competition for transport. Relative to the human cell lines used, murine cell lines were 80-1300-fold less sensitive to 1843U89 and the other benzoquinazolines tested. This decreased sensitivity appeared to be due, at least in part, to decreased transport or accumulation in murine cells. Ki values for inhibition of methotrexate transport for the benzoquinazolines were 5-17-fold higher in L1210 cells than in MOLT-4 cells. 1843U89, the benzoquinazoline which was transported most efficiently and which was the most potent inhibitor of the growth of human cells, exhibited the largest difference between binding to the MOLT-4 human and L1210 murine transporter. The V/K for L1210 transport was 80-fold less than that for MOLT-4. Initial antitumor studies, using the human thymidine kinase-deficient line GC3TK- to circumvent problems associated with murine transport as well as the high circulating thymidine levels in mice, indicated that 1843U89 had marked in vivo antitumor activity.

The voltage-dependent action of pentobarbital on batrachotoxin-modified human brain sodium channels.

The voltage-dependent action of the intravenous anesthetic pentobarbital on human brain sodium channels activated by batrachotoxin was examined using planar lipid bilayer methods. Fractional open time-data were fitted by Boltzmann functions to yield simple parameters characterizing the voltage-dependence of the fractional open time. Pentobarbital caused a dose-dependent reduction of the maximum fractional open time of the sodium channel and a shift of the potential of half-maximal open time towards hyperpolarized potentials, whereas the slope parameter of the Boltzmann-fits was unaffected. A statistically significant increase of the variability of these parameters was found only in the case of the maximum fractional open time, indicating a random fluctuation of pentobarbital-induced suppression of the sodium channels over time. The voltage-dependent action of pentobarbital probably results from either a pentobarbital-modification of channel activation gating and/or a modification of the pentobarbital action by the gating process itself.

Purified and unpurified sodium channels from eel electroplax in planar lipid bilayers.

Highly purified sodium channel protein from the electric eel, Electrophorus electricus, was reconstituted into liposomes and incorporated into planar bilayers made from neutral phospholipids dissolved in decane. The purest sodium channel preparations consisted of only the large, 260-kD tetrodotoxin (TTX)-binding polypeptide. For all preparations, batrachotoxin (BTX) induced long-lived single-channel currents (25 pS at 500 mM NaCl) that showed voltage-dependent activation and were blocked by TTX. This block was also voltage dependent, with negative potentials increasing block. The permeability ratios were 4.7 for Na+:K+ and 1.6 for Na+:Li+. The midpoint for steady state activation occurred around -70 mV and did not shift significantly when the NaCl concentration was increased from 50 to 1,000 mM. Veratridine-induced single-channel currents were about half the size of those activated by BTX. Unpurified, nonsolubilized sodium channels from E. electricus membrane fragments were also incorporated into planar bilayers. There were no detectable differences in the characteristics of unpurified and purified sodium channels, although membrane stability was considerably higher when purified material was used. Thus, in the eel, the large, 260-kD polypeptide alone is sufficient to demonstrate single-channel activity like that observed for mammalian sodium channel preparations in which smaller subunits have been found.

Grayanotoxin-I-modified eel electroplax sodium channels. Correlation with batrachotoxin and veratridine modifications.

To probe the structure-function relationships of voltage-dependent sodium channels, we have been examining the mechanisms of channel modification by batrachotoxin (BTX), veratridine (VTD), and grayanotoxin-I (GTX), investigating the unifying mechanisms that underlie the diverse modifications of this class of neurotoxins. In this paper, highly purified sodium channel polypeptides from the electric organ of the electric eel were incorporated into planar lipid bilayers in the presence of GTX for comparison with our previous studies of BTX (Recio-Pinto, E., D. S. Duch, S. R. Levinson, and B. W. Urban. 1987. J. Gen. Physiol. 90:375-395) and VTD (Duch, D. S., E. Recio-Pinto, C. Frenkel, S. R. Levinson, and B. W. Urban. 1989. J. Gen. Physiol. 94:813-831) modifications. GTX-modified channels had a single channel conductance of 16 pS. An additional large GTX-modified open state (40-55 pS) was found which occurred in bursts correlated with channel openings and closings. Two voltage-dependent processes controlling the open time of these modified channels were characterized: (a) a concentration-dependent removal of inactivation analogous to VTD-modified channels, and (b) activation gating similar to BTX-modified channels, but occurring at more hyperpolarized potentials. The voltage dependence of removal of inactivation correlated with parallel voltage-dependent changes in the estimated K1/2 of VTD and GTX modifications. Ranking either the single channel conductances or the depolarization required for 50% activation, the same sequence is obtained: unmodified > BTX > GTX > VTD. The efficacy of the toxins as activators follows the same ranking (Catterall, W. A. 1977. J. Biol. Chem. 252:8669-8676).

Veratridine modification of the purified sodium channel alpha-polypeptide from eel electroplax.

In the interest of continuing structure-function studies, highly purified sodium channel preparations from the eel electroplax were incorporated into planar lipid bilayers in the presence of veratridine. This lipoglycoprotein originates from muscle-derived tissue and consists of a single polypeptide. In this study it is shown to have properties analogous to sodium channels from another muscle tissue (Garber, S. S., and C. Miller. 1987. Journal of General Physiology. 89:459-480), which have an additional protein subunit. However, significant qualitative and quantitative differences were noted. Comparison of veratridine-modified with batrachotoxin-modified eel sodium channels revealed common properties. Tetrodotoxin blocked the channels in a voltage-dependent manner indistinguishable from that found for batrachotoxin-modified channels. Veratridine-modified channels exhibited a range of single-channel conductance and subconductance states. The selectivity of the veratridine-modified sodium channels for sodium vs. potassium ranged from 6-8 in reversal potential measurements, while conductance ratios ranged from 12-15. This is similar to BTX-modified eel channels, though the latter show a predominant single-channel conductance twice as large. In contrast to batrachotoxin-modified channels, the fractional open times of these channels had a shallow voltage dependence which, however, was similar to that of the slow interaction between veratridine and sodium channels in voltage-clamped biological membranes. Implications for sodium channel structure are discussed.

Hormonal regulation of guanosine triphosphate cyclohydrolase activity and biopterin levels in the rat adrenal cortex.

The regulation of GTP-cyclohydrolase (GTP-CH) activity and tetrahydrobiopterin (BH4) levels in the adrenal cortex were studied in intact and hypophysectomized rats. Treatment with a single dose of reserpine (5 mg/kg) or insulin-induced hypoglycemia (4 h) elevated adrenocortical BH4 3-fold by 10 h; BH4 levels remained elevated after 24 h and returned to control levels by 48-72 h. GTP-CH was significantly increased 24 h after hypoglycemic shock, and the increased enzyme activity preceded the changes in BH4 levels after reserpine treatment. Two and a half hours of stress by immobilization also increased GTP-CH activity and BH4 levels in the adrenal cortex. The activities of sepiapterin reductase and dihydrofolate reductase, putative enzymes in the biosynthetic pathway from GTP to BH4, were not increased by reserpine. Both reserpine and insulin increased the apparent maximum velocity for GTP, with no increase in the affinity of the enzyme for its substrate, further suggesting that the experimental treatments induce the synthesis of GTP-CH. Hypophysectomy completely blocked the reserpine-dependent increase in both cortical GTP-CH activity and BH4 content. The administration of purified porcine ACTH to intact and hypophysectomized rats elevated adrenocortical GTP-CH activity and cofactor levels. Synthetic ACTH-(1-24) also enhanced the enzyme activity and BH4 levels in the adrenal cortex. Thus, pituitary control of adrenal cortical GTP-CH synthesis and biopterin levels appears to be mediated through the secretion of ACTH. The changes in enzyme activity and cofactor levels after activation of the hypothalamo-hypophyseal axis or ACTH administration suggest that BH4, a cofactor for certain monooxygenases, has some function, as yet unknown, in the adaptive changes of the adrenal cortex in response to stress.

Regulation of tetrahydrobiopterin biosynthesis in cultured adrenal cortical tumor cells by adrenocorticotropin and adenosine 3',5'-cyclic monophosphate.

Y-1 adrenal cortical tumor cells in culture, which contain substantial amounts of tetrahydrobiopterin [6R-(L-erythro-1',2'-dihydroxypropyl)5,6,7,8-tetrahydropterin] (BH4) and GTP cyclohydrolase (GTP-CH), were used to study the regulation of BH4 biosynthesis by ACTH and cAMP. ACTH produced a dose-dependent increase in steroidogenesis, BH4 levels and GTP-CH activity. Maximal stimulation of BH4 biosynthesis occurred at the same concentration of ACTH that caused maximal stimulation of steroidogenesis. ACTH-(1-24) was more potent than ACTH-(1-39). The stimulation of BH4 biosynthesis by ACTH was dependent on cell density, being greater at lower cell densities, but was independent of time in culture. The lack of stimulation by ACTH at higher cell densities was due to an increase in the specific activity of GTP-CH in the control cells as density increased. This increase may be due in part to the increased release of steroids, since exogenous steroids added to low density cultures also resulted in an increase in the specific activity of the enzyme. Addition of steroids had no effect on ACTH-dependent stimulation of BH4 biosynthesis at low cell densities. (Bu)2cAMP, 8-bromo-cAMP, and forskolin all produced time- and dose-dependent increases in BH4 levels, GTP-CH activity, and steroidogenesis. Maximum increases in GTP-CH and BH4 occurred at concentrations similar to those required for maximal stimulation of steroidogenesis. In the Kin-8 mutant of Y-1 cells, which has a type 1 cAMP-dependent protein kinase with an altered regulatory subunit, ACTH was unable to increase BH4 levels or GTP-CH activity at a concentration that produced maximal stimulation of BH4 and steroid biosynthesis in the parent Y-1 line. These studies indicate that Y-1 cells in culture are useful for studying the regulation of BH4 biosynthesis in the adrenal cortex.

Inhibition of voltage-dependent sodium channels by Ro 31-8220, a 'specific' protein kinase C inhibitor.

We find that several protein kinase C (PKC) inhibitors, previously considered to be specific, directly inhibit voltage-dependent Na(+) channels at their useful concentrations. Bisindolylmaleimide I (GF 1092037), IX (Ro 31-8220) and V (an inactive analogue), but not H7 (a non-selective isoquinolinesulfonamide protein kinase inhibitor), inhibited Na(+) channels assessed by several independent criteria: Na(+) channel-dependent glutamate release and [(3)H]batrachotoxinin-A 20-alpha-benzoate binding in rat cortical synaptosomes, veratridine-stimulated 22Na(+) influx in CHO cells expressing rat CNaIIa Na(+) channels and Na(+) currents measured in isolated rat dorsal root ganglion neurons by whole cell patch-clamp recording. These findings limit the usefulness of the bisindolylmaleimide class PKC inhibitors in excitable cells.

Potentiation of radiation therapy by vinorelbine (Navelbine) in non-small cell lung cancer.

Vinorelbine (Navelbine; Burroughs Wellcome Co, Research Triangle Park, NC; Pierre Fabre Medicament, Paris, France), a semisynthetic vinca alkaloid that is a potent inhibitor of mitotic microtubule polymerization, was recently approved for the treatment of non-small cell lung cancer. Radiotherapy also has been widely used to treat this malignancy. Since other antitumor agents that act on microtubules, such as paclitaxel and estramustine, have been shown to act as radiosensitizers, we studied the ability of vinorelbine to potentiate radiation. The in vitro activity of this combination was evaluated in the human lung carcinoma cell lines NCI-H460 and A549. when NCI-H460 cells were exposed to vinorelbine for 24 hours and then irradiated (1 to 6 Gy) the drug potentiated radiation in a dose-dependent manner, with the ratio of fractional survival (radiation) to fractional survival (drug plus radiation) ranging from 1.7:1 at 1 Gy to 5.5:1 at 6 Gy. When the treatment sequence was reversed (ie, radiation was followed by drug exposure), similar survival ratios were obtained at concentrations of vinorelbine that were five to 10 times lower. In this cell line radiation produced a block in the G2/M phase of the cell cycle, with the maximum block (60% to 70%) occurring 10 hours after treatment. The greatest potentiation was seen when irradiated cells were exposed to vinorelbine after they had plateaued in the G2/M phase of the cycle. Vinorelbine given early after irradiation, when only 10% to 30% of the cells were in G2/M, produced survival ratios similar to those of controls treated with radiation alone. In A549 cells radiation induced a G1 block. In this case, vinorelbine was unable to potentiate the effects of radiation. These studies show that vinorelbine can potentiate the antitumor effects of radiation and that the potentiation is cell cycle-dependent, with the maximal effect being obtained when the cells are in the G2 phase.

Benzo[f]quinazoline inhibitors of thymidylate synthase: methyleneamino-linked aroylglutamate derivatives.

Syntheses of several new inhibitors of thymidylate synthase (TS) structurally related to folic acid are described in which the pterin portion of the folate molecule is replaced by a benzo[f]quinazoline moiety, but which retain the natural methyleneamino link to the benzoylglutamate side chain. The effect on enzyme activity and cytotoxicity of various changes in the structure of the (p-aminobenzoyl)glutamate side chain are reported. Replacement of the benzamide portion of the (p-aminobenzoyl)glutamate moiety with 2-fluorobenzamido, 2-isoindolinyl, 1,2-benzisothiazol-2-yl, and 2-thenamido moieties varied in effect from a 9-fold diminution of TS activity to a 5-fold enhancement, while cytotoxic potency on SW-480 and MCF-7 tumor lines showed increases ranging from 3.6- to 450-fold. The detrimental effect on enzyme activity and cytotoxicity of alkyl substitution on the PABA nitrogen is confirmed for these compounds, in contrast with several series of previously reported quinazoline antifolates (2). Substitution of a C3-methyl substituent for 3-amino had little effect on TS activity but was beneficial in terms of solubility and cytotoxicity. The excellent combination of TS inhibitory activity, FPGS substrate activity, and affinity for the reduced folate transport system in the most potent of these derivatives, 3e, resulted in IC50 values of 0.2-0.8 nM against these tumor lines.

Thienyl and thiazolyl acyclic analogues of 5-deazatetrahydrofolic acid.

Analogues of N-[4-[[3-(2,4-diamino-1,6-dihydro-6-oxo-5-pyrimidinyl)propyl]amino] benzoyl]-L-glutamic acid (5-DACTHF), in which the phenylene group is replaced by either a thienoyl or a thiazolyl group were synthesized. These compounds were prepared by reductive amination of suitably protected pyrimidinylpropionaldehyde with the aminoaroyl glutamates. These glutamates were in turn synthesized from the corresponding nitroaroyl carboxylic acids by condensation with protected glutamic acid followed by catalytic reduction. The compounds were tested as inhibitors of methotrexate uptake as a measure of binding to the reduced folate transport system, as inhibitors of glycinamide ribonucleotide transformylase, as substrates for folylpolyglutamate synthetase, and as inhibitors of tumor cell growth in cell culture. The thiophene analogue was found to be equal in activity to 5-DACTHF in the MCF-7 cell growth inhibition assay while the thiazole analogue was 9-fold more active. Indeed this thiazole was over 4 times more active in the MCF-7 cell line than the clinically investigated compound 5,10-dideaza-5,6,7,8-tetrahydrofolic acid (DDATHF).

Synthesis and antitumor activity of 2,4-diamino-6-(2,5-dimethoxybenzyl)-5-methylpyrido[2,3-d]pyrimidine.

The synthesis of 2,4-diamino-6-(2,5-dimethoxybenzyl)-5-methylpyrido[2,3-d]pyrimidine (BW301U, 7) by a route that has general applicability to the preparation of many 6-(substituted benzyl)-5-methylpyrido[2,3-d]pyrimidines is described. The key intermediate, 2,4-diamino-7,8-dihydro-6-(2,5-dimethoxybenzyl)-5-methyl-7-oxopyrido[2,3-d]pyrimidine (4), is converted to the 7-chloro compound 5 by treatment with a 1:1 complex of N,N-dimethylformamide--thionyl chloride, and 5 is hydrogenolyzed with palladium on charcoal in the presence of potassium hydroxide to yield 7. BW301U is a potent lipid-soluble inhibitor of mammalian dihydrofolate reductase and has significant activity against the Walker 256 carcinosarcoma in rats.

Folate analogues. 31. Synthesis of the reduced derivatives of 11-deazahomofolic acid, 10-methyl-11-deazahomofolic acid, and their evaluation as inhibitors of glycinamide ribonucleotide formyltransferase.

The Boon-Leigh procedure, involving condensation of a 6-chloro-5-nitropyrimidine (22) with an alpha-amino ketone (20 or 21) followed by reduction of the nitro group, cyclization, and L-glutamylation, led to the formation of 11-deazahomofolate (29) and its 10-methyl derivative (30). The corresponding (6R,S)-5,6,7,8-tetrahydro (4, 5) and 7,8-dihydro (31, 32) derivatives were prepared by catalytic hydrogenation. (6S)-11-Deazatetrahydrohomofolate was prepared from 29 by enzymatic reduction. Compounds 29 and 30 had little effect (IC50 greater than 2 x 10(-5) M) on Lactobacillus casei glycinamide ribonucleotide (GAR) formyltransferase but (6R,S)-11-deazatetrahydrohomofolate (4) is a potent inhibitor of this enzyme (IC50 = 5 x 10(-8) M). It is at least 100 times more inhibitory than 33, the 6S compound, indicating that the 6R component of the mixture having the unnatural configuration at C6 (34) is responsible for the potent inhibition. Compound 4 is a much weaker inhibitor of murine (L1210) and human (MOLT-4) leukemia cell GAR formyltransferases (IC50 greater than 1 x 10(-5) M). (6R,S)-11-Deaza-10-methyltetrahydrohomofolate (5) (IC50 = 1.1 x 10(-5) is 200 times weaker than 4 against L. casei GAR formyltransferase. However, 11-deaza-10-methyldihydrohomofolate (32) is more inhibitory (IC50 = 5.5 x 10(-7) M) than 5 or 30. None of the compounds showed inhibition of L. casei aminoimidazolecarboxamide ribonucleotide (AICAR) formyltransferase, dihydrofolate reductase, or thymidylate synthase. The dihydro derivatives 31 and 32 are 5% as active as dihydrofolate as substrates for L. casei dihydrofolate reductase. Compound 4 showed moderate inhibition of the growth of L. casei, Streptococcus faecium, MOLT-4 cells, and MCF-7 human breast adenocarcinoma cells.

Synthesis and biological activity of open-chain analogues of 5,6,7,8-tetrahydrofolic acid--potential antitumor agents.

This study describes the synthesis and in vitro antitumor activity of inhibitors of purine de novo biosynthesis that are analogues of N-[4-[[3-(2,4-diamino-1,6-dihydro-6-oxo-5-pyrimidinyl) propyl]amino]benzoyl-L-glutamic acid (5-DACTHF). Benzene ring substituted analogues were synthesized from a protected pyrimidinyl propionaldehyde and a substituted benzoyl glutamate moiety by a key reductive amination step. Pyrimidine and linking chain substituted analogues were built up stepwise from p-aminobenzoic acid or analogues. The compounds were tested as inhibitors of methotrexate uptake as a measure of binding to the reduced folate transport system, as inhibitors of glycinamide ribonucleotide transformylase, as substrates for folylpolyglutamate synthetase, and as inhibitors of tumor cell growth in cell culture. With the exception of 2'-F substituent, the ring-substituted analogues are less active than the parent compound. Replacement of the 10-nitrogen by carbon, sulfur, or oxygen produced less than 2-fold changes to biological activity in vitro. A four-atom linking chain and an amino group at the 2-position on the pyrimidine ring are important for good activity.

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.

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.