Synthesis and Characterization of cis-/trans-(±)-3-Alkyl-3,4-dihydro-6,7-dimethoxy-1-oxo-1H-isochromene-4-carboxylic Acids

Abstract

A series of new 3-alkyl substituted cis- and trans-(±)-3,4-dihydro-6,7-dimethoxy-1-oxo-1H-isochromene-4-carboxylic acids (cis-/trans-1–3) was synthesized through the reaction of 6,7-dimethoxyhomophthalic anhydride with aliphatic aldehydes of varying chain lengths. Their structure and configuration were elucidated using spectral methods, including ¹H, ¹³C, DEPT-135 NMR, FTIR, UV-Vis, and HRMS analyses. A deductive conformational analysis was performed for determining the preferred conformations in solution and to explain the observed vicinal coupling constants.

1. Introduction

Isocoumarins (1-oxo-1H-isochromenes) and their 3,4-dihydro derivatives (Figure 1) represent a class of natural and synthetic compounds [1,2] that exhibit a broad spectrum of biological activities. These include antimicrobial [3,4,5], antifungal [6,7], immunomodulatory [8], gastroprotective [9], antiallergic [10], antiulcer [11], anticancer [12,13,14], and anti-inflammatory [13,15,16] properties, to name just a few.

Numerous natural and synthetic 3,4-dihydroisocoumarins are 3,4-disubstituted, thus forming two sigma diastereomers: cis and trans, defined by the position of the substituents around the plane of the heterocyclic ring system. Their configuration can be established using the vicinal coupling constant value between the H-3 and H-4 atoms (³J_3,4) observed in the ¹H NMR spectra. Generally, coupling constants in the range 3 to 6 Hz indicate a cis configuration that favors a synclinal (sc or gauche) orientation of the protons, whereas those between 10 and 13 Hz suggest a trans configuration that favors an antiperiplanar (ap or trans) orientation. However, it is noteworthy that both diastereomers are conformationally flexible and exist as a mixture of conformers, with some of them being preferable due to steric, electronic, or solvent effects. This factor is crucial because the preferred conformations define the compound’s properties. For example, they could influence physicochemical characteristics, reactivity, and interactions with other molecules, such as enzymes. Furthermore, one might incorrectly designate the configuration of an isolated or synthesized single diastereomer if the conformational flexibility is not considered. Therefore, it can be recommended that a set of different experimental relationships should be considered and utilized to address this issue. An example is the title compounds cis- and trans-(±)-3,4-dihydro-6,7-dimethoxy-1-oxo-1H-isochromene-4-carboxylic acids (1–3), of which one of the isomers (trans-1–3) do not conform to the established relationship for the coupling constants, regardless of the solvent, due to the preferred conformation it adopts in solution.

2. Results and Discussion

The targeted compounds 1–3 were synthesized following a known procedure outlined for the preparation of cis- and trans-(±)-3,4-dihydro-1-oxo-1H-isochromene-4-carboxylic acids with an aromatic or heteroaromatic substituent at C-3 [19]. The synthetic pathway (Scheme 1) involves a reaction between a selected aliphatic aldehyde (octanal, decanal, undecanal) and 6,7-dimethoxyhomophthalic anhydride in dry chloroform, using 4-Dimethylaminopyridine (DMAP) as a catalyst at room temperature. This is followed by separating and purifying the resulting diastereomeric mixture via column chromatography. The diastereomeric ratios obtained from ¹H NMR analyses of the reaction mixtures and yields after the reaction work-up are listed in

Table 1. Yield and diastereoisomeric ratio of the synthesized compounds cis-/trans-1–3.

	R	Yield, %	cis, %	trans, %
1	–C7H15	92	40	60
2	–C9H19	85	40	60
3	–C10H21	77	41	59

. Notably, DMAP, a well-documented catalyst for speeding up acylation processes [20], was used for the first time in this reaction and proved to be effective.

Another important aspect is that the stereoselectivity of the reaction is less pronounced than that of aromatic aldehydes [19], resulting in a diastereomeric ratio of 60:40 for trans–cis. This reduction can be attributed to steric effects, as aliphatic aldehydes possess a smaller effective volume compared to their aromatic counterparts.

The newly synthesized compound structures were assigned using various spectral techniques, including ¹H, ¹³C, DEPT-135 NMR, FTIR, UV-Vis, and HRMS analysis. NMR spectra were recorded in two solvents with differing polarities: CDCl₃ (nonpolar) and DMSO-d₆ (polar). The interpretation of the ¹H NMR spectra is consistent with the existing literature for similar compounds [19,21,22,23,24]. All compound proton spectra exhibit distinct peaks, irrespective of their configuration or solvent. These comprise two singlets for the aromatic protons H-8 and H-5, two singlets for the methoxy groups, and multiplet signals for the alkyl chain’s methyl and methylene groups at C-3. Additional signals reflecting configuration differences suggest the presence of cis- and trans-diastereomers, with multiplets identified for H-3 and doublets for H-4. For clarity, a representative ¹H NMR spectrum of trans-1 in DMSO-d₆, with labeled protons, is shown in Figure 2. Additionally,

Table 2. ¹H NMR parameters of cis- and trans-1–3 at room temperature in different solvents. * Given is the multiplet center and range, in brackets; n.a.—not applicable.

Compd.	Chemical Shifts (ppm), Multiplicity, and Coupling Constants (Hz)
	H-8, s		H-5, s		H-4, d		H-3, m *		3J3,4
	CDCl3	DMSO	CDCl3	DMSO	CDCl3	DMSO	CDCl3	DMSO	CDCl3	DMSO
cis-1	7.57	7.39	6.73	7.02	3.78	3.86	4.59 (4.62–4.57)	4.62 (4.65–4.58)	3.2	3.3
cis-2	7.58	7.39	6.73	7.02	3.73	3.85	4.59 (4.61–4.56)	4.61 (4.66–4.56)	3.0	3.3
cis-3	7.58	7.38	6.73	7.02	3.73	3.86	4.58 (4.60–4.55)	4.61 (4.66–4.55)	3.2	3.3
Average (cis)	7.58	7.39	6.73	7.02	3.75	3.86	4.59	4.61	3.1	3.3
trans-1	7.57	7.37	6.73	6.99	3.74	3.93	4.91 (4.95–4.87)	4.85 (4.88–4.81)	4.4	3.3
trans-2	7.57	7.38	6.73	6.99	3.78	3.92	4.91 (4.94–4.88)	4.85 (4.89–4.80)	4.5	3.3
trans-3	7.58	7.37	6.73	6.99	3.78	3.92	4.92 (4.95–4.88)	4.84 (4.89–4.79)	4.5	3.3
Average (trans)	7.57	7.37	6.73	6.99	3.77	3.92	4.91	4.85	4.5	3.3
Δδ	0.01	0.02	n.a.	0.03	0.02	0.06	0.32	0.24	1.4	n.a.

lists the ¹H NMR parameters for key signals across all compounds, which are further discussed in the subsequent paragraphs. The IR spectra of cis- and trans-1–3 in solid form reveal almost no differences, with the strongest bands located at approximately 1720 and 1663 cm⁻¹, representing the stretching vibrations (ν_C=O) of the lactone and carboxyl groups. Additionally, several weaker bands in the 1600–1450 cm⁻¹ range indicate in-plane vibrations of the aromatic fragment [21]. The UV-Vis spectra also coincide, showcasing two peaks at 266 nm and 299 nm, related to the B-band of the aromatic system and the n → π* transition of the C=O functional group [21]. The HRMS spectra confirm the molecular weight of all compounds. All spectral characterization data are provided as Supplementary Materials.

As noted earlier, the configuration of cis and trans diastereomers is typically established by analyzing the vicinal coupling constant value (³J_3,4) of H-3 and H-4 observed in the ¹H NMR spectra. However, as shown in Table 2, this method proved inadequate for the spectra recorded in DMSO-d₆, as the substitution pattern influences conformational preferences, leading to nearly indistinguishable values of around 3.3 Hz for ³J_3,4 in both diastereomers.

To accurately assign the relative configuration, we referred to previously established correlations [19,22]. First, the base-catalyzed reactions of homophthalic anhydrides with aldehydes show stereoselectivity that favors the trans isomers (Table 1). Second, the chemical shifts of the H-3 and H-4 protons in trans isomers are shifted downfield compared to those in the cis isomer (Table 2). Third, the coupling constants ³J_3,4 in the trans isomer vary with solvent polarity. The first two relationships hold for the spectra in DMSO-d₆, while the third requires further experiments. To validate these relationships and unequivocally assign the configurations of compounds 1–3, we also recorded spectra in CDCl₃ (Table 2). As shown, the change in solvent slightly affects ³J_3,4 for the cis isomers, altering them from 3.3 Hz in DMSO-d₆ to 3.1 Hz in CDCl₃, while it significantly impacts the trans isomers, changing them from 3.3 Hz in DMSO-d₆ to 4.5 Hz in CDCl₃. We further employed a deductive approach and performed a conformational analysis to rationalize these results. For clarity, the two most preferred conformations of the isochromanone ring of both diastereomers are presented in perspective and as Newman projections along the C3-C4 bond in Figure 3. Based on the Karplus equation [25,26], ³J_3,4 in the cis isomers are supposed to be in the lower range (3 to 6 Hz), regardless of the conformation (torsional angle of 60° in both conformations), while in trans isomers, they may vary from 3–6 Hz to 10–13 Hz depending on the torsion angle (60° for trans-1–3b and 180° for trans-1–3a, respectively).

The data analysis allows us to conclude that the conformational equilibrium for the trans isomers predominantly favors conformers trans-1–3b, regardless of the solvent employed (³J_3,4 in the range 3 to 6 Hz). However, we observed that the solvent does influence the preferred conformation to some degree, which is evident from the corresponding coupling constants changes. The favored conformation shows axial and pseudoaxial substituents at C-3 and C-4, respectively. This preference arises from reduced steric hindrance between the alkyl substituent and the carboxyl group in this conformation. While this differs somewhat from the established trends in cyclohexane systems, it can be linked to the presence of three sp² carbon atoms in the ring structure, which eliminates 1,3-diaxial interactions and favors the antiperiplanar conformation of the larger substituents. Additionally, an intramolecular hydrogen bond between the carboxyl group and the lactone’s oxygen atom (Figure 3) may also have contributed to this conformation, as well as to conformation cis-1–3a for the cis isomer.

To evaluate further the behavior of the compounds studied, we conducted additional NMR analyses on a specific diastereomeric pair (cis-/trans-2) at various temperatures: 20, 40, 60, and 80 °C. The results are presented in Figure 4.

As can be seen, the temperature alteration prompts a similar change in the signals of both isomers, leading to a common pattern for the configuration-independent signals (H-5, H-8, and the two methoxy groups), and, intriguingly, for H-3 as well. In contrast, the behavior of the signal for H-4 differs, remaining at a constant position for the cis isomer while shifting upfield as the temperature rises for the trans isomer. Additionally, the ³J_3,4 coupling constant stays intact for the cis isomer (3.3 Hz), but it increases by 0.2 Hz for every 20 °C for the trans isomer, changing from 3.3 Hz at 20 °C to 3.9 Hz at 80 °C. These results align the above made conclusions and support the idea that external factors affecting a compound’s conformation can be a helpful tool for verifying the configuration of an unknown compound; however, this approach should only be considered for compounds that adopt conformations with distinct properties.

3. Materials and Methods

3.1. General

All chemicals used in this study were purchased from Sigma-Aldrich (FOT, Sofia, Bulgaria). The organic solvents were of analytical grade and were used without further purification. TLC was performed on pre-coated 0.2 mm aluminum plates with silica gel 60 with fluorescence indicator (Alugram^® SIL G/UV254, Macherey-Nagel, Merck, Darmstadt, Germany). Column chromatography was performed on Horizon High Performance FLASH chromatography system (HPFC) with cartridges filled with Silica gel 60 [particle size—0.06–0.2 mm (70–230 mesh), MACHEREY-NAGEL, Düren, Germany]. IR spectra were recorded in solid state on a Nicolet iS5 FT-IR Spectrometer equipped with iD5 ATR Accessory (Thermo Scientific, ACM 2, Sofia, Bulgaria). UV-Vis spectra were recorded on Evolution 60S UV-Visible Spectrophotometer (Thermo Scientific, ACM 2, Sofia, Bulgaria) using quartz cuvette. NMR spectra were recorded on a Bruker Avance III HD (500 MHz and 126 MHz for ¹H and ¹³C, respectively) using CDCl₃ or DMSO-d₆ as solvents. The chemical shifts (δ) are given in ppm and J values are reported in Hz. High-Resolution Mass Spectra (HRMS) were obtained on a Shimadzu LCMS-9050 (Shimadzu Handels GmbH., Korneuburg, Austria).

3.2. Synthesis

The corresponding aldehyde (1 equiv.) was added to a solution of 6,7-dimethoxyhomophtalic anhydride (1.1 equiv.) in 10 mL dry chloroform and DMAP (1 equiv.) was added. The resulting mixture was stirred for 1 h at r.t. (22–23 °C). At the end of the reaction (TLC monitoring), the obtained carboxylic acids were extracted with 10% NaHCO₃ and the aqueous layer was acidified (pH = 3) with 18% HCl and extracted with EtOAc. The organic layer was dried with Na₂SO₄. The solvent was evaporated and the diastereoisomers of the corresponding (±)-3-alkyl-3,4-dihydro-6,7-dimethoxy-1-oxo-1H-isochromene-4-carboxylic acid were isolated via column chromatography (mobile phase: petroleum ether/EtOAc = 1/1 + formic acid).

3.2.1. cis- and trans-(±)-3-Heptyl-3,4-dihydro-6,7-dimethoxy-1-oxo-1H-isochromene-4-carboxylic Acids (1)

6,7-Dimethoxyhomophtalic anhydride (2.00 g, 9.00 mmol) reacted with octanal (1.05 g, 8.18 mmol) in the presence of 1.00 g (8.18 mmol) DMAP to give white crystals of 1 (2.63 g, 92% yield). After purification and separation, cis and trans isomers were acquired:

cis-1, m.p. = 132–134 °C (from CH₂Cl₂: petroleum ether, b.p. = 35–60 °C); R_f = 0.37 (EtOAc: CH₂Cl₂ = 2:3); UV-Vis (MeCN) λ_max = 267 nm (ε = 9965 L mol⁻¹ cm⁻¹); 299 nm (ε = 5662 L mol⁻¹ cm⁻¹); FTIR (solid) cm⁻¹: ν = 1723 (s, C=O), 1663 (m, C=O), 1604 (w, ArCH), 1514 (m, ArCH); ¹H-NMR (500 MHz, CDCl₃): δ = 7.57 (1H, s, 8-CH), 6.73 (1H, s, 5-CH), 4.62–4.57 (1H, m, 3-CH), 3.94 (3H, s, 7-OCH₃), 3.93 (3H, s, 6-OCH₃), 3.78 (3H, d, ³J_3,4 = 3.2 Hz, 4-CH), 2.03–1.92 (1H, m, 1′-CH₂), 1.85–1.74 (1H, m, 1′-CH₂), 1.69–1.56 (1H, m, 2′-CH₂), 1.53–1.42 (1H, m, 2′-CH₂), 1.28 (8H, m, 3′-CH₂, 4′-CH₂, 5′-CH₂, 6′-CH₂), 0.88 (3H, ³J_6′,7′ = 6.7 Hz, 7′-CH₃); ¹³C-NMR (126 MHz, CDCl₃): δ = 173.63 (C, C=O, COOH), 164.79 (C, 1C), 153.66 (C, 6C), 149.61 (C, 7C), 130.50 (C, 4aC), 117.71 (C, 8aC), 112.17 (CH, 8C), 109.25 (CH, 5C), 78.61 (CH, 3C), 56.32 (CH₃, 6-OCH₃), 56.26 (CH₃, 7-OCH₃), 47.07 (CH, 4C), 32.69 (CH₂), 31.72 (CH₂), 29.19 (CH₂), 29.09 (CH₂), 25.34 (CH₂), 22.62 (CH₂), 14.08 (CH₃, 7′-CH₃). ¹H-NMR (500 MHz, DMSO): δ = 7.39 (1H, s, 8-CH), 7.02 (1H, s, 5-CH), 4.65–4.58 (1H, td, ³J_3,1′ = 7.0, ³J_3,4 = 3.3 Hz, 3-CH), 3.86 (1H, d, ³J_3,4 = 3.2 Hz, 4-CH), 3.84 (3H, s, 7-OCH₃), 3.81 (3H, s, 6-OCH₃), 1.81–1.68 (2H, m, 1′-CH₂), 1.55–1.37 (2H, m, 2′-CH₂), 1.37–1.20 (8H, m, 3′-CH₂, 4′-CH₂, 5′-CH₂, 6′-CH₂), 0.87 (3H, t, ³J_6′,7′ = 6.8 Hz, 7′-CH₃). ¹³C-NMR (126 MHz, DMSO): δ = 170.69 (C, C=O, COOH), 164.26 (C, 1C), 153.10 (C, 6C), 148.59 (C, 7C), 132.52 (C, 4aC), 117.19 (C, 8aC), 111.24 (CH, 8C), 110.14 (CH, 5C), 78.41 (CH, 3C), 55.97 (CH₃, 6-OCH₃), 55.68 (CH₃, 7-OCH₃), 46.41 (CH, 4C), 32.27 (CH₂), 31.16 (CH₂), 28.69 (CH₂), 28.57 (CH₂), 24.69 (CH₂), 22.08 (CH₂), 13.96 (7′-CH₃).

HRMS (ESI) m/z, calculated for [M − H]⁻ C₁₉H₂₅O₆⁻: 349.16566, found [M − H]⁻: 349.16430.

trans-1, m.p = 134–136 °C (from CH₂Cl₂: petroleum ether, b.p. = 35–60 °C); R_f = 0.33 (EtOAc: CH₂Cl₂ = 2:3); UV-Vis (MeCN) λ_max = 266 nm (ε = 10,803 L mol⁻¹ cm⁻¹); 299 nm (ε = 6160 L mol⁻¹ cm⁻¹); FTIR (solid) cm⁻¹: ν = 1722 (s, C=O), 1662 (m, C=O), 1604 (w, ArCH), 1514 (m, ArCH); ¹H NMR (500 MHz, CDCl₃): δ = 9.23 (1H, s, COOH), 7.57 (1H, s, 8-CH), 6.73 (1H, s, 5-CH), 4.95–4.87 (1H, dt, ³J_3,1‘ = 8.9, ³J_3,4 = 4.4 Hz, 3-CH), 3.94 (3H, s, 7-OCH₃), 3.92 (3H, s, 6-OCH₃), 3.74 (1H, d, ³J_3,4 = 4.4 Hz, 4-CH), 1.87–1.67 (1H, m, 1′-CH₂), 1.67–1.48 (2H, m, 1′-CH₂, 2′-CH₂), 1.48–1.38 (1H, m, 2′-CH₂), 1.38–1.15 (8H, m, 3′-CH₂, 4′-CH₂, 5′-CH₂, 6′-CH₂), 0.85 (3H, t, ³J_6′,7′ = 6.8 Hz, 7′-CH₃). ¹³C-NMR (126 MHz, CDCl₃): δ = 175.83 (C, C=O, COOH), 163.95 (C, 1C), 154.14 (C, 6C), 149.51 (C, 7C), 128.88 (C, 4aC), 117.17 (C, 8aC), 111.95 (CH, 8C), 110.08 (CH, 5C), 79.05 (CH, 3C), 56.43 (CH₃, 6-OCH₃), 56.35 (CH₃, 7-OCH₃), 47.42 (CH, 4C), 33.92 (CH₂), 31.81 (CH₂), 29.21 (CH₂), 29.16 (CH₂), 25.42 (CH₂), 22.70 (CH₂), 14.17 (CH₃, 7′-CH₃). ¹H-NMR (500 MHz, DMSO): δ = 7.37 (1H, s, 8-CH), 6.99 (1H, s, 5-CH), 4.88–4.81 (1H, ddd, ³J_3,1′ = 8.5, ³J_3,1′ = 5.2, ³J_3,4 = 3.3 Hz, 3-CH), 3.93 (1H, d, ³J_3,4 = 3.3 Hz, 4-CH), 3.84 (3H, s, 7-OCH₃), 3.81 (3H, s, 6-OCH₃), 1.64–1.46 (2H, m, 1′-CH₂), 1.45–1.30 (2H, m, 2′-CH₂), 1.30–1.15 (8H, m, 3′-CH₂, 4′-CH₂, 5′-CH₂, 6′-CH₂), 0.84 (3H, t, ³J_6′,7′ = 6.9 Hz, 7′-CH₃). ¹³C-NMR (126 MHz, DMSO): δ = 172.49 (C, C=O, COOH), 163.30 (C, 1C), 153.88 (C, 6C), 148.94 (C, 7C), 130.92 (C, 4aC), 116.98 (C, 8aC), 111.66 (CH, 8C), 111.33 (CH, 5C), 79.56 (CH, 3C), 56.34 (CH₃, 6-OCH₃), 56.10 (CH₃, 7-OCH₃), 47.01 (CH, 4C), 33.60 (CH₂), 31.58 (CH₂), 28.94 (CH₂), 28.91 (CH₂), 25.32 (CH₂), 22.50 (CH₂), 14.39 (CH₃, 7′-CH₃).

HRMS (ESI) m/z, calculated for [M − H]⁻ C₁₉H₂₅O₆⁻: 349.16566, found [M − H]⁻: 349.16366.

3.2.2. cis- and trans-(±)-3,4-Dihydro-6,7-dimethoxy-3-nonyl-1-oxo-1H-isochromene-4-carboxylic Acids (2)

6,7-dimethoxyhomophtalic anhydride (1.29 g, 5.8 mmol) reacted with decanal (0.83 g, 5.3 mmol) in the presence of 0.646 g (5.3 mmol) DMAP to give white crystals of 2 (1.71 g, 85% yield). After purification and separation, cis and trans isomers were acquired:

cis-2, m.p. = 137–139 °C (from CH₂Cl₂: petroleum ether, b.p. = 35–60 °C); R_f = 0.39 (EtOAc: CH₂Cl₂ = 2:3); UV-Vis (MeCN) λ_max = 266 nm (ε = 11,844 L mol⁻¹ cm⁻¹); 299 nm (ε = 6822 L mol⁻¹ cm⁻¹); FTIR (solid) cm⁻¹: ν = 1721 (s, C=O), 1663 (m, C=O), 1604 (w, ArCH), 1515 (m, ArCH); ¹H NMR (500 MHz, CDCl₃) δ 9.04 (1H, s, COOH), 7.58 (1H, s, 8-CH), 6.73 (1H, s, 5-CH), 4.61–4.56 (1H, m, 3-CH), 3.93 (3H, s, 7-OCH₃), 3.91 (3H, s, 6-OCH₃), 3.73 (1H, d, ³J_3,4 = 3.0 Hz, 4-CH), 2.01–1.91 (1H, m, 1′-CH₂), 1.84–1.73 (1H, m, 1′-CH₂), 1.66–1.54 (1H, m, 2′-CH₂), 1.51–1.39 (1H, m, 2′-CH₂), 1.37–1.17 (12H, m, 3′-CH₂, 4′-CH₂, 5′-CH₂, 6′-CH₂, 7′-CH₂, 8′-CH₂), 0.87 (3H, t, ³J_8′,9′ = 6.8 Hz, 9′-CH₃). ¹³C-NMR (126 MHz, CDCl₃): δ = 174.86 (C, C=O, COOH), 165.02 (C, 1C), 153.77 (C, 6C), 149.71 (C, 7C), 130.68 (C, 4aC), 117.80 (C, 8aC), 112.27 (CH, 8C), 109.40 (CH, 5C), 78.80 (CH, 3C), 56.43 (CH₃, 6-OCH₃), 56.37 (CH₃, 7-OCH₃), 47.24 (CH, 4C), 32.78 (CH₂), 31.99 (CH₂), 29.60 (CH₂), 29.58 (CH₂), 29.38 (CH₂), 29.34 (CH₂), 25.48 (CH₂), 22.78 (CH₂), 14.23 (CH₃, 9′-CH₃). ¹H-NMR (500 MHz, DMSO): δ = 7.39 (1H, s, 8-CH), 7.02 (1H, s, 5-CH), 4.66–4.56 (1H, td, ³J_3,1′ = 6.9, ³J_3,4 = 3.3 Hz, 3-CH), 3.85 (1H, d, ³J_3,4 = 3.2 Hz, 4-CH), 3.84 (3H, s, 7-OCH₃), 3.81 (3H, s, 6-OCH₃), 1.81–1.67 (2H, m, 1′-CH₂), 1.56–1.38 (2H, m, 2′-CH₂), 1.38–1.19 (12H, m, 3′-CH₂, 4′-CH₂, 5′-CH₂, 6′-CH₂, 7′-CH₂, 8′-CH₂), 0.86 (3H, t, ³J_8′,9′ = 6.9 Hz, 9′-CH₃). ¹³C NMR (126 MHz, DMSO): 171.15 (C, C=O, COOH), 164.72 (C, 1C), 153.57 (C, 6C), 149.06 (C, 7C), 132.98 (C, 4aC), 117.65 (C, 8aC), 111.70 (CH, 8C), 110.60 (CH, 5C), 78.87 (CH, 3C), 56.42 (CH₃, 6-OCH₃), 56.13 (CH₃, 7-OCH₃), 46.88 (CH, 4C), 32.74 (CH₂), 31.77 (CH₂), 29.40 (CH₂), 29.37 (CH₂), 29.20 (CH₂), 29.16 (CH₂), 25.16 (CH₂), 22.57 (CH₂), 14.43 (CH₃, 9′-CH₃).

HRMS (ESI) m/z, calculated for [M − H]⁻ C₂₁H₂₉O₆⁻: 377.19696, found [M − H]⁻: 377.19537;

trans-2, m.p. = 140–143 °C (from CH₂Cl₂: petroleum ether, 35–60 °C); R_f = 0.35 (EtOAc: CH₂Cl₂ = 2:3); UV-Vis (MeCN) λ_max = 266 nm (ε = 9493 L mol⁻¹ cm⁻¹); 299 nm (ε = 5378 L mol⁻¹ cm⁻¹); FTIR (solid) cm⁻¹: ν = 1719 (s, C=O), 1663 (m, C=O), 1604 (w, ArCH), 1515 (m, ArCH); ¹H-NMR (500 MHz, CDCl₃) δ 9.30 (1H, s, COOH), 7.57 (1H, s, 8-CH), 6.73 (1H, s, 5-CH), 4.94–4.88 (1H, dt, ³J_3,1‘ = 8.9, ³J_3,4 = 4.5 Hz, 3-CH), 3.94 (3H, s, 7-OCH₃), 3.92 (3H, s, 6-OCH₃), 3.78 (1H, d, ³J_3,4 = 4.5 Hz, 4-CH), 1.84–1.72 (1H, m, 1′-CH₂), 1.65–1.48 (2H, m, 1′-CH₂, 2′-CH₂), 1.46–1.36 (1H, m, 2′-CH₂), 1.30–1.20 (12H, m, 3′-CH₂, 4′-CH₂, 5′-CH₂, 6′-CH₂, 7′-CH₂, 8′-CH₂), 0.86 (3H, t, ³J_8′,9′ = 6.8 Hz, 9′-CH₃). ¹³C-NMR (126 MHz, CDCl₃): δ = 175.88 (C, C=O, COOH), 163.95 (C, 1C), 154.13 (C, 6C), 149.50 (C, 7C), 128.93 (C, 4aC), 117.18 (C, 8aC), 111.95 (CH, 8C), 110.05 (CH, 5C), 79.05 (CH, 3C), 56.42 (CH₃, 6-OCH₃), 56.34 (CH₃, 7-OCH₃), 47.45 (CH, 4C), 33.93 (CH₂), 31.95 (CH₂), 29.57 (CH₂), 29.52 (CH₂), 29.37 (CH₂), 29.27 (CH₂), 25.43 (CH₂), 22.76 (CH₂), 14.21 (CH₃, 9′-CH₃). ¹H-NMR (500 MHz, DMSO): δ = 7.38 (1H, s, 8-CH), 6.99 (1H, s, 5-CH), 4.89–4.80 (1H, ddd, ³J_3,1′ = 8.5, ³J_3,1′ = 5.2, ³J_3,4 = 3.3 Hz, 3-CH), 3.92 (1H, d, ³J_3,4 = 3.3 Hz, 4-CH), 3.84 (3H, s, 7-OCH₃), 3.81 (3H, s, 6-OCH₃), 1.64–1.46 (2H, m, 1′-CH₂), 1.44–1.30 (2H, m, 2′-CH₂), 1.28–1.17 (12H, m, 3′-CH₂, 4′-CH₂, 5′-CH₂, 6′-CH₂, 7′-CH₂, 8′-CH₂), 0.84 (3H, t, ³J_8′,9′ = 6.9 Hz, 9′-CH₃). ¹³C-NMR (126 MHz, DMSO): 172.48 (C, C=O, COOH), 163.28 (C, 1C), 153.88 (C, 6C), 148.95 (C, 7C), 130.90 (C, 4aC), 116.98 (C, 8aC), 111.66 (CH, 8C), 111.32 (CH, 5C), 79.55 (CH, 3C), 56.33 (CH₃, 6-OCH₃), 56.09 (CH₃, 7-OCH₃), 47.00 (CH, 4C), 33.60 (CH₂), 31.72 (CH₂), 29.30 (CH₂), 29.28 (CH₂), 29.12 (CH₂), 28.95 (CH₂), 25.30 (CH₂), 22.54 (CH₂), 14.39 (CH₃, 9′-CH₃).

HRMS (ESI) m/z, calculated for [M − H]⁻ C₂₁H₂₉O₆⁻: 377.19696, found [M − H]⁻: 377.19482.

3.2.3. cis- and trans-(±)-3-Decyl-3,4-dihydro-6,7-dimethoxy-1-oxo-1H-isochromene-4-carboxylic Acids (3)

6,7-dimethoxyhomophtalic anhydride (0.611 g, 2.8 mmol) reacted with undecanal (0.426 g, 2.5 mmol) in the presence of 0.306 g (2.5 mmol) DMAP to give white crystals of 2 (0.76 g, 77% yield). After purification and separation, cis and trans isomers were acquired:

cis-3, m.p. = 143–145 °C (from CH₂Cl₂: petroleum ether, b.p. = 35–60 °C); R_f = 0.41 (EtOAc: CH₂Cl₂ = 2:3); UV-Vis (MeCN) λ_max = 266 nm (ε = 10,940 L mol⁻¹ cm⁻¹); 299 nm (ε = 6290 L mol⁻¹ cm⁻¹); FTIR (solid) cm⁻¹: ν = 1722 (s, C=O), 1663 (m, C=O), 1603 (w, ArCH), 1516 (m, ArCH); ¹H-NMR (500 MHz, CDCl₃): δ = 9.44 (1H, s, COOH), 7.58 (1H, s, 8-CH), 6.73 (1H, s, 5-CH), 4.60–4.55 (1H, ddd, ³J_3,1‘ = 8.5, ³J_3,1′ = 5.4, ³J_3,4 = 3.5 Hz, 3-CH), 3.93 (3H, s, 7-OCH₃), 3.91 (3H, s, 6-OCH₃), 3.73 (1H, d, ³J_3,4 = 3.2 Hz, 4-CH), 2.00–1.90 (1H, m, 1′-CH₂), 1.83–1.73 (1H, m, 1′-CH₂), 1.65–1.54 (1H, m, 2′-CH₂), 1.51–1.41 (1H, m, 2′-CH₂), 1.35–1.20 (14H, m, 3′-CH₂, 4′-CH₂, 5′-CH₂, 6′-CH₂, 7′-CH₂, 8′-CH₂, 9′-CH₂), 0.87 (3H, t, ³J_9′,10′ = 6.8 Hz, 10′-CH₃). ¹³C-NMR (126 MHz, CDCl₃): δ = 174.90 (C, C=O, COOH), 165.04 (C, 1C), 153.77 (C, 6C), 149.70 (C, 7C), 130.68 (C, 4aC), 117.79 (C, 8aC), 112.26 (CH, 8C), 109.40 (CH, 5C), 78.79 (CH, 3C), 56.42 (CH₃, 6-OCH₃), 56.36 (CH₃, 7-OCH₃), 47.23 (CH, 4C), 32.77 (CH₂), 32.00 (CH₂), 29.68 (CH₂), 29.64 (CH₂), 29.58 (CH₂), 29.43 (CH₂), 29.34 (CH₂), 25.47 (CH₂), 22.79 (CH₂), 14.22 (CH₃, 10′-CH₃). ¹H-NMR (500 MHz, DMSO): δ = 7.38 (1H, s, 8-CH), 7.02 (1H, s, 5-CH), 4.66–4.55 (1H, td, ³J_3,1′ = 7.0, ³J_3,4 = 3.3 Hz, 3-CH), 3.86 (1H, d, J = 3.2 Hz, 4-CH), 3.85 (3H, s, 7-OCH₃), 3.82 (3H, s, 6-OCH₃), 1.80–1.69 (2H, m, 1′-CH₂), 1.56–1.38 (2H, m, 2′-CH₂), 1.37–1.17 (14H, m, 3′-CH₂, 4′-CH₂, 5′-CH₂, 6′-CH₂, 7′-CH₂, 8′-CH₂, 9′-CH₂), 0.86 (3H, t, ³J_9′,10′ = 6.9 Hz, 10′-CH₃). ¹³C-NMR (126 MHz, DMSO): 171.15 (C, C=O, COOH), 164.71 (C, 1C), 153.57 (C, 6C), 149.06 (C, 7C), 132.97 (C, 4aC), 117.65 (C, 8aC), 111.70 (CH, 8C), 110.59 (CH, 5C), 78.87 (CH, 3C), 56.42 (CH₃, 6-OCH₃), 56.13 (CH₃, 7-OCH₃), 46.88 (CH, 4C), 32.74 (CH₂), 31.77 (CH₂), 29.47 (CH₂), 29.42 (CH₂), 29.40 (CH₂), 29.20 (CH₂), 25.17 (CH₂), 22.57 (CH₂), 14.42 (CH₃, 10′-CH₃).

HRMS (ESI) m/z, calculated for [M − H]⁻ C₂₂H₃₁O₆⁻: 391.21261, found [M − H]⁻: 391.21064.

trans-3, m.p. = 148–150 °C (from CH₂Cl₂: petroleum ether, b.p. = 35–60 °C); R_f = 0.38 (EtOAc: CH₂Cl₂ = 2:3); UV-Vis (MeCN) λ_max = 266 nm (ε = 10,176 L mol⁻¹ cm⁻¹); 299 nm (ε = 5847 L mol⁻¹ cm⁻¹); FTIR (solid) cm⁻¹: ν = 1722 (s, C=O), 1663 (m, C=O), 1603 (w, ArCH), 1517 (m, ArCH); ¹H-NMR (500 MHz, CDCl₃) δ 7.58 (1H, s, 8-CH), 6.73 (1H, s, 5-CH), 4.95–4.88 (1H, dt, ³J_3,1′ = 8.9, ³J_3,4 = 4.5 Hz, 3-CH), 3.94 (3H, s, 7-OCH₃), 3.93 (3H, s, 6-OCH₃), 3.78 (1H, d, ³J_3,4 = 4.4 Hz, 4-CH), 1.78 (1H, m, 1′-CH₂), 1.57 (2H, m, 1′-CH₂, 2′-CH₂), 1.43 (1H, m, 2′-CH₂), 1.24 (14H, m, 3′-CH₂, 4′-CH₂, 5′-CH₂, 6′-CH₂, 7′-CH₂, 8′-CH₂, 9′-CH₂), 0.87 (3H, t, ³J_9′,10′ = 6.9 Hz, 10′-CH₃). ¹³C-NMR (126 MHz, CDCl₃): δ = 175.46 (C, C=O, COOH), 163.72 (C, 1C), 154.00 (C, 6C), 149.40 (C, 7C), 128.72 (C, 4aC), 117.10 (C, 8aC), 111.84 (CH, 8C), 109.91 (CH, 5C), 78.89 (CH, 3C), 56.30 (CH₃, 6-OCH₃), 56.23 (CH₃, 7-OCH₃), 47.28 (CH, 4C), 33.83 (CH₂), 31.87 (CH₂), 29.55 (CH₂), 29.51 (CH₂), 29.40 (CH₂), 29.29 (CH₂), 29.16 (CH₂), 25.32 (CH₂), 22.67 (CH₂), 14.11 (CH₃, 10′-CH₃). ¹H-NMR (500 MHz, DMSO): δ = 7.37 (1H, s, 8-CH), 6.99 (1H, s, 5-CH), 4.89–4.79 (1H, ddd, ³J_3,1′ = 8.5, ³J_3,1′ = 5.2, ³J_3,4 = 3.3 Hz, 3-CH), 3.92 (1H, d, J = 3.3 Hz, 4-CH), 3.84 (3H, s, 7-OCH₃), 3.81 (3H, s, 6-OCH₃), 1.63–1.44 (2H, m, 1′-CH₂), 1.44–1.30 (2H, m, 2′-CH₂), 1.30–1.14 (14H, m, 3′-CH₂, 4′-CH₂, 5′-CH₂, 6′-CH₂, 7′-CH₂, 8′-CH₂, 9′-CH₂), 0.86 (3H, t, ³J_9′,10′ = 6.9 Hz, 10′-CH₃). ¹³C NMR (126 MHz, DMSO): 172.48 (C, C=O, COOH), 163.28 (C, 1C), 153.88 (C, 6C), 148.95 (C, 7C), 130.90 (C, 4aC), 116.98 (C, 8aC), 111.66 (CH, 8C), 111.32 (CH, 5C), 79.55 (CH, 3C), 56.33 (CH₃, 6-OCH₃), 56.09 (CH₃, 7-OCH₃), 47.00 (CH, 4C), 33.60 (CH₂), 31.74 (CH₂), 29.42 (CH₂), 29.35 (CH₂), 29.28 (CH₂), 29.14 (CH₂), 28.95(CH₂), 25.31 (CH₂), 22.55 (CH₂), 14.40 (CH₃, 10′-CH₃).

HRMS (ESI) m/z, calculated for [M − H]⁻ C₂₂H₃₁O₆⁻: 391.21261, found [M − H]⁻: 391.21098.

4. Conclusions

Six new 3-alkyl substituted cis- and trans-(±)-3,4-dihydro-6,7-dimethoxy-1-oxo-1H-isochromene-4-carboxylic acids (cis-/trans-1–3) were synthesized and analyzed using ¹H, ¹³C, DEPT-135 NMR, FTIR, UV-Vis, and HRMS techniques. Conformational analysis was conducted to elucidate the preferred conformations in solution and clarify the observed vicinal coupling constants. The findings indicate that the compounds’ flexibility should always be considered when determining their configuration.