Naphthazarin Derivatives Research Articles

Identification of a biosynthetic gene cluster for a red pigment cristazarin produced by a lichen-forming fungus Cladonia metacorallifera.

Lichens are known to produce many novel bioactive metabolites. To date, approximately 1,000 secondary metabolites have been discovered, which are predominantly produced by the lichen mycobionts. However, despite the extensive studies on production of lichen secondary metabolites, little is known about the responsible biosynthetic gene clusters (BGCs). Here, we identified a putative BGC that is implicated in production of a red pigment, cristazarin (a naphthazarin derivative), in Cladonia metacorallifera. Previously, cristazarin was shown to be specifically induced in growth media containing fructose as a sole carbon source. Thus, we performed transcriptome analysis of C. metacorallifera growing on different carbon sources including fructose to identify the BGC for cristazarin. Among 39 polyketide synthase (PKS) genes found in the genome of C. metacorallifera, a non-reducing PKS (coined crz7) was highly expressed in growth media containing either fructose or glucose. The borders of a cristazarin gene cluster were delimited by co-expression patterns of neighboring genes of the crz7. BGCs highly conserved to the cristazarin BGC were also found in C. borealis and C. macilenta, indicating that these related species also have metabolic potentials to produce cristazarin. Phylogenetic analysis revealed that the Crz7 is sister to fungal PKSs that biosynthesize an acetylated tetrahydoxynaphthalene as a precursor of melanin pigment. Based on the phylogenetic placement of the Crz7 and putative functions of its neighboring genes, we proposed a plausible biosynthetic route for cristazarin. In this study, we identified a lichen-specific BGC that is likely involved in the biosynthesis of a naphthazarin derivative, cristazarin, and confirmed that transcriptome profiling under inducing and non-inducing conditions is an effective strategy for linking metabolites of interest to biosynthetic genes.

In silico molecular modelling studies and antibiofilm efficacy of shikonin against Candida albicans: mechanistic insight

In the recent past, the occurrence of fungal infections has increased drastically and candidiasis, caused prominently by Candida albicans, is foremost among them which has caused significant mortality and morbidity majorly in immune-compromised patients. Shikonin is a well-known natural naphthazarin derivative with promising antifungal efficacy, but it's mechanism of action is still unclear. Keeping this in view, present workwas designed to get a mechanistic insight of anti-candida efficacy of shikonin via in vitro experiments and in situ molecular modelling studies. The current exploratory study is based on research that uses both qualitative and quantitative techniques, including minimum inhibitory concentration, minimum biofilm inhibitory concentration, time kill assay, cell cycle analysis and apoptotic assays, static biofilm formation assays, microscopic biofilm assessment assays, ergosterol content estimation and molecular docking/simulation studies. The study revealed a notable effect of shikonin against Candida albicans, including retardation of biofilms. Shikonin, with its increasing concentration leads to candidal cell apoptosis and necrosis establishing its dose-dependent effect. Additionally, it exhibited fungicidal activity via a mechanism of action likely related to ergosterol complexation which was further corroborated by molecular docking and simulation studies.

Synthesis and molecular modeling studies of naphthazarin derivatives as novel selective inhibitors of α-glucosidase and α-amylase

Diabetes mellitus is known as one of the most life-threatening diseases and has attracted the attention of medicinal chemists. The design and synthesis of novel potential candidates for the inhibition of α-amylase and α-glucosidase enzymes which are responsible for the digestion of dietary carbohydrates are an important approach for the treatment of diabetes. In this current work, a range of novel mono and naphthazarin derivatives were successfully synthesized by the esterification reaction of naphthazarin with different benzoyl chlorides and heterocyclic acyl chlorides. The synthesized compounds were subjected to in vitro antidiabetic activities through α-glucosidase and α-amylase inhibition properties. Despite the limited inhibition potential against α-amylase enzymes, naphthazarin derivatives would be promising targets for antidiabetic study due to the specificity for α-glucosidase enzyme. The compound 18 revealed a dual inhibition behavior, however all the other active compounds were detected as specific for α-glucosidase inhibition and quite potent compared to the standard acarbose which is promising for eliminating the side effects associated with the non-selectivity of acarbose. The compound 19 was the best candidate for inhibition with IC50 value of 7.4 µM and resulted 150-fold better activity compared to the standard. Free energy calculations support the experimental binding affinity of compound 19 found as the most promising drug candidate for α-glucosidase among the synthesized compounds. The plausible binding mode and the interaction of compound 19 complexed with α-glucosidase has been revealed by MD simulations.

Synthesis, Structure Elucidation, Antibacterial Activities, and Synergistic Effects of Novel Juglone and Naphthazarin Derivatives Against Clinical Methicillin-Resistant Staphylococcus aureus Strains

Infections caused by drug-resistant bacteria are a serious threat to human and global public health. Moreover, in recent years, very few antibiotics have been discovered and developed by pharmaceutical companies. Therefore, there is an urgent need to discover and develop new antibacterial agents to combat multidrug-resistant bacteria. In this study, two novel series of juglone/naphthazarin derivatives (43 compounds) were synthesized and evaluated for their antibacterial properties against various clinical and reference Gram-positive MSSA, clinical Gram-positive MRSA, and clinical and reference Gram-negative bacteria E. coli and P. aeruginosa. These strains are of clinical importance because they belong to ESKAPE pathogens. Compounds 3al, 5ag, and 3bg showed promising activity against clinical and reference MSSA (MIC: 1–8 µg/ml) and good efficacy against clinical MRSA (MIC: 2–8 µg/ml) strains. 5am and 3bm demonstrated better activity on both MSSA (MIC: 0.5 µg/ml) and MRSA (MIC: 2 µg/ml) strains. Their MICs were similar to those of cloxacillin against clinical MRSA strains. The synergistic effects of active compounds 3al, 5ag, 5am, 3bg, and 3bm were evaluated with reference antibiotics, and it was found that the antibiotic combination with 3bm efficiently enhanced the antimicrobial activity. Compound 3bm was found to restore the sensitivity of clinical MRSA to cloxacillin and enhanced the antibacterial activity of vancomycin when they were added together. In the presence of 3bm, the MIC values of vancomycin and cloxacillin were lowered up to 1/16th of the original MIC with an FIC index of 0.313. Moreover, compounds 3al, 5ag, 5am, 3bg, and 3bm did not present hemolytic activity on sheep red blood cells. In silico prediction of ADME profile parameter results for 3bm is promising and encouraging for further development.

Naphthazarin Derivatives in the Light of Intra- and Intermolecular Forces.

Our long-term investigations have been devoted the characterization of intramolecular hydrogen bonds in cyclic compounds. Our previous work covers naphthazarin, the parent compound of two systems discussed in the current work: 2,3-dimethylnaphthazarin (1) and 2,3-dimethoxy-6-methylnaphthazarin (2). Intramolecular hydrogen bonds and substituent effects in these compounds were analyzed on the basis of Density Functional Theory (DFT), Møller–Plesset second-order perturbation theory (MP2), Coupled Clusters with Singles and Doubles (CCSD) and Car-Parrinello Molecular Dynamics (CPMD). The simulations were carried out in the gas and crystalline phases. The nuclear quantum effects were incorporated a posteriori using the snapshots taken from ab initio trajectories. Further, they were used to solve a vibrational Schrödinger equation. The proton reaction path was studied using B3LYP, B97XD and PBE functionals with a 6-311++G(2d,2p) basis set. Two energy minima (deep and shallow) were found, indicating that the proton transfer phenomena could occur in the electronic ground state. Next, the electronic structure and topology were examined in the molecular and proton transferred (PT) forms. The Atoms In Molecules (AIM) theory was employed for this purpose. It was found that the hydrogen bond is stronger in the proton transferred (PT) forms. In order to estimate the dimers’ stabilization and forces responsible for it, the Symmetry-Adapted Perturbation Theory (SAPT) was applied. The energy decomposition revealed that dispersion is the primary factor stabilizing the dimeric forms and crystal structure of both compounds. The CPMD results showed that the proton transfer phenomena occurred in both studied compounds, as well as in both phases. In the case of compound 2, the proton transfer events are more frequent in the solid state, indicating an influence of the environmental effects on the bridged proton dynamics. Finally, the vibrational signatures were computed for both compounds using the CPMD trajectories. The Fourier transformation of the autocorrelation function of atomic velocity was applied to obtain the power spectra. The IR spectra show very broad absorption regions between 700 cm–1700 cm and 2300 cm–3400 cm in the gas phase and 600 cm–1800 cm and 2200 cm–3400 cm in the solid state for compound 1. The absorption regions for compound 2 were found as follows: 700 cm–1700 cm and 2300 cm–3300 cm for the gas phase and one broad absorption region in the solid state between 700 cm and 3100 cm. The obtained spectroscopic features confirmed a strong mobility of the bridged protons. The inclusion of nuclear quantum effects showed a stronger delocalization of the bridged protons.

Synthesis of naphthazarin derivatives and identification of novel thioredoxin reductase inhibitor as potential anticancer agent

Mammalian thioredoxin reductase (TrxR) enzymes play a crucial role in regulating multiple redox-based signaling pathways and have attracted increasing attention as promising anticancer drug targets. We report here the synthesis of a panel of naphthazarin derivatives and discovery of 2-methyl-5,8-dihydroxy-1,4-naphthoquinone (3, 2-methylnaphthazarin) as a potent cytotoxic agent with a submicromolar half maximal inhibitory concentration to the human promyelocytic leukemia HL-60 cells. Mechanism studies reveal that the compound selectively inhibits TrxR to induce oxidative stress-mediated apoptosis of HL-60 cells. Knockdown of TrxR sensitizes the cells to 3 insults, while overexpression of the functional enzyme confers resistance to the compound treatment, underpinning the physiological significance of targeting TrxR by 3. Clarification of the interaction of compound 3 with TrxR unveils a mechanism underlying the cellular action of the compound, and sheds light in considering development of the compound as a potential cancer chemotherapeutic agent.

Rechargeable organic batteries using chloro-substituted naphthazarin derivatives as positive electrode materials

The use of redox active organic compounds as an alternative positive electrode material of rechargeable lithium batteries can be a solution for the resource issues of the current battery system. To satisfy both the high capacity and long cycle life of the batteries using organic active materials, naphthazarin (5,8-dihydroxy-1,4-naphthoquinone) derivatives, which potentially exhibit a four-electron transfer redox reaction, were investigated. While the unsubstituted naphthazarin lithium salt (1), having a high theoretical capacity of up to about 550 mAh g−1, showed only half the expected capacity and a short cycle life as a positive electrode active material, the chloro-substituted ones (1-Cl 2 , 1-Cl 4 ) exhibited improved properties in both their initial capacity utilization and cycle life. In addition, the high stability of a chloro-substituted naphthazarin salt (1-Cl 4 ) was supported by a reversible electrochromic behavior during the redox reaction. The substituent effect of the naphthazarin derivatives on the cycle stability was discussed with respect to the battery performance and electrochromic behavior. Also, a guide for designing a new organic active material which shows a high capacity and long cycle life is suggested.

Phytotoxic studies of naphthoquinone intermediates from the synthesis of the natural product Naphthotectone

1,4-Naphthoquinones exhibit strong activity as antimalarial, antibacterial, antifungal and anticancer agents. These kinds of compounds have also shown phytotoxic activity. The first example of an allelochemical to be described was naphthoquinone juglone (5-hydroxy-1,4-naphthoquinone). This fact led us to conduct a study of the phytotoxic activity and structure–activity relationships of 26 naphthazarin derivatives. The compounds belong to two main groups, 5,8-dioxygenated-1,4-naphthoquinones and 1,4,5,8-tetraoxygenated naphthalenes with different functionalizations and chains at positions 2, 4, 6 and 7. The results show that substitution with halogens and modification of the hydroxyl groups on the aromatic ring with methoxy or acetate groups seems to play an important role in enhancing the activity.

Rechargaeble Organic Batteries Using Naphthazarin Derivatives: The Effect of Chloro-Substituents on the Battery Performance

One of the recent requirements for the current rechargeable lithium battery systems is to reduce the amount of minor metal-based materials, especially from the positive-electrode. Our strategy to confront this issue is to replace the rare-metal oxides-based positive-electrode with redox active organic ones. So far, we have demonstrated that a series of low-molecular-weight quinone derivatives, which undergo multi-electron redox reactions, can be an alternative material category [1‒3]. To increase the discharge capacity of such organic compounds, we focused our attention on the naphthazarin (5,8-dihydroxy-1,4-naphthoquinone) skeleton as a new redox unit. This skeleton can exhibit a four-electron transfer redox reaction, which should lead to a large capacity of up to 550 mAh/g. This paper compares the battery performance of the electrodes incorporating the lithium salt of the naphthazarin skeleton (1)and some analogues carrying two or four peripheral chloro-substituents (1-Cl2 , 1-Cl4 ) (Fig. 1a). 5,8-Dihydroxy-1,4-naphthoquinone and 2,3-dichloro-5,8-dihydroxy-1,4-naphthoquinone were purchased. 2,3,6,7-Tetrachloro-5,8-dihydroxy-1,4-naphthoquinone was synthesized by the chlorination of 2,3,6,7-tetrachloro-1,4,5,8-naphthodiquinone using sulfuryl chloride. The active material salts, 1, 1-Cl2 , and 1-Cl4 , were prepared by neutralizing the above-mentioned naphthazarin derivatives with lithium hydroxide. Their electrochemical properties as the positive-electrode active materials were evaluated in a coin-type half-cell using a metallic lithium sheet negative electrode and the electrolyte solution of tetraglyme/lithium bis(trifluoromethanesulfonyl)amide equimolar mixture. Figure 1b shows the discharge curves of the prepared electrodes after the initial charge. In this figure, the capacities are expressed in the percentage of the theoretical values (1: 531; 1-Cl2 : 396; 1-Cl4 : 316 mAh/g) which assumes each four-electron transfer reactions. All discharge curves have complicated forms composed of some plateau potential regions, reflecting their multi-electron transfer reactions during the discharge processes. While the electrode of 1 was only about 50% of the theoretical value, the electrodes of 1-Cl2 and 1-Cl4 showed higher values of 88 and 87%, respectively. The actual specific discharge capacities of the electrodes of 1, 1-Cl2 , and 1-Cl4 were 259, 349, and 274 mAh/g, respectively. We consider that this difference in the initial utilization ratio results from the fact that the solubility of the charged (oxidized) state of 1 in the electrolyte solution might be higher than those of the chloro-substituted 1-Cl2 and 1-Cl4 . In the cycle test, the discharge capacity of the electrode using 1 quickly dropped upon cycling; it decreased to 13% of the theoretical value after 20 cycles as shown in Fig. 1c. On the other hand, the electrodes usingchloro-substituted ones exhibited better performance; the electrodes of 1-Cl2 and 1-Cl4 respectively maintained 28 and 57% of the theoretical value after 20 cycles. In addition to the battery tests, a potentiostatic polarization measurement combined with a UV-Vis spectroscopic apparatus was applied to solutions of 1, 1-Cl2 , and 1-Cl4 in a dimethyl sulfoxide/lithium bis(trifluoromethanesulfonyl)amide system. The measurement revealed that the solution state of the unsubstituted 1 is less stable than those of the chloro-substituted 1-Cl2 and 1-Cl4 during their redox reactions, and 1-Cl4 was most stable among them. As is often the case, the discharge capacity of low-molecular-weight organic active materials tend to decrease upon cycling. One of the reasons of this capacity decay is suggested to be the loss of the active materials from the electrodes by the dissolution of the organic molecules in the electrolyte solutions. In this study, there seems a correlation between the electrochemical stability of the monomer state and the cycle-life performance, i.e., higher the electrochemical stability, the longer the cycle-life. The chloro substituent is considered to work as a protecting group to suppress the undesired side reactions during the redox reaction. This study suggests that both enhancing the chemical durability and suppressing the solubility in the electrolyte solution are important to design a long cycle-life organic active material.

Computational Study of Electronic Effects from β-Substituents on the Tautomerism of Naphthazarin Derivatives

In this work, semi-empirical AM1 and DFT B3LYP/6-31G** calculations were applied in the study of the interconversion among tautomers of several naphthazarin and 5-amino-8-hydroxy-1,4-naphthoquinone β-substituted derivatives bearing electron-donor or electron-withdrawing groups. Using a semi-empirical method, detailed potential energy landscapes for proton transfers were built, from which four tautomers and four transition states of interconversions were identified for each compound. These structures were recalculated without restraints and, using the Boltzmann distribution, the populations for each of the four tautomers and their respective molar fractions were calculated. The calculations showed that the tautomeric equilibrium is shifted to the tautomer where the ring with the substituent has a quinonic nature and is more pronounced when the β-substituent is an electron donor group. For derivatives of 5-amino-8-hydroxy-1,4-naphthoquinone, an equilibrium between an aromatic and a 1,5-naphthoquinonic non-aromatic enamine was observed, being the former the most stable.

Total Synthesis of Hybocarpone, a Cytotoxic Naphthazarin Derivative from the Lichen Lecanora hybocarpa, and Related Compounds

The concise synthesis of the lichen-derived antitumor agent hybocarpone (1) and its analogs is described. A new synthetic approach is based on the direct oxidative dimerization of the available naphthazarin precursors in the formation of the binaphtho[2,3-b; 2,3-d]furantetraone structure. It was shown that the first step to tetrahydrofuran features is the bridging hindered S*,S* and R*,S* carbon-carbon bonds of the molecules, setting the relative configurations of the 5aS*,6aS*,12aS*,12bS* and 5aS*,6aR*,12aR*,12bS* diastereomers.

Chemistry of naphthazarin derivatives. Transetherification selectivity of naphthazarin methoxy derivatives

Transetherification of naphthazarin methoxy derivatives in basic conditions occurs only with primary monohydric alcohols. 1,3-Butanediol due to the assistance of the hydroxy group at the C1 atom gives rise to a mixture of transetherification products at the primary and secondary hydroxy groups in a ratio 3: 1. The reaction with methyl α-D-glucopyranoside affords predominantly methyl 6-O-(5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-α-D-glucopyranoside, previously unknown water-soluble naphthoquinone-carbohydrate conjugate.

Synthesis of water-soluble N-Acetyl-L-cysteine conjugates of naphtazarin (5,8-Dihydroxy-1,4-naphthoquinone) derivatives

Base-catalytic condensation of N-acetyl-L-cysteine with substituted naphthazarins was first obtained a series of mono-, di-, tri- and tetra-substituted S-aminoacid naphthazarin conjugates bearing alkyl (hydroxy) groups. It was shown that three- and tetra-N-acetyl-L-cysteine naphthazarin derivatives are readily soluble in water.

Chemistry of naphthazarin derivatives: XV. Bromination of naphthazarin and its derivatives

Bromination of a number of naphthazarin (5,8-dihydroxy-1,4-naphthoquinone) derivatives having different substituents in the aromatic ring with molecular bromine in carbon tetrachloride was studied. Preparative procedures for the synthesis of 2-bromo-5,8-dihydroxy-7-methoxy-1,4-naphthoquinone, 2-bromo-6,7-dichloro-5,8-dihydroxy-1,4-naphthoquinone, 2-bromo-3,5,8-trihydroxy-1,4-naphthoquinone, and 2-bromo-6,7-dichloro-3,5,8-trihydroxy-1,4-naphthoquinone were developed.

DNA damage induced by shikonin in the presence of Cu(II) ions: potential mechanism of its activity to apoptotic cell death

Shikonin (β-alkannin), a naphthazarin derivative, has shown a variety of abilities such as anti-inflammatory, antitumoral, cytotoxic, and antimicrobial activities. In the presence of Cu(II), shikonin caused breakage of supercoiled plasmid pBR322 DNA. Other metal ions tested [Mg(II), Ca(II), and Ni(II)] were ineffective and only Fe(II) has the same ability in the DNA breakage reaction. The involvement of active oxygen in the reaction was established by the inhibition of DNA breakage by superoxide dismutase, catalase, thiourea, sodium azide, potassium iodide, and sodium benzoate. Cu(I) was shown to be an essential intermediate using the Cu(I)-specific sequestering reagent neocuproine. Shikonin induced HeLa cell apoptosis involved in the mechanism of increasing intracellular reactive oxygen species (ROS). It was suggested that shikonin generated ROS as a pro-oxidant in the presence of Cu(II), and ROS resulted in DNA damage and apoptotic cell death in cells.

Determination of Naphthazarin Derivatives in 16 Alkanna Species by RP-LC Using UV and MS for Detection

As part of our ongoing studies, a new and validated method for analysis of isohexenyl-naphthazarines in Turkish Alkanna species was developed. By using reversed phase material (Synergi MAX RP) it was possible to determine four known isohexenylnaphthazarin-derivatives, namely alkannin, acetylalkannin, deoxyalkannin, and β,β-dimethylacrylalkannin within 25 min in n-hexane extracts of the roots. The quantification of these compounds in 16 Anatolian Alkanna species is described for the first time. In order to assure peak identity LC-MS experiments were performed. Quantitative results revealed that the most dominant compound in the extracts was β,β-dimethylacrylalkannin.

Cytotoxic activity of intermediates and side products of echinochrome synthesis

The cytotoxic activity of the principal intermediates and side products of echinochrome synthesis toward gametes of the sea urchin Strogylocentrotus intermedius was studied. Di-and trichloro naphthazarin derivatives with two vicinal Cl atoms were the most active.

Determination of Naphthazarin Derivatives in Endemic Turkish Alkanna Species by Reversed Phase High Performance Liquid Chromatography

The quantification of free and total alkannins in 13 endemic Anatolian Alkanna species is described for the first time. Extraction of the samples was performed by sonication with hexane, followed by hydrolysis in 1 N NaOH. For analysis, a new HPLC method, utilizing reversed phase material (Synergi Max RP), was developed and successfully validated. The obtained data confirmed that the assay is sensitive (LOD of 13 ng on-column for alkannin), accurate (the recovery rate was 92.3%) and precise (RSD<or=6.8% for intra-day precision; RSD<or=4.8% for inter-day precision). LC-MS experiments were performed to assure peak identity and purity. Quantitative results revealed that roots of Alkanna tubulosa showed the highest amount of total alkannins (0.84%), while Alkanna mughlae contained the highest percentage of free alkannins (0.04%). Compared to the commonly used A. TINCTORIA (total alkannins: 0.11 to 0.54%; free alkannins: 0.002 to 0.01%) these two species could therefore serve as alternative sources for medicinally important alkannin derivatives.

Chemistry of naphthazarin derivatives 13. Conformational analysis of 3-(alk-1-enyl)-2-hydroxy-1,4-naphthoquinones by quantum chemistry methods

The molecular structures of various conformers of 2-hydroxy-1,4-naphthoquinone; 3-(alk-1-enyl)-2-hydroxy-1,4-naphthoquinones; 2,5,8-trihydroxy-1,4-naphthoquinone; and 3-(alk-1-enyl)-2,5,8-trihydroxy-1,4-naphthoquinones were studied by density functional theory (B3LYP/6-31(d), B3LYP/6-31(d, p)) and ab initio (MP2/6-31G, MP2/6-31(d)) methods. The strengths of the intramolecular hydrogen bonds formed by the β-hydroxy group with the O atom at C(1) and with the double bond π-electrons of the alkenyl substituents in the quinonoid rings were estimated. The compounds studied mainly exist as rotamers with the former-type hydrogen bonds. The splitting of the quinonoid bands of the stretching vibrations of the β-hydroxy group in the IR spectra of 3-(alk-1-enyl)-2-hydroxy-1,4-naphthoquinones and 3-(alk-1-enyl)-2,5,8-trihydroxy-1,4-naphthoquinones in hexane solutions is due to the existence of rotamers formed upon internal rotation of the alkenyl substituent.

Cytotoxicity in vitro of naphthazarin derivatives from Onosma arenaria

The cytotoxicity of naphthazarin derivatives isolated from the roots of Onosma arenaria on human cervix adenocarcinoma cells (HeLa) and leukaemia K562 cells, as well on non-malignant peripheral blood mononuclear cells (PBMC) was studied. The results show that beta-hydroxyisovalerylalkannin, acetylalkannin and the pigment fraction exhibited high cytotoxicity in vitro against the tested cell lines, as well the healthy PBMC before or after activation with phytohaemagglutinin.