An asiatic acid derived trisulfamate acts as a nanomolar inhibitor of human carbonic anhydrase VA.

This investigation delves into the inhibitory capabilities of a specific set of triterpenoic acids on diverse isoforms of human carbonic anhydrase (hCA). Oleanolic acid (1), maslinic acid (2), betulinic acid (3), platanic acid (4), and asiatic acid (5) were chosen as representative triterpenoids for evaluation. The synthesis involved acetylation of parent triterpenoic acids 1-5, followed by sequential reactions with oxalyl chloride and benzylamine, de-acetylation of the amides, and subsequent treatment with sodium hydride and sulfamoyl chloride, leading to the formation of final compounds 21-25. Inhibition assays against hCAs I, II, VA, and IX demonstrated noteworthy outcomes. A derivative of betulinic acid, compound 23, exhibited a Ki value of 88.1 nM for hCA VA, and a derivative of asiatic acid, compound 25, displayed an even lower Ki value of 36.2 nM for the same isoform. Notably, the latter compound displayed enhanced inhibitory activity against hCA VA when compared to the benchmark compound acetazolamide (AAZ), which had a Ki value of 63.0 nM. Thus, this compound surpasses the inhibitory potency and isoform selectivity of the standard compound acetazolamide (AAZ). In conclusion, the research offers insights into the inhibitory potential of selected triterpenoic acids across diverse hCA isoforms, emphasizing the pivotal role of structural attributes in determining isoform-specific inhibitory activity. The identification of compound 25 as a robust and selective hCA VA inhibitor prompts further exploration of its therapeutic applications.

Madecassic Acid-A New Scaffold for Highly Cytotoxic Agents.

Due to their manifold biological activities, natural products such as triterpenoids have advanced to represent excellent leading structures for the development of new drugs. For this reason, we focused on the syntheses and cytotoxic evaluation of derivatives obtained from gypsogenin, hederagenin, and madecassic acid, cytotoxicity increased-by and large-from the parent compounds to their acetates. Another increase in cytotoxicity was observed for the acetylated amides (phenyl, benzyl, piperazinyl, and homopiperazinyl), but a superior cytotoxicity was observed for the corresponding rhodamine B conjugates derived from the (homo)-piperazinyl amides. In particular, a madecassic acid homopiperazinyl rhodamine B conjugate 24 held excellent cytotoxicity and selectivity for several human tumor cell lines. Thus, this compound was more than 10,000 times more cytotoxic than parent madecassic acid for A2780 ovarian cancer cells. We assume that the presence of an additional hydroxyl group at position C-6 in derivatives of madecassic, as well as the (2α, 3ß) configuration of the acetates in ring A, had a beneficial effect onto the cytotoxicity of the conjugates, as well as onto tumor/non-tumor cell selectivity.

Rhodamine 101 Conjugates of Triterpenoic Amides Are of Comparable Cytotoxicity as Their Rhodamine B Analogs.

Pentacyclic triterpenoic acids (betulinic, oleanolic, ursolic, and platanic acid) were selected and subjected to acetylation followed by the formation of amides derived from either piperazine or homopiperazine. These amides were coupled with either rhodamine B or rhodamine 101. All of these compounds were screened for their cytotoxic activity in SRB assays. As a result, the cytotoxicity of the parent acids was low but increased slightly upon their acetylation while a significant increase in cytotoxicity was observed for piperazinyl and homopiperazinyl amides. A tremendous improvement in cytotoxicity was observed; however, for the rhodamine B and rhodamine 101 conjugates, and compound 27, an ursolic acid derived homopiperazinyl amide holding a rhodamine 101 residue showed an EC50 = 0.05 µM for A2780 ovarian cancer cells while being less cytotoxic for non-malignant fibroblasts. To date, the rhodamine 101 derivatives presented here are the first examples of triterpene derivatives holding a rhodamine residue different from rhodamine B.

Synthesis of messagenin and platanic acid chalcone derivatives and their biological potential.

The chalcone derivatives of 20-oxo-lupanes have been synthesised and screened for some types of biological activity. Ozonolysis of lupanes afforded 20-oxo-derivatives with the following condensation using different aromatic aldehydes by Claisen‒Schmidt reaction to the target compounds. The E configuration of 19-[3-(pyridin-3-yl)-prop-2-en-1-one]-fragment was established by X-ray analysis. Screening of cytotoxic activity against NCI-60 cancer cell line panel revealed, that messagenin derivative 9 has the highest activity with GI50 value ranged from 0.304 to 0.804 µM. A colorimetric SRB assay revealed for the 2,30-bis-furfurylidene derivative 11 and 30-bromo-20-oxo-29-nor-3,28-diacetoxy-betulin 16 cytotoxic activity against breast carcinoma MCF-7 and ovarian carcinoma A2780 cell lines. Compounds 11 and 13 acted also as inhibitors of the enzyme α-glucosidase (from S. saccharomyces) with IC50 values of 1.76 µM and 3.3 µM thus being 97- and 52-fold more active than standard acarbose. Antiviral potency of compounds 12 and 14 against HCMV, HSV-1 and HPV is also discussed.[Formula: see text].

MSBA-S - A pentacyclic sulfamate as a new option for radiotherapy of human breast cancer cells.

Many pentacyclic triterpenoids show anti-cancer and anti-inflammatory properties. Recently, we detected a pronounced cytotoxicity and radiosensitivity of two betulinyl sulfamates in human breast cancer cells. Besides betulinic acid scaffold (BSBA-S), we synthesized several new sulfamate-coupled scaffolds from oleanolic acid (OSBA-S), ursolic acid (USBA-S), platanic acid (PSBA-S) and maslinic acid (MSBA-S). Highest cytotoxicity was monitored in breast cancer cell lines after MSBA-S treatment showing in SRB assays IC50 values between 3.7 µM and 5.8 µM. Other sulfamate/triterpene conjugates, however, were less cytotoxic holding IC50 values between 6.6 µM and >50 µM, respectively. MSBA-S-treated breast cancer cells displayed significantly reduced clonogenic survival and an increased rate of apoptosis as compared to the other conjugates. In addition, MSBA-S in combination with irradiation resulted in effects on radiosensitivity in MDA-MB-231 cells (DMF10 = 1.14). In particular, ROS formation was strongly assessed in MSBA-S-treated breast cancer cells. Our findings suggest that the sulfamate derivative of maslinic acid MSBA-S might be a new option for the radiation therapy in breast cancer cells.

n-Propyl 6-amino-2,6-dideoxy-2,2-difluoro-ß-d-glucopyranoside is a good inhibitor for the ß-galactosidase from E. coli.

A convenient route has been developed for the synthesis of novel 6-amino-2,2-(or 3,3-difluoro)-2-(or 3),6-dideoxy-hexopyranoses. Biological screening showed these compounds as good inhibitors for several glycosidases. Especially n-propyl 6-amino-2,6-dideoxy-2,2-difluoro-ß-d-glucopyranoside (8) was an excellent competitive inhibitor for the ß-galactosidase from E. coli holding a K i of 0.50 µM.

Cytotoxic triterpenoid-safirinium conjugates target the endoplasmic reticulum.

Safirinium P and Q fluorescence labels were synthesized and conjugated with spacered triterpenoic acids to access hybrid structures. While the parent safirinium compounds were not cytotoxic at all, many triterpenoid safirinium P and Q conjugates showed moderate cytotoxicity. An exception, however, was safirinium P derived compound 30 holding low EC50 = 5.4 µM (for A375 cells) to EC50 = 7.5 µM (for FaDu cells) as well as EC50 = 6.6 µM for non-malignant fibroblasts NIH 3T3. Fluorescence imaging showed that the safirinium core structures cannot enter the cells (not even after a prolonged incubation time of 24 h), while the conjugates (as exemplified for 30) are accumulating in the endoplasmic reticulum but not in the mitochondria. The development of safirinium-hybrids targeting the endoplasmic reticulum can be regarded as a promising strategy in the development of cytotoxic agents.

Mitocanic Di- and Triterpenoid Rhodamine B Conjugates.

The combination of the "correct" triterpenoid, the "correct" spacer and rhodamine B (RhoB) seems to be decisive for the ability of the conjugate to accumulate in mitochondria. So far, several triterpenoid rhodamine B conjugates have been prepared and screened for their cytotoxic activity. To obtain cytotoxic compounds with EC50 values in a low nano-molar range combined with good tumor/non-tumor selectivity, the Rho B unit has to be attached via an amine spacer to the terpenoid skeleton. To avoid spirolactamization, secondary amines have to be used. First results indicate that a homopiperazinyl spacer is superior to a piperazinyl spacer. Hybrids derived from maslinic acid or tormentic acid are superior to those from oleanolic, ursolic, glycyrrhetinic or euscaphic acid. Thus, a tormentic acid-derived RhoB conjugate 32, holding a homopiperazinyl spacer can be regarded, at present, as the most promising candidate for further biological studies.

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.

Synthesis of some steroidal mitocans of nanomolar cytotoxicity acting by apoptosis.

Several steroids (abiraterone, prednisone, testosterone, cholesterol) and the BCL-2 inhibitor bexarotene were used as starting materials to synthesize iperazinyl-spacered rhodamine B conjugates. The conjugates were screened for their cytotoxicity in SRB assays against several human tumor cell lines and found to be active in a low µM to nM range. The conjugate derived from testosterone held an EC50 = 59 nM against MCF-7 tumor cells and acted mainly by necrosis. The prednisone conjugate, however, was less cytotoxic but acted mainly by apoptosis and held a moderate selectivity against MCF-7 tumor cells.

In the Mists of a Fungal Metabolite: An Unexpected Reaction of 2,4,5-Trimethoxyphenylglyoxylic Acid.

The reactions of phenylglyoxylic acids during the synthesis and biological evaluation of fungal metabolites led to the discovery of hitherto unknown compounds with a p-quinone methide (p-QM) structure. The formation of these p-QMs using 13C-labelled starting materials revealed a key-step of this reaction being a retro-Friedel-Crafts alkylation.

Ureidobenzenesulfonamides as efficient inhibitors of carbonic anhydrase II.

Sulfonamides represent an important class of drugs because of their inhibitory effect on carbonic anhydrases (CAs). We therefore synthesized several ureidobenzenesulfonamides and evaluated their bCA II inhibition for their potential use as anti-glaucoma gents. Since these compounds must not show cytotoxic effects, their cytotoxic potential against several human tumor cell lines and non-malignant fibroblasts was investigated. Several fluorophenyl substituted sulfonamides were efficient inhibitors of bCA II. Only one benzylphenyl substituted sulfonamide, however, showed a remarkable selectivity for HT29 colorectal carcinoma cells while being significantly less cytotoxic to non-malignant fibroblasts.

Synthesis and Biological Evaluation of Structurally Varied 5'-/6'-Isonucleosides and Theobromine-Containing N-Isonucleosidyl Derivatives.

Isonucleosides are rather stable regioisomeric analogs of nucleosides with broad therapeutic potential. We have previously demonstrated the ability of 5' and 6'-isonucleosides to inhibit the activity of acetylcholinesterase, a major target for Alzheimer's disease therapy. Continuing with our research on this topic, we report herein on the synthesis and biological evaluation of a variety of novel terminal isonucleosides and theobromine isonucleotide analogs. Xylofuranose-based purine or uracil 5'-isonucleosides and xylofuranos-5'-yl or glucos-6'-yl theobromine derivatives were accessed via Mitsunobu coupling between partially protected xylofuranose or glucofuranose derivatives with a nucleobase using conventional or microwave-assisted heating conditions. Theobromine-containing N-isonucleosidyl sulfonamide and phosphoramidate derivatives were synthesized from isonucleosidyl acetate precursors. The most active compounds in the cholinesterase inhibition assays were a glucopyranose-based theobromine isonucleosidyl acetate, acting as a dual inhibitor of acetylcholinesterase (AChE, Ki = 3.1 µM) and butyrylcholinesterase (BChE, Ki = 5.4 µM), and a 2-O,4-O-bis-xylofuranos-5'-yl uracil derivative, which displayed moderate inhibition of AChE (Ki = 17.5 µM). Docking studies revealed that the active molecules are positioned at the gorge entrance and at the active site of AChE. None of the compounds revealed cytoxic activity to cancer cells as well as to non-malignant mouse fibroblasts.

2-O-(2-chlorobenzoyl) maslinic acid triggers apoptosis in A2780 human ovarian carcinoma cells.

Depending on the conditions of the reactions, maslinic acid can be converted into the corresponding 2-O-, 3-O-, or 2,3-di-O-acylated compounds in good yields. These compounds showed in SRB assays a significantly increased cytotoxicity as compared to the parent compound maslinic acid. For the most active compound of this series, i.e. 2-O-(2-chlorobenzoyl) maslinic acid (5), more detailed cell biological tests (i.e. AO/PI dye exclusion experiments, an annexin V assay, and microscopic investigations) on A2780 (human ovarian carcinoma cells) revealed that this compound triggers apoptosis.

Substituted cinnamic anhydrides act as selective inhibitors of acetylcholinesterase.

Cinnamic anhydrides have been shown to be more than reactive reagents, but they also act as inhibitors of the enzyme acetylcholinesterease (AChE). Thus, out of a set of 33 synthesised derivatives, several of them were mixed type inhibitors for AChE (from electric eel). Thus, (E)-3-(2,4-dimethoxyphenyl)acrylic anhydride (2c) showed Ki = 8.30 ±â€¯0.94 µM and Ki' = 9.54 ±â€¯0.38 µM, and for (E)-3-(3-chlorophenyl)acrylic anhydride (2u) Ki = 8.23 ±â€¯0.93 µM and Ki' = 13.07 ±â€¯0.46 µM were measured. While being not cytotoxic to many human cell lines, these compounds showed an unprecedented and noteworthy inhibitory effect for AChE but not for butyrylcholinesterase (BChE).

Caffeic acid phenethyl ester (CAPE)-derivatives act as selective inhibitors of acetylcholinesterase.

Unexpected inhibitory effects against eeAChE could be found for a newly synthesized class of caffeic acid phenethyl ester (CAPE) derivatives. Thus, phenethyl-(E)-3-(3,5-dimethoxy-4-phenethoxyphenyl)-acrylate (Ki = 1.97 ±â€¯0.38 µM, Ki´â€¯= 2.44 ±â€¯0.07 µM) and 4-(2-(((E)-3-(3,4-bis(benzyloxy)phenyl)acryloyl)oxy)ethyl)-1,2-phenylene (2E,2'E)-bis(3-(3,4-bis(benzyloxy)phenyl)acrylate) (Ki = 0.72 ±â€¯0.31 µM, Ki´â€¯= 1.80 ±â€¯0.21 µM) showed very good inhibition of eeAChE, while being non cytotoxic for malignant human cancer cells and non-malignant mouse fibroblasts. Also, they are weak inhibitors for BChE (from equine serum).

The cytotoxicity of oleanane derived aminocarboxamides depends on their aminoalkyl substituents.

Several oligo-methylene diamine derived carboxamides of oleanolic and maslinic acid have been prepared, and substitutions of the terminal primary amine as well as variations of the length of alkyl chain of the diamine moiety were made. Biological evaluation of their cytotoxic activity was performed using photometric sulforhodamin B assays employing a panel of different human cancer cell lines. These experiments showed most of the carboxamides to be cytotoxic with EC50 values below 10 µM. Prolongation of the alkyl chain length initially reduced EC50 values to a minimum, but a decrease in cytotoxicity was observed for longer alkyl chains. Variation of substituents at the terminal nitrogen atom, however, did not influence EC50 values at all. Noteworthy results were obtained particularly for compounds 4, 6 and 23 as indicated by EC50 values lower than 2 µM, and in case of a maslinic derivative 23 even an increased tumor/non-tumor cell selectivity was observed. These compounds were further investigated using fluorescence microscopy and flow cytometry analysis, which revealed 6 to show indications of apoptosis.

Triterpene-Based Carboxamides Act as Good Inhibitors of Butyrylcholinesterase.

A set of overall 40 carboxamides was prepared from five different natural occurring triterpenoids including oleanolic, ursolic, maslinic, betulinic, and platanic acid. All of which were derived from ethylene diamine holding an additional substituent connected to the ethylene diamine group. These derivatives were evaluated regarding their inhibitory activity of the enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) employing Ellman's assay. We further determined the type of inhibition and inhibition constants. Carboxamides derived from platanic acid have been shown to be potent and selective BChE inhibitors. Especially the mixed-type inhibitor (3ß)-N-(2-pyrrolidin-1-ylethyl)-3-acetyloxy-20-oxo-30-norlupan-28-amide (35) showed a remarkably low Ki of 0.07 ± 0.01 µM (Ki' = 2.38 ± 0.48 µM) for the inhibition of BChE.

An Improved Scalable Synthesis of α- and ß-Amyrin.

The synthesis of α- and ß-amyrin was accomplished starting from easily accessible starting materials, oleanolic, and ursolic acid. The procedures allow the preparation of ß-amyrin in an exceptionally short scalable manner via selective iodation and reduction. For α-amyrin, a different synthetic approach had to be chosen providing access to α-amyrin in medium-to-large scale.

Converting maslinic acid into an effective inhibitor of acylcholinesterases.

During the last decade, maslinic acid has been evaluated for many biological properties, e.g. as an anti-tumor or an anti-viral agent but also as a nutraceutical. The potential of maslinic acid and related derivatives to act as inhibitors of acetyl- or butyryl-cholinesterase was examined in this communication in more detail. Cholinesterases do still represent an interesting group of target enzymes with respect to the investigation and treatment of the Alzheimer's disease and other dementia illnesses as well. Although other triterpenoic acids have successfully been tested for their ability to act as inhibitors of cholinesterases, up to now maslinic acid has not been part of such studies. For this reason, three series of maslinic acid derivatives possessing modifications at different centers were synthesized and subjected to Ellman's assay to determine their inhibitory strength and type of inhibitory action. While parent compound maslinic acid was no inhibitor in these assays, some of the compounds exhibited an inhibition of acetylcholinesterase in the single-digit micro-molar range. Two compounds were identified as inhibitors of butyrylcholinesterase showing inhibition constants comparable to those of galantamine, a drug often used in the treatment of Alzheimer's disease. Furthermore, additional selectivity as well as cytotoxicity studies were performed underlining the potential of several derivatives and qualifying them for further investigations. Docking studies revealed that the different kinetic behavior within the same compound series may be explained by the ability of the compounds to enter the active site gorge of AChE.