A novel protocol for the one-pot borylation/Suzuki reaction provides easy access to hinge-binding groups for kinase inhibitors.

The one-pot borylation/Suzuki reaction is a very efficient means of accessing cross-coupling products of two aryl-halide partners that generally requires the use of specific catalysts or ligands and/or relatively long reaction times. This new microwave-assisted method provides a quick one-pot borylation/Suzuki reaction protocol that we applied to the synthesis of various bi- or poly-aryl scaffolds, including a variety of aryl and heteroaryl ring systems and the core frameworks of kinase inhibitors vemurafenib and GDC-0879.

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.

A novel vascular endothelial growth factor-directed therapy that selectively activates cytotoxic prodrugs.

We have generated fusion proteins between vascular endothelial growth factor (VEGF) and the bacterial enzyme carboxypeptidase G2 (CPG2) that can activate the prodrug 4-[(2-chloroethyl)(2-mesyloxyethyl)amino]benzoyl-L-glutamic acid (CMDA). Three asparagine residues of CPG2 were mutated to glutamine (CPG2(Q)3) to prevent glycosylation during secretion, and truncations of VEGF(165) were fused to either the C- or N-terminal of CPG2. The K(m) of the fusion proteins (37.5 microM) was similar to that of secreted CPG2(Q)3 (29.5 microM) but greater than that of wild-type CPG2 (8 microM). The affinity of the fusion proteins for VEGF receptor-2 (VEGFR2) (K(d)=0.5-1.1 nM) was similar to that of [(125)I]VEGF (K(d)=0.5 nM) (ELISA) or slightly higher (K(d)=1.3-9.6 nM) (competitive RIA). One protein, VEGF(115)-CPG2(Q)3-H(6), possessed 140% of the enzymic activity of secreted CPG2(Q)3, and had a faster half-maximal binding time for VEGFR2 (77 s), than the other candidates (330 s). In vitro, VEGF(115)-CPG2(Q)3-H(6) targeted CMDA cytotoxicity only towards VEGFR-expressing cells. The plasma half-life of VEGF(115)-CPG2(Q)3-H(6) in vivo was 3 h, comparable to equivalent values observed in ADEPT. We conclude that enzyme prodrug therapy using VEGF as a targeting moiety represents a promising novel antitumour therapy, with VEGF(115)-CPG2(Q)3-H(6) being a lead candidate.

Selective activation of anticancer prodrugs by monoclonal antibody-enzyme conjugates.

A great deal of interest has surrounded the activities of monoclonal antibodies (mAbs), and mAb-drug, toxin and radionuclide conjugates for the treatment of human cancers. In the last few years, a number of new mAb-based reagents have been clinically approved (Rituxan, Herceptin, and Panorex), and several others are now in advanced clinical trials. Successful therapeutic treatment of solid tumors with drug conjugates of such macromolecules must overcome the barriers to penetration within tumor masses, antigen heterogeneity, conjugated drug potency, and efficient drug release from the mAbs inside tumor cells. An alternative strategy for drug delivery involves a two-step approach to cancer therapy in which mAbs are used to localize enzymes to tumor cell surface antigens. Once the conjugate binds to the cancer cells and clears from the systemic circulation, antitumor prodrugs are administered that are catalytically converted to active drugs by the targeted enzyme. The drugs thus released are capable of penetrating within the tumor mass and eliminating both cells that have and have not bound the mAb-enzyme conjugate. Significant therapeutic effects have been obtained using a broad range of enzymes along with prodrugs that are derived from both approved anticancer drugs and highly potent experimental agents. This review focuses on the activities of several mAb-enzyme/prodrug combinations, with an emphasis on those that have provided mechanistic insight, clinical activity, novel protein constructs, and the potential for reduced immunogenicity.

Appropriate subcellular localisation of prodrug-activating enzymes has important consequences for suicide gene therapy.

Escherichia coli B nitroreductase (NR) has been expressed stably in MDA-MB-361 human breast adenocarcinoma cells either as the wild-type protein (wtNR), which is distributed evenly between the cytoplasmic and nuclear compartments, or targeted to the mitochondrion (mtNR). Whereas bacterial NR is active as a dimer, a proportion of wtNR is monomeric. In contrast, mtNR is mostly dimeric, suggesting that it adopts a more stable, native conformation. Despite this, when tested in gene-directed enzyme prodrug therapy cell cytotoxicity studies, cells expressing wtNR or mtNR had similar sensitivity to the prodrug CB1954 and mounted similar bystander killing effects. Furthermore, when short prodrug exposures were given, wtNR was more efficient at killing cells than mtNR. These data demonstrate that the site of enzyme expression and prodrug activation is an important variable that requires consideration in suicide gene therapy approaches.

In suicide gene therapy, the site of subcellular localization of the activating enzyme is more important than the rate at which it activates prodrug.

The bacterial enzyme carboxypeptidase G2 (CPG2) can be expressed both intracellularly (CPG2*) or tethered to the outer surface (stCPG2(Q)3) of mammalian cells, where it is able to activate mustard prodrugs for use in suicide gene therapy protocols. Here we compare the properties of CPG2 expressed in these two locations. CPG2 is active as a dimer, and one of the mutations required to block glycosylation of stCPG2(Q)3 destabilizes the dimers. Some of the mutations to this site partially correct the dimerization defect and recover a proportion of the activity. Surface tethering also recovers some enzyme activity, but through an unknown mechanism. The efficacy of CPG2 in these two locations is compared with the tumor cell lines A2780, SK-OV-3, and WiDr, which are sensitized to the prodrug 4-([2-chloroethyl][2-mesyloxyethyl]amino)benzoyl-L-glutamic acid (CMDA) by both CPG2* and stCPG2(Q)3 expression in suicide gene therapy protocols in vitro. We find that stCPG2(Q)3 is a more efficient mediator of CMDA-dependent cell killing than CPG2*. Lower levels of stCPG2(Q)3 activity are required to give cell killing that can only be achieved by higher levels of CPG2*. In bystander effect assays, low levels of stCPG2(Q)3 are required for efficient killing, whereas relatively high levels of CPG2* activity are required. Also, shorter exposures to prodrug are required for cell killing when stCPG2(Q)3 is expressed compared with when CPG2* is expressed. These data demonstrate that the location of the enzyme in the cell is more important than the enzyme activity as the determinant in mediating cytotoxicity.

Antibody-directed enzyme prodrug therapy: efficacy and mechanism of action in colorectal carcinoma.

In antibody-directed enzyme prodrug therapy, an enzyme conjugated to an antitumor antibody is given i.v. and localizes in the tumor. A prodrug is then given, which is converted to a cytotoxic drug selectively in the tumor. Ten patients with colorectal carcinoma expressing carcinoembryonic antigen received antibody-directed enzyme prodrug therapy with A5B7 F(ab')2 antibody to carcinoembryonic antigen conjugated to carboxypeptidase G2 (CPG2). A galactosylated antibody directed against the active site of CPG2 (SB43-gal) was given to clear and inactivate circulating enzyme. A benzoic acid mustard-glutamate prodrug was given when plasma enzyme levels had fallen to a predetermined safe level, and this was converted by CPG2 in the tumor into a cytotoxic form. Enzyme levels derived from quantitative gamma camera imaging and from direct measurements in plasma and tumor biopsies showed that the median tumor:plasma ratio of enzyme exceeded 10000:1 at the time of prodrug administration. Enzyme concentrations in the tumor (median, 0.47 units g(-1)) were sufficient to generate cytotoxic levels of active drug. The concentration of prodrug needed for optimal conversion (Km) of 3 microM was achieved. Prodrug conversion to drug was shown by finding detectable levels of drug in plasma. There was evidence of tumor response; one patient had a partial response, and six patients had stable disease for a median of 4 months after previous tumor progression (one of these six had a tumor marker response). Manageable neutropenia and thrombocytopenia occurred. Conditions for effective antitumor therapy were met, and there was evidence of tumor response in colorectal cancer.

Regressions of established breast carcinoma xenografts by carboxypeptidase G2 suicide gene therapy and the prodrug CMDA are due to a bystander effect.

The role of the bystander effect in the treatment of a human breast carcinoma xenograft was studied by suicide gene therapy with carboxypeptidase G2 (CPG2) and CMDA. Cells expressing enzymatically active surface-tethered bacterial CPG2 [stCPG2(Q)3] were mixed with control beta-galactosidase (beta-Gal)-expressing cells to give stCPG2(Q)3:beta-Gal ratios of, respectively: group 1, 0:100; group 2, 10:90; group 3, 50:50; and group 4, 100:0. Four days after injection of the cells into nude mice, the prodrug 4-[(2-chloroethyl)(2-mesyloxyethyl)amino]benzoyl-L-glutamic acid (CMDA) was administered. Tumor growth delay correlated well with the levels of stCPG2(Q)3 expression: group 1, 0 day delay; group 2, 10 days; group 3, 16 days; and group 4, 90 days. Similarly, the number of cures was strongly correlated to the levels of stCPG2(Q)3 activity: group 1, zero of six cured; group 2, one of six cured; group 3, three of six cured and group 4, four of six cured. There was a good correlation between CPG2 enzyme activity in the tumors and the number of cures. The majority of cells from groups 2 and 3 were apoptotic whereas those from group 1 were not, indicating a substantial bystander effect in the tumors. These results suggest that a bystander effect plays a major role in suicide gene therapy regimens with stCPG2(Q)3 and CMDA.

Enhancement of antibody-directed enzyme prodrug therapy in colorectal xenografts by an antivascular agent.

The irregular nature of solid tumor vasculature produces a heterogeneous distribution of antibody-targeted therapies within the tumor mass, which frequently results in reduced therapeutic efficacy. We have, therefore, combined two complementary therapies: Antibody-directed Enzyme Prodrug Therapy (ADEPT), which targets tumor cells, and an agent that selectively destroys tumor vasculature. A single i.p. dose (27.5 mg/kg) of the drug 5,6-dimethylxanthenone-4-acetic acid (DMXAA), given to nude mice bearing the LS174T colorectal xenograft, destroyed all but a peripheral rim of tumor cells, without enhancing survival. The ADEPT system, in which a pretargeted enzyme activates a prodrug, consisted of the F(ab')2 fragment of anti-carcinoembryonic antigen antibody A5B7 conjugated to the bacterial enzyme carboxypeptidase G2 and the prodrug 4-[(2-chloroethyl)(2-mesyloxyethyl)amino]benzoyl-L-glutamic acid, which was given i.p. in three doses of 500 mg/kg at 72, 84, and 96 h post-conjugate administration (25 units of carboxypeptidase G2). The antibody-enzyme conjugate could be selectively retained at approximately twice the control levels by administration of the antivascular agent at the time of optimal conjugate localization within the tumor (20 h post-conjugate administration), as demonstrated by gamma counting, phosphor plate image analysis, and active enzyme measurement. This resulted in significantly enhanced tumor growth inhibition in groups of six mice, compared to conventional ADEPT therapy, with no concomitant increase in systemic toxicity. In a separate experiment, aimed at trapping the prodrug within the tumor, a 16-fold increase over control values was produced (means, 44.8 versus 2.8 microg/g tumor) when DMXAA was given 4 h prior to 4-[(2-chloroethyl)(2-mesyloxyethyl)amino]benzoyl-L-glutamic acid. The therapeutic window was small, with no significant enhancement of prodrug retention when DMXAA was given at either earlier or later time points. This correlated with the time of vascular shut-down induced by the antivascular agent. We are currently investigating whether it is more advantageous to trap increased levels of conjugate or prodrug within the tumor for maximal enhancement of conventional ADEPT. These studies demonstrate that combined use of antibody-directed and antivascular therapies can significantly benefit the therapeutic outcome of either strategy alone.

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.

Serine and tyrosine phosphorylations cooperate in Raf-1, but not B-Raf activation.

The Raf family of serine/threonine protein kinases couple growth factor receptor stimulation to mitogen activated protein kinase activation, but their own regulation is poorly understood. Using phospho-specific antisera, we show that activated Raf-1 is phosphorylated on S338 and Y341. Expression of Raf-1 with oncogenic Ras gives predominantly S338 phosphorylation, whereas activated Src gives predominantly Y341 phosphorylation. Phosphorylation at both sites is maximal only when both oncogenic Ras and activated Src are present. Raf-1 that cannot interact with Ras-GTP is not phosphorylated, showing that phosphorylation is Ras dependent, presumably occurring at the plasma membrane. Mutations which prevent phosphorylation at either site block Raf-1 activation and maximal activity is seen only when both are phosphorylated. Mutations at S339 or Y340 do not block Raf-1 activation. While B-Raf lacks a tyrosine phosphorylation site equivalent to Y341 of Raf-1, S445 of B-Raf is equivalent to S338 of Raf-1. Phosphorylation of S445 is constitutive and is not stimulated by oncogenic Ras. However, S445 phosphorylation still contributes to B-Raf activation by elevating basal and consequently Ras-stimulated activity. Thus, there are considerable differences between the activation of the Raf proteins; Ras-GTP mediates two phosphorylation events required for Raf-1 activation but does not regulate such events for B-Raf.

Prodrugs for antibody- and gene-directed enzyme prodrug therapies (ADEPT and GDEPT).

Antibody- and gene-directed enzyme prodrug therapy are two-step targeting strategies designed to improve the selectivity of antitumour agents. The approaches are based on the activation of specially designed prodrugs by antibody-enzyme conjugates targeted to tumour-associated antigens (ADEPT) or by enzymes expressed by exogenous genes in tumour cells (GDEPT). Herein the design, synthesis, physico-chemical and biological properties, kinetics and clinical trials of the prodrugs and the enzymes carboxypeptidase G2 and nitroreductase are reviewed for ADEPT and GDEPT.

Recent developments in gene-directed enzyme prodrug therapy (GDEPT) for cancer.

Gene-directed enzyme prodrug therapy (GDEPT) is a promising two-step treatment for solid malignant tumors. In the first step, the gene for a foreign enzyme is administered and directed to the tumor, where it may be expressed using specific transcriptional elements. In the second step, prodrugs are administered and activated by the foreign enzyme expressed at the tumor. This review focuses on the progress from the end of 1997 to date. Important issues, such as viral and non-viral vectors, new enzyme/prodrug systems, new strategies, advances in the understanding of the bystander effects, the comparison of different systems used in GDEPT and clinical trials are outlined.

Self-immolative nitrogen mustard prodrugs for suicide gene therapy.

Four new potential self-immolative prodrugs derived from phenol and aniline nitrogen mustards, four model compounds derived from their corresponding fluoroethyl analogues and two new self-immolative linkers were designed and synthesized for use in the suicide gene therapy termed GDEPT (gene-directed enzyme prodrug therapy). The self-immolative prodrugs were designed to be activated by the enzyme carboxypeptidase G2 (CPG2) releasing an active drug by a 1, 6-elimination mechanism via an unstable intermediate. Thus, N-[(4-¿[4-(bis¿2-chloroethyl¿amino)phenoxycarbonyloxy]methyl¿pheny l)c arbamoyl]-L-glutamic acid (23), N-[(4-¿[4-(bis¿2-chloroethyl¿amino)phenoxycarbonyloxy]methyl¿pheno xy) carbonyl]-L-glutamic acid (30), N-[(4-¿[N-(4-¿bis[2-chloroethyl]amino¿phenyl)carbamoyloxy]methyl¿+ ++phen oxy)carbonyl]-L-glutamic acid (37), and N-[(4-¿[N-(4-¿bis[2-chloroethyl]amino¿phenyl)carbamoyloxy]methyl¿+ ++phen yl)carbamoyl]-L-glutamic acid (40) were synthesized. They are bifunctional alkylating agents in which the activating effects of the phenolic hydroxyl or amino functions are masked through an oxycarbonyl or a carbamoyl bond to a benzylic spacer which is itself linked to a glutamic acid by an oxycarbonyl or a carbamoyl bond. The corresponding fluoroethyl compounds 25, 32, 42, and 44 were also synthesized. The rationale was to obtain model compounds with greatly reduced alkylating abilities that would be much less reactive with nucleophiles compared to the corresponding chloroethyl derivatives. This enabled studies of these model compounds as substrates for CPG2, without incurring the rapid and complicated decomposition pathways of the chloroethyl derivatives. The prodrugs were designed to be activated to their corresponding phenol and aniline nitrogen mustard drugs by CPG2 for use in GDEPT. The synthesis of the analogous novel parent drugs (21b, 51) is also described. A colorectal cell line was engineered to express CPG2 tethered to the outer cell surface. The phenylenediamine compounds were found to behave as prodrugs, yielding IC50 prodrug/IC50 drug ratios between 20- and 33-fold (for 37 and 40) and differentials of 12-14-fold between CPG2-expressing and control LacZ-expressing clones. The drugs released are up to 70-fold more potent than 4-[(2-chloroethyl)(2-mesyloxyethyl)amino]benzoic acid that results from the prodrug 4-[(2-chloroethyl)(2-mesyloxyethyl)amino]benzoyl-L-glutamic acid (CMDA) which has been used previously for GDEPT. These data demonstrate the viability of this strategy and indicate that self-immolative prodrugs can be synthesized to release potent mustard drugs selectively by cells expressing CPG2 tethered to the cell surface in GDEPT.

Sensitization of colorectal and pancreatic cancer cell lines to the prodrug 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954) by retroviral transduction and expression of the E. coli nitroreductase gene.

Expression of genes encoding prodrug-activating enzymes can increase the susceptibility of tumor cells to prodrugs, and may ultimately achieve a better therapeutic index than conventional chemotherapy. CB1954 is a weak, monofunctional alkylating agent which can be activated by Escherichia coli nitroreductase to a potent dysfunctional alkylating agent which crosslinks DNA. We have inserted the nitroreductase gene into an LNCX-based retroviral vector, to allow efficient gene transfer and expression in colorectal (LS174T) and pancreatic (SUIT2, BxPC3, and AsPC1) cancer cell lines. A clone of LS174T cells expressing nitroreductase showed > 50-fold increased sensitivity to CB1954, and nitroreductase-expressing clones of pancreatic tumor lines were up to approximately 500-fold (SUIT2) more sensitive than parental cells. Concentrations of CB1954 minimally toxic to nontransduced cells achieved 100% cell death in a 50:50 mix of parental cells with SUIT2 cells expressing nitroreductase; and marked "bystander" cell killing was seen with just 10% of cells expressing nitroreductase. Significant bystander cell killing was dependent on a high cell density. In conjunction with regional delivery of vectors and tumor selectivity of cell entry and/or gene expression, nitroreductase and CB1954 may be an attractive combination for prodrug-activating enzyme gene therapy of colorectal and pancreatic cancer.

Gene-directed enzyme prodrug therapy: a review of enzyme/prodrug combinations.

Gene-directed enzyme prodrug therapy (GDEPT) is a promising, new, two-step treatment for cancer chemotherapy. In the first step, the gene for a foreign enzyme is administered and is directed to the tumour, where it is expressed by the use of specific promoters. In the second step, injected prodrugs are activated by the foreign enzyme. The design and synthesis of prodrugs able to undergo enzymatic activation in such systems is an essential component. This review focuses on the requirements which must be fulfilled by the components of GDEPT systems in order for this therapy to be considered a realistic possibility. A special emphasis is placed on the description of the prodrugs used in GDEPT protocols and the requirement for a bystander effect is also discussed.