G. J. Koomen

Probing FtsZ and Tubulin with C8-Substituted GTP Analogs Reveals Differences in Their Nucleotide Binding Sites

The cytoskeletal proteins, FtsZ and tubulin, play a pivotal role in prokaryotic cell division and eukaryotic chromosome segregation, respectively. Selective inhibitors of the GTP-dependent polymerization of FtsZ could constitute a new class of antibiotics, while several inhibitors of tubulin are widely used in antiproliferative therapy. In this work, we set out to identify selective inhibitors of FtsZ based on the structure of its natural ligand, GTP. We found that GTP analogs with small hydrophobic substituents at C8 of the nucleobase efficiently inhibit FtsZ polymerization, whereas they have an opposite effect on the polymerization of tubulin. The inhibitory activity of the GTP analogs on FtsZ polymerization allowed us to crystallize FtsZ in complex with C8-morpholino-GTP, revealing the binding mode of a GTP derivative containing a nonmodified triphosphate chain.

ABCG2 (breast cancer resistance protein/mitoxantrone resistance-associated protein) ATPase assay: a useful tool to detect drug-transporter interactions.

The ATPase assay using membrane preparations from recombinant baculovirus-infected Spodoptera frugiperda ovarian (Sf9) cells is widely used to detect the interaction of compounds with different ATP-binding cassette transporters. However, Sf9 membrane preparations containing the wild-type ABCG2 transporter show an elevated baseline vanadate-sensitive ATPase activity, which cannot be further stimulated by substrates of ABCG2. Therefore, this assay system cannot be used for the detection of ABCG2 substrates. To overcome this difficulty we 1) purified membranes from a selected human cell line expressing wild-type ABCG2, and 2) inhibited the baseline ATPase activity with different inhibitors. In our modified assay, ABCG2 substrates were able to stimulate the baseline ATPase activity of ABCG2 expressed in membranes of human cells. Furthermore, using the specific ABCG2 inhibitors Ko143 or Ko134 allowed us to suppress the baseline vanadate-sensitive ATPase activity. Substrates of ABCG2 could stimulate this suppressed baseline ATPase, resulting in a better signal-to-background ratio and a robust assay to detect substrates of the ABCG2 transporter. The ATPase assay and the direct vesicular transport measurements for estrone-3-sulfate were in good accordance.

Inter- and intramolecular addition of ester anions to nicotinium salts: A facile approach to nauclefine and ellipticine derivatives

Abstract Reaction of ester anions ( 3a-c ) with 1-(2 -indolyl-ethyl)nicotinium chloride ( 2a ) and 1-(1-methyl-2-indolylethyl)-nicotinium chloride ( 2b ) leads to 2,7-naphthyridine-1,3-diones 5a-c . Acid catalyzed cyclization of the latter yield pentacyclic napthyridocarboline derivatives 1a-c . In an analogous reaction sequence, addition of the anion of 2-carboethoxy-1,3-dithiacyclopentane ( 11 ) to 1-benzyl-3-[N-2(indolylethyl)]-carbamoylpyridinium bromide results in the corresponding naphthyridindione derivative 13 which, via subsequent desulfurization (Raney-Ni) and cyclization gives the alkaloid nauclefine ( 10 ). An approach to the pyridocarbazole system, involving the intramolecular addition of ester aniion of 1-benzyl-3-[1′ -methyl-2′ -(1″-ethoxycarbonylethyl)-3 -indolyl] carbonylpyridinium bromide to the 4-position of the pyridinium moiety of the molecule, has been developed. The reaction has been employed for the synthesis of olivacine and diverse ellipticine derivatives.

Unconventional nucleotide analogues: Reaction of carbenes with uracil and uridine derivatives

Abstract Carbenes 2a-d (:CCl 2 , :CBr 2 , :CFC1, :CHCOOC 2 H 5 ) add to the 5,6-double bond of uracil derivatives la-e to give adducts in modest to good yields The unsymmetrically substituted carbenes :CFC1 2c and CHCOOC 2 H 5 , 2d lead, as expected, to the formation of mixtures of endo and exo -isomers 3c-n which were separated and identified via their 1 H NMR spectra. Reaction of 2d with pyrimidine derivatives 5a,b resulted in products which can be rationalized in terms of addition at either the 5,6 or the 3,4 double bond. Addition of carbenes 2a-c to uridine derivatives gave diastereomeric adducts 12-16 which were isolated and identified. The two diastereomeric series, referred to as A and B, have been correlated and assigned the configurations 5 S , 6 S and 5 R , 6 R , respectively, on the basis of X-ray structure analysis of 12B The adducts of 2a,c and the uridine derivatives 11a,b have been deprotected to give 7,7-dihalocyclothymidines.

Fluorescent nucleoside analogues : Synthesis of fluorescent pyrido[2,1-i]purines, and their corresponding ribosides

Abstract Fluorescent pyrido[2,1-i]purines can in principle be obtained via Michael-addition of a suitable anion of a purine derivative to an acetylenic ester, followed by based-catalyzed cyclization, as depicted in Scheme II. 6-Substituted purine-derivatives are obtained via nucleophilic substitution of 6-chloro- and 6-methylsulfonylpurine ( 8a and 8b ). In the presence of methyl propiolate and sodium methoxide, before cyclization, two consecutive Michael-additions take place, leading to 13 and 14 . With substituted acetylenic esters, cyclization occurs after one Michael-addition. Michael-additions with ethylenic esters did not lead to expected cyclization products, except in cases where oxidation took place. -For the conversion of the pyrido[2,1-i]purines into the corresponding ribosides protection against nucleophilic attack was necessary.

Unconventional nucleotide analogues—XVII1: Ring-transformations of uracil dihalocarbene adducts

Abstract Halocarbene adducts of 1,3-disubstituted uracil (3a, b, d) undergo ring-enlargement to yield 1,3-diazepine derivatives (4a–d). The ring-opening of the cyclopropane system is controlled by the stereochemical configuration of the halogen atom, which can be eliminated as a halide ion. Reduction of the adducts with n-Bu3SnH leads to a variety of 1,3-diazepines. Details of the mechanism of formation of the diazepines and their further transformations are discussed.

Synthesis of polynuclear heterocycles via the reaction of α-ester carbanions with quaternized nicotinamide salts. A facile stereoselective synthesis of sesbanine

Abstract α-Ester carbanions add to N-benzylnicotinamide salts to give C-4 and C-6 substituted primary products. The C-4 substituted dihydronicotinamides undergo a further reaction involving nucleophilic attack of the amide nitrogen on the carbonyl group of the ester. The resulting cyclization products can be oxidized and debenzylated to 1,2-dioxo-1,2,3,4-tetrahydro-2,7-naphthyridine derivatives. The sequence of reactions has been utilized in the synthesis of several heterocycles and in the stereoselective synthesis of the alkaloid sesbanine.

Unconventional nucleotide analogues—XVIII1: Ring expansion of uridine halocarbene adducts. Synthesis of diazepine nucleosides

Abstract The diastereomeric adducts of dichlorocarbene and dibromocarbene with (protected) uridine react with alcohols to give diazepine nucleosides ( 4a – c ). The endo -chloro- exo -fluorocarbene adducts ( 1d and 2d ) also react analogously to yield diazepine nucleoside 4d . On the other hand, the corresponding exo -chloro- endo -fluoro isomers ( 1e and 2e ) are totally inert under the same reaction conditions. The adducts 1b and 2b yield, besides the ring-expanded product ( 4b ), uridine-5-aldehyde ( 6a ) in varying amounts which depend upon the conformation of the diastereomer. These results are explained on the basis of a possible role of the ring-oxygen of the ribose moiety.

Palladium-catalyzed amination of 6-chloropurine. Synthesis of N-6-substituted adenosine analogues.

Abstract Room-temperature treatment of persilylated 6-chloro-9-β-D-ribofuranosyl-purine with a variety of aliphatic and aromatic amines, in the presence of Pd2(dba)3, BINAP and base, leads to N6-substituted adenosine analogues in fair to good yields. Coupling of chloropurine with a chiral aziridinyl diester is applied in the synthesis of a potential adenylosuccinate lyase inhibitor.

New (1-deaza)purine derivatives via efficient c-2 nitration of the (1-deaza)purine ring

Nitration of substituted (1‐deaza)purines using a mixture of tetrabutylammonium nitrate (TBAN) and trifluoracetic acid anhydride (TFAA) was applied to prepare nitrosubstituted (1‐deaza)purines at low temperature. The nitro group influences the system twofold: 1) it activates other substituents towards nucleophilic aromatic substitution and 2) it can be substituted itself leading to a variety of di‐substituted (1‐deaza)purines, also via solid phase syntheses. Several of the molecules obtained were studied for their antiprotozoal activity and for interactions with the different human adenosine receptors.