Non-malignant Mouse Fibroblasts Research Articles

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.

Development of indanones and isatins as non-cytotoxic inhibitors of cholinesterases

A small library of indanone-amides and substituted isatin derivatives has been prepared; these compounds have been investigated for their ability to act as inhibitors for the enzymes acetyl- and butyrylcholinesterase (AChE, BChE). Several of them were moderate inhibitors for AChE and not cytotoxic for a variety of human tumor cell lines as well as for non-malignant mouse fibroblasts. In this library consisting of 49 derivatives, 5,7-dibromo-4-iodoisatin was shown to be a good mixed-type inhibitor for AChE (K­i = 2.52 ± 0.61 µM and Ki´ = 11.74 ± 1.31 µM) but this compound also acted as a dual inhibitor for BChE (Ki = 4.49 ± 0.32 µM and Ki´ = 6.56 ± 0.57 µM). Interestingly, N-hexyl-1-oxo-2,3-dihydro-1H-indene-2-carboxamide was cytotoxic especially for MCF-7 breast adenocarcinoma cells (EC50 = 4.28 ± 0.5 µM).

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.

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 ± 0.38 μM, Ki´ = 2.44 ± 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 ± 0.31 μM, Ki´ = 1.80 ± 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).

β-11-Keto-boswellic acid derived amides: synthesis and cytotoxicity

The aim of this study was to prepare 11-keto-β-boswellic acid derivatives modified at C-24 and to evaluate their in vitro cytotoxicity. Acetyl-11-keto-β-boswellic acid (AKBA) was isolated from frankincense and transformed into 11-keto-β-boswellic acid (KBA). Both compounds served as starting materials for the synthesis of several amides or hydrazides. The derivatives were fully characterized, and their cytotoxicity was evaluated in vitro using sulforhodamine B (SRB) assays employing two human tumor cell lines (A2780 and MCF7) as well as nonmalignant mouse fibroblasts (NIH 3T3). Nearly all of the compounds were more cytotoxic than their parent compounds. The highest cytotoxicity was observed for (3 α, 4 β) 3-acetyloxy-N-(3- aminopropyl)-11-oxo-urs-12-en-24-amide (15) and (3 α, 4 β) 3-acetyloxy-N-[4-(3-aminopropyl)piperazin-1-yl]- propyl-11-oxo-urs-12-en-24-amide (16) and the ovarian carcinoma cell line A2780. These compounds showed EC50 = 1.0-1.7 µM while being significantly less toxic for the mouse fibroblasts NIH 3T3 (EC50 = 9.3-16.3µM). Thus, compounds 15 and 16 have good antitumor effects and may serve as starting points for developing potential and selective antitumor agents

Synthesis and cytotoxic screening of beta-boswellic acid derivatives

Beta-boswellic acids are triterpenoids being generic to the plants of genus boswellia. Although they were shown to exhibit different biological activities, the cytotoxic potential of b-boswellic acid derivatives remained by and large unexploited. To expand the potential of these compounds we developed simple procedures for the interconversion of the most important b-boswellic acids 1-4 and prepared several other derivatives 5-48. These compounds were screened for their cytotoxic activity in sulforhodamine B assays employing several human tumor cell lines and nonmalignant mouse fibroblasts. One of these compounds, a difluoromethylester of 3-O-acetyl-11-keto-beta-boswellic acid 23, was cytotoxic for human breast adenocarcinoma cells MCF-7 (EC50 = 6.5 mM) while being significantly less cytotoxic for the mouse fibroblasts.

Rhodamine B conjugates of triterpenoic acids are cytotoxic mitocans even at nanomolar concentrations

Triterpenoic acids 1–6 exhibited very low or no cytotoxicity at all, but their corresponding 2,3-di-O-acetyl-piperazinyl amides 13–18 showed low EC50 values for several human tumor cell lines. Their cytotoxicity, however, was also high for the non-malignant mouse fibroblasts NIH 3T3. A significant improvement was achieved by preparing the rhodamine B derivatives 19–24. While rhodamine B is not cytotoxic (up to a concentration of 30μM – cut-off of the assay), the triterpenoid piperazine-spacered rhodamine B derivatives were cytotoxic in nano-molar concentration. Compound 24 (a diacetylated maslinic acid derivative) was most toxic for several human tumor cell lines but less toxic for mouse fibroblasts NIH 3T3. Staining and double-staining experiments revealed 24 to act as a mitocan.

Drotaverine - a Concealed Cytostatic!

Drotaverine (also known as dihydroperparine or No-Spa® ) is an antispasmodic drug closely related to papaverin. Drotaverin also acts as a cytostatic compound for several human tumor cell lines and nonmalignant mouse fibroblasts, and EC50 values as low as 3.0 μM were observed in SRB assays for HT-29 human colorectal carcinoma cells. Small structural changes (e.g., aromatization, benzylic oxidation) led to a reduced activity or a complete loss of cytotoxicity. Staining of the cells with acridine orange showed the cell membrane of the dead cells to be still intact, and a slight G1/G0 arrest in the treated cells was observed after 24 h. Extra annexin V-FITC/PI assays and flow cytometry revealed drotaverine mainly to act as a cytostatic and only to a minor extent as cytotoxic agent.

Selective killing of cancer cells with triterpenoic acid amides - The substantial role of an aromatic moiety alignment

2,3-Di-O-acetyl-triterpenoic acid derived amides possessing a (2β, 3β) configuration in ring A and two acetyl groups were previously shown to possess high cytotoxicity for human tumor cell lines but to exhibit low cytotoxicity for non-malignant mouse fibroblasts. In this study, augustic acid (1) and 2-epi-corosolic acid (2) were chosen as starting points for the synthesis of analogs. While augustic acid derived 3-quinolinyl amide 9 gave low EC50 values in SRB assays but was cytotoxic for all lines, the isomeric 4-isoquinolinyl amide 21 was very cytotoxic for the tumor cell lines but significantly less cytotoxic for the mouse fibroblasts NIH 3T3. In addition, a triacetylated 4-isoquinolinyl derivative of asiatic acid (28) gave EC50 = 80 nM (for A2780 ovarian cancer cells). As shown by additional experiments (acridine orange/propidium iodide staining, fluorescence spectroscopy and cell cycle investigations) these compounds act mainly by apoptosis.

Urea derivates of ursolic, oleanolic and maslinic acid induce apoptosis and are selective cytotoxic for several human tumor cell lines

2,3-Di-O-acetyl-maslinic acid benzylamide (5) has previously been shown to possess high cytotoxicity for a variety of human tumor cell lines while being of low cytotoxicity to non-malignant cells. Structural modifications performed on 5 revealed that the presence of these acetyl groups in 5 and the presence of (2β,3β)-configurated centers seems necessary for obtaining high cytotoxicity combined with best selectivity between malignant cells and non-malignant mouse fibroblasts. Compounds carrying an ursane skeleton showed weaker cytotoxicity than their oleanane derived analogs. In addition, the benzylamide function in compound 5 should be replaced by a phenylurea moiety to gain better cytotoxicity while retaining and improving the selectivity. Thus, maslinic acid derived N-[2β,3β-di-O-acetyl-17β-amino-28-norolean-12-en-17-yl]phenylurea (45) gave best results showing EC50 = 0.9 μM (for A2780 ovarian cancer cells) with EC50 > 120 μM for fibroblasts (NIH 3T3) and triggered apoptosis while caspase-3 was not activated by this compound.

Betulinic acid derived hydroxamates and betulin derived carbamates are interesting scaffolds for the synthesis of novel cytotoxic compounds

The betulinic acid-derived hydroxamates 5–18, the amides 19–24, and betulin-derived bis-carbamates 25–28 as well as the carbamates 31–40 and 44–48 were prepared and evaluated for their antiproliferative activity in a photometric sulforhodamine B (SRB) assay against several human cancer cell lines and nonmalignant mouse fibroblasts (NIH 3T3). While for 3-O-acetyl hydroxamic acid 5 EC50 values as low as EC50 = 1.3 μM were found, N,O-bis-alkyl substituted hydroxamates showed lowered cytotoxicity (EC50 = 16–20 μM). In general, hydroxamic acid derivatives showed only reduced selectivity for tumor cells, except for allyl substituted compound 13 (EC50 = 5.9 μM for A2780 human ovarian carcinoma cells and EC50 > 30 μM for nonmalignant mouse fibroblasts). The cytotoxicity of betulinic acid derived amides 19–24 and of betulin derived bis-carbamates 25–28 was low, except for N-ethyl substituted 25. Hexyl substituted 39 showed EC50 = 5.6 μM (518A2 cells) while for mouse fibroblasts EC50 > 30 was determined.

Chemoenzymatic synthesis and cytotoxicity of oenanthotoxin and analogues

We developed a synthetic scheme for the synthesis of naturally occurring (14R)-oenanthotoxin and several analogs. Key-steps of this synthesis were an efficient homo-coupling of alkynes and a chemoenzymatic resolution of racemic oenanthotoxin using novozyme 435 and vinyl acetate. The compounds were screened for their cytotoxic activity using a photometric sulforhodamine B assays and several human tumor cell lines. Oenanthotoxin and many derivatives thereof were cytotoxic to tumor cell lines as well as to non-malignant mouse fibroblasts. The highest activity was determined for human ovarian cancer cells A2780 with EC50=3.8μM.

Simple structural modifications confer cytotoxicity to allobetulin

A variety of allobetulin derivatives was synthesized from allobetulin or allobetulone. These compounds were screened for their cytotoxic activity using a photometric SRB assay employing six different human tumor cell lines. In summary, opening of ring A of allobetulin in general lowers the cytotoxicity, but the 2,3-seco diethyl ester was highly cytotoxic and remarkable selective for A549 lung carcinoma cells while being significantly less cytotoxic for non-malignant mouse fibroblasts. The introduction of an amino group at position C-3 in the allobetulin skeleton enhances cytotoxicity and furnishes highly cytotoxic compounds. Their selectivity to distinguish between cancer cell and non-malignant cell depends on the configuration at position C-3.

The DNA-Mismatch Repair Enzyme hMSH2 Modulates UV-B-Induced Cell Cycle Arrest and Apoptosis in Melanoma Cells

The mechanisms by which the post-replicative DNA mismatch repair (MMR) enzyme MSH2 is involved in the complex response mechanisms to UV damage are yet to be clarified. Here, we show increased levels of MSH2 mRNA in malignant melanoma, metastases of melanoma, and melanoma cell (MeWo) lines as compared with melanocytic nevi or primary cultured benign melanocytes. UV-B treatment modulated MSH2 expression and silencing of MSH2 gene expression using small interfering RNA technology regulated UV-B-induced cell cycle arrest and apoptosis in human MeWo. We show that MSH2-deficient non-malignant mouse fibroblasts (MEF-/-) are partially resistant against UV-B-induced apoptosis and show reduced S-Phase accumulation. In addition, we show that an Msh2 point mutation (MEFGA) that affects MMR does not affect UV-B-induced apoptosis. In conclusion, we demonstrate that MSH2 modulates in human melanocytes both UV-B-induced cell cycle regulation and apoptosis, most likely via independent, uncoupled mechanisms.

Radioprotective and antitumor activity evaluation of newly synthesized adamantyl tenocyclidines.

Several adamantyl derivatives of thienyl phencyclidine (tenocyclidine; TCP) were newly sythesized and characterized: adamantyl derivatives containing piperidine (TAPIP), pyrrolidine (TAPYR), and morpholine (TAMORPH) groups. Their biological activity was evaluated by in vitro testing of their effect on the proliferative and reproductive ability (cytotoxicity) of a human tumor cell strain and nonmalignant mouse fibroblasts in culture. We also tested them for their radioprotective effect after ionizing irradiation, and as anticancer agents on the same human tumor cell strain. Compared with TCP, adamantyl derivatives are less toxic and have outstanding radioprotective properties. These derivatives (especially TAMORPH) increase apoptotic death of human malignant cells. The radiation-modifying effect studied on C3Hf mice in vivo showed that the adamantyl derivatives of TCP have a more enhanced radioprotective effect and that they are less toxic than TCP itself. The present data are discussed and compared with those previously reported for structurally related phencyclidine derivatives.