Thermodynamic Stability of Flavylium Salts as a Valuable Tool to

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Thermodynamic Stability of Flavylium Salts as a Valuable Tool to Design the Synthesis of A-Type Proanthocyanidin Analogues Alfonso Alejo-Armijo, A. Jorge Parola, Fernando Pina, Joaquín Altarejos, and Sofia Salido J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b01780 • Publication Date (Web): 07 Sep 2018 Downloaded from http://pubs.acs.org on September 7, 2018

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The Journal of Organic Chemistry

Thermodynamic Stability of Flavylium Salts as a Valuable Tool to Design the Synthesis of A-Type Proanthocyanidin Analogues

Alfonso Alejo-Armijo,†,‡ A. Jorge Parola,*,‡ Fernando Pina,‡ Joaquín Altarejos,† and Sofía Salido*,†

†

Departamento de Química Inorgánica y Orgánica, Facultad de Ciencias Experimentales,

Universidad de Jaén, Campus de Excelencia Internacional Agroalimentario, ceiA3, 23071 Jaén, Spain

‡

LAQV, REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia,

Universidade NOVA de Lisboa, 2829-516, Caparica, Portugal

ACS Paragon Plus Environment

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ABSTRACT: A convenient method to synthesize A-type proanthocyanidin (PAC) analogues from flavylium salts and π-nucleophiles has been developed. It was found that the thermodynamic stability of the starting flavylium salt, assessed by the measurement of the apparent acidity constant (K'a), was the key parameter to design effective one-pot reactions between flavylium salts and nucleophiles such as phloroglucinol and (+)-catechin. When flavylium salts have a pK'a value of 1.7 or lower, the synthesis of the corresponding 2,8dioxabyciclo[3.3.1]nonane derivative was properly achieved.


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Proanthocyanidins (PACs) are phenolic compounds widely distributed in nature. PACs of the A-type present the unusual 2,8-dioxabicyclo[3.3.1]nonane skeleton (Figure 1) which affords conformational rigidity to the structure. This feature could play an important role in the main biological activities reported for A-type PACs: antiviral, anti-adhesion, and antitumoral properties.1−3 As an example, cinnamtannin B-1 (1) is an A-type PAC (Figure 1) isolated from Laurus nobilis L. by the authors,4 which exhibits a large number of cellular actions such as antiaggregant and antiapoptotic effects in human platelets,5,6 and antimicrobial and antibiofilm activities,7 among many others.8 OH HO

O

OH OH

OH OH

O

OH OH

O

O

OH

OH

HO OH

HO

OH

Cinnamtannin B-1 (1)

Figure 1. Biologically important 2,8-dioxabicyclo[3.3.1]nonane core of A-type proanthocyanidins.

Several attempts have been made in order to synthesize both A-type PACs9,10 and their synthetic analogues (2,8-dioxabi-cyclo[3.3.1]nonane derivatives).11−14 Moreover, synthetic analogues afford the opportunity to prepare a large series of compounds with similar or improved biological activities when compared with natural ones.15 Although almost all methods reported to date synthesize these bicyclic derivatives using 2-hydroxychalcones as starting materials,11−13 we consider that starting from flavylium salts could be a more interesting approach16,17 (Scheme 1). As far as we know, it remains unclear the electronic


3


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features that flavylium salts must fulfil to be suitable starting materials for the synthesis of 2,8-dioxa-bicyclo[3.3.1]nonane derivatives.17

SCHEME 1. Flavylium Salts as Starting Point to Synthesize 2,8-Dioxabicyclo[3.3.1]nonane Derivatives

The stability of flavylium compounds depends on several factors including pH and the nature of the substituents. These organic cations are involved in a complex network of chemical reactions that should be studied in terms of thermodynamics and kinetics in order to understand their reactivity with nucleophiles18 (Scheme 2). The overall thermodynamic stability of the flavylium cation may be measured using the apparent acidity constant K'a which characterizes the overall equilibrium between the flavylium cation and all other species (see below eqs 5−6).


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SCHEME 2. Flavylium Salt Chemical Equilibria and Global Thermodynamic Constant Exemplified for 3-Substituted 3',4',7-Trihydroxyflavylium Derivatives

When electron-donor substituents are present in the flavylium structure they increase the charge density in the pyrylium ring, making the flavylium cation more stable against hydration. In contrast, when electron-withdrawing groups are present the flavylium is easily attacked by nucleophiles, for instance, in aqueous solutions amino groups give rise to pK'a values between 4 and 6 while nitro groups give pK'a values close to zero.18 These factors should be taken into account during the design of this kind of molecule for their use in synthesis, and may be the reason why this kind of particular molecule has rarely been used in the synthesis of 2,8-dioxabicyclo[3.3.1] nonane derivatives.

With this idea in mind, our research groups have recently described the synthesis of several flavylium salts and studied their thermodynamic and kinetic properties.19 The analysis


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of the data obtained in this study allowed us to propose that the overall thermodynamic stability of flavylium salts, as measured by the apparent acidity constant K'a (Scheme 2),18 is related to the electronic density of the flavylium cation.19 On this basis we envisioned that electronic density of flavylium salts could be estimated by the experimental measurement of K'a, and therefore their applicability to the synthesis of 2,8-dioxabicyclo[3.3.1]nonanes could be predicted. For these reasons we herein report: (i) the synthesis of several flavylium cations, including two new ones, (ii) experimental determination of the K'a for a selection of flavylium salts, (iii) their reactivity with two different nucleophiles, phloroglucinol or (+)-catechin, to afford the corresponding 2,8-dioxabicyclo[3.3.1]nonane derivatives, ten of them being new. We

have

also

developed

a

new

microwave

methodology

to

synthesize

2,8-

dioxabicyclo[3.3.1]nonanes using flavylium salts with high electronic density to afford new dioxabicycles that could not be prepared by the standard method previously reported.17

Synthesis of flavylium salts (9–17). The synthesis of flavylium salts was performed by aldol condensation in acidic media according to reported procedures.16,20,21 The substitution pattern in rings A and C was designed to cover a range of electronic densities in the 1benzopyrylium moiety, using from OH electron donor groups to NO2 electron withdrawing ones. Ring B was kept constant throughout the series and was chosen to be 3',4'dihydroxybenzene to simulate the catechol moiety in natural cyanin. Table 1 presents the methodology followed to obtain flavylium salts 9–17.


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Table 1. Synthesis of Flavylium Salts and respective pK'a Values in MeOH/H2O 1:1 (v/v)

Entry 1 2 3 4 5 6 7 8 9 10 11 12 13 a

compd 9 10 11 11 12 13 14 15 16 16 17 17 17

R1 H H H H H H H H H H OH OH OH

R2 H H H H NO2 NO2 H H H H H H H

R3 OH OH OH OH H H H H H H OH OH OH

R4 H CH3 Cl Cl H CH3 H CH3 Cl Cl H H H

methoda i i i ii i i i i i ii i ii iii

pK'ab 3.1c 2.7d –0.65d –0.65d –1.7 –2.3 1.7 1.3 –1.9 –1.9 3.8e 3.8e 3.8e

Reaction conditions: i. H2SO4, HOAc; ii. HCl (g), EtOH; iii. HCl (g), MeOH; bSee Experimental section; cIn

water;22 dIn MeOH/H2O 1:1 (v/v);19 eIn MeOH/H2O 3:1 (v/v).23

Most flavylium salts have been synthesized following a similar procedure to that described by the group,21 involving condensation in CH3CO2H/conc. H2SO4 4:1 (v/v). However, these conditions failed with increased steric hindrance of the benzaldehyde or acetophenone derivatives (entries 3, 9 and 11 in Table 1). In these cases, other alternative synthetic methods should be used to solve these difficulties16,20 (entries 4, 10, 12 and 13 in Table 1), in particular by using alcohols saturated with gaseous hydrogen chloride.


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pK'a measurements. The flavylium cation is a stable species only at very acidic pH values. Raising the pH leads to the appearance of different species shown in Scheme 2 in a complex kinetic process involving 4 steps. The following set of equations account for the chemical equilibria of Scheme 2.

AH+ + H2O AH+ + 2 H2O B Cc

A + H3O+ B + H3O+

Cc Ct

Ka

Proton transfer

(1)

Kh

Hydration

(2)

Kt

Tautomerization

(3)

Ki

Isomerization

(4)

The system can be simplified by considering a single acid-base equilibrium involving the AH+ species (acid) and all other species collected under CB (conjugate base), eq 5, characterized by the apparent global acidity constant K'a: AH+ + 2 H2O

CB + H3O+

K'a

(5)

where

[CB] = [A] + [B] + [Cc] + [Ct]

(6)

In this sense, the K'a constant is related to the reactivity of the flavylium salt with a water molecule. Therefore, this parameter could be used to predict the reactivity of flavylium salts against other nucleophiles. The pK'a values in methanol/water 1:1 (v/v) were determined by UV-vis absorption spectrophotometry on equilibrated solutions, upon pH jumps from an acidic solution of the flavylium compound to other pH values. These results are presented in Table 1. Flavylium cations with hydroxyl electron-donor groups (9, 10, 17) present the


8

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highest pK'a values, while those containing the nitro or chloro electron-withdrawing groups (11–13, 16) present the lowest pK'a values, confirming that they are more easily hydrated to form the hemiketal and subsequent species rendering the flavylium less stable, i.e., more susceptible to nucleophilic attack.

Synthesis of 2,8-dioxabicyclo[3.3.1]nonane derivatives (18–33). The syntheses of 2,8dioxabicyclo[3.3.1]nonane derivatives were performed following a procedure similar to those described in the literature.16,17 Thus, the corresponding flavylium salts were combined with 1 equivalent of phloroglucinol or (+)-catechin using the standard conditions described by Kraus17 (method iv) as a starting point. Tables 2 and 3 present the compounds obtained upon reaction with phloroglucinol and (+)-catechin, respectively, while Figure 2 plots the dependence of reaction yields (using method iv) on the electron density of the flavylium salts as assessed by the pK'a value.

Table 2. Reaction between Flavylium Salts and Phloroglucinol

Entry

compd

R1

R2

R3

R4

methoda

1 2 3 4 5 6 7

18 18 19 20 21 22 23

H H H H H H H

NO2 NO2 NO2 H H H H

H H H H H H OH

H H CH3 Cl H CH3 Cl

iv v iv iv iv iv iv


yield (%)b 85 32 83 81 84 80 78

9


8 9 10 11 12 13 14 15 16 17 a

H H H H H H H OH OH OH

23 24 24 24 25 25 25 26 26 26

H H H H H H H H H H

OH OH OH OH OH OH OH OH OH OH

Cl H H H CH3 CH3 CH3 H H H

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v iv v vi iv v vi iv v vi

10 1 25 20 0 12 0 0 0 40

Reaction conditions: iv. MeOH, 50 °C, 24 h; v. MW, MeOH, 80 °C, 20 min; vi. MW, MeOH/aq buffer (pH:

5.8), 100 °C. bYield calculated by HPLC calibration.

Table 3. Reaction between Flavylium Salts and (+)-Catechin

Entry

compd

R1

R2

R3

R4

methoda

yield (%)b

1 2 3 4 5 6 7 8 9 10

27 28 29 30 31 32 33 33 34 34

H H H H H H H H H H

NO2 NO2 H H H H H H H H

H H H H H OH OH OH OH OH

H CH3 Cl H CH3 Cl H H CH3 CH3

iv iv iv iv iv iv iv v iv v

64 61 63 52 25 57 0 8 0 0


10

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a

b

Reaction conditions: iv. MeOH, 50 °C, 24 h; v. MW, MeOH, 80 °C, 20 min; Yield calculated by HPLC

calibration.

Flavylium salts with low electronic density (pK'a < 1.7) reacted properly with both nucleophiles (entries 1, 3–7 in Table 2; entries 1–4 and 6 in Table 3). For each flavylium salt, a higher yield was observed for the reaction with phloroglucinol which is a stronger nucleophile when compared with (+)-catechin. The weaker nucleophilic character of (+)catechin led to greater discrimination with respect to the effect of the pK'a of the flavylium salts in reaction yields, as shown in Figure 2.

Figure 2. Reaction yield for the formation of 2,8-dioxabicyclo[3.3.1]nonane adducts upon reaction of flavylium salts with phloroglucinol (red) and (+)-catechin (blue) using method iv as a function of the pK'a value of the flavylium cations (Tables 2 and 3).

Moreover, when the electronic density of the flavylium salts increased, the yield diminished to such an extent that the reaction did not occur (entries 9, 12 and 14–16 in Table 2; entries 7 and 9–10 in Table 3). The formation of compound 30 from flavylium 14 and (+)-


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catechin in 52% yield falls out of the trend (open blue circle in Figure 2) most likely due to the absence of the methyl group whose steric hindrance leads to the lower yield observed for the synthesis of compound 31 from flavylium 15 (25%) (entry 5 in Table 3). Although this steric effect is also expected for flavylia 12 and 13, in these strongly electron deficient substrates the electronic component prevails and the difference in yield is much smaller (∆yield = 3% for analogues formed from 12 and 13 (entries 1–2 in Table 3) while ∆yield = 27% for analogues formed from 14 and 15 (entries 4–5 in Table 3)).

For those flavylium salts with high electronic density, we have developed a new synthetic

methodology

to

achieve

the

synthesis

of

the

corresponding

2,8-

dioxabicyclo[3.3.1]nonane (method v in Tables 2 and 3). In these cases, the corresponding flavylium salt with high electronic density (pK'a > 1.7) was combined with 1 equivalent of phloroglucinol or (+)-catechin, dissolved in anhydrous methanol, and heated in a microwave at 80 ºC for 20 minutes. This microwave methodology allowed us to use high electronic density flavylium salts as starting materials to obtain adducts in low yields (entries 10 and 13 in Table 2; entry 8 in Table 3). However, when we attempted to apply this new microwave methodology to synthesize bicycle 26 (entry 16 in Table 2), the reaction failed, and other microwave methodology had to be used16 in order to achieve its synthesis (entry 17 in Table 2).

In conclusion, the thermodynamic stability of flavylium salts (determined by the constant K'a) depends primarily on the hydration reaction, and is related to their electronic density, making it a very interesting tool for predicting their reactivity with mild π-


12

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nucleophiles. Thus, it has been observed that when flavylium salts have a pK'a value of 1.7 or lower, standard conditions (method iv) may be useful to achieve the synthesis of bicyclic derivatives. For flavylium salts with higher pK'a values, microwave methods (v or vi) should be used in order to achieve the reaction between these flavylium salts and nucleophiles. This synthetic methodology has allowed us to synthesize nine flavylium salts (9–17), two of which were synthesized for the first time (15 and 16), and sixteen 2,8-dioxabyciclo[3.3.1]nonane derivatives (18–33), ten of which were synthesized for the first time (19, 20, 22–25, 29 and 31–33).

EXPERIMENTAL SECTION

Reagents, Techniques and Instruments. All reactions were carried out at room temperature or in oil baths with electronic temp control under nitrogen atmosphere with dry solvents and under anhydrous conditions, unless otherwise mentioned. Microwave assisted reactions were performed in a sealed vessel using a CEM Discover monomodal MW reactor with temp and pressure internal probes. All the chemicals were purchased and used without further purification. Anhydrous MeOH was dried according to a standard method.24 Yields refer to chromatographically pure compounds, unless otherwise stated. Reactions were monitored by thin-layer chromatography (TLC) on precoated aluminum sheets of silicagel (Merck 60 F254) and visualized by UV irradiation (254 nm; 365 nm) and by analytical HPLC performed on a C18 reverse-phase Spherisorb ODS-2 column, 250 mm × 3 mm i.d., 5 µm, at a flow rate of 0.7 mL/min. Separations were performed by size exclusion column chromatography (Sephadex LH-20; particle size 25–100 µm) and by semipreparative HPLC,


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the latter being performed on a RP-HPLC column (Spherisorb ODS-2 column, 250 mm × 10 mm i.d., 5 µm) at a flow rate of 5 mL/min. IR spectra were recorded by attenuated total reflection (ATR) method using neat compds. The wavelengths are reported in reciprocal centimeters (νmax/cm−1). UV/Vis absorption spectra were recorded on Varian Cary 100 Bio or Varian Cary 4000 spectrophotometers.1H and

13

C NMR spectra were acquired on a 400/100

MHz spectrometer. The solvent for NMR samples was CD3OD or CD3CN. Chemical shifts are reported in parts per million (δ, ppm). Internal reference for NMR spectra is TMS at 0.00 ppm. Coupling constants are reported in Hertz (J, Hz). Multiplicities of the signals are reported using the standard abbreviations: singlet (s), broad singlet (br s), doublet (d), broad doublet (br d), doublet of doublets (dd), doublet of doublet of doublets (ddd), triplet (t), doublet of triplets (dt) and multiplet (m). The complete assignment of 1H and 13C signals was performed by analysis of the correlated homonuclear H,H-COSY and heteronuclear H,CHMBC, H,C-HSQC spectra. High-resolution mass spectra (HRMS) were recorded on a quadrupole time-of-flight (Q-TOF) mass spectrometer.

General Procedure A for the Synthesis of Flavylium Salts (9, 10, 12–15). A mixture of the salicylic aldehyde derivative (2–4, 1 mmol), the 3',4'-dihydroxyacetophenone derivative (6–7, 1 mmol), 98% H2SO4 (0.3 mL; 5.4 mmol) and HOAc (1.3 mL) was stirred overnight at rt following a similar procedure to that described by Calogero et al.21 Then, Et2O (30 mL) was added and a reddish solid precipitated. The solid was filtered off, carefully washed with Et2O and vacuum dried. The structure and purity of the known starting flavylium salt materials (9 (261 mg, 74% yield);21 10 (260 mg, 71% yield);19 12 (293 mg, 77% yield);15


14

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14 (302 mg, 90% yield)21) were confirmed by comparison of their physical and spectral data (1H NMR and 13C NMR) with those reported in literature. 3',4'-Dihydroxy-3-methyl-6-nitroflavylium hydrogensulfate (13). Procedure A was followed by using aldehyde 3 (167 mg, 1 mmol) and ketone 7 (166 mg, 1 mmol). A blackish solid was obtained by treating the solution with Et2O. The solid was filtered off, washed with Et2O, and dried, yielding the compd 13 as a black solid (337 mg, 85%). Melting point: 230 oC (decomposes). 1H NMR (400 MHz, DCl/CD3CN, pD ≈ 1.0) δ 8.05 (m, 2H, H-5, H-7), 6.99 (d, J = 9.3 Hz, 1H, H-8), 6.88 (br s, 1H, H-2'), 6.77 (m, 3H, H-4, H-5', H-6'), 1.68 (s, 3H, CH3); 13

C NMR (100 MHz, DCl/CD3CN, pD ≈ 1.0) δ 158.8 (C-9), 146.9 (C-4'), 146.0 (C-3'), 143.1

(C-6), 134.8 (C-3), 133.7 (C-1'), 125.5 (C-7), 123.4 (C-4), 122.7 (C-5), 121.5 (C-10), 118.9 (C-6'), 116.5 (C-8), 115.7 (C-5') 114.6 (C-2'), 106.6 (C-2), 18.9 (CH3). FT-IR (ATR) νmax 3069, 1589, 1535, 1489, 1408, 1308, 1277, 1165, 1119, 1055, 943, 862, 824, 750, 706, 681. UV-vis (MeOH/HCl 1:1; pH = 1): λmax (log ε): 312 (4.51), 274 (4.86). HRMS (ESI/Q-TOF) m/z [M]+ calcd for C16H12NO5 298.0715, found 298.0710. 3',4'-Dihydroxy-3-methylflavylium hydrogensulfate (15). Procedure A was followed by using aldehyde 4 (0.1 mL, 1 mmol) and ketone 7 (166 mg, 1 mmol). An orange solid was obtained by treating the solution with Et2O. The solid was filtered off, washed with Et2O, and dried, yielding the compd 15 as an orange solid (316 mg, 90%). Melting point: 190 oC (decomposes). 1H NMR (400 MHz, DCl/CD3CN, pD ≈ 1.0,) δ 9.29 (s, 1H, H-4), 8.23 (m, 3H, H-5, H-7, H-8), 7.90 (m, 3H, H-6, H-2', H-6'), 7.18 (d, J = 9.1 Hz, 1H, H-5'), 2.88 (s, 3H, CH3); 13C NMR (100 MHz, DCl/ CD3CN, pD ≈ 1.0) δ 174.0 (C-2), 156.1 (C-4), 154.4 (C-9), 152.7 (C-4'), 144.5 (C-3'), 137.5 (C-7), 129.2 (C-3), 129.1 (C-6), 128.7 (C-5), 126.7 (C-6'), 122.7 (C-10), 120.9 (C-1'), 117.8 (C-8), 117.5 (C-2'), 115.8 (C-5'), 19.7 (CH3). FT-IR (ATR)


15


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νmax 3086, 1585, 1558, 1493, 1418, 1367, 1302, 1248, 1144, 1018, 916, 864, 808, 750, 696. UV-vis (MeOH/HCl 1:1; pH = 1): λmax (log ε): 465 (4.23), 277 (4.12). HRMS (ESI/Q-TOF) m/z [M]+ calcd for C16H13O3 253.0859, found 253.0855.

General Procedure B for the Synthesis of Flavylium Salts (11, 16, 17). A mixture of benzaldehyde derivative (2, 4–5, 4 mmol) and the acetophenone derivative (6, 8, 4 mmol) dissolved in absolute ethanol or methanol (40 mL) was saturated at 0 °C with dry HCl (g) for 1 h. The reaction mixture was stirred overnight at rt following a similar procedure to that described by Kraus et al.20 Then, the solvent was removed, Et2O was added and a solid precipitated. The solid was filtered off and carefully washed with Et2O and dried. The structure and purity of the known starting flavylium salts (11 (1.0 g, 77% yield);19 16 (1.18 g, 96% yield);15 17 (405 mg, 33% yield)16) were confirmed by comparison of their physical and spectral data (1H NMR and 13C NMR) with those reported in the literature.

General Procedure C for the Synthesis of 2,8-Dioxabicyclo[3.3.1]nonane (18–23, 27–32). A mixture of the flavylium salt (11–16) and phloroglucinol or (+)-catechin hydrate (0.5 mmol) in absolute methanol (8 mL) was stirred overnight at 50 °C following a similar procedure to that described by Kraus et al.17 Then, the solvent was removed and the crude was purified by semipreparative HPLC or size-exclusion chromatography (SEC). The structure and purity of the known dioxabicyclo[3.3.1]nonanes (18 (170 mg, 64% yield from aldehyde 3),17 20 (126 mg, 60% yield from aldehyde 4),15 21 (128 mg, 63% yield from aldehyde 4),17 27 (168 mg, 45% yield from aldehyde 3),17 28 (145 mg, 42% yield from


16

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aldehyde 3),17 30 (112 mg, 38% yield from aldehyde 4)17) were confirmed by comparison of their physical and spectral data (1H NMR and 13C NMR) with those reported in the literature. 2-(3,4-Dihydroxyphenyl)-3-methyl-6-nitrochromane-(4→4,2→O-5)-phloroglucinol (19). Procedure C was followed by using the flavylium salt 13 (200 mg) and phloroglucinol (63 mg, 0.5 mmol). Then, the solvent was removed and the crude was purified by sizeexclusion chromatography (SEC). Purification on Sephadex LH-20 eluting with MeOH/H2O 9:1 yielded pure analogue 19 as a pale yellow solid (170 mg, 63% from aldehyde 3). This compd decomposes at 220 ºC. 1H NMR (400 MHz, CD3CN) δ 8.22 (br s, 1H, H-5(A)), 8.02 (dd, J = 8.9 Hz, J = 2.2 Hz, 1H, H-7(A)), 7.12 (br s, 1H, H-2'(B)), 7.05 (m, 2H, H-6'(B), H8(A)), 6.90 (d, J = 8.3 Hz, 1H, H-5'(B)), 6.10* (br s, 1H, H-6(D)), 6.05* (br s, 1H, H-2(D), 4.25 (d, J = 1.6 Hz, 1H, H-4(C)), 2.47 (dd, J = 6.8 Hz, J = 1.6 Hz, 1H, H-3(C)), 0.78 (d, J = 6.8 Hz, 3H, CH3); 13C NMR (100 MHz, CD3CN) δ 158.1 (C-9(A)), 157.2 (C-1(D)), 155.8 (C3(D)), 153.8 (C-5(D)), 145.2 (C-4'(B)), 144.2 (C-3'(B)), 141.3 (C-6(A)), 131.1 (C-1'(B)), 130.4 (C-10(A)), 123.6 (C-7(A)), 122.7 (C-5(A)), 118.2 (C-6'(B)), 116.3 (C-8(A)), 114.7 (C5'(B)), 113.5 (C-2'(B)), 104.9 (C-4(D)), 102.6 (C-2(C)), 96.3 (C-2(D)), 94.8 (C-6(D)), 33.7 (C-3(C)), 32.6 (C-4(C)), 12.7 (CH3) (*these signals may be interchanged). FT-IR (ATR) νmax 3356, 2976, 1610, 1508, 1479, 1437, 1333, 1250, 1132, 1109, 1082, 999, 922, 893, 822, 781, 748, 690. UV-vis (MeOH): λmax (log ε): 313 (3.90), 287 (3.92), 253 (3.82). HRMS (ESI/QTOF) m/z [M+H]+ calcd for C22H18NO8 424.1027, found 424.1025. HRMS (ESI/Q-TOF) m/z [M+Cl]– calcd for C22H17NO8Cl 458.0645, found 458.0655. 2-(3,4-Dihydroxyphenyl)-3-methylchromane-(4→4,2→O-5)-phloroglucinol

(22).

Procedure C was followed by using the flavylium salt 15 (175 mg) and phloroglucinol (63 mg, 0.5 mmol). Then, the solvent was removed and the crude was purified by semipreparative


17


Page 18 of 33

HPLC. Separation was carried out with H2O/CH3CO2H (99.8:0.2, v/v, solvent A) and MeOH/CH3CO2H (99.8:0.2, v/v, solvent B) at a flow rate of 5 mL/min and linear gradient from 35% to 50% B for 10 min; 50% B for 5 min, linear gradient from 50% to 100% B for 5 min and 5 min to return to the initial conditions, yielding pure analogue 22 as a white semisolid (50 mg, 49% yield from aldehyde 4). 1H NMR (400 MHz, CD3CN) δ 7.32 (dd, J = 7.8 Hz, J = 1.8 Hz, 1H, H-5(A)), 7.04 (d, J = 2.3 Hz, 1H, H-2'(B)), 6.97 (m, 1H, H-7(A)), 6.92 (dd, J = 8.3 Hz, J = 2.3 Hz, 1H, H-6'(B)), 6.78 (m, 3H, H-5'(B) H-6(A), H-8(A)), 5.97* (d, J = 2.3 Hz, 1H, H-2(D)), 5.92* (d, J = 2.3 Hz, 1H, H-6(D)), 4.05 (d, J = 2.5 Hz, 1H, H-4(C)), 2.25 (dd, J = 7.0 Hz, J = 2.5 Hz, 1H, H-3(C)), 0.70 (d, J = 7.0 Hz, 3H, CH3); 13C NMR (100 MHz, CD3CN) δ 157.9 (C-1(D)), 157.3 (C-3(D)), 153.6 (C-9(A), C-5(D)), 145.7 (C-4'(B)), 145.5 (C-3'(B)), 133.5 (C-1'(B)), 131.0 (C-10(A)), 128.3 (C-5(A)), 128.2 (C-7(A)), 121.6* (C-8(A)), 119.1 (C-6'(B)), 116.4* (C-6(A)), 115.7 (C-5'(B)), 114.9 (C-2'(B)), 104.5 (C-4(D)), 102.8 (C2(C)), 97.1 (C-2(D)), 95.5 (C-6(D)), 36.1 (C-3(C)), 34.5 (C-4(C)), 14.1 (CH3) (*these signals may be interchanged). FT-IR (ATR) νmax 3373, 2980, 1609, 1516, 1485, 1456, 1302, 1219, 1134, 1082, 1053, 1001, 935, 870, 820, 754. UV-vis (MeOH): λmax (log ε): 280 (3.63). HRMS (ESI/Q-TOF) m/z [M+H]+ calcd for C22H19O6 379.1171, found 379.1172. HRMS (ESI/QTOF) m/z [M–H]– calcd for C22H17O6 377.1026, found 377.1040. 3-Chloro-2-(3,4-dihydroxyphenyl)-7-hydroxychromane-(4→4,2→O-5)-phloroglucinol (23). Procedure C was followed by using the flavylium salt 11 (162 mg) and phloroglucinol (63 mg, 0.5 mmol). Then, the solvent was removed and the crude was purified by semipreparative HPLC. Separation was carried out with H2O/CH3CO2H (99.8:0.2, v/v, solvent A) and MeOH/CH3CO2H (99.8:0.2, v/v, solvent B) at a flow rate of 5 mL/min and linear gradient from 35% to 50% B for 10 min; linear gradient from 50% to 100% B for 5 min


18

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and 5 min to return to the initial conditions, yielding pure analogue 23 as a white semisolid (100 mg, 45% from aldehyde 2). 1H NMR (400 MHz, CD3OD) δ 7.13 (d, J = 8.1 Hz, 1H, H5(A)), 7.10 (d, J = 2.3 Hz, 1H, H-2'(B)), 6.98 (dd, J = 8.3 Hz, J = 2.3 Hz, 1H, H-6'(B)), 6.77 (d, J = 8.3 Hz, 1H, H-5'(B)), 6.32 (dd, J = 8.1 Hz, J = 2.5 Hz, 1H, H-6(A)), 6.28 (d, J = 2.5 Hz, 1H, H-8(A)), 5.99* (d, J = 2.3 Hz, 1H, H-6(D)), 5.94* (d, J = 2.3 Hz, 1H, H-2(D)), 4.50 (d, J = 3.1 Hz, 1H, H-3(C)), 4.40 (d, J = 3.1 Hz, 1H, H-4(C)); 13C NMR (100 MHz, CD3OD) δ 158.4 (C-7(A)), 158.1 (C-1(D)), 157.1 (C-3(D)), 155.6 (C-9(A)), 153.1 (C-5(D)), 147.0 (C4'(B)), 145.5 (C-3'(B)), 132.0 (C-1'(B)), 129.1 (C-5(A)), 119.8 (C-6'(B)), 119.7 (C-10(A)), 115.6 (C-2'(B)), 115.5 (C-5'(B)), 109.7 (C-6(A)), 104.2 (C-4(D)), 103.4 (C-8(A)), 99.9 (C2(C)), 97.3 (C-6(D)), 95.5 (C-2(D)), 57.8 (C-3(C)), 36.6 (C-4(C)). FT-IR (ATR) νmax 3358, 1614, 1504, 1464, 1306, 1140, 1115, 1067, 1005, 982, 887, 825. UV-vis (MeOH): λmax (log ε): 280 (3.60). HRMS (ESI/Q-TOF) m/z [M+H]+ calcd for C21H16ClO7 415.0573, found 415.0575. HRMS (ESI/Q-TOF) m/z [M–H]– calcd for C21H14ClO7 413.0427, found 413.0447. 3-Chloro-2-(3,4-dihydroxyphenyl)chromane-(4→8,2→O-7)-catechin (29). Procedure C was followed by using the flavylium salt 16 (154 mg) and (+)-catechin hydrate (154 mg, 0.5 mmol). Then, the solvent was removed and the crude was purified by semipreparative HPLC. Separation was carried out with H2O/CH3CO2H (99.8:0.2, v/v, solvent A) and MeOH/CH3CO2H (99.8:0.2, v/v, solvent B) at a flow rate of 5 mL/min and linear gradient from 45% to 70% B for 15 min; 70% B for 3 min, linear gradient from 70% to 100% B for 2 min and 5 min to return to the initial conditions, yielding the pure analogue 29 as a 2:1 mixture of diastereomers (103 mg, 35% from aldehyde 4). Major isomer: 1H NMR (400 MHz, CD3OD) δ 7.11 (d, J = 2.1 Hz, 1H, H-2'(B)), 7.08 (dd, J = 7.6 Hz, J = 1.5 Hz, 1H, H-5(A)), 7.00 (m, 2H, H-7(A), H-6'(B)), 6.95 (br s, 1H, H-2'(E)), 6.80 (m, 5H, H-6(A), H-8(A), H-


19


Page 20 of 33

5'(B), H-5'(E), H-6'(E)), 6.13 (s, 1H, H-6(D)), 4.53 (m, 2H, H-3(C), H-2(F)), 4.47 (d, J = 3.1 Hz, 1H, H-4(C)), 4.02 (m, 1H, H-3(F)), 2.93 (dd, J = 16.3 Hz, J = 5.6 Hz, 1H, H-4α(F)), 2.50 (m, 1H, H-4β(F)). 13C NMR (100 MHz, CD3OD) δ 156.0 (C-5(D)), 153.8 (C-9(A)), 153.6 (C9(D)), 150.7 (C-7(D)), 147.0 (C-4'(B)), 146.4 (C-3'(B)), 146.3 (C-4'(E)), 145.5 (C-3'(E)), 132.2 (C-1'(E)), 131.9 (C-1'(B)), 129.0 (C-7(A)), 128.4 (C-5(A)), 128.2 (C-10(A)), 122.4* (C6(A)), 120.3 (C-6'(E)), 119.7 (C-6'(B)), 116.4 (C-8(A)), 116.2 (C-5'(E))), 115.6 (C-5'(B)), 115.5 (C-2'(B)), 115.4 (C-2'(E)), 103.5* (C-10(D)), 103.4* (C-8(D)), 100.0 (C-2(C)), 96.1 (C6(D)), 83.0 (C-2(F)), 68.5 (C-3(F)), 57.2 (C-3(C)), 37.1 (C-4(C)), 28.1 (C-4(F)) (*these signals may be interchanged). Minor isomer: 1H NMR (400 MHz, CD3OD) δ 7.11 (d, J = 2.1 Hz, 1H, H-2'(B)), 7.06 (dd, 1H, J = 7.6 Hz, J = 1.5 Hz, H-5(A)), 7.00 (m, 2H, H-7(A), H6'(B)), 6.95 (br s, 1H, H-2'(E)), 6.80 (m, 5H, H-6(A), H-8(A), H-5’(B), H-5'(E), H-6' (E)), 6.14 (s, 1H, H-6(D)), 4.67 (d, J = 7.9 Hz, 1H, H-2(F)), 4.50 (d, J = 3.1 Hz, 1H, H-3(C)), 4.40 (d, J = 3.1 Hz, 1H, H-4(C)), 3.91 (m, 1H, H-3(F)), 2.91 (dd, J = 16.3 Hz, J = 5.6 Hz, 1H, H4α(F)), 2.50 (m, 1H, H-4β(F)). 13C NMR (100 MHz, CD3OD) δ 156.6 (C-5(D)), 153.7 (C9(A)), 153.5 (C-9(D)), 150.8 (C-7(D)), 147.0 (C-4'(B)), 146.4 (C-3'(B)), 146.3 (C-4'(E)), 145.5 (C-3'(E)), 132.1 (C-1'(E)), 132.0 (C-1'(B)), 129.1 (C-7(A)), 129.0 (C-5(A)), 128.0 (C10(A)), 122.4 (C-6(A)), 120.5 (C-6'(E)), 119.7 (C-6'(B)), 116.4 (C-8(A)), 116.0 (C-5'(E)), 115.6 (C-5'(B)), 115.5 (C-2'(B)), 115.4 (C-2'(E)), 103.1* (C-10(D)), 103.0* (C-8(D)), 100.0 (C-2(C)), 96.7 (C-6(D)), 83.5 (C-2(F)), 68.6 (C-3(F)), 57.2 (C-3(C)), 37.2 (C-4(C)), 28.7 (C4(F)) (*these signals may be interchanged). FT-IR (ATR) νmax 3335, 1609, 1520, 1437, 1285, 1231, 1204, 1105, 1057, 964, 883, 818, 760. UV-vis (MeOH): λmax (log ε): 280 (3.90). HRMS (ESI/Q-TOF) m/z [M+H]+ calcd for C30H24ClO9 563.1096, found 563.1098. HRMS (ESI/QTOF) m/z [M–H]– calcd for C30H22ClO9 561.0953, found 561.0972.


20

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2-(3,4-Dihydroxyphenyl)-3-methylchromane-(4→8,2→O-7)-catechin (31). Procedure C was followed by using the flavylium salt 15 (175 mg) and (+)-catechin hydrate (154 mg, 0.5 mmol). Then, the solvent was removed and the crude was purified by semipreparative HPLC. Separation was carried out with H2O/CH3CO2H (99.8:0.2, v/v, solvent A) and MeOH/CH3CO2H (99.8:0.2, v/v, solvent B) at a flow rate of 5 mL/min and linear gradient from 55% to 100% B for 20 min and 5 min to return to the initial conditions, yielding the pure analogue 31 as a 2.3:1 mixture of diastereomers (22 mg, 15% from aldehyde 4). Major isomer: 1H NMR (400 MHz, CD3OD) δ 7.18 (dd, J = 7.6 Hz, J = 1.7 Hz, 1H, H-5(A)), 7.01 (m, 2H, H-7(A), H-2'(B)), 6.91 (m, 1H, H-6'(B)), 6.84 (d, J = 2.1 Hz, 1H, H-2'(E)), 6.75 (m, 5 H, H-6 (A), H-8 (A), H-5’(B), H-5'(E), H-6' (E)), 6.09 (s, 1H, H-6(D)), 4.65 (d, J = 8.1 Hz, 1H, H-2(F)), 3.96 (d, J = 2.5 Hz, 1H, H-4(C)), 3.90 (m, 1H, H-3(F)), 2.84 (dd, J = 16.1 Hz, J = 5.4 Hz, 1H, H-4α(F)), 2.52 (dd, J = 16.1 Hz, J = 8.5 Hz, 1H, H-4β(F)), 2.22 (m, 1H, H3(C)), 0.69 (d, J = 6.8 Hz, 3H, CH3); 13C NMR (100 MHz, CD3OD) δ 155.6 (C-5(D)), 153.7 (C-9(D)), 152.7 (C-9(D)), 152.2 (C-7(D)), 146.5 (C-4'(E)), 146.4 (C-4'(B)), 145.7 (C-3'(B), C-3'(E)), 133.5 (C-1'(B)), 132.2 (C-1'(E)), 130.9 (C-10(A)), 128.7 (C-5(A)), 128.2 (C-7(A)), 121.6* (C-8(A)), 120.5 (C-6'(E)), 119.1 (C-6'(B)), 116.3 (C-6(A)), 116.0* (C-5'(E)), 115.7 (C2’(E)), 115.6* (C-5'(B)), 114.9 (C-2’(B)), 104.0 (C-8(D)), 102.8 (C-2(C)), 102.7 (C-10(D)), 96.0 (C-6(D)), 83.5 (C-2(F)), 68.7 (C-3(F)), 36.1 (C-3(C)), 34.5 (C-4(C)), 27.9 (C-4(F)), 13.2 (CH3) (*these signals may be interchanged). Minor isomer: 1H NMR (400 MHz, CD3OD) δ 7.01 (m, 3H, H-7(A), H-2'(B), H-5(A)), 6.91 (m, 1H, H-6'(B)), 6.81 (d, J = 2.1 Hz, 1H, H2'(E)), 6.75 (m, 5 H, H-6 (A), H-8 (A), H-5’(B), H-5'(E), H-6' (E)), 6.09 (s, 1H, H-6(D)), 4.51 (d, J = 8.1 Hz, 1H, H-2(F)), 4.01 (d, J = 2.5 Hz, 1H, H-4(C)), 3.90 (m, 1H, H-3(F)), 2.85 (dd, J = 16.1 Hz, J = 5.4 Hz, 1H, H-4α(F)), 2.52 (dd, J = 16.1 Hz, J = 8.5 Hz, 1H, H-4β(F)), 2.22


21


(m, 1H, H-3(C)), 0.73 (d, J = 6.8 Hz, 3H, CH3);

Page 22 of 33

13

C NMR (100 MHz, CD3OD) δ 155.7 (C-

5(D)), 153.8 (C-9(A)), 153.7 (C-9(A)), 151.2 (C-7(D)), 146.3 (C-4'(B), (C-4'(E)), 145.7 (C3'(B), C-3'(E)), 133.5 (C-1'(B)), 132.2 (C-1'(E)), 131.1 (C-10(A)), 128.6 (C-5(A)), 128.1 (C7(A)), 121.6* (C-8(A)), 120.3 (C-6'(E)), 119.1 (C-6'(B)), 116.2* (C-6(A)), 115.9 (C-5'(E)), 115.7 (C-2’(E)), 115.5* (C-5'(B)), 114.9 (C-2’(B)), 104.4 (C-8(D)), 102.8 (C-2(C)), 102.7 (C10(D)), 96.1 (C-6(D)), 82.9 (C-2(F)), 68.6 (C-3(F)), 36.1 (C-3(C)), 34.5 (C-4(C)), 28.5 (C4(F)), 13.3 (CH3) (*these signals may be interchanged). FT-IR (ATR) νmax 3329, 2941, 1607, 1520, 1437, 1285, 1219, 1107, 1013, 934, 868, 752. UV-vis (MeOH): λmax (log ε): 281 (3.87). HRMS (ESI/Q-TOF) m/z [M+H]+ calcd for C31H27O9 543.1648, found 543.1646. HRMS (ESI/Q-TOF) m/z [M–H]– calcd for C31H25O9 541.1503, found 541.1519. 3-Chloro-2-(3,4-dihydroxyphenyl)-7-hydroxychromane-(4→8,2→O-7)-catechin (32). Procedure C was followed by using the flavylium salt 11 (162 mg) and (+)-catechin hydrate (154 mg, 0.5 mmol). Then, the solvent was removed and the crude was purified by semipreparative HPLC. Separation was carried out with H2O/CH3CO2H (99.8:0.2, v/v, solvent A) and MeOH/CH3CO2H (99.8:0.2, v/v, solvent B) at a flow rate of 5 mL/min and linear gradient from 45% to 70% B for 15 min; 70% B for 3 min, linear gradient from 70% to 100% B for 2 min and 5 min to return to the initial conditions, yielding compound 32a (major diastereomer, 66 mg, 21% from aldehyde 2) and compound 32b (minor diastereomer, 33 mg, 11% from aldehyde 2). Compound 32a: 1H NMR (400 MHz, CD3OD) δ 6.87 (d, J = 1.9 Hz, 1H, H-2'(B)), 6.80 (br s, 2H, H-5'(E), H-6'(E)), 6.77 (d, J = 8.5 Hz, 1H, H-5'(B)), 6.25 (d, J = 2.3 Hz, 1H, H-8(A)), 6.24 (dd, J = 8.3 Hz, J = 2.3 Hz, 1H, H-6(A)), 6.11 (s, 1H, H-6(D)), 4.55 (d, J = 7.9 Hz, 1H, H-2(F)), 4.49 (d, J = 3.1 Hz, 1H, H-3(C)), 4.37 (d, J = 3.1 Hz, 1H, H4(C)), 4.02 (m, 1H, H-3(F)), 2.94 (dd, J = 16.3 Hz, J = 5.6 Hz, 1H, H-4α(F)), 2.52 (dd, J =


22

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16.3 Hz, J = 8.5 Hz, 1H, H-4β(F));

13

C NMR (100 MHz, CD3OD) δ 158.5 (C-7(A)), 156.0

(C-5(D)), 153.8 (C-9(D)), 153.6 (C-9(A)), 150.7 (C-7(D)), 147.0 (C-4'(B)), 146.4 (C-3'(B), C4'(E)), 145.6 (C-3'(E)), 132.0 (C-1'(B), C-1'(E)), 128.9 (C-5(A)), 120.3 (C-6'(E)), 119.8 (C6'(B)), 119.7 (C-10(A)), 116.2 (C-5'(E)), 115.5 (C-5'(B), C-2'(E)), 115.4 (C-2’(B)), 109.8 (C6(A)), 104.2 (C-8(D)), 103.4 (C-10(D), C-8(A)), 100.0 (C-2(C)), 96.2 (C-6(D)), 83.0 (C2(F)), 68.6 (C-3(F)), 57.8 (C-3(C)), 36.7 (C-4(C)), 29.2 (C-4(F)). Compound 32b: 1H NMR (400 MHz, CD3OD) δ 7.07 (d, J = 2.1 Hz, 1H, H-2'(B)), 6.96 (m, 2H, H-5(A), H-6'(B)), 6.83 (d, J = 2.1 Hz, 1H, H-2'(E)), 6.75 (m, 2H, H-5'(E), H-5'(B)), 6.72 (dd, J = 8.3 Hz, J = 2.1 Hz, 1H, H-6'(E)), 6.30 (dd, J = 8.1 Hz, J = 2.3 Hz, 1H, H-6(A)), 6.28 (d, J = 2.3 Hz, 1H, H-8(A)), 6.11 (s, 1H, H-6(D)), 4.66 (d, J = 7.9 Hz, 1H, H-2(F)), 4.47 (d, J = 3.1 Hz, 1H, H-3(C)), 4.30 (d, J = 3.1 Hz, 1H, H-4(C)), 3.93 (m, 1H, H-3(F)), 2.91 (dd, J = 16.2 Hz, J = 5.5 Hz, 1H, H4α(F)), 2.55 (dd, J = 16.2 Hz, J = 8.7 Hz, 1H, H-4β(F));

13

C NMR (100 MHz, CD3OD) δ

158.5 (C-7(A)), 155.9 (C-5(D)), 153.8 (C-9(D)), 153.4 (C-9(A)), 150.8 (C-7(D)), 147.0 (C4'(B)), 146.5 (C-3'(B)), 146.4 (C-4'(E)), 145.6 (C-3'(E)), 132.2 (C-1'(B)), 132.0 (C-1'(E)), 129.5 (C-5(A)), 120.5 (C-6'(E)), 119.7 (C-6'(B)), 119.5 (C-10(A)), 116.0* (C-5'(E)), 115.6* (C-5'(B)), 115.5 (C-2'(E), C-2’(B)), 109.7 (C-6(A)), 103.8 (C-8(D)), 103.3 (C-8(A)), 103.1 (C-10(D)), 99.9 (C-2(C)), 96.0 (C-6(D)), 83.6 (C-2(F)), 68.7 (C-3(F)), 57.5 (C-3(C)), 36.6 (C4(C)), 28.8 (C-4(F)) (*these signals may be interchanged). FT-IR (ATR) νmax 3308, 2943, 1612, 1506, 1448, 1281, 1105, 984, 820, 646. UV-vis (MeOH): λmax (log ε): 280 (3.88). HRMS (ESI/Q-TOF) m/z [M+H]+ calcd for C30H24ClO10 579.1051, found 579.1044. HRMS (ESI/Q-TOF) m/z [M–H]– calcd for C30H22ClO10 577.0901, found 577.0920.


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General Procedure D for the Synthesis of Dioxabicyclo[3.3.1]nonanes through Flavylium Salts with High Electronic Density (24–26, 33). A mixture of flavylium salt derivative (9, 10, 17) and phloroglucinol or (+)-catechin hydrate (0.2 mmol) in absolute MeOH (6 mL) was irradiated in a microwave reactor at 80 °C, using a maximum power of 250 W (the power was stabilized at 20 W for most of the time), for 20 min, following a similar procedure to that described by Selenski & Pettus.16 Then, the solvent was removed and the crude was purified by semipreparative HPLC on a C18 reverse-phase Spherisorb ODS-2 column using methanol/water as the eluent. The structure and purity of the known dioxabicyclo[3.3.1]nonane 24 (10 mg, 10% yield from aldehyde 2)was confirmed by comparison of its physical and spectral data (1H NMR and 13C NMR) with the reported in the literature.15 The synthesis of dioxabicyclo[3.3.1]nonane 26 (12 mg, 20% yield from aldehyde 5) was achieved according to Selenski’s procedure.16 2-(3,4-Dihydroxyphenyl)-7-hydroxy-3-methylchromane-(4→4,2→O-5)-phloroglucinol (25). Procedure D was followed by using the flavylium salt 10 (73 mg) and phloroglucinol (25 mg, 0.2 mmol) in absolute MeOH (6 mL). Then, the solvent was removed and the crude was purified by semipreparative HPLC. Separation was carried out with H2O/CH3CO2H (99.8:0.2, v/v, solvent A) and MeOH/CH3CO2H (99.8:0.2, v/v, solvent B) at a flow rate of 5 mL/min and linear gradient from 35% to 50% B for 10 min; then from 50% B to 100% B for 5 min, and 5 min to return to the initial conditions, yielding pure compd 25 as a white foam (10 mg, 11% from aldehyde 2). 1H NMR (400 MHz, CD3OD) δ 7.11 (d, J = 8.1 Hz, 1H, H5(A)), 7.03 (d, J = 2.3 Hz, 1H, H-2’(B)), 6.91 (dd, J = 8.3 Hz, J = 2.3 Hz, 1H, H-6’(B)), 6.77 (d, J = 8.3 Hz, 1H, H-5’(B)), 6.25 (m, 2H, H-6(A), H-8(A)), 5.95* (d, J = 2.3 Hz, 1H, H6(D)), 5.91* (d, J = 2.3 Hz, 1H, H-2(D)), 3.95 (d, J = 2.6 Hz, 1H, H-4(C)), 2.24 (dd, J = 6.8 Hz, J = 2.6 Hz, 1H, H-3(C)), 0.66 (d, J = 6.8 Hz, 3H, CH3(C)); 13C NMR (100 MHz, CD3OD)


24

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δ 157.7 (C-1(D)), 157.6 (C-7(A)), 157.1 (C-3(D)), 154.3 (C-9(A)), 153.6 (C-5(D)), 146.7 (C4’(B)), 145.7 (C-3’(B)), 133.6 (C-1’(B)), 128.7 (C-5(A)), 122.6 (C-10(A)), 119.1 (C-6’(B)), 115.7 (C-5’(B)), 114.9 (C-2’(B)), 108.8& (C-8(A)), 105.1 (C-4(D)), 103.4& (C-6 (A)), 102.7 (C-2(C)), 97.1 (C-2(D)), 95.4* (C-6 (D)), 36.5 (C-3(C)), 33.8 (C-4(C)), 14.24 (CH3) (*these signals may be interchanged). FT-IR (ATR) νmax 3333, 2979, 1609, 1504, 1458, 1302, 1229, 1117, 1082, 1007, 976, 926, 885, 820. UV-vis (MeOH): λmax (log ε): 280 (3.66). HRMS (ESI/Q-TOF) m/z [M+H]+ calcd for C22H19O7 395.1125, found 395.1124. HRMS (ESI/QTOF) m/z [M–H]– calcd for C22H17O7 393.0995, found 393.0994. 2-(3,4-Dihydroxyphenyl)-7-hydroxychromane-(4→8,2→O-7)-catechin (33). Procedure D was followed by using the flavylium salt 9 (70 mg) and (+)-catechin (58 mg, 0.2 mmol) in absolute MeOH (6 mL). Then, the solvent was removed and the crude was purified by semipreparative HPLC. Separation was carried out with H2O/CH3CO2H (99.8:0.2, v/v, solvent A) and MeOH/CH3CO2H (99.8:0.2, v/v, solvent B) at a flow rate of 5 mL/min and linear gradient from 45% to 70% B for 15 min; 70% B for 3 min, linear gradient from 70% to 100% B for 2 min and 5 min to return to the initial conditions, yielding pure compd 33 as a 1.6:1 mixture of diastereoisomers (9 mg, 7% from aldehyde 2). Major isomer: 1H NMR (400 MHz, CD3OD) δ 7.09 (m, 1H, H-2'(B)), 6.96 (m, 1H, H-6'(B)), 6.91 (d, J = 8.2 Hz, 1H, H5(A)), 6.82 (d, J = 1.7 Hz, 1H, H-2'(E)), 6.78 (m, 3H, H-5'(E), H-6'(E), H-5'(B), 6.32 (d, J = 2.3 Hz, 1H, H-8(A)), 6.23 (dd, J = 8.2 Hz, J = 2.3 Hz, 1H, H-6(A)), 6.04 (s, 1H, H-6(D)), 4.58 (d, J = 7.6 Hz, 1H, H-2(F)), 4.23 (t, J = 2.9 Hz, 1H, H-4(C)), 3.98 (m, 1H, H-3(F)), 2.91 (dd, J = 16.2 Hz, J = 5.3 Hz, 1H, H-4α(F)), 2.55 (dd, J = 16.2 Hz, J = 8.2 Hz, 1H, H-4β(F)), 2.13 (m, 2H, H-3(C)); 13C NMR (100 MHz, CD3OD) δ 157.7 (C-7(A)), 155.6 (C-5(D)), 154.4 (C-9(D)), 152.3 (C-9(A)), 152.0 (C-7(D)), 146.6 (C-4'(B)), 146.4& (C-3'(B)), 146.3& (C-


25


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4'(E)), 145.9 (C-3'(E)), 135.3 (C-1'(B)), 132.2 (C-1'(E)), 128.9 (C-5(A)), 120.6 (C-10(A)), 120.2 (C-6'(E)), 118.3 (C-6'(B)), 116.2* (C-5'(E)), 115.8* (C-5'(B)), 115.4 (C-2'(E)), 114.3 (C2’(B)), 109.2 (C-6(A)), 107.7 (C-8(D)), 103.7 (C-8(A)), 102.6 (C-10(D)), 99.9 (C-2(C)), 96.2 (C-6(D)), 82.9 (C-2(F)), 68.8 (C-3(F)), 35.2 (C-3(C)), 29.2 (C-4(F)), 27.4 (C-4(C)) (*,&these signals may be interchanged). Minor isomer: 1H NMR (400 MHz, CD3OD) δ 7.10 (d, J = 2.2 Hz, 1H, H-2'(B)), 7.04 (d, J = 8.2 Hz, 1H, H-5(A)), 6.98 (dd, J = 8.4 Hz, J = 2.2 Hz, 1H, H6'(B)), 6.87 (d, J = 1.7 Hz, 1H, H-2'(E)), 6.78 (m, 3H, H-5'(E), H-6'(E), H-5'(B)), 6.36 (d, J = 2.3 Hz, 1H, H-8(A)), 6.31 (dd, J = 8.2 Hz, J = 2.3 Hz, 1H, H-6(A)), 6.06 (s, 1H, H-6(D)), 4.70 (d, J = 7.6 Hz, 1H, H-2(F)), 4.19 (t, J = 2.9 Hz, 1H, H-4(C)), 3.98 (m, 1H, H-3(F)), 2.87 (dd, J = 16.2 Hz, J = 5.3 Hz, 1H, H-4α(F)), 2.55 (dd, J = 16.2 Hz, J = 8.2 Hz, 1H, H-4β(F)), 2.13 (m, 2H, H-3(C)); 13C NMR (100 MHz, CD3OD) δ 157.8 (C-7(A)), 155.5 (C-5(D)), 154.4 (C-9(A)), 152.6 (C-9(D)), 152.1 (C-7(D)), 146.6 (C-4'(B)), 146.4& (C-3'(B)), 146.3& (C4'(E)), 145.9 (C-3'(E)), 135.3 (C-1'(B)), 132.3 (C-1'(E)), 129.5 (C-5(A)), 120.4 (C-10(A)), 120.3 (C-6'(E)), 118.3 (C-6'(B)), 116.0# (C-5'(E)), 115.8# (C-5'(B)), 115.4 (C-2'(E)), 114.3 (C2’(B)), 109.2 (C-6(A)), 107.6 (C-8(D)), 103.7 (C-5(A)), 102.3 (C-10(D)), 99.9 (C-2(C)), 96.1 (C-6(D)), 83.4 (C-2(F)), 68.8 (C-3(F)), 35.3 (C-3(C)), 28.5 (C-4(F)), 27.4 (C-4(C)) (#,&these signals may be interchanged). FT-IR (ATR) νmax 3333, 2933, 1610, 1506, 1443, 1290, 1117, 972, 816. UV-vis (MeOH): λmax (log ε): 280 (3.84). HRMS (ESI/Q-TOF) m/z [M+H]+ calcd for C30H25O10 545.1440, found 545.1440. HRMS (ESI/Q-TOF) m/z [M–H]– calcd for C30H23O10 543.1295, found 543.1318.

Thermodynamic Studies and pK'a Determinations. These studies were performed in MeOH/H2O 1:1 (v/v) solutions. The pH jumps were carried out in two different ways: (i) by


26

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adding a stock solution of flavylium salt in 1:1 MeOH/HCl 0.2M (0.5 mL) to a quartz cuvette containing a solution of 1:1 MeOH/NaOH 0.2M (0.5 mL), MeOH (0.25 mL), and universal buffer of Theorell and Stenhagen (0.25 mL)25 at the desired final pH or, for the more acidic solutions, (ii) by adding a stock solution of flavylium salt in 1:1 MeOH/HCl (37%) (0.5 mL) to a quartz cuvette containing MeOH (0.5 mL), and HCl at the desired concentration (from 12M to 1M) (0.25 mL). The final pH of the solutions was measured in a Crison GLP 212 pH meter (i) or calculated from the concentration of acid and corrected by the Hammett’s acidity function (H0) (ii).26 In Figures S1–S5 (Supporting Information) are shown the spectral variations of flavylium salts 12–16 after a direct pH jump.


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ASSOCIATED CONTENT

Supporting Information Copies of 1H and 13C NMR spectra, and figures with spectral variations of flavylium salts 12– 16 after direct pH jumps (PDF).

AUTHOR INFORMATION

Corresponding Authors *E-mail: [email protected] *E-mail: [email protected]

ORCID Alfonso Alejo-Armijo: 0000-0001-8691-4628 A. Jorge Parola: 0000-0002-1333-9076 Fernando Pina: 0000-0001-8529-6848 Joaquín Altarejos: 0000-0002-5532-8535 Sofía Salido: 0000-0003-2319-7873

Notes The authors declare no competing financial interest.


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ACKNOWLEDGMENTS

This study was supported by the University of Jaén (UJA081605) and by the Associate Laboratory for Green Chemistry-LAQV, which is financed by national funds from FCT/MCTES (UID/QUI/50006/2013) and co-financed by the ERDF under the PT2020 Partnership Agreement (POCI-01-0145-FEDER-007265). A. A.-A. thanks the University of Jaén for a pre-doctoral fellowship and also the Fundación Alfonso Martín Escudero for its current post-doctoral fellowship. Part of the work was supported by the Centro de Instrumentación Científico-Técnica of the University of Jaén.


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(23) Melo, M. J.; Moura, S.; Roque; A.; Maestri, M.; Pina, F. Photochemistry of Luteolinidin “Write-Lock-Read-Unlock-Erase” with a Natural Compound. J. Photochem. Photobiol., A 2000, 135, 33. (24) Armarego, W. L. F.; Chai, C. L. L. Purification of Laboratory Chemicals. Butterworth Heinemann: Burlington, 2003. (25) Küster, F. W.; Thiel, A.; Brückner, E. Tabelle per Le Analisi Chimiche e Chimico-fisiche. Hoepli: Milano, 1985; 157–160. The universal buffer used was prepared in the following way: 85% (w/w) phosphoric acid (2.3 mL), monohydrated citric acid (7.00 g), and boric acid (3.54 g) were dissolved in water; 1M NaOH (343 mL) was then added, and the solution was diluted to 1 L with water. (26) Bell, R. P. The Proton in Chemistry. Chapman and Hall: London, 1973.


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Thermodynamic Stability of Flavylium Salts as a Valuable Tool to

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