Workup procedure optimisation towards a flow process

With optimised conditions for all the photoredox couplings in hand, we started to investigate the next step, consisting of successive deprotection, hydrolysis, and decarboxylation. At this stage, the treatment of the crude mixture after the photoredox coupling have to be defined.

4.1.4.1 Telescoped process investigation

To best mimic a flow process, a telescoped approach was initially tested by directly adding an aqueous 6 M HCl solution to the crude photoredox coupling mixture (without intermediate treatment, Scheme 44). After refluxing the mixture for 24 h, the solvents were removed under reduced pressure. The residue was suspended in water and the aqueous layer was extracted with chloroform (Ia) to remove non-water-soluble impurities (target product 365 is insoluble in chloroform). Removal of water in vacuo afforded a yellow (residual Ir-4) sticky residue, whose composition was determined by HRMS and NMR analysis. While the product (365) was the main component of the mixture (crude A), it was contaminated with DMAP and traces of a DMAP-adduct (369) side-product

(Scheme 44). This side-product originates from the attack of the DMAP-derived α-amino

radical (cf. 1.2.2) on the olefin (competitive DMAP attack).

To remove DMAP and the side-product (369) from the aqueous layer, both acidic (Ib) and basic (Ic) workup procedures were tested. With a pKa of 9.7,[252] the concentration of

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same time, most of the product (365) was lost in the organic layer. Under acidic conditions, DMAP was completely protonated (370) and therefore only found in the aqueous layer, but the protonated amino-group (pKa = 10.2) of the coupling product (365)

prevented it from being sufficiently extracted into the organic layer. Because of the multiple acidities of the product (365), none of the tested aqueous workup conditions showed an efficient separation.

Scheme 44 – Fully telescoped process impurities and tested purification methods.

We next attempted to separate the impurities by solid-supported scavenging with ion- exchange resins (II).[253] These recyclable, polymer-based materials can reversibly bind compounds by ionic interactions and have been widely used in continuous processes to remove trace metals, switch between different solvent systems, or act as solid-supported reagents.[254,255]

An acidic QP-TSA (TsOH) resin was initially used (Ia). We rationalised that the sulfonate groups could favourably interact with protonated amino residue. The product (365) was successfully bound to the resin under acidic conditions, as it was not detected in the obtained eluent fraction. However, some product was already released during the following neutral wash with water. The residual material was released with aqueous 1 M

ammonia wash and contained product (365), side-product (369), and residual DMAP (370), which could also interact in their protonated form with the cation-exchanger.

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To circumvent this issue, we then used a basic anion-exchanger Amberlite IRA-400 (NR4+) (IIb). This material carries quaternary ammonium functionalities on its surface,

which can potentially interact with the negatively charged carboxylate groups (pKa = 4.2).

After the immobilisation on the ion-exchange resin under basic (pH = 9.8) or neutral conditions (pH = 7.0), the product (365) did not bind strongly enough to the material and was partially released during the aqueous wash. Changing the washing solvent from water to THF did not result in any improvement. Although DMAP was not present in the release fraction (using 3 M HCl), the product (365) was still contaminated with the side- product (369), due to their structural similarities.

4.1.4.2 Intermediate treatment investigation

Since, it appeared that purification is a problem at the final product stage, a fully telescoped process cannot provide high-purity product. We decided to test an intermediate aqueous workup to remove the problematic DMAP-impurities before engaging the mixture in the second step. In contrast to chromatographic separation, an aqueous workup could still be integrated into a continuous process by using an in-line phase separator.[256,257]

Initial investigations showed that DMAP was effectively removed by treating the crude mixture with aqueous ammonium chloride solution (pH 5-6) and extracting the product with ethyl acetate.

Table 26 – NMR yields for drug precursor coupling products after acidic aqueous workup.

Reactions carried out by adapting GP(IX) to the conditions described. *Yield determined by 1_{H-NMR analysis}

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The yields in coupling products after the aqueous workup (Table 26) match those obtained without any treatment (cf. Table 24). This observation suggests that the Boc-amino coupling products are not deprotected using this mildly acidic treatment.

We then turned our attention to the second step using baclofen as the model system

(Scheme 45). The residue was dissolved in acetone and 6 M HCl before to be refluxed

overnight. After solvents removal, the residue was dissolved in water and washed with chloroform to remove non-polar impurities. The 1H-NMR was clean, with only the side- product (369 < 5%) as a minor impurity (crude B). Despite its higher purity than in crude A, the amino acid (365) did not crystallise from the mixture. Several drying and crystallisation conditions in water, methanol, 2-propanol, CH2Cl2 and combinations

thereof were tested, but none of them resulted in a crystalline product.

Scheme 45 – Intermediate workup approach and tested purification methods.

Thus, we decided to subject the crude B to a chromatographic separation. Due to their high polarity, both product (365) and side-product (369) interact strongly with silica, which makes them unlikely to be separated by regular phase silica chromatography. To be able to visualise product-containing fractions from chromatographic separations with modified silica stationary phases, we tested different polar solvent systems on TLC plates. Neither highly polar solvent mixtures of chloroform and methanol, nor pure methanol or mixtures of n-BuOH and acetic acid could elute the compounds on silica gel. Only a complex eluent mixture containing EtOAc, CHCl3, MeOH, water and formic acid

(40:30:20:5:5) resulted in an acceptable separation.[258] _{However, this separation was not}

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We further tested conditions for the purification on reverse phase silica (C-18, IIIa). Because of the insolubility of the amino acid product in acetonitrile, we used different concentrations of water in methanol as the mobile phase, but neither highly polar (H2O:MeOH 80:20) eluent systems, nor pure methanol showed sufficient separation.

Instead, the compound mixture was found unseparated in the first few fractions. We concluded that the interaction of the charged compounds with the non-polar phase was too weak in comparison to their high solubility in the solvent system.

We therefore decided to try a more polar solid phase for the separation by hydrophilic interaction liquid chromatography (HILIC).[259] These materials allow the separation of products with regular phase solvent systems, which are strongly retained on silica and weakly retained under reversed phase conditions. The modified stationary phase strongly interacts with the polar component of the solvent system (usually water), which forms a thin hydrophilic layer on the surface. Hence polar compounds in the mobile phase are retained by both ionic and hydrophilic liquid-liquid interactions.[260]

Initial tests with EtOAc and MeOH (1:1) as mobile phase on spherical modified silica (Claricep-HILIC®, Agela) showed promising separation. Further optimisation revealed a mixture of acetonitrile, methanol, and water (90:10:3) as a suitable solvent system and afforded the product 365 (purity > 90% by 1_H-NMR).

Although suitable conditions for purifying these compounds were finally obtained, we also had to keep the cost efficiency in mind. Besides the valuable separation capacity and recyclability of the HILIC stationary phase, its high price (one thousand pounds per kilogram) limits its larger scale applications.

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