Upscaling possibilities of RR97a and derivatives

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chromatography. High amounts of pyridocarbazole were however also isolated in smaller scales.

Due to the raised number of by-products and a general smearing of both diastereoisomers, as well as the by-products, on silica during purification, the compound could only be obtained in a pure fashion after multiple flash columns using a gradient of CH2Cl2/MeOH (1:0 to 200:1 to 10:1).

Hence, this approach was rejected due to the extensive purification procedure and the decreased yield.

Since the yield for the half-scale was obviously larger compared to higher or smaller scales, four separate reactions of 125 µmol pyridocarbazole each were performed separately and two reactions each were pooled for purification. Flash chromatography separation of the diastereoisomers were performed over 40 g silica using the slow gradient of CH2Cl2/MeOH described above. The different diastereomers were further repeatedly purified over 1.5-3.0 g of silica. An exact yield for these reactions however could not be obtained, since part of the compound got lost during purification. The total yield of all reactions described in this chapter combined was 12% for 201a.

Altogether this second approach of splitting the reactions and pooling them for work-up meant again a huge amount of work for purification. Though a smaller extent of by-products was qualitatively determined on TLC, compared to the first approach using a larger scale. Therefore, the second option was not further pursued and other solutions for the upscaling problem were investigated, as described in the following chapter.

3.3.2. Optimisation of reaction conditions

Since the yields in the complex reactions were generally very low, which is most probably based on the sluggish chemistry,^[295] a study for improved reaction conditions was performed. The low yields in the original reaction are presumably based on a fast deprotection of the pyridocarbazole, deactivating it towards coordination. Moreover, this may also cause the formation of numerous by-products in the reaction, as indicated earlier by TLC analysis.

Based on this experience, another approach towards the synthesis of RR97a and RR97b as well as its derivatives was required. Conveniently, STEFAN MOLLIN showed in his PhD thesis^[296] that upon reacting structurally distantly related kinase inhibiting ligands (e.g. phenylquinoline derived ligand) first with RhCl3 and afterwards with N-methyl-N-(pyridine-2-ylmethyl)glycine as tridentate ligand, the cis-isomer (carboxylate of the ligand is coordinated cis to the phenylquinoline nitrogen) was formed to a greater extent than the trans-isomer (~2:1). Reacting the glycine ligand first with RhCl3

and then adding a benzyl-protected pyridocarbazole, a ratio of cis to trans isomers (carboxylate of the ligand is coordinated cis or trans to the indole nitrogen) of 1:3 was obtained. Based on these

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results, a similar approach was pursued for the given system to obtain RR97a and RR97b (Scheme 8).

Scheme 8: Proposed reaction sequence for improvement of the cis/trans ratio of the formed complexes 201a and 201b.

To test the hypothesis of positively influencing the ratio of 201a (trans-isomer) to 201b (cis-isomer) obtained in the reaction, the proline ligand 206 (38.0 µmol, 1.00 eq) was reacted in a small-scale with RhCl3 (38.0 µmol, 1.00 eq) in a 1:1 mixture of CH3CN/H2O (3.8 mL) for 16 h at 90 °C, as indicated for the glycine ligand in STEFAN’s thesis. A complex precursor is thereby expected to be formed in a way where the rhodium centre of RhCl3 coordinates three acetonitrile ligands before one chlorido and two acetonitrile ligands were exchanged by the carboxylate of 206 to form 207 (Scheme 8). ¹H NMR analysis of the complex precursor 207 after flash chromatography (CH2Cl2/MeOH) however showed at least two sets of signals in the aromatic region. These might be based on either monodentate ligand exchange in the complex 207, two proline ligands coordinated to one rhodium centre, or unreacted ligand in the spectrum. Without further purification, the precursor mixture was reacted in CH3CN/H2O 1:1 at 90 °C for 16 h with the TBS-protected pyridocarbazole. Hereby, no reaction was observed by TLC analysis. Therefore, the solvent was removed and replaced with EtOH/H2O 1:1 and the reaction mixture was heated to 90 °C for 72 h. TLC analysis indeed showed the formation of the two expected diastereomers with the trans signal giving a more intense colouring, indicating that the reaction conditions shift the cis/trans ratio. It was attempted to drive the reaction to completion by adding pyridocarbazole at different time points (~0.5 eq directly after precursor synthesis, another 0.5 eq 3 h after the start, and extra 0.1 eq at time point 5 h and 0.1 eq after 15 h). However, a completion of the reaction could not be achieved.

Based on these positive results, a screening for the optimal solvent for the second reaction step was performed. Especially the high amounts of unreacted pyridocarbazole were of great concern.

Therefore, 50 µmol of each the ligand and RhCl3 were reacted under stirring in 50 mL CH3CN/H2O 1:1 at 90 °C for 16 h. Afterwards, the solvent was removed and either 5 mL EtOH,

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DMF or EtOH/H2O 1:1 were added together with 1.0 eq of pyridocarbazole 205. All solvents are polar and therefore able to coordinate and stabilise possible charged intermediates. The reactions were stirred at 90 °C for 65 h and the results were analysed qualitatively via TLC analysis. While the reaction with pure EtOH contained large amounts of unreacted pyridocarbazole, some complex and various by-products, the reaction using DMF showed hardly any reaction to the desired complexes as well as hardly any unreacted pyridocarbazole left (probably based on cleavage of the TBS-protecting group and subsequent abolishment of the reactivity of the pyridocarbazole).

Both reaction conditions showed much lower product formation compared to the original solvent mixture, which might be associated to the missing water as aqueous solutions are known to promote the substitution of a chlorido-ligand^[294] as mentioned earlier. Another possibility for the low yields is a faster deprotection of the pyridocarbazole in the changed solvent system. Therefore, for both reactions the solvent was removed, replaced by EtOH/H2O 1:1 and stirred for 24 h at 95 °C. TLC analysis after that reaction time showed a small improvement of the original EtOH reaction and no improvement of the DMF reaction, indicating indeed the loss of the TBS-protection group of the pyridocarbazole. Therefore, the precursor approach was generally dismissed for the unsubstituted pyridocarbazole.

In parallel to the studies using unmodified pyridocarbazole, a comparison study of the original one pot synthesis reaction conditions and the precursor approach was done by BENEDIKT HEINRICH

using an indole-5-ol modified pyridocarbazole (208). This investigation was supposed to help understanding the influence of the electronic properties of the ligand.^[297]

Scheme 9: Different synthetic approaches towards the synthesis of 209a and 209b. On the left-hand side, the reaction conditions of the one pot synthesis are shown. On the right-hand side the conditions of the precursor approach.

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Starting with very small reaction scales, both reaction pathways (Scheme 9) were confirmed to result into the desired complexes 209a and 209b as shown qualitatively by TLC analysis. The complex formation using the precursor approach was subsequently tested in both solvent systems, CH3CN/H2O 1:1 and EtOH/H2O 1:1, for the second reaction step and evaluated qualitatively via TLC. Minimal reaction was thereby detected in the original solvent (CH3CN/H2O), even with a temperature increase up to 105 °C. The use of EtOH/H2O resulted in the formation of a spot for the a isomer 209a that has been much more intense compared to the one of the b isomer 209b.

When scaling-up the precursor approach (152 µmol pyridocarbazole), ¹H-NMR analysis of the two isomers after purification via silica flash chromatography using CH2Cl2/MeOH (1:0 to 100:1 to 10:1) showed an extensive number of by-products, probably unreacted precursor and/or unreacted ligands. These by-products were not easily removable by flash chromatography over silica since the complexes and by-products have similar retention factors (Rfs) in the given solvents and tend to smear on silica. An isolated spot from the 5-hydroxy pyridocarbazole reaction, which was suggested to belong to the precursor 207, was analysed by ¹H-NMR spectroscopy in DMSO-d6, where it showed all signals of the ligand but the expected CH3CN signal. A high-resolution mass spectrum however showed the exact mass of LRhCl(DMSO) (with L representing the deprotonated proline derived ligand 206), so that the CH3CN ligand might have been exchanged in the NMR tube due to its reactivity and excess DMSO present. The missing chlorido ligand in the exact mass was probably lost during ionisation. These results indicate a formation of metal precursor as suggested.

Interestingly, a similar species as the precursor was also found when scaling-up the one-pot approach, indicating a lower reactivity of the pyridocarbazole-ligand and therefore leftover RhCl3

which might react with the later added proline ligand. However, this side product was present to a much smaller extent in comparison to the by-products detected in the precursor approach, which is probably based on the changed solvent system. Also, the a isomer 209a, which is the isomer of interest, could be obtained in a pure fashion, while only the b isomer 209b was inseparable from this side product, which was not the case for the precursor approach. Still 209a was formed to a much smaller extent, compared to the precursor approach, deeming both reaction pathways as not very suitable to form larger amounts of 209a.

To sum up, the precursor approach indeed leads to the formation of a higher ratio of a (trans) to b (cis) isomer, however with the draw-back that also more and partly inseparable by-products were formed. Therefore, this variant seems not to offer a suitable possibility to enhance the reaction yield, for scale-up or save time in work-up. All in all, no approach helped to scale the reactions or yield up to a significant extent.

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