Spirocyclic dimer SpiD7 activates the unfolded protein response to selectively inhibit growth and induce apoptosis of cancer cells.

The unfolded protein response (UPR) is an adaptation mechanism activated to resolve transient accumulation of unfolded/misfolded proteins in the endoplasmic reticulum. Failure to resolve the transient accumulation of such proteins results in UPR-mediated programmed cell death. Loss of tumor suppressor gene or oncogene addiction in cancer cells can result in sustained higher basal UPR levels; however, it is not clear if these higher basal UPR levels in cancer cells can be exploited as a therapeutic strategy. We hypothesized that covalent modification of surface-exposed cysteine (SEC) residues could simulate unfolded/misfolded proteins to activate the UPR, and that higher basal UPR levels in cancer cells would provide the necessary therapeutic window. To test this hypothesis, here we synthesized analogs that can covalently modify multiple SEC residues and evaluated them as UPR activators. We identified a spirocyclic dimer, SpiD7, and evaluated its effects on UPR activation signals, that is, XBP1 splicing, phosphorylation of eIF2α, and a decrease in ATF 6 levels, in normal and cancer cells, which were further confirmed by RNA-Seq analyses. We found that SpiD7 selectively induced caspase-mediated apoptosis in cancer cells, whereas normal cells exhibited robust XBP1 splicing, indicating adaptation to stress. Furthermore, SpiD7 inhibited the growth of high-grade serous carcinoma cell lines ~3-15-fold more potently than immortalized fallopian tube epithelial (paired normal control) cells and reduced clonogenic growth of high-grade serous carcinoma cell lines. Our results suggest that induction of the UPR by covalent modification of SEC residues represents a cancer cell vulnerability and can be exploited to discover novel therapeutics.

Synthesis of Highly Functionalized Spirocycles and Pentafulvene-Containing Dyes Involving 2-(2'-ketoalkyl)-1,3-indandiones.

Synthesis of highly functionalized spiro[4.4]nonane and spiro[4.5]decane motifs by the reaction of dimethylacetylenedicarboxylate (DMAD) with 2-(2'-ketoalkyl)-1,3-indandiones and 2-(3'-ketoalkyl)-1,3-indandiones, respectively, has been developed by utilizing a catalytic amount of DABCO. The tertiary hydroxy-containing spiro[4.4]nonane products were converted into fully conjugated pentafulvene π-systems in an acidic medium through dehydration and unprecedented C-C bond rearrangement.

Immunosuppressive diarylpropane dimer and spirocyclic-monomers from Horsfieldia kingii.

Horsfiequinone G (1), a dimeric diarylpropane featuring an unprecedentedly oxo-6/7/6 fused ring system, a new flavane, horsfielenide F (2), three naturally occurring spirocyclic monomers containing all-carbon quaternary centers, horspirotone A (3), horspirotone B (4), and methyl spirobroussonin B (5), along with horsfiequinone A (6) were isolated from Horsfieldia kingii. Their structures and absolute configurations were determined by the inspection of extensive spectroscopic data and electronic circular dichroism (ECD) calculations. Biological evaluations of these isolates revealed that compounds 1 - 3 and 5 - 6 exhibited specifically immunosuppressive activities against Con A-induced T lymphocytes with IC50 values ranging from 2.07 to 12.34 µM (selectivity indices = 2.3-25.2). Compound 1 also suppressed the secretion of inflammatory factors like IL-1ß and IL-6 in RAW264.7 cells which could present a new class of nonsteroidal anti-inflammatory agent. Finally, the primary structure-activity relationship (SAR) was also discussed.

A Theoretical Investigation of Novel Sila- and Germa-Spirocyclic Imines and Their Relevance for Electron-Transporting Materials and Drug Discovery.

A new class of spirocyclic imines (SCIs) has been theoretically investigated by applying a variety of quantum chemical methods and basis sets. The uniqueness of these compounds is depicted by various peculiarities, e.g., the incidence of planar six-membered rings each with two imine groups (two π bonds) and the incorporation of the isosteres carbon, silicon, or germanium spiro centers. Additional peculiarities of these novel SCIs are mirrored by their three-dimensionality, the simultaneous occurrence of nucleophilic and electrophilic centers, and the cross-hyperconjugative (spiro-conjugation) interactions, which provoke charge mobility along the spirocyclic scaffold. Substitution of SCIs with strong electron-withdrawing substituents, like the cyano group or fluorine, enhances their docking capability and impacts their reactivity and charge mobility. To gain thorough knowledge about the molecular properties of these SCIs, their structures have been optimized and various quantum chemical concepts and models were applied, e.g., full NBO analysis and the frontier molecular orbitals (FMOs) theory (HOMO-LUMO energy gap) and the chemical reactivity descriptors derived from them. For the assessment of the charge density distribution along the SCI framework, additional complementary quantum chemical methods were used, e.g., molecular electrostatic potential (MESP) and Bader's QTAIM. Additionally, using the aromaticity index NICS (nuclear independent chemical shift) and other criteria, it could be shown that the investigated cross-hyperconjugated sila and germa SCIs are spiro-aromatics of the Heilbronner Craig-type Möbius aromaticity.

Chiral Auxiliary Approach for Gold(I)-Catalyzed Cyclizations.

Two different classes of stereoselective cyclizations have been developed using a chiral auxiliary approach with commercially available [JohnPhosAu(MeCN)SbF6 ] as catalyst. First, a stereoselective cascade cyclization of 1,5-enynes was achieved using the Oppolzer camphorsultam as chiral auxiliary. In this case, a one-pot cyclization-hydrolysis sequence was developed to directly afford enantioenriched spirocyclic ketones. Then, the stereoselective alkoxycyclization of 1,6-enynes was mediated by an Evans-type oxazolidinone. A reduction-hydrolysis sequence was selected to remove the auxiliary to give enantioenriched ß-tetralones. DFT studies confirmed that the steric clash between the chiral auxiliary and alkene accounts for the experimentally observed diastereoselective cyclization through the Si face.

Design and synthesis of novel spirocyclic carboxylic acids as potent and orally bioavailable DGAT1 inhibitors and their biological evaluation.

A series of novel spirocyclic DGAT1 inhibitors containing the oxadiazole motif were designed and synthesized for biological evaluation. Several compounds exhibited potent diacylglycerol acyltransferase 1 (DGAT1) inhibitory activity. Optimization of the series led to the identification of five lead compounds 8, 9, 10, 11 and 12 that showed excellent in-vitro activity with IC50 values ranging from 7 to 20 nM against human DGAT1. All compounds demonstrated good druggability as well as microsomal stability and safety profiles such as hERG and CYP. Compound 12 significantly reduced plasma triglyceride levels in-vivo in the mouse model of acute lipid challenge. Significant reduction in plasma TG excursion was observed, thus indicating DGAT1 inhibition in-vivo.

Dimers of isatin derived α-methylene-γ-butyrolactone as potent anti-cancer agents.

The IKK-NFκB complex is a key signaling node that facilitates activation of gene expression in response to extracellular signals. The kinase IKKß and the transcription factor RELA have been targeted by covalent modifiers that bind to surface exposed cysteine residues. A common feature in well characterized covalent modifiers of RELA and IKKß is the Michael acceptor containing α-methylene-γ-butyrolactone functionality. Through synthesis and evaluation of a focused set of α-methylene-γ-butyrolactone containing spirocyclic dimers (SpiDs) we identified SpiD3 as an anticancer agent with low nanomolar potency. Using cell-free and cell-based studies we show that SpiD3 is a covalent modifier that generates stable RELA containing high molecular weight complexes. SpiD3 inhibits TNFα-induced IκBα phosphorylation resulting in the blockade of RELA nuclear translocation. SpiD3 induces apoptosis, inhibits colony formation and migration of cancer cells. The NCI-60 cell line screen revealed that SpiD3 potently inhibits growth of leukemia cell lines, making it a suitable pre-therapeutic lead for hematological malignancies.

Structure-activity and structure-property relationship studies of spirocyclic chromanes with antimalarial activity.

Malaria is a prevalent and lethal disease. The fast emergence and spread of resistance to current therapies is a major concern and the development of a novel line of therapy that could overcome, the problem of drug resistance, is imperative. Screening of a set of compounds with drug/natural product-based sub-structural motifs led to the identification of spirocyclic chroman-4-one 1 with promising antimalarial activity against the chloroquine-resistant Dd2 and chloroquine-sensitive 3D7 strains of the parasite. Extensive structure-activity and structure-property relationship studies were conducted to identify the essential features necessary for its activity and properties.

Design, Synthesis, Antiviral Evaluation, and Molecular Dynamics Simulation Studies of New Spirocyclic Thiopyrimidinones as Anti HCoV-229E.

The current pandemic threat presented by viral pathogens like SARS-CoV-2 (COVID-19) suggests that virus emergence and dissemination are not geographically confined. As a result, the quest for antiviral agents has become critical to control this pandemic. In the current study, we provide a novel family of spirocyclic thiopyrimidinone derivatives whose cytotoxicity and antiviral efficacy were investigated against human coronavirus 229E (HCoV-229E) as a model for the Coronaviridae family. We utilized MTT and cytopathic effect (CPE) inhibitory tests on green monkey kidney (vero-E6) cell lines. The new molecules showed varied degrees of antiviral activity against the vero-E6 cell lines with minimal cytotoxicity. With a high level of a selective index (SI=14.8), compound 9 showed outstanding inhibitory ability and could effectively suppress the human coronavirus 229E. Molecular dynamics simulation (MD) studies were performed to measure the interaction and stability of the protein-ligand complex in motion. The MD results for the most active compound 9 explored remarkable interactions with the binding pockets of the main protease (Mpro) of SARS-CoV-2 enzyme confirming the results gained from in vitro experiments. ADMET properties were also predicted for all the tested compounds. All these results demonstrated that the novel spirocyclic thiopyrimidinone derivatives would have the potential to be safe, low-cost chemical compounds that might be used as a novel therapeutic option for Coronaviridae viruses like COVID-19.

Exploration of Spirocyclic Derivatives of Ciprofloxacin as Antibacterial Agents.

The previously reported as well as newly synthesized derivatives of the 1-oxa-9-azaspiro[5.5]undecane were employed in the synthesis of thirty-six derivatives of ciprofloxacin using commercially available 7-chloro-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid and the literature protocol involving the preparation of boron chelate complex to facilitate nucleophilic aromatic substitution. All new fluoroquinolone derivatives were tested against two gram-positive as well as three gram-negative strains of bacteria. With the activity spectrum of the new derivatives being substantially narrower than that of ciprofloxacin, compounds were distinctly active against two of the five strains: gram-negative Acinetobacter baumannii 987® and gram-positive Bacillus cereus 138®. Towards these two strains, a large group of compounds displayed equal or higher potency than ciprofloxacin.

Arteannuin-B and (3-Chlorophenyl)-2-Spiroisoxazoline Derivative Exhibit Anti-Inflammatory Effects in LPS-Activated RAW 264.7 Macrophages and BALB/c Mice-Induced Proinflammatory Responses via Downregulation of NF-κB/P38 MAPK Signaling.

Host inflammatory responses are key to protection against injury; however, persistent inflammation is detrimental and contributes to morbidity and mortality. Herein, we demonstrated the anti-inflammatory role of Arteannuin-B (1) and its new spirocyclic-2-isoxazoline derivative JR-9 and their side effects in acute inflammatory condition in vivo using LPS-induced cytokines assay, carrageenan-induced paw edema, acetic acid-induced writhing and tail immersion. The results show that the spirocyclic-2-isoxazoline derivative is a potent anti-inflammatory agent with minimal cell toxicity as compared to Arteannuin-B. In addition, the efficacies of these compounds were also validated by flow cytometric, computational, and histopathological analysis. Our results show that the anti-inflammatory response of JR-9 significantly reduces the ability of mouse macrophages to produce NO, TNF-α, and IL-6 following LPS stimulation. Therefore, JR-9 is a prospective candidate for the development of anti-inflammatory drugs and its molecular mechanism is likely related to the regulation of NF-κB and MAPK signaling pathway.

Natural spirocyclic alkaloids and polyphenols as multi target dementia leads.

The U rhynchophylla, U tomentosa, Isatis indigotica Fortune, Voacanga Africana, herbal constituents, fungal extracts from Aspergillus duricaulis culture media, include spirooxindoles, polyphenols or bridged spirocyclic alkaloids. Their constituents exhibit specific and synergistic multiple neuroprotective properties including inhibiting of Aß fibril induced cytotoxicity, NMDA receptor inhibition in mice models of Alzheimer's disease (AD). The pioneering research from Woodward to Waldmann has advanced the synthesis of spirocyclic alkaloids. Furthermore, the elucidation of the genetic analysis, biochemical pathways that links strictosidine to the alkaloids akuammicine, stemmadenine, tabersonine, catharanthine, will now enable the biotechnological generation, also stimulate synthesis of related bridged spirocyclic alkaloids for medicinal investigations. From the value of spirocyclic structures as multi target dementia leads, we hypothesise that simpler Lipinski-like natural/synthetic alkaloid analogues may likewise be discovered that provide neurocognitive enhancing activities against dementia and AD.

Synthesis and pharmacological characterization of mitochondrial KATP channel openers with enhanced mitochondriotropic effects.

Mitochondria play a key role for deciding fate of cells and thus are considered an attractive target for pharmacological interventions focused on containment of myocardial ischemia/reperfusion (I/R) injury. Notably, the activation of mitochondrial potassium (mitoK) channels produces a mild depolarization of mitochondrial membrane, that contributes to reduce the driving force to calcium uptake and prevents the formation of mitochondrial transition membrane pore (MPTP); these events underlie anti-ischemic cardioprotection. However, an ideal mitoK channel opener should possess the fundamental requirement to be delivered at mitochondrial level; therefore, to improve the mitochondrial delivery of a previously characterized spirocyclic benzopyrane F81, new compounds have been developed. The three original triphenylphosphonium (TPP+)-derivatives of F81 (1-3), were evaluated for their cardioprotective activity on both isolated cardiac mitochondria and cardiac H9c2 cell line. Compound 1 was further investigated in an in vivo infarct model. This work confirms that the TPP+ strategy applied to mitoKATP openers could foster mitochondrial delivery and enhance the cardioprotective effects of mitochondrial activators of potassium channels.

Development of 2'-aminospiro [pyrano[3,2-c]quinoline]-3'-carbonitrile derivatives as non-ATP competitive Src kinase inhibitors that suppress breast cancer cell migration and proliferation.

Src kinase activity controls diverse cellular functions, including cell growth, migration, adhesion, and survival. It is de-regulated in several cancers, including breast cancer, where it is highly expressed and phosphorylated. Thus, targeting Src by a small molecule is a feasible strategy for managing different breast cancer types. Several Src kinase inhibitors are available, including the FDA-approved drug (dasatinib). However, they are primarily ATP-competitive inhibitors that have been reported to lack specificity towards Src. We have a long-time interest in discovering protein kinase inhibitors that are non-competitive for ATP. In this project, three groups of 2'-aminospiro[pyrano[3,2-c]quinoline]-3'-carbonitrile derivatives were designed and synthesized, hypothesizing that small molecules with a spiro scaffold appended to a pyrano[3,2-c]quinoline analog could act as non-ATP competitive Src kinase inhibitors. 3b, 3c, and 3d inhibited Src kinase activity with IC50s of 4.9, 5.9, and 0.9 µM, respectively. At the same time, they did not impact the MDM2/p53 interaction in HEK293 cells, which has been reported to be affected by some spirocyclic compounds. 25 µM of 3b, 3c, or 3d did not inhibit the kinase activity of ERK2, JNK1, or p38-alpha in an in-vitro kinase assay. Steady-state kinetic studies for the effect of 3d on the ability of recombinant Src to phosphorylate its substrate (Srctide) revealed a non-ATP competitive inhibition mechanism. 1.6 µM of 3d was enough to diminish Src, Fak, and paxillin phosphorylation in the breast cancer cell lines MDA-MB-231 and MCF7. In the NCI screening, 3d induced broad tumor cytotoxicity for the NCI-60 cell lines, including all the breast cancer cell lines. The potency of 3b, 3c, and 3d to inhibit migration, proliferation, and colony formation of MDA-MB-231 and proliferation of MCF7 cells correlates with their potency to suppress Src kinase activity in the same cell line. Noticeably, the cell growth suppression and apoptosis induction in the tested cell lines can be attributed to the ability of the new derivatives to suppress the ERK and Akt survival pathways downstream of Src.

Synthesis of spirocyclic ß- and γ-sultams by one-pot reductive cyclization of cyanoalkylsulfonyl fluorides.

One-pot intramolecular cyclization of novel sp3-enriched cyanoalkylsulfonyl fluorides into spirocyclic ß- or γ-sultams is disclosed. The method relies on nitrile group reduction followed by sulfonylation of amino group thus formed upon mild conditions (NaBH4, NiCl2·6H2O in MeOH). Cyclization proceeds smoothly with considerable efficiency (48-84%, 10 examples) on up to 30 g scale. The cyanoalkylsulfonyl fluoride intermediates can be obtained via S-nucleophilic substitution in ß-functionalized alkanenitriles or double alkylation of α-alkylthioacetonitrile, followed by oxidative chlorination with Cl2 and further reaction with KHF2. The title mono- and bifunctional sultams are advanced sp3-enriched building blocks for drug discovery and organic synthesis providing novel substitution patterns and frameworks mimicking saturated nitrogen heterocycles such as pyrrolidine/pyrrolidone.

Synthesis and Cytotoxicity Evaluation of Spirocyclic Bromotyrosine Clavatadine C Analogs.

Marine-originated spirocyclic bromotyrosines are considered as promising scaffolds for new anticancer drugs. In a continuation of our research to develop potent and more selective anticancer compounds, we synthesized a library of 32 spirocyclic clavatadine analogs by replacing the agmatine, i.e., 4-(aminobutyl)guanidine, side chain with different substituents. These compounds were tested for cytotoxicity against skin cancer using the human melanoma cell line (A-375) and normal human skin fibroblast cell line (Hs27). The highest cytotoxicity against the A-375 cell line was observed for dichloro compound 18 (CC50 0.4 ± 0.3 µM, selectivity index (SI) 2). The variation of selectivity ranged from SI 0.4 to reach 2.4 for the pyridin-2-yl derivative 29 and hydrazide analog of 2-picoline 37. The structure-activity relationships of the compounds in respect to cytotoxicity and selectivity toward cancer cell lines are discussed.

One new spirocyclic lactone and one new benzopyran derivative from Aspergillus terreus.

One new spirocyclic lactone, terreinlactone C (1), and one new benzopyran derivative, 2,2-dimethyl-3-hydroxychroman-6-aldehyde (2), were discovered from the fungus Aspergillus terreus. The chemical structures of compounds 1 and 2 were elucidated by detailedly analyzing NMR and HRESIMS data. Compound 1 is the first natural product with a 1-oxaspiro[4.5]decan-2-one ring system and a possible biogenetic pathway is proposed. Two compounds were tested for their cytotoxic activities against five human cancer cell lines.[Formula: see text].

Spirocyclic Nitroxides as Versatile Tools in Modern Natural Sciences: From Synthesis to Applications. Part I. Old and New Synthetic Approaches to Spirocyclic Nitroxyl Radicals.

Spirocyclic nitroxyl radicals (SNRs) are stable paramagnetics bearing spiro-junction at a-, b-, or g-carbon atom of the nitroxide fragment, which is part of the heterocyclic system. Despite the fact that the first representatives of SNRs were obtained about 50 years ago, the methodology of their synthesis and their usage in chemistry and biochemical applications have begun to develop rapidly only in the last two decades. Due to the presence of spiro-function in the SNRs molecules, the latter have increased stability to various reducing agents (including biogenic ones), while the structures of the biradicals (SNBRs) comprises a rigid spiro-fused core that fixes mutual position and orientation of nitroxide moieties that favors their use in dynamic nuclear polarization (DNP) experiments. This first review on SNRs will give a glance at various strategies for the synthesis of spiro-substituted, mono-, and bis-nitroxides on the base of six-membered (piperidine, 1,2,3,4-tetrahydroquinoline, 9,9'(10H,10H')-spirobiacridine, piperazine, and morpholine) or five-membered (2,5-dihydro-1H-pyrrole, pyrrolidine, 2,5-dihydro-1H-imidazole, 4,5-dihydro-1H-imidazole, imidazolidine, and oxazolidine) heterocyclic cores.

Enantioselective α-Carbonylative Arylation for Facile Construction of Chiral Spirocyclic ß,ß'-Diketones.

We herein describe the first enantioselective α-carbonylative arylation, providing a diverse set of chiral spiro ß,ß'-diketones bearing various ring sizes and functionalities in high yields and good to excellent enantioselectivities. Calculations suggest the transformation proceeds through reductive elimination instead of nucleophilic addition pathway.

Recent Progress in Small Spirocyclic, Xanthene-Based Fluorescent Probes.

The use of fluorescent probes in a multitude of applications is still an expanding field. This review covers the recent progress made in small molecular, spirocyclic xanthene-based probes containing different heteroatoms (e.g., oxygen, silicon, carbon) in position 10'. After a short introduction, we will focus on applications like the interaction of probes with enzymes and targeted labeling of organelles and proteins, detection of small molecules, as well as their use in therapeutics or diagnostics and super-resolution microscopy. Furthermore, the last part will summarize recent advances in the synthesis and understanding of their structure-behavior relationship including novel computational approaches.