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.

Prodrugs of thymidylate synthase inhibitors: potential for antibody directed enzyme prodrug therapy (ADEPT).

Prodrugs of quinazoline antifolate thymidylate synthase (TS) inhibitors have been designed and synthesized for use in antibody-directed enzyme prodrug therapy (ADEPT). The syntheses of the alpha-linked dipeptides of two potent thymidylate synthase inhibitors, ZD1694 [N-[5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6- ylmethyl)-N-methylamino]-2-thenoyl]-L-glutamic acid] and ICI198583 ¿N-[4-[N-[(2-methyl-3,4-dihydro-4-oxo-6-quinazolinyl) methyl]-N-prop-2-ynylamino]benzoyl]-L-glutamic acid¿ are described. The alpha-carboxyl of the glutamic acid has been linked through an amide bond to an L-alanine or an L-glutamic acid. The alpha-linked L-dipeptide prodrugs were designed to be activated to their corresponding thymidylate synthase inhibitors at a tumour site by prior administration of a monoclonal antibody conjugated to the enzyme carboxypeptidase A (CPA). The viability of a colorectal cell line was monitored with the potential prodrugs in the presence or absence of CPA or with the parent drugs alone. All the dipeptides had greatly decreased cytotoxicity, with a deactivation of approximately 100-fold for the ZD1694 prodrugs and approximately 20-200-fold for the ICI198583 prodrugs. Activation of the alpha-linked L-alanine dipeptides with CPA led to a cytotoxicity enhancement of approximately 10-100 fold.

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.

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.

The synthesis and thymidylate synthase inhibitory activity of L-gamma-L-linked dipeptide and L-gamma-amide analogues of 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI 198583).

Sixteen gamma-linked dipeptide and four L-Glu-gamma-amide analogues of 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI 198583) have been synthesized and evaluated as inhibitors of thymidylate synthase (TS). Z-blocked L-Glu-gamma-L-linked dipeptides and L-Glu-gamma-amides were prepared by condensing alpha-tert-butyl-N-(benzyloxycarbonyl)-L-glutamic acid with the appropriate tert-butyl-protected L-amino acid or amine. The Z group was removed by catalytic hydrogenolysis, and the resulting dipeptides or L-Glu-gamma-amides were condensed with the appropriate pteroic acid analogue trifluoroacetate salt using diethyl cyanophosphoridate as coupling reagent. Deprotection with trifluoroacetic acid in the final step gave the desired quinazoline gamma-linked dipeptides and L-Glu-gamma-amides as their trifluoroacetate salts. Nearly all the dipeptide analogues were potent inhibitors of TS, the best being ICI 198583-gamma-L-2-aminoadipate (IC50 = 2 nM). Several of these dipeptides were found to be susceptible to enzymatic hydrolysis in mice. The quinazoline monocarboxylate L-Glu-gamma-amides, lacking an alpha'-carboxyl group, are less active against TS and L1210 cell growth but are also not susceptible to enzymatic hydrolysis in mice.

The in vivo metabolic stability of dipeptide analogues of the quinazoline antifolate, ICI 198583, in mice.

In the search for quinazoline thymidylate synthase inhibitors that are not subject to intracellular polyglutamation, a class of dipeptide analogues of the diglutamate of 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI 198583-gamma-L-glu) has been evaluated for their stability to in vivo hydrolysis. Replacement of the second glutamate with another amino acid, e.g. alanine, prevented polyglutamation in vitro but such compounds were subject to hydrolysis when injected into mice. The extent of hydrolysis was measured in plasma, liver and kidney by HPLC analysis of tissue removed from mice 1 hr after i.p. injection. The enzyme responsible for this hydrolysis is thought to be a gamma-glutamyl hydrolase which hydrolyses the amide bond, releasing ICI 198583 which may then be polyglutamated. Development of stable dipeptide compounds was achieved by structural modification in two principal ways: either by replacement of the second amino acid (e.g. glutamate or alanine) with its D-enantiomer or removal of the carboxyl on the alpha-carbon of the second amino acid (alpha'-COOH). In this second approach two series of compounds were investigated. Monocarboxylate-derived dipeptides, e.g. ICI 198583-gamma-L-phenylalanine or ICI 198583-gamma-phenylglycine, resulted in stable compounds after removal of the alpha'-COOH (to give -ethylamide and -benzylamide derivatives, respectively). However, for the dicarboxylic amino acids a less clear picture emerged. Although removal of the alpha'-COOH from ICI198583-gamma-L-glutamate to give ICI 198583-gamma-gamma-aminobutyric acid resulted in a stable compound, the corresponding aspartate analogue (-beta-alanine) was subject to hydrolysis.

The measurement of polyglutamate metabolites of the thymidylate synthase inhibitor, ICI D1694, in mouse and human cultured cells.

A method is described for the measurement of the polyglutamates of the quinazoline thymidylate synthase inhibitor, N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin- 6-ylmethyl)-N-methylamino]-2-theonyl)-L-glutamic acid (ICI D1694). This involved incubation of cells with [5-3H]ICI D1694, extraction of the polyglutamates and their analysis by HPLC using an ion-pairing method. Co-chromatography with ICI D1694 and its synthetic di-hexaglutamate standards (UV detection) aided identification of the [3H]polyglutamates in the fractions recovered from the HPLC. Recovery of the polyglutamates at each stage of extraction and analysis was very good (77-84% overall recovery). Polyglutamates readily accumulated as the tri-, tetra and penta forms and occasionally a small amount of hexaglutamate was found. After mouse L1210 leukemia or human W1L2 lymphoblastoid cells were incubated for 30 min with 0.1 microM [3H]ICI D1694 there was a approximately 6-fold concentration effect intracellularly with most of the 3H associated with polyglutamate forms (approximately 75% and 96% for the L1210 and W1L2, respectively). Even some of the higher chain length tetra- and pentaglutamates could be detected at this time. After 4 hr incubation the total level of intracellular 3H had risen to 2-3 microM, greater than 96% of which was associated with polyglutamates (mainly tetra- and pentaglutamates). Four other human cell lines, two ovarian (CH1 and 41M), the MCF-7 breast and the HT-29 colon, were examined for their ability to form intracellular polyglutamates. A 4 hr incubation with 0.1 microM [3H]ICI D1694 resulted in a substantial intracellular accumulation of the drug (20-100-fold) in its polyglutamate forms with only 2-20% remaining as the parent monoglutamate, depending on the cell line. The major polyglutamate was again cell line dependent, ranging from the tri to the penta form. Prolonging the incubation time to 24 hr allowed a further accumulation of drug with a larger percentage appearing as tri- to hexaglutamates. Although cell lines differed in the total level of polyglutamates formed and the pattern of chain length observed, rapid and extensive polyglutamation of ICI D1694 occurred in all the cell types examined.

Gamma-linked dipeptide analogues of 2-desamino-2-methyl-N10-propargyl-5,8- dideazafolate as antitumour agents.

Our search for water-soluble quinazoline TS inhibitors that are transported into cells via the RFC, but are not substrates for FPGS, led us to the synthesis of dipeptide analogues of ICI 198583 diglutamate. Although a number of dipeptide analogues were active against isolated TS and L1210 cells in vitro, lack of in vivo stability was a problem. This was circumvented by the synthesis of modified dipeptides where either the alpha-carboxyl of the second amino acid was removed (alpha'-COOH) e.g. -L-glu-GABA or where the second amino acid was the unnatural D-enantiomer e.g.-L-glu-D-glu. Further studies were performed with the -L-glu-D-glu and its 7-CH3, 2'F modified analogue, demonstrating that they use the RFC for cell entry but are not active through polyglutamate formation. The latter compound was tested against experimental tumour models and found to have good activity.

Electrospray ionization mass spectrometry for analysis of low-molecular-weight anticancer drugs and their analogues.

In this study, several anticancer drugs and their analogues consisting of organic and organometallic compounds were analyzed by electrospray ionization mass spectrometry (ESI/MS) using a quadrupole mass spectrometer. Protonated molecular ions [M+H](+) were observed for all of the compounds studied, and in the case of the two steroid sulfates, deprotonated molecular ions [M-H](-) were obtained. Tandem mass spectrometry was performed on these quasimolecular ions, and the product ions formed provided useful fragmentation patterns that were characteristic for the compounds. This study provides evidence that ESI/MS is a sensitive technique for structure confirmation and identification of small organic and organometallic molecules.

Syntheses and thymidylate synthase inhibitory activity of the poly-gamma-glutamyl conjugates of N-[5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino ]-2-thenoyl]-L-glutamic acid (ICI D1694) and other quinazoline antifolates.

Thirteen poly-gamma-glutamates derived from several novel antifolates have been synthesized by a convergent route. The syntheses of poly-gamma-glutamyl conjugates of N-[5-[N-(3,4-dihydro-2- methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino]-2-theno yl]-L-glutamic acid (8) (ICI D1694), 2-desamino-N10-propargyl-5,8-dideazafolic acid (6), 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (7), 2-desamino-2-methyl-N10-propargyl-2'-fluoro-5,8-dideazafolic acid (9), and 2-desamino-2-methyl-4-chloro-N10-propargyl-2'-fluoro-3,5,8-trideazafo lic acid (11) are described. A key step in the route involves coupling of an alpha-tert-butyl-protected poly-gamma-glutamate of the required chain length to the appropriate 5,8-dideazapteroic acid, obtained by carboxypeptidase G2 cleavage of the parent monoglutamate, if available, or by chemical synthesis. Deprotection with trifluoroacetic acid in the final step gave the desired poly-gamma-glutamyl antifolates as their trifluoroacetate salts. As inhibitors of thymidylate synthase, these polyglutamates were more potent in every case than the corresponding non-polyglutamylated drug.

Quinazoline antifolate thymidylate synthase inhibitors: benzoyl ring modifications in the C2-methyl series.

The synthesis of nine new 2-methyl-10-propargylquinazoline antifolates with substituents in the p-aminobenzoyl ring is described. In general the synthetic route involved the coupling of the appropriate ring-substituted diethyl N-[4-(prop-2-ynylamino)benzoyl]-L-glutamate with 6-(bromomethyl)-3,4-dihydro-2-methyl-4-oxoquinazoline followed by deprotection using mild alkali. The compounds were tested as inhibitors of partially purified L1210 thymidylate synthase (TS). They were also examined for their inhibition of the growth L1210 cells in culture. Compared to the parent compound 1a the 2'-fluoro analogue 2a exhibited enhanced potency in both systems whereas the 3'-fluoro analogue 3a showed enhanced growth inhibitory properties against L1210 cells despite being a poorer inhibitor of the isolated enzyme. Chloro, hydroxy, methoxy, and nitro substituents in the 2'-position were also well tolerated by the enzyme but failed to give enhanced growth inhibition. The series was extended to cover analogues of the 2'-fluoro, 3'-fluoro, 2'-chloro, 2'-methyl, 2'-amino, 2'-methoxy, and 2'-nitro derivatives with modified alkyl substituents at N10.

Pharmacokinetic studies with the antifolate C2-desamino-C2-methyl-N10-propargyl-2'-trifluoromethyl-5,8-dideazafolic acid (CB3988) in mice and rats using in vivo 19F-NMR spectroscopy.

In vivo 19F-NMR spectroscopy has been used to study the pharmacokinetics of the experimental antifolate drug CB3988 (C2-desamino-C2-methyl-N10-propargyl-2'trifluoromethyl-5,8-dideazafolic acid) in mice and rats. NMR results have been compared to those obtained by HPLC and the effect of the inclusion of the CF3 group evaluated by comparing the pharmacokinetics of CB3988 and ICI 198583 (C2-desamino-C2-methyl-N10-propargyl-5,8-dideazafolic acid) in rats. In mice, following the administration of CB3988 (500 mg kg-1 i.v.), drug could be detected in both the upper and the lower abdomen. NMR signal from the upper abdomen reached maximum intensity 10-40 min after administration, declining thereafter with a half life of 28 min. Signal detected in the lower abdomen reached maximum intensity 60-90 min after treatment. HPLC analyses indicated that CB3988 was present at appreciable concentrations (about 20-30 mg ml-1) in both bile and urine which is consistent with the signal from the upper and lower abdomen being derived from the gall bladder and urinary bladder, respectively. Studies in rats also indicated that CB3988 (100 mg kg-1 i.v.) rapidly entered and was cleared from the upper abdomen. Comparison of data from rats with intact and cannulated bile ducts suggested that 19F-NMR could detect CB3988 undergoing enterohepatic circulation. Furthermore, comparison of the plasma half life of CB3988 with the half life for the decline of the NMR signal from the upper abdomen suggested that NMR measurements may reflect the plasma clearance of CB3988. When the pharmacokinetics of CB3988 and ICI 198583 were compared the only significant difference was in the alpha phase half life which was 2-fold faster for CB3988. These data demonstrate that CB3988 is cleared rapidly by both biliary and urinary excretion. This is in contrast to N10-propargyl-5,8-dideazafolic acid, where delayed excretion is associated with hepatic and renal toxicities. The ability to study CB3988 pharmacokinetics non-invasively by 19F-NMR spectroscopy confirms the utility of the technique and, since 19F-NMR can be applied directly to clinical investigations, it may be possible to obtain similar information in humans.

Novel prodrugs which are activated to cytotoxic alkylating agents by carboxypeptidase G2.

The synthesis of three novel prodrugs, 4-[bis[2-(mesyloxy)ethyl]amino]benzoyl-L-glutamic acid (7), 4-[(2-chloroethyl)[2-(mesyloxy)ethyl]amino]benzoyl-L-glutamic acid (8), and 4-[bis(2-chloroethyl)amino]benzoyl-L-glutamic acid (9), for use as anticancer agents, is described here. Each is a bifunctional alkylating agent in which the activating effect of the ionized carboxyl function is masked through an amide bond to the glutamic acid residue. These relatively inactive prodrugs are designed to be activated to their corresponding nitrogen alkylating agents (10, 11, and 12, respectively) at a tumor site by prior administration of a monoclonal antibody conjugated to the bacterial enzyme carboxypeptidase G2 (CPG2). The viability of two different tumor cell lines was monitored with each prodrug in the presence of CPG2. All three compounds showed substantial prodrug activity--with conversion to the corresponding active drug leading to greatly increased cytotoxicity.

Mechanism of photooxidation of bacteriochlorophyll c derivatives. A possible model for natural chlorophyll breakdown.

The photooxidation of methyl pheophorbides, derived from bacteriochlorophyll c, yields ring-opened acetylbilitrienes which are structurally related to mammalian bile pigments. The mechanism of this photooxidation, and that of certain mesomethyl-substituted model compounds, has been studied using 18O labeling with molecular oxygen. In all cases, the methine bridge carbon atoms were retained in the ring-opened photoproducts and both oxygen atoms incorporated into the acetylbilitriene photoproducts were derived from a single oxygen molecule. In this respect, ring opening of bacteriochlorophyll c derivatives and mesomethyl-substituted model compounds differs from the nonphotochemical (enzymatic) ring opening of heme which involves elimination of the methine bridge carbon atom as carbon monoxide and insertion of two oxygen atoms, each from a different molecule of molecular oxygen. The process of photooxidation of bacteriochlorophyll c and mesomethyl-substituted model compounds studied may have implications for the mechanism of natural degradation of chlorophyll a and b during leaf senescence and grain ripening.

Antihypertensive ureidopiperidines.

The synthesis of a series of 1-aralkyl-4-ureidopiperidines is reported. These compounds are related to the benzamidopiperidines exemplified by indoramin. Some of the ureidopiperidines are more potent antihypertensive agents than their benzamidopiperidine counterparts. Two examples, 1-(2-thenoyl)-3-[1-[2-(3-indolyl)ethyl]piperid-4-yl]urea and 1-(2-thenoyl)-3-[1-[4-(4-fluorophenyl)-4-oxobutyl]piperid-4-yl]urea (19 and 58), emerged as the most potent antihypertensive agents in this series.