Structure-property relationships in dicyanopyrazinoquinoxalines and their hydrogen-bonding-capable dihydropyrazinoquinoxalinedione derivatives.

Presented here is the design, synthesis, and study of a variety of novel hydrogen-bonding-capable π-conjugated N-heteroacenes, 1,4-dihydropyrazino[2,3-b]quinoxaline-2,3-diones (DPQDs). The DPQDs were accessed from the corresponding weakly hydrogen-bonding dicyanopyrazinoquinoxaline (DCPQ) suspensions with excess potassium hydroxide, resulting in moderate to good yields. Both families of compounds were analyzed by UV-vis and NMR spectroscopy, where the consequences of hydrogen bonding capability could be assessed through the structure-property studies. Conversion of the DCPQs into hydrogen-bonding capable DPQDs results in modulation of frontier MO energies, higher molar extinction coefficients, enhanced crystallinity, and on-average higher thermal stability (where in some cases the 5% weight loss temperature is increased by up to 100 °C). Single crystal X-ray diffraction data could be obtained for three DPQDs. One reveals pairwise hydrogen bonding in the solid state as well as a herringbone packing arrangement rendering it a promising candidate for additional studies in the context of organic optoelectronic devices.

Dicyanorhodanine-Pyrrole Conjugates for Visible Light-Driven Quantitative Photoswitching in Solution and the Solid State.

Small molecule photoswitches capable of toggling between two distinct molecular states in response to light are versatile tools to monitor biological processes, control photochemistry, and design smart materials. In this work, six novel dicyanorhodanine-based pyrrole-containing photoswitches are reported. The molecular design avails both the Z and E isomers from synthesis, where each can be isolated using chromatographic techniques. Inter- and intramolecular hydrogen bonding (H-bonding) interactions available to the E and Z isomers, respectively, uniquely impart thermal stability to each isomer over long time periods. Photoisomerization could be assessed by solution NMR and UV-vis spectroscopic techniques along with complementary ground- and excited-state computational studies, which show good agreement. Quantitative E → Z isomerization occurs upon 523 nm irradiation of the parent compound (where R = H) in solution, whereas Z → E isomerization using 404 nm irradiation offers a photostationary state (PSS) ratio of 84/16 (E/Z). Extending the π-conjugation of the pyrrole unit (where R = p-C6H4-OMe) pushes the maximum absorption to the yellow-orange region of the visible spectrum and allows bidirectional quantitative isomerization with 404 and 595 nm excitation. Comparator molecules have been prepared to report how the presence or absence of H-bonding affects the photoswitching behavior. Finally, studies of the photoswitches in neat films and photoinactive polymer matrices reveal distinctive structural and optical properties of the Z and E isomers and ultimately afford reversible photoswitching to spectrally unique PSSs using visible light sources including the Sun.

DR5 disulfide bonding as a sensor and effector of protein folding stress.

New agents are needed that selectively kill cancer cells without harming normal tissues. The TRAIL ligand and its receptors, DR5 and DR4, exhibit cancer-selective toxicity, but TRAIL analogs or agonistic antibodies targeting these receptors have not received FDA approval for cancer therapy. Small molecules for activating DR5 or DR4 independently of protein ligands may bypass some of the pharmacological limitations of these protein drugs. Previously described Disulfide bond Disrupting Agents (DDAs) activate DR5 by altering its disulfide bonding through inhibition of the Protein Disulfide Isomerases (PDIs) ERp44, AGR2, and PDIA1. Work presented here extends these findings by showing that disruption of single DR5 disulfide bonds causes high-level DR5 expression, disulfide-mediated clustering, and activation of Caspase 8-Caspase 3 mediated pro-apoptotic signaling. Recognition of the extracellular domain of DR5 by various antibodies is strongly influenced by the pattern of DR5 disulfide bonding, which has important implications for the use of agonistic DR5 antibodies for cancer therapy. Disulfide-defective DR5 mutants do not activate the ER stress response or stimulate autophagy, indicating that these DDA-mediated responses are separable from DR5 activation and pro-apoptotic signaling. Importantly, other ER stressors, including Thapsigargin and Tunicamycin also alter DR5 disulfide bonding in various cancer cell lines and in some instances, DR5 mis-disulfide bonding is potentiated by overriding the Integrated Stress Response (ISR) with inhibitors of the PERK kinase or the ISR inhibitor ISRIB. These observations indicate that the pattern of DR5 disulfide bonding functions as a sensor of ER stress and serves as an effector of proteotoxic stress by driving extrinsic apoptosis independently of extracellular ligands.

Thin-Film Morphology and Optical Properties of Photoisomerizable Donor-Acceptor Oligothiophenes.

It was recently reported that the most popular electron-accepting units introduced to π-conjugated oligomers studied for organic photovoltaic applications are susceptible to unwanted and even destructive photochemical reactions. The consequences of Z/E photoisomerization of the popular 2-(1,1-dicyanomethylene)rhodanine (RCN) unit on the optical and morphological properties of a homologous series of RCN-functionalized oligothiophenes are studied here. Oligomers consisting of one, two, or three thiophene units were studied as pure Z isomers and with E isomer compositions of 25, 53, and 45%, respectively, for Z/E mixtures. Solutions of Z isomers and Z/E mixtures were characterized by UV-vis and photoluminescence spectroscopy, wherein changes to optical properties were evaluated on the basis of E isomer content. X-ray diffraction of thin-film Z/E mixtures reveals crystalline domains of both Z and E forms after thermal annealing for mono- and bithiophene oligomers, with greater interplanar spacing for E crystalline domains than the Z counterparts along the substrate normal direction. The surface morphology viewed by atomic force microscopy also shows fiberlike structures for the E form with a much larger aspect ratio than for the Z domains in the bithiophene oligomer. Optical characterization reveals drastic changes in the solid state upon introduction of the E form for the mono- and bithiophene derivatives, whereas subtle consequences are noted for the terthiophene analogue. Most notably, a 132 nm redshift in maximum absorption occurs for the bithiophene oligomer films containing 53% E isomer compared to the pure Z counterpart. Finally, although solid-state photoisomerization experiments find no evidence of Z → E isomerization in polycrystalline Z films, E → Z isomerization is observed and becomes more restrictive in films with higher crystallinity (i.e., after thermal annealing). This structure-property study, which elucidates the consequences of the RCN configuration on solid-state packing and optical properties, is expected to guide the development of more efficient and stable organic optoelectronic devices.

Anticancer Agents Derived from Cyclic Thiosulfonates: Structure-Reactivity and Structure-Activity Relationships.

Reported are structure-property-function relationships associated with a class of cyclic thiosulfonate molecules-disulfide-bond disrupting agents (DDAs)-with the ability to downregulate the Epidermal Growth Factor Receptor (HER) family in parallel and selectively induce apoptosis of EGFR+ or HER2+ breast cancer cells. Recent findings have revealed that the DDA mechanism of action involves covalent binding to the thiol(ate) from the active site cysteine residue of members of the protein disulfide isomerase (PDI) family. Reported is how structural modifications to the pharmacophore can alter the anticancer activity of cyclic thiosulfonates by tuning the dynamics of thiol-thiosulfonate exchange reactions, and the studies reveal a correlation between the biological potency and thiol-reactivity. Specificity of the cyclic thiosulfonate ring-opening reaction by a nucleophilic attack can be modulated by substituent addition to a parent scaffold. Lead compound optimization efforts are also reported, and have resulted in a considerable decrease of the IC50 /IC90 values toward HER-family overexpressing breast cancer cells.

Inhibitors of ERp44, PDIA1, and AGR2 induce disulfide-mediated oligomerization of Death Receptors 4 and 5 and cancer cell death.

Breast cancer mortality remains unacceptably high, indicating a need for safer and more effective therapeutic agents. Disulfide bond Disrupting Agents (DDAs) were previously identified as a novel class of anticancer compounds that selectively kill cancers that overexpress the Epidermal Growth Factor Receptor (EGFR) or its family member HER2. DDAs kill EGFR+ and HER2+ cancer cells via the parallel downregulation of EGFR, HER2, and HER3 and activation/oligomerization of Death Receptors 4 and 5 (DR4/5). However, the mechanisms by which DDAs mediate these effects are unknown. Affinity purification analyses employing biotinylated-DDAs reveal that the Protein Disulfide Isomerase (PDI) family members AGR2, PDIA1, and ERp44 are DDA target proteins. Further analyses demonstrate that shRNA-mediated knockdown of AGR2 and ERp44, or expression of ERp44 mutants, enhance basal DR5 oligomerization. DDA treatment of breast cancer cells disrupts PDIA1 and ERp44 mixed disulfide bonds with their client proteins. Together, the results herein reveal DDAs as the first small molecule, active site inhibitors of AGR2 and ERp44, and demonstrate roles for AGR2 and ERp44 in regulating the activity, stability, and localization of DR4 and DR5, and activation of Caspase 8.

Tuning Supramolecular Polymer Assembly through Stereoelectronic Interactions.

The supramolecular polymerization of 2,11-dithia[3.3]paracyclophanes through self-complementary intermolecular and transannular amide hydrogen bonding is presented. An n → π* interaction between the amide hydrogen bonding units and the central bridging atom results from the single-point exchange of a carbon atom for a sulfur atom. This orbital donor-acceptor interaction can be strengthened by oxidizing the sulfide to a sulfone which acts to shorten the donor···acceptor distance and increase orbital overlap. Experimental signatures of the increased n → π* interaction include larger isodesmic polymerization elongation constants in solution, changes in characteristic bond stretching frequencies, and geometric/structural changes evaluated by X-ray crystallography. The experimental data are supported by extensive computational investigations of both assembling and nonassembling 2,11-dithia[3.3]paracyclophanes as well as a rationally designed model system to confirm the role of stereoelectronic effects on supramolecular polymer assembly.

Repurposing Tranexamic Acid as an Anticancer Agent.

Tranexamic Acid (TA) is a clinically used antifibrinolytic agent that acts as a Lys mimetic to block binding of Plasminogen with Plasminogen activators, preventing conversion of Plasminogen to its proteolytically activated form, Plasmin. Previous studies suggested that TA may exhibit anticancer activity by blockade of extracellular Plasmin formation. Plasmin-mediated cleavage of the CDCP1 protein may increase its oncogenic functions through several downstream pathways. Results presented herein demonstrate that TA blocks Plasmin-mediated excision of the extracellular domain of the oncoprotein CDCP1. In vitro studies indicate that TA reduces the viability of a broad array of human and murine cancer cell lines, and breast tumor growth studies demonstrate that TA reduces cancer growth in vivo. Based on the ability of TA to mimic Lys and Arg, we hypothesized that TA may perturb multiple processes that involve Lys/Arg-rich protein sequences, and that TA may alter intracellular signaling pathways in addition to blocking extracellular Plasmin production. Indeed, TA-mediated suppression of tumor cell viability is associated with multiple biochemical actions, including inhibition of protein synthesis, reduced activating phosphorylation of STAT3 and S6K1, decreased expression of the MYC oncoprotein, and suppression of Lys acetylation. Further, TA inhibited uptake of Lys and Arg by cancer cells. These findings suggest that TA or TA analogs may serve as lead compounds and inspire the production of new classes of anticancer agents that function by mimicking Lys and Arg.

Influence of Amide Connectivity on the Hydrogen-Bond-Directed Self-Assembly of [n.n]Paracyclophanes.

Reported here is the synthesis and self-assembly characterization of [n.n]paracyclophanes ([n.n]pCps, n=2, 3) equipped with anilide hydrogen bonding units. These molecules differ from previous self-assembling [n.n]paracyclophanes ([n.n]pCps) in the connectivity of their amide hydrogen bonding units (C-centered/carboxamide vs. N-centered/anilide). This subtle change results in a ≈30-fold increase in the elongation constant for the [2.2]pCp-4,7,12,15-tetraanilide ([2.2]pCpNTA) compared to previously reported [2.2]pCp-4,7,12,15-tetracarboxamide ([2.2]pCpTA), and a ≈300-fold increase in the elongation constant for the [3.3]pCp-5,8,14,17-tetraanilide ([3.3]pCpNTA) compared to previously reported [3.3]pCp-5,8,14,17-tetracarboxamide ([3.3]pCpTA). The [n.n]pCpNTA monomers also represent the reversal of a previously reported trend in solution-phase assembly strength when comparing [2.2]pCpTA and [3.3]pCpTA monomers. The origins of the assembly differences are geometric changes in the association between [n.n]pCpNTA monomers-revealed by computations and X-ray crystallography-resulting in a more favorable slipped stacking of the intermolecular π-surfaces ([n.n]pCpNTA vs. [n.n]pCpTA), and a more complementary H-bonding geometry ([3.3]pCpNTA vs. [2.2]pCpNTA).

Photoisomerization of dicyanorhodanine-functionalized thiophenes.

π-Conjugated oligomers functionalized with the popular dicyanorhodanine (RCN) electron acceptor are shown to be susceptible to photo-induced Z/E isomerization. The stereochemistry of two model RCN-functionalized thiophenes is confirmed by single crystal X-ray analysis and 2D NMR, and shown to be the thermodynamically stable Z form. Relative energies, Z/E configurations, and conformational preferences are modelled using density functional theory (DFT). The photophysical properties of the model compounds are explored experimentally and computationally; the Z and E isomers display similar absorption profiles with significant spectral overlap and are inseparable upon irradiation to a photostationary state. The well-behaved photoisomerization process is routinely observable by thin-layer chromatography, UV-vis, and NMR, and the photochemical behavior of the two RCN-functionalized thiophenes is characterized under varying wavelengths of irradiation. Ultraviolet (254 nm) irradiation results in photostationary state compositions of 56/44 and 69/31 Z-isomer/E-isomer for substrates functionalized with one thiophene and two thiophenes, respectively. Ambient laboratory lighting results in excess of 10 percent E-isomer for each species in solution, an important consideration for processing such materials, particularly for organic photovoltaic applications. In addition, a photoswitching experiment is conducted to demonstrate the reversible nature of the photoreaction, where little evidence of fatigue is observed over numerous switching cycles. Overall, this work showcases an approach to characterize the stereochemistry and photochemical behavior of dicyanorhodanine-functionalized thiophenes, widely used components of functional molecules and materials.

Self-Assembling [n.n]Paracyclophanes: A Structure-Property Relationship Study.

Reported here is the synthesis, characterization, and isodesmic supramolecular polymerization of [3.3]paracyclophane-5,8,14,17-tetracarboxamide ([3.3]pCpTA). The self-assembling monomer, a bridge-expanded homolog of [2.2]paracyclophane-4,7,12,15-tetracarboxamide ([2.2]pCpTA), forms homochiral assemblies in nonpolar solution and the solid state through double-helical intermolecular and transannular hydrogen bonding. The additional methylene unit in the [3.3]paracyclophane bridge results in a weakened supramolecular assembly for [3.3]pCpTA compared to [2.2]pCpTA in solution. Likely origins of the change in assembly strength, revealed through X-ray crystallography, computational analysis, and solution-phase spectroscopy, are an increase in (a) the intramolecular and intermolecular deck-to-deck spacing compared to [2.2]paracyclophane resulting from larger amide dihedral angles accompanying transannular hydrogen bonding in the [3.3]paracyclophane and (b) monomer entropy associated with the scissoring motion of the [3.3]paracyclophane bridge.

Disulfide bond-disrupting agents activate the tumor necrosis family-related apoptosis-inducing ligand/death receptor 5 pathway.

Disulfide bond-disrupting agents (DDAs) are a new chemical class of agents recently shown to have activity against breast tumors in animal models. Blockade of tumor growth is associated with downregulation of EGFR, HER2, and HER3 and reduced Akt phosphorylation, as well as the induction of endoplasmic reticulum stress. However, it is not known how DDAs trigger cancer cell death without affecting nontransformed cells. As demonstrated here, DDAs are the first compounds identified that upregulate the TRAIL receptor DR5 through transcriptional and post-transcriptional mechanisms to activate the extrinsic cell death pathway. At the protein level, DDAs alter DR5 disulfide bonding to increase steady-state DR5 levels and oligomerization, leading to downstream caspase 8 and 3 activation. DDAs and TRAIL synergize to kill cancer cells and are cytotoxic to HER2+ cancer cells with acquired resistance to the EGFR/HER2 tyrosine kinase inhibitor Lapatinib. Investigation of the mechanisms responsible for DDA selectivity for cancer cells reveals that DDA-induced upregulation of DR5 is enhanced in the context of EGFR overexpression. DDA-induced cytotoxicity is strongly amplified by MYC overexpression. This is consistent with the known potentiation of TRAIL-mediated cell death by MYC. Together, the results demonstrate selective DDA lethality against oncogene-transformed cells, DDA-mediated DR5 upregulation, and protein stabilization, and that DDAs have activity against drug-resistant cancer cells. Our results indicate that DDAs are unique in causing DR5 accumulation and oligomerization and inducing downstream caspase activation and cancer cell death through mechanisms involving altered DR5 disulfide bonding. DDAs thus represent a new therapeutic approach to cancer therapy.

Differentiating the mechanism of self-assembly in supramolecular polymers through computation.

The mechanism by which monomers in solution, beyond a certain concentration or below a certain temperature, self-assemble to form one dimensional supramolecular polymers determines much of the bulk properties of the polymer. The two distinct pathways of assembly, namely isodesmic and cooperative, can be experimentally identified using spectroscopy and in simulations via a determination of the dependence of the association constant on the oligomer size. Employing large scale free energy calculations, we have been able to show the independence of the free energy change of oligomerization on size in the self-assembly of a [2.2]paracyclophane-tetracarboxamide ([2.2]pCpTA) derivative ([2.2]pCpTA-hex), which is experimentally shown to follow the isodesmic pathway. In contrast, simulations show the free energy change in the case of benzene-1,3,5-tricarboxamide (BTA) to depend on the oligomer size which is a signature of its cooperative nature of self-assembly. These observations are rationalized through the development of a macrodipole moment in BTA oligomers and lack thereof in the [2.2]pCpTA system.

A novel proteotoxic combination therapy for EGFR+ and HER2+ cancers.

While HER2 and EGFR are overexpressed in breast cancers and multiple other types of tumors, the use of EGFR and/or HER2 inhibitors have failed to cure many cancer patients, largely because cancers acquire resistance to HER2/EGFR-specific drugs. Cancers that overexpress the HER-family proteins EGFR, HER2, and HER3 are uniquely sensitive to agents that disrupt HER2 and EGFR protein folding. We previously showed that disruption of disulfide bond formation by Disulfide Disrupting Agents (DDAs) kills HER2/EGFR overexpressing cells through multiple mechanisms. Herein, we show that interference with proline isomerization in HER2/EGFR overexpressing cells also induces cancer cell death. The peptidyl-prolyl isomerase inhibitor Cyclosporine A (CsA) selectively kills EGFR+ or HER2+ breast cancer cells in vitro by activating caspase-dependent apoptotic pathways. Further, CsA synergizes with the DDA tcyDTDO to kill HER2/EGFR overexpressing cells in vitro and the two agents cooperate to kill HER2+ tumors in vivo. There is a critical need for novel strategies to target HER2+ and EGFR+ cancers that are resistant to currently available mechanism-based agents. Drugs that target HER2/EGFR protein folding, including DDAs and CsA, have the potential to kill cancers that overexpress EGFR or HER2 through the induction of proteostatic synthetic lethality.

Synthesis, Optoelectronic Properties, Self-Association, and Base Pairing of Nucleobase-Functionalized Oligothiophenes.

Device-relevant π-conjugated oligothiophenes with the canonical nucleobases directly embedded into the π-framework have been designed, synthesized, and characterized. These oligomers offer the ability to tune optoelectronic properties via the intimate merging of the nucleobase molecular electronic structure with base-pairing fidelity. Analysis of their optical and electronic properties in a hydrogen-bond-disrupting solvent (DMF) indicates that the nucleobase identity influences the intrinsic electronic properties of the semiconductors. These differences are supported by DFT calculations which demonstrate that the HOMO/LUMO orbitals are distributed differently for each compound. The solubility and competition between self-association and base pairing in a hydrogen-bond-supporting solvent (chloroform) was studied to better understand the oligomer behavior under conditions relevant for downstream solution processing into thin-film devices. These solution studies reveal that in each case base-pairing is preferred to self-aggregation; the relatively weak heteroassociation of 1A-1U (35 ± 5 M-1) should be amenable to facile solution processing and successive hydrogen bond formation in the solid state, while the strong heteroassociation between 1G and 1C (>104 M-1) should enable assemblies to be preformed in solution. These results are expected to enable the synthesis of more complex π-conjugated architectures and facilitate their extension to optoelectronic devices.

The unfolded protein response as a target for anticancer therapeutics.

The endoplasmic reticulum (ER) is an essential organelle in eukaryotic cells, responsible for protein synthesis, folding, sorting, and transportation. ER stress is initiated when the unfolded or misfolded protein load exceeds the capacity of the ER to properly fold protein. Tumor microenvironmental conditions, such as nutrient deprivation, hypoxia, and oxidative stress perturb protein folding and trigger chronic ER stress. Cancer cells can tolerate mild ER stress, however, persistent and severe ER stress kills cancer cells by inducing their autophagy, apoptosis, necroptosis, or immunogenic cell death. Based on this rationale, many drugs have been developed for triggering irremediable ER stress in cancer cells by targeting various processes in the secretory pathway. This review discusses the mechanisms of protein targeting to the ER, the key signaling cassettes that are involved in the ER stress response, and their correlation with cancer formation and progression. Importantly, this review discusses current experimental and FDA approved anti-cancer drugs that induce ER stress, and emerging targets within the secretory pathway for the development of new anticancer drugs.

Benzotrifuran (BTFuran): a building block for π-conjugated systems.

Reported here is the first synthesis, X-ray crystal structure, and derivatization of benzotrifuran (BTFuran). Single crystal X-ray analysis of BTFuran shows a tight hexagonal packing stabilized by π-stacking interactions and C-H···O contacts. α-Lithiation of BTFuran enables the preparation of reactive intermediates suitable for cross-coupling reactions, allowing access to representative BTFuran-containing π-conjugated systems.