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

Article abstract of DOI:10.1021/acs.joc.8b01780

A convenient method to synthesize A-type proanthocyanidin analogues from flavylium salts and ?€-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 2,8-dioxabyciclo[3.3.1]nonane derivative was properly achieved.

Full text of DOI:10.1021/acs.joc.8b01780

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Note
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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Thermodynamic Stability of Flavylium Salts as a Valuable Tool to Design
the Synthesis of A-Type Proanthocyanidin Analogues
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Alfonso AlejoꢀArmijo,†,‡ 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, 2829ꢀ516, Caparica, Portugal
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ABSTRACT: A convenient method to synthesize Aꢀtype proanthocyanidin (PAC) analogues
from flavylium salts and πꢀnucleophiles 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 oneꢀpot 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 2,8ꢀ
dioxabyciclo[3.3.1]nonane derivative was properly achieved.
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Proanthocyanidins (PACs) are phenolic compounds widely distributed in nature. PACs of the
Aꢀtype present the unusual 2,8ꢀdioxabicyclo[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 Aꢀtype PACs: antiviral, antiꢀadhesion, and antitumoral
properties.1−3 As an example, cinnamtannin Bꢀ1 (1) is an Aꢀtype 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
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antibiofilm activities,7 among many others.8
OH
HO
O
OH
OH
O
OH
OH
OH
OH
OH
O
O
OH
HO
OH
HO
OH
Cinnamtannin B-1 (1)
Figure 1. Biologically important 2,8ꢀdioxabicyclo[3.3.1]nonane core of Aꢀtype proanthocyanidins.
Several attempts have been made in order to synthesize both Aꢀtype PACs9,10 and their
synthetic analogues (2,8ꢀdioxabiꢀcyclo[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 2ꢀhydroxychalcones 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
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features that flavylium salts must fulfil to be suitable starting materials for the synthesis of
2,8ꢀdioxaꢀbicyclo[3.3.1]nonane derivatives.17
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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
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When electronꢀdonor 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 electronꢀwithdrawing 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 2,8ꢀdioxabicyclo[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 2,8ꢀdioxabicyclo[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 2,8ꢀdioxabicyclo[3.3.1]nonane derivatives, ten of them being new.
We have also developed a new microwave methodology to synthesize 2,8ꢀ
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
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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 1ꢀ
benzopyrylium 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)
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b
Entry compd R1
R2
H
H
R3
OH
OH CH3
OH Cl
OH Cl
R4 methoda
pK'a
3.1c
1
2
H
H
H
i
i
9
2.7d
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17
3
4
H
H
H
H
i
–0.65d
–0.65d
–1.7
–2.3
1.7
ii
i
i
5
6
H
H
NO2
NO2
H
H
H
H
CH3
H
7
8
H
H
H
H
i
H
CH3
Cl
i
1.3
9
H
H
H
H
H
H
i
–1.9
–1.9
3.8e
3.8e
3.8e
10
11
12
13
Cl
ii
i
OH
OH
OH
H
H
OH
OH
OH
H
H
ii
iii
H
H
aReaction conditions: i. H2SO4, HOAc; ii. HCl (g), EtOH; iii. HCl (g), MeOH; See Experimental section; In
b
c
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.
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AH+ + H2O
AH+ + 2 H2O
A + H3O+
B + H3O+
Ka
Kh
Kt
Ki
Proton transfer
Hydration
(1)
(2)
(3)
(4)
B
Cc
Ct
Tautomerization
Isomerization
Cc
The system can be simplified by considering a single acidꢀbase 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 UVꢀvis 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 electronꢀdonor groups (9, 10, 17) present the
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highest pK'a values, while those containing the nitro or chloro electronꢀwithdrawing 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.
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Synthesis of 2,8ꢀdioxabicyclo[3.3.1]nonane derivatives (18–33). The syntheses of 2,8ꢀ
dioxabicyclo[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
yield
Entry compd R1
R2
R3
R4 methoda
(%)b
85
1
2
3
4
5
6
7
H
H
H
H
H
H
H
NO2
NO2
NO2
H
H
H
H
H
H
H
H
H
iv
v
18
18
19
20
21
22
23
32
CH3
Cl
iv
iv
iv
iv
iv
83
81
H
H
84
H
CH3
80
H
OH Cl
78
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H
H
H
H
H
H
H
H
H
H
H
H
OH Cl
v
iv
v
10
1
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26
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OH
OH
OH
H
H
H
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H
25
20
0
H
vi
iv
v
H
OH CH3
OH CH3
OH CH3
H
12
0
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H
vi
iv
v
OH
OH
OH
OH
OH
OH
H
H
H
0
0
vi
40
a
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
yield
(%)b
Entry compd
R1
R2
R3
R4 methoda
1
2
H
H
H
H
H
H
H
H
H
H
NO2
NO2
H
H
H
H
CH3
Cl
iv
iv
iv
iv
iv
iv
iv
v
64
61
63
52
25
57
0
27
28
29
30
31
32
33
33
34
34
3
H
4
H
H
H
5
H
H
CH3
Cl
6
H
OH
OH
OH
7
H
H
8
H
H
8
9
H
OH CH3
OH CH3
iv
v
0
10
H
0
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a
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Reaction conditions: iv. MeOH, 50 °C, 24 h; v. MW, MeOH, 80 °C, 20 min; Yield calculated by HPLC
5
6
calibration.
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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 2,8ꢀdioxabicyclo[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
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= 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 2,8ꢀ
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 πꢀ
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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 2,8ꢀdioxabyciclo[3.3.1]nonane
derivatives (18–33), ten of which were synthesized for the first time (19, 20, 22–25, 29 and
31–33).
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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 thinꢀlayer 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 reverseꢀphase Spherisorb ODSꢀ2 column, 250 mm × 3 mm i.d., 5 ꢁm, at
a flow rate of 0.7 mL/min. Separations were performed by size exclusion column
chromatography (Sephadex LHꢀ20; particle size 25–100 ꢁm) and by semipreparative HPLC,
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the latter being performed on a RPꢀHPLC column (Spherisorb ODSꢀ2 column, 250 mm × 10
mm i.d., 5 ꢁm) 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
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Varian Cary 4000 spectrophotometers.1H and 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,HꢀCOSY and heteronuclear H,Cꢀ
HMBC, H,CꢀHSQC spectra. Highꢀresolution mass spectra (HRMS) were recorded on a
quadrupole timeꢀofꢀflight (QꢀTOF) 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
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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.
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3',4'ꢀDihydroxyꢀ3ꢀmethylꢀ6ꢀnitroflavylium 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, Hꢀ5, Hꢀ7), 6.99 (d,
J = 9.3 Hz, 1H, Hꢀ8), 6.88 (br s, 1H, Hꢀ2'), 6.77 (m, 3H, Hꢀ4, Hꢀ5', Hꢀ6'), 1.68 (s, 3H, CH3);
13C NMR (100 MHz, DCl/CD3CN, pD ≈ 1.0) δ 158.8 (Cꢀ9), 146.9 (Cꢀ4'), 146.0 (Cꢀ3'), 143.1
(Cꢀ6), 134.8 (Cꢀ3), 133.7 (Cꢀ1'), 125.5 (Cꢀ7), 123.4 (Cꢀ4), 122.7 (Cꢀ5), 121.5 (Cꢀ10), 118.9
(Cꢀ6'), 116.5 (Cꢀ8), 115.7 (Cꢀ5') 114.6 (Cꢀ2'), 106.6 (Cꢀ2), 18.9 (CH3). FTꢀIR (ATR) νmax
3069, 1589, 1535, 1489, 1408, 1308, 1277, 1165, 1119, 1055, 943, 862, 824, 750, 706, 681.
UVꢀvis (MeOH/HCl 1:1; pH = 1): λmax (log ε): 312 (4.51), 274 (4.86). HRMS (ESI/QꢀTOF)
m/z [M]+ calcd for C16H12NO5 298.0715, found 298.0710.
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3',4'ꢀDihydroxyꢀ3ꢀmethylflavylium 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
o
dried, yielding the compd 15 as an orange solid (316 mg, 90%). Melting point: 190 C
(decomposes). 1H NMR (400 MHz, DCl/CD3CN, pD ≈ 1.0,) δ 9.29 (s, 1H, Hꢀ4), 8.23 (m, 3H,
Hꢀ5, Hꢀ7, Hꢀ8), 7.90 (m, 3H, Hꢀ6, Hꢀ2', Hꢀ6'), 7.18 (d, J = 9.1 Hz, 1H, Hꢀ5'), 2.88 (s, 3H,
CH3); 13C NMR (100 MHz, DCl/ CD3CN, pD ≈ 1.0) δ 174.0 (Cꢀ2), 156.1 (Cꢀ4), 154.4 (Cꢀ9),
152.7 (Cꢀ4'), 144.5 (Cꢀ3'), 137.5 (Cꢀ7), 129.2 (Cꢀ3), 129.1 (Cꢀ6), 128.7 (Cꢀ5), 126.7 (Cꢀ6'),
122.7 (Cꢀ10), 120.9 (Cꢀ1'), 117.8 (Cꢀ8), 117.5 (Cꢀ2'), 115.8 (Cꢀ5'), 19.7 (CH3). FTꢀIR (ATR)
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max 3086, 1585, 1558, 1493, 1418, 1367, 1302, 1248, 1144, 1018, 916, 864, 808, 750, 696.
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UVꢀvis (MeOH/HCl 1:1; pH = 1): λmax (log ε): 465 (4.23), 277 (4.12). HRMS (ESI/QꢀTOF)
m/z [M]+ calcd for C16H13O3 253.0859, found 253.0855.
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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 sizeꢀexclusion 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
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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.
2ꢀ(3,4ꢀDihydroxyphenyl)ꢀ3ꢀmethylꢀ6ꢀnitrochromaneꢀ(4→4,2→Oꢀ5)ꢀphloroglucinol
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(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 sizeꢀ
exclusion chromatography (SEC). Purification on Sephadex LHꢀ20 eluting with MeOH/H2O
9:1 yielded pure analogue 19 as a pale yellow solid (170 mg, 63% from aldehyde 3). This
1
compd decomposes at 220 ºC. H NMR (400 MHz, CD3CN) δ 8.22 (br s, 1H, Hꢀ5(A)), 8.02
(dd, J = 8.9 Hz, J = 2.2 Hz, 1H, Hꢀ7(A)), 7.12 (br s, 1H, Hꢀ2'(B)), 7.05 (m, 2H, Hꢀ6'(B), Hꢀ
8(A)), 6.90 (d, J = 8.3 Hz, 1H, Hꢀ5'(B)), 6.10* (br s, 1H, Hꢀ6(D)), 6.05* (br s, 1H, Hꢀ2(D),
4.25 (d, J = 1.6 Hz, 1H, Hꢀ4(C)), 2.47 (dd, J = 6.8 Hz, J = 1.6 Hz, 1H, Hꢀ3(C)), 0.78 (d, J =
6.8 Hz, 3H, CH3); 13C NMR (100 MHz, CD3CN) δ 158.1 (Cꢀ9(A)), 157.2 (Cꢀ1(D)), 155.8 (Cꢀ
3(D)), 153.8 (Cꢀ5(D)), 145.2 (Cꢀ4'(B)), 144.2 (Cꢀ3'(B)), 141.3 (Cꢀ6(A)), 131.1 (Cꢀ1'(B)),
130.4 (Cꢀ10(A)), 123.6 (Cꢀ7(A)), 122.7 (Cꢀ5(A)), 118.2 (Cꢀ6'(B)), 116.3 (Cꢀ8(A)), 114.7 (Cꢀ
5'(B)), 113.5 (Cꢀ2'(B)), 104.9 (Cꢀ4(D)), 102.6 (Cꢀ2(C)), 96.3 (Cꢀ2(D)), 94.8 (Cꢀ6(D)), 33.7
(Cꢀ3(C)), 32.6 (Cꢀ4(C)), 12.7 (CH3) (*these signals may be interchanged). FTꢀIR (ATR) νmax
3356, 2976, 1610, 1508, 1479, 1437, 1333, 1250, 1132, 1109, 1082, 999, 922, 893, 822, 781,
748, 690. UVꢀvis (MeOH): λmax (log ε): 313 (3.90), 287 (3.92), 253 (3.82). HRMS (ESI/Qꢀ
TOF) m/z [M+H]+ calcd for C22H18NO8 424.1027, found 424.1025. HRMS (ESI/QꢀTOF) m/z
[M+Cl]– calcd for C22H17NO8Cl 458.0645, found 458.0655.
2ꢀ(3,4ꢀDihydroxyphenyl)ꢀ3ꢀmethylchromaneꢀ(4→4,2→Oꢀ5)ꢀ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
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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, Hꢀ5(A)), 7.04 (d, J = 2.3 Hz, 1H, Hꢀ2'(B)), 6.97 (m, 1H, Hꢀ7(A)), 6.92
(dd, J = 8.3 Hz, J = 2.3 Hz, 1H, Hꢀ6'(B)), 6.78 (m, 3H, Hꢀ5'(B) Hꢀ6(A), Hꢀ8(A)), 5.97* (d, J =
2.3 Hz, 1H, Hꢀ2(D)), 5.92* (d, J = 2.3 Hz, 1H, Hꢀ6(D)), 4.05 (d, J = 2.5 Hz, 1H, Hꢀ4(C)), 2.25
(dd, J = 7.0 Hz, J = 2.5 Hz, 1H, Hꢀ3(C)), 0.70 (d, J = 7.0 Hz, 3H, CH3); 13C NMR (100 MHz,
CD3CN) δ 157.9 (Cꢀ1(D)), 157.3 (Cꢀ3(D)), 153.6 (Cꢀ9(A), Cꢀ5(D)), 145.7 (Cꢀ4'(B)), 145.5
(Cꢀ3'(B)), 133.5 (Cꢀ1'(B)), 131.0 (Cꢀ10(A)), 128.3 (Cꢀ5(A)), 128.2 (Cꢀ7(A)), 121.6* (Cꢀ8(A)),
119.1 (Cꢀ6'(B)), 116.4* (Cꢀ6(A)), 115.7 (Cꢀ5'(B)), 114.9 (Cꢀ2'(B)), 104.5 (Cꢀ4(D)), 102.8 (Cꢀ
2(C)), 97.1 (Cꢀ2(D)), 95.5 (Cꢀ6(D)), 36.1 (Cꢀ3(C)), 34.5 (Cꢀ4(C)), 14.1 (CH3) (*these signals
may be interchanged). FTꢀIR (ATR) νmax 3373, 2980, 1609, 1516, 1485, 1456, 1302, 1219,
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1134, 1082, 1053, 1001, 935, 870, 820, 754. UVꢀvis (MeOH): λmax (log ε): 280 (3.63). HRMS
(ESI/QꢀTOF) m/z [M+H]+ calcd for C22H19O6 379.1171, found 379.1172. HRMS (ESI/Qꢀ
TOF) m/z [M–H]– calcd for C22H17O6 377.1026, found 377.1040.
3ꢀChloroꢀ2ꢀ(3,4ꢀdihydroxyphenyl)ꢀ7ꢀhydroxychromaneꢀ(4→4,2→Oꢀ5)ꢀ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
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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, Hꢀ
5(A)), 7.10 (d, J = 2.3 Hz, 1H, Hꢀ2'(B)), 6.98 (dd, J = 8.3 Hz, J = 2.3 Hz, 1H, Hꢀ6'(B)), 6.77
(d, J = 8.3 Hz, 1H, Hꢀ5'(B)), 6.32 (dd, J = 8.1 Hz, J = 2.5 Hz, 1H, Hꢀ6(A)), 6.28 (d, J = 2.5
Hz, 1H, Hꢀ8(A)), 5.99* (d, J = 2.3 Hz, 1H, Hꢀ6(D)), 5.94* (d, J = 2.3 Hz, 1H, Hꢀ2(D)), 4.50
(d, J = 3.1 Hz, 1H, Hꢀ3(C)), 4.40 (d, J = 3.1 Hz, 1H, Hꢀ4(C)); 13C NMR (100 MHz, CD3OD)
δ 158.4 (Cꢀ7(A)), 158.1 (Cꢀ1(D)), 157.1 (Cꢀ3(D)), 155.6 (Cꢀ9(A)), 153.1 (Cꢀ5(D)), 147.0 (Cꢀ
4'(B)), 145.5 (Cꢀ3'(B)), 132.0 (Cꢀ1'(B)), 129.1 (Cꢀ5(A)), 119.8 (Cꢀ6'(B)), 119.7 (Cꢀ10(A)),
115.6 (Cꢀ2'(B)), 115.5 (Cꢀ5'(B)), 109.7 (Cꢀ6(A)), 104.2 (Cꢀ4(D)), 103.4 (Cꢀ8(A)), 99.9 (Cꢀ
2(C)), 97.3 (Cꢀ6(D)), 95.5 (Cꢀ2(D)), 57.8 (Cꢀ3(C)), 36.6 (Cꢀ4(C)). FTꢀIR (ATR) νmax 3358,
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1614, 1504, 1464, 1306, 1140, 1115, 1067, 1005, 982, 887, 825. UVꢀvis (MeOH): λmax (log
ε): 280 (3.60). HRMS (ESI/QꢀTOF) m/z [M+H]+ calcd for C21H16ClO7 415.0573, found
415.0575. HRMS (ESI/QꢀTOF) m/z [M–H]– calcd for C21H14ClO7 413.0427, found 413.0447.
3ꢀChloroꢀ2ꢀ(3,4ꢀdihydroxyphenyl)chromaneꢀ(4→8,2→Oꢀ7)ꢀ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, Hꢀ2'(B)), 7.08 (dd, J = 7.6 Hz, J = 1.5 Hz, 1H, Hꢀ5(A)),
7.00 (m, 2H, Hꢀ7(A), Hꢀ6'(B)), 6.95 (br s, 1H, Hꢀ2'(E)), 6.80 (m, 5H, Hꢀ6(A), Hꢀ8(A), Hꢀ
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5'(B), Hꢀ5'(E), Hꢀ6'(E)), 6.13 (s, 1H, Hꢀ6(D)), 4.53 (m, 2H, Hꢀ3(C), Hꢀ2(F)), 4.47 (d, J = 3.1
Hz, 1H, Hꢀ4(C)), 4.02 (m, 1H, Hꢀ3(F)), 2.93 (dd, J = 16.3 Hz, J = 5.6 Hz, 1H, Hꢀ4α(F)), 2.50
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9
(m, 1H, Hꢀ4β(F)). 13C NMR (100 MHz, CD3OD) δ 156.0 (Cꢀ5(D)), 153.8 (Cꢀ9(A)), 153.6 (Cꢀ
9(D)), 150.7 (Cꢀ7(D)), 147.0 (Cꢀ4'(B)), 146.4 (Cꢀ3'(B)), 146.3 (Cꢀ4'(E)), 145.5 (Cꢀ3'(E)),
132.2 (Cꢀ1'(E)), 131.9 (Cꢀ1'(B)), 129.0 (Cꢀ7(A)), 128.4 (Cꢀ5(A)), 128.2 (Cꢀ10(A)), 122.4* (Cꢀ
6(A)), 120.3 (Cꢀ6'(E)), 119.7 (Cꢀ6'(B)), 116.4 (Cꢀ8(A)), 116.2 (Cꢀ5'(E))), 115.6 (Cꢀ5'(B)),
115.5 (Cꢀ2'(B)), 115.4 (Cꢀ2'(E)), 103.5* (Cꢀ10(D)), 103.4* (Cꢀ8(D)), 100.0 (Cꢀ2(C)), 96.1 (Cꢀ
6(D)), 83.0 (Cꢀ2(F)), 68.5 (Cꢀ3(F)), 57.2 (Cꢀ3(C)), 37.1 (Cꢀ4(C)), 28.1 (Cꢀ4(F)) (*these
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signals may be interchanged). Minor isomer: H NMR (400 MHz, CD3OD) δ 7.11 (d, J = 2.1
Hz, 1H, Hꢀ2'(B)), 7.06 (dd, 1H, J = 7.6 Hz, J = 1.5 Hz, Hꢀ5(A)), 7.00 (m, 2H, Hꢀ7(A), Hꢀ
6'(B)), 6.95 (br s, 1H, Hꢀ2'(E)), 6.80 (m, 5H, Hꢀ6(A), Hꢀ8(A), Hꢀ5’(B), Hꢀ5'(E), Hꢀ6' (E)),
6.14 (s, 1H, Hꢀ6(D)), 4.67 (d, J = 7.9 Hz, 1H, Hꢀ2(F)), 4.50 (d, J = 3.1 Hz, 1H, Hꢀ3(C)), 4.40
(d, J = 3.1 Hz, 1H, Hꢀ4(C)), 3.91 (m, 1H, Hꢀ3(F)), 2.91 (dd, J = 16.3 Hz, J = 5.6 Hz, 1H, Hꢀ
4α(F)), 2.50 (m, 1H, Hꢀ4β(F)). 13C NMR (100 MHz, CD3OD) δ 156.6 (Cꢀ5(D)), 153.7 (Cꢀ
9(A)), 153.5 (Cꢀ9(D)), 150.8 (Cꢀ7(D)), 147.0 (Cꢀ4'(B)), 146.4 (Cꢀ3'(B)), 146.3 (Cꢀ4'(E)),
145.5 (Cꢀ3'(E)), 132.1 (Cꢀ1'(E)), 132.0 (Cꢀ1'(B)), 129.1 (Cꢀ7(A)), 129.0 (Cꢀ5(A)), 128.0 (Cꢀ
10(A)), 122.4 (Cꢀ6(A)), 120.5 (Cꢀ6'(E)), 119.7 (Cꢀ6'(B)), 116.4 (Cꢀ8(A)), 116.0 (Cꢀ5'(E)),
115.6 (Cꢀ5'(B)), 115.5 (Cꢀ2'(B)), 115.4 (Cꢀ2'(E)), 103.1* (Cꢀ10(D)), 103.0* (Cꢀ8(D)), 100.0
(Cꢀ2(C)), 96.7 (Cꢀ6(D)), 83.5 (Cꢀ2(F)), 68.6 (Cꢀ3(F)), 57.2 (Cꢀ3(C)), 37.2 (Cꢀ4(C)), 28.7 (Cꢀ
4(F)) (*these signals may be interchanged). FTꢀIR (ATR) νmax 3335, 1609, 1520, 1437, 1285,
1231, 1204, 1105, 1057, 964, 883, 818, 760. UVꢀvis (MeOH): λmax (log ε): 280 (3.90). HRMS
(ESI/QꢀTOF) m/z [M+H]+ calcd for C30H24ClO9 563.1096, found 563.1098. HRMS (ESI/Qꢀ
TOF) m/z [M–H]– calcd for C30H22ClO9 561.0953, found 561.0972.
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2ꢀ(3,4ꢀDihydroxyphenyl)ꢀ3ꢀmethylchromaneꢀ(4→8,2→Oꢀ7)ꢀ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
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isomer: H NMR (400 MHz, CD3OD) δ 7.18 (dd, J = 7.6 Hz, J = 1.7 Hz, 1H, Hꢀ5(A)), 7.01
(m, 2H, Hꢀ7(A), Hꢀ2'(B)), 6.91 (m, 1H, Hꢀ6'(B)), 6.84 (d, J = 2.1 Hz, 1H, Hꢀ2'(E)), 6.75 (m, 5
H, Hꢀ6 (A), Hꢀ8 (A), Hꢀ5’(B), Hꢀ5'(E), Hꢀ6' (E)), 6.09 (s, 1H, Hꢀ6(D)), 4.65 (d, J = 8.1 Hz,
1H, Hꢀ2(F)), 3.96 (d, J = 2.5 Hz, 1H, Hꢀ4(C)), 3.90 (m, 1H, Hꢀ3(F)), 2.84 (dd, J = 16.1 Hz, J
= 5.4 Hz, 1H, Hꢀ4α(F)), 2.52 (dd, J = 16.1 Hz, J = 8.5 Hz, 1H, Hꢀ4β(F)), 2.22 (m, 1H, Hꢀ
3(C)), 0.69 (d, J = 6.8 Hz, 3H, CH3); 13C NMR (100 MHz, CD3OD) δ 155.6 (Cꢀ5(D)), 153.7
(Cꢀ9(D)), 152.7 (Cꢀ9(D)), 152.2 (Cꢀ7(D)), 146.5 (Cꢀ4'(E)), 146.4 (Cꢀ4'(B)), 145.7 (Cꢀ3'(B),
Cꢀ3'(E)), 133.5 (Cꢀ1'(B)), 132.2 (Cꢀ1'(E)), 130.9 (Cꢀ10(A)), 128.7 (Cꢀ5(A)), 128.2 (Cꢀ7(A)),
121.6* (Cꢀ8(A)), 120.5 (Cꢀ6'(E)), 119.1 (Cꢀ6'(B)), 116.3 (Cꢀ6(A)), 116.0* (Cꢀ5'(E)), 115.7 (Cꢀ
2’(E)), 115.6* (Cꢀ5'(B)), 114.9 (Cꢀ2’(B)), 104.0 (Cꢀ8(D)), 102.8 (Cꢀ2(C)), 102.7 (Cꢀ10(D)),
96.0 (Cꢀ6(D)), 83.5 (Cꢀ2(F)), 68.7 (Cꢀ3(F)), 36.1 (Cꢀ3(C)), 34.5 (Cꢀ4(C)), 27.9 (Cꢀ4(F)), 13.2
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(CH3) (*these signals may be interchanged). Minor isomer: H NMR (400 MHz, CD3OD) δ
7.01 (m, 3H, Hꢀ7(A), Hꢀ2'(B), Hꢀ5(A)), 6.91 (m, 1H, Hꢀ6'(B)), 6.81 (d, J = 2.1 Hz, 1H, Hꢀ
2'(E)), 6.75 (m, 5 H, Hꢀ6 (A), Hꢀ8 (A), Hꢀ5’(B), Hꢀ5'(E), Hꢀ6' (E)), 6.09 (s, 1H, Hꢀ6(D)), 4.51
(d, J = 8.1 Hz, 1H, Hꢀ2(F)), 4.01 (d, J = 2.5 Hz, 1H, Hꢀ4(C)), 3.90 (m, 1H, Hꢀ3(F)), 2.85 (dd,
J = 16.1 Hz, J = 5.4 Hz, 1H, Hꢀ4α(F)), 2.52 (dd, J = 16.1 Hz, J = 8.5 Hz, 1H, Hꢀ4β(F)), 2.22
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(m, 1H, Hꢀ3(C)), 0.73 (d, J = 6.8 Hz, 3H, CH3); C NMR (100 MHz, CD3OD) δ 155.7 (Cꢀ
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5(D)), 153.8 (Cꢀ9(A)), 153.7 (Cꢀ9(A)), 151.2 (Cꢀ7(D)), 146.3 (Cꢀ4'(B), (Cꢀ4'(E)), 145.7 (Cꢀ
3'(B), Cꢀ3'(E)), 133.5 (Cꢀ1'(B)), 132.2 (Cꢀ1'(E)), 131.1 (Cꢀ10(A)), 128.6 (Cꢀ5(A)), 128.1 (Cꢀ
7(A)), 121.6* (Cꢀ8(A)), 120.3 (Cꢀ6'(E)), 119.1 (Cꢀ6'(B)), 116.2* (Cꢀ6(A)), 115.9 (Cꢀ5'(E)),
115.7 (Cꢀ2’(E)), 115.5* (Cꢀ5'(B)), 114.9 (Cꢀ2’(B)), 104.4 (Cꢀ8(D)), 102.8 (Cꢀ2(C)), 102.7 (Cꢀ
10(D)), 96.1 (Cꢀ6(D)), 82.9 (Cꢀ2(F)), 68.6 (Cꢀ3(F)), 36.1 (Cꢀ3(C)), 34.5 (Cꢀ4(C)), 28.5 (Cꢀ
4(F)), 13.3 (CH3) (*these signals may be interchanged). FTꢀIR (ATR) νmax 3329, 2941, 1607,
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1520, 1437, 1285, 1219, 1107, 1013, 934, 868, 752. UVꢀvis (MeOH): λmax (log ε): 281 (3.87).
HRMS (ESI/QꢀTOF) m/z [M+H]+ calcd for C31H27O9 543.1648, found 543.1646. HRMS
(ESI/QꢀTOF) m/z [M–H]– calcd for C31H25O9 541.1503, found 541.1519.
3ꢀChloroꢀ2ꢀ(3,4ꢀdihydroxyphenyl)ꢀ7ꢀhydroxychromaneꢀ(4→8,2→Oꢀ7)ꢀ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,
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11% from aldehyde 2). Compound 32a: H NMR (400 MHz, CD3OD) δ 6.87 (d, J = 1.9 Hz,
1H, Hꢀ2'(B)), 6.80 (br s, 2H, Hꢀ5'(E), Hꢀ6'(E)), 6.77 (d, J = 8.5 Hz, 1H, Hꢀ5'(B)), 6.25 (d, J =
2.3 Hz, 1H, Hꢀ8(A)), 6.24 (dd, J = 8.3 Hz, J = 2.3 Hz, 1H, Hꢀ6(A)), 6.11 (s, 1H, Hꢀ6(D)),
4.55 (d, J = 7.9 Hz, 1H, Hꢀ2(F)), 4.49 (d, J = 3.1 Hz, 1H, Hꢀ3(C)), 4.37 (d, J = 3.1 Hz, 1H, Hꢀ
4(C)), 4.02 (m, 1H, Hꢀ3(F)), 2.94 (dd, J = 16.3 Hz, J = 5.6 Hz, 1H, Hꢀ4α(F)), 2.52 (dd, J =
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16.3 Hz, J = 8.5 Hz, 1H, Hꢀ4β(F)); C NMR (100 MHz, CD3OD) δ 158.5 (Cꢀ7(A)), 156.0
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(Cꢀ5(D)), 153.8 (Cꢀ9(D)), 153.6 (Cꢀ9(A)), 150.7 (Cꢀ7(D)), 147.0 (Cꢀ4'(B)), 146.4 (Cꢀ3'(B), Cꢀ
4'(E)), 145.6 (Cꢀ3'(E)), 132.0 (Cꢀ1'(B), Cꢀ1'(E)), 128.9 (Cꢀ5(A)), 120.3 (Cꢀ6'(E)), 119.8 (Cꢀ
6'(B)), 119.7 (Cꢀ10(A)), 116.2 (Cꢀ5'(E)), 115.5 (Cꢀ5'(B), Cꢀ2'(E)), 115.4 (Cꢀ2’(B)), 109.8 (Cꢀ
6(A)), 104.2 (Cꢀ8(D)), 103.4 (Cꢀ10(D), Cꢀ8(A)), 100.0 (Cꢀ2(C)), 96.2 (Cꢀ6(D)), 83.0 (Cꢀ
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2(F)), 68.6 (Cꢀ3(F)), 57.8 (Cꢀ3(C)), 36.7 (Cꢀ4(C)), 29.2 (Cꢀ4(F)). Compound 32b: H NMR
(400 MHz, CD3OD) δ 7.07 (d, J = 2.1 Hz, 1H, Hꢀ2'(B)), 6.96 (m, 2H, Hꢀ5(A), Hꢀ6'(B)), 6.83
(d, J = 2.1 Hz, 1H, Hꢀ2'(E)), 6.75 (m, 2H, Hꢀ5'(E), Hꢀ5'(B)), 6.72 (dd, J = 8.3 Hz, J = 2.1 Hz,
1H, Hꢀ6'(E)), 6.30 (dd, J = 8.1 Hz, J = 2.3 Hz, 1H, Hꢀ6(A)), 6.28 (d, J = 2.3 Hz, 1H, Hꢀ8(A)),
6.11 (s, 1H, Hꢀ6(D)), 4.66 (d, J = 7.9 Hz, 1H, Hꢀ2(F)), 4.47 (d, J = 3.1 Hz, 1H, Hꢀ3(C)), 4.30
(d, J = 3.1 Hz, 1H, Hꢀ4(C)), 3.93 (m, 1H, Hꢀ3(F)), 2.91 (dd, J = 16.2 Hz, J = 5.5 Hz, 1H, Hꢀ
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4α(F)), 2.55 (dd, J = 16.2 Hz, J = 8.7 Hz, 1H, Hꢀ4β(F)); C NMR (100 MHz, CD3OD) δ
158.5 (Cꢀ7(A)), 155.9 (Cꢀ5(D)), 153.8 (Cꢀ9(D)), 153.4 (Cꢀ9(A)), 150.8 (Cꢀ7(D)), 147.0 (Cꢀ
4'(B)), 146.5 (Cꢀ3'(B)), 146.4 (Cꢀ4'(E)), 145.6 (Cꢀ3'(E)), 132.2 (Cꢀ1'(B)), 132.0 (Cꢀ1'(E)),
129.5 (Cꢀ5(A)), 120.5 (Cꢀ6'(E)), 119.7 (Cꢀ6'(B)), 119.5 (Cꢀ10(A)), 116.0* (Cꢀ5'(E)), 115.6*
(Cꢀ5'(B)), 115.5 (Cꢀ2'(E), Cꢀ2’(B)), 109.7 (Cꢀ6(A)), 103.8 (Cꢀ8(D)), 103.3 (Cꢀ8(A)), 103.1
(Cꢀ10(D)), 99.9 (Cꢀ2(C)), 96.0 (Cꢀ6(D)), 83.6 (Cꢀ2(F)), 68.7 (Cꢀ3(F)), 57.5 (Cꢀ3(C)), 36.6 (Cꢀ
4(C)), 28.8 (Cꢀ4(F)) (*these signals may be interchanged). FTꢀIR (ATR) νmax 3308, 2943,
1612, 1506, 1448, 1281, 1105, 984, 820, 646. UVꢀvis (MeOH): λmax (log ε): 280 (3.88).
HRMS (ESI/QꢀTOF) m/z [M+H]+ calcd for C30H24ClO10 579.1051, found 579.1044. HRMS
(ESI/QꢀTOF) 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 reverseꢀphase Spherisorb
ODSꢀ2 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
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2ꢀ(3,4ꢀDihydroxyphenyl)ꢀ7ꢀhydroxyꢀ3ꢀmethylchromaneꢀ(4→4,2→Oꢀ5)ꢀ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, Hꢀ
5(A)), 7.03 (d, J = 2.3 Hz, 1H, Hꢀ2’(B)), 6.91 (dd, J = 8.3 Hz, J = 2.3 Hz, 1H, Hꢀ6’(B)), 6.77
(d, J = 8.3 Hz, 1H, Hꢀ5’(B)), 6.25 (m, 2H, Hꢀ6(A), Hꢀ8(A)), 5.95* (d, J = 2.3 Hz, 1H, Hꢀ
6(D)), 5.91* (d, J = 2.3 Hz, 1H, Hꢀ2(D)), 3.95 (d, J = 2.6 Hz, 1H, Hꢀ4(C)), 2.24 (dd, J = 6.8
Hz, J = 2.6 Hz, 1H, Hꢀ3(C)), 0.66 (d, J = 6.8 Hz, 3H, CH3(C)); 13C NMR (100 MHz, CD3OD)
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δ 157.7 (Cꢀ1(D)), 157.6 (Cꢀ7(A)), 157.1 (Cꢀ3(D)), 154.3 (Cꢀ9(A)), 153.6 (Cꢀ5(D)), 146.7 (Cꢀ
4’(B)), 145.7 (Cꢀ3’(B)), 133.6 (Cꢀ1’(B)), 128.7 (Cꢀ5(A)), 122.6 (Cꢀ10(A)), 119.1 (Cꢀ6’(B)),
115.7 (Cꢀ5’(B)), 114.9 (Cꢀ2’(B)), 108.8& (Cꢀ8(A)), 105.1 (Cꢀ4(D)), 103.4& (Cꢀ6 (A)), 102.7
(Cꢀ2(C)), 97.1 (Cꢀ2(D)), 95.4* (Cꢀ6 (D)), 36.5 (Cꢀ3(C)), 33.8 (Cꢀ4(C)), 14.24 (CH3) (*these
signals may be interchanged). FTꢀIR (ATR) νmax 3333, 2979, 1609, 1504, 1458, 1302, 1229,
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1117, 1082, 1007, 976, 926, 885, 820. UVꢀvis (MeOH): λmax (log ε): 280 (3.66). HRMS
(ESI/QꢀTOF) m/z [M+H]+ calcd for C22H19O7 395.1125, found 395.1124. HRMS (ESI/Qꢀ
TOF) m/z [M–H]– calcd for C22H17O7 393.0995, found 393.0994.
2ꢀ(3,4ꢀDihydroxyphenyl)ꢀ7ꢀhydroxychromaneꢀ(4→8,2→Oꢀ7)ꢀ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
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1.6:1 mixture of diastereoisomers (9 mg, 7% from aldehyde 2). Major isomer: H NMR (400
MHz, CD3OD) δ 7.09 (m, 1H, Hꢀ2'(B)), 6.96 (m, 1H, Hꢀ6'(B)), 6.91 (d, J = 8.2 Hz, 1H, Hꢀ
5(A)), 6.82 (d, J = 1.7 Hz, 1H, Hꢀ2'(E)), 6.78 (m, 3H, Hꢀ5'(E), Hꢀ6'(E), Hꢀ5'(B), 6.32 (d, J =
2.3 Hz, 1H, Hꢀ8(A)), 6.23 (dd, J = 8.2 Hz, J = 2.3 Hz, 1H, Hꢀ6(A)), 6.04 (s, 1H, Hꢀ6(D)),
4.58 (d, J = 7.6 Hz, 1H, Hꢀ2(F)), 4.23 (t, J = 2.9 Hz, 1H, Hꢀ4(C)), 3.98 (m, 1H, Hꢀ3(F)), 2.91
(dd, J = 16.2 Hz, J = 5.3 Hz, 1H, Hꢀ4α(F)), 2.55 (dd, J = 16.2 Hz, J = 8.2 Hz, 1H, Hꢀ4β(F)),
2.13 (m, 2H, Hꢀ3(C)); 13C NMR (100 MHz, CD3OD) δ 157.7 (Cꢀ7(A)), 155.6 (Cꢀ5(D)), 154.4
(Cꢀ9(D)), 152.3 (Cꢀ9(A)), 152.0 (Cꢀ7(D)), 146.6 (Cꢀ4'(B)), 146.4& (Cꢀ3'(B)), 146.3& (Cꢀ
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4'(E)), 145.9 (Cꢀ3'(E)), 135.3 (Cꢀ1'(B)), 132.2 (Cꢀ1'(E)), 128.9 (Cꢀ5(A)), 120.6 (Cꢀ10(A)),
120.2 (Cꢀ6'(E)), 118.3 (Cꢀ6'(B)), 116.2* (Cꢀ5'(E)), 115.8* (Cꢀ5'(B)), 115.4 (Cꢀ2'(E)), 114.3 (Cꢀ
2’(B)), 109.2 (Cꢀ6(A)), 107.7 (Cꢀ8(D)), 103.7 (Cꢀ8(A)), 102.6 (Cꢀ10(D)), 99.9 (Cꢀ2(C)), 96.2
(Cꢀ6(D)), 82.9 (Cꢀ2(F)), 68.8 (Cꢀ3(F)), 35.2 (Cꢀ3(C)), 29.2 (Cꢀ4(F)), 27.4 (Cꢀ4(C)) (*,&these
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signals may be interchanged). Minor isomer: H NMR (400 MHz, CD3OD) δ 7.10 (d, J = 2.2
Hz, 1H, Hꢀ2'(B)), 7.04 (d, J = 8.2 Hz, 1H, Hꢀ5(A)), 6.98 (dd, J = 8.4 Hz, J = 2.2 Hz, 1H, Hꢀ
6'(B)), 6.87 (d, J = 1.7 Hz, 1H, Hꢀ2'(E)), 6.78 (m, 3H, Hꢀ5'(E), Hꢀ6'(E), Hꢀ5'(B)), 6.36 (d, J =
2.3 Hz, 1H, Hꢀ8(A)), 6.31 (dd, J = 8.2 Hz, J = 2.3 Hz, 1H, Hꢀ6(A)), 6.06 (s, 1H, Hꢀ6(D)),
4.70 (d, J = 7.6 Hz, 1H, Hꢀ2(F)), 4.19 (t, J = 2.9 Hz, 1H, Hꢀ4(C)), 3.98 (m, 1H, Hꢀ3(F)), 2.87
(dd, J = 16.2 Hz, J = 5.3 Hz, 1H, Hꢀ4α(F)), 2.55 (dd, J = 16.2 Hz, J = 8.2 Hz, 1H, Hꢀ4β(F)),
2.13 (m, 2H, Hꢀ3(C)); 13C NMR (100 MHz, CD3OD) δ 157.8 (Cꢀ7(A)), 155.5 (Cꢀ5(D)), 154.4
(Cꢀ9(A)), 152.6 (Cꢀ9(D)), 152.1 (Cꢀ7(D)), 146.6 (Cꢀ4'(B)), 146.4& (Cꢀ3'(B)), 146.3& (Cꢀ
4'(E)), 145.9 (Cꢀ3'(E)), 135.3 (Cꢀ1'(B)), 132.3 (Cꢀ1'(E)), 129.5 (Cꢀ5(A)), 120.4 (Cꢀ10(A)),
120.3 (Cꢀ6'(E)), 118.3 (Cꢀ6'(B)), 116.0# (Cꢀ5'(E)), 115.8# (Cꢀ5'(B)), 115.4 (Cꢀ2'(E)), 114.3 (Cꢀ
2’(B)), 109.2 (Cꢀ6(A)), 107.6 (Cꢀ8(D)), 103.7 (Cꢀ5(A)), 102.3 (Cꢀ10(D)), 99.9 (Cꢀ2(C)), 96.1
(Cꢀ6(D)), 83.4 (Cꢀ2(F)), 68.8 (Cꢀ3(F)), 35.3 (Cꢀ3(C)), 28.5 (Cꢀ4(F)), 27.4 (Cꢀ4(C)) (#,&these
signals may be interchanged). FTꢀIR (ATR) νmax 3333, 2933, 1610, 1506, 1443, 1290, 1117,
972, 816. UVꢀvis (MeOH): λmax (log ε): 280 (3.84). HRMS (ESI/QꢀTOF) m/z [M+H]+ calcd
for C30H25O10 545.1440, found 545.1440. HRMS (ESI/QꢀTOF) 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
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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
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Copies of 1H and 13C NMR spectra, and figures with spectral variations of flavylium salts 12–
16 after direct pH jumps (PDF).
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AUTHOR INFORMATION
Corresponding Authors
*Eꢀmail: ssalido@ujaen.es
*Eꢀmail: ajp@fct.unl.pt
ORCID
Alfonso AlejoꢀArmijo: 0000ꢀ0001ꢀ8691ꢀ4628
A. Jorge Parola: 0000ꢀ0002ꢀ1333ꢀ9076
Fernando Pina: 0000ꢀ0001ꢀ8529ꢀ6848
Joaquín Altarejos: 0000ꢀ0002ꢀ5532ꢀ8535
Sofía Salido: 0000ꢀ0003ꢀ2319ꢀ7873
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
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This study was supported by the University of Jaén (UJA081605) and by the Associate
Laboratory for Green ChemistryꢀLAQV, which is financed by national funds from
FCT/MCTES (UID/QUI/50006/2013) and coꢀfinanced by the ERDF under the PT2020
Partnership Agreement (POCIꢀ01ꢀ0145ꢀFEDERꢀ007265). A. A.ꢀA. thanks the University of
Jaén for a preꢀdoctoral fellowship and also the Fundación Alfonso Martín Escudero for its
current postꢀdoctoral fellowship. Part of the work was supported by the Centro de
Instrumentación CientíficoꢀTécnica of the University of Jaén.
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