Structure of human glycolate oxidase in complex with the inhibitor 4-carboxy-5-[(4-chlorophenyl)sulfanyl]-1,2,3-thiadiazole.

Glycolate oxidase, a peroxisomal flavoenzyme, generates glyoxylate at the expense of oxygen. When the normal metabolism of glyoxylate is impaired by the mutations that are responsible for the genetic diseases hyperoxaluria types 1 and 2, glyoxylate yields oxalate, which forms insoluble calcium deposits, particularly in the kidneys. Glycolate oxidase could thus be an interesting therapeutic target. The crystal structure of human glycolate oxidase (hGOX) in complex with 4-carboxy-5-[(4-chlorophenyl)sulfanyl]-1,2,3-thiadiazole (CCPST) has been determined at 2.8 A resolution. The inhibitor heteroatoms interact with five active-site residues that have been implicated in catalysis in homologous flavodehydrogenases of L-2-hydroxy acids. In addition, the chlorophenyl substituent is surrounded by nonconserved hydrophobic residues. The present study highlights the role of mobility in ligand binding by glycolate oxidase. In addition, it pinpoints several structural differences between members of the highly conserved family of flavodehydrogenases of L-2-hydroxy acids.

New potent acetylcholinesterase inhibitors in the tetracyclic triterpene series.

A new highly selective inhibitor of acetylcholinesterase (AChE) was discovered by high-throughput screening. Compound 1 was synthesized from a natural product, the N-3-isobutyrylcycloxobuxidine-F 2. A new extraction protocol of this compound is described. The hemisynthesis and optimization of 1 are reported. The analogs of 1 were tested in vitro for the inhibition of both cholinesterases (AChE and BuChE). These compounds selectively inhibited AChE. Extensive molecular docking studies were performed with 2 and AChE employing Discover Biosym software to rationalize the binding interaction. The results suggested that ligand 2 binds simultaneously to both catalytic and peripheral sites of AChE.

Semisynthesis and biological evaluation of a novel D-seco docetaxel analogue.

[reaction: see text] A 4-methyl-5-oxo docetaxel analogue has been prepared starting from 10-deacetylbaccatin III. This new D-seco docetaxel analogue is slightly less potent than docetaxel at microtubule stabilization in vitro and has about 1/1000th the cytotoxicity of docetaxel. The lack of improved activity for this compound compared to other D-modified taxoids confirms that a C-5 oxygen atom is not required for biological activity.

Echinosulfonic acid D: an ESI MSn evaluation of a new cytotoxic alkaloid from the New-Caledonian sponge Psammoclemma sp.

A new bromoindolesulfonic acid derivative, echinosulfonic acid D (1) was isolated from the New-Caledonian sponge Psammoclemma sp. in a minute quantity. The structure of the alkaloid was established by spectroscopic methods and, in particular, by ESI MSn experiments. Echinosulfonic acid D was cytotoxic to KB cells (IC50 2 microg/mL).

Synthesis of C2-C3'N-linked macrocyclic taxoids. Novel docetaxel analogues with high tubulin activity.

Novel C2-C3'N-linked macrocyclic taxoids 4 bearing an aromatic ring at position C2 were synthesized. These compounds, tethered between N3' and the C2-aromatic ring at the ortho, meta, or para position, were constructed by ring-closing metathesis. The para-substituted derivatives were unable to stabilize microtubules, whereas the ortho- and meta-substituted compounds show significant activity in cold-induced microtubule disassembly assay. The meta derivative 4c is the first C2-C3'-linked cyclic analogue to be equipotent to paclitaxel in this assay and to show significant cytotoxicity. Computational studies of the conformational behavior of these compounds indicate that they can adopt several conformations including mainly the "T-shaped" forms. Docking experiments have shown that the "T-shaped" form is preferred for a good interaction of these compounds with the beta-tubulin binding pocket.

Synthesis of novel macrocyclic docetaxel analogues. Influence of their macrocyclic ring size on tubulin activity.

This work describes the synthesis of a series of novel macrocyclic taxoids 3 and 3(H) designed to mimic the docetaxel solid-state ("nonpolar") conformation. These compounds, bearing 18-, 20-, 21-, and 22-membered rings connecting the C-2 OH and C-3' NH moieties, were constructed by ring-closing olefin metathesis of the taxoid-omega,omega'-dienes 4. Biological evaluation of these new taxoids showed that activity is dependent on the ring size, and only the 22-membered ring taxoid 3d exhibits significant tubulin binding. Synthesis of the open-chain analogues 7 and 7(H) and comparison of their biological activities with macrocyclic taxoids show that the carbon tether between C-2 OH and C-3' NH does not hamper tubulin binding. Computational studies of the conformational behavior of the macrocyclic taxoids 3 indicate that the 18-, 20-, and 21-membered-ring 3a-c adopt mainly conformations that are not recognized by tubulin. The most active taxoid 3d appears to adopt a conformation that is between the "nonpolar" and T-shaped forms.

Deletion of the oxetane ring in docetaxel analogues: synthesis and biological evaluation.

Two new docetaxel analogues have been prepared starting from 10-deacetylbaccatin III. Both derivatives lack the oxetane D-ring but possess the 4alpha-acetoxy group, which is important for biological activity. The influence of a more or less constrained C-ring was evaluated by adding, or not adding, a double bond in this ring. Both compounds were found to be equally less active than docetaxel in biological assays. [reaction: see text]

Isolation of cytotoxic chondropsins, macrolide lactams from the New-Caledonian marine sponge Psammoclemma sp. and electrospray ion trap multiple stage MS study of these macrolides.

Psammoclemma sp. (Phoriospongiidae) was selected for study through biological screening carried out in New Caledonia. The crude extract of Psammoclemma sp. was highly active on KB cells and its fractionation led to the isolation of two macrolides chondropsin A 1 and 73-deoxychondropsin A 2. The complex structures were elucidated by a combination of spectroscopic methods (NMR, ESI-MS/MS). These known compounds were evaluated for their cytotoxic activity towards several tumor cell lines and exhibited an IC50 of approximately 10(-10) M. Flow-cytometric analysis revealed that chondropsin A 1 and 73-deoxychondropsin A 2 blocked the entry of HL 60 and KB cells into G2/M phase, leading to cell death by apoptosis.

High resolution crystal structure of rat long chain hydroxy acid oxidase in complex with the inhibitor 4-carboxy-5-[(4-chlorophenyl)sulfanyl]-1, 2, 3-thiadiazole. Implications for inhibitor specificity and drug design.

Long chain hydroxy acid oxidase (LCHAO) is responsible for the formation of methylguanidine, a toxic compound with elevated serum levels in patients with chronic renal failure. Its isozyme glycolate oxidase (GOX), has a role in the formation of oxalate, which can lead to pathological deposits of calcium oxalate, in particular in the disease primary hyperoxaluria. Inhibitors of these two enzymes may have therapeutic value. These enzymes are the only human members of the family of FMN-dependent l-2-hydroxy acid-oxidizing enzymes, with yeast flavocytochrome b(2) (Fcb2) among its well studied members. We screened a chemical library for inhibitors, using in parallel rat LCHAO, human GOX and the Fcb2 flavodehydrogenase domain (FDH). Among the hits was an inhibitor, CCPST, with an IC(50) in the micromolar range for all three enzymes. We report here the crystal structure of a complex between this compound and LCHAO at 1.3 Å resolution. In comparison with a lower resolution structure of this enzyme, binding of the inhibitor induces a conformational change in part of the TIM barrel loop 4, as well as protonation of the active site histidine. The CCPST interactions are compared with those it forms with human GOX and those formed by two other inhibitors with human GOX and spinach GOX. These compounds differ from CCPST in having the sulfur replaced with a nitrogen in the five-membered ring as well as different hydrophobic substituents. The possible reason for the ∼100-fold difference in affinity between these two series of inhibitors is discussed. The present results indicate that specificity is an issue in the quest for therapeutic inhibitors of either LCHAO or GOX, but they may give leads for this quest.

Synthesis and biological evaluation of vinca alkaloids and phomopsin hybrids.

Ten hybrids of vinca alkaloids and phomopsin A have been synthesized by linking the octahydrophomopsin lateral chain to the tertiary amine of the cleavamine moiety of anhydrovinblastine (AVLB) and vinorelbine. These compounds have been elaborated in order to obtain original products that may interfere with both binding sites of vinblastine (VLB) and phomopsin in tubulin. Although NMR and molecular modeling studies have shown that the orientation of the added peptide chains of these hybrids is not the same as those of phomopsin A, most of them are very potent inhibitors of microtubules assembly and they present good cytotoxicity against KB cell line. These interesting biological activities may eventually be explained by the fact that their lateral chain resides in a pocket distinct from that of the phomopsin A peptide, at the interface of tubulins beta and alpha.

Novel C2-C3' N-peptide linked macrocyclic taxoids. Part 2: synthesis and biological activities of docetaxel analogues with a peptide side chain at C2 and their macrocyclic derivatives.

The synthesis of a series of novel docetaxel analogues possessing a peptide side chain at the C2 position as well as peptide macrocyclic taxoids is described. These compounds were designed to mimic a region of the alpha-tubulin loop equivalent to the paclitaxel binding pocket of beta-tubulin. Fifteen new peptide taxoids were obtained and evaluated as inhibitors of microtubule disassembly as well as cell proliferation. The relationships between these new taxoids and the tau protein motif interacting with microtubules are discussed.

Synthesis and biological evaluation of B-ring analogues of (-)-rhazinilam.

Three macrocyclic analogues of rhazinilam 1 having a 11- or 12-membered B-ring with an endocyclic carbamate group or an amino-acid residue were synthesized from the natural product. These analogues 3 and 4 displayed a very low activity on tubulin. Thirty N-1 and C-16 substituted analogues of rhazinilam were also synthesized regioselectively from rhazinilam. Stereochemical analyses showed that N-1 and C-16alpha analogues have the same conformation as rhazinilam, whereas C-16beta analogues adopt a different conformation for rings B and D. All N-1 and C-16 analogues were less active than rhazinilam on tubulin, though analogues 5a, 6aalpha, 6balpha, and 6f having the less bulky substituents retained close affinities. A few analogues either active (like 6f) or inactive (like 5o) on tubulin showed significant inhibition of the growth of KB cancer cells.

Synthesis and biological evaluation of (3,4,5-trimethoxyphenyl)indol-3-ylmethane derivatives as potential antivascular agents.

Combretastatin A-4 (CSA-4), a stilbene derivative, is a potent vascular disrupting agent (VDA) with the structural requirement of a cis-configuration to maintain a molecular geometry and a correct orientation of both phenyl groups. A series of indolic analogues of CSA-4 was synthesized by means of an efficient strategy. Six compounds (20b, 25b-27b, 32b, and 35b) were identified as potent inhibitors of tubulin polymerization and also displayed cytotoxic activities on B16 melanoma cells at a nanomolar level. Both activities were well correlated with the ability to induce morphological changes of EA.hy 926 endothelial cells. In conclusion, the cis-stilbene skeleton of CSA-4 could conveniently be replaced by the 3-aroylindolic moiety, thus avoiding any isomerization leading to inactive trans compounds.

NMR study of quinolizidine alkaloids: relative configurations, conformations.

Extracts of fruits and leaves of Connarus paniculatus afford six quinolizidine alkaloids which were identified as piptanthine, 18-epipiptanthine, ormosanine, homoormosanine, podopetaline (monohydrochloride) and homopodopetaline on the basis of high-field NMR studies. 1D and 2D NMR experiments provide complete assignments of the (1)H and (13)C spectra. In conjunction with detection of nuclear Overhauser effects (NOESY), these results allow detailed structure characterization including determination of relative configurations for the chiral sites and conformational analysis. Exchange phenomena involving nitrogen inversion were detected.

Novel C2-C3' N-peptide linked macrocyclic taxoids. Part 1: Synthesis and biological activities of docetaxel analogues with a peptide side chain at C3'.

A series of novel docetaxel analogues possessing a peptide side chain at the C3'-N position was synthesized. These compounds were designed to mimic a region of the alpha-tubulin loop that is equivalent to the paclitaxel binding pocket in beta-tubulin. Eight new peptidic taxoids were obtained and evaluated as inhibitors of microtubule disassembly, as well as for their cytotoxicity.

New antitubulin derivatives in the combretastatin A4 series: synthesis and biological evaluation.

Two series of combretastatin A4 derivatives (acrylamide=carboxamide and carbamate) were synthesized in order to improve the water solubility and stabilize the cis-configuration of the double bond. Their cytotoxic effects were evaluated against MCF-7, KB-3-1 and IGROV human cancer cell lines, as well as their inhibitory activity on tubulin polymerization. Results were compared to those of carboxamide 1, chosen as reference. Potent inhibitions were observed on both tests in the carboxamide series, particularly for compound 4d bearing a fluorine group in replacement of the 3-hydroxyl of CA4. In contrast, most of the carbamates were either inactive or displayed only moderate cytotoxicities. Interestingly, a submicromolar IC(50) was measured on MCF-7 cells for 6g, although this compound was totally devoid of antitubulin activity.

Two novel series of allocolchicinoids with modified seven membered B-rings: design, synthesis, inhibition of tubulin assembly and cytotoxicity.

Two new attractive series of allocolchicinoids were designed as inhibitors of tubulin assembly using the potent ketone 4 and the tetracyclic, pyrazole annulated NCME variant 7 (NCME = N-acetyl colchinol-O-methylether (2)) as lead structures. The first group of inhibitors of type 6 with novel oxepine and azepine B-ring structures belongs to the NCME-series and was synthesized via a multistep total synthesis starting from simple and cheap 3-methoxybenzaldehyde (12) and 3,4,5-trimethoxybenzaldehyde (13). Biaryl-coupling of the starting materials 12 and 13 was accomplished via Ziegler-Ullmann-reaction to furnish the biphenyl 11 equipped with two carbaldehyde functions. The subsequent Cannizzaro reaction of this dicarbaldehyde 11 proceeded with high regioselectivity to yield almost exclusively the key compound, the hydroxymethyl carboxylic acid 9. Ring closure to the o,o'-bridged biphenyls was accomplished by two routes: on the one hand, treatment of 9 with aqueous hydrochloric acid yielded the lactone 15. On the other hand, a four step sequence starting from the isomeric mixture 9/10 furnished the constitutionally isomeric lactams 23 and 24; these could be converted to the corresponding thiolactams 25 and 26 and to the tetrazole annulated NCME-type derivatives 27 and 28. The second series of bioactive compounds are congeners of allocolchicine (3). The well known desacetyl allocolchicine (29) was easily oxidized to the oxime 30, which was further transformed to the corresponding ketone 31. This served as key precursor for the syntheses of various tetracyclic allocolchicine modifications 33-36 annulated with a pyrazole, isoxazole, pyrimidine or 2-aminopyrimidine heterocycle, respectively. Unexpectedly, all the NCME-variants with a substituent in position 7 like in NCME (2) inhibited the tubulin assembly only moderately. In contrast, the new series of allocolchicine modifications proved to be highly potent antimicrotubule agents. Inhibition of tubulin assembly occurred at lower concentrations compared to those measured for the reference colchicine (1). Surprisingly, these promising results could not be confirmed in the cytotoxicity tests against the human MCF-7 breast cancer cell line, where an unexpected loss of effectiveness compared to the corresponding NCME-derivatives was observed.

Application of the palladium-catalyzed borylation/Suzuki coupling (BSC) reaction to the synthesis of biologically active biaryl lactams.

The palladium-catalyzed, two-step, one-pot borylation/Suzuki coupling (BSC) reaction was developed to synthesize sterically hindered 2,2'-disubstituted biphenyl and phenyl-indole compounds in a short, simple, and efficient manner from two easily accessible aryl halides. High yields can be obtained by choosing properly both components according to their rough electronic properties. The illustration of the utility of this method was provided by the solution and solid-phase synthesis of seven- or eight-membered biphenyl lactams 5a-e, as well as paullone 3a. These compounds exhibit moderate albeit significant cytotoxicities and may serve as structural models for future medicinal chemistry developments.

Synthesis and biological evaluation of A-ring biaryl-carbamate analogues of rhazinilam.

An improvement of the synthesis of biphenyl-carbamate 2a, the most active analogue of rhazinilam 1 so far, was performed using the Pd-catalyzed borylation/Suzuki coupling (BSC) method developed in our laboratories. The preparation of A-ring analogues of 2a bearing electron-withdrawing or donating groups is reported according to this new synthetic scheme. The antitubulin properties as well as the cytotoxicity of these compounds toward human cancer cell lines were evaluated in comparison with rhazinilam and 2a.