Endoperoxide Group Research Articles

Ring-Contracted Artemisinin Derivatives as Novel CDK 4/6 Inhibitors: Synthesis and Anti-Breast Cancer Evaluation.

The endoperoxide group of artemisinins is universally accepted an essential group for their anti-cancer effects. In this study, a series of D-ring-contracted artemisinin derivatives were constructed by combining ring-contracted artemisinin core with fragments of functional heterocyclic molecules or classical CDK4/6 inhibitors to identify more efficacious breast cancer treatment agents. Twenty-six novel hybridized molecules were synthesized and characterized by HRMS, IR, 1H-NMR and 13C NMR. In antiproliferative activities and kinase inhibitory effects assays, we found that the antiproliferative effects of B01 were close to those of the positive control Palbociclib, with GI50 values of 4.87±0.23 μM and 9.97±1.44 μM towards T47D cells and MDA-MB-436 cells respectively. In addition, the results showed that B01 was the most potent compound against CDK6/cyclin D3 kinase, with an IC50 value of 0.135±0.041 μM, and its activity was approximately 1/3 of the positive control Palbociclib.

Artesunate ameliorates ligature-induced periodontitis by attenuating NLRP3 inflammasome-mediated osteoclastogenesis and enhancing osteogenic differentiation

Periodontitis, arguably the greatest common infective chronic inflammatory disease, is characterized by an imbalance of the host immune system and excessive osteoclastogenesis activity with severe alveolar bone loss. Nevertheless, in consideration of the harmful effects of repeated treatment, more sensible intervention drugs for periodontitis need to be developed. Artesunate (ART), derived from Artemisia annua L., has shown remarkable pharmacokinetic and clinical value, as well as anti-inflammatory and immunomodulatory effects in various immune and chronic diseases due to its endoperoxide group. However, the role of ART in mediating periodontitis-induced alveolar bone resorption has not been examined. In this study, ART treatment effectively ameliorated ligature-induced periodontitis via attenuating osteoclast formation in a dose-dependent manner. Mechanistically, RNA-seq revealed that ART dramatically reduced the enrichment of NLRP3 inflammasome-related genes. Concordant with our study, MCC950, a specific inhibitor of NLRP3 inflammasome, also greatly restrained osteoclastogenesis, suggesting that ART suppressed osteoclast formation by blocking NLRP3 inflammasome activation. In addition to regulating osteoclastogenesis, ART significantly enhanced osteogenic differentiation by alleviating the expression of cytokines in inflammatory conditions. Our data shed light on the probably potential mechanism of ART treatment for the intervention of periodontitis.

PH-dependent rearrangement determines the iron-activation and antitumor activity of artemisinins

The action mechanisms of artemisinins remains elusive for decades, and one long-standing question is whether the indispensable peroxide group is activated by iron or heme. Although heme usually reacts faster with artemisinins than iron does, we have found that rearrangement of dihydroartemisinin (DHA) into monoketo-aldehyde-peroxyhemiacetal (MKA) under physiological conditions can significantly enhance its reaction towards iron. The rearrangement is pH-dependent and the derived MKA is identified by LC-MS in the cellular metabolites of DHA in cancer cells. MKA reacts quickly with ferrous irons to afford reactive carbon-centered radicals and can inhibit enzyme activities in vitro. Moreover, MKA oxidizes ferrous irons to ferric irons, which may explain the effect of DHA on decreasing cellular labile iron pool (LIP). Both addition of exogenous iron and increase in LIP via triggering ferroptosis can enhance the cytotoxicity of DHA against cancer cells. While artesunate (ATS) can also decompose to MKA after hydrolyzing into DHA, the other artemisinins of lower antitumor activity, e.g. artemisinin (ART), artemether (ATM) and arteether (ATE), exhibit negligible hydrolysis and rearrangement under the same conditions. Our study reveals the vital role of molecular rearrangement to the activation and activity of artemisinins and provides a new strategy for designing antitumor molecules containing endoperoxide group.

Artesunate activation by heme in an aqueous medium

The reaction between the antimalarial drug artesunate (ATS) and ferriprotoporphyrin_(IX) (FPIX) in the presence of glutathione (GSH) has been monitored by nuclear magnetic resonance (NMR) spectroscopy. By following the disappearance of resonances of protons near the endoperoxide group in ATS, the rate at which the drug is activated can be directly measured. In an aqueous medium, the rate of ATS activation is limited by the rate of reduction of the FPIX Fe(III) center by GSH. The reaction is observed to slow dramatically in the presence of other heme binding antimalarial drugs. These findings explain the long observed antagonism between artemisinin derivatives and quinoline-based drugs. This discovery suggests that combination therapy that involves artemisinin or any of its derivatives and a quinoline-based drug may be compromised.

Tehranolide inhibits cell proliferation via calmodulin inhibition, PDE, and PKA activation.

Tehranolide, natural sesquiterpene lactone with an endoperoxide group, has been shown to inhibit cell growth in cancer cells. Tehranolide was purified from Artemisia diffusa. To detect cell viability and proliferation, MTT assay was performed. In order to determine the role of tehranolide on calmodulin (CaM) structure and activity, its effects were evaluated with fluorescence emission spectra and CaM-mediated activation of phosphodiesterase (PDE1), in comparison with artemisinin. In fact, PDE1 inhibition, cAMP accumulation, and cAMP-dependent protein kinase A (PKA) activation were examined. The inhibitory effect of tehranolide on CaM structure is more than artemisinin. The kinetic analysis of tehranolide–CaM interaction has shown that this agent competitively inhibited the activation of PDE1 without affecting Vmax. Tehranolide increased Km value in higher amounts compared with artemisinin. Moreover, tehranolide had a cytotoxic effect on K562 cell line but not on normal human lymphocytes. Additionally, PDE inhibition and consequent cAMP accumulation and PKA activity were required for inhibiting cancer cell growth by tehranolide. Our results show that tehranolide significantly reduces cell proliferation in a time and dose-dependent manner in K562 cells via CaM inhibition, following PDE inhibition, cAMP accumulation, and consequent PKA activity.

Synthesis of artemisinin dimers using the Ugi reaction and their in vitro efficacy on breast cancer cells

The Ugi four-component reaction was used to prepare a series of artemisinin monomers and dimers. We found that the endoperoxide group in artemisinin remains intact during the reaction. The new artemisinin dimers showed potent anti-cancer activity against two human breast cancer cell lines, MDA-MB-231 and BT-474. One of the Ugi artemisinin dimers showed an IC50 value of 12nM when tested on BT474 cells, more than 600 times more potent than artesunate. Furthermore, the same Ugi artemisinin dimer showed a low toxicity when tested on MCF10A, a nontumorigenic cell line, resulting in a selectivity index of more than 8000.

Tehranolide inhibits proliferation of MCF-7 human breast cancer cells by inducing G0/G1 arrest and apoptosis

Tehranolide, a novel natural sesquiterpene lactone with an endoperoxide group, bears a structural similarity to artemisinin and has been shown to inhibit cell growth. However, the underlying mechanisms of these activities remain obscure. The purpose of this study was to investigate the fundamental mechanisms by which tehranolide inhibits growth in MCF-7 cells. Cell growth was determined by using the MTT viability assay and counting cells. Apoptosis and cell-cycle progression were evaluated by means of Hoechst 33258 staining, flow cytometry with annexin-V/propidium iodide double staining, and ROS formation. The protein expression of Bax and Bcl-2 was demonstrated by Western blotting. Moreover, to determine the molecular mechanism whereby tehranolide mediates G0/G1 arrest, the expression of PI3K, p-PI3K, Akt, p-Akt, p27kip1, cyclin D1, and CDK4 was monitored. Cell proliferation was significantly inhibited by tehranolide in a dose- and time-dependent manner. This compound inhibited cell proliferation and induced G0/G1 arrest through the PI3K/Akt/cyclin D1 pathway. It also induced apoptosis and an increase in ROS. In addition, an increase in cytochrome c and Bax, as well as a decrease in Bcl-2, was observed. Moreover, blocking the CD95 receptor with an anti-CD95 antibody (ZB4) had no effect on tehranolide-mediated apoptosis. This study has yielded promising results, which show for the first time that tehranolide does inhibit the growth of cancer cells. The selective inhibition of cancer cell growth, the apoptosis induction via the mitochondrial pathway, and the G0/G1 arrest by modulating the PI3K/AKT signaling pathway and downregulating cyclin D1, which leads to the release of p27kip1 and the association of this inhibitor with the cyclin E/CDK2 complex, ultimately preventing cell-cycle progression from G1 to S phase, all serve to provide support for further studies of tehranolide as a possible anticancer drug in the clinical treatment of cancer.

Chemical constituents and biological activities of Iranian Artemisia species

Acyclic monoterpenes, monoterpene hydroperoxide, bicyclic monoterpene glycosides and unusual monoterpenes have been found in some Iranian Artemisia species. The volatile oils of Artemisia are chemically complex mixtures, often containing in excess of 100 individual components for instance in the oil of Artemisia siberi more than 160 constituents were identified. Cyclic sesquiterpenes, namely bisabolene derivatives and a salsolene keton have also been in the A. siberi described. Reports dealing with isolation and structure elucidation of sesquiterpene lactones in Artemisia species have increased dramatically. The extract of the aerial parts of Artemisia diffusa collected in the province of Khorassan (Iran) afforded, in addition to several eudesmanolides, a new type of sesquiterpene lactone with the endoperoxide group that probably has the same effect of Artemisinin as the antimalarial agent. Penta methoxylated flavone from Artemisia kulbadica has been also reported. Iranian Artemisia species are reviewed in this paper.

The Role of Heme and the Mitochondrion in the Chemical and Molecular Mechanisms of Mammalian Cell Death Induced by the Artemisinin Antimalarials

The artemisinin compounds are the frontline drugs for the treatment of drug-resistant malaria. They are selectively cytotoxic to mammalian cancer cell lines and have been implicated as neurotoxic and embryotoxic in animal studies. The endoperoxide functional group is both the pharmacophore and toxicophore, but the proposed chemical mechanisms and targets of cytotoxicity remain unclear. In this study we have used cell models and quantitative drug metabolite analysis to define the role of the mitochondrion and cellular heme in the chemical and molecular mechanisms of cell death induced by artemisinin compounds. HeLa ρ(0) cells, which are devoid of a functioning electron transport chain, were used to demonstrate that actively respiring mitochondria play an essential role in endoperoxide-induced cytotoxicity (artesunate IC(50) values, 48 h: HeLa cells, 6 ± 3 μM; and HeLa ρ(0) cells, 34 ± 5 μM) via the generation of reactive oxygen species and the induction of mitochondrial dysfunction and apoptosis but do not have any role in the reductive activation of the endoperoxide to cytotoxic carbon-centered radicals. However, using chemical modulators of heme synthesis (succinylacetone and protoporphyrin IX) and cellular iron content (holotransferrin), we have demonstrated definitively that free or protein-bound heme is responsible for intracellular activation of the endoperoxide group and that this is the chemical basis of cytotoxicity (IC(50) value and biomarker of bioactivation levels, respectively: 10β-(p-fluorophenoxy)dihydroartemisinin alone, 0.36 ± 0.20 μM and 11 ± 5%; and with succinylacetone, >100 μM and 2 ± 5%).

Using the Rotaxane Mechanical Bond to Enhance Chemical Reactivity

Rates of cycloreversion for squaraine rotaxane mono(endoperoxides) were enhanced by structural modifications that increased cross-component steric destabilization of the inward directed 9,10-anthracene endoperoxide group. The largest rate enhancements were obtained when the surrounding macrocycle contained two 2,6-pyridine dicarboxamide bridging units, which induced a cavity contraction effect. The precursor fluorescent, near-IR, squaraine rotaxanes are effectively photostable because the mono(endoperoxide) products, formed by reaction with photogenerated singlet oxygen, rapidly cyclorevert back to the original squaraine rotaxane.

Theoretical studies on model reaction pathways of prostaglandin H2 isomerization to prostaglandin D2/E2

Model reaction mechanisms in the biosynthesis of prostaglandin D2 (PGD2) and prostaglandin E2 (PGE2) from prostaglandin H2 with PGD2/E2 synthase were examined using the ab initio second-order Moller–Plesset perturbation method and density functional theory. The reaction was modeled similar to the isomerization of 2,3-dioxabicyclo[2.2.1]heptane to 3-hydroxycyclopentanone in the presence of MeS−. An explicit solvation of two H2O molecules was also considered, and two probable types of reaction mechanisms were demonstrated. One mechanism starts with proton abstraction from an oxygen-bound carbon at the endoperoxide by a thiolate ion and the other is stepwise and involves attack of a thiolate anion on an oxygen of the endoperoxide group in the first step with protonation of the other oxygen, followed by deprotonation from a carbon-attached oxygen to break an O–S bond to yield PGD2 or PGE2. We also found that the mPW1LYP hybrid method was superior to the B3LYP functional for systems with respect to the state-of-the-art CCSD(T) energetics.

Tehranolide molecule modulates the immune response, reduce regulatory T cell and inhibits tumor growth in vivo

Tehranolide named as new type of a sesquiterpene lactone with an endoperoxide group which purified from Artemisia diffusa and having similar structure to Artemisinin. Tehranolide is hypothesized to possess effects akin to Artemisinin, which is active against cancer cells. In the present research we emphasized on the direct correlation between the tumor sizes, immune response; including cytokin network, T regulatory cells and Tehranolide intraperitoneally injected Tehranolide. In this study, Tehranolide was purified from Artemisia difussa. First we evaluated the effects of Tehranolide on cell growth inhibition (in vitro) by MTT assay and second investigated the immune responses, these include measuring tumor growth in the female Balb/c mice transplanted with spontaneous mouse mammary tumor and treated with Tehranolide, splenocyte proliferation detected by using the BrdU kit, measurement of cytokine profile by ELISA and analysis of T-lymphocytes subpopulation in spleen by Flow cytometry. Our results showed a significant ( p < 0.05) decrease in the tumor volume and the level of IL-4 in the animals treated with Tehranolide, compared to untreated group. In addition, a significant ( p < 0.05) increase in the lymphocytes proliferation and the level of IFN-γ in the animals treated with Tehranolide in comparison with control group. Furthermore, we regulate the regulatory T cells in order to improve the outcome of cancer immunotherapy. The measurement of splenic CD4 +CD25 +Foxp3 + T lymphocytes indicated that Tehranolide significantly ( p < 0.05) decreased the number of these lymphocytes. These findings show that the use of Tehranolide molecule represents a novel strategy with major suggestions for cancer therapy approaches.

Tehranolide could shift the immune response towards Th1 and modulate the intra-tumor infiltrated T regulatory cells.

Artemisia diffusa contains a new type of sesquiterpene lactone with an endoperoxide group (Tehranolide). Due to the existing similarity between the structures of Tehranolide and Artemisinin, it was hypothesized that Tehranolide would have similar effects as Artemisinin. In this study, the immunotherapeutic effectiveness of Tehranolide was investigated by direct intra-tumoral injection. Tehranolide was purified from Artemisia diffusa, and its effect on the tumor volume was investigated. The splenocyte proliferation, shifting of cytokine profile, and the presence of naturally-occurring CD4+CD25+Foxp3+ Treg cells were assessed to describe the anti-tumor immune response. Analysis of immune response showed that, intra-tumoral injection of Tehranolide decreased the rate of tumor growth compared to control group. Furthermore, the proliferative response of mice treated with Tehranolide was enhanced. In comparison with the control group, production of both IL-4 and IFN-gamma was induced (p<0.05). The results indicated a decrease in tumor CD4+CD25+Foxp3+ T lymphocytes in the Tehranolide-treated group compared to the control group. Treatment of tumors with Tehranolide attenuated CD4+CD25+Foxp3+ Treg cell-mediated immune suppression and elicited a persistent anti-tumor immunity against cancer.

Structural Basis of the Catalytic Mechanism Operating in Open-Closed Conformers of Lipocalin Type Prostaglandin D Synthase

Lipocalin type prostaglandin D synthase (L-PGDS) is a multifunctional protein acting as a somnogen (PGD2)-producing enzyme, an extracellular transporter of various lipophilic ligands, and an amyloid-β chaperone in human cerebrospinal fluid. In this study, we determined the crystal structures of two different conformers of mouse L-PGDS, one with an open cavity of the β-barrel and the other with a closed cavity due to the movement of the flexible E-F loop. The upper compartment of the central large cavity contains the catalytically essential Cys65 residue and its network of hydrogen bonds with the polar residues Ser45, Thr67, and Ser81, whereas the lower compartment is composed of hydrophobic amino acid residues that are highly conserved among other lipocalins. SH titration analysis combined with site-directed mutagenesis revealed that the Cys65 residue is activated by its interaction with Ser45 and Thr67 and that the S45A/T67A/S81A mutant showed less than 10% of the L-PGDS activity. The conformational change between the open and closed states of the cavity indicates that the mobile calyx contributes to the multiligand binding ability of L-PGDS.

Tehranolide, A sesquiterpene lactone with an endoperoxide group that probably has the same effect as the antimalarial agent Artemisinin

The malaria situation is aggravated by the appearance of strains of Plasmodium falciparum resistant to antimalarial drugs as well as by the resistance of vector Anopheles mosquitoes to DDT and other insecticides. Nearly 300–500 million people are infected by malaria and the incidence of this disease is dramatically increasing, since many strains of Plasmodium falciparum, the parasite responsible for the majority of fatal malaria infections, have become resistant to chloroquine and other traditional antimalarial drugs. Fortunately, these strains are still susceptible to the artemisinin derivatives. All of the artemisinin compounds contain stable endoperoxide bridges. The extract of the aerial parts of A. diffusa collected in the Province of Khorassan (Iran) afforded, in addition to several eudesmanolides, a new type of sesquiterpene lactone (Tehranolide) with an endoperoxide group. Previously, we reported antimalarial effect of extract of A. diffusa against P. berghei. Since the endoperoxide group is an essential requirement for the antimalarial activity of artemisinin, we have presumed the antimalarial properties of the crude extract are attributed to Tehranolide. We report here the in vivo laboratory evaluation of anti-malarial effects of Tehranolide, was done on P. berghei infected NMRI mice. The antimalarial effects of Tehranolide in 27mg/ml concentration (high dose) were injected s.c. every day for 12 days after infection in malaria mice. Three groups of mice (n=5) were investigated for antimalarial efficacy, the degree of parasitaemia, assessment of pathology including body weight, physiological activities, hepatomegaly and splenomegaly. Parasitaemia was measured every other day by counting Geimsa-stained blood smears which were taken from end tail cutting.

Transferrin receptor-dependent cytotoxicity of artemisinin–transferrin conjugates on prostate cancer cells and induction of apoptosis

Artemisinin, a natural product isolated from Artemisia annua, contains an endoperoxide group that can be activated by intracellular iron to generate toxic radical species. Cancer cells over-express transferrin receptors (TfR) for iron uptake while most normal cells express nearly undetectable levels of TfR. We prepared a series of artemisinin-tagged transferrins (ART-Tf) where different numbers of artemisinin units are attached to the N-glycoside chains of transferrin (Tf). The Tf bearing ∼16 artemisinins retains the functionality of both Tf and artemisinin. Reduction of TfRs by TfR siRNA transfection significantly impaired the ability of ART-Tf, but not dihydroartemisinin, to kill cells. We also demonstrate that the ART-Tf conjugate kills the prostate carcinoma cell line DU 145 by the mitochondrial pathway of apoptosis.

Theoretical investigation of anthracene‐9,10‐endoperoxide vertical singlet and triplet excitation spectra

The electronic absorption spectrum of anthracene-9,10-endoperoxide (APO) has been investigated by means of multiconfigurational multi-state second order perturbation theory on complete active space self-consistent field wavefunctions (MS-CASPT2/CASSCF) and two single reference methods: time-dependent density functional theory (TD-DFT) and coupled cluster of second order (CC2). After testing several active spaces and basis sets, a CAS (14,12) active space together with an ANO-S basis set was found an appropriate choice to describe the vertical singlet and triplet electronic states of APO. Unfortunately, TD-DFT and CC2 methods cannot reproduce the MS-CASPT2 and experimental spectrum. Our MS-CASPT2//CASSCF(14,12)/ANO-S calculations predict a predominant pi*(OO)sigma*(OO) character for the lowest singlet excited state S(1) at 3.85 eV. Accordingly, the lowest singlet state of APO should be responsible for homolysis of the endoperoxide group. The next two absorbing excited states, experimentally proposed to be responsible for singlet oxygen production and therefore connected to the biological interest of APO, have been computed vertically at 4.34 and 4.59 eV and assigned to pi(CC)pi*(CC) and pi*(OO)pi*(CC) transitions, respectively. The vertical triplet electronic spectrum follows the singlet vertical spectrum ordering. The high density of triplet and singlet excited states of different nature within few eV points to the possibility of intersystem crossings between potential energy surfaces of different multiplicity.

Raman and infrared characterization of the vibrational properties of the antimalarial drug artemisinin

AbstractArtemisinin is one of the most powerful new generation antimalarial drugs. The active center is the peroxide group of the caged trioxane heterocycle, which either blocks the free heme released by the Plasmodium falciparum parasite or acts on key proteins causing the parasite death. Both processes involve the activation of the 1,2,4‐trioxanic ring through interaction with the Fe(II)–heme complex. Since considerable effort was made to synthesize antimalarial drugs containing the endoperoxide group, it becomes fundamental to know the structural and dynamical properties of artemisinin. For this reason, Raman and Fourier transform (FT) infrared studies have been undertaken here on the vibrational properties of artemisinin, its derivative artemether and δ‐valerolactone, the latter being the molecule mimicking the lactonic ring of artemisinin. The vibrational data of the three molecules have been analyzed through the comparison with ab initio calculations based on DFT/B3‐LYP/cc‐pVDZ procedures. Modes of artemisinin have been correlated with those of component units. Copyright © 2008 John Wiley &amp; Sons, Ltd.

Molecular Characterization of a Novel Type of Prostamide/Prostaglandin F Synthase, Belonging to the Thioredoxin-like Superfamily

Prostaglandin F (PGF) ethanolamide (prostamide F) synthase, which catalyzed the reduction of prostamide H(2) to prostamide F(2alpha), was found in mouse and swine brain. The enzyme was purified from swine brain, and its amino acid sequence was defined. The mouse enzyme consisted of a 603-bp open reading frame coding for a 201-amino acid polypeptide with a molecular weight of 21,669. The amino acid sequence placed the enzyme in the thioredoxin-like superfamily with Cys(44) being the active site. The enzyme expressed in Escherichia coli as well as the native enzyme catalyzed not only the reduction of prostamide H(2) to prostamide F(2alpha) but also that of PGH(2) to PGF(2alpha). The V(max) and K(m) values for prostamide H(2) were about 0.25 micromol/min.mg of protein and 7.6 microm, respectively, and those for PGH(2) were about 0.69 micromol/min.mg of protein and 6.9 microm, respectively. Neither PGE(2) nor PGD(2) served as a substrate for this synthase. Based on these data, we named the enzyme prostamide/PGF synthase. Although the enzyme showed a broad specificity for reductants, reduced thioredoxin preferentially served as a reducing equivalent donor for this enzyme. Moreover, Northern and Western blot analyses in addition to the prostamide F synthase activity showed that the enzyme was mainly distributed in the brain and spinal cord, and the immunohistochemical study in the spinal cord showed that the enzyme was found mainly in the cytosol. These results suggest that prostamide/PGF synthase may play an important functional role in the central nervous system.

Radiolytically induced one-electron reduction of artemisinin in H2O/ethanol (1:1 v/v) solution: a pulse radiolysis study.

The aim was to obtain information on the one-electron reduction of the antimalarial natural drug artemisinin (ART). The pulse radiolysis of ART in H2O/ethanol (EtOH) (1:1 v/v) solution was studied in the absence and presence of the so-called redox indicators N,N,N’,N’-tetramethyl-p-phenylenediamine (TMPD), Fe(CN)64 − , 1,1′-dimethyl-4,4′-bipyridinium dichloride (methyl viologen, MV2 + ) and Fe(CN)63 − . In an argon-purged solution, ART reacts with solvated electrons (es−) with k = 4.4 × 109 dm3 mol − 1 s − 1 generating an absorption band rising in the ultraviolet region similar to the spectrum of the CH3•CHOH radical. The species originating from the reaction between ART and es− do not show any appreciable reactivity toward Fe(CN)64 − , TMPD, MV2 + and Fe(CN)63 − . The experiments performed in the presence of ART and MV2 + have provided strong support to the idea that the first species obtained from the addition of the electron, which is believed to occur at the endoperoxide group level, undergoes a rapid (k on the order of 108 s − 1 or higher) intramolecular rearrangement to give species, most likely carbon-centred radicals, that show some reactivity towards the methyl viologen radical cation (MV• + ).