Synthesis and antiproliferative activity of (-)-cleistenolide, (6S)-cleistenolide and 4-substituted cleistenolide analogues.

A new total synthesis of the natural δ-lactone cleistenolide (1) and its (6S)-stereoisomer 2 was achieved starting from d-glucose. Key steps in the synthesis of 1 involved: oxidative cleavage of the C1-C2 bond in partially protected d-glucose derivative (20), and chain extension of resulting aldehyde 20a with a single C2 fragment using (Z)-selective Wittig olefination. Synthesis of 2 involves the following key steps: periodate cleavage of the C5-C6 bond in the commercially available monoacetone d-glucose (24), followed by C2 chain elongation by using the (Z)-selective Wittig olefination. This new approach is also applied to prepare a few new 4-substituted cleistenolide analogues (3 - 18). Compounds 3 - 7 were designed using molecular hybridization, while the remaining eleven analogues were designed using the bioisosterism method. MTT assay showed that most analogues were more active than lead 1 against several malignant cells, but were completely inactive in the culture of normal foetal lung fibroblasts (MRC-5). The K562 cells appeared to be the most sensitive to the synthesized analogues. The strongest antiproliferative activity against this cell line was shown by 4-O-cinnamoyl derivative 3 and 4,6-di-O-benzyl derivative 17, with submicromolar IC50 values (0.76 and 0.67 µM, respectively). Structural features important for the activity of this class of compounds were identified by SAR analysis.

First total synthesis of asperilactone B. Revision of absolute stereochemistry of asperilactones B and C.

The first total synthesis and absolute configuration assignment of asperilactone B (I) have been accomplished. Additionally, a revision of the absolute stereochemistry of asperilactone C has been done. The first total synthesis of the opposite enantiomer of asperilactone B (ent-I) has also been achieved, as well as that of C-7 epimers of both asperilactones B (8) and C (9).

Synthesis and biological activity of thiophene bioisosteres of natural styryl lactone goniofufurone and related compounds.

Ten new thiophene derivatives related to goniofufurone have been obtained by multistep synthesis starting from d-glucose. The critical step of the synthesis was the Grignard reaction of 2-thienyl magnesium bromide with a protected dialdose, yielding the C-5 epimeric thiophene derivatives 9 and 10. The mixture was oxidized to the 5-keto derivative 11, which after deprotection was converted to the corresponding keto-lactone 14. Stereoselective reduction of 14 afforded the thiophene mimic of goniofufurone 3. Esterification of 3 with cinnamic or 4-fluorocinnamic acid gave hybrids 5-7. Synthesized analogues were evaluated for their in vitro cytotoxicity against several tumour cell lines. The vast majority of them showed better activity than lead 1. In the culture of K562 cells, compound 3 was more active than the commercial antitumour drug doxorubicin. Structural features of analogues important for their antiproliferative activities were identified by SAR analysis. Pro-apoptotic potential examination of compound 3 on the K562 cell line was performed using flow cytometry, double fluorescence staining and apoptotic morphology screening. Results show that this derivative induces cell membrane disruptions attributable to apoptosis and induces the apoptotic morphology, but decreasing simultaneously the population of cells in the subG1 phase of the cell cycle. The results further suggest that analogue 3 achieves strong cytotoxicity without causing DNA fragmentation. This is clearly indicated by the relatively low incidence of micronuclei, as well as the SAR analysis of all biological effects.

The first total synthesis and revision of absolute stereochemistry of natural cytotoxic lactone cleistanolate.

Development of a synthetic route applicable to d-ribose and d-xylose enabled the synthesis of cleistanolate putative structure, its five stereoisomers, and led to revision and confirmation of absolute stereochemistry of the natural product. Key steps of the synthesis included zinc-mediated THF ring-opening and stereoselective dihydroxylation under the Upjohn conditions. The first total synthesis of cleistanolate was completed in eight steps starting from d-xylose. The C-5 stereocenter of the natural product was assigned the correct (5S)-stereochemistry. Cytotoxicity of natural product was briefly investigated.

Natural product protulactone A: Total synthesis from D-galactose, X-ray analysis and biological evaluation.

Synthesis of protulactone A (PLA, 1) and twelve of its analogues have been achieved starting from d-galactose. PLA was isolated in the crystalline state, and its crystal structure was determined utilizing X-ray crystallography, which confirmed the assumed stereochemistry at all stereocenters. All tested compounds displayed antiproliferative activity against a panel of tumour cell lines, and all of them were non-cytotoxic toward the normal cells (MRC-5). Natural product PLA (1) was the most active against the K562 and MCF-7 cell lines (IC50 6.52 and 2.20 µM, respectively). Some of the synthesized derivatives showed very potent cytotoxicity, especially analogues 11, 13 and 15 (IC50 1.08-1.14 µM against MCF-7), and 9 and 14 (IC50 1.29 and 1.64 µM against K562). SAR analysis indicated important structural motifs for antiproliferative activity. Unfortunately, PLA (1), its C-7 epimer (2) and demethylated analogue (3) did not display a significant antimicrobial activity (two Gram-positive and two Gram-negative bacteria and one fungal strain) and they also cannot affect the ability to modulate bacterial communication.

Design, synthesis, and biological evaluation of thiazole bioisosteres of goniofufurone through in vitro antiproliferative activity and in vivo toxicity.

The synthesis of several new goniofufurone bioisosteres was achieved in which the phenyl residue was replaced by a thiazole ring. The key steps of the synthesis included the initial condensation of cyanohydrin benzoates with cysteine ethyl ester hydrochloride, followed by the subsequent reaction of resulting C-4' epimeric thiazolines with DBU, to introduce 5-deoxy functionality and to elaborate the thiazole ring in one step. Synthesized compounds showed potent growth inhibitory effects against selected human tumour cell lines, especially bioisostere 4, which in the culture of MCF-7 cells displayed the highest activity (IC50 = 0.19 nM) of all compounds under evaluation. This molecule exhibited 64474-fold higher antiproliferative activity than lead 2 and was1053-fold more active than the commercial antitumour agent doxorubicin in the culture of MCF-7 cells. The structural features of the tested compounds responsible for their antiproliferative activity have been identified by preliminary SAR analysis. The toxicity of the most active compound 4 was assessed by an in vivo experiment in a zebrafish model (Danio rerio), whereupon it was found non-toxic at any of the tested concentrations up to 125 µM.

Synthesis and antimicrobial activity of (-)-cleistenolide and analogues.

Using the "chiral pool" approach, two modified total syntheses of the biologically active δ-lactone cleistenolide (1) have been achieved starting from d-glucose. These approaches also enabled the preparation of novel analogues and derivatives of natural product 1. The applied strategy for the synthesis of 1 involves: the initial degradation of the chiral precursor for a single C-atom, C2-fragment chain extension using Z-selective Wittig reaction, and the final δ-lactonization. All tested cleistenolide analogues displayed antimicrobial activity against a panel of nine microbial strains, most of them superseding the activity of cleistenolide itself, and, in some cases, coming close in value to the observed minimal inhibitory concentrations of chloramphenicol. Increased lipophilicity of the derivatives and the non-sterically congested conjugated lactone moiety were a prerequisite for analogues with high inhibitory activity against S. aureus and, in general, Gram-positive bacteria.

Synthesis, antiproliferative activity and SAR analysis of (-)-cleistenolide and analogues.

A new, modified total synthesis of (-)-cleistenolide (1) and sixteen new analogues or derivatives was achieved starting from commercially available 1,2-O-isopropylidene-α-d-glucofuranose. The synthesis of 1 proceeds in six steps and 67% overall yield, using single-carbon atom degradation of a protected chiral precursor, (Z)-selective Wittig olefination, and acid catalyzed δ-lactonization. A new Lewis acid promoted procedure for one-pot O-debenzylation/O-acylation has been developed to complete the synthesis of natural product 1 and selected analogues. The synthesized compounds were tested in vitro to evaluate their cytotoxicity against K562, HL-60, Jurkat, Raji, MCF-7, MDA-MB 231, HeLa, A549, and MRC-5 cell lines. All (-)-cleistenolide analogues exhibited significantly higher cytotoxicity than lead 1 against the majority of cell lines tested. Most of the synthesized compounds are more active than doxorubicin on at least one malignant cell line, but were almost completely inactive against normal MRC-5 cells. The structural features of the tested compounds responsible for their antiproliferative activity have been identified by preliminary SAR analysis.

Structure based design, synthesis and in vitro antitumour activity of tiazofurin stereoisomers with nitrogen functions at the C-2' or C-3' positions.

Three novel tiazofurin analogues having d-arabino stereochemistry and nitrogen functionalities at the C-2' position (5-7) have been designed and synthesized in multistep sequences, starting from d-glucose. The known d-xylo stereoisomer of 1 (compound 2) along with two new analogues bearing nitrogen functions at the C-3' (3 and 4) has also been synthesized from the same sugar precursor. The synthetic sequence consisted of the following three stages: (i) the multistep synthesis of suitably protected pentofuranosyl cyanides, (ii) the construction of ethyl thiazole-4-carboxylate part by cyclocondensation of thus obtained glycofuranosyl cyanides with l-cysteine ethyl ester followed by dehydrogenation, and (iii) the final transformation of the ethyl thiazole-4-carboxylates into the target tiazofurin analogues using the esters ammonolysis. The tiazofurin analogues were evaluated for their antitumour activities in cell-culture-based assays. Compounds 3, 4 (d-xylo) and 7 (d-arabino), showed remarkable antitumour activities, with IC50 values in the range of 4-7 nM. Preliminary structure-activity relationship allowed identification of two analogues with antiproliferative activities exceeding that of the parent compound 1 for several orders of magnitude (e.g. 4: 1354-fold against Raji, 7: 309-fold against K562). Flow cytometry data and Western blot analysis suggested that cytotoxic effects of d-xylo stereoisomers in the culture of K562 cells caused changes in the cell cycle distribution, as well as the induction of apoptosis in caspase-dependent way. The increase of apoptotic cells percentage in treated samples is also confirmed with fluorescent double-staining method. Genotoxicity testing showed that the analogues with the xylo-configuration (2-4) are far less genotoxic than tiazofurin.

In vitro antitumor activity, ADME-Tox and 3D-QSAR of synthesized and selected natural styryl lactones.

Prostate cancer is a common cause of death in men and a novel treating methods should be developed. In order to find a new drug for prostate cancer, a series of novel conformationally constrained analogues of (+)-goniofufurone and 7-epi-(+)-goniofufurone, as well as the newly synthesized styryl lactones containing the cinnamic acid ester groups were evaluated for in vitro cytotoxicity against prostate cancer cell (PC-3). Furthermore, prediction of physicochemical characteristics and drugability as well as in silico ADME-Tox tests of investigated compounds were performed. The 3D-QSAR model was established using the comparative molecular field analysis method. According to obtained results, the tricyclic compounds 9 and 10 had the highest potency with IC50 < 20 µM. Evaluation of structural features through 3D-QSAR model identified steric field feature on the cinnamic acid ester groups at C-7 as a crucial for the cytotoxic activity. This research suggests that most of the analysed compounds have desirable properties for drug candidates and high potential in drug development, which recommend them for further research in treatment of prostate cancer. Furthermore, obtained 3D-QSAR model is able to successfully identify styryl lactones that have significant cytotoxic activity and provide information for screening and design of novel inhibitors against PC-3 cell line that could be used as drugs in treatment of the prostate cancer.

Novel O-methyl goniofufurone and 7-epi-goniofufurone derivatives: synthesis, in vitro cytotoxicity and SAR analysis.

Novel goniofufurone (1) and 7-epi-goniofufurone (2) derivatives bearing a methoxy group at the C-5 and/or C-7 positions were prepared and their in vitro antitumour activity against some human tumour cell lines was evaluated. Some of the analogues displayed powerful antiproliferative effects against the studied tumour cells, but almost all of them were non-cytotoxic toward the normal cells (MRC-5). A SAR study reveals that the introduction of a methoxy group at the C-7 position may increase the antiproliferative effects of the analogues. The most active compounds are 7-O-methyl derivatives of goniofufurone (3) and 7-epi-(+)-goniofufurone (6), which exhibited 1177- and 451-fold higher potencies than the leads 1 and 2 toward the MDA-MB 231 cell line. At the same time, compound 3 is almost 1.5-fold more active than the commercial drug doxorubicin (DOX) against the same cell line. Flow cytometry data confirmed that the cytotoxic effects of these analogues are mediated by apoptosis, additionally revealing that these molecules induced changes in the K562 cell cycle distribution.

A comparative study of chromatographic behavior and lipophilicity of selected natural styryl lactones, their derivatives and analogues.

This study is based on the analyses of the retention behavior of selected natural styryl lactones and their synthetic analogues in reversed-phase high-performance liquid chromatography. Chromatographic separations were achieved applying ZORBAX SB-C18 column and two different mobile phases: methanol-water and acetonitrile-water. Chromatographic lipophilicity of the analyzed compounds was defined by logk0 constant and correlated with in silico molecular descriptors. According to the statistical validation parameters, obtained results indicate that the presented linear and multiple quantitative structure-retention relationship models can successfully predict the chromatographic lipophilicity of structurally similar compounds. Hierarchical cluster analyses (HCA) was applied in order to group similar compounds according to their chromatographic and in silico lipophilicity. It can be concluded that chromatographic systems with methanol-water were better for modelling of logk0. Modelling was performed in order to characterize compounds regarding their lipophilicity profiles as future drug candidates.

Synthesis and in vitro antitumour activity of crassalactone D, its stereoisomers and novel cinnamic ester derivatives.

Naturally occurring styryl lactone, crassalactone D (1), unnatural 4-epi-crassalactone D (2), and the corresponding 7-epimers (3 and 4) have been synthesized starting from d-glucose. The key step of the synthesis is a new one-pot sequence that commenced with a Z-selective Wittig olefination of suitably functionalized sugar lactols with a stabilized ylide, (methoxycarbonylmethylene)-triphenylphosphorane, in dry methanol, to afford 1 or 3, in the mixtures with the corresponding 4-epimers (2 or 4, respectively). A number of 6-O-cinnamoyl derivatives of styryl lactones 1-4 have been prepared, bearing electron donating or electron withdrawing functionalities in the C-4 position of cinnamic acid residue. The synthesized products were evaluated for their in vitro antiproliferative activity against selected human tumour cell lines, whereupon very potent cytotoxicities have been recorded in many cases. SAR analysis indicated some important structural features responsible for biological activity, such as stereochemistry at the C-4 and C-7 positions, as well as the nature of a substituent at the C-4 position in the aromatic ring of cinnamoate moiety. Flow cytometry and Western blot analysis data gave insight in the mechanism underlying antiproliferative effects of the synthesized compounds.

Design, synthesis and in vitro antitumour activity of new goniofufurone and 7-epi-goniofufurone mimics with halogen or azido groups at the C-7 position.

A series of new antitumour lactones containing the [3.3.0] bicyclic furano-lactone core and the halogen or azido group at the C-7 position have been designed, synthesized, and evaluated for their in vitro antitumour activity against a panel of human tumour cell lines. Some of the analogues displayed powerful antiproliferative effects to certain human tumour cells, but all of them were devoid of any cytotoxicity towards the normal foetal lung fibroblasts (MRC-5). A SAR study reveals the structural features of these lactones that may affect their antiproliferative activity. These are: the nature of substituent present at the C-7 position, stereochemistry at the C-7 position, the absence of phenyl group at the C-7 position. Flow cytometry data indicate that the cytotoxic effects of the synthesized analogues in a culture of K562 cells are mediated by apoptosis, additionally revealing that these molecules induced changes in cell cycle distribution of these cells. Results of Western blot analysis suggested that the most of synthesized compounds induce apoptosis in K562 cells in caspase-dependent way.

New antitumour agents with α,ß-unsaturated δ-lactone scaffold: Synthesis and antiproliferative activity of (-)-cleistenolide and analogues.

A stereoselective total synthesis of (-)-cleistenolide (1) from d-glucose has been achieved. This new approach for the synthesis of (-)-cleistenolide and analogues involves a one-C-atom degradation of the chiral precursor, (Z)-selective Wittig olefination, followed by the final δ-lactonisation. Synthesized compounds showed potent growth inhibitory effects against selected human tumour cell lines, especially 2,4,6-trichlorobenzoyl derivative 12, which in the culture of MDA-MB 231 cells displayed the highest activity (IC50 0.02µM) of all compounds under evaluation. A preliminary SAR study reveals the structural features that are beneficial for antiproliferative activity of synthesized δ-lactones, such as presence of either electron-withdrawing or electron-donating substituents in the aromatic ring, as well as the presence of cinnamoyl functionality instead of benzoyl group at the O-7 position.

Synthesis and in vitro antitumour activity of tiazofurin analogues with nitrogen functionalities at the C-2' position.

Synthesis of three tiazofurin (1) isosteres with nitrogen functionalities at the C-2' position (N3, NH2 and NH3(+)Cl(-)) has been achieved, in multistep sequences, starting from monoacetone d-glucose. A number of potential bioisosteres of 1 bearing acylamido functions at the C-2' position have also been synthesized from the same sugar precursor. In vitro cytotoxicities of target molecules against a number of human tumour cell lines were recorded and compared with those observed for lead molecule 1. Some of the synthesized compounds showed potent in vitro antitumour activity, such as 2'-azido derivative 2, which is the most potent of all molecules under evaluation (IC50 0.004 µM against MCF-7 cells). Flow cytometry data suggest that cytotoxic effects of these compounds in the culture of K562 cells might be mediated by apoptosis, additionally revealing that these molecules induced changes in cell cycle distribution of these cells. Results of Western blot analysis indicate that the synthesized tiazofurin analogues induce apoptosis in a caspase-dependent way.

Synthesis and antiproliferative activity of goniobutenolides A and B, 5-halogenated crassalactone D derivatives and the corresponding 7-epimers.

A new synthesis of goniobutenolides A (1) and B (2) and the corresponding 7-epimers has been achieved starting from diacetone d-glucose. The key step of the synthesis is a new one-pot sequence that commenced with Z-selective Wittig (or Horner-Wadsworth-Emmons) olefination, followed by successive γ-lactonisation and ß-elimination. The above-mentioned unsaturated lactones were then converted to the corresponding 5-halogenated crassalactone D derivatives by using the appropriate haloetherification protocol. The most of synthesized compounds exhibited potent cytotoxic activities against a panel of tumour cell lines. The main structural features responsible for their antitumour potency have been revealed by means of SAR analysis. Flow cytometry data suggested that cytotoxic effects of these compounds in the culture of K562 cells might be mediated by apoptosis, additionally revealing that these molecules induced changes in cell cycle distribution of these cells. Results of semi-quantitative Western blot analysis indicate that the most of synthesized compounds induce apoptosis in a caspase-dependent manner.

Design, synthesis and SAR analysis of antitumour styryl lactones related to (+)-crassalactones B and C.

A series of styryl lactones containing the cinnamic acid ester groups such as (+)-crassalactones B (3a) and C (4a), 5,7-di-O-cinamoyl derivative 6, the corresponding 7-epimers and 7-deoxy derivatives have been synthesized, characterized and evaluated for their in vitro antitumour activity against a panel of several human tumour cell lines. Twelve new analogues such as 5-O- or 7-O-(4-methoxycinnamoyl), 5-O- or 7-O-(4-nitrocinnamoyl) and 5-O- or 7-O-(4-fluorocinnamoyl) esters of (+)-goniofufurone (3b-d), 7-epi-(+)-goniofufurone (epi-3b-d), as well as 7-deoxy derivatives 5b-d have been prepared to correlate all compounds in a SAR study. Some of the analogues displayed powerful antiproliferative effects on selected human tumour cell lines, but none of them demonstrated cytotoxicity towards the normal foetal lung fibroblasts (MRC-5). Thus, for the 7-epi-crassalactone B (epi-3a) was found to be a potent inhibitor of HL-60 cells growth, with an IC50 value that is approximately 46-fold lower than that observed for the commercial antitumour drug doxorubicin in the culture of the same cells. A SAR analysis performed on these lactones reveals the main structural features that affect their antiproliferative activity, such as nature of the substituents at the C-4 in the aromatic rings of cinnamoyl moieties, the absolute stereochemistry, as well as the presence of a deoxy function at the C-7 position.

Conformationally constrained goniofufurone mimics as inhibitors of tumour cells growth: Design, synthesis and SAR study.

Synthesis of conformationally restricted (+)-goniofufurone (1) and 7-epi-(+)-goniofufurone (2) analogues, with embedded O-isopropylidene, O-methylidene or cyclic carbonate functions is disclosed starting from d-glucose. A number of potential bioisosteres of 1 and 2 bearing both 5,7-O-methylidene and 4-substituted cinnamoyloxy functions at the C-7 position have also been synthesized. In vitro cytotoxicity of target molecules against a number of human tumour cell lines were recorded and compared with those observed for the parent molecules 1 and 2. Some of the analogues displayed powerful antiproliferative effects on selected human tumour cell lines, but all of them were devoid of any cytotoxicity towards the normal foetal lung fibroblasts (MRC-5). A SAR study reveals the structural features of these lactones that may increase their antiproliferative activity.