Synthesis of Rhodamine-Conjugated Lupane Type Triterpenes of Enhanced Cytotoxicity.

Various conjugates with rhodamines were prepared by starting with betulinic acid (BA) and platanic acid (PA). The molecules homopiperazine and piperazine, which were identified in earlier research, served as linkers between the rhodamine and the triterpene. The pentacyclic triterpene's ring A was modified with two acetyloxy groups in order to possibly boost its cytotoxic activity. The SRB assays' cytotoxicity data showed that conjugates 13-22, derived from betulinic acid, had a significantly higher cytotoxicity. Of these hybrids, derivatives 19 (containing rhodamine B) and 22 (containing rhodamine 101) showed the best values with EC50 = 0.016 and 0.019 µM for A2780 ovarian carcinoma cells. Additionally, based on the ratio of EC50 values, these two compounds demonstrated the strongest selectivity between malignant A2780 cells and non-malignant NIH 3T3 fibroblasts. A375 melanoma cells were used in cell cycle investigations, which showed that the cells were halted in the G1/G0 phase. Annexin V/FITC/PI staining demonstrated that the tumor cells were affected by both necrosis and apoptosis.

Developing an Amide-Spacered Triterpenoid Rhodamine Hybrid of Nano-Molar Cytotoxicity Combined with Excellent Tumor Cell/Non-Tumor Cell Selectivity.

Asiatic acid, a pentacyclic triterpene, was converted into a series of piperazinyl, homopiperazinyl, and 1,5-diazocinyl spacered rhodamine conjugates, differing in the type of spacer and the substitution pattern on the rhodamine moiety of the hybrids. The compounds were tested for cytotoxic activity in SRB assays and compound 12, holding an EC50 of 0.8 nM, was the most cytotoxic compound of this series, but compound 18 (containing a ring expanded 1,5-diazocinyl moiety and n-propyl substituents on the rhodamine) was the most selective compound exhibiting a selectivity factor of almost 190 while retaining high cytotoxicity (EC50 = 1.9 nM, for A2780 ovarian carcinoma).

The Finally Rewarding Search for A Cytotoxic Isosteviol Derivative.

Acid hydrolysis of stevioside resulted in a 63% yield of isosteviol (1), which served as a starting material for the preparation of numerous amides. These compounds were tested for cytotoxic activity, employing a panel of human tumor cell lines, and almost all amides were found to be non-cytotoxic. Only the combination of isosteviol, a (homo)-piperazinyl spacer and rhodamine B or rhodamine 101 unit proved to be particularly suitable. These spacered rhodamine conjugates exhibited cytotoxic activity in the sub-micromolar concentration range. In this regard, the homopiperazinyl-spacered derivatives were found to be better than those compounds with piperazinyl spacers, and rhodamine 101 conjugates were more cytotoxic than rhodamine B hybrids.

Synthesis and Characterization of Phenylalanine Amides Active against Mycobacterium abscessus and Other Mycobacteria.

Nα-2-thiophenoyl-d-phenylalanine-2-morpholinoanilide [MMV688845, Pathogen Box; Medicines for Malaria Venture; IUPAC: (2R)-N-(1-((2-morpholinophenyl)amino)-1-oxo-3-phenylpropan-2-yl)thiophene-2-carboxamide)] is a hit compound, which shows activity against Mycobacterium abscessus (MIC90 6.25-12.5 µM) and other mycobacteria. This work describes derivatization of MMV688845 by introducing a thiomorpholine moiety and the preparation of the corresponding sulfones and sulfoxides. The molecular structures of three analogs are confirmed by X-ray crystallography. Conservation of the essential R configuration during synthesis is proven by chiral HPLC for an exemplary compound. All analogs were characterized in a MIC assay against M. abscessus, Mycobacterium intracellulare, Mycobacterium smegmatis, and Mycobacterium tuberculosis. The sulfone derivatives exhibit lower MIC90 values (M. abscessus: 0.78 µM), and the sulfoxides show higher aqueous solubility than the hit compound. The most potent derivatives possess bactericidal activity (99% inactivation of M. abscessus at 12.5 µM), while they are not cytotoxic against mammalian cell lines.

Small Structural Differences Govern the Carbonic Anhydrase II Inhibition Activity of Cytotoxic Triterpene Acetazolamide Conjugates.

Acetylated triterpenoids betulin, oleanolic acid, ursolic acid, and glycyrrhetinic acid were converted into their succinyl-spacered acetazolamide conjugates. These conjugates were screened for their inhibitory activity onto carbonic anhydrase II and their cytotoxicity employing several human tumor cell lines and non-malignant fibroblasts. As a result, the best inhibitors were derived from betulin and glycyrrhetinic acid while those derived from ursolic or oleanolic acid were significantly weaker inhibitors but also of diminished cytotoxicity. A betulin-derived conjugate held a Ki = 0.129 µM and an EC50 = 8.5 µM for human A375 melanoma cells.

Asiatic acid as a leading structure for derivatives combining sub-nanomolar cytotoxicity, high selectivity, and the ability to overcome drug resistance in human preclinical tumor models.

Amides and rhodamine B conjugates of different pentacyclic triterpene acids have been shown outstanding cytotoxicity for human tumor cells. Starting from asiatic acid, a new rhodamine B hybrid has been synthesized, and its cytotoxic activity was investigated employing several human tumor cell lines (A375 (melanoma), HT29 (colorectal carcinoma), MCF7 (breast adenocarcinoma), A2780 (ovarian carcinoma), HeLa (cervical carcinoma), (NIH 3T3 (non-malignant murine fibroblasts). For these conjugates of this kind it has been established that the spacer attached to the carboxyl group at ring E governs the magnitude of the cytotoxicity. These asiatic acid - rhodamine B conjugates were highly cytotoxic for human tumor cell lines but also selective. For example, 7, an acetylated homopiperazinyl spacered rhodamine B conjugate, held an EC50 = 0.8 nM for A2780 ovarian carcinoma cells. Additional staining experiments showed the rhodamine B conjugates to act as mitocans and to effect apoptosis. In further tests using 3D spheroid models of colorectal- and mamma carcinoma, 7 demonstrated activity in the lower nanomolar range and the ability to overcome resistance to clinically used standard chemotherapeutic drugs. Therefore 7 induces cytotoxic effects leading to an equal response in the chemotherapy of both sensitive and resistant tumor models. Analyses of mitochondrial function and glycolysis and respiration derived ATP production confirmed compound 7 to act as mitocan but also revealed a rapid perturbation of the cellular energy metabolism as the primary mechanism of action, which is completely different to conventional chemotherapeutic drugs and thereby explains the ability of compound 7 to overcome chemotherapeutic drug resistance.

Converting bile acids into mitocans.

Cholic acid (1, CD), deoxycholic (3, DCA), chenodeoxycholic acid (5, CDCA), ursodeoxycholic acid (7, UDCA), and lithocholic acid (9, LCA) were acetylated and converted into their piperazinyl spacered rhodamine B conjugates 16-20. While the parent bile acids showed almost no cytotoxic effects for several human tumor cell lines, the piperazinyl amides were cytostatic but an even superior effect was observed for the rhodamine B conjugates. Extra staining experiments showed these compounds as mitocans; they led to a cell arrest in the G1 phase.

New amides derived from sclareolide as anticholinesterase agents.

A series of 25 amides (15 new) derived from (3aR)-(+)-sclareolide were prepared and subjected to Ellman's assay to determine their efficacies as inhibitors for AChE or BuChE. Five amides (9, 13, 14, 15 and 17) caused inhibition of one of the enzymes greater than 60%; thereby those that inhibited BuChE were more active than positive control galantamine, and they showed better Ki values (1.07 to 8.49). In general, it was found that molecules holding a meta-substituted phenyl group showed a higher percentage of enzymatic inhibition. Molecular modelling calculations indicated the putative interactions of compounds with the amino acids residues of both enzymes AChE and BuChE. The cytotoxicity of compounds 9, 13, 14, 15 and 17 was evaluated against a non-malignant murine embryonic fibroblast cell line (NIH 3T3). Of special note is compound 15, as it presented the second-best Ki value for BuChE (1.71), was not cytotoxic (EC50 > 30 µM). Compound 15 also does not violate Lipinski rules, and showed permeability in the blood brain barrier, indicating that it can be considered a lead for the development of new drugs to treat Alzheimer's disease.

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 and cytotoxicity of betulin and betulinic acid derived 30-oxo-amides.

Betulin and betulinic acid derived 30-oxo-amides were prepared by hydroboration, subsequent oxidation and amidation; these novel compounds were screened for their cytotoxic activity by SRB assays. All of the compounds showed significant cytotoxic activity for different human tumor cell lines. Small changes in the structure, however, resulted in significant changes in the cytotoxicity of the compounds. Of special interest were compounds 11 and 12, each holding an extra ethylenediamine moiety. These C-30 amides which showed low EC50 values, and both of them acted mainly by apoptosis.

Structure-Activity Relationship of Anti-Mycobacterium abscessus Piperidine-4-carboxamides, a New Class of NBTI DNA Gyrase Inhibitors.

Mycobacterium abscessus causes difficult-to-cure pulmonary infections. The bacterium is resistant to most anti-infective agents, including first line antituberculosis (anti-TB) drugs. MMV688844 (844) is a piperidine-4-carboxamide (P4C) with bactericidal properties against M. abscessus. We recently identified DNA gyrase as the molecular target of 844. Here, we present in silico docking and genetic evidence suggesting that P4Cs display a similar binding mode to DNA gyrase as gepotidacin. Gepotidacin is a member of the Novel Bacterial Topoisomerase Inhibitors (NBTIs), a new class of nonfluoroquinolone DNA gyrase poisons. Thus, our work suggests that P4Cs present a novel structural subclass of NBTI. We describe structure-activity relationship studies of 844 leading to analogues showing increased antibacterial activity. Selected derivatives were tested for their inhibitory activity against recombinant M. abscessus DNA gyrase. Further optimization of the lead structures led to improved stability in mouse plasma and increased oral bioavailability.

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].

Synthesis and In Silico Docking of New Pyrazolo[4,3-e]pyrido[1,2-a]pyrimidine-based Cytotoxic Agents.

To explore a new set of anticancer agents, a novel series of pyrazolo[4,3-e]pyrido[1,2-a]pyrimidine derivativeshave been designed and synthesized viacyclocondensation reactions of pyrazolo-enaminone with a series of arylidenemalononitriles; compound 5 was obtained from 5-amino-4-cyanopyrazole. The structures of the target compounds were investigated by spectral techniques and elemental analysis (IR, UV-Vis, 1H NMR, 13C NMR and ESI-MS). All compounds were evaluated for their in vitro cytotoxicity employing a panel of different human tumor cell lines, A375, HT29, MCF7, A2780, FaDu as well as non-malignant NIH 3T3 and HEK293 cells. It has been found that the pyrazolo-pyrido-pyrimidine analog bearing a 4-Br-phenyl moiety was the most active toward many cell lines with EC50 values ranging between 9.1 and 13.5 µM. Moreover, in silico docking studies of the latter with six anticancer drug targets, i.e., DHFR, VEGFR2, HER-2/neu, hCA-IX, CDK6 and LOX5, were also performed, in order to gain some insights into their putative mode of binding interaction and to estimate the free binding energy of this bioactive molecule.

Racemization-free synthesis of Nα-2-thiophenoyl-phenylalanine-2-morpholinoanilide enantiomers and their antimycobacterial activity.

Nα-2-thiophenoyl-D-phenylalanine-2-morpholinoanilide (MMV688845, IUPAC: N-(1-((2-morpholinophenyl)amino)-1-oxo-3-phenylpropan-2-yl)thiophene-2-carboxamide) from the Pathogen Box® library (Medicines for Malaria Ventures, MMV) is a promising lead compound for antimycobacterial drug development. Two straightforward synthetic routes to the title compound starting from phenylalanine or its Boc-protected derivative are reported. Employing Boc-phenylalanine as starting material and the T3P® and PyBOP® amide coupling reagents enables racemization-free synthesis, avoiding the need for subsequent separation of the enantiomers. The crystal structure of the racemic counterpart gives insight into the molecular structure and hydrogen bonding interactions in the solid state. The R-enantiomer of the title compound (derived from D-phenylalanine) exhibits activity against non-pathogenic and pathogenic mycobacterial strains, whereas the S-enantiomer is inactive. Neither of the enantiomers and the racemate of the title compound shows cytotoxicity against various mammalian cells.

Type and position of linkage govern the cytotoxicity of oleanolic acid rhodamine B hybrids.

Oleanolic acid/rhodamine B hybrids exhibit different cytotoxicity depending on the way these two structural elements are linked. While a hybrid holding a piperazinyl spacer at C-28 proved to be cytotoxic in the nano-molar concentration range, hybrids with a direct linkage of the Rho B residue to C-3 of the triterpenoid skeleton are cytotoxic only in the low micro-molar concentration range without any selectivity. This once again underlines the importance of selecting the right spacer and the most appropriate position on the skeleton of the triterpene to achieve the most cytotoxic hybrids possible.

A simple but unusual rearrangement of an oleanane to a taraxerane-28,14 ß -olide.

Reaction of 3-O-acetyl-oleanolic acid (3) with formic acid/hydrogen peroxide at 100 °C for several hours provides an extraordinary but simple pathway to a taraxeran-28,14 ß -olide type triterpenoid while the same reaction at 0 °C occurred without re-arrangement of the carbon skeleton, and an oleanane-28,13 ß -olide was obtained instead. The products from these reactions were subjected to a cytotoxicity screening employing several human tumor cell lines showing the latter compound not cytotoxic while the former was cytotoxic especially for MCF-7 (breast adenocarcinoma), and FaDu (hypopharyngeal carcinoma) cells. The highest cytotoxicity, however, was observed for 3 ß, 12α, 13 ß -trihydroxy-oleanan-28-oic acid (6) holding with EC50 = 4.2 µM for MCF-7 tumor cells.

The Presence of a Cyclohexyldiamine Moiety Confers Cytotoxicity to Pentacyclic Triterpenoids.

Pentacyclic triterpenoids oleanolic acid, ursolic acid, betulinic acid, and platanic acid were acetylated and converted into several amides 9-31; the cytotoxicity of which has been determined in sulforhodamine B assays employing seral human tumor cell lines and nonmalignant fibroblasts. Thereby, a betulinic acid/trans-1,4-cyclohexyldiamine amide showed excellent cytotoxicity (for example, EC50 = 0.6 µM for HT29 colon adenocarcinoma cells).

Cytotoxic Potential of a-Azepano- and 3-Amino-3,4-SeCo-Triterpenoids.

Semi-synthetic triterpenoids, holding an amino substituted seven-membered A-ring (azepano-ring), which could be synthesized from triterpenic oximes through a Beckmann type rearrangement followed by a reduction of lactame fragment, are considered to be novel promising agents exhibiting anti-microbial, alpha-glucosidase, and butyrylcholinesterase inhibitory activities. In this study, in an attempt to develop new antitumor candidates, a series of A-ring azepano- and 3-amino-3,4-seco-derivatives of betulin, oleanolic, ursolic, and glycyrrhetinic acids were evaluated for their cytotoxic activity against five human cancer cell lines and non-malignant mouse fibroblasts by means of a colorimetric sulforhodamine assay. Azepanoallobetulinic acid amide derivative 11 was the most cytotoxic compound of this series but showed little selectivity between the different human tumor cell lines. Flow cytometry experiments showed compound 11 to act mainly by apoptosis (44.3%) and late apoptosis (21.4%). The compounds were further screened at the National Cancer Institute towards a panel of 60 cancer cell lines. It was found that compounds 3, 4, 7, 8, 9, 11, 15, 16, 19, and 20 showed growth inhibitory (GI50) against the most sensitive cell lines at submicromolar concentrations (0.20-0.94 µM), and their cytotoxic activity (LC50) was also high (1-6 µM). Derivatives 3, 8, 11, 15, and 16 demonstrated a certain selectivity profile at GI50 level from 5.16 to 9.56 towards K-562, CCRF-CEM, HL-60(TB), and RPMI-8226 (Leukemia), HT29 (Colon cancer), and OVCAR-4 (Ovarian cancer) cell lines. Selectivity indexes of azepanoerythrodiol 3 at TGI level ranged from 5.93 (CNS cancer cell lines SF-539, SNB-19 and SNB-75) to 14.89 for HCT-116 (colon cancer) with SI 9.56 at GI50 level for the leukemia cell line K-562. The present study highlighted the importance of A-azepano-ring in the triterpenic core for the development of novel antitumor agents, and a future aim to increase the selectivity profile will thus lie in the area of modifications of azepano-triterpenic acids at their carboxyl group.

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.