New MKLP-2 inhibitors in the paprotrain series: Design, synthesis and biological evaluations.

Members of the kinesin superfamily are involved in key functions during intracellular transport and cell division. Their involvement in cell division makes certain kinesins potential targets for drug development in cancer chemotherapy. The two most advanced kinesin targets are Eg5 and CENP-E with inhibitors in clinical trials. Other mitotic kinesins are also being investigated for their potential as prospective drug targets. One recently identified novel potential cancer therapeutic target is the Mitotic kinesin-like protein 2 (MKLP-2), a member of the kinesin-6 family, which plays an essential role during cytokinesis. Previous studies have shown that inhibition of MKLP-2 leads to binucleated cells due to failure of cytokinesis. We have previously identified compound 1 (paprotrain) as the first selective inhibitor of MKLP-2. Herein we describe the synthesis and biological evaluation of new analogs of 1. Our structure-activity relationship (SAR) study reveals the key chemical elements in the paprotrain family necessary for MKLP-2 inhibition. We have successfully identified one MKLP-2 inhibitor 9a that is more potent than paprotrain. In addition, in vitro analysis of a panel of kinesins revealed that this compound is selective for MKLP-2 compared to other kinesins tested and also does not have an effect on microtubule dynamics. Upon testing in different cancer cell lines, we find that the more potent paprotrain analog is also more active than paprotrain in 10 different cancer cell lines. Increased selectivity and higher potency is therefore a step forward toward establishing MKLP-2 as a potential cancer drug target.

Synthesis and biological evaluation of C-13' substituted 7'-homo-anhydrovinblastine derivatives.

Recent publications highlighted that vinca derivatives either functionalized on C-12' or enlarged on cycle C' could be more cytotoxic than vinblastine or vinorelbine, both used in anti-cancer therapy. By combining these two results, nine new 7'-homo-anhydrovinblastine derivatives functionalized on C-13' were elaborated. The synthesis of key intermediates, their one-step transformation into final products in mild conditions and their biological activities are presented.

Synthesis of polysubstituted benzofuran derivatives as novel inhibitors of parasitic growth.

A series of polysubstituted benzofuran derivatives was easily and rapidly prepared using a tandem Sonogashira coupling/cyclization reaction. Subsequent acylation afforded a small library of 39 new compounds that were assayed in cellulo on Plasmodium falciparum and Trypanosoma brucei parasites. Some of them exhibited good inhibitory activity on T. brucei proliferation.

Synthesis and biological evaluation of novel heterocyclic derivatives of combretastatin A-4.

A novel series of combretastatin A-4 heterocyclic analogues was prepared by replacement of the B ring with indole, benzofurane or benzothiophene, attached at the C2 position. These compounds were evaluated for their abilities to inhibit tubulin assembly: derivative cis3b, having a benzothiophene, showed an activity similar to those of colchicine or deoxypodophyllotoxine. The antiproliferative and antimitotic properties of cis3b against keratinocyte cancer cell lines were also evaluated and the intracellular organization of microtubules in the cells after treatment with both stereoisomers of 3b was also determined, using confocal microscopy.

Influence of the skeleton on the cytotoxicity of flavonoids.

Analogs of 3'-amino-5-hydroxy-3,6,7,8,4'-pentamethoxy-flavone, a strongly cytotoxic and antimitotic semisynthetic flavone, were synthesized in the aurone, isoflavone and isoflavanone series. Comparison of the biological activity of these new compounds with the reference showed a potent cytotoxicity only in the flavone series. Influence of the hydroxy group (at C-5 in flavones, at C-4 in aurones) on the cytotoxicity, known to be favorable in flavones, was found to be detrimental in aurones. This observation was related to the hydrogen bonding formed with the carbonyl group, strong in the flavones, but of weak intensity in the aurones.

Synthesis of novel 7-oxo and 7-hydroxy trifluoroallocolchicinoids with cytotoxic effect.

The synthesis of 7-oxo and 7-hydroxy trifluoroallocolchicinoids was achieved through the intramolecular cyclization of o-phenyl-ß-phenylalanines. The resulting compounds were evaluated for their cytotoxic activity against KB cells and their inhibitory effect towards the polymerization of tubulin. The results yielded some potent cytotoxic compounds with correlated partial antitubulin effect.

Synthesis of antiproliferative flavones from calycopterin, major flavonoid of Calycopteris floribunda Lamk.

Eighteen new analogues of 5,3'-dihydroxy-3,6,7,8,4'-pentamethoxy-flavone, a potent natural cytotoxic and antimitotic flavone, were synthesized from calycopterin, the major flavonoid of Calycopteris floribunda Lamk., a traditional Asian medicinal plant. One of them, the 3'-amino substituted analogue, displayed almost the same activity as the reference compound. Pharmacomodulation at C-3' on the B-ring, and at C-5,6,7 and 8 on the A-ring allowed to refine structure-activity relationships within the cytotoxic flavones series.

Semisynthesis of natural flavones inhibiting tubulin polymerization, from hesperidin.

Semisynthesis of 5,3'-dihydroxy-3,6,7,8,4'-pentamethoxyflavone (1), a natural flavone that binds with high affinity to tubulin, was performed from hesperidin, the very abundant Citrus flavanone, by a five-step sequence. The last step of the synthesis also gave rise to 5,3'-dihydroxy-3,6,7,4'-tetramethoxyflavone (= casticin or vitexicarpin) (10), 5,3'-dihydroxy-3,7,8,4'-tetramethoxyflavone (= gossypetin 3,7,8,4'-tetramethyl ether) (11), and, unexpectedly, 5,7,3'-trihydroxy-3,6,8,4'-tetramethoxyflavone (12) and 5,3'-dihydroxy-8-dimethylamino-3,6,7,4'-tetramethoxyflavone (= 8-dimethylaminocasticin) (13). Cytotoxicity and antitubulin activity of these five flavones, as well as 5,3'-dihydroxy-3,7,4'-trimethoxyflavone (= ayanin) (14) and intermediate 6,8-dibromo-ayanin (8), were evaluated. Comparison of the responses confirmed and clarified the influence of the A-ring substitution pattern on the biological activity.

Synthesis and tubulin-binding properties of new allocolchicinoids.

Allocolchicinoids with B- and C-ring variations were synthesized using sequential enyne-metathesis/ Diels-Alder reactions (A-->AB-->ABC approach) and evaluated for their inhibitory effect on tubulin assembly in vitro. (-)-Allocolchicine 11 with methyl ester at C10 and (+/-)-cyclopropyl allocolchicinoid 32 exhibit similar activity than (-)-colchicine (1), probably derived from a similar flexibility in the biphenyl system. The presence of methyl ester at C10 led to a little loss in potency in comparison with the series with methyl ester at C9. A complete loss of activity was observed for allocolchicine 9 with methyl ester at C11.

Semisynthesis and antiproliferative evaluation of a series of 3'-aminoflavones.

A series of 3'-aminoflavones 5,6,7,8-tetra- or 5,7-dioxygenated on the A-ring was synthesized from tangeretin or naringin, two natural Citrus flavonoids. These flavones were evaluated for antiproliferative activity, activation of apoptosis, and inhibition of tubulin assembly. The most antiproliferative flavones exhibit a common 5-hydroxy-6,7,8-trimethoxy substitution pattern on the A-ring.

Synthesis and biological evaluation of a series of tangeretin-derived chalcones.

A series of chalcones polyoxygenated on the ring A (with pentamethoxy or 2'-hydroxy-3',4',5',6'-tetramethoxy substitution patterns) was synthesized from tangeretin, a natural Citrus flavonoid. These chalcones were evaluated for their antiproliferative, activation of apoptosis, inhibition of tubulin assembly and antileishmanial activities. Comparison with the reference analogous 3',4',5'-trimethoxylated chalcones showed that such peroxygenated substitution patterns on the ring A were less beneficial to these activities.

Synthesis and biological evaluation of cis-locked vinylogous combretastatin-A4 analogues: derivatives with a cyclopropyl-vinyl or a cyclopropyl-amide bridge.

A series of novel combretastatin A4 analogues, in which the cis-olefinic bridge is replaced by a cyclopropyl-vinyl or a cyclopropyl-amide moiety, were synthesized and evaluated for inhibition of tubulin polymerization and antiproliferative activity. The derivative 9a with a (cis,E)-cyclopropyl-vinyl unit is the most promising compound. As expected, molecular docking of 9a has shown that only one of the cis-cyclopropyl enantiomers is a good ligand for tubulin.

New C5-alkylated indolobenzazepinones acting as inhibitors of tubulin polymerization: cytotoxic and antitumor activities.

A series of 5-alkylindolobenzazepin-7-ones was synthesized by Suzuki coupling between 3-iodoindole-2-carboxylates and the appropriate alpha-alkylbenzylamino o-boronic acids followed by cyclization to the lactam. Derivatives having a linear alkyl chain at C5 were found to be highly cytotoxic to KB cells with IC50 values in the 30-80 nM range. These compounds also inhibited the polymerization of tubulin with IC50's of 1-2 microM. Compound 4f (( S)-5-ethyl) showed comparable antiproliferative activities (IC50's of 30-70 nM) in a variety of cancer cell lines, cell growth being arrested at the G2/M phase. Compound 4f induced apoptosis in a dose-dependent manner in three different cancer cell lines and was shown to affect cell morphology in a manner consistent with its inhibitory action on tubulin polymerization. Using the experimental model of glioma grafted on the chick chorio-allantoic membrane, local treatment with compound 4f markedly reduced tumor progression.

Synthesis and antitumor activity of benzils related to combretastatin A-4.

A series of benzil derivatives related to combretastatin A-4 (CA-4) have been synthesized by oxidation of diarylalkynes promoted by PdI(2) in DMSO. Using this new protocol, 14 benzils were prepared in good to excellent yields and their biological activity has been delineated. Several benzils exhibited excellent antiproliferative activity: for example, 4j and 4k bearing the greatest resemblance to CA-4 and AVE-8062, respectively, were found to inhibit cell growth at the nanomolar level (20-50nM) on four human tumor cell lines. Flow cytometric analysis indicates that these compounds act as antimitotics and arrest the cell cycle in G(2)/M phase. A cell-based assay indicated that compounds 4j and 4k displayed a similar inhibition of tubulin assembly with an IC(50) value similar to CA-4. These results clearly demonstrated that the Z-double bond of CA-4 can be replaced by a 1,2-diketone unit without significant loss of cytotoxicity and inhibition of tubulin assembly potency.

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.

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]

One-pot synthesis of vinca alkaloids-phomopsin hybrids.

Hybrids of vinca alkaloids and phomopsin A have been elaborated with the aim of interfering with the "vinca site" and the "peptide site" of the vinca domain in tubulin. They were synthesized by an efficient one-pot procedure that directly links the octahydrophomopsin lateral chain to the velbenamine moiety of 7'-homo-anhydrovinblastine. In their modeled complexes with tubulin, these hybrids were found to superimpose nicely on the tubulin-bound structures of vinblastine and phomopsin A. This good matching can account for the fact that two of them are very potent inhibitors of microtubules assembly and are cytotoxic against four cancer cell lines.

Synthesis and biological evaluation of a new series of highly functionalized 7'-homo-anhydrovinblastine derivatives.

Sixteen new 7'-homo-anhydrovinblastine derivatives were prepared in one or two steps from vinorelbine by means of an original and regiospecific rearrangement and subsequent diastereoselective reduction. This strategy has allowed fast access to a family of vinca alkaloid derivatives with an enlarged and functionalized ring C'. Their synthesis and biological evaluation are reported. One compound (compound 35) is 1.7 times more active than vinorelbine as a tubulin assembly inhibitor. Moreover, some of these compounds are highly cytotoxic, and two of them are more potent than vinorelbine on HCT116 and K562 cell lines. Molecular modeling studies, carried out with two of the new vinca derivatives, provide useful hints about how a given functionalization introduced at positions 7' and 8' of the C' ring results in improved binding interactions between one of the new derivatives and the interdimer interface when compared to the parent compound vinblastine.