Enhancement of the Antitumor and Antimetastatic Effect of Topotecan and Normalization of Blood Counts in Mice with Lewis Carcinoma by Tdp1 Inhibitors-New Usnic Acid Derivatives.

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is an important DNA repair enzyme and one of the causes of tumor resistance to topoisomerase 1 inhibitors such as topotecan. Inhibitors of this Tdp1 in combination with topotecan may improve the effectiveness of therapy. In this work, we synthesized usnic acid derivatives, which are hybrids of its known derivatives: tumor sensitizers to topotecan. New compounds inhibit Tdp1 in the micromolar and submicromolar concentration range; some of them enhance the effect of topotecan on the metabolic activity of cells of various lines according to the MTT test. One of the new compounds (compound 7) not only sensitizes Krebs-2 and Lewis carcinomas of mice to the action of topotecan, but also normalizes the state of the peripheral blood of mice, which is disturbed in the presence of a tumor. Thus, the synthesized substances may be the prototype of a new class of additional therapy for cancer.

Terpene-Containing Analogues of Glitazars as Potential Therapeutic Agents for Metabolic Syndrome.

Metabolic syndrome is a complex of abnormalities involving impaired glucose and lipid metabolism, which needs effective pharmacotherapy. One way to reduce lipid and glucose levels associated with this pathology is the simultaneous activation of nuclear PPAR-alpha and gamma. For this purpose, we synthesized a number of potential agonists based on the pharmacophore fragment of glitazars with the inclusion of mono- or diterpenic moiety in the molecular structure. The study of their pharmacological activity in mice with obesity and type 2 diabetes mellitus (C57Bl/6Ay) revealed one substance that was capable of reducing the triglyceride levels in the liver and adipose tissue of mice by enhancing their catabolism and expressing a hypoglycemic effect connected with the sensitization of mice tissue to insulin. It has also been shown to have no toxic effects on the liver.

Natural Products and Their Derivatives as Inhibitors of the DNA Repair Enzyme Tyrosyl-DNA Phosphodiesterase 1.

Tyrosyl-DNA phosphodiesterase 1 (TDP1) is an important repair enzyme that removes various covalent adducts from the 3' end of DNA. Particularly, covalent complexes of topoisomerase 1 (TOP1) with DNA stabilized by DNA damage or by various chemical agents are an examples of such adducts. Anticancer drugs such as the TOP1 poisons topotecan and irinotecan are responsible for the stabilization of these complexes. TDP1 neutralizes the effect of these anticancer drugs, eliminating the DNA adducts. Therefore, the inhibition of TDP1 can sensitize tumor cells to the action of TOP1 poisons. This review contains information about methods for determining the TDP1 activity, as well as describing the inhibitors of these enzyme derivatives of natural biologically active substances, such as aminoglycosides, nucleosides, polyphenolic compounds, and terpenoids. Data on the efficiency of combined inhibition of TOP1 and TDP1 in vitro and in vivo are presented.

Novel TDP1 Inhibitors: Disubstituted Thiazolidine-2,4-Diones Containing Monoterpene Moieties.

Tyrosyl-DNA-phosphodiesterase 1 (TDP1) is a promising target for antitumor therapy; the use of TDP1 inhibitors with a topoisomerase 1 poison such as topotecan is a potential combination therapy. In this work, a novel series of 3,5-disubstituted thiazolidine-2,4-diones was synthesized and tested against TDP1. The screening revealed some active compounds with IC50 values less than 5 µM. Interestingly, compounds 20d and 21d were the most active, with IC50 values in the submicromolar concentration range. None of the compounds showed cytotoxicity against HCT-116 (colon carcinoma) and MRC-5 (human lung fibroblasts) cell lines in the 1-100 µM concentration range. Finally, this class of compounds did not sensitize cancer cells to the cytotoxic effect of topotecan.

Synthesis and Evaluation of Hypoglycemic Activity of Structural Isomers of ((Benzyloxy)phenyl)propanoic Acid Bearing an Aminobornyl Moiety.

Free fatty acid receptor-1 (FFAR1) agonists are promising candidates for therapy of type 2 diabetes because of their ability to normalize blood sugar levels during hyperglycemia without the risk of hypoglycemia. Previously, we synthesized compound QS-528, a FFA1 receptor agonist with a hypoglycemic effect in C57BL/6NCrl mice. In the present work, structural analogs of QS-528 based on (hydroxyphenyl)propanoic acid bearing a bornyl fragment in its structure were synthesized. The seven novel compounds synthesized were structural isomers of compound QS-528, varying the positions of the substituents in the aromatic fragments as well as the configuration of the asymmetric center in the bornyl moiety. The studied compounds were shown to have the ability to activate FFAR1 at a concentration of 10 µM. The cytotoxicity of the compounds as well as their effect on glucose uptake in HepG2 cells were studied. The synthesized compounds were found to increase glucose uptake by cells and have no cytotoxic effect. Two compounds, based on the meta-substituted phenylpropanoic acid, 3-(3-(4-(((1R,2R,4R)-1,7,7-trimethylbicyclo-[2.2.1]heptan-2-ylamino)methyl)benzyloxy)phenyl)propanoic acid and 3-(3-(3-(((1R,2R,4R)-1,7,7-trimethylbicyclo [2.2.1]heptan-2-ylamino)methyl)benzyloxy)phenyl)propanoic acid, were shown to have a pronounced hypoglycemic effect in the oral glucose tolerance test with CD-1 mice.

Transcriptomic Analysis of CRISPR/Cas9-Mediated PARP1-Knockout Cells under the Influence of Topotecan and TDP1 Inhibitor.

Topoisomerase 1 (TOP1) is an enzyme that regulates DNA topology and is essential for replication, recombination, and other processes. The normal TOP1 catalytic cycle involves the formation of a short-lived covalent complex with the 3' end of DNA (TOP1 cleavage complex, TOP1cc), which can be stabilized, resulting in cell death. This fact substantiates the effectiveness of anticancer drugs-TOP1 poisons, such as topotecan, that block the relegation of DNA and fix TOP1cc. Tyrosyl-DNA phosphodiesterase 1 (TDP1) is able to eliminate TOP1cc. Thus, TDP1 interferes with the action of topotecan. Poly(ADP-ribose) polymerase 1 (PARP1) is a key regulator of many processes in the cell, such as maintaining the integrity of the genome, regulation of the cell cycle, cell death, and others. PARP1 also controls the repair of TOP1cc. We performed a transcriptomic analysis of wild type and PARP1 knockout HEK293A cells treated with topotecan and TDP1 inhibitor OL9-119 alone and in combination. The largest number of differentially expressed genes (DEGs, about 4000 both up- and down-regulated genes) was found in knockout cells. Topotecan and OL9-119 treatment elicited significantly fewer DEGs in WT cells and negligible DEGs in PARP1-KO cells. A significant part of the changes caused by PARP1-KO affected the synthesis and processing of proteins. Differences under the action of treatment with TOP1 or TDP1 inhibitors alone were found in the signaling pathways for the development of cancer, DNA repair, and the proteasome. The drug combination resulted in DEGs in the ribosome, proteasome, spliceosome, and oxidative phosphorylation pathways.

Hepatoprotective Effect of a New FFAR1 Agonist-N-Alkylated Isobornylamine.

Free fatty acid receptor-1 (FFAR1) is one of the possible therapeutic targets in the search for new hepatoprotective drugs. FFAR1 agonists were found to have hypolipidemic, antifibrotic, anti-inflammatory, antiproliferative and antioxidant effects in addition to hypoglycemic action. In this work, we conducted a study of the hepatoprotective effect of the compound QS-528 (previously discovered as an agonist of FFAR1) at doses of 60, 90, 120 and 150 mg/kg on carbon tetrachloride (CCl4)-induced liver injury. At the end of the experiment, a biochemical blood assay demonstrated that the introduction of QS-528 dose-dependently reduces the levels of liver enzymes (AST, ALT and ALKP). Histological and morphometric studies of animals' livers treated with QS-528 at doses of 120 and 150 mg/kg showed a decrease in degenerative/necrotic changes in hepatocytes and an increase in the regenerative activity of the liver. In addition, no toxicity at a single oral dose of 1000 mg/kg and an increase in HepG2 cell viability in vitro were found. Thus, the compound QS-528 was found to exhibit a hepatoprotective effect against CCl4-induced toxic liver damage.

Monoterpene substituted thiazolidin-4-ones as novel TDP1 inhibitors: Synthesis, biological evaluation and docking.

Tyrosyl-DNA phosphodiesterase 1(TDP1) is a promising target for a new therapy in oncological disease as an adjunct to topoisomerase 1 (TOP1) drugs. In this paper, novel thiazolidin-4-one derivatives with a benzyl and monoterpene substituents were synthesized. Compounds with a monoterpene fragment attached via a phenyloxy linker were active against TDP1 with IC50 values in the 1 ÷ 3 µM range, while direct attachment of monoterpene moiety to the thiazolidin-4-one fragment had no activity. Molecular modelling predicted two plausible binding modes of the active compounds both effectively blocking access to the catalytic site of TDP. At non-toxic concentrations the active ligands potentiated the efficacy of the TOP1 poison topotecan in human cervical cancer HeLa cells, but not in non-cancerous HEK293A cells.

Study of Hypoglycemic Activity of Novel 9-N-alkyltetrahydroberberine Derivatives.

Novel 9-N-alkyltetrahydroberberine derivatives were synthesized, among which, based on the results of OGTT, one compound containing the longest aliphatic substituent was selected for study in mice C57BL/6Ay, which demonstrate obesity, impaired glucose tolerance, and concomitant liver non-alcoholic fatty disease. Administration of this substance at a dose of 15 mg/kg for four weeks improved the insulin sensitivity of mice, which resulted in a decrease in fasting glucose levels and improved the tolerance of mice to OGTT glucose loading. A decrease in the level of lactate in the blood and a decrease in the amount of glucokinase in the liver were also found. The introduction of compound 3c did not have a toxic effect on animals based on biochemical data, histological analysis, and measurements of general parameters such as body weight and feed intake. Thus, the 9-N-heptyltetrahydroberberine derivative showed prominent hypoglycemic effects, which makes it promising to obtain and study other derivatives with longer substituents.

Carbonylation of Polyfluorinated 1-Arylalkan-1-ols and Diols in Superacids.

We describe the carbonylation of a series of mono and dihydroxy derivatives of polyfluorinated alkylbenzenes and benzocycloalkenes with OH groups at benzylic positions using carbon monoxide in the presence of a superacid (TfOH, a TfOH-SbF5 mixture, or a FSO3H-SbF5 mixture). It was shown that the superacid-catalyzed addition of CO to various primary and secondary polyfluorinated alcohols and diols gives the corresponding mono- and dicarboxylic acids or lactones. The efficiency of various superacids depending on alcohol structure was evaluated, and FSO3H-SbF5 yielded the best results in most transformations. The addition of CO to secondary 1-arylalkan-1-ols containing vicinal fluorine atoms was found to be accompanied by elimination of HF with the formation of α,ß-unsaturated aryl-carboxylic acids. In contrast to primary and secondary alcohols, conversion of tertiary perfluoro-1,1-diarylalkan-1-ols into carbonylation products is not complete, and the resulting carboxylic acids are easily decarboxylated after water treatment of the reaction mixture.

The Study of Hypoglycemic Activity of 7-Terpenylcoumarins.

Natural and synthetic coumarins are often considered privileged scaffolds for obtaining pharmacological agents with hypoglycemic activity. Chemical modification of coumarins often leads to antidiabetic agents with greater efficacy. In the present work, twenty monoterpene-substituted 7-hydroxycoumarins were synthesized. A new approach using the Mitsunobu reaction was shown to be effective for the synthesis of target compounds. All of the synthesized compounds were evaluated in an oral glucose tolerance test, and two of them containing geranyl and (-)-myrtenyl substituents showed in vivo hypoglycemic action. A possible mechanism of action of these compounds may include inhibition of DPP IV, which was proved in an in vitro test.

Triterpenic Acid Amides as Potential Inhibitors of the SARS-CoV-2 Main Protease.

Although the incidence and mortality of SARS-CoV-2 infection has been declining during the pandemic, the problem related to designing novel antiviral drugs that could effectively resist viruses in the future remains relevant. As part of our continued search for chemical compounds that are capable of exerting an antiviral effect against the SARS-CoV-2 virus, we studied the ability of triterpenic acid amides to inhibit the SARS-CoV-2 main protease. Molecular modeling suggested that the compounds are able to bind to the active site of the main protease via non-covalent interactions. The FRET-based enzyme assay was used to reveal that compounds 1e and 1b can inhibit the SARS-CoV-2 main protease at micromolar concentrations.

New Hybrid Compounds Combining Fragments of Usnic Acid and Thioether Are Inhibitors of Human Enzymes TDP1, TDP2 and PARP1.

Tyrosyl-DNA phosphodiesterase 1 (TDP1) catalyzes the cleavage of the phosphodiester bond between the tyrosine residue of topoisomerase 1 (TOP1) and the 3' phosphate of DNA in the single-strand break generated by TOP1. TDP1 promotes the cleavage of the stable DNA-TOP1 complexes with the TOP1 inhibitor topotecan, which is a clinically used anticancer drug. This article reports the synthesis and study of usnic acid thioether and sulfoxide derivatives that efficiently suppress TDP1 activity, with IC50 values in the 1.4-25.2 µM range. The structure of the heterocyclic substituent introduced into the dibenzofuran core affects the TDP1 inhibitory efficiency of the compounds. A five-membered heterocyclic fragment was shown to be most pharmacophoric among the others. Sulfoxide derivatives were less cytotoxic than their thioester analogs. We observed an uncompetitive type of inhibition for the four most effective inhibitors of TDP1. The anticancer effect of TOP1 inhibitors can be enhanced by the simultaneous inhibition of PARP1, TDP1, and TDP2. Some of the compounds inhibited not only TDP1 but also TDP2 and/or PARP1, but at significantly higher concentration ranges than TDP1. Leader compound 10a showed promising synergy on HeLa cells in conjunction with the TOP1 inhibitor topotecan.

Discovery of Novel Sultone Fused Berberine Derivatives as Promising Tdp1 Inhibitors.

A new type of berberine derivatives was obtained by the reaction of berberrubine with aliphatic sulfonyl chlorides. The new polycyclic compounds have a sultone ring condensed to C and D rings of a protoberberine core. The reaction conditions were developed to facilitate the formation of sultones with high yields without by-product formation. Thus, it was shown that the order of addition of reagents affects the composition of the reaction products: when sulfochlorides are added to berberrubine, their corresponding 9-O-sulfonates are predominantly formed; when berberrubine is added to pre-generated sulfenes, sultones are the only products. The reaction was shown to proceed stereo-selectively and the cycle configuration was confirmed by 2D NMR spectroscopy. The inhibitory activity of the synthesized sultones and their 12-brominated analogs against the DNA-repair enzyme tyrosyl-DNA phosphodiesterase 1 (Tdp1), an important target for a potential antitumor therapy, was studied. All derivatives were active in the micromolar and submicromolar range, in contrast to the acyclic analogs and 9-O-sulfonates, which were inactive. The significance of the sultone cycle and bromine substituent in binding with the enzyme was confirmed using molecular modeling. The active inhibitors are mostly non-toxic to the HeLa cancer cell line, and several ligands show synergy with topotecan, a topoisomerase 1 poison in clinical use. Thus, novel berberine derivatives can be considered as candidates for adjuvant therapy against cancer.

Usnic Acid Conjugates with Monoterpenoids as Potent Tyrosyl-DNA Phosphodiesterase 1 Inhibitors.

Hybrid molecules created from different pharmacophores of natural and synthetic equivalents are successfully used in pharmaceutical practice. One promising target for anticancer therapy is tyrosyl-DNA phosphodiesterase 1 (Tdp1) because it can repair DNA lesions caused by DNA-topoisomerase 1 (Top1) inhibitors, resulting in drug resistance. In this study, new hybrid compounds were synthesized by combining the pharmacophoric moiety of a set of natural compounds with inhibitory properties against Tdp1, particularly, phenolic usnic acid and a set of different monoterpenoid fragments. These fragments were connected through a hydrazinothiazole linker. The inhibitory properties of the new compounds mainly depended on the structure of the terpenoid moieties. The two most potent compounds, 9a and 9b, were synthesized from citral and citronellal, which contain acyclic fragments with IC50 values in the range of 10-16 nM. Some synthesized derivatives showed low cytotoxicity against HeLa cells and increased the effect of the Top1 inhibitor topotecan in vitro by three to seven times. These derivatives may be considered as potential agents for the development of anticancer therapies when combined with Top1 inhibitors.

Exploring bulky natural and natural-like periphery in the design of p-(benzyloxy)phenylpropionic acid agonists of free fatty acid receptor 1 (GPR40).

Six derivatives of 3-phenylpropionic acid bearing various natural and natural-like, spatially defined peripheral motifs have been synthesized and evaluated in vitro for free fatty acid receptor 1 (FFA1) activation. Two frontrunner compounds (bearing a bornyl and cytosine groups) were evaluated in an oral glucose tolerance test in mice where both demonstrated the ability to sustain blood glucose levels following a glucose challenge. The bornyl compound displayed a somewhat superior, dose-dependent efficacy and, therefore, can be regarded as a lead compounds for further development as a therapeutic agent for type 2 diabetes mellitus. Its high affinity to FFA1 was rationalized by docking experiments.

The First Berberine-Based Inhibitors of Tyrosyl-DNA Phosphodiesterase 1 (Tdp1), an Important DNA Repair Enzyme.

A series of berberine and tetrahydroberberine sulfonate derivatives were prepared and tested against the tyrosyl-DNA phosphodiesterase 1 (Tdp1) DNA-repair enzyme. The berberine derivatives inhibit the Tdp1 enzyme in the low micromolar range; this is the first reported berberine based Tdp1 inhibitor. A structure-activity relationship analysis revealed the importance of bromine substitution in the 12-position on the tetrahydroberberine scaffold. Furthermore, it was shown that the addition of a sulfonate group containing a polyfluoroaromatic moiety at position 9 leads to increased potency, while most of the derivatives containing an alkyl fragment at the same position were not active. According to the molecular modeling, the bromine atom in position 12 forms a hydrogen bond to histidine 493, a key catalytic residue. The cytotoxic effect of topotecan, a clinically important topoisomerase 1 inhibitor, was doubled in the cervical cancer HeLa cell line by derivatives 11g and 12g; both displayed low toxicity without topotecan. Derivatives 11g and 12g can therefore be used for further development to sensitize the action of clinically relevant Topo1 inhibitors.

Inhibitory Effect of New Semisynthetic Usnic Acid Derivatives on Human Tyrosyl-DNA Phosphodiesterase 1.

Usnic acid, a lichen secondary metabolite produced by a whole number of lichens, has attracted the interest of researchers owing to its broad range of biological activity, including antiviral, antibiotic, anticancer properties, and it possessing a certain toxicity. The synthesis of new usnic acid derivatives and the investigation of their biological activity may lead to the discovery of compounds with better pharmacological and toxicity profiles. In this context, a series of new usnic acid derivatives comprising a terpenoid moiety were synthesized, and their ability to inhibit the catalytic activity of the human DNA repair enzyme tyrosyl-DNA phosphodiesterase 1 was investigated. The most potent compounds (15A, 15B, 15G: , and 16A, 16B, 16G: ) had IC50 values in the range of 0.33 - 2.7 µM. The inhibitory properties were mainly dependent on the flexibility and length of the terpenoid moiety, but not strongly dependent on the configuration of the asymmetric centers. The synthesized derivatives showed low cytotoxicity against human cell lines in an MTT assay. They could be used as a basis for the development of more effective anticancer therapies when combined with topoisomerase 1 inhibitors.

New Hydrazinothiazole Derivatives of Usnic Acid as Potent Tdp1 Inhibitors.

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a promising therapeutic target in cancer therapy. Combination chemotherapy using Tdp1 inhibitors as a component can potentially improve therapeutic response to many chemotherapeutic regimes. A new set of usnic acid derivatives with hydrazonothiazole pharmacophore moieties were synthesized and evaluated as Tdp1 inhibitors. Most of these compounds were found to be potent inhibitors with IC50 values in the low nanomolar range. The activity of the compounds was verified by binding experiments and supported by molecular modeling. The ability of the most effective inhibitors, used at non-toxic concentrations, to sensitize tumors to the anticancer drug topotecan was also demonstrated. The order of administration of the inhibitor and topotecan on their synergistic effect was studied, suggesting that prior or simultaneous introduction of the inhibitor with topotecan is the most effective.

Synthesis and evaluation of aryliden- and hetarylidenfuranone derivatives of usnic acid as highly potent Tdp1 inhibitors.

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a repair enzyme for stalled DNA-topoisomerase 1 (Top 1) cleavage complexes and other 3'-end DNA lesions. Tdp1 is a promising target for anticancer therapy, since it can repair DNA lesions caused by Top1 inhibitors leading to drug resistance. Hence, Tdp1 inhibition should result in synergistic effect with Top1 inhibitors. Twenty nine derivatives of (+)-usnic acid were tested for in vitro Tdp1 inhibitory activity using a fluorescent-based assay. Excellent activity was obtained, with derivative 6m demonstrating the lowest IC50 value of 25 nM. The established efficacy was verified using a gel-based assay, which gave close results to that of the fluorescent assay. In addition, molecular modeling in the Tdp1 substrate binding pocket suggested plausible binding modes for the active analogues. The synergistic effect of the Tdp1 inhibitors with topotecan, a Top1 poison in clinical use, was tested in two human cell lines, A-549 and HEK-293. Compounds 6k and 6x gave very promising results. In particular, 6x has a low cytotoxicity and an IC50 value of 63 nM, making it a valuable lead compound for the development of potent Tdp1 inhibitors for clinical use.