Total synthesis of cryptophycin-24 (Arenastatin A) amenable to structural modifications in the C16 side chain.

Two efficient protocols for the synthesis of tert-butyl (5S,6R,2E, 7E)-5-[(tert-butyldimethylsilyl)oxy]-6-methyl-8-phenyl-2, 7-octadienoate, a major component of the cryptophycins, are reported. The first utilized the Noyori reduction and Frater alkylation of methyl 5-benzyloxy-3-oxopentanoate to set two stereogenic centers, which became the C16 hydroxyl and C1' methyl of the cryptophycins. The second approach started from 3-p-methoxybenzyloxypropanal and a crotyl borane reagent derived from (-)-alpha-pinene to set both stereocenters in a single step and provided the dephenyl analogue, tert-butyl (5S,6R,2E)-5-[(tert-butyldimethylsilyl)oxy]-6-methyl-2, 7-octadienoate, in five steps. This compound was readily converted to the 8-phenyl compound via Heck coupling. The silanyloxy esters were efficiently deprotected and coupled to the C2-C10 amino acid fragment to provide desepoxyarenastatin A and its dephenyl analogue. The terminal olefin of the latter was further elaborated via Heck coupling. Epoxidation provided cryptophycin-24 (arenastatin A).

The oxetane conformational lock of paclitaxel: structural analysis of D-secopaclitaxel.

Analysis of the 1H NMR data of paclitaxel in comparison with its oxetane ring-opened analogue D-secopaclitaxel suggests that the oxetane moiety (D-ring) of paclitaxel serves as a conformational lock for the diterpene moiety and the C13 side chain.

Conformationally restricted paclitaxel analogues: macrocyclic mimics of the "hydrophobic collapse" conformation.

Conformationally restricted macrocyclic analogues of paclitaxel were prepared, by connecting the 3'-phenyl group and the 2-benzoate moiety with two-atom tethers to mimic the "hydrophobic collapse" paclitaxel conformation. The analogues did not show activity in a tubulin assembly assay.

Potent inhibition of cytochrome P-450 2D6-mediated dextromethorphan O-demethylation by terbinafine.

Cytochrome P-450 (CYP) 2D6 is responsible for the biotransformation of over 35 pharmacologic agents. In the process of studying CYP2D6 we identified phenotype-genotype discordance in two individuals receiving terbinafine. This prompted evaluation of the potential for terbinafine to inhibit CYP2D6 in vitro. Human hepatic microsomes and heterologously expressed CYP2D6 were incubated with terbinafine or quinidine and the formation of dextrorphan from dextromethorphan was determined by HPLC. Additionally, preliminary conformational analyses were conducted to determine the fit of terbinafine into a previously described pharmacophore model for CYP2D6 inhibitors. The apparent Km and Vmax of dextrorphan formation from four human hepatic microsome samples ranged from 5.8 to 6.8 microM and from 172 to 300 pmol/min/mg protein, respectively. Values of Km and Vmax in the heterologously expressed CYP2D6 system averaged 6.5 +/- 2.1 microM and 1342 +/- 147 pmol/min/mg protein, respectively. Terbinafine inhibited dextromethorphan O-demethylation with an apparent Ki ranging from 28 to 44 nM in human hepatic microsomes and averaging 22.4 +/- 0.6 nM for the heterologously expressed enzymes. Results of quinidine in these systems produced values for Ki ranging from 18 to 43 nM. Such strong inhibition of CYP2D6 by terbinafine would not have been predicted by the previously proposed pharmacophore model of CYP2D6 inhibitors based on molecular structure. Terbinafine is a potent inhibitor of CYP2D6 with apparent Ki values well below plasma and tissue concentrations typically achieved during a therapeutic course. This agent needs to be evaluated in vivo to determine the impact of CYP2D6 inhibition by terbinafine on the metabolism of concomitantly administered CYP2D6 substrates.

Probing the environment of tubulin-bound paclitaxel using fluorescent paclitaxel analogues.

To determine the environment of different positions in the paclitaxel molecule when bound to tubulin, we have synthesized six fluorescent analogues in which a (dimethylamino)benzoyl group has been introduced into the 7- and 10-positions, and the benzoyl groups at the 2- and N- as well as the 3'-phenyl ring have been modified with dimethylamino functions. In a tubulin assembly assay, the N-m- and N-p-(dimethylamino)benzoyl derivatives had activities comparable to the activity of paclitaxel. The 2-, 3'-, and 10-analogues had slightly reduced activity, and the 7-derivative was about 5% as active as paclitaxel. On the basis of the results of studies of the effect of solvents on the fluorescence emission spectra, it is proposed that the unbound analogues form hydrogen bonds with protic solvents. But the 7- and 10-substituted analogues appear to be more affected by protic solvents than the other analogues. Previously, we studied the binding of the N-meta derivative to tubulin and microtubules [Sengupta, S., et al. (1995) Biochemistry 34, 11889-11894]. In this study, we extended the studies to include the 2-, 7-, and 10-derivatives. Similar to the N-substituted analogue, binding of the 2-derivative to tubulin was accompanied by a large blue shift, whereas a very small shift occurred when the 7- and 10-substituted derivatives bound. The 2- and N-substituted analogues bind to microtubules with an increase in fluorescence intensity over that which was observed with tubulin, whereas binding of the 7- and 10-substituted analogues was accompanied by a large quenching in fluorescence. This quenching may be due to the presence of charged residues in the protein near the 7- and 10-(dimethylamino)benzoyl groups or to pi stacking of the groups with an aromatic side chain. The presence of paclitaxel with microtubules prevented the fluorescence increase of the 2- and N-derivatives and quenching of the 7- and 10-derivatives. The difference in behavior of the fluorescent analogues upon binding to polymerized tubulin, coupled with the solvent studies on the free drugs, suggests that the 2- and N-benzoyl groups of paclitaxel bind in a hydrophobic pocket of tubulin but could participate in hydrogen bonding, and the 7- and 10-positions are in a more hydrophilic environment.

Preparation of phenolic paclitaxel metabolites.

The synthesis and biological evaluation of the two known phenolic metabolites of paclitaxel are described. The C3'-phenolic metabolite 2 of paclitaxel was prepared from 7-(triethylsilyl)-baccatin III (8) and enantioenriched N-benzoyl-2-azetidinone 7. The C2-phenolic metabolite 3 was synthesized from paclitaxel (1a) via selective C2 debenzoylation and reacylation.

Interaction of a fluorescent paclitaxel analogue with tubulin.

To study the mechanism of binding of the antitumor agent paclitaxel to microtubules and tubulin, we have synthesized a fluorescent analogue of the drug. A dimethylamino group was introduced onto the 3'-N-benzoyl group of paclitaxel. This compound was synthesized from N-debenzoylpaclitaxel and 3-(dimethylamino)benzoyl chloride in 67% yield. N-Debenzoyl-N-[3-(dimethylamino)benzoyl]-paclitaxel has activity similar to paclitaxel in inducing microtubule assembly and binds to tubulin at the paclitaxel-binding site. Under assembly conditions, binding of this paclitaxel analogue to tubulin occurs in a time-dependent manner and is accompanied by a large increase in fluorescence intensity, as well as a large blue shift in the emission maximum. In addition, evidence is presented to show that this compound also binds to tubulin in the dimeric state, but the binding affinity is much lower (Kd = 49 +/- 8 microM at 25 degrees C) than that reported for polymeric tubulin. The fluorescent paclitaxel analogue, with a high quantum yield, will be a useful tool in studying the mechanism of paclitaxel binding to tubulin and the environment of the paclitaxel-binding site on tubulin.

The effect of the aromatic rings of taxol on biological activity and solution conformation: synthesis and evaluation of saturated taxol and taxotere analogues.

The synthesis and biological evaluation of novel cyclohexyl analogues of taxol and taxotere are detailed. 2-(Cyclohexylcarbonyl)-2-debenzoylbaccatin III (6) was prepared from baccatin III by hydrogenation. Subsequent coupling of 6 with N-t-BOC-3-[(tert-butyldimethylsilyl)oxy]-4-phenyl-2-azetidinone (7), followed by removal of the protecting groups, afforded 2-(cyclohexylcarbonyl)-2-debenzoyltaxotere (9). In a similar synthetic sequence, 3'-cyclohexyl-3'-dephenyltaxol (14) was prepared from N-benzoyl-3-[(tert-butyldimethylsilyl)oxy]-4-cyclohexyl-2-azetidinone (12) and (triethylsilyl)baccatin III. The taxol analogue 15, in which all three taxol phenyl groups are substituted by a cyclohexyl moiety, was synthesized in one step from taxol via hydrogenation. All three analogues (9, 14, and 15) exhibited strong activity in the microtubule assembly assay and cytotoxicity comparable to taxol against B16 melanoma cells. It was also shown that 9, like taxol and taxotere, has an extended side chain in chloroform, but in DMSO/water mixtures preferentially adopts a different conformation in which the 2-(cyclohexylcarbonyl), 3'-phenyl, and 4-acetyl groups cluster. However, this behavior does not appear to occur for 3'-cyclohexyl analogues 14 and 15, in which the side chain conformation remains extended independent of solvent. These results suggest the aromaticity of the 3'-phenyl ring significantly stabilizes the clustered conformation.