Bifunctionality and Antitumor Efficacy of ZG-126, a Vitamin D Receptor Agonist/Histone Deacetylase Inhibitor Hybrid Molecule.

Analogues of hormonal vitamin D, 1,25-dihydroxyvitamin D (1,25D), signal through the nuclear vitamin D receptor (VDR). They have potential in combination therapies with other anticancer agents such as histone deacetylase inhibitors (HDACi's). Here, we characterize the ZG series of hybrid compounds that combine HDACi within the backbone of a VDR agonist. All display improved solubility, with ZG-126 being the most robustly bifunctional molecule in multiple cell lines. ZG-126 is well tolerated and strongly induces VDR target gene expression in vivo at therapeutic doses. Its antitumor efficacy is superior to 1,25D and the HDACi SAHA, separately or together, in mouse models of melanoma and triple-negative breast cancer (TNBC). Notably, ZG-126 treatment reduces metastases almost 4-fold in an aggressive TNBC model. ZG-126 also reduces total macrophage infiltration and the proportion of immunosuppressive M2-polarized macrophages in TNBC tumors by 2-fold. ZG-126 thus represents a bifunctional and efficacious anticancer agent with improved physicochemical properties.

Design, synthesis and antiproliferative activity of raloxifene/histone deacetylase inhibitor hybrids in breast cancer.

Antiestrogen/histone deacetylase inhibitor (HDACi) hybrids were designed by merging structures of raloxifene with suberoylanilide hydroxamic acid, incorporating the HDACi unit into the phenolic ring of the antiestrogen. These hybrids were synthesized with a range of HDACi chain lengths and assessed for bifunctionality. Four hybrids, 21 (YW471), 22 (YW490), 27(YW486), and 28 (YW487) showed good potency both as antiestrogens in a BRET assay and in a fluorometric HDACi assay. The antiproliferative activity of the hybrids was demonstrated in both ER+ MCF7 and ER- MDA-MB-231 breast cancer cell lines.

Mimicking enzymatic cation-π interactions in hydrazide catalyst design: access to trans-decalin frameworks.

Chiral bicyclic hydrazide organocatalysts have previously been shown to catalyze the cyclization of (Z)-polyene substrates with high enantioselectivity, but with poor selectivity for the corresponding (E)-polyenes. Here we demonstrate that diazapane carboxylates bearing terphenyl groups efficiently catalyze (E)-polyene bicyclization with enantioselectivities up to 94 : 6 er and with high diastereoselectivity for trans-decalin formation. The catalysts function by simultaneously initiating the cyclization via iminium ion formation and stabilizing intermediates/transition states by cation-π interactions.

An Organocatalytic Oxy-Cope/Michael Cascade Reaction.

Ethyl diazepane carboxylate catalyzes the oxy-Cope rearrangement of 4-hydroxy- and 4-alkoxy-1,5-hexadiene-2-carboxaldehydes via iminium ion activation. The resulting intermediate undergoes an intramolecular Michael reaction to furnish cyclopentane-containing products. The reaction proceeds with a range of substrates, including both cyclic and acyclic substrates, and tolerates substitution on the vinyl substituent. Substrates fused on a cycloalkane framework undergo net ring expansion/cyclopentannulation with a high degree of stereocontrol via chairlike transition states. The reaction extends iminium organocatalysis to the oxy-Cope rearrangement, embedded within a complexity-generating cascade transformation.

Do Antarafacial Cycloadditions Occur? Cycloaddition of Heptafulvalene with Tetracyanoethylene.

The cycloaddition of heptafulvalene (1) with tetracyanoethylene (TCNE) was previously described as an example of an antarafacial cycloaddition, a [π14 a +π2 s ] process that afforded only the trans cycloadduct by virtue of the edge-to-face approach of TCNE, facilitated by the S shape of 1. The reaction has been investigated in depth and found not to be a concerted antarafacial process. At low temperature, the reaction is observed to give a mixture of cis and trans cycloadducts as well as a [4+2] cycloadduct. The mixture of products is converted to the trans cycloadduct by equilibration upon warming to room temperature. Studies with diethyl 2,3-dicyanofumarate and -maleate confirmed the formation of cis cycloadducts. DFT studies at the M06-2X/6-311+G(2d,p) SCRF=acetone level of theory show that the originally proposed edge-to-face approach of TCNE to 1 is highly disfavored, whereas a stepwise mechanism involving the addition of TCNE at C2 to form a zwitterion followed by collapse at either C2' or C7' is energetically accessible. The Diels-Alder adduct is also formed in a stepwise reaction by competitive addition of TCNE at C4 of 1. These studies suggest that edge-to-face interactions are prohibitive in even the most favorable cases.

Molecular mechanisms of bifunctional vitamin D receptor agonist-histone deacetylase inhibitor hybrid molecules in triple-negative breast cancer.

The active form of vitamin D, 1,25-dihydroxyvitamin D (1,25D), and its analogues signal through the nuclear vitamin D receptor (VDR), a ligand-regulated transcription factor, and have been extensively investigated as anticancer agents. 1,25D and its analogs have potential in combination therapies because they exhibit synergistic activities with other anticancer agents such as histone deacetylase inhibitors (HDACi). We have developed a series of hybrid molecules that combine HDACi within the backbone of a VDR agonist and thus represent fully integrated bifunctional molecules. They exhibit anti-tumor efficacy in reducing tumor growth and metastases in an aggressive model of triple-negative breast cancer. However, their solubility is limited by their hydrophobic diarylpentane cores. Our goals here were two-fold: (1) to improve the solubility of hybrids by introducing nitrogen into diarylpentane cores, and (2) to investigate the molecular mechanisms underlying their anti-tumor efficacy by performing comparative gene expression profiling studies with 1,25D and the potent HDACi suberoylanilide hydroxamic acid (SAHA). We found that substituting aryl with pyrydyl rings did not sacrifice bifunctionality and modestly improved solubility. Notably, one compound, AM-193, displayed enhanced potency as a VDR agonist and in cellular assays of cytotoxicity. RNAseq studies in triple negative breast cancer cells revealed that gene expression profiles of hybrids were very similar to that of 1,25D, as was that observed with 1,25D and SAHA combined. The effects of SAHA alone on gene expression were limited and distinct from those 1,25D or hybrids. The combined results suggest that efficacy of hybrids arises from targeting HDACs that do not have a direct role in gene regulation. Moreover, pathways analysis revealed that hybrids regulate numerous genes controlling immune cell infiltration into tumors and suppress the expression of several secreted molecules that promote breast cancer growth and metastasis.

Total Synthesis of (-)-3-Oxoisotaxodione.

The first total synthesis of the abietaquinone methide diterpenoid (-)-3-oxoisotaxodione is reported. The key enabling step is the use of a chiral bicyclic hydrazide as an organocatalyst for the enantioselective polyene cyclization of a (Z)-polyene substrate to form the cis-decalin core of the natural product. The α-oxo-para-quinone methide unit is formed by a two-step oxidation from a phenol, enabling an efficient synthesis of the natural product.

Dual-function antiandrogen/HDACi hybrids based on enzalutamide and entinostat.

The combination of androgen receptor antagonists with histone deacetylase inhibitors (HDACi) has been shown to be more effective than antiandrogens alone in halting growth of prostate cancer cell lines. Here we have designed, synthesized and assessed a series of antiandrogen/HDACi hybrids by combining structural features of enzalutamide with either SAHA or entinostat. The hybrids are demonstrated to maintain bifunctionality using a fluorometric HDAC assay and a bioluminescence resonance energy transfer (BRET) antiandrogen assay. Antiproliferative assays showed that hybrids bearing o-aminoanilide-based HDACi motifs outperformed hydroxamic acid based HDACi's. The hybrids demonstrated selectivity for epithelial cell lines vs. stromal cell lines, suggesting a potentially useful therapeutic window.

Mechanism of an Organocatalytic Cope Rearrangement Involving Iminium Intermediates: Elucidating the Role of Catalyst Ring Size.

The mechanism of the organocatalytic Cope rearrangement is elucidated through a combined computational and experimental approach. As reported previously, hydrazides catalyze the Cope rearrangement of 1,5-hexadiene-2-carboxaldehydes via iminium ion formation, and seven- and eight-membered ring catalysts are more active than smaller ring sizes. In the present work, quantum mechanical computations and kinetic isotope effect experiments demonstrate that the Cope rearrangement step, rather than iminium formation, is rate-limiting. The computations further explain how the hydrazide catalyst lowers the free-energy barrier of the Cope rearrangement via an associative transition state that is stabilized by enehydrazine character. The computations also explain the catalyst ring size effect, as larger hydrazide rings are able to accommodate optimal transition-state geometries that minimize the unfavorable lone-pair repulsion between neighboring nitrogen atoms and maximize the favorable hyperconjugative donation from each nitrogen atom into neighboring electron-poor sigma bonds, with the seven-membered catalyst achieving a nearly ideal transition-state geometry that is comparable to that of an unconstrained acyclic catalyst. Experimental kinetics studies support the computations, showing that the seven-membered and acyclic hydrazide catalysts react 10 times faster than the six-membered catalyst. Unraveling the mechanism of this reaction is an important step in understanding other reactions catalyzed by hydrazides, and explaining the ring size effect is critical because cyclic catalysts provide a constrained scaffold, enabling the development of asymmetric variants of these reactions.

Ostrich eggshell bead strontium isotopes reveal persistent macroscale social networking across late Quaternary southern Africa.

Hunter-gatherer exchange networks dampen subsistence and reproductive risks by building relationships of mutual support outside local groups that are underwritten by symbolic gift exchange. Hxaro, the system of delayed reciprocity between Ju/'hoãn individuals in southern Africa's Kalahari Desert, is the best-known such example and the basis for most analogies and models of hunter-gatherer exchange in prehistory. However, its antiquity, drivers, and development remain unclear, as they do for long-distance exchanges among African foragers more broadly. Here we show through strontium isotope analyses of ostrich eggshell beads from highland Lesotho, and associated strontium isoscape development, that such practices stretch back into the late Middle Stone Age. We argue that these exchange items originated beyond the macroband from groups occupying the more water-stressed subcontinental interior. Tracking the emergence and persistence of macroscale, transbiome social networks helps illuminate the evolution of social strategies needed to thrive in stochastic environments, strategies that in our case study show persistence over more than 33,000 y.

Hydrazide-Catalyzed Polyene Cyclization: Asymmetric Organocatalytic Synthesis of cis-Decalins.

Polyene cyclizations offer rapid entry into terpenoid ring systems. Although enantioselective cyclizations of (E)-polyenes to form trans-decalin ring systems are well precedented, highly enantioselective cyclizations of (Z)-polyenes to form the corresponding cis-decalins have not been reported. Here, we describe the first application of iminium catalysis to the initiation of polyene cyclizations. Ethyl 1,2-diazepane-1-carboxylate catalyzes the cyclization of polyenes bearing enal initiators. Moreover, chiral bicyclic hydrazides catalyze the cyclizations of (Z)-polyene substrates to form cis-decalins with enantioselectivities of up to 97:3 er. DFT calculations suggest the catalysts promote the reaction by stabilizing positive charge as it develops during the bicyclization.

Biogenic carbonate mercury and marine temperature records reveal global influence of Late Cretaceous Deccan Traps.

The climate and environmental significance of the Deccan Traps large igneous province of west-central India has been the subject of debate in paleontological communities. Nearly one million years of semi-continuous Deccan eruptive activity spanned the Cretaceous-Paleogene boundary, which is renowned for the extinction of most dinosaur groups. Whereas the Chicxulub impactor is acknowledged as the principal cause of these extinctions, the Deccan Traps eruptions are believed to have contributed to extinction patterns and/or enhanced ecological pressures on biota during this interval of geologic time. We present the first coupled records of biogenic carbonate clumped isotope paleothermometry and mercury concentrations as measured from a broad geographic distribution of marine mollusk fossils. These fossils preserve evidence of simultaneous increases in coastal marine temperatures and mercury concentrations at a global scale, which appear attributable to volcanic CO2 and mercury emissions. These early findings warrant further investigation with additional records of combined Late Cretaceous temperatures and mercury concentrations of biogenic carbonate.

A Spiroalkylation Method for the Stereoselective Construction of α-Quaternary Carbons and Its Application to the Total Synthesis of (R)-Puraquinonic Acid.

Cyclic α-quaternary carbon stereocenters were prepared from biselectrophillic substrates and an easily prepared chiral bicyclic sulfonyl lactam. This was achieved in two steps by spiroalkylation, employing biphasic reaction conditions with a phase-transfer catalyst, followed by reduction and alkylation with a series of alkyl halide electrophiles. The products of this method were isolated in good yields with with high levels of diastereoselectivity. This methodology was employed in the enantioselective total synthesis of (R)-puraquinonic acid (1) for a late-stage installation of the α-quaternary carbon stereocenter. This enabled the shortest synthesis of 1 to date, an eight-pot sequence providing an overall yield of 14%.

Aminoalkylation of [1.1.1]Propellane Enables Direct Access to High-Value 3-Alkylbicyclo[1.1.1]pentan-1-amines.

Bicyclo[1.1.1]pentanes are effective bioisoteres for aromatic rings, tert-butyl groups, and alkynes. Here we report the first method to synthesize 3-alkylbicyclo[1.1.1]pentan-1-amines directly from [1.1.1]propellane via sequential addition of magnesium amides and alkyl electrophiles. The mild reaction conditions tolerate a variety of important functional groups and enable efficient incorporation of several pharmaceutically relevant amines onto the bicyclo[1.1.1]pentane scaffold. This method's utility is highlighted by its ability to significantly streamline the syntheses of several important bicyclo[1.1.1]pentan-1-amine building blocks.

Author Correction: Allogenic and Autogenic Signals in the Stratigraphic Record of the Deep-Sea Bengal Fan.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Incorporation of histone deacetylase inhibitory activity into the core of tamoxifen - A new hybrid design paradigm.

Hybrid antiestrogen/histone deacetylase (HDAC) inhibitors were designed by appending zinc binding groups to the 4-hydroxystilbene core of 4-hydroxytamoxifen. The resulting hybrids were fully bifunctional, and displayed high nanomolar to low micromolar IC50 values against both the estrogen receptor α (ERα) and HDACs in vitro and in cell-based assays. The hybrids were antiproliferative against ER+ MCF-7 breast cancer cells, with hybrid 28b possessing an improved activity profile compared to either 4-hydroxytamoxifen or SAHA. Hybrid 28b displayed gene expression patterns that reflected both ERα and HDAC inhibition.

Homozygous Calcium-Sensing Receptor Polymorphism R544Q Presents as Hypocalcemic Hypoparathyroidism.

Context: Autosomal dominant hypocalcemia type 1 (ADH1) is caused by heterozygous activating mutations in the calcium-sensing receptor gene (CASR). Whether polymorphisms that are benign in the heterozygous state pathologically alter receptor function in the homozygous state is unknown. Objective: To identify the genetic defect in an adolescent female with a history of surgery for bilateral cataracts and seizures. The patient has hypocalcemia, hyperphosphatemia, and low serum PTH level. The parents of the proband are healthy. Methods: Mutation testing of PTH, GNA11, GCM2, and CASR was done on leukocyte DNA of the proband. Functional analysis in transfected cells was conducted on the gene variant identified. Public single nucleotide polymorphism (SNP) databases were searched for the presence of the variant allele. Results: No mutations were identified in PTH, GNA11, and GCM2 in the proband. However, a germline homozygous variant (c.1631G>A; p.R544Q) in exon 6 of the CASR was identified. Both parents are heterozygous for the variant. The variant allele frequency was near 0.1% in SNP databases. By in vitro functional analysis, the variant was significantly more potent in stimulating both the Ca2+i and MAPK signaling pathways than wild type when transfected alone (P < 0.05) but not when transfected together with wild type. The overactivity of the mutant CaSR is due to loss of a critical structural cation-π interaction. Conclusions: The patient's hypoparathyroidism is due to homozygosity of a variant in the CASR that normally has weak or no phenotypic expression in heterozygosity. Although rare, this has important implications for genetic counseling and clinical management.

Allogenic and Autogenic Signals in the Stratigraphic Record of the Deep-Sea Bengal Fan.

The Himalayan-sourced Ganges-Brahmaputra river system and the deep-sea Bengal Fan represent Earth's largest sediment-dispersal system. Here we present detrital zircon U-Pb provenance data from Miocene to middle Pleistocene Bengal Fan turbidites, and evaluate the influence of allogenic forcing vs. autogenic processes on signal propagation from the Himalaya to the deep sea. Our data record the strong tectonic and climatic forcing characteristic of the Himalayan system: after up to 2500 km of river transport, and >1400 km of transport by turbidity currents, the U-Pb record faithfully represents Himalayan sources. Moreover, specific U-Pb populations record Miocene integration of the Brahmaputra drainage with the Asian plate, as well as the rapid Plio-Pleistocene incision through, and exhumation of, the eastern Himalayan syntaxis. The record is, however, biased towards glacial periods when rivers were extended across the shelf in response to climate-forced sea-level fall, and discharged directly to slope canyons. Finally, only part of the record represents a Ganges or Brahmaputra provenance end-member, and most samples represent mixing from the two systems. Mixing or the lack thereof likely represents the fingerprint of autogenic delta-plain avulsions, which result in the two rivers delivering sediment separately to a shelf-margin canyon or merging together as they do today.

Efficacy of hybrid vitamin D receptor agonist/histone deacetylase inhibitors in vitamin D-resistant triple-negative 4T1 breast cancer.

Hormonal 1,25-dihydroxyvitamin D (1,25D) and its analogues have shown efficacy in some preclinical models of cancer. However, many models are resistant to the antiproliferative effects of 1,25D or its analogues in vitro or in vivo, and such compounds have failed in the clinic as monotherapies because of tumor resistance. Given the observed synergism between 1,25D analogues and histone deacetylase inhibitors (HDACi) in 1,25D-resistant cells, we previously developed a series of hybrid secosteroidal and easily assembled non-secosteroidal analogues that combined agonism for the vitamin D receptor and HDACi in a single backbone. These compounds displayed enhanced efficacy against 1,25D-resistant malignant cells in vitro. Structure/function studies led to synthesis of several non-secosteroidal variants in which HDACi potency was optimized without substantially sacrificing VDR agonism. Here, we present the first studies of efficacy in vivo of two of these compounds, DK-366 and DK-406, in the aggressive mouse 4T1 model of triple-negative breast cancer, a form of the disease for which treatment options are limited. 4T1 cells are resistant in vitro to the cytostatic and cytotoxic effects of 1,25D and the potent HDACi SAHA individually up to concentrations of 1µM and 50µM, respectively, whereas combinations of the two are efficacious. In vitro, DK-366 or -406 induced dose-dependent arrest of cell proliferation and cytotoxicity at 10-20µM. In vivo, the maximum tolerated dose (MTD) of DK-366 and DK-406 were 2.5 and 5.0mg/kg, respectively. Although the compounds induced hypercalcemia at elevated doses, consistent with VDR agonism in vivo, they both reduced tumor burden at doses below their MTD's. Moreover, in a separate experiment, DK-406 at 5mg/kg reduced 4T1 lung metastases by at least 50%. Under the same conditions, 1,25D (0.25µg/kg) and SAHA (25mg/kg) combined had no effect on tumor burden or on lung metastases. These experiments show that hybrid compounds are bioavailable and efficacious against a particularly aggressive model of metastatic breast cancer, providing strong support for the therapeutic potential of the hybrid concept.