Design, synthesis and biological evaluation of fluorescent ligands for MT₁ and/or MT₂ melatonin receptors

Abstract

Fluorescent melatoninergic ligands have been designed by associating the 4-azamelatonin ligands with different fluorophores. The ligands show good affinities for MT₁ and/or MT₂ receptors and substitution of the fluorophore at positions 2 or 5 of the azamelatonin core had a direct impact on the MT receptors selectivity while grafting the fluorophores on position N1 produced fluorescent ligands with good affinities for both MT₁/MT₂ receptors. The optimal position N-1, C-2 or C-5 on the 4-azamelatonin ligand appeared strongly dependent upon the nature of the fluorophore itself.

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Introduction

Melatonin (N-acetyl-5-methoxytryptamine, Scheme 1) is a neurohormone synthesized by the pineal gland during the dark period in all animal species and immediately released. The rhythm of melatonin synthesis is directly controlled by photoperiodism. Melatonin secretion both in blood and in cerebrospinal fluid transduces the photoperiodic message to all central or peripheral structures expressing melatoninergic receptors or binding sites, allowing synchronization of several cellular and physiological events to a given daylength.¹ Melatonin rhythm synchronizes a wide range of physiological functions including among others circadian rhythms or seasonal reproduction.² Anarchic melatonin secretion also accounts for disorders of sleep/wake rhythms associated or not with depression. Melatonin receptors play an important role in the mode of action of a few drugs such as ramelteon (Rozerem®), a melatonin agonist, employed to treat insomnia and nocturia³ and agomelatine (Valdoxan®), a melatonin agonist and 5-HT_2C antagonist, indicated for the treatment of depression.⁴

Scheme 1 Design of the MT receptors fluorescent ligands.

This hormone targets two high affinity receptors, MT₁ and MT₂. The melatoninergic receptor subfamily which includes MT₁, MT₂ and the orphan receptor GPR50, belongs to the G protein-coupled receptor (GPCR) superfamily. MT₁ and MT₂ receptors share specific short sequences of amino acids and exhibit an amino acid sequence identity of approximately 55%. However, this sequence identity is larger within transmembrane segments, which explains the difficulty in obtaining selective ligands.⁵

Almost 20 years after the cloning of melatonin receptor subtypes, their pharmacology and their respective roles are still poorly known. This difficulty lies mainly on the lack of selective pharmacological markers for the study of different receptors. Indeed, the bulk of research on melatonin mechanisms of action is mainly performed on using a single radioligand, 2-[¹²⁵I]-iodomelatonin. This tool used in many autoradiography or displacement studies allowed the demonstration of melatoninergic binding sites at the central level and in many peripheral structures. The tissue distribution of melatonin binding sites has been only partially elucidated, firstly for the lack of selective radioligands for MT₁ or MT₂ receptors which makes discrimination between melatonin receptors impossible, and secondly, for the absence specific MT₁- or MT₂-directed primary antibodies.

In addition, the growth of fluorescence techniques for the study of ligand–receptor interactions has led to a boom in the design of new fluorescent ligands. The discovery of ligands with a high affinity for one receptor, easily discernible, even at the cellular level, has given fresh impetus to investigate GPCRs. The advantages of this approach are its safety and lower cost compared to traditional radioligand approaches. To the best of our knowledge, only a very few fluorescent ligands of melatonin receptors have been described to date based on coumarin,⁶ bodipy⁷ and 7-azaindole⁸ derivatives. In the present paper, we report the design, synthesis, spectroscopic characterisation and biological evaluation of fluorescent melatoninergic ligands.

Results and discussion

For many years, our team has been involved in the development of innovative molecules able to interact with melatonin receptors (MT₁ and MT₂) for the development of new therapeutic indication.⁹ These data combined with others¹⁰ have led to the determination on the melatonin core of three main sites in terms of activity and selectivity. The methoxy group at position 5 and the N-ethylacetamide chain at position C3 are necessary for the recognition of MT₁ and MT₂ receptors, the indole ring acting as a tensor with a defined length between both sites. The acyl group appears essential for the affinity to the receptors while the methoxy (or alkoxy) group at position 5 is involved in the intrinsic activity.

Relying on the endogenous hormone, numerous analogs have been synthesized in order to improve the pharmacological profile and more specifically the catabolic stability of the ligands. From the diversity of ligands generated, structure–activity relationships have been revealed (Scheme 1). Bulky substituents at position C2 on the indole or analogs scaffold maintain MT₂ affinity and decrease MT₁ affinity, leading to selective ligands towards MT₂ receptors (up to 900 towards MT₁).¹¹

Conversely, huge substituents in place of the methoxy group allow the ligand to keep MT₁ affinity while MT₂ affinity is decreased, leading to selective ligands towards MT₁ receptors (up to 220 towards MT₂).¹²

In the light of these results, it clearly appeared to us that it was possible to introduce fluorophores at one or the other position without losing affinity and furthermore by increasing selectivity towards MT₁ or MT₂ receptors. Various fluorescent probes have been developed, however, when linked close to the pharmacophore, these probes can interfere with the ligand binding domain. For this reason, the choice of both the fluorophore and linker between the fluorophore and the pharmacophore is crucial and has to be optimized in order to hopefully keep the biological activity and allow the insertion of the ligands in the binding site of GPCRs. Our choice went to six families of fluorophores ie, nitrobenzoxadiazole (NBD), dansyl, phthalimide, BODIPY Fl®,¹³ fluorescein, rhodamine which have been selected (i) for their reported efficiency in GPCRs probes¹⁴ and (ii) for the complementary range of emission wavelength (from 450 to 600 nm). Most of those fluorophores are commonly coupled through an activated ester with an amine linked to the pharmacophore. This amine function is also absolutely adapted for NBD and phthalimide grafting. In this context, we designed our fluorescent probes based on the 4-azamelatonin pharmacophore 1 previously developed in our laboratory due to its very high affinity over MT₁ and MT₂ receptors.¹⁵ Six amine-containing pharmacophore 2–7 were considered for conjugation with the fluorophores and evaluation as MT receptors ligands (Fig. 1).

Fig. 1 Amine containing 4-azamelatonin precursors.

The first set of molecules was oriented to the functionalisation of the alkyl acetyl chain at the C-3 position. 4-Azamelatonine 1¹⁵ was deacetylated by potassium hydroxyde to afford free amine 2 which was directly engaged to a S_NAr reaction with NBD-Cl to give fluorescent probe 8 in good yield. The amidation reaction between 2 and activated BODIPY FL® dye gave ligand 9 in 33% yield (Scheme 2).

Scheme 2 Synthesis of amine analogue 2 and its “C3 tagging” derivatives 8 and 9.

We then focused on the C-2 tagged ligands with the objective to obtain MT₂ selective probes. Iodination of the 4-azamelatonine 1 occurred at position C-2 in the presence of N-iodosuccinimide in 74% yield. The iodinated compound 10 was committed in a Suzuki–Miyaura reaction with the corresponding cyanophenylboronic acid with catalytic amount of palladium tetrakis to afford product 11 and 12 in 97% and 73% yield, respectively. Cyano groups were then reduced to amines 3 and 4 under RANEY® nickel catalysed hydrogenation in very good yields (Scheme 3).

Scheme 3 Synthesis of C2 functionalized analogues 3 and 4.

Amines 3 and 4 were then tagged with NDB and BODIPY dyes. The four probes 13, 14, 15 and 16 were isolated in moderate to good yields (Scheme 4).

Scheme 4 Synthesis of probes 13 to 16 “C2 tagging”.

In this promising series of C2 tagged ligands, we also wanted to investigate the effect of small chromophores such as NBD and phthalimide types, located closed to the azamelatonin pharmacophore in analogy with melatonin analogues substituted at C2 with conformationally flexible N-methylindoline and N-methylisoindoline groups.¹⁶ For this purpose, 2-cyano-4-azamelatonin 17 was obtained via a palladium catalysed cyanation reaction of 2-iodomelatonin 10. The key amine 5 was synthesized by hydride reduction in 70% yield (Scheme 5). Condensation of 5 with the 4-(dimethylamino)phthalic acid lead to the phthalimide probe 18. Amine 5 was also used in a methylation/S_NAr sequence to introduce the NBD fluorophore in position C2 and isolate compound 20.

Scheme 5 Synthesis of probes 18 and 20.

In order to obtain C5 functionalized fluorescent ligands, we have developed an original synthetic pathway from the commercially available 2-chloro-5-nitropyridine 21. In situ formed sodium 2-(allyloxycarbonylamino)ethanolate was reacted with 21 in an S_NAr process to give product 22 in 68% yield (Scheme 6). The nitro group was then reduced using Béchamp reduction and aniline 23 was converted into the corresponding hydrazine 24 by reduction of the intermediate diazonium salt. Aza-Fischer indolisation¹⁵ followed by palladium catalysed allylcarbamate deprotection gave the expected amine 6 in good yield.

Scheme 6 Synthesis of the C5 amino functionalized analogue 6.

From amine 6, a series of six new fluorescent probes was synthesized (Scheme 7). The amine 6 was reacted with NBD-Cl and activated BODIPY to provide probes 26 and 27 in 33% yield in both cases. Amine 6 was also engaged in reaction with dansyl-Cl, fluorescein isothiocyanate (FITC) and 4-(dimethylamino)phthalic acid to afford desired fluorescent probes 28, 29 and 30 in 66%, 50% and 42% yield, respectively.

Scheme 7 Synthesis of probes 26 to 30 “C5 tagging”.

Amine 6 was also coupled with isonipecotic acid, using BOP reagent followed by a N-Boc deprotection in the presence of trifluoroacetic acid to give piperidine 31. This secondary amine was used for rhodamine 6G tagging using HBTU as coupling reagent. The desired fluorescent probe 32 was isolated in 41% yield (Scheme 8).

Scheme 8 Synthesis of probe 32 “C5 tagging”.

Finally, we focused on the N1 tagged ligands. 4-Azamelatonin 1 was alkylated with 1,2-dibromoethane and the desired product 33 was isolated in 65% yield. Nucleophilic substitution of bromide with sodium azide, followed by RANEY® nickel catalysed hydrogenation afforded the expected amine 7 in 64% yield for two steps (Scheme 9).

Scheme 9 Synthesis of N1 functionalized analogue 7.

Once this amine obtained, five new probes were synthesized. As previously, amine 7 reacted with NBD-Cl, FITC and activated acid of BODIPY FL® and was functionalized for reacting with rhodamine 6G to provide compounds 34 to 37 in moderate to good yield (Scheme 10).

Scheme 10 Synthesis of probes 34 to 37 “N1 tagging”.

The photophysical properties of these new fluorescent ligands were measured in dimethylsulfoxide (DMSO). Absorption, emission wavelengths, molar extinction coefficient, quantum yields and brightness of compounds 8, 9, 13–16, 18, 20, 26–30, 32, 34–37 are reported in Table 1 (see ESI† for absorption and emission spectra).

Table 1 Photophysical properties of compounds 8, 9, 13–16, 18, 20, 26–30, 32, 34–37 in DMSO except for compound 36 in NaOH 0.1 N

Entry	Product	λmax (nm)	λem (nm)	ελmax (L mol^−1 cm^−1)	Quantum yield Φ	Brightness ε Φ
1	8	488	541	16 500	<0.01	<500
2	9	508	516	30 100	0.78	23 445
3	13	490	539	57 300	<0.01	<500
4	14	475	536	25 000	0.01	<500
5	15	508	516	89 200	0.46	41 030
6	16	508	516	79 100	0.61	48 250
7	18	401	525	5400	0.04	<500
8	20	492	537	36 300	0.01	<500
9	26	478	538	19 900	0.01	<500
10	27	507	516	109 500	1	109 500
11	28	338	528	6200	0.54	3350
12	29	523	538	15 000	0.72	10 800
13	30	400	516	5800	0.09	520
14	32	542	564	81 200	0.88	71 450
15	34	478	539	25 500	<0.01	<500
16	35	508	516	112 400	0.46	51 700
17	36	490	517	68 600	0.12	8230
18	37	542	563	64 300	0.84	54 000

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The binding affinities of the final ligands 8, 9, 13–16, 18, 20, 26–30, 32, 34–37 were determined in competition radioligand binding assays using 2-[¹²⁵I]-iodomelatonin on membranes of CHO cells expressing human MT₁ or MT₂ receptors. Results for binding affinities and MT₁/MT₂ selectivity ratios are reported in Table 2.

Table 2 Binding affinity of compounds 8, 9, 13–16, 18, 20, 26–30, 32, 34–37 on human MT₁ and MT₂ receptors

Entry	Product	Ki hMT1 (nM)	Ki hMT2 (nM)	MT1/MT2
a Not significant.
1	8	2200 ± 500	2900 ± 900	^a
2	9	703 ± 60	344 ± 36	2
3	13	139 ± 20	25 ± 15	5.6
4	14	23 ± 7	0.2 ± 0.01	115
5	15	144 ± 12	105 ± 14	1.4
6	16	112 ± 9	121 ± 14	0.9
7	18	8 ± 0.1	16 ± 3	0.5
8	20	>1000	7 ± 0.8	>140
9	26	3.8 ± 1.3	44 ± 23	0.01
10	27	169 ± 27	1770 ± 700	0.1
11	28	80 ± 6	>1000	< 0.08
12	29	420 ± 10	65 ± 1	6.5
13	30	180 ± 30	>1000	<0.18
14	32	580 ± 30	>1000	^a
15	34	217 ± 25	75 ± 4	0.7
16	35	12 ± 7	13 ± 5	0.9
17	36	900 ± 130	>1000	^a
18	37	190 ± 25	170 ± 30	1.1

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4-Azamelatonin analogues 8 and 9, bearing the fluorophore at position C3, did not show high affinity for both MT receptors which was not very surprising considering literature data.¹⁷ More interestingly, analogues 13–16, 18 and 20, tagged with a fluorophore at position C-2, showed a much higher affinity for MT receptors. As expected, those ligands were more selective for the MT₂ receptor with the puzzling exceptions of compounds 16 and 18. The most promising MT₂ selective fluorescent probes, 14 and 20, were both composed with the NBD fluorophores. Unfortunately, the brightness intensity for both compounds was very low (Table 1, entries 4 and 8) which made these compounds unsuitable for a future use on biological samples which commonly display a persistent residual autofluorescence. Compounds 15 with a BODIPY FL® fluorophore was brighter but unfortunately not selective for one of the MT receptors (Tables 1 and 2, entry 5).

On the other hand, “C5 tagged” 4-azamelatonin analogues 26–30 and 32 showed as expected good affinities and selectivity for the MT₁ receptor (Table 2, entries 9–14). One exception was observed with the fluorescein tagged compound 29 (Table 2, entry 12) which had a better affinity for the MT₂ receptor. Once again, the most active and selective probes with small fluorophores (NBD, dansyl and phthalimide, Table 1, entries 9, 11 and 13) were not bright enough for cellular imaging. The rhodamine analogue 32 (Table 2, entry 14) showed only a moderate affinity for MT₁. Compound 27 appeared to be a MT₁ selective fluorescent probe with a moderate affinity and a very good brightness (Tables 1 and 2, entry 10) thanks to the BODIPY FL® fluorophore.

Finally, with the N1 tagged analogues, a very good fluorescent probe for MT₁ and MT₂ receptors was obtained and characterized. In this case, compounds 35 comprising BODIPY FL® showed the best affinity for both MT receptors without any selectivity (Table 2, entry 16) but with a very good brightness (Table 1, entry 16). However this probe was not specific towards MT receptors expressing cells. NBD, fluorescein and rhodamine derivatives 34, 36 and 37 were not selective either and much less active on MT receptors (Table 2, entries 15, 17 and 18).

Conclusion

We have shown in this paper that the strategy associating the 4-azamelatonin ligands with different fluorescent probes produced fluorescent melatoninergic ligands with good affinities for MT₁ and/or MT₂ receptors. Binding experiments on MT₁ and MT₂ human receptors confirmed our starting hypothesis that substitution at positions 2 or 5 of the azamelatonin core had a direct impact on the MT receptors selectivity while grafting the fluorophores on position N1 produced fluorescent ligands with good affinities for both MT₁/MT₂ receptors.

In a few examples, small fluorophores produced ligands with the best affinities for MT receptors but their limited brightnesses represented a strong limitation for further use on biological samples. Despite no evidence obtained for obvious structure–activity relationships, the optimal position N-1, C-2 or C-5 on the 4-azamelatonin ligand appeared strongly dependent upon the nature of the fluorophore itself. Work is underway to demonstrate the interest of such fluorescent melatonin ligands as tools for a better understanding of the role of MT receptors.

Experimental

General information

Organic synthesis. All reagents were purchased from commercial suppliers and were used without further purification.

Tetrahydrofuran was purified with a dry station GT S100, dichloromethane, triethylamine, diisopropylethylamine and dimethylformamide were distillated over CaH₂.

The reactions were monitored by thin-layer chromatography (TLC) analysis using silica gel (60 F254) plates. Compounds were visualized by UV irradiation and/or spraying with a solution of cerium molybdate, or phosphomolybdic acid, followed by heating at 200 °C. Flash column chromatography was performed on silica gel 60 (230–400 mesh, 0.040–0.063 mm). Reversed-phase column flash-chromatographies were performed on octadecyl-functionalised silica gel (mean pore size 60 Å) from Aldrich.

¹H and ¹³C NMR spectra were recorded on a spectrometer at 250 MHz (¹³C, 62.9 MHz) or 400 MHz (¹³C, 100 MHz). Chemical shifts are given in parts per million from tetramethylsilane (TMS) as internal standard. The following abbreviations are used for the proton spectra multiplicities: s: singlet, d: doublet, t: triplet, q: quartet, qt: quintuplet, m: multiplet, br: broad. Coupling constants (J) are reported in Hertz (Hz). Signals were assigned as far as possible by means of two-dimensional NMR spectroscopy: ¹H–¹H–COSY, ¹H–¹³C–COSY (HSQC: Heteronuclear Single Quantum Coherence).

Ionspray methodology was used to record mass spectra. HRMS spectra were recorded on a Maxis Bruker 4G.

The infrared spectra of compounds were recorded on a Thermo Scientific Nicolet iS10.

Melting points (mp [°C]) were taken on open capillary tubes and are uncorrected, performed on a Electrothermal IA 9100.

Photophysical measurements. UV-visible spectra were obtained on a Varian Cary 50 scan spectrophotometer by using a rectangular quartz micro cell (Hellma, 104-QS, light path: 10 mm, 1.0 mL). Fluorescence spectroscopic studies (emission/excitation spectra) were performed with a Varian Cary Eclipse spectrophotometer with a fluorescence quartz ultra-micro cell (Hellma, 105.250-QS, light path: 10 × 2 mm, 50 μL). Emission spectra were recorded under the same conditions after excitation at the corresponding wavelength (see Table S1,† excitation and emission filters: auto, excitation and emission slit = 5 nm) in DMSO. Relative quantum yields were measured in DMSO at 25 °C by a relative method using a suitable standard (see Table S1†). The following equation was used to determine the relative fluorescence quantum yield:

Φ_F(x) = (A_S/A_X)(F_X/F_S)(n_X/n_S)²Φ_F(s)

where A is the absorbance (in the range 0.01–0.1 A.U.), F is the area under the emission curve, n is the refractive index of the solvents (at 25 °C) used in measurements, and the subscripts S and X represent standard and unknown, respectively.

Procedures and characterization data for the products

2-(5-Methoxy-1H-pyrrolo[3,2-b]pyridin-3-yl)ethanamine 2. To a solution of 4-azamelatonin (500 mg, 2.14 mmol, 1 eq.) in abs EtOH (30 mL) were added KOH (4 g) and water (15 mL). The resulting mixture was stirred at reflux for 48 h then diluted with water (30 mL) and extracted with DCM (3 × 40 mL). The combined organic layers were washed with water, brine, dried over MgSO₄ and evaporated to dryness to afford the crude amine 2 as colorless oil (398 mg, 97% yield). The crude product was reacted without further purification.

¹H NMR (400 MHz, CDCl₃): δ = 9.27 (bs, 1H, NH), 7.47 (d, J = 8.8 Hz, 1H), 7.08 (s, 1H), 6.55 (d, J = 8.8 Hz, 1H), 3.96 (s, 3H), 3.05–3.15 (m, 2H), 2.92 (t, J = 6.2 Hz, 2H), 1.86 (bs, 2H, NH₂). ¹³C NMR (101 MHz, CDCl₃): δ = 159.6, 141.8, 125.0, 124.9, 121.8, 113.7, 105.1, 53.3, 42.6, 28.7. IR: ν_max (cm⁻¹) = 1576, 1402, 1257, 1030. MS (ESI): m/z = 192.1 [M + H]⁺.

N-(2-(2-(4-(Aminomethyl)phenyl)-5-methoxy-1H-pyrrolo[3,2-b]pyridin-3-yl)ethyl)acetamide 3. A mixture of 11 (380 mg, 1.13 mmol, 1 eq.) and Ni-RANEY® in MeOH (25 mL) was stirred at room temperature under H₂ (100 psi) atmosphere of for 7 hours. Thereafter, the mixture was filtered under celite and the filtrate was evaporated to dryness to afford the crude product 3 as colorless oil (315 mg, 82% yield). The crude product was reacted without further purification.

¹H NMR (400 MHz, CDCl₃): δ = 8.23 (bs, 1H, NH), 7.60 (d, J = 7.5 Hz, 1H), 7.52–7.37 (m, 4H), 6.64 (d, J = 7.5 Hz, 1H), 4.05 (s, 3H), 3.98–3.90 (m, 2H), 3.68–3.61 (m, 2H), 3.10 (t, J = 5.0 Hz, 2H), 1.91 (s, 3H). IR: ν_max (cm⁻¹) = 825, 1029, 1112, 1240, 1294, 1400, 1584, 1651, 2362, 2921. HRMS (ESI): m/z = 339.1821 [M + H]⁺ calculated for C₁₉H₂₃N₄O₂, found: m/z = 339.1833.

N-(2-(2-(3-(Aminomethyl)phenyl)-5-methoxy-1H-pyrrolo[3,2-b]pyridin-3-yl)ethyl)acetamide 4. A mixture of 12 (365 mg, 1.09 mmol, 1 eq.) and Ni-RANEY® in MeOH (20 mL) was stirred at room temperature under an atmosphere of H₂ (100 psi) for 7 hours. Thereafter, the mixture was filtered under celite and the filtrate was evaporated to dryness to afford the crude product 4 as colorless oil (350 mg, 94% yield). The crude product was reacted without further purification.

¹H NMR (400 MHz, CDCl₃): δ = 8.18 (bs, 1H), 7.61 (d, J = 7.5 Hz, 1H), 7.52–7.37 (m, 4H), 6.65 (d, J = 7.5 Hz, 1H), 4.06 (s, 3H), 3.96 (s, 2H), 3.68–3.60 (m, 2H), 3.12 (t, J = 5 Hz, 2H), 1.91 (s, 3H). IR: ν_max (cm⁻¹) = 798, 1028, 1240, 1287, 1404, 1489, 1578, 1648, 2930, 3270. HRMS (ESI): m/z = 339.1821 [M + H]⁺ calculated for C₁₉H₂₃N₄O₂, found: m/z = 339.1829.

N-(2-(5-(2-Aminoethoxy)-1H-pyrrolo[3,2-b]pyridin-3-yl)ethyl)acetamide 6. A solution of 4-azamelatonine 25 (100 mg, 0.29 mmol, 1 eq.) and dimethylbarbituric acid (90 mg, 0.58 mmol, 2 eq.) in dry DCM was degassed 15 min with argon. Thereafter Pd(PPh₃)₄ (15 mg, 14 μmol, 0.05 eq.) was added. The mixture was stirred 1 h at room temperature. An oily compound appeared. The solvent was removed. The residue was washed twice with DCM and dried under vacuum to afford the expected compound 6 as an oily orange solid (75 mg, 98% yield).

¹H NMR (400 MHz, MeOD): δ = 7.65 (d, J = 8.0 Hz, 1H), 7.26 (s, 1H), 6.62 (d, J = 8.0 Hz, 1H), 4.57 (t, J = 5.0 Hz, 2H), 3.53 (t, J = 7.5 Hz, 2H), 3.33–3.28 (m, 2H), 2.94 (t, J = 7.5 Hz, 2H), 1.88 (s, 3H). ¹³C NMR (101 MHz, MeOD): δ = 173.4, 159.5, 142.1, 127.9, 127.4, 124.5, 112.9, 105.4, 64.2, 41.4, 41.0, 25.4, 22.9. IR: ν_max (cm⁻¹) = 775, 1018, 1237, 1296, 1369, 1568, 1645, 2931, 3293. HRMS (ESI): m/z = 263.1508 [M + H]⁺ calculated for C₁₃H₁₉N₄O₂, found: m/z = 263.1499.

N-(2-(1-(2-Aminoethyl)-5-méthoxy-1H-pyrrolo[3,2-b]pyridin-3-yl)ethyl)acetamide 7. To a solution of compound 33a (420 mg, 1.39 mmol, 1 eq.) in AcOEt (20 mL) was added a catalytic amount of RANEY® nickel. The resulting mixture was stirred 3 h under hydrogen atmosphere at room temperature. The solution was then filtered under celite and the residue was purified by flash chromatography with a mixture of DCM/MeOH (9/1) as eluent to afford the expected compound 7 as a colorless oil (278 mg, 64% yield).

¹H NMR (400 MHz, DMSO): δ = 1.80 (s, 3H), 2.84 (t, J = 6.8 Hz, 2H), 3.18–3.22 (m, 2H), 3.31–3.37 (m, 2H), 4.02 (s, 3H), 4.55 (t, J = 6.0 Hz, 2H), 6.95 (d, J = 8.4 Hz, 1H), 7.72 (s, 1H), 8.11 (bs, 1H, NH), 8.45 (d, J = 8.4 Hz, 1H). IR: ν_max (cm⁻¹) = 1609, 1523, 1344, 1293, 1037. MS (ESI): m/z = 277.0 [M + H]⁺.

N-(2-(5-Methoxy-1H-pyrrolo[3,2-b]pyridin-3-yl)ethyl)-7-nitrobenzo[c][1,2,5]oxadiazol-4-amine 8. A solution of the amine 2 (15 mg, 76 μmol, 1.08 eq.) and NBD-Cl (14 mg, 70 μmol, 1 eq.) in abs. ethanol (1 mL) was irradiated in microwave 15 min at 70 °C. Thereafter, 100 μL of Et₃N was added and the mixture stirred 15 min at room temperature. After concentration under reduced pressure, the residue was purified by flash chromatography with a step gradient AcOEt (of 0 to 70%) in petroleum ether to afford the expected compound as a yellow solid (20 mg, 80% yield).

¹H NMR (400 MHz, DMSO): δ = 10.95 (bs, 1H, NH), 9.65 (bs, 1H, NH), 8.49 (d, J = 8.8 Hz, 1H), 7.67 (d, J = 8.7 Hz, 1H), 7.41 (s, 1H), 6.61 (d, J = 8.8 Hz, 1H), 6.54 (d, J = 8.7 Hz, 1H), 3.95–3.80 (m, 5H), 3.11 (t, J = 7.4 Hz, 2H). ¹³C NMR (101 MHz, DMSO): δ 159.2, 145.7, 144.9, 144.6, 141.6, 138.2, 126.3, 125.1, 122.9, 121.0, 110.9, 105.0, 99.6, 53.0, 44.3, 40.4, 40.2, 40.0, 39.8, 39.6, 23.5. IR: ν_max (cm⁻¹) = 3268, 1617, 1576, 1528, 1487, 1292, 1115, 806. HRMS (ESI): m/z = 355.1155 [M + H]⁺ calculated for C₁₆H₁₅N₆O₄, found: m/z = 355.1169. Melting point: 207 °C.

(Z)-3-(1-(Difluoroboryl)-5-((3,5-dimethyl-2H-pyrrol-2-ylidene)methyl)-1H-pyrrol-2-yl)-N-(2-(5-methoxy-1H-pyrrolo[3,2-b]pyridin-3-yl)ethyl)propanamide 9. Dry DIEA (6 μL, 4.4 mg, 34 μmol, 2 eq.) was added to a solution of BODIPY FL® (5 mg, 17 μmol, 1 eq.) and TSTU (5 mg, 17 μmol, 1 eq.) in dry DMF (500 μL). The resulting solution was protected from the light and stirred 2 h at room temperature under an atmosphere of argon. Thereafter a solution of the amine 2 (9 mg, 51 μmol, 3 eq.) in dry DMF (500 μL) was added and the mixture was stirred overnight at room temperature. After removing of the DMF, the residue was purified by flash chromatography with a step gradient of AcOEt (10 to 80%) in petroleum ether to yield an orange solid (3 mg, 33% yield).

¹H NMR (400 MHz, CDCl₃): δ = 7.93 (bs, 1H, NH), 7.52 (d, J = 8.7 Hz, 1H), 7.11 (d, J = 2.5 Hz, 1H), 7.03 (s, 1H), 6.92–6.70 (m, 2H), 6.57 (d, J = 8.7 Hz, 1H), 6.24 (d, J = 3.8 Hz, 1H), 6.10 (s, 1H), 3.96 (s, 3H), 3.60 (dd, J = 11.8 and 5.5 Hz, 2H), 3.25 (t, J = 7.4 Hz, 2H), 2.96 (t, J = 6.2 Hz, 2H), 2.68–2.41 (m, 5H), 2.25 (s, 3H). ¹³C NMR (101 MHz, CDCl₃): δ 171.9, 159.8, 159.7, 157.9, 143.6, 141.5, 135.0, 133.3, 128.2, 125.1, 124.9, 123.7, 122.2, 120.2, 117.2, 113.4, 105.2, 77.3, 77.0, 76.7, 53.4, 41.0, 35.7, 29.7, 24.1, 14.9, 11.3. IR: ν_max (cm⁻¹) = 1028, 1053, 1081, 1258, 1406, 1601, 1654, 2339, 2360, 2884, 2964. HRMS (ESI): m/z = 466.2226 [M + H]⁺ calculated for C₂₄H₂₇BF₂N₅O₂, found: m/z = 466.2206. Melting point: 94 °C.

N-(2-(2-Iodo-5-methoxy-1H-pyrrolo[3,2-b]pyridin-3-yl)ethyl)acetamide 10. To a solution of the azamelatonin 1 (200 mg, 0.86 mmol, 1.00 eq.) in THF (10 mL) was added portionwise N-iodosuccinimide (290 mg, 1.29 mmol, 1.50 eq.) then stirred 3 hours at room temperature. After completion of the reaction, the resulting mixture was extracted with ethyl acetate (3 × 40 mL). The combined organic layers were washed with sodium thiosulfate (20 mL), brine, dried over MgSO₄ and evaporated to dryness. The residue was purified by flash chromatography with ethyl acetate to afford the expected compound 10 as a yellow solid (228 mg, 74% yield).

¹H NMR (400 MHz, CDCl₃): δ = 9.88 (bs, 1H, NH), 7.53 (d, J = 8.8 Hz, 1H), 7.31 (bs, 1H, NH), 6.53 (d, J = 8.8 Hz, 1H), 3.99 (s, 3H), 3.55–3.60 (m, 2H), 2.94 (t, J = 6.0 Hz, 2H), 1.95 (s, 3H). ¹³C NMR (101 MHz, CDCl₃): δ = 170.8, 159.8, 141.0, 128.3, 121.8, 118.9, 105.6, 82.0, 53.5, 41.2, 25.8, 23.6. IR: ν_max (cm⁻¹) = 1027, 1237, 1391, 1556, 1608, 3202. MS (ESI): m/z = 360.0 [M + H]⁺. Melting point: 143 °C.

N-(2-(2-(4-Cyanophenyl)-5-methoxy-1H-pyrrolo[3,2-b]pyridin-3-yl)ethyl)acetamide 11. A solution of 10 (200 mg, 0.55 mmol, 1 eq.), 4-cyanophenylboronic acid (102 mg, 0.69 mmol, 1.25 eq.) and K₂CO₃ (462 mg, 3.34 mmol, 6 eq.) in dioxane (5 mL) and water (2 mL) was degassed with argon 30 min and Pd(PPh₃)₄ (29 mg, 28 μmol, 0.05 eq.) was added thereafter. The mixture was refluxed for 3 hours. Then, AcOEt was added and the resulting solution was washed twice with H₂O. The organic layer was dried over MgSO₄ and evaporated to dryness. The crude product was purified by flash chromatography with a step gradient of AcOEt (50–90%) in petroleum ether to afford the expected product as a beige solid (180 mg, 97% yield).

¹H NMR (400 MHz, CDCl₃): δ = 8.65 (bs, 1H, NH), 7.71 (d, J = 8.3 Hz, 2H), 7.66–7.60 (m, 3H), 7.32 (bs, 1H, NH), 6.70 (d, J = 8.7 Hz, 1H), 4.05 (s, 3H), 3.63 (dd, J = 11.4 and 5.4 Hz, 2H), 3.10 (t, J = 8.0 Hz, 2H), 1.89 (s, 3H). ¹³C NMR (101 MHz, CDCl₃): δ = 170.5, 160.5, 143.2, 137.0, 135.3, 132.9, 128.3, 125.5, 122.5, 118.7, 113.2, 111.5, 107.5, 53.6, 41.8, 23.4, 23.2. IR: ν_max (cm⁻¹) = 1023, 1107, 1239, 1287, 1410, 1543, 1649, 2225, 3231, 3367. HRMS (ESI): m/z = 335.1508 [M + H]⁺ calculated for C₁₉H₁₉N₄O₂, found: m/z = 335.1523.

N-(2-(2-(3-Cyanophenyl)-5-methoxy-1H-pyrrolo[3,2-b]pyridin-3-yl)ethyl)acetamide 12. A solution of 10 (200 mg, 0.55 mmol, 1 eq.), 3-cyanophenylboronic acid (102 mg, 0.69 mmol, 1.25 eq.) and K₂CO₃ (462 mg, 3.34 mmol, 6 eq.) in dioxane (5 mL) and water (2 mL) was degassed with argon 30 min and Pd(PPh₃)₄ (29 mg, 28 μmol, 0.05 eq.) was added thereafter. The mixture was refluxed for 3 hours. Then, AcOEt was added and the resulting solution was washed twice with H₂O. The organic layer was dried over MgSO₄ and evaporated to dryness. The crude product was purified by flash chromatography with a step gradient of AcOEt (50–90%) in petroleum ether to afford the expected product as a beige solid (135 mg, 73% yield).

¹H NMR (400 MHz, CDCl₃): δ = 9.00 (s, 1H, NH), 7.81 (s, 1H), 7.74 (d, J = 7.8 Hz, 1H), 7.64–7.61 (m, 2H), 7.54 (t, J = 7.8 Hz, 1H), 7.46 (bs, 1H, NH), 6.67 (d, J = 8.7 Hz, 1H), 4.04 (s, 3H), 3.63 (dd, J = 11.5, 5.2 Hz, 2H), 3.08 (t, J = 5.2 Hz, 2H), 1.90 (s, 3H). ¹³C NMR (101 MHz, CDCl₃): δ = 170.6, 160.4, 143.0, 138.1, 134.0, 132.5, 131.5, 131.2, 130.0, 122.5, 118.5, 113.4, 112.4, 107.1, 53.5, 41.9, 23.5, 23.1. IR: ν_max (cm⁻¹) = 1027, 1236, 1259, 1292, 1434, 1463, 1490, 1544, 1651, 2233, 2929, 3179. HRMS (ESI): m/z = 335.1508 [M + H]⁺ calculated for C₁₉H₁₉N₄O₂, found: m/z = 335.1505.

N-(2-(5-Methoxy-2-(4-((7-nitrobenzo[c][1,2,5]oxadiazol-4-ylamino)methyl)phenyl)-1H-pyrrolo[3,2-b]pyridin-3-yl)ethyl)acetamide 13. A mixture of 3 (50 mg, 0.15 mmol, 1.08 eq.) and NBD-Cl (27 mg, 0.14 mmol, 1 eq.) in abs. EtOH was stirred 15 min at 70 °C in microwave. Thereafter the mixture was evaporated to dryness. The residue was purified by flash chromatography using a mixture of AcOEt/petroleum ether as eluent (1/1) to afford the expected compound as a brown solid (20 mg, 29% yield).

¹H NMR (400 MHz, MeOD): δ = 8.45 (d, J = 8.9 Hz, 1H), 7.68 (d, J = 8.2 Hz, 2H), 7.64 (d, J = 8.6 Hz, 1H), 7.55 (d, J = 8.2 Hz, 2H), 6.56 (d, J = 8.6 Hz, 1H), 6.30 (d, J = 8.4 Hz, 1H), 4.80 (bs, 2H), 3.97 (s, 3H), 3.53 (dd, J = 14.8, 6.1 Hz, 2H), 3.10 (t, J = 6.1 Hz, 2H), 1.80 (s, 3H). ¹³C NMR (101 MHz, MeOD): δ 171.8, 159.7, 144.9, 144.6, 144.0, 142.8, 137.1, 136.7, 136.1, 132.5, 132.4, 128.0, 127.5, 125.1, 122.5, 121.5, 109.2, 104.4, 99.1, 52.3, 46.4, 40.0, 23.3, 21.2. IR: ν_max (cm⁻¹) = 807, 1022, 1238, 1290, 1403, 1438, 1576, 2928, 3272. HRMS (ESI): m/z = 502.1839 [M + H]⁺ calculated for C₂₅H₂₄N₇O₅, found: m/z = 502.1830. Melting point: 126 °C.

N-(2-(5-Methoxy-2-(3-((7-nitrobenzo[c][1,2,5]oxadiazol-4-ylamino)methyl)phenyl)-1H-pyrrolo[3,2-b]pyridin-3-yl)ethyl)acetamide 14. A mixture of 4 (50 mg, 0.15 mmol, 1.08 eq.) and NBD-Cl (27 mg, 0.14 mmol, 1 eq.) in abs. EtOH was stirred 15 min at 70 °C in microwave. Thereafter the mixture was evaporated to dryness. The residue was purified by flash chromatography using a mixture of AcOEt/petroleum ether as eluent (1/1) to afford the expected compound as a brown solid (40 mg, 59% yield).

¹H NMR (400 MHz, CDCl₃): δ = 8.54, (s, 1H), 8.40–8.36 (m, 2H), 7.76 (s, 1H), 7.56 (d, J = 8.6 Hz, 1H), 7.45–7.25 (m, 3H), 6.83 (bs, 1H, NH), 6.59 (d, J = 8.6 Hz, 1H), 6.22 (d, J = 9.0 Hz, 1H), 4.77 (bs, 2H), 3.99 (s, 3H), 3.56 (dd, J = 15.2 and 6.1 Hz, 2H), 3.09 (t, J = 6.2 Hz, 2H), 2.05 (s, 3H). ¹³C NMR (101 MHz, CDCl₃): δ 171.4, 160.1, 144.4, 144.0, 143.2, 136.9, 136.5, 133.4, 129.5, 127.6, 127.3, 126.4, 124.8, 121.8, 110.9, 106.0, 53.4, 47.6, 41.2, 23.7, 23.4. IR: ν_max (cm⁻¹) = 804, 1023, 1047, 1112, 1252, 1287, 2227, 2392, 2924, 2959. HRMS (ESI): m/z = 502.1839 [M + H]⁺ calculated for C₂₅H₂₄N₇O₅, found: m/z = 502.1831. Melting point: 147 °C.

(Z)-N-(4-(3-(2-Acetamidoethyl)-5-methoxy-1H-pyrrolo[3,2-b]pyridin-2-yl)benzyl)-3-(1-(difluoroboryl)-5-((3,5-dimethyl-2H-pyrrol-2-ylidene)methyl)-1H-pyrrol-2-yl)propanamide 15. A mixture of 3 (30 mg, 88 μmol, 1 eq.), BODIPY-FL® (31 mg, 100 μmol, 1.2 eq.), DCC (22 mg, 100 μmol, 1.2 eq.) and DMAP (13 mg, 100 μmol, 1.2 eq.) in DCM (1.5 mL) was stirred 5 hours at room temperature. Thereafter, the mixture was evaporated to dryness. The residue was purified by flash chromatography using a step gradient of MeOH (0 to 5%) in DCM to afford the expected compound as an orange solid (20 mg, 37% yield).

¹H NMR (400 MHz, CDCl₃): δ = 8.70 (bs, 1H, NH), 7.63–7.58 (m, 2H), 7.37 (d, J = 7.9 Hz, 2H), 7.19 (d, J = 7.7 Hz, 2H), 7.10 (s, 1H), 6.86 (d, J = 3.9 Hz, 1H), 6.63 (d, J = 8.7 Hz, 1H), 6.43–6.21 (m, 2H), 6.10 (s, 1H), 4.40 (d, J = 5.7 Hz, 2H), 4.04 (s, 3H), 3.60 (d, J = 5.6 Hz, 2H), 3.29 (t, J = 7.3 Hz, 2H), 3.15–2.98 (m, 2H), 2.71 (t, J = 7.4 Hz, 2H), 2.53 (s, 3H), 2.22 (s, 3H), 1.90 (s, 3H). ¹³C NMR (101 MHz, CDCl₃): δ 171.8, 170.4, 160.4, 160.0, 156.9, 144.1, 143.2, 138.4, 137.6, 135.2, 133.4, 131.3, 128.2, 128.1, 128.0, 124.7, 123.9, 122.1, 120.5, 117.4, 110.9, 105.7, 53.4, 43.1, 42.2, 35.9, 24.8, 23.3, 22.8, 14.9, 11.3. IR: ν_max (cm⁻¹) = 750, 969, 1061, 1081, 1135, 1172, 1247, 1332, 1370, 1404, 1524, 1602, 1653. HRMS (ESI): m/z = 613.2910 [M + H]⁺ calculated for C₃₃H₃₆BF₂N₆O₃, found: m/z = 613.2903. Melting point: 78 °C.

(Z)-N-(3-(3-(2-Acetamidoethyl)-5-methoxy-1H-pyrrolo[3,2-b]pyridin-2-yl)benzyl)-3-(1-(difluoroboryl)-5-((3,5-dimethyl-2H-pyrrol-2-ylidene)methyl)-1H-pyrrol-2-yl)propanamide 16. A mixture of 4 (30 mg, 88 μmol, 1 eq.), BODIPY-FL® (31 mg, 100 μmol, 1.2 eq.), DCC (22 mg, 100 μmol, 1.2 eq.) and DMAP (13 mg, 100 μmol, 1.2 eq.) in DCM (1.5 mL) was stirred 5 hours at room temperature. Thereafter, the mixture was evaporated to dryness. The residue was purified by flash chromatography using a step gradient of MeOH (0 to 5%) in DCM to afford the expected compound as an orange solid (38 mg, 70% yield).

¹H NMR (400 MHz, CDCl₃): δ = 8.97 (s, 1H, NH), 7.57 (d, J = 8.7 Hz, 1H), 7.41 (s, 1H), 7.39–7.13 (m, 4H), 7.00 (bs, 1H, NH), 6.85 (s, 1H), 6.62 (d, J = 4.0 Hz, 1H), 6.60 (d, J = 8.7 Hz, 1H), 6.15 (d, J = 4.0 Hz, 1H), 6.08 (s, 1H), 4.40 (d, J = 6.0 Hz, 2H), 4.01 (s, 3H), 3.51 (dd, J = 11.8 and 6.2 Hz, 2H), 3.27 (t, J = 7.4 Hz, 2H), 3.02 (t, J = 6.7 Hz, 2H), 2.67 (t, J = 7.4 Hz, 2H), 2.49 (s, 3H), 2.17 (s, 3H), 1.87 (s, 3H). ¹³C NMR (101 MHz, CDCl₃): δ 172.1, 170.6, 160.0, 159.9, 157.4, 143.8, 143.1, 139.4, 137.6, 135.0, 133.3, 132.7, 128.9, 128.2, 127.3, 126.8, 126.4, 124.7, 123.7, 122.0, 120.3, 117.3, 110.6, 105.5, 53.3, 43.1, 41.6, 35.6, 24.8, 23.2, 23.2, 14.9, 11.2. IR: ν_max (cm⁻¹) = 969, 1082, 1134, 1173, 1245, 1405, 1430, 1526, 1602, 1648. HRMS (ESI): m/z = 613.2910 [M + H]⁺ calculated for C₃₃H₃₆BF₂N₆O₃, found: m/z = 613.2910. Melting point: 84 °C.

N-(2-(2-Cyano-5-methoxy-1H-pyrrolo[3,2-b]pyridin-3-yl)ethyl)acetamide 17. To a degassed solution of iodomelatonin 10 (573 mg, 1.60 mmol, 1.0 eq.) in THF (30 mL), KCN (311 mg, 4.80 mmol, 3.0 eq.) and Pd(PPh₃)₄ (184 mg, 0.16 mmol, 0.1 eq.) were added. The resulting mixture was stirred at reflux for 3 h and H₂O (10 mL) was added. The aqueous phase was extracted with EtOAc (3 × 20 mL) and the combined organic layers were washed with brine (30 mL), dried over MgSO₄ and concentrated under reduce pressure. Thereafter, the residue was purified by flash chromatography using a step gradient of MeOH (0 to 5%) in EtOAc to afford the expected compound as a white solid (531 mg, 93% yield).

¹H NMR (400 MHz, MeOD): δ 7.65 (d, J = 9.0 Hz, 1H), 6.76 (d, J = 8.9 Hz, 1H), 3.94 (s, 3H), 3.56 (t, 2H), 3.07 (t, J = 6.6 Hz, 2H), 1.87 (s, 3H). ¹³C NMR (101 MHz, MeOD): δ 171.9, 160.7, 139.0, 126.7, 123.5, 123.0, 113.5, 110.2, 106.2, 52.3, 39.0, 23.6, 21.3. IR: ν_max (cm⁻¹) = 3378, 3305, 2212, 1586, 1403, 1280, 1250, 1107, 1030, 807. HRMS (ESI): m/z = 259.1189 [M + H]⁺ calculated for C₁₃H₁₅N₄O₂, found: m/z = 259.1195. Melting point: 205 °C.

N-(2-(2-((5-(Dimethylamino)-1,3-dioxoisoindolin-2-yl)methyl)-5-methoxy-1H-pyrrolo[3,2-b]pyridin-3-yl)ethyl)acetamide 18. To a solution of 4-(dimethylamino)phthalic acid (90 mg, 0.43 mmol, 1.0 eq.) in THF (3 mL) was added carbonyldiimidazole (160 mg, 0.99 mmol, 2.2 eq.). The mixture was stirred to reflux for 30 min and a solution of amine 5 (111 mg, 0.43 mmol, 1.0 eq.) in THF (3 mL) was added. The resulting mixture was stirred 4 h at reflux and H₂O (10 mL) was added. The aqueous phase was extracted with EtOAc (3 × 20 mL) and the combined organic layers were washed with brine (30 mL), dried over MgSO₄ and concentrated under reduce pressure. Thereafter, the residue was purified by flash chromatography using a step gradient of MeOH (1 to 2%) in DCM to afford the expected compound as a yellow solid (22 mg, 12% yield).

¹H NMR (400 MHz, CDCl₃): δ 7.54 (d, J = 8.6 Hz, 1H), 7.47 (d, J = 8.8 Hz, 1H), 6.97 (d, J = 2.5 Hz, 1H), 6.79–6.64 (m, 1H), 6.51 (d, J = 8.7 Hz, 1H), 4.82 (d, J = 2.8 Hz, 2H), 3.90 (d, J = 2.7 Hz, 3H), 3.55–3.44 (m, 2H), 3.34 (s, 2H), 3.14–2.97 (m, 8H), 1.81 (s, 3H). ¹³C NMR (101 MHz, CDCl₃): δ 171.0, 169.1, 168.9, 159.8, 154.5, 141.6, 134.4, 132.7, 125.1, 124.4, 122.1, 116.6, 114.7, 111.5, 105.9, 105.7, 53.3, 41.4, 40.3, 32.4, 22.8, 22.1. IR: ν_max (cm⁻¹) = 1701, 1647, 1386, 1286, 1026, 819, 760. HRMS (ESI): m/z = 436.1979 [M + H]⁺ calculated for C₂₃H₂₆N₅O₄, found: m/z = 436.1978. Melting point: 261 °C.

Ethyl-(3-(2-acetamidoethyl)-5-methoxy-1H-pyrrolo[3,2-b]pyridin-2-yl)methylcarbamate 19. To a solution of cyanide 17 (190 mg, 0.74 mmol, 1.0 eq.) in THF (13 mL) was added LiAlH₄ (84 mg, 2.2 mmol, 3.0 eq.). The resulting mixture was stirred at reflux for 1 h and an aqueous solution of NaOH (1 N, 0.1 mL) was added. The mixture was then dried over MgSO₄ and concentrated under reduced pressure to dryness and the crude amine 5 (135 mg, 70% yield) was used in the next step without further purification. To a solution of amine 5 (111 mg, 0.43 mmol, 1.0 eq.) in DCM (20 mL) were added Et₃N (0.14 mL, 1.06 mmol, 2.5 eq.) and ethyl chloroformate (40 μL, 0.43 mmol, 1.0 eq.). The resulting mixture was stirred at room temperature for 3 h and H₂O (10 mL) was added. The aqueous phase was extracted with DCM (3 × 20 mL) and the combined organic layers were washed with brine (30 mL), dried over MgSO₄ and concentrated under reduce pressure. Thereafter the residue was purified by flash chromatography using AcOEt 100% to afford the expected compound as a white solid (50 mg, 58% yield).

¹H NMR (400 MHz, CDCl₃) δ 9.18 (s, 1H), 7.51 (d, J = 8.7 Hz, 1H), 7.28 (s, 1H), 6.59 (d, J = 8.7 Hz, 1H), 5.91 (s, 1H), 4.41 (d, J = 6.1 Hz, 2H), 4.14 (d, J = 7.1 Hz, 2H), 4.01 (s, 3H), 3.59–3.52 (m, 2H), 2.98 (t, J = 6.8, 5.4 Hz, 2H), 1.90 (s, 3H), 1.24 (t, J = 7.1 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 170.6, 159.8, 158.1, 142.0, 135.9, 124.2, 122.0, 110.3, 105.4, 61.3, 53.3, 41.6, 36.0, 23.2, 22.4, 14.6. IR: ν_max (cm⁻¹) = 3307, 1073, 1634, 1272, 1249, 1228, 1096, 1033, 795. HRMS (ESI): m/z = 335.1714 [M + H]⁺ calculated for C₁₆H₂₃N₄O₄, found: m/z = 335.1712. Melting point: 147 °C.

N-(2-(5-Methoxy-2-((methyl(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)methyl)-1H-pyrrolo[3,2-b]pyridin-3-yl)ethyl)acetamide 20. To a solution of carbamate 19 (50 mg, 0.15 mmol, 1.0 eq.) in THF (5 mL) was added LiAlH₄ (17 mg, 0.45 mmol, 3.0 eq.). The resulting mixture was stirred at 60 °C for 6 h and an aqueous solution of 1 N NaOH (0.1 mL) was added. The mixture was then dried over MgSO₄ and concentrated under reduce pressure to dryness and the crude methylamine was used in the next step without further purification. A mixture of methylamine (25 mg, 0.09 mmol, 1.0 eq.) and NBD-Cl (35 mg, 0.18 mmol, 2.0 eq.) in abs. EtOH (5 mL) was stirred at reflux for 2 h. Thereafter the mixture was evaporated to dryness and the residue was purified by flash chromatography using a step gradient of MeOH (1 to 5%) in DCM to afford the expected compound as an orange solid (10 mg, 24% yield over 2 steps).

¹H NMR (400 MHz, DMSO): δ 10.84 (s, 1H), 8.54 (d, J = 9.1 Hz, 1H), 7.92 (s, 1H), 7.54 (d, J = 8.6 Hz, 1H), 6.53 (dd, J = 9.0, 2.6 Hz, 2H), 5.66–5.33 (m, 2H), 3.88 (s, 3H), 3.60–3.20 (m, 5H), 2.90 (d, J = 7.2 Hz, 2H), 1.77 (s, 3H). ¹³C NMR (101 MHz, DMSO): δ 169.4, 169.4, 159.2, 146.8, 145.5, 145.3, 142.0, 136.9, 132.2, 125.0, 122.5, 121.4, 111.4, 105.3, 103.5, 53.0, 50.6, 23.7, 23.1, 23.1. IR: ν_max (cm⁻¹) = 1545, 1285, 1253, 1228, 1211, 1073, 1003, 820, 806. HRMS (ESI): m/z = 440.1677 [M + H]⁺ calculated for C₂₀H₂₂N₇O₅, found: m/z = 440.1675. Melting point: 234 °C.

Allyl-2-(5-nitropyridin-2-yloxy)ethylcarbamate 22. A solution of 2-chloro-5-nitropyridine 21 (500 mg, 3.15 mmol, 1 eq.) and N-(allyloxycarbonyl)ethanolamine (570 μL, 641 mg, 4.41 mmol, 1.40 eq.) in dry THF (12 mL) was cooled at 0 °C. NaH 60% in oil (176 mg, 4.41 mmol, 1.40 eq.) was then portionwise added and the resulting mixture was stirred 1 h at 0 °C. Thereafter, the mixture was hydrolysed with water and the pH was allowed to 2 with an aq. solution of HCl. The solution was extracted three times with AcOEt. The organic layers were dried over MgSO₄ and concentrated under reduced pressure. The residue was purified by flash chromatography with a step gradient of AcOEt (0 to 30%) in petroleum ether to give a yellow solid as the expected compound 22 (570 mg, 68% yield).

¹H NMR (400 MHz, CDCl₃): δ = 9.06 (d, J = 2.8 Hz, 1H), 8.37 (dd, J = 2.8 and 8.8 Hz, 1H), 6.84 (d, J = 8.8 Hz, 1H), 5.95–5.88 (m, 1H), 5.30 (d, J = 10.4 Hz, 1H), 5.22 (d, J = 10.4 Hz, 1H), 5.10 (bs, 1H), 4.58 (d, J = 10.8 Hz, 2H), 4.52 (t, J = 5.2 Hz, 2H), 3.63 (dd, J = 5.2 and 10.8 Hz, 2H). ¹³C NMR (101 MHz, CDCl₃): δ = 166.9, 156.4, 145.0, 140.5, 134.4, 132.9, 118.1, 111.5, 66.8, 66.0, 40.5. IR: ν_max (cm⁻¹) = 834, 947, 998, 1027, 1109, 1151, 1257, 1306, 1345, 1506, 1600, 1690, 1727, 2940, 3318. HRMS (ESI): m/z = 290.0753 [M + Na]⁺ calculated for C₁₁H₁₃N₃O₅Na, found: m/z = 290.0765.

Allyl-2-(5-aminopyridin-2-yloxy)ethylcarbamate 23. A 6 M aq. solution of HCl (3.5 mL) and iron powder (1.41 g) was added to a solution of nitropyridine 22 (450 mg, 1.68 mmol, 1 eq.) in abs. EtOH (10 mL). The resulting mixture was refluxed 1 h and then cooled at room temperature. The pH of the mixture was allowed to 8 with a saturated aqueous solution of Na₂CO₃ and the mixture was filtered through celite. EtOH of the filtrate was evaporated under reduced pressure and the residue was extracted three times with DCM. The organic layers were washed with brine, dried over MgSO₄ and concentrated to afford the expected compound 23 without any further purification as brown oil (335 mg, 84% yield).

¹H NMR (400 MHz, CDCl₃): δ = 7.61 (d, J = 3.2 Hz, 1H), 7.02 (dd, J = 3.2 and 8.8 Hz, 1H), 6.58 (d, J = 8.8 Hz, 1H), 5.95–5.88 (m, 1H), 5.32–5.28 (m, 2H), 5.20 (d, J = 10.4 Hz, 1H), 4.56 (d, J = 5.6 Hz, 2H), 4.29 (t, J = 4.8 Hz, 2H), 3.55 (dd, J = 5.2 and 10.4 Hz, 2H), 3.39 (ls, 2H). ¹³C NMR (101 MHz, CDCl₃): δ = 157.0, 156.1, 136.9, 132.7, 132.6, 127.4, 117.4, 110.6, 65.4, 64.6, 40.6. IR: ν_max (cm⁻¹) = 826, 927, 1055, 1150, 1245, 1418, 1489, 1528, 1700, 2944, 3338. HRMS (ESI): m/z = 238.1192 [M + H]⁺ calculated for C₁₁H₁₆N₃O₃, found: m/z = 238.1197.

Allyl-2-(5-hydrazinylpyridin-2-yloxy)ethylcarbamate 24. A solution of aminopyridine 23 (330 mg, 1.39 mmol, 1 eq.) in a 6 M aq. HCl solution (3.5 mL) was cooled at 0 °C and a solution of NaNO₂ (96 mg, 1.39 mmol, 1 eq.) in water (3.5 mL) was added dropwise. The resulting mixture was stirred 30 min at 0 °C. Thereafter a solution of SnCl₂·2H₂O (788 mg, 3.49 mmol, 2.5 eq.) in water (3.5 mL) was added dropwise at 0 °C. The mixture was stirred 3 h at 0 °C and then the pH of the mixture was allowed to 10 with a 40% mass. aq. solution of KOH. The resulting mixture was extracted three times with AcOEt. The organic layers were dried over MgSO₄ and evaporated to dryness to yield the expected compound 24 without any purification as a brown oil (295 mg, 84% yield).

¹H NMR (400 MHz, CDCl₃): δ = 7.74 (d, J = 3.2 Hz, 1H), 7.19 (dd, J = 3.2 and 8.8 Hz, 1H), 6.65 (d, J = 8.8 Hz, 1H), 5.92–5.87 (m, 1H), 5.25–5.18 (m, 3H), 4.56 (d, J = 8.4 Hz, 2H), 4.29 (t, J = 8.8 Hz, 2H) 3.59–3.52 (m, 4H).

Allyl-2-(3-(2-acetamidoethyl)-1H-pyrrolo[3,2-b]pyridin-5-yloxy)ethylcarbamate 25. A solution of hydrazine 24 (255 mg, 1.01 mmol, 1 eq.) and N-(4,4-diethoxybutyl)acetamide (250 mg, 1.22 mmol, 1.2 eq.) in 4% vol. aq. H₂SO₄ solution (5 mL) was refluxed for 3 h. After cooling at room temperature, the pH of the solution was allowed to 8 with a saturated aq. solution of NaHCO₃. The resulting mixture was extracted three times with AcOEt. The organic layers were washed with brine, dried over MgSO₄ and evaporated to dryness. The residue was purified by flash chromatography with a step gradient of MeOH (0 to 5%) in DCM to afford the expected compound 25 as yellow oil (100 mg, 40% yield).

¹H NMR (400 MHz, CDCl₃): δ = 8.72 (bs, 1H), 7.57 (d, J = 8.0 Hz, 1H), 7.13 (s, 1H), 6.86 (ls, 1H), 6.59 (d, J = 8.0 Hz, 1H), 5.92–5.88 (m, 1H), 5.64 (ls, 1H), 5.29 (d, J = 12.0 Hz, 1H), 5.19 (d, J = 12.0 Hz, 1H), 4.56 (d, J = 4.0 Hz, 2H), 4.46 (t, J = 4.0 Hz, 2H), 3.63–3.56 (m, 4H), 2.97 (t, J = 4.0 Hz, 2H), 1.91 (s, 3H). ¹³C NMR (101 MHz, CDCl₃): δ = 170.4, 159.2, 156.5, 141.7, 133.0, 125.4, 125.3, 122.5, 117.9, 113.6, 105.6, 65.7, 65.1, 41.3, 41.1, 24.2, 23.5. IR: ν_max (cm⁻¹) = 775, 804, 993, 1053, 1244, 1413, 1537, 1574, 1646, 1700, 2936, 3291. HRMS (ESI): m/z = 347.1719 [M + H]⁺ calculated for C₁₇H₂₃N₄O₄, found: m/z = 347.1706.

N-(2-(5-(2-(7-Nitrobenzo[c][1,2,5]oxadiazol-4-ylamino)ethoxy)-1H-pyrrolo[3,2-b]pyridin-3-yl)ethyl)acetamide 26. A solution of the amine 6 (20 mg, 76 μmol, 1.08 eq.) and NBD-Cl (14 mg, 70 μmol, 1 eq.) in abs. ethanol (1 mL) was irradiated in microwave 15 min at 70 °C. Thereafter, 100 μL of Et₃N was added and the mixture stirred 15 min at room temperature. After concentration under reduced pressure, the residue was purified by flash chromatography with a step gradient of MeOH (0 to 5%) in DCM to afford the expected compound as a yellow solid (10 mg, 33% yield).

¹H NMR (400 MHz, DMSO): δ = 10.89 (bs, 1H, NH), 9.64 (bs, 1H, NH), 8.51 (d, J = 8.5 Hz, 1H), 7.89 (bs, 1H, NH), 7.64 (d, J = 8.7 Hz, 1H), 7.31 (s, 1H), 6.56–6.52 (m, 2H), 4.60 (t, J = 5.7 Hz, 2H), 3.34–3.30 (m, 4H), 2.79 (t, J = 6.5 Hz, 2H), 1.78 (s, 3H). ¹³C NMR (101 MHz, DMSO): δ 169.4, 158.2, 145.8, 144.9, 144.4, 141.4, 138.2, 126.1, 125.2, 122.9, 121.4, 112.0, 104.8, 100.0, 62.8, 46.2, 43.4, 24.7, 23.1. IR: ν_max (cm⁻¹) = 1015, 1145, 1241, 1268, 1413, 1531, 1583, 1624, 2920, 3399. HRMS (ESI): m/z = 426.1526 [M + H]⁺ calculated for C₁₉H₂₀N₇O₅, found: m/z = 426.1534. Melting point: 223 °C.

(Z)-N-(2-(3-(2-Acetamidoethyl)-1H-pyrrolo[3,2-b]pyridin-5-yloxy)ethyl)-3-(1-(difluoroboryl)-5-((3,5-dimethyl-2H-pyrrol-2-ylidene)methyl)-1H-pyrrol-2-yl)propanamide 27. Dry DIEA (6 μL, 4.4 mg, 34 μmol, 2 eq.) was added to a solution of BODIPY FL® (5 mg, 17 μmol, 1 eq.) and TSTU (5 mg, 17 μmol, 1 eq.) in dry DMF (500 μL). The resulting solution was protected from the light and stirred 2 h at room temperature under an atmosphere of argon. Thereafter a solution of the amine 6 (14 mg, 51 μmol, 3 eq.) in dry DMF (500 μL) was added and the mixture was stirred overnight at room temperature. After removing of the DMF, the residue was purified by flash chromatography with a step gradient of MeOH (0 to 5%) in DCM as eluent to afford an orange solid (3 mg, 33% yield).

¹H NMR (400 MHz, CDCl₃): δ = 7.97 (bs, 1H, NH), 7.54 (d, J = 8.7 Hz, 1H), 7.16 (s, 1H), 6.98 (s, 1H), 6.82–6.78 (m, 2H), 6.56 (d, J = 8.7 Hz, 1H), 6.36 (bs, 1H, NH), 6.27 (d, J = 3.9 Hz, 1H), 6.10 (s, 1H), 4.40 (t, J = 5.4 Hz, 2H), 3.68–3.64 (m, 2H), 3.59–3.55 (m, 2H), 3.27 (t, J = 7.4 Hz, 2H), 2.97 (t, J = 6.4 Hz, 2H), 2.65 (t, J = 7.4 Hz, 2H), 2.54 (s, 3H), 2.22 (s, 3H), 1.89 (s, 3H). ¹³C NMR (101 MHz, CDCl₃): δ 171.9, 170.2, 160.0, 159.1, 157.4, 143.8, 141.6, 135.0, 133.4, 128.3, 125.0, 125.0, 123.7, 122.1, 120.3, 117.5, 113.7, 105.6, 64.6, 41.0, 39.2, 35.9, 24.8, 24.2, 23.3, 14.9, 11.3. IR: ν_max (cm⁻¹) = 973, 1085, 1137, 1176, 1252, 1414, 1606, 2923, 3291. HRMS (ESI): m/z = 537.2597 [M + H]⁺ calculated for C₂₇H₃₂BN₆O₃F₂, found: m/z = 537.2606. Melting point: 99 °C.

N-(2-(5-(2-(5-(Dimethylamino)naphthalene-1-sulfonamido)ethoxy)-1H-pyrrolo[3,2-b]pyridin-3-yl)ethyl)acetamide 28. To a solution of amine 6 (50 mg, 0.19 mmol, 1 eq.) in 6 mL of a mixture of CH₃OH–CH₃CN (1/5) were added dansyl-chloride (60 mg, 0.22 mmol, 1.2 eq.) and Et₃N (0.1 mL, 0.72 mmol, 3.7 eq.). The resulting mixture was stirred 3 h at room temperature. Thereafter, the mixture was evaporated to dryness and the residue was purified by flash chromatography using a step gradient of EtOAc (50 to 80%) in petroleum ether to afford the expected compound as a white solid (62 mg, 66% yield).

¹H NMR (250 MHz, CDCl₃): δ 9.03 (d, J = 2.8 Hz, 1H), 8.49 (d, J = 8.5 Hz, 1H), 8.23 (dd, J = 7.3, 1.3 Hz, 1H), 8.15 (d, J = 8.6 Hz, 1H), 7.83 (s, 1H), 7.59–7.42 (m, 2H), 7.19–6.95 (m, 3H), 6.76 (s, 1H), 6.42 (d, J = 8.7 Hz, 1H), 4.47–4.34 (m, 2H), 3.60 (s, 2H), 3.41–3.21 (m, 2H), 3.07 (d, J = 7.3 Hz, 2H), 2.82 (s, 6H), 1.85 (s, 3H). ¹³C NMR (63 MHz, CDCl₃): δ 170.6, 159.1, 151.8, 141.1, 134.5, 130.4, 129.8, 129.5, 129.4, 127.8, 125.7, 125.4, 123.1, 122.7, 118.7, 115.0, 112.7, 105.6, 65.9, 45.4, 44.4, 40.6, 24.3, 23.4. IR: ν_max (cm⁻¹) = 3270, 2937, 1651, 1574, 1412, 1307, 1248, 1199, 1092, 788, 681. HRMS (ESI): m/z = 495, 1940 [M + H]⁺ calculated for C₂₅H₃₀N₅O₄S, found: m/z = 495, 1946. Melting point: 87 °C.

5-(3-(2-(3-(2-Acetamidoethyl)-1H-pyrrolo[3,2-b]pyridin-5-yloxy)ethyl)thioureido)-2-(3-hydroxy-6-oxo-6H-xanthen-9-yl)benzoic acid 29. To a solution of amine 6 (50 mg, 0.19 mmol, 1.5 eq.) in 6 mL of a mixture of CH₃OH–CH₃CN (1/5) were added FITC (50 mg, 0.13 mmol, 1 eq.) and DIEA (0.1 mL, 0.57 mmol, 4.4 eq.). The resulting mixture was stirred 12 h at room temperature in the dark. Thereafter, the mixture was evaporated to dryness and the residue was purified by flash chromatography using a step gradient of MeOH (5 to 10%) in DCM to afford the expected compound as an orange solid (42 mg, 50% yield).

¹H NMR (400 MHz, MeOD): δ 8.08 (d, J = 2.0 Hz, 1H), 7.67 (s, 1H), 7.60 (s, 1H), 7.20 (s, 1H), 7.08 (d, J = 8.1 Hz, 1H), 6.88 (d, J = 8.9 Hz, 2H), 6.72–6.48 (m, 5H), 4.59 (d, J = 5.4 Hz, 1H), 4.05 (s, 2H), 3.54–3.48 (m, 2H), 2.96–2.92 (m, 2H), 1.91 (s, 3H). ¹³C NMR (101 MHz, MeOD): δ 181.5, 171.8, 170.5, 158.7, 154.9, 141.4, 130.0, 125.4, 125.3, 122.0, 115.7, 112.0, 111.9, 104.3, 102.3, 63.6, 44.0, 40.1, 23.8, 21.4. IR: ν_max (cm⁻¹) = 3250, 2923, 1573, 1432, 1289, 1205, 1106, 911, 789, 670. HRMS (ESI): m/z = 652.1860 [M + H]⁺ calculated for C₃₄H₃₀N₅O₇S, found: m/z = 652.1859. Melting point: 249 °C.

N-(2-(5-(2-(5-(Dimethylamino)-1,3-dioxoisoindolin-2-yl)ethoxy)-1H-pyrrolo[3,2-b]pyridin-3-yl)ethyl)acetamide 30. To a solution of 4-(dimethylamino)phthalic acid (40 mg, 0.19 mmol, 1.0 eq.) in THF (2 mL) was added carbonyldiimidazole (70 mg, 0.43 mmol, 2.2 eq.). The mixture was stirred to reflux for 30 min and a solution of amine 6 (50 mg, 0.19 mmol, 1.0 eq.) in THF (2 mL) was added. The resulting mixture was stirred 4 h at reflux and H₂O (10 mL) was added. The aqueous phase was extracted with EtOAc (3 × 20 mL) and the combined organic layers were washed with brine (30 mL), dried over MgSO₄ and concentrated under reduce pressure. Thereafter, the residue was purified by flash chromatography using a step gradient of MeOH (2 to 5%) in DCM to afford the expected compound as a yellow solid (35 mg, 42% yield).

¹H NMR (400 MHz, CDCl₃): δ 8.50 (s, 1H), 7.62 (d, J = 8.5 Hz, 1H), 7.51 (d, J = 8.7 Hz, 1H), 7.17–7.00 (m, 3H), 6.77 (dd, J = 8.5, 2.4 Hz, 1H), 6.55 (d, J = 8.7 Hz, 1H), 4.60 (t, J = 6.1 Hz, 2H), 4.11 (t, J = 6.1 Hz, 2H), 3.62–3.47 (m, 2H), 3.09 (s, 6H), 2.94 (t, J = 6.4 Hz, 2H), 1.91 (s, 3H). ¹³C NMR (101 MHz, CDCl₃): δ 170.4, 169.1, 168.7, 158.9, 154.3, 141.6, 134.7, 125.0, 125.0, 124.8, 122.2, 117.6, 114.6, 113.8, 105.8, 105.7, 62.7, 41.4, 40.4, 37.0, 29.7, 24.1, 23.4. IR: ν_max (cm⁻¹) = 3271, 2918, 1754, 1697, 1651, 1574, 1413, 1282, 1196, 1084, 1015, 971, 775. HRMS (ESI): m/z = 436.1979 [M + H]⁺ calculated for C₂₃H₂₆N₅O₄, found: m/z = 436.1977. Melting point: 79 °C.

tert-Butyl-4-(2-(3-(2-acetamidoethyl)-1H-pyrrolo[3,2-b]pyridin-5-yloxy)ethylcarbamoyl)piperidine-1-carboxylate 31a. To a solution of amine 6 (100 mg, 0.38 mmol, 1.0 eq.) in 6 mL of a mixture of CH₃OH–CH₃CN (1/5) were added 1-(tert-butoxycarbonyl)piperidine-4-carboxylic acid (88 mg, 0.38 mmol, 1.0 eq.), BOP reagent (168 mg, 0.38 mmol, 1.0 eq.) and DIEA (0.2 mL, 1.14 mmol, 3.0 eq.). The resulting mixture was stirred 12 h at room temperature. Thereafter, the mixture was evaporated to dryness and the residue was purified by flash chromatography using a step gradient of MeOH (5 to 10%) in DCM to afford the expected compound as a colorless oil (90 mg, 50% yield).

¹H NMR (400 MHz, CDCl₃): δ 8.81 (s, 1H), 7.57 (d, J = 8.7 Hz, 1H), 7.13 (d, J = 2.6 Hz, 1H), 6.81 (d, J = 4.6 Hz, 1H), 6.56 (d, J = 8.7 Hz, 1H), 6.49 (s, 1H), 4.45 (t, J = 5.4 Hz, 2H), 4.10 (d, J = 7.2 Hz, 2H), 3.68 (d, J = 5.5 Hz, 2H), 3.57 (d, J = 6.1 Hz, 2H), 2.94 (t, J = 6.7 Hz, 2H), 2.69 (s, 2H), 2.24 (s, 1H), 1.91 (s, 3H), 1.73 (d, J = 3.6 Hz, 2H), 1.61 (d, J = 4.4 Hz, 2H), 1.43 (s, 9H). ¹³C NMR (101 MHz, CDCl₃): δ 174.8, 170.3, 159.0, 154.7, 141.6, 125.3, 125.2, 122.4, 113.1, 105.2, 79.7, 64.5, 43.2, 40.9, 39.2, 28.6, 28.4, 24.3, 23.4. IR: ν_max (cm⁻¹) = 3294, 2938, 1652, 1532, 1442, 1364, 1254, 1163, 1132, 1024, 731. HRMS (ESI): m/z = 474.2711 [M + H]⁺ calculated for C₂₄H₃₆N₅O₅, found: m/z = 474.2713.

(Z)-N-(2-(3-(2-Acetamidoethyl)-1H-pyrrolo[3,2-b]pyridin-5-yloxy)ethyl)-1-(2-(3-(ethylamino)-6-(ethylimino)-2,7-dimethyl-6H-xanthen-9-yl)benzoyl)piperidine-4-carboxamide 32. To a solution of 31a (90 mg, 0.19 mmol, 1.0 eq.) in 10 mL of DCM was added trifluoroacetic acid (1 mL). The resulting mixture was stirred 2 h at room temperature. Thereafter, the mixture was evaporated to dryness. The crude amine 31 was used in the next step without further purification. To a solution of amine 31 (35 mg, 0.09 mmol, 1.0 eq.) in DMF (2 mL) were added R6G-acid (40 mg, 0.09 mmol, 1.0 eq.), HBTU (40 mg, 0.10 mmol, 1.1 eq.) and DIEA (0.1 mL, 0.57 mmol, 5.7 eq.). The resulting mixture was stirred 12 h at room temperature and HCl 1 N (10 mL) was added. The aqueous phase was extracted with DCM/iPrOH (1/1) (3 × 20 mL) and the combined organic layers were washed with brine (30 mL), dried over MgSO₄ and concentrated under reduce pressure. Thereafter, the residue was purified by flash chromatography using a step gradient of MeOH (10 to 20%) in DCM to afford the expected compound as a red solid (30 mg, 41% yield over 2 steps).

¹H NMR (400 MHz, MeOD): δ 8.06 (s, 1H), 7.82–7.68 (m, 2H), 7.67–7.55 (m, 2H), 7.53–7.41 (m, 1H), 7.22 (s, 1H), 7.00 (d, J = 1.3 Hz, 2H), 6.87 (s, 2H), 6.53 (d, J = 8.7 Hz, 1H), 4.38 (t, J = 5.6 Hz, 2H), 4.15 (d, J = 13.0 Hz, 1H), 3.81 (d, J = 13.2 Hz, 1H), 3.62–3.41 (m, 8H), 2.92 (s, 3H), 2.54 (s, 1H), 2.36 (t, J = 3.8 Hz, 1H), 2.19–2.12 (m, 6H), 1.90 (s, 3H), 1.59 (s, 2H), 1.38–1.28 (m, 8H). ¹³C NMR (101 MHz, MeOD): δ 175.5, 171.7, 167.8, 158.7, 157.5, 156.4, 156.3, 154.9, 141.3, 135.6, 131.0, 130.2, 129.7, 129.6, 127.3, 125.3, 125.3, 125.1, 121.9, 113.5, 111.7, 104.1, 93.6, 63.7, 41.9, 40.9, 40.0, 38.8, 38.1, 28.6, 27.9, 23.8, 21.3, 16.3, 16.1, 12.6. IR: ν_max (cm⁻¹) = 3249, 1604, 1558, 1366, 1242, 1180, 1126, 1020, 798, 673. HRMS (ESI): m/z = 770.4024 [M + H]⁺ calculated for C₄₅H₅₂N₇O₅, found: m/z = 770.4019. Melting point: 190 °C.

N-(2-(1-(2-Bromoethyl)-5-methoxy-1H-pyrrolo[3,2-b]pyridin-3-yl)ethyl)acetamide 33. To a solution of 4-azamelatonine 1 (1.0 g, 4.30 mmol, 1 eq.) and tetra-n-butylammonium iodide (95 mg, 0.25 mmol, 0.05 eq.) in 1,2-dibromoethane (12 mL) was added an aqueous solution of NaOH (30 mL, 50% w/w) at 0 °C. The mixture was then stirred 2 h at room temperature. After water (30 mL) was added and the resulting mixture was extracted three times with DCM. The organic layers were washed with brine, dried over MgSO₄ and evaporated to dryness. The residue was purified by flash chromatography with a step gradient step of AcOEt (50 to 100%) in petroleum ether to afford the expected compound 33 as a white solid (950 mg, 65% yield).

¹H NMR (250 MHz, CDCl₃): δ = 7.56 (d, J = 8.8 Hz, 1H), 7.18 (bs, 1H, NH), 7.09 (s, 1H), 6.65 (d, J = 8.8 Hz, 1H), 4.44 (t, J = 6.6 Hz, 2H), 4.02 (s, 3H), 3.53–3.63 (m, 4H), 2.97 (t, J = 6.0 Hz, 2H), 1.93 (s, 3H). ¹³C NMR (62.5 MHz, CDCl₃): δ = 170.1, 160.0, 142.2, 128.2, 125.3, 120.3, 113.7, 105.7, 53.5, 48.3, 41.5, 30.2, 24.1, 23.5, IR: ν_max (cm⁻¹) = 2940, 1636, 1560, 1433, 1249, 1022. Melting point: 97 °C.

N-(2-(1-(2-Azidoethyl)-5-méthoxy-1H-pyrrolo[3,2-b]pyridin-3-yl)ethyl)acetamide 33a. A solution of compound 33 (400 mg, 1.17 mmol, 1 eq.) and sodium azide (459 mg, 7.02 mmol, 6 eq.) in a mixture of dioxane/water (20 mL/10 mL) was stirred 48 h at reflux. After, water (30 mL) was added and the resulting mixture was extracted three times with EtOAc. The organic layers were washed with brine, dried over MgSO₄ and evaporated to dryness. The product 33a was obtained without purification as brown oil (353 mg, 99%).

¹H NMR (250 MHz, CDCl₃): δ = 7.56 (d, J = 8.8 Hz, 1H), 7.10 (bs, 1H, NH), 7.08 (s, 1H), 6.65 (d, J = 8.8 Hz, 1H), 4.21 (t, J = 5.6 Hz, 2H), 4.02 (s, 3H), 3.55–3.64 (m, 4H), 2.98 (t, J = 6.0 Hz, 2H), 1.93 (s, 3H). ¹³C NMR (CDCl₃, 62.5 MHz): δ = 170.2, 160.1, 142.3, 128.1, 125.5, 120.4, 114.1, 105.8, 53.5, 51.6, 46.1, 41.5, 24.1, 23.5. IR: ν_max (cm⁻¹) = 3297, 2943, 2145, 1636, 1562, 1436, 1249. MS (ESI) m/z = 303.0 (M + H)⁺.

N-(2-(5-Methoxy-1-(2-(7-nitrobenzo[c][1,2,5]oxadiazol-4-ylamino)ethyl)-1H-pyrrolo[3,2-b]pyridin-3-yl)ethyl)acetamide 34. A solution of the amine 7 (70 mg, 0.25 mmol, 1.2 eq.) and NBD-Cl (42 mg, 0.21 mmol, 1 eq.) in abs. ethanol (3 mL) was irradiated in microwave 15 min at 70 °C. Thereafter, the precipitate was filtered and washed with cold EtOH to afford the expected compound 34 as a yellow solid (40 mg, 43% yield).

¹H NMR (400 MHz, DMSO): δ = 9.33 (bs, 1H), 8.35 (d, J = 8.3 Hz, 1H), 7.87 (bs, 1H), 7.73 (d, J = 8.8 Hz, 1H), 7.34 (s, 1H), 6.43 (d, J = 8.0 Hz, 1H), 6.20 (d, J = 8.4 Hz, 1H), 4.42 (t, J = 5.6 Hz, 2H), 3.83 (s, 3H), 3.33–3.28 (m, 4H), 2.72 (t, J = 7.4 Hz, 2H), 1.77 (s, 3H). ¹³C NMR (101 MHz, DMSO): δ 169.4, 159.1, 145.3, 144.8, 144.3, 141.8, 138.0, 129.1, 125.6, 121.4, 121.2, 112.4, 104.7, 99.6, 53.0, 44.9, 44.3, 39.5, 24.5, 23.1. IR: ν_max (cm⁻¹) = 796, 1027, 1116, 1260, 1437, 1489, 1535, 1586, 1623, 1654, 2944, 3258. HRMS (ESI): m/z = 440.1682 [M + H]⁺ calculated for C₂₀H₂₂N₇O₅, found: m/z = 440.1699. Melting point: 260 °C.

(Z)-N-(2-(3-(2-Acetamidoethyl)-5-methoxy-1H-pyrrolo[3,2-b]pyridin-1-yl)ethyl)-3-(1-(difluoroboryl)-5-((3,5-dimethyl-2H-pyrrol-2-ylidene)methyl)-1H-pyrrol-2-yl)propanamide 35. Dry DIEA (6 μL, 4.4 mg, 34 μmol, 2 eq.) was added to a solution of BODIPY FL® (5 mg, 17 μmol, 1 eq.) and TSTU (5 mg, 17 μmol, 1 eq.) in dry DMF (500 μL). The resulting solution was protected from the light and stirred 2 h at room temperature under an atmosphere of argon. Thereafter a solution of the amine 7 (14 mg, 51 μmol, 3 eq.) in dry DMF (500 μL) was added and the mixture was stirred overnight at room temperature. After removing of the DMF, the residue was purified by flash chromatography with 0 to 5% of MeOH in DCM as eluent to afford an orange solid (3 mg, 33% yield).

¹H NMR (400 MHz, CDCl₃): δ = 7.53 (d, J = 8.8 Hz, 1H), 7.16 (bs, 1H, NH), 7.08 (s, 1H), 6.94–6.80 (m, 2H), 6.60 (d, J = 8.8 Hz, 1H), 6.26 (d, J = 4.0 Hz, 1H), 6.14 (s, 1H), 5.84 (bs, 1H, NH), 4.08 (t, J = 5.9 Hz, 2H), 4.00 (s, 3H), 3.53–3.48 (m, 4H), 3.23 (t, 7.3 Hz, 2H), 2.86 (t, J = 7.0 Hz, 2H), 2.59–2.56 (m, 5H), 2.27 (s, 3H), 1.87 (s, 3H). ¹³C NMR (101 MHz, CDCl₃): δ 172.2, 170.1, 160.6, 159.7, 156.8, 144.2, 141.9, 135.2, 133.2, 128.1, 128.1, 128.1, 125.5, 123.8, 120.6, 120.5, 117.3, 113.2, 105.3, 53.3, 45.9, 41.6, 40.0, 35.6, 24.6, 23.9, 23.3, 14.9, 11.3. IR: ν_max (cm⁻¹) = 727, 970, 1080, 1132, 1174, 1250, 1424, 1486, 1529, 1601, 1650, 2360, 2930, 3287. HRMS (ESI): m/z = 551.2754 [M + H]⁺ calculated for C₂₈H₃₄BN₆O₃F₂, found: m/z = 551.2758. Melting point: 86 °C.

5-(3-(2-(3-(2-Acetamidoethyl)-5-methoxy-1H-pyrrolo[3,2-b]pyridin-1-yl)ethyl)thioureido)-2-(3-hydroxy-6-oxo-6H-xanthen-9-yl)benzoic acid 36. To a solution of amine 7 (50 mg, 0.21 mmol, 1.5 eq.) in 6 mL of a mixture of CH₃OH–CH₃CN (1/5) were added FITC (55 mg, 0.14 mmol, 1 eq.) and DIEA (0.1 mL, 0.57 mmol, 4.0 eq.). The resulting mixture was stirred 12 h at room temperature and in the dark. Thereafter, the mixture was evaporated to dryness and the residue was purified by flash chromatography using a step gradient of MeOH (5 to 10%) in DCM to afford the expected compound as an orange solid (52 mg, 56% yield).

¹H NMR (400 MHz, MeOD): δ 8.07 (s, 1H), 7.87 (d, J = 8.8 Hz, 1H), 7.55 (d, J = 8.3 Hz, 1H), 7.30 (s, 1H), 7.08 (d, J = 8.2 Hz, 1H), 6.74–6.49 (m, 8H), 4.45 (d, J = 6.1 Hz, 2H), 3.99 (t, J = 6.2 Hz, 2H), 3.94 (s, 3H), 3.52 (t, J = 7.0 Hz, 2H), 2.95 (t, J = 6.9 Hz, 2H), 1.90 (s, 3H). ¹³C NMR (101 MHz, MeOD): δ 181.9, 171.7, 169.8, 159.7, 152.9, 141.8, 140.6, 130.5, 130.0, 128.4, 126.1, 124.4, 120.7, 119.1, 112.5, 112.1, 110.2, 103.9, 102.1, 52.5, 44.6, 44.2, 40.1, 23.6, 21.4. IR: ν_max (cm⁻¹) = 3423, 2928, 1739, 1605, 1573, 1434, 1298, 1207, 1175, 1109, 1074, 848, 671. HRMS (ESI): m/z = 666.2017 [M + H]⁺ calculated for C₃₅H₃₁N₅O₇S, found: m/z = 666.2014. Melting point: 291 °C.

(Z)-N-(2-(3-(2-Acetamidoethyl)-5-methoxy-1H-pyrrolo[3,2-b]pyridin-1-yl)ethyl)-1-(2-(3-(ethylamino)-6-(ethylimino)-2,7-dimethyl-6H-xanthen-9-yl)benzoyl)piperidine-4-carboxamide 37. To a solution of amine 6 (100 mg, 0.36 mmol, 1.0 eq.) in 6 mL of a mixture of CH₃OH–CH₃CN (1/5) were added 1-(tert-butoxycarbonyl)piperidine-4-carboxylic acid (83 mg, 0.36 mmol, 1.0 eq.), PyBOP reagent (187 mg, 0.36 mmol, 1.0 eq.) and DIEA (0.19 mL, 1.08 mmol, 3.0 eq.). The resulting mixture was stirred 12 h at room temperature. Thereafter, the mixture was evaporated to dryness and the residue was purified by flash chromatography using a step gradient of MeOH (5 to 10%) in DCM to afford the carbamate protected compound as colorless oil (80 mg, 46% yield).

¹H NMR (400 MHz, CDCl₃): δ 7.56 (d, J = 8.8 Hz, 1H), 7.07 (s, 1H, NH), 7.03 (s, 1H), 6.63 (d, J = 8.8 Hz, 1H), 5.90 (s, 1H, NH), 4.21 (t, J = 5.7 Hz, 2H), 4.09 (d, J = 12.0 Hz, 2H), 4.02 (s, 3H), 3.56 (dd, J = 12.7, 6.2 Hz, 4H), 2.96 (t, J = 6.0 Hz, 2H), 2.69–2.65 (m, 1H), 2.21–2.09 (m, 1H), 1.84 (s, 3H), 1.71–1.60 (m, 2H), 1.60–1.50 (m, 2H), 1.46 (s, 9H), 1.34–1.21 (m, 1H). ¹³C NMR (101 MHz, CDCl₃): δ 175.0, 170.3, 159.8, 154.6, 141.9, 127.9, 125.7, 120.5, 113.4, 105.3, 79.7, 53.4, 45.7, 43.0, 41.6, 40.2, 28.4, 28.4, 24.0, 23.3. IR: ν_max (cm⁻¹) = 3304, 2941, 1651, 1541, 1423, 1365, 1256, 1163, 1128, 1027, 732. HRMS (ESI): m/z = 488.2867 [M + H]⁺ calculated for C₂₅H₃₈N₅O₅, found: m/z = 488.2864.

To a solution of the previous carbamate (60 mg, 0.12 mmol, 1.0 eq.) in 10 mL of DCM was added trifluoroacetic acid (1 mL). The resulting mixture was stirred 2 h at room temperature. Thereafter, the mixture was evaporated to dryness. The crude amine was used in the next step without further purification. To a solution of this amine (45 mg, 0.12 mmol, 1.0 eq.) in DMF (2 mL) were added R6G-acid (40 mg, 0.09 mmol, 1.0 eq.), HBTU (44 mg, 0.12 mmol, 1.1 eq.) and DIEA (0.1 mL, 0.57 mmol, 4.7 eq.). The resulting mixture was stirred 12 h at room temperature and HCl 1 N (10 mL) was added. The aqueous phase was extracted with DCM/iPrOH (1/1) (3 × 20 mL) and the combined organic layers were washed with brine (30 mL), dried over MgSO₄ and concentrated under reduce pressure to dryness and the residue was purified by flash chromatography using a step gradient of MeOH (10 to 20%) in DCM to afford the expected compound as a pink solid (28 mg, 30% yield over 2 steps).

¹H NMR (400 MHz, MeOD): δ 7.75–7.73 (m, 2H), 7.65–7.56 (m, 2H), 7.43–7.40 (m, 1H), 7.13 (s, 1H), 6.98 (s, 2H), 6.85 (s, 2H), 6.56 (d, J = 8.7 Hz, 1H), 4.18 (t, J = 5.6 Hz, 2H), 4.15 (d, J = 13.0 Hz, 1H), 3.93 (s, 3H), 3.72–3.69 (m, 3H), 3.53–3.45 (m, 8H), 3.22–3.15 (m, 2H), 2.92–2.88 (m, 2H), 2.48–2.42 (m, 1H), 2.16 (s, 6H), 1.90 (s, 3H), 1.43–1.39 (m, 2H), 1.38–1.35 (m, 9H). ¹³C NMR (101 MHz, MeOD): δ. 175.3, 171.7, 167.8, 159.6, 157.5, 156.4, 156.3, 154.9, 141.7, 135.5, 130.9, 130.2, 129.8, 129.7, 128.4, 127.3, 126.0, 120.5, 113.6, 112.0, 104.0, 93.7, 54.5, 52.7, 45.0, 42.5, 41.8, 41.0, 10.2, 39.6, 38.2, 29.4, 28.4, 27.7, 23.7, 21.7, 17.6, 16.1, 12.9. IR: ν_max (cm⁻¹) = 3422, 1650, 1605, 1529, 1498, 1434, 1302, 1184, 1021, 834, 739. HRMS (ESI): m/z = 784.4181 [M + H]⁺ calculated for C₄₆H₅₄N₇O₅, found: m/z = 784.4173. Melting point: 226 °C.

Biological assays

2-[¹²⁵I]-Iodomelatonin binding assay conditions were essentially as previously described.¹⁸ Briefly, binding was initiated by addition of membrane preparations from stable CHO cells expressing human MT1 or MT2 receptors (4 μg mL⁻¹) diluted in binding buffer (50 mM Tris HCl buffer, pH 7.4 containing 5 mM MgCl₂) to 2-[¹²⁵I]-iodomelatonin (0.025 and 0.2 nM respectively for MT1 and MT2 receptors due to a MT1/MT2 ratio of approximately 0.125 for cold 2-iodomelatonin) and the tested drug. Nonspecific binding was defined in the presence of 1 μM melatonin. After a 120 min incubation at 37 °C, reaction was stopped by rapid filtration through GF/B filters presoaked in 0.5% (v/v) polyethylenimine. Filters were washed three times with 1 mL of ice-cold 50 mM Tris HCl buffer, pH 7.4. Data from the dose response curves (seven concentrations in duplicate) were analyzed using the program PRISM (Graph Pad Software Inc., San Diego, CA, USA) to yield IC₅₀ (inhibitory concentration 50). Results are expressed as Ki = IC₅₀/1 + ([L]/KD), where [L] is the concentration of radioligand used in the assay and KD, the dissociation constant of the radioligand characterizing the membrane preparation.

Acknowledgements

This work was supported by La Région Centre (SP; SM, SD, APR2009-LOIREMEL; GV, CL, APR2012-LIFERMEL) and the Labex SynOrg (ANR-11-LABX-0029).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra10812a

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