Hederagenin amide derivatives as potential antiproliferative agents.

In this study, a series of C-28 amides derivatives of hederagenin with or without the presence of an acetyl group at positions 3 and 23 in ring A, were synthetized aiming to develop potent cytotoxic agents. Their structures were confirmed by MS, IR, 1H NMR and 13C NMR spectroscopic analyses and their cytotoxic activities were screened in SRB assays using a panel of six human cancer cell lines. The majority of the amide derivatives were cytotoxic for a variety of human tumor cell lines. In general, the hydroxylated derivatives (1a-1d; EC50 in the range 1.2-22.5 µM) were less active than the acetylated derivatives (2a-2n; EC50 in the range 0.4-9.0 µM). Hydroxylated derivative bearing pyrrolidinyl substituent 1c, was the most active for HT29 human line cells (EC50 = 1.2 µM), however their acetylated derivative 2c was the most potent and selective against A2780, FaDu, SW1736 cells, showing EC50 values between 0.4 and 1.7 µM and SI between 5.6 and 24. Staining experiments combined with fluorescence microscopy indicate that the cell membrane became permeable, and finally a process of secondary necrosis was observed. In addition, the docking results showed that acetylated compounds display more affinity to HER2 than to USP7, indicating that HER2 is a most probable receptor, both proteins found in tumor cell line A2780.

Ethylenediamine Derived Carboxamides of Betulinic and Ursolic Acid as Potential Cytotoxic Agents.

Two easily accessible, natural occurring triterpenoids, betulinic and ursolic acid, were used as starting materials for the synthesis of novel cytotoxic agents. A set of 28 ethylenediamine-spacered carboxamides was prepared holding an additional substituent connected to the ethylenediamine group. The compounds were screened in SRB assays to evaluate their cytotoxic activity employing several human tumor cell lines. Betulinic acid-derived carboxamides 17⁻30 showed significantly higher cytotoxicity than their ursolic acid analogs 3⁻16. In particular, compounds 25 and 26 were highly cytotoxic, as indicated by EC50 values lower than 1 µM.

Ugi multicomponent-reaction: Syntheses of cytotoxic dehydroabietylamine derivatives.

Isocyanide-based multicomponent reactions - especially the standard four component Ugi reaction - provide an easy and powerful access to compounds with an auspicious pharmacological potential. Therefore, a set of 16 novel derivatives of the diterpene dehydroabietylamine was synthesized by the Ugi-4CR. The subsequent screening of the synthesized α-acylamino carboxamides in colorimetric sulforhodamine B assays revealed an in vitro cytotoxicity towards several human tumor cell lines. Particularly, the rhodamine B conjugates 14-16 showed a remarkable cytotoxic activity, characterized by EC50 values in a low three-digit nanomolar range. The screening of rhodamine B amide 17 that was obtained for comparison by a Schotten-Baumann reaction showed that the linkage of the rhodamine B moiety and the diterpene influences significantly its cytotoxic potency. While 14 was highly cytotoxic and acted as a mitocan, compound 17 was not cytotoxic at all. This observation underlines the importance of the type of coupling between the diterpene and the rhodamine part. The presence of a rhodamine B moiety in the molecules doesn't necessarily guarantee that the compound is cytotoxic.

Targeting mitochondria: Esters of rhodamine B with triterpenoids are mitocanic triggers of apoptosis.

Triterpenoic acids, ursolic acid (1), oleanolic acid (2), glycyrrhetinic acid (3) and betulinic acid (4) were converted into their corresponding methyl 5-8 and benzyl esters 9-12 or benzyl amides 21-24. These derivatives served as starting materials for the synthesis of pink colored rhodamine B derivatives 25-36 which were screened for cytotoxicity in colorimetric SRB assays. All of the compounds were cytotoxic for a variety of human tumor cell lines. The activity of the benzyl ester derivatives 29-32 was lower than the cytotoxicity of the methyl esters 25-28. The benzyl amides 33-36 were the most cytotoxic compounds of this series. The most potential compound was a glycyrrhetinic acid rhodamine B benzyl amide 35. This compound showed activity against the different cancer cell lines in a two-digit to low three-digit nano-molar range. Staining experiments combined with fluorescence microscopy showed that this compound triggered apoptosis in A2780 ovarian carcinoma cells and acted as a mitocan.

An access to a library of novel triterpene derivatives with a promising pharmacological potential by Ugi and Passerini multicomponent reactions.

The promising combination of natural product leads and their derivatization by isocyanide-based multicomponent reactions (IMCRs) has gained interest in accessing diversity-oriented libraries with auspicious pharmacological potential. Therefore, a set of 34 Ugi and 3 Passerini products was successfully synthesized starting from naturally occurring triterpenoids, i.e. oleanolic acid (OA) and maslinic acid (MA), followed by a biological evaluation of the novel α-acylamino carboxamides and the α-acyloxy carboxamides in colorimetric SRB assays to determine their cytotoxic potential. Especially, the MA-Ugi products 6a, 6b and 7b showed a remarkable cytotoxicity for A2780 ovarian carcinoma cells in a low µM range. Compounds 6a and 7b induced programmed cell death in part through the apoptosis pathway.

Synthesis of new triterpenic monomers and dimers as potential antiproliferative agents and their molecular docking studies.

In the current investigation, new monomers of myrrhanone B and lupeolic acid were prepared via reaction of triterpenic acids with linkers in the presence of K2CO3. In addition, new bis-myrrhanone B homodimers, myrrhanone B-myrrhanol B heterodimers, and bis-myrrhanone ß-boswellic acids heterodimer were prepared. Evaluation of these compounds on the proliferation of four different human cancer cell lines, viz., FaDu (pharynx carcinoma), A2780 (ovarian carcinoma), HT29 (colon adenocarcinoma) and A375 (malignant melanoma) has been performed. It is worth mentioning that compounds 4, 7, 8, 10, and 11 possess potent antiproliferative effect towards HT29 cancer cells with IC50 values of 8.1 µM, 5.4 µM, 8.8 µM, 6.8 µM, and 8.2 µM, respectively. In addition, these compounds display good to moderate antiproliferative activities towards A2780 and A375 with IC50 values ranging from 10.4 to 24.2 µM. Moreover, the molecular docking studies of most active compounds (4, 7, 8, 10 and 11) with six anti-cancer drug targets DHFR, VEGFR2, HER-2/neu, CDK6, hCA-IX and LOX also carried, in order to know the mode of binding interaction and energy of this class of compounds.

Platanic acid: A new scaffold for the synthesis of cytotoxic agents.

Thirty-seven different derivatives (2-38) have been prepared from platanic acid, a natural occurring triterpenoid. Main emphasis was the introduction of several N-containing functional groups such as amines, amides and oximes and their screening for cytotoxic activity employing several human tumor cell lines using SRB assays. In these SRB assays, nearly all compounds showed good cytotoxicity for these human tumor cell lines. Two compounds (17 and 38), however, were submitted to extended biological testing and investigated with respect to their mode of action using fluorescence microscopy and FACS analysis. Compound 17, a methyl (3ß, 20R) 3-acetyloxy-20-amino-30-norlupan-28-oate, induced apoptosis in A2780 ovarian carcinoma cells.

Leishmanicidal and cytotoxic activity of hederagenin-bistriazolyl derivatives.

Aiming to obtain new potent leishmanicidal and cytotoxic compounds from natural sources, the triterpene hederagenin was converted into several new 1,2,3-triazolyl derivatives tethered at C-23 and C-28. For this work hederagenin was isolated from fruits of Sapindus saponaria and reacted with propargyl bromide to afford as a major product bis-propargylic derivative 1 in 74%. Submitting this compound to Huisgen 1,3-dipolar cycloaddition reactions with several azides afforded the derivatives 2-19 with yields in the range of 40-87%. All compounds have been screened for in vitro cytotoxic activity in a panel of five human cancer cell lines by a SRB assay. The bioassays showed that compound 19 was the most cytotoxic against all human cancer cell lines with EC50 = 7.4-12.1 µM. Moreover, leishmanicidal activity was evaluated through the in vitro effect in the growth of Leishmania infantum, and derivatives 1, 2, 5 and 17 were highly effective preventing proliferation of intracellular amastigote forms of L. infantum (IC50 = 28.8, 25.9, 5.6 and 7.4 µM, respectively). All these compounds showed a higher selectivity index and low toxicity against two strains of kidney BGM and liver HepG2 cells. Compound 5 has higher selectivity (1780 times) in comparison with the commercial antimony drug and is around 8 times more selective than the most active compound previously reported hederagenin derivative. Such high activity associated with low toxicities make the new bis-traiazolyl derivatives promising candidates for the treatment of leishmaniasis. In addition, hederagenin and some derivatives (2, 5 and 17) showed interaction in the binding site of the enzyme CYP51Li.

Piperlongumine B and analogs are promising and selective inhibitors for acetylcholinesterase.

Piperlongumine B (19), an alkaloid previously isolated from long pepper (Piper longum) has been synthesized for the first time in a short sequence and in good yield together with 19 analogs. Screening of these compounds in Ellman's assays showed several of them to be good inhibitors of acetylcholinesterase while being less active for butyrylcholinesterase. Activity of the compounds increased with the ring size of the heterocycle, and a maximum of activity was observed for an analog holding 12 methylene groups in the aliphatic side chain. These compounds may be regarded as promising candidates for the development of efficient inhibitors of acetylcholinesterase being useful for the treatment of Alzheimer's disease.

Natural abenquines and synthetic analogues: Preliminary exploration of their cytotoxic activity.

In this study, we explore the cytotoxic activity of four natural abenquines (2a-d) and fourteen synthetic analogues (2e-j and 3a-h) against a panel of six human cancer cell lines using a SRB assay. It was found that most of the compounds revealed higher levels of cytotoxic activities than naturally occurring abenquines. The analogues carrying ethylpyrrolidinyl and ethylpyrimidinyl with either an acetyl group (2h-i) or a benzoyl group (3f-g), were the most potent against all human cancer cell lines and displayed EC50 between a range of 0.6-3.4µM. Notably, of the compounds tested, compound 2i proved the most cytotoxic against both ovarian (A2780) and breast (MCF7) cells, showing EC50=0.6 and 0.8µM respectively. Likewise, the analogues 2i, 3f and 3g showed strong activity against cell HT29 with EC50=0.9µM for these compounds.

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.

Amino derivatives of platanic acid act as selective and potent inhibitors of butyrylcholinesterase.

A set of thirtyfive 30-norlupan derivatives (2-36) was prepared from the natural triterpenoid platanic acid (PA), and the hydroxyl group at C-3, the carboxyl group at C-17 and the carbonyl group at C-20 were modified. These derivatives were tested for their inhibitory activity for the enzymes acetylcholinesterase (AChE, from electric eel) and butyrylcholinesterase (BChE, from equine serum) using Ellman's assay. Extra enzyme kinetic studies were performed. The most active compound was (3ß, 20R)-3-acetyloxy-20-amino-30-norlupan-28-oate (32) showing a Ki value of 0.01 ± 0.003 µM for BChE. This compound proved to be a selective (FB = 851), mixed-type inhibitor for BChE.

Synthesis and proapoptotic activity of oleanolic acid derived amides.

Thirty-one different 3-O-acetyl-OA derived amides have been prepared and screened for their cytotoxic activity. In the SRB assays nearly all the carboxamides displayed good cytotoxicity in the low µM range for several human tumor cell lines. Low EC50 values were obtained especially for the picolinylamides 14-16, for a N-[2-(dimethylamino)-ethyl] derivative 27 and a N-[2-(pyrrolinyl)-ethyl] carboxamide 28. These compounds were submitted to an extensive biological testing and proved compound 15 to act mainly by an arrest of the tumor cells in the S phase of the cell cycle. Cell death occurred by autophagy while compounds 27 and 28 triggered apoptosis.

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 (2ß, 3ß) 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 (2ß,3ß)-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-[2ß,3ß-di-O-acetyl-17ß-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.

Amino(oxo)acetate moiety: A new functional group to improve the cytotoxicity of betulin derived carbamates.

While 3-O-acetylated betulin derivatives carrying a carbamate moiety at position C-28 are of rather low cytotoxicity for human tumor cell lines, the corresponding C-3 amino(oxo) acetates show good cytotoxicity. For example, an EC50 as low as 2.0µM was found for (3ß) 28-{[(hexylamino)carbonyl]oxy}lup-20(29)-en-3-yl amino(oxo)acetate (16) employing the ovarian cancer cell line A2780.

Novel hederagenin-triazolyl derivatives as potential anti-cancer agents.

A series of novel aryl-1H-1,2,3-triazol-4-yl methylester and amide derivatives of the natural product hederagenin was synthesized aiming to develop new antitumor agents, using Huisgen 1,3-dipolar cycloaddition reactions, with yields between 35% and 95%. The structures of all derivatives (2-31) were confirmed by MS, IR, (1)H NMR and (13)C NMR spectroscopic data. The cytotoxic activities of all compounds were screened against a panel of six human cancer cell lines using SRB assay. It was found that most of the compounds displayed higher levels of antitumor activities as compared to parent hederagenin. Compounds 4, 8 and 15 were the most potent against all human cancer cell lines. Furthermore, compound 11 was the most cytotoxic against cell HT29 showing EC50 = 1.6 µM and a selectivity index of 5.4.

Targeting cancer cells with oleanolic and ursolic acid derived hydroxamates.

Oleanolic and ursolic acid derived hydroxamates were easily obtained from their parent compounds; they were screened for their cytotoxicity applying SRB assays employing several human tumor cell lines. Low EC50 values were determined for compounds in which the nitrogen as well as the oxygen in the hydroxamic acid part still holds acidic hydrogens. Thus, ursolic acid derived compounds having at least an OH and/or NH moiety in the hydroxamate part of the molecule showed good cytotoxicity but they are significantly less selective for the tumor cells than oleanolic acid derived compounds. Good results were determined for oleanolic acid derived 7 for tumor cell lines 518A2 (melanoma, EC50=3.3 µM), A2780 (ovarian carcinoma, EC50=3.4 µM) and HT29 (colon adenocarcinoma, EC50=5.6 µM) while being significantly less cytotoxic for fibroblasts (EC50=20.4 µM).

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.