Synthesis of 6-amino-6-deoxyhyaluronan as an intermediate for conjugation with carboxylate-containing compounds: application to hyaluronan-camptothecin conjugates.

A novel methodology for making drug conjugates using hyaluronan as a carrier was developed. This strategy involves a completely regioselective two-step synthesis of 6-amino-6-deoxyhyaluronan, which is then easily functionalized with drugs through a suitable linker. The case of hyaluronan-camptothecin conjugates is described, making use of a simple succinate linker. The antitumor activity of new hyaluronan derivatives prepared is at present under evaluation.

Synthesis of 6-O-methotrexylhyaluronan as a drug delivery system.

Selective halogenation of hyaluronan and partial halogen substitution by methotrexate led to 6-chloro-6-deoxy-6-O-methotrexylhyaluronan, a potential antitumor drug. The remaining halogen could be further substituted by a second organic carboxylate, leading to mixed esters. 6-O-Acetyl-6-O-methotrexylhyaluronan and 6-O-butyryl-6-O-methotrexylhyaluronan were thus synthesized and characterized by NMR spectroscopy.

Small-molecule inhibitors of the MDM2-p53 protein-protein interaction based on an isoindolinone scaffold.

From a set of weakly potent lead compounds, using in silico screening and small library synthesis, a series of 2-alkyl-3-aryl-3-alkoxyisoindolinones has been identified as inhibitors of the MDM2-p53 interaction. Two of the most potent compounds, 2-benzyl-3-(4-chlorophenyl)-3-(3-hydroxypropoxy)-2,3-dihydroisoindol-1-one (76; IC(50) = 15.9 +/- 0.8 microM) and 3-(4-chlorophenyl)-3-(4-hydroxy-3,5-dimethoxybenzyloxy)-2-propyl-2,3-dihydroisoindol-1-one (79; IC(50) = 5.3 +/- 0.9 microM), induced p53-dependent gene transcription, in a dose-dependent manner, in the MDM2 amplified, SJSA human sarcoma cell line.

Isoindolinone-based inhibitors of the MDM2-p53 protein-protein interaction.

A series of 2-N-alkyl-3-aryl-3-alkoxyisoindolinones has been synthesised and evaluated as inhibitors of the MDM2-p53 interaction. The most potent compound, 3-(4-chlorophenyl)-3-(4-hydroxy-3,5-dimethoxybenzyloxy)-2-propyl-2,3-dihydroisoindol-1-one (NU8231), exhibited an IC50 of 5.3 +/- 0.9 microM in an ELISA assay, and induced p53-dependent gene transcription in a dose-dependent manner, in the SJSA human sarcoma cell line.

Synthesis of a Val-Pro diaminodiol dipeptide isostere by epoxyamine cyclization.

[reaction: see text] The stereoselective synthesis of a novel proline-containing dipeptide isostere is described. Starting from l-valine, three new contiguous stereocenters are generated by asymmetric induction and epoxide chemistry, while the pyrrolidine ring of proline is introduced in the final step via intramolecular ring opening of the amino acid derived epoxyamine. Proline-containing peptidomimetics are potentially attractive as selective inhibitors of proline-specific enzymes, such as PPIases and retroviral proteases, and as analogues of bioactive peptides.

Small hydroxyethylene-based peptidomimetics inhibiting both HIV-1 and C. albicans aspartic proteases.

We have extended a highly flexible method for rapidly assembling aspartic protease inhibitors to produce symmetric and asymmetric monohydroxyethylene peptidomimetics. This method is based on the prior synthesis of the central non-cleavable peptide-bond isostere [NH(2)-P(1)psiP1'-NH(2); psi=hydroxyethylene isostere, HNCH(Bz)CHOHCH(2)CH(Bz)NH], with the possibility of accurately controlling its stereochemistry (S,S,S or S,R,S), and subsequently adding appropriate flanking units, chosen from commercially available amino acids, aromatic carboxylic acids, or phenoxyacetic acid (Poa) derivatives. The method was used to make asymmetric inhibitors of general formula Kyn-Xaa-PhepsiPhe-dmPoa, (Kyn=kynurenic acid, Xaa=Val, Thr or D-thienylglycine, M(r)=716-754) and symmetric inhibitors of formula xPoa-PhepsiPhe-xPoa (xPoa=Poa or dimethyl-, hydroxy-, formyl- or acetyl-Poa, M(r)=553-609), with logP(o/w) values ranging from 4.1 to 7.6. Inhibition of HIV-PR did not depend on the stereochemistry of the hydroxyl group, while it depended markedly on the substituents present on the Poa residues, with dmPoa being preferred over Poa or its more hydrophilic derivatives. Conversely, inhibition of Candida albicans Sap2 was higher for the S,S,S epimers, and Poa or its hydrophilic derivatives were preferred over dmPoa.

A catalytic antibody programmed for torsional activation of amide bond hydrolysis.

Amidase antibody 312d6, obtained against the sulfonamide hapten 4 a that mimics the transition state for hydrolysis of a distorted amide, accelerates the hydrolysis of the corresponding amides 1 a-3 a by a factor of 10(3) at pH 8. The mechanisms of both the uncatalyzed and antibody-catalyzed reactions were studied. Between pH 8 and 12 the uncatalyzed hydrolysis of N-toluoylindoles 1 a and 3 a shows a simple first-order dependence on [OH(-)], while hydrolysis of 3 a is zeroth-order in [OH(-)] below pH 8. The pH profile for hydrolysis of the corresponding tryptophan amide 2 a is more complex due to the dissociation of the zwitterion into an anion with pK(a) 9.74; hydrolysis of the zwitterionic and the anionic form of 2 a both show simple first-order dependence on [OH(-)]. Absence of (18)O exchange between H(2) (18)O/(18)OH(-) and the substrate, a normal SKIE for both 1 a (k(H)/k(D)=1.12) and 3 a (k(H)/k(D)=1.24) and the value of the Hammett constant rho for hydrolysis of p-substituted amides 3 a-e are consistent with an ester-like mechanism in which formation of the tetrahedral intermediate is rate-determining and the amine departs as anion. The 312d6-catalyzed hydrolysis of 3 a was studied between pH 7.5 and 9, and its independence of pH in this range indicates that water is the reacting nucleophile. Hydrolysis of 3 a is only partially inhibited by the sulfonamide hapten, and this indicates that non-specific catalysis by the protein accompanies the specific process. Only the nonspecific process is observed in the hydrolysis of amides 3 with para substituents other than methyl. Binding studies on the corresponding series of p-substituted sulfonamides 5 a-e confirm the high specificity of antibody 312d6 for p-methyl substituted substrates.

Epoxyalcohol route to hydroxyethylene dipeptide isosteres. Stereodivergent synthesis of the diamino alcohol core of ritonavir and its C-2 epimer.

A stereoselective synthesis of hydroxyethylene dipeptide isosteres based on the 1,4-diamino-2-hydroxybutane structure is described. Horner-Emmons olefination of phosphonates derived from alpha-amino acids, stereoselective reduction of the resulting enones to allylic alcohols, and syn epoxidation of the latter lead to enantiomerically pure 1-amino-2-hydroxy-3,4-epoxybutanes, key intermediates in the synthesis. Reductive cleavage of the epoxy alcohols with Red-Al proceeds in a highly regioselective way, giving 1-amino-2,4-dihydroxybutanes, from which diamino alcohol hydroxyethylene isosteres are obtained by selective protection of the secondary 2-hydroxy group, via cyclization to 1,3-oxazolidinone, and further elaboration of the 4-hydroxy. Both C-2 epimers of 1,4-diamino-2-hydroxybutanes are accessible by appropriate choice of the conditions for cyclization. The approach is demonstrated by the synthesis of a series of six hydroxyethylene dipeptide isosteres, including the diamino alcohol core of potent HIV-protease inhibitor ritonavir 18 and its C-2 epimer 11a.