Synthesis of diversely substituted pyridin-2(1H)-ones and in vivo evaluation of their anti-allodynic effect on cutaneous inflammatory mechanical allodynia

Théo Frazier; Pauline Murail; Arthur Boulangé; Nazim Chalane; Francis Giraud; Alain Artola; Radhouane Dallel; Fabrice Anizon; Pascale Moreau

doi:10.1039/d4md00375f

. 2024 Jul 3;15(8):2900–2921. doi: 10.1039/d4md00375f

Synthesis of diversely substituted pyridin-2(1H)-ones and in vivo evaluation of their anti-allodynic effect on cutaneous inflammatory mechanical allodynia^†

Théo Frazier ^a,^‡, Pauline Murail ^b,^‡, Arthur Boulangé ^a, Nazim Chalane ^a, Francis Giraud ^a, Alain Artola ^b,^✉, Radhouane Dallel ^b,^✉, Fabrice Anizon ^a,^✉, Pascale Moreau ^a,^✉

PMCID: PMC11324054 PMID: 39149104

Abstract

A series of 3,5-disubtituted pyridin-2(1H)-ones was synthesized. As part of a structure–activity relationship study performed in this series, structural modifications were based on 3-(indol-4-yl)-5-(pyridin-4-ylamino)pyridin-2(1H)-one B, which exhibited a potent anti-allodynic effect in a rat model of inflammatory pain. In this study, new compounds were assessed for their ability to inhibit cutaneous mechanical allodynia in rats by using the capsaicin pain model. Compound 36, a 2-methoxypyridine derivative, yielded the most promising outcome, demonstrating an enhanced analgesic effect.

New compounds were assessed for their ability to inhibit cutaneous mechanical allodynia in rats by using the capsaicin pain model.

1. Introduction

Chronic pain is a worldwide health problem that affects a significant portion of the population. Mechanical allodynia (MA), a common symptom of chronic pain caused by normally non-painful mechanical stimuli, remains incompletely understood, although some biological targets involved have been identified.¹ Furthermore, there are limited effective medical treatments for alleviating pain without side-effects or risk of abuse potential, as illustrated by opioid analgesics. Therefore, there is still an urgent need to develop new chemical agents that could enhance our understanding of the physiological mechanisms underlying pain and facilitate the identification of novel therapeutical approaches.

As part of our research on the development of new heteroaromatic compounds with biological applications, we recently reported on a pyridin-2(1H)-one series with potent analgesic properties. Indeed, we initially reported that intracisternal injection of compound A (5 μL at 100 μM) rapidly and strongly prevented the development of inflammatory MA on the facial complete Freund's adjuvant (CFA) model in rats, as well as reversed neuropathic MA induced by the chronic constriction injury of the rat's infraorbital nerve (IoN-CCI).² Further development in this series led to compound B that completely prevented MA on the rat facial inflammatory model at 10-fold lower concentration (i.c., 5 μL at 10 μM) (Fig. 1).³ Accordingly, compound B was selected as a starting point for further development, to expand the structure–activity relationship (SAR) study performed around this pyridin-2(1H)-one scaffold. One of the objectives was to highlight the structural requirements essential to analgesic activity. Thus, based on compound B structure, new derivatives were synthesized in which the nitrogen atom linking the pyridine and pyridinone rings was replaced by either an oxygen atom or an amide function (Fig. 1). In addition, to get an insight into the role of the hydrogen donor positions, the corresponding heteroatoms were methylated. This included pyridinone oxygen, considering the hydroxypyridine tautomer of this heterocycle. Finally, substitution of the indolic nitrogen atom by aminoalkyl groups was undertaken with the objective of improving water solubility in this series (Fig. 1). New compounds were evaluated for their potency to inhibit cutaneous MA in rats.

2. Results and discussion

2.1. Synthesis of diversely substituted pyridin-2(1H)-ones

The synthesis of amide analogues of compound B was achieved from either intermediate 2 or 3 which were prepared from commercially available 1 (Scheme 1).³ First, O-benzylation of compound 1 and Suzuki–Miyaura coupling with indole-4-boronic acid pinacol ester led to 2 in similar yields to those we previously reported.³ Improved reduction of the nitro group by catalytic hydrogenation led to 3 in 84% yield, instead of 70% yield by using Fe/NH₄Cl.³ From compound 2, amides 6 and 7 were straightforwardly synthesized after nitro reduction/debenzylation by catalytic hydrogenation and acylation steps. However, this procedure did not allow us to prepare pyridinyl derivative 8 due to difficulties in the acylation step. Therefore, this analogue was prepared from 3, after acylation to give 4 and debenzylation using BBr₃. Interestingly, catalytic hydrogenation of 4 using palladium hydroxide did not provide 8, possibly due to a catalyst poisoning effect of the pyridine moiety. In addition, when the hydrochloride salt of 4 was prepared first, reduction of the pyridine moiety led to piperidine 9.⁴ Finally, compound 10, the nitro-reduced analogue of 7, was similarly prepared from 3, after acylation leading to 5, and nitro reduction/debenzylation under catalytic hydrogenation.^2,3

Preparation of ether analogues of compound B took advantage of an Ullmann-type coupling using aryl/heteroaryl iodides from compound 14 (Scheme 2). The latter was prepared in three steps from previously reported compound 11.³ Firstly, the Sandmeyer reaction produced iodopyridine derivative 12 that was then easily and regioselectively converted to hydroxypyridine 14.^5,6 Ullmann ether synthesis with aryl/heteroaryl iodides produced compounds 15–17.⁷ It should be noted that our attempts to perform an Ullmann-type coupling directly from 12 and phenol derivatives was not satisfying. Only compound 15 could be obtained through this method, with 66% yield. From 15–17, subsequent steps were introduction of the indol-4-yl moiety via Suzuki–Miyaura cross-coupling to give 18–20 and then debenzylation leading to 21–23. As for the amide series depicted in Scheme 1, reduced analogues 24 and 25 were also synthesized. The first was prepared from 19 by catalytic hydrogenation to reduce the nitro group and cleave the benzyl protecting group. Regarding 25, the pyridine moiety of compound 23 could not be easily hydrogenated. Therefore 25 was prepared from 12: coupling of benzyl 4-hydroxypiperidine-1-carboxylate under copper catalysis led to compound 26,⁶ then the indol-4-yl moiety was introduced before cleavage of the protecting groups.

The next part of this synthetic work was to introduce a methyl group on heteroatoms of compound B, except for the nitrogen atom of the pyridine ring. First, commercially available 1 was methylated using CH₃I and silver carbonate in acetonitrile under microwave irradiation to give regioselectively N-methylated 29, in 95% yield (Scheme 3). However, O-methylated regioisomer 30 was isolated in only 28% yield under similar alkylation conditions in toluene, together with 29 in 67% yield.⁸ Next steps were those already reported:² reduction of the nitro group, Buchwald–Hartwig coupling with 4-iodopyridine and Suzuki–Miyaura reaction with indole-4-boronic acid pinacol ester, to give compounds 35 and 36.

Methylation of the nitrogen atom linking the two heterocycles started from compound 11 with a 3-step monomethylation of the amino group: 11 was trifluoroacetylated, methylated and then the trifluoroacetyl group was removed to give 37.⁹ Introduction of the pyridinyl and indolyl substituents and final benzyl cleavage produced compound 40.

Finally, the N-methylated indole derivative was obtained from previously reported compound 41.² Suzuki–Miyaura cross-coupling with boronic acid pinacol ester 43, prepared by borylation of commercially available 42,¹⁰ gave 44 that was subsequently deprotected to obtain 45.

Analogues substituted on the indole nitrogen by aminoalkyl chains were prepared in a similar pathway to the one used for synthesizing 45 (Scheme 4). Thus, starting from 4-bromoindole, N-alkylation with either 1,3-dibromopropane or 1,4-dibromobutane led to compounds 47 and 48.¹¹ Nucleophilic substitution with various secondary amines (Et₂NH, pyrrolidine, piperidine, morpholine, N-methylpiperazine) gave aminopropyl and aminobutyl compounds 49–58.¹² Further borylation,¹⁰ Suzuki coupling with 41 and final deprotection provided derivatives 79–88.

2.2. In vivo assessment of the anti-allodynic effect

We evaluated in vivo the analgesic efficacy of new compounds 6–10, 21–25, 35, 36, 40, 45, 79, and 80 in rats by using the hindpaw capsaicin model, where animals were administered a subcutaneous injection of capsaicin (25 μg in 25 μL) into the plantar surface of the right hindpaw to induce cutaneous MA. All the compounds were intrathecally administered at a concentration of 5 μM, 30 min before the capsaicin injection. Their analgesic efficacy is compared with that of compound B, which was selected as a starting point in this SAR study enlargement³ (Fig. 1).

Substitution of the indole nitrogen of compound B by a methyl (45), diethylaminopropyl (79) or diethylaminobutyl (80) group led to decreased activity toward capsaicin-induced MA (Fig. 2, left), suggesting that substitution at this position is detrimental to the desired biological activity. This indicates that the indole NH may act as a hydrogen bond donor in the interactions of compound B with its biological target. Analogues 81–88 substituted at the indole nitrogen by aminoalkyl groups were not behaviorally assessed to avoid unnecessary use of animals, and because some of the other prepared analogues showed improved activity. Indeed, among the other tested compounds, seven showed effective analgesic effects. Notably, compared with compound B (56% MA inhibition), three (compounds 7, 25, and 36) exhibited similar or superior MA inhibition (Fig. 2, left).

When the NH of the (pyridin-4-yl)amino group in compound B was methylated (40) or replaced by an amide function (8) or an oxygen atom (23), the MA inhibitory activity was reduced. However, in the amide and ether series, the 4-nitrophenyl (7, 22) and piperidinyl derivatives (9, 25) led to similar or slightly superior activities to that of compound B. The best results were obtained for 7 and 25, with 58% and 64% MA inhibition, respectively. These findings show that the replacement of the NH group at the pyridinone 5-position by an amide function or an oxygen atom is not detrimental to the analgesic activity. In addition, in line with our previous results, the nature of the ring attached to the amide or ether function has a significant effect, with 4-nitrophenyl and piperidinyl being the best substituents.

Finally, only one of these newly synthesized compounds, O-methylated compound 36, demonstrated a larger MA inhibition (85% inhibition) (red bar in Fig. 2, left and right). This suggests that methylation at the pyridinone oxygen atom produces a striking positive effect, while N-methylation only leads to slightly reduced potency.

3. Conclusion

In conclusion, the initial SAR study around 3,5-disubstituted pyridine-2(1H)-one derivatives was extended. We show that the NH linking group at the 5-position can be replaced by an aminocarbonyl group or an oxygen atom, with good activity reached for compounds 7 and 25. On the contrary, methylation or aminoalkylation of the indole nitrogen produces compounds with lower potency. The best result within the newly prepared compounds is obtained with O-methylated derivative 36. Methoxypyridine 36 exhibited 85% inhibition of capsaicin-induced mechanical allodynia, instead of 56% for previously reported compound B.

In addition to previously reported inhibition of inflammatory pain on the facial CFA model in rats, and neuropathic pain induced by the IoN-CCI of the rat's infraorbital nerve, we demonstrate in this work the potency of this series toward capsaicin-induced inflammatory mechanical allodynia. These results confirm that 3,5-disubstituted pyridin-2(1H)-ones and their derivatives are of high interest for the development of novel analgesics.

4. Experimental section

4.1. Chemistry

4.1.1. General

Starting materials were obtained from commercial suppliers and used without further purification. IR spectra were recorded on a Perkin-Elmer Spectrum 65 FT-IR spectrometer ( in cm⁻¹). NMR spectra, recorded on a Bruker AVANCE 400 III HD (¹H: 400 MHz, ¹³C: 101 MHz) or Bruker AVANCE 500 III HD (¹H: 500 MHz, ¹³C: 126 MHz), are reported in ppm using the solvent residual peak as an internal standard; the following abbreviations are used: singlet (s), doublet (d), triplet (t), quadruplet (q), quintuplet (quint), doublet of doublets (dd), multiplet (m), broad signal (br s). Coupling constants are expressed in hertz. High resolution mass spectra were obtained on a Thermo Scientific Q Exactive Q-Orbitrap apparatus (UCA Partner, Université Clermont Auvergne, Clermont-Ferrand, France). Chromatographic purifications were performed by column chromatography using 40–63 μm silica gel or by flash column chromatography using puriFlash® XS 520 Plus with IR-50SI-F0004, IR-50SI-F0012, IR-50SI-F0025 or IR-50SI-F0040 columns. Reactions were monitored by TLC using fluorescent silica gel plates (60 F254 from Macherey Nagel). Melting points were measured on a Stuart SMP3 apparatus and are uncorrected.

The purity of compounds 6–10, 21–25, 35, 36, 40, 45, 79–88 was established by HPLC analysis using a VWR Hitachi chromatograph with a DAD detector and a Macherey Nagel Nucleodur C18 Gravity column (4.6 mm × 250 mm, 5 μm). The flow rate was 0.5 mL min⁻¹ and analysis was performed at 25 °C. Detection wavelength is indicated for each compound. Solvents were (A) water/0.1% trifluoroacetic acid, (B) acetonitrile and (C) water. Method A: gradient was 95 : 5 A/B for 5 min, then 95 : 5 A/B to 5 : 95 A/B in 20 min and then 5 : 95 A/B for 10 min. Method B: gradient was 95 : 5 C/B for 5 min, then 95 : 5 C/B to 5 : 95 C/B in 20 min and then 5 : 95 C/B for 10 min. Method C: 70 : 30 A/B for 10 min. Method D: 60 : 40 A/B for 10 min.

4.1.2. General procedures

Procedure A for the preparation of compounds 6 and 7

To a solution of 2 (100 mg, 0.290 mmol) in MeOH (3 mL) and CH₂Cl₂ (0.5 mL) degassed with argon was added 20% Pd(OH)₂/C (3.3 mg, 4.7 μmol, 0.02 eq.). The mixture was then hydrogenated at room temperature for 6 h in the dark. After filtration through a pad of Celite, the solid was washed with MeOH. The filtrate was evaporated under reduced pressure to give crude 5-amino-3-(1H-indol-4-yl)pyridin-2(1H)-one as a green-dark solid (R_f = 0.16 (CH₂Cl₂/MeOH 95 : 5 + 0.5% Et₃N)) that was used for the next step without further purification.

To a solution under argon, in the dark, of crude 5-amino-3-(1H-indol-4-yl)pyridin-2(1H)-one in a 3 : 1 THF/DMF mixture (amount for 0.072 M starting material concentration) was added Et₃N (1.2 eq.). The mixture was cooled to 0 °C before the addition of the acid chloride derivative (1.1 eq.). The solution was stirred for 1 h in an ice bath in the dark. Water was added and the resulting mixture was extracted three times with ethyl acetate. The organic phase was washed with a saturated aqueous solution of Na₂CO₃ and with brine, then dried over MgSO₄ and filtered. After evaporation under reduced pressure, the crude was purified by column chromatography.

Procedure B. Debenzylation using BBr₃, for the preparation of compounds 8, 22, 23, 79, 83 and 85

A solution under argon of the benzylated derivative in dry dichloromethane (amount for 0.02 M starting material concentration) was cooled to −10 °C (for 8, 22, 23) or to 0 °C (for 79, 83, 85). A solution of BBr₃ in dichloromethane (1 M, 4 eq.) was added dropwise. The mixture was stirred at 0 °C (unless otherwise indicated) until reaction completion. The reaction mixture was then quenched by addition of excess of Et₃N and then methanol. After evaporation under reduced pressure, the crude mixture was purified by column chromatography.

Procedure C. Hydrogenation/hydrogenolysis, for the preparation of compounds 10, 21, 24, 25

To a solution under argon of the benzylated derivative in MeOH or CH₂Cl₂/MeOH (1 : 1) degassed with argon was added 20% Pd(OH)₂/C (0.02 eq.). The mixture was then hydrogenated at room temperature for several hours. The mixture was filtered through a pad of Celite which was then washed with methanol. The filtrate was evaporated under reduced pressure and the crude was purified by column chromatography.

Procedure D. Ullmann ether synthesis, for the preparation of compounds 15–17

A screw-cap tube was charged with 14, CuI (0.05 eq.), and Cs₂CO₃ (2 eq.). The tube was flushed with argon and 1,4-dioxane (amount for 0.25 M starting material concentration), 2,2,6,6-tetramethylheptane-3,5-dione (0.1 eq.) and iodide derivative (2 eq.) were added. The tube was then sealed with a screw cap and heated in an oil bath at 60 °C for 24 h. The resulting mixture was filtered through a pad of Celite which was then washed with EtOAc. The filtrate was evaporated under reduced pressure and the crude was purified by column chromatography.

Procedure E. Suzuki–Miyaura cross-coupling, for the preparation of compounds 18–20, 27, 35, 36, 39, 44 and 69–78

To a solution under argon of the brominated derivative in 1,4-dioxane (amount for 0.1 M starting material concentration) were added indole-4-boronic acid pinacol ester or derivatives (1.5 eq. of indole-4-boronic acid pinacol ester or 1–2 eq. based on the starting material of the borylation procedure) and 2 M aqueous solution of Na₂CO₃ (5 eq.). The mixture was degassed with argon for 10 min before the addition of PdCl₂(PPh₃)₂ (0.05 eq.). The solution was refluxed overnight. The resulting mixture was filtered through a pad of Celite which was then washed with EtOAc. The organic layer was washed with water and then with brine, dried over MgSO₄, filtered and concentrated under reduced pressure. The crude was purified by column chromatography.

Procedure F. Reduction of nitro derivatives, for the preparation of compounds 31 and 32

To a solution of the nitro derivative (1 eq.) in a 10 : 1 propan-2-ol/water mixture (amount for 0.045 M starting material concentration) were added Fe powder (6 eq.) and NH₄Cl (0.4 eq.). The mixture was refluxed for several hours. Then, the mixture was filtered through a pad of Celite which was then washed with ethyl acetate. The filtrate was washed with water, and the aqueous phase was extracted with ethyl acetate. Combined organic phases were dried over MgSO₄, filtered and then evaporated. The obtained crude was purified by column chromatography.

Procedure G. Buchwald–Hartwig cross-coupling, for the preparation of compounds 33, 34 and 38

A screw-cap tube under argon was charged with the amine derivative (1 eq.), Pd(OAc)₂ (0.05 eq.), Xantphos (0.05 eq.) and Cs₂CO₃ (2 eq.). Then, anhydrous 1,4-dioxane (amount for 0.2 M starting material concentration) degassed with argon and 4-iodopyridine (1 eq.) were added. The tube was sealed and the mixture was stirred at 100 °C for several hours. The resulting suspension was filtered through a pad of Celite which was then washed with ethyl acetate. After evaporation of the filtrate, the brown residue was purified by column chromatography.

Procedure H. Debenzylation by catalytic hydrogenolysis, for preparation of compounds 45, 80–82, 84 and 86–88

To a solution under argon of the benzylated derivative (1 eq.) and acetic acid (5 eq.) in CH₂Cl₂/MeOH 1 : 1 degassed with argon was added 20% Pd(OH)₂/C (0.2 eq.). The mixture was then hydrogenated at room temperature overnight. Et₃N (200 μL) was added and the mixture was filtered through a pad of Celite which was then washed with methanol. The filtrate was evaporated under reduced pressure and the crude was purified by column chromatography.

Procedure I. Amination of bromoalkylindoles 47 and 48, for the preparation of compounds 49–58

To a solution under argon of 47 or 48 (1 eq.) in DMF (amount for 0.25 M starting material concentration) was added the amine derivative (5.0 eq.). The mixture was stirred for 1.5 h at room temperature. The resulting mixture was diluted in water and extracted three times with CH₂Cl₂. Combined organic phases were dried over MgSO₄, filtered, and evaporated under reduced pressure. After evaporation, the crude was purified by column chromatography.

Procedure J. Borylation of 4-bromo-1H-indole derivatives, for the preparation of compounds 59–68

A microwave tube under argon was charged with the 4-bromo-1H-indole derivative (1 eq., ∼100 mg per 10 mL tube), Pd(dppf)Cl₂ (0.1 eq.), KOAc (3.0 eq.) and bis(pinacolato)diboron (1.1 eq.). Then, anhydrous 1,4-dioxane (1 mL for 100 mg of starting material) degassed with argon was added. The tube was sealed and the mixture was heated under microwave irradiation for 30 min (dynamic control, P_max = 150 W, T = 80 °C). The resulting mixture was concentrated under reduced pressure and the crude was purified by column chromatography.

4.1.3. 4-(2-(Benzyloxy)-5-nitropyridin-3-yl)-1H-indole 2

To a solution under argon of crushed 1 (1.90 g, 8.68 mmol, 1 eq.) in anhydrous toluene (26 mL), benzyl bromide (2.2 mL, 18.5 mmol, 2.1 eq.) was added. The mixture was stirred at room temperature for 5 min. Then, the mixture was stirred at 70 °C and crushed Ag₂CO₃ was added in three portions of 0.35 eq. every hour (3 × 840 mg, 3 × 3.05 mmol, 3 × 0.35 eq.). After 3 h 30 min at 70 °C, the mixture was filtered through a pad of Celite which was then washed with ethyl acetate. The obtained yellow solid was purified by column chromatography (SiO₂, cyclohexane/EtOAc 100 : 0 to 5 : 95) to give the desired product with a residual impurity which was removed by washing with cyclohexane, to give 2-(benzyloxy)-3-bromo-5-nitropyridine (2.51 g, 8.12 mmol, 94%) as a white solid. R_f = 0.40 (cyclohexane/EtOAc 98 : 2). For characterization data, see ref. 3.

To a solution under argon of 2-(benzyloxy)-3-bromo-5-nitropyridine (500 mg, 1.62 mmol) in 1,4-dioxane (16 mL) were added the indole-4-boronic acid pinacol ester (587 mg, 2.41 mmol, 1.5 eq.) and 2 M aqueous solution of Na₂CO₃ (4 mL, 8 mmol, 5 eq.). The mixture was degassed with argon for 10 min before the addition of PdCl₂(PPh₃)₂ (56 mg, 0.08 mmol, 0.05 eq.). The solution was refluxed overnight. Ethyl acetate was added and the resulting mixture was washed with water. The organic phase was dried over MgSO₄ and filtered. After evaporation under reduced pressure, the crude oil was purified by column chromatography (SiO₂, cyclohexane/EtOAc 95 : 5 to 80 : 20) to give 2 (472 mg, 1.37 mmol, 84%) as a yellow solid. R_f = 0.25 (cyclohexane/EtOAc 8 : 2). For characterization data, see ref. 3.

4.1.4. 6-(Benzyloxy)-5-(1H-indol-4-yl)pyridin-3-amine 3

To a solution of compound 2 (465 mg, 1.35 mmol) in a 10 : 1 propan-2-ol/water mixture (33 mL) were added Fe powder (455 mg, 8.15 mmol, 6 eq.) and NH₄Cl (29 mg, 0.54 mmol, 0.4 eq.). The mixture was refluxed for 2 h and then filtered through a Celite pad. The solid was washed with EtOAc, and the filtrate was evaporated. The obtained oil was purified by column chromatography (SiO₂, cyclohexane/EtOAc 7 : 3 to 5 : 5) to give 3 (358 mg, 1.14 mmol, 84%) as a brown solid. R_f = 0.13 (cyclohexane/EtOAc 7 : 3). For characterization data, see ref. 3.

4.1.5. N-[6-Benzyloxy-5-(1H-indol-4-yl)pyridin-3-yl]pyridine-4-carboxamide 4

To a solution of 4-isonicotinoyl chloride hydrochloride (51 mg, 0.286 mmol, 1.5 eq.) in dichloromethane (3 mL) under argon atmosphere was added triethylamine (32 μL, 0.230 mmol, 1.2 eq.) and then dropwise a second solution of 3 (60 mg, 0.190 mmol) and triethylamine (32 μL, 0.230 mmol, 1.2 eq.) in dichloromethane (2 mL). The resulting solution was stirred for 30 min at rt. The solvent was removed and water was added. The aqueous phase was extracted three times with ethyl acetate and combined organic extracts were washed with a saturated aqueous solution of Na₂CO₃ and then with brine. The organic phase was dried over MgSO₄, filtered and evaporated under reduced pressure. The obtained yellow solid was purified by column chromatography (Et₃N-treated SiO₂, EtOAc 100%) to give 4 (72 mg, 0.171 mmol, 90%) as a yellow solid. R_f = 0.29 (EtOAc 100%); Mp > 205 °C (decomposition); IR (ATR) 3261, 1611, 1588, 1488, 1324, 1220, 853, 748, 729, 690 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 11.22 (s, 1H), 10.64 (s, 1H), 8.80 (m, 2H), 8.59 (d, J = 2.6, 1H), 8.25 (d, J = 2.6, 1H), 7.89 (m, 2H), 7.42 (dd, J₁ = 7.1, J₂ = 1.7, 1H), 7.39 (t, J = 2.8, 1H), 7.37–7.22 (m, 5H), 7.20–7.13 (m, 2H), 6.39 (m, 1H), 5.41 (s, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 164.0 (C), 156.5 (C), 150.3 (2CH), 141.5 (C), 137.5 (C), 137.2 (CH), 136.1 (C), 132.9 (CH), 129.7 (C), 128.2 (2CH), 127.47 (2CH), 127.44 (C), 127.43 (CH), 126.5 (C), 125.7 (CH), 123.2 (C), 121.5 (2CH), 120.7 (CH), 120.1 (CH), 111.2 (CH), 100.6 (CH), 67.1 (CH₂); HRMS (ESI+) m/z calcd for C₂₆H₂₁N₄O₂ (M + H)⁺ 421.1659, found 421.1650.

4.1.6. N-[6-Benzyloxy-5-(1H-indol-4-yl)pyridin-3-yl]-4-nitrobenzamide 5

To a solution of 4-nitro benzoyl chloride (48 mg, 0.259 mmol, 1.6 eq.) in dichloromethane (3 mL) under argon atmosphere was added a solution of triethylamine (23 μL, 0.165 mmol, 1.04 eq.) and 3 (50 mg, 0.159 mmol) in dichloromethane (2 mL). The resulting solution was stirred for 2 h at rt. The solvent was removed under reduced pressure and water was added. The aqueous phase was extracted three times with ethyl acetate and combined organic extracts were washed with a saturated aqueous solution of Na₂CO₃ and then with brine. The organic phase was dried over MgSO₄, filtered and evaporated under reduced pressure to give 5 (73 mg, 0.157 mmol, 99%) as a yellow solid. R_f = 0.12 (cyclohexane/EtOAc 8 : 2); Mp > 220 °C (decomposition); IR (ATR) 3432, 3342, 2922, 1654, 1514, 1341, 1266, 1214, 755, 718, 695 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 11.22 (s, 1H), 10.70 (s, 1H), 8.60 (d, J = 2.4 Hz, 1H), 8.39 (d, J = 8.7 Hz, 2H), 8.26 (d, J = 2.4 Hz, 1H), 8.22 (d, J = 8.7 Hz, 2H), 7.45–7.38 (m, 2H), 7.37–7.22 (m, 5H), 7.20–7.13 (m, 2H), 6.39 (m, 1H), 5.41 (s, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 163.9 (C), 156.5 (C), 149.2 (C), 140.1 (C), 137.5 (C), 137.2 (CH), 136.1 (C), 132.9 (CH), 129.9 (C), 129.2 (2CH), 128.2 (2CH), 127.48 (2CH), 127.45 (C), 127.43 (CH), 126.5 (C), 125.7 (CH), 123.6 (2CH), 123.2 (C), 120.7 (CH), 120.1 (CH), 111.2 (CH), 100.6 (CH), 67.1 (CH₂); HRMS (ESI+) m/z calcd for C₂₇H₂₁N₄O₄ (M + H)⁺ 465.1557, found 465.1547.

4.1.7. N-[5-(1H-Indol-4-yl)-6-oxo-1,6-dihydropyridin-3-yl]benzamide 6

Compound 6 was prepared according to general procedure A, starting from 2 (100 mg, 0.290 mmol). The crude oil was purified by column chromatography (Et₃N-treated SiO₂, CH₂Cl₂/MeOH 98 : 2 to 9 : 1 + 0.5% Et₃N). The obtained solid was washed with CH₂Cl₂ to give 6 (43 mg, 0.131 mmol, 45%) as a purple solid. R_f = 0.35 (CH₂Cl₂/MeOH 95 : 5 + 0.5% Et₃N); Mp > 250 °C; IR (ATR) 3241, 1647, 1351, 1254, 1119, 747, 684, 607 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 11.60 (br s, 1H), 11.15 (s, 1H), 10.12 (s, 1H), 8.01 (d, J = 2.6, 1H), 7.99 (d, J = 2.7, 1H), 7.97–7.92 (m, 2H), 7.61–7.56 (m, 1H), 7.55–7.50 (m, 2H), 7.40–7.36 (m, 2H), 7.32 (d, J = 7.2, 1H), 7.11 (t, J = 7.7, 1H), 6.51 (m, 1H); ¹³C NMR (101 MHz, DMSO-d₆) δ 165.2 (C), 159.5 (C), 136.2 (C), 135.6 (CH), 134.4 (C), 131.6 (CH), 129.9 (C), 128.4 (2CH), 128.2 (C), 127.5 (2CH), 126.2 (C), 125.3 (CH), 125.2 (CH), 120.4 (CH), 119.9 (CH), 119.7 (C), 110.9 (CH), 100.8 (CH); HRMS (ESI+) m/z calcd for C₂₀H₁₆N₃O₂ (M + H)⁺ 330.1237, found 330.1230. HPLC purity ≥99%, method A: t_R = 22.53 min, λ = 280 nm.

4.1.8. N-[5-(1H-Indol-4-yl)-6-oxo-1,6-dihydropyridin-3-yl]-4-nitrobenzamide 7

Compound 7 was prepared according to general procedure A, starting from 2 (100 mg, 0.290 mmol). The crude oil was purified by column chromatography (Et₃N-treated SiO₂, CH₂Cl₂/MeOH 98 : 2 to 92 : 8 + 0.5% Et₃N). The obtained solid was washed with CH₂Cl₂ to give 7 (31 mg, 0.0828 mmol, 29%) as a beige solid. R_f = 0.40 (CH₂Cl₂/MeOH 95 : 5 + 0.5% Et₃N); Mp > 250 °C; IR (ATR) 3434, 1568, 1524, 1465, 1341, 853, 751, 712, 619 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 11.69 (br s, 1H), 11.17 (s, 1H), 10.45 (s, 1H), 8.38 (d, J = 8.7, 2H), 8.18 (d, J = 8.8, 2H), 8.03 (d, J = 2.7 1H), 7.99 (d, J = 2.7, 1H), 7.43–7.35 (m, 2H), 7.32 (d, J = 7.2, 1H), 7.11 (t, J = 7.7, 1H), 6.51 (s, 1H); ¹³C NMR (101 MHz, DMSO-d₆) δ 163.5 (C), 159.6 (C), 149.2 (C), 140.0 (C), 136.2 (C), 135.3 (CH), 130.0 (C), 129.1 (2CH), 128.0 (C), 126.1 (C), 125.5 (CH), 125.4 (CH), 123.6 (2CH), 120.4 (CH), 119.9 (CH), 119.4 (C), 111.0 (CH), 100.8 (CH); HRMS (ESI+) m/z calcd for C₂₀H₁₅N₄O₄ (M + H)⁺ 375.1088, found 375.1077. HPLC purity ≥96%, method A: t_R = 23.40 min, λ = 280 nm.

4.1.9. N-[5-(1H-Indol-4-yl)-6-oxo-1,6-dihydropyridin-3-yl]pyridine-4-carboxamide 8

Compound 8 was prepared according to general procedure B, starting from 4 (100 mg, 0.238 mmol). The mixture was stirred for 7.5 h at 0 °C. The crude oil was purified by column chromatography (Et₃N-treated SiO₂, CH₂Cl₂/MeOH 93 : 7 to 90 : 10). The obtained solid was washed with CH₂Cl₂ to give 8 (42 mg, 0.127 mmol, 53%) as an ochre solid. R_f = 0.15 (CH₂Cl₂/MeOH 95 : 5 + 0.5% Et₃N); Mp > 250 °C; IR (ATR) 3276, 1611, 1588, 1488, 1324, 1220, 853, 748, 690 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 11.66 (br s, 1H), 11.16 (s, 1H), 10.37 (s, 1H), 8.78 (d, J = 4.0, 2H), 8.01 (s, 1H), 7.98 (s, 1H), 7.85 (d, J = 4.0, 2H), 7.41–7.35 (m, 2H), 7.32 (d, J = 7.1, 1H), 7.11 (t, J = 7.4, 1H), 6.50 (s, 1H); ¹³C NMR (101 MHz, DMSO-d₆) δ 163.6 (C), 159.6 (C), 150.3 (2CH), 141.4 (C), 136.1 (C), 135.3 (CH), 130.0 (C), 128.0 (C), 126.1 (C), 125.5 (CH), 125.4 (CH), 121.4 (2CH), 120.4 (CH), 119.9 (CH), 119.2 (C), 111.0 (CH), 100.8 (CH); HRMS (ESI+) m/z calcd for C₁₉H₁₅N₄O₂ (M + H)⁺ 331.1190, found 331.1194. HPLC purity ≥99%, method C: t_R = 3.63 min, λ = 346 nm.

4.1.10. N-[5-(1H-Indol-4-yl)-6-oxo-1,6-dihydropyridin-3-yl]piperidine-4-carboxamide 9

To compound 4 (100 mg, 0.238 mmol) was added a solution of 37% HCl (80 μL) in MeOH (5 mL). The mixture was concentrated under reduced pressure to obtained the corresponding hydrochloride salt. The salt was solubilized in MeOH (5 mL) and the solution was degassed with argon. 20% Pd(OH)₂/C (33 mg, 0.05 mmol, 0.2 eq.) was added and the mixture was hydrogenated at room temperature for 24 h. Et₃N (0.2 mL, 1.43 mmol, 6.0 eq.) was added and the mixture was filtered through a pad of Celite and the solid was washed with methanol. After evaporation under reduced pressure, the crude solid was purified by column chromatography (SiO₂, CH₂Cl₂/MeOH 1 : 1 + 2% Et₃N). The obtained solid was solubilized in acetonitrile and filtered to remove SiO₂. After evaporation, the solid was washed with CH₂Cl₂ to give 9 (27 mg, 0.080 mmol, 34%) as a white solid. R_f = 0.06 (CH₂Cl₂/MeOH 1 : 1 + 2% Et₃N); Mp > 241 °C (decomposition); IR (ATR) 3253, 2927, 2849, 1652, 1623, 1562, 1535, 1339, 1189, 1113, 958 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 11.14 (s, 1H), 9.63 (s, 1H), 7.88 (d, J = 2.6, 1H), 7.73 (d, J = 2.7, 1H), 7.39–7.34 (m, 2H), 7.28 (d, J = 7.2, 1H), 7.09 (t, J = 7.7 Hz, 1H), 6.43 (m, 1H), 3.00–2.92 (m, 2H), 2.51–2.41 (m, 2H), 2.38–2.27 (m, 1H), 1.70–1.61 (m, 2H), 1.56–1.43 (m, 2H), two exchangeable NH protons were not observed; ¹³C NMR (101 MHz, DMSO-d₆) δ 173.4 (C), 159.3 (C), 136.1 (C), 134.6 (CH), 129.9 (C), 128.2 (C), 126.1 (C), 125.3 (CH), 123.8 (CH), 120.4 (CH), 120.0 (C), 119.9 (CH), 110.9 (CH), 100.7 (CH), 45.5 (2CH₂), 43.4 (CH), 29.4 (2CH₂); HRMS (ESI+) m/z calcd for C₁₉H₂₁N₄O₂ (M + H)⁺ 337.1659, found 337.1652. HPLC purity ≥99%, method A: t_R = 17.91 min, λ = 280 nm.

4.1.11. 4-Amino-N-[5-(1H-indol-4-yl)-6-oxo-1,6-dihydropyridin-3-yl]benzamide 10

Compound 10 was prepared according to general procedure C in MeOH, starting from 5 (45 mg, 0.097 mmol) for 3 h. The crude was purified by column chromatography (Et₃N-treated SiO₂, CH₂Cl₂/MeOH 93 : 7 + 0.5% Et₃N to 90 : 10 + 0.5% Et₃N). The obtained solid was washed with chloroform to give 10 (31 mg, 0.090 mmol, 93%) as a beige solid. R_f = 0.19 (CH₂Cl₂/MeOH 92 : 8 + 0.5% Et₃N); Mp > 199 °C (decomposition); IR (ATR) 3215, 1599, 1498, 1337, 1268, 1178, 838, 752 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 11.53 (br s, 1H), 11.13 (s, 1H), 9.64 (s, 1H), 7.96 (d, J = 2.6, 1H), 7.93 (d, J = 2.5, 1H), 7.68 (d, J = 8.3, 2H), 7.41–7.33 (m, 2H), 7.30 (d, J = 7.2, 1H), 7.10 (t, J = 7.7 Hz, 1H), 6.59 (d, J = 8.4 Hz, 2H), 6.50 (s, 1H), 5.73 (s, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 165.3 (C), 159.5 (C), 152.2 (C), 136.2 (C), 136.0 (CH), 129.7 (C), 129.2 (2CH), 128.4 (C), 126.3 (C), 125.3 (CH), 124.8 (CH), 120.7 (C), 120.5 (CH), 120.3 (C), 120.0 (CH), 112.6 (2CH), 110.9 (CH), 101.0 (CH); HRMS (ESI+) m/z calcd for C₂₀H₁₇N₄O₂ (M + H)⁺ 345.1346, found 345.1338. HPLC purity ≥99%, method A: t_R = 19.11 min, λ = 280 nm.

4.1.12. 2-(Benzyloxy)-3-bromo-5-iodopyridine 12

To a solution of 11 (100 mg, 0.358 mmol) in acetonitrile (1.4 mL) and water (0.25 mL) in an ice bath was added p-toluenesulfonic acid monohydrate (204 mg, 1.07 mmol, 3 eq.). The resulting solution was stirred for 10 min and then a solution of sodium nitrite (49 mg, 0.71 mmol, 2 eq.) and KI (149 mg, 0.90 mmol, 2.5 eq.) in water (0.25 mL) was added dropwise. The mixture was stirred for 10 min in an ice bath and then at room temperature. The reaction was monitored by release of N₂. After addition of water, saturated aqueous solutions of NaHCO₃ and Na₂S₂O₃ were added. The aqueous phase was extracted three times with CH₂Cl₂. Combined organic phases were dried over MgSO₄, filtered and evaporated. The crude was purified by column chromatography (SiO₂, cyclohexane 100%) to give the desired product 12 (94 mg, 0.241 mmol, 67%) as a white solid. R_f = 0.19 (toluene/cyclohexane 1 : 9); Mp 83 °C; IR (ATR) 2923, 1559, 1466, 1428, 1352, 1309, 1300, 1238, 1048, 1014, 891, 730, 719, 692 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 8.40 (d, J = 2.0, 1H), 8.38 (d, J = 2.0, 1H), 7.47–7.43 (m, 2H), 7.42–7.36 (m, 2H), 7.36–7.30 (m, 1H), 5.40 (s, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 158.5 (C), 151.2 (CH), 148.6 (CH), 136.4 (C), 128.4 (2CH), 127.9 (CH), 127.6 (2CH), 107.8 (C), 83.5 (C), 68.1 (CH₂); HRMS (ESI+) m/z calcd for C₁₂H₁₀BrINO (M + H)⁺ 389.8985, found 389.8986.

4.1.13. 2-(Benzyloxy)-3-bromo-5-[2-(trimethylsilyl)ethoxy]pyridine 13

A screw-cap tube was charged with CuI (17 mg, 0.089 mmol, 0.1 eq.), 1,10-phenanthroline (33 mg, 0.183 mmol, 0.2 eq.), Cs₂CO₃ (593 mg, 1.82 mmol, 2 eq.), and 12 (355 mg, 0.91 mmol). The tube was flushed with argon and 2-(trimethylsilyl)ethanol (391 μL, 2.73 mmol, 3 eq.) and dry toluene (450 μL) were added. The tube was then sealed with a screw cap and placed in a pre-heated oil bath at 110 °C and stirred overnight. The resulting suspension was cooled to room temperature and concentrated under reduced pressure. The crude was purified by column chromatography (SiO₂, toluene/cyclohexane 1 : 1) to give the desired product 13 (328 mg, 0.86 mmol, 95%) as a white solid. R_f = 0.30 (toluene/cyclohexane 4 : 6); Mp 82 °C; IR (ATR) 3052, 2940, 1426, 1351, 1310, 1301, 1240, 731, 720, 692 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 7.87 (d, J = 2.4, 1H), 7.79 (d, J = 2.4, 1H), 7.44 (d, J = 7.4, 2H), 7.38 (t, J = 7.4, 2H), 7.31 (t, J = 7.1, 1H), 5.35 (s, 2H), 4.09 (t, J = 8.1, 2H), 1.05 (t, J = 8.1, 2H), 0.05 (s, 9H); ¹³C NMR (101 MHz, DMSO-d₆) δ 153.1 (C), 150.4 (C), 137.1 (C), 131.7 (CH), 129.7 (CH), 128.4 (2CH), 127.7 (CH), 127.4 (2CH), 105.9 (C), 67.7 (CH₂), 66.8 (CH₂), 17.2 (CH₂), −1.3 (3CH₃); HRMS (ESI+) m/z calcd for C₁₇H₂₃BrNO₂Si (M + H)⁺ 380.0676, found 380.0674.

4.1.14. 6-(Benzyloxy)-5-bromopyridin-3-ol 14

To a solution of 13 (525 mg, 1.38 mmol) in dry DMF (2.8 mL) under argon was added cesium fluoride (630 mg, 4.15 mmol, 3 eq.) and heated at 60 °C for 2 h 30 min. Reaction mass was diluted with water and extracted three times with ethyl acetate. Combined organic phases were washed with brine, dried over MgSO₄ and concentrated under reduced pressure. The crude was purified by column chromatography (SiO₂, cyclohexane/EtOAc 9 : 1 to 8 : 2) to give the desired product 14 (338 mg, 1.21 mmol, 87%) as a yellow solid. R_f = 0.38 (cyclohexane/EtOAc 8 : 2); Mp 145 °C; IR (ATR) 3250–2500, 1434, 1344, 1229, 1208, 1059, 1022, 736, 694 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 9.74 (s, 1H), 7.71 (d, J = 2.6, 1H), 7.51 (d, J = 2.6, 1H), 7.45–7.41 (m, 2H), 7.40–7.35 (m, 2H), 7.33–7.28 (m, 1H), 5.32 (s, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 152.1 (C), 149.2 (C), 137.3 (C), 131.7 (CH), 129.9 (CH), 128.4 (2CH), 127.6 (CH), 127.4 (2CH), 105.6 (C), 67.6 (CH₂); HRMS (ESI+) m/z calcd for C₁₂H₁₁BrNO₂ (M + H)⁺ 279.9968, found 279.9970.

4.1.15. 2-(Benzyloxy)-3-bromo-5-phenoxypyridine 15

Compound 15 was prepared according to general procedure D, starting from 14 (60 mg, 0.143 mmol). The mixture was heated for 24 h. The crude was purified by column chromatography (SiO₂, toluene/cyclohexane 2 : 8) to give 15 (31 mg, 0.087 mmol, 61%) as a colorless oil.

Alternatively, compound 15 can be prepared from 12: a screw-cap tube was charged with 12 (100 mg, 0.256 mmol), CuI (2.5 mg, 0.013 mmol, 0.05 eq.), Cs₂CO₃ (167 mg, 0.513 mmol, 2 eq.) and phenol (25 mg, 0.266 mmol, 1 eq.). The tube was flushed with argon and 1,4-dioxane (1 mL) and 2,2,6,6,6-tetramethyl-3,5-heptanedione (5 μL, 0.024 mmol, 0.1 eq.) were added. The tube was then sealed with a screw cap and heated in an oil bath at 100 °C for 41 h. The resulting mixture was diluted with water and extracted three times with EtOAc. Combined organic phases were washed with brine, dried over MgSO₄, filtered and evaporated under reduced pressure. The crude oil was purified by column chromatography (SiO₂, toluene/cyclohexane 1 : 9 to 2 : 8) to give 15 (60 mg, 0.168 mmol, 66%) as a colorless oil. R_f = 0.25 (toluene/cyclohexane 2 : 8); aspect: colorless oil; IR (ATR) 1587, 1436, 1212, 855, 729, 691 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 8.04 (d, J = 2.6, 1H), 7.93 (d, J = 2.6, 1H), 7.48 (d, J = 7.4, 2H), 7.43–7.31 (m, 5H), 7.14 (t, J = 7.3, 1H), 7.02 (d, J = 8.0, 2H), 5.41 (s, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 157.3 (C), 155.4 (C), 147.5 (C), 136.8 (1C + 1CH), 134.5 (CH), 130.1 (2CH), 128.4 (2CH), 127.9 (CH), 127.6 (2CH), 123.5 (CH), 117.4 (2CH), 106.2 (C), 68.2 (CH₂); HRMS (ESI+) m/z calcd for C₁₈H₁₅BrNO₂ (M + H)⁺ 356.0281, found 356.0272.

4.1.16. 2-Benzyloxy-3-bromo-5-(4-nitrophenoxy)pyridine 16

Compound 16 was prepared according to general procedure D, starting from 14 (60 mg, 0.214 mmol). The mixture was heated for 24 h. The crude was purified by column chromatography (SiO₂, toluene/cyclohexane 1 : 1) to give 16 (62 mg, 0.155 mmol, 72%) as a yellow-orange solid. R_f = 0.17 (toluene/cyclohexane 1 : 1); Mp 130 °C; IR (ATR) 1427, 1347, 1311, 1240, 1044, 891, 731, 721, 692, 605 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 8.25 (d, J = 9.1, 2H), 8.19 (d, J = 2.4, 1H), 8.18 (d, J = 2.4, 1H), 7.49 (d, J = 7.2, 2H), 7.41 (t, J = 7.3, 2H), 7.35 (t, J = 7.0, 1H), 7.19 (d, J = 9.1, 2H), 5.44 (s, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 163.0 (C), 156.5 (C), 145.6 (C), 142.5 (C), 138.1 (CH), 136.6 (C), 135.8 (CH), 128.5 (2CH), 127.9 (CH), 127.6 (2CH), 126.2 (2CH), 117.0 (2CH), 106.5 (C), 68.4 (CH₂); HRMS (ESI+) m/z calcd for C₁₈H₁₄BrN₂O₄ (M + H)⁺ 401.0131, found 401.0126.

4.1.17. 2-Benzyloxy-3-bromo-5-[(pyridin-4-yl)oxy]pyridine 17

Compound 17 was prepared according to general procedure D, starting from 14 (100 mg, 0.357 mmol). The mixture was heated for 24 h. The crude was purified by column chromatography (SiO₂, cyclohexane/EtOAc 8 : 2 to 7 : 3) to give 17 (105 mg, 0.294 mmol, 82%) as a white solid. R_f = 0.18 (cyclohexane/EtOAc 7 : 3); Mp 99 °C; IR (ATR) 3027, 3004, 2939, 2924, 1576, 1466, 1433, 1358, 1251, 1207, 1056, 901, 865, 816 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 8.48 (br s, 2H), 8.17 (s, 2H), 7.49 (d, J = 6.9, 2H), 7.41 (t, J = 7.1, 2H), 7.35 (t, J = 6.8, 1H), 6.98 (d, J = 4.3, 2H), 5.44 (s, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 164.2 (C), 156.5 (C), 151.5 (2CH), 144.9 (C), 138.2 (CH), 136.6 (C), 135.9 (CH), 128.4 (2CH), 127.9 (CH), 127.6 (2CH), 111.6 (2CH), 106.5 (C), 68.4 (CH₂); HRMS (ESI+) m/z calcd for C₁₇H₁₄BrN₂O₂ (M + H)⁺ 357.0233, found 357.0228.

4.1.18. 4-(2-Benzyloxy-5-phenoxypyridin-3-yl)-1H-indole 18

Compound 18 was prepared according to general procedure E, starting from 15 (114 mg, 0.32 mmol). The mixture was refluxed overnight. The crude was purified by column chromatography (SiO₂, cyclohexane/EtOAc 95 : 5 to 90 : 10) to give 18 (126 mg, 0.32 mmol, quantitative) as a white-green solid. R_f = 0.38 (cyclohexane/EtOAc 8 : 2); Mp 116 °C; IR (ATR) 3427, 3308, 1423, 1353, 1217, 731, 718, 686, 666, 624, 611 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 11.21 (s, 1H), 8.06 (d, J = 2.9, 1H), 7.50 (d, J = 2.9, 1H), 7.43–7.22 (m, 9H), 7.16–7.06 (m, 5H), 6.25 (m, 1H), 5.39 (s, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 157.6 (C), 156.3 (C), 147.6 (C), 137.3 (C), 136.01 (C), 135.98 (CH), 131.3 (CH), 130.1 (2CH), 128.2 (2CH), 127.53 (2CH), 127.47 (CH), 127.0 (C), 126.4 (C), 125.7 (CH), 124.7 (C), 123.2 (CH), 120.7 (CH), 120.1 (CH), 117.5 (2CH), 111.4 (CH), 100.3 (CH), 67.3 (CH₂); HRMS (ESI+) m/z calcd for C₂₆H₂₁N₂O₂ (M + H)⁺ 393.1598, found 393.1592.

4.1.19. 4-[2-Benzyloxy-5-(4-nitrophenoxy)pyridin-3-yl]-1H-indole 19

Compound 19 was prepared according to general procedure E, starting from 16 (170 mg, 0.424 mmol). The mixture was refluxed overnight. The crude was purified by column chromatography (SiO₂, cyclohexane/EtOAc 9 : 1 to 8 : 2) to give impure 19 (196 mg) as a brown solid. 19 was used in the next step (preparation of 22 and 24) without further purification. R_f = 0.28 (cyclohexane/EtOAc 8 : 2); ¹H NMR (400 MHz, DMSO-d₆) δ 11.22 (s, 1H), 8.27 (d, J = 9.3, 2H), 8.19 (d, J = 2.9, 1H), 7.70 (d, J = 2.9, 1H), 7.44–7.11 (m, 11H), 6.31 (m, 1H), 5.42 (s, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 163.3 (C), 157.4 (C), 145.7 (C), 142.3 (C), 137.3 (CH), 137.2 (C), 136.0 (C), 132.7 (CH), 128.2 (2CH), 127.6 (2CH), 127.5 (CH), 126.8 (C), 126.4 (C), 126.3 (2CH), 125.8 (CH), 125.2 (C), 120.7 (CH), 120.2 (CH), 116.9 (2CH), 111.5 (CH), 100.5 (CH), 67.5 (CH₂); HRMS (ESI+) m/z calcd for C₂₆H₂₀N₃O₄ (M + H)⁺ 437.1448, found 438.1435.

4.1.20. 4-{2-Benzyloxy-5-[(pyridin-4-yl)oxy]pyridin-3-yl}-1H-indole 20

Compound 20 was prepared according to general procedure E, starting from 17 (80 mg, 0.224 mmol). The mixture was refluxed for 1 h 30 min. The crude was purified by column chromatography (SiO₂, cyclohexane/EtOAc 5 : 5 to 6 : 4). The obtained solid was solubilized in EtOAc and the solution was washed with a 0.1 M aqueous solution of HCl and then with a saturated aqueous solution of Na₂CO₃ to remove a residual impurity. The organic layer was dried over MgSO₄, filtered and concentrated under reduced pressure to give 20 (80 mg, 0.203 mmol, 91%) as a grey solid. R_f = 0.23 (cyclohexane/EtOAc 1 : 1); Mp > 162 °C (decomposition); IR (ATR) 3055, 2919, 2853, 1577, 1438, 1418, 1242, 1206, 989, 825 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 11.22 (s, 1H), 8.48 (d, J = 5.1, 2H), 8.17 (s, 1H), 7.68 (s, 1H), 7.41 (d, J = 7.1, 1H), 7.39–7.22 (m, 6H), 7.19–7.10 (m, 2H), 7.04 (d, J = 5.2, 2H), 6.30 (s, 1H), 5.41 (s, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 164.5 (C), 157.4 (C), 151.6 (2CH), 145.0 (C), 137.4 (CH), 137.2 (C), 136.0 (C), 132.8 (CH), 128.2 (2CH), 127.6 (2CH), 127.5 (CH), 126.8 (C), 126.5 (C), 125.8 (CH), 125.1 (C), 120.7 (CH), 120.2 (CH), 111.6 (2CH), 111.5 (CH), 100.4 (CH), 67.5 (CH₂); HRMS (ESI+) m/z calcd for C₂₅H₂₀N₃O₂ (M + H)⁺ 394.1550, found 394.1541.

4.1.21. 3-(1H-Indol-4-yl)-5-phenoxypyridin-2(1H)-one 21

Compound 21 was prepared according to general procedure C in CH₂Cl₂/MeOH (1 : 1), starting from 18 (100 mg, 0.255 mmol). The mixture was stirred for 3 h 30 min. The crude was purified by column chromatography (Et₃N-treated SiO₂, cyclohexane/EtOAc 3 : 7 to 0 : 10) to give 21 (73 mg, 0.241 mmol, 95%) as a white solid. R_f = 0.10 (EtOAc 100% + 0.5% Et₃N); Mp > 165 °C (decomposition); IR (ATR) 3285, 1652, 1613, 1588, 1557, 1488, 1464, 1323, 1220, 873, 852, 747, 726, 689, 616 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 11.68 (br s, 1H), 11.15 (s, 1H), 7.48–7.30 (m, 6H), 7.26 (d, J = 6.9, 1H), 7.14–7.02 (m, 4H), 6.28 (s, 1H); ¹³C NMR (101 MHz, DMSO-d₆) δ 159.7 (C), 158.2 (C), 137.5 (C), 136.1 (C), 134.6 (CH), 130.8 (C), 130.0 (2CH), 127.8 (C), 126.1 (C), 125.7 (CH), 125.5 (CH), 122.7 (CH), 120.4 (CH), 120.0 (CH), 116.5 (2CH), 111.1 (CH), 100.3 (CH); HRMS (ESI+) m/z calcd for C₁₉H₁₅N₂O₂ (M + H)⁺ 303.1128, found 303.1122. HPLC purity ≥96%, method A: t_R = 25.43 min, λ = 280 nm.

4.1.22. 3-(1H-Indol-4-yl)-5-(4-nitrophenoxy)pyridin-2(1H)-one 22

Compound 22 was prepared according to general procedure B, starting from 19 (80 mg). The mixture was stirred for 1 h at 0 °C. The crude was purified by column chromatography (Et₃N-treated SiO₂, CH₂Cl₂/MeOH 99 : 1 to 97 : 3). The obtained solid was washed with CH₂Cl₂ to give 22 (33 mg, 0.095 mmol, 55% in two steps from 16) as a pale-yellow solid. R_f = 0.31 (CH₂Cl₂/MeOH 95 : 5); Mp > 245 °C (decomposition); IR (ATR) 3258, 1607, 1512, 1467, 1331, 1239, 1109, 845 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 11.88 (br s, 1H), 11.15 (s, 1H), 8.27 (d, J = 8.5, 2H), 7.60 (s, 1H), 7.53 (s, 1H), 7.38 (d, J = 7.5, 1H), 7.34 (s, 1H), 7.30–7.22 (m, 3H), 7.10 (t, J = 7.5, 1H), 6.32 (s, 1H); ¹³C NMR (101 MHz, DMSO-d₆) δ 163.6 (C), 160.0 (C), 142.1 (C), 136.1 (C), 135.9 (C), 134.5 (CH), 131.4 (C), 127.7 (C), 127.0 (CH), 126.2 (2CH), 126.1 (C), 125.5 (CH), 120.4 (CH), 120.0 (CH), 116.4 (2CH), 111.2 (CH), 110.5 (CH); HRMS (ESI+) m/z calcd for C₁₉H₁₄N₃O₄ (M + H)⁺ 348.0979, found 348.0970. HPLC purity ≥98%, method D: t_R = 4.33 min, λ = 318 nm.

4.1.23. 3-(1H-Indol-4-yl)-5-[(pyridin-4-yl)oxy]pyridin-2(1H)-one 23

Compound 23 was prepared according to general procedure B, starting from 20 (57 mg, 0.145 mmol). The mixture was stirred for 5 h 30 min at 0 °C. The crude was purified by column chromatography (Et₃N-treated SiO₂, CH₂Cl₂/MeOH 97 : 3 to 94 : 6). The obtained solid was washed with CH₂Cl₂ to give 23 (24 mg, 0.079 mmol, 55%) as a pale-yellow solid. R_f = 0.22 (CH₂Cl₂/MeOH 95 : 5); Mp > 250 °C; IR (ATR) 3200–2700, 1615, 1586, 1473, 1326, 1255, 1237, 1208, 875, 828 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 11.84 (br s, 1H), 11.15 (s, 1H), 8.47 (m, 2H), 7.57 (d, J = 2.5 Hz, 1H), 7.50 (d, J = 3.0, 1H), 7.37 (d, J = 8.0, 1H), 7.33 (t, J = 2.6, 1H), 7.25 (d, J = 7.0, 1H), 7.10 (t, J = 7.7, 1H), 7.05 (m, 2H), 6.31 (s, 1H); ¹³C NMR (101 MHz, DMSO-d₆) δ 164.7 (C), 160.0 (C), 151.5 (2CH), 136.1 (C), 135.3 (C), 134.6 (CH), 131.2 (C), 127.7 (C), 127.0 (CH), 126.1 (C), 125.5 (CH), 120.4 (CH), 120.0 (CH), 111.3 (2CH), 111.2 (CH), 100.4 (CH); HRMS (ESI+) m/z calcd for C₁₈H₁₄N₃O₂ (M + H)⁺ 304.1081, found 304.1075. HPLC purity ≥99%, method C: t_R = 2.75 min, λ = 346 nm.

4.1.24. 5-(4-Aminophenoxy)-3-(1H-indol-4-yl)pyridin-2(1H)-one 24

Compound 24 was prepared according to general procedure C in CH₂Cl₂/MeOH (1 : 1), starting from 19 (80 mg). The mixture was stirred for 24 h. The crude was purified by column chromatography (Et₃N-treated SiO₂, CH₂Cl₂/MeOH 96 : 4 to 93 : 7). The obtained solid was washed with CH₂Cl₂ to give 24 (35 mg, 0.110 mmol, 64% in two steps from 16) as a beige solid. R_f = 0.22 (CH₂Cl₂/MeOH 95 : 5); Mp > 152 °C (decomposition); IR (ATR) 3379, 3332, 3106, 2849, 1619, 1502, 1468, 1329, 1209, 852 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 11.48 (br s, 1H), 11.14 (s, 1H), 7.40 (d, J = 2.6, 1H), 7.36 (d, J = 8.0, 1H), 7.32 (m, 1H), 7.23 (d, J = 7.2, 1H), 7.17 (d, J = 2.6, 1H), 7.09 (t, J = 7.7, 1H), 6.80 (d, J = 8.5, 2H), 6.56 (d, J = 8.4, 2H), 6.26 (s, 1H), 4.89 (s, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 159.3 (C), 147.7 (C), 144.8 (C), 140.3 (C), 136.1 (C), 133.6 (CH), 130.3 (C), 128.0 (C), 126.1 (C), 125.4 (CH), 123.3 (CH), 120.4 (CH), 119.9 (CH), 118.8 (2CH), 114.8 (2CH), 111.0 (CH), 100.3 (CH); HRMS (ESI+) m/z calcd for C₁₉H₁₆N₃O₂ (M + H)⁺ 318.1237, found 318.1230. HPLC purity ≥98%, method B: t_R = 21.32 min, λ = 240 nm.

4.1.25. 3-(1H-Indol-4-yl)-5-[(piperidin-4-yl)oxy]pyridin-2(1H)-one 25

Compound 25 was prepared according to general procedure C in CH₂Cl₂/MeOH (1 : 1), starting from 27 (120 mg, 0.225 mmol). The mixture was stirred for 24 h. The crude was purified by column chromatography (Et₃N-treated SiO₂, CH₂Cl₂/MeOH 85 : 15 + 1% Et₃N). The obtained solid was washed with CH₂Cl₂ to give 25 (45 mg, 0.145 mmol, 65%) as a white solid. R_f = 0.10 (CH₂Cl₂/MeOH 8 : 2 + 1% Et₃N); Mp > 167 °C (decomposition); IR (ATR) 3240, 2944, 1650, 1604, 1559, 1465, 1334, 1208, 1152, 1000, 819 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 11.13 (s, 1H), 7.43 (d, J = 2.9, 1H), 7.39–7.33 (m, 2H), 7.20 (d, J = 7.2, 1H), 7.14 (d, J = 2.7, 1H), 7.09 (t, J = 7.7, 1H), 6.31 (s, 1H), 4.12–4.04 (m, 1H), 2.98–2.89 (m, 2H), 2.50 (2H under solvent residual signal), 1.93–1.84 (m, 2H), 1.48–1.37 (m, 2H), two exchangeable NH protons were not observed; ¹³C NMR (101 MHz, DMSO-d₆) δ 159.0 (C), 139.6 (C), 136.1 (C), 134.3 (CH), 130.2 (C), 128.4 (C), 126.2 (C), 125.3 (CH), 122.0 (CH), 120.4 (CH), 119.9 (CH), 110.9 (CH), 100.6 (CH), 76.4 (CH), 43.5 (2CH₂), 32.2 (2CH₂); HRMS (ESI+) m/z calcd for C₁₈H₂₀N₃O₂ (M + H)⁺ 310.1550, found 310.1541. HPLC purity ≥98%, method A: t_R = 18.21 min, λ = 280 nm.

4.1.26. Benzyl 4-[(6-benzyloxy-5-bromopyridin-3-yl)oxy]piperidine-1-carboxylate 26

A screw-cap tube was charged with CuI (5 mg, 0.026 mmol, 0.1 eq.), 1,10-phenanthroline (10 mg, 0.055 mmol, 0.2 eq.), Cs₂CO₃ (167 mg, 0.51 mmol, 2 eq.), 12 (100 mg, 0.256 mmol) and benzyl 4-hydroxy-1-piperidinecarboxylate (181 mg, 0.77 mmol, 3 eq.). The tube was flushed with argon and dry toluene (1 mL) was added. The tube was then sealed with a screw cap and placed in a pre-heated oil bath at 110 °C and stirred for 24 h. The resulting mixture was filtered through a pad of Celite which was then washed with EtOAc. The filtrate was evaporated under reduced pressure. The crude was purified by column chromatography (SiO₂, cyclohexane/EtOAc 9 : 1 to 8 : 2) to give the desired product 26 (85 mg, 0.171 mmol, 67%) as a white wax. R_f = 0.35 (cyclohexane/EtOAc 8 : 2); IR (ATR) 2948, 2868, 1693, 1430, 1357, 1221, 1196, 1046, 1026 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 7.94 (d, J = 2.6, 1H), 7.90 (d, J = 2.6, 1H), 7.44 (d, J = 7.3, 2H), 7.41–7.29 (m, 8H), 5.36 (s, 2H), 5.08 (s, 2H), 4.60–4.52 (m, 1H), 3.76–3.67 (m, 2H), 3.32–3.19 (m, 2H), 1.94–1.85 (m, 2H), 1.59–1.48 (m, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 154.4 (C), 153.5 (C), 148.7 (C), 137.05 (C), 136.96 (C), 133.9 (CH), 131.5 (CH), 128.42 (2CH), 128.38 (2CH), 127.8 (CH), 127.7 (CH), 127.53 (2CH), 127.47 (2CH), 105.9 (C), 73.9 (CH), 67.8 (CH₂), 66.2 (CH₂), 40.7 (2CH₂), 30.1 (2CH₂); HRMS (ESI+) m/z calcd for C₂₅H₂₆BrN₂O₄ (M + H)⁺ 497.1070, found 497.1070.

4.1.27. Benzyl 4-{[6-benzyloxy-5-(1H-indol-4-yl)pyridin-3-yl]oxy}piperidine-1-carboxylate 27

Compound 27 was prepared according to general procedure E, starting from 26 (200 mg, 0.402 mmol). The mixture was refluxed overnight. The crude was purified by column chromatography (SiO₂, cyclohexane/EtOAc 85 : 5 to 75 : 25) to give 27 (195 mg, 0.365 mmol, 91%) as a white solid. R_f = 0.36 (cyclohexane/EtOAc 7 : 3); Mp 62 °C; IR (ATR) 3421, 2928, 2861, 1678, 1421, 1222, 1198, 1024 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 11.19 (s, 1H), 7.95 (d, J = 2.5, 1H), 7.50 (d, J = 2.5, 1H), 7.43–7.20 (m, 12H), 7.17–7.08 (m, 2H), 6.26 (s, 1H), 5.32 (s, 2H), 5.08 (s, 2H), 4.61–4.53 (m, 1H), 3.79–3.67 (m, 2H), 3.36–3.19 (m, 2H), 1.99–1.88 (m, 2H), 1.66–1.53 (m, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 154.6 (C), 154.4 (C), 148.5 (C), 137.6 (C), 137.0 (C), 136.0 (C), 133.3 (CH), 129.0 (CH), 128.4 (2CH), 128.1 (2CH), 127.8 (CH), 127.7 (C), 127.5 (2CH), 127.4 (2CH), 127.3 (CH), 126.6 (C), 125.6 (CH), 124.2 (C), 120.7 (CH), 120.1 (CH), 111.1 (CH), 100.6 (CH), 73.4 (CH), 67.0 (CH₂), 66.2 (CH₂), 40.7 (2CH₂), 30.3 (2CH₂); HRMS (ESI+) m/z calcd for C₃₃H₃₂N₃O₄ (M + H)⁺ 534.2387, found 534.2379.

4.1.28. 3-Bromo-1-methyl-5-nitropyridin-2(1H)-one 29

To a microwave tube (CEM, 10 mL) charged with 1 (126 mg, 0.58 mmol) in acetonitrile (3 mL) under argon atmosphere were added Ag₂CO₃ (189 mg, 0.68 mmol, 1.2 eq.) and then MeI (70 μL, 1.12 mmol, 2 eq.). The tube was sealed and the mixture was irradiated for 2 × 1 h (dynamic control type, P_max = 150 W, T = 60 °C). Then, the mixture was filtered through a pad of Celite which was then washed with ethyl acetate. After evaporation under reduced pressure, the crude was purified by column chromatography (SiO₂, cyclohexane/EtOAc 98 : 2 to 7 : 3) to give 29 (128 mg, 0.55 mmol, 95%) as a white solid. R_f = 0.11 (cyclohexane/EtOAc 8 : 2); for characterization data, see ref. 8.

4.1.29. 3-Bromo-2-methoxy-5-nitropyridine 30

To a solution of 1 (1.26 g, 5.75 mmol) in anhydrous toluene (57 mL) under argon atmosphere were added crushed Ag₂CO₃ (2.15 g, 7.8 mmol, 1.4 eq.) and MeI (3.52 mL, 57 mmol, 10 eq.). The mixture was stirred at room temperature overnight. The mixture was filtered through a pad of Celite which was then washed with ethyl acetate. After evaporation under reduced pressure, the crude was purified by column chromatography (SiO₂, cyclohexane/EtOAc 95 : 5 to 6 : 4) to give the desired O-methylated product 30 (378 mg, 1.62 mmol, 28%) as a pale-yellow solid but also the N-methylated product 29 (897 mg, 3.85 mmol, 67%) as a pale-yellow solid. R_f = 0.55 (cyclohexane/EtOAc 9 : 1); for characterization data, see ref. 8.

4.1.30. 5-Amino-3-bromo-1-methylpyridin-2(1H)-one 31

Compound 31 was prepared according to general procedure F, starting from 29 (478 mg, 2.05 mmol). The mixture was refluxed for 4 h. The crude was purified by column chromatography (SiO₂, CH₂Cl₂/MeOH 98 : 2 to 97 : 3) to give 31 (305 mg, 1.50 mmol, 73%) as a green solid. R_f = 0.73 (CH₂Cl₂/MeOH 95 : 5); Mp > 162 °C (decomposition); IR (ATR) 3313, 3185, 1565, 1532, 1408, 1256, 1121, 771, 743 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 7.56 (d, J = 2.8, 1H), 6.95 (d, J = 2.8, 1H), 4.39 (br s, 2H), 3.40 (s, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 155.3 (C), 136.2 (CH), 129.0 (C), 120.8 (CH), 114.8 (C), 38.1 (CH₃); HRMS (ESI+) m/z calcd for C₆H₈BrN₂O (M + H)⁺ 202.9815, found 202.9814.

4.1.31. 5-Bromo-6-methoxypyridin-3-amine 32

Compound 32 was prepared according to general procedure F, starting from 30 (473 mg, 2.03 mmol). The mixture was refluxed for 1.5 h. The crude was purified by column chromatography (SiO₂, cyclohexane/EtOAc 7 : 3) to give 32 (413 mg, 2.03 mmol, quantitative) as a yellow solid. R_f = 0.34 (cyclohexane/EtOAc 7 : 3); Mp 58 °C; IR (ATR) 3413, 3333, 3217, 2933, 1474, 1403, 1246, 1219, 1052, 1011, 864 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 7.50 (d, J = 2.5, 1H), 7.30 (d, J = 2.5, 1H), 4.98 (s, 2H), 3.77 (s, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 151.0 (C), 140.9 (C), 130.1 (CH), 128.2 (CH), 105.4 (C), 53.6 (CH₃); HRMS (ESI+) m/z calcd for C₆H₈BrN₂O (M + H)⁺ 202.9815, found 202.9815.

4.1.32. 3-Bromo-1-methyl-5-[(pyridin-4-yl)amino]pyridin-2(1H)-one 33

Compound 33 was prepared according to general procedure G, starting from 31 (150 mg, 0.74 mmol). The mixture was heated for 6 h. The crude was purified by column chromatography (SiO₂, CH₂Cl₂/MeOH 98 : 2 + 0.5% Et₃N) to give 33 (169 mg, 0.60 mmol, 82%) as a green solid. R_f = 0.22 (CH₂Cl₂/MeOH 98 : 2 + 0.5% Et₃N); Mp > 198 °C (decomposition); IR (ATR) 3273, 1663, 1571, 1512, 1211, 990, 806 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 8.20 (s, 1H), 8.11 (m, 2H), 7.85 (d, J = 2.7, 1H), 7.81 (d, J = 2.7, 1H), 6.60 (m, 2H), 3.51 (s, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 157.1 (C), 152.4 (C), 149.8 (2CH), 141.8 (CH), 135.7 (CH), 118.9 (C), 114.6 (C), 108.2 (2CH), 38.3 (CH₃); HRMS (ESI+) m/z calcd for C₁₁H₁₁BrN₃O (M + H)⁺ 280.0080, found 280.0078.

4.1.33. 5-Bromo-6-methoxy-N-(pyridin-4-yl)pyridin-3-amine 34

Compound 34 was prepared according to general procedure G, starting from 32 (300 mg, 1.48 mmol). The mixture was heated for 2 h. The crude was purified by column chromatography (SiO₂, EtOAc + 0.5% Et₃N) to give 34 (303 mg, 1.08 mmol, 73%) as a yellow solid. R_f = 0.17 (EtOAc + 0.5% Et₃N); Mp 172 °C; IR (ATR) 3280–2700, 1602, 1593, 1524, 1472, 1416, 1290, 1214, 1054, 990, 810, 738 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 8.68 (s, 1H), 8.17 (m, 2H), 8.07 (d, J = 2.4, 1H), 7.91 (d, J = 2.4, 1H), 6.75 (m, 2H), 3.92 (s, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 155.7 (C), 150.7 (C), 150.1 (2CH), 139.0 (CH), 136.3 (CH), 131.9 (C), 108.6 (2CH), 105.8 (C), 54.3 (CH₃); HRMS (ESI+) m/z calcd for C₁₁H₁₁BrN₃O (M + H)⁺ 280.0080, found 280.0083.

4.1.34. 3-(1H-Indol-4-yl)-1-methyl-5-[(pyridin-4-yl)amino]pyridin-2(1H)-one 35

Compound 35 was prepared according to general procedure E, starting from 33 (100 mg, 0.357 mmol). The mixture was refluxed overnight. The crude was purified by column chromatography (SiO₂, CH₂Cl₂/MeOH 97 : 3 + 0.5% Et₃N) to give 35 (110 mg, 0.348 mmol, 97%) as a beige solid. R_f = 0.30 (CH₂Cl₂/MeOH 95 : 5 + 0.5% Et₃N); Mp > 230 °C (decomposition); IR (ATR) 3261, 1586, 1574, 1519, 999, 802, 760 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 11.15 (s, 1H), 8.30 (s, 1H), 8.12 (d, J = 5.8, 2H), 7.76 (d, J = 2.6, 1H), 7.47 (d, J = 2.8, 1H), 7.37 (d, J = 8.0, 1H), 7.34 (t, J = 2.5, 1H), 7.24 (d, J = 7.2, 1H), 7.10 (t, J = 7.7, 1H), 6.68 (d, J = 6.0, 2H), 6.33 (s, 1H), 3.54 (s, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 159.6 (C), 152.8 (C), 149.4 (2CH), 137.8 (CH), 136.1 (C), 134.0 (CH), 130.3 (C), 128.3 (C), 126.2 (C), 125.4 (CH), 120.5 (CH), 120.0 (CH), 118.5 (C), 111.0 (CH), 108.2 (2CH), 100.6 (CH), 37.6 (CH₃); HRMS (ESI+) m/z calcd for C₁₉H₁₇N₄O (M + H)⁺ 317.1397, found 317.1395. HPLC purity ≥98%, method A: t_R = 18.43 min, λ = 280 nm.

4.1.35. 5-(1H-Indol-4-yl)-6-methoxy-N-(pyridin-4-yl)pyridin-3-amine 36

Compound 36 was prepared according to general procedure E, starting from 34 (100 mg, 0.357 mmol). The mixture was refluxed overnight. The crude was purified by two column chromatography (SiO₂, EtOAc + 0.5% Et₃N, then CH₂Cl₂/MeOH 99 : 1 + 0.5% Et₃N) to give 36 (97 mg, 0.307 mmol, 86%) as a beige solid. R_f = 0.15 (EtOAc + 0.5% Et₃N); Mp 132 °C; IR (ATR) 3300–2700, 1605, 1591, 1465, 1396, 1213, 996, 808, 748, 731 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 11.21 (s, 1H), 8.67 (s, 1H), 8.15 (m, 2H), 8.11 (d, J = 2.7, 1H), 7.60 (d, J = 2.7, 1H), 7.42 (d, J = 8.0, 1H), 7.37 (m, J = 2.8, 1H), 7.15 (t, J = 7.7, 1H), 7.08 (dd, J = 7.2, 0.9, 1H), 6.79 (m, 2H), 6.25 (m, 1H), 3.84 (s, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 157.0 (C), 151.2 (C), 150.1 (2CH), 138.6 (CH), 136.0 (C), 133.9 (CH), 130.9 (C), 127.6 (C), 126.5 (C), 125.7 (CH), 124.0 (C), 120.8 (CH), 120.1 (CH), 111.2 (CH), 108.5 (2CH), 100.4 (CH), 53.3 (CH₃); HRMS (ESI+) m/z calcd for C₁₉H₁₇N₄O (M + H)⁺ 317.1397, found 317.1393. HPLC purity ≥99%, method A: t_R = 20.67 min, λ = 280 nm.

4.1.36. 6-Benzyloxy-5-bromo-N-methylpyridin-3-amine 37

To a solution of 11 (100 mg, 0.358 mmol) in CH₂Cl₂ (1.2 mL) were added Et₃N (91 μL, 0.65 mmol, 1.8 eq.) and trifluoroacetic acid anhydride (100 μL, 0.72 mmol, 2 eq.) at 0 °C. The mixture was stirred for 30 min at 0 °C and then concentrated under reduced pressure. The residue was solubilized in acetone (1.15 mL), then K₂CO₃ (247 mg, 1.790 mmol, 5 eq.) and MeI (110 μL, 1.77 mmol, 5 eq.) were added. The mixture was stirred at 40 °C for 2 h. After cooling, the suspension was filtered and the filtrate was concentrated under reduced pressure to give a white solid. The intermediate was dissolved in MeOH (1.15 mL) and ACN (0.5 mL), and NaOH (1 M, 537 μL, 0.537 mmol, 1.5 eq.) was added. The mixture was stirred for 1.5 h, diluted in water and extracted three times with ethyl acetate. Combined organic phases were dried over MgSO₄, filtered and concentrated under reduced pressure. The crude oil was purified by column chromatography (SiO₂, cyclohexane/EtOAc 9 : 1) to give 37 (97 mg, 0.331 mmol, 92% in three steps from 11) as a colorless oil. R_f = 0.16 (cyclohexane/EtOAc 9 : 1); IR (ATR) 3413, 2927, 2881, 2814, 1490, 1472, 1445, 1424, 1358, 1286, 1229, 1044, 992, 862, 728, 694 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 7.47 (d, J = 2.6, 1H), 7.44–7.40 (m, 2H), 7.39–7.34 (m, 2H), 7.32–7.27 (m, 1H), 7.30 (d, J = 2.6, 1H), 5.59 (q, J = 5.0, 1H), 5.29 (s, 2H), 2.65 (d, J = 5.2, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 150.2 (C), 142.6 (C), 137.6 (C), 128.3 (2CH), 127.8 (CH), 127.5 (CH), 127.3 (2CH), 126.2 (CH), 106.2 (C), 67.3 (CH₂), 30.1 (CH₃); HRMS (ESI+) m/z calcd for C₁₃H₁₄BrN₂O (M + H)⁺ 293.0284, found 293.0282.

4.1.37. 6-Benzyloxy-5-bromo-N-methyl-N-(pyridin-4-yl)pyridin-3-amine 38

Compound 38 was prepared according to general procedure G, starting from 37 (171 mg, 0.58 mmol). The mixture was heated for 4 h. The crude was purified by column chromatography (SiO₂, EtOAc + 0.5% Et₃N) to give 38 (192 mg, 0.52 mmol, 89%) as an orange oil. R_f = 0.18 (EtOAc + 0.5% Et₃N); IR (ATR) 3029, 1587, 1442, 1222, 1049, 985, 806, 732, 695 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 8.17–8.14 (m, 3H), 8.12 (d, J = 2.3, 1H), 7.52–7.47 (m, 2H), 7.44–7.39 (m, 2H), 7.38–7.32 (m, 1H), 6.58 (m, 2H), 5.45 (s, 2H), 3.26 (s, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 157.0 (C), 153.5 (C), 149.7 (2CH), 144.3 (CH), 141.3 (CH), 137.3 (C), 136.7 (C), 128.4 (2CH), 127.9 (CH), 127.6 (2CH), 108.1 (CH), 106.4 (C), 68.2 (CH₂), 39.4 (CH₃); HRMS (ESI+) m/z calcd for C₁₈H₁₇BrN₃O (M + H)⁺ 370.0550, found 370.0551.

4.1.38. 6-Benzyloxy-5-(1H-indol-4-yl)-N-methyl-N-(pyridin-4-yl)pyridin-3-amine 39

Compound 39 was prepared according to general procedure E, starting from 38 (173 mg, 0.467 mmol). The mixture was refluxed overnight. The crude was purified by column chromatography (SiO₂, EtOAc + 0.5% Et₃N) to give 39 (162 mg, 0.399 mmol, 85%) as a beige solid. R_f = 0.17 (EtOAc + 0.5% Et₃N); Mp > 224 °C (decomposition); IR (ATR) 3250–2980, 2940, 1591, 1506, 1456, 1237, 1220, 988, 818, 749, 736, 723, 693 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 11.21 (s, 1H), 8.17 (d, J = 2.6, 1H), 8.16 (m, 2H), 7.71 (d, J = 2.6, 1H), 7.42–7.23 (m, 7H), 7.18–7.11 (m, 2H), 6.64 (m, 2H), 6.29 (m, 1H), 5.43 (s, 2H), 3.32 (s, 3H, under H₂O signal); ¹³C NMR (101 MHz, DMSO-d₆), not recorded due to low solubility in DMSO-d₆; HRMS (ESI+) m/z calcd for C₂₆H₂₃N₄O (M + H)⁺ 407.1866, found 407.1870.

4.1.39. 3-(1H-Indol-4-yl)-5-[methyl(pyridin-4-yl)amino]pyridin-2(1H)-one 40

To a solution under argon of 39 (25 mg, 0.062 mmol) and acetic acid (23 μL, 0.40 mmol, 6.5 eq.) in MeOH (1.6 mL) degassed with argon was added 20% Pd(OH)₂/C (11 mg, 0.016 mmol, 0.25 eq.). The mixture was then hydrogenated (balloon) at room temperature overnight. The mixture was evaporated under reduced pressure and the crude was purified by column chromatography (SiO₂, CH₂Cl₂/MeOH 96 : 4 + 0.5% Et₃N). After evaporation, the solid was washed with chloroform to give 40 (7 mg, 0.022 mmol, 36%) as a beige solid. R_f = 0.11 (CH₂Cl₂/MeOH 96 : 4 + 0.5% Et₃N); Mp > 250 °C; IR (ATR) 3150–2600, 1648, 1624, 1597, 997, 821, 754 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 11.88 (br s, 1H), 11.14 (s, 1H), 8.16 (d, J = 5.3, 2H), 7.49–7.46 (m, 2H), 7.36 (d, J = 8.1, 1H), 7.33 (t, J = 2.7, 1H), 7.26 (d, J = 7.3, 1H), 7.10 (t, J = 7.7, 1H), 6.63 (d, J = 5.7, 2H), 6.31 (m, 1H), 3.23 (s, 3H); ¹³C NMR (126 MHz, DMSO-d₆) δ 160.3 (C), 154.0 (C), 149.7 (2CH), 139.8 (CH), 136.1 (C), 132.9^a (CH), 131.6^a (C), 128.0 (C), 126.1 (C), 125.4 (CH), 125.3^a (C), 120.4 (CH), 119.9 (CH), 111.0 (CH), 107.9 (2CH), 100.5 (CH), 39.5 (CH₃), ^a determined from ¹H–¹³C HSQC and HMBC experiments; HRMS (ESI+) m/z calcd for C₁₉H₁₇N₄O (M + H)⁺ 317.1397, found 317.1394. HPLC purity ≥97%, method A: t_R = 18.44 min, λ = 280 nm.

4.1.40. 6-(Benzyloxy)-5-(1-methyl-1H-indol-4-yl)-N-(pyridin-4-yl)pyridin-3-amine 44

A microwave tube under argon was charged with 42 (50 mg, 0.238 mmol), Pd(dppf)Cl₂ (17 mg, 0.023 mmol, 0.1 eq.), KOAc (70 mg, 0.71 mmol, 3 eq.) and bis(pinacolato)diboron (91 mg, 0.36 mmol, 1.5 eq.). Then, anhydrous 1,4-dioxane (1 mL) degassed with argon was added. The tube was sealed and the mixture was heated under microwave irradiation for 30 min (dynamic control, P_max = 150 W, T = 80 °C). The resulting mixture was concentrated under reduced pressure and the crude was purified by column chromatography (SiO₂, cyclohexane/acetone 8 : 2 to 5 : 5), but some of the starting material remained in the product 43 (60 mg). R_f = 0.73 (cyclohexane/acetone 1 : 1). Compound 43 was used for the next step without further purification.

Compound 44 was prepared according to general procedure E, starting from 41 (76 mg, 0.213 mmol) in 1.5 mL of 1,4-dioxane and 43 (60 mg). The solution was refluxed overnight. After Celite filtration, the solution was washed with water and then with brine, dried over MgSO₄, filtered and concentrated under reduced pressure. The crude was purified by column chromatography (SiO₂, cyclohexane/acetone 95 : 5 + 0.5% Et₃N to 50 : 50 + 0.5% Et₃N) to give 44 (48 mg, 0.118 mmol, 55%) as a white solid. R_f = 0.25 (cyclohexane/acetone 1 : 1 + 0.5% Et₃N); Mp 85 °C; IR (ATR) 3029, 2921, 2848, 1591, 1425, 1413, 1287, 1213, 992, 813 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 8.70 (s, 1H), 8.15 (d, J = 5.9, 2H), 8.11 (d, J = 2.6, 1H), 7.62 (d, J = 2.5, 1H), 7.45 (d, J = 7.5, 1H), 7.38–7.17 (m, 8H), 6.80 (d, J = 5.9, 2H), 6.29 (d, J = 2.9, 1H), 5.38 (s, 2H), 3.81 (s, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 156.3 (C), 151.0 (C), 150.1 (2CH), 138.4 (CH), 137.5 (C), 136.5 (C), 134.0 (CH), 131.2 (C), 129.9 (CH), 128.2 (2CH), 127.7 (C), 127.5 (2CH), 127.4 (CH), 126.8 (C), 123.8 (C), 120.8 (CH), 120.3 (CH), 109.5 (CH), 108.5 (2CH), 99.7 (CH), 67.1 (CH₂), 32.6 (CH₃); HRMS (ESI+) m/z calcd for C₂₆H₂₃N₄O (M + H)⁺ 407.1866, found 407.1859.

4.1.41. 3-(1-Methyl-1H-indol-4-yl)-5-(pyridin-4-ylamino)pyridin-2(1H)-one 45

Compound 45 was prepared according to general procedure H, starting from 44 (80 mg, 0.197 mmol) in CH₂Cl₂/MeOH 1 : 1 (4 mL). The crude was purified by column chromatography (SiO₂, CH₂Cl₂/MeOH 95 : 5 + 0.5% Et₃N to 90 : 10 + 0.5% Et₃N). After evaporation, the solid was washed with CH₂Cl₂ to give 45 (45 mg, 0.142 mmol, 72%) as a pale-yellow solid. R_f = 0.34 (CH₂Cl₂/MeOH 9 : 1 + 0.5% Et₃N); Mp 183 °C; IR (ATR) 3300–2150, 1592, 1470, 1353, 1284, 1043, 1000 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 11.73 (br s, 1H), 8.22 (s, 1H), 8.11 (d, J = 6.0, 2H), 7.49 (d, J = 2.7, 1H), 7.41 (d, J = 8.1, 1H), 7.35–7.32 (m, 2H), 7.30 (d, J = 7.2, 1H), 7.17 (t, J = 7.7, 1H), 6.64 (d, J = 6.0, 2H), 6.34 (d, J = 2.8, 1H), 3.80 (s, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 159.9 (C O), 152.5 (C), 149.9 (2CH), 138.7 (CH), 136.6 (C), 130.6 (C), 129.7 (CH), 129.5 (CH), 128.3 (C), 126.5 (C), 120.6 (CH), 120.1 (CH), 119.5 (C), 109.2 (CH), 108.1 (2CH), 99.7 (CH), 32.6 (CH₃); HRMS (ESI+) m/z calcd C₁₉H₁₇ON₄ (M + H)⁺ 317.1397, found 317.1390. HPLC purity ≥99%, method A: t_R = 18.96 min, λ = 280 nm.

4.1.42. 4-Bromo-1-(3-bromopropan-1-yl)-1H-indole 47

To a solution of NaOH (3.8 g) in water (5 mL) were added toluene (10 mL), TBAB (5.9 g, 18.3 mmol, 3.6 eq.), 4-bromoindole (1.00 g, 5.1 mmol) and 1,3-dibromopropane (3.10 mL, 30.5 mmol, 6 eq.). The solution was stirred at room temperature for 16 h. The resulting mixture was diluted with water and extracted three times with dichloromethane. Combined organic phases were dried over MgSO₄, filtered and concentrated under reduced pressure. The crude oil was purified by column chromatography (SiO₂, cyclohexane/CH₂Cl₂ 100 : 0 to 95 : 5) to give 47 (1.20 g, 3.78 mmol, 74%) as a colorless oil. R_f = 0.26 (cyclohexane/CH₂Cl₂ 8 : 2); IR (ATR) 2935, 1432, 1244, 1165, 888 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 7.55 (d, J = 8.2, 1H), 7.51 (d, J = 3.2, 1H), 7.25 (d, J = 7.6, 1H), 7.09 (t, J = 7.8, 1H), 6.42 (dd, J = 3.2, 0.7, 1H), 4.32 (t, J = 6.8, 2H), 3.42 (t, J = 6.5, 2H), 2.29 (quint, J = 6.7, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 136.1 (C), 129.7 (CH), 128.3 (C), 122.4 (CH), 121.8 (CH), 113.7 (C), 109.5 (CH), 100.7 (CH), 44.3 (CH₂), 32.8 (CH₂), 31.5 (CH₂); HRMS (ESI+) m/z calcd for C₁₁H₁₂Br₂N (M + H)⁺ 315.9331, found 315.9325.

4.1.43. 4-Bromo-1-(4-bromobutan-1-yl)-1H-indole 48

To a solution of NaOH (5.7 g) in water (7.5 mL) were added toluene (15 mL), TBAB (8.85 g, 27.5 mmol, 3.6 eq.), 4-bromoindole (1.50 g, 7.65 mmol) and 1,4-dibromobutane (5.48 mL, 45.9 mmol, 6 eq.). The solution was stirred at room temperature for 16 h. The resulting mixture was diluted with water and extracted three times with dichloromethane. Combined organic phases were dried over MgSO₄, filtered and concentrated under reduced pressure. The crude oil was purified by column chromatography (SiO₂, cyclohexane/CH₂Cl₂ 100 : 0 to 95 : 5) to give 48 (1.60 g, 4.82 mmol, 63%) as a colorless oil. R_f = 0.26 (cyclohexane/CH₂Cl₂ 8 : 2); IR (ATR) 2939, 2871, 1478, 1433, 1336, 1282, 1131, 888 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 7.56 (d, J = 8.2, 1H), 7.52 (d, J = 3.1, 1H), 7.24 (d, J = 7.6, 1H), 7.07 (t, J = 7.9, 1H), 6.40 (d, J = 2.9, 1H), 4.24 (t, J = 6.8, 2H), 3.53 (t, J = 6.5, 2H), 1.92–1.82 (m, 2H), 1.78–1.69 (m, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 136.1 (C), 129.7 (CH), 128.3 (C), 122.2 (CH), 121.6 (CH), 113.6 (C), 109.6 (CH), 100.4 (CH), 45.0 (CH₂), 34.5 (CH₂), 29.5 (CH₂), 28.5 (CH₂); HRMS (ESI+) m/z calcd for C₁₂H₁₄Br₂N (M + H)⁺ 329.9488, found 329.9481.

4.1.44. 4-Bromo-1-[3-(diethylamino)propan-1-yl]-1H-indole 49

Compound 49 was prepared according to general procedure I, starting from 47 (144 mg, 0.454 mmol). The crude oil was purified by column chromatography (SiO₂, CH₂Cl₂/MeOH 1 : 0 to 9 : 1) to give 49 (106 mg, 0.343 mmol, 75%) as an orange oil. R_f = 0.54 (cyclohexane/acetone 1 : 1 + 0.5% Et₃N); IR (ATR) 2966, 2802, 1433, 1336, 1162, 1069, 889 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 7.51 (d, J = 8.3, 1H), 7.49 (d, J = 3.2, 1H), 7.23 (d, J = 7.5, 1H), 7.07 (t, J = 7.9, 1H), 6.39 (d, J = 2.8, 1H), 4.20 (t, J = 6.9, 2H), 2.41 (q, J = 7.1, 4H), 2.29 (t, J = 6.9, 2H), 1.85 (quint, J = 6.9, 2H), 0.89 (t, J = 7.1, 6H); ¹³C NMR (101 MHz, DMSO-d₆) δ 136.1 (C), 129.8 (CH), 128.3 (C), 122.1 (CH), 121.6 (CH), 113.6 (C), 109.5 (CH), 100.3 (CH), 49.2 (CH₂), 46.1 (2CH₂), 44.0 (CH₂), 27.4 (CH₂), 11.5 (2CH₃); HRMS (ESI+) m/z calcd for C₁₅H₂₂BrN₂ (M + H)⁺ 309.0961, found 309.0958.

4.1.45. 4-Bromo-1-[4-(diethylamino)butan-1-yl]-1H-indole 50

Compound 50 was prepared according to general procedure I, starting from 48 (527 mg, 1.59 mmol). The mixture was stirred for 3 h at room temperature. The crude oil was purified by column chromatography (SiO₂, CH₂Cl₂/MeOH 100 : 0 + 0.5% Et₃N to 98 : 2 + 0.5% Et₃N) to give 50 (359 mg, 1.11 mmol, 70%) as a brown oil. R_f = 0.35 (CH₂Cl₂/MeOH 98 : 2 + 0.5% Et₃N); IR (ATR) 2966, 2798, 1434, 1336, 1290, 888 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 7.53 (d, J = 8.2, 1H), 7.50 (d, J = 3.1, 1H), 7.23 (d, J = 7.5, 1H), 7.06 (t, J = 8.0, 1H), 6.39 (d, J = 3.1, 1H), 4.19 (t, J = 6.9, 2H), 2.36 (q, J = 7.1, 4H), 2.31 (t, J = 7.2, 2H), 1.73 (quint, J = 7.3, 2H), 1.32 (quint, J = 7.2, 2H), 0.88 (t, J = 7.1, 6H); ¹³C NMR (101 MHz, DMSO-d₆) δ 136.1 (C), 129.7 (CH), 128.3 (C), 122.1 (CH), 121.5 (CH), 113.6 (C), 109.6 (CH), 100.2 (CH), 51.7 (CH₂), 46.2 (2CH₂), 45.9 (CH₂), 27.8 (CH₂), 24.0 (CH₂), 11.7 (2CH₃); HRMS (ESI+) m/z calcd for C₁₆H₂₄BrN₂ (M + H)⁺ 323.1117, found 323.1115.

4.1.46. 4-Bromo-1-[3-(pyrrolidin-1-yl)propan-1-yl]-1H-indole 51

Compound 51 was prepared according to general procedure I, starting from 47 (395 mg, 1.25 mmol). The crude oil was purified by column chromatography (SiO₂, CH₂Cl₂/MeOH 1 : 0 to 97 : 3) to give 51 (349 mg, 1.14 mmol, 91%) as an orange oil. R_f = 0.21 (CH₂Cl₂/MeOH 95 : 5); IR (ATR) 2954, 2791, 1435, 1346, 1172, 1127, 890 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 7.52 (d, J = 8.2, 1H), 7.48 (d, J = 3.1, 1H), 7.23 (d, J = 7.5, 1H), 7.06 (t, J = 7.8, 1H), 6.39 (d, J = 3.1, 1H), 4.22 (t, J = 6.8, 2H), 2.40–2.31 (m, 4H), 2.27 (t, J = 7.0, 2H), 1.89 (quint, J = 6.8, 2H), 1.72–1.60 (m, 4H); ¹³C NMR (101 MHz, DMSO-d₆) δ 136.2 (C), 129.8 (CH), 128.2 (C), 122.1 (CH), 121.5 (CH), 113.5 (C), 109.5 (CH), 100.3 (CH), 53.4 (2CH₂), 52.3 (CH₂), 43.9 (CH₂), 29.1 (CH₂), 23.1 (2CH₂); HRMS (ESI+) m/z calcd for C₁₅H₂₀BrN₂ (M + H)⁺ 307.0804, found 307.0796.

4.1.47. 4-Bromo-1-[4-(pyrrolidin-1-yl)butan-1-yl]-1H-indole 52

Compound 52 was prepared according to general procedure I, starting from 48 (527 mg, 1.59 mmol). The crude oil was purified by column chromatography (SiO₂, CH₂Cl₂/MeOH 1 : 0 to 98 : 2) to give 52 (409 mg, 1.27 mmol, 80%) as an orange oil. R_f = 0.26 (CH₂Cl₂/MeOH 95 : 5); IR (ATR) 2932, 2787, 1433, 1336, 1172, 1128, 888 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 7.53 (d, J = 8.2, 1H), 7.50 (d, J = 3.2, 1H), 7.23 (d, J = 7.6, 1H), 7.06 (t, J = 7.9, 1H), 6.39 (d, J = 3.0, 1H), 4.19 (t, J = 7.0, 2H), 2.39–2.28 (m, 6H), 1.77 (quint, J = 7.3, 2H), 1.68–1.57 (m, 4H), 1.36 (quint, J = 7.5, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 136.1 (C), 129.8 (CH), 128.3 (C), 122.1 (CH), 121.6 (CH), 113.6 (C), 109.6 (CH), 100.2 (CH), 54.9 (CH₂), 53.5 (2CH₂), 45.8 (CH₂), 27.8 (CH₂), 25.4 (CH₂), 23.0 (2CH₂); HRMS (ESI+) m/z calcd for C₁₆H₂₂BrN₂ (M + H)⁺ 321.0961, found 321.0957.

4.1.48. 4-Bromo-1-[3-(piperidin-1-yl)propan-1-yl]-1H-indole 53

Compound 53 was prepared according to general procedure I, starting from 47 (395 mg, 1.25 mmol). The crude oil was purified by column chromatography (SiO₂, CH₂Cl₂/MeOH 100 : 0 to 98 : 2) to give 53 (368 mg, 1.15 mmol, 92%) as an orange oil. R_f = 0.10 (CH₂Cl₂/MeOH 98 : 2); IR (ATR) 2923, 2771, 1429, 1302, 1172, 1118, 891 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 7.53 (d, J = 8.2, 1H), 7.48 (d, J = 3.1, 1H), 7.22 (dd, J = 7.5, 0.7, 1H), 7.06 (t, J = 7.9, 1H), 6.39 (dd, J = 3.1, 0.8, 1H), 4.21 (t, J = 6.8, 2H), 2.28–2.18 (br s, 4H), 2.10 (t, J = 6.8, 2H), 1.88 (quint, J = 6.8, 2H), 1.52–1.45 (m, 4H), 1.40–1.32 (m, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 136.2 (C), 129.8 (CH), 128.2 (C), 122.1 (CH), 121.5 (CH), 113.5 (C), 109.6 (CH), 100.3 (CH), 55.0 (CH₂), 53.9 (2CH₂), 43.8 (CH₂), 27.0 (CH₂), 25.6 (2CH₂), 24.1 (CH₂); HRMS (ESI+) m/z calcd for C₁₆H₂₂BrN₂ (M + H)⁺ 321.0961, found 321.0958.

4.1.49. 4-Bromo-1-[4-(piperidin-1-yl)butan-1-yl]-1H-indole 54

Compound 54 was prepared according to general procedure I, starting from 48 (450 mg, 1.36 mmol). The crude oil was purified by column chromatography (SiO₂, CH₂Cl₂/MeOH 100 : 0 to 98 : 2) to give 54 (422 mg, 1.26 mmol, 93%) as an orange oil. R_f = 0.21 (CH₂Cl₂/MeOH 95 : 5); IR (ATR) 2930, 2850, 2799, 2763, 1433, 1336, 1298, 1167, 1121, 888 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 7.54 (d, J = 8.2, 1H), 7.50 (d, J = 3.2, 1H), 7.23 (dd, J = 7.5, 0.7, 1H), 7.06 (t, J = 7.9, 1H), 6.39 (dd, J = 3.1, 0.8, 1H), 4.19 (t, J = 7.1, 2H), 2.32–2.15 (m, 6H), 1.74 (quint, J = 7.4, 2H), 1.48–1.40 (m, 4H), 1.40–1.30 (m, 4H); ¹³C NMR (101 MHz, DMSO-d₆) δ 136.1 (C), 129.8 (CH), 128.3 (C), 122.1 (CH), 121.5 (CH), 113.6 (C), 109.6 (CH), 100.2 (CH), 57.7 (CH₂), 53.9 (2CH₂), 45.8 (CH₂), 27.7 (2CH₂), 25.5 (CH₂), 24.1 (CH₂), 23.4 (CH₂); HRMS (ESI+) m/z calcd for C₁₇H₂₄BrN₂ (M + H)⁺ 335.1117, found 335.1115.

4.1.50. 4-Bromo-1-[3-(morpholin-4-yl)propan-1-yl]-1H-indole 55

Compound 55 was prepared according to general procedure I, starting from 47 (144 mg, 0.454 mmol). The crude oil was purified by column chromatography (SiO₂, CH₂Cl₂/MeOH 1 : 0 to 95 : 5) to give 55 (144 mg, 0.446 mmol, 98%) as a colorless oil. R_f = 0.66 (cyclohexane/acetone 1 : 1 + 1% Et₃N); aspect: colorless oil; IR (ATR) 2947, 2851, 2809, 1433, 1269, 1115 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 7.54 (d, J = 8.2, 1H), 7.49 (d, J = 3.0, 1H), 7.23 (d, J = 7.5, 1H), 7.06 (t, J = 7.9, 1H), 6.39 (d, J = 3.1, 1H), 4.22 (t, J = 6.6, 2H), 3.61–3.51 (m, 4H), 2.32–2.22 (br s, 4H), 2.15 (t, J = 6.8, 2H), 1.89 (quint, J = 6.7, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 136.2 (C), 129.8 (CH), 128.2 (C), 122.1 (CH), 121.5 (CH), 113.5 (C), 109.6 (CH), 100.3 (CH), 66.2 (2CH₂), 54.8 (CH₂), 53.2 (2CH₂), 43.7 (CH₂), 26.5 (CH₂); HRMS (ESI+) m/z calcd for C₁₅H₂₀BrN₂O (M + H)⁺ 323.0754, found 323.0749.

4.1.51. 4-Bromo-1-[4-(morpholin-4-yl)butan-1-yl]-1H-indole 56

Compound 56 was prepared according to general procedure I, starting from 48 (160 mg, 0.483 mmol). The crude oil was purified by column chromatography (SiO₂, CH₂Cl₂/MeOH 1 : 0 to 95 : 5) to give 56 (157 mg, 0.466 mmol, 96%) as a colorless oil. R_f = 0.45 (CH₂Cl₂/MeOH 98 : 2 + 0.5% Et₃N); IR (ATR) 2941, 2852, 2807, 1434, 1115 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 7.53 (d, J = 8.2, 1H), 7.49 (d, J = 3.1, 1H), 7.23 (d, J = 7.5, 1H), 7.06 (t, J = 7.9, 1H), 6.38 (d, J = 2.9, 1H), 4.19 (t, J = 7.0, 2H), 3.53–3.48 (m, 4H), 2.29–2.19 (m, 6H), 1.75 (quint, J = 7.2, 2H), 1.35 (quint, J = 7.2, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 136.1 (C), 129.8 (CH), 128.3 (C), 122.1 (CH), 121.6 (CH), 113.6 (C), 109.6 (CH), 100.2 (CH), 66.2 (2CH₂), 57.4 (CH₂), 53.2 (2CH₂), 45.8 (CH₂), 27.5 (CH₂), 23.0 (CH₂); HRMS (ESI+) m/z calcd for C₁₆H₂₂BrN₂O (M + H)⁺ 337.0910, found 337.0908.

4.1.52. 4-Bromo-1-[3-(4-methylpiperazin-1-yl)propan-1-yl]-1H-indole 57

Compound 57 was prepared according to general procedure I, starting from 47 (395 mg, 1.25 mmol). The crude oil was purified by column chromatography (SiO₂, CH₂Cl₂/MeOH 100 : 0 to 98 : 2) to give 57 (377 mg, 1.12 mmol, 90%) as a colorless oil. R_f = 0.08 (CH₂Cl₂/MeOH 98 : 2); IR (ATR) 2935, 2792, 1433, 1282, 1173, 1162, 1139, 1013, 889, 817 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 7.53 (d, J = 8.2, 1H), 7.48 (d, J = 3.2, 1H), 7.23 (dd, J = 7.6, 0.6, 1H), 7.06 (t, J = 7.9, 1H), 6.39 (dd, J = 3.1, 0.7, 1H), 4.21 (t, J = 6.8, 2H), 2.40–2.20 (br s, 8H), 2.16–2.12 (m, 2H), 2.14 (s, 3H), 1.88 (quint, J = 6.8, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 136.2 (C), 129.8 (CH), 128.2 (C), 122.1 (CH), 121.5 (CH), 113.5 (C), 109.6 (CH), 100.3 (CH), 54.7 (2CH₂), 54.3 (CH₂), 52.5 (2CH₂), 45.7 (CH₃), 43.7 (CH₂), 26.9 (CH₂); HRMS (ESI+) m/z calcd for C₁₆H₂₃BrN₃ (M + H)⁺ 336.1070, found 336.1067.

4.1.53. 4-Bromo-1-[4-(4-methylpiperazin-1-yl)butan-1-yl]-1H-indole 58

Compound 58 was prepared according to general procedure I, starting from 48 (527 mg, 1.6 mmol). The crude oil was purified by column chromatography (SiO₂, CH₂Cl₂/MeOH 100 : 0 + 0.5% Et₃N to 98 : 2 + 0.5% Et₃N) to give 58 (564 mg, 1.6 mmol, quantitative) as a yellow oil. R_f = 0.26 (CH₂Cl₂/MeOH 98 : 2 + 0.5% Et₃N); IR (ATR) 2927, 2815, 2794, 1436, 1339, 1371, 1282, 1165, 1010, 812 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 7.54 (d, J = 8.2, 1H), 7.50 (d, J = 3.1, 1H), 7.23 (d, J = 7.5, 1H), 7.06 (t, J = 7.9, 1H), 6.38 (d, J = 3.1, 1H), 4.19 (t, J = 7.0, 2H), 2.37–2.15 (m, 8H), 2.22 (t, J = 7.2, 1H), 2.11 (s, 3H), 1.74 (quint, J = 7.3, 2H), 1.35 (quint, J = 7.4, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 136.1 (C), 129.8 (CH), 128.3 (C), 122.1 (CH), 121.5 (CH), 113.6 (C), 109.6 (CH), 100.2 (CH), 57.0 (CH₂), 54.7 (2CH₂), 52.6 (2CH₂), 45.8 (CH₂), 45.7 (CH₃), 27.6 (CH₂), 23.4 (CH₂); HRMS (ESI+) m/z calcd for C₁₇H₂₅BrN₃ (M + H)⁺ 350.1226, found 350.1222.

4.1.54. 6-(Benzyloxy)-5-{1-[3-(diethylamino)propan-1-yl]-1H-indol-4-yl}-N-(pyridin-4-yl)pyridin-3-amine 69

Compound 59 was first prepared according to general procedure J, starting from 49 (100 mg, 0.323 mmol). The crude was purified by column chromatography (SiO₂, cyclohexane/acetone 10 : 0 + 0.5% Et₃N to 5 : 5 + 0.5% Et₃N), but some of the starting material remained in the obtained brown oil 59 (104 mg). R_f = 0.47 (cyclohexane/acetone 1 : 1 + 0.5% Et₃N). Compound 59 was used for the next step without further purification.

Compound 69 was prepared according to general procedure E, starting from 41 (168 mg, 0.472 mmol) and 59 (185 mg). The crude was purified by column chromatography (SiO₂, CH₂Cl₂/MeOH 95 : 5 to 85 : 15). Evaporation gave compound 69 (170 mg, 0.336 mmol, 71%) as a beige solid. R_f = 0.19 (CH₂Cl₂/MeOH 9 : 1); Mp 89 °C; IR (ATR) 2962, 2931, 1592, 1445, 1418, 1213, 988, 816 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 8.80 (s, 1H), 8.16 (d, J = 5.6, 2H), 8.13 (d, J = 2.6, 1H), 7.64 (d, J = 2.5, 1H), 7.52 (d, J = 7.5, 1H), 7.42 (d, J = 3.0, 1H), 7.35–7.17 (m, 7H), 6.82 (d, J = 6.0, 2H), 6.33 (d, J = 3.0, 1H), 5.38 (s, 2H), 4.25 (t, J = 6.9, 2H), 2.76–2.56 (m, 6H), 1.97 (quint, J = 6.9, 2H), 1.00 (t, J = 7.1, 6H); ¹³C NMR (101 MHz, DMSO-d₆) δ 156.4 (C), 151.3 (C), 149.7 (2CH), 138.6 (CH), 137.4 (C), 135.8 (C), 134.1 (CH), 131.1 (C), 129.0 (CH), 128.1 (2CH), 127.9 (C), 127.5 (2CH), 127.4 (CH), 126.9 (C), 123.7 (C), 120.9 (CH), 120.3 (CH), 109.6 (CH), 108.5 (2CH), 100.1 (CH), 67.1 (CH₂), 48.8 (CH₂), 46.3 (2CH₂), 43.4 (CH₂), 26.2 (CH₂), 10.4 (2CH₃); HRMS (ESI+) m/z calcd for C₃₂H₃₇N₅O (M + 2H)²⁺ 253.6494, found 253.6489.

4.1.55. 6-(Benzyloxy)-5-{1-[4-(diethylamino)butan-1-yl]-1H-indol-4-yl}-N-(pyridin-4-yl)pyridin-3-amine 70

Compound 60 was first prepared according to general procedure J, starting from 50 (340 mg, 1.05 mmol). The crude was purified by column chromatography (SiO₂, cyclohexane/acetone 9 : 1 + 0.5% Et₃N to 7 : 3 + 0.5% Et₃N), but some of the starting material remained in the obtained brown oil 60 (331 mg). R_f = 0.44 (cyclohexane/acetone 1 : 1 + 0.5% Et₃N). Compound 60 was used for the next step without further purification.

Compound 70 was prepared according to general procedure E, starting from 41 (326 mg, 0.92 mmol) and 60 (330 mg). The crude was purified by column chromatography (SiO₂, acetone + 0.5% Et₃N) to give 70 (224 mg, 0.43 mmol, 47%) as a yellow-white solid. R_f = 0.14 (acetone + 0.5% Et₃N); Mp 122–124 °C; IR (ATR) 2964, 2929, 2801, 1591, 1417, 1213 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 8.70 (s, 1H), 8.16 (d, J = 6.0, 2H), 8.12 (d, J = 2.6, 1H), 7.64 (d, J = 2.5, 1H), 7.50 (d, J = 7.6, 1H), 7.41 (d, J = 3.0, 1H), 7.34–7.15 (m, 7H), 6.80 (d, J = 6.0, 2H), 6.31 (d, J = 3.0, 1H), 5.38 (s, 2H), 4.21 (t, J = 6.9, 2H), 2.46–2.30 (m, 6H), 1.77 (quint, J = 7.0, 2H), 1.37 (quint, J = 7.2, 2H), 0.90 (t, J = 7.0, 6H); ¹³C NMR (101 MHz, DMSO-d₆) δ 156.3 (C), 151.1 (C), 150.1 (2CH), 138.5 (CH), 137.4 (C), 135.8 (C), 134.0 (CH), 131.2 (C), 129.0 (CH), 128.2 (2CH), 127.9 (C), 127.5 (2CH), 127.4 (CH), 126.9 (C), 123.8 (C), 120.8 (CH), 120.2 (CH), 109.7 (CH), 108.5 (2CH), 99.9 (CH), 67.1 (CH₂), 51.8 (CH₂), 46.2 (2CH₂), 45.6 (CH₂), 27.8 (CH₂), 24.0 (CH₂), 11.6 (2CH₃); HRMS (ESI+) m/z calcd for C₃₃H₃₉N₅O (M + 2H)²⁺ 260.6572, found 260.6572.

4.1.56. 6-(Benzyloxy)-5-{1-[3-(pyrrolidin-1-yl)propan-1-yl]-1H-indol-4-yl}-N-(pyridin-4-yl)pyridin-3-amine 71

Compound 61 was first prepared according to general procedure J, starting from 51 (752 mg, 2.45 mmol). The crude was purified by column chromatography (SiO₂, cyclohexane/acetone 9 : 1 + 0.5% Et₃N to 7 : 3 + 0.5% Et₃N), but some of the starting material remained in the obtained brown oil 61 (702 mg). R_f = 0.53 (CH₂Cl₂/MeOH 8 : 2 + 0.5% Et₃N). Compound 61 was used for the next step without further purification.

Compound 71 was prepared according to general procedure E, starting from 41 (320 mg, 0.898 mmol) with 6.5 mL of dioxane and 61 (350 mg). The crude was purified by column chromatography (SiO₂, cyclohexane/EtOAc 50 : 50 + 0.5% Et₃N to 0 : 100 + 0.5% Et₃N) to give 71 (339 mg, 0.673 mmol, 75%) as a brown solid. R_f = 0.08 (acetone + 0.5% Et₃N); Mp 84–85 °C; IR (ATR) 2927, 2791, 1590, 1459, 1407, 1238, 1211, 993, 818 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 8.71 (s, 1H), 8.16 (d, J = 6.0, 2H), 8.12 (d, J = 2.6, 1H), 7.64 (d, J = 2.6, 1H), 7.49 (d, J = 7.5, 1H), 7.39 (d, J = 3.0, 1H), 7.35–7.15 (m, 7H), 6.81 (d, J = 6.1, 2H), 6.31 (d, J = 2.9, 1H), 5.38 (s, 2H), 4.24 (t, J = 6.7, 2H), 2.44–2.37 (m, 4H), 2.34 (t, J = 6.9, 2H), 1.92 (quint, J = 6.7, 2H), 1.74–1.64 (4H); ¹³C NMR (101 MHz, DMSO-d₆) δ 156.3 (C), 151.0 (C), 150.1 (2CH), 138.5 (CH), 137.4 (C), 135.8 (C), 134.0 (CH), 131.2 (C), 129.1 (CH), 128.1 (2CH), 127.8 (C), 127.5 (2CH), 127.4 (CH), 126.8 (C), 123.8 (C), 120.8 (CH), 120.2 (CH), 109.6 (CH), 108.5 (2CH), 99.9 (CH), 67.1 (CH₂), 53.5 (2CH₂), 52.5 (CH₂), 43.6 (CH₂), 29.1 (CH₂), 23.1 (2CH₂); HRMS (ESI+) m/z calcd for C₃₂H₃₅N₅O (M + 2H)²⁺ 252.6415, found 252.6412.

4.1.57. 6-(Benzyloxy)-5-{1-[4-(pyrrolidin-1-yl)butan-1-yl]-1H-indol-4-yl}-N-(pyridin-4-yl)pyridin-3-amine 72

Compound 62 was first prepared according to general procedure J, starting from 52 (358 mg, 1.11 mmol). The crude was purified by column chromatography (SiO₂, cyclohexane/acetone 9 : 1 + 0.5% Et₃N to 7 : 3 + 0.5% Et₃N), but some of the starting material remained in the obtained yellow oil 62 (292 mg). R_f = 0.23 (cyclohexane/acetone 7 : 3 + 0.5% Et₃N). Compound 62 was used for the next step without further purification.

Compound 72 was prepared according to general procedure E, starting from 41 (100 mg, 0.281 mmol) and 62 (114 mg). The crude was purified by column chromatography (SiO₂, cyclohexane/EtOAc 50 : 50 + 0.5% Et₃N to 0 : 100 + 0.5% Et₃N) to give 72 (110 mg, 0.212 mmol, 76%) as a white powder. R_f = 0.09 (acetone/MeOH 95 : 5 + 0.5% Et₃N); Mp 109 °C; IR (ATR) 2925, 1593, 1424 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 8.70 (s, 1H), 8.16 (d, J = 5.9, 2H), 8.12 (d, J = 2.5, 1H), 7.64 (d, J = 2.5, 1H), 7.50 (d, J = 7.3, 1H), 7.41 (d, J = 3.0, 1H), 7.34–7.14 (m, 7H), 6.81 (d, J = 6.1, 2H), 6.31 (d, J = 2.7, 1H), 5.38 (s, 2H), 4.21 (t, J = 6.7, 2H), 2.41–2.31 (m, 6H), 1.79 (quint, J = 7.0, 2H), 1.67–1.59 (4H), 1.41 (quint, J = 7.3, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 156.3 (C), 151.0 (C), 150.1 (2CH), 138.4 (CH), 137.4 (C), 135.8 (C), 134.0 (CH), 131.2 (C), 129.0 (CH), 128.1 (2CH), 127.8 (C), 127.43 (2CH), 127.38 (CH), 126.9 (C), 123.8 (C), 120.7 (CH), 120.2 (CH), 109.7 (CH), 108.5 (2CH), 99.8 (CH), 67.1 (CH₂), 55.0 (CH₂), 53.5 (2CH₂), 45.5 (CH₂), 27.8 (CH₂), 25.6 (CH₂), 23.0 (2CH₂); HRMS (ESI+) m/z calcd for C₃₃H₃₇N₅O (M + 2H)²⁺ 259.6494, found 259.6489.

4.1.58. 6-(Benzyloxy)-5-{1-[3-(piperidin-1-yl)propan-1-yl]-1H-indol-4-yl}-N-(pyridin-4-yl)pyridin-3-amine 73

Compound 63 was first prepared according to general procedure J, starting from 53 (350 mg, 1.09 mmol). The crude was purified by column chromatography (SiO₂, cyclohexane/acetone 9 : 1 + 0.5% Et₃N), but some of the starting material remained in the obtained brown solid 63 (406 mg). R_f = 0.37 (cyclohexane/acetone 7 : 3 + 0.5% Et₃N). Compound 63 was used for the next step without further purification.

Compound 73 was prepared according to general procedure E, starting from 41 (390 mg, 1.09 mmol) and 63 (404 mg). The crude was purified by column chromatography (SiO₂, acetone + 0.5% Et₃N) to give 73 (329 mg, 0.64 mmol, 58%) as a white solid. R_f = 0.19 (acetone + 0.5% Et₃N); Mp 77 °C; IR (ATR) 2931, 1591, 1446, 1419, 1214, 993 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 8.70 (s, 1H), 8.16 (d, J = 6.0, 2H), 8.12 (d, J = 2.5, 1H), 7.64 (d, J = 2.5, 1H), 7.50 (d, J = 7.5, 1H), 7.38 (d, J = 3.0, 1H), 7.34–7.14 (m, 7H), 6.81 (d, J = 6.1, 2H), 6.31 (d, J = 2.9, 1H), 5.38 (s, 2H), 4.22 (t, J = 6.6, 2H), 2.33–2.21 (br s, 4H), 2.17 (t, J = 6.6, 2H), 1.90 (quint, J = 6.8, 2H), 1.55–1.45 (m, 2H), 1.42–1.33 (m, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 156.3 (C), 151.0 (C), 150.1 (2CH), 138.5 (CH), 137.4 (C), 135.8 (C), 134.0 (CH), 131.2 (C), 129.1 (CH), 128.1 (2CH), 127.8 (C), 127.44 (2CH), 127.39 (CH), 126.8 (C), 123.8 (C), 120.7 (CH), 120.2 (CH), 109.6 (CH), 108.5 (2CH), 99.9 (CH), 67.1 (CH₂), 55.1 (CH₂), 53.9 (2CH₂), 43.5 (CH₂), 27.0 (CH₂), 25.6 (2CH₂), 24.1 (CH₂); HRMS (ESI+) m/z calcd for C₃₃H₃₇N₅O (M + 2H)²⁺ 259.6494, found 259.6489.

4.1.59. 6-(Benzyloxy)-5-{1-[4-(piperidin-1-yl)butan-1-yl]-1H-indol-4-yl}-N-(pyridin-4-yl)pyridin-3-amine 74

Compound 64 was first prepared according to general procedure J, starting from 54 (135 mg, 0.40 mmol). The tube was sealed and the mixture was heated under microwave irradiation for 60 min. The crude was purified by column chromatography (SiO₂, cyclohexane/acetone 9 : 1 + 0.5% Et₃N to 7 : 3 + 0.5% Et₃N), but some of the starting material remained in the obtained brown solid 64 (135 mg). R_f = 0.32 (cyclohexane/acetone 7 : 3 + 0.5% Et₃N). Compound 64 was used for the next step without further purification.

Compound 74 was prepared according to general procedure E, starting from 41 (143 mg, 0.40 mmol) and 64 (135 mg). The crude was purified by column chromatography (SiO₂, acetone + 0.5% Et₃N) to give 74 (56 mg, 0.105 mmol, 26%) as a white solid. R_f = 0.16 (acetone + 0.5% Et₃N); Mp 71 °C; IR (ATR) 2927, 1591, 1445, 1418, 1358, 1285, 1213, 993, 746, 695 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 8.70 (s, 1H), 8.16 (d, J = 5.6, 2H), 8.12 (d, J = 2.5, 1H), 7.64 (d, J = 2.6, 1H), 7.51 (d, J = 7.5, 1H), 7.41 (d, J = 3.1, 1H), 7.34–7.15 (m, 7H), 6.81 (d, J = 5.6, 2H), 6.30 (d, J = 3.0, 1H), 5.38 (s, 2H), 4.21 (t, J = 6.9, 2H), 2.29–2.18 (m, 6H), 1.76 (quint, J = 7.1, 2H), 1.48–1.30 (m, 8H); ¹³C NMR (101 MHz, DMSO-d₆) δ 156.3 (C), 151.0 (C), 150.1 (2CH), 138.4 (CH), 137.4 (C), 135.7 (C), 133.9 (CH), 131.2 (C), 129.0 (CH), 128.1 (2CH), 127.8 (C), 127.42 (2CH), 127.37 (CH), 126.9 (C), 123.8 (C), 120.7 (CH), 120.2 (CH), 109.7 (CH), 108.5 (2CH), 99.8 (CH), 67.1 (CH₂), 57.9 (CH₂), 54.0 (2CH₂), 45.5 (CH₂), 27.7 (CH₂), 25.5 (2CH₂), 24.1 (CH₂), 23.5 (CH₂); HRMS (ESI+) m/z calcd for C₃₄H₃₉N₅O (M + 2H)²⁺ 266.6572, found 266.6564.

4.1.60. 6-(Benzyloxy)-5-{1-[3-(morpholin-4-yl)propan-1-yl]-1H-indol-4-yl}-N-(pyridin-4-yl)pyridin-3-amine 75

Compound 65 was first prepared according to general procedure J, starting from 55 (500 mg, 1.55 mmol). The crude was purified by column chromatography (SiO₂, cyclohexane/acetone 100 : 0 + 0.5% Et₃N to 50 : 50 + 0.5% Et₃N), but some of the starting material remained in the obtained grey powder 65 (391 mg). R_f = 0.71 (cyclohexane/acetone 1 : 1 + 1% Et₃N). Compound 65 was used for the next step without further purification.

Compound 75 was prepared according to general procedure E, starting from 41 (466 mg, 1.31 mmol) and 65 (532 mg). The crude was purified by column chromatography (SiO₂, CH₂Cl₂/MeOH 95 : 5 + 0.5% Et₃N to 90 : 10 + 0.5% Et₃N) to give 75 (554 mg, 1.07 mmol, 81%) as a pink solid. R_f = 0.46 (CH₂Cl₂/MeOH 9 : 1 + 0.5% Et₃N); Mp 88 °C; IR (ATR) 2944, 2807, 1591, 1417, 1213, 1115, 988, 814 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 8.70 (s, 1H), 8.16 (d, J = 5.8, 2H), 8.12 (d, J = 2.5, 1H), 7.64 (d, J = 2.5, 1H), 7.50 (d, J = 7.6, 1H), 7.40 (d, J = 3.0, 1H), 7.34–7.14 (m, 7H), 6.80 (d, J = 6.0, 2H), 6.31 (d, J = 2.9, 1H), 5.38 (s, 2H), 4.24 (t, J = 6.7, 2H), 3.60–3.54 (m, 4H), 2.34–2.26 (br s, 4H), 2.20 (t, J = 6.8, 2H), 1.92 (quint, J = 6.6, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 156.3 (C), 151.0 (C), 150.1 (2CH), 138.5 (CH), 137.4 (C), 135.8 (C), 134.0 (CH), 131.2 (C), 129.1 (CH), 128.1 (2CH), 127.8 (C), 127.45 (2CH), 127.40 (CH), 126.8 (C), 123.8 (C), 120.7 (CH), 120.2 (CH), 109.6 (CH), 108.5 (2CH), 99.9 (CH), 67.1 (CH₂), 66.2 (2CH₂), 54.9 (CH₂), 53.2 (2CH₂), 43.4 (CH₂), 26.6 (CH₂); HRMS (ESI+) m/z calcd for C₃₂H₃₅N₅O₂ (M + 2H)²⁺ 260.6390, found 260.6386.

4.1.61. 6-(Benzyloxy)-5-{1-[4-(morpholin-4-yl)butan-1-yl]-1H-indol-4-yl}-N-(pyridin-4-yl)pyridin-3-amine 76

Compound 66 was first prepared according to general procedure J, starting from 56 (829 mg, 2.46 mmol). The crude was purified by column chromatography (SiO₂, cyclohexane/acetone 100 : 0 + 0.5% Et₃N to 50 : 50 + 0.5% Et₃N), but some of the starting material remained in the obtained brown powder 66 (513 mg). R_f = 0.65 (cyclohexane/acetone 1 : 1 + 1% Et₃N). Compound 66 was used for the next step without further purification.

Compound 76 was prepared according to general procedure E, starting from 41 (432 mg, 1.21 mmol) and 66 (513 mg). The crude was purified by column chromatography (SiO₂, acetone + 0.5% Et₃N) to give 76 (431 mg, 0.81 mmol, 67%) as a beige–orange solid. R_f = 0.18 (acetone + 0.5% Et₃N); Mp 93 °C; IR (ATR) 2929, 2856, 2807, 1591, 1418, 1213, 1115, 991, 814 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 8.74 (s, 1H), 8.16 (d, J = 6.2, 2H), 8.12 (d, J = 2.6, 1H), 7.64 (d, J = 2.7, 1H), 7.51 (d, J = 7.5, 1H), 7.41 (d, J = 3.1, 1H), 7.35–7.15 (m, 7H), 6.81 (d, J = 6.3, 2H), 6.31 (d, J = 2.9, 1H), 5.38 (s, 2H), 4.21 (t, J = 6.9, 2H), 3.52 (t, J = 4.5, 4H), 2.30–2.22 (m, 6H), 1.79 (quint, J = 7.2, 2H), 1.40 (quint, J = 7.4, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 156.3 (C), 151.2 (C), 149.8 (2CH), 138.5 (CH), 137.4 (C), 135.7 (C), 134.0 (CH), 131.1 (C), 129.0 (CH), 128.1 (2CH), 127.8 (C), 127.43 (2CH), 127.39 (CH), 126.9 (C), 123.8 (C), 120.7 (CH), 120.2 (CH), 109.7 (CH), 108.5 (2CH), 99.8 (CH), 67.1 (CH₂), 66.2 (2CH₂), 57.5 (CH₂), 53.2 (2CH₂), 45.5 (CH₂), 27.5 (CH₂), 23.1 (2CH₂); HRMS (ESI+) m/z calcd for C₃₃H₃₇N₅O₂ (M + 2H)²⁺ 267.6468, found 267.6469.

4.1.62. 6-(Benzyloxy)-5-{1-[3-(4-methylpiperazin-1-yl)propan-1-yl]-1H-indol-4-yl}-N-(pyridin-4-yl)pyridin-3-amine 77

Compound 67 was first prepared according to general procedure J, starting from 57 (350 mg, 1.04 mmol). The crude was purified by column chromatography (SiO₂, cyclohexane/acetone 9 : 1 + 0.5% Et₃N to 3 : 7 + 0.5% Et₃N), but some of the starting material remained in the obtained colorless oil 67 (310 mg). R_f = 0.21 (cyclohexane/acetone 6 : 4 + 0.5% Et₃N). Compound 67 was used for the next step without further purification.

Compound 77 was prepared according to general procedure E, starting from 41 (370 mg, 1.04 mmol) and 67 (308 mg). The crude was purified by column chromatography (SiO₂, acetone/MeOH 100 : 0 + 0.5% Et₃N to 85 : 15 + 0.5% Et₃N) to give 77 (268 mg, 0.503 mmol, 48%) as a beige solid. R_f = 0.13 (acetone/MeOH 85 : 15 + 0.5% Et₃N); Mp 94 °C; IR (ATR) 2935, 2792, 1591, 1446, 1418, 1359, 1282, 1214, 1164, 993, 814 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 8.69 (s, 1H), 8.16 (d, J = 6.0, 2H), 8.12 (d, J = 2.6, 1H), 7.64 (d, J = 2.6, 1H), 7.49 (d, J = 7.4, 1H), 7.38 (d, J = 3.1, 1H), 7.34–7.14 (m, 7H), 6.80 (d, J = 6.2, 2H), 6.31 (d, J = 3.0, 1H), 5.38 (s, 2H), 4.22 (t, J = 6.6, 2H), 2.41–2.22 (br s, 8H), 2.19 (t, J = 6.8, 2H), 2.14 (s, 3H), 1.90 (quint, J = 6.5, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 156.3 (C), 151.0 (C), 150.1 (2CH), 138.5 (CH), 137.4 (C), 135.8 (C), 134.0 (CH), 131.2 (C), 129.1 (CH), 128.1 (2CH), 127.8 (C), 127.44 (2CH), 127.39 (CH), 126.8 (C), 123.8 (C), 120.7 (CH), 120.2 (CH), 109.6 (CH), 108.5 (2CH), 99.9 (CH), 67.1 (CH₂), 54.8 (2CH₂), 54.4 (CH₂), 52.6 (2CH₂), 45.7 (CH₃), 43.4 (CH₂), 27.0 (CH₂); HRMS (ESI+) m/z calcd for C₃₃H₃₈N₆O (M + 2H)²⁺ 267.1548, found 267.1547.

4.1.63. 6-(Benzyloxy)-5-{1-[4-(4-methylpiperazin-1-yl)butan-1-yl]-1H-indol-4-yl}-N-(pyridin-4-yl)pyridin-3-amine 78

Compound 68 was first prepared according to general procedure J, starting from 58 (540 mg, 1.54 mmol). The crude was purified by column chromatography (SiO₂, cyclohexane/acetone 9 : 1 + 0.5% Et₃N to 4 : 6 + 0.5% Et₃N), but some of the starting material remained in the obtained colorless oil 68 (470 mg). R_f = 0.24 (cyclohexane/acetone 6 : 4 + 0.5% Et₃N). Compound 68 was used for the next step without further purification.

Compound 78 was prepared according to general procedure E, starting from 41 (548 mg, 1.54 mmol) and 68 (469 mg). The crude was purified by column chromatography (SiO₂, acetone/MeOH 100 : 0 + 0.5% Et₃N to 85 : 15 + 0.5% Et₃N) to give 78 (370 mg, 0.68 mmol, 44%) as a beige solid. R_f = 0.23 (acetone/MeOH 8 : 2 + 0.5% Et₃N); Mp 86 °C; IR (ATR) 2932, 2792, 1591, 1445, 1418, 1358, 1281, 1213, 1163, 991, 813 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 8.70 (s, 1H), 8.17 (d, J = 6.1, 2H), 8.13 (d, J = 2.6, 1H), 7.65 (d, J = 2.6, 1H), 7.52 (d, J = 7.4, 1H), 7.41 (d, J = 3.1, 1H), 7.36–7.15 (m, 7H), 6.81 (d, J = 6.2, 2H), 6.31 (d, J = 3.0, 1H), 5.39 (s, 2H), 4.21 (t, J = 6.9, 2H), 2.39–2.17 (br s, 8H), 2.25 (t, J = 7.1, 2H), 2.12 (s, 3H), 1.78 (quint, J = 7.1, 2H), 1.40 (quint, J = 7.2, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 156.3 (C), 151.0 (C), 150.1 (2CH), 138.4 (CH), 137.4 (C), 135.7 (C), 134.0 (CH), 131.2 (C), 129.0 (CH), 128.1 (2CH), 127.9 (C), 127.42 (2CH), 127.38 (CH), 126.9 (C), 123.8 (C), 120.7 (CH), 120.2 (CH), 109.7 (CH), 108.5 (2CH), 99.8 (CH), 67.1 (CH₂), 57.1 (CH₂), 54.8 (2CH₂), 52.6 (2CH₂), 45.8 (CH₃), 45.5 (CH₂), 27.6 (CH₂), 23.5 (CH₂); HRMS (ESI+) m/z calcd for C₃₄H₄₀N₆O (M + 2H)²⁺ 274.1626, found 274.1619.

4.1.64. 3-{1-[3-(Diethylamino)propan-1-yl]-1H-indol-4-yl}-5-(pyridin-4-ylamino)pyridin-2(1H)-one 79

Compound 79 was prepared according to general procedure B, starting from 89 (90 mg, 0.178 mmol). The mixture was stirred at 0 °C for 1 h and then at room temperature for 6 h. The crude was purified by column chromatography (SiO₂, CH₂Cl₂/MeOH 90 : 10 + 0.5% Et₃N to 85 : 15 + 0.5% Et₃N). After evaporation, the solid was solubilized in CH₂Cl₂ and washed with a saturated aqueous solution of Na₂CO₃, dried over MgSO₄ and filtered. After evaporation, compound 79 (22 mg, 0.053 mmol, 30%) was obtained as a beige solid. R_f = 0.19 (CH₂Cl₂/MeOH 9 : 1 + 0.5% Et₃N); Mp > 128 °C (decomposition); IR (ATR) 2965, 2810, 1595, 1513, 1468, 1345, 1209, 997, 817 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 11.70 (br s, 1H), 8.22 (s, 1H), 8.11 (m, 2H), 7.49 (d, J = 2.9, 1H), 7.44 (d, J = 8.2, 1H), 7.37 (d, J = 3.1, 1H), 7.33 (d, J = 2.6, 1H), 7.28 (dd, J = 7.4, 0.9, 1H), 7.15 (dd, J = 8.1, 7.4, 1H), 6.64 (m, 2H), 6.35 (dd, J = 3.2, 0.8, 1H), 4.20 (t, J = 6.9, 2H), 2.43 (q, J = 7.1, 4H), 2.34 (t, J = 7.0, 2H), 1.86 (quint, J = 6.9, 2H), 0.92 (t, J = 7.1, 6H); ¹³C NMR (101 MHz, DMSO-d₆) δ 159.9 (C O), 152.4 (C), 149.9 (2CH), 138.7 (CH), 135.9 (C), 130.6 (C), 129.5 (CH), 128.8 (CH), 128.4 (C), 126.5 (C), 120.5 (CH), 120.0 (CH), 119.5 (C), 109.3 (CH), 108.1 (2CH), 99.8 (CH), 49.4 (CH₂), 46.1 (2CH₂), 43.7 (CH₂), 27.5 (CH₂), 11.6 (2CH₃); HRMS (ESI+) m/z calcd for C₂₅H₃₁N₅O (M + 2H)²⁺ 208.6259, found 208.6254. HPLC purity ≥98%, method A: t_R = 17.78 min, λ = 280 nm.

4.1.65. 3-{1-[4-(Diethylamino)butan-1-yl]-1H-indol-4-yl}-5-(pyridin-4-ylamino)pyridin-2(1H)-one 80

Compound 80 was prepared according to general procedure H, starting from 70 (120 mg, 0.231 mmol) in 5 mL of solvent. The crude was purified by column chromatography (SiO₂, CH₂Cl₂/MeOH 90 : 10 + 0.5% Et₃N to 85 : 15 + 0.5% Et₃N). The obtained solid was solubilized in CH₂Cl₂ and washed with a saturated aqueous solution of Na₂CO₃, dried over MgSO₄ and filtered. After evaporation compound 80 (55 mg, 0.128 mmol, 55%) was obtained as a beige solid. R_f = 0.32 (CH₂Cl₂/MeOH 85 : 15 + 0.5% Et₃N); Mp > 119 °C; IR (ATR) 2963, 2929, 2800, 1595, 1511, 1345, 1210, 993, 817 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 11.71 (br s, 1H), 8.22 (s, 1H), 8.12 (d, J = 5.7, 2H), 7.49 (d, J = 2.6, 1H), 7.46 (d, J = 8.2, 1H), 7.38 (d, J = 2.9, 1H), 7.33 (d, J = 2.5, 1H), 7.28 (d, J = 7.2, 1H), 7.15 (t, J = 7.8, 1H), 6.64 (d, J = 5.8, 2H), 6.35 (d, J = 2.8, 1H), 4.19 (t, J = 6.8, 2H), 2.38 (q, J = 7.1, 4H), 2.33 (t, J = 7.2, 2H), 1.75 (quint, J = 7.1, 2H), 1.36 (quint, J = 7.3, 2H), 0.90 (t, J = 7.0, 6H); ¹³C NMR (101 MHz, DMSO-d₆) δ 160.0 (C O), 152.4 (C), 149.9 (2CH), 138.6 (CH), 135.9 (C), 130.5 (C), 129.7 (CH), 128.7 (CH), 128.5 (C), 126.5 (C), 120.5 (CH), 120.0 (CH), 119.5 (C), 109.3 (CH), 108.1 (2CH), 99.8 (CH), 51.8 (CH₂), 46.2 (2CH₂), 45.5 (CH₂), 27.8 (CH₂), 24.1 (CH₂), 11.7 (2CH₃); HRMS (ESI+) m/z calcd for C₂₆H₃₃N₅O (M + 2H)²⁺ 215.6337, found 215.6337. HPLC purity ≥99%, method A: t_R = 18.02 min, λ = 240 nm.

4.1.66. 3-{1-[3-(Pyrrolidin-1-yl)propan-1-yl]-1H-indol-4-yl}-5-(pyridin-4-ylamino)pyridin-2(1H)-one 81

Compound 81 was prepared according to general procedure H, starting from 71 (120 mg, 0.238 mmol) in 5 mL of solvent. The crude was purified by column chromatography (SiO₂, CH₂Cl₂/MeOH 95 : 5 + 0.5% Et₃N to 90 : 10 + 0.5% Et₃N). After evaporation, the solid was solubilized in CH₂Cl₂ and the solution was washed with a saturated aqueous solution of Na₂CO₃, dried over MgSO₄ and filtered. After evaporation, the solid was washed with CH₂Cl₂ to give compound 81 (52 mg, 0.126 mmol, 53%) as a pale-yellow solid. R_f = 0.31 (CH₂Cl₂/MeOH 85 : 15 + 0.5% Et₃N); Mp > 139 °C; IR (ATR) 2925, 2791, 1591, 1510, 1344, 1213, 994, 814 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 11.69 (br s, 1H), 8.24 (s, 1H), 8.12 (d, J = 5.1, 2H), 7.49 (d, J = 2.8, 1H), 7.45 (d, J = 8.2, 1H), 7.37 (d, J = 3.1, 1H), 7.34 (d, J = 2.7, 1H), 7.28 (d, J = 7.3, 1H), 7.16 (t, J = 7.8, 1H), 6.64 (d, J = 5.8, 2H), 6.35 (d, J = 3.0, 1H), 4.23 (t, J = 6.7, 2H), 2.47–2.40 (m, 4H), 2.36 (t, J = 6.8, 2H), 1.92 (quint, J = 6.8, 2H), 1.65–1.73 (4H, m); ¹³C NMR (101 MHz, DMSO-d₆) δ 159.9 (C O), 152.5 (C), 149.9 (2CH), 138.7 (CH), 135.9 (C), 130.6 (C), 129.5 (CH), 128.8 (CH), 128.4 (C), 126.5 (C), 120.5 (CH), 120.0 (CH), 119.5 (C), 109.3 (CH), 108.1 (2CH), 99.9 (CH), 53.5 (2CH₂), 52.5 (CH₂), 43.6 (CH₂), 29.0 (CH₂), 23.1 (2CH₂); HRMS (ESI+) m/z calcd for C₂₅H₂₉N₅O (M + 2H)²⁺ 207.6181, found 207.6181. HPLC purity ≥98%, method A: t_R = 17.70 min, λ = 280 nm.

4.1.67. 3-{1-[4-(Pyrrolidin-1-yl)butan-1-yl]-1H-indol-4-yl}-5-(pyridin-4-ylamino)pyridin-2(1H)-one 82

Compound 82 was prepared according to general procedure H, starting from 72 (120 mg, 0.232 mmol) in 5 mL of solvent. The crude was purified by column chromatography (SiO₂, CH₂Cl₂/MeOH 90 : 10 + 0.5% Et₃N to 85 : 15 + 0.5% Et₃N). After evaporation, the solid was washed with CH₂Cl₂ to give 82 (53 mg, 0.124 mmol, 53%) as a yellow solid. R_f = 0.08 (CH₂Cl₂/MeOH 9 : 1 + 0.5% Et₃N); Mp > 127 °C (decomposition); IR (ATR) 2927, 2788, 1594, 1511, 1345, 1209, 995, 816 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 11.71 (br s, 1H), 8.22 (s, 1H), 8.12 (d, J = 5.2, 2H), 7.49 (d, J = 2.6, 1H), 7.45 (d, J = 8.2, 1H), 7.38 (d, J = 2.9, 1H), 7.33 (d, J = 2.5, 1H), 7.28 (d, J = 7.2, 1H), 7.15 (t, J = 7.7, 1H), 6.64 (d, J = 5.7, 2H), 6.35 (d, J = 2.8, 1H), 4.19 (t, J = 6.8, 2H), 2.43–2.34 (m, 6H), 1.78 (quint, J = 7.0, 2H), 1.69–1.59 (4H), 1.40 (quint, J = 7.3, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 159.9 (C O), 152.5 (C), 149.9 (2CH), 138.7 (CH), 135.9 (C), 130.7 (C), 129.5 (CH), 128.8 (CH), 128.5 (C), 126.5 (C), 120.5 (CH), 120.0 (CH), 119.6 (C), 109.4 (CH), 108.1 (2CH), 99.8 (CH), 55.0 (CH₂), 53.5 (2CH₂), 45.4 (CH₂), 27.8 (CH₂), 25.5 (CH₂), 23.0 (2CH₂); HRMS (ESI+) m/z calcd for C₂₆H₃₁N₅O (M + 2H)²⁺ 214.6259, found 214.6258. HPLC purity ≥99%, method A: t_R = 17.89 min, λ = 240 nm.

4.1.68. 3-{1-[3-(Piperidin-1-yl)propan-1-yl]-1H-indol-4-yl}-5-(pyridin-4-ylamino)pyridin-2(1H)-one 83

Compound 83 was prepared according to general procedure B, starting from 73 (120 mg, 0.232 mmol). The mixture was stirred at 0 °C for 1 h and then at room temperature for 3 h. The crude was purified by column chromatography (SiO₂, acetone/MeOH 90 : 10 + 0.5% Et₃N to 85 : 15 + 1% Et₃N). After evaporation, the solid was washed with cyclohexane to give compound 83 (46 mg, 0.108 mmol, 46%) as an orange solid. R_f = 0.19 (acetone/MeOH 85 : 15 + 1% Et₃N); Mp > 142 °C (decomposition); IR (ATR) 2930, 1594, 1511, 1211, 996, 816 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 11.71 (br s, 1H), 8.22 (s, 1H), 8.11 (d, J = 5.5, 2H), 7.49 (d, J = 2.7, 1H), 7.45 (d, J = 8.2, 1H), 7.36 (d, J = 3.0, 1H), 7.33 (m, 1H), 7.28 (d, J = 7.3, 1H), 7.15 (t, J = 7.7, 1H), 6.64 (d, J = 5.7, 2H), 6.35 (d, J = 2.9, 1H), 4.20 (t, J = 6.6, 2H), 2.32–2.21 (br s, 4H), 2.16 (t, J = 6.7, 2H), 1.89 (quint, J = 6.7, 2H), 1.54–1.46 (m, 4H), 1.42–1.33 (m, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 159.9 (C), 152.5 (C), 149.9 (2CH), 138.7 (CH), 135.9 (C), 130.6 (C), 129.5 (CH), 128.8 (CH), 128.4 (C), 126.5 (C), 120.5 (CH), 120.0 (CH), 119.5 (C), 109.3 (CH), 108.1 (2CH), 99.9 (CH), 55.2 (CH₂), 53.9 (2CH₂), 43.5 (CH₂), 27.1 (CH₂), 25.6 (2CH₂), 24.1 (CH₂); HRMS (ESI+) m/z calcd for C₂₆H₃₁N₅O (M + 2H)²⁺ 214.6259, found 214.6258. HPLC purity ≥95%, method A: t_R = 17.49 min, λ = 280 nm.

4.1.69. 3-{1-[4-(Piperidin-1-yl)butan-1-yl]-1H-indol-4-yl]-5-(pyridin-4-ylamino)pyridin-2(1H)-one 84

Compound 84 was prepared according to general procedure H, starting from 74 (75 mg, 0.141 mmol) in 4 mL of solvent. The crude was purified by column chromatography (SiO₂, CH₂Cl₂/MeOH 95 : 5 + 0.5% Et₃N to 90 : 10 + 0.5% Et₃N). After evaporation, the solid was solubilized in CH₂Cl₂ and the solution was washed with a saturated aqueous solution of Na₂CO₃, dried over MgSO₄ and filtered. After evaporation, compound 84 (31 mg, 0.070 mmol, 50%) was obtained as a pale-yellow solid. R_f = 0.33 (CH₂Cl₂/MeOH 9 : 1 + 0.5% Et₃N); Mp > 128 °C (decomposition); IR (ATR) 2930, 1598, 1512, 1347, 1210, 997, 817 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 11.68 (br s, 1H), 8.21 (s, 1H), 8.11 (m, 2H), 7.48 (d, J = 2.9, 1H), 7.45 (d, J = 8.2, 1H), 7.37 (d, J = 3.1, 1H), 7.34 (d, J = 2.8, 1H), 7.28 (dd, J = 7.3, 0.9, 1H), 7.15 (dd, J = 8.1, 7.4, 1H), 6.63 (m, 2H), 6.34 (dd, J = 3.1, 0.8, 1H), 4.18 (t, J = 7.0, 2H), 2.28–2.18 (br s, 4H), 2.20 (t, J = 7.3, 2H), 1.75 (quint, J = 7.3, 2H), 1.48–1.30 (m, 8H); ¹³C NMR (101 MHz, DMSO-d₆) δ 160.1 (C O), 152.5 (C), 149.9 (2CH), 138.6 (CH), 135.8 (C), 130.4 (C), 129.9 (CH), 128.7 (CH), 128.6 (C), 126.5 (C), 120.5 (CH), 120.0 (CH), 119.5 (C), 109.3 (CH), 108.1 (2CH), 99.8 (CH), 55.9 (CH₂), 54.0 (2CH₂), 45.5 (CH₂), 27.8 (CH₂), 25.6 (2CH₂), 24.2 (CH₂), 23.6 (CH₂); HRMS (ESI+) m/z calcd for C₂₇H₃₃N₅O (M + 2H)²⁺ 221.6337, found 221.6332. HPLC purity ≥96%, method A: t_R = 17.99 min, λ = 240 nm.

4.1.70. 3-{1-[3-(Morpholin-4-yl)propan-1-yl]-1H-indol-4-yl}-5-(pyridin-4-ylamino)pyridin-2(1H)-one 85

Compound 85 was prepared according to general procedure B, starting from 75 (100 mg, 0.192 mmol). The mixture was stirred at 0 °C for 1 h and then at room temperature for 5 h. The crude was purified by column chromatography (SiO₂, CH₂Cl₂/MeOH 95 : 5 + 0.5% Et₃N to 90 : 10 + 0.5% Et₃N). After evaporation, the solid was washed with CH₂Cl₂ to give compound 85 (20 mg, 0.047 mmol, 24%) as a pale-yellow solid. R_f = 0.40 (CH₂Cl₂/MeOH 9 : 1 + 0.5% Et₃N); Mp > 132 °C; IR (ATR) 2923, 2853, 2807, 1593, 1511, 1210, 1114, 995, 816 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 11.71 (br s, 1H), 8.23 (s, 1H), 8.12 (d, J = 5.1, 2H), 7.49 (d, J = 2.6, 1H), 7.46 (d, J = 8.2, 1H), 7.38 (d, J = 3.0, 1H), 7.34 (br s, 1H), 7.28 (d, J = 7.2, 1H), 7.15 (t, J = 7.7, 1H), 6.64 (d, J = 5.4, 2H), 6.35 (d, J = 2.8, 1H), 4.22 (t, J = 6.6, 2H), 3.60–3.55 (m, 4H), 2.34–2.25 (br s, 4H), 2.20 (t, J = 6.8, 2H), 1.91 (quint, J = 6.6, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 159.9 (C O), 152.5 (C), 149.9 (2CH), 138.7 (CH), 135.9 (C), 130.6 (C), 129.5 (CH), 128.8 (CH), 128.4 (C), 126.5 (C), 120.5 (CH), 120.0 (CH), 119.5 (C), 109.3 (CH), 108.1 (2CH), 99.9 (CH), 66.2 (2CH₂), 55.0 (CH₂), 53.2 (2CH₂), 43.4 (CH₂), 26.7 (CH₂); HRMS (ESI+) m/z calcd for C₂₅H₂₉N₅O₂ (M + 2H)²⁺ 215.6155, found 215.6151. HPLC purity ≥98%, method A: t_R = 17.49 min, λ = 280 nm.

4.1.71. 3-{1-[4-(Morpholin-4-yl)butan-1-yl]-1H-indol-4-yl}-5-(pyridin-4-ylamino)pyridin-2(1H)-one 86

Compound 86 was prepared according to general procedure H, starting from 76 (100 mg, 0.187 mmol) in 5 mL of solvent. The crude was purified by column chromatography (SiO₂, CH₂Cl₂/MeOH 95 : 5 + 0.5% Et₃N to 90 : 10 + 0.5% Et₃N). After evaporation, the solid was solubilized in CH₂Cl₂ and washed with a saturated aqueous solution of Na₂CO₃, dried over MgSO₄ and filtered. After evaporation, compound 86 (46 mg, 0.104 mmol, 55%) was obtained as a pale-yellow solid. R_f = 0.45 (CH₂Cl₂/MeOH 9 : 1 + 0.5% Et₃N); Mp > 128 °C (decomposition); IR (ATR) 2927, 2860, 2807, 1591, 1510, 1211, 1114, 994, 863, 815 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 11.70 (br s, 1H), 8.21 (s, 1H), 8.11 (m, 2H), 7.48 (d, J = 2.9, 1H), 7.46 (d, J = 8.2, 1H), 7.38 (d, J = 3.2, 1H), 7.33 (d, J = 2.6, 1H), 7.28 (d, J = 7.2, 1H), 7.15 (t, J = 7.8, 1H), 6.63 (m, 2H), 6.34 (d, J = 3.0, 1H), 4.19 (t, J = 6.9, 2H), 3.55–3.48 (m, 4H), 2.31–2.22 (br s, 4H), 2.25 (t, J = 7.3, 2H), 1.77 (quint, J = 7.2, 2H), 1.39 (quint, J = 7.4, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 160.1 (C O), 152.5 (C), 149.9 (2CH), 138.6 (CH), 135.8 (C), 130.5 (C), 129.8 (CH), 128.7 (CH), 128.6 (C), 126.6 (C), 120.5 (CH), 120.0 (CH), 119.5 (C), 109.3 (CH), 108.1 (2CH), 99.8 (CH), 66.2 (2CH₂), 57.5 (CH₂), 53.2 (2CH₂), 45.4 (CH₂), 27.6 (CH₂), 23.2 (CH₂); HRMS (ESI+) m/z calcd for C₂₆H₃₁N₅O₂ (M + 2H)²⁺ 222.6233, found 222.6230. HPLC purity ≥98%, method A: t_R = 17.63 min, λ = 240 nm.

4.1.72. 3-{1-[3-(4-Methylpiperazin-1-yl)propan-1-yl]-1H-indol-4-yl]-5-(pyridin-4-ylamino)pyridin-2(1H)-one 87

Compound 87 was prepared according to general procedure H, starting from 77 (120 mg, 0.225 mmol) in 5 mL of solvent. The crude was purified by column chromatography (SiO₂, CH₂Cl₂/MeOH 95 : 5 + 0.5% Et₃N to 85 : 15 + 0.5% Et₃N). After evaporation, the solid was washed with CH₂Cl₂ to 87 (67 mg, 0.151 mmol, 67%) as a white solid. R_f = 0.40 (CH₂Cl₂/MeOH 85 : 15 + 0.5% Et₃N); Mp > 150 °C (decomposition); IR (ATR) 2934, 2792, 1602, 1517, 1275, 1214, 998, 818 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 11.71 (br s, 1H), 8.22 (s, 1H), 8.11 (d, J = 5.9, 2H), 7.49 (d, J = 2.8, 1H), 7.45 (d, J = 8.2, 1H), 7.36 (d, J = 3.0, 1H), 7.33 (d, J = 2.4, 1H), 7.28 (d, J = 7.3, 1H), 7.15 (t, J = 7.8, 1H), 6.63 (d, J = 6.1, 2H), 6.35 (d, J = 2.9, 1H), 4.20 (t, J = 6.5, 2H), 2.40–2.22 (br s, 8H), 2.18 (t, J = 6.8, 2H), 2.14 (s, 3H), 1.89 (quint, J = 6.7, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 159.9 (C O), 152.4 (C), 149.9 (2CH), 138.7 (CH), 135.9 (C), 130.6 (C), 129.5 (CH), 128.8 (CH), 128.4 (C), 126.5 (C), 120.5 (CH), 120.0 (CH), 119.5 (C), 109.3 (CH), 108.1 (2CH), 99.9 (CH), 54.8 (2CH₂), 54.5 (CH₂), 52.6 (2CH₂), 45.7 (CH₃), 43.4 (CH₂), 27.1 (CH₂); HRMS (ESI+) m/z calcd for C₂₆H₃₂N₆O (M + 2H)²⁺ 222.1313, found 222.1308. HPLC purity ≥99%, method A: t_R = 17.74 min, λ = 240 nm.

4.1.73. 3-{1-[4-(4-Methylpiperazin-1-yl)butan-1-yl]-1H-indol-4-yl]-5-(pyridin-4-ylamino)pyridin-2(1H)-one 88

Compound 88 was prepared according to general procedure H, starting from 78 (120 mg, 0.219 mmol) in 5 mL of solvent. The crude was purified by column chromatography (SiO₂, CH₂Cl₂/MeOH 90 : 10 + 0.5% Et₃N to 85 : 15 + 0.5% Et₃N). After evaporation, the solid was solubilized in CH₂Cl₂ and the solution was washed with a saturated aqueous solution of Na₂CO₃, dried over MgSO₄ and filtered. After evaporation, compound 88 (79 mg, 0.173 mmol, 79%) was obtained as a pale-yellow solid. R_f = 0.30 (CH₂Cl₂/MeOH 85 : 15 + 0.5% Et₃N); Mp > 135 °C (decomposition); IR (ATR) 2934, 2794, 1596, 1512, 1461, 1281, 1211, 995, 817 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 11.67 (br s, 1H), 8.21 (s, 1H), 8.11 (d, J = 5.9, 2H), 7.47 (d, J = 2.8, 1H), 7.45 (d, J = 8.6, 1H), 7.37 (d, J = 3.0, 1H), 7.34 (d, J = 2.4, 1H), 7.28 (d, J = 7.2, 1H), 7.14 (t, J = 7.7, 1H), 6.63 (d, J = 6.0, 2H), 6.34 (d, J = 2.9, 1H), 4.18 (t, J = 7.0, 2H), 2.38–2.16 (br s, 8H), 2.24 (t, J = 7.1, 2H), 2.11 (s, 3H), 1.75 (quint, J = 7.0, 2H), 1.38 (quint, J = 7.3, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 160.0 (C O), 152.5 (C), 149.9 (2CH), 138.6 (CH), 135.8 (C), 130.5 (C), 129.8 (CH), 128.7 (CH), 128.6 (C), 126.5 (C), 120.5 (CH), 120.0 (CH), 119.5 (C), 109.4 (CH), 108.1 (2CH), 99.8 (CH), 57.1 (CH₂), 54.8 (2CH₂), 52.6 (2CH₂), 45.7 (CH₃), 45.4 (CH₂), 27.7 (CH₂), 23.5 (CH₂); HRMS (ESI+) m/z calcd for C₂₇H₃₄N₆O (M + 2H)²⁺ 229.1392, found 229.1391. HPLC purity ≥99%, method A: t_R = 17.41 min, λ = 240 nm.

4.2. In vivo assessment of the anti-allodynic effect

Behavioral study in rat

Study approval

Animal protocols were approved by the Committee of Animal Research at the University of Clermont Auvergne, France, and authorized by the French Superior Education and Research Ministry (#41113). They were in accordance with the International Association for the Study of Pain (https://www.iasp-pain.org/resources/guidelines/iasp-guidelines-for-the-use-of-animals-in-research/) and the European Directive 2010/63/EU and ARRIVE guidelines.

Rat capsaicin model

Capsaicin (Sigma-Aldrich) was dissolved in an endolipid solution and conserved at 4 °C. For behavioral testing, animals received a subcutaneous injection of capsaicin (25 μg in 25 μL) solution into the right hindpaw using a 27-gauge needle coupled to a 100 μL Hamilton syringe, as described previously.¹³

Intrathecal injection

For investigation of the effects of newly synthesized compounds upon capsaicin-induced cutaneous mechanical allodynia, animals were briefly (about 3 min) anesthetized using a mask with 2% isoflurane and received an intrathecal injection of either the compound (5 μM in 10 μL saline) or vehicle alone (saline + 0.01% DMSO) using a 10 μL Hamilton syringe. After recovery (about 2 min), rats were placed in an observation field (0.6 × 0.6 m²) under red light for a 30 min habituation session. During this period, rats were adapted to the observation field and red light. Immediately after the 30 min habituation period, animals received the subcutaneous injection of capsaicin.

Behavioral responses

Behavioral sensory testing was performed as previously described.¹⁴ Briefly, rats were first allowed to acclimatize to 0.6 × 0.6 m² glass chambers used for behavioral testing as well as to innocuous mechanical stimulation for 3 days before the start of the behavioral sensory testing. The mechanical withdrawal threshold was determined using von Frey (VF) filaments (Bioseb, France) applied to the plantar surface of the right hindpaw via the descending-ascending method. Each filament was applied to the skin 5 times for 3 s (at 10 s intervals). The VF filament threshold (VFT, in grams) was equal to the filament evoking a hindpaw withdrawal response in three out of five trials. After the injection of capsaicin, the VFT was recorded at 10-minute intervals for 65 minutes. The level of mechanical allodynia inhibition of a given compound was computed as follows: [(AUC vehicle − AUC compound)/AUC vehicle] × 100 (AUC is the area under the curve).

Data availability

The data supporting this article (¹H NMR, ¹³C NMR and HRMS spectra of all new compounds) have been included as part of the ESI.†

Conflicts of interest

The authors declare no conflict of interest.

Supplementary Material

MD-015-D4MD00375F-s001

MD-015-D4MD00375F-s001.pdf^{(12.6MB, pdf)}

Acknowledgments

This research was financed by the French government IDEX-ISITE initiative 16-IDEX-0001 (CAP 20-25), Région Auvergne-Rhône-Alpes (Pack Ambition Recherche), Université Clermont Auvergne, Inserm and CNRS. The authors (TF, AB, FG, FA, PM) also thank WeylChem InnoTec (Frankfurt, Germany) for financial support and Aurélie Job for HPLC analysis.

^†

Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4md00375f

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