Total Synthesis and Biological Evaluation of Tubulysin Analogues.

We report a second-generation synthesis of the exceedingly potent antimitotic agent N14-desacetoxytubulysin H (1) as well as the preparation of nine analogues of this lead structure. Highlights of our synthetic efforts include an efficient late-stage functionalization that allows for the preparation of new side-chain- and backbone-modified analogues. We also discovered C-terminal modifications that preserve the exquisite biological activity of acid 1 and offer the opportunity for effective conjugation to cell type-targeting moieties. All analogues had antiproliferative activities in the high picomolar to low nanomolar range and caused apoptosis and mitotic arrest as measured in a high content nuclear morphology assay. The ten synthetic agents described herein spanned a range of almost 4 orders of magnitude in biological activity and illustrate the continued potential to discover extraordinarily potent antiproliferative compounds based on natural product leads.

The anti-promyelocytic leukemia mode of action of two endophytic secondary metabolites unveiled by a proteomic approach.

As a result of a program to find antitumor compounds of endophytes from medicinal Asteraceae, the steroid (22E,24R)-8,14-epoxyergosta-4,22-diene-3,6-dione (a) and the diterpene aphidicolin (b) were isolated from the filamentous fungi Papulaspora immersa and Nigrospora sphaerica, respectively, and exhibited strong cytotoxicity against HL-60 cells. A proteomic approach was used in an attempt to identify the drugs' molecular targets and their respective antiproliferative mode of action. Results suggested that the (a) growth inhibition effect occurs by G2/M cell cycle arrest via reduction of tubulin alpha and beta isomers and 14-3-3 protein gamma expression, followed by a decrease of apoptotic and inflammatory proteins, culminating in mitochondrial oxidative damage that triggered autophagy-associated cell death. Moreover, the decrease observed in the expression levels of several types of histones indicated that (a) might be disarming oncogenic pathways via direct modulation of the epigenetic machinery. Effects on cell cycle progression and induction of apoptosis caused by (b) were confirmed. In addition, protein expression profiles also revealed that aphidicolin is able to influence microtubule dynamics, modulate proteasome activator complex expression, and control the inflammatory cascade through overexpression of thymosin beta 4, RhoGDI2, and 14-3-3 proteins. Transmission electron micrographs of (b)-treated cells unveiled dose-dependent morphological characteristics of autophagy- or oncosis-like cell death.

Synthesis and biological properties of C-2 triazolylinosine derivatives.

O(6)-(Benzotriazol-1H-yl)guanosine and its 2'-deoxy analogue are readily converted to the O(6)-allyl derivatives that upon diazotization with t-BuONO and TMS-N(3) yield the C-2 azido derivatives. We have previously analyzed the solvent-dependent azide·tetrazole equilibrium of C-6 azidopurine nucleosides, and in contrast to these, the O(6)-allyl C-2 azido nucleosides appear to exist predominantly in the azido form, relatively independent of solvent polarity. In the presently described cases, the tetrazole appears to be very minor. Consistent with the presence of the azido functionality, each neat C-2 azide displayed a prominent IR band at 2126-2130 cm(-1). A screen of conditions for the ligation of the azido nucleosides with alkynes showed that CuCl in t-BuOH/H(2)O is optimal, yielding C-2 1,2,3-triazolyl nucleosides in 70-82% yields. Removal of the silyl groups with Et(3)N·3HF followed by deallylation with PhSO(2)Na/Pd(PPh(3))(4) gave the C-2 triazolylinosine nucleosides. In a continued demonstration of the versatility of O(6)-(benzotriazol-1H-yl)purine nucleosides, one C-2 triazolylinosine derivative was converted to two adenosine analogues via these intermediates, under mild conditions. Products were desilylated for biological assays. The two C-2 triazolyl adenosine analogues demonstrated pronounced antiproliferative activity in human ovarian and colorectal carcinoma cell cultures. When evaluated for antiviral activity against a broad spectrum of DNA and RNA viruses, some of the C-2 triazolylinosine derivatives showed modest inhibitory activity against cytomegalovirus.

Synthesis and biological evaluation of neopeltolide and analogs.

The synthesis of neopeltolide analogues that contain variations in the oxazole-containing side chain and in the macrolide core are reported along with the GI(50) values for these compounds against MCF-7, HCT-116, and p53 knockout HCT-116 cell lines. Although biological activity is sensitive to changes in the macrocycle and the side chain, several analogues displayed GI(50) values of <25 nM. Neopeltolide and several of the more potent analogues were significantly less potent against p53 knockout cells, suggesting that p53 plays an auxiliary role in the activity of these compounds.

Total synthesis and biological evaluation of pederin, psymberin, and highly potent analogs.

The potent cytotoxins pederin and psymberin have been prepared through concise synthetic routes (10 and 14 steps in the longest linear sequences, respectively) that proceed via a late-stage multicomponent approach to construct the N-acyl aminal linkages. This route allowed for the facile preparation of a number of analogs that were designed to explore the importance of the alkoxy group in the N-acyl aminal and functional groups in the two major subunits on biological activity. These analogs, including a pederin/psymberin chimera, were analyzed for their growth inhibitory effects, revealing several new potent cytotoxins and leading to postulates regarding the molecular conformational and hydrogen bonding patterns that are required for biological activity. Second generation analogs have been prepared based on the results of the initial assays and a structure-based model for the binding of these compounds to the ribosome. The growth inhibitory properties of these compounds are reported. These studies show the profound role that organic chemistry in general and specifically late-stage multicomponent reactions can play in the development of unique and potent effectors for biological responses.

A simplified synthesis of novel dictyostatin analogues with in vitro activity against epothilone B-resistant cells and antiangiogenic activity in zebrafish embryos.

The natural product (--)-dictyostatin is a microtubule-stabilizing agent that potently inhibits the growth of human cancer cells, including paclitaxel-resistant clones. Extensive structure-activity relationship studies have revealed several regions of the molecule that can be altered without loss of activity. The most potent synthetic dictyostatin analogue described to date, 6-epi-dictyostatin, has superior in vivo antitumor activity against human breast cancer xenografts compared with paclitaxel. In spite of their encouraging activities in preclinical studies, the complex chemical structure of the dictyostatins presents a major obstacle for their development into novel antineoplastic therapies. We recently reported a streamlined synthesis of 16-desmethyl-25,26-dihydrodictyostatins and found several agents that, when compared with 6-epi-dictyostatin, retained nanomolar activity in cellular microtubule-bundling assays but had lost activity against paclitaxel-resistant cells with mutations in ß-tubulin. Extending these studies, we applied the new, highly convergent synthesis to generate 25,26-dihydrodictyostatin and 6-epi-25,26-dihydrodictyostatin. Both compounds were potent microtubule-perturbing agents that induced mitotic arrest and microtubule assembly in vitro and in intact cells. In vitro radioligand binding studies showed that 25,26-dihydrodictyostatin and its C6-epimer were capable of displacing [3H]paclitaxel and [14C]epothilone B from microtubules with potencies comparable to (--)-dictyostatin and discodermolide. Both compounds inhibited the growth of paclitaxel- and epothilone B-resistant cell lines at low nanomolar concentrations, synergized with paclitaxel in MDA-MB-231 human breast cancer cells, and had antiangiogenic activity in transgenic zebrafish larvae. These data identify 25,26-dihydrodictyostatin and 6-epi-25,26-dihydrodictyostatin as candidates for scale-up synthesis and further preclinical development.

Characterization and detection of cellular and proteomic alterations in stable stathmin-overexpressing, taxol-resistant BT549 breast cancer cells using offgel IEF/PAGE difference gel electrophoresis.

Stathmin/oncoprotein 18, a protein that regulates microtubule dynamics, is highly expressed in a number of tumors including leukemia, lymphoma, neuroblastoma, breast, ovarian, and prostate cancers. High stathmin levels have been associated with the development of resistance to the widely used anti-cancer drug taxol ((®)Taxol, paclitaxel). The mechanisms of stathmin-mediated taxol resistance are not well-understood at the molecular level. To better understand the role of stathmin in taxol resistance, we stably overexpressed stathmin twofold in BT549 human breast cancer cells and characterized several cell processes involved in the mechanism of action of taxol. After stable overexpression of stathmin, neither the cell doubling time nor the mitotic index was altered and the microtubule polymer mass was reduced only modestly (by 18%). Unexpectedly, microtubule dynamicity was reduced by 29% after stathmin overexpression, resulting primarily from reduction in the catastrophe frequency. Sensitivity to taxol was reduced significantly (by 44%) in a clonogenic assay, and stathmin appeared to protect the cells from the spindle-damaging effects of taxol. The results suggest that in the stably stathmin-overexpressing clones, compensatory gene expression occurred that resulted in normal rates of cell proliferation and prevented the increase in catastrophe frequency expected in response to stathmin. Stathmin overexpression protected the cells from taxol-induced abnormal mitoses, and thus induced taxol resistance. Using offgel IEF/PAGE difference gel electrophoresis, we identified a number of proteins whose expression is reduced in the taxol-resistant stathmin-overexpressing cell lines, including proteins involved in the cytoskeleton and cell structure, the stress response, protein folding, glycolysis, and catalysis.

Streamlined syntheses of (-)-dictyostatin, 16-desmethyl-25,26-dihydrodictyostatin, and 6-epi-16-desmethyl-25,26-dihydrodictyostatin.

The dictyostatins are a promising class of potential anti-cancer drugs because they are powerful microtubule-stabilizing agents, but the complexity of their chemical structures is a severe impediment to their further development. On the basis of both synthetic and medicinal chemistry analyses, 16-desmethyl-25,26-dihydrodictyostatin and its C6 epimer were chosen as potentially potent yet accessible dictyostatin analogues, and three new syntheses were developed. A relatively classical synthesis involving vinyllithium addition and macrocyclization gave way to a newer and more practical approach based on esterification and ring-closing metathesis reaction. Finally, aspects of these two approaches were combined to provide a third new synthesis based on esterification and Nozaki-Hiyama-Kishi reaction. This was used to prepare the target dihydro analogues and the natural product. All of the syntheses are streamlined because of their high convergency. The work provided several new analogues of dictyostatin, including a truncated macrolactone and a C10 E-alkene, which were 400- and 50-fold less active than (-)-dictyostatin, respectively. In contrast, the targeted 16-desmethyl-25,26-dihydrodictyostatin analogues retained almost complete activity in preliminary biological assays.

Azide-tetrazole equilibrium of C-6 azidopurine nucleosides and their ligation reactions with alkynes.

Facile syntheses of C-6 azidopurine ribonucleosides and 2'-deoxyribonucleosides have been developed. For silyl- and acetyl-protected as well as unprotected nucleosides, access to the azido derivatives could be readily attained via displacement of BtO(-) from the O(6)-(benzotriazol-1-yl)inosine nucleosides by azide anion. Use of diphenylphosphoryl azide/DBU as a simple route to the acetyl-protected azido nucleosides was also evaluated, but this proved to be inferior. Since these azido nucleosides can exist in an azide.tetrazole equilibrium, the effect of solvent polarity on this equilibrium was investigated. Subsequently, a detailed analysis of Cu-mediated azide-alkyne ("click") ligation was undertaken. Biphasic CH(2)Cl(2)/H(2)O medium proved to be best for the ligation reactions, suppressing the undesired azide reduction that was competing. Interestingly, although the tetrazolyl isomer predominates (ca. 80%) in CD(2)Cl(2) and in CD(2)Cl(2)/D(2)O, the Cu-catalyzed click reactions proceed smoothly with the silyl-protected ribo- and 2'-deoxyribonucleosides, leading to the C-6 triazolyl products in good to excellent yields. Thus, depletion of the azido form from the reaction mixture shifts the azide.tetrazole equilibrium, eventually resulting in complete consumption of azide and tetrazole. In several cases, major and minor azide-alkyne ligation products were observed, and characterization data are provided for both. In order to confirm the regiochemistry leading to the major isomer, one product was crystallized and evaluated by X-ray crystallography. The Cu-catalyzed azide-alkyne ligation is clearly efficient and significantly superior to thermal reactions, which were slow. Biological evaluation showed low cytotoxicities for the agents, suggesting their usefulness as biological probes.

High-content analysis of cancer-cell-specific apoptosis and inhibition of in vivo angiogenesis by synthetic (-)-pironetin and analogs.

The natural product (-)-pironetin is a structurally simple small molecule microtubule-perturbing agent whose biological activities appear to be exquisitely dependent on defined stereochemistry and the presence of an eletrophilic alpha,beta-unsaturated lactone moiety. We used alkaloid-catalyzed acyl halide-aldehyde cyclocondensation reactions in asymmetric total syntheses of (-)-pironetin and three synthetic analogs, and evaluated their biological activities by high-content analysis in cell culture and in a zebrafish model. Synthetic (-)-pironetin and 2,3-dihydro-3-hydroxypironetin caused mitotic arrest and programmed cell death in human lung cancer cells but not in normal lung fibroblasts, had nanomolar growth inhibitory activity in multi-drug resistant cells, and inhibited neovascularization in zebrafish embryos. Synthetic (-)-pironetin delayed the onset but increased the extent of tubulin assembly in vitro. The data illustrate the power of acyl halide-aldehyde cyclocondensation to generate biologically active synthetic analogs of stereochemically complex targets and suggest that (-)-pironetin and 2,3-dihydro-3-hydroxypironetin possess unique properties that may bestow them with advantages over existing microtubule-perturbing agents in the context of a whole organism or under conditions of multi-drug resistance.

Select pyrimidinones inhibit the propagation of the malarial parasite, Plasmodium falciparum.

Plasmodium falciparum, the Apicomplexan parasite that is responsible for the most lethal forms of human malaria, is exposed to radically different environments and stress factors during its complex lifecycle. In any organism, Hsp70 chaperones are typically associated with tolerance to stress. We therefore reasoned that inhibition of P. falciparum Hsp70 chaperones would adversely affect parasite homeostasis. To test this hypothesis, we measured whether pyrimidinone-amides, a new class of Hsp70 modulators, could inhibit the replication of the pathogenic P. falciparum stages in human red blood cells. Nine compounds with IC(50) values from 30 nM to 1.6 micrOM were identified. Each compound also altered the ATPase activity of purified P. falciparum Hsp70 in single-turnover assays, although higher concentrations of agents were required than was necessary to inhibit P. falciparum replication. Varying effects of these compounds on Hsp70s from other organisms were also observed. Together, our data indicate that pyrimidinone-amides constitute a novel class of anti-malarial agents.

Microtubule binding and disruption and induction of premature senescence by disorazole C(1).

Disorazoles comprise a family of 29 macrocyclic polyketides isolated from the fermentation broth of the myxobacterium Sorangium cellulosum. The major fermentation product, disorazole A(1), was found previously to irreversibly bind to tubulin and to have potent cytotoxic activity against tumor cells, possibly because of its highly electrophilic epoxide moiety. To test this hypothesis, we synthesized the epoxide-free disorazole C(1) and found it retained potent antiproliferative activity against tumor cells, causing prominent G(2)/M phase arrest and inhibition of in vitro tubulin polymerization. Furthermore, disorazole C(1) produced disorganized microtubules at interphase, misaligned chromosomes during mitosis, apoptosis, and premature senescence in the surviving cell populations. Using a tubulin polymerization assay, we found disorazole C(1) inhibited purified bovine tubulin polymerization, with an IC(50) of 11.8 +/- 0.4 microM, and inhibited [3H]vinblastine binding noncompetitively, with a K(i) of 4.5 +/- 0.6 microM. We also found noncompetitive inhibition of [3H]dolastatin 10 binding by disorazole C(1), with a K(i) of 10.6 +/- 1.5 microM, indicating that disorazole C(1) bound tubulin uniquely among known antimitotic agents. Disorazole C(1) could be a valuable chemical probe for studying the process of mitotic spindle disruption and its relationship to premature senescence.

Tubulin-perturbing naphthoquinone spiroketals.

Several natural and synthetic naphthoquinone spiroketals are potent inhibitors of the thioredoxin-thioredoxin reductase redox system. Based on the antimitotic and weak antitubulin actions noted for SR-7 ([8-(furan-3-ylmethoxy)-1-oxo-1,4-dihydronaphthalene-4-spiro-2'-naphtho[1'',8''-de][1',3'][dioxin]), a library of related compounds was screened for tubulin-perturbing properties. Two compounds, TH-169 (5'-hydroxy-4'H-spiro[1,3-dioxolane-2,1'-naphthalen]-4'-one) and TH-223 (5'-methoxy-4'H-spiro[1,3-dioxane-2,1'-naphthalen]-4'-one), had substantial effects on tubulin assembly and were antiproliferative at low micromolar concentrations. TH-169 was the most potent at blocking GTP-dependent polymerization of 10 mum tubulin in vitro with a remarkable 50% inhibitory concentration of ca. 400 nm. It had no effect on paclitaxel-induced microtubule assembly and did not cause microtubule hypernucleation. TH-169 failed to compete with colchicine for binding to beta-tubulin. The 50% antiproliferative concentration of TH-169 against human cancer cells was at or slightly below 1 mum. Flow cytometry showed that 1 mum TH-169 caused an increase in G(2)/M and hypodiploid cells. TH-169 eliminated the PC-3 cells' polyploid population and increased their expression of p21(WAF1) and Hsp70 in a concentration-dependent manner. The antiproliferative effect of TH-169 was irreversible and independent of changes in caspases, actin, tubulin, glyceraldehyde phosphate dehydrogenase or Bcl-x(S/L). This structurally simple naphthoquinone spiroketal represents a small molecule, tubulin-interactive agent with a novel apoptotic pathway and attractive biological function.

Cell-based and biochemical structure-activity analyses of analogs of the microtubule stabilizer dictyostatin.

Compounds that bind to microtubules (MTs) and alter their dynamics are highly sought as a result of the clinical success of paclitaxel and docetaxel. The naturally occurring compound (-)-dictyostatin binds to MTs, causes cell cycle arrest in G(2)/M at nanomolar concentrations, and retains antiproliferative activity in paclitaxel-resistant cell lines, making dictyostatin an attractive candidate for development as an antineoplastic agent. In this study, we examined a series of dictyostatin analogs to probe biological and biochemical structure-activity relationships. We used a high-content multiparameter fluorescence-based cellular assay for MT morphology, chromatin condensation, mitotic arrest, and cellular toxicity to identify regions of dictyostatin that were essential for biological activity. Four analogs (6-epi-dictyostatin, 7-epi-dictyostatin, 16-normethyldictyostatin, and 15Z,16-normethyldictyostatin) retained low nanomolar activity in the cell-based assay and were chosen for analyses with isolated tubulin. All four compounds were potent inducers of MT assembly. Equilibrium binding constant (K(i)) determinations using [(14)C]epothilone B, which has a 3-fold higher affinity for the taxoid binding site than paclitaxel, indicated that 6-epi-dictyostatin and 7-epi-dictyostatin displaced [(14)C]epothilone B with K(i) values of 480 and 930 nM, respectively. 16-Normethyldictyostatin and 15Z,16-normethyldictyostatin had reduced affinity (K(i) values of 4.55 and 4.47 muM, respectively), consistent with previous reports showing that C16-normethyldictyostatin loses potency in paclitaxel-resistant cell lines that have a Phe270-to-Val mutation in the taxoid binding site of beta-tubulin. Finally, we developed a set of quantitative structure-activity relationship equations correlating structures with antiproliferative activity. The equations accurately predicted biological activity and will help in the design of future analogs.

Structure-activity and high-content imaging analyses of novel tubulysins.

The synthesis and biological evaluation of three tubulysin analogs provides the first structure-activity relationship in this family of potent cytotoxic myxobacteria metabolites. Most importantly, the labile N,O-acetal at N(14) is not essential for biological activity. Further, structural simplifications are possible without abolishing biological activities. The N-terminal amino acid can be replaced with N-methylsarcosine, and the configuration at the acetoxy-bearing stereocenter at C(11) is important but not critical for almost all aspects of the biological profile. Our data encourage further development of these compounds as potential therapeutic agents in cancer treatment.

Total synthesis and biological evaluation of C16 analogs of (-)-dictyostatin.

The structure-activity relationship of the crucial C16 region of (-)-dictyostatin was established through total synthesis of analogs followed by detailed biological characterization. A versatile synthetic strategy was used to prepare milligram quantities of 16-normethyldictyostatin, 16-epi-dictyostatin, and the C16-normethyl-C15Z isomer. Along the way, a number of other E/Z isomers and epimers were prepared, and a novel lactone ring contraction to make iso-dictyostatins with 20-membered macrolactones (instead of 22-membered macrolactones) was discovered. The synthesis of 16-normethyl-15,16-dehydrodictyostatin is the first of any dictyostatin by a maximally convergent route in which three main fragments are assembled, coupled in back-to-back steps, and then processed through refunctionalization and macrolactonization. Cell-based and biochemical evaluations showed 16-normethyl-15,16-dehydrodictyostatin and 16-normethyldictyostatin to be the most potent of the new agents, only 2- and 5-fold less active than (-)-dictyostatin itself. This data and that from previously generated dictyostatin analogs are combined to produce a picture of the structure-activity relationships in this series of anticancer agents.

Synthesis and biological evaluation of (-)-dictyostatin and stereoisomers.

Total syntheses of (-)-dictyostatin, 6,16-bis-epi-dictyostatin, 6,14,19-tris-epi-dictyostatin and a number of other isomers and analogs are reported. Three main fragments-top, middle and bottom-were first assembled and then joined by olefination or anionic addition reactions. After appending the two dienes at either end of the molecule, macrolactonization and deprotection completed the syntheses. The work proves both the relative and absolute configurations of (-)-dictyostatin. The compounds were evaluated by cell-based measurements of increased microtubule mass and antiproliferative activity, and in vitro tubulin polymerization assays as well as competitive assays with paclitaxel for its binding site on microtubules. These assays showed dictyostatin to be the most potent of the agents and further showed that the structural alterations caused from 20- to >1000-fold decreases in activity.

Synthesis and biological evaluation of purealin and analogues as cytoplasmic dynein heavy chain inhibitors.

Cytoplasmic dynein plays important roles in membrane transport, mitosis, and other cellular processes. A few small-molecule inhibitors of cytoplasmic dynein have been identified. We report here the first synthesis of purealin, a natural product isolated from the sea sponge Psammaplysilla purea, which is known to inhibit axonemal dynein. Also described are the first syntheses, by modular amide coupling reactions, of the natural product purealidin A (a component of purealin) and a small library of analogues. The library was examined for inhibition of cytoplasmic dynein heavy chain and cell growth. The compounds showed effective antiproliferative activity against a mouse leukemia cell line but selective activities against human carcinoma cell lines. Purealin and some of the analogues inhibited the microtubule-stimulated ATPase activity of recombinant cytoplasmic dynein heavy chain motor domain. The inhibitory effect of purealin was concentration dependent and uncompetitive, supporting the hypothesis that it does not compete with the binding of ATP.

Fluorous mixture synthesis of (-)-dictyostatin and three stereoisomers.

[reaction: see text] A mixture of four stereoisomers whose configurations are encoded by fluorous silyl protecting groups has been prepared and converted over 22 steps to a mixture of protected dictyostatins. Demixing by fluorous HPLC followed by removal of the fluorous protecting groups (detagging) provides dictyostatin and three C6,C7 stereoisomers. Biological evaluation showed that the monoepimers of the natural product retained highly potent activity.

Tubulin assembly, taxoid site binding, and cellular effects of the microtubule-stabilizing agent dictyostatin.

(-)-Dictyostatin is a sponge-derived, 22-member macrolactone natural product shown to cause cells to accumulate in the G2/M phase of the cell cycle, with changes in intracellular microtubules analogous to those observed with paclitaxel treatment. Dictyostatin also induces assembly of purified tubulin more rapidly than does paclitaxel, and nearly as vigorously as does dictyostatin's close structural congener, (+)-discodermolide (Isbrucker et al. (2003), Biochem. Pharmacol. 65, 75-82). We used synthetic (-)-dictyostatin to study its biochemical and cytological activities in greater detail. The antiproliferative activity of dictyostatin did not differ greatly from that of paclitaxel or discodermolide. Like discodermolide, dictyostatin retained antiproliferative activity against human ovarian carcinoma cells resistant to paclitaxel due to beta-tubulin mutations and caused conversion of cellular soluble tubulin pools to microtubules. Detailed comparison of the abilities of dictyostatin and discodermolide to induce tubulin assembly demonstrated that the compounds had similar potencies. Dictyostatin inhibited the binding of radiolabeled discodermolide to microtubules more potently than any other compound examined, and dictyostatin and discodermolide had equivalent activity as inhibitors of the binding of both radiolabeled epothilone B and paclitaxel to microtubules. These results are consistent with the idea that the macrocyclic structure of dictyostatin represents the template for the bioactive conformation of discodermolide.