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PDF file - 48K,Histological analysis of lungs from NOD-SCID mice bearing human melanoma xenografts following vehicle or dasatinib treatment

PDF file - 124K, Contains extended Materials and Methods for the manuscript

PDF file - 64K, Western blotting for phospho-EGFR in tumour xenografts

PDF file - 77K,Supplementary Figure Legends for Supplementary Figures S1 and S2

In order to identify new bioactive glasses (BGs) with optimal antioxidant properties, we carried out an evaluation of a series of cerium-doped BGs [Ce-BGs─H, K, and mesoporous bioactive glasses (MBGs)] loaded with different biomolecules, namely, gallic acid, polyphenols (POLY), and anthocyanins. Quantification of loading at variable times highlighted POLY on MBGs as the system with the highest loading. The ability to dismutate hydrogen peroxide (catalase-like activity) of the BGs evaluated is strongly correlated with cerium doping, while it is marginally decreased compared to the parent BG upon loading with biomolecules. Conversely, unloaded Ce-BGs show only a marginal ability to dismutate the superoxide anion (SOD)-like activity, while upon loading with biomolecules, POLY in particular, the SOD-like activity is greatly enhanced for these materials. Doping with cerium and loading with biomolecules give complementary antioxidant properties to the BGs investigated; combined with the persistent bioactivity, this makes these materials prime candidates for upcoming studies on biological systems.

Functionalized pyrazole-urea scaffolds are a common type II chemotype for the inhibition of protein kinases (PKs), binding simultaneously into the ATP-binding pocket with an ATP bioisostere and into a vicinal allosteric pocket with a pyrazole group. Standard approaches to the scaffold require multi-step synthesis of the ATP bioisostere followed by phosgene or triphosgene-mediated coupling with the substituted pyrazole group. Here we report an expedient approach to the chemotype, characterized by an optimized MW-assisted Suzuki coupling on easily accessed bromo-phenyl pyrazole ureas. The new protocol allowed quick access a large library of target analogues covering a broad chemical space of putative protein kinases inhibitors (PKIs).

Oncogenic BRAF is a critical driver of proliferation and survival and is thus a validated therapeutic target in cancer. We have developed a potent inhibitor, termed 1t (CCT239065), of the mutant protein kinase, V600EBRAF. 1t inhibits signaling downstream of V600EBRAF in cancer cells, blocking DNA synthesis, and inhibiting proliferation. Importantly, we show that 1t is considerably more selective for mutated BRAF cancer cell lines compared with wild-type BRAF lines. The inhibitor is well tolerated in mice and exhibits excellent oral bioavailability (F = 71%). Suppression of V600EBRAF-mediated signaling in human tumor xenografts was observed following oral administration of a single dose of 1t. As expected, the growth rate in vivo of a wild-type BRAF human tumor xenograft model is unaffected by inhibitor 1t. In contrast, 1t elicits significant therapeutic responses in mutant BRAF–driven human melanoma xenografts. Cancer Res; 70(20); 8036–44. ©2010 AACR.

Supplementary Figure 1 from A Novel, Selective, and Efficacious Nanomolar Pyridopyrazinone Inhibitor of V600EBRAF

Supplementary Table 1 from A Novel, Selective, and Efficacious Nanomolar Pyridopyrazinone Inhibitor of V600EBRAF

Supplementary Table 2 from A Novel, Selective, and Efficacious Nanomolar Pyridopyrazinone Inhibitor of V600EBRAF

Bioactive glasses (BGs) for biomedical applications are doped with therapeutic inorganic ions (TIIs) in order to improve their performance and reduce the side effects related to the surgical implant. Recent literature in the field shows a rekindled interest toward rare earth elements, in particular cerium, and their catalytic properties. Cerium-doped bioactive glasses (Ce-BGs) differ in compositions, synthetic methods, features, and in vitro assessment. This review provides an overview on the recent development of Ce-BGs for biomedical applications and on the evaluation of their bioactivity, cytocompatibility, antibacterial, antioxidant, and osteogenic and angiogenic properties as a function of their composition and physicochemical parameters.

Background KRAS is mutated in ∼90% of pancreatic ductal adenocarcinomas, ∼35% of colorectal cancers and ∼20% of non-small-cell lung cancers. There has been recent progress in targeting G12CKRAS specifically, but therapeutic options for other mutant forms of KRAS are limited, largely because the complexity of downstream signaling and feedback mechanisms mean that targeting individual pathway components is ineffective. Design The protein kinases RAF and SRC are validated therapeutic targets in KRAS-mutant pancreatic ductal adenocarcinomas, colorectal cancers and non-small-cell lung cancers and we show that both must be inhibited to block growth of these cancers. We describe CCT3833, a new drug that inhibits both RAF and SRC, which may be effective in KRAS-mutant cancers. Results We show that CCT3833 inhibits RAF and SRC in KRAS-mutant tumors in vitro and in vivo, and that it inhibits tumor growth at well-tolerated doses in mice. CCT3833 has been evaluated in a phase I clinical trial (NCT02437227) and we report here that it significantly prolongs progression-free survival of a patient with a G12VKRAS spindle cell sarcoma who did not respond to a multikinase inhibitor and therefore had limited treatment options. Conclusions New drug CCT3833 elicits significant preclinical therapeutic efficacy in KRAS-mutant colorectal, lung and pancreatic tumor xenografts, demonstrating a treatment option for several areas of unmet clinical need. Based on these preclinical data and the phase I clinical unconfirmed response in a patient with KRAS-mutant spindle cell sarcoma, CCT3833 requires further evaluation in patients with other KRAS-mutant cancers., Highlights • We synthesized new drug, CCT3833, that inhibits both RAF and SRC, and so may be effective in KRAS-mutant cancers. • CCT3833 inhibits both signaling pathways in vitro and in vivo, inhibits tumor growth and leads to regressions in mice. • CCT3833 significantly prolonged progression-free survival of a patient with a G12VKRAS spindle cell sarcoma. • CCT3833 is a potential treatment option for several areas of unmet clinical need.

The scale-up of a chiral bicyclic homopiperazine of pharmaceutical interest was investigated. The outcome and safety profile of a key batch ring-expansion step via Schmidt rearrangement was improved using continuous-flow chemistry. The selectivity of nitrogen insertion for the ring expansion was improved via an alternative photochemical oxaziridine rearrangement under mild conditions, which when converted to continuous-flow in a simple and efficient flow reactor allowed the first photochemical scale-up of a homopiperazine.

The anaplastic lymphoma kinase (ALK) is abnormally expressed and hyperactivated in a number of tumors and represents an ideal therapeutic target. Despite excellent clinical responses to ALK inhibition, drug resistance still represents an issue and novel compounds that overcome drug-resistant mutants are needed. We designed, synthesized, and evaluated a large series of azacarbazole inhibitors. Several lead compounds endowed with submicromolar potency were identified. Compound 149 showed selective inhibition of native and mutant drug-refractory ALK kinase in vitro as well as in a Ba/F3 model and in human ALK+ lymphoma cells. The three-dimensional (3D) structure of a 149:ALK-KD cocrystal is reported, showing extensive interaction through the hinge region and the catalytic lysine 1150.

Cerium-doped bioactive glasses (Ce-BGs) are implant materials that present high biocompatibility, modulate the levels of reactive oxygen species, and exert antimicrobial activity. The potential of BGs, 45S5, and K50S derived glasses doped with CeO2 (1.2, 3.6, and 5.3 mol%) to inhibit the growth of pathogen microbes was thoroughly investigated according to the ISO 22196:2011 method properly adapted. A significant reduction of the E. coli charge was detected in all glasses, including the BGs without cerium. The evolution of pH of the medium not inoculated following the immersion of the Ce-BGs was monitored. The presence of cerium did not affect markedly the pH trend, which increased rapidly for both compositions. The change of pH was strongly mitigated by the presence of 200 mM phosphate buffer pH 7.0 (PB) in the medium. In media buffered by PB, the growth of E. coli, Pseudomonas aeruginosa, Listeria monocytogenes, Staphylococcus aureus, and C. albicans was not affected by the presence of BGs doped or not with cerium, suggesting that the antibacterial activity of Ce-BGs is linked to the increase of environmental pH rather than to specific ion effects. However, Ce-BGs resulted promising biomaterials that associate low toxicity to normal cells to a considerable antimicrobial effect, albeit the latter is not directly associated with the presence of cerium.

The Anaplastic Lymphoma Kinase (ALK) is a therapeutic target for personalized medicine in selected cancers. Despite excellent clinical responses to ALK inhibitors, most patients develop drug resistance and relapse. New compounds with alternative binding modes are needed to overcome resistant mutants. Here we describe a medicinal chemistry effort to the design and development of novel ALK inhibitors based on a 4,6-substituted α-carboline scaffold. Active compounds were able to inhibit the gatekeeper L1196M mutant, in several cases better than the wild-type enzyme. Compound 43 showed potent non-ATP-competitive inhibition of wild-type and mutant ALK, including G1202R, in biochemical and cellular assays, as well as in xenograft mouse models.

Unprotected exposure to solar UV is the main recognized cause of the spreading of skin cancer that has occurred in the last decades. Organic and inorganic UV filters are the main defense against UV exposure and have become ubiquitous in personal-care products, packaging, plastics, dyes, and many other sectors. UV filters still have some stability and efficacy issues making it important to find stable and broad-spectrum UV absorbers, safer for human health and the environment. In this work UV filters octinoxate and avobenzone were encapsulated into zeolites with different chemistry and topology leading to the production of hybrid zeolites/organic filter sunscreens. The obtained materials were deeply characterized and their filtering capacity evaluated. The encapsulation of UV filters in cationic zeolites resulted in an enhanced UV filtering capacity, whereas that in acid zeolites seem less effective in absorbing UV radiation, with emerging visible coloring. The enhancement of UV filtering power is of paramount importance for the future development and exploitation of ZEOfilters, possibly being the key to reduce the quantity of actives, stabilizers, and co-formulants employed, with benefits for humankind and the environment.

Aphanizomenon flos-Aquae (AFA) cyanobacteria from Klamath Lake (Oregon) are considered a "superfood" due to their broad nutritional profile that has proved to have health-enhancing properties. The AFA metabolome is quite complex. Here, we present a study that, combining multinuclear 1H, 31P, and 13C Nuclear Magnetic Resonance (NMR) spectroscopy and high-resolution mass spectrometry, led to the detection of uncommon phosphorylated metabolites in AFA. We focused our attention on 31P NMR signals at 20 ppm, a chemical shift that usually points to the presence of phosphonates. The molecules contributing to 20 ppm 31P NMR signals revealed, instead, to be nucleoside 2′,3′-cyclic monophosphates. These metabolites were fully characterized by multinuclear 1H, 31P, and 13C NMR spectroscopy and high-resolution mass spectrometry.

We report the synthesis and preliminary evaluation of (1S,2S,4R)-7,7-dimethyl-1-phenylbicyclo[2.2.1]heptan-2-ol 1a, which was obtained in 3 steps from inexpensive starting materials, as a chiral auxiliary. The potential for asymmetric induction was investigated by carrying out aza-Diels-Alder reactions with cyclopentadiene and imine derivatives of 1a from (R)-(+)-1-phenylethylamine, (S)-(−)-1-phenylethylamine and benzylamine. The results showed marked exo selectivity and good diastereoisomeric excess when 1a was combined with (R)-(+)-1-phenylethylamine. These results are comparable with those reported using (−)-8-phenylmenthol, suggesting that 1a can represent an economically viable alternative to current chiral auxiliaries.

Asbestos is a commercial term indicating six natural silicates with asbestiform crystal habit. Of these, five are double-chain silicates (amphibole) and one is a layer silicate (serpentine asbestos or chrysotile). Although all species are classified as human carcinogens, their degree of toxicity is still a matter of debate. Amphibole asbestos species are biopersistent in the human lungs and exert their chronic toxic action for decades, whereas chrysotile is not biopersistent and transforms into an amorphous silica structure prone to chemical/physical clearance when exposed to the acidic environment created by the alveolar macrophages. There is evidence in the literature of the toxicity of chrysotile, but its limited biopersistence is thought to explain the difference in toxicity with respect to amphibole asbestos. To date, no comprehensive model describing the toxic action of chrysotile in the lungs is available, as the structure and toxic action of the product formed by the biodissolution of chrysotile are unknown. This work is aimed at fulfilling this gap and explaining the toxic action in terms of structural, chemical, and physical properties. We show that chrysotile's fibrous structure induces cellular damage, mainly through physical interactions. Based on our previous work and novel findings, we propose the following toxicity model: inhaled chrysotile fibers exert their toxicity in the alveolar space by physical and biochemical action. The fibers are soon leached by the intracellular acid environment into a product with residual toxicity, and the dissolution process liberates toxic metals in the intracellular and extracellular environment.

The cytocompatibility of potential bioactive cerium-containing (Ce3+/Ce4+) glasses is here investigated by preparing three different glasses with increasing amount of doping CeO2 (1.2, 3.6 and 5.3 mol% of CeO2, called BG_1.2, BG_3.6 and BG_5.3, respectively) based on 45S5 Bioglass&reg, (called BG). These materials were characterized by Environmental Scanning Electron Microscopy (ESEM) and infrared spectroscopy (FTIR) after performing bioactivity tests in Dulbecco&rsquo, s Modified Eagle Medium (DMEM) solution, and the ions released in solution were determined by Inductively Coupled Plasma Mass Spectrometry (ICP-MS) and Optical Emission Spectrometry (ICP-OES). The data obtained clearly show that the glass surfaces of BG, BG_1.2 and BG_3.6 were covered by hydroxyapatite (HA), while BG_5.3 favored the formation of a cerium phosphate crystal phase. The cytotoxicity tests were performed using both murine long bone osteocyte-like (MLO-Y4) and mouse embryonic fibroblast (NIH/3T3) cell lines. The cerium-containing bioactive glasses show an increment in cell viability with respect to BG, and at long times, no cell aggregation and deformation were observed. The proliferation of NIH/3T3 cells increased with the cerium content in the glasses, in particular, BG_3.6 and BG_5.3 showed a higher proliferation of cells than the negative control. These results highlight and enforce the proposal of cerium-doped bioactive glasses as a new class of biomaterials for hard-tissue applications.

In this work, we investigate the ability of mesoporous bioactive glasses (MBGs) modified with cerium ions (Ce 3+ /Ce 4+ ) to act as catalase and superoxide dismutase (SOD) mimetic materials. We have previously reported that the catalytic properties of bioactive Ce-containing glasses based on 45S5 Bioglass ® and synthesized via melting are influenced by: i) composition (presence/absence of P 2 O 5 ); ii) Ce 3+ /Ce 4+ molar ratio. The introduction of cerium species drastically decreased the bioactivity in terms of Hydroxyapatite formation during bioactivity tests in vitro . We thus decided to add cerium to MBGs, a class of glasses with improved bioactivity with respect to classical molten glasses. MBGs exhibit a high surface area and their reactivity is increased with respect to the molten glasses, and they are able to induce the formation of Hydroxyapatite over the surface within shorter times with respect to the 45S5. The catalase and SOD mimetic activity tests revealed that the Ce-MGB are able to act as mimetic materials for the two enzymes. Both Infrared Spectroscopy and X-ray diffraction analysis have confirmed the presence of Hydroxyapatite over both 80SiO 2 –15CaO–5P 2 O 5 , and 80SiO 2 –20CaO samples modified by 5.3% mol of CeO 2 ; simultaneously the glasses maintain a good catalase activity. Moreover, the 80SiO 2 –15CaO–5P 2 O 5 potential bioactive glasses showed SOD mimetic activity. These results highlight that it is possible to obtain a glass with both antioxidant and bioactivity properties.

Summary BRAF and MEK inhibitors are effective in BRAF mutant melanoma, but most patients eventually relapse with acquired resistance, and others present intrinsic resistance to these drugs. Resistance is often mediated by pathway reactivation through receptor tyrosine kinase (RTK)/SRC-family kinase (SFK) signaling or mutant NRAS, which drive paradoxical reactivation of the pathway. We describe pan-RAF inhibitors (CCT196969, CCT241161) that also inhibit SFKs. These compounds do not drive paradoxical pathway activation and inhibit MEK/ERK in BRAF and NRAS mutant melanoma. They inhibit melanoma cells and patient-derived xenografts that are resistant to BRAF and BRAF/MEK inhibitors. Thus, paradox-breaking pan-RAF inhibitors that also inhibit SFKs could provide first-line treatment for BRAF and NRAS mutant melanomas and second-line treatment for patients who develop resistance., Graphical Abstract, Highlights • pan-RAF inhibitors also inhibit SRC family kinases • The compounds do not induce paradoxical activation of ERK in RAS mutant cells • The compounds are active in BRAF and NRAS mutant melanomas • The compounds are active in PDXs resistant to BRAF or BRAF plus MEK inhibitors, Girotti et al. describe two pan-RAF inhibitors that also inhibit SRC-family kinases. These compounds do not drive paradoxical MEK/ERK activation and can inhibit MEK in NRAS mutant cells. Moreover, the agents can overcome resistance to clinical BRAF or combination BRAF/MEK inhibitors in patient-derived xenografts.

We generated cell lines resistant to BRAF inhibitors and show that the EGF receptor (EGFR)–SRC family kinase (SFK)–STAT3 signaling pathway was upregulated in these cells. In addition to driving proliferation of resistant cells, this pathway also stimulated invasion and metastasis. EGFR inhibitors cooperated with BRAF inhibitors to block the growth of the resistant cells in vitro and in vivo, and monotherapy with the broad specificity tyrosine kinase inhibitor dasatinib blocked growth and metastasis in vivo. We analyzed tumors from patients with intrinsic or acquired resistance to vemurafenib and observed increased EGFR and SFK activity. Furthermore, dasatinib blocked the growth and metastasis of one of the resistant tumors in immunocompromised mice. Our data show that BRAF inhibitor-mediated activation of EGFR–SFK–STAT3 signaling can mediate resistance in patients with BRAF-mutant melanoma. We describe 2 treatments that seem to overcome this resistance and could deliver therapeutic efficacy in patients with drug-resistant BRAF-mutant melanoma. Significance: Therapies that target the driver oncogenes in cancer can achieve remarkable responses if patients are stratified for treatment. However, as with conventional therapies, patients often develop acquired resistance to targeted therapies, and a proportion of patients are intrinsically resistant and fail to respond despite the presence of an appropriate driver oncogene mutation. We found that the EGFR/SFK pathway mediated resistance to vemurafenib in BRAF-mutant melanoma and that BRAF and EGFR or SFK inhibition blocked proliferation and invasion of these resistant tumors, providing potentially effective therapeutic options for these patients. Cancer Discov; 3(2); 158–67. ©2012 AACR. This article is highlighted in the In This Issue feature, p. 125

After its identification as an oncogene in 2002, mutant BRAF has become the target of a number of drug discovery programmes, primarily aimed at the treatment of late stage or unresectable melanoma. Some of the drugs thus developed, such as vemurafenib and dabrafenib, show impressive responses in melanoma patients harbouring a BRAF mutation.This review summarises the patent literature on BRAF from 2006 to 2012, focusing on the specific areas of inhibitors of mutant BRAF, drug combinations including BRAF inhibitors, diagnostic methods for use with mutant BRAF inhibitorsdiagnosis and treatment of mutant BRAF cancers resistant to BRAF inhibitors.Whilst these first-generation BRAF inhibitors initially mediate excellent responses in late stage or unresectable melanoma patients bearing the V600 mutation, resistance usually occurs and patients eventually relapse. The patent literature for new BRAF inhibitors and therapies reflects the desire to develop second-generation drugs able to overcome this resistance and combination treatments that increase the efficiency of current mutant BRAF inhibitors.

An one-pot sequence of borylation and Suzuki reaction is used for the preparation of 20 bi- and polyarenes, e.g. vemurafenib precursor (IIId) and analogues (V) of kinase inhibitor GDC-0879.

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.

Cutaneous squamous-cell carcinomas and keratoacanthomas are common findings in patients treated with BRAF inhibitors.We performed a molecular analysis to identify oncogenic mutations (HRAS, KRAS, NRAS, CDKN2A, and TP53) in the lesions from patients treated with the BRAF inhibitor vemurafenib. An analysis of an independent validation set and functional studies with BRAF inhibitors in the presence of the prevalent RAS mutation was also performed.Among 21 tumor samples, 13 had RAS mutations (12 in HRAS). In a validation set of 14 samples, 8 had RAS mutations (4 in HRAS). Thus, 60% (21 of 35) of the specimens harbored RAS mutations, the most prevalent being HRAS Q61L. Increased proliferation of HRAS Q61L-mutant cell lines exposed to vemurafenib was associated with mitogen-activated protein kinase (MAPK)-pathway signaling and activation of ERK-mediated transcription. In a mouse model of HRAS Q61L-mediated skin carcinogenesis, the vemurafenib analogue PLX4720 was not an initiator or a promoter of carcinogenesis but accelerated growth of the lesions harboring HRAS mutations, and this growth was blocked by concomitant treatment with a MEK inhibitor.Mutations in RAS, particularly HRAS, are frequent in cutaneous squamous-cell carcinomas and keratoacanthomas that develop in patients treated with vemurafenib. The molecular mechanism is consistent with the paradoxical activation of MAPK signaling and leads to accelerated growth of these lesions. (Funded by Hoffmann-La Roche and others; ClinicalTrials.gov numbers, NCT00405587, NCT00949702, NCT01001299, and NCT01006980.).

Over the last few years, BRAF has emerged as a validated target in melanoma. This review summarises recent advances in the development of BRAF inhibitors, focussing on agents that have been assessed clinically.

Oncogenic BRAF is a critical driver of proliferation and survival and is thus a validated therapeutic target in cancer. We have developed a potent inhibitor, termed 1t (CCT239065), of the mutant protein kinase, V600EBRAF. 1t inhibits signaling downstream of V600EBRAF in cancer cells, blocking DNA synthesis, and inhibiting proliferation. Importantly, we show that 1t is considerably more selective for mutated BRAF cancer cell lines compared with wild-type BRAF lines. The inhibitor is well tolerated in mice and exhibits excellent oral bioavailability (F = 71%). Suppression of V600EBRAF-mediated signaling in human tumor xenografts was observed following oral administration of a single dose of 1t. As expected, the growth rate in vivo of a wild-type BRAF human tumor xenograft model is unaffected by inhibitor 1t. In contrast, 1t elicits significant therapeutic responses in mutant BRAF–driven human melanoma xenografts. Cancer Res; 70(20); 8036–44. ©2010 AACR.

Mutated BRAF serine/threonine kinase is implicated in several types of cancer, with particularly high frequency in melanoma and colorectal carcinoma. We recently reported on the development of BRAF inhibitors based on a tripartite A-B-C system featuring an imidazo[4,5]pyridin-2-one group hinge binder. Here we present the design, synthesis, and optimization of a new series of inhibitors with a different A-B-C system that has been modified by the introduction of a range of novel hinge binders (A ring). The optimization of the hinge binding moiety has enabled the development of compounds with low nanomolar potencies in both BRAF inhibition and cellular assays. These compounds display optimal pharmacokinetic properties that warrant further in vivo investigations.

The fluxional behaviour of [Pd(η 2 -fn)(N-SMe)] ( 2 ) (fn = fumaronitrile, N-SMe = 2-methylthiomethylpyridine) and of [Pd(η 2 -tmetc)( N - N ′-4-anisyl)] ( 3 ) (tmetc = tetramethylethylenetetracarboxylate, N - N ′-4-anisyl = 2-(4-methoxyphenyliminomethane)pyridine) were monitored by 1 H NMR spectroscopy and quantitatively determined by line-shape analyses (for 2 ) and selective inversion recovery experiments (for 3 ). The coalescence of the AB multiplet of fn hydrogens of 2 is concentration dependent and presents a strongly negative Δ S ≠ , suggesting the intermediacy of a dimeric complex and ruling out the hypothesis of olefin rotation. The accurate evaluation of all spectral features also allows determination of the approaching mode of the monomeric units. The inversion transfer between the tmetc methyls of 3 reveals a true propeller-like olefin rotation. The presence of a nucleophilic electron pair at sulfur in 2 triggers the formation of the dimeric intermediate.

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.

Background: Resistance to imatinib is an important clinical issue in the treatment of Philadelphia chromosomepositive leukemias which is being tackled by the development of new, more potent drugs, such as the dual Src/Abl tyrosine kinase inhibitors dasatinib and bosutinib and the imatinib analog nilotinib. In the current study we describe the design, synthesis and biological properties of an imatinib analog with a chlorine-substituted benzamide, namely compound 584 (cmp-584). Design and Methods: To increase the potency, we rationally designed cmp-584, a compound with enhanced shape complementarity with the kinase domain of Abl. cmp-584 was synthesized and characterized in vitro against a panel of 67 serine/threonine and tyrosine kinases using radioactive and enzyme-linked immunosorbent kinase assays. We studied inhibitory cellular activity using Bcr/Abl-positive human cell lines, murine transfectants in proliferation experiments, and a murine xenotransplanted model. Kinase assays on isolated Bcr/Abl protein were also performed. Finally, we used a wash-out approach on whole cells to study the binding kinetics of the inhibitor. Results: cmp-584 showed potent anti-Abl activity both on recombinant protein (IC50: 8 nM) and in cell-based assays (IC50: 0.1-10 nM). The drug maintained inhibitory activity against platelet-derived growth factor receptors and c-KIT and was also active against Lyn (IC50: 301 nM). No other kinase of the panel was inhibited at nanomolar doses. cmp-584 was 20- to 300-fold more active than imatinib in cells. This superior activity was evident in intact cells, in which full-length Bcr-Abl is present. In vivo experiments confirmed the activity of cmp-584. Wash-out experiments showed that short exposure to the drug impaired cell proliferation and Bcr-Abl phosphorylation for a substantially longer period of time than imatinib. Conclusions: The present results suggest a slower off-rate (dissociation rate) of cmp-584 compared to imatinib as an explanation for the increased cellular activity of the former. ©2008 Ferrata Storti Foundation.

Well-defined. rigid molecular domes are obtained via Substitution of the chlorine atoms of hexachlorobenzotrinorbornadiene syn-3. efficiently synthesized in 1:2 ratio along with its anti-isomer by CuTC-promoted cyclotrimerization of 3-bromo-2-trimethylstannylnorbornadiene 8. The three dichlorovinyl functions at the edge of syn-3 are displaced by sulfur nucleophiles and Grignard reagents with Ni(II)Cl(2)dppe as catalyst, gaining the persubstituted vinyl sulfides 9 and 10, and persubstituted methyl 12 and phenylethynyl 13.

Generally, the bimolecular nucleophilic substitution at the saturated carbon (SN2) and the mechanistically similar substitution at the vinyl carbon (SN2-Vin) cannot be quantitatively compared, because of the many interfering steric and electronic factors. The cis and trans rearrangements of trans c-2,t-3-di-tert-butyl-r-1-methylthiiranium ion 5a into thietanium ions 6a and 6b and that of 2,3-di-tert-butyl-1-methylthiirenium ion 7 into thietium ion 8 can be be compared both experimentally and computationally. They occur with intramolecular SN2 and SN2-Vin mechanisms respectively and are both almost exclusively governed by the nucleofugality of the sulfonium leaving group.

Copper(I) thiophen-2-carboxylate was successfully employed in the trimerization of [2.2.1] bicyclic vic-bromotrimethyltin olefins (in their racemic composition), bearing different functionalities, to invariably obtain almost quantitative yields of the syn and anti tris-annelated benzenes. The two isomers come in different ratios, smaller than or equal to the statistical 1:3 ratio, depending on the steric hindrance opposed by the functionalities. In the case of enantiopure (3-bromo-4,7,7-trimethylbicyclo[2.2.1]hept-2-en-2-yl)trimethylstannane, the 1:9 ratio found with Cu(NO(3))(2).3H(2)O increases to 1:6.

Melanoma is a highly metastatic and lethal form of skin cancer. The protein kinase BRAF is mutated in about 40% of melanomas, and BRAF inhibitors improve progression-free and overall survival in these patients. However, after a relatively short period of disease control, most patients develop resistance because of reactivation of the RAF-ERK (extracellular signal-regulated kinase) pathway, mediated in many cases by mutations in RAS. We found that BRAF inhibition induces invasion and metastasis in RAS mutant melanoma cells through a mechanism mediated by the reactivation of the MEK (mitogen-activated protein kinase kinase)-ERK pathway, increased expression and secretion of interleukin 8, and induction of protease-dependent invasion. These events were accompanied by a cell morphology switch from predominantly rounded to predominantly elongated cells. We also observed similar responses in BRAF inhibitor-resistant melanoma cells. These data show that BRAF inhibitors can induce melanoma cell invasion and metastasis in tumors that develop resistance to these drugs.

The protein kinase BRAF is mutated ∼40% of human melanomas. BRAF is a component of the RAS/RAF/MEK/ERK pathway and BRAF or MEK inhibitors increase progression-free and overall survival in melanoma patients with BRAF mutations. However, most patients relapse with acquired resistance and ∼20% of patients present intrinsic resistance and do not respond to these drugs. We describe here two novel compounds that target mutant BRAF and wild-type CRAF. Our compounds inhibited the growth of melanoma cells that were resistant to BRAF-selective inhibitors. ERK pathway reactivation is responsible for resistance to BRAF targeted therapies in ∼60% of the patients and in ∼25% of patients resistance is driven by acquisition of mutations in NRAS. We show that our compounds inhibited the growth of melanoma cells that were resistant to BRAF-selective inhibitors due to pathway reactivation mediated by different mechanisms. We show that the drugs were active against patient derived xenografts (PDXs) from patients with acquired or intrinsic resistance to BRAF-selective inhibitors and in whose tumors resistance was associated with ERK pathway reactivation. Further, our compounds are active in a PDX from a patient whose tumor developed acquired resistance to a combination of a BRAF-selective plus a MEK inhibitor and associated with acquisition of an NRAS mutation. Thus, our panRAF inhibitors can inhibit melanomas with different mechanisms of acquired or intrinsic resistance to BRAF-selective and BRAF-selective/MEK inhibitor combinations, potentially providing first-line treatment for naïve patients and second-line treatments for a range of relapsed patients. Citation Format: Maria R. Girotti, Filipa Lopes, Natasha Preece, Dan Niculescu-Duvaz, Alfonso Zambon, Lawrence Davies, Steven Whittaker, Grazia Saturno, Amaya Viros, Malin Pedersen, Bart MJM Suijkerbuijk, Delphine Menard, Robert Mcleary, Louise Johnson, Laura Fish, Sarah Ejiama, Berta Sanchez-Laorden, Neil Carragher, Kenneth Macleod, Garry Ashton, Anna Marusiak, Alberto Fusi, John Brognard, Margaret Frame, Paul Lorigan, Caroline J. Springer, Richard Marais. Novel panRAF inhibitors active in melanomas that are resistant to BRAF-selective, or BRAF-selective/MEK inhibitor combinations. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3704. doi:10.1158/1538-7445.AM2014-3704