From propenolysis to enyne metathesis: tools for expedited assembly of 4a,8a-azaboranaphthalene and extended polycycles with embedded BN.

The synthesis of BN-containing molecules, which have an interesting isosteric relationship to their parent all-C cores, has drawn a great deal of attention as an avenue to alter and tune molecular function. Nevertheless, many cores with embedded BN are still hard to synthesize, and thus, further effort is required in this direction. Herein, we present an integrated approach to BN-containing polycycles rooted in an exceptionally clean B-N condensation of amines with a tri-allylborane. Having released propene as the only byproduct, the resulting BN precursors are seamlessly telescoped into BN-containing polycyclic cores via a set of additional methodologies, either developed here ad-hoc or applied for the first time for the synthesis of BN-cycles. As the "sharpening stone" of the process, BN-embedded naphthalene, which has previously only been obtained in low yield, can now be synthesized efficiently through propenolysis, ring-closing metathesis and a new high-yielding aromatization. As a more advanced application, an analogously obtained BN-containing bis-enyne is readily converted to BN-containing non-aromatic tetra-, penta- and hexacyclic structures via ring-closing enyne metathesis, followed by the Diels-Alder cycloaddition. The resulting air-sensitive structures are easily handled by preventive hydration (quaternization) of their B-N bridge; reverting this hydration restores the original Bsp2-Nsp2 structure. In the future, these structures may pave the way to BN-anthracenes and other π-extended BN-arenes.

When Unsuspected Crystallinity Ruins Biological Testing in Early Discovery: A Case Study.

The impact of the crystalline or amorphous structure of a solid on the solubility and pharmacokinetic properties of a drug candidate is always considered by the pharmaceutical industry during the development of a new drug; however, it is not so frequently considered during the early drug discovery process by organic and medicinal chemists, particularly those working in academia. We want to share, as an example, the false negative obtained in the biological testing of a solid sample of a tyrosine kinase inhibitor due to its unexpected crystallinity and lower solubility with respect to a solid amorphous batch of the same compound and the experimentation carried out to establish the origin of such a discrepancy.

1H-Pyrazolo[3,4-b]pyridines: Synthesis and Biomedical Applications.

Pyrazolo[3,4-b]pyridines are a group of heterocyclic compounds presenting two possible tautomeric forms: the 1H- and 2H-isomers. More than 300,000 1H-pyrazolo[3,4-b]pyridines have been described which are included in more than 5500 references (2400 patents) up to date. This review will cover the analysis of the diversity of the substituents present at positions N1, C3, C4, C5, and C6, the synthetic methods used for their synthesis, starting from both a preformed pyrazole or pyridine, and the biomedical applications of such compounds.

Expanding the Diversity at the C-4 Position of Pyrido[2,3-d]pyrimidin-7(8H)-ones to Achieve Biological Activity against ZAP-70.

Pyrido[2,3-d]pyrimidin-7(8H)-ones have attracted widespread interest due to their similarity with nitrogenous bases found in DNA and RNA and their potential applicability as tyrosine kinase inhibitors. Such structures, presenting up to five diversity centers, have allowed the synthesis of a wide range of differently substituted compounds; however, the diversity at the C4 position has mostly been limited to a few substituents. In this paper, a general synthetic methodology for the synthesis of 4-substituted-2-(phenylamino)-5,6-dihydropyrido[2,3-d]pyrimidin-7(8H)-ones is described. By using cross-coupling reactions, such as Ullmann, Buchwald-Hartwig, Suzuki-Miyaura, or Sonogashira reactions, catalyzed by Cu or Pd, we were able to describe new potential biologically active compounds. The resulting pyrido[2,3-d]pyrimidin-7(8H)-ones include N-alkyl, N-aryl, O-aryl, S-aryl, aryl, and arylethynyl substituents at C4, which have never been explored in connection with the biological activity of such heterocycles as tyrosine kinase inhibitors, in particular as ZAP-70 inhibitors.

1,6-Naphthyridin-2(1H)-ones: Synthesis and Biomedical Applications.

Naphthyridines, also known as diazanaphthalenes, are a group of heterocyclic compounds that include six isomeric bicyclic systems containing two pyridine rings. 1,6-Naphthyridines are one of the members of such a family capable of providing ligands for several receptors in the body. Among such structures, 1,6-naphthyridin-2(1H)-ones (7) are a subfamily that includes more than 17,000 compounds (with a single or double bond between C3 and C4) included in more than 1000 references (most of them patents). This review will cover the analysis of the diversity of the substituents present at positions N1, C3, C4, C5, C7, and C8 of 1,6-naphthyridin-2(1H)-ones, the synthetic methods used for their synthesis (both starting from a preformed pyridine or pyridone ring), and the biomedical applications of such compounds.

The Versatile Reaction Chemistry of an Alpha-Boryl Diazo Compound.

The first α-boryl diazo compound that is capable of engaging in classic synthetic organic diazo reaction chemistry is described. The diazomethyl-1,2-azaborine 1, which is a BN isostere of phenyldiazomethane, is significantly more stable than phenyldiazomethane; its reaction chemistry ranges from C-H activation, O-H activation, [3+2] cycloaddition, and halogenation, to Ru-catalyzed carbonyl olefination. The demonstrated broad range of reactivity of diazomethyl-1,2-azaborine 1 makes it an exceptionally versatile synthetic building block for the 1,2-azaborine heterocyclic motif.

A captured room temperature stable Wheland intermediate as a key structure for the orthogonal decoration of 4-amino-pyrido[2,3-d]pyrimidin-7(8H)-ones.

Wheland intermediates are usually unstable compounds and only a few have been isolated at very low temperatures. During our work on tyrosine kinase inhibitors, we studied the bromination of 7 in order to obtain a dibromo substituted pyrido[2,3-d]pyrimidin-7(8H)-one which could be orthogonally decorated. Surprisingly, treatment of 7 with 3 equiv. of Br2 in acetic acid (AcOH) afforded 12, a captured room temperature stable Wheland bromination intermediate stabilized by the bromination of the imino tautomer of the amino group at C4 of the pyridopyrimidine skeleton. The structure was confirmed by crystal structure determination from powder X-ray diffraction data. Treatment of 12 with DMSO afforded the dibromo substituted compound 13 presenting a bromine atom at C6 and C5-C6 unsaturation. 13 was directly accessed by treating 7 with N-bromosuccinimide (NBS), a protocol extended to other compounds using NBS or N-iodosuccinimide (NIS) to afford 6-halo substituted systems. 26, bearing an iodine at C6 and a p-bromophenylamino at C2, allows the orthogonal decoration of pyridopyrimidines.

Pyrido[2,3-d]pyrimidin-7(8H)-ones: Synthesis and Biomedical Applications.

Pyrido[2,3-d]pyrimidines (1) are a type of privileged heterocyclic scaffolds capable of providing ligands for several receptors in the body. Among such structures, our group and others have been particularly interested in pyrido[2,3-d]pyrimidine-7(8H)-ones (2) due to the similitude with nitrogen bases present in DNA and RNA. Currently there are more than 20,000 structures 2 described which correspond to around 2900 references (half of them being patents). Furthermore, the number of references containing compounds of general structure 2 have increased almost exponentially in the last 10 years. The present review covers the synthetic methods used for the synthesis of pyrido[2,3-d]pyrimidine-7(8H)-ones (2), both starting from a preformed pyrimidine ring or a pyridine ring, and the biomedical applications of such compounds.

Co-activation of STAT3 and YES-Associated Protein 1 (YAP1) Pathway in EGFR-Mutant NSCLC.

Background: The efficacy of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in EGFR-mutant non-small cell lung cancer (NSCLC) is limited by adaptive activation of cell survival signals. We hypothesized that both signal transducer and activator of transcription 3 (STAT3) and Src-YES-associated protein 1 (YAP1) signaling are dually activated during EGFR TKI treatment to limit therapeutic response. Methods: We used MTT and clonogenic assays, immunoblotting, and quantitative polymerase chain reaction to evaluate the efficacy of EGFR TKI alone and in combination with STAT3 and Src inhibition in three EGFR-mutant NSCLC cell lines. The Chou-Talalay method was used for the quantitative determination of drug interaction. We examined tumor growth inhibition in one EGFR-mutant NSCLC xenograft model (n = 4 mice per group). STAT3 and YAP1 expression was evaluated in tumors from 119 EGFR-mutant NSCLC patients (64 in an initial cohort and 55 in a validation cohort) by quantitative polymerase chain reaction. Kaplan-Meier and Cox regression analyses were used to assess the correlation between survival and gene expression. All statistical tests were two-sided. Results: We discovered that lung cancer cells survive initial EGFR inhibitor treatment through activation of not only STAT3 but also Src-YAP1 signaling. Cotargeting EGFR, STAT3, and Src was synergistic in two EGFR-mutant NSCLC cell lines with a combination index of 0.59 (95% confidence interval [CI] = 0.54 to 0.63) for the PC-9 and 0.59 (95% CI = 0.54 to 0.63) for the H1975 cell line. High expression of STAT3 or YAP1 predicted worse progression-free survival (hazard ratio [HR] = 3.02, 95% CI = 1.54 to 5.93, P = .001, and HR = 2.57, 95% CI = 1.30 to 5.09, P = .007, respectively) in an initial cohort of 64 EGFR-mutant NSCLC patients treated with firstline EGFR TKIs. Similar results were observed in a validation cohort. Conclusions: Our study uncovers a coordinated signaling network centered on both STAT3 and Src-YAP signaling that limits targeted therapy response in lung cancer and identifies an unforeseen rational upfront polytherapy strategy to minimize residual disease and enhance clinical outcomes.

Design, synthesis and biological evaluation of pyrido[2,3-d]pyrimidin-7-(8H)-ones as HCV inhibitors.

The design and selection of a combinatorial library of pyrido[2,3-d]pyrimidin-7(8H)-ones (4) has allowed the synthesis of 121 compounds, using known and new synthetic methodologies, and the evaluation of the inhibitory activity against hepatitis C virus (HCV) genotype 1b replicon. Among these compounds, 21{4,10} and 24{2,10} presented very high activities [EC50 = 0.027 µM (CC50 = 5.3 µM) and EC50 = 0.034 µM (CC50 = 13.5 µM), respectively] and high selectivity indexes, 196 and 397. These values are similar to the EC50 reported for sofosbuvir (2) (0.048 µM) using a similar methodological approach and the same virus subtype. 21{4,10} and 24{2,10} are obtained through shorter synthetic itineraries than sofosbuvir and 24{2,10} is achiral contrary to sofosbuvir which presents 4 stereogenic centers. In silico studies suggest that 21{4,10} and 24{2,10} inhibits NS5B polymerase through allosteric site binding.

Investigating molecular dynamics-guided lead optimization of EGFR inhibitors.

The epidermal growth factor receptor (EGFR) is part of an extended family of proteins that together control aspects of cell growth and development, and thus a validated target for drug discovery. We explore in this work the suitability of a molecular dynamics-based end-point binding free energy protocol to estimate the relative affinities of a virtual combinatorial library designed around the EGFR model inhibitor 6{1} as a tool to guide chemical synthesis toward the most promising compounds. To investigate the validity of this approach, selected analogs including some with better and worse predicted affinities relative to 6{1} were synthesized, and their biological activity determined. To understand the binding determinants of the different analogs, hydrogen bonding and van der Waals contributions, and water molecule bridging in the EGFR-analog complexes were analyzed. The experimental validation was in good qualitative agreement with our theoretical calculations, while also a 6-dibromophenyl-substituted compound with enhanced inhibitory effect on EGFR compared to the reference ligand was obtained.

Nitrogen positional scanning in tetramines active against HIV-1 as potential CXCR4 inhibitors.

The paradigm, derived from bicyclams and other cyclams, by which it is necessary to use the p-phenylene moiety as the central core in order to achieve high HIV-1 antiviral activities has been reexamined for the more flexible and less bulky structures 4, previously described by our group as potent HIV-1 inhibitors. The symmetrical compounds 7{x,x} and the non-symmetrical compounds 8{x,y} were designed, synthesized and biologically evaluated in order to explore the impact on the biological activity of the distance between the phenyl ring and the first nitrogen atom of the side chains. EC50 exactly followed the order 7{x,x} < 8{x,x} < 4{x,x} indicating that, for such flexible tetramines, the presence of two methylene units on each side of the central phenyl ring increases the biological activity contrary to AMD3100. A computational study of the interactions of 4{3,3}, 7{3,3} and 8{3,3} with CXCR4 revealed interactions in the same pocket region with similar binding modes for 4{3,3} and 7{3,3} but a different one for 8{3,3}.

4-Amino-2-arylamino-6-(2,6-dichlorophenyl)-pyrido[2,3-d]pyrimidin-7-(8H)-ones as BCR kinase inhibitors for B lymphoid malignancies.

A new family of 4-aminopyrido[2,3-d]pyrimidines active against non-Hodgkin's lymphomas (NHLs) is described. Among these compounds, 19 inhibits the most upstream tyrosine kinases in the B cell receptor (BCR) signaling pathway which are involved in the mature B cell neoplasms. Thus, 19 showed antiproliferative activity at 24 h and 48 h against a panel of 20 NHLs cell lines with GI50 ranging from 1.3 to 6.9 µM at 24 h, and 1.4-7.2 µM at 48 h, being this effect related to a significant (20-90%) inhibition of the phosphorylation of the BCR-related kinases Btk, Syk, and Lyn. Most importantly, 19 was able to induce a 63% reduction in Rec-1 cell proliferation, which was significantly greater than the 31% and 3% blockade of proliferation observed after cell treatment with R406, a Syk inhibitor, and ibrutinib, a Btk inhibitor, respectively. The computational blind docking and ligand binding within the pockets of Btk, Syk and Lyn kinases showed that compound 19 presents the same kind of interactions of described cocrystallized inhibitors.

A new and practical method for the synthesis of 6-aryl-5,6-dihydropyrido[2,3-d]pyrimidine-4,7(3Η,8Η)-diones.

A one step general synthetic methodology for the synthesis of 6-aryl-5,6-dihydropyrido[2,3-d]pyrimidine-4,7(3H,8H)-diones (17{[Formula: see text]} ([Formula: see text]) and 20{[Formula: see text]} ([Formula: see text])) is described. This methodology is based on reacting a 2-aryl-substituted acrylate (16{[Formula: see text]}) with the corresponding 6-aminopyrimidin-4(3[Formula: see text]-one (13 ([Formula: see text]; 19 ([Formula: see text])) in presence of a base under microwave irradiation. The resulting pyrido[2,3-[Formula: see text]]pyrimidines present an aryl substituent at position C6, precisely the one directly related to the biological activity of such heterocycles. These protocols have been extended to other 2-alkyl-substituted and 3-alkyl (or aryl)-substituted acrylates but with lower yields.

ALK and ROS1 as a joint target for the treatment of lung cancer: a review.

Rearrangements of the anaplastic lymphoma kinase (ALK) have been described in multiple malignancies, including non-small cell lung cancer (NSCLC). ALK fusions have gain of function properties while activating mutations in wild-type ALK can also occur within the tyrosine kinase domain. ALK rearrangements define a new molecular subtype of NSCLC that is exquisitely sensitive to ALK inhibition. Crizotinib, an orally available small molecule ATP-mimetic compound which was originally designed as a MET inhibitor, was recognized to have "off-target" anti-ALK activity and has been approved in the USA for the treatment of patients with ALK-positive NSCLC. Chromosomal rearrangements involving the ROS1 receptor tyrosine kinase have also been recently described in NSCLC, while crizotinib is currently under clinical trial in this molecular subset of NSCLC patients. The basic approaches of any computer aided drug design work in terms of structure and ligand based drug design. Details of each of these approaches should be covered with an emphasis on utilizing both in order to develop multi-targeted small-molecule kinase inhibitors. Such multi-targeted tyrosine kinase inhibitors can have antiproliferative activity against both ROS1and ALK rearranged NSCLC. Herein, we highlight the importance of targeting these proteins and the advances in optimizing more potent and selective ALK and ROS1 kinase inhibitors.

Synthesis of 2-arylamino substituted 5,6-dihydropyrido[2,3-d]pyrimidine-7(8H)-ones from arylguanidines.

A practical protocol was developed for the synthesis of 2-arylamino substituted 4-amino-5,6-dihydropyrido[2,3-d]pyrimidin-7(8H)-ones from α,ß-unsaturated esters, malononitrile, and an aryl substituted guanidine via the corresponding 3-aryl-3,4,5,6- tetrahydropyrido[2,3-d]pyrimidin-7(8H)-ones. Such compounds are formed upon treatment of 2-methoxy-6-oxo-1,4,5,6-tetrahydropyridine-3-carbonitriles with an aryl substituted guanidine in 1,4-dioxane and are converted to the desired 4-aminopyridopyrimidines with NaOMe/MeOH through a Dimroth rearrangement. The overall yields of this three-step protocol are, generally speaking, higher than the multicomponent reaction, previously developed by our group, between an α,ß-unsaturated ester, malononitrile, and an aryl substituted guanidine.

Diverse combinatorial design, synthesis and in vitro evaluation of new HEPT analogues as potential non-nucleoside HIV-1 reverse transcription inhibitors.

New analogues of 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) were synthesized and evaluated for their in vitro activities against HIV-1 in MT-4 cell cultures. Chemical diversity was introduced in 4 of the six positions of the core and the influence of each substituent was studied. This library was built on the basis of a rational diversity analysis with the objective of maximizing diversity and thus, the activity range with a minimum number of synthesized compounds. Among them, 2{1,2,3,1} and 2{1,2,3,4} exhibited the most potent anti-HIV-1 activities (EC(50)=0.015 µg/mL; 0.046 µM, SI >1667) and (EC(50)=0.025 µg/mL; 0.086 µM, SI >1000), respectively, which were about 71-fold and 38-fold more active than the reference compound HEPT (EC(50)=1.01 µg/mL; 3.27 µM, SI >25).

Discovery of novel non-cyclam polynitrogenated CXCR4 coreceptor inhibitors.

HIV cell fusion and entry have been validated as targets for therapeutic intervention against infection. Bicyclams were the first low-molecular-weight compounds to show specific interaction with CXCR4. The most potent bicyclam was AMD3100, in which the two cyclam moieties are tethered by a 1,4-phenylenebis(methylene) bridge. It was withdrawn from clinical trials owing to its lack of oral bioavailability and cardiotoxicity. We have designed a combinatorial library of non-cyclam polynitrogenated compounds by preserving the main features of AMD3100. At least two nitrogen atoms on each side of the p-phenylene moiety, one in the benzylic position and the other(s) in the heterocyclic system were maintained, and the distances between them were similar to the nitrogen atom distances in cyclam. A selection of diverse compounds from this library were prepared, and their in vitro activity was tested in cell cultures against HIV strains. This led to the identification of novel potent CXCR4 coreceptor inhibitors without cytotoxicity at the tested concentrations.