Phenotypic Plasticity in Circulating Tumor Cells Is Associated with Poor Response to Therapy in Metastatic Breast Cancer Patients.

Circulating tumor cells (CTCs) are indicators of metastatic spread and progression. In a longitudinal, single-center trial of patients with metastatic breast cancer starting a new line of treatment, a microcavity array was used to enrich CTCs from 184 patients at up to 9 timepoints at 3-month intervals. CTCs were analyzed in parallel samples from the same blood draw by imaging and by gene expression profiling to capture CTC phenotypic plasticity. Enumeration of CTCs by image analysis relying primarily on epithelial markers from samples obtained before therapy or at 3-month follow-up identified the patients at the highest risk of progression. CTC counts decreased with therapy, and progressors had higher CTC counts than non-progressors. CTC count was prognostic primarily at the start of therapy in univariate and multivariate analyses but had less prognostic utility at 6 months to 1 year later. In contrast, gene expression, including both epithelial and mesenchymal markers, identified high-risk patients after 6-9 months of treatment, and progressors had a shift towards mesenchymal CTC gene expression on therapy. Cross-sectional analysis showed higher CTC-related gene expression in progressors 6-15 months after baseline. Furthermore, patients with higher CTC counts and CTC gene expression experienced more progression events. Longitudinal time-dependent multivariate analysis indicated that CTC count, triple-negative status, and CTC expression of FGFR1 significantly correlated with inferior progression-free survival while CTC count and triple-negative status correlated with inferior overall survival. This highlights the utility of protein-agnostic CTC enrichment and multimodality analysis to capture the heterogeneity of CTCs.

Autoimmunity affecting the biliary tract fuels the immunosurveillance of cholangiocarcinoma.

Cholangiocarcinoma (CCA) results from the malignant transformation of cholangiocytes. Primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC) are chronic diseases in which cholangiocytes are primarily damaged. Although PSC is an inflammatory condition predisposing to CCA, CCA is almost never found in the autoimmune context of PBC. Here, we hypothesized that PBC might favor CCA immunosurveillance. In preclinical murine models of cholangitis challenged with syngeneic CCA, PBC (but not PSC) reduced the frequency of CCA development and delayed tumor growth kinetics. This PBC-related effect appeared specific to CCA as it was not observed against other cancers, including hepatocellular carcinoma. The protective effect of PBC was relying on type 1 and type 2 T cell responses and, to a lesser extent, on B cells. Single-cell TCR/RNA sequencing revealed the existence of TCR clonotypes shared between the liver and CCA tumor of a PBC host. Altogether, these results evidence a mechanistic overlapping between autoimmunity and cancer immunosurveillance in the biliary tract.

Davis-Beirut Reaction Inspired Nitroso Diels-Alder Reaction.

A Davis-Beirut reaction inspired nitroso Diels-Alder protocol is reported. The starting material for the procedure is a nitrophenyl moiety with the para position appropriately substituted with a 2°-amine (see 5) or 2°-alcohol (see 6). Deprotonation at the benzylic position followed by concomitant oxidation of the benzylic position and reduction of the nitro moiety delivers a nitrosophenyl intermediate, which subsequently undergoes a nitroso Diels-Alder reaction. This one-pot procedure delivers aryldihydro-1,2-oxazines in moderate yields.

1-BENZYLSPIRO[PIPERIDINE-4,1'-PYRIDO[3,4-b]indole] 'co-potentiators' for minimal function CFTR mutants.

We previously identified a spiro [piperidine-4,1-pyrido [3,4-b]indole] class of co-potentiators that function in synergy with existing CFTR potentiators such as VX-770 or GLGP1837 to restore channel activity of a defined subset of minimal function cystic fibrosis transmembrane conductance regulator (CFTR) mutants. Here, structure-activity studies were conducted to improve their potency over the previously identified compound, 20 (originally termed CP-A01). Targeted synthesis of 37 spiro [piperidine-4,1-pyrido [3,4-b]indoles] was generally accomplished using versatile two or three step reaction protocols with each step having high efficiency. Structure-activity relationship studies established that analog 2i, with 6'-methoxyindole and 2,4,5-trifluorobenzyl substituents, had the greatest potency for activation of N1303K-CFTR, with EC50 ∼600 nM representing an ∼17-fold improvement over the original compound identified in a small molecule screen.

Colloidal Palladium Nanoparticles for Selective Hydrogenation of Styrene Derivatives with Reactive Functional Groups.

This article presents the catalysis investigation of octanethiolate-capped palladium nanoparticles (C8 PdNP) and phenylethanethiolate-capped palladium nanoparticles (PhC2 PdNP) for chemoselective catalytic hydrogenation reactions of styrene derivatives in the presence of other reducible functionalities. The results show that the C8 PdNP is highly active under mild reaction conditions (room temperature and atmospheric pressure) and selective for hydrogenating monosubstituted alkene groups without reducing other reactive functional groups such as nitro, halo, carbonyls, and so forth. In comparison, the noncovalent interactions between surface phenyl ligands and aromatic substrates are found to hinder the hydrogenation activity of PhC2 PdNP.

A Redox Isomerization Strategy for Accessing Modular Azobenzene Photoswitches with Near Quantitative Bidirectional Photoconversion.

Photoswitches capable of accessing two geometric states are highly desirable, especially if their design is modular and incorporates a pharmacophore tethering site. We describe a redox isomerization strategy for synthesizing p-formylazobenzenes from p-nitrobenzyl alcohol. The resulting azo-aldehydes can be readily converted to photoswitchable compounds with excellent photophysical properties using simple hydrazide click chemistry. As a proof of principle, we synthesized a photoswitchable surfactant enabling the photocontrol of an emulsion with exceptionally high spatiotemporal precision.

Synthesis and evaluation of tetrahydropyrazolopyridine inhibitors of anion exchange protein SLC26A4 (pendrin).

Pendrin is a transmembrane chloride/anion antiporter that is strongly upregulated in the airways in rhinoviral infection, asthma, cystic fibrosis and chronic rhinosinusitis. Based on its role in the regulation of airway surface liquid depth, pendrin inhibitors have potential indications for treatment of inflammatory airways diseases. Here, a completely regioselective route to tetrahydro-pyrazolopyridine pendrin inhibitors based on 1,3-diketone and substituted hydrazine condensation was been developed. Structure-activity relationships at the tetrahydropyridyl nitrogen were investigated using a focused library, establishing the privileged nature of N-phenyl ureas and improving inhibitor potency by greater than 2-fold.

Davis-Beirut Reaction: A Photochemical Brønsted Acid Catalyzed Route to N-Aryl 2H-Indazoles.

The Davis-Beirut reaction provides access to 2H-indazoles from aromatic nitro compounds. However, N-aryl targets have been traditionally challenging to access due to competitive alternate reaction pathways. Previously, the key nitroso imine intermediate was generated under alkaline conditions, but as reported here, the photochemistry of o-nitrobenzyl alcohols empowered Brønsted acid catalyzed conditions for accessing N-aryl targets. Anilines and alkyl amines give different outcomes under optimized conditions; the proposed mechanism was studied using quantum chemical calculations.

Davis-Beirut Reaction: Diverse Chemistries of Highly Reactive Nitroso Intermediates in Heterocycle Synthesis.

Indazoles are an important class of nitrogen heterocycles because of their excellent performance in biologically relevant applications, such as in chemical biology and medicinal chemistry. In these applications, convenient synthesis using commercially available and diverse building blocks is highly desirable. Within this broad class, 2H-indazoles are relatively underexploited when compared to 1H-indazole, perhaps because of regioselectivity issues associated with the synthesis of 2H-indazoles. This Account describes our unfolding of the synthetic utility of the Davis-Beirut reaction (DBR) for the construction of 2H-indazoles and their derivatives; parallel unfoldings of mechanistic models for these interrelated N-N bond forming reactions are also summarized. The Davis-Beirut reaction is a robust method that exploits the diverse chemistries of a key nitroso imine or nitroso benzaldehyde intermediate generated in situ under redox neutral conditions. The resulting N-N bond-forming heterocyclization between nucleophilic and electrophilic nitrogens can be leveraged for the synthesis of multiple classes of indazoles and their derivatives, such as simple or fused indazolones, thiazolo-indazoles, 3-alkoxy-2H-indazoles, 2H-indazole N-oxides, and 2H-indazoles with various substitutions on the ring system or the nitrogens. These diverse products can all be synthesized under alkaline conditions and the various strategies for accessing these heterocycles are discussed. Alternatively, we have also developed methods involving mild photochemical conditions for the nitrobenzyl → aci-nitro → nitroso imine sequence. Solvent consideration is especially important for modulating the chemistry of the reactive intermediates in these reactions; the presence of water is critically important in some cases, but water's beneficial effect has a ceiling because of the alternative reaction pathways it enables. Fused 2H-indazoles readily undergo ring opening reactions to give indazolones when treated with nucleophiles or electrophiles. Furthermore, palladium-catalyzed cross coupling, the Sonagashira reaction, EDC amide coupling, 1,3-dipolar cycloadditions with nitrile oxides, copper-catalyzed alkyne-azide cycloadditions (click reaction), as well as copper-free click reactions, can all be used late-stage to modify 2H-indazoles and indazolones. The continued development and applications of the Davis-Beirut reaction has provided many insights for taming the reactivity of highly reactive nitro and nitroso groups, which still has a plethora of underexplored chemistries and challenges. For example, there is currently a limited number of nonfused 2H-indazole examples containing an aryl substitution at nitrogen. This is caused by relatively slow N-N bond formation between N-aryl imine and nitroso reactants, which allows water to add to the key nitroso imine intermediate causing imine bond cleavage to be a competitive reaction pathway rather than proceeding through the desired N-N bond-forming heterocyclization.

A phase Ib study of entinostat plus lapatinib with or without trastuzumab in patients with HER2-positive metastatic breast cancer that progressed during trastuzumab treatment.

BACKGROUND: Human epidermal growth factor 2 (HER2) is an effective therapeutic target in breast cancer; however, resistance to anti-HER2 agents such as trastuzumab and lapatinib develops. In a preclinical model, an HDAC inhibitor epigenetically reversed the resistance of cancer cells to trastuzumab and showed synergistic efficacy with lapatinib in inhibiting growth of trastuzumab-resistant HER2-positive (HER2+) breast cancer. METHODS: A phase 1b, dose escalation study was performed to assess maximum tolerated dose, safety/toxicity, clinical efficacy and explored pharmacodynamic biomarkers of response to entinostat combined with lapatinib with or without trastuzumab. RESULTS: The combination was safe. The MTD was lapatinib, 1000 mg daily; entinostat, 12 mg every other week; trastuzumab, 8 mg/kg followed by 6 mg/kg every 3 weeks. Adverse events included diarrhoea (89%), neutropenia (31%), and thrombocytopenia (23%). Neutropenia, thrombocytopenia and hypokalaemia were noted. Pharmacodynamic assessment did not yield conclusive results. Among 35 patients with evaluable response, PR was observed in 3 patients and CR in 3 patients, 1 maintained SD for over 6 months. DISCUSSION: This study identified the MTD of the entinostat, lapatinib, and trastuzumab combination that provided acceptable tolerability and anti-tumour activity in heavily pre-treated patients with HER2+ metastatic breast cancer, supporting a confirmatory trial.

Novel 2-(5-Imino-5H-isoquinolones[3,4-b]quinoxalin-7-ylmethyl)-benzonitrile (DIQ3) and Other Related Derivatives Targeting Colon Cancer Cells: Syntheses and in Vitro Models.

Chemotherapy has been shown to be effective in reducing the progression and development of cancer in metastatic patients. However, drug selectivity is still a major issue for most chemotherapeutics. In this study, we synthesized four novel heterocyclic compounds having similarity in structure with quinone systems whereby nitrogen atoms replace the oxygen atoms. The anticancer activity of these compounds (DIQ3-6) was tested against HCT116 human colon cancer cells. We showed that all four heterocycles caused significant reduction in colon cancer cell viability at doses as low as 4 µM, a concentration that was not cytotoxic to normal human FHs74Int intestinal cell lines. Interestingly, these heterocycles inhibited colon sphere formation in 3D cultures at first generation (G1), mainly because of inhibition of proliferation as evidenced by Ki67 staining. Thus, DIQ3 causes sufficient eradication of the self-renewal ability of the highly resistant cancer stem cells. This study represents the first documentation of the activity of these novel heterocyclic compounds, particularly compound DIQ3, and their potential therapeutic use in targeting colon cancer self-renewal capacity. Our findings provide the basis for proposing these nontoxic and stable compounds for additional testing against cancer.

Accessing Multiple Classes of 2 H-Indazoles: Mechanistic Implications for the Cadogan and Davis-Beirut Reactions.

The Cadogan cyclization is a robust but harsh method for the synthesis of 2 H-indazoles, a valuable class of nitrogen heterocycles. Although nitrene generation by exhaustive deoxygenation is widely accepted as the operating mechanism in the reductive cyclization of nitroaromatics, non-nitrene pathways have only been theorized previously. Here, 2 H-indazole N-oxides were synthesized through an interrupted Cadogan/Davis-Beirut reaction and are presented as direct evidence of competent oxygenated intermediates; mechanistic implications for both reactions are discussed. Isolation and characterization of these N-oxides enabled a formal Cadogan cyclization at room temperature for 2 H-indazole synthesis.

Photochemical Preparation of 1,2-Dihydro-3 H-indazol-3-ones in Aqueous Solvent at Room Temperature.

o-Nitrosobenzaldehyde is a reactive intermediate useful in the synthesis of nitrogen heterocycles. Previous strategies for using o-nitrosobenzaldehyde involve its isolation via chromatography and/or formation under harsh conditions. Herein, this intermediate was photochemically generated in situ from o-nitrobenzyl alcohols in a mild, efficient manner for the construction of 1,2-dihydro-3 H-indazol-3-ones using an aqueous solvent at room temperature. This convenient reaction offers several advantages over reported methods. The commercially available photoreactor employed 3 × 18 W bulbs outputting broad emission above 365 nm.

Distinct epidemiological profiles associated with inflammatory breast cancer (IBC): A comprehensive analysis of the IBC registry at The University of Texas MD Anderson Cancer Center.

BACKGROUND: To date, studies on inflammatory breast cancer (IBC) lack comprehensive epidemiological data. We analyzed detailed prospectively collected clinical and epidemiological data from the IBC Registry at The University of Texas MD Anderson Cancer Center. METHODS: Patients with IBC (n = 248) were consecutively diagnosed and prospectively enrolled between November 2006 and April 2013. All patients were newly diagnosed and at least 18 years old. Secondary IBC was excluded. Overall 160 variables were collected and evaluated including sociodemographics, anthropometrics, tobacco and alcohol consumption, reproductive variables, and family history data. RESULTS: Mean age at diagnosis was 51.6 (±11.5 SD) years, and the majority of patients were White (77.8%). A mean BMI ≥ 25 kg/m2, irrespective of menopausal status, was observed in 80.2% of all patients, with 82.6% of African Americans being obese. Approximately 42.2% of patients were ever smokers, and 91% reported ever being pregnant. A history of breastfeeding was reported in 54% of patients, with significant differences between ethnic groups in favor of White women (P<0.0001). Other reproductive factors such as use of birth control pills & hormone replacement therapy were also more frequently associated with White women compare to other ethnic groups (P < 0.05). In the multivariate Cox proportional hazard analysis, African American or Hispanic ethnicity, not having breastfed, higher clinical stage, and TNBC subtype were associated with shorter survival. CONCLUSION: Our data suggest that IBC is associated with distinct epidemiological profiles. This information could assist in targeting patients with specific preventive strategies based on their modifiable behavioral patterns.

N-N Bond Formation between Primary Amines and Nitrosos: Direct Synthesis of 2-Substituted Indazolones with Mechanistic Insights.

A concise, one-step route to indazolones from primary alkyl amines and o-nitrobenzyl alcohols is reported. The key step in this readily scalable indazolone forming process involves base-mediated in situ o-nitrobenzyl alcohol → o-nitrosobenzaldehyde conversion. Although this functional group interconversion is known to be useful for 2 H-indazole synthesis, its reactivity was modulated for indazolone formation.

Safety and Efficacy of Panitumumab Plus Neoadjuvant Chemotherapy in Patients With Primary HER2-Negative Inflammatory Breast Cancer.

Importance: Combining conventional chemotherapy with targeted therapy has been proposed to improve the pathologic complete response (pCR) rate in patients with inflammatory breast cancer (IBC). Epidermal growth factor receptor (EGFR) expression is an independent predictor of low overall survival in patients with IBC. Objective: To evaluate the safety and efficacy of the anti-EGFR antibody panitumumab plus neoadjuvant chemotherapy in patients with primary human epidermal growth factor receptor 2 (HER2)-negative IBC. Design, Setting, and Participants: Women with primary HER2-negative IBC were enrolled from 2010 to 2015 and received panitumumab plus neoadjuvant chemotherapy. Median follow-up time was 19.3 months. Tumor tissues collected before and after the first dose of panitumumab were subjected to immunohistochemical staining and RNA sequencing analysis to identify biomarkers predictive of pCR. Intervention: Patients received 1 dose of panitumumab (2.5 mg/kg) followed by 4 cycles of panitumumab (2.5 mg/kg), nab-paclitaxel (100 mg/m2), and carboplatin weekly and then 4 cycles of fluorouracil (500 mg/m2), epirubicin (100 mg/m2), and cyclophosphamide (500 mg/m2) every 3 weeks. Main Outcomes and Measures: The primary end point was pCR rate; the secondary end point was safety. The exploratory objective was to identify biomarkers predictive of pCR. Results: Forty-seven patients were accrued; 7 were ineligible. The 40 enrolled women had a median age of 57 (range, 23-68) years; 29 (72%) were postmenopausal. Three patients did not complete therapy because of toxic effects (n = 2) or distant metastasis (n = 1). Nineteen patients had triple-negative and 21 had hormone receptor-positive IBC. The pCR and pCR rates were overall, 11 of 40 (28%; 95% CI, 15%-44%); triple-negative IBC, 8 of 19 (42%; 95% CI, 20%-66%); and hormone receptor-positive/HER2-negative IBC, 3 of 21 (14%; 95% CI, 3%-36%). During treatment with panitumumab, nab-paclitaxel, and carboplatin, 10 patients were hospitalized for treatment-related toxic effects, including 5 with neutropenia-related events. There were no treatment-related deaths. The most frequent nonhematologic adverse event was skin rash. Several potential predictors of pCR were identified, including pEGFR expression and COX-2 expression. Conclusions and Relevance: This combination of panitumumab and chemotherapy showed the highest pCR rate ever reported in triple-negative IBC. A randomized phase 2 study is ongoing to determine the role of panitumumab in patients with triple-negative IBC and to further validate predictive biomarkers. Trial Registration: ClinicalTrials.gov Identifier: NCT01036087.

Davis-Beirut Reaction: Alkoxide versus Hydroxide Addition to the Key o-Nitrosoimine Intermediate.

Reaction options, alkoxide vs hydroxide vs amine addition to the key intermediate (o-nitrosoimine) generated in the Davis-Beirut reaction of an o-nitrobenzylamine substrate, are reported to explain the nucleophilic addition selectivity of this one-pot indazole-forming process. The hydroxide addition/deprotection pathway as well as the fate of the resulting o-nitrosobenzaldehyde were both uncovered with several o-nitrobenzylamine substrates, and design elements required for an efficient double Davis-Beirut reaction, inspired by new mechanistic insights, were defined.

Diverting Reactive Intermediates Toward Unusual Chemistry: Unexpected Anthranil Products from Davis-Beirut Reaction.

The discovery of a new variation on the Davis-Beirut reaction is described in which an atypical heterocyclic framework (the anthranil or benzo[c]isoxazole framework) is formed as the result of diversion of a key reactive intermediate away from its expected reactivity-a potentially general approach to reaction design and development. Experimental and computational support for the proposed mechanism and origins of altered reactivity are described.

High-Potency Phenylquinoxalinone Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Activators.

We previously identified phenylquinoxalinone CFTRact-J027 (4) as a cystic fibrosis transmembrane conductance regulator (CFTR) activator with an EC50 of ∼200 nM and demonstrated its therapeutic efficacy in mouse models of constipation. Here, structure-activity studies were done on 36 synthesized phenylquinoxalinone analogs to identify compounds with improved potency and altered metabolic stability. Synthesis of the phenylquinoxalinone core was generally accomplished by condensation of 1,2-phenylenediamines with substituted phenyloxoacetates. Structure-activity studies established, among other features, the privileged nature of a properly positioned nitro moiety on the 3-aryl group. Synthesized analogs showed improved CFTR activation potency compared to 4 with EC50 down to 21 nM and with greater metabolic stability. CFTR activators have potential therapeutic indications in constipation, dry eye, cholestatic liver diseases, and inflammatory lung disorders.