Correction: Duncan, K.R., Suzuki, Y.J. Vitamin E Nicotinate. Antioxidants 2017, 6, 20.

In the original version of our article [1], three lines were omitted from the α-tocopherol and α-tocopheryl nicotinate structures in Figure 1 during the manuscript processing[...].

Subdiaphragmatic Vagotomy With Pyloroplasty Ameliorates the Obesity Caused by Genetic Deletion of the Melanocortin 4 Receptor in the Mouse.

Background/Objectives: We tested the hypothesis that abolishing vagal nerve activity will reverse the obesity phenotype of melanocortin 4 receptor knockout mice (Mc4r-/-). Subjects/Methods: In two separate studies, we examined the efficacy of bilateral subdiaphragmatic vagotomy (SDV) with pyloroplasty in the prevention and treatment of obesity in Mc4r-/- mice. Results: In the first study, SDV prevented >20% increase in body weight (BW) associated with this genotype. This was correlated with a transient reduction in overall food intake (FI) in the preventative arm of the study. Initially, SDV mice had reduced weekly FI; however, FI normalized to that of controls and baseline FI within the 8-week study period. In the second study, the severe obesity that is characteristic of the adult Mc4r-/- genotype was significantly improved by SDV with a magnitude of 30% loss in excess BW over a 4-week period. Consistent with the first preventative study, within the treatment arm, SDV mice also demonstrated a transient reduction in FI relative to control and baseline levels that normalized over subsequent weeks. In addition to the accompanying loss in weight, mice subjected to SDV showed a decrease in respiratory exchange ratio (RER), and an increase in locomotor activity (LA). Analysis of the white fat-pad deposits of these mice showed that they were significantly less than the control groups. Conclusions: Altogether, our data demonstrates that SDV both prevents gain in BW and causes weight loss in severely obese Mc4r-/- mice. Moreover, it suggests that an important aspect of weight reduction for this type of monogenic obesity involves loss of signaling in vagal motor neurons.

Vitamin E Nicotinate.

Vitamin E refers to a family of compounds that function as lipid-soluble antioxidants capable of preventing lipid peroxidation. Naturally occurring forms of vitamin E include tocopherols and tocotrienols. Vitamin E in dietary supplements and fortified foods is often an esterified form of α-tocopherol, the most common esters being acetate and succinate. The vitamin E esters are hydrolyzed and converted into free α-tocopherol prior to absorption in the intestinal tract. Because its functions are relevant to many chronic diseases, vitamin E has been extensively studied in respect to a variety of diseases as well as cosmetic applications. The forms of vitamin E most studied are natural α-tocopherol and the esters α-tocopheryl acetate and α-tocopheryl succinate. A small number of studies include or focus on another ester form, α-tocopheryl nicotinate, an ester of vitamin E and niacin. Some of these studies raise the possibility of differences in metabolism and in efficacy between vitamin E nicotinate and other forms of vitamin E. Recently, through metabolomics studies, we identified that α-tocopheryl nicotinate occurs endogenously in the heart and that its level is dramatically decreased in heart failure, indicating the possible biological importance of this vitamin E ester. Since knowledge about vitamin E nicotinate is not readily available in the literature, the purpose of this review is to summarize and evaluate published reports, specifically with respect to α-tocopheryl nicotinate with an emphasis on the differences from natural α-tocopherol or α-tocopheryl acetate.

Design and synthesis of Cyclopenta[g]quinazoline-based antifolates as inhibitors of thymidylate synthase and potential antitumor agents(,).

Following the development of raltitrexed, the synthesis of nonpolyglutamatable inhibitors of TS that do not use the reduced folate carrier (RFC) for cellular entry should provide compounds which overcome mechanisms of resistance to folate-based inhibitors of TS that are associated with decreased/altered folylpolyglutamate synthetase (FPGS) expression and/or an impaired RFC. Examination of a computer graphics model of the humanized Escherichia coli TS enzyme with quinazoline inhibitors of TS, such as 1 bound in the active site of the enzyme, suggested that conformational restriction introduced by bridging the C9 with C7 to form a pentacycle may be beneficial for binding to TS. That led to the synthesis of a series of potent cyclopenta[g]quinazoline-based inhibitors of the enzyme in which the glutamyl residue associated with classical antifolates was replaced with a variety of glutamate-derived ligands; the most potent inhibitor being the L-Glu-gamma-D-GluT(alpha) derivative 7j. In the mouse L1210:1565 cell line (mutant RFC), the majority of these compounds had activity equal or only slightly greater compared with the parental L1210 cell line, indicating a reduced dependence on the RFC for cellular uptake in the L1210 cell line.

Design and synthesis of potent non-polyglutamatable quinazoline antifolate thymidylate synthase inhibitors.

The synthesis is described of a series of analogues of the potent thymidylate synthase (TS) inhibitor, N-[4-[N-[(3,4-dihydro-2, 7-dimethyl-4-oxo-6-quinazolinyl)methyl]-N-prop-2-ynylamino]-2-f luorob enzoyl]-L-glutamic acid (4, ZM214888), in which the glutamic acid moiety is replaced by homologous amino acids and alpha-amino acids where the omega-carboxylate is replaced by acylsulfonamides and acidic heterocycles. In general these modifications when compared to 4 gave compounds with increased potency as inhibitors of isolated TS and as cytotoxic agents against murine tumor cell lines. The new compounds require transport by the reduced folate carrier for entry into cells but are not converted intracellularly into polyglutamated species. Agents with this profile are expected to show activity against tumors that are resistant to classical antifolates due to low expression of folylpolyglutamate synthetase. The analogue (S)-2-[4-[N-[(3,4-dihydro-2, 7-dimethyl-4-oxo-6-quinazolinyl)methyl]-N-prop-2-ynylamino]-2-f luorob enzamido]-4-(1H-1,2,3,4-tetrazol-5-yl)butyric acid (35, ZD9331) has been selected as a clinical development candidate and is currently undergoing phase I studies.

A novel class of lipophilic quinazoline-based folic acid analogues: cytotoxic agents with a folate-independent locus.

Three lipophilic quinazoline-based aminomethyl pyridine compounds, which differ only in the position of the nitrogen in their pyridine ring, are described. CB300179 (2-pyridine), CB300189 (4-pyridine) and CB30865 (3-pyridine) all inhibited isolated mammalian TS with IC50 values of 508, 250 and 156 nM respectively. CB30865 was the most potent growth inhibitory agent (IC50 values in the range 1-100 nM for several mouse and human cell types). CB300179 and CB300189 were active in the micromolar range. Against W1L2 cells, CB300179 and CB300189 demonstrated reduced potency in the presence of exogenous thymidine (dThd), and against a W1L2:C1 TS overproducing cell line. In contrast, CB30865 retained activity in these systems. Furthermore, combinations of precursors and end products of folate metabolism, e.g. dThd/hypoxanthine (HX) or leucovorin (LV), did not prevent activity. CB30865 did not interfere with the incorporation of tritiated dThd, uridine or leucine after 4 h. A cell line was raised with acquired resistance to CB30865 (W1L2:R865; > 200-fold), which was not cross-resistant to CB300179 or CB300189. In addition, W1L2:R865 cells were as sensitive as parental cells to agents from all the major chemotherapeutic drug classes. CB300179 and CB300189 induced an S phase accumulation (preventable by co-administration of dThd). No cell cycle redistribution was observed following exposure (4-48 h) to an equitoxic concentration of CB30865. In the NCI anticancer drug-discovery screen, CB30865 displayed a pattern of activity which was not consistent with known anti-tumour agents. These data suggest that CB30865 represents a class of potent potential anti-tumour agents with a novel mechanism of action.

Combination of raltitrexed with other cytotoxic agents: rationale and preclinical observations.

Agents for use in combination therapy should be effective as monotherapy in the tumour type of interest, have different mechanisms of action or pharmacology, and preferably non-overlapping toxicity profiles. Raltitrexed is effective as monotherapy in a number of tumour types, but it is hoped that combining it with other cytotoxic agents will lead to enhanced efficacy. Raltitrexed and 5-fluorouracil (5-FU) are specific and non-specific inhibitors, respectively, of thymidylate synthase, a critical enzyme in the de novo synthesis of DNA. Preclinical studies have indicated that raltitrexed and 5-FU have an incompletely overlapping spectrum of antitumour activity and may have additive or synergistic effects on colon carcinoma cells. These interactions are schedule-dependent (raltitrexed should precede 5-FU). Pre-treatment of colon carcinoma cells with raltitrexed has also been shown to increase intracellular levels of phosphoribosyl pyrophosphate resulting in increased incorporation of 5-FU nucleotides into RNA. Raltitrexed has a different mechanism of action from two other new agents active in colorectal cancer, irinotecan and oxaliplatin, and tumours are therefore not necessarily cross-resistant. Short pre-exposure of colon carcinoma cells to the irinotecan active metabolite, 7-ethyl-10-hydroxy-camptothecin (SN-38), prior to exposure to raltitrexed has consistently resulted in synergistic cell kill, whereas the reverse sequence is antagonistic. Preliminary results indicate that equitoxic doses of raltitrexed and cisplatin, or oxaliplatin, are antagonistic in two colon carcinoma cell lines. However, because there are major difficulties in translating preclinical drug combination results to the clinical settings, these results should be interpreted with caution.

Cellular pharmacology and in vivo activity of a new anticancer agent, ZD9331: a water-soluble, nonpolyglutamatable, quinazoline-based inhibitor of thymidylate synthase.

ZD9331 is a drug that was developed from a potent class of water-soluble, C7-methyl-substituted, quinazoline-based inhibitors of thymidylate synthase (TS) that are transported into cells via a saturable, carrier-mediated system (reduced folate carrier, or RFC) but are not substrates for folylpolyglutamate synthetase. ZD9331 is the gamma-tetrazole analogue of 2-desamino-2, 7-dimethyl-N10-propargyl-2'fluoro-5,8-dideaza folate (ZM214888), with a TS Ki of approximately 0.4 nM. ZD9331 exhibits potent growth inhibitory and cytotoxic activity; e.g., IC50 for the inhibition of human W1L2 lymphoblastoid cell line was 7 nM. The addition of thymidine to the culture medium increased the IC50 in W1L2 cells >10, 000-fold, demonstrating the high specificity of the drug for TS. ZD9331 is transported into cells predominantly via the RFC. Accordingly, it competes with methotrexate (MTX) and folinic acid for cellular uptake and has reduced activity against two cell lines with low expression of the RFC (L1210:1565 and CEM/MTX). In addition, a cell line with acquired resistance to ZD9331 displays reduced uptake of both ZD9331 and MTX. A mouse cell line (L1210:RD1694), with acquired resistance to ZD1694 due to reduced folylpolyglutamate synthetase activity, was not significantly cross-resistant to ZD9331. The flux through TS, as measured by 3H release from 5-[3H]deoxyuridine, was rapidly inhibited when cells were incubated with ZD9331. However, because ZD9331 cannot form polyglutamates, TS activity recovered rapidly once cells were placed in drug-free medium. The minimum curative dose of ZD9331 in the i.m. L5178Y TK-/- tumor model was approximately 3 mg/kg when given by 24-h continuous infusion, and it was 25-50 mg/kg when given by a single i.p. or i.v. injection. ZD9331 had antitumor activity against the L5178Y TK+/- tumor when administered by 7-day continuous infusion; growth delays of more than 5 days (and some cures) were seen at doses of 25-50 mg/kg/day. At higher doses, significant weight loss (gastrointestinal toxicity) and myelosuppression (neutropenia and thrombocytopenia) were observed, suggesting that these may be dose-limiting toxicities in the Phase I clinical studies.

Folate-based inhibitors of thymidylate synthase: synthesis and antitumor activity of gamma-linked sterically hindered dipeptide analogues of 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI 198583).

In an effort to synthesize inhibitors of thymidylate synthase (TS) that do not undergo polyglutamation, a series of gamma-linked sterically hindered dipeptide analogues of 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI 198583) was prepared. A methyl, ethyl, or propargyl group was incorporated into the gamma-glutamyl amide bond of gamma-linked L,L dipeptide derivatives of ICI 198583, such as ICI 198583-gamma-L-Glu. In addition, steric bulk was introduced on either side of the gamma-glutamyl bond of ICI 198583-gamma-L-Glu or ICI 198583-gamma-L-Ala. The resulting dipeptide analogues, e.g., ICI 198583-gamma-MeGlu and ICI 198583-gamma-Aib, were apparently stable to in vivo hydrolysis but poorer inhibitors of TS and L1210 cell growth. However, introduction of 7-Me, 2'-F substitution into the quinazoline nucleus gave significant improvement in the inhibitory activity against thymidylate synthase. Compounds 28-30, the 7-Me, 2'-F derivatives of ICI 198583-gamma-MeGlu, ICI 198583-gamma-EtGlu, and ICI 198583-gamma-PgGlu, respectively, were potent inhibitors of TS (K(iapp) = 0.21-1.1 nM) and L1210 cell growth (IC50 = 0.05-0.34 microM) and were similar to that seen with the most potent gamma-linked L,D dipeptide derivatives of ICI 198583 previously synthesized. Furthermore, the low cross-resistance ratios for the L1210:R(D1694)/L1210 cell line indicated that 28-30 do not undergo polyglutamation.

Biological activity and intracellular metabolism of ZD1694 in human leukemia cell lines with different resistance mechanisms to antifolate drugs.

The biological activity and cellular metabolism of ZD1694, a novel folate-based thymidylate synthase (TS) inhibitor, were analyzed in a human leukemia cell line, MOLT-3, and its antifolate-resistant sublines with different mechanisms of resistance to methotrexate (MTX), trimetrexate (TMQ) and N10-propargyl-5,8-dideazafolic acid (CB3717). MOLT-3/CB3717(40), which was selected for CB3717 resistance, demonstrated impaired membrane drug transport via reduced folate carrier (RFC) and lower accumulation of [3H]ZD1694-polyglutamates in the cells with a shift in the polyglutamate distribution profile to shorter chain length polyglutamates, indicating an alteration in polyglutamation capacity in this subline. Impaired RFC and reduced rate of polyglutamation could explain the cross-resistance (12-fold) of this subline to ZD1694. On the other hand, there was little or no cross-resistance to this drug in a subline (MOLT-3/TMQ800) reportedly resistant to TMQ through impaired membrane transport for TMQ and an increase in dihydrofolate reductase (DHFR) activity. Total amount of ZD1694 polyglutamated to a level higher than diglutamate was approximately 1.7-fold higher in the TMQ-resistant cells than that in the parent cells, but a low degree of increase in TS activity in the cells counteracted the supposed increase in sensitivity to ZD1694. MOLT-3/TMQ800-MTX10000 cells, which were established by sequential exposure of the TMQ-resistant cells to MTX and were previously shown to amplify mutated DHFR with low affinity for MTX, showed a decreased accumulation of polyglutamated ZD1694 as compared with the parent line and this was consistent with cross-resistance to ZD1694 in this subline. Overproduction of variant DHFR scarcely influenced the sensitivity to this drug. These results indicate that ZD1694 could overcome antifolate resistance through a mechanism such as amplified DHFR activity, and the biological activity of this drug against the cells paralleled the amount of polyglutamated drug inside the cells. Determination of polyglutamation capacity in tumor cells may allow prediction of sensitivity to this drug.

The influence of drug-exposure conditions on the development of resistance to methotrexate or ZD1694 in cultured human leukaemia cells.

The influence of drug exposure conditions on the development of resistance to methotrexate (MTX) or ZD1694 was studied by treating MOLT-3 human lymphoblastic-leukaemia cells in a continuous or a pulsatile (high-dose, short term) drug-exposure schedule. Continuous exposure of the cells to MTX with stepwise escalation of the drug concentrations resulted in a MTX-resistant sub-line (MOLT-3/MTX(10000)) with impaired reduced-folate carrier (RFC) and increased dihydro-folate-reductase (DHFR) activity. Conversely, a MTX-resistant clone (MOLT-3/MTX. P-9) with unaltered RFC and DHFR activity, but with decreased cellular accumulation of anti-folates, was selected by high-dose short-term treatment of the cells with MTX. MTX resistance in the latter cells was pronounced after short-term rather than continuous-exposure incubation with MTX, suggesting defective polyglutamation of the drug. On the other hand, 2 ZD1694-resistant sub-lines which were established by continuous (MOLT-3/ZD1694. C) or by pulsatile drug-exposure schedule (MOLT-3/ZD1694.P-9) demonstrated extremely low accumulation and poor retention of [3H]ZD1694, with no change in initial drug uptake and little or no increase of thymidylate-synthase (TS) activity irrespective of drug exposure conditions for their establishment. HPLC analysis displayed a virtual absence of ZD1694 polyglutamates in both ZD1694-resistant sub-lines and low accumulation in MOLT-3/MTX.p-9 as compared to the parent line. However, folylpolyglutamate-synthetase(FPGS) mRNA was only moderately decreased in the 2 ZD1694-resistant sub-lines and to an even lesser extent in MOLT-3/MTX.p-9. In addition, gamma-glutamyl-hydrolase(GGH) activity was not increased, but was slightly down-regulated in the polyglutamation-defective sub-lines. These results indicate that the mechanism(s) of the resistance developed may depend not only on drug-exposure conditions while raising resistance but also on the biochemical properties of the drug.

Quinazoline antifolates thymidylate synthase inhibitors: lipophilic analogues with modification to the C2-methyl substituent.

Modification of the potent thymidylate synthase (TS) inhibitor 1-[[N-[4-[N-[(3,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl]-N- prop-2-ynylamino]benzoyl]amino]methyl]-3-nitrobenzene (4a) has led to the synthesis of quinazolinone antifolates bearing functionalized alkyl substituents at C2. A general synthetic route was developed which involved coupling the appropriate 1-[[N-[4-(alkylamino)benzoyl)amino]methyl]-3-nitrobenzene 20-22 with a 6-(bromomethyl)-2-(acetoxymethyl)-3,4-dihydro-4-oxoquinazoline 9 or 10. Replacement of the 2-acetoxy group by a chlorine atom followed by the displacement of the halogen of 25a-c by various nucleophiles led to compounds 26-40. Good TS (IC50 < 1 microM) and growth inhibition (IC50 0.1-1 microM) were found with most of these new antifolates. TS inhibitors in this series do not apparently require the reduced folate carrier (RFC) for cell entry (they most likely penetrate the cell membrane by passive diffusion) and are not polyglutamated. N, O, S, Cl, and CN as well as large amino and mercapto substituents were tolerated by the enzyme. The simultaneous incorporation of 7-methyl and 2'-F substituents gave a series of highly potent agents inhibiting cell growth at concentrations < 1 microM (24, 27bc; 30-32b, 35b). The incorporation of suitable C2 substituents has overcome the decrease in aqueous solubility observed with lipophilic quinazoline antifolates. This is best illustrated by compound 31a, where up to a 54-fold increase in solubility has been achieved by the incorporation of an N-methylpiperazine nucleus into the C2-methyl group of 4a.

Quinazoline antifolate thymidylate synthase inhibitors: gamma-linked L-D, D-D, and D-L dipeptide analogues of 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI 198583).

The syntheses of gamma-linked L-D, D-D, and D-L dipeptide analogues of 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI 198583) are described. The general methodology for the synthesis of these molecules involved the preparation of the dipeptide derivatives employing solution phase peptide synthesis followed by condensation of the dipeptide free bases with the appropriate pteroic acid analogue via diethyl cyanophosphoridate (DEPC) activation. In the final step, tert-butyl esters were removed by trifluoroacetic acid (TFA) hydrolysis. Z-L-Glu-OBut-gamma-D-Ala-OBut, for example, was prepared from alpha-tert-butyl N-(benzyloxycarbonyl)-L-glutamate and tert-butyl D-alaninate via isobutyl-mixed anhydride coupling. The Z-group was removed by catalytic hydrogenolysis and the resulting dipeptide free base condensed with 2-desamino-2-methyl-N10-propargyl-5,8-dideazapteroic acid via DEPC coupling. Finally, tert-butyl esters were removed by TFA hydrolysis to give ICI 198583-gamma-D-Ala. The compounds were tested as inhibitors of thymidylate synthase and L1210 cell growth. Good enzyme and growth inhibitory activity were found with gamma-linked L-D dipeptides, the best examples being the Glu-gamma-D-Glu derivative 35 (Ki = 0.19 nM, L1210 IC50 = 0.20 +/- 0.017 microM) and the Glu-gamma-D-alpha-aminoadipate derivative 39 (Ki = 0.12 nM, L1210 IC50 = 0.13 +/- 0.063 microM). In addition, ICI 198583 L-gamma-D-linked dipeptides were resistant to enzymatic degradation in mice.

Cellular pharmacokinetics of ZD1694 in cultured human leukaemia cells sensitive, or made resistant, to this drug.

We have analysed the cellular metabolism of a novel thymidylate synthase (TS) inhibitor, ZD1694, in MOLT-3 and K562 human leukaemia cell lines sensitive to or made resistant to ZD1694 by continuous exposure of the cells to ZD1694 with stepwise escalation of the drug concentration. The initial cellular uptake of [3H]ZD1694 was greater in K562 cells than in MOLT-3 cells and the drug accumulated approximately 3-fold more in the former cells following incubation with 0.1 microM ZD1694 at 37 degrees C for 24 h. TS and dihydrofolate reductase activities were not significantly different between the two cell lines. After a 30-min incubation with the drug at 37 degrees C, 85% of the total drug (2.3 pmol/mg protein) in K562 cells was found as tri- to pentaglutamates, whereas MOLT-3 cells accumulated less drug in this time (0.83 pmol/mg protein) and polyglutamates of chain length greater than triglutamate were not found to a significant extent. When the incubation time was extended to 24 h, the polyglutamate profile in K562 cells was progressively shifted towards those of long glutamate chain length and 59% of the total cellular drug (204 pmol/mg protein) was identified as the penta form. In contrast, even distribution between tri- and pentaglutamate was observed in MOLT-3 cells. Total cellular polyglutamates were approximately 3-fold higher in K562 cells than in MOLT-3 cells, and this may explain the 2.5-fold difference in the sensitivity to ZD1694 between the two cell lines. Continuous exposure of MOLT-3 and K562 cells to ZD1694 up to 1 microM or 0.1 microM resulted in 1600- and 4200-fold resistant sublines, respectively (MOLT-3/ZD1694.C and K562/ZD1694.C). The resistant MOLT-3 cells showed a markedly lower cellular accumulation and poor retention of [3H]ZD1694 with no significant change of initial drug uptake by 10 min and with a little increase of TS activity. HPLC analysis demonstrated that more than 90% of the 3H co-eluted with the monoglutamate (parent drug) in the resistant MOLT-3 cells, indicating extremely diminished polyglutamation in the cells. On the other hand, cellular uptake of [3H]ZD1694 was extensively impaired in K562/ZD1694.C cells and cellular accumulation of the drug was only 2.5% of that in the parent cells following 24 h incubation with the drug. Neither an increase of TS or dihydrofolate reductase activity nor a change in the polyglutamate formation profile was observed in the resistant K562 cells. These results indicate that the cellular ability to produce the polyglutamate metabolites of ZD1694 must influence the sensitivity of the tumour cells to this drug, and development of mechanisms involved in the ZD1694 resistance may relate to the intrinsic biochemical properties of the cells.

Quinazoline thymidylate synthase inhibitors: methods for assessing the contribution of polyglutamation to their in vitro activity.

Many quinazoline thymidylate synthase (TS) inhibitors undergo intracellular metabolism to polyglutamate forms which can significantly alter their activity and pharmacodynamics through improved TS inhibition and drug retention. When a series of quinazolines was tested for inhibitory activity towards TS (IC50 0.001-2 microM) and the growth of L1210 cells (IC50 0.005-10 microM), no direct correlation was observed. However, a very good correlation was apparent if a L1210 variant cell line (L1210: RD1694) was used. This line is deficient in its ability to form antifolate polyglutamates. A number of other intact cell methods have also been developed which estimate the contribution that intracellular polyglutamation makes to a compound's activity. These assays were validated using a series of quinazoline-based TS inhibitors with well-defined activity for TS, folypolyglutamate synthetase (FPGS) and the reduced-folate cell membrane carrier (RFC). Short-exposure growth-inhibition assays or the measurement of TS activity in situ after various incubation times, followed by different lengths of time in drug-free medium, can indicate both the speed and extent of appearance of retentive forms (usually polyglutamates). Continuous-exposure growth-inhibition assays, in the presence of leucovorin (LV), are also useful, since only the growth-inhibitory potency of polyglutamated analogues is significantly decreased by LV. Highly polyglutamated compounds, e.g. ZD1694, are virtually inactive in the presence of a high concentration of LV. It is proposed that these methods, when considered together, provide a greater degree of information concerning the rate and extent of polyglutamation of a particular compound than isolated FPGS assays alone.

Quinazoline-based thymidylate synthase inhibitors: relationship between structural modifications and polyglutamation.

Quinazoline-based analogues of folic acid are a group of thymidylate synthase (TS) inhibitors that display a wide spectrum of activity for cultured tumour cells, partly due to their differential ability to form polyglutamate metabolites that are (i) more potent TS inhibitors and (ii) not readily effluxed from cells. The rate of cell membrane transport and folylpolyglutamate synthetase substrate activity influence compound polyglutamation. A series of intact-cell assays has been used to determine how specific modifications of 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolate (ICI 198583) affect compound polyglutamation. Those containing the 'classical' glutamate structure were usually, but not always, well polyglutamated intracellularly. Replacement of N10 propargyl with smaller aliphatic substituents, particularly when combined with replacement of the benzene ring with thiophene or thiazole heterocycles, was beneficial for antitumour activity through polyglutamate formation. Fluorination of the benzene, particularly if a F was adjacent to the 'bridge region' (3'F or 2',5'diF), also gave compounds with a high dependence on polyglutamation for activity. Those analogues with 2-CH2OH or NH2 substituents were poor substrates for the reduced-folate cell membrane carrier which can account for their reduced polyglutamation rate and hence growth-inhibitory activity. A large decrease or prevention of polyglutamation was achieved by the introduction of CH3, CH2CH3, Br or C1 on C7. The concomitant enhancement in TS inhibition by these modifications gave compounds active under continuous-exposure cell culture conditions. Some ICI 198583 analogues had the glutamate moiety replaced with unnatural amino acids or dipeptides. Only the L-gamma-L-glu analogue (a polyglutamate metabolite of ICI 198583) gave activity entirely attributable to polyglutamate formation.

ZD1694 (Tomudex): a new thymidylate synthase inhibitor with activity in colorectal cancer.

ZD1694 (Tomudex) is a new antifolate which is a specific inhibitor of thymidylate synthase (TS). Evidence suggests that ZD1694 has a spectrum of activity that only partially overlaps with 5-fluorouracil (modulated with leucovorin) against colon tumours in vitro. Potent cytotoxic activity is dependent upon active uptake into cells via the reduced folate/methotrexate cell membrane carrier (RFC) and subsequent metabolism to polyglutamated forms (tri, tetra and pentaglutamates). These polyglutamates are approximately 60-fold more active as TS inhibitors and are not effluxed readily from cells. Extensive polyglutamation also occurs in various mouse tissues (e.g. small intestinal epithelium, liver and kidney), resulting in high tissue/plasma drug ratios which persist for a prolonged period. ZD1694 has antitumour activity in mice, although the high plasma thymidine in this species complicates: (1) the interpretation of therapeutic index; (2) tumour types in which activity is likely to be observed; and (3) translation of doses and schedules for clinical evaluation. ZD1694 entered clinical study and has completed Phase I and II evaluation, with activity observed in several tumour types. Appreciable activity in the Phase II colorectal study (29% objective response rate on interim analysis) led to the current Phase III study, randomised against 5-fluorouracil/leucovorin.

Relationships between resistance to cisplatin and antifolates in sensitive and resistant tumour cell lines.

Possible relationships between tumour resistance to cisplatin and the folate-based thymidylate synthase (TS) inhibitors, CB3717 and ZD1694 (tomudex), have been investigated in vitro using a panel of tumour cell lines (predominantly human ovarian), either parental or possessing acquired resistance to cisplatin or ZD1694. Across eight parent human tumour cell lines, ZD1694 was the most potent drug (mean IC50 of 1.9 x 10(-8) M), being over 250 times as potent as its prototype CB3717 (mean IC50 of 4.8 x 10(-6) M). In five pairs of acquired cisplatin-resistant human tumour cell lines (three ovarian, one cervical and one testicular) which encompass all of the main known mechanisms of platinum drug resistance, ZD1694, CB3717 and the DHFR inhibitor, methotrexate, all exhibited non-cross-resistance. The cervical line, HX/155cisR, showed collateral sensitivity to ZD1694, CB3717, 5-fluorouracil (FUra) and fluorodeoxyuridine (FdUrd). One cell line, A2780cisR, showed a low level of cross-resistance to FUra (resistance factor, RF, of 1.5) and FdUrd (RF of 3.8). A2780cisR, in common with two other cisplatin-resistant lines, did not possess elevated TS activity compared with its parent. Cisplatin retained activity in four acquired ZD1694-resistant cell lines (encompassing reduced folate transport, elevated TS and defective polyglutamation mechanisms of resistance). Furthermore, combinations of ZD1694 with each of the platinum-based drugs, cisplatin, carboplatin and the recently introduced orally administrable, JM216, all showed additive growth inhibitory effects by median effect analysis. These data suggest that the tumour inhibitory properties of the recently introduced highly potent TS inhibitor, ZD1694, and cisplatin, and, moreover, their respective mechanisms of resistance, do not overlap. Therefore, these drugs may be considered for combination in the clinic.

Mechanisms of acquired resistance to the quinazoline thymidylate synthase inhibitor ZD1694 (Tomudex) in one mouse and three human cell lines.

Four cell lines, the mouse L1210 leukaemia, the human W1L2 lymphoblastoid and two human ovarian (CH1 and 41M) cell lines, were made resistant to ZD1694 (Tomudex) by continual exposure to incremental doses of the drug. A 500-fold increase in thymidylate synthase (TS) activity is the primary mechanism of resistance to ZD1694 in the W1L2:RD1694 cell line, which is consequently highly cross-resistant to other folate-based TS inhibitors, including BW1843U89, LY231514 and AG337, but sensitive to antifolates with other enzyme targets. The CH1:RD1694 cell line is 14-fold resistant to ZD1694, largely accounted for by the 4.2-fold increase in TS activity. Cross-resistance was observed to other TS inhibitors, including 5-fluorodeoxyuridine (FdUrd). 41M:RD1694 cells, when exposed to 0.1 microM [3H]ZD1694, accumulated approximately 20-fold less 3H-labelled material over 24 h than the parental line. Data are consistent with this being the result of impaired transport of the drug via the reduced folate/methotrexate carrier. Resistance was therefore observed to methotrexate but not to CB3717, a compound known to use this transport mechanism poorly. The mouse L1210:RD1694 cell line does not accumulate ZD1694 or Methotrexate (MTX) polyglutamates. Folylpolyglutamate synthetase substrate activity (using ZD1694 as the substrate) was decreased to approximately 13% of that observed in the parental line. Cross-resistance was found to those compounds known to be active through polyglutamation.

Quinazoline antifolate thymidylate synthase inhibitors: replacement of glutamic acid in the C2-methyl series.

The synthesis of a series of analogues of the potent thymidylate synthase (TS) inhibitor N-[4-[N-[(3,4-dihydro-2-methyl-4-oxo-6- quinazolinyl)methyl]-N-prop-2-ynylamino]benzoyl]-L-glutamic acid (ICI 198583, 1) is described in which the glutamic acid residue has been replaced by other alpha-amino acids. Most of these analogues were prepared by coupling of tert-butyl-4-(prop-2-ynylamino)benzoate (37) with 6-(bromomethyl)-3,4-dihydro-2-methyl-4-oxoquinazoline (34) followed by deprotection of the tert-butyl ester to the acid and azide-mediated coupling to the appropriate amino acid or amino acid ester. In cases where the amino acid ester was unreactive with the acid azide, a modification was used in which the quinazolinone moiety was protected as its 3-(pivaloyloxy)methyl derivative. This permitted the generation of the more reactive acid chloride of the p-aminobenzoate unit. In general these modifications result in compounds that have equivalent potency to 1 as inhibitors of isolated TS except where the amino acid lacks a lipophilic alpha-substituent. These compounds appear to require the reduced folate carrier (RFC) for transport into cells, but since they are not converted intracellularly into polyglutamated forms, they have a lower level of cytotoxicity compared to 1. The removal of the alpha-carboxylic acid has given a second set of analogues of 1 which contain simple alkyl amide, benzyl, substituted benzyl, and heterocyclic benzyl amide derivatives. These are considerably less potent than 1 as TS inhibitors but display 1-10 microM cytotoxicities due to the fact that they do not require RFC transport and can presumably readily enter cells by passive diffusion through the cell membrane. Molecular modeling and NMR studies indicated that the incorporation of, respectively, 7-methyl and 2'-fluoro substituents would favor the optimum conformation of these molecules for interaction with the TS enzyme. Accordingly, these substituents were incorporated into selected examples to give the series of analogues 47-55. These all show enhanced (approximately 10-fold) inhibition of TS compared to their unsubstituted counterparts. In the substituted benzylamides (51, 52) and heterocyclic benzyl amides (53-55) the ability to enter cells by passive diffusion results in highly potent (< 1 microM) cytotoxic agents.