Modifications of (pyridine-2-ylmethyl)- D -proline in the 4-pyridine position

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3.4.4.1.1. Synthesis of ((4-chloropyridine-2-yl)methyl)-D-proline

Since the 4-chloro modified pyridine-2-yl methanol was now available, the 4-chloro modified derivative of the original proline derived ligand 206 was synthesised in addition to the planned ligands and complexes to investigate whether a change in polarity and/or possible formation of halogen-bridges might influence binding to the kinase active site, as well as solubility and permeability. Interestingly, chloride groups are hydrophobic and therefore associated with a reduction in solubility, but in a significant number of examples in small molecules, the addition of a F, Cl, CF3 or even CH3 group leads to an improvement in solubility.^[309] So the chance to improve either solubility or permeability by adding a chloride group seemed quite promising.

The synthesis was partially performed by JOHANNA PLAG during her internship,^[319] according to a modified procedure of the original ligand, as reported by STEFAN MOLLIN.^[298] The hydroxy group of (4-chloropyridine-2-yl) methanol (232) was first chlorinated using thionyl chloride and the resulting HCl salt 233 was directly further converted with D-proline methyl ester (234) in a FINKELSTEIN reaction (Scheme 12). The obtained methyl ((4-chloropyridine-2-yl)methyl)-D -prolinate (40% yield over 2 steps) was further reacted by a simple basic ester hydrolysis using sodium hydroxide to obtain the desired ligand 236 in 63% yield.

Scheme 12: Synthesis of ((4-chloropyridine-2-yl)methyl)-D-proline after a modified literature procedure.^[298] First the alcohol was chlorinated using thionyl chloride and further converted using a FINKELSTEIN reaction. The final ligand was obtained by basic ester cleavage.

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3.4.4.1.2. Synthesis of ((4-(3-morpholinopropoxy)pyridine-2-yl)methyl)-D-proline and ((4-((3-morpholinopropyl)amino)pyridine-2-yl)- D-proline

3.4.4.1.2.1. Synthesis of (4-((3-morpholinopropyl)amino)pyridine-2-yl)methanol

For ligands containing solubilising morpholine-based out-of-plane appendages, nucleophilic reactions were planned using 232, for both introducing an hydroxy group or amine in the 4-pyridine position. To introduce a structurally complex N-nucleophile, different reaction conditions can be found in literature, including the use of N,N-diisopropylethylamine (DIPEA, HÜNIG's base) as base in toluene^[320] or a simple neat reaction e.g. with pyrrolidine as nucleophile.^[317]

Both conditions were used to introduce N-(3-aminopropyl)-morpholine to the (4-chloropyridine-2-yl) methanol in para-position (Scheme 13). In the first reaction, the resulting product could not be isolated from the HÜNIG's base, its salts or other impurities using flash column chromatography or different extraction methods. Any further reaction towards the final ligand yielded in no product.

For the second reaction conditions, JOHANNA PLAG was able to show during her internship^[319] that the desired (4-((3-morpholinopropyl)amino)pyridine-2-yl)methanol (238) was formed under neat reaction conditions as proven via HRMS and ¹H-NMR. However, it was found to be inseparable from the starting material 237 by silica flash column chromatography with a CH2Cl2/MeOH gradient or reversed phase HPLC (C18 CH3CN/H2O 1:1, 0.5% TFA).

Scheme 13: Reaction conditions, based on a modified literature methods^[317,320] to synthesise (4-((3-morpholinopropyl)amino)pyridine-2-yl)methanol.

Using the same reaction conditions, JOHANNA tried to isolate 238 by distilling the starting material 237 of (boiling point of 224 °C according to the supplier’s information). However, even under fine vacuum 238 would decompose (at around 180 °C) before the starting material starts to evaporate.

The logical consequence seemed to consume all leftover 237 to get a pure product. However, even when reacting 232/237 in a 3:1 mixture, the morpholine was not fully converted. Therefore, the introduction of various protecting groups was pursued, to influence the retention factors of both

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starting materials, to enhance chromatographical work-up. These included a TBS group for the hydroxy group of 238 (using TBSCl, imidazole, I2), an acetyl group to both the primary and secondary amine in all species (using Ac2O and 18-crown-6 for selectivity over the alcohol), a Boc group to the secondary amine (using Boc2O and 4-DMAP) or a Cbz group to protect the primary amine (using CbzCl and NaH). All reactions were monitored by TLC analysis for conversion and a change of the Rf values of the starting materials and products. For both, the TBS and acetyl protection groups no change in Rf values was observed in the most polar combination CH2Cl2/MeOH 10:1, so that these groups were excluded. With the Boc group, a single protection of the starting material and a double protection of the product was achieved, accomplishing a change in Rf values. However, separation on silica using silica flash chromatography with a CH2Cl2/MeOH gradient was not possible. The same holds true for the Cbz protection group, where both starting material and product were protected. Since all these affords did not succeed in obtaining 238 in a pure fashion, the product and therefore ligand and final complex 211 were not further pursued.

3.4.4.1.2.2. Synthesis of (4-(3-morpholinopropoxy)pyridine-2-yl)methanol

Similar effort as for (4-((3-morpholinopropyl)amino)pyridine-2-yl)methanol was invested into the synthesis of (4-(3-morpholinopropoxy)pyridine-2-yl)methanol by JOHANNA,^[319] successfully.

Scheme 14: Reaction conditions derived from modified literature methods^[321] to synthesise (4-((3-morpholinopropyl)amino)pyridine-2-yl)methanol.

The best procedure was modified from a literature known nucleophilic aromatic substitution^[321]

using NaH to first prepare sodium 3-morpholinopropan-1-olate before adding 232 at low temperatures (0 °C). After reacting further at 100 °C for 16 h, 240 was isolated by doubly silica flash chromatography (5 g/3 g silica, CH2Cl2 to CH2Cl2/MeOH 10:1) with a yield of 23%.

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3.4.4.1.3. Synthesis of ((4-(3-morpholinopropoxy)pyridine-2-yl)methyl)-D-proline

The final ligand ((4-(3-morpholinopropoxy)pyridine-2-yl)methyl)-D-proline (243) was prepared analogous to the previously described ligand by chlorination of the hydroxy group, a subsequent FINKELSTEIN reaction with D-proline methyl ester (234, 38% over both steps) and basic ester hydrolysis (95%) using aqueous sodium hydroxide (Scheme 15).

Scheme 15: Synthesis of ((4-(3-morpholinopropoxy)pyridine-2-yl)methyl)-D-proline after a modified literature procedure.^[298] First the hydroxy group was chlorinated using thionyl chloride and further converted with methyl-D-prolinate using a FINKELSTEIN reaction. The final ligand was obtained by basic ester cleavage.

3.4.4.1.4. Synthesis of ((4-((dimethylamino)methyl)pyridine-2-yl)methyl)-D-proline The synthesis of the dimethylamino-ligand was performed by HENRIK LÖW and JOHANNA PLAG

during their internships in the group.^[319,322] They both started with a radical hydroxymethylation of 4-cyanopyridine (244) to 2-hydroxymethyl-4-cyanopyridine (245) according to a reported procedure (Scheme 16).^[323] Mechanistically, this is a radical substitution where a nucleophilic hydroxymethyl radical is added to the heterocycle.^[323,324] The lower yield (20%) in comparison to the literature (28%) can be explained by aged starting materials. However, the yield was still much better in comparison to a radical hydroxymethylation using ammonium persulfate and methanol in aqueous sulphuric acid (6% as determined by HENRIK LÖW).^[325]

Scheme 16: Radical hydroxymethylation of 4-cyanopyridine using HOSA and Fe(II) in H2SO4 as catalyst.

The cyanopyridine 245 was further reduced using hydrogen and Pd on carbon (10%) in acetic acid (Scheme 17).^[326] Based on contained acetic acid in the raw ¹H-NMR spectrum with an unknown

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stoichiometry to the product (probably due to salt formation), the (aminomethyl)pyridine 246 was directly used for the next step without further purification. N-alkylation using paraformaldehyde in formic acid in a microwave mediated reaction, according to a modified literature procedure,^[327]

resulted in 28% of 247 as a yellow oil. Cleaner reaction paths, like reduction of 245 with lithium aluminium hydride in THF,^[325] did not lead to any yield in the reaction, based on the need for aqueous work-up and a high solubility of 246in water, from which the product cannot be extracted or isolated.

Scheme 17: Synthesis of 2-hydroxymethyl-4-((dimethylamino)methyl)pyridine from 2-hydroxymethyl-4-cyanopyridine by reduction using H2 and 10% Pd/C and subsequent N-alkylation using paraformaldehyde in a microwave (MW) mediated synthesis.

The final ligand ((4-((dimethylamino)methyl)pyridine-2-yl)methyl)-D-proline (250) was prepared analogously to the previously described ligands by chlorination of the hydroxy group (50%), a subsequent FINKELSTEIN reaction with D-proline methylester (234, 11%) and basic ester hydrolysis (65%) using aqueous sodium hydroxide (Scheme 18).

Scheme 18: Synthesis of ((4-((dimethylamino)methyl)pyridine-2-yl)methyl)-D-proline after a modified literature procedure.^[298] First the hydroxy group was chlorinated using thionyl chloride and further converted using a FINKELSTEIN reaction. The final ligand was obtained by basic ester cleavage.

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3.4.4.1.5. Synthesis of methyl ((4-hexylpyridine-2-yl) methyl)-D-proline

To explore the possibilities of a pure aliphatic side chain, a modified ligand was synthesised by MATTHIAS TRIPP during his bachelor thesis.^[328] Originally starting from 4-picoline (251), he was able to synthesis and 4-hexylpyridine (87%, Scheme 19) according to a modified literature synthesis using lithium diisopropylamine to deprotonate 4-picoline and the according iodoalkane in a SN2 reaction.^[329]

Scheme 19: Synthesis of 4-hexylpyridine using a literature derived SN2 reaction^[329] was performed by MATTHIAS TRIPP during his bachelor thesis.^[328]

The 4-alkylpyridine 252 was further converted to the N-oxide using hydrogen peroxide according to literature procedures (Scheme 20).^[330] A subsequent 2-hydroxymethylation was conducted using trimethyloxonium tetrafluoroborate and ammonium persulfate in methanol.^[331] Mechanistically, it is thought that (CH3)3OBF4 methylates the oxygen of the N-oxide, while the persulfate is homolytically cleaved in the heat resulting in a radical which reacts with the solvent and forms a methanol radical. These methanol radicals might then react with the pyridines and are directed into ortho position, based on the electron donating methoxy group. The radical from the resulting compound is finally passed on to another methanol molecule in the solvent, after cleavage of the N-methanol group or by directly cleaving a methanol radical from the pyridine nitrogen. The hydroxymethylated product, in which the pyridine nitrogen is no longer oxidised, is thereby less reactive compared to the starting material, making a follow up reaction improbable.^[331]

Scheme 20: N-oxidation using hydrogen peroxide^[330] and subsequent 2-hydroxymethylation of 4-hexylpyridine according to a literature described radical reaction mechanism^[331] was performed by MATTHIAS TRIPP.^[328]

This reaction however resulted (4-hexylpyridine-2yl)-methanol (254) only in a low yield of 18%

over both steps (Scheme 20).

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The final ligand, methyl ((4-hexylpyridine-2-yl)methyl)-D-proline (257), was prepared according to a modified literature procedure (Scheme 21).^[332] Analogous to the previously described ligands, first the hydroxy group was chlorinated (50%), followed by a FINKELSTEIN reaction with D-proline methylester (74%) and basic ester hydrolysis (73%) using aqueous sodium hydroxide to get to the final ligand (Scheme 21) in 11% yield over six steps.^[328]

Scheme 21: Synthesis of methyl ((4-hexylpyridine-2-yl) methyl)-D-proline after a modified literature procedure.^[332] First the hydroxy group was chlorinated using thionyl chloride and further converted using a FINKELSTEIN reaction. The final ligand was obtained by basic ester cleavage.

The final ligand however contained some ethanol, which could not be removed by extensive drying, the yield is hence based on the ethanol free ligand according to ¹H-NMR.^[328] As the following complex reactions were conducted in a mixture of EtOH/H2O 1:1, this posed however no problem.

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3.4.4.2. Complex synthesis with modified (pyridine-2-ylmethyl)-D-proline ligands