The benzimidazole system was substituted with a hydroxyethyl group to attach the targeting moiety. The synthesis of the western part of the new epothilone analogues was based on an aldol reaction between ketoester A-5 and α-chiral aldehyde A-6. The key step in the synthesis of the Western building block for these analogs was again an aldol reaction, this time between Schinzer ketone A-18 and α-chiral aldehyde A-17 (Scheme A-2).
The initial steps in the synthesis of the eastern fragment up to the Brown allylation to install the C15 stereocenter were identical to the procedure described for the above trans-cyclopropyl analogs.
Introduction
Microtubule-Stabilizing Agents as Anticancer Drugs
- Epothilones
100 % Control: GTP MT at 37 °C or in the absence of calcium; the concentrations of the respective compounds are indicated in italics). The data suggested that the tubulin binding site could fit larger groups in the northeastern part of the epothilone structure. The data suggest that the stiffening of the side chain increases the cytotoxic activity of the corresponding compounds.
Sagopilone (see chapter) has been evaluated in a large number of clinical trials [67, 80], but the development of the drug has been interrupted.
Tumor Targeting
- Peptides as Targeting Moieties
- Tumor Targeting Through EGFR
This leads to an inhibition of the phosphorylation and thereby an inhibition of further signal transduction. This leads to an inhibition of the phosphorylation and thereby an inhibition of further signal transduction. It could be demonstrated that the binding of the hybrid peptide correlated with the EGFR expression level.
Activation of the EGFR signaling cascade was measured by quantification of ERK phosphorylation [175].
Aims & Scope
We want to investigate this phenomenon by comparing the biological activity of 12S,13S and 12R,13R cyclopropane epothilone analogs. The hydroxyethyl side chain is equally suitable for conjugation of the targeting moiety, and its introduction is further supported by findings after the initiation of this project in our laboratory, demonstrating superior cytotoxic activity of hydroxyethyl compared to aminoethyl methylbenzimidazole bearing epothilone. Tumor targeting of EGFR-overexpressing tumors (see 1.2.2) should be achieved by conjugation of the appropriate epothilone analog to the GE11 peptide (section 1.2.4.2) via a reductively cleavable disulfide bond.
The hydroxyethyl side chain is equally suitable for conjugation of a targeting moiety, and its introduction is further supported by this project in our laboratory showing a superior cytotoxic activity of hydroxyethyl versus aminoethylmethylbenzimidazole-bearing epothilone.
Results & Discussion
Attempted Synthesis of an Epothilone Analog with an Amine Side-Chain
- C9-C10 Disconnection: Still-Gennari Approach
- C9-C10 Disconnection: Nozaki Hiyama-Kishi Approach to the Eastern Building Block
- C10-C11 Disconnection
- C9-C10 Disconnection: Modified First Generation Approach
Oxidative cleavage of the double bond in osmium tetroxide 20 (catalytic) and sodium metaperiodate afforded aldehyde 23 in excellent (quantitative) yield. However, the diastereomeric ratio was poor, varying from 6:1 to 2:1 in favor of the desired isomer. The growth of the reaction under optimized conditions was also associated with safety issues, due to the strong exothermic formation of the cyclopropanating reagent.
The next steps in this strategy would have been a Grieko-Sharpless olefination and cleavage of the silyl ether. The weak reactivity of the hydroxyl group is probably due to the steric hindrance of the pseudoneopentyl position [198]. However, crude iodide 33 was used for side chain elongation via nitrile formation (Scheme 8).
The reaction of the crude mesylate with sodium cyanide again gave a complex mixture of products. A Wittig reaction with MeOCHPPh3, followed by acid hydrolysis of the resulting enol ether, was planned to provide the cyclopropyl building block. The yield of the desired diastereomer 68 was 74%, the combined total yield of both diastereomers was 95%.
One equivalent of texylborane must coordinate with benzimidazole, leading to the disappearance of the starting material on TLC. In some cases, this also resulted in the deprotection of the TBS-protected primary alcohol and traces of the diol.
Synthesis of a 12R,13S Cyclopropyl Epothilone B Analog with a Hydroxyethyl-Benzimidazole
- Forward Synthesis
- TBS / TBS / TBDPS Strategy
Instead of using a PMB protecting group, the terminal hydroxyl group in 94 was then protected as a TES ether (Scheme 37). Indeed, the standard oxidative workup, which allows cleavage and degradation of the ligand, also led to cleavage of the TES ether; thus, only weakly acidic conditions could be used, which did not allow removal of all the ligand. Due to the difficulties in the purification of the TES-protected intermediates depicted in Scheme 37, it was decided to switch to an alternative orthogonal protection strategy.
As illustrated in Scheme 38, this alternative approach involved in a first step the change of the TBS ether moiety in 92 to a TBDPS ether group to give olefin 106 in 63% yield (2 steps) 2 . Olefin 106 was hydroborated and the resulting alcohol 107 was protected as a TBS ether in good yield (93%). Cyclopropane 110 was obtained in 78% yield with a diastereomeric excess of 80% in favor of the desired epimer.
The diastereomers could not be separated and the diastereomeric mixture was used as such in the following steps. However, the synthesis of 4 (section 3.3) describes the synthesis directly with a TBDPS ether and can be used for it. TBS-benzyl ether can be readily cleaved with CSA in 1:1 MeOH/DCM to give Eastern building block 115 in 90% yield.
Traces of another compound could be separated (less than 1 mg vs. 34 mg for the desired compound); this byproduct may have the inverted configuration at C6 and C7, as a consequence of the imperfect stereochemical purity of the starting acid 5. Silyl ether 117 was then deprotected with TASF [210] followed by HF-pyridine to give epothilone 3 in 17% yield by HPLC .
Synthesis of a 12S,13R Cyclopropyl Epothilone B Analog with a Hydroxyethyl-Benzimidazole
- Forward Synthesis
The terminal double bond of the resulting dienoic ester 106 was then selectively hydroborated with 9-BBN in excellent yield (93%) without reduction of the trisubstituted double bond. Allyl alcohol 109 was cyclopropanated under Charette conditions using the R,R ligand 25 to give cyclopropane 126 in 67% yield with a de of 67% (Scheme 42). The diastereomers could not be separated at this step and the following reactions were performed with the mixture of diastereomers.
The hydroxymethyl group in 126 was then converted to a methyl group by iodination and subsequent reduction with sodium borohydride to give 129. Moderate yield of 58% from 128 to 129 resulted in some cleavage of the primary TBS ether to give 130 under acidic action. after iodide reduction. Cleavage of the primary TBS ether into 129 by CSA followed by Grieco–Sharpless olefination afforded olefin 131 in moderate yield of 49%.
Macrolactone 134 was then obtained by RCM of diene 133 with Grubb's 2nd generation catalyst (72%, single isomer). The macrolactone originating from the minor diastereomer of acid 5 could be separated at this step.
Synthesis of trans-cyclopropyl Epothilone Analog 135
- Retrosynthesis
- Forward Synthesis of the Western Building Block
- Tubulin Binding Affinity & Tubulin Critical Concentration
- Cell Cycle Analysis
- Immunofluorescence
- Cytotoxicity Assessment
Both HEK293 and MCF-7 cells showed fluorescence intensities at the level of the isotype control. The reductive cleavage of the conjugate was analyzed under conditions mimicking those found in the endosome and cytoplasm of cancer cells [221]. Thus, cells were treated with serum-free medium containing 1 µM of the selected compound or vehicle.
Therefore, the change in fluorescence anisotropy is a suitable tool for determining the binding affinity of the epothilone analogues. It must therefore be assumed that there has been a change in the plant's heat capacity during the measurements. The most plausible reason for a change in heat capacity is a restructuring of the microtubule structure upon MSA binding, as has already been described.
These unplanned compounds were then subjected to an additional displacement assay to rule out chemical modifications of the ligand during the binding experiments. Cc was determined by treating different concentrations of tubulin with a 1.25 excess of the compound in DMSO. Cells were treated with serial dilutions of the compounds and vehicle and stained with propidium iodide (PI) after 20 hours of incubation.
For both vehicle controls, most cells were in the G0/G1 phase, with a circle. The IC50 was determined by reading the luminescence of cells exposed to compound dilutions after 72 hours.
Conclusions & Outlook
Conjugate 154 showed differential cytotoxic activity in cells expressing EGFR to a greater or lesser extent (87 nM in A431 and 209 nM in SW480). The compound showed the lowest IC50 in HEK293 cells not overexpressing EGFR. This was most likely due to the concentration of reductive species secreted by these cells that cleaved the disulfide bond in 154 and released the epothilone thiol 156. 155 was shown not to be an optimal negative control for the specificity of 154 because it was also subject to reduction.
The optimal negative control in this case would be the conjugate of 156 with the encoded CysGE11 peptide. The trans-cyclopropyl analog 135 is of particular interest because it would allow the determination of the bioactive (i.e., bound to the tubulin dimer and microtubule) conformation of trans-cyclopropyl epothilone analogs, which is unknown to date. Analysis of STD and TR-NOESY spectra will allow modeling of 135 in the paclitaxel binding site of tubulin in the near future.
The tubulin binding affinity was analyzed for all new epothilone analogues and the effects on the cell cycle, microtubule structure and nucleus were investigated for analogues 3, 4, 135 and 155. This is presumably due to the absence of reductive species in the tubulin binding affinity assay. 3 and 135 were the most potent analogs synthesized within this thesis and showed cytotoxic activity in the range of Epo A, while having a stronger tubulin binding affinity; especially 3 with a 10 times higher Ka.
Cell cycle arrest in G2/M was observed in A549 cells treated with analog 3, 4, 135 and 155 at concentrations also found to be cytotoxic. The cells were mostly poly- or micronucleated, even at lower concentrations of the respective compounds.
Experimental
General Procedures
Reactions
- Reactions Described in Section 3.1.1.2
- Reactions Described in Section 3.1.2.2
- Reactions Described in Section 3.1.2.3
- Reaction Described in Section 3.1.3.2
- Reactions Described in Section 3.1.3.3
- Reactions Described in Section 3.1.4.2
- Reactions Described in Section 3.2.1
- Reactions Described in Section 3.2.2
- Reactions Described in Section 3.3.1
- Reactions Described in Section 3.4.3
- Reactions Described in Section 3.5
The solution was washed with saturated aqueous NH4Cl, extracted with DCM, dried over magnesium sulfate and concentrated. NH4Cl was added, extracted with EtOAc, dried over magnesium sulfate and concentrated. Brine was added and extracted with EtOAc, dried over magnesium sulfate and concentrated.
Brine was added and extracted with DCM, dried over magnesium sulfate and concentrated. It was washed with NH4Cl, extracted with EtOAc, dried over magnesium sulfate and concentrated. It was washed with sodium thiosulfate, extracted with DCM, dried over magnesium sulfate and concentrated.
The solution was washed with saturated aqueous NaHCO 3 , extracted with EtOAc, dried over MgSO 4 and concentrated.
Biological Profiling
Bibliography
Annex
