Synthesis of Functionalized Isoquinolone Derivatives via Rh(III)-Catalyzed [4+2]-Annulation of Benzamides with Internal Acetylene-Containing α-CF3-α-Amino Carboxylates

A convenient pathway to a new series of α-CF3-substituted α-amino acid derivatives bearing pharmacophore isoquinolone core in their backbone has been developed. The method is based on [4+2]-annulation of N-(pivaloyloxy) aryl amides with orthogonally protected internal acetylene-containing α-amino carboxylates under Rh(III)-catalysis. The target annulation products can be easily transformed into valuable isoquinoline derivatives via a successive aromatization/cross-coupling operation.

On the other hand, modern peptide-based drug design very often aims on selective derivatizing of the peptide backbone through the introduction of additional functional groups or proven heterocyclic pharmacophores in order to modulate the required properties [35][36][37][38][39][40][41]. In this respect, α-fluoroalkyl-containing α-amino acids are of particular interest since they find widespread bio-organic applications as biological tracers, mechanistic probes, and enzyme inhibitors as well as medical applications including blood pressure control, allergies, and tumor growth [42][43][44][45][46]. The incorporation of fluorinated α-amino acids into key positions of bioactive peptides is one of the most common strategies to improve their pharmacokinetic profiles, conformational and proteolytic stability, and membrane permeability [43][44][45][46][47][48][49][50]. Therefore, the development of new representatives of α-fluoromethyl-α-amino acids, including those decorated with pharmacophore heterocycle rings, is of high interest.

Scheme 1.
Previous and present work. Scheme 1. Previous and present work.

Results and Discussion
We commenced our study by examining a model reaction between phenyl hydroxamate 1a and the readily available internal phenyl acetylene 2a [56] bearing an N-Boc-protected α-CF 3 -α-amino ester group for the screening of optimal conditions for the annulation. The rhodium catalytic system [Cp*RhCl 2 ] 2 /CsOAc, as the most competent catalyst for such type of transformation, was initially tested to activate the process. As a result, the reaction was found to smoothly proceed in the presence of 5 mol% [Cp*RhCl 2 ] 2 and 2.0 equiv. of cesium acetate in methanol at room temperature for 4 h to give the corresponding isoquinolone derivative 3a in 70% NMR yield ( Table 1, entry 1), along with noticeable amounts of starting materials. Encouraged by this result, we screened some solvents and bases for the reaction (entries 2-5). The best conversion of the starting materials and isolated yield of 3a (measured by 19 F NMR spectroscopy) were achieved by the usage of 2,2,2-trifluoroethanol (TFE) and CsOAc (entry 2). Subsequent reduction of catalyst and additive loading (entries 2-6) has revealed the optimal reaction conditions: [Cp*RhCl 2 ] 2 (3 mol%) and CsOAc (1 equiv.), r.t., 2 h in MeOH (entry 8). Iridium-, cobalt-and ruthenium-based complexes have proved to be absolutely inactive in the process (entries 10-12). The reaction does not take place in the absence of any catalyst or base, expectedly (entries 13, 14). In continuation of our current research on metal-catalyzed C-H bond activation [52][53][54][55], here we want to disclose a convenient regio-selective approach to new isoquinolone-containing α-amino acid derivatives derived from internal aryl acetylenes bearing protected α-CF3-α-amino carboxylate framework under rhodium(III)-catalysis, and their further synthetic transformations into highly functionalized isoquinolines (Scheme 1).

Results and Discussion
We commenced our study by examining a model reaction between phenyl hydroxamate 1a and the readily available internal phenyl acetylene 2a [56] bearing an N-Boc-protected α-CF3-α-amino ester group for the screening of optimal conditions for the annulation. The rhodium catalytic system [Cp*RhCl2]2/CsOAc, as the most competent catalyst for such type of transformation, was initially tested to activate the process. As a result, the reaction was found to smoothly proceed in the presence of 5 mol% [Cp*RhCl2]2 and 2.0 equiv. of cesium acetate in methanol at room temperature for 4 h to give the corresponding isoquinolone derivative 3a in 70% NMR yield ( Table 1, entry 1), along with noticeable amounts of starting materials. Encouraged by this result, we screened some solvents and bases for the reaction (entries 2-5). The best conversion of the starting materials and isolated yield of 3a (measured by 19 F NMR spectroscopy) were achieved by the usage of 2,2,2-trifluoroethanol (TFE) and CsOAc (entry 2). Subsequent reduction of catalyst and additive loading (entries 2-6) has revealed the optimal reaction conditions: [Cp*RhCl2]2 (3 mol%) and CsOAc (1 equiv.), r.t., 2 h in MeOH (entry 8). Iridium-, cobaltand ruthenium-based complexes have proved to be absolutely inactive in the process (entries 10-12). The reaction does not take place in the absence of any catalyst or base, expectedly (entries 13, 14). With these found conditions in hand, different aryl hydroxamates were involved in [4+2]-annulation with different internal acetylene-containing amino ester 2a-e (Scheme 2). The latter were easily synthesized via an addition of Grignard reagent (CH 2 =C=CH-MgBr), generated from propargyl bromide, to orthogonally protected α-CF 3 -α-imino carboxy-lates followed by Sonogashira coupling with aryl halogenides according to the previously described protocol [56]. As a result, a series of the corresponding isoquinolinonecontaining α-CF 3 -amino carboxylates 3a-r were obtained in good yields and high degree of regio-selectivity. The observed selectivity of the [4+2]-annulation process of 1 with α-(arylpropargyl)-α-amino esters 2a-e bearing donor substituents in aryl group could be probably explained by alkyne insertion into initially formed 5-membered rhodacycle intermediate [26,27], according to its inherent polarity (for proposed mechanism see Supplementary Materials, Scheme S1). The nature of the substituents in hydroxamate component did not significantly affect the outcome of the reaction in all investigated cases (Scheme 2). 2). The latter were easily synthesized via an addition of Grignard reagent (CH2=C=CH-MgBr), generated from propargyl bromide, to orthogonally protected α-CF3-α-imino carboxylates followed by Sonogashira coupling with aryl halogenides according to the previously described protocol [56]. As a result, a series of the corresponding isoquinolinone-containing α-CF3-amino carboxylates 3a-r were obtained in good yields and high degree of regio-selectivity. The observed selectivity of the [4+2]-annulation process of 1 with α-(arylpropargyl)-α-amino esters 2a-e bearing donor substituents in aryl group could be probably explained by alkyne insertion into initially formed 5-membered rhodacycle intermediate [26,27], according to its inherent polarity (for proposed mechanism see Supporting Info, Scheme S1). The nature of the substituents in hydroxamate component did not significantly affect the outcome of the reaction in all investigated cases (Scheme 2).
However, the presence of an electron-withdrawing nitro group in para-position of aryl substituent of acetylene component leads to a mixture of the corresponding regioisomers 3s and 3t in a ratio of 3:2 respectively, which were easily separated by column chromatography on silica gel. The absence of selectivity in this case can be likely addressed to a change in the polarity of the triple bond due to the influence of strong acceptor group. The structure of each regio-isomer was assigned by 2D NOESY experiments (see Supporting Information). Thus, an intensive cross peak between proton of CH2 group of amino acid residue and ortho-proton of phenyl ring was found in the spectrum of isomer 3t that has not been observed for compound 3s; instead, a cross peak between the ortho-protons of close located phenyl moieties appeared (Scheme 3).

Scheme 2. Synthesis of isoquinolone-containing α-amino carboxylates 3.
However, the presence of an electron-withdrawing nitro group in para-position of aryl substituent of acetylene component leads to a mixture of the corresponding regioisomers 3s and 3t in a ratio of 3:2 respectively, which were easily separated by column chromatography on silica gel. The absence of selectivity in this case can be likely addressed to a change in the polarity of the triple bond due to the influence of strong acceptor group. The structure of each regio-isomer was assigned by 2D NOESY experiments (see Supplementary Materials). Thus, an intensive cross peak between proton of CH 2 group of amino acid residue and ortho-proton of phenyl ring was found in the spectrum of isomer 3t that has not been observed for compound 3s; instead, a cross peak between the ortho-protons of close located phenyl moieties appeared (Scheme 3). All synthesized compounds were fully characterized by physicochemical methods. In addition, the structure of isoquinolone 3a was confirmed by X-ray crystallographic analysis ( Figure 2). Considering the fact that isoquinolines are key structural elements of many bioactive compounds including drugs [57][58][59] (see Figure 1), we were interested in further investigation of synthetic potential of obtained isoquinolones as universal precursors of the corresponding isoquinoline derivatives decorated with amino acid residues. Thus, we found that the isoquinolones 3 could easily undergo an aromatization into the isoquinolines under treatment with triflic anhydride in the presence of pyridine. The reactions proceed in CH2Cl2 under mild conditions and go to completion within 15 min at ambient temperature furnishing the corresponding 1-OTf-substituted isoquinolines 4a-o in good to high yields (Scheme 4). All synthesized compounds were fully characterized by physicochemical methods. In addition, the structure of isoquinolone 3a was confirmed by X-ray crystallographic analysis ( Figure 2).
All synthesized compounds were fully characterized by physicochemical m In addition, the structure of isoquinolone 3a was confirmed by X-ray crystall analysis ( Figure 2). Considering the fact that isoquinolines are key structural elements of many b compounds including drugs [57][58][59] (see Figure 1), we were interested in furth tigation of synthetic potential of obtained isoquinolones as universal precurso corresponding isoquinoline derivatives decorated with amino acid residues. T found that the isoquinolones 3 could easily undergo an aromatization into the i lines under treatment with triflic anhydride in the presence of pyridine. The r proceed in CH2Cl2 under mild conditions and go to completion within 15 min at temperature furnishing the corresponding 1-OTf-substituted isoquinolines 4a-o to high yields (Scheme 4). Considering the fact that isoquinolines are key structural elements of many bioactive compounds including drugs [57][58][59] (see Figure 1), we were interested in further investigation of synthetic potential of obtained isoquinolones as universal precursors of the corresponding isoquinoline derivatives decorated with amino acid residues. Thus, we found that the isoquinolones 3 could easily undergo an aromatization into the isoquinolines under treatment with triflic anhydride in the presence of pyridine. The reactions proceed in CH 2 Cl 2 under mild conditions and go to completion within 15 min at ambient temperature furnishing the corresponding 1-OTf-substituted isoquinolines 4a-o in good to high yields (Scheme 4).
The well-known pseudo-halogen nature of the TfO group pushed us to explore some further useful transformations of the synthesized isoquinoline derivatives 4, such as Pdcatalysed cross-coupling reactions. As a result, it turned out that the compounds 4 could serve as suitable cross-coupling partners in Suzuki reaction with various aryl boronic acids. It was revealed that the reactions of 4d,f,l with 4-metoxy phenyl boronic acid readily proceeded in dioxane-water mixture under catalysis with Pd(PPh 3 ) 2 Cl 2 /NaHCO 3 system at 100 • C, leading to the formation of the expected cross-coupling products 5a-c in high yields (Scheme 5a).
The same isoquinoline-containing α-amino acid derivatives 4d,f,m were included in coupling with phenyl acetylene under the classical Sonogashira reaction conditions to afford the corresponding products 6a-c in good yields (Scheme 5b). Finally, we found that the OTf group could be successfully removed in the presence of catalytic amounts of PdCl 2 (dppf) complex and excess of formic acid to give isoquinolines 7a,b in acceptable yields (Scheme 5c). In addition, in order to demonstrate a feasibility for the further synthetic applications of the compounds obtained, e.g., in peptide synthesis or other useful derivatizations, we have performed selective deprotections of the N-PG-α-amino esters. Thus, the ester 3a was saponified using 5% solution of potassium hydroxide in methanol to get free carboxylic acid 8 in high yield. The Boc-protective group of compound 3q was selectively removed by the treatment of its solution in methylene chloride with excess of trifluoroacetic acid at room temperature furnishing free amino ester 9 in 85% yield (Scheme 6). In addition, in order to demonstrate a feasibility for the further synthetic applications of the compounds obtained, e.g., in peptide synthesis or other useful derivatizations, we have performed selective deprotections of the N-PG-α-amino esters. Thus, the ester 3a was saponified using 5% solution of potassium hydroxide in methanol to get free carboxylic acid 8 in high yield. The Boc-protective group of compound 3q was selectively removed by the treatment of its solution in methylene chloride with excess of trifluoroacetic acid at room temperature furnishing free amino ester 9 in 85% yield (Scheme 6). Molecules 2022, 27, x FOR PEER REVIEW 8 of 23 Scheme 6. Removal of protective groups.

General Information
All solvents used in the reactions were freshly distilled from appropriate drying agents before use. All other reagents were distilled as necessary. The corresponding starting acetylenes were easily synthesized via the previously described protocol [56]. Analytical TLC was performed with Merck silica gel 60 F254 plates; visualization was accomplished with UV light or spraying with Ce(SO4)2 solution in 5% H2SO4. Chromatography was carried out using Merck silica gel (Kieselgel 60, 0.063-0.200 mm) and petroleum ether/ethyl acetate as an eluent. The NMR spectra were obtained with Bruker AV-300, AV-400, AV-500 and Inova-400 spectrometers operating at 300, 400, and 500 MHz, respectively, for 1 H (TMS reference), at 101 and 126 for 13  To a solution of the corresponding isoquinolone (0.1 g, 0.2 mmol, 1 equiv.) in dry dichloromethane (15 mL), pyridine (1.5 equiv.) and Tf2O (1.5 equiv.) were added at 0 °C. After having been stirred at 0 °C for 30 min, the reaction mixture was treated with water and extracted with dichloromethane. The organic layer was washed with saturated NaHCO3 (aq.), dried over anhydrous MgSO4, filtered, and evaporated to dryness. The residue was purified by column chromatography on silica gel (eluent petroleum ether/ethyl acetate = 15/1) to give the desired product.

General Information
All solvents used in the reactions were freshly distilled from appropriate drying agents before use. All other reagents were distilled as necessary. The corresponding starting acetylenes were easily synthesized via the previously described protocol [56]. Analytical TLC was performed with Merck silica gel 60 F 254 plates; visualization was accomplished with UV light or spraying with Ce(SO 4 ) 2 solution in 5% H 2 SO 4 . Chromatography was carried out using Merck silica gel (Kieselgel 60, 0.063-0.200 mm) and petroleum ether/ethyl acetate as an eluent. The NMR spectra were obtained with Bruker AV-300, AV-400, AV-500 and Inova-400 spectrometers operating at 300, 400, and 500 MHz, respectively, for 1 H (TMS reference), at 101 and 126 for 13

General Procedure for C-H Activation/Annulation of Aryl Hydroxamate with Acetylenes. Synthesis of the Compounds 3a-r
A dried 10 mL Shlenk tube equipped with a magnetic stirring bar was subsequently charged with a corresponding acetylene (0.1 g, 0.27 mmol, 1.0 equiv.), TFE (2 mL), corresponding aryl hydroxamate (0.06 g, 0.27 mmol, 1.0 equiv.), [Cp * RhCl 2 ] 2 (4.9 mg, 8.0 µmol, 3 mol%) and CsOAc (0.05 g, 0.27 mmol, 1.0 equiv.) under Ar. The reaction mixture was stirred at room temperature for 4 h until the completion of the reaction, monitored by TLC and 19 F NMR. By this time, the product precipitate had been formed and then was isolated from the reaction mixture by filtration.

General Procedure for the Sonogashira Reaction. Synthesis of the Compounds 6a-c
A dried 10 mL Shlenk tube was charged with a magnetic stir bar, DMF (5.5 mL) and corresponding triflate derivative (0.1 g, 0.15 mmol, 1.0 equiv.), and the solution was degassed three times. Then, (Ph 3 P) 2 PdCl 2 (5.2 mg, 7.5 µmol, 5 mol%) was added and the degassing procedure repeated. After that, Et 3 N (0.28 mL) and phenyl acetylene (0.02 g, 0.22 mmol, 1.5 equiv.) was added and the mixture was degassed. Then, copper iodide (2.8 mg, 0.01 mmol, 10 mol%) was added and the reaction was stirred at room temperature overnight. After the completion (monitored by TLC) the reaction mixture was poured into 1M HCl (20 mL) and extracted with ethyl acetate (3 × 10 mL). The organic layers were dried over anhydrous MgSO 4 , filtered and evaporated to dryness. The crude product was purified by column chromatography (eluent petroleum ether/ethyl acetate = 10/1) to give the desired product.

General Procedure for Ester Hydrolysis. Synthesis of the Compound 8
The corresponding isoquinolone 3a (0.3 g, 0.6 mmol) was dissolved in 5% KOH/MeOH-H 2 O (1:1) (13 mL) and stirred at room temperature for 2 h. After evaporation of solvents under reduced pressure, water (15 mL) was added to a residue, and the suspension was washed with diethyl ester (3 × 10 mL) before being acidified with HCl conc. until pH 3-4 and extracted with ethyl acetate (3 × 7 mL). The ethyl acetate extracts were combined and dried over anhydrous MgSO 4 , filtered, and evaporated to dryness.

General Procedure for the Removing of the Boc-Protecting Group. Synthesis of the Compound 9
A solution of Boc-protected isoquinolone 3q (0.25 g, 0.42 mmol) in a biphasic mixture of trifluoroacetic acid/dichlormethane (4 mL/10 mL) was stirred at room temperature for 1.5 h. After evaporation of solvents under reduced pressure, water (15 mL) was added to the residue and the resulting water solution was neutralized with saturated solution of sodium bicarbonate until pH 7. Then the mixture was extracted with diethyl ether (3 × 10 mL). The organic layer was dried over anhydrous MgSO 4 and evaporated to dryness.   164.8, 161.9, 159.2, 138.3 and 137.1, 137.1,  135.3 and 135.0, 133.5 and 133.1, 132.2 and 132.1, 131.3, 130.6 and 130.5, 129.8 and 129.5,  128.9 and 128.8, 128.4 and 128.2, 127.1 and 127.0, 125.6 and 125.5, 125.3 and 125    Methyl 2-(tert-butoxycarbonylamino)-3,3,3-trifluoro-2-((1-oxo-4-p-tolyl-6-(trifluoromethyl)-1,2-dihydroisoquinolin-3-yl)methyl)propanoate (3o). matrix least-squares technique against F 2 ,with the anisotropic thermal parameters for all non-hydrogen atoms using the SHELXL [60] program package. Hydrogen atoms of the NH groups were located in the different Fourier maps and freely refined without constraints. The remaining hydrogen atoms were placed in calculated positions and refined using a riding model with U iso (H) = 1.5U eq (C) for hydrogen atoms of methyl groups and U iso (H) = 1.2U eq (C) for other carbon atoms. The crystal data and structure refinement details are presented in Supplementary Materials (Table S1). Single-crystal X-ray diffraction analysis was performed using the equipment of the JRC PMR IGIC RAS.

Conclusions
In conclusion, we have elaborated a convenient pathway to a new series of α-CF 3substituted α-amino acid derivatives bearing a pharmacophore isoquinolone core in their backbone. The method is based on [4+2]-annulation of N-(pivaloyloxy) aryl amides with orthogonally protected internal acetylene-containing α-amino carboxylates under Rh(III)catalysis. The reaction smoothly proceeds at an ambient temperature in trifluoroethanol in the presence of 3 mol/% of rhodium dimer complex (Cp*RhCl 2 ) 2 and 1 equiv. of cesium acetate to afford the target products in good yields. The latter compounds proved to be suitable substrates for further conversion to valuable isoquinoline derivatives via a subsequent aromatization/cross-coupling synthetic operation. The biological activity of the obtained compounds is currently being studied.