Intramolecular C-N Bond Formation via Thermal Arene C-H Bond Activation Supported by Au(III) Complexes

Abstract

One of the main tactics to access C-N bonds from inactivated C-H functionalities is direct transition metal-supported aminations. Due to the often harsh reaction conditions, the current goal in the field is the search for more mild and sustainable transformations. Herein, we present the first solvent-free thermally induced C-N bond formation driven by Au(III) salts. The general structure of the products was confirmed by ¹H, ¹³C, ¹⁵N NMR, TGA-DTA and ATR/FT-IR analysis. Additionally, all derivatives were tested as catalysts in a three-component coupling reaction between phenylacetylene, benzaldehyde and piperidine and as anticancer agents on HL-60 and MCF-7 cell lines.

1. Introduction

Direct functionalization of an inactive and highly stable C-H bond, feasible in mild reaction conditions, remains a challenge in the field of organometallic reactions. Over the years, transition metals such as Pd, Pt, Rh, Ir and Ru have been widely utilized as metal centers in catalytic systems and have enabled the design of various transformation protocols [1,2]. Among various transition metals Au also started to be recognized as a promising constituent in organic synthesis. Particularly Au(III), isoelectronic to the widely used Pd(II) and Pt(II), can be easily applied in constructing analogous metal catalysts, additionally more available due to its higher abundance [3]. Gold complexes had been applied in numerous reactions enabling the formation of new C-C and C-X (X=O, N, S, halogen) bonds [4,5,6,7,8]. Most of these procedures rely on the Lewis acidity of Au, resulting in the activation of carbon–carbon $\pi$-bonds towards nucleophiles. More recently, the ability of Au(I)/Au(III) catalytic systems to perform cross-coupling transformations and arene C-H activation were also highlighted. Au(III), in the form of AuBr₃, HAuCl₄ and NaAuCl₄ can be efficiently applied in nitrogen-assisted C_ary-H activation through cyclometalation [9,10,11]. Cleavage and functionalization of the C-H bonds can be facilitated by prior nitrogen functional group coordination that directs the regiochemistry of the process. Several examples of N-donor ligands, like monodentate phenylpyridines [12], N, C, N-tridentate 1,3-(2-pirydyl) benzene [13] and N-confused porphirines [14], can undergo facile cycloauration to form various stable cyclometalated Au(III) species [10]. In this process, the nitrogen donor first coordinates the Au center to form the σ-complex 1. Further intramolecular C_aryl-H bond activation leads to the metallacycle 2. If the nitrogen donor is also the coupling partner towards the pseudo-nucleophilic metallated carbon, it enables, through the reductive elimination of Au(I) species, a straightforward transformation of a sp²C-H bond to a sp²C-N bond (structure 3, Scheme 1).

An ideal direct sp²C-N bond formation is one that can be performed through a coupling of C-H with N-H with no pre-activation of either reaction partner, defined as a cross dehydrogenative coupling (CDC) amination [15]. Until now, several research groups constructed various N-heterocycles by intramolecular CDC amination using Cu, Pd or Ru catalysts (Scheme 2) [16,17,18,19,20].

In this field, the applicability of Au-catalytic systems is limited and just starting to develop. Recently, Kim and co-workers [21] presented the first direct intramolecular sp²C-sp²N bond formation through carbene-induced reductive elimination from two Au(III) dichloride organometallics containing C(2′)-deprotonated, N(1),C(2′)-chelating 2-benzylpyridine* or 2-benzoylpyridine* anionic ligands ([Au^III(2-benzylpyridine*)Cl₂]—4x; [Au^III(2-benzoylpyridine*)Cl₂]—4y). Heating the 4x or 4y metallacycles with various imidazolium-derived cations 5 (1,3-bis(2,4,6-trimethylphenyl)-, 1,3-bis(2,6-diisopropylphenyl)-, 1,3-bis(4-hydroxophenyl)-, 1,3-di-tert-butyl- and 1,3-dicyclohexyl imidazolium; all in the chloride or tetrafluoroborate salts) in 1,4-dioxane and upon the presence of NBu₄(acac), resulted in the formation of heterocyclic cations 6x or 6y (with acac^- counterions) and the respective Au(I)-(imidazol-2-ylidene) monochloride complexes 7, with simultaneous HCl elimination (Scheme 3). The X-ray structure of polymeric (6y)_nⁿ⁺[AgCl₂]_nⁿ⁻ salt (ROFWUH) [22], as well as the ¹H and ¹³C NMR spectra (but not the ¹⁵N NMR ones) of 6x and 6y were also reported [21].

2. Materials and Methods

2.1. General

HAuCl₄ was prepared by dissolving gold in aqua regia, while NaAuCl₄ by the reaction of HAuCl₄ with NaHCO₃, followed by solvent evaporation. Benzaldehyde was purchased from Ubichem, phenylacetylene from TCI, and all other reagents and solvents from Sigma-Aldrich. Column chromatography was performed using Merck 40-63D 60 Å silica gel.

Elemental analyses were performed on a Vario MACRO CHN analyzer (Elementar Analysensysteme GmbH, Langenselbold, Germany). Melting points were measured with a Büchi Tottoli SPM-20 heating unit (Büchi Labortechnik AG, Flawil, Switzerland) and remained uncorrected. Thermogravimetric analysis was performed on SDT 2960 thermoanalizer (TA Instruments, New Castle, DE, USA). Far-IR spectra were measured on FT-IR Vertex 70V spectrometer (Bruker, Karlsruhe, Germany). Mass spectrum was collected on a Shimadzu High Performance Liquid Chromatograph/Mass Spectrometer LCMS-8030 (Shimadzu, Kyoto, Japan). ¹H and ¹³C NMR spectra (including ¹H-¹³C HSQC and HMBC) were measured by a Bruker Avance III 400 MHz NMR spectrometer, while the ¹H-¹⁵N HMBC ones by a Bruker Avance III 700 MHz spectrometer, at 295–300 K, in CDCl₃ or DMSO-d₆ (Bruker, Karlsruhe, Germany). The ¹H and ¹³C chemical shifts were referenced to TMS (with residual ¹H and ¹³C solvent signals as primary references—CDCl₃: 7.24 ppm and 77.2 ppm; DMSO-d₆: 2.50 ppm and 39.5 ppm), while those of ¹⁵N—to external neat nitromethane (CH₃NO₂). All NMR spectra were carried out using ACD/NMR Processor Academic Edition (product version 12.01), and the most important are presented in Supplementary Materials (Figures S1–S43).

Human promyelocytic leukemia cell line (HL-60) and a solid tumor-derived human breast adenocarcinoma cell line (MCF-7) were obtained from the European Collection of Cell Cultures (ECACC, Porton Down, UK). Leukemia cells were grown in RPMI 1640 plus GlutaMax I medium (Gibco/Life Technologies, Carlsbad, CA, USA). MCF-7 cells were maintained in Minimum Essential Medium Eagle (Sigma Aldrich, St. Louis, MO, USA) and supplemented with 2 mM glutamine and Men Non-essential amino acid solution (Sigma Aldrich, St. Louis, MO, USA). Both media were supplemented with 10% heat-inactivated fetal bovine serum (Biological Industries, Beit-Haemek, Israel) and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin) (Sigma-Aldrich, St. Louis, MO, USA). The nontumorigenic mammary gland/breast MCF-10A cell line was purchased from the American Type Culture Collection (ATCC). For MCF-10A cells MEGM Mammary Epithelial Bullet Kit was used. Cells were maintained at 37 °C in a 5% CO₂ humidified atmosphere and were grown until 80% confluent.

2.2. Procedures

2.2.1. General Procedure for the Synthesis of Ligands 8–15

This general procedure can be exemplified by the synthesis of 8. A mixture of 2-bromopyridine (5.0 mmol), benzenethiol (5.0 mmol) and K₂CO₃ (10.0 mmol) in dimethyl sulfoxide (DMSO; 4 mL) was stirred at 110 °C for 24 h. It was cooled to room temperature, diluted with water (10 mL) and extracted with dichloromethane (4 × 5 mL). The organic phase was washed with water (3 × 20 mL), dried over anhydrous MgSO₄, filtered and concentrated under vacuum. The residue was purified by column chromatography on silica gel with dichloromethane/hexane 8:2 as eluent to afford the sulfide. The 9–15 heterocycles were obtained similarly, using various derivatives of 2-bromopyridine (6-methyl-, 5-methyl-, 4-methyl- for 9, 10, 11, respectively) or benzenethiol (4-methyl-, 4-tert-butyl-, 4-bromo-, 4-nitro- for 12, 13, 14, 15, respectively). The yields were as follows: 8—78%, 9—82%, 10—75%, 11—88%, 12—79%, 13—77%, 14—72%, 15—70%. The ¹H, ¹³C and ¹⁵N NMR spectra of 8 and 12–15 were already reported and assigned in our recent paper [23], while those of 9–11 are described in Supplementary Data (Figures S1–S12).

2.2.2. General Procedure for the Synthesis of Au(III) Trichloride Complexes 8a–15a

A total of 1 mmol of NaAuCl₄ in 18 mL of water was added to 1 mmol of the corresponding ligand 8–15 in 30 mL of methanol and stirred at 50 °C for 24 h. The yellow/orange/red crystals were filtered on a Büchner funnel and dried on air. This method produced Au(III) trichloride complexes 8a–15a with the following yields: 8a—78%, 9a—86%, 10a—88%, 11a—86%, 12a—69%, 13a—79%, 14a—77%, 15a—72%. The ¹H, ¹³C and ¹⁵N NMR spectra of 8a and 12a–15a were already reported and assigned in our recent paper [23], while those of 9a–11a are given in Supplementary Data (Figures S13–S18).

2.2.3. General Procedure for the Synthesis of Tetrachloroaurate(III) Salts 8b–15b

The 0.20 mmol of the corresponding Au(III) trichloride complex 8a–15a was placed in one-neck RBF under vacuum (0.1 mm Hg) and heated at certain temperature (8a—180 °C, 9a—150 °C, 10a—190 °C, 11a—180 °C, 12a—165 °C, 13a—170 °C, 14a—195 °C, 15a—190 °C) for 6 h. Then, the resulting solid mixture was allowed to cool to room temperature and washed with CHCl₃. The solid residue was dissolved in MeOH and filtered, and the solvent removed under reduced pressure. This method produced tetrachloroaurate(III) salts 8b-15b with the following yields: 8b—69%, 9b—54%, 10b—71%, 11b—82%, 12b—66%, 13b—75%, 14b—53%, 15b—65%. The ¹H, ¹³C and ¹⁵N NMR spectra of 8b–15b are given in Supplementary Data (Figures S19–S43).

2.3. Studies Of Catalytic Properties of 8b–15b

A mixture of catalyst (8b–15b, 0.01 mmol), benzaldehyde 19 (106 mg, i.e., 1.0 mmol), piperidine 20 (93.7 mg, i.e., 1.1 mmol) and phenylacetylene 18 (153 mg, i.e., 1.5 mmol) was stirred at 40–55 °C for 24–48 h. The yields of 19 conversions were determined by ¹H NMR analysis of crude reaction mixtures, using 10.05 and 4.83 ppm signals integration areas.

2.4. Metabolic Activity Assay

The MTT (3-(4,5-dimethyldiazol-2-yl)-2,5 diphenyl tetrazolium bromide) assay, which measures activity of cellular dehydrogenases, was used to determine the cytotoxicity of new compounds [22]. In brief, cells were seeded on 96-well plates in 100 µL of culture medium and left to grow for 24 h. Tested compounds were dissolved in DMSO and diluted with the complete culture medium. To each well, 100 µL of such prepared dilution was added to obtain concentration range from 10⁻⁷ to 10⁻³ M. After 24 h of treatment, MTT (5 mg/mL in PBS) was added, and cells were incubated for an additional 2 h. Then, the medium was removed and insoluble blue formazan crystals were dissolved in 100 µL of DMSO. The absorbance of the formazan product was measured at 540 nm using FlexStation 3 Multi-Mode Microplate Reader (Molecular Devices, LLC., San Jose, CA, USA). The untreated cells were used as a control. The data were expressed as mean ± SEM of three independent experiments.

3. Results and Discussion

3.1. Synthesis and Characterization

Herein, we report that the C-H activation with the formation of intramolecular sp²C-sp²N bond can be conducted in solvent-free conditions, just by heating the Au(III) σ-complexes 8a–15a below the particular melting point.

In the first step, we have prepared a series of 2-phenylsulfanylpyridine ligands 8–15 and transformed them into the Au(III) trichloride complexes 8a–15a by the methodology presented previously by Fuchita et al. [24], and later modified in our research group (Scheme 4) [23]; the details of these syntheses are given below.

The species 8, 12–15 and 8a, 12a–15a were already spectroscopically (¹H, ¹³C, ¹⁵N NMR), and structurally (single crystal X-ray study of 8a—DUCYEI and 14a—DUCYIM) [21] characterized [22], while the data of 9–11 and 9a–11a are described in Supplementary Data (Figures S1–S18).

In the second step, the Au(III) trichloride complexes 8a–15a were converted via heating to the corresponding tetrachloroaurate(III) salts 8b–15b, which contain heterocyclic cations analogous to those of 6x and 6y, described by Kim et al. [21]. This procedure was effective for the simplest complex 8a and its analogues, based on substrates functionalized on either the pyridine or the phenyl ring (9a–11a and 12a–15a, respectively; Scheme 5). It was successfully applied in the gram-scale synthesis of 8b (1 mmol), leading to 86% yield of the corresponding product. The spectroscopic characterization (IR, ¹H, ¹³C, ¹⁵N NMR) of 8b–15b is given in Supplementary Data (Figures S19–S43). We assume that the mechanism of 8a → 8b reaction includes the formation of the intermediate Au(III) dichloride organometallic 16 (following HCl elimination), which is transformed to the heterocyclic cation present in the tetrachloroaurate(III) salt 8b. Moreover, a part of 8a is also decomposed to the 2-phenylsulfanylpyridine ligand 8.

The elimination of compound 8 was confirmed by thermogravimetric analysis TGA-DTA. Upon heating when the temperature reached ca. 180 °C, the complex 8a decomposed in two steps. The first degradation step, ca. 31% decrease of weight, indicated the 8a → 8 reaction. Next, at elevated temperature, the second step revealed the elimination of HAuCl₄ species (Figure 1).

The analogous reactions 9a–15a → 9b–15b occur probably according to the same mechanism.

The assumed structural formulae of the cations present in the 8b–15b salts are in agreement with the fully assigned (by ¹H-¹³C and ¹H-¹⁵N HMQC/HMBC techniques) NMR spectra. The respective ¹H, ¹³C and ¹⁵N chemical shifts, and the comparison of these chemical shifts to those for the corresponding parent heterocycles 8–15, allows us to determine the relevant differences, which reveal characteristic patterns similar for all studied cations thus, confirming their analogous structures (Tables S7–S9, part S2, Supplementary Data).

The most characteristic phenomenon is large deshielding (by ca. 0.4–2.2 ppm) of all heterocyclic protons, expressed especially for the H(3) and H(3′) atoms (Δ^1H = ca. 1.6–2.2 ppm and ca. 1.3–1.7 ppm, respectively), as well as for the nitrogen-adjacent H(6) proton (Δ^1H = ca. 1.6–1.9 ppm); in the latter case this effect results in unusually high δ_cat^1H values (>10 ppm).

The heterocyclic carbons are either deshielded (C(3), C(4), C(5), C(2′)) or shielded (C(2), C(6), C(1′), C(3′), C(6′)), but also nearly unaffected (C(4′), C(5′)). The heterocyclic nitrogens are significantly shielded (by ca. 89–92 ppm), this effect reflecting their quaternary character (=N<⁺).

The ¹H–¹³C long-range (via two or three bonds) correlations inside the pyridine or phenyl moieties, exhibited by the ¹H–¹³C HMBC spectra of the Au(III) trichloride complexes 8a–15a and the cations present in the tetrachloroaurate(III) salts 8b–15b, are typical for such six-membered aromatic rings, and are analogous to those recently reported for the heterocycles 8 and 12–15 [23] (and presently for 9–11).

However, in case of 8b and 10b–15b also, another correlation between H(6) and C(2′) atoms (via three bonds) is observed, exhibiting the presence of the new N(1)–C(2′) bonding (obviously, it is not observed for 9b which has no H(6) proton); otherwise, i.e., if this additional connection between pyridine and phenyl rings was absent, there would be no possibility for any ¹H–³C spin-spin interactions involving H(6) and C(2′). This H(6) –C(2′) correlation, being a crucial proof for the tricyclic structure of the cations present in 8b–15b salts, has been graphically emphasized (with a circle and an arrow) at the respective ¹H-¹³C HMBC spectra, reprinted in Supplementary Data (Figures S21, S28, S31, S34, S37, S40, S43).

Additionally, the presence of tetrachloroaurate(III) counterions in the salts 8b–15b was confirmed by far-IR spectra. One strong band at 350–355 cm⁻¹ (exemplified for 8b at Figure 2; see also Supplementary Data, Schemes S7–S14) and corresponds to the well-known asymmetric Au-Cl stretching vibration in the AuCl₄⁻ anion [25].

Moreover, the mass spectrum of the tetrachloroaurate(III) salt 8b exhibits the main peak at 186.1, which corresponds well to that calculated for the respective heterocyclic cation C₁₁H₈AuCl₄NS⁺ (186.0; see Supplementary Data, Figure S22).

3.2. Catalytic Properties

In the past, some Au(III)–salen complexes and the [Au^III(2-phenylpyridine*)Cl₂] organometallic were exhibited to catalyze three-component coupling reactions between aldehydes, amines and alkynes with high yields; when chiral prolinol derivatives were used as substrates, excellent diastereoselectivities were reached (up to 99:1) [26,27]. Similarly, we have recently observed high catalytic activity of the Au(III) trichloride complexes 8a and 12a–15a in the coupling reaction between phenylacetylene 18, benzaldehyde 19 and piperidine 20, to form 1-(1,3-diphenylprop-2-yn-1-yl)piperidine 21 (yields 84–99%) (Scheme 6) [23].

The above facts prompted us to evaluate the catalytic properties of the presently studied tetrachloroaurate(III) salts 8b–15b, in the same 18 + 19 + 20 three-component reaction; the yields at 55 °C were as high as 97–99% (

Table 1. Yields of three-component reaction between phenylacetylene, benzaldehyde and piperidine catalyzed by tetrachloroaurate(III) salts 8b–15b.

Catalyst	t [h]	T [°C]	Yield [%]
8b	24	55	99
9b	24	55	97
10b	24	55	97
11b	24	55	99
12b	24	55	99
13b	24	55	99
14b	24	55	98
15b	24	40	58
15b	48	40	66
15b	24	55	99
NaAuCl4	24	55	84

). It is worth noting, that lowering the temperature to 40 °C resulted in a significant yield decrease, below 70%—as exemplified by the species 15b. The same reaction catalyzed by sodium tetrachloroaurate(III) enabled us to obtain the final piperidine derivative, yet the yield was lower than for the used catalysts 8b–15b. This indicates that the presence of the organic cation enhances the reactions efficiency.

In the next step, using catalyst 8b, we tested various aldehydes (p-anisaldehyde 22, 4-(trifluoromethyl) benzaldehyde 23, cyclohexanecarboaldehyde 24) and amines (pyrrolidine 25, morpholine 26, dibutylamine 27) for three-component reactions. Corresponding products 28–33 were obtained with very high yields (Scheme 7).

3.3. Biological Activity

Due to the known anticancer properties of Au(I) and Au(III) complexes, or organometallics, e.g., with thiolates, thioureas, N-heterocyclic carbines, phosphanes and alkynes [28], the in vitro cytotoxicity of the tetrachloroaurate(III) salts 8b–15b was tested against leukemia HL-60 and breast cancer MCF-7 cell lines. The cells were exposed to the increasing concentrations of analogues for 48 h and investigated using conventional MTT assay [29]. Cisplatin was used as a positive control. The most cytotoxic on both cell lines was 13b, with the half maximal inhibitory concentration (IC₅₀) of 20.9 ± 0.9 and 16.9 ± 0.8 µM on HL-60 and MCF-7 cells, respectively (

Table 2. Cytotoxic activity of tetrachloroaurate(III) salts 8b–15b.

Compound	IC50 [μM] a
	HL-60	MCF-7	MCF-10A	Selectivity Index MCF-10A/MCF-7
8b	>100	>100
9b	42.7 ± 1.4	76.9 ± 4.4
10b	>100	71.6 ± 6.7
11b	36.5 ± 2.2	74.0 ± 0.3
12b	41.2 ± 1.8	21.3 ± 2.0	21.8 ± 2.3	1.02
13b	20.9 ± 0.9	16.9 ± 0.8	20.7 ± 0.5	1.20
14b	75.8 ± 6.8	41.5 ± 1.7
15b	>100	>100
Cisplatin	4.9 ± 0.1	23.6 ± 1.3

^a Compound concentration required to inhibit metabolic activity by 50%. Data are expressed as the mean ± SEM from the concentration response curves of at least three experiments.

). In MCF-7 cells this compound exhibited 1.4-fold higher inhibitory activity than Cisplatin. Two most cytotoxic for MCF-7 cells compounds, 12b and 13b, were also tested on a non-tumorigenic MCF-10A cell line, to evaluate their influence on normal cells. Analogue 12b was equally active on MCF-7 and MCF-10A cells, while 13b was 1.2-fold less toxic for normal than for breast cancer cells.

4. Conclusions

In this article we present the first thermal C-N bond formation supported by Au(III) salts, performed in solvent-free conditions. This type of cross dehydrogenative coupling amination is one of the first examples efficiently conducted using gold complexes. The structure of the obtained compounds was confirmed by ¹H, ¹³C, ¹⁵N NMR, TGA-DTA, and ATR/FT-IR analysis with a detailed discussion concerning the differences in the chemical shifts of cations in salts and their parent ligands. The mechanism of the reaction was also investigated and indicated the formation of a metallacycle as an intermediate. Additionally, all Au(III) salts were tested as catalysts in a multicomponent synthesis of propargylic amine. The reaction was almost quantitative in each case. We observed that organic cation enhanced the efficiency of the reaction. The anticancer activity was evaluated, showing a promising antiproliferative potential of the tert-butyl salt.