Specificity of Photochemical Energy Conversion in Photosystem I from the Green Microalga Chlorella ohadii.

Primary excitation energy transfer and charge separation in photosystem I (PSI) from the extremophile desert green alga Chlorella ohadii grown in low light were studied using broadband femtosecond pump-probe spectroscopy in the spectral range from 400 to 850 nm and in the time range from 50 fs to 500 ps. Photochemical reactions were induced by the excitation into the blue and red edges of the chlorophyll Qy absorption band and compared with similar processes in PSI from the cyanobacterium Synechocystis sp. PCC 6803. When PSI from C. ohadii was excited at 660 nm, the processes of energy redistribution in the light-harvesting antenna complex were observed within a time interval of up to 25 ps, while formation of the stable radical ion pair P700+A1- was kinetically heterogeneous with characteristic times of 25 and 120 ps. When PSI was excited into the red edge of the Qy band at 715 nm, primary charge separation reactions occurred within the time range of 7 ps in half of the complexes. In the remaining complexes, formation of the radical ion pair P700+A1- was limited by the energy transfer and occurred with a characteristic time of 70 ps. Similar photochemical reactions in PSI from Synechocystis 6803 were significantly faster: upon excitation at 680 nm, formation of the primary radical ion pairs occurred with a time of 3 ps in ~30% complexes. Excitation at 720 nm resulted in kinetically unresolvable ultrafast primary charge separation in 50% complexes, and subsequent formation of P700+A1- was observed within 25 ps. The photodynamics of PSI from C. ohadii was noticeably similar to the excitation energy transfer and charge separation in PSI from the microalga Chlamydomonas reinhardtii; however, the dynamics of energy transfer in C. ohadii PSI also included slower components.

Acetylcholinesterase Inhibitory Activity of Modified Lupane, Oleanane, and Ursane A-seco-Triterpenoids.

A series of new lupane, ursane, and oleanane type triterpenic A-seco-derivatives containing bromo-, azido-, alkyne-, 1H-tetrazol-5-yl-, 5-methyloxazol-2-yl-, N-(4-(4-methylpiperazin-1-yl)but-2-yn-1-yl), and a carbonyl group at C2, C24, C28, C30 positions has been synthesized. The bioactivity was evaluated by Ellman's method, and the results showed that most of the compounds displayed moderate acetylcholinesterase inhibitory activities in vitro. Among them, A-seco-derivatives of 28-oxo-allobetuline and betulinic acid with bromo- and azido-groups exhibited the most potent inhibitory activity against AChE. Extra experiments showed methyl 2-cyano-3,4-seco-dibromo- and 2-cyano-3,4-seco-diazido-derivatives of betulinic acid as mixed-type inhibitors, with Ki values as low as Ki =0.18 µM and Ki =0.21 µM, respectively.

Synthesis, Anti-Influenza H1N1 and Anti-Dengue Activity of A-Ring Modified Oleanonic Acid Polyamine Derivatives.

A series of sixteen A-ring modified (2,3-indolo-, 2-benzylidene) oleanonic acid derivatives, holding some cyclic amines, linear polyamines and benzylaminocarboxamides at C28, has been synthesized and screened for antiviral activity against influenza A/PuertoRico/8/34 (H1N1) and Dengue virus serotypes of DENV-1, -2, -3, -4. It was found that 28-homopiperazine 2 and 3-N-phthalyl 22 amides of oleanonic acid demonstrated high potency with selectivity index SI 27 (IC50 21 µM) and 42 (IC50 12 µM). Oleanonic acid aminoethylpiperazine amide 6 and C-azepano-erythrodiol 23 appeared to be the most effective compounds against DENV-1 (IC50's 67 and 107 µM) and -2 (IC50's 86 and 68 µM correspondingly) serotypes.

Novel A-Ring Chalcone Derivatives of Oleanolic and Ursolic Amides with Anti-Proliferative Effect Mediated through ROS-Triggered Apoptosis.

A series of A-ring modified oleanolic and ursolic acid derivatives including C28 amides (3-oxo-C2-nicotinoylidene/furfurylidene, 3ß-hydroxy-C2-nicotinoylidene, 3ß-nicotinoyloxy-, 2-cyano-3,4-seco-4(23)-ene, indolo-, lactame and azepane) were synthesized and screened for their cytotoxic activity against the NCI-60 cancer cell line panel. The results of the first assay of thirty-two tested compounds showed that eleven derivatives exhibited cytotoxicity against cancer cells, and six of them were selected for complete dose-response studies. A systematic study of local SARs has been carried out by comparative analysis of potency distributions and similarity relationships among the synthesized compounds using network-like similarity graphs. Among the oleanane type triterpenoids, C2-[4-pyridinylidene]-oleanonic C28-morpholinyl amide exhibited sub-micromolar potencies against 15 different tumor cell lines and revealed particular selectivity for non-small cell lung cancer (HOP-92) with a GI50 value of 0.0347 µM. On the other hand, superior results were observed for C2-[3-pyridinylidene]-ursonic N-methyl-piperazinyl amide 29, which exhibited a broad-spectrum inhibition activity with GI50 < 1 µM against 33 tumor cell lines and <2 µM against all 60 cell lines. This compound has been further evaluated for cell cycle analysis to decipher the mechanism of action. The data indicate that compound 29 could exhibit both cytostatic and cytotoxic activity, depending on the cell line evaluated. The cytostatic activity appears to be determined by induction of the cell cycle arrest at the S (MCF-7, SH-SY5Y cells) or G0/G1 phases (A549 cells), whereas cytotoxicity of the compound against normal cells is nonspecific and arises from apoptosis without significant alterations in cell cycle distribution (HEK293 cells). Our results suggest that the antiproliferative effect of compound 29 is mediated through ROS-triggered apoptosis that involves mitochondrial membrane potential depolarization and caspase activation.

New Investigations with Lupane Type A-Ring Azepane Triterpenoids for Antimycobacterial Drug Candidate Design.

Twenty lupane type A-ring azepano-triterpenoids were synthesized from betulin and its related derivatives and their antitubercular activity against Mycobacterium tuberculosis, mono-resistant MTB strains, and nontuberculous strains Mycobacterium abscessus and Mycobacterium avium were investigated in the framework of AToMIc (Anti-mycobacterial Target or Mechanism Identification Contract) realized by the Division of Microbiology and Infectious Diseases, NIAID, National Institute of Health. Of all the tested triterpenoids, 17 compounds showed antitubercular activity and 6 compounds were highly active on the H37Rv wild strain (with MIC 0.5 µM for compound 7), out of which 4 derivatives also emerged as highly active compounds on the three mono-resistant MTB strains. Molecular docking corroborated with a machine learning drug-drug similarity algorithm revealed that azepano-triterpenoids have a rifampicin-like antitubercular activity, with compound 7 scoring the highest as a potential M. tuberculosis RNAP potential inhibitor. FIC testing demonstrated an additive effect of compound 7 when combined with rifampin, isoniazid and ethambutol. Most compounds were highly active against M. avium with compound 14 recording the same MIC value as the control rifampicin (0.0625 µM). The antitubercular ex vivo effectiveness of the tested compounds on THP-1 infected macrophages is correlated with their increased cell permeability. The tested triterpenoids also exhibit low cytotoxicity and do not induce antibacterial resistance in MTB strains.

3-Pyridinylidene Derivatives of Chemically Modified Lupane and Ursane Triterpenes as Promising Anticancer Agents by Targeting Apoptosis.

Cancer persists as a global challenge due to the extent to which conventional anticancer therapies pose high risks counterbalanced with their therapeutic benefit. Naturally occurring substances stand as an important safer alternative source for anticancer drug development. In the current study, a series of modified lupane and ursane derivatives was subjected to in vitro screening on the NCI-60 cancer cell line panel. Compounds 6 and 7 have been identified as highly active with GI50 values ranging from 0.03 µM to 5.9 µM (compound 6) and 0.18-1.53 µM (compound 7). Thus, these two compounds were further assessed in detail in order to identify a possible antiproliferative mechanism of action. DAPI (4',6-diamidino-2-phenylindole) staining revealed that both compounds induced nuclei condensation and overall cell morphological changes consistent with apoptotic cell death. rtPCR analysis showed that both compounds induced upregulation of proapoptotic Bak and Bad genes while downregulating Bcl-XL and Bcl-2 antiapoptotic genes. Molecular docking analysis revealed that both compounds exhibited high scores for Bcl-XL inhibition, while compound 7 showed higher in silico Bcl-XL inhibition potential as compared to the native inhibitor ATB-737, suggesting that compounds may induce apoptotic cell death through targeted antiapoptotic protein inhibition, as well.

Synthesis and cholinesterase inhibiting potential of A-ring azepano- and 3-amino-3,4-seco-triterpenoids.

In this study, a series of A-ring azepano- and 3-amino-3,4-seco-derivatives were synthesized from betulin, oleanolic, ursolic and glycyrrhetinic acids aiming to develop new cholinesterase inhibitors. Azepanobetulin, azepanoerythrodiol and azepanouvaol were modified to give amide and tosyl derivatives, while azepano-anhydrobetulines and azepano-glycyrrhetols were obtained for the first time. Oleanane and ursane type 3-amino-3,4-seco-4(23)-en triterpenic alcohols were synthesized by reducing the corresponding 2-cyano-derivatives accessible from Beckmann type 2 rearrangements. The compounds were screened in colorimetric Ellman's assays to determine their ability to act as inhibitors for the enzymes acetylcholinesterase (AChE, from electric eel) and butyrylcholinesterase (BChE, from equine serum). While most of these compounds were only moderate inhibitors for AChE, several of them were shown to be inhibitors for BChE acting as mixed-type inhibitors. Azepanobetulin 1, its C28-amide derivatives 7 and 8, azepano-11-deoxo-glycyrrhetol 12 and azepanouvaol 18 held inhibition constants Ki ranging between 0.21 ± 0.06 to 0.68 ± 0.19 µM. Thus, they were approximately 4 to 10 times more active than standard galantamine hydrobromide. For all of the compounds reasonably high docking scores for BChE were obtained being in good agreement with the experimental results from the enzymatic studies. As a result, A-ring azepano-triterpenoids were found to be new scaffolds for the development of BChE inhibitors.

Diastereoselective Synthesis of Triterpenoid 1,2,4-Trioxolanes by Griesbaum Co-ozonolysis.

Diastereoselective synthesis of triterpenoid 1,2,4-trioxolanes by Griesbaum co-ozonolysis was shown for the first time. Ozonolysis of 2-methoxyoximes (syn-anti-isomers mixture) of allobetulin or methyl oleanoate with CF3-ketones resulted in asymmetrical spiro-1,2,4-trioxolanes as mixtures of diastereomers in yields up to 80-85%. The configuration of the spiro-C-2 center of individual ozonides was determined by 2D NMR spectra and X-ray crystallographic analysis. The products of ozonolysis of triterpenoid 3-methoxyoximes were mixtures of regioisomeric N-methoxylactams. Thus, the fundamental differences in the oxidation of homologous triterpenoid 2- or 3-methoxyoximes with ozone have been established. These results may afford a new stage in the development of the Griesbaum method as applied to natural compounds and biologically active peroxides.

Structural modifications of 2,3-indolobetulinic acid: Design and synthesis of highly potent α-glucosidase inhibitors.

A series of nineteen nitrogen-containing lupane triterpenoids was obtained by modification of C2, C3, C20 and C28 positions of betulonic acid and their α-glucosidase inhibiting activity was investigated. Being a leader compound from our previous study, 2,3-indolo-betulinic acid was used as the main template for different modifications at C-(28)-carboxyl group to obtain cyano-, methylcyanoethoxy-, propargyloxy- and carboxamide derivatives. 20-Oxo- and 29-hydroxy-20-oxo-30-nor-analogues of 2,3-indolo-betulinic acid were synthesized by ozonolysis of betulonic acid followed by Fischer indolization reaction. To compare the influence of the fused indole or the seven-membered A-ring on the inhibitory activity, lupane A-azepanones with different substituents at C28 were synthesized. The structure-activity relationships revealed that the enzyme inhibition activity dramatically increased (up to 4730 times) when the carboxylic group of 2,3-indolo-betulinic acid was converted to the corresponding amide. Thus, the IC50 values for glycine amide and L-phenylalanine amides were 0.04 and 0.05 µM, respectively. This study also revealed that 2,3-indolo-platanic acid is 4.5 times more active than the parent triterpenoid with IC50 of 0.4 µM. Molecular modeling suggested that improved potency is due to additional polar interactions formed between C28 side chain and a sub-pocket of the α-glucosidase allosteric site.

Antiviral potency of lupane and oleanane alkynyl-derivatives against human cytomegalovirus and papillomavirus.

A library of 18 structurally diverse semisynthetic lupane, oleanane, and ursane types triterpenoids, including C19- or C28-(1,2,3-triazolyl)- and aminomethylated derivatives obtained by the «click¼ reaction with various aromatic and sugar azides or by Mannich reaction with secondary amines, were tested for antiviral activity against HCMV, HSV-1, and HPV-11 types. C28-Triazolyl-derivative with a benzyl substituent of 2,3-indolo-oleanolic acid was the most active against the HCMV virus with EC50 < 0.05 (SI > 81). Lupane 3,28-diacetoxy-triazolyl derivatives with phenyl- and fluorophenyl-fragments possess the highest activity among all screened compounds toward HPV-11 type virus with EC50 values of 2.97 µM and 1.20 µM, SI90 values of 28 and >125, respectively. One can see that modification of triterpenic alkynes to Mannich bases was more efficient in increasing an activity against HSV-1 than their conversion to triazoles.

Diethoxyphosphoryloxy-oleanolic acid is a nanomolar-inhibitor of butyrylcholinesterase.

A series of new betulin, lupeol, erythrodiol, and oleanolic acid phosphoryloxy- and furoyloxy-derivatives has been synthesized and their structure was confirmed by NMR spectroscopy. Synthesized compounds were subjected to Ellman's assays to determine their ability to inhibit the enzymes AChE and BChE. Among them, diethoxyphosphoryloxy-oleanolic acid inhibited BChE with a value of 99%, thereby acting as a mixed-type inhibitor holding very low Ki values of Ki = 6.59 nM and Ki ' = 1.97 nM, respectively.

Antiviral opportunities of Mannich bases derived from triterpenic N-propargylated indoles.

Oleanolic and glycyrrhetic acids alkyne derivatives were synthesized as a result of propargylation of the indole NH-group condensed with the triterpene A-ring, the following aminomethylation led to a series of Mannich bases. The synthesized compounds were tested for their potential inhibition of influenza A/PuertoRico/8/34 (H1N1) virus in Madin-Darby canine kidney (MDCK) cell culture and SARS-CoV-2 pseudovirus in baby hamster kidney-21-human angiotensin-converting enzyme 2 (BHK-21-hACE2) cells. Mannich bases of oleanolic and glycyrrhetic acids N-propargylated indoles 7, 8, and 12 were the most efficacious against influenza virus A with IC50 7-10 µM together with a low toxicity (CC50 > 145 µM) and high selectivity index SI value 20. Indolo-oleanolic acid morpholine amide Mannich base holding N-methylpiperazine moiety 9 showed anti-SARS-CoV-2 pseudovirus activity with EC50 value of 14.8 µM. Molecular docking and dynamics modeling investigated the binding mode of the compounds 7 and 12 into the binding pocket of influenza A virus M2 protein and compound 9 into the RBD domain of SARS-CoV-2 spike glycoprotein.

Allobetulone rearrangement to l8αH,19ßH-ursane triterpenoids with antiviral activity.

Allobetulone E-ring rearrangement under treating with HClO4 in Ac2O under reflux afforded new triterpenoids: 3,28-diacetoxy-21-acetyl-2(3),20(21)-18α,19ßH-ursandiene 3 and 3,28-diacetoxy-2(3),18(19)-oleandiene 4. 18α,19ßH-Ursanes were transformed at A- and E-rings into indolo- and bis-furfurylidene 7 derivatives. Structure elucidation was performed using COSY, NOESY, HSQC and HMBC experiments, and X-Ray analysis for 3. The potential of newly obtained 18α,19ßH-ursanes was evaluated against HCMV and HPV-11, the NCI-60 cancer cell panel and inhibition of α-glucosidase. All of the compounds have shown viral inhibition towards HCMV compared to standard drug Acyclovir. 3ß-Acetoxy-21ß-acetyl-20ß,28-epoxy-18α,19ßН-ursane 1 showed moderate activity (EC50 4.87 µM) towards the HCMV-resistant isolate (GDGr K17) compared to standard drug Cidofovir and was four times more potent than Ganciclovir. Compound 7 inhibited the cell growth of the three melanoma and one colon cancer cell. 3-Oxo-21ß-acetyl-20ß,28-epoxy-18α,19ßН-ursane 5 and compound 7 inhibited α-glucosidase with IC50 28.0 µM and 4.0 µM being from 6 to 44 times more active than acarbose.

Azepanodipterocarpol is potential candidate for inhibits influenza H1N1 type among other lupane, oleanane, and dammarane A-ring amino-triterpenoids.

A series of lupane-, oleanane- and dammarane-based triterpenoids with 3ß-amino, A-ring azepano- and 3,4-seco-fragments has been synthesized and evaluated for antiviral activity against influenza A(H1N1) virus. It was found that azepanodipterocarpol 8 and 3ß-amino-28-oxoallobetulin 11 showed antiviral activity with IC50 1.1 and 2.6 µg ml-1, and selectivity index of 19 and 10, respectively.

Synthesis of erythrodiol C-ring derivatives and their activity against Chlamydia trachomatis.

To develop new potential agents against Chlamydia trachomatis among oleanane type triterpenoids the synthesis, spectral and X-ray analysis as well as antimicrobial screening of C-12 oxygen and nitrogen derivatives of erythrodiol is presented. The reduction of methyl 3ß-acetoxy-12-oxo-oleanoate with LiAlH4 led to isomeric erythrodiol 12ß- and 12α-hydroxy-derivatives, their stereochemistry with respect to the position of hydroxyl-group at C-12 was determined based on the multiplets splitting patterns, the magnitude of the spin-spin interaction, and NOESY interactions. Methyl 3ß-acetoxy-12-oxo-oleanoate was transformed to 12E-hydroxyimino- and 12E-methoxyimino-derivatives by the interaction with NH2OH∙HCl or CH3ONH2∙HCl, respectively. By Beckmann rearrangement with SOCl2 in dioxane 12E-oxime was converted to C-lactame and its following reduction with LiAlH4 in THF or dioxane led to erythrodiol C-azepanone or C-azepane derivatives. The structure 3-O,12-N-bis-acetyl-derivative of C-azepane-erythrodiol was confirmed by the single crystal X-ray analysis. Erythrodiol 12ß-hydroxy- and C-azepane derivatives were found to be lead compounds with significant activity against C. trachomatis with MIC 1.56 and 3.125 µg/mL. Molecular docking was employed to suggest potential binding interaction, the tested compounds are likely to act as Cdu1 protein inhibitors while 12ß-hydroxy-erythrodiol exhibited the highest affinity towards this respective target protein. These results indicated that C-ring oxygen and nitrogen erythrodiol derivatives might be considered for further research in the design of antibacterial agents against Chlamydia trachomatis.

Newly synthesised oxime and lactone derivatives from Dipterocarpus alatus dipterocarpol as anti-diabetic inhibitors: experimental bioassay-based evidence and theoretical computation-based prediction.

Dipterocarpus alatus-derived products are expected to exhibit anti-diabetes properties. Natural dipterocarpol (1) was isolated from Dipterocarpus alatus collected in Quang Nam province, Vietnam; afterwards, 20 derivatives including 13 oxime esters (2 and 3a-3m) and 7 lactones (4, 5, 6a-6e) were semi-synthesised. Their inhibitory effects towards diabetes-related proteins were investigated experimentally (α-glucosidase) and computationally (3W37, 3AJ7, and PTP1B). Except for compound 2, the other 19 compounds (3a-3m, 4, 5, and 6a-6d) are reported for the first time, which were modified at positions C-3, C-24 and C-25 of the dipterocarpol via imidation, esterification, oxidative cleavage and lactonisation reactions. A framework based on docking-QSARIS combination was proposed to predict the inhibitory behaviour of the ligand-protein complexes. Enzyme assays revealed the most effective α-glucosidase inhibitors, which follow the order 5 (IC50 of 2.73 ± 0.05 µM) > 6c (IC50 of 4.62 ± 0.12 µM) > 6e (IC50 of 7.31 ± 0.11 µM), and the computation-based analysis confirmed this, i.e., 5 (mass: 416.2 amu; polarisability: 52.4 Å3; DS: -14.9 kcal mol-1) > 6c (mass: 490.1 amu; polarisability: 48.8 Å3; DS: -13.7 kcal mol-1) > 6e (mass: 549.2 amu; polarisability: 51.6 Å3; DS: -15.2 kcal mol-1). Further theoretical justifications predicted 5 and 6c as versatile anti-diabetic inhibitors. The experimental results encourage next stages for the development of anti-diabetic drugs and the computational strategy invites more relevant work for validation.

Silicon ameliorates iron deficiency of cucumber in a pH-dependent manner.

Strategy I plants may respond to iron (Fe) deficiency by rhizosphere acidification. Here, the role of medium pH-values in silicon (Si)-induced mitigation Fe deficiency in Strategy I plants (Cucumis sativus) was investigated, particularly the metabolites regulated by a lack of Fe, using a target metabolomics approach. Plants were grown hydroponically, either with (+Fe) or in Fe-free (-Fe) nutrient solution, with (+Si) or without (-Si) a Si supply. The nutrient solution was adjusted to pH 5.0 or 6.0 and checked daily. Leaf metabolites potentially involved in Fe transport were determined. The typical Fe responses of cucumber (e.g., decrease in leaf chlorophyll, Fe imbalance) were more pronounced when plants were grown at pH 6.0 than 5.0, during long-term Fe deficiency (15 days). Major metabolites up-regulated by Fe deficiency and found in young leaf were succinic, citric and glutamic acids, respectively; their maximal concentrations occurred in Fe-starved plants grown at pH 6.0 without Si supply. Silicon (Si)-induced effects accompanied with alleviation chlorosis symptoms, were most distinct in plants grown at pH 6.0 for an extended period without Fe. Changes in abundance of metabolites specifically up-regulated by a lack of Fe may be manifested before any Si-induced changes in plant Fe content were apparent, suggesting that metabolite responses are highly sensitive to a Fe-dependent signal altered by Si treatments under Fe deficiency. The results indicate that Si supply was more evident when plants were more stressed by an increase in nutrient solution pH under Fe-limited conditions.

Interactions between aluminium, iron and silicon in Cucumber sativus L. grown under acidic conditions.

Aluminium (Al) is one of the major stressors for plants in acidic soils, negatively affecting plant growth and nutrient balances. Significant efforts have been undertaken to understand mechanisms of Al tolerance in plants. However, little is known of the relevance of iron (Fe) and silicon (Si) nutrition under Al stress conditions. The objectives of this study were to determine whether effects induced by Fe and Si are of importance for limitation of Al moving via xylem in plants (Cucumis sativus L.). Cucumber plants (cv. Phoenix and Solovei) were grown (i) hydroponically in a complete nutrient solution at pH 4.0, either with (+Fe) or in Fe-free (-Fe) nutrient solution, without (-Si) or with (+Si) supply of Si, without (-Al) or with (+Al) exposure of Al and (ii) in soil. Xylem sap concentrations of Al, Fe and Si were measured. To characterise the pattern of xylem sap transport of Al and Fe, metabolomic changes of root tissues were investigated. Although the growth of cucumber plants was not significantly affected by Al3+ (Al-tolerant), Al exposure decreased xylem sap Fe (+Fe plants) and increased ferric chelate reductase (FC-R) activity of roots (-Fe plants). On the other hand, Fe supply greatly mitigated the Al-induced increase in xylem sap Al. The ameliorative effect of Fe depended on plant genotypes and was more pronounced in the more Fe-efficient cultivar Phoenix, which presented the highest level of xylem sap Fe. Xylem sap Fe was positively correlated with root serine, succinic and fumaric acids, suggesting that a probable underlying mechanism of Al tolerance might involve the chelation of Fe by biosynthesis of these chelating compounds. The Si-modulated root succinate increase appears to be of great importance for facilitating long-distance transport of Fe, thereby hindering Al transport from roots to shoots. The results highlight for the first time the importance of both Fe and Si supply in plant exclusion of Al under acidic conditions.