New positive allosteric modulators of the metabotropic glutamate receptor 2 (mGluR2). Identification and synthesis of N-propyl-5 -substituted isoquinolones

Andrés A. Trabanco; Guillaume Duvey; José María Cid; Gregor J. Macdonald; Philippe Cluzeau; Vanthea Nhem; Rocco Furnari; Nadia Behaj; Géraldine Poulain; Terry Finn; Sonia Poli; Hilde Lavreysen; Alexandre Raux; Yves Thollon; Nicolas Poirier; David D'Addona; José Ignacio Andrés; Robert Lutjens; Emmanuel Le Poul; Hassan Imogai; Jean-Philippe Rocher

doi:10.1039/C0MD00200C

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DOI: 10.1039/C0MD00200C (Concise Article) Med. Chem. Commun., 2011, 2, 132-139

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New positive allosteric modulators of the metabotropic glutamate receptor 2 (mGluR2). Identification and synthesis of N-propyl-5-substituted isoquinolones

Andrés A. Trabanco *^a, Guillaume Duvey ^b, José María Cid ^a, Gregor J. Macdonald ^c, Philippe Cluzeau ^b, Vanthea Nhem ^b, Rocco Furnari ^b, Nadia Behaj ^b, Géraldine Poulain ^b, Terry Finn ^b, Sonia Poli ^b, Hilde Lavreysen ^d, Alexandre Raux ^b, Yves Thollon ^b, Nicolas Poirier ^b, David D'Addona ^b, José Ignacio Andrés ^a, Robert Lutjens ^b, Emmanuel Le Poul ^b, Hassan Imogai ^b and Jean-Philippe Rocher ^b
^aMedicinal Chemistry, Janssen Research & Development, Janssen-Cilag S.A., Polígono Industrial, Calle Jarama 75, Toledo, 45007, Spain. E-mail: atrabanc@its.jnj.com
^bAddex Pharmaceuticals, 12 chemin des Aulx, Plan-les-Ouates, 1228, Geneva, Switzerland
^cMedicinal Chemistry, Janssen Research & Development, Janssen Pharmaceutica N.V., Turnhoutseweg 30, B-2340, Beerse, Belgium
^dNeuroscience, Janssen Research & Development, Janssen Pharmaceutica N.V., Turnhoutseweg 30, B-2340, Beerse, Belgium

Received 3rd November 2010 , Accepted 30th November 2010

First published on 17th December 2010

Abstract

A series of N-propyl-5-substituted isoquinolones was identified as positive allosteric modulators (PAM) of metabotropic glutamate receptor 2 (mGluR2) via high-throughput screening (HTS). The subsequent synthesis and preliminary SAR exploration that led to the identification of compound 20 are described.

Introduction

Activation of group II metabotropic glutamate receptors (mGluR) mGluR2 and mGluR3 may provide anxiolytic and/or antipsychotic effects.^1,2,3 The mixed mGluR2/mGluR3 agonist LY354740 (1, Fig. 1) has shown anxiolytic potential in healthy human volunteers, showing activity in fear-potentiated startle and panic induction after CO₂ challenge.^4,5 A related prodrug LY2140023 (2) demonstrated improvements in positive and negative symptoms compared to placebo in schizophrenic patients.⁶ There is evidence from mice knock-out studies that preclinical anti-psychotic effects may be mediated via the mGluR2 receptor.⁷


	Fig. 1 Structures of mixed mGluR2/3 agonists LY354740 (1), LY2140023 (2), and recent mGluR2 PAM chemotypes (3–5).

There is increasing interest in identifying positive allosteric modulators (PAMs) of mGluR2 which bind at an alternative site to the orthosteric endogenous agonist.⁸ We have recently reviewed the currently known mGluR2 PAM chemotypes⁹ and new reports are appearing on a frequent basis.^10,11,12 The general structures of three of those recently disclosed mGuR2PAM chemotypes 3–5 are shown in Fig. 1. In this letter we present the discovery and preliminary SAR exploration of N-propyl-5-substituted isoquinolones 6 as a new series of mGluR2 PAMs.

Results and discussion

High-throughput screening of the Addex Pharmaceuticals compound collection in an mGluR2 PAMFLIPR (fluorometric imaging plate reader) assay resulted in the discovery of a singleton isoquinolone7 which possessed an interesting functional potency (FLIPR pEC₅₀ = 6.8). The potentiating activity of 7 was confirmed via a subsequent mGluR2 [³⁵S]-GTPγS assay (Fig. 2). Two different read outs were obtained from the GTPγS assay: (a) pEC₅₀ for potentiation of the glutamate signal, and (b) the maximal response obtained using the test compound plus an EC₂₀ of glutamate normalized to the maximal response obtained with glutamate alone (% E_MAX)¹³ Compound 7 had a GTPγS pEC₅₀ of 6.3 and an E_MAX of 123%. 7 was tested for single point microsomal stability in human liver microsomes (HLM) and showed moderate metabolic stability (52% metabolized after 15 min incubation). We considered the singleton 7 as a potentially attractive starting point for an initial limited CNS focused SAR exploration aiming to confirm activity within the isoquinolone chemotype 6.


	Fig. 2 Isoquinolone hit 7 identified from a FLIPR mGluR2 PAM high-throughput screen.

The initial set of compounds 7–20 covered variation of the R¹group at position C-5 of the isoquinolone ring while maintaining the R² substituent constant and equal to hydrogen (R² = H). Some examples where R² = Cl (21–23) were also designed to study the similarity of this series with the reported isoindolone derivatives 5 (Fig. 1).

Chemistry

The target compounds 7–23 were prepared following the synthetic strategies shown in Schemes 1–3.


	Scheme 1 Preparation of 6-aminoisoquinolones. Reagents and conditions: (i) NaH, 1-bromopropane, DMF, 0 °C to rt, 12 h. (ii) R'R′′NH, t-BuONa, BINAP, Pd₂(dba)₃, toluene, μW, 180 °C, 1 h, 12–98%.


	Scheme 2 Preparation of 6-alkoxyisoquinolones. Reagents and conditions: (i) 1-(2-chloroethyl)-4-methoxybenzene, K₂CO₃, CH₃CN, 180 °C μW, 15 min. (ii) a) Ac₂O, 120 °C, 3 h; b) NaOH 2 M, 50 °C, 1 h. (iii) NaH, 1-bromopropane, DMF, 0 °C to rt, 12 h.


	Scheme 3 Preparation of 8-chloro-substituted isoquinolones. Reagents and conditions: (i) NCS, Pd(OAc)₂, DMF, 110 °C, μW, 15 min. (ii) N-propylamine, EDCI, HOBt, DCM, 50 °C, 8 h. (iii) LDA, DMF, −78 °C to −10 °C, 12 h. (iv) RNH₂, t-BuONa, BINAP, Pd₂(dba)₃, toluene, 110 °C, 2 h, 40–72%.

The majority of final compounds were synthesized from commercially available 5-bromoisoquinolone24 following the synthesis steps shown in Scheme 1. Thus N-alkylation of 24 with N-bromopropane in the presence of sodium hydride afforded compound 25 in 56% yield. Microwave-assisted Buchwald–Hartwig type cross-coupling reaction of 25 with several amines yielded the corresponding coupling products in variable yield (12–98%) depending on the nature of the coupled amine.

Compound 13, bearing an ether linker, was prepared as shown in Scheme 2. O-alkylation of the N-oxide derivative 26 with 4-methoxyphenethyl bromide afforded the N-oxide27 in moderate yield.¹⁴ Heating of compound 27 in acetic anhydride gave an acyl derivative that was not isolated but converted directly to the isoquinolone28 by treatment with NaOH.¹⁵N-alkylation of 28 with 1-bromopropane afforded the final compound 13 in 17% yield.

Finally, compounds 21–23 bearing a chlorine substituent at position C-8 were prepared as shown in Scheme 3. Commercially available 3-bromo-2-methylbenzoic acid29 was chlorinated by treatment with N-chlorosuccinimide (NCS) in the presence of 0.3 to 0.8 equivalents of Pd(OAc)₂ to yield compound 30,¹⁶ which was then transformed into the amide31 under standard amide formation conditions. Ring cyclisation of 31 by reaction with LDA and subsequest quenching of the benzylic organolithium with DMF afforded 8-chloro-5-bromo-N-propylisoquinolone32 in good yield.¹⁷ Buchwald–Hartwig type coupling of 32 with the corresponding amines afforded the targeted compounds 21–23.

Pharmacology

The variations around the R¹ and R²groups and the functional activity and metabolic stability data in human liver microsomes (HLM) of N-propylisoquinolones 7–23 are listed in Table 1.¹⁸

Table 1 GTPγS functional activity and HLM stability data of representative mGluR2 PAMs 7–23

Compound	R²	GTPγS pEC₅₀^a	GTPγS E_MAX (%)	HLM (%)^b
a Values are means of three experiments. b HLM data refer to % of compound metabolized after 15 min at 5 μM concentration.
7	H	6.3	123	52
8	H	6.9	137	91
9	H	6.6	110
10	H	6.6	92	97
11	H	6.7	134
12	H	6.4	93
13	H	6.6	123
14	H	6.8	112	57
15	H	5.7	80	38
16	H	6.5	129
17	H	6.3	81
18	H	5.9	65
19	H	6.7	162	34
20	H	6.7	163	26
21	Cl	6.6	68
22	Cl	4.3	24	32
23	Cl	6.30	59	43

Compounds 7–20 were the result of the exploration around R¹ while keeping constant R² = H. Firstly, the effect of different substituents on the distal phenyl ring was explored. Thus, a methoxy substituent (8, 9 and 10) resulted in a slight increase in potency, particularly pronounced when placed at the ortho position (pEC₅₀ = 6.9, E_MAX = 137%). Unfortunately, the stability of 8 and 10 in HLM worsened and both compounds showed a high metabolic turnover (8: 91% and 10 : 97% metabolized after 15 min in HLM). A 4-methyl substituent 11 was also beneficial for potency (pEC₅₀ = 6.7, E_MAX = 134%). This potency increase obtained with compounds 8–11 gave us confidence on the viablity of the singleton hit 7.

The role of the NH group at position C-5 of the isoquinolone ring was studied with the preparation of compounds 12 and 13. The activity found for both compounds was in the same range as the one of compound 7, indicating that a hydrogen donorgroup is not essential for activity at position C-5.

The distance between the distal phenyl group and the isoquinolone core was then explored with compounds 14 and 15. A direct relationship between the length of the alkyl chain and the increase in potency was observed. Thus, phenylpropyl derivative 14 turned out to be half a log unit more potent than the phenethyl hit 7, with the shorter benzyl derivative 15, being only weakly active. Unfortunately, this potency increase observed with the propyl spacer was not accompanied by an improvement on the metabolic stability (14: 57% metabolized), and only the shorter benzyl was found to be metabolically more stable (15: 38% metabolized).

In view of the better metabolic profile obtained with the benzyl analogue, further mapping of the phenyl group in 15 was performed in an attempt to find molecules that would combine metabolic stability with high potency (16–20). Overall, the SAR on the aryl closely paralleled that of the phenethyl analogues. Thus the methoxy- derivatives 16 and 17 showed comparable potency to their corresponding phenethyl pairs 9 and 10. Compound 18, bearing the liphophilic but bulky phenoxy substituent was less active and had a much lower E_MAX value. The best results were obtained with the small and lipophilic 3-trifluoromethoxy- (19: pEC₅₀ = 6.7, E_MAX = 162%) and chloro-analogues (20: pEC₅₀ = 6.70, E_MAX = 163%). Remarkably, metabolic stability was also improved with 19 and 20.

Given the structural analogy of our isoquinolone series with reported mGluR2 PAM isoindolones, analogues 21–23 having a chloro atom at position C-8 (R² = Cl) were prepared. Unlike with the isoindolone series, the presence of the 8-chloro in the isoquinolone core was not beneficial for activity, and compounds 21–23 showed activity in the same range as their analogues 7, 15 and 16, however a significantly lower E_MAX was measured in all cases.

Metabolite identification studies indicated that in most cases oxidative N-dealkylation at position C-5 of the isoquinolone ring is the main metabolic pathway. Initial SAR results showed that this metabolic pathway could be disfavored by shortening of the N-alkyl side chain as shown with some benzyl derivatives (15, 19–20 and 22–23).

After this initial exploration, compounds 19 and 20 were identified which combined good potency and metabolic stability. Compound 20 was further explored for its ability to potentiate the in vitro concentration response curve (CRC) of glutamate on cloned human mGluR2.

Results indicative of positive allosteric modulation were observed. As shown in Fig. 3, the CRC of glutamate shifts to the left and upwards with increasing concentrations of compound 20. A 6.6-fold shift in the glutamate EC₅₀ was seen in the presence of 3 μM of 20(glutamate pEC₅₀ was 4.9 and 5.8 in the absence or presence of 20, respectively).


	Fig. 3 Glutamate (glu) concentration response curve in presence of varying concentration of compound 20, demonstrating a 6.6-fold-shift in glu EC₅₀ at 3 μM concentration of 20 (experiment performed with CHOcells expressing cloned human mGluR2).

Conclusions

In summary, a novel series of 5-substituted isoquinolones with mGluR2 PAM activity has been identified. Initial SAR studies from the HTS singleton 7 confirmed the isoquinolone series 6 as a novel and viable mGluR2 PAM chemotype. Preliminary SAR data suggest that (a) lipophilicity on the distal phenyl ring seems to be good for activity (b) a hydrogen bond donor at position C-5 is not required for activity, (c) the distance between the core and the phenyl ring is important, with a 3 carbon spacer optimal for activity. At present, metabolic stability is the main issue identified for this novel chemotype, nevertheless the shorter benzyl derivatives may allow to circumvent this issue. From our first round exploration, we identified compounds (19 and 20) with good potency in the GTPγS assay and improved metabolic stability in human liver microsomes. Compound 20 proved to be a positive allosteric modulator of mGluR2 by its ability to potentiate the in vitro CRC of glutamate, deserving further optimization. Further evaluation of this compound as well as a broader SAR exploration are underway and will be reported in due course.

Experimental

Biology

Membrane preparation. CHO-cells were cultured to pre-confluence and stimulated with 5 mM butyrate for 24 h, prior to washing in PBS, and then collection by scraping in homogenisationbuffer (50 mM Tris-HCl buffer, pH 7.4, 4 °C). Cell lysates were homogenized briefly (15 s) using an ultra-turrax homogenizer. The homogenate was centrifuged at 23 500 × g for 10 min and the supernatant discarded. The pellet was resuspended in 5 mM Tris-HCl, pH 7.4 and centrifuged again (30 000 × g, 20 min, 4 °C). The final pellet was resuspended in 50 mM HEPES, pH 7.4 and stored at −80 °C in appropriate aliquots before use. Protein concentration was determined by the Bradford method (Bio-Rad, USA) with bovine serum albumin as standard.

[³⁵S]GTPγS binding assay. Measurement of mGluR2 positive allosteric modulatory activity of test compounds in membranes containing human mGluR2 was performed using frozen membranes that were thawed and briefly homogenised prior to pre-incubation in 96-well microplates (15 μg/assay well, 30 min, 30 °C) in assay buffer (50 mM HEPES pH 7.4, 100 mM NaCl, 3 mM MgCl₂, 50 μM GDP, 10 μg ml⁻¹saponin) with increasing concentrations of positive allosteric modulator (from 0.3 nM to 50 μM) and either a minimal pre-determined concentration of glutamate (PAM assay), or no added glutamate. For the PAM assay, membranes were pre-incubated with glutamate at EC₂₅ concentration, i.e. a concentration that gives 25% of the maximal response glutamate. After addition of [³⁵S]GTPγS (0.1 nM, f.c.) to achieve a total reaction volume of 200 μl, microplates were shaken briefly and further incubated to allow [³⁵S]GTPγS incorporation on activation (30 min, 30 °C). The reaction was stopped by rapid vacuum filtration over glass-fibre filter plates (Unifilter 96-well GF/B filter plates, Perkin-Elmer, Downers Grove, USA) microplate using a 96-well plate cell harvester (Filtermate, Perkin-Elmer, USA), and then by washing three times with 300 μl of ice-cold wash buffer (Na₂PO₄·2H₂O 10 mM, NaH₂PO₄·H₂O 10 mM, pH = 7.4). Filters were then air-dried, and 40 μl of liquid scintillation cocktail (Microscint-O) was added to each well, and membrane-bound [³⁵S]GTPγS was measured in a 96-well scintillation plate reader (Top-Count, Perkin-Elmer, USA). Non-specific [³⁵S]GTPγS binding is determined in the presence of cold 10 μM GTP. Each curve was performed at least three times using duplicate sample per data point and at 11 concentrations.

Data analysis. The concentration-response curves in the presence of added EC₂₅ of mGluR2 agonistglutamate to determine positive allosteric modulation (PAM), were generated using the Prism GraphPad software (Graph Pad Inc, San Diego, USA). The curves were fitted to a four-parameter logistic equation (Y = Bottom + (Top-Bottom)/(1 + 10^((LogEC₅₀-X)*Hill Slope) allowing determination of EC₅₀ values. The EC₅₀ is the concentration of a compound that causes a half-maximal potentiation of the glutamate response. This is calculated by subtracting the maximal responses of glutamate in presence of a fully saturating concentration of a positive allosteric modulator from the response of glutamate in absence of a positive allosteric modulator. The concentration producing the half-maximal effect is then calculated as EC₅₀. The pEC₅₀ values below are calculated as the −logEC₅₀ (wherein EC₅₀ is expressed in mol L⁻¹).

Chemistry general. All chemicals and solvents were obtained from commercial suppliers and used without further purification. Reactions were monitored by thin layer chromatography and/or liquid chromatography-mass spectrometry (LCMS). Flash column chromatography: silica gel (220–440 mesh, Fluka). ¹H-NMR: Brucker 500MHz. Chemical shifts δ are repoted in parts per million (ppm) against the reference compound tetramethylsilane and calculated using the chemical shift of the signal of the residual non-deuterated solvent. Melting point: melting point apparatus B-540 (Buchi), uncorrected. MS: ESI, electrospray inonisation: Waters Micromass ZQ 2996 system, peaks are given in m/z (% of basis peak). Flow rate: 1mL min⁻¹; injection volume. 3 μM; column: XTerra RP C-18 (5 μM) peaks are given in m/z (% of basis peak); solvents: A: water with 0.1% (v/v) formic acid; B. Acetonitrile with 0.07% (v/v) formic acid: gradient elution: (A%): 0–0.5 min 95%, 0.5–6.0 min 0%, 6.0–6.5 min 95%, 6.5–7 min 0%. UV detection: diode array: λ =200–400nm. All screening compounds were prepared to >95%purity by LCMS and/or ¹H-NMR analysis. In the case of exploratory library synthesis, all compounds were analysed by LCMS and representative examples were characterized by ¹H-NMR.

General procedures

5-(4-Methoxyphenethylamino)-2-propylisoquinolin-1(2H)-one (10). To a solution of 5-chloroisoquinolin-1(2H)-one24 (596 mg, 2.66 mmol) in DMF (10 mL) at 0 °C was added sodium hydride (67 mg, 2.8 mmol) and the mixture was stirred for 15 min. 1-Bromopropane (0.27 mL, 2.93 mmol) was added and the mixture was stirred at room temperature for 12 h. The reaction mixture was poured into water and extracted with EtOAc (2 × 25 mL). The combined organic layers were dried (MgSO₄), filtered and concentrated in vacuo. The residue was purified flash chromatography (AIT Flashsmart prepacked column 25 g SiO₂, CH₂Cl₂) to afford 25: orange solid, yield 396. mg (56%). ¹H- NMR: (300MHz, CDCl₃) δ 1.00 (t, J = 7.4 Hz, 3H), 1.80 (m, 2H), 3.91 (t, J = 7.2 Hz, 2H), 6.82 (d, J = 7.7 Hz, 1H), 7.10 (d, J = 7.7 Hz, 1H), 7.29 (d, J = 7.7 Hz, 1H), 7.81 (dd, J = 7.7 Hz, 1H), 8.33 (d, J = 7.7 Hz, 1H). ESI-MS: m/z 266.9 [M + H]⁺.

To a mixture of 5-bromo-2-propylisoquinolin-1(2H)-one25 (287 mg, 1.08 mmol), t-BuONa (160 mg, 1.62 mmol), Pd₂(dba)₃ (50 mg, 0.054 mmol), BINAP (34 mg, 0.054 mmol) in degassed dry toluene (4 mL) was added 4-methoxyphenetyl amine (250 mg, 1.62 mmol). The reaction mixture was heated (sealed tube) at 180 °C for 1 h under microwave irradiation. The mixture was cooled to room temperature and filtered through Celite^®. The reaction mixture was poured into water and extracted with CH₂Cl₂ (2 × 25 mL). The combined organic layers were dried (Na₂SO₄), filtered and concentrated in vacuo. The crude product was purified by flash chromatography (AIT Flashsmart prepacked column 25 g, SiO₂, cyclohexane/AcOEt 90/10) to afford 10: white solid. mp.: 110 °C. ¹H- NMR: (300MHz, CDCl₃) δ 0.96 (t, J = 7.2 Hz, 3H), 1.02 (t, J = 7.2 Hz, 2H), 1.80 (m, 2H), 2.97 (d, J = 6.9 Hz, 2H), 3.43–3.49 (m, 3H), 3.80 (s, 3H), 3.94 (t, J = 7.5 Hz, 2H), 6.26 (d, J = 7.8 Hz, 1H), 6.87 (d, J = 8.7 Hz, 3H), 7.00 (d, J = 7.5 Hz, 1H), 7.17 (d, J = 8.7 Hz, 2H), 7.36 (t, J = 8.1 Hz, 1H), 7.85 (d, J = 8.1 Hz, 1H). ESI-MS: m/z 337.4 [M + H]⁺.

The procedure used for compound 10 was further used to prepare the following compounds:

5-Phenethylamino-2-propylisoquinolin-1(2H)-one (7). Obtained from phenethylamine. 7: orange solid. mp.: 120 °C. ¹H-NMR: (300MHz, CDCl₃) δ 0.96 (t, J = 7.2 Hz, 3H), 1.79 (m, 2H), 3.05 (t, J = 6.9 Hz, 2H), 3.51 (t, J = 6.9 Hz, 2H), 3.95 (t, J = 7.2 Hz, 2H), 6.32 (m, 1H), 7.02 (d, J = 7.5 Hz, 1H), 7.23–7.27 (m, 3H), 7.36 (m, 4H), 7.90 (m, 1H). ESI-MS: m/z 307.3 [M + H]⁺.

5-(2-Methoxyphenethylamino)-2-propylisoquinolin-1(2H)-one (8). Obtained from 2-methoxyphenethylamine. 8: off-white solid. mp.: 98 °C. ¹H-NMR: (300MHz, CDCl₃) δ 0.96 (t, J = 7.4 Hz, 3H), 1.8 (m, 2H), 3.05 (t, J = 6.7 Hz, 2H), 3.4 (t, J = 6.7 Hz, 2H), 3.9 (t, J = 7.4 Hz, 2H), 4.0 (t, J = 7.4 Hz, 2H), 6.3 (d, J = 7.8 Hz, 1H), 6.92 (d, J = 8.4 Hz, 2H), 6.95 (td, J = 7.4 Hz, J = 1.0 Hz, 1H), 7.02 (d, J = 7.8 Hz, 1H), 7.2 (dd, J = 7.4 Hz, J = 1.8 Hz, 1H), 7.25 (dd, J = 7.4 Hz, J = 1.8 Hz, 1H), 7.3 (d, 1H), 7.38 (t, J = 7.8 Hz, 1H), 7.8 (d, J = 8.2 Hz, 1H). ESI-MS: m/z 337.3 [M + H]⁺.

5-(3-Methoxyphenethylamino)-2-propylisoquinolin-1(2H)-one (9). Obtained from 3-methoxyphenethylamine. 9: white solid. mp.: 124 °C ¹H-NMR: (300MHz, CDCl₃) δ 0.98 (t, J = 7.2 Hz, 3H), 1.82 (m, 2H), 3.02 (t, J = 7.0 Hz, 2H), 3.51 (t, J = 6 Hz, 2H), 3.81 (s, 3H), 3.97 (t, J = 7.8 Hz, 2H), 6.27 (d, J = 7.8 Hz, 1H), 6.85 (m, 4H), 7.02 (t, J = 7.4 Hz, 1H), 7.28 (m, 1H), 7.38 (t, J = 8.4 Hz, 1H), 7.85 (d, J = 8.4 Hz, 1H). ESI-MS: m/z 337.4 [M + H]⁺.

5-(4-Methylphenethylamino)-2-propylisoquinolin-1(2H)-one (11). Obtained from 2-methoxyphenethylamine. 11: white solid, mp.: 127 °C. ¹H- NMR: (300MHz, CDCl₃) δ 0.96 (t, J = 7.4 Hz, 3H), 1.81 (m, 2H), 2.20 (s, 3H), 2.97 (t, J = 6.8 Hz, 2H), 3.47 (t, J = 6.8 Hz, 2H), 3.97 (t, J = 7.4 Hz, 2H), 6.26 (d, J = 7.2 Hz, 1H), 6.82 (d, J = 7.2 Hz, 1H), 6.92 (d, J = 7.6 Hz, 1H), 7.05 (m, 3H), 7.15 (s, 1H), 7.31 (t, J = 7.9 Hz, 1H), 7.80 (d, J = 7.9 Hz, 1H). ESI-MS: m/z 320.4 [M + H]⁺.

5-[N-(4-Methoxyphenethyl)-N-methylamino]-2-propylisoquinolin-1(2H)-one (12). Obtained from N-(4-Methoxyphenethyl)-N-methylamine. 12: yellow oil. ¹H-NMR: (300MHz, CDCl₃) δ 1.00 (t, J = 7.4 Hz, 3H), 1.80 (m, 2H), 2.83 (t, J = 7.4 Hz, 2H), 2.87 (s, 3H), 3.25 (t, J = 7.6 Hz, 2H), 3.81 (s, 3H), 4.01 (t, J = 7.4 Hz, 2H), 6.71 (d, J = 7.6 Hz, 2H), 6.80 (dd, J = 6.5 Hz, 2H), 7.01 (d, J = 7.6 Hz, 1H), 7.10 (d, J = 8.7 Hz, 2H), 7.35 (d, J = 7.4 Hz, 1H), 7.43 (t, J = 8.0 Hz, 1H), 8.16 (d, J = 8.0 Hz, 1H). ESI-MS: m/z 350.5 [M + H]⁺.

5-(3-Phenylpropylamino)-2-propylisoquinolin-1(2H)-one (14). Obtained from 3-phenylpropylamine. 14: white solid. mp.:118 °C. ¹H-NMR: (300MHz, CDCl₃) δ 0.97 (t, J = 7.5 Hz, 3H), 1.80 (s, J = 7.5 Hz, 2H), 2.08 (q, J = 7.2 Hz, 2H), 2.79 (t, J = 7.5 Hz, 2H), 3.26 (t, J = 7.2 Hz, 2H), 3.95 (t, J = 7.2 Hz, 2H), 6.29 (d, J = 7.5 Hz, 1H), 6.83 (m, 1H), 7.02 (d, J = 7.8 Hz, 1H), 7.28 (m, 7H), 7.85 (d, J = 8.1 Hz, 1H). ESI-MS: m/z 320.3 [M + H]⁺.

5-Benzylamino-2-propylisoquinolin-1(2H)-one (15). Obtained from benzylamine. 15: orange solid. mp.: 111 °C. ¹H-NMR: (300MHz, CDCl₃) δ 0.97 (t, J = 7.2 Hz, 3H), 1.81 (m, 2H), 3.96 (t, J = 7.2 Hz, 2H), 4.44 (s, 2H), 7.05 (d, J = 7.5 Hz, 1H), 6.49 (m, 1H), 7.36 (m, 7H), 7.91 (m, 1H). ESI-MS: m/z 293.4 [M + H]⁺.

5-(3-Methoxybenzylamino)-2-propylisoquinolin-1(2H)-one (16). Obtained from 3-methoxybenzylamine. 16: yellow solid. mp.: 140 °C. ¹H-NMR: (300MHz, CDCl₃) δ 0.99 (t, J = 7.2 Hz, 3H), 1.61 (s, 2H), 1.83 (m, 2H), 3.83 (s, 3H), 3.98 (t, J = 7.2 Hz, 2H), 4.42 (s, 3H), 6.45 (d, J = 7.9 Hz, 1H), 6.85 (m, 2H), 7.07 (m, 3H), 7.35 (m, J = 7.9 Hz, 2H), 7.87 (d, J = 4.0 Hz, 1H). ESI-MS: m/z 323.4 [M + H]⁺.

5-(4-Methoxybenzylamino)-2-propylisoquinolin-1(2H)-one (17). Obtained from 4-methoxybenzylamine. 17: white solid. mp.:140 °C. ¹H-NMR: (300MHz, CDCl3) δ 0.95 (t, J = 7.2 Hz, 3H), 1.81 (m, 2H), 3.80 (s, 3H), 3.98 (t, J = 7.4 Hz, 2H), 4.41 (s, 2H), 6.88 (m, J = 6.7 Hz, 2H), 7.08 (m, J = 7.4 Hz, 1H), 7.31 (m, 3H), 7.35 (m, J = 7.9 Hz, 3H). ESI-MS: m/z 323.4 [M + H] +.

5-(3-Phenoxybenzylamino)-2-propylisoquinolin-1(2H)-one (18). Obtained from 3-phenoxybenzylamine. 18: beige solid. mp.: 128 °C. ¹H-NMR: (300MHz, CDCl₃) δ 1.00 (t, J = 7.4 Hz, 3H), 1.80 (q, J = 7.4 Hz, 2H), 3.99 (t, J = 8.4 Hz, 2H), 4.41 (s, 2H), 6.46 (d, J = 7.7 Hz, 1H), 6.77 (d, J = 7.7 Hz, 1H), 7.05 (m, 7H), 7.28 (m, 4H), 7.86 (d, J = 7.7 Hz, 1H). ESI-MS: m/z 385.5 [M + H]⁺.

2-Propyl-5-(3-trifluoromethoxybenzylamino)isoquinolin-1(2H)-one (19). Obtained from 3-trifluoromethoxybenzylamine. 19: beige solid. ¹H-NMR: (300MHz, CDCl₃) δ 0.98 (t, J = 7.4 Hz, 3H), 1.80 (q, J = 7.4 Hz, 2H), 3.96 (t, J = 7.4 Hz, 2H), 4.55 (s, 2H), 6.90 (d, J = 7.4 Hz, 1H), 7.22 (m, 6H), 7.60 (m, 1H), 8.45 (d, J = 7.2 Hz, 1H). ESI-MS: m/z 377.4 [M + H]⁺.

5-(2-Chlorobenzylamino)-2-propylisoquinolin-1(2H)-one (20). Obtained from 2-chlorobenzylamine. 20: light yellow. mp.:140 °C. ¹H-NMR: (300MHz, CDCl3) δ 0.98 (t, J = 7.4 Hz, 3H), 1.81 (q, J = 7.4 Hz, 2H), 3.99 (t, J = 8.4 Hz, 2H), 4.55 (s, 2H), 6.46 (d, J = 7.7 Hz, 1H), 6.77 (d, J = 7.7 Hz, 1H), 7.08 (m, 1H), 7.29 (m, 3H), 7.42 (m, 2H), 7.86 (d, J = 8.2 Hz, 1H). ESI -MS: m/z 327.8 [M + H]⁺.

5-(4-Methoxyphenetoxy)isoquinoline-N-oxyde (27). To a solution of 5-hydroxyisoquinolin-N-oxyde26 (500 mg, 3.1 mmol) in acetonitrile (15 mL) at room temperature were added K₂CO₃ (1.1 g, 6 mmol) and 1-(2-chloroethyl)-4-methoxybenzene (1.06 g, 6.2 mmol). The mixture was heated (sealed tube) at 180 °C for 15 min under microwave irradiation. The mixture was cooled to room temperature and filtered through Celite^®. The filtrate was evaporated to dryness and the residue was purified flash chromatography (AIT Flashsmart prepacked column 70 g SiO₂, CH₂Cl₂) to afford 25: brown solid, yield 0.405 mg (44%). ESI-MS: m/z 296 [M + H]⁺.

5-(4-Methoxyphenetoxy)isoquinolin-1(2H)-one (28). A solution of 5-chloroisoquinoline N-oxyde27 (810 mg, 2.73 mmol) in acetic anhydride (20 mL) was stirred for 3 h at reflux temperature. The mixture was allowed to cool to room temperature and then further stirred overnight. The anhydride acetic was then evaporated under reduced pressure and a solution of NaOH (2 M, 10 mL) was added to the residue. The reaction mixture was stirred for 1 h at 50 °C. Then the reaction mixture was acidified (pH = 6) with citric acid (5% in water) and a brown precipitate formed. The precipitate was filtered, washed with cold water and dried under vacuum. The resulting solid was taken up in CH₂Cl₂ (20 mL), washed with brine, dried (MgSO₄), filtered and evaporated to yield 28 which was used in the next reaction step without further purification: brown solid, yield 620 mg, (77%).

5-(4-Methoxyphenetoxy)2-propylisoquinolin-1(2H)-one (13). To a solution of 5-(4-methoxyphenetoxy)isoquinolin-1(2H)-one28 (100 mg, 0.34 mmol) in DMF (10 mL) at 0 °C was added sodium hydride (10 mg, 0.41 mmol) and the mixture was stirred for 15 min. 1-Bromopropane (0.035 mL, 0.38 mmol) was added The mixture was heated (sealed tube) at 180 °C for 15 min under microwave irradiation. The mixture was cooled to room temperature and filtered through Celite^®. The reaction mixture was poured into water and extracted with EtOAc (2 × 25 mL). The combined organic layers were dried (MgSO₄), filtered and concentrated in vacuo. The residue was purified by flash chromatography (AIT Flashsmart prepacked column 25 g SiO₂, 2% MeOH in CH₂Cl₂) to afford 13: orange oil, yield 20. mg (17%). ¹H-NMR: (300MHz, CDCl3) δ 0.97 (t, J = 7.5 Hz, 3H), 1.81 (m, 2H), 3.13 (t, J = 6.3 Hz, 2H), 3.80 (s, 3H), 3.96 (t, J = 7.5 Hz, 2H), 4.24 (t, J = 7.2 Hz, 2H), 6.85 (m, 3H), 7.02 (m, 2H), 7.24 (d, J = 8.7 Hz, 2H), 7.36 (t, J = 8.4 Hz, 1H), 7.99 (d, J = 8.1 Hz, 1H). ESI-MS: m/z 338 [M + H]⁺.

3-Bromo-6-chloro-2-methylbenzoic acid (30). A solution of 3-bromo-2-methylbenzoic acid29 (10 g, 47 mmol), NCS (7.5 g, 56 mmol) and Pd(OAc)₂ (4.2 g, 19 mmol) in DMF (90 mL) was heated at 110 °C for 15 min under microwave irradiation. The mixture was cooled to room temperature and NaOH 6.0 N was slowly added, then extracted with AcOEt (3 × 25 mL). The aqueous phase was acidified at pH∼1 with HCL 6.0 N and then extracted with AcOEt (3 × 25 mL). The organic layer was dried over MgSO₄, filtered and evaporated till dryness to afford 30 which was used without further purification: beige solid, yield 12 g (quant.). ESI-MS: m/z 347–251 [M − H]⁻.

3-Bromo-6-chloro-2-methyl-N-propylbenzamide (31). To a solution of 3-bromo-6-chloro-2-methylbenzoic acid30 (10 g, 40.1 mmol) and HOBt (12.3 g, 80.2 mmol) in dichloromethane (150 mL) at room temperature was added EDCI.HCl (15.4 g, 80.2 mmol) and stirred for 15 min before adding propylamine (7.11 g, 120 mmol). The reaction mixture was heated at 50 °C overnight, then cooled to room temperature and water (100 mL) was added. The aqueous layer was extracted with DCM (2 × 50 mL) and combined organic layers were washed with a saturated solution of NaHCO₃ and brine, then dried over MgSO₄, filtered and concentrated in vacuo. The residue was purified flash chromatography (AIT Flashsmart prepacked column 90 g SiO₂, cyclohexane/AcOEt 80 [thin space (1/6-em)]

20) to afford 31: yellow solid, yield 10.5 g (90%). ESI-MS: m/z 290–294 [M + H]⁺.

5-Bromo-8-chloro-2-propylisoquinolin-1(2H)-one (32). To a solution of 3-bromo-6-chloro-2-methyl-N-propylbenzamide31 (1.77 g, 6.09 mmol) in dry THF (100 mL) at −78 °C and under nitrogen atmosphere, was added a 1.8M solution of lithium diisopropylamide (8.46 mL, 15.2 mmol) over 20 min. The reaction mixture was stirred at −78 °C for 2 h, then dry DMF (2.35 mL, 30.5 mmol) was added dropwise over 10 min. After 10 more minutes, the reaction mixture was allowed to warm up untill −10 °C and was hydrolysed slowly with a 6.0 N solution of HCl. The reaction mixture was extracted with AcOEt (3 × 25 mL), dried over MgSO₄, filtered and concentrated in vacuo. The residue was triturated in diisopropylether, filtered and dried, to afford 32: brown solid, yield 1.3 g (71%). ESI-MS: m/z 300–304 [M + H]⁺.

8-Chloro-5-(phenethylamino)-2-propylisoquinolin-1(2H)-one (21). To a mixture of 5-bromo-8-chloro-2-propylisoquinolin-1(2H)-one32 (200 mg, 0.665 mmol), t-BuONa (96 mg, 0.998 mmol), Pd₂(dba)₃ (31 mg, 0.033 mmol), BINAP (41 mg, 0.067 mmol) in degassed dry toluene (7 mL) was added phenethylamine (81 mg, 0.665 mmol). The reaction mixture was heated at 110 °C for 2 h. The mixture was cooled to room temperature and filtered through Celite^®. The reaction mixture was poured into water and extracted with EtOAc (2 × 10 mL). The combined organic layers were dried (Na₂SO₄), filtered and concentrated in vacuo. The crude product was purified by flash chromatography (AIT Flashsmart prepacked column 15 g, SiO₂, DCM–AcOEt 98 [thin space (1/6-em)]

2) to afford 21: beige solid. mp.: 107 °C. ¹H-NMR: (300MHz, CDCl₃) δ 0.96 (t, J = 7.4 Hz, 3H), 1.78 (m, 2H), 2.99 (d, J = 7.3 Hz, 2H), 3.45 (t, J = 7.4 Hz, 3H), 3.80 (t, J = 7.4 Hz, 3H), 6.26 (s, 1H), 6.87–7.42 (m, 8H). ESI-MS: m/z 341–343 [M + H]⁺.

The procedure used for compound 21 was further used to prepare the following compounds:

5-(Benzylamino)-8-chloro-2-propylisoquinolin-1(2H)-one (22). Obtained from benzylamine. 22: brown solid. mp.: 152 °C. ¹H-NMR: (300MHz, CDCl₃) δ 0.95 (t, J = 7.4 Hz, 3H), 1.73 (m, 2H), 3.87 (t, J = 7.4 Hz, 2H), 4.38 (s, 2H), 6.34 (s, 1H), 6.88 (m, 1H), 7.02 (m, 1H), 7.23 (m, 6H). ESI-MS: m/z 327–329 [M + H]⁺.

8-Chloro-5-(3-methoxybenzylamino)-2-propylisoquinolin-1(2H)-one (23). Obtained from (3-methoxyphenyl)methanamine. 23: white solid. mp.: 152 °C. ¹H-NMR: (300MHz, CDCl₃) δ 0.98 (t, J = 7.4 Hz, 3H), 1.82 (q, J = 7.4 Hz, 2H), 3.89 (s, 3H), 3.94 (t, J = 7.7 Hz, 2H), 4.41 (s, 2H), 6.34 (d, J = 7.7 Hz, 1H), 6.84 (d, J = 8.4 Hz, 1H), 6.86–6.98 (m, 4H), 7.05 (m, 1H). ESI-MS: m/z 357–359 [M + H]⁺.

Notes and references

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