Synthesis of Unsymmetrical Squaramides as Allosteric GSK-3ß Inhibitors Promoting ß-Catenin-Mediated Transcription of TCF/LEF in Retinal Pigment Epithelial Cells.

The glycogen synthase kinase 3ß (GSK-3ß) is a ubiquitous enzyme that is a validated target for the development of potential therapeutics useful in several diseases including retinal degeneration. Aiming at developing an innovative class of allosteric inhibitors of GSK-3ß potentially useful for retinal degeneration, we explored the class of squaramides. The developed compounds (6 a-l) were obtained through a nontoxic one-pot synthetic protocol, which employs low-cost goods and avoids any purification step. Ethanol was used as the reaction solvent, simultaneously allowing the pure reaction products' recovery (by precipitation). Out of this set of squaramides, 6 j stood out, from computational and enzymatic converging data, as an ATP non-competitive inhibitor of GSK-3ß of micromolar potency. When engaged in cellular studies using retinal pigment epithelial cells (ARPE-19) transfected with a luciferase reporter gene under the control of T-cell factor/lymphoid enhancer factor (TCF/LEF) binding sites, 6 j was able to dose-dependently induce ß-catenin nuclear accumulation, as shown by the increased luciferase activity at a concentration of 2.5 µM.

Synthesis and evaluation of squaramide and thiosquaramide inhibitors of the DNA repair enzyme SNM1A.

SNM1A is a zinc-dependent nuclease involved in the removal of interstrand crosslink lesions from DNA. Inhibition of interstrand crosslink repair enzymes such as SNM1A is a promising strategy for improving the efficacy of crosslinking chemotherapy drugs. Initial studies have demonstrated the feasibility of developing SNM1A inhibitors, but the full potential of this enzyme as a drug target has yet to be explored. Herein, the synthesis of a family of squaramide- and thiosquaramide-bearing nucleoside derivatives and their evaluation as SNM1A inhibitors is reported. A gel electrophoresis assay was used to identify nucleoside derivatives bearing an N-hydroxysquaramide or squaric acid moiety at the 3'-position, and a thymidine derivative bearing a 5'-thiosquaramide, as candidate SNM1A inhibitors. Quantitative IC50 determination showed that a thymidine derivative bearing a 5'-thiosquaramide was the most potent inhibitor, followed by a thymidine derivative bearing a 3'-squaric acid. UV-Vis titrations were carried out to evaluate the binding of the (thio)squaramides to zinc ions, allowing the order of inhibitory potency to be rationalised. The membrane permeability of the active inhibitors was investigated, with several compounds showing promise for future in vivo applications.

Mechanochemical synthesis insights and solid-state characterization of quininium aspirinate, a glass-forming drug-drug salt.

Quinine (an antimalarial) and aspirin (a nonsteroidal anti-inflammatory drug) were combined into a new drug-drug salt, quininium aspirinate, C20H25N2O2+·C9H7O4-, by liquid-assisted grinding using stoichiometric amounts of the reactants in a 1:1 molar ratio, and water, EtOH, toluene, or heptane as additives. A tetrahydrofuran (THF) solution of the mechanochemical product prepared using EtOH as additive led to a single crystal of the same material obtained by mechanochemistry, which was used for crystal structure determination at 100 K. Powder X-ray diffraction ruled out crystallographic phase transitions in the 100-295 K interval. Neat mechanical treatment (in a mortar and pestle, or in a ball mill at 20 or 30 Hz milling frequencies) gave rise to an amorphous phase, as shown by powder X-ray diffraction; however, FT-IR spectroscopy unambiguously indicates that a mechanochemical reaction has occurred. Neat milling the reactants at 10 and 15 Hz led to incomplete reactions. Thermogravimetry and differential scanning calorimetry indicate that the amorphous and crystalline mechanochemical products form glasses/supercooled liquids before melting, and do not recrystallize upon cooling. However, the amorphous material obtained by neat grinding crystallizes upon storage into the salt reported. The mechanochemical synthesis, crystal structure analysis, Hirshfeld surfaces, powder X-ray diffraction, thermogravimetry, differential scanning calorimetry, FT-IR spectroscopy, and aqueous solubility of quininium aspirinate are herein reported.

Squaramides as Bioisosteres in Contemporary Drug Design.

Squaramides represent a class of vinylogous amides that are derived from the squarate oxocarbon dianion. While they have been known since the 1950s, squaramides have only recently emerged (in the last 10-20 years) as particularly useful chemical entities in a variety of applications. They have found particular use as bioisosteric replacements of several heteroatomic functional groups, notably ureas, thioureas, guanidines, and cyanoguanidines, owing in part to their similar capacity toward hydrogen bonding and ability to reliably engender defined conformations in drug ligands. This Review aims to provide a comprehensive overview of the deployment of squaramides as bioisosteres within the drug design landscape. Their utility in this space is further rationalized through an examination of the physicochemical properties of squaramides in contrast to other functional groups. In addition, we consider the deployment of related cyclic oxocarbanion derivatives as potential bioisosteric replacements of ureas and related functional groups.

Synthesis and biological evaluation of novel urea, thiourea and squaramide diastereomers possessing sugar backbone.

A series of novel chiral 14 urea, thiourea and squaramide stereoisomers possessing carbohydrate backbones as well as amide functional groups was synthesized and characterized by their, 1H NMR, 13C NMR, FT-IR, HRMS, optical rotation, and melting points. Their antiproliferative activities were investigated against HeLa and PC3 cell lines. The compounds 9, 11 and 12 showed better activities at 25 µM against PC3 cell line with respect to the standard 5-fluorouracil (5-FU). Especially, the compounds 9 and 11 showed higher activities than the standard 5-FU even at low concentration (5 µM) against HeLa cell line. IC50 results also confirm these activities. The compounds 9, 10 and 11 have the IC50 values of 1.10 µM, 1.51 µM and 1.02 µM, respectively while 5-FU has 2.51 µM. Moreover, their cytotoxicity tests have proven that their viabilities were in between 50% and 100%.

Combinatorial Synthesis of Novel 9R-Acyloxyquinine Derivatives as Insecticidal Agents.

BACKGROUND: It is one of the effective ways for pesticide innovation to develop new insecticides from natural products as lead compounds. Quinine, the main alkaloid in the bark of cinchona tree as well as in plants in the same genus, is recognized as a safe and potent botanical insecticide to many insects. The structural modification of quinine into 9R-acyloxyquinine derivatives is a potential approach for the development of novel insecticides, which showed more toxicity than quinine. However, there are no reports on the insecticidal activity of 9Racyloxyquinine derivatives to control Mythimna separata. METHODS: Endeavor to discover biorational natural products-based insecticides, 20 novel 9Racyloxyquinine derivatives were prepared and assessed for their insecticidal activity against M. separata in vivo by the leaf-dipping method at 1 mg/mL. RESULTS: Among all the compounds, especially derivatives 5i, 5k and 5t exhibited the best insecticidal activity with final mortality rates of 50.0%, 57.1%, and 53.6%, respectively. CONCLUSION: Overall, a free 9-hydroxyl group is not a prerequisite for insecticidal activity and C9- substitution is well tolerated; modification of out-ring double-bond is acceptable, and hydrogenation of double-bond enhances insecticidal activity; Quinine ring is essential and open of it is not acceptable. These preliminary results will pave the way for further modification of quinine in the development of potential new insecticides.

Potent HIV-1 protease inhibitors incorporating squaramide-derived P2 ligands: Design, synthesis, and biological evaluation.

We describe the design, synthesis, and biological evaluation of novel HIV-1 protease inhibitors containing a squaramide-derived scaffold as the P2 ligand in combination with a (R)-hydroxyethylamine sulfonamide isostere. Inhibitor 3h with an N-methyl-3-(R)-aminotetrahydrofuranyl squaramide P2-ligand displayed an HIV-1 protease inhibitory Ki value of 0.51 nM. An energy minimized model of 3h revealed the major molecular interactions between HIV-1 protease active site and the tetrahydrofuranyl squaramide scaffold that may be responsible for its potent activity.

Synthesis and biological evaluation of aza-crown ether-squaramide conjugates as anion/cation symporters.

Aim: Anion/cation symport across cellular membranes may lead to cell apoptosis and be developed as a strategy for new anticancer drug discovery. Methodology: Four aza-crown ether-squaramide conjugates were synthesized and characterized. Their anion recognition, anion/cation symport, cytotoxicity and probable mechanism of action were investigated in details. Conclusion: These conjugates are able to form ion-pairing complexes with chloride anions and facilitate the transmembrane transport of anions via an anion/cation symport process. They can disrupt the cellular homeostasis of chloride anions and sodium cations and induce the basification of acidic organelles in live cells. These conjugates exhibit moderate cytotoxicity toward the tested cancer cells and trigger cell apoptosis by mediating the influx of chloride anions and sodium cations into live cells.

Squaramides as novel class I and IIB histone deacetylase inhibitors for topical treatment of cutaneous t-cell lymphoma.

A series of squaramide-based hydroxamic acids were designed, synthesized and evaluated against human HDAC enzyme. Squaramides were found to be potent in the Hut78 cell line, but initially suffered from low solubility. Leads with improved solubility and metabolic profiles were shown to be class I, IIB and IV selective.

Squaramide-Based Supramolecular Materials for Three-Dimensional Cell Culture of Human Induced Pluripotent Stem Cells and Their Derivatives.

Synthetic hydrogel materials can recapitulate the natural cell microenvironment; however, it is equally necessary that the gels maintain cell viability and phenotype while permitting reisolation without stress, especially for use in the stem cell field. Here, we describe a family of synthetically accessible, squaramide-based tripodal supramolecular monomers consisting of a flexible tris(2-aminoethyl)amine (TREN) core that self-assemble into supramolecular polymers and eventually into self-recovering hydrogels. Spectroscopic measurements revealed that monomer aggregation is mainly driven by a combination of hydrogen bonding and hydrophobicity. The self-recovering hydrogels were used to encapsulate NIH 3T3 fibroblasts as well as human-induced pluripotent stem cells (hiPSCs) and their derivatives in 3D. The materials reported here proved cytocompatible for these cell types with maintenance of hiPSCs in their undifferentiated state essential for their subsequent expansion or differentiation into a given cell type and potential for facile release by dilution due to their supramolecular nature.

Synthesis of novel quinine analogs and evaluation of their effects on Trypanosoma cruzi.

AIM: Chagas disease is a tropical disease caused by the hemoflagellate protozoan Trypanosoma cruzi. There is no vaccine for Chagas disease and available drugs (e.g., benznidazole) are effective only during the acute phase, displaying a variable curative activity in the established chronic form of the disease. New leads with high efficacy and better toxicity profiles are urgently required. Materials & methods: A library of novel quinine derivatives was synthesized using Heck chemistry and evaluated against the various developmental forms of T. cruzi. RESULTS AND CONCLUSION: Several novel quinine analogs with trypanocidal activity have been identified with the para-nitro-substituted derivative displaying a submicromolar IC50, which is 83-times lower than quinine and three-times lower than benznidazole. Transmission electron microscopy analysis demonstrated that these compounds induced a marked vacuolization of the kinetoplast of intracellular amastigotes and cell-derived trypomastigotes.

Piperidine and octahydropyrano[3,4-c] pyridine scaffolds for drug-like molecular libraries of the European Lead Factory.

We report short and efficient scalable syntheses of enantiomerically pure (3R,4S)-3-(hydroxymethyl4-(hydroxyethyl))-piperidine and 1-hydroxymethyl-octahydro-1H-pyrano[3,4-c]pyridine scaffolds. The alkaloid core was readily synthesized from naturally occurring quinine and can serve as a valued starting point for drug-discovery. Cleavage of a terminal 1,2-diol and acid catalysed epoxide opening cyclization are the key steps involved. A number of members of a projected small-molecular library is synthesized for each scaffold.

Development of Chiral, Bifunctional Thiosquaramides: Enantioselective Michael Additions of Barbituric Acids to Nitroalkenes.

We report a general method for the synthesis of chiral thiosquaramides, a class of bifunctional catalysts not previously described in the literature. Thiosquaramides are found to be more acidic and significantly more soluble in nonpolar solvents than their oxosquaramide counterparts, and they are excellent catalysts for the unreported, enantioselective conjugate addition reaction of the barbituric acid pharmacaphore to nitroalkenes, delivering the chiral barbiturate derivatives in high yields and high enantioselectivities, even with catalyst loadings as low as 0.05 mol%.

A novel complexity-to-diversity strategy for the diversity-oriented synthesis of structurally diverse and complex macrocycles from quinine.

Recent years have witnessed a global decline in the productivity and advancement of the pharmaceutical industry. A major contributing factor to this is the downturn in drug discovery successes. This can be attributed to the lack of structural (particularly scaffold) diversity and structural complexity exhibited by current small molecule screening collections. Macrocycles have been shown to exhibit a diverse range of biological properties, with over 100 natural product-derived examples currently marketed as FDA-approved drugs. Despite this, synthetic macrocycles are widely considered to be a poorly explored structural class within drug discovery, which can be attributed to their synthetic intractability. Herein we describe a novel complexity-to-diversity strategy for the diversity-oriented synthesis of novel, structurally complex and diverse macrocyclic scaffolds from natural product starting materials. This approach exploits the inherent structural (including functional) and stereochemical complexity of natural products in order to rapidly generate diversity and complexity. Readily-accessible natural product-derived intermediates serve as structural templates which can be divergently functionalized with different building blocks to generate a diverse range of acyclic precursors. Subsequent macrocyclisation then furnishes compounds that are each based around a distinct molecular scaffold. Thus, high levels of library scaffold diversity can be rapidly achieved. In this proof-of-concept study, the natural product quinine was used as the foundation for library synthesis, and six novel structurally diverse, highly complex and functionalized macrocycles were generated.

Underexplored Opportunities for Natural Products in Drug Discovery.

The importance of natural products in the treatment of human disease is well documented. While natural products continue to have a profound impact on human health, chemists have succeeded in generating semisynthetic analogues that sometimes overshadow the original natural product in terms of clinical significance. Synthetic efforts based on natural products have primarily focused on improving their drug-like features while targeting utility in the same biological space. A less documented phenomenon is that natural products can serve as powerful starting materials to generate drug substances with novel therapeutic utility that is unrelated to the biological space of the natural product starting material. In this Perspective, examples of natural product derived marketed drugs with therapeutic utility in clinical space that is different from the biological profile of the starting material are presented, demonstrating that this is not merely a theoretical concept but both a clinical reality and an underexplored opportunity.

Historical Review: Problematic Malaria Prophylaxis with Quinine.

Quinine, a bitter-tasting, short-acting alkaloid drug extracted from cinchona bark, was the first drug used widely for malaria chemoprophylaxis from the 19th century. Compliance was difficult to enforce even in organized groups such as the military, and its prophylaxis potential was often questioned. Severe adverse events such as blackwater fever occurred rarely, but its relationship to quinine remains uncertain. Quinine prophylaxis was often counterproductive from a public health viewpoint as it left large numbers of persons with suppressed infections producing gametocytes infective for mosquitoes. Quinine was supplied by the first global pharmaceutical cartel which discouraged competition resulting in a near monopoly of cinchona plantations on the island of Java which were closed to Allied use when the Japanese Imperial Army captured Indonesia in 1942. The problems with quinine as a chemoprophylactic drug illustrate the difficulties with medications used for prevention and the acute need for improved compounds.

Novel squaramides with in vitro liver stage antiplasmodial activity.

A structure-activity relationship study was performed with ten 8-aminoquinoline-squaramides compounds active against liver stage malaria parasites, using human hepatoma cells (Huh7) infected by Plasmodium berghei parasites. In addition, their blood-schizontocidal activity was assessed against chloroquine-resistant W2 strain Plasmodium falciparum. Compound 3 was 7.3-fold more potent than the positive control primaquine against liver-stage parasites, illustrating the importance of the squarate moiety to activity.

Analytics of Quinine and its Derivatives.

The objective of this study was to perform a synthesis and analysis of the most important information on quinine and its derivatives, which are still very important in the treatment of malaria. The analysis of stereoisomers of quinine and its derivatives was conducted using two techniques, high-performance liquid chromatography and capillary electrophoresis. Particularly noteworthy is the technique used for the determination of isotachophoresis, referred to as one of the so-called green chemistry techniques. Particular attention was paid to properties and the use of quinine and its derivatives in the treatment of malaria. The analytical part will supplement knowledge about quinidine, quinine, and cinchonidine, and will contribute to the growth of research on the so-much-needed drugs against malaria.

Structure-affinity relationship of the cocaine-binding aptamer with quinine derivatives.

In addition to binding its target molecule, cocaine, the cocaine-binding aptamer tightly binds the alkaloid quinine. In order to understand better how the cocaine-binding aptamer interacts with quinine we have used isothermal titration calorimetry-based binding experiments to study the interaction of the cocaine-binding aptamer to a series of structural analogs of quinine. As a basis for comparison we also investigated the binding of the cocaine-binding aptamer to a set of cocaine metabolites. The bicyclic aromatic ring on quinine is essential for tight affinity by the cocaine-binding aptamer with 6-methoxyquinoline alone being sufficient for tight binding while the aliphatic portion of quinine, quinuclidine, does not show detectable binding. Compounds with three fused aromatic rings are not bound by the aptamer. Having a methoxy group at the 6-position of the bicyclic ring is important for binding as substituting it with a hydrogen, an alcohol or an amino group all result in lower binding affinity. For all ligands that bind, association is driven by a negative enthalpy compensated by unfavorable binding entropy.