Synthesis and hLDH Inhibitory Activity of Analogues to Natural Products with 2,8-Dioxabicyclo[3.3.1]nonane Scaffold

Abstract

Human lactate dehydrogenase (hLDH) is a tetrameric enzyme present in almost all tissues. Among its five different isoforms, hLDHA and hLDHB are the predominant ones. In the last few years, hLDHA has emerged as a therapeutic target for the treatment of several kinds of disorders, including cancer and primary hyperoxaluria. hLDHA inhibition has been clinically validated as a safe therapeutic method and clinical trials using biotechnological approaches are currently being evaluated. Despite the well-known advantages of pharmacological treatments based on small-molecule drugs, few compounds are currently in preclinical stage. We have recently reported the detection of some 2,8-dioxabicyclo[3.3.1]nonane core derivatives as new hLDHA inhibitors. Here, we extended our work synthesizing a large number of derivatives (42–70) by reaction between flavylium salts (27–35) and several nucleophiles (36–41). Nine 2,8-dioxabicyclo[3.3.1]nonane derivatives showed IC₅₀ values lower than 10 µM against hLDHA and better activity than our previously reported compound 2. In order to know the selectivity of the synthesized compounds against hLDHA, their hLDHB inhibitory activities were also measured. In particular, compounds 58, 62a, 65b, and 68a have shown the lowest IC₅₀ values against hLDHA (3.6–12.0 µM) and the highest selectivity rate (>25). Structure–activity relationships have been deduced. Kinetic studies using a Lineweaver–Burk double-reciprocal plot have indicated that both enantiomers of 68a and 68b behave as noncompetitive inhibitors on hLDHA enzyme.

1. Introduction

Lactate dehydrogenase (LDH; EC 1.1.1.27) is an enzyme widely distributed in nature that belongs to the 2-hydroxyacid oxidoreductases family. It is a tetrameric molecule (140 kDa) mainly formed by two different kinds of subunits of 35 kDa each: the M-type (muscle) subunit encoded by Ldha gene and the H-type (heart) encoded by Ldhb gene. The combination of both subunits provides up to five different LDH isoforms: H₄ (also named LDH-1 or LDHB), H₃M (LDH-2), H₂M₂ (LDH-3), HM₃ (LDH-4), and M₄ (LDH-5 or LDHA). All of them show different kinetic behaviors and tissue distributions. The homotetramers LDHA and LDHB are the major isozymes that are present in human cells and are mainly located in (i) skeletal muscle and liver, and (ii) in heart and brain tissues, respectively [1,2].

This enzyme plays an important role in several metabolic pathways where it regulates the homeostasis of pyruvate/lactate, hydroxypyruvate/glycerate, and oxalate/glyoxylate, using NADH as cofactor. One of the main and most studied roles of LDH enzymes is the regulation of the energy supply mechanism in cancer and normal cells (Warburg effect) [3].

Non-cancer cells obtain their energy demand by the complete metabolism of glucose, which is performed in two subsequent steps: (i) conversion of glucose into pyruvate (glycolysis), and (ii) degradation of the generated pyruvate to CO₂ (tricarboxylic acid cycle (TCA)). The complete catabolism of glucose allows a sustainable energy production system due to the generation of ATP molecules and the regeneration of NAD⁺ cofactor during the oxidative phosphorylation (OXPHOS).

LDH and pyruvate dehydrogenase kinase (PDHK) enzymes are in charge of regulating the flux of pyruvate into TCA cycle [4]. The LDH function becomes very important when oxygen supply is reduced and/or cells increase their glucose demand and, therefore, the fermentation pathway is activated. In this case, pyruvate is reduced into lactate, regenerating NAD⁺ and producing ATP molecules but in a less efficient way. This last reaction is catalyzed by LDH enzymes and allows the maintenance of the equilibrium when glucose uptake is increased.

On the opposite hand, cancer cells need precursors to build up macromolecules in a quicker way. In that sense, and trying to fulfill this requirement, they increase their glucose uptake and adjust its metabolism in favor of a quicker but less efficient process of obtaining energy [5], in which lactic acid fermentation and LDH enzymes play the main role even in the presence of oxygen (aerobic glycolysis). As a result, high levels of glycolytic metabolites are formed, which can be used for the quick development of cancer cells [6]. This change in the metabolic glucose pathway causes the need for overexpressed LDH enzymes in cancer tissues, turning these enzymes, and particularly LDHA isozyme, into a possible therapeutic target for the development of new anticancer therapies [7].

Moreover, apart from its relevance in cancer, human LDHA isozyme (hLDHA) has recently attracted great interest among scientists, due to its proven important role in molecular mechanism of different kind of disorders [8], such as vascular diseases [9,10], epilepsy [11,12], tuberculosis [13], pulmonary fibrosis [14], arthritis [15] and other inflammatory diseases [16,17], and in primary hyperoxalurias (PHs) [18]. Recent advances in the understanding of the molecular mechanisms of several diseases have led to the development of novel therapeutic approaches, such as enzyme deletion using in vivo CRISPR/Cas9 [19,20,21] or mRNA silencing using siRNA [22,23,24,25,26]. However, pharmacological treatments based on small-molecule drugs could present some advantages compared to the use of biotechnological approaches. The cost of production is generally lower, and they can be administered orally and present better ADME (absorption, distribution, metabolism, and excretion) properties. In any case, a combined treatment with both types of pharmacological agents could have synergistic effect and benefits, such as lowering the required doses or the frequency of administration. Actually, approaches based on attenuated expression of LDHA or inhibition of LDHA activity by small-molecule drugs appear as emerging strategies for the treatment of these diseases [27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43]. Pharmaceutical companies and researchers in academia are investing significant efforts to identify natural or synthetic hLDHA inhibitors with a huge structural variability. Although some of them have shown EC₅₀ values in cancer cell line at nanomolar range [33], limitations related to ADME and pharmacokinetic properties have reduced the number of compounds evaluated in vivo and, to the best of our knowledge, none of them have progressed towards clinical trial [27,28].

Our research group is also interested in the development of hLDHA inhibitors as a pharmacological option for the treatment of PHs, a rare life-threatening genetic disease [34,35]. We recently reported the detection of (±)-2,8-dioxabicyclo[3.3.1]nonane derivatives, analogues to A-type proanthocyanidin natural products, as a new family of hLDHA inhibitors [35]. That research was inspired by the results obtained by Li et al., who performed LDHA inhibition assays on procyanidin-enriched fractions from Spatholobus suberectus extract [44], and by our experience in the synthesis of compounds with this bicyclic scaffold [45,46,47]. Thus, we have reported the synthesis of twenty (±)-2,8-dioxabicyclo[3.3.1]nonane derivatives (1–20) (Figure 1), their hLDHA and hLDHB inhibitory activities, and the ability of three of them (2, 3 and 9) to reduce the quantity of oxalate generated by hyperoxaluric mouse hepatocytes (PH1, PH2, and PH3) in vitro [35].

These results encouraged us to continue exploring this family of bicyclic compounds and to synthesize and evaluate a more complete collection of 2,8-dioxabicyclo[3.3.1]nonane derivatives in order to establish structure−activity relationships that allow to understand the main features that bicyclic core should fulfil to ensure high potency and selectivity towards hLDHA.

2. Results and Discussion

2.1. Chemical Synthesis

Our research group previously synthesized (±)-2,8-dioxabicyclo[3.3.1]nonane derivatives by reaction of flavylium salts with phenolic nucleophiles, such as phloroglucinol or resorcinol [45,47]. The fact that these compounds have emerged as a new family of hLDHA selective inhibitors [35], encouraged us to systematically design a series of 2,8-dioxabicyclo[3.3.1]nonane compounds in order to gain insight into the structural requirements that are responsible for hLDHA activity and hLDHA/hLDHB selectivity. In that sense, 44 bicyclic derivatives (1–6; 42–70) (35 are new compounds) have been prepared by reaction between 9 different flavylium salts (27–35) (3 are new compounds), with modifications at A- and B-rings, and phloroglucinol (36) and other non-phenolic π-nucleophiles (37–41) (Scheme 1). Nucleophiles 36–38 are commercially available, but pyrone-type compounds 39–41 have been prepared as racemic mixtures according to a previously reported methodology by reaction of the proper benzaldehyde derivative and ethyl acetoacetate [48,49]. The yields obtained and the spectroscopic data of these pyrone derivatives are consistent with the reported ones (39 (88%) [48], 40 (80%) [48], and 41 (74%) [48,50]).

Flavylium salts were synthesized by an aldol condensation under acidic conditions between salicylic aldehyde (21) or derivatives (22 and 23) and acetophenone derivatives (24–26) according to procedures previously used by us [35,45,51]. Three different substituents have been selected at position 6 of the A-ring (R₁ at Scheme 1) with different electronic effects on the bicyclic core. Strong and weak electron withdrawing groups (EWGs: NO₂ and Cl, respectively) along with the absence of a substituent in A-ring have been employed. For the B-ring, OH and OMe groups have been selected at position 3′ and/or 4′ (R₂ and R₃ at Scheme 1), taking into account that hydroxyl groups always appear in polyphenolic scaffold-based LDHA inhibitors (LDHAi’s) [31]. All flavylium salts were obtained with moderate to quantitative yields (63–99%;

Table 1. Reaction yields in the synthesis of flavylium salts 27−35 and (±)-dioxabicycles 1-6 and 42−70.

Entry	Compound	R1	R2	R3	R4	Yield 1
1	27	H	OH	OH	-	90%
2	28	NO2	OH	OH	-	77%
3	29	Cl	OH	OH	-	79%
4	30	H	OMe	OMe	-	96%
5	31	NO2	OMe	OMe	-	99%
6	32	Cl	OMe	OMe	-	99%
7	33	H	OH	H	-	79%
8	34	NO2	OH	H	-	76%
9	35	Cl	OH	H	-	63%
10	(±)-1 2	H	OH	OH	-	34%
11	(±)-2 2	NO2	OH	OH	-	64%
12	(±)-3 2	Cl	OH	OH	-	43%
13	(±)-42	H	OMe	OMe	-	38%
14	(±)-43	NO2	OMe	OMe	-	52%
15	(±)-44	Cl	OMe	OMe	-	62%
16	(±)-4 2	H	OH	H	-	22%
17	(±)-5 2	NO2	OH	H	-	45%
18	(±)-6 2	Cl	OH	H	-	42%
19	(±)-45	H	OH	OH	-	72%
20	(±)-46	NO2	OH	OH	-	79%
21	(±)-47	Cl	OH	OH	-	81%
22	(±)-48	H	OMe	OMe	-	55%
23	(±)-49	NO2	OMe	OMe	-	54%
24	(±)-50	Cl	OMe	OMe	-	81%
25	(±)-51	H	OH	H	-	56%
26	(±)-52	NO2	OH	H	-	58%
27	(±)-53	Cl	OH	H	-	57%
28	(±)-54	H	OH	OH	-	10%
29	(±)-55	NO2	OH	OH	-	47%
30	(±)-56	Cl	OH	OH	-	19%
31	(±)-57	H	OMe	OMe	-	23%
32	(±)-58	NO2	OMe	OMe	-	42%
33	(±)-59	Cl	OMe	OMe	-	67%
34	(±)-60	NO2	OH	H	-	46%
35	(±)-61	Cl	OH	H	-	15%
36	(±)-62a	H	OMe	OMe	H	5%
	(±)-62b	H	OMe	OMe	H	9%
37	(±)-63a	H	OMe	OMe	Cl	12%
	(±)-63b	H	OMe	OMe	Cl	20%
38	(±)-64a	H	OMe	OMe	OMe	17%
	(±)-64b	H	OMe	OMe	OMe	21%
39	(±)-65a	NO2	OMe	OMe	H	6%
	(±)-65b	NO2	OMe	OMe	H	33%
40	(±)-66a	NO2	OMe	OMe	Cl	19%
	(±)-66b	NO2	OMe	OMe	Cl	26%
41	(±)-67a	NO2	OMe	OMe	OMe	14%
	(±)-67b	NO2	OMe	OMe	OMe	24%
42	(±)-68a	Cl	OMe	OMe	H	12%
	(±)-68b	Cl	OMe	OMe	H	29%
43	(±)-69a	Cl	OMe	OMe	Cl	10%
	(±)-69b	Cl	OMe	OMe	Cl	33%
44	(±)-70a	Cl	OMe	OMe	OMe	12%
	(±)-70b	Cl	OMe	OMe	OMe	29%

¹ Yields calculated from the starting aldehydes 21–23 (see Scheme 1). ² These compounds have been previously synthesized by us [35] and their yields are included here for comparative purposes.

). It is worth noting that the three new flavylium salts with two OMe groups in B-ring (30–32) were obtained with the highest yields (96–99%).

The synthesis of (±)-2,8-dioxabicyclo[3.3.1]nonane derivatives was carried out by a sequential double addition of the nucleophilic moiety (36–41, Scheme 1) to the electrophilic moiety of the flavylium salt (27–35, Scheme 1), according to procedures previously used and optimized by us [35,45]. Although the addition of commercially available nucleophiles, phloroglucinol (36) [35,45,52], 4-hydroxycoumarin (37) [53], or 2-hydroxy-1,4-naphthoquinone (38) [54] as nucleophilic moiety to achieve the synthesis of (±)-2,8-dioxabicyclo[3.3.1]nonane have been previously reported, to the best of our knowledge, it is the first time that pyrone derivatives, such as compounds 39–41, have been employed to obtain the corresponding bicyclic structures (compounds 62–70, Scheme 1).

As gathered in Table 1, the highest yields obtained in these nucleophilic addition reactions (54–81%) corresponded to the synthesis of (±)-2,8-dioxabicyclo[3.3.1]nonane derivatives with a 4-hydroxycoumarin moiety (45–53), the presence or absence of electron-withdrawing groups (EWGs) at A-ring (NO₂, Cl) being not clearly reflected in the yields. However, the nature of the group at A-ring does influence the yields of derivatives with a phloroglucinol moiety. Thus, compounds 2, 3, 5, 6, 43, and 44, containing EWGs (NO₂ or Cl) at A-ring have been obtained with moderate yields (42–64%) versus compounds 1, 4, and 42, with no substituents at A-ring, which have been obtained with lower yields (22–38%). This behavior had already been observed previously by us [35] and the same trend was noticed when 2-hydroxy-1,4-naphthoquinone (38) was used as a nucleophile (derivatives 54–61). On the other hand, (±)-2,8-dioxabicyclo[3.3.1]nonane derivatives with pyrone-type moieties (62–70) have been synthesized as mixtures of two diastereomers since racemic 4-hydroxy-6-phenyl-5,6-dihydro-2H-pyran-2-one (39), 6-(4-chlorophenyl)-4-hydroxy-5,6-dihydro-2H-pyran-2-one (40), or 4-hydroxy-6-(4-methoxyphenyl)-5,6-dihydro-2H-pyran-2-one (41) were used as nucleophiles. These mixtures of diastereomers have been obtained with moderate yields (32–45%), except for derivative 62 (14%). Chromatographic separations of these mixtures led to pure (±)-2,8-dioxabicyclo[3.3.1]nonane derivatives 62a–70a and 62b–70b (Figure 2).

A diastereomeric excess has been observed for compounds 62–70 in all cases, in favor of diastereomers 62b–70b (1:1.2–1:5.5, entries 37–45, Table 1) that have a “trans” relative arrangement of rings B and E (Figure 2). A rationale of these results can be found in the less favored approach of one of the enantiomers of the nucleophile to the flavylium salt to give the “cis” diastereomer versus the more favored approach of the other enantiomer to the same face of the salt to give the “trans” diastereomer (Figure 3).

All synthesized compounds were totally characterized using spectroscopic and spectrometric techniques such as ¹H NMR, ¹³C NMR, 2D NMR, and HRMS (¹H NMR and ¹³C NMR spectra are included in the Supplementary Material Figures S1–S82). Moreover, their purities were previously checked by HPLC (chromatograms of all byciclic compounds are included in the Supplementary Material Figures S83–S132). Flavylium salts 27 [55], 28 [46], 29 [35], 33 [56], 34 [57], and 35 [35], and (±)-dioxabicyclic derivatives 1–2 [52], 3–6 [35], 48 [53], 51 [53], and 57 [54] were described previously and their spectroscopic data agreed with those reported in the literature. On the other hand, flavylium salts 30–32 and bicycles 42–47, 49–50, 52–56, and 58–70 are new compounds and their structural characterization is reported for the first time

2.2. Inhibitory Activity of (±)-2,8-Dioxabicyclo[3.3.1]Nonanes 1–6, 42–70 against hLDHA and hLDHB

The inhibitory activity of all synthetized byciclic derivatives (1–6, 42–70) against both enzymes hLDHA and hLDHB have been measured by a kinetic spectrofluorimetric assay [58]. Briefly, the decrease in the coenzyme β-NADH fluorescence (λ_emission = 460 nm) was measured for 10 min, at different concentration of inhibitors, and its slope was compared with the one obtained when there were not inhibitors added (100% enzymatic activity). The inhibitory activity of the synthetized compounds is expressed as the concentration needed to reduce the enzymatic activity of hLDHA or hLDHB up to 50% (IC₅₀). The dose response curves against hLDHA and hLDHB of all new byciclic compounds are included in the Supplementary Material (Figures S133–S198).

All synthesized bicyclic compounds, except seven of them (1, 4, 42, 63b, 64a, 64b, and 70a), showed IC₅₀ values against hLDHA in the low micromolar range (3.6–50 μM), and it is also worth highlighting that the inhibitory activity of ten bicyclic derivatives (2, 43, 44, 47, 58, 60, 62a, 65b, 66a, and 66b) showed IC₅₀ values lower than 10 µM (

Table 2. Inhibition activity of (±)-2,8-dioxabicyclo[3.3.1]nonane derivatives against hLDHA and hLDHB.

Compound	hLDHA IC50 (μM) 2	R2	hLDHB IC50 (μM) 2	R2	Selectivity Rate (IC50 hLDHB/hLDHA)
(±)-1 1	73.1 ± 2.5	0.87	35.4 ± 0.8	0.93	0.5
(±)-2 1	9.7 ± 1.1	0.93	28.6 ± 1.9	0.97	3.0
(±)-3 1	15.7 ± 2.7	0.94	39.7 ± 2.8	0.98	2.5
(±)-42	153.6 ± 11.3	0.93	293.1 ± 2.4	0.98	1.9
(±)-43	8.2 ± 0.2	0.97	65.3 ± 2.1	0.96	8.0
(±)-44	9.6 ± 0.3	0.98	23.1 ± 1.1	0.95	2.4
(±)-4 1	60.0 ± 4.7	0.92	45.9 ± 1.5	0.96	0.8
(±)-5 1	24.4 ± 2.7	0.94	69.7 ± 4.7	0.95	2.9
(±)-6 1	26.7 ± 0.8	0.94	85.5 ± 2.5	0.98	3.2
(±)-45	21.4 ± 0.5	0.98	28.6 ± 0.6	0.95	1.3
(±)-46	28.5 ± 0.4	0.99	14.1 ± 0.3	0.96	0.5
(±)-47	9.1 ± 0.5	0.97	14.3 ± 0.9	0.99	1.6
(±)-48	49.6 ± 1.7	0.97	>300	-	>6.0
(±)-49	25.7 ± 0.3	0.97	212.3 ± 1.4	0.98	8.3
(±)-50	13.2 ± 0.8	0.94	>300	-	>22.7
(±)-51	34.4 ± 0.8	0.91	31.5 ± 3.0	0.97	0.9
(±)-52	36.9 ± 1.2	0.97	66.2 ± 1.0	0.93	1.8
(±)-53	11.2 ± 0.6	0.97	18.3 ± 1.0	0.97	1.6
(±)-54	20.7 ± 0.5	0.99	13.8 ± 0.2	0.98	0.7
(±)-55	27.4 ± 0.6	0.93	29.0 ± 2.4	0.94	1.1
(±)-56	13.7 ± 0.6	0.98	118.9 ± 2.7	0.99	8.7
(±)-57	42.2 ± 2.4	0.91	>300	-	>7.1
(±)-58	8.5 ± 1.0	0.96	>300	-	>35.3
(±)-59	20.4 ± 1.2	0.98	266.1 ± 4.8	0.96	13.0
(±)-60	4.1 ± 0.4	0.96	13.2 ± 1.3	0.97	3.2
(±)-61	20.1 ± 1.6	0.97	22.8 ± 1.4	0.96	1.1
(±)-62a	3.6 ± 0.3	0.94	150.3 ± 3,9	0.97	41.8
(±)-62b	39.4 ± 1.2	0.97	>300	-	>7.6
(±)-63a	17.4 ± 0.3	0.97	250.6 ± 10.5	0.97	14.4
(±)-63b	50.8 ± 0.4	0.98	296.1 ± 2.7	0.99	5.8
(±)-64a	52.8 ± 5.3	0.98	>300	-	>5.7
(±)-64b	96.7 ± 9.0	0.95	>300	-	>3.1
(±)-65a	12.5 ± 1.1	0.97	269.2 ± 11.0	0.97	21.5
(±)-65b	8.5 ± 0.4	0.98	>300	-	>35.3
(±)-66a	5.8 ± 0.2	0.98	28.1 ± 0.4	0.99	4.8
(±)-66b	6.0 ± 0.1	0.97	20.4 ± 0.7	0.98	3.4
(±)-67a	14.8 ± 0.6	0.93	>300	-	>20.3
(±)-67b	14.6 ± 0.3	0.98	53.7 ± 0.9	0.99	3.7
(±)-68a	12.0 ± 0.9	0.99	>300	-	>25.0
(±)-68b	28.5 ± 0.5	0.97	>300	-	>10.5
(±)-69a	20.0 ± 0.6	0.99	140.9 ± 2.5	0.99	7.1
(±)-69b	14.2 ± 0.8	0.98	77.0 ± 9.7	0.98	5.4
(±)-70a	112.2 ± 3.0	0.99	>300	-	>2.7
(±)-70b	20.2 ± 0.7	0.95	292.3 ± 4.1	0.98	14.5

¹ These compounds have been synthesized and evaluated previously by us [35] and their inhibition data are included here for comparative purposes. ² IC₅₀ data are presented as the mean ± SD of n = 3 replicates.

). Among the 2,8-dioxabicyclo[3.3.1]nonane derivatives with phloroglucinol moiety, compounds 43 and 44 have shown the highest activity, being similar to compound 2, one of the most active compounds previously reported by us [35]. In the case of derivatives with 4-hydroxycoumarin and 2-hydroxy-1,4-naphthoquinone moieties, only one compound (47) and two compounds (58 and 60), respectively, showed better activity than 2. However, four derivatives with pyrone-type moiety (62a, 65b, 66a, and 66b) showed better activity than 2. In fact, the most active hLDHA inhibitor synthesized is 62a (IC₅₀ = 3.6 µM), a 2,8-dioxabicyclo[3.3.1]nonane derivative with a 4-hydroxy-6-phenyl-5,6-dihydro-2H-pyran-2-one moiety (39), and two methoxy groups at B-ring as substituents (Table 2).

Although inhibition potency (IC₅₀ against hLDHA) is an important parameter that characterize an inhibitor, its selectivity through the target enzyme governs its applicability and it is a feature that should be also taken into account. In order to know the selectivity of the synthesized compounds against hLDHA, their hLDHB inhibitory activities were also measured. In Figure 4, the inverse of IC₅₀ for each compound against both enzymes is represented for the sake of clarity. All of them showed higher inhibitory activity (higher 1/IC₅₀ values) against hLDHA than against hLDHB, except for compounds 1, 4, 46, 51, and 54.

Figure 5 illustrates the selectivity rate, defined as the ratio between IC₅₀ values against hLDHB and IC₅₀ values against hLDHA (Table 2), for every 2,8-dioxabicyclo[3.3.1]nonane derivative synthetized (1–6, 42–70). High selectivity rate values (>10) have been observed for eleven compounds, one with 4-hydroxycoumarin (50), two with 2-hydroxy-1,4-naphthoquinone (58 and 59), and eight with a pyrone-type moieties (62a, 63a, 65a, 65b, 67a, 68a, 68b, and 70b). In particular, compounds 58, 62a, 65b, and 68a have shown the lowest IC₅₀ values against hLDHA (3.6–12.0 µM) and the highest selectivity rate (>25), which allow us to select them as hits for future structural optimization. These data seem to demonstrate that the use of pyrone derivatives as nucleophilic moiety is an important structural feature for ensuring high potency and selectivity against hLDHA.

Regarding the behavior of both diastereomers of compounds 62–70, all of them showed better inhibition activity against hLDHA than against hLDHB (Figure 4), and in terms of selectivity (Figure 5), a slightly higher selectivity is observed for the “cis” than for the “trans” isomers, with the exception of compounds 65 and 70 (Figure 5). This means that a clear influence of the relative arrangement of rings B and E of compounds 62–70 cannot be established yet with the current data.

According to these inhibitory activity and selectivity results, a preliminary structure–activity analysis can be made. Figure 6 shows the number of compounds (in percentage) with a specific structural moiety (substituents at A- and B-rings and nucleophilic moiety) that meet the requirement of a selected inhibition activity (blue line, hLDHA inhibition activity lower than 30 μM; yellow line, hLDHB inhibition activity higher than 30 μM). It is deduced from the graphic that the presence of EWGs (NO₂ and Cl) at A-ring is an important feature to ensure higher inhibition activity against hLDHA. Percentagewise, the number of compounds with a nitro or chloro substituent at A-ring and IC₅₀ values against hLDHA lower than 30 µM is much higher than those without a substituent at A-ring. In addition, the number of compounds with chloro at A-ring and hLDHB inhibition activity higher than 30 μM is also higher than those with a nitro substituent. This means that derivatives with Cl at A-ring show better selectivity rate.

Regarding B-ring substituents, the percentage of compounds with two oxygenated groups (R₂=R₃=OH or OMe) and IC₅₀ values against hLDHA lower than 30 µM is higher than those with just one group (R₂=OH; R₃=H). Within this subgroup of deoxygenated compounds, the percentage of cathecol derivatives (R₂=R₃=OH) with hLDHB inhibition activity higher than 30 μM is lower, pointing out a decrease in the selectivity when free hydroxyl groups are present.

Finally, the influence of the nucleophilic moiety on the inhibitory activity against hLDHA and on the selectivity rate is remarkable (Figure 6). Percentagewise, the highest number of compounds with low IC₅₀ values against hLDHA corresponds to those with 4-hydroxycoumarin (37), 4-hydroxy-6-phenyl-5,6-dihydro-2H-pyran-2-one (39), or 6-(4-chlorophenyl)-4-hydroxy-5,6-dihydro-2H-pyran-2-one (40) moieties. In particular, the substitution pattern presented in nucleophile 39 favors a greater number of compounds with IC₅₀ values against hLDHB higher than 30 μM, thereby increasing its selectivity as happen for compounds 62, 65, or 68.

2.3. Resolution of Racemates and Inhibitory Activity of 2,8-Dioxabicyclo[3.3.1]nonanes (+)-66a, (−)-66a, (+)-68a, (−)-68a, (+)-68b, and (−)-68b against hLDHA and hLDHB

Some of the most active and selective racemic compounds synthesized (66a, 68a, and 68b) were purified by chiral HPLC, using a Chiralpak IC column and hexane and dicholomethane (30:70, v/v) as mobile phase. The proper purity of each enantiomer has been also checked by HPLC (chromatograms of each pure enantiomer are included in the Supplementary Material Figures S127–S132), using a Chiralpak IC analytical column and by the measurement of their optical rotation. The inhibition activity of pure enantiomers has been determined against both enzymes, hLDHA and hLDHB (

Table 3. Inhibition activity (hLDHA and hLDHB) of pure enantiomers of a selection of the most active and selective 2,8-dioxabicyclo[3.3.1]nonane derivatives.

Compound	hLDHA IC50 (μM)	R2	hLDHB IC50 (μM)	R2	Selectivity Rate (IC50 hLDHB/hLDHA)
(+)-66a	9.1 ± 0.5	0.99	43.2 ± 1.7	0.99	4.8
(−)-66a	14.9 ± 0.8	0.96	40.8 ± 0.2	0.98	2.7
(+)-68a	10.2 ± 0.3	0.98	>300	-	>29.4
(−)-68a	15.8 ± 0.6	0.99	>300	-	>19.0
(+)-68b	27.3 ± 0.2	0.99	>300	-	>11.0
(−)-68b	27.6 ± 1.8	0.96	>300	-	>11.0

). The dose response curves against hLDHA and hLDHB of each pure enantiomer are included in the Supplementary Material (Figures S199–S206).

According to the results obtained, it seems that the spatial influence of the substituents in the selected 2,8-dioxabicyclo[3.3.1]nonane derivatives for the enzymatic inhibition assays is not very high due to the similar values of IC₅₀ obtained for each pair of enantiomers. Moreover, the inhibition activity of each single enantiomer was also very similar to the one obtained by its corresponding racemic mixture. For the hLDHA inhibition test, it seems that a better behavior is observed for dextrorotarory enantiomers, especially for compounds 66a and 68a (1.5 times more potent). For the hLDHB inhibition test, only compound 66a was active and both enantiomers showed also similar potency.

It seems that the relative “cis–trans” arrangement of B and E ring in pyrone-type bicycle derivatives (62a–70a vs. 62b–70b) plays a more important role in the enzymatic inhibition assays performed than the different absolute configurations observed for pure enantiomers.

2.4. Mechanism of hLDHA and hLDHB Inhibition

In order to explore the mechanism of inhibition of some of the most active compounds on hLDHA, both enantiomers of the racemate 68a and 68b have been selected. Assays measuring the enzymatic activity of both compounds in the presence of four different inhibitor concentrations and without an inhibitor and eight different substrate (pyruvate) concentrations have been measured by a similar kinetic spectrofluorimetric procedure to that described to calculate IC₅₀ values against hLDHA (Section 3.4). The initial velocity (V₀) was determined as the maximum slope per minute calculated in the linear interval of the graph representing the progression of the enzymatic reaction at each inhibitor and substrate concentration. The progression of the enzymatic reaction was monitored by the decrease in fluorescence, due to NADH conversion into NAD⁺, registered at λ_ex 340 nm and λ_em 460 nm every 60 s during 10 min. Nonlinear fits of V₀ versus substrate concentration (Michaelis–Menten type) were performed for each inhibitor concentration and without inhibitor to determine V_max and K_M at each curve (values of V_max and K_M are included in the Supplementary Material Tables S1–S4). A decrease in apparent V_max and no effect on the apparent K_M were observed (with increasing concentrations of both enantiomers of racemate 68a and 68b), which is indicative of a noncompetitive inhibition. In addition, their corresponding Lineweaver–Burk double-reciprocal graphs showed intersection on the x axis, which is also favorable to this type of inhibition (graphs included in the Supplementary Material Figures S207–S210). Finally, a noncompetitive inhibition fit of V₀ versus substrate concentration allowed us to obtain the K_i values, 4.1 and 16.1 μM for dextrorotatory 68a and levorotatory 68a, respectively, and 35.9 and 76.9 μM for dextrorotatory 68b and levorotatory 68b, respectively. Noncompetitive inhibition is not affected by the substrate concentration and this mechanism could suggest an allosteric binding to the enzyme, which has already been suggested by others [59].

3. Materials and Methods

3.1. General Experimental Methods

All solvents and reagents used in the synthesis or biological evaluations were purchased and were used as received, except MeOH, which was previously dried, using standard methodologies [60].

All reactions were conducted under inert conditions at room temperature or at 50 °C. Reactions were monitored by analytical thin-layer chromatography (TLC) on silica gel 60 F₂₅₄ precoated aluminum sheets (0.25 mm, Merck Chemicals, Darmsdadt, Germany) and spots were detected using UV light (λ = 254 nm). Purification steps of synthesized racemic compounds were carried out by column chromatography (CC) using Sephadex LH-20 or Silica gel 60 (particle size 0.040–0.063 mm) (Merck Chemicals, Darmsdadt, Germany). Purity grade of synthesized racemic compounds were performed on a Waters 600E analytical HPLC (Waters Chromatography Division, Milford, MA, USA) that had a diode array detector (DAD), operating in the range of 190–800 nm (Waters CapLC 2996 Photodiode Array Detector, Waters Chromatography Division, Milford, MA, USA), at 30 °C and using a C18 reversed-phase Spherisorb ODS-2 column, 250 mm × 3 mm i.d., 5 μm (Waters Chromatography Division, Milford, MA, USA). The best separation conditions were accomplished with H₂O:CH₃COOH, 99.8:0.2, v/v (solvent A) and CH₃CN:CH₃COOH, 99.8:0.2, v/v (solvent B) at a flow rate of 0.7 mL/min: linear gradient from 20% to 80% B for 20 min; 80% B for 20 min; and 5 min to come back to the initial conditions. Purity data of the compounds were measured at 280 nm and are included in the Supplementary Material section. Chiral HPLC purifications were conducted on the same HPLC equipment previously described using a Chiralpak IC column, (250 mm × 10 mm i.d., 5 μm (Daicel Corporation, Illkrich, France). The best separation was achieved with 30% of hexane (solvent A) and 70% of dichloromethane (solvent B) at a flow rate of 8 mL/min for 30 min. The purity of the separated enantiomers has been checked by analytical HPLC using a Chiralpak IC column, (250 mm × 3 mm i.d., 5 μm (Daicel Corporation, Illkrich, France). Optical rotations ([α]_D) were recorded on a Jasco P-2000 automatic polarimeter (Jasco Analytical Instruments, Easton, MD, USA), using quartz cells of 1 dm path length, in methanol solutions.

¹H NMR and ¹³C NMR spectra were measured on a Bruker Avance 400 spectrometer (Bruker Daltonik GmbH, Bremen, Germany) operating at 400 and 100 MHz for ¹H and ¹³C, respectively. Deuterated methanol (CD₃OD), deuterated chloroform (CDCl₃), or deuterated dimethylsulfoxide ((CD₃)₂SO) was used as solvents. For flavylium salts a drop of TFA-d (trifluoroacetic acid deuterated) was added to obtain acidic conditions. Chemical shifts (in ppm) were referenced to solvent peaks as internal reference. The coupling constants (J) are expressed in hertz (Hz). The coupling system is described by using the following abbreviations: d, doublet; t, triplet; m, multiplet; br s, broad singlet; br d, broad doublet; dd, doublet of doublets; ddd, doublet of doublet of doublets; td, triplet of doublets; dq, doublet of quartets. The total assignment of ¹H and ¹³C signals was made using 2D NMR spectra such as COSY, HSQC, and HMBC. High-resolution mass spectra (HRMS) were conducted on an Agilent 6520B Quadrupole time-of-flight (QTOF) mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) with an electrospray ionization (ESI) interface operating in positive or negative mode.

3.2. General Methodology A Applied to Synthesize Flavylium Salts (27–35)

In a round flask was added the salicylic aldehyde derivative (2 mmol), the acetophenone derivative (2 mmol), 98% H₂SO₄ (0.6 mL; 10.8 mmol), and HOAc (2.6 mL). The mixture was stirred during one night at room temperature according to procedures previously used and described by us [35,45,46,51]. Then, 30 mL of Et₂O was slowly added and a reddish solid precipitated. This solid was filtered off and carefully washed with more Et₂O and dried. The flavylium salts 27 (0.605 g, 90% yield), 28 (0.586 g, 77% yield), 29 (0.608 g, 79%), 33 (0.506 g, 79% yield), 34 (0.555 g, 76% yield), and 35 (0.447 g, 63% yield) were previously described with comparable yields and their structures could be confirmed by analogy to their spectral data with those reported in the literature: 27 [55], 28 [46], 29 [35], 33 [56], 34 [57], and 35 [35].

3.2.1. 3′,4′-Dimethoxyflavylium Hydrogen Sulfate (30)

General methodology A was applied using 2-hydroxybenzaldehyde (21, 0.217 mL, 2 mmol) and 3′,4′-dimethoxyacetophenone (25, 0.360 g, 2 mmol). Pure compound 30 was synthesized as a red solid (0.700 g, 96% yield). Melting point: 193–195 °C; ¹H NMR (400 MHz, (CD₃)₂SO/TFA-d, pD ≈ 1.0) δ 9.45 (d, J = 9.1 Hz, 1H, H-4), 8.99 (d, J = 9.1 Hz, 1H, H-3), 8.51–8.38 (m, 2H, H-8, H-6′), 8.33 (dd, J = 8.2, 1.6 Hz, 1H, H-5), 8.26 (ddd, J = 8.7, 7.2, 1.6 Hz, 1H, H-7), 8.07 (d, J = 2.3 Hz, 1H, H-2′), 7.92 (t, J = 7.5 Hz, 1H, H-6), 7.38 (d, J = 8.9 Hz, 1H, H-5′), 4.01 (s, 3H, 4′-OMe) 3.98 (s, 3H, 3′-OMe); ¹³C NMR (100 MHz, DMSO/TFA-d, pD ≈ 1.0) δ 174.7 (C-2), 158.6 (C-4′), 155.6 (C-9), 154.9 (C-4), 150.3 (C-3′), 138.8 (C-7), 130.6 (C-5), 130.3 (C-6), 128.9 (C-6′), 124.1 (C-10), 121.5 (C-1′), 119.8 (C-8), 118.7 (C-3), 113.6 (C-5′), 112.5 (C-2′), 57.0 (C-4′-OMe), 56.5 (C-3′-OMe); HRMS (ESI-TOF) m/z [M]⁺ Calcd. For C₁₇H₁₅O₃ 267.1021, found 267.1023.

3.2.2. 3′,4′-Dimethoxy-6-nitroflavylium Hydrogen Sulfate (31)

General methodology A was applied using 2-hydroxy-5-nitrobenzaldehyde (22, 0.334 g, 2 mmol) and 3′,4′-dimethoxyacetophenone (25, 0.360 g, 2 mmol). Pure compound 31 was synthesized as a red solid (0.810 g, 99% yield). Melting point: 105–107 °C; ¹H NMR (400 MHz, (CD₃)₂SO/TFA-d, pD ≈ 1.0; mixture of trans-chalcone:flavylium cation 1:0.3) Major compound: δ 8.25 (d, J = 2.8 Hz, 1H, H-5), 8.02 (dd, J = 9.0, 2.9 Hz, 1H, H-7), 7.60 (dd, J = 8.4, 2.0 Hz, 1H, H-6′), 7.40 (d, J = 2.0 Hz, 1H, H-2′), 7.08 (ov, 1H, H-4), 7.00 (ov, 1H, H-5′), 6.99 (ov, 1H, H-3), 6.96 (ov, 1H, H-8), 3.80 (s, 3H, 4′-OMe) 3.77 (s, 3H, 3′-OMe); ¹³C NMR (100 MHz, DMSO/TFA-d, pD ≈ 1.0) δ 191.7 (C-2), 161.8 (C-9), 153.5 (C-4′), 148.9 (C-3′), 139.1 (C-6), 132.7 (C-4), 130.1 (C-1′), 128.4 (C-3), 126.1 (C-5), 125.8 (C-7), 123.7 (C-6′), 123.3 (C-10), 115.6 (C-8), 110.8 (C-5′), 110.4 (C-2′), 55.7 (C-4′-OMe), 55.3 (C-3′-OMe); HRMS (ESI-TOF) m/z [M]⁺ Calcd. For C₁₇H₁₄ClO₃ 301.0631, found 301.0635.

3.2.3. 3′,4′-Dimethoxy-6-chloroflavylium Hydrogen Sulfate (32)

General methodology A was applied using 2-hydroxy-5-chlorobenzaldehyde (23, 0.313 g, 2 mmol) and 3′,4′-dimethoxyacetophenone (25, 0.360 g, 2 mmol). Pure compound 32 was synthesized as a red solid (0.790 g, 99% yield). Melting point: 190 °C (decomposes); ¹H NMR (400 MHz, (CD₃)₂SO/TFA-d, pD ≈ 1.0; mixture of trans-chalcone:flavylium cation 1:0.7) Major compound: δ 7.58 (dd, J = 8.4, 2.0 Hz, 1H, H-6′), 7.40 (d, J = 2.0 Hz, 1H, H-2′), 7.26 (d, J = 2.7 Hz, 1H, H-5), 7.10 (dd, J = 8.7, 2.5 Hz, 1H, H-7), 7.04 (d, J = 8.4 Hz, 1H, H-5′), 7.07 (d, J = 6.4 Hz, 1H, H-4), 6.82 (d, J = 8.7, 1H, H-8), 6.81 (d, J = 6.4 Hz, 1H, H-3), 3.81 (s, 3H, 4′-Ome) 3.78 (s, 3H, 3′-Ome); ¹³C NMR (100 MHz, DMSO/TFA-d, pD ≈ 1.0) δ 191.8 (C-2), 154.5 (C-9), 153.4 (C-4′), 148.5 (C-3′), 133.3 (C-4), 130.1 (C-1′), 129.3 (C-5, C-7), 127.5 (C-3), 124.4 * (C-6), 123.6 (C-6′), 122.0 * (C-10), 117.0 (C-8), 111.1 (C-5′), 110.6 (C-2′), 55.9 (C-4′-Ome), 55.5 (C-3′-Ome); *these signals may be interchangeable. HRMS (ESI-TOF) m/z [M]⁺ Calcd. For C₁₇H₁₄ClO₃ 301.0631, found 301.0635.

3.3. General Methodology B Applied to Synthesize 2,8-Dioxabicyclo[3.3.1]nonanes (1–6, 42–70)

In a round flask was added the flavylium salt species (27–35) (0.5 mmol), phloroglucinol (36), 4-hydroxycoumarin (37), 2-hydroxy-1,4-naphthoquinone (38), 4-hydroxy-6-phenyl-5,6-dihydro-2H-pyran-2-one (39), 6-(4-chlorophenyl)-4-hydroxy-5,6-dihydro-2H-pyran-2-one (40) or 4-hydroxy-6-(4-methoxyphenyl)-5,6-dihydro-2H-pyran-2-one (41) (0.5 mmol), and absolute MeOH (8 mL). The mixture was stirred overnight at 50 °C in an oil bath using the method previously described by Kraus et al. [52] and also used by us [35,45,46]. Finally, the solvent was removed and the crude was purified by CC using Silica gel 60 as stationary phase.

The dioxabicyclic derivatives 1 (0.069 g, 34% yield from 21), 2 (0.170 g, 64% from 22), 3 (0.084 g, 43% from 23), 4 (0.032 g, 22% from 21), 5 (0.088 g, 45% from 22), 6 (0.077 g, 42% from 23), 48 (0.123 g, 55% from 21), 51 (0.129 g, 56% from 21), and 57 (0.053 g, 23% from 21) were previously described by us [35] and others [52,53,54] with comparable yields and their structures could be confirmed by analogy to their spectral data with those reported in the literature: 1-2 [35,52], 3-6 [35], 48 [53], 51 [53], and 57 [54]. Analytical HPLC (λ = 280 nm): compound 1 (purity: 98%; t_R = 15.9 min); compound 2 (purity: 97%; t_R = 21.2 min); compound 3 (purity: 98%; t_R = 18.8 min); compound 4 (purity: 97%; t_R = 18.0 min); compound 5 (purity: >99%; t_R = 14.9 min); compound 6 (purity: 98%; t_R = 20.7 min); compound 48 (purity 98%; t_R = 21.2 min); compound 51 (purity >99%; t_R = 19.0 min); and compound 57 (purity 97%; t_R = 21.6 min).

3.3.1. 2-(3′,4′-Dimethoxyphenyl)-chromane-(4→4″, 2→O-5″)-phloroglucinol (42)

General methodology B was applied using the flavylium salt 30 (0.182 g) and phloroglucinol (36, 0.063 g, 0.5 mmol). The purification step was performed by CC using mixtures of hexane-EtOAc (75:25) as mobile phase. Pure compound 42 was obtained as a pale orange amorphous solid (0.07 g, 38% from 21). Melting point: 77–80 °C (decomposes); Compound 42: ¹H NMR (400 MHz, CDCl₃) δ 7.28 (dd, J = 7.5, 1.7 Hz, 1H, H-5), 7.15 (ov, 1H, H-5′), 7.13 (br s, 1H, H-2′), 6.97 (ddd, J = 8.1, 7.5, 1.7 Hz, 1H, H-7), 6.87 (d, J = 8.9 Hz, 1H, H-6′), 6.81 (dd, J = 8.1, 1.2 Hz, 1H, H-8), 6.75 (td, J = 7.5, 1.2 Hz, 1H, H-6), 5.84 (br s, 2H, H-2″, H-6″), 4.26 (t, J = 3.0 Hz, 1H, H-4), 3.74 (s, 6H, 3′-OMe, 4′-OMe), 2.29 (d, J = 3.0 Hz, 2H, H-3); ¹³C NMR (100 MHz, CDCl₃) δ 158.2 * (C-1″ (D)), 156.3 * (C-3″ (D)), 154.6 (C-9 (A)), 153.7 (C-5″ (D)), 150.8 (C-4′ (B)), 150.2 (C-3′ (B)), 136.4 (C-1′ (B)), 129.3 (C-10 (A)), 129.0 (C-5 (A)), 128.5 (C-7(A)), 122.1 (C-6(A)), 119.7 (C-6′ (B)), 117.0 (C-8 (A)), 112.5 (C-5′ (B)), 111.0 (C-2′ (B)), 107.3 (C-4″ (D)), 100.0 (C-2 (C)), 96.9 ^# (C-2″ (D)), 95.8 ^# (C-6″ (D)), 56.6 (3′-OMe, 4′-OMe), 34.8 (C-3 (C)), 28.2 (C-4 (C)); ^{*, #} these signals may be interchangeable. HRMS (ESI-TOF) m/z [M+H]⁺ Calcd. For C₂₃H₂₀O₆ 392.126, found 392.1265. Analytical HPLC (λ = 280 nm): purity: >99%; t_R = 15.9 min.

3.3.2. 2-(3′,4′-Dimethoxyphenyl)-6-nitrochromane-(4→4″, 2→O-5″)-phloroglucinol (43)

General methodology B was applied using the flavylium salt 31 (0.205 g) and phloroglucinol (36, 0.063 g, 0.5 mmol). The purification step was performed by CC using mixtures of DCM-acetone (95:5) as mobile phase. Pure compound 43 was obtained as a pale yellow amorphous solid (0.116 g, 52% from 22). Melting point: 193–195 °C; Compound 43: ¹H NMR (400 MHz, DMSO-d₆) δ 8.20 (d, J = 2.8 Hz, 1H, H-5), 8.04 (dd, J = 9.0, 2.8 Hz, 1H, H-7), 7.03 (d, J = 8.3 Hz, 1H, H-5′), 7.27–7.22 (ov, 2H, H-2′, H-6′), 7.17 (d, J = 9.0 Hz, 1H, H-8), 5.97 (d, J = 2.2 Hz, 1H, H-2″), 5.89 (d, J = 2.2 Hz, H-6″), 4.44 (br s, 1H, H-4), 3.80 (s, 3H, 3′-OMe), 3.78 (s, 3H, 4′-OMe), 2.45–2.36 (m, 2H, H-3); ¹³C NMR (100 MHz, DMSO-d₆) δ 158.5 (C-9 (A)), 158.0 (C-1″ (D)), 155.5 (C-3″ (D)), 152.8 (C-5″ (D)), 149.9 (C-4′ (B)), 149.1 (C-3′ (B)), 141.4 (C-6(A)), 133.4 (C-1′ (B)), 129.6 (C-10 (A)), 124.2 (C-7(A)), 123.4 (C-5 (A)), 118.6 (C-6′ (B)), 117.5 (C-8 (A)), 111.9 (C-5′ (B)), 111.1 (C-2′ (B)), 104.3 (C-4″ (D)), 100.1 (C-2 (C)), 96.8 (C-2″ (D)), 95.0 (C-6″ (D)), 56.2 (3′-OMe, 4′-OMe), 32.0 (C-3 (C)), 26.6 (C-4 (C)). HRMS (ESI-TOF) m/z [M+H]⁺ Calcd. For C₂₃H₁₉NO₈ 437.1111, found 437.1108. Analytical HPLC (λ = 280 nm): purity: 98%; t_R = 16.7 min.

3.3.3. 2-(3′,4′-Dimethoxyphenyl)-6-chlorochromane-(4→4″, 2→O-5″)-phloroglucinol (44)

General methodology B was applied using the flavylium salt 32 (0.199 g) and phloroglucinol (36, 0.063 g, 0.5 mmol). The purification step was performed by CC using mixtures of DCM-MeOH (97:3) as mobile phase. Pure compound 44 was obtained as a pale pink amorphous solid (0.135 g, 62% from 23). Melting point: 257–259 °C; Compound 44: ¹H NMR (400 MHz, MeOD) δ 7.37 (d, J = 2.6 Hz, 1H, H-5), 7.26–7.23 (ov, 2H, H-2′, H-6′), 7.07 (dd, J = 8.6, 2.6 Hz, 1H, H-7), 7.00 (d, J = 8.9 Hz, 1H, H-5′), 6.90 (d, J = 8.6 Hz, 1H, H-8), 5.98-5.96 (ov, 2H, H-2″, H-6″), 4.365 (t, J = 3.0 Hz, 1H, H-4), 3.87 (s, 3H, 4′-OMe), 3.86 (s, 3H, 3′-OMe), 2.30–2.19 (m, 2H, H-3); ¹³C NMR (100 MHz, MeOD) δ 158.5 (C-3″ (D)), 156.3 (C-1″ (D)), 154.5 (C-5″ (D)), 152.5 (C-9 (A)), 150.9 (C-4′ (B)), 150.2 (C-3′ (B)), 136.0 (C-1′ (B)), 131.2 (C-10 (A)), 128.4 (C-5 (A)), 128.2 (C-7(A)), 126.7 (C-6(A)), 119.7 (C-6′ (B)), 118.5 (C-8 (A)), 112.4 (C-5′ (B)), 111.0 (C-2′ (B)), 106.5 (C-4″ (D)), 100.2 (C-2 (C)), 97.0 (C-2″ (D)), 95.9 (C-6″ (D)), 55.1 (3′-OMe), 55.0 (4′-OMe), 32.8 (C-3 (C)), 26.6 (C-4 (C)). HRMS (ESI-TOF) m/z [M+H]⁺ Calcd. For C₂₃H₁₉ClO₆ 426.087, found 426.0866. Analytical HPLC (λ = 280 nm): purity: >99%; t_R = 17.6 min.

3.3.4. 2-(3′,4′-Dihydroxyphenyl)-chromane-(4→3″, 2→O-4″)-4″-hydroxycoumarin (45)

General methodology B was applied using the flavylium salt 27 (0.168 g) and 4-hydroxycoumarin (37, 0.081g, 0.5 mmol). The purification step was performed by CC using mixtures of DCM-acetone (95:5) as mobile phase. Pure compound 45 was obtained as a pink amorphous solid (0.159 g, 72% from 21). Melting point: 128–130 °C (decomposes); Compound 45: ¹H NMR (400 MHz, DMSO-d₆) δ 7.79 (dd, J = 7.9, 1.6 Hz, 1H, H-5″), 7.62 (ddd, J = 8.5, 7.3, 1.6, 1H, H-7″), 7.43–7.33 (ov, 3H, H-5, H-6″, H-8″), 7.18 (ov, 2H, H-7, H-2′), 7.05 (dd, J = 8.3, 2.3 Hz, 1H, H-6′), 7.00 (dd, J = 8.2, 1.2 Hz, 1H, H-8), 6.96 (td, J = 7.4, 1.2 Hz, 1H, H-6), 6.85 (d, J = 8.3 Hz, 1H, H-5′), 4.21 (d, J = 3.0 Hz, 1H, H-4), 2.54–2.38 (m, 2H, H-3); ¹³C NMR (100 MHz, DMSO-d₆) δ 160.5 (C-2″ (D)), 157.5 (C-4″ (D)), 151.8 (C-9″ (D)), 151.2 (C-9 (A)), 146.3 (C-4′ (B)), 145.1 (C-3′ (B)), 132.5 (C-7″ (D)), 130.3 (C-1′ (B)), 128.3 (C-7(A)), 127.8 * (C-6″ (D)), 125.6 (C-10 (A)), 124.6 (C-5 (A)), 122.4 (C-5″ (D)), 121.7 (C-6(A)), 116.6 (C-8″ (D), C-6′ (B)), 116.1 (C-8 (A)), 115.4 (C-5′ (B)), 114.4 (C-10″ (D)), 113.3 (C-2′ (B)), 105.9 (C-3″ (D)), 100.5 (C-2 (C)), 31.6 (C-3 (C)), 26.8 (C-4 (C)). HRMS (ESI-TOF) m/z [M+H]⁺ Calcd. For C₂₆H₂₀O₆ 428.126, found 428.1261. Analytical HPLC (λ = 280 nm): purity: >99%; t_R = 17.2 min.

3.3.5. 2-(3′,4′-Dihydroxyphenyl)-6-nitrochromane-(4→3″, 2→O-4″)-4″-hydroxycoumarin (46)

General methodology B was applied using the flavylium salt 28 (0.191 g) and 4-hydroxycoumarin (37, 0.081g, 0.5 mmol). The purification step was performed by CC using mixtures of DCM-acetone (95:5). Pure compound 46 was obtained as a pale grey amorphous solid (0.228 g, 79% from 22). Melting point: 128–130 °C (decomposes); Compound 46: ¹H NMR (400 MHz, MeOD) δ 8.23 (d, J = 2.8 Hz, 1H, H-5), 7.99 (dd, J = 9.0, 2.8 Hz 1H, H-7), 7.74 (dd, J = 8.5, 1.6 Hz, 1H, H-5″), 7.50 (ddd, J = 8.2, 7.5, 1.6, 1H, H-7″), 7.26–7.20 (ov, 2H, H-6″, H-8″), 7.11 (d, J = 2.3 Hz, 1H, H-2′), 7.05 (d, J = 9.0 Hz, 1H, H-8), 7.00 (dd, J = 8.3, 2.3 Hz, 1H, H-6′), 6.78 (d, J = 8.3 Hz, 1H, H-5′), 4.30 (t, J = 3.0 Hz, 1H, H-4), 2.50 (dd, J = 14.0, 3.0 Hz, 1H, H-3), 2.39 (dd, J = 14.0, 3.0 Hz, 1H, H-3); ¹³C NMR (100 MHz, MeOD) δ 163.3 (C-2″ (D)), 159.9 (C-4″ (D)), 158.5 (C-9 (A)), 153.9 (C-9″ (D)), 148.0 (C-4′ (B)), 146.7 (C-3′ (B)), 143.6 (C-6(A)), 134.0 (C-7″ (D)), 131.6 (C-1′ (B)), 128.3 (C-10 (A)), 125.9 (C-6″ (D)), 125.5 (C-7(A)), 124.8 (C-5 (A)), 123.9 (C-5″ (D)), 118.5 (C-6′ (B)), 118.3 (C-8 (A)), 117.9 (C-8″ (D)), 116.4 (C-5′ (B)), 116.1 (C-10″ (D)), 114.2 (C-2′ (B)), 106.4 (C-3″ (D)), 102.6 (C-2 (C)), 33.0 (C-3 (C)), 28.8 (C-4 (C)). HRMS (ESI-TOF) m/z [M+H]⁺ Calcd. For C₂₄H₁₅NO₈ 445.0798, found 445.0795. Analytical HPLC (λ = 280 nm): purity: 99%; t_R = 17.8 min.

3.3.6. 2-(3′,4′-Dihydroxyphenyl)-6-chlorochromane-(4→3″, 2→O-4″)-4″-hydroxycoumarin (47)

General methodology B was applied using the flavylium salt 29 (0.185 g) and 4-hydroxycoumarin (37, 0.081g, 0.5 mmol). The purification step was performed by CC using mixtures of DCM-acetone (95:5). Pure compound 47 was obtained as a pale pink amorphous solid (0.225 g, 81% from 23). Melting point: 98–100 °C (decomposes); Compound 47: ¹H NMR (400 MHz, DMSO-d₆) δ 7.81 (dd, J = 7.9, 1.6 Hz, 1H, H-5″), 7.65 (ddd, J = 8.7, 7.3, 1.6, 1H, H-7″), 7.44–7.37 (ov, 2H, H-6″, H-8″), 7.36 (d, J = 2.7 Hz, 1H, H-5), 7.24 (dd, J = 8.7, 2.6 Hz 1H, H-7), 7.17 (d, J = 2.3 Hz, 1H, H-2′), 7.05 (ov, 2H, H-8, H-6′), 6.85 (d, J = 8.3 Hz, 1H, H-5′), 4.23 (br t, J = 3.0 Hz, 1H, H-4), 2.55 (dd, J = 13.9, 3.2 Hz, 1H, H-3), 2.44 (dd, J = 13.9, 2.9 Hz, 1H, H-3); ¹³C NMR (100 MHz, DMSO-d₆) δ 160.5 (C-2″ (D)), 157.5 (C-4″ (D)), 151.8 (C-9″ (D)), 150.2 (C-9 (A)), 146.4 (C-4′ (B)), 145.1 (C-3′ (B)), 132.7 (C-7″ (D)), 129.9 (C-1′ (B)), 128.0 (C-7(A)), 127.6 (C-10 (A)), 127.1 (C-5 (A)), 125.1 (C-6(A)), 124.6 (C-8″ (D)), 122.4 (C-5″ (D)), 117.9 (C-8 (A)), 116.8 (C-6′ (B)), 116.6 (C-6″ (D)), 115.4 (C-5′ (B)), 114.3 (C-10″ (D)), 113.2 (C-2′ (B)), 105.3 (C-3″ (D)), 100.6 (C-2 (C)), 30.7 (C-3 (C)), 26.7 (C-4 (C)). HRMS (ESI-TOF) m/z [M+H]⁺ Calcd. For C₂₄H₁₅ClO₆ 434.0557, found 434.0557. Analytical HPLC (λ = 280 nm): purity: 98%; t_R = 19.0 min.

3.3.7. 2-(3′,4′-Dimethoxyphenyl)-6-nitrochromane-(4→3″, 2→O-4″)-4″-hydroxycoumarin (49)

General methodology B was applied using the flavylium salt 31 (0.205 g) and 4-hydroxycoumarin (37, 0.081g, 0.5 mmol). The purification step was performed by CC using mixtures of DCM-acetone (97:3). Pure compound 49 was obtained as a white amorphous solid (0.126 g, 54% from 22). Melting point: 142–144 °C; Compound 49: ¹H NMR (400 MHz, MeOD) δ 8.43 (d, J = 2.7 Hz, 1H, H-5), 8.08 (dd, J = 9.0, 2.7 Hz 1H, H-7), 7.84 (dd, J = 7.9, 1.6 Hz, 1H, H-5″), 7.54 (ddd, J = 8.7, 7.3, 1.6, 1H, H-7″), 7.33-7.26 (ov, 3H, H-6′, H-6″, H-8″), 7.21 (d, J = 2.2 Hz, 1H, H-2′), 7.11 (d, J = 9.0 Hz, 1H, H-8), 6.98 (d, J = 8.5 Hz, 1H, H-5′), 4.48 (t, J = 3.0 Hz, 1H, H-4), 3.95 (s, 3H, 3′-OMe), 3.94 (s, 3H, 4′-OMe), 2.51 (qd, J = 13.8, 3.0 Hz, 2H, H-3); ¹³C NMR (100 MHz, MeOD) δ 161.3 (C-2″ (D)), 158.3 (C-4″ (D)), 156.9 (C-9 (A)), 152.8 (C-9″ (D)), 152.8 (C-4′ (B)), 150.5 (C-3′ (B)), 142.6 (C-6(A)), 132.8 (C-7″ (D)), 131.2 (C-1′ (B)), 126.4 (C-10 (A)), 124.7 * (C-8″ (D)), 124.6 (C-7(A)), 124.3 (C-5 (A)), 122.9 (C-5″ (D)), 118.4 * (C-6′ (B)), 117.3 (C-8 (A)), 117.3 * (C-6″ (D)), 114.9 (C-10″ (D)), 111.1 (C-5′ (B)), 109.0 (C-2′ (B)), 105.3 (C-3″ (D)), 100.8 (C-2 (C)), 56.4 (3′-OMe), 56.3 (4′-OMe), 32.4 (C-3 (C)), 27.4 (C-4 (C)). HRMS (ESI-TOF) m/z [M+H]⁺ Calcd. For C₂₆H₁₉NO₈ 473.1110, found 473.1111. Analytical HPLC (λ = 280 nm): purity: 97%; t_R = 21.3 min.

3.3.8. 2-(3′,4′-Dimethoxyphenyl)-6-chlorochromane-(4→3″, 2→O-4″)-4″-hydroxycoumarin (50)

General methodology B was applied using the flavylium salt 32 (0.199 g) and 4-hydroxycoumarin (37, 0.081g, 0.5 mmol). The purification step was performed by CC using mixtures of DCM-MeOH (98:2). Pure compound 50 was obtained as a white amorphous solid (0.168 g, 81% from 23). Melting point: 130–133 °C; Compound 50: ¹H NMR (400 MHz, CDCl₃) δ 7.84 (dd, J = 7.9, 1.3 Hz, 1H, H-5″), 7.53–7.49 (ov, 2H, H-5, H-8″), 7.28–7.25 (ov, 3H, H-6′, H-6″, 7″), 7.21 (d, J = 2.2 Hz, 1H, H-2′), 7.13 (dd, J = 8.7, 2.5 Hz 1H, H-7), 6.96 (d, J = 8.5 Hz, 2H, H-8, H-5′), 4.33 (t, J = 3.0 Hz, 1H, H-4), 3.93 (s, 6H, 3′-OMe, 4′-OMe), 2.42 (d, J = 3.0 Hz, 2H, H-3); ¹³C NMR (100 MHz, CDCl₃) δ 161.6 (C-2″ (D)), 158.6 (C-9″ (D)), 152.7 (C-4″ (D)), 150.2 (C-9 (A), C-4′ (B)), 149.2 (C-3′ (B)), 124.3 (C-7″ (D)), 132.6 (C-8″ (D)), 132.0 * (C-1′ (B)), 128.6 (C-7(A)), 128.1 (C-5 (A)), 127.2 * (C-6(A)), 126.9 * (C-10 (A)), 122.9 (C-5″ (D)), 118.4 (C-6′ (B)), 117.8 (C-8 (A)), 117.2 (C-6″ (D)), 115.7 (C-3″ (D)), 115.1 (C-10″ (D)), 111.1 (C-5′ (B)), 109.1 (C-2′ (B)), 100.5 (C-2 (C)), 56.3 (3′-OMe), 56.2 (4′-OMe), 32.8 (C-3 (C)), 27.4 (C-4 (C)). HRMS (ESI-TOF) m/z [M+H]⁺ Calcd. For C₂₆H₁₉ClO₆ 462.087, found 462.0868. Analytical HPLC (λ = 280 nm): purity: >99%; t_R = 22.9 min.

3.3.9. 2-(4′-Hydroxyphenyl)-6-nitrochromane-(4→3″, 2→O-4″)-4″-hydroxycoumarin (52)

General methodology B was applied using the flavylium salt 34 (0.183 g) and 4-hydroxycoumarin (37, 0.081g, 0.5 mmol). The purification step was performed by CC using mixtures of DCM-acetone (95:5). Pure compound 52 was obtained as a white amorphous solid (0.158 g, 58% from 22). Melting point: 275–278 °C; Compound 52: ¹H NMR (400 MHz, DMSO-d₆) δ 8.23 (d, J = 2.9 Hz, 1H, H-5), 8.10 (dd, J = 9.0, 2.9 Hz 1H, H-7), 7.84 (dd, J = 8.0, 1.7 Hz, 1H, H-5″), 7.68–7.63 (ov, 3H, H-2′, H-6′, H-7″), 7.43 (dd, J = 8.5, 1.1 Hz, 1H, H-8″), 7.39 (td, J = 7.6, 1.1 Hz, 1H, H-6″), 7.26 (d, J = 9.0 Hz, 1H, H-8), 6.95-6.89 (m, 2H, H-3′, H-5′), 4.42 (t, J = 3.0 Hz, 1H, H-4), 2.67 (dd, J = 13.9, 3.0 Hz, 1H, H-3), 2.58 (dd, J = 13.9, 3.0 Hz, 1H, H-3); ¹³C NMR (100 MHz, DMSO-d₆) δ 160.4 (C-2″ (D)), 158.6 (C-4′ (B)), 157.3 (C-4″ (D)), 156.9 (C-9 (A)), 151.9 (C-9″ (D)), 141.4 (C-6(A)), 132.8 (C-7″ (D)), 128.1 (C-1′ (B)), 127.2 (C-2′ (B), C-6′ (B)), 126.7 (C-10 (A)), 124.7 (C-6″ (D)), 124.4 (C-7(A)), 123.3 (C-5 (A)), 122.6 (C-5″ (D)), 117.3 (C-8 (A)), 116.7 (C-8″ (D)), 115.2 (C-3′ (B), C-5′ (B)), 114.2 (C-10″ (D)), 105.3 (C-3″ (D)), 101.1 (C-2 (C)), 30.3 (C-3 (C)), 26.6 (C-4 (C)). HRMS (ESI-TOF) m/z [M+H]⁺ Calcd. For C₂₄H₁₅ClO₆ 429.0849, found 429.0847. Analytical HPLC (λ = 280 nm): purity: 98%; t_R = 19.3 min.

3.3.10. 2-(4′-Hydroxyphenyl)-6-chlorochromane-(4→3″, 2→O-4″)-4″-hydroxycoumarin (53)

General methodology B was applied using the flavylium salt 35 (0.177 g) and 4-hydroxycoumarin (37, 0.081g, 0.5 mmol). The purification step was performed by CC using mixtures of DCM-acetone (95:5). Pure compound 53 was obtained as a white amorphous solid (0.190 g, 57% from 23). Melting point: 250–252 °C (decomposes); Compound 53: ¹H NMR (400 MHz, DMSO-d₆) δ 7.81 (dd, J = 7.9, 1.6 Hz, 1H, H-5″), 7.67–7.59 (ov, 3H, H-2′, H-6′, H-7″), 7.42 (dd, J = 8.4, 1.0 Hz, 1H, H-8″), 7.40–7.35 (ov, 2H, H-5, H-6″), 7.24 (dd, J = 8.7, 2.6 Hz 1H, H-7), 7.06 (d, J = 8.7 Hz, 1H, H-8), 6.92–6.87 (m, 2H, H-3′, H-5′), 4.25 (t, J = 3.0 Hz, 1H, H-4), 2.59 (dd, J = 13.8, 3.2 Hz, 1H, H-3), 2.49–2.42 (ov, 1H, H-3); ¹³C NMR (100 MHz, DMSO-d₆) δ 160.5 (C-2″ (D)), 158.4 (C-4′ (B)), 157.6 (C-4″ (D)), 151.8 (C-9″ (D)), 150.2 (C-9 (A)), 132.7 (C-7″ (D)), 129.4 (C-1′ (B)), 128.0 (C-7(A)), 127.6 (C-10 (A)), 127.1 (C-2′ (B), C-6′ (B)), 127.0 (C-5 (A)), 125.1 (C-6(A)), 124.6 (C-6″ (D)), 122.5 (C-5″ (D)), 118.0 (C-8 (A)), 116.6 (C-8″ (D)), 115.1 (C-3′ (B), C-5′ (B)), 114.3 (C-10″ (D)), 105.2 (C-3″ (D)), 100.7 (C-2 (C)), 30.8 (C-3 (C)), 26.7 (C-4 (C)). HRMS (ESI-TOF) m/z [M+H]⁺ Calcd. For C₂₄H₁₅ClO₆ 418.0608, found 418.0606. Analytical HPLC (λ = 280 nm): purity: 98%; t_R = 20.7 min.

3.3.11. 2-(3′,4′-Dihydroxyphenyl)-chromane-(4→3″, 2→O-2″)-2″-hydroxy-1″,4″-naphthoquinone (54)

General methodology B was applied using the flavylium salt 27 (0.168 g) and 2-hydroxy-1,4-naphthoquinone (38, 0.087g, 0.5 mmol). The purification step was performed by CC using mixtures of DCM-acetone (90:10). Pure compound 54 was obtained as a yellow amorphous solid (0.023 g, 10% from 21). Melting point: 190–192 °C (decomposes); Compound 54: ¹H NMR (400 MHz, DMSO-d₆) δ 8.01–7.94 (m, 2H, H-5″, H-8″), 7.86–7.76 (m, 2H, H-6″, H-7″), 7.35 (dd, J = 7.6, 1.7 Hz, 1H, H-5), 7.02–6.99 (m, 2H, H-8, H-6′), 7.14 (d, J = 2.3 Hz, 1H, H-2′), 7.18 (ddd, J = 8.2, 7.4, 1.7 Hz, 1H, H-7), 6.95 (td, J = 7.4, 1.2 Hz, 1H, H-6), 6.83 (d, J = 8.3 Hz, 1H, H-5′), 4.41 (t, J = 3.0 Hz, 1H, H-4), 2.43–2.34 (m, 2H, H-3); ¹³C NMR (100 MHz, DMSO-d₆) δ 182.2 (C-1″ (D)), 178.5 (C-4″ (D)), 152.8 (C-3″ (D)), 151.6 (C-9 (A)), 146.3 (C-4′ (B)), 145.0 (C-3′ (B)), 134.5 ^# (C-7″ (D)), 133.9 ^# (C-6″ (D)), 131.1^$ (C-9″ (D)), 130.6^$ (C-10″ (D)), 130.3 (C-1′ (B)), 128.3 (C-5 (A), C-7 (A)), 126.0 * (C-8″ (D)), 125.8 * (C-5″ (D)), 124.5 (C-2″ (D)), 124.8 (C-10 (A)), 121.6 (C-6(A)), 116.8 (C-6′ (B)), 116.2 (C-8 (A)), 115.2 (C-5′ (B)), 113.4 (C-2′ (B)), 100.1 (C-2 (C)), 30.6 (C-3 (C)), 25.7 (C-4 (C)). HRMS (ESI-TOF) m/z [M+H]⁺ Calcd. For C₂₅H₁₆O₆ 412.0947, found 412.0942. Analytical HPLC (λ = 280 nm): purity: 97%; t_R = 18.6 min.

3.3.12. 2-(3′.4′-Dihydroxyphenyl)-6-nitrochromane-(4→3″, 2→O-2″)-2″-hydroxy-1″,4″-naphthoquinone (55)

General methodology B was applied using the flavylium salt 28 (0.191 g) and 2-hydroxy-1,4-naphthoquinone (38, 0.087g, 0.5 mmol). The purification step was performed by CC using mixtures of DCM-acetone (95:5). Pure compound 55 was obtained as a yellow amorphous solid (0.140 g, 47% from 22). Melting point: 247–250 °C; Compound 55: ¹H NMR (400 MHz, DMSO-d₆) δ 8.21 (d, J = 2.8 Hz, 1H, H-5), 8.08 (dd, J = 9.0, 2.8 Hz 1H, H-7), 8.02–7.96 (m, 2H, H-5″, H-8″), 7.86–7.78 (m, 2H, H-6″, H-7″), 7.24 (d, J = 9.0 Hz, 1H, H-8), 7.17 (d, J = 2.3 Hz, 1H, H-2′), 7.03 (dd, J = 8.3, 2.2 Hz 1H, H-6′), 6.85 (d, J = 8.2 Hz, 1H, H-5′), 4.58 (t, J = 2.9 Hz, 1H, H-4), 2.54-2.46 (m, 2H, H-3); ¹³C NMR (100 MHz, CDCl₃) δ 182.2 (C-1″ (D)), 178.2 (C-4″ (D)), 157.3 (C-9 (A)), 152.5 (C-3″ (D)), 146.6 (C-4′ (B)), 145.1 (C-3′ (B)), 141.3 (C-6(A)), 134.5 ^# (C-6″ (D)), 134.1 ^# (C-7″ (D)), 131.1 (C-10″ (D)), 130.7 (C-9″ (D)), 129.2 (C-1′ (B)), 126.4 (C-2″ (D)), 126.0 * (C-10 (A), C-8″ (D)), 125.8 * (C-5″ (D)) 124.3 (C-7(A)), 123.9 (C-5 (A)), 117.3 (C-8 (A)), 116.8 (C-6′ (B)), 115.3 (C-5′ (B)), 113.4 (C-2′ (B)), 100.6 (C-2 (C)), 29.7 (C-3 (C)), 25.6 (C-4 (C)). HRMS (ESI-TOF) m/z [M+H]⁺ Calcd. For C₂₅H₁₅NO₈ 457.0798, found 457.0803. Analytical HPLC (λ = 280 nm): purity: 98%; t_R = 18.2 min.

3.3.13. 2-(3′,4′-Dihydroxyphenyl)-6-chlorochromane-(4→3″, 2→O-2″)-2″-hydroxy-1″,4″-naphthoquinone (56)

General methodology B was applied using the flavylium salt 29 (0.185 g) and 2-hydroxy-1,4-naphthoquinone (38, 0.087g, 0.5 mmol). The purification step was performed by CC using mixtures of DCM-acetone (90:10). Pure compound 56 was obtained as a yellow amorphous solid (0.052 g, 19% from 23). Melting point: 300–302 °C (decomposes); Compound 56: ¹H NMR (400 MHz, DMSO-d₆) δ 8.03-7.95 (m, 2H, H-5″, H-8″), 7.87–7.77 (m, 2H, H-6″, H-7″), 7.35–7.33 (m, 1H, H-5), 7.22 (dd, J = 8.7, 2.6 Hz 1H, H-7), 7.13 (d, J = 2.3 Hz, 1H, H-2′), 7.04 (d, J = 8.7 Hz, 1H, H-8), 7.00 (dd, J = 8.3, 2.3 Hz 1H, H-6′), 6.85–6.81 (m, 1H, H-5′), 4.41 (t, J = 3.1 Hz, 1H, H-4), 2.40 (ov, 1H, H-3); ¹³C NMR (100 MHz, DMSO-d₆) δ 182.2 (C-1″ (D)), 178.3 (C-4″ (D)), 152.9 (C-3″ (D)), 150.6 (C-9 (A)), 146.4 (C-4′ (B)), 145.1 (C-3′ (B)), 134.5 ^# (C-7″ (D)), 133.9 ^# (C-6″ (D)), 131.1^$ (C-9″ (D)), 130.7^$ (C-10″ (D)), 129.9 (C-1′ (B)), 128.0 (C-7(A)), 127.6 (C-5 (A)), 126.8 (C-10 (A)), 126.0 * (C-8″ (D)), 125.8 * (C-5″ (D)), 125.0 (C-6(A)), 123.8 (C-2″ (D)), 118.0 (C-8 (A)), 116.8 (C-6′ (B)), 115.2 (C-5′ (B)), 113.4 (C-2′ (B)), 100.2 (C-2 (C)), 30.1 (C-3 (C)), 25.7 (C-4 (C)). HRMS (ESI-TOF) m/z [M+H]⁺ Calcd. For C₂₅H₁₅ClO₆ 446.0557, found 446.0557. Analytical HPLC (λ = 280 nm): purity: 95%; t_R = 19.7 min.

3.3.14. 2-(3′.4′-Dimethoxyphenyl)-6-nitrochromane-(4→3″, 2→O-2″)-2″-hydroxy-1″,4″-naphthoquinone (58)

General methodology B was applied using the flavylium salt 31 (0.205 g) and 2-hydroxy-1,4-naphthoquinone (38, 0.087g, 0.5 mmol). The purification step was performed by CC using mixtures of DCM-acetone (98:2). Pure compound 58 was obtained as a yellow amorphous solid (0.100 g, 42% from 22). Melting point: 148–150 °C; Compound 58: ¹H NMR (400 MHz, CDCl₃) δ 8.38 (d, J = 2.7 Hz, 1H, H-5), 8.13-8.04 (ov, 3H, H-7, H-5″, H-8″), 7.77–7.64 (m, 2H, H-6″, H-7″), 7.27 (dd, J = 8.3, 2.1 Hz 1H, H-6′), 7.24 (d, J = 2.1 Hz, 1H, H-2′), 7.10 (d, J = 9.0 Hz, 1H, H-8), 6.94 (d, J = 8.3 Hz, 1H, H-5′), 4.63 (t, J = 2.7 Hz, 1H, H-4), 3.94 (s, 3H, 3′-OMe), 3.92 (s, 3H, 3′-OMe), 2.49 (dd, J = 13.8, 3.0 Hz, 1H, H-3), 2.40 (dd, J = 13.8, 3.0 Hz, 1H, H-3); ¹³C NMR (100 MHz, CDCl₃) δ 182.5 (C-1″ (D)), 178.7 (C-4″ (D)), 157.4 (C-9 (A)), 153.0 (C-3″ (D)), 150.4 (C-4′ (B)), 149.2 (C-3′ (B)), 142.5 (C-6(A)), 134.6 ^# (C-6″ (D)), 134.0 ^# (C-7″ (D)), 131.7 (C-10″ (D)), 131.4 (C-1′ (B)), 130.8 (C-9″ (D)), 126.8 * (C-5″ (D)), 126.7 * (C-8″ (D)), 125.4 (C-10 (A)), 123.4 (C-2″ (D)), 124.7 (C-5 (A), C-7 (A)), 118.6 (C-6′ (B)), 117.4 (C-8 (A)), 111.1 (C-5′ (B)), 109.1 (C-2′ (B)), 100.5 (C-2 (C)), 56.3 (3′-OMe), 56.2 (4′-OMe), 31.5 (C-3 (C)), 26.3 (C-4 (C)); ^{*, #}these signals may be interchangeable. HRMS (ESI-TOF) m/z [M+H]⁺ Calcd. For C₂₇H₁₉NO₈ 485.1111, found 485.1112. Analytical HPLC (λ = 280 nm): purity: >99%; t_R = 21.3 min.

3.3.15. 2-(3′,4′-Dimethoxyphenyl)-6-chlorochromane-(4→3″, 2→O-2″)-2″-hydroxy-1″,4″-naphthoquinone (59)

General methodology B was applied using the flavylium salt 32 (0.199 g) and 2-hydroxy-1,4-naphthoquinone (38, 0.087g, 0.5 mmol). The purification step was performed by CC using mixtures of DCM-MeOH (98:2). Pure compound 59 was obtained as a yellow amorphous solid (0.150 g, 67% from 23). Melting point: 124–127 °C.; Compound 59: ¹H NMR (400 MHz, DMSO-d₆) δ 8.06-7.99 (m, 2H, H-5″, H-8″), 7.71–7.64 (m, 2H, H-6″, H-7″), 7.42 (d, J = 2.6 Hz, 1H, H-5), 7.29 (dd, J = 8.4, 2.1 Hz 1H, H-6′), 7.23 (d, J = 2.1 Hz, 1H, H-2′), 7.10 (dd, J = 8.7, 2.6 Hz 1H, H-7), 6.94 (d, J = 8.7 Hz, 1H, H-8), 6.91 (d, J = 8.4 Hz, 1H, H-5′), 4.48 (t, J = 3.0 Hz, 1H, H-4), 3.92 (s, 3H, 3′-OMe), 3.90 (s, 3H, 4′-OMe), 2.43 (dd, J = 13.7, 3.0 Hz, 1H, H-3), 2.29 (dd, J = 13.7, 3.0 Hz, 1H, H-3); ¹³C NMR (100 MHz, CDCl₃) δ 182.4 (C-1″ (D)), 178.6 (C-4″ (D)), 153.2 (C-3″ (D)), 150.5 (C-9 (A)), 149.8 (C-4′ (B)), 148.9 (C-3′ (B)), 134.2 (C-6″ (D)), 133.5 (C-7″ (D)), 131.5 (C-10″ (D)), 131.4 (C-1′ (B)), 130.9 (C-9″ (D)), 128.3 (C-7(A)), 128.0 (C-5 (A)), 126.7 (C-2″ (D)), 126.5 * (C-8″ (D)), 126.3 * (C-5″ (D)), 125.8 (C-6(A)), 124.1 (C-10 (A)), 118.3 (C-6′ (B)), 117.9 (C-8 (A)), 110.8 (C-5′ (B)), 108.9 (C-2′ (B)), 100.0 (C-2 (C)), 56.0 (3′-OMe, 4′-OMe)31.6 (C-3 (C)), 26.1 (C-4 (C)). HRMS (ESI-TOF) m/z [M+H]⁺ Calcd. For C₂₇H₁₉ClO₆ 474.087, found 474.087. Analytical HPLC (λ = 280 nm): purity: 98%; t_R = 23.2 min.

3.3.16. 2-(4′-Hydroxyphenyl)-6-nitrochromane-(4→3″, 2→O-2″)-2″-hydroxy-1″,4″-naphthoquinone (60)

General methodology B was applied using the flavylium salt 34 (0.183 g) and 2-hydroxy-1,4-naphthoquinone (38, 0.087 g, 0.5 mmol). Compound 60 was recovered by filtration as a yellow amorphous solid (0.134 g, 46% from 22). Melting point: 270–273 °C (decomposes); Compound 60: ¹H NMR (400 MHz, DMSO-d₆) δ 8.21 (d, J = 2.8 Hz, 1H, H-5), 8.08 (dd, J = 9.0, 2.9 Hz 1H, H-7), 8.00–7.95 (m, 2H, H-5″, H-8″), 7.86–7.79 (m, 2H, H-6″, H-7″), 7.59 (d, J = 8.3 Hz, 2H, H-2′, H-6′), 7.25 (d, J = 9.0 Hz, 1H, H-8), 6.89 (d, J = 8.3 Hz, 2H, H-3′, H-5′), 4.60 (t, J = 3.0 Hz, 1H, H-4), 2.53-2.49 (m, 2H, H-3); ¹³C NMR (100 MHz, CDCl₃) δ 182.2 (C-1″ (D)), 178.0 (C-4″ (D)), 158.5 (C-4′ (B)), 157.3 (C-9 (A)), 152.6 (C-3″ (D)), 141.3 (C-6(A)), 134.6 ^# (C-7″ (D)), 134.0 ^# (C-6″ (D)), 131.1 * (C-9″ (D)), 130.7 * (C-10″ (D)), 128.8 (C-1′ (B)), 127.2 (C-2′ (B), C-6′ (B)), 126.1 (C-5″ (D)), 126.0 (C-10 (A)), 125.9 (C-8″ (D)), 124.3 (C-7(A)), 123.9 (C-5 (A)), 123.4 (C-2″ (D)), 117.3 (C-8 (A)), 115.1 (C-3′ (B), C-5′ (B)), 100.6 (C-2 (C)), 29.5 (C-3 (C)), 25.6 (C-4 (C)); *these signals may be interchangeable. HRMS (ESI-TOF) m/z [M+H]⁺ Calcd. For C₂₅H₁₅NO₇ 441.0849, found 441.0855. Analytical HPLC (λ = 280 nm): purity: >99%; t_R = 19.6 min.

3.3.17. 2-(4′-Hydroxyphenyl)-6-chlorochromane-(4→3″, 2→O-2″)-2″-hydroxy-1″,4″-naphthoquinone (61)

General methodology B was applied using the flavylium salt 35 (0.177 g) and 2-hydroxy-1,4-naphthoquinone (38, 0.087g, 0.5 mmol). The purification step was performed by CC using mixtures of DCM-acetone (98:2). Pure compound 61 was obtained as a red-orange amorphous solid (0.050 g, 15% from 23). Melting point: 249–251 °C; Compound 61: ¹H NMR (400 MHz, DMSO-d₆) δ 8.03-7.96 (m, 2H, H-5″, H-8″), 7.89–7.78 (m, 2H, H-6″, H-7″), 7.59–7.53 (m, 2H, H-2′, H-6′), 7.34 (d, J = 2.6 Hz, 1H, H-5), 7.23 (dd, J = 8.7, 2.6 Hz 1H, H-7), 7.06 (d, J = 8.7 Hz, 1H, H-8), 6.92-6.85 (m, 2H, H-3′, H-5′), 4.45-4.42 (m, 1H, H-4), 2.48-2.39 (m, 2H, H-3); ¹³C NMR (100 MHz, CDCl₃) δ 182.2 (C-1″ (D)), 178.3 (C-4″ (D)), 158.3 (C-4′ (B)), 152.9 (C-3″ (D)), 150.0 (C-9 (A)), 134.5 ^# (C-7″ (D)), 133.9 ^# (C-6″ (D)), 131.1 * (C-9″ (D)), 130.7 * (C-10″ (D)), 129.4 (C-1′ (B)), 128.0 (C-7(A)), 127.6 (C-5 (A)), 127.1 (C-2′ (B), C-6′ (B)), 126.7 (C-10 (A)), 126.0 (C-5″ (D)), 125.8 (C-8″ (D)), 125.0 (C-6(A)), 123.7 (C-2″ (D)), 118.1 (C-8 (A)), 115.2 (C-3′ (B), C-5′ (B)), 100.3 (C-2 (C)), 30.0 (C-3 (C)), 25.6 (C-4 (C)); *these signals may be interchangeable. HRMS (ESI-TOF) m/z [M+H]⁺ Calcd. For C₂₅H₁₅ClO₅ 430.0608, found 430.0615. Analytical HPLC (λ = 280 nm): purity: 99%; t_R = 21.3 min.

3.3.18. 2-(3′,4′-Dimethoxyphenyl)-chromane-(4→3″, 2→O-4″)-4″-hydroxy-6″-phenyl-5″,6″-dihydro-2H-pyran-2″-one (62a and 62b)

General methodology B was applied using the flavylium salt 30 (0.182 g) and 4-hydroxy-6-phenyl-5,6-dihydro-2H-pyran-2-one (39, 0.095 g, 0.5 mmol), previously prepared according to the methodology described by Andersh et al. [48,49]. The purification step was performed by CC using mixtures of hexane-EtOAc (75:25). Pure compound 62a was obtained as a pale yellow amorphous solid (0.011 g, 5.0% from 21) and pure compound 62b as a white amorphous solid (0.019 g, 9.0% from 21); Compound 62a: ¹H NMR (400 MHz, CDCl₃) δ 7.38–7.24 (m, 5H, H-5, H-2‴, H-3‴), 7.23–7.16 (ov, 3H, H-7, H-6′, H-4‴), 7.13 (d, J = 2.2 Hz, 1H, H-2′), 7.06 (dd, J = 8.1, 1.2 Hz, 1H, H-8), 6.97 (td, J = 7.4, 1.2 Hz, 1H, H-6), 6.90 (d, J = 8.5 Hz, 1H, H-5′), 5.31 (dd, J = 12.3, 4.0 Hz, 1H, H-6″), 4.30 (t, J = 3.0 Hz, 1H, H-4), 3.90 (s, 6H, 3′-OMe, 4′-OMe), 3.03-2.96 (m, 1H, H-5″), 2.70 (dd, J = 17.2, 4.0 Hz, 1H, H-5″), 2.35-2.22 (m, 2H, H-3); ¹³C NMR (100 MHz, CDCl₃) δ 165.8 (C-2″ (D)), 163.3 (C-4″ (D)), 151.6 (C-9 (A)), 150.0 (C-4′ (B)), 148.0 (C-3′ (B)), 138.4 (C-1‴ (E)), 132.2 (C-1′ (B)), 128.9 (C-5 (A), C-3‴ (E)), 128.3 * (C-7(A)), 128.1 * (C-4‴ (E)), 126.6 (C-2‴ (E)), 126.4 (C-10 (A)), 122.2 (C-6(A)), 118.3 (C-6′ (B)), 116.5 (C-8 (A)), 110.9 (C-5′ (B)), 109.1 (C-2′ (B)), 106.6 (C-3″ (D)), 100.6 (C-2 (C)), 76.6 (C-6″ (D)), 56.0 (3′-OMe, 4′-OMe), 34.3 (C-5″ (D)), 33.2 (C-3 (C)), 26.5 (C-4 (C)); *these signals could be interchangeable. Melting point: 94–96 °C. HRMS (ESI-TOF) m/z [M+H]⁺ Calcd. For C₂₈H₂₄O₆ 456.1573, found 456.1571. Analytical HPLC (λ = 280 nm): purity: 96%; t_R = 20.7 min. Compound 62b: ¹H NMR (400 MHz, CDCl₃) δ 7.56 (dd, J = 7.5, 1.6 Hz, 1H, H-5), 7.35–7.30 (m, 5H, H-2‴, H-3‴, H-4‴), 7.21 (dd, J = 8.4, 2.1 Hz, 1H, H-6′), 7.19-7.16 (ov, 2H, H-2′, H-7), 7.01 (dd, J = 8.2, 1.1 Hz, 1H, H-8), 7.01 (td, J = 7.4, 1.1 Hz, 1H, H-6), 6.91 (d, J = 8.4, 1H, H-5′), 5.49 (dd, J = 12.2, 4.1 Hz, 1H, H-6″), 4.04 (t, J = 3.1 Hz, 1H, H-4), 3.92 (s, 3H, 4′-OMe), 3.90 (s, 3H, 3′-OMe), 2.84 (dd, J = 17.5, 12.3 Hz, 1H, H-5″), 2.68 (ddd, J = 17.5, 4.1, 1.1 Hz, 1H, H-5″), 2.38 (qd, J = 13.5, 3.1, 2H, H-3); ¹³C NMR (100 MHz, CDCl₃) δ 165.7 (C-2″ (D)), 163.6 (C-4″ (D)), 151.5 (C-9 (A)), 150.0 (C-4′ (B)), 149.0 (C-3′ (B)), 138.3 (C-1‴ (E)), 132.4 (C-1′ (B)), 129.1 (C-5 (A)), 128.9 (C-3‴ (E), C-4‴ (E)), 128.2 (C-7(A)), 126.2 (C-2‴ (E)), 126.0 (C-10 (A)), 122.0 (C-6(A)), 118.3 (C-6′ (B)), 116.5 (C-8 (A)), 110.9 (C-5′ (B)), 109.1 (C-2′ (B)), 106.7 (C-3″ (D)), 100.4 (C-2 (C)), 77.5 (C-6″ (D)), 56.3 (4′-OMe), 56.2 (3′-OMe), 34.3 (C-5″ (D)), 33.3 (C-3 (C)), 27.6 (C-4 (C)). Melting point: 172–174 °C. HRMS (ESI-TOF) m/z [M+H]⁺ Calcd. For C₂₈H₂₄O₆ 456.1573, found 456.1573. Analytical HPLC (λ = 280 nm): purity: >99%; t_R = 20.7 min.

3.3.19. 2-(3′,4′-Dimethoxyphenyl)-chromane-(4→3″, 2→O-4″)-6″-(4‴-chlorophenyl)-4″-hydroxy-5″,6″-dihydro-2H-pyran-2″-one (63a and 63b)

General methodology B was applied using the flavylium salt 30 (0.182 g) and 6-(4-chlorophenyl)-4-hydroxy-5,6-dihydro-2H-pyran-2-one (40, 0.122 g, 0.5 mmol), previously prepared according to the methodology described by Andersh et al. [48,49]. The purification step was performed by CC using mixtures of hexane-EtOAc (75:25). Pure compound 63a was obtained as a pale yellow amorphous solid (0.031 g, 12% from 21) and pure compound 63b as a pale yellow amorphous solid (0.050 g, 20% from 21); Compound 63a: ¹H NMR (400 MHz, CDCl₃) δ 7.37–7.28 (ov, 5H, H-5, H-2‴, H-3‴), 7.28–7.16 (ov, 2H, H-7, H-6′), 7.12 (d, J = 2.1 Hz, 1H, H-2′), 7.05 (dd, J = 7.7, 1.1 Hz, 1H, H-8), 6.96 (td, J = 7.4, 1.2 Hz, 1H, H-6), 6.90 (d, J = 8.5 Hz, 1H, H-5′), 5.29 (dd, J = 12.3, 4.1 Hz, 1H, H-6″), 4.28 (t, J = 2.8 Hz, 1H, H-4), 3.90 (s, 6H, 3′-OMe, 4′-OMe), 2.84 (ddd, J = 17.2, 12.3, 1.0 Hz, 1H, H-5″), 2.68 (dd, J = 17.2, 4.1 Hz, 1H, H-5″), 2.30 (qd, J = 13.5, 2.8 Hz, 2H, H-3); ¹³C NMR (100 MHz, CDCl₃) δ 165.5 (C-2″ (D)), 163.1 (C-4″ (D)), 151.6 (C-9 (A)), 150.0 (C-4′ (B)), 149.0 (C-3′ (B)), 136.1 (C-1‴ ©), 134.7 (C-4‴ (E)), 132.1 (C-1′ (B)), 129.1 (C-3‴ (E)), 128.4 (C-7(A)), 128.1 (C-5 (A)), 127.6 (C-2‴ (E)), 126.3 (C-10 (A)), 122.2 (C-6(A)), 118.3 (C-6′ (B)), 116.5 (C-8 (A)), 110.9 (C-5′ (B)), 109.0 (C-2′ (B)), 106.6 (C-3″ (D)), 100.6 (C-2 (C)), 76.0 (C-6″ (D)), 56.2 (3′-OMe, 4′-OMe), 34.3 (C-5″ (D)), 33.1 (C-3 (C)), 26.8 (C-4 (C)). Melting point: 110–112 °C HRMS (ESI-TOF) m/z [M+H]⁺ Calcd. for C₂₈H₂₃ClO₆ 490.1183, found 490.1177. Analytical HPLC (λ = 280 nm): purity: 97%; t_R = 21.8 min. Compound 63b: ¹H NMR (400 MHz, CDCl₃) δ 7.53 (dd, J = 7.5, 1.2 Hz, 1H, H-5), 7.31–7.25 (m, 4H, H-2‴, H-3‴), 7.22-7.16 (ov, 3H, H-7, H-2′, H-6′), 7.01 (dd, J = 8.2, 1.2 Hz, 1H, H-8), 6.97 (td, J = 7.4, 1.2 Hz, 1H, H-6), 6.91 (d, J = 8.4 Hz, 1H, H-5′), 5.47 (dd, J = 12.0, 4.4 Hz, 1H, H-6″), 4.04 (t, J = 3.1 Hz, 1H, H-4), 3.92 (s, 3H, 3′-OMe), 3.90 (s, 3H, 4′-OMe), 2.79 (ddd, J = 17.5, 12.2, 0.8 Hz, 1H, H-5″), 2.67 (ddd, J = 17.5, 4.4, 1.2 Hz, 1H, H-5″), 2.39 (qd, J = 13.5, 3.1 Hz, 2H, H-3); ¹³C NMR (100 MHz, CDCl₃) δ 165.4 (C-2″ (D)), 163.4 (C-4″ (D)), 151.5 (C-9 (A)), 150.1 (C-4′ (B)), 149.1 (C-3′ (B)), 136.8 (C-1‴ (E)), 134.7 (C-4‴ (E)), 132.3 (C-1′ (B)), 129.1 (C-3‴ (E)), 128.9 (C-5 (A)), 128.3 (C-7(A)), 127.6 (C-2‴ (E)), 125.9 (C-10 (A)), 122.1 (C-6(A)), 118.3 (C-6′ (B)), 116.2 (C-8 (A)), 110.9 (C-5′ (B)), 109.1 (C-2′ (B)), 106.8 (C-3″ (D)), 100.5 (C-2 (C)), 76.7 (C-6″ (D)), 56.3 (3′-OMe), 56.0 (4′-OMe), 34.2 (C-5″ (D)), 33.3 (C-3 (C)), 27.5 (C-4 (C)). Melting point: 173–176 °C HRMS (ESI-TOF) m/z [M+H]⁺ Calcd. for C₂₈H₂₃ClO₆ 490.1183, found 490.1164. Analytical HPLC (λ = 280 nm): purity: >99%; t_R = 21.9 min.

3.3.20. 2-(3′,4′-Dimethoxyphenyl)-chromane-(4→3″, 2→O-4″)-4″-hydroxy-6″-(4‴-methoxyphenyl)-5″,6″-dihydro-2H-pyran-2″-one (64a and 64b)

General methodology B was applied using the flavylium salt 30 (0.182 g) and 4-hydroxy-6-(4-methoxyphenyl)-5,6-dihydro-2H-pyran-2-one (41, 0.110 g, 0.5 mmol), previously prepared according to the methodology described by Andersh et al. [48,49] (spectroscopic data agree with those reported in the literature [50]). The purification step was performed by CC using mixtures of hexane-EtOAc (75:25). Pure compound 64a was obtained as a white amorphous solid (0.044 g, 17% from 21) and pure compound 64b as a white amorphous solid (0.052 g, 21% from 21); Compound 64a: ¹H NMR (400 MHz, CDCl₃) δ 7.36 (dd, J = 7.5, 1.6 Hz, 1H, H-5), 7.30–7.26 (m, 2H, H-2‴), 7.23–7.16 (ov, 2H, H-7, H-6′), 7.14 (d, J = 2.2 Hz, 1H, H-2′), 7.05 (dd, J = 8.2, 1.2 Hz, 1H, H-8), 6.96 (td, J = 7.4, 1.1 Hz, 1H, H-6), 6.90 (d, J = 8.4 Hz, 1H, H-5′), 6.89-6.85 (m, 2H, H-3‴), 5.25 (dd, J = 12.3, 4.0 Hz, 1H, H-6″), 4.28 (t, J = 3.1 Hz, 1H, H-4), 3.92 (s, 3H, 4′-OMe), 3.90 (s, 3H, 3′-OMe), 3.81 (s, 3H, 4‴-OMe), 3.00 (ddd, J = 17.2, 12.4, 4.0 Hz, 1H, H-5″), 2.64 (dd, J = 17.2, 4.0 Hz, 1H, H-5″), 2.29 (qd, J = 13.5, 3.2 Hz, 2H, H-3); ¹³C NMR (100 MHz, CDCl₃) δ 165.9 (C-2″ (D)), 163.3 (C-4″ (D)), 160.0 (C-4‴ (E)), 151.6 (C-9 (A)), 150.0 (C-4′ (B)), 149.0 (C-3′ (B)), 132.3 (C-1′ (B)), 130.4 (C-1‴ (E)), 128.3 (C-7(A)), 128.1 (C-5 (A)), 128.0 (C-2‴ (E)), 126.5 (C-10 (A)), 122.2 (C-6(A)), 118.3 (C-6′ (B)), 116.5 (C-8 (A)), 114.2 (C-3‴ (E)), 110.9 (C-5′ (B)), 109.1 (C-2′ (B)), 106.5 (C-3″ (D)), 100.5 (C-2 (C)), 76.7 (C-6″ (D)), 56.2 (3′-OMe, 4′-OMe), 55.4 (4‴-OMe), 34.2 (C-5″ (D)), 33.1 (C-3 (C)), 26.7 (C-4 (C)). Melting point: 172–175 °C. HRMS (ESI-TOF) m/z [M+H]⁺ Calcd. For C₂₉H₂₆O₇ 486.1679, found 486.1658. Analytical HPLC (λ = 280 nm): purity: 98%; t_R = 20.7 min. Compound 64b: ¹H NMR (400 MHz, CDCl₃) δ 7.59 (dd, J = 7.6, 1.7 Hz, 1H, H-5), 7.32–7.28 (m, 2H, H-2‴), 7.28-7.20 (ov, 3H, H-7, H-2′, H-6′), 7.06 (dd, J = 8.2, 1.2 Hz, 1H, H-8), 7.01 (td, J = 7.5, 1.2 Hz, 1H, H-6), 6.95 (d, J = 8.4 Hz, 1H, H-5′), 6.92-6.86 (m, 2H, H-3‴), 5.48 (dd, J = 12.4, 4.0 Hz, 1H, H-6″), 4.08 (t, J = 3.2 Hz, 1H, H-4), 3.97 (s, 3H, 3′-Ome), 3.95 (s, 3H, 4′-OMe), 3.81 (s, 3H, 4‴-OMe), 2.89 (dd, J = 17.2, 12.4 Hz, 1H, H-5″), 2.68 (ddd, J = 17.2, 4.0, 1.2 Hz, 1H, H-5″), 2.42 (qd, J = 13.5, 3.2 Hz, 2H, H-3); ¹³C NMR (100 MHz, CDCl₃) δ 165.8 (C-2″ (D)), 163.6 (C-4″ (D)), 160.0 (C-4‴ (E)), 151.5 (C-9 (A)), 149.9 (C-4′ (B)), 149.0 (C-3′ (B)), 132.4 (C-1′ (B)), 130.3 (C-1‴ (E)), 128.9 (C-7(A)), 128.1 (C-5 (A)), 127.8 (C-2‴ (E)), 126.0 (C-10 (A)), 121.9 (C-6(A)), 118.3 (C-6′ (B)), 116.1 (C-8 (A)), 114.1 (C-3‴ (E)), 110.9 (C-5′ (B)), 109.1 (C-2′ (B)), 106.6 (C-3″ (D)), 100.3 (C-2 (C)), 77.4 (C-6″ (D)), 56.2 (3′-OMe, 4′-OMe), 55.4 (4‴-OMe), 34.2 (C-5″ (D)), 33.3 (C-3 (C)), 27.5 (C-4 (C)). Melting point: 170–173 °C (decomposes). HRMS (ESI-TOF) m/z [M+H]⁺ Calcd. For C₂₉H₂₆O₇ 486.1679, found 486.1663. Analytical HPLC (λ = 280 nm): purity: 98%; t_R = 20.6 min.

3.3.21. 2-(3′,4′-Dimethoxyphenyl)-6-nitrochromane-(4→3″, 2→O-4″)-4″-hydroxy-6″-phenyl-5″,6″-dihydro-2H-pyran-2″-one (65a and 65b)

General methodology B was applied using the flavylium salt 31 (0.205 g) and 4-hydroxy-6-phenyl-5,6-dihydro-2H-pyran-2-one (39, 0.095 g, 0.5 mmol), previously prepared according to the methodology described by Andersh et al. [48,49]. The purification step was performed by CC using mixtures of hexane-EtOAc (75:25). Pure compound 65a was obtained as a pale yellow amorphous solid (0.015 g, 6% from 22) and pure compound 65b as a pale orange amorphous solid (0.081 g, 33% from 22); Compound 65a: ¹H NMR (400 MHz, CDCl₃) δ 8.28 (d, J = 2.7 Hz, 1H, H-5), 8.11 (dd, J = 9.0, 2.8 Hz, 1H, H-7), 7.37–7.33 (m, 5H, H-2‴, H-3‴, H-4‴), 7.16 (dd, J = 8.5, 2.2 Hz, 1H, H-6′), 7.12 (d, J = 9.0 Hz, 1H, H-8), 7.10 (d, J = 2.2 Hz, 1H, H-2′), 6.92 (d, J = 8.5 Hz, 1H, H-5′), 5.32 (dd, J = 12.0, 4.0 Hz, 1H, H-6″), 4.40 (t, J = 3.2 Hz, 1H, H-4), 3.91 (s, 6H, 3′-OMe, 4′-OMe), 3.03 (ddd, J = 17.2, 12.0, 1.1 Hz, 1H, H-5″), 2.74 (dd, J = 17.2, 4.1 Hz, 1H, H-5″), 2.36 (d, J = 3.2 Hz, 2H, H-3); ¹³C NMR (100 MHz, CDCl₃) δ 165.5 (C-2″ (D)), 163.2 (C-4″ (D)), 157.0 (C-9 (A)), 150.4 (C-4′ (B)), 149.3 (C-3′ (B)), 142.5 (C-6(A)), 138.0 (C-1‴ (E)), 130.9 (C-1′ (B)), 129.0 (C-3‴ (E)), 129.1 (C-4‴ (E)), 127.3 (C-10 (A)), 126.2 (C-2‴ (E)), 124.5 (C-7(A)), 123.4 (C-5 (A)), 118.2 (C-6′ (B)), 117.1 (C-8 (A)), 111.0 (C-5′ (B)), 108.9 (C-2′ (B)), 106.1 (C-3″ (D)), 100.8 (C-2 (C)), 76.9 (C-6″ (D)), 56.3 (3′-OMe), 56.2 (4′-OMe), 34.0 (C-5″ (D)), 32.3 (C-3 (C)), 26.9 (C-4 (C)). Melting point: 198–200 °C. HRMS (ESI-TOF) m/z [M+H]⁺ Calcd. for C₂₈H₂₃NO₈ 501.1424, found 501.1425. Analytical HPLC (λ = 280 nm): purity: 99%; t_R = 20.4 min. Compound 65b: ¹H NMR (400 MHz, CDCl₃) δ 8.44 (d, J = 2.7 Hz, 1H, H-5), 8.07 (dd, J = 9.0, 2.8 Hz, 1H, H-7), 7.34–7.31 (m, 5H, H-2‴, H-3‴, H-4‴), 7.19 (dd, J = 8.4, 2.2 Hz, 1H, H-6′), 7.14 (d, J = 2.2 Hz, 1H, H-2′), 7.07 (d, J = 9.0 Hz, 1H, H-8), 6.92 (d, J = 8.5 Hz, 1H, H-5′), 5.51 (dd, J = 12.3, 4.1 Hz, 1H, H-6″), 4.14 (br s, 1H, H-4), 3.93 (s, 3H, 3′-OMe), 3.91 (s, 3H, 4′-OMe), 2.85 (dd, J = 17.7, 12.3 Hz, 1H, H-5″), 2.71 (ddd, J = 17.6, 4.1, 1.1 Hz, 1H, H-5″), 2.53 (dd, J = 13.7, 3.3 Hz, 1H, H-3), 2.38 (dd, J = 13.7, 2.8 Hz, 1H, H-3); ¹³C NMR (100 MHz, CDCl₃) δ 165.3 (C-2″ (D)), 163.5 (C-4″ (D)), 156.8 (C-9 (A)), 150.3 (C-4′ (B)), 149.2 (C-3′ (B)), 142.3 (C-6(A)), 137.9 (C-1‴ (E)), 131.0 (C-1′ (B)), 129.0 (C-4‴ (E)), 128.9 (C-3‴ (E)), 126.9 (C-10 (A)), 126.1 (C-2‴ (E)), 124.8 (C-5 (A)), 124.4 (C-7(A)), 118.2 (C-6′ (B)), 116.7 (C-8 (A)), 111.0 (C-5′ (B)), 108.9 (C-2′ (B)), 106.1 (C-3″ (D)), 100.7 (C-2 (C)), 77.5 (C-6″ (D)), 56.3 (3′-OMe), 56.2 (4′-OMe), 34.2 (C-5″ (D)), 32.4 (C-3 (C)), 27.5 (C-4 (C)). Melting point: 143–145 °C. HRMS (ESI-TOF) m/z [M+H]⁺ Calcd. for C₂₈H₂₃NO₈ 501.1424, found 501.1421. Analytical HPLC (λ = 280 nm): purity: >99%; t_R = 20.6 min.

3.3.22. 2-(3′,4′-Dimethoxyphenyl)-6-nitrochromane-(4→3″, 2→O-4″)-6″-(4‴-chlorophenyl)-4″-hydroxy-5″,6″-dihydro-2H-pyran-2″-one (66a and 66b)

General methodology B was applied using the flavylium salt 31 (0.205 g) and 6-(4-chlorophenyl)-4-hydroxy-5,6-dihydro-2H-pyran-2-one (40, 0.122 g, 0.5 mmol), previously prepared according to the methodology described by Andersh et al. [48,49]. The purification step was performed by CC using mixtures of hexane-EtOAc (75:25). Pure compound 66a was obtained as a white amorphous solid (0.051 g, 19 % from 22) and pure compound 66b as a pale yellow amorphous solid (0.071 g, 26% from 22); Compound 66a: ¹H NMR (400 MHz, CDCl₃) δ 8.27 (d, J = 2.7 Hz, 1H, H-5), 8.10 (dd, J = 9.0, 2.7 Hz, 1H, H-7), 7.37–7.28 (m, 4H, H-2‴, H-3‴), 7.16 (dd, J = 8.5, 2.2 Hz, 1H, H-6′), 7.12 (d, J = 9.0 Hz, 1H, H-8), 7.09 (d, J = 2.22 Hz, 1H, H-2′), 6.91 (d, J = 8.5 Hz, 1H, H-5′), 5.30 (dd, J = 12.1, 4.1 Hz, 1H, H-6″), 4.38 (t, J = 3.1 Hz, 1H, H-4), 3.91 (s, 3H, 3′-OMe), 3.90 (s, 3H, 4′-OMe), 2.98 (ddd, J = 17.2, 12.1, 1.0 Hz, 1H, H-5″), 2.72 (dd, J = 17.2, 4.1 Hz, 1H, H-5″), 2.36 (d, J = 3.1 Hz, 2H, H-3); ¹³C NMR (100 MHz, CDCl₃) δ 164.9 (C-2″ (D)), 163.0 (C-4″ (D)), 156.9 (C-9 (A)), 150.4 (C-4′ (B)), 149.2 (C-3′ (B)), 142.5 (C-6(A)), 136.5 (C-1‴ (E)), 134.9 (C-4‴ (E)), 130.7 (C-1′ (B)), 129.2 (C-3‴ (E)), 127.1 (C-2‴ (E), C-10 (A)), 124.5 (C-7(A)), 123.9 (C-5 (A)), 118.2 (C-6′ (B)), 117.1 (C-8 (A)), 111.0 (C-5′ (B)), 108.9 (C-2′ (B)), 106.1 (C-3″ (D)), 100.9 (C-2 (C)), 76.1 (C-6″ (D)), 56.3 (3′-OMe), 56.2 (4′-OMe), 34.0 (C-5″ (D)), 32.5 (C-3 (C)), 26.6 (C-4 (C)). Melting point: 194–196 °C. HRMS (ESI-TOF) m/z [M+H]⁺ Calcd. for C₂₈H₂₂NO₈ 535.1034, found 535.1032. Analytical HPLC (λ = 280 nm): purity: 97%; t_R = 21.7 min. Compound (+)-66a: Chiral HPLC (λ = 280 nm): purity: >99%; t_R = 19.7 min. [α]_D: +34 (c. 0.4, CHCl₃). Compound (−)-66a: Chiral HPLC (λ = 280 nm): purity: >99%; t_R = 25.5 min. [α]_D: −26 (c. 0.2, CHCl₃). Compound 66b: ¹H NMR (400 MHz, CDCl₃) δ 8.42 (d, J = 2.7 Hz, 1H, H-5), 8.08 (dd, J = 8.9, 2.7 Hz, 1H, H-7), 7.31–7.26 (m, 4H, H-2‴, H-3‴), 7.18 (dd, J = 8.4, 2.1 Hz, 1H, H-6′), 7.13 (d, J = 2.1 Hz, 1H, H-2′), 7.06 (d, J = 9.0 Hz, 1H, H-8), 6.91 (d, J = 8.5 Hz, 1H, H-5′), 5.49 (dd, J = 12.1, 4.2 Hz, 1H, H-6″), 4.12 (br s, 1H, H-4), 3.92 (s, 3H, 3′-OMe), 3.91 (s, 3H, 4′-OMe), 2.80 (dd, J = 17.6, 12.1 Hz, 1H, H-5″), 2.70 (dd, J = 17.6, 4.2 Hz, 1H, H-5″), 2.52 (dd, J = 13.8, 3.3 Hz, 1H, H-3), 2.38 (dd, J = 13.8, 2.8 Hz, 1H, H-3); ¹³C NMR (100 MHz, CDCl₃) δ 165.1 (C-2″ (D)), 163.4 (C-4″ (D)), 156.8 (C-9 (A)), 150.4 (C-4′ (B)), 149.2 (C-3′ (B)), 142.4 (C-6(A)), 136.4 (C-1‴ (E)), 134.9 (C-4‴ (E)), 130.9 (C-1′ (B)), 129.1 (C-3‴ (E)), 127.6 (C-2‴ (E)), 126.8 (C-10 (A)), 124.8 (C-5 (A)), 124.4 (C-7(A)), 118.2 (C-6′ (B)), 116.8 (C-8 (A)), 111.0 (C-5′ (B)), 108.9 (C-2′ (B)), 106.1 (C-3″ (D)), 100.7 (C-2 (C)), 76.7 (C-6″ (D)), 56.3 (3′-OMe), 56.2 (4′-OMe), 34.1 (C-5″ (D)), 32.4 (C-3 (C)), 27.5 (C-4 (C)). Melting point: 154–157 °C. HRMS (ESI-TOF) m/z [M+H]⁺ Calcd. for C₂₈H₂₂NO₈ 535.1034, found 535.1030. Analytical HPLC (λ = 280 nm): purity: >99%; t_R = 21.8 min.

3.3.23. 2-(3′,4′-Dimethoxyphenyl)-6-nitrochromane-(4→3″, 2→O-4″)-4″-hydroxy-6″-(4‴-methoxyphenyl)-5″,6″-dihydro-2H-pyran-2″-one (67a and 67b)

General methodology B was applied using the flavylium salt 31 (0.205 g) and 4-hydroxy-6-(4-methoxyphenyl)-5,6-dihydro-2H-pyran-2-one (41, 0.110 g, 0.5 mmol), previously prepared according to the methodology described by Andersh et al. [48,49] (spectroscopic data agree with those reported in the literature [50]). The purification step was performed by CC using mixtures of hexane-EtOAc (75:25). Pure compound 67a was obtained as a yellow foam (0.040 g, 14% from 22) and pure compound 67b as a pale yellow amorphous solid (0.064 g, 24% from 22); Compound 67a: ¹H NMR (400 MHz, CDCl₃) δ 8.28 (d, J = 2.7 Hz, 1H, H-5), 8.10 (dd, J = 9.0, 2.7 Hz, 1H, H-7), 7.30–7.25 (m, 2H, H-2‴), 7.16 (dd, J = 8.5, 2.2 Hz, 1H, H-6′), 7.13ߝ7.09 (ov, 2H, H-8, H-2′), 6.92 (d, J = 8.5 Hz, 1H, H-5′), 6.90–6.84 (m, 2H, H-3‴), 5.26 (dd, J = 12.1, 4.1 Hz, 1H, H-6″), 4.39 (t, J = 3.0 Hz, 1H, H-4), 3.91 (s, 6H, 3′-OMe, 4′-OMe), 3.78 (s, 3H, 4‴-OMe), 3.03 (ddd, J = 17.3, 12.2, 1.0 Hz, 1H, H-5″), 2.69 (dd, J = 17.3, 4.1 Hz, 1H, H-5″), 2.40-2.32 (m, 2H, H-3); ¹³C NMR (100 MHz, CDCl₃) δ 165.3 (C-2″ (D)), 163.3 (C-4″ (D)), 160.1 (C-4‴ (E)), 156.9 (C-9 (A)), 150.4 (C-4′ (B)), 149.2 (C-3′ (B)), 142.5 (C-6(A)), 138.0 (C-1‴ (E)), 130.9 (C-1′ (B)), 127.7 (C-2‴ (E)), 127.0 (C-10 (A)), 124.4 (C-7(A)), 123.7 (C-5 (A)), 118.2 (C-6′ (B)), 117.1 (C-8 (A)), 114.3 (C-3‴ (E)), 111.1 (C-5′ (B)), 108.9 (C-2′ (B)), 105.9 (C-3″ (D)), 100.8 (C-2 (C)), 76.7 (C-6″ (D)), 56.3 (3′-OMe), 56.2 (4′-OMe), 55.5 (4‴-OMe), 33.9 (C-5″ (D)), 32.3 (C-3 (C)), 26.6 (C-4 (C)). HRMS (ESI-TOF) m/z [M+H]⁺ Calcd. for C₂₉H₂₅NO₉ 531.1529, found 531.1532. Analytical HPLC (λ = 280 nm): purity: >99%; t_R = 20.6 min. Compound 67b: ¹H NMR (400 MHz, CDCl₃) δ 8.44 (d, J = 2.7 Hz, 1H, H-5), 8.08 (dd, J = 8.9, 2.8 Hz, 1H, H-7), 7.27–7.24 (m, 2H, H-2‴), 7.19 (dd, J = 8.4, 2.2 Hz, 1H, H-6′), 7.13 (d, J = 2.2 Hz, 1H, H-2′), 7.07 (d, J = 8.9 Hz, 1H, H-8), 6.92 (d, J = 8.5 Hz, 1H, H-5′), 6.87-6.82 (m, 2H, H-3‴), 5.45 (dd, J = 12.5, 4.0 Hz, 1H, H-6″), 4.13 (br s, 1H, H-4), 3.93 (s, 3H, 3′-OMe), 3.91 (s, 3H, 4′-OMe), 3.77 (s, 3H, 4‴-OMe), 2.86 (dd, J = 17.4, 12.5 Hz, 1H, H-5″), 2.67 (ddd, J = 17.4, 4.0, 1.2 Hz, 1H, H-5″), 2.51 (dd, J = 13.7, 3.3 Hz, 2H, H-3); ¹³C NMR (100 MHz, CDCl₃) δ 165.5 (C-2″ (D)), 163.6 (C-4″ (D)), 160.1 (C-4‴ (E)), 156.9 (C-9 (A)), 150.4 (C-4′ (B)), 149.2 (C-3′ (B)), 142.4 (C-6(A)), 131.0 (C-1′ (B)), 129.9 (C-1‴ (E)), 127.8 (C-2‴ (E)), 126.9 (C-10 (A)), 125.0 (C-5 (A)), 124.4 (C-7(A)), 118.2 (C-6′ (B)), 116.7 (C-8 (A)), 114.2 (C-3‴ (E)), 111.0 (C-5′ (B)), 108.9 (C-2′ (B)), 106.1 (C-3″ (D)), 100.6 (C-2 (C)), 77.6 (C-6″ (D)), 56.3 (3′-OMe), 56.2 (4′-OMe), 55.6 (4‴-OMe), 34.1 (C-5″ (D)), 32.5 (C-3 (C)), 27.5 (C-4 (C)). Melting point: 162–164 °C. HRMS (ESI-TOF) m/z [M+H]⁺ Calcd. for C₂₉H₂₅NO₉ 531.1529, found 531.1529. Analytical HPLC (λ = 280 nm): purity: >99%; t_R = 20.6 min.

3.3.24. 2-(3′,4′-Dimethoxyphenyl)-6-chlorochromane-(4→3″, 2→O-4″)-4″-hydroxy-6″-phenyl-5″,6″-dihydro-2H-pyran-2″-one (68a and 68b)

General methodology B was applied using the flavylium salt 32 (0.199 g) and 4-hydroxy-6-phenyl-5,6-dihydro-2H-pyran-2-one (39, 0.095 g, 0.5 mmol), previously prepared according to the methodology described by Andersh et al. [48,49]. The purification step was performed by CC using mixtures of hexane-EtOAc (75:25). Pure compound 68a was obtained as a white amorphous solid (0.026 g, 12% from 23) and pure compound 68b as a white amorphous solid (0.066 g, 29% from 23); Compound 68a: ¹H NMR (400 MHz, CDCl₃) δ 7.38–7.30 (m, 6H, H-5, H-2‴, H-3‴, H-4‴), 7.17–7.13 (ov, 2H, H-7, H-6′), 7.10 (d, J = 2.2 Hz, 1H, H-2′), 6.97 (d, J = 8.7 Hz, 1H, H-8), 6.90 (d, J = 8.5 Hz, 1H, H-5′), 5.32 (dd, J = 12.2, 4.0 Hz, 1H, H-6″), 4.25 (t, J = 2.8 Hz, 1H, H-4), 3.90 (s, 6H, 3′-OMe, 4′-OMe), 3.00 (ddd, J = 17.2, 12.3, 1.1 Hz, 1H, H-5″), 2.71 (dd, J = 17.2, 4.1 Hz, 1H, H-5″), 2.30 (qd, J = 13.5, 2.8 Hz, 2H, H-3); ¹³C NMR (100 MHz, CDCl₃) δ 165.5 (C-2″ (D)), 163.4 (C-4″ (D)), 150.3 (C-9 (A)), 150.1 (C-4′ (B)), 149.1 (C-3′ (B)), 138.8 (C-1‴ (E)), 131.8 (C-1′ (B)), 128.9 (C-5 (A), C-3‴ (E)), 128.3 (C-7(A)), 127.9 (C-10 (A)), 127.7 (C-4‴ (E)), 127.0 (C-6(A)), 126.2 (C-2‴ (E)), 118.3 (C-6′ (B)), 117.8 (C-8 (A)), 110.9 (C-5′ (B)), 109.0 (C-2′ (B)), 106.1 (C-3″ (D)), 100.6 (C-2 (C)), 76.9 (C-6″ (D)), 56.2 (3′-OMe, 4′-OMe), 34.2 (C-5″ (D)), 32.7 (C-3 (C)), 26.7 (C-4 (C)). Melting point: 106–108 °C HRMS (ESI-TOF) m/z [M+H]⁺ Calcd. for C₂₈H₂₃ClO₆ 490.1182, found 490.1183. Analytical HPLC (λ = 280 nm): purity: >99%; t_R = 21.9 min. Compound (+)-68a: Chiral HPLC (λ = 280 nm): purity: >99%; t_R = 10.9 min. [α]_D: + 17 (c. 0.5, CHCl₃). Compound (−)-68a: Chiral HPLC (λ = 280 nm): purity: > 99%; t_R = 14.1 min. [α]_D: −16 (c. 0.2, CHCl₃). Compound 68b: ¹H NMR (400 MHz, CDCl₃) δ 7.63 (d, J = 2.5 Hz, 1H, H-5), 7.47–7.40 (m, 5H, H-2‴, H-3‴, H-4‴), 7.28 (dd, J = 8.4, 2.2 Hz, 1H, H-6′), 7.25-7.19 (ov, 1H, H-7), 7.24 (d, J = 2.2 Hz, 1H, H-2′), 7.04-6.98 (ov, 1H, H-8, H-5′), 5.59 (dd, J = 12.3, 4.1 Hz, 1H, H-6″), 4.25 (br s, 1H, H-4), 3.90 (s, 6H, 3′-OMe, 4′-OMe), 2.94 (dd, J = 17.5, 12.3 Hz, 1H, H-5″), 2.78 (ddd, J = 17.5, 4.1, 1.1 Hz, 1H, H-5″), 2.46 (qd, J = 13.5, 2.8 Hz, 2H, H-3); ¹³C NMR (100 MHz, CDCl₃) δ 165.5 (C-2″ (D)), 163.7 (C-4″ (D)), 150.2 (C-9 (A)), 150.1 (C-4′ (B)), 149.1 (C-3′ (B)), 138.1 (C-1‴ (E)), 131.9 (C-1′ (B)), 129.0 (C-4‴ (E)), 128.9 (C-3‴ (E)), 128.6 (C-5 (A)), 128.2 (C-7(A)), 127.5 (C-10 (A)), 126.8 (C-6(A)), 126.2 (C-2‴ (E)), 118.2 (C-6′ (B)), 117.4 (C-8 (A)), 110.9 (C-5′ (B)), 109.0 (C-2′ (B)), 106.2 (C-3″ (D)), 100.4 (C-2 (C)), 77.5 (C-6″ (D)), 56.3 (3′-Ome, 4′-Ome), 34.3 (C-5″ (D)), 32.9 (C-3 (C)), 27.5 (C-4 (C)). Melting point: 178–180 °C. HRMS (ESI-TOF) m/z [M+H]⁺ Calcd. For C₂₈H₂₃ClO₆ 490.1182, found 490.1183. Analytical HPLC (λ = 280 nm): purity: 97%; t_R = 21.8 min. Compound (+)-68b: Chiral HPLC (λ = 280 nm): purity: >99%; t_R = 8.2 min. [α]_D: + 236 (c. 0.5, CHCl₃). Compound (−)-68b: Chiral HPLC (λ = 280 nm): purity: > 99%; t_R = 20.9 min. [α]_D: −237 (c. 0.9, CHCl₃).

3.3.25. 2-(3′,4′-Dimethoxyphenyl)-6-chlorochromane-(4→3″, 2→O-4″)-6″-(4‴-chlorophenyl)-4″-hydroxy-5″,6″-dihydro-2H-pyran-2″-one (69a and 69b)

General methodology B was applied using the flavylium salt 32 (0.199 g) and 6-(4-chlorophenyl)-4-hydroxy-5,6-dihydro-2H-pyran-2-one (40, 0.122 g, 0.5 mmol), previously prepared according to the methodology described by Andersh et al. [48,49]. The purification step was performed by CC using mixtures of hexane-EtOAc (75:25). Pure compound 69a was obtained as a yellow amorphous solid (0.025 g, 10% from 23) and pure compound 69b as a pale orange amorphous solid (0.082 g, 33% from 23); Compound 69a: ¹H NMR (400 MHz, CDCl₃) δ 7.36–7.29 (m, 5H, H-5, H-2‴, H-3‴), 7.17–7.13 (ov, 2H, H-7, H-6′), 7.09 (d, J = 2.2 Hz, 1H, H-2′), 6.97 (d, J = 8.7 Hz, 1H, H-8), 6.90 (d, J = 8.5 Hz, 1H, H-5′), 5.30 (dd, J = 12.3, 4.1 Hz, 1H, H-6″), 4.24 (br s, 1H, H-4), 3.90 (s, 6H, 3′-OMe, 4′-OMe), 2.95 (dd, J = 17.2, 12.3 Hz, 1H, H-5″), 2.69 (dd, J = 17.2, 4.1 Hz, 1H, H-5″), 2.34-2.23 (m, 2H, H-3); ¹³C NMR (100 MHz, CDCl₃) δ 165.3 (C-2″ (D)), 163.3 (C-4″ (D)), 150.2 * (C-4′ (B)), 150.1 * (C-9 (A)), 149.1 (C-3′ (B)), 136.7 (C-1‴ (E)), 134.8 (C-4‴ (E)), 131.7 (C-1′ (B)), 129.2 (C-3‴ (E)), 128.3 (C-7(A)), 127.8 (C-6(A)), 127.6 (C-2‴ (E)), 127.7 (C-5 (A)), 127.1 (C-10 (A)), 118.2 (C-6′ (B)), 117.8 (C-8 (A)), 110.9 (C-5′ (B)), 109.0 (C-2′ (B)), 106.2 (C-3″ (D)), 100.6 (C-2 (C)), 76.1 (C-6″ (D)), 56.2 (3′-OMe, 4′-OMe), 34.2 (C-5″ (D)), 32.7 (C-3 (C)), 26.6 (C-4 (C)); *these signals could be interchangeable. Melting point: 172–175 °C. HRMS (ESI-TOF) m/z [M+H]⁺ Calcd. for C₂₈H₂₂Cl₂O₆ 524.0793, found 524.0793. Analytical HPLC (λ = 280 nm): purity: 97%; t_R = 22.9 min. Compound 69b: ¹H NMR (400 MHz, CDCl₃) δ 7.51 (d, J = 2.5 Hz, 1H, H-5), 7.34–7.24 (m, 4H, H-2‴, H-3‴), 7.17 (dd, J = 8.4, 2.2 Hz, 1H, H-6′), 7.16-7.09 (ov, 2H, H-2′, H-7), 6.93-6.91 (ov,2H, H-8, H-5′), 5.47 (dd, J = 12.2, 4.3 Hz, 1H, H-6″), 4.00 (d, J = 3.0 Hz, 1H, H-4), 3.92 (s, 3H, 3′-OMe), 3.90 (s, 3H, 4′-OMe), 2.80 (dd, J = 17.5, 12.2 Hz, 1H, H-5″), 2.68 (ddd, J = 17.5, 4.3, 1.0 Hz, 1H, H-5″), 2.37 (qd, J = 13.6, 3.0, 2H, H-3); ¹³C NMR (100 MHz, CDCl₃) δ 166.2 (C-2″ (D)), 163.3 (C-4″ (D)), 150.1 (C-4′ (B), C-9 (A)), 149.1 (C-3′ (B)), 136.7 (C-1‴ (E)), 134.8 (C-4‴ (E)), 131.8 (C-1′ (B)), 129.1 (C-3‴ (E)), 128.5 (C-5 (A)), 128.2 (C-7(A)), 127.5 (C-2‴ (E)), 127.4 (C-10 (A)), 126.9 (C-6(A)), 118.2 (C-6′ (B)), 117.4 (C-8 (A)), 110.9 (C-5′ (B)), 109.2 (C-2′ (B)), 106.3 (C-3″ (D)), 100.5 (C-2 (C)), 76.7 (C-6″ (D)), 56.3 (3′-OMe), 56.2 (4′-OMe), 34.2 (C-5″ (D)), 32.8 (C-3 (C)), 27.4 (C-4 (C)). Melting point: 110–113 °C. HRMS (ESI-TOF) m/z [M+H]⁺ Calcd. for C₂₈H₂₂Cl₂O₆ 524.0793, found 524.0794. Analytical HPLC (λ = 280 nm): purity: 98%; t_R = 23.2 min.

3.3.26. 2-(3′.4′-Dimethoxyphenyl)-6-chlorochromane-(4→3″, 2→O-4″)-4″-hydroxy-6″-(4‴-methoxyphenyl)-5″,6″-dihydro-2H-pyran-2″-one (70a and 70b)

General methodology B was applied using the flavylium salt 32 (0.199 g) and 4-hydroxy-6-(4-methoxyphenyl)-5,6-dihydro-2H-pyran-2-one (41, 0.110 g, 0.5 mmol), previously prepared according to the methodology described by Andersh et al. [48,49] (spectroscopic data agree with those reported in the literature [50]). The purification step was performed by CC using mixtures of hexane-EtOAc (75:25). Pure compound 70a was obtained as a white foam (0.032 g, 12% from 23) and pure compound 70b as a yellow amorphous solid (0.075 g, 29% from 23); Compound 70a: ¹H NMR (400 MHz, CDCl₃) δ 7.35 (d, J = 2.5 Hz, 1H, H-5), 7.29 (d, J = 8.7 Hz, 2H, H-2‴), 7.17–7.13 (ov, 2H, H-7, H-6′), 7.10 (d, J = 2.1 Hz, 1H, H-2′), 6.97 (d, J = 8.7 Hz, 1H, H-8), 6.92-6.87 (ov, 3H, H-5′, H-3‴), 5.26 (dd, J = 12.4, 4.0 Hz, 1H, H-6″), 4.24 (br s, 1H, H-4), 3.90 (s, 6H, 3′-OMe, 4′-OMe), 3.78 (s, 3H, 4‴-OMe), 3.00 (dd, J = 17.0, 12.6 Hz, 1H, H-5″), 2.65 (dd, J = 17.2, 4.0 Hz, 1H, H-5″), 2.36-2.20 (m, 2H, H-3); ¹³C NMR (100 MHz, CDCl₃) δ 165.7 (C-2″ (D)), 163.5 (C-4″ (D)), 160.0 (C-4‴ (E)), 150.3 (C-9 (A)), 150.1 (C-4′ (B)), 149.1 (C-3′ (B)), 131.8 (C-1′ (B)), 130.2 (C-1‴ (E)), 127.8 (C-2‴ (E)), 128.2 (C-7(A)), 127.9 (C-10 (A)), 127.7 (C-5 (A)), 127.0 (C-6(A)), 118.2 (C-6′ (B)), 117.8 (C-8 (A)), 114.2 (C-3‴ (E)), 110.9 (C-5′ (B)), 109.0 (C-2′ (B)), 106.1 (C-3″ (D)), 100.5 (C-2 (C)), 76.7 (C-6″ (D)), 56.2 (3′-OMe, 4′-OMe), 55.3 (4‴-OMe), 34.1 (C-5″ (D)), 32.7 (C-3 (C)), 26.6 (C-4 (C)). HRMS (ESI-TOF) m/z [M+H]⁺ Calcd. for C₂₉H₂₅ClO₇ 520.1289, found 520.1280. Analytical HPLC (λ = 280 nm): purity: 98%; t_R = 21.9 min. Compound 70b: ¹H NMR (400 MHz, CDCl₃) δ 7.53 (d, J = 2.5 Hz, 1H, H-5), 7.27 (d, J = 8.7 Hz, 2H, H-2‴), 7.18 (dd, J = 8.4, 2.1 Hz, 1H, H-6′), 7.14-7.11 (ov, 2H, H-7, H-2′), 6.94-6.90 (ov, 2H, H-8, H-5′), 6.85 (d, J = 8.7 Hz, 2H, H-3‴), 5.44 (dd, J = 12.3, 3.8 Hz, 1H, H-6″), 4.00 (br s, 1H, H-4), 3.92 (s, 3H, 3′-OMe), 3.90 (s, 3H, 4′-OMe), 3.78 (s, 3H, 4‴-OMe), 2.85 (dd, J = 17.5, 12.5 Hz, 1H, H-5″), 2.64 (dd, J = 17.5, 3.8 Hz, 1H, H-5″), 2.36 (qd, J = 13.6, 3.2 Hz, 2H, H-3); ¹³C NMR (100 MHz, CDCl₃) δ 165.7 (C-2″ (D)), 163.8 (C-4″ (D)), 160.1 (C-4‴ (E)), 150.2 (C-9 (A)), 150.1 (C-4′ (B)), 149.1 (C-3′ (B)), 132.0 (C-1′ (B)), 130.2 (C-1‴ (E)), 127.8 (C-2‴ (E)), 128.6 (C-5 (A)), 128.2 (C-7(A)), 127.5 (C-10 (A)), 126.8 (C-6(A)), 118.2 (C-6′ (B)), 117.4 (C-8 (A)), 114.2 (C-3‴ (E)), 110.9 (C-5′ (B)), 109.0 (C-2′ (B)), 106.2 (C-3″ (D)), 100.4 (C-2 (C)), 77.4 (C-6″ (D)), 56.3 (3′-OMe), 56.2 (4′-OMe), 55.5 (4‴-OMe), 34.2 (C-5″ (D)), 32.9 (C-3 (C)), 27.5 (C-4 (C)). Melting point: 108-111 °C HRMS (ESI-TOF) m/z [M+H]⁺ Calcd. for C₂₉H₂₅ClO₇ 520.1289, found 520.1289. Analytical HPLC (λ = 280 nm): purity: 98%; t_R = 21.9 min.

3.4. Human Lactate Dehydrogenase A Enzymatic Activity Assay

The ability of the synthesized compounds to inhibit the hLDHA enzyme was measured using recombinant human LDHA (95%, specific activity >300 units/mg and concentration of 0.5 mg/mL, Abcam, Cambridge, United Kingdom) with sodium pyruvate (96%, Merck) as substrate and β-NADH (≥97%, Merck) as cofactor in a potassium phosphate buffer (100 mM, pH 7.3). The enzymatic assay was conducted on 96-well microplates and the decrease in the β-NADH fluorescence (λ_excitation = 340 nm; λ_emission = 460 nm) was detected in a TECAN Infinite 200 Pro M Plex fluorescent plate reader (Tecan Instrument, Inc.) at 28 °C. The activity was determined according to our previously described protocol [34,35]. The final volume in each well was set to 200 µL using 100 mM potassium phosphate buffer, 0.041 units/mL LDHA, 155 µM β-NADH, 1 mM pyruvate (saturated conditions), and DMSO solutions (5%, v/v) of pure compounds at concentrations in the range of 1–600 µM. The addition of pyruvate allowed the beginning of the reaction and fluorescence was registered every 60 s during 10 min. A lineal time interval was selected to calculate the slope at every single concentration. The establishment of the 0% and 100% enzymatic activity was performed by controls and by the use of the inhibitor 3-[[3[(cyclopropylamino)sulfonyl]-7-(2,4-dimethoxy-5-pyrimidinyl)-4-quinolinyl]amino]-5-(3,5-difluorophenoxy)benzoic acid (GSK 2,837,808 A, Tocris, MN, USA) at 1 µM [61]. The slope obtained at each concentration was compared to the one obtained for the 100% enzymatic activity control to determine the corresponding enzymatic activity. A nonlinear regression analysis in GraphPad Prism version 9.00 for Windows (GraphPad Software, La Jolla, CA, USA) was used for dose response curve, fitting of logarithm of inhibitor concentration vs. normalized enzymatic activity, to calculate IC₅₀ values (Supplementary Material section). All measurements were made in triplicate and data were expressed as the mean ± SD.

3.5. Human Lactate Dehydrogenase B Enzymatic Activity Assay

The ability of the synthesized compounds to inhibit the hLDHB enzyme was measured using recombinant human LDHB (95%, specific activity >300 units/mg and concentration of 1.0 mg/mL, Abcam, Cambridge, UK) following the same fluorimetric protocol described in Section 3.4.

3.6. Method for Determining the Mechanism of Inhibition on hLDHA

For each compound evaluated, three replicates of four different concentrations of the compounds in the presence of eight substrate concentrations were included in the kinetic fluorometric assay. The preparation of 96-well microplates and the reaction mixture in each well is the same as that described in Section 3.4. After the addition of the substrate, the maximum slope per minute, determined in a linear interval for each well, was used to establish the initial velocity (V₀) corresponding to each inhibitor and substrate concentration. Therefore, values of V₀ were obtained as a mean of three replicates. Nonlinear regression fits of V₀ versus substrate concentration have been performed in GraphPad Prism 9.00 for Windows (GraphPad Software, La Jolla, California, USA) to calculate V_max, K_M and K_i. Linear Lineweaver–Burk double-reciprocal plots were created using Microsoft Excel 2019 (Microsoft Office Professional Plus 2019) to propose a mechanism of inhibition on hLDHA.

4. Conclusions

The synthesis of 38 compounds (42–70) with a 2,8-dioxabicyclo[3.3.1]nonane core has been carried out following a two-step methodology previously used by us. The yields of these syntheses are significantly affected by the substituents of the electrophilic flavylium salt (27–35) and by the type of nucleophile added in the second stage (36–41). As expected, the highest yields were obtained when the more nucleophilic unit (4-hydroxycoumarin, 37) and/or when EWGs (NO₂ and Cl) in the flavylium salt unit were used (42–81%).

Most of the synthesized (±)-2,8-dioxabicyclo[3.3.1]nonane compounds showed higher inhibitory activity against hLDHA than against hLDHB. Nine of the new compounds (43, 44, 47, 58, 60, 62a, 65b, 66a, and 66b) and the reference compound 2, previously synthesized, showed IC₅₀ values against hLDHA lower than 10 µM. On the other hand, all of the synthesized compounds showed IC₅₀ values against hLDHB higher than 10 µM. In fact, only three of the new compounds (46, 51 and 54) presented a slightly higher inhibitory activity against hLDHB than against hLDHA. Quiral HPLC was used to separate enantiomers of a selection of the most active and selective compounds synthesized (66a, 68a, and 68b). The inhibition assays performed with these pure enantiomers revealed that the influence of the absolute spatial configurations in the inhibition behaviors of the selected 2,8-dioxabicyclo[3.3.1]nonane derivatives is not very high, although it seems that it is always favorable to dextrorotatory enantiomers on hLDHA. Moreover, the study of the inhibition kinetics of enantiomers of 68a and 68b seems to indicate a noncompetitive inhibition behavior for all cases and a slight preference for the dextrorotatory enantiomer (2–4 times more potent) during the inhibition kinetic process on hLDHA enzyme.

All these studies seem to indicate that 2,8-dioxabicyclo[3.3.1]nonane derivatives are promising inhibitors in terms of potency and selectivity against hLDHA enzyme. However, some additional tests have to be performed in order to reduce possible unspecific inhibition due to protein aggregation induced by the tested compounds (using a BSA/Triton enriched medium).

A structure–activity analysis has been conducted taking into account potency and selectivity against hLDHA and hLDHB enzymes, concluding that the presence of electron–withdrawing groups (NO₂ and Cl) at the A-ring, two methoxy groups at the B-ring, and preferably a pyrone moiety in the final compounds seems to be important features that 2,8-dioxabicyclo[3.3.1]nonane derivatives should fulfil in order to ensure achieving hLDHA inhibitors with high activity and selectivity (62a, 65b and 68a).