Novel tricyclic pyrazole BRAF inhibitors with imidazole or furan central scaffolds.

V-RAF murine sarcoma viral oncogene homolog B1 (BRAF) is a serine/threonine-specific protein kinase that is mutated with high frequency in cutaneous melanoma, and many other cancers. Inhibition of mutant BRAF is an attractive therapeutic approach for the treatment of melanoma. A triarylimidazole BRAF inhibitor bearing a phenylpyrazole group (dimethyl-[2-(4-{5-[4-(1H-pyrazol-3-yl)-phenyl]-4-pyridin-4-yl-1H-imidazol-2-yl}-phenoxy)-ethyl]-amine, 1a) was identified as an active BRAF inhibitor. Based on this starting point, we synthesized a series of analogues leading to the discovery of 6-{2-[4-(4-methyl-piperazin-1-yl)-phenyl]-5-pyridin-4-yl-3H-imidazol-4-yl}-2,4-dihydro-indeno[1,2-c]pyrazole (1j), with nanomolar activity in three assays: inhibition of purified mutant BRAF activity in vitro; inhibition of oncogenic BRAF-driven extracellular regulated kinase (ERK) activation in BRAF mutant melanoma cell lines; and inhibition of proliferation in these cells.

Development of novel, highly potent inhibitors of V-RAF murine sarcoma viral oncogene homologue B1 (BRAF): increasing cellular potency through optimization of a distal heteroaromatic group.

We describe the design, synthesis, and optimization of a series of new inhibitors of V-RAF murine sarcoma viral oncogene homologue B1 (BRAF), a kinase whose mutant form (V600E) is implicated in several types of cancer, with a particularly high frequency in melanoma. Our previously described inhibitors with a tripartite A-B-C system (where A is a hinge binding pyrido[4,5-b]imidazolone system, B is an aryl spacer group, and C is a heteroaromatic group) were potent against purified (V600E)BRAF in vitro but were less potent in accompanying cellular assays. Substitution of different aromatic heterocycles for the phenyl based C-ring is evaluated herein as a potential means of improving the cellular potencies of these inhibitors. Substituted pyrazoles, particularly 3-tert-butyl-1-aryl-1H-pyrazoles, increase the cellular potencies without detrimental effects on the potency on isolated (V600E)BRAF. Thus, compounds have been synthesized that inhibit, with low nanomolar concentrations, (V600E)BRAF, its downstream signaling in cells [as measured by the reduction of the phosphorylation of extracellular regulated kinase (ERK)], and the proliferation of mutant BRAF-dependent cells. Concomitant benefits are good oral bioavailability and high plasma concentrations in vivo.

BRAF inhibitors based on an imidazo[4,5]pyridin-2-one scaffold and a meta substituted middle ring.

We recently reported on the development of a novel series of BRAF inhibitors based on a tripartite A-B-C system characterized by a para-substituted central aromatic core connected to an imidazo[4,5]pyridin-2-one scaffold and a substituted urea linker. Here, we present a new series of BRAF inhibitors in which the central phenyl ring connects to the hinge binder and substrate pocket of BRAF with a meta-substitution pattern. The optimization of this new scaffold led to the development of low-nanomolar inhibitors that permits the use of a wider range of linkers and terminal C rings while enhancing the selectivity for the BRAF enzyme in comparison to the para series.

Novel potent BRAF inhibitors: toward 1 nM compounds through optimization of the central phenyl ring.

BRAF, a serine/threonine specific protein kinase that is part of the MAPK pathway and acts as a downstream effector of RAS, is a potential therapeutic target in melanoma. We have developed a series of small-molecule BRAF inhibitors based on a 1H-imidazo[4,5-b]pyridine-2(3H)-one scaffold (ring A) as the hinge binding moiety and a number of substituted phenyl rings C that interact with the allosteric binding site. The introduction of various groups on the central phenyl ring B combined with appropriate A- and C-ring modifications afford very potent compounds that inhibit (V600E)BRAF kinase activity in vitro and oncogenic BRAF signaling in melanoma cells. Substitution on the central phenyl ring of a 3-fluoro, a naphthyl, or a 3-thiomethyl group improves activity to yield compounds with an IC(50) of 1 nM for purified (V600E)BRAF and nanomolar activity in cells.

Pyridoimidazolones as novel potent inhibitors of v-Raf murine sarcoma viral oncogene homologue B1 (BRAF).

BRAF is a serine/threonine kinase that is mutated in a range of cancers, including 50-70% of melanomas, and has been validated as a therapeutic target. We have designed and synthesized mutant BRAF inhibitors containing pyridoimidazolone as a new hinge-binding scaffold. Compounds have been obtained which have low nanomolar potency for mutant BRAF (12 nM for compound 5i) and low micromolar cellular potency against a mutant BRAF melanoma cell line, WM266.4. The series benefits from very low metabolism, and pharmacokinetics (PK) that can be modulated by methylation of the NH groups of the imidazolone, resulting in compounds with fewer H-donors and a better PK profile. These compounds have great potential in the treatment of mutant BRAF melanomas.

A novel technique to monitor carboxypeptidase G2 expression in suicide gene therapy using 19F magnetic resonance spectroscopy.

Development and evaluation of new anticancer drugs are expedited when minimally invasive biomarkers of pharmacokinetic and pharmacodynamic behaviour are available. Gene-directed enzyme prodrug therapy (GDEPT) is a suicide gene therapy in which the anticancer drug is activated in the tumor by an exogenous enzyme previously targeted by a vector carrying the gene. GDEPT has been evaluated in various clinical trials using several enzyme/prodrug combinations. The key processes to be monitored in GDEPT are gene delivery and expression, as well as prodrug delivery and activation. {4-[bis(2-chloroethyl)amino]-3,5-difluorobenzoyl}-L-glutamic acid, a prodrug for the GDEPT enzyme carboxypeptidase-G2 (CPG2; K(m) = 1.71 microM; k(cat) = 732 s(-1)), was measured with (19)F magnetic resonance spectroscopy (MRS). The 1 ppm chemical shift separation found between the signals of prodrug and activated drug (4-[bis(2-chloroethyl)amino]-3,5-difluorobenzoic acid) is sufficient for the detection of prodrug activation in vivo. However, these compounds hydrolyze rapidly, and protein binding broadens the MR signals. A new CPG2 substrate was designed with hydroxyethyl instead of chloroethyl groups (K(m) = 3.5 microM, k(cat) = 747 s(-1)). This substrate is nontoxic and stable in solution, has a narrow MRS resonance in the presence of bovine and foetal bovine albumin, and exhibits a 1.1 ppm change in chemical shift upon cleavage by CPG2. In cells transfected to express CPG2 in the cytoplasm (MDA MB 361 breast carcinoma cells and WiDr colon cancer cells), well-resolved (19)F MRS signals were observed from clinically relevant concentrations of the new substrate and its nontoxic product. The MRS conversion half-life (470 min) agreed with that measured by HPLC (500 min). This substrate is, therefore, suitable for evaluating gene delivery and expression prior to administration of the therapeutic agent.

Attenuated Salmonella targets prodrug activating enzyme carboxypeptidase G2 to mouse melanoma and human breast and colon carcinomas for effective suicide gene therapy.

PURPOSE: We engineered the oncolytic Salmonella typhimurium-derived bacterium VNP20009 as a vector to target delivery to tumors of the prodrug-activating enzyme carboxypeptidase G2 (CPG2) and to show enhanced antitumor efficacy on administration of different prodrugs. EXPERIMENTAL DESIGN: We characterized CPG2 expression in vectors by immunoblotting, immunofluorescence, and enzyme activity. We assessed prodrug activation by high-performance liquid chromatography. Target human tumor cell and bacterial vector cell cytotoxicity was measured by flow cytometry and colony-forming assays. Therapy was shown in two human tumor xenografts and one mouse allograft with postmortem analysis of bacterial and CPG2 concentration in the tumors. RESULTS: CPG2 is expressed within the bacterial periplasm. It activates prodrugs and induces cytotoxicity in human tumor cells but not in host bacteria. Following systemic administration, bacteria multiply within xenografts reaching 2 x 10(7)/g to 2 x 10(8)/g at 40 days postinoculation. The concentration of CPG2 in these tumors increases steadily to therapeutic levels of 1 to 6 units/g. The bacteria alone reduce the growth of the tumors. Subsequent administration of prodrugs further reduces significantly the growth of the xenografts. CONCLUSIONS: The bacteria multiply within tumors, resulting in a selective expression of CPG2. The CPG2-expressing bacteria alone reduce the growth of tumors. However, in the presence of prodrugs activated by CPG2, this oncolytic effect is greatly increased. We conclude that bacterial oncolytic therapy, combined with CPG2-mediated prodrug activation, has great potential in the treatment of a range of cancers.

Novel inhibitors of the v-raf murine sarcoma viral oncogene homologue B1 (BRAF) based on a 2,6-disubstituted pyrazine scaffold.

BRAF, a serine/threonine kinase, plays a key role in the development of certain types of cancer, particularly melanoma. 2-(3,4,5-Trimethoxyphenylamino)-6-(3-acetamidophenyl)-pyrazine, 1, was identified as a low micromolar (IC 50 = 3.5 microM) BRAF inhibitor from a high-throughput screen of a library of 23000 compounds. This compound was chosen as the starting point of a program aimed at developing inhibitors of mutant (V600E)BRAF. We have already reported on the optimization of the trimethoxyphenylamino moiety of 1. In this paper, we describe the synthesis of a series of compounds derived from 1 with the purpose of optimization of the pyrazine central core and the phenylacetamido moiety in order to increase the potency against (V600E)BRAF compared to CRAF. The biological activity of the new inhibitors was assessed against mutant (V600E)BRAF in vitro. Several compounds were identified with IC 50s of 300-500 nM for (V600E)BRAF, and all compounds that were assessed showed selectivity for (V600E)BRAF compared to CRAF by 5-->86-fold.

Suicide gene therapy of human colon carcinoma xenografts using an armed oncolytic adenovirus expressing carboxypeptidase G2.

We have designed a targeted systemic suicide gene therapy that combines the advantages of tumor-selective gene expression, using the human telomerase promoter (hTERT), with the beneficial effects of an oncolytic adenovirus to deliver the gene for the prodrug-activating enzyme carboxypeptidase G2 (CPG2) to tumors. Following delivery of the vector (AdV.hTERT-CPG2) and expression of CPG2 in cancer cells, the prodrug ZD2767P was administered for conversion by CPG2 to a cytotoxic drug. This system is sometimes termed gene-directed enzyme prodrug therapy (GDEPT). Here, we have shown that it is applicable to 10 human colorectal carcinoma cell lines with a direct correlation between viral toxicity and CPG2 production. SW620 xenografts were selected for analysis and were significantly reduced or eradicated after a single administration of AdV.hTERT-CPG2 followed by a prodrug course. The oncolytic effect of adenovirus alone did not result in DNA cross-links or apoptosis, whereas DNA cross-links and apoptosis occurred following prodrug administration, showing the combined beneficial effects of the GDEPT system. The apoptotic regions extended beyond the areas of CPG2 expression in the tumors, indicative of significant bystander effects in vivo. Higher concentrations of vector particles and CPG2 were found in the AdV.hTERT-CPG2 plus prodrug-treated tumors compared with the virus alone, showing an unexpected beneficial and cooperative effect between the vector and GDEPT. This is the first time that a tumor-selective GDEPT vector has been shown to be effective in colorectal carcinoma and that apoptosis and significant bystander effects have been identified as the mechanisms of cytotoxicity within the tumor.

Novel inhibitors of B-RAF based on a disubstituted pyrazine scaffold. Generation of a nanomolar lead.

B-RAF, a serine/threonine kinase, plays an important role in the development of certain classes of cancer, especially melanoma. As a result of high-throughput screening of a 23,000 compound library, 2-(3,4,5-trimethoxyphenylamino)-6-(3-acetamidophenyl)pyrazine, 1, was identified as a low micromolar (IC(50) = 3.5 microM) B-RAF inhibitor. This compound was chosen as the starting point of a program aimed at producing potent inhibitors of B-RAF. We have synthesized a series of 40 novel compounds, which involved extensive modifications to the 2-(3,4,5-trimethoxyphenylamino) moiety (ring A) of 1. Their biological profiles against isolated B-RAF and mutated B-RAF in a cellular assay have been determined. These efforts led to the identification of two compounds exhibiting activities lower than 800 nM against B-RAF.

Activated B-RAF is an Hsp90 client protein that is targeted by the anticancer drug 17-allylamino-17-demethoxygeldanamycin.

Hsp90 is a ubiquitously expressed molecular chaperone that folds, stabilizes, and functionally regulates many cellular proteins. The benzoquinone ansamysin 17-allylamino-17-demethoxygeldanamycin (17-AAG) is an anticancer drug that disrupts Hsp90 binding to its clients, causing their degradation through the ubiquitin-dependent proteasomal pathway. The protein kinase B-RAF is mutated in approximately 7% of human cancers. The most common mutation (approximately 90%) is (V600E)B-RAF, which has constitutively elevated kinase activity, stimulates cancer cell proliferation, and promotes survival. Here, we show that (V600E)B-RAF is an Hsp90 client protein that requires Hsp90 for its folding and stability. (V600E)BRAF is more sensitive to degradation by 17-AAG treatment than (WT)B-RAF and we show that the majority of the other mutant forms of B-RAF are also sensitive to 17-AAG-mediated proteasomal degradation. Our data show that B-RAF is an important target for 17-AAG in human cancer.

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.

Systemic gene-directed enzyme prodrug therapy of hepatocellular carcinoma using a targeted adenovirus armed with carboxypeptidase G2.

Hepatocellular carcinoma is the fifth most common cancer worldwide, and there is no effective therapy for unresectable disease. We have developed a targeted systemic therapy for hepatocellular carcinoma. The gene for a foreign enzyme is selectively expressed in the tumor cells and a nontoxic prodrug is then given, which is activated to a potent cytotoxic drug by the tumor-localized enzyme. This approach is termed gene-directed enzyme prodrug therapy (GDEPT). Adenoviruses have been used to target cancer cells, have an intrinsic tropism for liver, and are efficient gene vectors. Oncolytic adenoviruses produce clinical benefits, particularly in combination with conventional anticancer agents and are well tolerated. We rationalized that such adenoviruses, if their expression were restricted to telomerase-positive cancer cells, would make excellent gene vectors for GDEPT therapy of hepatocellular carcinoma. Here we use an oncolytic adenovirus to deliver the prodrug-activating enzyme carboxypeptidase G2 (CPG2) to tumors in a single systemic administration. The adenovirus replicated and produced high levels of CPG2 in two different hepatocellular carcinoma xenografts (Hep3B and HepG2) but not other tissues. GDEPT enhanced the adenovirus-alone therapy to elicit tumor regressions in the hepatocellular carcinoma models. This is the first time that CPG2 has been targeted and expressed intracellularly to effect significant therapy, showing that the combined approach holds enormous potential as a tumor-selective therapy for the systemic treatment of hepatocellular carcinoma.

B-RAF is a therapeutic target in melanoma.

B-RAF is a serine/threonine-specific protein kinase that is mutated in approximately 70% of human melanomas. However, the role of this signalling molecule in cancer is unclear. Here, we show that ERK is constitutively activated in melanoma cells expressing oncogenic B-RAF and that this activity is required for proliferation. B-RAF depletion by siRNA blocks ERK activity, whereas A-RAF and C-RAF depletion do not affect ERK signalling. B-RAF depletion inhibits DNA synthesis and induces apoptosis in three melanoma cell lines and we show that the RAF inhibitor BAY43-9006 also blocks ERK activity, inhibits DNA synthesis and induces cell death in these cells. BAY43-9006 targets B-RAF signalling in vivo and induces a substantial growth delay in melanoma tumour xenografts. Our data demonstrate that oncogenic B-RAF activates ERK signalling, induces proliferation and protects cells from apoptosis, demonstrating that it is an important therapeutic target and thus provides novel strategies for clinical management of melanoma and other cancers.

Self-immolative nitrogen mustards prodrugs cleavable by carboxypeptidase G2 (CPG2) showing large cytotoxicity differentials in GDEPT.

Nineteen novel potential self-immolative prodrugs and their corresponding drugs have been synthesized for gene-directed enzyme prodrug therapy (GDEPT) with carboxypeptidase G2 (CPG2) as the activating enzyme. The compounds are derived from o- and p-amino and p-methylamino aniline nitrogen mustards. Their aqueous stability, kinetics of drug release by CPG2, and cytotoxicity in the colon carcinoma cell line WiDr, expressing either surface-tethered CPG2 (stCPG2(Q)3) or control beta-galactosidase, are assessed. The effect of various structural features on stability, kinetics of activation, and biological activity is discussed. The p-methylamino prodrugs are the most stable compounds from this series, with the largest cytotoxicity differentials between CPG2-expressing and nonexpressing cells. The most potent compounds in all series are prodrugs of bis-iodo nitrogen mustards. 4-[N-[4'-Bis(2' '-iodoethyl)aminophenyl]-N'-methylcarbamoyloxymethyl]phenylcarbamoyl-l-glutamic acid, compound 39b, is 124-fold more cytotoxic to WiDr cells expressing CPG2 than to cells expressing beta-galactosidase. An additional six compounds show better cytotoxicity differential than the published N-[4-[(2-chloroethyl)(2-mesyloxyethyl)amino]benzoyl]-l-glutamic acid (CMDA) prodrug.

Three new prodrugs for suicide gene therapy using carboxypeptidase G2 elicit bystander efficacy in two xenograft models.

Three new prodrugs, [prodrug 1: 4-[bis(2-iodoethyl)amino]-phenyloxycarbonyl-L-glutamic acid; prodrug 2: 3-fluoro-4-[bis(2-chlorethyl)amino]benzoyl-L-glutamic acid; and prodrug 3: 3,5-difluoro-4-[bis(2-iodoethyl)amino]benzoyl-L-glutamic acid] have been assessed for use with a mutant of carboxypeptidase G2 (CPG2, glutamate carboxypeptidase, EC 3.4.17.11,) engineered to be tethered to the outer tumor cell surface (stCPG2(Q)3) as the activating enzyme in suicide gene therapy systems. All three of the prodrugs produce much greater cytotoxicity differentials between stCPG2(Q)3- and control beta-galactosidase (beta-gal)-expressing breast carcinoma MDA MB 361 and colon carcinoma WiDr cells (70- to 450-fold) than was previously observed (19- to 27-fold) with 4-[(2-chloroethyl)(2-mesyloxyethyl)amino]benzoyl-L-glutamic acid (CMDA). Prodrug 1 is the most effective antitumor agent in xenografts in mice inoculated with 100% stCPG2(Q)3-expressing MDA MB 361 cells, whereas prodrugs 2 and 3 are most effective when the percentage of stCPG2(Q)3-expressing cells is 50% or 10%. In nude mice bearing xenografts arising from inocula of 100% stCPG2(Q)3-expressing WiDr cells, prodrug 2 is the most effective antitumor agent. All three of the prodrugs produced histological evidence of substantial bystander cell killing in WiDr xenografts in which only 10% or 50% of the cells inoculated were expressing stCPG2(Q)3. We conclude that all three of the prodrugs are more effective therapeutically with stCPG2(Q)3 than is the previously described prodrug CMDA and, also, that the optimal choice of prodrug varies among different tumor types and that prodrugs, optimized for their bystander effect, are effective when only low percentages of cells in a tumor express CPG2.