Maytansinol Derivatives: Side Reactions as a Chance for New Tubulin Binders

Abstract

Maytansinol is a valuable precursor for the preparation of maytansine derivatives (known as maytansinoids). Inspired by the intriguing structure of the macrocycle and the success in targeted cancer therapy of the derivatives, we explored the maytansinol acylation reaction. As a result, we were able to obtain a series of derivatives with novel modifications of the maytansine scaffold. We characterized these molecules by docking studies, by a comprehensive biochemical evaluation, and by determination of their crystal structures in complex with tubulin. The results shed further light on the intriguing chemical behavior of maytansinoids and confirm the relevance of this peculiar scaffold in the scenario of tubulin binders.

Keywords: maytansine binding site; maytansinol; microtubules; tubulin; tubulin binders.

Scheme 1

Structure of maytansine (1 a), maytansinol (1 b), and the generic acylation reaction of maytansinol.

Figure 1

General structures of the new maytansinol derivatives obtained.

Figure 2

Structures of the acyl groups and acylating agents used.

Figure 3

Representative HPLC chromatogram. ZORBAX SB C₈ column (3.5 μm ×4.6×150 mm). Pressure: 85 bar; Flow rate: 1 mL/min. UV: 254 and 210 nm with DAD detection. Mobile phase: H₂O/ACN 1 min isocratic at 50 % ACN, then gradient to 90 % ACN over 10 min. Retention times: 1 b, 3.10 min; 3, 4.08 min; 4 a, 5.20 min; 5 a, 6.90 min; 6 a, 7.20 min; 7 a, 9.40 min.

Figure 4

Inhibition of tubulin assembly activity by selected compounds. All experiments were performed in triplicate. Time courses of assembly of 25 μM tubulin in GAB in the presence of vehicle (DMSO; black lines), 27.5 μM maytansine 1 a (red lines), or maytansinol 1 b (green lines), or A) 2 (yellow), 6 a (cyan), 7 a (blue); or B) 3 (dark yellow), 4 a (dark red); or C) 4 b (pink), 4 c (orange), 5 a (dark blue), 5 b (dark pink), 5 c (dark cyan).

Figure 5

Determination of the binding constant of the ligands. Displacement of Fc‐maytansine assays for the ligands. A) Maytansine 1 a (–•–), maytansinol 1 b (–•–), 2 (–•–), 3 (–•–), 4 a (‐ ‐▪‐ ‐), 4 b (‐ ‐▪‐ ‐). (B) Maytansine 1 a (–•–), 4 c (–•–), 5 a (–•–), 5 b (–•–), 5 c (–•–), 6 a (‐ ‐▪‐ ‐), 7 A (‐ ‐▪‐ ‐). The data are from three independent experiments and represent mean±SEM. The solid lines represent fits to the data (see the Methods in the Supporting information).

Figure 6

Effects of maytansinoids on cells in interphase and mitosis. The effect of these compounds on the microtubule network and mitotic spindle was characterized in A549 tumor cells by fluorescence microscopy. Cells were treated for 24 h with the different compounds analyzed: A)–C’’) control (DMSO 0.5 %), D)–E’’) 1 a 5 nM, F)–G’’) 1 b 100 nM, and H)–I’’) 4 a 5 nM. Cells were immunostained for A)–I) α‐tubulin or A’)–I’) DNA, and A’’)–I’’) the images obtained were merged (tubulin in green and DNA in magenta). A)–A’’) Interphase cell treated with DMSO; notice the regular microtubule network evenly distributed in the cytoplasm. B)–B’’) Control metaphase cell with a normally distributed bipolar mitotic spindle in which all chromosomes are positioned in the metaphase plate. C)–C’’) Control late‐anaphase cell in which sister chromatids are observed segregating to the daughter poles through a bipolar anaphase spindle; note that no anaphases are later observed in treated cells. D)–D’’, F)–F’’, H)–H’’) Interphase cells displaying a range of less‐dense microtubule networks and reduction of the microtubule mass compared with A–A’’ (multinucleated heteroploid cells ‐D’, H’‐ or single‐nucleated cells ‐F’‐ may appear in any of the treatments with the three compounds). E)–E’’, G)–G’’, I)–I’’) Blocked mitotic cells with condensed chromosomes showing less microtubular mass (when compared with B–B’’) organized in star‐ or comet‐shaped pseudopoles (one cell shown in E–E’’, three in G–G’’ and two in I–I’’). All images are shown at the same scale; scale bar: 10 μm.

Figure 7

X‐ray analysis of the T₂R‐TTL‐maytansinoid complexes. A) Overall view of the interaction between the maytansinoids (blue) and tubulin (gray) in relation to the bound nucleotides (purple). The maytansine binding site is located on the β‐tubulin surface in close proximity to the bound GDP molecule. The tubulin molecule is in ribbon representation (gray), and the interacting maytansinoid and nucleotides are in surface and stick representations, respectively. B) Close‐up of the interaction between the maytansinoid 4 a (PDB ID: 5SB9, blue) and tubulin (gray). The interacting residues and ligand are represented as sticks. Oxygen atoms are in red, nitrogens are blue, and the chlorine atom is in green. Hydrogen bonds are displayed as black dashed lines. C) Superposition of the T₂R‐TTL‐maytansine structure (PDB ID: 4TV8, maytansine in gray) and the 4 a‐T₂R‐TTL structure (4 a in blue). The dashed lines indicate the hydrogen‐bond interactions established by the maytansine molecule. 4 a adopts the same binding pose as its parent compound, except for the interaction of the acyl group with Asn101, which is less pronounced and thus not displayed in the 4 a structure, all interactions are conserved. D) The superposition of the T₂R‐TTL‐4 a (4 a in blue) and the T₂R‐TTL‐5 a (5 a in orange) structures shows that the elimination of the C8‐OH group in 5 a has no major effect on the coordination of the ligand. Although the heterocycle moiety is flattened by the double bond in 5 a, the position of the ring is anchored at its position by coordination to Asn102 and Lys105.

Figure 8

Superimposition of the crystallographic binding mode of compound 4 a (blue) and the best conformer predicted by AutoDock Vina (light yellow) when bound to β‐tubulin (gray).