Significant differences in biological parameters between prodrugs cleavable by carboxypeptidase G2 that generate 3,5-difluoro-phenol and -aniline nitrogen mustards in gene-directed enzyme prodrug therapy systems

Nine new nitrogen mustard compounds derived from 2,6-difluoro-4-hydroxy- (3a-e) and 2,6-difluoro-4-amino- (4a-d) aniline were synthesized as potential prodrugs. They were designed to be activated to their corresponding 3,5-difluorophenol and -aniline (4)-nitrogen mustards by the enzyme carboxypeptidase G2 (CPG2) in gene-directed enzyme prodrug therapy (GDEPT) models. The compounds were tested for cytotoxicity in the MDA MB-361 breast adenocarcinoma. The cell line was engineered to express stably either CPG2 tethered to the cell surface stCPG2-(Q)3 or beta-galactosidase (beta-Gal) as control. The cytotoxicity differentials were calculated between CPG 2-expressing and -nonexpressing cells and yielded different results for the two series of prodrugs despite their structural similarities. While the phenol compounds are ineffective as prodrugs, their aniline counterparts exhibit outstanding activity in the tumor cell lines expressing CPG2. [3,5-Difluoro-4-[bis(2-chloroethyl)amino]phenyl]carbamoyl-l-glutamic acid gave a differential of >227 in MDA MB361 cells as compared with 19 exhibited by 4-[(2-chloroethyl)(2-mesyloxyethyl)amino]benzoyl-l-glutamic acid, 1a, which has been in clinical trials.     

Novel fluorinated prodrugs for activation by carboxypeptidase G2 showing good in vivo antitumor activity in gene-directed enzyme prodrug therapy

Sixteen novel polyfluorinated benzoic acid mustards have been synthesized for use in gene-directed enzyme prodrug therapy (GDEPT). Eight of these were benzoic acid L-glutamate mustards for evaluation as prodrugs and the other eight were the active drugs formed by the action of the bacterial enzyme carboxypeptidase G2 (CPG2). All of the di- and trifluorinated prodrugs were efficiently cleaved by the enzyme. In contrast, the tetrafluorinated prodrugs were found to be competitive inhibitors of CPG2, the first such inhibitors to have been described. The di- and trifluorinated prodrugs were differentially cytotoxic to human breast carcinoma cells (MDA MB 361) expressing CPG2, compared to control cells that did not express the enzyme. The difluorinated prodrug {4-[bis(2-bromoethyl)amino]-3,5-difluorobenzoyl}-L-glutamic acid and its iodoethylamino analogue were effective substrates for the enzyme and showed excellent therapeutic activity in CPG2-expressing MDA MB 361 xenografts, either curing or greatly inhibiting tumor growth and extending the life of the animals.     

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.     

Self-immolative anthracycline prodrugs for suicide gene therapy.

Four novel potential prodrugs derived from daunorubicin (8, 10) and doxorubicin (12, 14) were designed and synthesized. They are self-immolative prodrugs for suicide gene therapy activation by the enzyme carboxypeptidase G2 (CPG2) subsequently releasing the corresponding anthracyclines, by a 1,6-elimination mechanism. A mammary carcinoma cell line (MDA MB 361) was engineered to express CPG2 intracellularly (CPG2) or extracellularly, tethered to the outer cell membrane (stCPG2(Q)3). The prodrugs derived from doxorubicin showed prodrug/drug cytotoxicity differentials of 21-fold (compound 12) and 23-fold (compound 14). Prodrug 12 underwent an 11-fold activation when assayed in the cell line expressing externally surface-tethered CPG2.     

Design, synthesis, and biological evaluation of cyclic and acyclic nitrobenzylphosphoramide mustards for E. coli nitroreductase activation

In efforts to obtain anticancer prodrugs for antibody-directed or gene-directed enzyme prodrug therapy using E. coli nitroreductase, a series of nitrobenzylphosphoramide mustards were designed and synthesized incorporating a strategically placed nitro group in a position para to the benzylic carbon for reductive activation. All analogues were good substrates of E. coli nitroreductase with half-lives between 2.9 and 11.9 min at pH 7.0 and 37 degrees C. Isomers of the 4-nitrophenylcyclophosphamide analogues 3 and 5 with a benzylic oxygen para to the nitro group showed potent selective cytotoxicity in nitroreductase (NTR) expressing cells, while analogues 4 and 6 with a benzylic nitrogen para to the nitro group showed little selective cytotoxicity despite their good substrate activity. These results suggest that good substrate activity and the benzylic oxygen are both required for reductive activation of 4-nitrophenylcyclophosphamide analogues by E. coli nitroreductase. Isomers of analogue 3 showed 23,000-29,000x selective cytotoxicity toward NTR-expressing V79 cells with an IC(50) as low as 27 nM. They are about as active as and 3-4x more selective than 5-aziridinyl-2,4-dinitrobenzamide (CB1954). The acyclic 4-nitrobenzylphosphoramide mustard ((+/-)-7) was found to be the most active and most selective compound for activation by NTR with 170,000x selective cytotoxicity toward NTR-expressing V79 cells and an IC(50) of 0.4 nM. Compound (+/-)-7also exhibited good bystander effect compared to 5-aziridinyl-2,4-dinitrobenzamide. The low IC(50), high selectivity, and good bystander effects of nitrobenzylphosphoramide mustards in NTR-expressing cells suggest that they could be used in combination with E. coli nitroreductase in enzyme prodrug therapy.     

Synthesis and structure-activity relationships for 2,4-dinitrobenzamide-5-mustards as prodrugs for the Escherichia coli nfsB nitroreductase in gene therapy

A series of 2,4-dinitrobenzamide mustards were prepared from 5-chloro-2,4-dinitrobenzoic acid or the corresponding 5-dimesylate mustard as potential prodrugs for gene-directed enzyme prodrug therapy (GDEPT) with the E. coli nfsB nitroreductase (NTR). The compounds, including 32 new examples, were evaluated in four pairs of NTR+ve/-ve cell lines for selective cytotoxicity (IC50 and IC50 ratios), in multicellular layer (MCL) cultures for bystander effects, and for in vivo activity against tumors grown from stably NTR transfected EMT6 and WiDr cells in nude mice. Multivariate regression analysis of the IC50 results was undertaken using a partial least-squares projection to latent structures model. In NTR-ve lines, cytotoxicity correlated positively with logP, negatively with hydrogen bond acceptors (HA) and donors (HD) in the amide side chain, and positively with the reactivity of the less-reactive leaving group of the mustard function, likely reflecting toxicity due to DNA monoadducts. Potency and selectivity for NTR+ve lines was increased by logP and HD, decreased by HA, and was positively correlated with the leaving group efficiency of the more-reactive group, likely reflecting DNA crosslinking. NTR selectivity was greatest for asymmetric chloro/mesylate and bromo/mesylate mustards. Bystander effects in the MCL assay also correlated positively with logP and negatively with leaving group reactivity, presumably reflecting the transcellular diffusion/reaction properties of the activated metabolites. A total of 18 of 22 mustards showed equal or greater bystander efficiencies in MCLs than the aziridinylbenzamide CB 1954, which is currently in clinical trial for NTR-GDEPT. The dibromo and bromomesylate mustards were surprisingly well tolerated in mice. High MTD/IC50 (NTR+ve) ratios translated into curative activity of several compounds against NTR+ve tumors. A bromomesylate mustard showed superior activity against WiDr tumors grown from 1:9 mixtures of NTR+ve and NTR-ve cells, indicating a strong bystander effect in vivo.     

Synthesis and evaluation of nitroheterocyclic carbamate prodrugs for use with nitroreductase-mediated gene-directed enzyme prodrug therapy

A variety of nitroheterocyclic carbamate prodrugs of phenylenediamine mustard and 5-amino-1-(chloromethyl)-3-[(5,6,7-trimethoxyindol-2-yl)carbonyl]-1,2-dihydro-3H-benz[e]indoline (amino-seco-CBI-TMI), covering a wide range of reduction potential, were prepared and evaluated for use in gene-directed enzyme prodrug therapy (GDEPT) using a two-electron nitroreductase (NTR) from Escherichia coli B. The carbamate prodrugs and corresponding amine effectors were tested in a cell line panel comprising parental and NTR-transfected human (SKOV3/SKOV3-NTR(neo), WiDr/WiDr-NTR(neo)), Chinese hamster (V79(puro)/V79-NTR(puro)), and murine (EMT6/EMT6-NTR(puro)) cell line pairs and were compared with the established NTR substrates CB1954 (an aziridinyl dinitrobenzamide) and the analogous dibromomustard. The 1-methyl-2-nitroimidazol-5-ylmethyl carbamate of phenylenediamine mustard was metabolized rapidly by EMT6-NTR(neo) but not EMT6 cells, demonstrating that it is an efficient substrates for NTR. Despite this, the carbamates of phenylenediamine mustards show relatively low differential cytotoxicity for NTR+ve cells in IC(50) assays, apparently because they retain sufficient alkylating reactivity that most of the prodrug reacts with nucleophiles during the drug exposure period. In contrast, the corresponding amino-seco-CBI-TMI prodrugs were less efficient NTR substrates but had greater chemical stability, were more potent, and showed substantial NTR-ve/NTR+ve ratios in the cell line panel, with ratios of 15-100-fold for the 1-methyl-2-nitro-1H-imidazol-5-ylmethyl and 1-methyl-5-nitro-1H-imidazol-2-ylmethyl carbamates of amino-seco-CBI-TMI. The activity of these two prodrugs was evaluated against NTR-expressing EMT6 tumors comprising ca. 10% NTR+ve cells. Small but not statistically significant killing of NTR+ve cells was observed, with no effect against NTR-ve target cells. The lack of activity against NTR+ve cells in tumors, despite potent and selective activity in culture, indicates that pharmacokinetic optimization will be required if in vivo efficacy against solid tumors is to be achieved with this new class of NTR prodrugs.     

Phthalimide analogs of CB 1954: synthesis and bioactivation

Four novel 4-substituted 5-nitrophthalimides (5-substituted-6-nitro-1,3-dihydro-isoindol-1,3-diones), 6, 7, 10 and 11, and the known 5 are prepared as analogs of the dinitrobenzamide prodrug CB 1954, 1, and considered as potential candidates for gene-directed enzyme prodrug therapy. All the phthalimides are poor substrates for Escherichia coli nitroreductase compared to CB 1954. However, 6, 7, 10 and 11 are reduced by both the human and rat forms of DT-diaphorase; 10 is a particularly good substrate but 7 decomposes in phosphate buffer. A cell-line panel consisting of V79 cells that have been engineered to express various levels of either the human or rat forms of DT-diaphorase in an identical cellular background was used to evaluate these compounds as prodrugs activated by this enzyme. The cytotoxic effect of CB 1954 is proportional to the activity of either the rat or human enzyme but cells expressing the rat enzyme were much more sensitive (10000-fold at higher levels of DT-diaphorase activity) than cells expressing comparable levels of the human enzyme. These results demonstrate that the resistance of human tumors to CB 1954 can be accounted for solely by the kinetic properties of the enzyme for this prodrug. The nitrophthalimide analogs overcome this kinetic failing of CB 1954. However, these compounds are not activated to produce cytotoxicity in these DT-diaphorase-expressing cell lines. It is postulated their reduction products fail to undergo an acylation reaction in a manner analogous to CB 1954. Thus, reduction by DT-diaphorase is not predictive of cytotoxicity in this class of prodrugs.     

Quinoline antifolate thymidylate synthase inhibitors: variation of the C2- and C4-substituents.

Modifications to the bicyclic ring system of the potent thymidylate synthase (TS) inhibitor N-[4-[N-[(2-amino-3,4-dihydro-4-oxo-6- quinazolinyl)methyl]-N-prop-2-ynylamino]benzoyl]-L-glutamic acid (1, CB3717) have led to the synthesis of a series of quinoline antifolates bearing a variety of substituents at the C2 and C4 positions. In general the synthetic route involved the coupling of the appropriate diethyl N-[4-(prop-2-ynylamino)benzoyl]-L-glutamate with a disubstituted 6-(bromomethyl)quinoline followed by deprotection using mild alkali. The compounds were tested as inhibitors of partially purified L1210 TS. As a measure of cytotoxicity, the compounds were tested for their inhibition of the growth of L1210 cells in culture. Good enzyme inhibition and cytotoxicity were found for compounds containing chloro, amino, or methyl substituents at the C2 position with chloro or bromo substituents at C4. The effect on enzyme inhibition of varying the N10 substituent of 2h was similar to that observed in the quinazolinone-containing antifolates, indicating that the quinoline compounds may be interacting with the enzyme in a similar way to the quinazolinones. Also, the introduction of a 2'-fluoro substituent into the benzoyl ring of several of the quinoline antifolates led to an increase in both TS inhibition and the inhibition of L1210 cell growth. These data demonstrate that the N3-H of the pyrimidine ring of the quinazolinone antifolates is not required for binding to TS if appropriate substituents are placed at the C2 and C4 positions of the bicyclic ring system.     

Quinazoline antifolate thymidylate synthase inhibitors: alkyl, substituted alkyl, and aryl substituents in the C2 position

Modification of the potent thymidylate synthase (TS) inhibitor N-[4-[N-[(2-amino-3,4-dihydro-4-oxo-6-quinazolinyl)methyl]-N-prop-2- ynylamino]benzoyl]-L-glutamic acid (1a) has led to the synthesis of quinazoline antifolates bearing alkyl, substituted alkyl, and aryl substituents at C2. In general the synthetic route involved the coupling of the appropriate diethyl N-[4-(alkylamino)benzoyl]-L-glutamate with a C2-substituted 6-(bromo-methyl)-3,4-dihydro-4-oxoquinazoline followed by deprotection using mild alkali. Good enzyme inhibition and cytotoxicity were found with compounds containing small nonpolar groups in the C2 position with the 2-desamino-2-methyl analogue 3a being the most potent. Larger C2 substituents were tolerated by the enzyme, but cytotoxicity was reduced. Highly potent series were followed up by the synthesis of a number of analogues in which the N10 substituent was varied. In this manner a number of interesting TS inhibitors have been prepared. Although none of these was more potent than 1a against the isolated enzyme, over half of the compounds prepared were more potent as cytotoxic agents against L1210 cells in culture. The potential of such compounds as useful antitumor agents was further enhanced by the finding that the improved aqueous solubilities of compounds such as 3a over 1a were reflected in vivo in that 3a was at least 5 times less toxic to mice than 1a.     

Cross-linking of the DNA repair protein Omicron6-alkylguanine DNA alkyltransferase to DNA in the presence of antitumor nitrogen mustards

The antitumor activity of chemotherapeutic nitrogen mustards including chlorambucil, cyclophosphamide, and melphalan is commonly attributed to their ability to induce DNA-DNA cross-links by consecutive alkylation of two nucleophilic sites within the DNA duplex. DNA-protein cross-linking by nitrogen mustards is not well characterized, probably because of its inherent complexity and the insufficient sensitivity of previous methodologies. If formed, DNA-protein conjugates are likely to contribute to both target and off-target cytotoxicity of nitrogen mustard drugs. Here, we show that the DNA repair protein, O (6)-alkylguanine DNA alkyltransferase (AGT), can be readily cross-linked to DNA in the presence of nitrogen mustards. Both chlorambucil and mechlorethamine induced the formation of covalent conjugates between (32)P-labeled double-stranded oligodeoxynucleotides and recombinant human AGT protein, which were detected by SDS-PAGE. Capillary HPLC-electrospray ionization mass spectrometry (ESI-MS) analysis of AGT that had been treated with the guanine half-mustards of chlorambucil or mechlorethamine revealed the ability of the protein to form either one or two cross-links to guanine. C145A AGT (a variant containing a single point mutation in the protein's active site) was found capable of forming a single guanine conjugate, while cross-linking was virtually abolished upon treatment of the C145A/C150S AGT double mutant with the guanine half-mustards. HPLC-ESI (+)-MS/MS sequencing of tryptic peptides obtained from the wild-type AGT protein that had been treated with nitrogen mustards in the presence of DNA confirmed that the cross-linking took place between the N7 position of guanine in DNA and two active site residues within the AGT protein (Cys (145) and Cys (150)). The exact chemical structures of AGT-DNA cross-links induced by chlorambucil and mechlorethamine were identified as N-(2-[ S-cysteinyl]ethyl)- N-(2-[guan-7-yl]ethyl)- p-aminophenylbuyric acid and N-(2-[ S-cysteinyl]ethyl)- N-(2-[guan-7-yl]ethyl)methylamine, respectively, based upon HPLC-MS/MS analysis of protein hydrolysates in parallel with the corresponding amino acid conjugates prepared synthetically. Mechlorethamine-induced AGT-DNA conjugates were isolated from protein extracts of AGT-expressing CHO cells but not control cells, demonstrating that nitrogen mustards can cross-link the AGT protein to DNA in the presence of other nuclear proteins. Because AGT is overexpressed in many tumor types, further investigations of the potential role of AGT-DNA cross-linking in the antitumor and mutagenic activity of antitumor nitrogen mustards are warranted.     

Structure-activity relationships for 4-nitrobenzyl carbamates of 5-aminobenz[e]indoline minor groove alkylating agents as prodrugs for GDEPT in conjunction with E. coli nitroreductase

Twelve substituted 4-nitrobenzyl carbamate prodrugs of the 5-aminobenz[e]indoline class of DNA minor groove alkylating agents were prepared and tested as prodrugs for gene-directed enzyme prodrug therapy (GDEPT) using a two-electron nitroreductase (NTR) from E. coli B. The prodrugs and effectors were tested in a cell line panel comprising parental and transfected human (SKOV/Skov-NTR(neo), WiDr/WiDr-NTR(neo)), Chinese hamster (V79(puro)/V79-NTR(puro)), and murine (EMT6/EMT6-NTR(puro)) cell line pairs. In the human cell line pairs, several analogues bearing neutral methoxyethoxy-, 2-hydroxyethoxy-, or 3-hydroxypropoxy-substituted side chains were good substrates for NTR as measured by cytotoxicity ratios, with NTR-ve/NTR+ve ratios similar to the established NTR substrates CB1954 (an aziridinyl dinitrobenzamide) and the analogous bromomustard. Selectivity for NTR decreased with increasing side-chain size or the presence of a basic amine group. Low to modest selectivity was observed in the Chinese hamster-derived cell line pair; however, in the murine EMT6/EMT6-NTR(puro) cell line pair, the above hydroxyalkoxy analogues again showed significant selectivity for NTR. The activity of the 2-hydroxyethoxy analogue was evaluated against NTR-expressing EMT6 tumors comprising ca. 10% NTR+ve cells at the time of tumor treatment. A small decrease in NTR+ve cells was observed after treatment, with a lesser effect against NTR-ve target cells, but these effects were not statistically significant and were much less than for the dinitrobenzamides. These results suggest that useful GDEPT prodrugs based on the 4-nitrobenzyl carbamate and 5-aminobenz[e]indoline motifs may be developed if optimization of pharmacokinetics can be addressed.     

Hypoxia-selective antitumor agents. 5. Synthesis of water-soluble nitroaniline mustards with selective cytotoxicity for hypoxic mammalian cells.

Nitroaniline mustards have potential as hypoxia-selective cytotoxic agents, with reductive metabolism activating the nitrogen mustard by converting the electron-withdrawing nitro group to an electron-donating hydroxylamine or amine. However, the parent compounds have poor aqueous solubility, and their potencies are limited by low reduction potentials (E1/2 ca. -600 mV versus the normal hydrogen electrode) and corresponding slow rates of nitro reduction. To address these limitations, a series of 4-nitroaniline mustards bearing hydrophilic side chains attached via an electron-withdrawing carboxamide group was prepared and evaluated for hypoxia-selective cytotoxicity against Chinese hamster cell lines. The N-[(N,N-dimethylamino)ethyl]carboxamide derivatives proved to have excellent aqueous solubility and improved cytotoxic potency, but their reduction potentials, while higher than the non-carboxamide compounds, were still low and little selectivity for hypoxic cells were observed. A series of carboxamides of 2,4-dinitroaniline mustard was also prepared. These compounds had reduction potentials in the desired range (E1/2 ca. -450 mV by cyclic voltammetry) and were more toxic to hypoxic than aerobic UV4 cells. The most selective compounds were 5-[N,N-bis(2-chloroethyl)amino]-2,4-dinitrobenzamide (20, SN 23862) and its water-soluble N-[(N,N-dimethylamino)ethyl]carboxamide analogue. These showed selectivities of 60- to 70-fold for hypoxic UV4 cells. The selectivity of 20 was much superior to that of its aziridine analogue (23, CB 1954), which was only 3.6-fold more toxic to hypoxic than oxic cells in the same system. Compound 20 is a much less efficient substrate than CB 1954 for the major aerobic nitroreductase from rat Walker tumor cells, NAD(P)H:quinone oxidoreductase (DT diaphorase). Lack of aerobic bioactivation of 20 by DT diaphorases may be responsible for its higher hypoxic selectivity than that of 23.     

Comparison of half-lives and cytotoxicity of N-chloroethyl-4-amino and N-mesyloxyethyl-benzoyl compounds, products of prodrugs in antibody-directed enzyme prodrug therapy (ADEPT).

The synthesis of two novel drugs, 4-[bis[2-(mesyloxy)ethyl]amino]benzoic acid (7) and 4-[(2-chloroethyl)[2-(mesyloxy)ethyl]amino]benzoic acid (8) is described here. They are the active drugs of two prodrugs (9 and 10) designed for use as anti-cancer agents. The prodrugs (9, 10 and 11) were made as a series of compounds which are bifunctional alkylating agents in which the activating effect of the ionized carboxyl function is masked through an amide bond to a glutamic acid residue. These relatively inactive prodrugs were designed to be activated to their corresponding alkylating agent active drugs (7, 8 and 12 respectively) at a tumour site by prior administration of a monoclonal antibody conjugated to a bacterial enzyme. This system is called antibody-directed enzyme prodrug therapy (ADEPT). The chemical half-lives of the prodrugs and their active drugs were measured in order to determine their relative reactivities. The half-lives ranged from 21 to 324 min for the active drugs and from 42 to 1158 min for the prodrugs. The viability of two different tumour cell lines was monitored with each active drug and prodrug. The IC50 values varied from 65 to 625 microM for the active drugs: no IC50 values could be obtained for the prodrugs, using a rapid incubation procedure. Each in vitro technique demonstrated the ability of the glutamic acid moiety to deactivate the drugs, forming effective prodrugs.     

Intensely cytotoxic anthracycline prodrugs: galactosides

We have reported the synthesis of a series of anthracycline analog prodrugs that give rise to intensely cytotoxic metabolites in the presence of carboxylate esterases and beta-glucuronidases. We now report structurally related prodrugs that are converted to similar potent metabolites in the presence of beta-galactosidases. The prototypical compound, N-[(4"RS)-4"-ethoxy-4"(1'"-O-beta-D-galactopyranosyl)butyl]daunorubicin, 8a, was prepared by reductive condensation of daunomycin with 1-O-[(1'RS)-1'-ethoxy-4'-oxobutyl]-2, 3, 4, 6-tetra-O-acetyl-beta-D-galactopyranoside in the presence of sodium cyanoborohydride, followed by deacetylation of the galactoside moiety with sodium methoxide. A related prodrug (8b) with enhanced lipophilicity (the 4'-hexoxy analog of 8a) and 8c (the propyldaunomycin analog of 8a) were prepared for comparative studies. 8a and 8b were isolated after chromatography on silica as a mixture of 4'R and 4'S diastereomers; 8c, on the other hand, was resolved into its component 3' diastereomers, 8c(R) and 8c(S). 8a, 8c(R) and 8c(S) showed no evidence of decomposition when incubated at 37 degrees C in 0.05 M phosphate buffer, pH 7.4, for 2 weeks; 8b, under the same conditions, was degraded with a half-life of 49 h. In the presence of two units of Escherichia coli beta-galactosidase per pmol of substrate, the half-lives of 8a, 8b, 8c(R) and 8c(S) were 1.98, 1.06, 3.5 and 2.4 h, respectively. HPLC analysis of the incubation mixtures showed that 8a and 8b gave rise to a single, chromatographically identical metabolite. 8c(R) and 8c(S) also gave rise to a single, identical metabolite. 8a and 8b were nearly one million-fold more toxic to human A375 melanoma cells in culture in the presence of E. coli beta-galactosidase than in the absence of the enzyme. The activation products of 8c(R) and 8c(S) were approximately 1000-fold less potent. These beta-galactoside prodrugs have chemotherapeutic potential for use in conjunction with tissue-targeting strategies such as antibody-directed enzyme prodrug therapy (ADEPT) and gene-directed enzyme prodrug therapy (GDEPT).     

Cardiotonic agents. 7. Prodrug derivatives of 4-ethyl-1,3-dihydro- 5-[4-(2-methyl-1H-imidazol-1-yl)benzoyl]-2H-imidazol-2-one.

The cardiotonic agent 4-ethyl-1,3-dihydro-5-4-(2-methyl-1H-imidazol-1-yl)benzoyl]-2H- imidazol-2-one (1) was found to have low bioavailability when administered orally to rats and dogs. A series of N-acyl derivatives, an underutilized prodrug of acidic NH compounds, has been synthesized and tested for their ability to improve the oral bioavailability of 1. Reaction of the monosodium salt of 1 with various anhydrides afforded the N-1 monoacylimidazolones with surprisingly high regioselectivity. In addition to the prodrugs, acylation of 1 with propionic or phenylacetic anhydride led to the novel 3H-pyrrolo[1,2-c]imidazole-3,5(2H)-diones 6. The prodrugs showed a significant increase in the partition coefficients with a minor decrease in the aqueous solubility. The benzoyl derivative 4b exhibited the highest stability in both pH 1.5 and 7.4 buffer solutions. Further evaluation of 4b showed rapid conversion to 1 in canine plasma (t1/2 = 38 min), and human plasma (t1/2 = 10 min). Oral studies indicated that the bioavailability of 4b was increased to greater than 75% (compared to less than 20% for 1), and hemodynamic studies demonstrated that the selective inotropic profile of 1 was retained.     

Enzyme specific activation of benzoquinone ansamycin prodrugs using HuCC49DeltaCH2-beta-galactosidase conjugates

To activate prodrugs for cancer treatment, an anti-TAG-72 antibody (HuCC49DeltaCH2) was used for delivery of an activation enzyme (beta-galactosidase) to specifically activate a geldanamycin prodrug (17-AG-C2-Gal) against colon cancer. The geldanamycin prodrug 17-AG-C2-Gal was synthesized by coupling a galactose-amine derivative with geldanamycin at the C-17 position. Molecular docking with two different programs (Affinity and Autodock) showed that the prodrug (17-AG-C2-Gal) was unable to bind to Hsp90; however, the product (17-AG-C2), enzymatically cleaved by beta-galactosidase conjugate, bound to Hsp90 in a similar way as geldanamycin and 17-AG. The computational docking results were further confirmed in experimental testing by the tetrazolium [3-(4,5-dimethythiazol-2-yl)]-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay and mass spectrometry. HuCC49DeltaCH2 was chemically conjugated to beta-galactosidase. The antibody-enzyme conjugate was able to target tumor antigen TAG-72 with the well-preserved enzymatic activity to activate 17-AG-C2-Gal prodrug. The released active drug 17-AG-C2 was demonstrated to induce up to 70% AKT degradation and enhance anticancer activity by more than 25-fold compared to the prodrug.     

Synthesis and biological activity of N-2,3-dihydroxypropyl-N-4-chlorobutyl nucleoside phosphoramidate prodrugs

N-2,3-dihydroxypropyl-N-4-chlorobutyl phosphoramidate prodrugs of thymidine and 5-fluoro-2'-deoxyuridine (5-FdU) 2-4 were synthesized as analogues of the known prodrugs 1a,b to assess the effects of the dihydroxypropyl moiety on prodrug activation, log P, and growth inhibitory activity in vitro. The thymidine analogues 2 and 3 were prepared as model compounds for kinetics and log P studies. 31P NMR kinetics following hydrogenolysis of 2 showed that the thymidine N-dihydroxypropyl phosphoramidate released thymidine monophosphate with a half-life of 212 min under model physiologic conditions compared to approximately 2 min for the corresponding N-methyl phosphoramidate reported previously. The measured log P for compound 3 was 1.1 log units lower than that of the analogous 1b, confirming that the dihydroxypropyl group significantly decreased prodrug lipophilicity. The dihydroxypropyl prodrug 4 showed cell growth inhibition activity in the NCI 60 cell line panel similar to that of the N-methyl analogue 1a previously reported.     

Synthesis and antimelanoma activity of analogues of N-acetyl-4-S-cysteaminylphenol

N-Acetyl-4-S-cysteaminylphenol (1) has been shown by Jimbow and co-workers to possess useful antimelanoma activity. It is an analogue of a biosynthetic intermediate in the pathway to melanin and probably acts as a prodrug, being oxidized to an o-quinone which reacts with essential enzymes containing sulphydryl groups resulting in interference with cell growth and proliferation. We have synthesized a range of analogues of 1 by varying the acyl portion of the amide with the intention of increasing the lipophilicity of the compounds. A modest increase in melanoma activity against six melanoma cell lines for these analogues could be correlated with increased lipophilicity. The most active of these compounds, N-[2-[(4-hydroxyphenyl)thiol]ethyl]cyclohexanecarboxamide (9), showed promising selectivity (lack of cytotoxicity) on the non-melanotic cell line SK-Mel-24 and on an ovarian cell line. A significant improvement in antimelanoma activity was observed with a new type of analogue containing three carbon atoms between the sulphur and nitrogen. The most active of these new analogues, N-[3-[(4-hydroxyphenyl)-thiolpropyl]-1-cyclohexanecarboxamide (15), had activity comparable to cisplatin against several cell lines and low cytotoxicity towards the non-melanotic cell line.     

Targeted Sialic Acid–Doxorubicin Prodrugs for Intracellular Delivery and Cancer Treatment

Purpose To evaluate a novel targeted anticancer prodrug consisting of several copies of sialic acid (SA, targeting moiety), doxorubicin (DOX), citric acid (multifunctional spacer) and poly(ethylene glycol) (PEG, carrier). Methods α, ω bis carboxyl PEG was covalently conjugated with multiple copies of SA and DOX through a citric acid spacer and characterized by proton nuclear magnetic resonance (¹HNMR), matrix-assisted laser desorption/ionization-time of flight (MALDI/TOF), and high-performance liquid chromatography (HPLC). The molecular models of conjugates were established using ChemDraw software. Stability, spontaneous and esterase-stimulated drug release was analyzed by HPLC. Cellular internalization (fluorescence microscopy) and cytotoxicity [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay] of free DOX and prodrugs were evaluated. Results ¹HNMR, MALDI/TOF, and HPLC showed the formation of the PEG prodrug conjugates. More than 40% of the drug was released from its conjugate in the presence of esterase enzyme, whereas the conjugate was stable at pH 7.4 in the absence of enzyme. Molecular modeling studies showed stable conformations of conjugates. The targeted prodrug conjugates with two copies of SA and DOX showed enhanced cytotoxicity when compared with non-targeted prodrugs and free DOX. Conclusions Targeting of the conjugate to cancer cells by SA with increased copies of targeting moiety and anticancer drug enhanced prodrug uptake by cancer cells and cytotoxicity of the prodrug.