The utility of this method is illustrated by way of an XP Screen against CYP121A1, a cytochrome P450 enzyme from Mycobacterium tuberculosis (Mtb) championed as a validated drug discovery target. A focused screening set was synthesized and tested by such means, with several members of the set showing promising activity against Mtb strain H37Rv. One compound was observed as an X-ray hit against CYP121A1 and showed improved activity against Mtb strain H37Rv under multiple assay conditions (pan-assay activity). Data obtained during X-ray crystallographic screening were utilized in a structure-based campaign to design a limited number of analogues (less than twenty), many of which also showed pan-assay activity against Mtb strain H37Rv. These included the benzo[b]oxazine derivative (MIC90 6.25 μM), a novel hit compound suitable as a starting point for a more involved hit to lead candidate medicinal chemistry campaign.There is a pressing need for new drugs against tuberculosis (TB) to combat the growing resistance to current antituberculars. Herein a novel strategy is described for hit generation against promising TB targets involving X-ray crystallographic screening in combination with phenotypic screening. This combined approach (XP Screen) affords both a validation of target engagement as well as determination of   •M.tuberculosis has been previously shown to be a crucial target for the survival of the mycobacteria.CYP121 from •M.tuberculosis.Strategies previously employed have identified high affinity inhibitors however these have lacked activity on •The strategy reported here uses a combination of X-ray crystallography and phenotypic screening (XP Screen) to identify compounds.•M.tuberculosis (up to 6.25 μM).The XP screen approach identified a number of compounds which show good affinity (up to 3.2 μM) and MIC against  Compounds 51–65 were screened for in vitro activity against CYP121A1 using UV–Vis spectroscopy. As with the initial X-ray hit 14, none of these direct analogues showed any discernible effect (no Soret band shift), suggesting that, like 14, they bind to CYP121A1 without direct contact to the heme. Likewise, a number of the compounds were examined by ITC, but it proved difficult to generate affinity data for these molecules using ITC due to inadequate solubility in aqueous buffers.Compounds 14 were screened against Mtb  were moderate inhibitors, and like 14, showed activity in all three of the growth media tested. Bridged bicyclic derivative 58 was active, but showed much reduced potency in BSA rich medium. N-Acetyl derivative 64 had essentially lost all activity, whereas the corresponding N-methyl derivative 57 was one of the compounds that exhibited pan-assay activity. It was noted that the most potent derivative against Mtb was 61 (MIC90 6.25 μM), a compound that had been designed based upon the overlap of X-ray crystal structures for 14 and 31, where the ligand in 31 had been positioned in an alternative orientation, with the ligand rotated 180° in the active site, so suggesting the idea of appending an aromatic ring onto the morpholine moiety.Despite this, a number of analogues of inst Mtb . Severalin cellulo active compounds is perhaps not too surprising. Despite there being numerous CYP121A1 crystal structures deposited in the PDB, the majority are with ligands that either have low cLogP or make direct contact with the iron atom of the heme group. As the substrate for the enzyme (cYY) has a cLogP of 0.82, the enzyme is clearly designed to accommodate substrate and product that are highly hydrophilic in nature. Recent studies recording imidazolyl- and triazolyl-derived pyrazoles as inhibitors of CYP121A1 have shown that, whilst compounds with higher lipophilicity (cLogP >4) showed better activity in cellulo, only some of those with much lower cLogP (1.44 and 2.68) afforded useable crystal structures pyrimidine or pyridopyrimidine. This scaffold morphing approach entailed the relocation of a pyrimidine nitrogen to the alternative ring position where it was suitably positioned to be able to interact with the sidechain hydroxyl group of Thr77 of the protein via an additional hydrogen bond, thereby potentially gaining additional affinity -mixture. When prepared via an alternative route over five steps from indole using known chemistry -isomer (olefinic coupling constant: J = 19 Hz). The added length of synthesis required to generate 74 resulted in only the three derivatives 81–83 with lowest cLogP values (1.22–1.77) being targeted. Thus chlorides 66–68 were reacted with 74  to afford 75–77 as the (E)-isomers (olefinic coupling constants: J = 16–17 Hz). Reduction of 75–77 with hydrogen over palladium on carbon in ethyl acetate yielded 78–80 (after chromatography on silica to remove over-reduced by-products) which, upon deprotection under acidic conditions, gave 81–83  , 74 wase salts) .Scheme 281–83 behaved, as expected, like all of the pyrimidine derivatives in this series in that we were unable to determine the affinities for these compounds against CYP121A1 by UV–Vis spectroscopy. This was reassuring, as it effectively ruled out the possibility of a change of binding mode in which interaction with the heme iron atom might conceivably occur via the cyclic secondary amine functionalities. Likewise, determination of potency by ITC proved difficult for these derivatives (in common with a number of the pyrimidines described above). Despite a significant reduction in cLogP values, it again proved impossible to collect satisfactory X-ray structures for 81–83. Nevertheless, the much improved solubility of 81–83 in aqueous media allowed the use of differential scanning fluorimetry to probe the binding affinities of 81–83 . Addition of all three bicyclic derivatives to solutions of CYP121A1 in aqueous buffer (1 mM ligand with 5 μM protein) resulted in increases to the melting temperature (Tm) of the protein  and 82 (+2.4 °C), with the ring expanded homologue 83 (+1.0 °C) showing a lesser effect (akin to that of aminopyrimidine 45 used as positive control). The original screening set, together with the set of advanced derivatives (as outlined above), were also subsequently screened by these means. Most compounds gave inconclusive results as a result of aggregation, due to limited ligand solubility in the aqueous buffer. Compounds with lower cLogP proved to be less problematic, with both 10 (an X-ray hit) and 55 (the 5-hydroxy derivative of the key X-ray hit 14) also showing evidence of activity (protein melting temperature shifts of +3.5 °C and +0.5 °C respectively).Fused bicyclic compounds  protein . This su7–10, 14, 21, 31, 33, 41–44, 51, 53, 55, 57, 60, 61, 63, 81–83) were tested in an LC-MS based activity assay, to determine their ability to inhibit the turnover of cYY into mycocyclosin relative to clotrimazole, a known azole derived inhibitor of CYP121A1. All compounds tested were either X-ray crystallographic hits, or had yielded encouraging data in UV–Visible spectroscopy, differential scanning fluorimetry, isothermal titration calorimetry or antimycobacterial activity assays (as described above). Under the conditions used, no CYP121A1 mediated turnover of the experimental compounds was observed, indicating that none of the compounds were substrates for CYP121A1. As expected, in samples containing both cYY and clotrimazole, a large amount of cYY (>50-fold over the positive control) remained present following incubation , a general trend towards inhibition of CYP121A1 was noted, thus confirming inhibitory activity of CYP121A1 as a general property of a number of key derivatives within the series of compounds. In particular, 14  and 61 (with pan-assay activity against whole cell Mtb) both markedly reduced the production of mycocyclosin  under the assay conditions.Given their respective positions within the medicinal chemistry campaign, the propensity of compounds on assay . In the  control . The pre3in cellulo or in vivo activity, with compounds being progressed that lack acceptable cell permeability, or poor ADMET properties.X-ray crystallographic screening is a frequently used technique for hit generation in the field of fragment-based drug discovery ,28 with Mtb have involved open-source initiatives utilizing the diverse array of compounds residing within corporate collections to provide lead compounds. These efforts have identified a series of 2-thiophenyl morpholines oxazine derivative 61, is a novel lead compound of moderate molecular mass (Mr = 356) suitable as a starting point for a more thorough lead-to-clinical candidate phase, that inhibits the CYP121A1 mediated turnover of cYY to mycocyclosin, and shows favorable activity against Mtb strain H37Rv  in comparison with a number of azole anti-fungal drugs  4,   4,   4, 4, 4, + 257, 259 . HRMS: [M + H]+ 257.0837. Calcd for C15H1435ClN2+: 257.0840. Δ = −1.2.od for 8  from 2-v5-Chloro-3-(2-(2-chloropyrimidin-4-yl)ethyl)-1H-indole (12). Prepared as per the method for 11 + 292, 294, 296 . HRMS: [M + H]+ 292.0403. Calcd for C14H1235Cl2N3+: 292.0403. Δ = 0.d for 11  from 2-cd for 11  and 5-ch5-Bromo-3-(2-(2-chloropyrimidin-4-yl)ethyl)-1H-indole (13). Prepared as per the method for 11 + 336, 338, 340 . HRMS: [M + Na]+ 357.9716. Calcd for C14H1179Br35ClN3Na+: 357.9717. Δ = −0.3.H-indole . Prepared for 11  and 5-br4-(4-(2-(1H-Indol-3-yl)ethyl)pyrimidin-2-yl)morpholine (14). A mixture of 3-(2-(2-chloropyrimidin-4-yl)ethyl)-1H-indole 11  and morpholine (1 mL) in ethanol (6 mL) was stirred and held at reflux for 4 h and allowed to cool to room temperature. The solvent was removed in vacuo and the residues partitioned between dichloromethane and water. The organic layer was separated, the solvent removed in vacuo and the residues subjected to column chromatography on silica. Elution with 0–100% ethyl acetate in petroleum ether (b.p. 40–60 °C) afforded 4-(4-(2-(1H-indol-3-yl)ethyl)pyrimidin-2-yl)morpholine 14  as a pale yellow solid. 1H NMR: δ 10.75 , 8.24 , 7.53 , 7.33 , 7.11 , 7.06 , 6.97 , 6.61 , 3.70 , 3.66 , 3.08 , 2.93 . 13C NMR: δ 170.9 (C), 161.8 (C), 158.0 (CH), 136.7 (C), 127.5 (C), 122.8 (CH), 121.3 (CH), 118.7 (CH), 118.6 (CH), 114.2 (C), 111.8 (CH), 110.0 (CH), 66.5 (CH2), 44.3 (CH2), 38.2 (CH2), 23.9 (CH2). LC-MS: [M + H]+ 309 . HRMS: [M + H]+ 309.1714. Calcd for C18H21N4O+: 309.1710. Δ = +1.2.4-(4-(2-(1H-Indol-3-yl)ethyl)pyrimidin-2-yl)thiomorpholine (15). Prepared as per the method for 14 from 3-(2-(2-chloropyrimidin-4-yl)ethyl)-1H-indole 11 and thiomorpholine. Yield 74%. Colorless solid. 1H NMR: δ 10.77 , 8.23 , 7.51 , 7.32 , 7.11 , 7.06 , 6.97 , 6.58 , 4.07 , 3.07 , 2.94 , 2.59 . 13C NMR: δ 171.0 (C), 161.2 (C), 158.1 (CH), 136.7 (C), 127.5 (C), 122.8 (CH), 121.3 (CH), 118.7 (CH), 118.6 (CH), 114.1 (C), 111.8 (CH), 109.6 (CH), 46.3 (CH2), 38.1 (CH2), 26.4 (CH2), 24.0 (CH2). LC-MS: [M + H]+ 325 . HRMS: [M + H]+ 325.1478. Calcd for C18H21N432S+: 325.1481. Δ = −1.0.3-(2-(2-(4-Methylpiperazin-1-yl)pyrimidin-4-yl)ethyl)-1H-indole (16). A mixture of 3-(2-(2-chloropyrimidin-4-yl)ethyl)-1H-indole 11  and N-methylpiperazine (1 mL) in ethanol (6 mL) was stirred and held at reflux for 4 h and allowed to cool to room temperature. The solvent was removed in vacuo and the residues partitioned between dichloromethane and water. The organic layer was separated, the solvent removed in vacuo and the residues triturated with a mixture of n-hexane and dichloromethane. The solids were collected by suction filtration to afford 3-(2-(2-(4-methylpiperazin-1-yl)pyrimidin-4-yl)ethyl)-1H-indole 16  as a pale yellow solid. 1H NMR: δ 10.75 , 8.20 , 7.52 , 7.32 , 7.11 , 7.06 , 6.97 , 6.56 , 3.74 , 3.07 , 2.92 , 2.35 , 2.21 . 13C NMR: δ 170.8 (C), 161.7 (C), 158.0 (CH), 136.7 (C), 127.5 (C), 122.8 (CH), 121.3 (CH), 118.7 (CH), 118.6 (CH), 114.2 (C), 111.8 (CH), 109.6 (CH), 54.9 (CH2), 46.4 (CH3), 43.7 (CH2), 38.2 (CH2), 23.9 (CH2). LC-MS: [M + H]+ 322 . HRMS: [M + H]+ 322.2023. Calcd for C19H24N5+: 322.2026. Δ = −1.0.3-piperazin-1-yl)pyrimidin-4-yl)ethyl)-1H-indole (17). Prepared as per the method for 31 from 3-(2-(2-chloropyrimidin-4-yl)ethyl)-1H-indole 11 and 1-piperazine dihydrochloride. Yield 67%. Colorless solid. 1H NMR: δ 10.75 , 8.22 , 7.53 , 7.33 , 7.12 , 7.06 , 6.97 , 6.58 , 3.75 , 3.22 , 3.07 , 2.92 , 2.67 . 13C NMR: δ 170.9 (C), 161.6 (C), 158.0 (CH), 136.7 (C), 127.5 (C), 126.5 , 122.8 (CH), 121.3 (CH), 118.7 (CH), 118.6 (CH), 114.2 (C), 111.8 (CH), 109.7 (CH), 57.4 , 53.4 (CH2), 43.8 (CH2), 38.2 (CH2), 24.0 (CH2). 19F NMR: δ −67.8. LC-MS: [M + H]+ 390 . HRMS: [M + H]+ 390.1902. Calcd for C20H23F3N5+: 390.1900. Δ = +0.5.4-(4-(2-(5-Chloro-1H-indol-3-yl)ethyl)pyrimidin-2-yl)morpholine (18). Prepared as per the method for 14 from 5-chloro-3-(2-(2-chloropyrimidin-4-yl)ethyl)-1H-indole 12 and morpholine. Yield 44%. Pale yellow solid. 1H NMR: δ 10.97 , 8.22 , 7.52 , 7.33 , 7.20 , 7.04 , 6.59 , 3.70 , 3.65 , 3.06 , 2.91 . 13C NMR: δ 170.7 (C), 161.8 (C), 158.0 (CH), 135.1 (C), 128.7 (C), 124.8 (CH), 123.4 (C), 121.2 (CH), 118.1 (CH), 114.2 (C), 113.3 (CH), 110.1 (CH), 66.5 (CH2), 44.3 (CH2), 38.2 (CH2), 23.6 (CH2). LC-MS: [M + H]+ 343, 345 . HRMS: [M + H]+ 343.1318. Calcd for C18H2035ClN4O+: 343.1320. Δ = −0.2.4-(4-(2-(5-Chloro-1H-indol-3-yl)ethyl)pyrimidin-2-yl)thiomorpholine (19). Prepared as per the method for 14 from 5-chloro-3-(2-(2-chloropyrimidin-4-yl)ethyl)-1H-indole 12 and thiomorpholine. Yield 66%. Pale orange solid. 1H NMR: δ 10.97 , 8.21 , 7.50 , 7.33 , 7.20 , 7.04 , 6.55 , 4.07 , 3.06 , 2.90 , 2.58 . 13C NMR: δ 170.9 (C), 161.2 (C), 158.1 (CH), 135.1 (C), 128.7 (C), 124.8 (CH), 123.4 (C), 121.2 (CH), 118.0 (CH), 114.2 (C), 113.3 (CH), 109.7 (CH), 46.3 (CH2), 38.2 (CH2), 26.4 (CH2), 23.6 (CH2). LC-MS: [M + H]+ 359, 361 . HRMS: [M + H]+ 359.1088. Calcd for C18H2035ClN432S+: 359.1092. Δ = −1.2.5-Chloro-3-(2-(2-(4-methylpiperazin-1-yl)pyrimidin-4-yl)ethyl)-1H-indole (20). Prepared as per the method for 16 from 5-chloro-3-(2-(2-chloropyrimidin-4-yl)ethyl)-1H-indole 12 and N-methylpiperazine. Yield 70%. Pale yellow solid. 1H NMR: δ 10.97 , 8.19 , 7.52 , 7.33 , 7.20 , 7.04 , 6.54 , 3.73 , 3.03 , 2.89 , 2.34 , 2.21 . 13C NMR: δ 170.6 (C), 161.7 (C), 158.0 (CH), 135.1 (C), 128.7 (C), 124.8 (CH), 123.4 (C), 121.2 (CH), 118.1 (CH), 114.3 (C), 113.3 (CH), 109.7 (CH), 54.9 (CH2), 46.3 (CH3), 43.7 (CH2), 38.2 (CH2), 23.6 (CH2). LC-MS: [M + H]+ 356, 358 . HRMS: [M + H]+ 356.1631. Calcd for C19H2335ClN5+: 356.1636. Δ = −1.5.4-(4-(2-(5-Bromo-1H-indol-3-yl)ethyl)pyrimidin-2-yl)morpholine (21). Prepared as per the method for 14 from 5-bromo-3-(2-(2-chloropyrimidin-4-yl)ethyl)-1H-indole 13 and morpholine. Yield 65%. Colorless solid. 1H NMR: δ 10.98 , 8.22 , 7.66 , 7.29 , 7.19 , 7.15 , 6.59 , 3.71 , 3.65 , 3.06 , 2.90 . 13C NMR: δ 170.7 (C), 161.8 (C), 158.0 (CH), 135.3 (C), 129.4 (C), 124.6 (CH),. 123.8 (CH), 121.1 (CH), 114.2 (C), 113.8 (CH), 111.3 (C), 110.1 (CH), 66.5 (CH2), 44.3 (CH2), 38.2 (CH2), 23.5 (CH2). LC-MS: [M + H]+ 387, 389 . HRMS: [M + H]+ 387.0818. Calcd for C18H2079BrN4O+: 387.0815. Δ = +0.7.4-(4-(2-(5-Bromo-1H-indol-3-yl)ethyl)pyrimidin-2-yl)thiomorpholine (22). Prepared as per the method for 14 from 5-bromo-3-(2-(2-chloropyrimidin-4-yl)ethyl)-1H-indole 13 and thiomorpholine. Yield 47%. Off-white solid. 1H NMR: δ 10.98 , 8.21 , 7.63 , 7.29 , 7.18 , 7.15 , 6.55 , 4.07 , 2.58 , 3.05 , 2.90 . 13C NMR: δ 170.8 (C), 161.2 (C), 158.1 (CH), 135.3 (C), 129.5 (C), 124.6 (CH), 123.8 (CH), 121.1 (CH), 114.2 (C), 113.8 (CH), 111.3 (C), 109.7 (CH), 46.2 (CH2), 38.2 (CH2), 26.4 (CH2), 23.6 (CH2). LC-MS: [M + H]+ 403, 405 . HRMS: [M + H]+ 403.0581. Calcd for C18H2079BrN432S+: 403.0586. Δ = −1.3.5-Bromo-3-(2-(2-(4-methylpiperazin-1-yl)pyrimidin-4-yl)ethyl)-1H-indole (23). Prepared as per the method for 16 from 5-bromo-3-(2-(2-chloropyrimidin-4-yl)ethyl)-1H-indole 13 and N-methylpiperazine. Yield 63%. Orange oil. 1H NMR: δ 10.98 , 8.19 , 7.65 , 7.29 , 7.18 , 7.15 , 6.54 , 3.73 , 3.05 , 2.89 , 2.34 , 2.21 . 13C NMR: δ 170.6 (C), 161.7 (C), 158.0 (CH), 135.3 (C), 129.5 (C), 124.6 (CH), 123.8 (CH), 121.1 (CH), 114.2 (C), 113.8 (CH), 111.3 (C), 109.7 (CH), 54.9 (CH2), 46.4 (CH3), 43.7 (CH2), 38.2 (CH2), 23.6 (CH2). LC-MS: [M + H]+ 400, 402 . HRMS: [M + H]+ 400.1123. Calcd for C19H2379BrN5+: 400.1132. Δ = +2.2.5-Bromo-3-piperazin-1-yl)pyrimidin-4-yl)ethyl)-1H-indole (24). Prepared as per the method for 31 from 5-bromo-3-(2-(2-chloropyrimidin-4-yl)ethyl)-1H-indole 13 and 1-piperazine dihydrochloride. Yield 75%. Pale yellow solid. 1H NMR: δ 10.98 , 8.20 , 7.66 , 7.29 , 7.19 , 7.16 , 6.56 , 3.75 , 3.25 , 3.06 , 2.90 , 2.67 . 13C NMR: δ 170.8 (C), 161.6 (C), 158.0 (CH), 135.3 (C), 129.5 (C), 126.5 , 124.6 (CH), 123.8 (CH), 121.1 (CH), 114.2 (C), 113.8 (CH), 111.3 (C), 109.8 (CH), 57.4 , 53.4 (CH2), 43.8 (CH2), 38.2 (CH2), 23.6 (CH2). 19F NMR: δ −67.8. LC-MS: [M + H]+ 468, 470 . HRMS: [M + H]+ 468.0996. Calcd for C20H2279BrF3N5+: 468.1005. Δ = −2.0.tert-Butyl 4-(4-(2-(5-bromo-1H-indol-3-yl)ethyl)pyrimidin-2-yl)piperazine-1-carboxylate (25). Prepared as per the method for 16 from 5-bromo-3-(2-(2-chloropyrimidin-4-yl)ethyl)-1H-indole 13 and tert-butyl piperazine-1-carboxylate. Yield 77%. Pale yellow solid. 1H NMR: δ 10.98 , 8.22 , 7.66 , 7.29 , 7.19 , 7.15 , 6.58 , 3.72 , 3.39 , 3.06 , 2.91 , 1.43 . 13C NMR: δ 170.8 (C), 161.6 (C), 158.0 (CH), 154.4  scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 480 0 480 0 0 40 0 40 -480 0 -480 0 0 -40z M0 280 l0 -40 480 0 480 0 0 40 0 40 -480 0 -480 0 0 -40z"/></g></svg>O), 135.3 (C), 129.5 (C), 124.7 (CH), 123.8 (CH), 121.1 (CH), 114.2 (C), 113.8 (CH), 111.3 (C), 110.1 (CH), 79.5 (C), 43.6 (CH2), 40.7 (CH2), 38.2 (CH2), 28.6 (CH3), 23.6 (CH2). LC-MS: [M + H]+ 486, 488 . HRMS: [M + Na]+ 508.1318. Calcd for C23H2879BrN5O2Na+: 508.1310. Δ = 0.5-Bromo-3-(2-(2-(piperazin-1-yl)pyrimidin-4-yl)ethyl)-1H-indole hydrochloride (26). Prepared as per the method for 37 from tert-butyl 4-(4-(2-(5-bromo-1H-indol-3-yl)ethyl)pyrimidin-2-yl)piperazine-1-carboxylate 25. Yield 70%. Pale orange solid. 1H NMR: δ 11.07 , 9.39 , 8.28 , 7.66 , 7.30 , 7.19 , 7.16 , 6.70 , 3.99 , 3.15 , 3.07 , 2.95 . 13C NMR: δ 171.6 (C), 160.7 (C), 157.5 (CH), 135.4 (C), 129.5 (C), 124.7 (CH), 123.8 (CH), 121.1 (CH), 114.0 (C), 113.8 (CH), 111.3 (C), 110.8 (CH), 42.8 (CH2), 40.8 (CH2), 38.2 (CH2), 23.5 (CH2). LC-MS: [M + H]+ 386, 388 . HRMS: [M + H]+ 386.0979. Calcd for C18H2179BrN5+: 386.0975. Δ = +1.0.4-(4-(2-(5-Bromo-1H-indol-3-yl)ethyl)pyrimidin-2-yl)piperazin-2-one (27). Prepared as per the method for 16 from 5-bromo-3-(2-(2-chloropyrimidin-4-yl)ethyl)-1H-indole 13 and piperazin-2-one. Yield 50%. Colorless solid. 1H NMR: δ 10.99 , 8.25 , 8.08 , 7.66 , 7.29 , 7.19 , 7.16 , 6.62 , 4.19 , 3.92 , 3.27 , 3.07 , 2.93 . 13C NMR: δ 170.9 (C), 167.6 (CO), 160.9 (C), 158.1 (CH), 135.3 (C), 129.4 (C), 124.6 (CH), 123.8 (CH), 121.1 (CH), 114.1 (C), 113.8 (CH), 111.3 (C), 110.3 (CH), 48.2 (CH2), 40.7 (CH2), 40.6 (CH2), 38.2 (CH2), 23.6 (CH2). LC-MS: [M + H]+ 400, 402 . HRMS: [M + H]+ 400.0757. Calcd for C18H1979BrN5O+: 400.0768. Δ = −2.8.2-(4-(2-(1H-Indol-3-yl)ethyl)pyrimidin-2-yl)isoxazolidine (28). Prepared as per the method for 31 from 3-(2-(2-chloropyrimidin-4-yl)ethyl)-1H-indole 11 and isoxazolidine hydrochloride. Yield 82%. Off-white solid. 1H NMR: δ 10.76 , 8.37 , 7.50 , 7.32 , 7.11 , 7.06 , 6.96 , 6.89 , 3.81 , 3.09 , 3.00 , 2.14 . 13C NMR: δ 171.6 (C), 165.9 (C), 158.3 (CH), 136.7 (C), 127.5 (C), 122.9 (CH), 121.4 (CH), 118.7 (CH), 118.6 (CH), 113.9 (C), 113.8 (CH), 111.8 (CH), 67.2 (CH2), 49.6 (CH2), 38.2 (CH2), 27.8 (CH2), 24.2 (CH2). LC-MS: [M + H]+ 295 . HRMS: [M + H]+ 295.1553. Calcd for C17H19N4O+: 295.153. Δ = 0.2-(4-(2-(5-Chloro-1H-indol-3-yl)ethyl)pyrimidin-2-yl)isoxazolidine (29). Prepared as per the method for 31 from 5-chloro-3-(2-(2-chloropyrimidin-4-yl)ethyl)-1H-indole 12 and isoxazolidine hydrochloride. Yield 76%. Colorless solid. 1H NMR: δ 10.98 , 8.37 , 7.49 , 7.33 , 7.20 , 7.05 , 6.89 , 3.81 , 3.07 , 2.98 , 2.14 . 13C NMR: δ 171.4 (C), 165.9 (C), 158.2 (CH), 135.1 (C), 128.7 (C), 124.9 (CH), 123.4 (C), 121.3 (CH), 118.0 (CH), 114.0 (C), 113.9 (CH), 113.3 (CH), 67.2 (CH2), 49.6 (CH2), 38.2 (CH2), 27.8 (CH2), 23.9 (CH2). LC-MS: [M + H]+ 329, 331 . HRMS: [M + H]+ 329.1159. Calcd for C17H1835ClN4O+: 329.1163. Δ = −1.3.2-(4-(2-(5-Bromo-1H-indol-3-yl)ethyl)pyrimidin-2-yl)isoxazolidine (30). Prepared as per the method for 31 from 5-bromo-3-(2-(2-chloropyrimidin-4-yl)ethyl)-1H-indole 13 and isoxazolidine hydrochloride. Yield 73%. Off-white solid. 1H NMR: δ 10.99 , 8.37 , 7.63 , 7.29 , 7.18 , 7.15 , 6.89 , 3.81 , 3.07 , 2.98 , 2.14 . 13C NMR: δ 171.4 (C), 165.9 (C), 158.2 (CH), 135.3 (C), 129.4 (C), 124.7 (CH), 123.8 (CH), 121.0 (CH), 113.9 (CH), 113.8 (CH), 111.4 (C), 67.2 (CH2), 49.6 (CH2), 38.2 (CH2), 27.8 (CH2), 23.9 (CH2). LC-MS: [M + H]+ 373, 375 . HRMS: [M + Na]+ 395.0473. Calcd for C17H1779BrN4ONa+: 395.0478. Δ = −1.3.3-(2-(2-(3-Methoxyazetidin-1-yl)pyrimidin-4-yl)ethyl)-1H-indole (31). A mixture of 3-(2-(2-chloropyrimidin-4-yl)ethyl)-1H-indole 11 , 3-methoxyazetidine hydrochloride  and anhydrous sodium carbonate  in ethanol (3 mL) was stirred and held at reflux for 8 h and allowed to cool to room temperature. The solvent was removed in vacuo and the residues partitioned between dichloromethane and water. The organic layer was separated, the solvent removed in vacuo and the residues subjected to column chromatography on silica. Elution with 0–100% ethyl acetate in petroleum ether (b.p. 40–60 °C) afforded 3-(2-(2-(3-methoxyazetidin-1-yl)pyrimidin-4-yl)ethyl)-1H-indole 31  as an off-white solid. 1H NMR: δ 10.76 , 8.20 , 7.52 , 7.32 , 7.12 , 7.06 , 6.97 , 6.62 , 4.30 , 4.21 , 3.83 , 3.25 , 3.05 , 2.91 . 13C NMR: δ 171.0 (C), 163.3 (C), 158.0 (CH), 136.7 (C), 127.5 (C), 122.8 (CH), 121.4 (CH), 18.7 (CH), 118.6 (CH), 114.1 (C), 111.8 (CH), 110.1 (CH), 69.9 (CH), 57.3 (CH2), 55.8 (CH3), 38.1 (CH2), 24.1 (CH2). LC-MS: [M + H]+ 309 . HRMS: [M + H]+ 309.1720. Calcd for C18H21N4O+: 309.1710. Δ = +3.2.5-Chloro-3-(2-(2-(3-methoxyazetidin-1-yl)pyrimidin-4-yl)ethyl)-1H-indole (32). Prepared as per the method for 31 from 5-chloro-3-(2-(2-chloropyrimidin-4-yl)ethyl)-1H-indole 12 and 3-methoxyazetidine hydrochloride. Yield 72%. Pale yellow solid. 1H NMR: δ 10.97 , 8.19 , 7.53 , 7.33 , 7.20 , 7.05 , 6.61 , 4.29 , 4.20 , 3.83 , 3.26 , 3.03 , 2.89 . 13C NMR: δ 170.9 (C), 163.3 (C), 158.0 (CH), 135.1 (C), 128.7 (C), 124.8 (CH), 123.4 (C), 121.3 (CH), 118.1 (CH), 114.2 (C), 113.3 (CH), 110.2 (CH), 69.9 (CH), 57.3 (CH2), 55.8 (CH3), 38.0 (CH2), 23.7 (CH2). LC-MS: [M + H]+ 343, 345 . HRMS: [M + H]+ 343.1312. Calcd for C18H2035ClN4O+: 343.1320. Δ = −2.4.5-Bromo-3-(2-(2-(3-methoxyazetidin-1-yl)pyrimidin-4-yl)ethyl)-1H-indole (33). Prepared as per the method for 31 from 5-bromo-3-(2-(2-chloropyrimidin-4-yl)ethyl)-1H-indole 13 and 3-methoxyazetidine hydrochloride. Yield 82%. Off-white solid. 1H NMR: δ 10.99 , 8.19 , 7.66 , 7.29 , 7.19 , 7.16 , 6.61 , 4.30 , 4.20 , 3.83 , 3.25 , 3.03 , 2.89 . 13C NMR: δ 170.9 (C), 163.3 (C), 157.9 (CH), 135.3 (C), 129.5 (C), 124.6 (CH), 123.8 (CH), 121.1 (CH), 114.1 (C), 113.8 (CH), 111.3 (C), 110.2 (CH), 69.9 (CH), 57.3 (CH2), 55.8 (CH3), 38.1 (CH2), 23.7 (CH2). LC-MS: [M + H]+ 387, 389 . HRMS: [M + H]+ 387.0802. Calcd for C18H2079BrN4O+: 387.0815. Δ = −3.4.3-(2-(2-(4-(Trifluoromethoxy)phenyl)pyrimidin-4-yl)ethyl)-1H-indole (34). A mixture of 3-(2-(2-chloropyrimidin-4-yl)ethyl)-1H-indole 11 , (4-(trifluoromethoxy)phenyl)-boronic acid , -dichloropalladium(II)  and anhydrous sodium carbonate  in water (0.5 mL), ethanol (1 mL) and toluene (2 mL) was degassed with nitrogen for 5 min and stirred and held at 90 °C in a sealed tube for 6 h and allowed to cool to room temperature. The mixture was diluted with ethyl acetate and water, the organic layer was separated, the solvent removed in vacuo and the residues subjected to column chromatography on silica. Elution with 0–50% ethyl acetate in petroleum ether (b.p. 40–60 °C) afforded 3-(2-(2-(4-(trifluoromethoxy)phenyl)pyrimidin-4-yl)ethyl)-1H-indole 34  as a pale yellow solid. 1H NMR: δ 10.77 , 8.76 , 8.53 , 7.57 , 7.52 , 7.37 , 7.33 , 7.14 , 7.07 , 6.97 , 3.21 . 13C NMR: δ 171.0 (C), 162.2 (C), 157.9 (CH), 150.6 , 137.0 (C), 136.7 (C), 130.2 (CH), 127.5 (C), 122.9 (CH), 121.5 (CH), 121.4 (CH), 120.5 , 119.6 (CH), 118.8 (CH), 118.7 (CH), 113.9 (C), 111.8 (CH), 38.2 (CH2), 24.1 (CH2). 19F NMR: δ −56.6 (OCF3). LC-MS: [M + H]+ 384 . HRMS: [M − H]- 382.1164. Calcd for C21H15F3N3O−: 382.1172. Δ = −2.1.2-Chloro-3-(2-(2-(4-(trifluoromethoxy)phenyl)pyrimidin-4-yl)ethyl)-1H-indole (35). A stirred solution of 3-(2-(2-(4-(trifluoromethoxy)phenyl)pyrimidin-4-yl)ethyl)-1H-indole 34  in tetrahydrofuran (2 mL) was treated with N-chlorosuccinimide  and the resulting mixture was stirred and held at reflux for 5 h and allowed to cool to room temperature. The solvent was removed in vacuo and the residues partitioned between dichloromethane and water. The organic layer was separated, the solvent removed in vacuo and the residues subjected to column chromatography on silica. Elution with 0–30% ethyl acetate in petroleum ether (b.p. 40–60 °C) afforded 2-chloro-3-(2-(2-(4-(trifluoromethoxy)phenyl)-pyrimidin-4-yl)ethyl)-1H-indole 35  as a pale orange solid. 1H NMR: δ 11.59 , 8.73 , 8.49 , 7.52 , 7.51 , 7.26 , 7.25 , 7.10 , 7.00 , 3.19 , 3.12 . 13C NMR: δ 170.3 (C), 162.2 (C), 157.9 (CH), 150.6 , 137.0 (C), 135.0 (C), 130.2 (CH), 127.2 (C), 122.0 (CH), 121.4 (CH), 121.1 (C), 120.5 , 119.7 (CH), 119.6 (CH), 118.4 (CH), 111.3 (CH), 109.6 (C), 37.6 (CH2), 23.0 (CH2). 19F NMR: δ −56.6 (OCF3). LC-MS: [M + H]+ 418, 420 . HRMS: [M − H]- 416.0781. Calcd for C21H1435ClF3N3O−: 416.0783. Δ = −0.5.2-Bromo-3-(2-(2-(4-(trifluoromethoxy)phenyl)pyrimidin-4-yl)ethyl)-1H-indole (36). A stirred solution of 3-(2-(2-(4-(trifluoromethoxy)phenyl)pyrimidin-4-yl)ethyl)-1H-indole 34  in dichloromethane (2 mL) was treated with N-bromosuccinimide  and the resulting mixture was stirred and held at room temperature for 4 h. The mixture was diluted with dichloromethane and water, the organic layer was separated, the solvent removed in vacuo and the residues subjected to column chromatography on silica. Elution with 0–30% ethyl acetate in petroleum ether (b.p. 40–60 °C) afforded 2-bromo-3-(2-(2-(4-(trifluoromethoxy)phenyl)pyrimidin-4-yl)ethyl)-1H-indole 36  as a pale pink solid. 1H NMR: δ 11.59 , 8.73 , 8.50 , 7.52 , 7.51 , 7.27 , 7.24 , 7.08 , 6.98 , 3.18 , 3.11 . 13C NMR: δ 170.3 (C), 162.2 (C), 157.9 (CH), 150.6 , 137.0 (C), 136.6 (C), 130.3 (CH), 127.4 (C), 122.0 (CH), 121.4 (CH), 120.5 , 119.7 (CH), 119.6 (CH), 118.3 (CH), 112.8 (C), 111.2 (CH), 109.1 (C), 37.8 (CH2), 24.0 (CH2). 19F NMR: δ −56.6 (OCF3). LC-MS: [M + H]+ 462, 464 . HRMS: [M − H]- 460.0270. Calcd for C21H1479BrF3N3O−: 460.0278. Δ = −1.8.3-(2-(2-(Pyridin-2-yl)pyrimidin-4-yl)ethyl)-1H-indole hydrochloride (37). A mixture of 3-(2-(2-chloropyrimidin-4-yl)ethyl)-1H-indole 11 , 4-methyl-8-(pyridin-2-yl)dihydro-4λ [4],8λ [4]-oxazaborolooxazaborole-2,6-dione , tris(dibenzylideneacetone)dipalladium(0) , 2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl (XPhos) , copper(II) acetate  and anhydrous potassium carbonate  in N,N-dimethylformamide (3 mL) and isopropanol (0.8 mL) + 401 . HRMS: [M + H]+ 401.1972. Calcd for C24H25N4O2+: 401.1972. Δ = 0.tert-butyl 3-(2-(2-(pyridin-2-yl)pyrimidin-4-yl)ethyl)-1H-indole-1-carboxylate 38  in diethyl ether (2 mL) and methanol (1 mL) and the mixture was stirred and held at 45 °C for 3 h and allowed to cool to room temperature. The solvent was removed in vacuo and the residues triturated with diethyl ether to afford 3-(2-(2-(pyridin-2-yl)pyrimidin-4-yl)ethyl)-1H-indole hydrochloride 37  as a brown solid. 1H NMR: δ 10.82 , 8.95 , 8.92 , 8.77 , 8.54 , 8.01 , 7.64 , 7.58 , 7.34 , 7.16 , 7.07 , 6.98 , 3.28 . 13C NMR: δ 172.3 (C), 158.2 (CH), 157.7 (C), 148.9 (C), 145.7 (CH), 145.2 (CH), 136.7 (C), 128.4 (CH), 127.5 (C), 125.7 (CH), 123.1 (CH), 122.4 (CH), 121.4 (CH), 118.8 (CH), 118.7 (CH), 113.6 (C), 111.9 (CH), 38.1 (CH2), 24.1 (CH2). LC-MS: [M + H]+ 301 . HRMS: [M + Na]+ 323.1271. Calcd for C19H16N4Na+: 323.1267. Δ = +1.2.Hydrogen chloride (2 M in diethyl ether) (0.5 mL) was added to a stirred solution of 5-Fluoro-3-(2-(pyridin-2-yl)ethyl)-1H-indole (39). Prepared as per the method for 8 + 241 (1.25 100). HRMS: [M + Na]+ 263.0953. Calcd for C15H13FN2Na+: 263.0955. Δ = −0.8.od for 8  from 2-v5-Iodo-3-(2-(pyridin-2-yl)ethyl)-1H-indole (40). Prepared as per the method for 8 + 349 . HRMS: [M + H]+ 349.0183. Calcd for C15H14IN2+: 349.0196. Δ = −3.8.od for 8  from 2-v5-Iodo-3-(2-(pyridin-4-yl)ethyl)-1H-indole (42). Prepared as per the method for 8 + 349 . HRMS: [M + H]+ 349.0207. Calcd for C15H14IN2+: 349.0196. Δ = +3.1.od for 8  from 4-v3-(2-(Pyridin-4-yl)ethyl)-5-(trifluoromethoxy)-1H-indole (44). Prepared as per the method for 8 + 307 . HRMS: [M + H]+ 307.1061. Calcd for C16H14F3N2O+: 307.1053. Δ = +2.6.od for 8  from 4-v3-(2-(2-Chloropyrimidin-4-yl)ethyl)-5-fluoro-1H-indole: (46). Prepared as per the method for 11 - 274, 276 . HRMS: [M − H]- 274.0544. Calcd for C14H1035ClFN3−: 274.0553. Δ = −3.3.d for 11  from 2-cd for 11  and 5-fl3-(2-(2-Chloropyrimidin-4-yl)ethyl)-5-methoxy-1H-indole (47). Prepared as per the method for 11 - 286, 288 . HRMS: [M + Na]+ 310.0718. Calcd for C15H1435ClN3ONa+: 310.0717. Δ = +0.3.d for 11  from 2-cd for 11  and 5-me3-(2-(2-Chloropyrimidin-4-yl)ethyl)-5-(trifluoromethoxy)-1H-indole (48). Prepared as per the method for 11 - 340, 342 . HRMS: [M − H]- 340.0457. Calcd for C15H1035ClF3N3O−: 340.0470. Δ = −3.9.d for 11  from 2-cd for 11  and 5-(t5-(Benzyloxy)-3-(2-(2-chloropyrimidin-4-yl)ethyl)-1H-indole (49). Prepared as per the method for 11 + 364, 366 . HRMS: [M + Na]+ 386.1031. Calcd for C21H1835ClN3ONa+: 386.1031. Δ = 0.d for 11  from 2-cd for 11  and 5-(b3-(2-(2-Chloropyrimidin-4-yl)ethyl)-1-methyl-1H-indole (50). Prepared as per the method for 11 + 272, 274 . HRMS: [M + Na]+ 294.0767. Calcd for C15H1435ClN3Na+: 294.0769. Δ = −0.7.d for 11  from 2-cd for 11  and 1-me4-(4-(2-(5-Fluoro-1H-indol-3-yl)ethyl)pyrimidin-2-yl)morpholine (51). Prepared as per the method for 14 from 3-(2-(2-chloropyrimidin-4-yl)ethyl)-5-fluoro-1H-indole 46 and morpholine. Yield 83%. Pale yellow solid. 1H NMR: δ 10.86 , 8.23 , 7.31 , 7.26 , 7.20 , 6.89 , 6.60 , 3.70 , 3.65 , 3.05 , 3.05 . 13C NMR: δ 170.8 (C), 161.8 (C), 158.0 (CH), 157.1 , 133.3 (C), 127.8 , 125.0 (CH), 114.5 , 112.7 , 110.1 (CH), 109.4 , 103.4 , 66.5 (CH2), 44.3 (CH2), 38.1 (CH2), 23.8 (CH2). 19F NMR: δ −125.7. LC-MS: [M + H]+ 327 . HRMS: [M + H]+ 327.1618. Calcd for C18H20FN4O+: 327.1616. Δ = +0.6.4-(4-(2-(5-Methoxy-1H-indol-3-yl)ethyl)pyrimidin-2-yl)morpholine (52). Prepared as per the method for 14 from 3-(2-(2-chloropyrimidin-4-yl)ethyl)-5-methoxy-1H-indole 47 and morpholine. Yield 81%. Yellow solid. 1H NMR: δ 10.59 , 8.23 , 7.21 , 7.07 , 6.94 , 6.70 , 6.60 , 3.75 , 3.70 , 3.65 , 3.05 , 2.92 . 13C NMR: δ 171.1 (C), 161.9 (C), 158.0 (CH), 153.4 (C), 131.9 (C), 127.9 (C), 123.5 (CH), 114.0 (C), 112.4 (CH), 111.5 (CH), 110.1 (CH), 100.6 (CH), 66.5 (CH2), 55.8 (CH3), 44.3 (CH2), 38.2 (CH2), 24.0 (CH2). LC-MS: [M + H]+ 339 . HRMS: [M + H]+ 339.1810. Calcd for C19H23N4O2+: 339.1815. Δ = −1.5.4-(4-(2-(5-(Trifluoromethoxy)-1H-indol-3-yl)ethyl)pyrimidin-2-yl)morpholine (53). Prepared as per the method for 14 from 3-(2-(2-chloropyrimidin-4-yl)ethyl)-5-(trifluoromethoxy)-1H-indole 48 and morpholine. Yield 77%. Pale yellow solid. 1H NMR: δ 11.06 , 8.22 , 7.44 , 7.40 , 7.27 , 7.02 , 6.59 , 3.69 , 3.65 , 3.08 , 2.92 . 13C NMR: δ 170.7 (C), 161.8 (C), 158.0 (CH), 141.8 , 135.1 (C), 127.7 (C), 125.4 (CH), 120.9 , 115.0 (C), 114.9 (CH), 112.8 (CH), 111.2 (CH), 110.1 (CH), 66.5 (CH2), 44.3 (CH2), 38.2 (CH2), 23.6 (CH2). 19F NMR: δ −56.8. LC-MS: [M + H]+ 393 . HRMS: [M + H]+ 393.1534. Calcd for C19H20F3N4O2+: 393.1533. Δ = +0.2.4-(4-(2-(5-(Benzyloxy)-1H-indol-3-yl)ethyl)pyrimidin-2-yl)morpholine (54). Prepared as per the method for 14 from 5-(benzyloxy)-3-(2-(2-chloropyrimidin-4-yl)ethyl)-1H-indole 44 and morpholine. Yield 72%. Off-white solid. 1H NMR: δ 10.62 , 8.24 , 7.48 , 7.40 , 7.32 , 7.22 , 7.08 , 7.06 , 6.78 , 6.59 , 5.07 , 3.70 , 3.64 , 3.03 , 2.90 . 13C NMR: δ 171.0 (C), 161.8 (C), 158.0 (CH), 152.4 (C), 138.3 (C), 132.0 (C), 128.8 (CH), 128.1 (CH), 128.0 (CH), 127.8 (C), 123.6 (CH), 114.0 (C), 112.4 (CH), 112.1 (CH), 110.1 (CH), 102.2 (CH), 70.3 (CH2), 66.5 (CH2), 44.3 (CH2), 38.3 (CH2), 24.0 (CH2). LC-MS: [M + H]+ 415 . HRMS: [M + H]+ 415.2133. Calcd for C25H27N4O2+: 415.2129. Δ = +0.9.3-(2-(2-Morpholinopyrimidin-4-yl)ethyl)-1H-indol-5-ol (55). A mixture of 4-(4-(2-(5-(benzyloxy)-1H-indol-3-yl)ethyl)pyrimidin-2-yl)morpholine 54 , 10% palladium on carbon (50 mg) in methanol (5 mL) and water (0.5 mL) was degassed with nitrogen for 5 min and then stirred and held at room temperature under an atmosphere of hydrogen gas for 2 h. The mixture was filtered, the catalyst rinsed with methanol (5 mL) and the organic solvent removed in vacuo. The residues were partitioned between dichloromethane and water, the organic layer separated, and the solvent removed in vacuo to afford 3-(2-(2-morpholinopyrimidin-4-yl)ethyl)-1H-indol-5-ol 55  as a colorless foam. 1H NMR: δ 10.44 , 8.59 , 8.25 , 7.11 , 7.01 , 6.83 , 6.60 , 6.59 , 3.71 , 3.66 , 2.98 , 2.89 . 13C NMR: δ 171.0 (C), 161.8 (C), 158.0 (CH), 150.6 (C), 131.3 (C), 128.2 (C), 123.3 (CH), 113.2 (C), 112.2 (CH), 111.7 (CH), 110.0 (CH), 102.7 (CH), 66.5 (CH2), 44.3 (CH2), 38.1 (CH2), 24.1 (CH2). LC-MS: [M + H]+ 325 . HRMS: [M + H]+ 325.1658. Calcd for C18H21N4O2+: 325.1659. Δ = −0.4.4-(4-(2-(1H-Indol-3-yl-5-d)ethyl)pyrimidin-2-yl)morpholine (56). A mixture of 4-(4-(2-(5-bromo-1H-indol-3-yl)ethyl)pyrimidin-2-yl)morpholine 21 , 10% palladium on carbon (50 mg) and 3 M aqueous potassium hydroxide  in methanol (5 mL) was degassed with nitrogen for 5 min and then stirred and held at room temperature under an atmosphere of deuterium gas for 1 h. The mixture was filtered, the catalyst rinsed with methanol (10 mL) and the organic solvent removed in vacuo. The residues were partitioned between dichloromethane and water, the organic layer separated, the solvent removed in vacuo and the residues subjected to column chromatography on silica. Elution with 0–100% ethyl acetate in petroleum ether (b.p. 40–60 °C) afforded 4-(4-(2-(1H-indol-3-yl-5-d)ethyl)pyrimidin-2-yl)morpholine 56  as a colorless solid. 1H NMR: δ 10.75 , 8.24 , 7.53 , 7.33 , 7.11 , 7.06 , 6.61 , 3.70 , 3.66 , 3.08 , 2.93 . 13C NMR: δ 170.9 (C), 161.8 (C), 158.0 (CH), 136.7 (C), 127.5 (C), 122.8 (CH), 121.3 (CH), 118.6 (CH), 114.2 (C), 111.8 (CH), 110.0 (CH), 66.5 (CH2), 44.3 (CH2), 38.2 (CH2), 23.9 (CH2) [one signal missing]. LC-MS: [M + H]+ 310 . HRMS: [M + H]+ 310.1774. Calcd for C18H20DN4O+: 310.1772. Δ = +0.6.4-(4-(2-(1-Methyl-1H-indol-3-yl)ethyl)pyrimidin-2-yl)morpholine (57). Prepared as per the method for 14 from 3-(2-(2-chloropyrimidin-4-yl)ethyl)-1-methyl-1H-indole 50 and morpholine. Yield 84%. Off-white solid. 1H NMR: δ 8.24 , 7.54 , 7.37 , 7.13 , 7.11 , 7.01 , 6.61 , 3.72 , 3.70 , 3.65 , 3.07 , 2.92 . 13C NMR: δ 170.8 (C), 161.8 (C), 158.0 (CH), 137.1 (C), 127.9 (C), 127.3 (CH), 121.5 (CH), 119.0 (CH), 118.7 (CH), 113.6 (C), 110.0 (CH), 110.0 (CH), 66.5 (CH2), 44.4 (CH2), 38.3 (CH2), 32.7 (CH3), 23.8 (CH2). LC-MS: [M + H]+ 323 . HRMS: [M + H]+ 323.1864. Calcd for C19H23N4O+: 323.1866. Δ = −0.7.3-(4-(2-(1H-Indol-3-yl)ethyl)pyrimidin-2-yl)-8-oxa-3-azabicyclo[3.2.1]octane (58). Prepared as per the method for 31 from 3-(2-(2-chloropyrimidin-4-yl)ethyl)-1H-indole 11 and 8-oxa-3-azabicyclo[3.2.1]octane hydrochloride. Yield 60%. Off-white solid. 1H NMR: δ 10.75 , 8.20 , 7.52 , 7.32 , 7.11 , 7.06 , 6.96 , 6.58 , 4.40 , 4.22 , 3.07 , 3.03 , 2.92 , 1.81 , 1.63 . 13C NMR: δ 170.7 (C), 162.6 (C), 157.8 (CH), 136.7 (C), 127.6 (C), 122.8 (CH), 121.3 (CH), 118.7 (CH), 118.6 (CH), 114.2 (C), 111.8 (CH), 109.9 (CH), 73.4 (CH), 49.9 (CH2), 38.2 (CH2), 28.0 (CH2), 23.9 (CH2). LC-MS: [M + H]+ 335 . HRMS: [M + H]+ 335.1873. Calcd for C20H23N4O+: 335.1867. Δ = +1.8.3-(4-(2-(5-Fluoro-1H-indol-3-yl)ethyl)pyrimidin-2-yl)-8-oxa-3-azabicyclo[3.2.1]octane (59). Prepared as per the method for 31 from 3-(2-(2-chloropyrimidin-4-yl)ethyl)-5-fluoro-1H-indole 46 and 8-oxa-3-azabicyclo[3.2.1]octane hydrochloride. Yield 92%. Yellow oil. 1H NMR: δ 10.86 , 8.19 , 7.30 , 7.26 , 7.20 , 6.89 , 6.58 , 4.40 , 4.22 , 3.04 , 3.02 , 2.90 , 1.80 , 1.63 . 13C NMR: δ 170.6 (C), 162.6 (C), 157.8 (CH), 157.0 , 133.3 (C), 127.8 , 125.0 (CH), 114.5 , 112.6 , 109.9 (CH), 109.4 , 103.4 , 73.4 (CH), 49.9 (CH2), 38.1 (CH2), 28.0 (CH2), 23.8 (CH2). 19F NMR: δ −125.7. LC-MS: [M + H]+ 353 . HRMS: [M + H]+ 353.1775. Calcd for C20H22FN4O+: 353.1772. Δ = +0.8.4-(4-(2-(1H-Indol-3-yl)ethyl)pyrimidin-2-yl)-1,4-oxazepane (60). Prepared as per the method for 31 from 3-(2-(2-chloropyrimidin-4-yl)ethyl)-1H-indole 11 and 1,4-oxazepane. Yield 88%. Colorless solid. 1H NMR: δ 10.75 , 8.19 , 7.51 , 7.32 , 7.10 , 7.06 , 6.96 , 6.53 , 3.85 , 3.70 , 3.59 , 3.07 , 2.92 , 1.85 . 13C NMR: δ 170.8 (C), 161.5 (C), 158.0 (CH), 136.7 (C), 127.6 (C), 122.7 (CH), 121.3 (CH), 118.7 (CH), 118.6 (CH), 114.2 (C), 111.8 (CH), 109.1 (CH), 69.6 (CH2), 69.5 (CH2), 49.3 (CH2), 45.6 (CH2), 38.3 (CH2), 29.6 (CH2), 24.0 (CH2). LC-MS: [M + H]+ 323 . HRMS: [M + H]+ 323.1869. Calcd for C19H23N4O+: 323.1866. Δ = +0.9.4-(4-(2-(1H-Indol-3-yl)ethyl)pyrimidin-2-yl)-3,4-dihydro-2H-benzo[b]oxazine (61). A mixture of 3-(2-(2-chloropyrimidin-4-yl)ethyl)-1H-indole 11  and 3,4-dihydro-2H-benzo[b]oxazine (0.1 mL) in ethanol (0.5 mL) was stirred and held at 120 °C in a sealed tube for 3 h and allowed to cool to room temperature. The solvent was removed in vacuo and the residues partitioned between dichloromethane and water. The organic layer was separated, the solvent removed in vacuo and the residues subjected to column chromatography on silica. Elution with 0–100% ethyl acetate in petroleum ether (b.p. 40–60 °C) afforded 4-(4-(2-(1H-Indol-3-yl)ethyl)pyrimidin-2-yl)-3,4-dihydro-2H-benzo[b]oxazine 61  as a colourless solid. 1H NMR: δ 10.76 , 8.38 , 7.97 , 7.53 , 7.33 , 7.10 , 7.07 , 6.98 , 6.94 , 6.87 , 6.85 , 6.81 , 4.24 , 4.19 , 3.13 , 3.02 . 13C NMR: δ 171.2 (C), 159.6 (C), 158.0 (CH), 146.4 (C), 136.8 (C), 127.9 (C), 127.5 (C), 124.3 (CH), 123.9 (CH), 122.8 (CH), 121.4 (CH), 119.8 (CH), 118.7 (CH), 118.6 (CH), 117.0 (CH), 114.0 (C), 112.7 (CH), 111.8 (CH), 65.9 (CH2), 42.4 (CH2), 38.1 (CH2), 24.0 (CH2). LC-MS: [M + H]+ 357 . HRMS: [M + H]+ 357.1528. Calcd for C22H21N4O+: 357.1529. Δ = −0.3.4-(4-(2-(5-Fluoro-1H-indol-3-yl)ethyl)pyrimidin-2-yl)-3,4-dihydro-2H-benzo[b]oxazine (62). Prepared as per the method for 61 from 3-(2-(2-chloropyrimidin-4-yl)ethyl)-5-fluoro-1H-indole 62 and 3,4-dihydro-2H-benzo[b]oxazine. Yield 58%. Yellow oil. 1H NMR: δ 10.87 , 8.38 , 7.95 , 7.32 , 7.26 , 7.18 , 6.94 , 6.90 , 6.87 , 6.85 , 6.81 , 4.24 , 4.19 , 3.09 , 3.00 . 13C NMR: δ 171.1 (C), 159.5 (C), 158.0 (CH), 157.1 , 146.3 (C), 133.4 (C), 127.9 (C), 127.8 , 125.1 (CH), 124.3 (CH), 123.9 (CH), 119.7 (CH), 117.0 (CH), 114.3 , 112.7 , 112.6 (CH), 109.4 , 103.4 , 65.9 (CH2), 42.4 (CH2), 38.0 (CH2), 23.8 (CH2). 19F NMR: δ −125.6. LC-MS: [M + H]+ 375 . HRMS: [M + H]+ 375.1616. Calcd for C22H20FN4O+: 375.1616. Δ = 0.4-(4-(2-(1H-Indol-3-yl)ethyl)pyrimidin-2-yl)-6-bromo-3,4-dihydro-2H-benzo[b]oxazine (63). A mixture of 3-(2-(2-chloropyrimidin-4-yl)ethyl)-1H-indole 11  and 6-bromo-3,4-dihydro-2H-benzo[b]oxazine  in ethanol (0.5 mL) was stirred and held at 160 °C with microwave irradiation for 1 h and allowed to cool to room temperature. The solvent was removed in vacuo and the residues partitioned between dichloromethane and water. The organic layer was separated, the solvent removed in vacuo and the residues subjected to column chromatography on silica. Elution with 0–60% ethyl acetate in petroleum ether (b.p. 40–60 °C) afforded 4-(4-(2-(1H-indol-3-yl)ethyl)pyrimidin-2-yl)-6-bromo-3,4-dihydro-2H-benzo[b]oxazine 63  as a pale orange solid. 1H NMR: δ 10.77 , 8.45 , 8.44 , 7.57 , 7.33 , 7.13 , 7.11 , 7.06 , 6.97 , 6.91 , 6.87 , 4.27 , 4.21 , 3.16 , 3.05 . 13C NMR: δ 171.2 (C), 159.2 (C), 158.2 (CH), 145.6 (C), 136.7 (C), 129.4 (C), 127.5 (C), 126.1 (CH), 126.0 (CH), 122.8 (CH), 121.4 (CH), 118.9 (CH), 118.8 (CH), 118.7 (CH), 114.0 (C), 113.3 (CH), 111.8 (CH), 111.0 (C), 65.8 (CH2), 42.2 (CH2), 38.1 (CH2), 23.9 (CH2). LC-MS: [M + H]+ 435, 437 . HRMS: [M + H]+ 435.0802. Calcd for C22H2079BrN4O+:435.0815. Δ = −3.0.1-(3-(2-(2-Morpholinopyrimidin-4-yl)ethyl)-1H-indol-1-yl)ethan-1-one (64). A solution of 4-(4-(2-(1H-indol-3-yl)ethyl)pyrimidin-2-yl)morpholine 14  in acetic anhydride (2 mL) was stirred and held at reflux for 32 h and allowed to cool to room temperature. The solvent was removed in vacuo and the residues partitioned between dichloromethane and an aqueous solution of sodium carbonate. The organic layer was separated, the solvent removed in vacuo and the residues subjected to column chromatography on silica. Elution with 0–100% ethyl acetate in petroleum ether (b.p. 40–60 °C) followed by rinsing of the solids with 25% diethyl ether in petroleum ether (b.p. 40–60 °C) afforded 1-(3-(2-(2-morpholinopyrimidin-4-yl)ethyl)-1H-indol-1-yl)ethan-1-one 64  as a pale yellow solid. 1H NMR: δ 8.31 , 8.27 , 7.68 , 7.63 , 7.33 , 7.28 , 6.66 , 3.70 , 3.65 , 3.07 , 2.99 , 2.61 . 13C NMR: δ 170.3 (C), 169.6 (CO), 161.8 (C), 158.2 (CH), 135.6 (C), 130.8 (C), 125.2 (CH), 124.1 (CH), 123.6 (CH), 121.4 (C), 119.5 (CH), 116.4 (CH), 109.9 (CH), 66.5 (CH2), 44.3 (CH2), 36.9 (CH2), 24.3 (CH3), 23.4 (CH2). LC-MS: [M + H]+ 351 . HRMS: [M + H]+ 351.1816. Calcd for C20H23N4O2+: 351.1816. Δ = 0.4-(4-(2-(1-(Methylsulphonyl)-1H-indol-3-yl)ethyl)pyrimidin-2-yl)morpholine (65). Methanesulphonyl chloride  in benzene (0.6 mL) was added dropwise to a rapidly stirred mixture of 4-(4-(2-(1H-indol-3-yl)ethyl)pyrimidin-2-yl)morpholine 14  and tetra-n-butylammonium hydrogen sulphate  in benzene (0.6 mL) and 50% w/w aqueous sodium hydroxide solution (0.6 mL) + 387 . HRMS: [M + H]+ 387.1481. Calcd for C19H23N4O332S+: 387.1486. Δ = −1.3.(0.6 mL)  and the tert-Butyl 4-morpholino-2-vinyl-5,7-dihydro-6H-pyrrolopyrimidine-6-carboxylate (70). A mixture of tert-butyl 2-chloro-4-morpholino-5,7-dihydro-6H-pyrrolopyrimidine-6-carboxylate 66 pyrimidine-6-carboxylate 70  as a colorless solid. 1H NMR: δ 6.60 , 6.41 , 5.62 , 4.73 , 4.39 , 4.37 , 3.67 , 1.46 . 13C NMR: δ 166.6 (C), 166.1 (C), 162.5 (C), 158.1 (CO), 137.3 (CH), 123.0 (CH2), 108.9 (C), 79.8 (C), 66.5 (CH2), 45.4 (CH2), 28.6 (CH3) [two signals missing]. LC-MS: [M + H]+ 333 . HRMS: [M + Na]+ 355.1741. Calcd for C17H24N4O3Na+: 355.1740. Δ = +0.2.ylate 66  -2-vinyl-5,7-dihydro-6H-pyrrolopyrimidine-6-carboxylate (71). Prepared as per the method for 70 from tert-butyl 4-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)-2-chloro-5,7-dihydro-6H-pyrrolopyrimidine-6-carboxylate 67 . LC-MS: [M + H]+ 359 . HRMS: [M + H]+ 359.2077. Calcd for C19H27N4O3+: 359.2078. Δ = −0.3.ylate 67  and 4,4,tert-Butyl 4-morpholino-2-vinyl-5,8-dihydropyridopyrimidine-7(6H)-carboxylate (72). Prepared as per the method for 70 from tert-butyl 2-chloro-4-morpholino-5,8-dihydropyridopyrimidine-7(6H)-carboxylate 68 . LC-MS: [M + H]+ 347 . HRMS: [M + H]+ 347.2069. Calcd for C18H27N4O3+: 347.2078. Δ = −2.6.ylate 68  and 4,4,tert-Butyl 4-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)-2-vinyl-5,8-dihydropyridopyrimidine-7(6H)-carboxylate (73). Prepared as per the method for 70 from tert-butyl 4-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)-2-chloro-5,8-dihydropyridopyrimidine-7(6H)-carboxylate 69 . LC-MS: [M + H]+ 373 . HRMS: [M + H]+ 373.2231. Calcd for C20H29N4O3+: 373.2234. Δ = −0.9.ylate 69  and 4,4,tert-Butyl (E)-3-vinyl)-1H-indole-1-carboxylate (74) + 370 . HRMS: [M + H–C4H8]+ 314.1552. Calcd for C17H21BNO4+: 314.1564. Δ = −3.9.ate (74) . Prepareborolane  from terboxylate  and 4,4,tert-Butyl (E)-2-(2-(1-(tert-butoxycarbonyl)-1H-indol-3-yl)vinyl)-4-morpholino-5,7-dihydro-6H-pyrrolopyrimidine-6-carboxylate (75). Prepared as per the method for 70 from tert-butyl 2-chloro-4-morpholino-5,7-dihydro-6H-pyrrolopyrimidine-6-carboxylate 66 and tert-butyl (E)-3-vinyl)-1H-indole-1-carboxylate 74. Yield 73%. Colorless solid. 1H NMR: δ 8.17 , 8.15 , 8.01 , 8.00 , 7.42 , 7.37 , 7.12 , 4.75 , 4.42 , 4.40 , 3.72 , 1.66 , 1.48 . 13C NMR: δ 166.6 (C), 166.1 (C), 163.3 (C), 158.2 (CO), 149.3 (CO), 136.0 (C), 128.6 (CH), 128.3 (C), 128.0 (CH), 127.8 (CH), 125.5 (CH), 124.0 (CH), 120.8 (CH), 117.9 (C), 117.8 (C), 115.5 (CH), 84.8 (C), 74.0 (C), 66.6 (CH2), 45.6 (CH2), 28.2 (CH3), 25.4 (CH3) [two signals missing]. LC-MS: [M + H]+ 548 . HRMS: [M + H]+ 548.2870. Calcd for C30H38N5O5+: 548.2873. Δ = −0.6.tert-Butyl (E)-4-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)-2-(2-(1-(tert-butoxycarbonyl)-1H-indol-3-yl)vinyl)-5,7-dihydro-6H-pyrrolopyrimidine-6-carboxylate (76). Prepared as per the method for 70 from tert-butyl 4-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)-2-chloro-5,7-dihydro-6H-pyrrolopyrimidine-6-carboxylate 67 and tert-butyl (E)-3-vinyl)-1H-indole-1-carboxylate 74. Yield 35%. Pale yellow solid. 1H NMR: δ 8.17 , 8.15 , 8.00 , 7.99 , 7.43 , 7.38 , 7.12 , 4.76 , 4.45 , 4.40 , 4.05 , 3.24 , 1.82 , 1.66 , 1.48 . 13C NMR: δ 167.5 (C), 166.2 (C), 163.5 (C), 159.4 (CO), 149.3 (CO), 136.1 (C), 128.7 (CH), 128.6 (CH), 128.0 (C), 127.8 (CH), 125.5 (CH), 124.0 (CH), 120.8 (CH), 117.9 (C), 115.5 (CH), 107.8 (C), 84.8 (C), 79.7 (C), 73.5 (CH), 50.8 (CH2), 28.6 (CH3), 28.2 (CH3) 27.9 (CH2) [two signals missing]. LC-MS: [M + H]+ 574 . HRMS: [M + H]+ 574.3029. Calcd for C32H40N5O5+: 574.3029. Δ = 0.tert-Butyl (E)-2-(2-(1-(tert-butoxycarbonyl)-1H-indol-3-yl)vinyl)-4-morpholino-5,8-dihydropyridopyrimidine-7(6H)-carboxylate (77). Prepared as per the method for 70 from tert-butyl 2-chloro-4-morpholino-5,8-dihydropyridopyrimidine-7(6H)-carboxylate 68 and tert-butyl (E)-3-vinyl)-1H-indole-1-carboxylate 74. Yield 35%. Tan solid. 1H NMR: δ 8.18 , 8.16 , 8.00 , 7.99 , 7.43 , 7.38 , 7.14 , 4.45 , 3.75 , 3.53 , 3.47 , 2.67 , 1.66 , 1.46 . 13C NMR: δ 164.6 (C), 161.4 (C), 161.0 (C), 154.2 (CO), 149.3 (CO), 136.0 (C), 128.4 (CH), 128.3 (C), 128.0 (CH), 127.8 (CH), 125.5 (CH), 124.1 (CH), 120.8 (CH), 117.8 (C), 115.5 (CH), 110.1 (C), 84.9 (C), 79.8 (C), 66.6 (CH2), 48.4 (CH2), 28.5 (CH3), 28.2 (CH3) [three signals missing]. LC-MS: [M + H]+ 562 . HRMS: [M + H]+ 562.3024. Calcd for C31H40N5O5+: 562.3029. Δ = −0.9.tert-Butyl 2-(2-(1-(tert-butoxycarbonyl)-1H-indol-3-yl)ethyl)-4-morpholino-5,7-dihydro-6H-pyrrolopyrimidine-6-carboxylate (78). A mixture of tert-butyl (E)-2-(2-(1-(tert-butoxycarbonyl)-1H-indol-3-yl)vinyl)-4-morpholino-5,7-dihydro-6H-pyrrolopyrimidine-6-carboxylate 75 , 10% palladium on carbon (30 mg) in ethyl acetate (5 mL) was degassed with nitrogen for 2 min and then stirred and held at room temperature under an atmosphere of hydrogen gas for 4 h. The mixture was filtered, the catalyst rinsed with ethyl acetate (5 mL) and the solvent removed in vacuo. The residues were partitioned between dichloromethane and water, the organic layer separated, the solvent removed in vacuo and the residues subjected to column chromatography on silica. Elution with 0–80% ethyl acetate in petroleum ether (b.p. 40–60 °C) afforded tert-butyl 2-(2-(1-(tert-butoxycarbonyl)-1H-indol-3-yl)ethyl)-4-morpholino-5,7-dihydro-6H-pyrrolopyrimidine-6-carboxylate 78  as a colorless solid. 1H NMR: δ 8.03 , 7.60 , 7.43 , 7.32 , 7.25 , 4.70 , 4.37 , 4.34 , 3.64 , 3.61 , 3.11 , 3.06 , 1.62 , 1.46 . 13C NMR: δ 168.5 (C), 166.5 (C), 166.1 (C), 158.2 (CO), 149.6 (CO), 135.2 (C), 130.7 (C), 124.8 (CH), 123.0 (CH), 122.9 (CH), 120.8 (C), 120.7 (C), 119.6 (CH), 115.1 (CH), 83.8 (C), 79.8 (C), 66.5 (CH2), 45.4 (CH2), 38.1 (CH2), 23.0 (CH2), 28.6 (CH3), 28.2 (CH3) [two signals missing]. LC-MS: [M + H]+ 550 . HRMS: [M + H]+ 550.3026. Calcd for C30H40N5O5+: 550.3029. Δ = −0.6.tert-Butyl 4-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)-2-(2-(1-(tert-butoxycarbonyl)-1H-indol-3-yl)ethyl)-5,7-dihydro-6H-pyrrolopyrimidine-6-carboxylate (79). Prepared as per the method for 78 from tert-butyl (E)-4-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)-2-(2-(1-(tert-butoxycarbonyl)-1H-indol-3-yl)vinyl)-5,7-dihydro-6H-pyrrolopyrimidine-6-carboxylate 76. Yield 52%. Off-white solid. 1H NMR: δ 8.02 , 7.59 , 7.43 , 7.32 , 7.24 , 4.70 , 4.37 , 4.33 , 3.92 , 3.12 , 3.04 , 1.78 , 1.66 , 1.62 , 1.46 . 13C NMR: δ 168.3 (C), 168.1 (C), 166.1 (C), 159.3 (CO), 149.6 (CO), 135.2 (C), 130.7 (C), 124.8 (CH), 123.1 (CH), 122.9 (CH), 119.7 (CH), 115.1 (CH), 107.7 (C), 107.3 (C), 83.9 (C), 79.7 (C), 73.3 (CH), 50.7 (CH2), 38.1 (CH2), 28.6 (CH3), 28.2 (CH3), 27.7 (CH2), 23.0 (CH2) [two signals missing]. LC-MS: [M + H]+ 576 . HRMS: [M + H]+ 576.3192. Calcd for C32H42N5O5+: 576.3186. Δ = +1.0.tert-Butyl 2-(2-(1-(tert-butoxycarbonyl)-1H-indol-3-yl)ethyl)-4-morpholino-5,8-dihydropyridopyrimidine-7(6H)-carboxylate80). Prepared as per the method for 78 from tert-butyl (E)-2-(2-(1-(tert-butoxycarbonyl)-1H-indol-3-yl)vinyl)-4-morpholino-5,8-dihydropyridopyrimidine-7(6H)-carboxylate 77. Yield 43%. Colorless solid. 1H NMR: δ 8.02 , 7.58 , 7.44 , 7.31 , 7.24 , 4.37 , 3.66 , 3.48 , 3.36 , 3.12 , 3.06 , 2.61 , 1.62 , 1.44 . 13C NMR: δ 166.1 (C), 166.0 (C), 164.4 (C), 154.1 (CO), 149.5 (CO), 135.2 (C), 130.8 (C), 124.8 (CH), 123.0 (CH), 122.9 (CH), 120.8 (C), 119.7 (CH), 115.2 (CH), 113.1 (C), 83.9 (C), 79.8 (C), 66.6 (CH2), 48.3 (CH2), 38.1 (CH2), 28.5 (CH3), 28.2 (CH3), 23.1 (CH2) [three signals missing]. LC-MS: [M + H]+ 564 . HRMS: [M + H]+ 564.3181. Calcd for C31H42N5O5+: 564.3186. Δ = −0.9.4-(2-(2-(1H-Indol-3-yl)ethyl)-6,7-dihydro-5H-pyrrolopyrimidin-4-yl)morpholine hydrochloride (81). Prepared as per the method for 37 from tert-butyl 2-(2-(1-(tert-butoxycarbonyl)-1H-indol-3-yl)ethyl)-4-morpholino-5,7-dihydro-6H-pyrrolopyrimidine-6-carboxylate 78. Yield 78%. Pale tan solid. 1H NMR: δ 10.84 , 10.57 , 7.55 , 7.34 , 7.15 , 7.07 , 6.98 , 4.70 , 4.44 , 3.74 , 3.68 , 3.18 . 13C NMR: δ 160.8 (C), 157.7 (C), 146.9 (C), 136.8 (C), 127.5 (C), 123.0 (CH), 121.4 (CH), 118.7 (CH), 118.7 (CH), 113.5 (C), 111.9 (CH), 106.8 (C), 66.4 (CH2), 49.4 (CH2), 48.4 (CH2), 46.2 (CH2), 37.1 (CH2), 23.2 (CH2). LC-MS: [M + H]+ 350 . HRMS: [M + H]+ 350.1980. Calcd for C20H24N5O+: 350.1981. Δ = −0.3.3-(2-(2-(1H-Indol-3-yl)ethyl)-6,7-dihydro-5H-pyrrolopyrimidin-4-yl)-8-oxa-3-azabicyclo[3.2.1]octane hydrochloride (82). Prepared as per the method for 37 from tert-butyl 4-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)-2-(2-(1-(tert-butoxycarbonyl)-1H-indol-3-yl)ethyl)-5,7-dihydro-6H-pyrrolopyrimidine-6-carboxylate 79. Yield 70%. Brown solid. 1H NMR: δ 10.83 , 10.47 , 7.54 , 7.33 , 7.14 , 7.07 , 6.98 , 4.70 , 4.41 , 4.03 , 3.29 , 3.16 , 1.80 , 1.62 . 13C NMR: δ 159.2 (C), 159.1 (C), 159.1 (C), 136.7 (C), 127.5 (C), 122.9 (CH), 121.4 (CH), 118.7 (CH), 118.7 (CH), 113.5 (C), 111.9 (CH), 106.8 (C), 73.4 (CH), 51.4 (CH2), 49.5 (CH2), 48.4 (CH2), 27.5 (CH2), 23.2 (CH2) [one signal missing]. LC-MS: [M + H]+ 376 . HRMS: [M + H]+ 376.2137. Calcd for C22H26N5O+: 376.2137. Δ = 0.4-(2-(2-(1H-Indol-3-yl)ethyl)-5,6,7,8-tetrahydropyridopyrimidin-4-yl)morpholine hydrochloride (83). Prepared as per the method for 37 from tert-butyl 2-(2-(1-(tert-butoxycarbonyl)-1H-indol-3-yl)ethyl)-4-morpholino-5,8-dihydropyridopyrimidine-7(6H)-carboxylate 80. Yield 75%. Tan solid. 1H NMR: δ 10.87 , 10.07 , 7.54 , 7.34 , 7.18 , 7.08 , 6.99 , 4.32 , 3.74 , 3.69 , 3.27 , 3.18 , 2.94 . 13C NMR: δ 159.1 (C), 155.6 (C), 153.1 (C), 136.7 (C), 127.4 (C), 123.1 (CH), 121.5 (CH), 118.9 (C), 118.8 (CH), 118.7 (CH), 111.9 (CH), 109.9 (C), 66.5 (CH2), 48.4 (CH2), 40.2 (CH2), 23.1 (CH2), 23.0 (CH2) [two signals missing]. LC-MS: [M + H]+ 364 . HRMS: [M + H]+ 364.2138. Calcd for C21H26N5O+: 364.2137. Δ = +0.2.4.26-tagged construct of CYP121A1 was also expressed and purified to homogeneity as reported previously [via a superloop onto a HisTrap FF affinity column  that had been equilibrated in 50 mM potassium phosphate, 50 mM KCl, 10% (v/v) glycerol, pH 8. After washing the column with twenty column volumes of equilibration buffer, CYP121A1 was eluted at 5 mL/min with equilibration buffer containing imidazole (using a stepped gradient of 0–200 mM imidazole in 40 mM steps) and collected as 4 mL fractions with monitoring for both protein and heme (respective absorbances at A280 and A417). The purity of fractions collected was assessed by SDS-PAGE gels. Pure fractions were combined and dialyzed overnight at 4 °C into fresh buffer  using a Slide-A-Lyser™ dialysis cassette , concentrated to around 500 μM in a bench top centrifuge  using a Vivaspin  ultrafiltration column , apportioned into small aliquots, flash frozen with liquid nitrogen and stored at −80 °C until required. Final protein concentrations were determined using a NanoDrop 2000c spectrophotometer  by monitoring for heme absorbance at 417 nm .Untagged CYP121A1 was expressed and purified to homogeneity as reported previously ,38. A Hieviously  with min4.3Untagged CYP121A1 was expressed and purified to homogeneity as described above with an additional final polishing step utilizing a HiLoad Superdex 75 preparative grade 16/600 . The column was equilibrated with 10 mM Tris, 100 mM KCl, pH 7.8 at 4 °C (adjusted with KOH/HCl) and CYP121A1 was eluted isocratically according to the manufacturer's instructions. Fractions with a calculated Reinheitszahl (Rz) ratio of ∼2 were pooled and concentrated using a Vivaspin 20 10,000 MWCO centrifugal concentrator . A modified form of the Rz ratio can be derived as follows:4)2SO4  as the precipitant and 1 M MES pH 5–6.5  as the buffer system in three lens microplates . Precipitant concentration and pH ranged between 1.5 and 2.5 M and 5–6.5 respectively. Sitting drops were set up with a Mosquito Crystal  using 20 mg/mL CYP121A1 in 1 μL drops with a 1:1 ratio of protein to mother liquor. Plates were incubated at 4 °C and crystallogenesis took between 3 and 7 days to occur. Mature crystals were approximately 1 mm in length with an arrowhead morphology. Crystals were either soaked with saturated compound solutions made up in fresh DMSO or solid compound was added directly to the drops. In both cases, 1 μL of mother liquor was removed from the reservoir and transferred to the drop containing crystals to prevent desiccation. During soaks with DMSO-solubilized compound, 1 μL of compound solution was introduced to the reservoir and thoroughly mixed before 0.5 μL was transferred to the drop, mixed and removed. The transfer process was performed a total of three times. Soaked crystals were periodically removed (up to ∼ 6 months), preserved in Parabar 10312  and flash-cooled in liquid nitrogen. Crystals were irradiated at the Diamond Light Source  using the i03, i04 or i04-1 beamlines using standard collection parameters. Data were automatically indexed, integrated, scaled and merged using the xia2 dials [The crystallization conditions identified previously  were usea2 dials  and xia2a2 dials ,42. Dataa2 dials , molecula2 dials  , 14 (7NQN) and 21 (7NQO) have been deposited with the RCSB Protein Data Bank (www.rcsb.org) and will be released upon publication.Accession codes and atomic coordinates for the X-ray structures of complexes of CYP121A1 with compounds 4.4Mycobacterium tuberculosis laboratory strain H37Rv was routinely cultured in Middlebrook 7H9 broth  supplemented with 10% (v/v) Albumin Dextrose Catalase (ADC) enrichment , 0.05% (v/v) tyloxapol and 0.02% (v/v) glycerol (c7H9). Liquid cultures were grown at 37 °C in 50 mL centrifugation tubes with rotation at 40 rpm until mid-exponential phase (OD600 ∼1). For drug susceptibility testing, two additional broth-based media were used: Middlebrook 7H9 broth with low BSA supplemented with 10% (v/v) of ADN enrichment (0.05% (w/v) Albumin, 2% (w/v) Dextrose, and 0.85% (w/v) NaCl), 0.05% (v/v) tyloxapol and 0.02% (v/v) glycerol (7H9-Low BSA) and Mycobacterial Minimal Medium with cholesterol consisting of 0.5 g/L l-asparagine, 1 g/L KH2PO4, 2.5 g/L Na2HPO4, 50 mg/L ferric ammonium citrate, 0.5 g/L MgSO47H2O, 0.5 mg/L CaCl2, 0.1 mg/mL ZnSO4, 0.2% (v/v) tyloxapol, 0.2% (v/v) ethanol and 0.01% (v/v) cholesterol (MMM-Ch). Prior to the antimycobacterial activity testing, Mtb cultures were pre-adapted in the different test conditions by washing 1 mL of culture suspension twice with c7H9, 7H9-Low BSA or MMM-Ch and resuspended in 10 mL of the same medium. Bacterial cultures were then incubated at 37 °C in 30 mL square bottles  and allowed to stand for eight days. Compounds in powder form were dissolved in DMSO at 50 mM prior to assay. A modified resazurin based colorimetric assay was performed in a 96 well plate format in the three different media . Compounds were serially diluted column-wise to give a range of testing concentrations from 400 μM to 0.78 μM. Quality controls of drug only and bacteria only wells were also included with every plate. Rifampicin (1st line anti-TB drug) and DMSO controls were also included. Plates were inoculated with about 105 CFUs of bacteria, sealed and incubated at 37 °C for one week (for c7H9 and 7H9-Low BSA media) or two weeks (for MMM-Ch medium). Following incubation, a solution containing 0.02% (w/v) resazurin dye  was added to all the wells and incubated for a further day. Minimum inhibition concentrations (MIC90) were calculated using visual determination of the lowest concentration of drug at which there was no change in the resazurin colour.4.5Interactions of compounds with untagged CYP121A1 were analyzed by UV–Visible spectroscopy. Protein fractions with Rz ratio of ∼2 were used for titrations. Compound stock solutions were made up to 30 mM in fresh DMSO. Titrations with clear colorless compound solutions were performed at 28 °C using either a single-beam Cary 60 UV–Visible spectrophotometer  or a dual-beam Cary 300 Bio UV–Visible spectrophotometer  recording between 240 and 800 nm. Temperature control was achieved using a Cary single or dual cell peltier accessory  and a Julabo AWC100 recirculating cooling bath . Titrations were performed in 1 cm path length quartz cuvettes  using a matched pair when necessary. A solution of CYP121A1  was used per titration in sterile-filtered 100 mM HEPES, 100 mM KCl, 0.005% Tween-20, pH 7.8 at 28 °C (adjusted with KOH/HCl) to a final volume of 1 mL. Final DMSO concentrations were kept below 1% v/v. Prior to each titration, a baseline correction and zero was performed with buffer alone. Following this, CYP12A1 was added and the cuvette was incubated for 5 min to allow for temperature equilibration. A compound-free absorbance spectrum was recorded and then small volumes of compound  were introduced using a Hamilton syringe fitted with a syringe guide . Following each compound addition an absorbance spectrum was recorded and this was repeated until no further spectral changes were observed. The data were baseline corrected and the difference plot  at 25 °C. Ligands were initially prepared as 25 mM stock solutions in DMSO‑d6 (and further diluted into DMSO‑d6 when necessary). Both ligands and CYP121A1 were diluted into identical buffer  to generate mixtures containing 10% (v/v) DMSO‑d6, a final ligand concentration of either 0.5 or 2.5 mM (depending on ligand solubility) and a CYP121A1 concentration of 50 μM. Ligand titrations consisted of a small (0.2 μL) initial injection (that was discarded during data processing) followed by nineteen further injections (each of 2 μL) at 120 s intervals. Control titrations were also performed (adding ligand into buffer in the absence of protein) to measure any heats of dilution or buffer mismatch and were subtracted from ligand titrations during data processing. Titration isotherms were integrated to afford the enthalpy change of each injection and were plotted against the molar ratio of added ligand. Titrations were fitted using a one-site binding model using Origin Analysis Software by setting the stoichiometry (N) to one (for weak binding compounds) or allowing stoichiometry to vary (for more potent compounds).ITC experiments to measure binding affinity of ligands to 4.7d6 or 4% (v/v) of 25 mM stock solutions of ligands in DMSO‑d6 . Experiments for samples giving a positive shift in protein melting temperature were repeated in triplicate under identical conditions and the collected data were averaged (over the four runs).DSF was performed using a Bio-Rad CFX Connect system , scanning from 25 °C to 95 °C in 0.5 °C increments each of 30 s duration. Samples were run in 96-well plates, with each well containing a final volume of 25 μL. Screening was conducted in 100 mM potassium phosphate pH 6.9, 2.5 × Sypro Orange, and with 5 μM Mtb CYP121A1 containing either 4% (v/v) DMSO‑4.8Escherichia coli flavodoxin NADP + oxidoreductase (FLDR) was expressed and purified as previously reported [Spinacia oleracea ferredoxin (FDX), Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase (G6PDH), glucose-6-phosphate (G6P) and NADPH were purchased from Sigma–Aldrich (UK). Reaction mixtures comprising of 5 μM CYP121A1, 4.6 μM FLDR, 10 μM FDX, 2 units of G6PDH, 10 mM G6P and 2 mM NADPH were run in 50 mM Tris-base, 150 mM KCl, pH 7.6 at 28 °C in a final volume of 500 μL. Reactions were run in amber, silanized glass vials . Positive and negative controls were prepared containing either 100 μM cYY  or with none respectively. For experimental samples, either 100 μM of compound or 100 μM compound and 100 μM cYY were used. The amount of DMSO was normalized across all samples to 1.6% v/v. For the positive control and experimental samples, compound and/or cYY was introduced prior to CYP121A1 to allow for proper equilibration. Reactions were started with the introduction of a mastermix comprising G6PDH, G6P and NADPH. Samples were incubated at 28 °C and 220 rpm for 2 h before reactions were stopped by the addition of 1 mL DCM via glass pipette. Samples were then briefly vortexed and then centrifuged for 10 min (466×g), the resulting lower organic phase was extracted using a glass pipette. This process was repeated once more, the organic phases were pooled and dried overnight in a fumehood. Following this, samples were further dried for 20 min in an EZ-2 centrifugal evaporator  set to aqueous mode with the lamp off. Samples were resuspended in 150 μL ACN  supplemented with 0.1% formic acid . LC–MS analysis was performed on an Agilent 1290 uHPLC system coupled to an Agilent 6545XT LC-QTOF controlled by MassHunter 10 . Columns used were either an EclipsePlus C18 RRHD 1.8 μm 2.1 mm × 150 mm  or a BonusRP RRHD 1.8 μm 2.1 mm × 50 mm  eluting at 0.45 mL/min at 60 °C. Mobile phases were either water supplemented with 0.1% formic acid (A) or acetonitrile supplemented with 0.1% formic acid (B). A gradient of 3–40% B over 6 min was used for separation. Signal acquisition was achieved using MS1 mode scanning from 100 to 3000 Da at 4 Hz. Data were analyzed on MassHunter Qualitative 10 and Quantitative 10 .Untagged CYP121A1 was expressed and purified to homogeneity as previously reported ,38. Eschreported ,51. Spin4.914 and 61 at 100 mM and a 30 mM cYY stock solution were prepared in fresh DMSO for competition assays. A titration with cYY alone was performed as a control using a stock solution at 15 mM prepared in fresh DMSO. Assays were performed on a dual-beam Cary 300 UV–Visible spectrophotometer . Following introduction of 5 μM CYP121A1, 100 μM of either compound 14 or 61 was introduced in two equal additions and spectra recorded. The second spectrum taken was used as the starting point for a titration with cYY. Reverse competition assays were performed using a spectrum with 100 μM cYY present as the starting point for a titration with compound 14 or 61. A 100 mM cYY stock solution and 30 mM compound stock solutions were prepared in fresh DMSO for the reverse competition assay. Final DMSO concentrations were kept below 1% v/v. Titrations were performed and data processed as previously described. Data were fit to the Hill equation to derive the Compound stock solutions of both In the Hill equation, One of the authors, Professor Chris Abell, died suddenly during the preparation of this manuscript. His fellow authors wish to dedicate this paper to his memory.Chemistry , expression and purification of CYP121A1 , X-ray crystallography , antimycobacterial activity assays , UV-vis spectroscopy (IRS), DSF and ITC (MF), LC-MS activity assay and competition assay , manuscript preparation , concpts and experimental design , project management and supervision .The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Sense of coherence (SOC), a concept that refers to individuals’ abilities to manage, comprehend, and find meaning in their lives and the world around them, has been shown to be an important predictor of health outcomes. While SOC was initially hypothesized to be static after early-adulthood, there is growing evidence that health interventions can strengthen SOC. In this study, we accordingly examined whether SOC could be strengthened among adults in the context of a physical activity intervention.0) and after eighteen months (T3), with information on 117 participants in both of these waves. To assess the change in SOC between T0 and T3, ordered logistic regressions were performed, as well as mixed ordered logistic regressions with random intercepts for group and program location.This intervention, Communities on the Move, was conducted in the Netherlands, and was primarily targeted at older adults from socially vulnerable backgrounds. Four cohorts were followed for 18 months each, between 2012 and 2016. The SOC-3 questionnaire was used to collect data on SOC at baseline , while those with moderate or strong SOC at baseline reported a median change of zero points between T0 and T3. Further, based on the results of the regression analyses, those with weaker SOC scores were most likely to have stronger SOC at T3: having a weak SOC at baseline was associated with a 76% probability of stronger SOC, and a 4% probability of weaker SOC at T3. These results indicated that SOC may be strengthened in vulnerable older adults, particularly when their SOC is initially low.This study found evidence that SOC significantly changed from T ResearcHow might SOC be strengthened? According to the salutogenic perspective of health, this occurs via Generalized Resistance Resources (GRRs). GRRs are social and individual resources that ‘help to manage stress and to thrive, moving towards the positive end of an ease/dis-ease continuum’ . GRRs caOne pathway to strengthen GRRs, and consequently SOC, is experiential learning, as argued by However, it is not yet clear what type of interventions are most effective at engaging in experiential learning, and ultimately at strengthening SOC. To date, interventions have ranged from those focused on nutrition ; care faIn this study, we therefore investigated whether SOC may have strengthened in the context of a Dutch community-based physical activity intervention aimed at vulnerable older adults. We hypothesized that this was indeed the case, particularly among individuals whose SOC at baseline was weak.22.1This study focuses on the intervention, Communities on the Move. In the intervention, participants were, via purposive sampling, recruited in collaboration with the Knowledge Center for Sport & Physical Activity Netherlands, and with representatives from local programs. These local program representatives were approached through the Knowledge Center for Sport & Physical Activity Netherlands network, information meetings, training sessions, field visits and snowball procedures. Participation was on a voluntary basis. Most participants in the intervention were from low SES backgrounds, and/or were immigrants to the Netherlands . MoreoveExperiential learning was embedded in Communities on the Move. Participants gave input into recruitment, program design, and tailoring physical activities to their needs. Participants practiced what they learned, and actively involved their social and physical environments, in order to sustain their behavior change . This meThe data used in this study came from the evaluation study of Communities on the Move . Partici0, T1 at six months, T2 at twelve months, and T3 at 18 months. At T3, there were 117 participants with complete covariate information. Data were collected via pen and paper questionnaires and were in Dutch, the working language of Communities on the Move. Socio-demographic factors and measurements of health, including SOC, were measured at baseline. SOC was measured again only at T3.The structure and duration of the programs varied. While some lasted for a fixed duration (10–13 weeks), other programs took the form of ongoing physical education classes. These exercises included outdoor activities  and indoor activities  . At base2.22.2.10). We derived SOC scores from the SOC-3 questionnaire, comprised of three questions, with one each aimed at manageability, comprehensibility, and meaningfulness (ibid.). Each question was scored by the participant from 1 to 3, whereby a score of 1 was associated with a strong SOC, and a score of 3 with a weak SOC. The combined SOC score therefore had a minimum of 3 (very strong SOC) and a maximum of 9 (very weak SOC). However, given this study’s small sample size, the condensed, three-category version of SOC-3 at T0 was used as the key predictor in regressions. Here, a score of 3 was considered strong, a score of 4 or 5 was considered moderate, and a score of 6 through 9 was considered weak (ibid.).Our key predictor was SOC at baseline (Tgfulness . These q3) and at baseline (T0). This more extended scale was used in 0 and T3.The outcome of this study was change in SOC score. This was measured by differencing SOC at the final wave of the study  for those who dropped out of Communities on the Move. Out of the initial 268 participants, only 117 finished the program. It may be that those who dropped out were systematically different from those who finished the intervention. To assess whether this was the case, logistic regressions were performed, in order to test if SOC at baseline was associated with an increased odds of dropping out.We did not have information on SOC at T33.10), 16% of participants had strong SOC scores (scores of 3). Fifty-six percent of participants had moderate SOC scores (scores of 4 or 5) at T0. Further, 27% of participants had weak SOC scores (scores of 6 through 9). At T3, the largest share of participants (65%) reported no change in SOC. This is followed by 21% reporting stronger SOC scores. An additional 14% reported weaker SOC scores.Sample characteristics are presented in 0 (with scores between 6 and 9) experienced the largest strengthening of SOC: these individuals’ SOC scores strengthened (decreased) by a median score of one point. In comparison, participants with strong SOC scores (with scores of 3) or moderate SOC scores (with scores of 4 or 5) at baseline reported a median change of zero points between T0 and T3.3.23, relative to the reference group of having a moderate SOC score (scores of 4 or 5) at T0. Conversely, having a weak SOC score at baseline (scores of 6 through 9) was strongly, significantly associated with a decreased odds of SOC weakening at T3. 3, and a 4% probability of weaker SOC at T3. There was therefore evidence in support of this study’s hypothesis that SOC score strengthened during Communities on the Move.Regarding the results of the mixed ordinal regression, the variance component parameters of group and program location were both 0.000. Also, based on the results of a likelihood-ratio test, the mixed model was not a better fit than the ordinal logistic regression. Therefore, group and program location do not appear to have played a role in the change in SOC score.3.3After adjusting for covariates, SOC score at baseline was not significantly related to the odds of dropping out of Communities on the Move. However, receiving income assistance, and giving no response to this question were significantly associated with an increased odds of dropping out of Communities on the Move. Being older and being born in the Netherlands were associated with lower odds of dropping out of Communities on the Move. These results are available on request.43, and that program group and location did not explain the variance in changes in SOC. These findings chimed with existing research, which has argued that there is more potential to change SOC among vulnerable groups, because these groups have the most to gain  compared other indicators collected during Communities on the Move, including physical activity levels, health-related quality of life, self-efficacy and enjoyment outcomes, measured at 12 months in the program, between drop-outs and non-drop outs. This previous study found that, when comparing other indicators of well-being, those who dropped out tended to score less positively. Moreover, in the present study, receiving income assistance (or not reporting a response) and being born abroad were significantly associated with the likelihood of dropping out. Ultimately, we found some evidence that more vulnerable participants were more likely to drop out.Similarly, this study had a high rate of drop-outs: 43% of participants at TThis pattern of drop-outs is by no means unique to Communities on the Move: in general, more vulnerable individuals are both less likely to be recruited for health promotion interventions, and are less likely to complete interventions once they are involved . Communi5In this study, we offered further insights into how and why SOC may be strengthened during a physical activity intervention. We found that SOC strengthened over the course of Communities on the Move, with those with the weakest SOC scores at baseline experiencing the largest strengthening in SOC. This study therefore provided evidence that SOC may be possible to strengthen among adults, particularly among those whose SOC scores are initially low. Based on the fact that change in SOC did not vary across groups and programs with different physical activity content, we argued that the intervention itself – rather than program-specific factors – played a larger role in strengthening SOC. Ultimately, SOC, as a subjective measures of well-being, may be an important complementary indicator to health promotion interventions.The completed evaluation study of Communities on the Move was funded by the ZonMW project, “Effectiveness and cost-effectiveness of the Communities on the Move program” (project number: 200130010).Kristina Thompson: Methodology, Formal analysis, Writing – original draft, Writing – review & editing, Visualization. Marion Herens: Conceptualization, Project administration, Data collection, Writing – original draft, Writing – review & editing. Johan van Ophem: Conceptualization, Writing – review & editing. Annemarie Wagemakers: Conceptualization, Project administration, Supervision, Writing – review & editing.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Knowledge of the level and duration of protective immunity against SARS‐CoV‐2 after primary infection is of crucial importance for preventive approaches. Currently, there is a lack of evidence on the persistence of specific antibodies. We investigated the generation and maintenance of neutralizing antibodies of convalescent SARS‐CoV‐2‐afflicted patients over a ten‐month period post‐primary infection using an immunofluorescence assay, a commercial chemiluminescent immunoassay and an in‐house enzyme‐linked neutralization assay. We present the successful application of an improved version of the plaque‐reduction neutralization assay which can be analysed optometrically to simplify data interpretation. Based on the results of the enzyme‐linked neutralization assay, neutralizing antibodies were maintained in 77.4% of convalescent individuals without relevant decay over ten months. Furthermore, a positive correlation between severity of infection and antibody titre was observed. In conclusion, SARS‐CoV‐2‐afflicted individuals have been proven to be able to develop and maintain neutralizing antibodies over a period of ten months after primary infection. Findings suggest long‐lasting presumably protective humoral immune responses after wild‐type infection. A year after the pandemic began and there is still a lack of data on the persistence of the immunologic footprint left in a convalescent SARS‐CoV‐2 afflicted patient. Moreover, the first publications reported discrepancies concerning the persistence of specific IgG antibodies  post‐infection.To obtain a reliable overview of the development of antibodies over time, we chose to compare three different serologic methods with different diagnostic targets. Specifically, we detected IgG antibodies targeting Spike 1 and 2 via chemiluminescent immunoassay, polyclonal IgG and IgM antibodies via immunofluorescence assay, and neutralizing antibodies using an in‐house enzyme‐linked neutralization assay (ELNA). A technique similiar to ELNA was recently used for SARS‐CoV‐2 serology by different research groups  were followed over 10 months post‐infection. Ethical approval to use residual routinely taken serum samples for retrospective antibody analyses was obtained by the Ethics Committee of the University Hospital Wuerzburg (no. 20201105_01). All participants provided written informed consent and the study was performed according to the principles of the declaration of Helsinki 2013.Following the initial baseline data collection, we conducted serological follow‐up examinations to evaluate seroconversion rates. First blood draw occurred 21–43 days  after the onset of symptoms in the first week of April 2020 , T3 (three months post‐infection), at T5 (five months post‐infection) and a final blood draw at 10 months post‐infection in February 2021, called T10. Clinical data were obtained using a standardized data collection form.Disease severity for each patient was assessed clinically using a standardized questionnaire including age, gender, pre‐existing as well as acute physical condition  and enzyme‐linked neutralization assay (ELNA).The presence of different types of antibodies was analysed in follow‐up serum samples by three different serologic methods to ensure validity of results: IgG in‐house immunofluorescence assay (IFA), IgM in‐house IFA, CLIA IGG2.2Nasopharyngeal swabs were taken by trained healthcare personnel. Immediately after collection, viral RNA was extracted using the Indimag Pathogen kit  and tested for SARS‐CoV‐2 by RT‐qPCR using the Bio‐Rad CFX96 system (Roche) with a LightMix Modular Assay kit in accordance with the modified Charité guidelines  method  was used for the neutralization assay.Isolation of SARS‐CoV‐2 was attempted from RT‐qPCR positive nasopharyngeal swabs by inoculation on VeroB4  in T25 tissue culture flasks for 1 hr at 35°C. After incubation, the sample was removed and Medium199 (Gibco) with 2.5% foetal calf serum  and a mixture of antibiotics  was added. We monitored virus cultures daily for cytopathic effects and tested for specific viral RNA every three days. Isolation was considered successful when the cytopathic effect was 80%–100% in passage 0 as well as passage 1 and/or Ct value in qPCR was lower than 20. Highly positive supernatants were harvested, centrifuged at 3,400 In‐house immunofluorescence assays (IFA) were assembled as described elsewhere  (LIAISON) is a CLIA (Chemiluminescent Immunoassay) which detects IgG antibodies reactive with the spike protein (S1/S2 domain). The assay was performed on the LIAISON® XL Analyzer according to the manufacturer's instructions. The diagnostic sensitivity was 97.9%  according to the manufacturer, the specificity in laboratory routine was 99.0% .The LIAISON2.46 cells/ml to give a confluent monolayer. Next day, an infectivity titration was carried out to determine 100 tissue culture infectious dose 50% (100 TCID50)  were seeded in flat‐bottom 96 well plates with Medium199 (Thermo Scientific Gibco) and 10% foetal calf serum (Thermo Scientific Gibco) at a density of about 10) Vihay, . Sera we) Vihay, . To eval5 TCID50) and serum dilutions in Medium199 were mixed and subsequently incubated for 1 hr at 35°C in U‐bottom 96 well plates . After incubation, a pre‐seeded flat‐bottom 96 well plate with confluent VeroB4 cells was used, medium was discarded, the incubated mixture of patient's serum and defined virus solution was transferred to each corresponding well of the flat‐bottom plate and the plate was incubated for 24 hr at 35°C. Incubation was stopped by discarding supernatant, cells were washed in PBS twice, fixed with ice‐cold acetone‐methanol (1:1) and frozen for at least 15 min. All steps were performed under strict observation and in compliance with biosafety level 3. The analysis was carried out like an enzyme‐linked immunosorbent assay using a BEP III (Siemens) according to the following steps: blocking , washing 3× , anti‐SARS‐CoV‐2 nucleocapsid protein IgG , washing 3×, adding of horseradish‐peroxidase‐conjugated goat anti‐mouse IgG , washing 3×, adding substrate tetramethylbenzidine (TMB) and stop solution (Siemens). The cut‐off titre was set by titrating defined negative human sera from volunteers out of healthy Tyrolean blood donors from the year 2009 and was set at 1:4 in combination with the viral dose of 1 × 105 TCID50 and calculated as median optic density minus the standard deviation. A sample was considered positive when the given optic density was higher than the cut‐off titre.With these evaluated sera, we adapted the PRNT to an ELNA without the need of an apparent cytopathic effect (CPE) and a shorter incubation period of <24 hr. For ELNA, sera were titrated in duplicate in twofold dilution steps, starting at a dilution of 1:4 in Medium199 containing 3% foetal calf serum. Equal volumes of virus  and time point 10 (T10) was defined as an increase or decrease of the titre. In the IgG CLIA assay, the development of antibodies was defined as a change in chemoluminescence of more than 50%.2.6n < 60) . A two‐sided significance level of p = .05 was used for determining statistical significance. The Spearman's rank correlation coefficient was used to analyse correlation of titres between CLIA and ELNA . To determine the predominant titres in a group of patients, the median was calculated  according to McHugh, Dichotomous data were tested by a chi‐squared test or Fisher's exact test in the case of small group size  were asymptomatically infected with SARS‐CoV‐2, 20 patients (58.8%) showed a mild, six (17.6%) a moderate and five (14.7%) a severe course of the disease.p = .11), although only one (5.6%) female had a severe course compared to four male patients (25.0%).In total, the group consisted of 18 women and 16 men. There were no gender‐related differences in the course of disease (SD = 16.4). Due to expected differences in disease course  of SARS‐CoV‐2‐positive patients in the convalescent phase (>21 days after symptom onset) tested had seroconverted at T1 in April 2020 and in total, 77.4% of the patients (24/31 samples) had seroconverted until February 2021 (T10), as determined by ELNA  of patients seroconverted one month post‐infection and 81.4% (26/31) stayed positive in this test method ten months later without a significant decrease in measurable units of IgG, as shown in Figure 3.5p = .003) and to 33.3% (10/30 tested patients) after 10 months (p < .0001).The highest number of positive individuals was found in the IFA IgG one month post‐infection , which dropped significantly to 65.6% (21/32 tested patients) within the study period of 5 months (3.6Both methods showed that specific antibodies stayed constant over the observation period and that greater disease severity led to a more stable antibody response.CLIA IgG recognized two sera from the first time point as positive (5.9%), which turned out as negative in ELNA. This result is aligned with the manufacturer's reported diagnostic accuracy . Repeated serologic investigations at T5 resulted in one patient (3.0%) false negative in CLIA IgG compared to the ELNA method. Ten months post‐infection, the CLIA IgG method recognized 83.9% positive samples (26/31 samples). Two samples that proved positive in CLIA but negative in ELNA had low titres of specific antibodies in CLIA.3.7n = 18). Two of the seven sera, which proved negative in IFA, were also negative in ELNA, the others were borderline positive with titres around 1:4 in ELNA, as shown in Table Immunofluorescence assay gave 94.0% positive results at T1 and the strongest decrease of antibodies until T10 with a decrease of positive patients of 60.7% , the lowest number of seroconversions was found in CLIA IgG . In 11 cases (32.4%), IgM was detectable by IFA 28–41 days (T1) after the onset of symptoms.SD = 12.4) with a mild course of disease did not develop SARS‐CoV‐2 specific antibody responses over the study period and eleven patients developed only weak antibody responses five months post‐infection.Four patients  of patients, the neutralizing antibody titres against SARS‐CoV‐2 stayed constant and did not change significantly during the ten‐month follow‐up period. Two patients were negative in the CLIA IgG assay but showed weak neutralizing activities with antibody titres of 1:4.3.9p < .05) (Mann–Whitney U‐test).Symptomatic patients showed significantly higher AU/ml at T1 and T3 (p = .07).Our data also revealed a relationship between the severity of infection and neutralizing activity by ELNA. Neutralizing titres were compared in a group of 3 asymptomatic versus 31 symptomatic patients, the symptomatic group had higher neutralizing titres approaching significance (p = .03). There was no significant difference in neutralization activities between asymptomatic and mild courses of disease (p = .17). Interestingly, 60% of the patients who experienced severe infections (3/5) developed low (titres of 1:4) of neutralizing antibodies  or neutralizing antibodies and disease severeity (k = −.0951), nor did we observe a significant predominance of severe cases in oder age groups or male patients (p = .53). The mean age of severe cases was 46.0  versus 49.0 years  in cases of other severity categories. All patients with severe infections had neutralizing antibodies at T1. However, only 2 (40%) maintained neutralizing antibodies until T5, and 60% (n = 3) in this group showed low titres (<1:8) 5 months post‐infection (T5).There was no correlation between age and disease severity , three (T3), five (T5) and ten months (T10) post‐PCR‐confirmed infection using three different serologic tools ELNA, IFA and CLIA. Our data showed the persistence of specific IgG antibodies during the follow‐up period of ten months and a significant positive correlation between disease severity at initial presentation and neutralization activity. Overall, 77.4% of patients (24/31) maintained specific neutralizing antibodies in ten months post‐infection. More than three‐quarters  of symptomatic patients and a third  of asymptomatic patients maintained neutralizing antibodies over the ten‐month observation period. Investigations of IgG levels and neutralizing antibodies in the early phase of convalescence gave a similar result in earlier work  were significantly higher than those in the asymptomatic group . Two of these asymptomatic patients were negative in the early stage of the convalescent phase four weeks post‐infection (T1). One patient had a mild course of disease and stayed negative in all tests, whereas another patient with asymptomatic infection developed a weak neutralization response in the late convalescent phase (T5). These findings are aligned with published data, where milder courses of disease may require longer periods to generate specific antibodies and in a low number of cases, patients did not seroconvert at all after infection with SARS‐CoV‐2 . Indeed, ELNA can be carried out 24 hr post‐infection by enzyme‐link using the structural protein N as a target due to its adequate abundance in infected cells, which is an approach that has also been used by others  as the assay's high binding possibilities to the polyclonal reconvalescent serum and the rapid decline of specific IgG antibodies in IFA with the lower sensitivity of this test system .