Synthesis of Benzopyrans and Quinolines with Nitrogenated Chain and Their Cytotoxicity Against Human Cancer Cell Lines

Abstract

Cancer caused 9.9 million deaths in 2020, and natural products and/or their structural analogs represent the greatest number of approved small‐molecules antitumor agents. Benzopyran and quinoline heterocycles have demonstrated cytotoxic activity against different cancer cell lines. Due to its high therapeutic potential, we report the synthesis of 2‐propanamide‐ and 2‐propanamine‐dihydrobenzopyrans bearing different amine moieties in the side chain, as well as the 7‐carbon prenylated derivatives (analogous to natural polyalthidin). Next, we synthesized the 2‐substituted and 2,3‐disubstituted quinolines as benzopyran analogs. We evaluated the cytotoxic activity of all nitrogenated derivatives against human cancer cell lines, including A549 (lung cancer), A2058 (melanoma), HepG2 (hepatocellular carcinoma), MCF‐7 (breast cancer), and Mia PaCa‐2 (pancreas cancer) by the MTT assay. Structure–activity relationship analysis revealed: (i) the benzopyran core was twofold more cytotoxic than quinoline analogs and reached ED₅₀ values in the low micromolar range (ED₅₀ < 10 μM) against A2058, HepG2, and MCF‐7; (ii) benzopyran amides showed higher cytotoxicity than benzopyran amines against MCF‐7, and afforded better results for studied lines except for Mia PaCa‐2; and (iii) the amine moiety introduced at 2‐position played a key role for activity; (iv) benzyl and p‐fluorobenzyl substituents protecting phenol group at 6‐position afforded a similar cytotoxicity.

Natural products and/or their structural analogs represent the greatest number of approved small‐molecules as antitumor agents. We report the synthesis of 2‐propanamide and 2‐propanamine dihydrobenzopyrans and 2‐ and 2,3‐disubstituted quinolines to evaluate their cytotoxicity against human A549 (lung), A2058 (melanoma), HepG2 (liver), MCF‐7 (breast), and Mia Paca‐2 (pancreas) cancer cell lines.

1. Introduction

Cancer is the first cause of premature death worldwide, and an increase is expected over the next decades [1]. In 2020, 19.3 million new cancer cases were reported and provided the cause of death of 9.9 million population [2]. This affection can be classified in more than a hundred different diseases depending on the affected body tissue [3]. Despite they might be considered as independent disorders basing on their causes, evolution, and treatments, all cancers have a factor in common: the cellular overgrowth and the ability to spread throughout the body [4]. Breast, lung, colorectal, prostate, and stomach cancer were the most prevalent ones, meanwhile the major rates of mortality were in lung, colorectal, liver, stomach, and breast [5]. The main approaches to treat different types of cancer include surgery, radiotherapy, targeted therapy, and chemotherapy, which are mostly used in combination for effective therapeutic treatment. However, cells develop resistance to anticancer drugs, and consequently, treatment success decreases [6]. Chemotherapy consists of using chemical drugs to kill tumor cells and/or inhibit their growth and proliferation. Depending on their mechanism of action, these drugs are classified as alkylating agents (cis‐platin), antimetabolites (5‐fluoroacil), antitumor antibiotics (doxorubicin), topoisomerase inhibitors (topotecan), and tubulin‐binding drugs (paclitaxel) [7]. Since 1946, about 259 small‐molecule antitumor agents have been approved in Western medicine. Among them, 79% were natural products, synthetic derivatives or drugs inspired by natural product structures [8]. Nowadays, curing cancer is still a challenge, and finding new natural products or structural analogs with an affordable cost‐effectiveness ratio, low toxicity, and capable of overcoming the cells’ resistance continues to attract the attention of pharmaceutical researchers. Benzopyran and quinoline heterocycle are widely found as the core structural motif of different bioactive compounds. These scaffolds manifest multiple kinds of pharmacological properties as anti‐inflammation [9], metabolic disorders regulation [10, 11], antibacterial [12], anti‐HIV [13], antioxidant [14], cytotoxic [15], and antiarthritic [16], among others.

Our research group demonstrated that polyalthidin, a 2‐prenylated benzopyran derived from the bark of Polyalthia cerasoides stems, exhibited cytotoxic activity through inhibition of the mammalian mitochondrial respiratory chain [17]. Compounds such as quercetin and genistein, flavonoid, and isoflavonoid (benzopyran core), displayed antitumour activity by antioxidant and antiproliferative effects, respectively. Quercetin at high concentrations (>30 μM) in combination with cisplatin revealed an antineoplastic effect by decreasing levels of reactive oxygen species in an in vitro and in vivo model of ovarian cancer (cell line C13*). Genistein was effective in vitro and in vivo models for the breast carcinoma cell line MCF‐7 with overexpression of the ERβ1 receptor by blocking cell cycle progression [18]. Among the alkaloids with a quinoline core, the following can be identified: camptothecin (obtained from the bark of the Chinese tree Camptotheca acuminata, Nyssaceae) [19]; cryptolepine (isolated from plants of the genus Cryptolepis, Apocynaceae) [20]; and berberine (isolated from the stem of Berberis aristata, Berberidaceae) [21], all of them as topoisomerase‐inhibiting antitumour agents (Figure 1).

FIGURE 1.

Natural cytotoxic benzopyrans and quinolines.

Due to its high therapeutic potential, a great effort has been made in the synthesis of bioactive agents containing these nuclei. Benzopyrans have demonstrated potent cancer cell growth inhibition in preclinical studies, such as 3‐amino‐1‐aryl‐9‐methoxy‐1H‐benzo[f]chromene‐2‐carbonitriles, 3‐acrylamido‐4H‐benzopyran‐4‐ones, and 3,7‐disubstituted benzopyran‐4‐ones [22, 23, 24]. Quinolines also exhibited cytotoxic effect, such as (E)‐1‐(arylsubstituted)‐3‐(2‐(quinolin‐4‐yl)oxazol‐5‐yl)prop‐2‐en‐1‐ones, 6‐amino‐substituted‐11‐methyl‐indolo‐[3,2‐c]‐quinolines, and N‐(2‐(dimethylamino)ethyl)‐2‐substituted‐7,8,9,10‐tetrahydro‐benzo[h]quinoline‐4‐carboxamide (Figure 2) [25, 26, 27]. Furthermore, synthetic derivatives of camptothecin (quinoline nucleus) are used in clinics as topoisomerase inhibitors [28]. These studies suggest that the presence of a nitrogen atom located in the side chain [23] and the presence of a small lipophilic electron donating substituent in para‐position of phenolic ring [24, 27] are essential features in cytotoxic benzopyrans. In this regard, we have recently proven that 2‐aminopropyl dihydrobenzopyrans were active against triple negative breast cancer such as MDA‐MB‐231 and MDA‐MB‐436 [29]. Here, we describe the synthesis of novel 2‐propanamide and 2‐propanamine dihydrobenzopyrans and their cytotoxic activity evaluation against five human cancer cell lines, including A549 (lung cancer), A2058 (melanoma), HepG2 (hepatocellular carcinoma), MCF‐7 (breast cancer), and Mia PaCa‐2 (pancreas cancer). We also explored the effect of the elongation of the side chain bearing an isoprenoid unit at 2‐position of the benzopyran nucleus of the most promising derivatives. Next, we have synthesized 2‐substituted and 2,3‐disubstituted quinolines [30] as benzopyran analogs and their cytotoxic activity was also studied. ED₅₀ (µM) results allowed to discover new hits as potential antitumor agents and establish their structure–activity relationship (SAR) taking into account different key structural features: (i) benzopyran and quinoline nucleus; (ii) the nitrogen functional group (amine or amide); (iii) the different amines on the side chain: (a) 2,2‐diphenylethylamine, (b) 2‐Cl‐3‐CF₃‐benzylamine, (c) p‐MeO‐phenethylamine, or (d) methylhistidinate; and (iv) the protective group at 6‐position: benzyl or p‐fluorobenzyl substituents.

FIGURE 2.

Synthetic benzopyrans and quinolines with cytotoxic activity in preclinical assays.

2. Results and Discussion

2.1. Chemistry

The synthetic route to obtain the benzopyran derivatives is outlined in Schemes 1 and 2. As previously reported, the O‐heterocycle was obtained by an aldol oxa‐Michael condensation between 2,5‐dihydroxyacetophenone and ethyl levulinate, and the benzo‐γ‐pyrone intermediate reduced under modified Clemensen conditions using zinc powder in acid medium to afford the ester dihydrobenzopyran 1 [10, 11, 31]. The free phenolic group was then treated with benzyl chloride or p‐fluorobenzyl chloride under basic conditions to obtain the lipophilic intermediates 2 and 3, respectively. The amide function was introduced by a sequence of reactions, including the hydrolysis of the ester group to carboxylic acid using potassium hydroxide followed by a chlorination with thionyl chloride to form the acid chloride, which was treated with the corresponding amines (2,2‐diphenylethylamine, 2‐chloro‐3‐(trifluoromethyl)‐benzylamine, p‐MeO‐phenethylamine, or methylhistidinate) in the presence of 4‐dimethylaminopyridine and triethylamine [11] to afford the benzopyran propanamides 4a‐4d and 5a‐5d. Next, amine derivatives were obtained from the ester intermediate 3 under controlled conditions using diisobutylaluminium hydride (DIBAL‐H) reagent at low temperature (−78ºC) to give the aldehyde 6, which was submitted to a reductive amination. The condensation between the aldehyde 6 with the appropriate amine such as 2,2‐diphenylethylamine or 2‐chloro‐3‐(trifluoromethyl) benzylamine provided the imines (Schiff base), which were immediately reduced with sodium triacetoxyborohydride to give benzopyran propanamines 7a and 7b, respectively [32].

SCHEME 1.

Synthesis of nitrogenated benzopyrans with a 3‐carbon length side chain. Reagents and conditions: (i) benzyl chloride or p‐fluorobenzyl chloride, K₂CO₃, EtOH, reflux, 3 h (76%–79%); (ii) KOH, MeOH, reflux, 2 h; (iii) SOCl₂/ CH₂Cl₂, reflux, 3 h; (iv) (a) 2,2‐diphenylethylamine, (b) 2‐Cl‐3‐CF₃‐benzylamine, (c) p‐MeO‐phenethylamine, or (d) methylhistidinate, 4‐DMAP, Et₃N, rt, overnight, N₂ (11%–52%); (v) DIBAL‐H, CH₂Cl₂, −78ºC, 15 min, N₂ (92%); (vi) (a) 2,2‐diphenylethylamine or (b) 2‐Cl‐3‐CF₃‐benzylamine, NaBH(OAc)₃, AcOH, dichloroethane, rt, 1.5 h, N₂ (75%–81%).

SCHEME 2.

Synthesis of nitrogenated benzopyrans with a 7‐carbon length side chain. Reagents and conditions: (i) CH₃C(MgBr)—CH₂, THF, −78ºC, 3 h, N₂; MeC(OEt)₃, isobutyric acid, 140ºC, 2 h (48%); (ii) DIBAL‐H, CH₂Cl₂, −78ºC, 15 min, N₂ (80%); (iii) (a) 2,2‐diphenylethylamine or (b) 2‐Cl‐3‐CF₃‐benzylamine, NaBH(OAc)₃, AcOH, dichloroethane, rt, 1.5 h, N₂ (69‐80%); (iv) TsCl, Et₃N, CH₂Cl₂, 0ºC, 2 h, N₂ (32%).

In addition, aldehyde 6 was subjected to a reaction sequence of Grignard followed by Johnson–Claisen rearrangement to afford benzopyran amines bearing a 7‐carbon side chain with isoprenyl moiety, analogous to natural polycerasoidol [33]. The ester 8 at 2‐position of benzopyran nucleus was partially reduced to obtain aldehyde intermediate 9. The aldehyde 9 was condensed via reductive amination with the appropriate amines and their corresponding imines immediately reduced as abovementioned to achieve amines 10a and 10b bearing a 7‐carbon length with isoprenyl moiety. Finally, amine 10a was N‐tosylated to afford the sulfonamide 11a [34].

Quinoline derivatives were prepared by the following reactions (Scheme 3). First, the free hydroxyl group of 5‐hydroxy‐2‐nitrobenzaldehyde was protected using benzyl chloride in the presence of potassium carbonate to give intermediate 12 and the nitro function was reduced to an amine in acid medium using iron powder [35]. The quinoline nucleus was obtained via Friedländer condensation by reaction of the intermediate with ethyl levulinate in a one pot reaction [36]. Two quinolines were obtained in a 1:1 ratio, the 2,3‐disubstituted quinoline 13 and the 2‐substituted quinoline 14, and then, the ester function was reduced to aldehyde using DIBAL‐H reagent, to attain aldehydes 15 and 16, respectively, as previously described. The condensation between the aldehyde and the proper amine gave amine derivatives 17a‐b and 18a‐b. N‐Tosylation of compound 18b afforded compound 19b.

SCHEME 3.

Synthesis of 2‐substituted and 2,3‐disubstituted quinolines containing amine moiety. Reagents and conditions: (i) benzyl chloride, K₂CO₃, DMF, reflux, 4 h (75%); (ii) 1. Fe²⁺, 0.1 M HCl, EtOH, reflux, 30 min, N₂; 2. pyrrolidine, ethyl levulinate, reflux, 3 h, N₂ (40%); (iii) DIBAL‐H, CH₂Cl₂, −78ºC, 15 min, N₂ (50%–75%); (iv) (a) 2,2‐diphenylethylamine or (b) 2‐Cl‐3‐CF₃‐benzylamine, NaBH(OAc)₃, AcOH, Cl(CH₂)₂Cl, rt, 1.5 h, N₂ (55%–92%); (v) p‐toluensulfonyl chloride, Et₃N, CH₂Cl₂, 0ºC, 2 h, N₂ (62%).

2.2. Cytotoxicity Bioassays

All synthesized compounds were tested by MTT method to evaluate their cytotoxicity against a panel of five human cancer cell lines: lung cancer cell line (A549), human melanoma cell line (A2058), human hepatoma cell line (HepG2), breast cancer cell line (MCF‐7), and pancreas cancer cell line (Mia PaCa‐2). These cell lines were selected due to their clinical relevance, representing cancers with high incidence and mortality worldwide. ED₅₀ (50% effective dose) values are summarized in Table 1 for benzopyrans and Table 2 for quinolines. In A549 lung cancer cells, benzopyran amides with p‐MeO‐phenethylamine moiety (4c, 5c) exhibited the highest cytotoxicity (ED₅₀ = 8.5 and 8.8 μM, respectively), whereas benzopyran amide 5b, benzopyran amines 7a, 7b, 10a, 10b, and quinoline 18a showed weak activity. In A2058 melanoma cells, most of the compounds displayed a remarkable cytotoxic activity. Benzopyran amides 4a, 4b, and 5a‐c reached ED₅₀ values in the low micromolar range (ED₅₀ < 10 μM). It was noticeable that benzopyran amines 7a, 10a and quinolines 17a, 18a, overall bearing a 2,2‐diphenylethylamine moiety showed greater cytotoxicity (ED₅₀ < 12.0 μM) than their 2‐chloro‐3‐trifluoromethylbenzyl benzopyran (7b, 10b) and quinoline (17b, 18b) analogs. In HepG2 cells, same behavior as in A549 was observed with doubled cytotoxicity as a trend. In breast cancer MCF‐7 cell line, benzopyran amides 4b, 4c and 5a‐c exhibited the best results (ED₅₀ < 10 μM), followed by benzopyran amines 7a, 10a, 10b (ED₅₀ = 10–15 μM), while most of quinoline analogs were noncytotoxic (ED₅₀ > 40 μM). In Mia PaCa‐2 pancreas cells, weak cytotoxicity was exhibited by benzopyran amines 7a, 7b, 10a, 10b (ED₅₀ = 10–25 μM) while benzopyran amides and quinolines derivatives were noncytotoxic. In general, amine derivatives can be protonated at physiological pH, which reduces their permeability across the membrane and potentially limits their intracellular accumulation and interaction with their target. However, benzopyran derivatives bearing an amine side chain exhibited greater cytotoxicity against Mia PaCa‐2 cells than their amide analogs. This enhanced potency may arise from the free amino group promoting direct target engagement through hydrogen bonding (e.g., DNA intercalation or enzyme inhibition) or facilitating cellular uptake via organic cation transporters overexpressed in Mia PaCa‐2 cells (e.g., OCT1//2) [37, 38]. Results also evidenced that methylhistidinate derivatives (4d, 5d), N‐tosylated derivatives (11a, 19b), and quinoline derivatives (17b, 18b) showed no cytotoxicity for all cell lines of the panel (ED₅₀ > 40 μM), except for 4d, 5d, and 17b against A2058 and 18b against HepG2 which gave weak cytotoxicity. Dose‐response curves for benzopyran and quinoline compounds are shown in Figures 3 and 4, respectively.

TABLE 1.

ED₅₀ (μM) of cytotoxicity for benzopyran derivatives in human lung, melanoma, liver, breast, and pancreas cancer cells.

Compound	A549a (lung)	A2058a (melanoma)	HepG2a (liver)	MCF‐7a (breast)	Mia PaCa‐2a (pancreas)
4a	>40.0	6.5 ± 0.5	>40.0	>40.0	>40.0
4b	>40.0	7.1 ± 1.2	>40.0	9.8 ± 0.9	>40.0
4c	8.5 ± 1.5	>40.0	4.9 ± 0.7	7.5 ± 1.4	>40.0
4d	>40.0	28.3 ± 1.5	>40.0	>40.0	>40.0
5a	>40.0	4.8 ± 0.5	>40.0	6.1 ± 0.5	>40.0
5b	14.6 ± 1.7	4.5 ± 0.6	6.3 ± 1.2	6.0 ± 0.5	>40.0
5c	8.8 ± 1.3	4.2 ± 0.5	4.8 ± 0.5	6.3 ± 0.3	>40.0
5d	>40.0	16.9 ± 0.8	>40.0	>40.0	>40.0
7a	12.9 ± 1.5	3.0 ± 0.2	5.1 ± 1.8	11.0 ± 1.1	17.0 ± 3.9
7b	16.4 ± 2.2	>40.0	11.5 ± 0.8	>40.0	12.3 ± 0.7
10a	13.6 ± 1.4	8.7 ± 1.0	6.8 ± 0.7	12.1 ± 1.5	13.1 ± 0.5
10b	21.5 ± 1.7	>40.0	14.7 ± 1.2	14.1 ± 1.8	22.3 ± 1.2
11a	>40.0	>40.0	>40.0	>40.0	>40.0

^a ED₅₀ values are expressed as mean ±95% confidence interval (CI) based on triplicate measurements (n = 3).

TABLE 2.

ED₅₀ (μM) of cytotoxicity for quinoline derivatives in human lung, melanoma, liver, breast, and pancreas cancer cells.

Compound	A549a (lung)	A2058a (melanoma)	HepG2a (liver)	MCF‐7a (breast)	Mia PaCa‐2a (pancreas)
17a	>40.0	11.9 ± 0.7	18.9 ± 1.1	>40.0	>40.0
17b	>40.0	26.8 ± 1.6	>40.0	>40.0	>40.0
18a	22.5 ± 2.1	7.7 ± 1.6	9.7 ± 2.4	25.5 ± 2.4	>40.0
18b	>40.0	>40.0	17.9 ± 3.9	>40.0	>40.0
19b	>40.0	>40.0	>40.0	>40.0	>40.0

^a ED₅₀ values are expressed as mean ±95% confidence interval (CI) based on triplicate measurements (n = 3).

FIGURE 3.

Dose‐response curves for A549, A2058, HepG2, MCF‐7, and Mia PaCa‐2 for active benzopyrans (4a‐d, 5a‐d, 7a, 7b, 10a, 10b). Normalized data are presented as mean ± SEM activity values plotted against concentration, along with dose–response curves for each compound and cell line, based on triplicate measurements (n = 3).

FIGURE 4.

Dose‐response curves for A549, A2058, HepG2, and MCF‐7 for active quinolines (17a, 17b, 18a, 18b). Normalized data are presented as mean ± SEM activity values plotted against concentration, along with dose–response curves for each compound and cell line, based on triplicate measurements (n = 3).

2.3. SAR

Based on the activity results obtained for the compounds studied, we can establish the following SAR (Figure 5):

• i.Concerning the heterocycle nucleus, the benzopyran derivatives demonstrated twofold greater cytotoxicity than their quinoline analogs (7a, 7b vs. 18a, 18b) across tested cell lines. In addition, the elongation of the side chain at 2‐position to obtain 7‐carbon prenylated derivatives instead of 3‐carbon nonprenylated benzopyrans, barely showed differences in cytotoxicity (7a‐b vs. 10a‐b). For quinolines, 2‐substituted derivatives displayed greater activity than their 2,3‐disubstituted analogs (18 vs. 17).
• ii.Regarding the functional group on the nitrogen (amine or amide), the amide function showed higher cytotoxicity than their amine analogs (5a, 5b vs. 7a, 7b) against breast MCF‐7 cells. By contrast, benzopyran amines showed certain cytotoxicity against pancreas Mia PaCa‐2 cell line, and their amide analogs were completely inactive.
• iii.For benzopyran amides, cytotoxicity showed the following trend: p‐MeO‐phenetylamide derivatives (4c, 5c) > 2,2‐diphenylethylamides (4a, 5a) and 2‐chloro‐3‐trifluoro‐benzylamides (4b, 5b) >> methylhistidinate derivatives (4d, 5d). For benzopyran amines and quinolines, the 2,2‐diphenylethylamine moiety (7a, 10a, 17a, 18a) exhibited greater cytotoxicity than 2‐chloro‐3‐trifluoro‐benzylamine derivatives (7b, 10b, 17b, 18b), while methylhistidinate derivatives (4d, 5d) and sulfonamide derivatives (11a, 19b) did not show toxicity.
• iv.The protection of the phenol group at 6‐position of benzopyran amides with a p‐fluorobenzyl substituent, which increases the lipophilicity compared to benzyl substituent, afforded a similar cytotoxicity (5a‐d vs. 4a‐d).

FIGURE 5.

Structure–activity relationship (SAR) for nitrogenated derivatives of benzopyrans and quinolines.

2.4. Physicochemical Properties

The drug‐like properties of all active benzopyrans and quinolines were evaluated using SwissADME server (free online website, http://www.swissadme.ch/index.php) [39] to provide physicochemical and predicted pharmacokinetic parameters. Among benzopyran derivatives, benzopyran amides 4c and 5c satisfied all Lipinski's rule of five, which includes molecular weight (< 500 g/mol), hydrogen bond donors (< 10), hydrogen bond acceptors (< 10), and lipophilicity (cLogP < 5). Both 4c and 5c showed an appropriate topological polar surface area (< 140 Ų), which is consistent with a favorable absorption through the gastrointestinal absorption tract. Furthermore, benzopyrans 4c and 5c, as well as quinolines 17a and 17b (which only violated the cLogP criterion) showed a predicted bioavailability score of 0.55, supporting the assumption of favorable oral absorption (Supporting Information). It is worth noting that most of tested compounds slightly exceeded the recommended parameters, resulting in minor violation of Lipinski's filters. Although the compounds displayed moderately acceptable physicochemical properties, they still required in‐depth in vivo validation.

3. Conclusion

We have synthesized 18 benzopyran and quinoline derivatives containing a nitrogen atom in the side chain with different motifs in order to explore their potential cytotoxicity in human cancer cell lines, and to establish a SAR study. It was noteworthy that the benzopyran nucleus bearing an amide function in the side chain gave the most promising cytotoxic agents. By contrast, only 2‐substituted quinoline 18a with a 2,2‐diphenylethylamine was active against most of the tested cell lines. The human melanoma cell line A2058 resulted the most sensitive to the synthesized compounds, while Mia PaCa‐2 (pancreas) the least. Therefore, benzopyran amide derivatives emerge as new hits in the development of cytotoxic compounds that may be useful in human cancer therapy. Furthermore, SwissADME predicted favorable physicochemical properties for oral absorption, most notably in the 2‐propanamide benzopyrans containing the p‐methoxyphenethylamine moiety (4c, 5c).

4. Experimental Section

4.1. General Information

All reactions were monitored by analytical thin‐layer chromatography with silica gel 60 F₂₅₄ (Merck 5554; Merck Group, Darmstadt, Germany). Reaction residues were purified by silica gel column chromatography (40–63 μm, Merck Group). Solvents and reagents were purchased from the commercial sources (Scharlab S.L., Barcelona, Spain; Sigma–Aldrich, St. Louis, MO), and used without further purification unless otherwise stated. Dry and freshly distilled solvents were used in those reactions performed under N₂. Quoted yields are of purified material. Final compounds were purified to ≥95% as assessed by Nuclear magnetic resonance (NMR) of ¹H NMR and LC‐MS/MS analysis. NMR of (¹H, and ¹³C, COrrelation SpectroscopY (COSY) and Heteronuclear Single Quantum Coherence (HSQC) spectra were recorded on a Bruker AC‐300, AC‐400 or AC‐500 spectrometer (Bruker Instruments, Kennewick, WA). Chemical shifts (δ) are reported in ppm and referenced to the internal deuterated solvent, with multiplicities indicated as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), or br (broad). Coupling constants (J) are reported in Hertz (Hz). High‐resolution electrospray ionization mass spectrometry (ESIMS) was achieved with a VG Auto Spec Fisons spectrometer (Fisons, Loughborough, UK). Liquid chromatography‐MS detection was performed on an ultrahigh performance liquid chromatography system (Shimadzu, LCMS‐8040) coupled to a tandem MS (MS/MS) triple quadrupole mass spectrometer equipped with an ESI source (Shimadzu, Kyoto, Japan).

4.2. General Procedure for Synthesis of 2‐Propanamide Benzopyrans (4a‐d, 5a‐d)

Benzopyran esters 2 (95.7 mg, 0.27 mmol) or 3 (100.6 mg, 0.27 mmol) were hydrolyzed with a 20% aqueous KOH solution for 2 h under reflux. The mixture was evaporated in vacuum to give the corresponding benzopyran acid, which was treated with SOCl₂ (0.15 mL, 2.06 mmol) in dry CH₂Cl₂ (1 mL) and refluxed for 3 h. The solvent was removed to obtain the acid chlorides which were used in the next step without further purification. The corresponding acid chloride was dissolved in dry CH₂Cl₂ and added dropwise to a solution of the proper amine (2,2‐diphenylethylamine, 2‐chloro‐3‐trifluoromethylbenzylamine, p‐methoxyphenethylamine, or methylhistidinate), 4‐DMAP, and Et₃N in a cooling bath at 0ºC for 1 h under N₂. Then, 5% aqueous HCl (1.5 mL) solution was added and extracted with CH₂Cl₂ (3 × 10 mL). The organic layers were combined, washed with 5% aqueous NaHCO₃ solution (3 × 10 mL), brine (3 × 10 mL), dried over anhydrous Na₂SO₄, and evaporated to dryness. The residue was subjected to silica gel column chromatography (CH₂Cl₂/MeOH, 98:2) to obtain amides 4a‐d and 5a‐d.

4.3. 6‐(Benzyloxy)‐2‐methyl‐2‐[3^′‐(2,2‐diphenylethyl)propanamide]dihydrobenzopyran (4a)

Reagents and conditions: Acid chloride obtained from ester 2 (95.7 mg, 0.27 mmol), dry CH₂Cl₂ (1 mL), 2,2‐diphenylethylamine (0.35 mmol), 4‐DMAP (2.4 mg, 0.02 mmol), Et₃N (19 μL, 0.14 mmol). The benzopyran amide 4a was obtained in a 52% yield as a colorless oil.¹H NMR (300 MHz, CDCl₃) δ 7.50–7.20 (m, 15H, 2xPh, OCH₂Ph), 6.74 (dd, J = 8.7, 2.9 Hz, 1H, CH‐7), 6.68 (d, J = 2.9 Hz, 1H, CH‐5), 6.62 (d, J = 8.7 Hz, 1H, CH‐8), 5.53–5.49 (m, 1H, NHCO), 5.01 (s, 2H, OCH₂Ph), 4.19 (t, J = 8.0 Hz, 1H, CH‐5^′), 3.96–3.86 (m, 2H, CH₂‐4^′), 2.76–2.69 (m, 2H, CH₂‐4), 2.23 (t, J = 7.9 Hz, 2H, CH₂‐2^′), 1.97–1.78 (m, 2H, CH₂‐1^′), 1.75–1.65 (m, 2H, CH₂‐3), 1.19 (s, 3H, CH₃‐2). ¹³C NMR (75 MHz, CDCl₃) δ 173.3 (NHCO), 152.2 (C‐6), 147.7 (C‐8a), 141.7 (2C, C‐Ph), 137.4 (C‐1″), 128.4, 128.0, 127.9 and 126.7 (10C, CH‐Ph), 128.6, 127.7 and 127.4 (5C, CH‐Ph), 121.3 (C‐4a), 117.7 (CH‐5), 115.0 (CH‐7), 114.4 (CH‐8), 74.5 (C‐2), 70.6 (OCH₂Ph), 50.4 (CH‐5^′), 43.7 (CH₂‐4^′), 34.3 (CH₂‐1^′), 31.0 (CH₂‐3), 28.4 (CH₂‐2^′), 23.6 (CH₃‐2), 22.2 (CH₂‐4). HRMS(ESI) m/z calcd for C₃₄H₃₆NO₃ [M+H]⁺ 506.2690, found: 506.2693.

4.4. 6‐(Benzyloxy)‐2‐methyl‐2‐[3^′‐(2‐chloro‐3‐(trifluoromethyl)benzyl)propanamide)]dihydrobenzopyran (4b)

Reagents and conditions: Acid chloride obtained from ester 2 (95.7 mg, 0.27 mmol), dry CH₂Cl₂ (1 mL), 2‐chloro‐3‐trifluoromethylbenzylamine (0.35 mmol), 4‐DMAP (2.4 mg, 0.02 mmol), Et₃N (19 μL, 0.14 mmol). The benzopyran amide 4b was obtained in a 52% yield as a colorless oil. ¹H NMR (300 MHz, CDCl₃) δ 7.65–7.57 (m, 2H, CH‐5‴, CH‐6‴), 7.45–7.28 (m, 6H, OCH₂Ph, CH‐4‴), 6.74 (dd, J = 8.7, 2.9 Hz, 1H, CH‐7), 6.68 (d, J = 2.9 Hz, 1H, CH‐5), 6.63 (d, J = 8.7 Hz, 1H, CH‐8), 6.09–6.07 (m, 1H, NHCO), 4.98 (s, 2H, OCH₂Ph), 4.57 (d, J = 6.1 Hz, 2H, CH₂‐4^′), 2.86–2.63 (m, 2H, CH₂−4), 2.41 (t, J = 7.9 Hz, 2H, CH₂‐2^′), 2.06–1.88 (m, 2H, CH₂‐1^′), 1.88–1.69 (m, 2H, CH₂‐3), 1.24 (s, 3H, CH₃‐2). ¹³C NMR (75 MHz, CDCl₃) δ 173.1 (NHCO), 152.3 (C‐6), 147.7 (C‐8a), 138.2 (C‐1‴), 137.4 (C‐1″), 133.7 (CH‐6‴), 131.7 (C‐2‴), 129.0 (d, J _CF = 29.5 Hz, C‐3‴), 128.5, 127.8 and 127.4 (5C, CH‐Ph), 126.9 (CH‐5‴), 126.8 (CH‐4‴), 122.9 (q, J _CF = 272.8 Hz, CF₃), 121.5 (C‐4a), 117.6 (CH‐5), 115.2 (CH‐7), 114.5 (CH‐8), 74.9 (C‐2), 70.6 (OCH₂Ph), 41.5 (CH₂‐4^′), 35.1 (CH₂‐1^′), 31.2 (CH₂‐3), 30.6 (CH₂‐2^′), 23.6 (CH₃‐2), 22.3 (CH₂‐4). HRMS(ESI) m/z calcd for C₂₈H₂₈ClF₃NO₃ [M+H]⁺ 518.1704, found: 518.1702.

4.5. 6‐(Benzyloxy)‐2‐methyl‐2‐[3^′‐(p‐methoxyphenethyl)propanamide)]dihydrobenzopyran (4c)

Reagents and conditions: Acid chloride obtained from ester 2 (95.7 mg, 0.27 mmol), dry CH₂Cl₂ (1 mL), p‐methoxyphenethylamine (0.35 mmol), 4‐DMAP (2.4 mg, 0.02 mmol), Et₃N (19 μL, 0.14 mmol). The benzopyran amide 4c was obtained in 11% yield as a colorless oil. ¹H NMR (300 MHz, CDCl₃) δ 7.49–7.28 (m, 5H, OCH₂Ph), 7.09 (d, J = 8.6 Hz, 2H, CH‐2‴, CH‐6‴), 6.83 (d, J = 8.6 Hz, 2H, CH‐3‴, CH‐5‴), 6.73 (dd, J = 8.7, 2.9 Hz, 1H, CH‐7), 6.69 (d, J = 2.9 Hz, 1H, CH‐5), 6.63 (d, J = 8.7 Hz, 1H, CH‐8), 5.79 (brs, 1H, NHCO), 4.98 (s, 2H, OCH₂Ph), 3.78 (s, 3H, OCH₃), 3.50–3.44 (m, 2H, CH₂‐4^′), 2.82–2.67 (m, 4H, CH₂‐4, CH₂‐5^′), 2.34 (t, J = 7.8 Hz, 2H, CH₂‐2^′), 1.93–1.77 (m, 2H, CH₂‐1^′), 1.75–1.62 (m, 2H, CH₂‐3), 1.23 (s, 3H, CH₃‐2). ¹³C NMR (75 MHz, CDCl₃) δ 173.3 (NHCO), 158.3 (C‐4t^′), 152.3 (C‐6), 147.7 (C‐8a), 137.4 (C‐1″), 130.7 (C‐1‴), 129.7 (2C, CH‐2‴, CH‐6‴), 128.5, 127.8 and 127.4 (5C, CH‐Ph), 121.6 (C‐4a), 117.6 (CH‐5), 115.2 (CH‐7), 114.5 (CH‐8), 114.0 (2C, CH‐3‴, CH‐5‴), 75.0 (C‐2), 70.6 (OCH₂Ph), 55.2 (OCH₃), 41.0 (CH₂‐4^′), 35.2 (CH₂‐1^′), 34.6 (CH₂‐5^′), 31.1 (CH₂‐3), 30.6 (CH₂‐2^′), 23.6 (CH₃‐2), 22.3 (CH₂‐4). HRMS(ESI) m/z calcd for C₂₉H₃₄NO₄ [M+H]⁺ 460.2482, found: 460.2480.

4.6. 6‐(Benzyloxy)‐2‐methyl‐2‐[3^′‐(methylhistidinate)propanamide)]dihydrobenzopyran (4d)

Reagents and conditions: Acid chloride obtained from ester 2 (95.7 mg, 0.27 mmol), dry CH₂Cl₂ (1 mL), methylhistidinate (0.35 mmol), 4‐DMAP (2.4 mg, 0.02 mmol), Et₃N (19 μL, 0.14 mmol). The benzopyran amide 4d was obtained in 24% yield as a colorless oil. ¹H NMR (300 MHz, CDCl₃) δ 7.47–7.28 (m, 7H, CH‐4‴, CH‐6‴, OCH₂Ph), 6.79–6.63 (m, 3H, CH‐5, CH‐7, CH‐8), 4.98 (s, 2H, OCH₂Ph), 3.71 (s, 3H, OCH₃), 3.33–2.93 (m, 3H, CH‐1‴, CH₂‐2‴), 2.80–2.66 (m, 2H, CH₂−4), 2.53 (t, J = 7.9 Hz, 2H, CH₂‐2^′), 2.06–1.70 (m, 4H, CH₂‐3, CH₂‐1^′), 1.26 (s, 3H, CH₃‐2). ¹³C NMR (75 MHz, CDCl₃) δ 172.9 (NHCO), 152.3 (C‐6), 147.7 (C‐8a), 137.4 (C‐1″), 134.7 (CH‐6‴), 127.8, 127.5 and 127.5 (5C, CH‐Ph), 121.4 (C‐4a), 118.9 (CH‐4‴), 117.7 (CH‐5), 115.1 (CH‐7), 114.5 (CH‐8), 74.6 (C‐2), 70.6 (OCH₂Ph), 60.9 (CH‐1‴), 52.9 (OCH₃), 34.3 (CH₂‐1^′), 31.1 (CH₂‐3), 30.0 (CH₂‐2‴), 28.4 (CH₂‐2^′), 23.6 (CH₃‐2), 22.3 (CH₂‐4). HRMS(ESI) m/z calcd for C₂₇H₃₂N₃O₅ [M+H]⁺ 478.2342, found: 478.2349.

4.7. 6‐(p‐Fluorobenzyloxy)‐2‐methyl‐2‐[3^′‐(2,2‐diphenylethyl)propanamide]dihydrobenzopyran (5a)

Reagents and conditions: Acid chloride obtained from ester 3 (100.6 mg, 0.27 mmol) in dry CH₂Cl₂ (1 mL), 2,2‐diphenylethylamine (0.35 mmol), 4‐DMAP (2.4 mg, 0.02 mmol), Et₃N (19 μL, 0.14 mmol). The benzopyran amide 5a was obtained in a 25% yield as a colorless oil. ¹H NMR (300 MHz, CDCl₃) δ 7.47–7.38 (m, 2H, CH‐2″, CH‐6″), 7.36–7.20 (m, 10H, 2xPh), 7.15–7.02 (m, 2H, CH‐3″, CH‐5″), 6.74 (dd, J = 8.7, 2.9 Hz, 1H, CH‐7), 6.69 (d, J = 2.9 Hz, 1H, CH‐5), 6.63 (d, J = 8.7 Hz, 1H, CH‐8), 5.66 (br s, 1H, NHCO), 4.97 (s, 2H, OCH₂Ph‐p‐F), 4.19 (t, J = 7.9 Hz, 1H, CH‐5^′), 3.96–3.86 (m, 2H, CH₂‐4^′), 2.77–2.63 (m, 2H, CH₂‐4), 2.28 (t, J = 7.7 Hz, 2H, CH₂‐2^′), 1.92–1.78 (m, 2H, CH₂‐1^′), 1.76–1.67 (m, 2H, CH₂‐3), 1.22 (s, 3H, CH₃‐2). ¹³C NMR (75 MHz, CDCl₃) δ 173.2 (NHCO), 162.5 (d, J _CF = 246.0 Hz, C‐4″), 152.1 (C‐6), 147.8 (C‐8a), 141.7 (2C, C‐Ph), 133.2 (d, J _CF = 3.3 Hz, C‐1″), 129.3 (d, 2C, J _CF = 8.3 Hz, CH‐2″, CH‐6″), 128.7, 128.0, 128.0 and 126.8 (10C, CH‐Ph), 121.6 (C‐4a), 117.6 (CH‐5), 115.4 (d, 2C, J _CF = 21.5 Hz, CH‐3″, CH‐5″), 115.2 (CH‐7), 114.5 (CH‐8), 74.9 (C‐2), 70.0 (OCH₂Ph‐p‐F), 50.5 (CH‐5^′), 43.8 (CH₂‐4^′), 35.1 (CH₂‐1^′), 31.1 (CH₂‐3), 30.6 (CH₂‐2^′), 23.6 (CH₃‐2), 22.3 (CH₂‐4). HRMS(ESI) m/z calcd for C₃₄H₃₅FNO₃ [M+H]⁺ 524.2595, found: 524.2583.

4.8. 6‐(p‐Fluorobenzyloxy)‐2‐methyl‐2‐[3^′‐(2‐chloro‐3‐(trifluoromethyl)benzyl)propanamide)] dihydrobenzopyran (5b)

Reagents and conditions: The acid chloride obtained from ester 3 (100.6 mg, 0.27 mmol) in dry CH₂Cl₂ (1 mL), 2‐chloro‐3‐trifluoromethylbenzylamine (0.35 mmol), 4‐DMAP (2.4 mg, 0.02 mmol), Et₃N (19 μL, 0.14 mmol). The benzopyran amide 5b was obtained in a 24% yield as a colorless oil. ¹H NMR (300 MHz, CDCl₃) δ 7.65–7.56 (m, 2H, CH‐5‴, CH‐6‴), 7.44–7.31 (m, 3H, CH‐2″, CH‐6″, CH‐4‴), 7.10–6.99 (m, 2H, CH‐3″, CH‐5″), 6.71–6.63 (m, 3H, CH‐7, CH‐5, CH‐8), 6.15–6.13 (m, 1H, NHCO), 4.93 (s, 2H, OCH₂Ph‐p‐F), 4.56 (d, J = 6.1 Hz, 2H, CH₂‐4^′), 2.80–2.69 (m, 2H, CH₂‐4), 2.41 (t, J = 7.9 Hz, 2H, CH₂‐2^′), 2.06–1.85 (m, 2H, CH₂‐1^′), 1.85–1.69 (m, 2H, CH₂‐3), 1.24 (s, 3H, CH₃‐2). ¹³C NMR (75 MHz, CDCl₃) δ 173.1 (NHCO), 162.5 (d, J _CF = 246.0 Hz, C‐4″), 152.1 (C‐6), 147.8 (C‐8a), 138.2 (C‐1‴), 133.7 (CH‐6‴), 133.2 (d, J _CF = 3.2 Hz, C‐1″), 131.3 (C‐2^′’’), 129.3 (d, 2C, J _CF = 8.1 Hz, CH‐2″, CH‐6″), 129.0 (d, J _CF = 30.8 Hz, C‐3‴), 126.9 (CH‐5‴), 126.8 (CH‐4‴), 122.9 (q, J _CF = 271.7 Hz, CF₃), 121.6 (C‐4a), 117.8 (CH‐5), 115.4 (d, 2C, J _CF = 21.4 Hz, CH‐3″, CH‐5″), 115.2 (CH‐7) 114.5 (CH‐8), 74.9 (C‐2), 70.0 (OCH₂Ph‐p‐F), 41.5 (CH₂‐4^′), 35.1 (CH₂‐1^′), 31.2 (CH₂−3), 30.6 (CH₂‐2^′), 23.6 (CH₃‐2), 22.3 (CH₂‐4). HRMS (ESI) m/z calcd for C₂₈H₂₇ClF₄NO₃ [M+H]⁺ 536.1610, found: 536.1616.

4.9. 6‐(p‐Fluorobenzyloxy)‐2‐methyl‐2‐[3^′‐(p‐methoxyphenethyl)propanamide)]dihydrobenzopyran (5c)

Reagents and conditions: The acid chloride obtained from ester 3 (100.6 mg, 0.27 mmol) in dry CH₂Cl₂ (1 mL), p‐methoxyphenethylamine (0.35 mmol), 4‐DMAP (2.4 mg, 0.02 mmol), Et₃N (19 μL, 0.14 mmol). The benzopyran amide 5c was obtained in 36% yield as a colorless oil. ¹H NMR (300 MHz, CDCl₃) δ 7.44–7.33 (m, 2H, CH‐2″, CH‐6″), 7.13–7.00 (m, 4H, CH‐3″, CH‐5″, CH‐2‴, CH‐6‴), 6.84 (d, J = 8.6 Hz, 2H, CH‐3‴, CH‐5‴), 6.72 (dd, J = 8.7, 2.9 Hz, 1H, CH‐7), 6.67 (d, J = 2.9 Hz, 1H, CH‐5), 6.64 (d, J = 8.7 Hz, 1H, CH‐8), 5.54 (brs, 1H, NHCO), 4.94 (s, 2H, OCH₂Ph‐p‐F), 3.79 (s, 3H, OCH₃), 3.53–3.40 (m, 2H, CH₂‐4^′), 2.79–2.68 (m, 4H, CH₂‐4, CH₂‐5^′), 2.35–2.23 (m, 2H, CH₂‐2^′), 2.02–1.86 (m, 2H, CH₂‐1^′), 1.82–1.71 (m, 2H, CH₂‐3), 1.23 (s, 3H, CH₃‐2). ¹³C NMR (75 MHz, CDCl₃) δ 172.9 (NHCO), 162.5 (d, J _CF = 245.9 Hz, CH‐4″), 158.2 (C‐4t^′), 152.1 (C‐6), 147.8 (C‐8a), 133.2 (d, J _CF = 3.1 Hz, C‐1″), 130.8 (C‐1‴), 129.7 (2C, CH‐2‴, CH‐6‴), 129.3 (d, 2C, J J _CF = 8.2 Hz, CH‐2″, CH‐6″), 121.6 (C‐4a), 117.7 (CH‐5), 115.4 (d, 2C, J _CF = 21.4 Hz, CH‐3″, CH‐5″), 115.2 (CH‐7), 114.5 (CH‐8), 114.0 (2C, CH‐3‴, CH‐5‴), 75.0 (C‐2), 70.0 (OCH₂Ph‐p‐F), 55.2 (OCH₃), 40.7 (CH₂‐4^′), 35.1 (CH₂‐1^′), 34.7 (CH₂‐5^′), 31.1 (CH₂‐3), 30.8 (CH₂‐2^′), 23.7 (CH₃‐2), 22.3 (CH₂‐4). HRMS(ESI) m/z calculated for C₂₉H₃₃FNO₄ [M+H]⁺ 478.2393, found: 478.2347.

4.10. 6‐(p‐Fluorobenzyloxy)‐2‐methyl‐2‐[3^′‐(methylhistidinate)propanamide)]dihydrobenzopyran (5d)

Reagents and conditions: The acid chloride obtained from ester 3 (100.6 mg, 0.27 mmol) in dry CH₂Cl₂ (1 mL), methylhistidinate (0.35 mmol), 4‐DMAP (2.4 mg, 0.02 mmol), Et₃N (19 μL, 0.14 mmol). The benzopyran amide 5d was obtained in 25% yield as a colorless oil. ¹H NMR (300 MHz, CDCl₃) δ 7.55–7.44 (m, 1H, CH‐6‴), 7.44–7.32 (m, 2H, CH‐2″, CH‐6″), 7.11–6.93 (m, 3H, CH‐3″, CH‐5″, CH‐4‴), 6.78–6.51 (m, 3H, CH‐5, CH‐7, CH‐8), 4.92 (s, 2H, OCH₂Ph‐p‐F),), 3.70 (s, 3H, OCH₃), 3.34–2.97 (m, 3H, CH‐1‴, CH₂‐2‴), 2.84–2.62 (m, 2H, CH₂‐4), 2.53–2.34 (m, 2H, CH₂‐2^′), 1.99–1.55 (m, 4H, CH₂‐3, CH₂‐1^′), 1.20 (s, 3H, CH₃‐2). ¹³C NMR (75 MHz, CDCl₃) δ 173.6 (NHCO), 162.5 (d, J _CF = 245.9 Hz, CH‐4^′’), 152.0 (C‐6), 147.9 (C‐8a), 134.2 (CH‐6‴), 133.1 (d, J _CF = 3.1 Hz, C‐1″), 129.3 (d, 2C, J _CF = 8.2 Hz, CH‐2″, CH‐6″), 121.7 (C‐4a), 118.8 (CH‐4‴), 117.7 (CH‐5), 115.4 (d, 2C, J _CF = 21.4 Hz, CH‐3″, CH‐5″), 115.3 (CH‐7), 114.5 (CH‐8), 75.0 (C‐2), 70.0 (OCH₂Ph‐p‐F), 61.1 (CH‐1‴), 52.6 (OCH₃), 35.0 (CH₂‐1^′), 31.0 (CH₂−3), 30.5 (CH₂‐2^′), 29.7 (CH₂‐2‴), 23.6 (CH₃‐2), 22.3 (CH₂‐4). HRMS(ESI) m/z calcd for C₂₇H₃₁FN₃O₅ [M+H]⁺ 496.2242, found: 496.2252.

4.11. General Procedure for Synthesis Benzopyran Amines (7a, 7b, 10a, 10b)

A mixture of 0.9 mmol of aldehyde 6 or 9 and the corresponding amine (2,2‐diphethylamine or 2‐chloro‐3‐(trifluoromethyl)‐benzylamine) in dry dichloroethane was stirred for 15 min at room temperature under N₂. Next, NaBH(OAc)₃ and one drop of acetic acid were added and stirred at room temperature for 1 h. Reaction mixture was diluted with EtOAc (15 mL) and washed with H₂O (3 × 10 mL), brine (3 × 10 mL), dried over anhydrous Na₂SO₄, and evaporated to dryness. The residue obtained was subjected to silica gel column chromatography (CH₂Cl₂/MeOH, 95:5) to afford compounds 7a, 7b, 10a, and 10b. Benzopyran amines 7a and 7b obtained in 75% and 81% yields, respectively, were previously described [29].

4.12. 6‐(p‐Fluorobenzyloxy)‐2‐methyl‐2‐[7^′‐(2,2‐diphenylethylamine)‐4‐(methyl‐hept‐4‐enal)]‐dihydrobenzopyran (10a)

Reagents and conditions: Aldehyde 9 (357 mg, 0.9 mmol), 2,2‐diphethylamine (1.5 mmol), dry dichloroethane (2 mL), NaBH(OAc)₃ (1.95 mmol), acetic acid (one drop). The benzopyran amine 10a was obtained in a 69% yield as a colorless oil.¹H NMR (300 MHz, CDCl₃) δ 7.43–7.33 (m, 2H, CH‐2″, CH‐6″), 7.35–7.14 (m, 10H, 2xPh), 7.11–6.99 (m, 2H, CH‐3″, CH‐5″), 6.75–6.63 (m, 3H, CH‐5, CH‐7, CH‐8), 5.01 (t, J = 6.8 Hz, 1H, CH‐3^′), 4.93 (s, 2H, OCH₂Ph‐p‐F), 4.28 (t, J = 7.7 Hz, 1H, CH‐9^′), 3.27 (d, J = 7.7 Hz, 2H, CH2‐8^′), 2.72 (t, J = 6.8 Hz, 2H, CH₂‐4), 2.66–2.48 (m, 2H, CH₂‐7^′), 2.05 (q, J = 8.0 Hz, 2H, CH₂‐2^′), 1.94–1.85 (m, 2H, CH₂‐5^′), 1.85–1.63 (m, 2H, CH₂‐3), 1.64–1.44 (m, 7H, CH₂‐1^′, CH₃‐4^′, CH₂‐6^′), 1.28 (s, 3H, CH₃−2). ¹³C NMR (75 MHz, CDCl₃) δ 162.4 (d, J _CF = 245.9 Hz, C‐4″), 151.9 (C‐6), 148.2 (C‐8a), 142.4 (2C, C‐Ph), 134.4 (C‐4^′), 133.3 (d, J _CF = 3.0 Hz, C‐1″), 129.3 (d, 2C, J _CF = 8.2 Hz, CH‐2″, CH‐6″), 128.7, 128.0 and 126.7 (10C, CH‐Ph), 124.6 (CH‐3^′), 121.7 (C‐4a), 117.6 (CH‐5), 115.4 (d, 2C, J _CF = 21.3 Hz, CH‐3″, CH‐5″), 115.2 (CH‐7) 114.4 (CH‐8), 75.6 (C‐2), 70.0 (OCH₂Ph‐p‐F), 53.9 (CH₂‐8^′), 50.5 (CH‐9^′), 49.0 (CH₂‐7^′), 39.3 (CH₂‐1^′), 37.0 (CH₂‐5^′), 30.9 (CH₂‐3), 27.1 (CH₂‐6^′), 24.1 (CH₃‐2), 22.4 (CH₂‐4), 22.1 (CH₂‐2^′), 15.7 (CH₃‐4^′). HRMS(ESI) m/z calcd for C₃₉H₄₅FNO₂ [M+H]⁺ 578.3429, found: 578.3438.

4.13. 6‐(p‐Fluorobenzyloxy)‐2‐methyl‐2‐[7^′‐(2‐chloro‐3‐(trifluoromethyl)benzyl‐amine)‐4‐(methyl‐hept‐4‐enal)]‐dihydrobenzopyran (10b)

Reagents and conditions: Aldehyde 9 (357 mg, 0.9 mmol) and 2‐chloro‐3‐(trifluoromethyl)‐benzylamine (1.5 mmol), dry dichloroethane (2 mL), NaBH(OAc)₃ (1.95 mmol), acetic acid (one drop). The benzopyran amine 10b was obtained in 80% yield as a colorless oil. ¹H NMR (300 MHz, CDCl₃) δ 7.65–7.52 (m, 2H, CH‐5″’, CH‐6‴), 7.43–7.32 (m, 3H, CH‐2″, CH‐6″, CH‐4‴), 7.13–7.01 (m, 2H, CH‐3″, CH‐5″), 6.73–6.66 (m, 3H, CH‐5, CH‐7, CH‐8), 5.12 (t, J = 6.6 Hz, 1H, CH‐3^′), 4.94 (s, 2H, OCH₂Ph‐p‐F), 4.00 (s, 2H, CH₂‐8^′), 2.72 (t, J = 6.8 Hz, 2H, CH₂‐4), 2.64 (t, J = 7.2 Hz, 2H, CH₂‐7^′), 2.15–1.96 (m, 4H, CH₂‐2^′, CH₂‐5^′), 1.86–1.71 (m, 2H, CH₂‐3), 1.71–1.50 (m, 7H, CH₂‐1^′, CH₃‐4^′, CH₂‐6^′), 1.27 (s, 3H, CH₃‐2). ¹³C NMR (75 MHz, CDCl₃) δ 162.4 (d, J _CF = 246.0 Hz, C‐4″), 151.9 (C‐6), 148.2 (C‐8a), 139.1 (C‐1‴), 134.4 (C‐4^′), 133.6 (CH‐6‴), 133.3 (d, J _CF = 3.2 Hz, C‐1″), 131.9 (C‐2‴), 129.3 (d, 2C, J _CF = 8.2 Hz, CH‐2″, CH‐6″), 128.6 (C‐3‴), 126.6 (2C, CH‐4‴, CH‐5‴), 124.8 (CH‐3^′), 122.9 (q, J _CF = 272.5 Hz, CF₃), 121.7 (C‐4a), 117.8 (CH‐5), 115.4 (d, 2C, J _CF = 21.5 Hz, CH‐3″, CH‐5″), 115.2 (CH‐7), 114.4 (CH‐8), 75.6 (C‐2), 70.0 (OCH₂Ph‐p‐F), 50.4 (CH₂‐8^′), 48.5 (CH₂‐7^′), 39.3 (CH₂‐1^′), 37.0 (CH₂‐5^′), 30.9 (CH₂−3), 27.4 (CH₂‐6^′), 24.1 (CH₃−2), 22.4 (CH₂−4), 22.1 (CH₂‐2^′), 15.7 (CH₃‐4^′). HRMS(ESI) m/z calcd for C₃₃H₃₇ClF₄NO₂ [M+H]⁺ 590.2443, found: 590.2446.

4.14. 6‐(p‐Fluorobenzyloxy)‐2‐methyl‐2‐[7^′‐(2,2‐diphenylethyl‐p‐methylbenzene‐sulfonamide)‐4‐(methyl‐hept‐4‐enal)]‐dihydrobenzopyran (11a)

A mixture of amine 10a (75 mg, 0.13 mmol) and 4‐toluensulfonyl chloride (24.8 mg, 0.13 mmol) was dissolved in dry CH₂Cl₂ and Et₃N (0.1 mL). The solution was stirred in a cooling ice bath for 2 h under N₂. The organic layer was washed with water (3 × 10 mL), dried over anhydrous Na₂SO₄, and evaporated to dryness. The residue was subjected to purification by silica gel column chromatography (Hexane/EtOAc, 80:20) to afford N‐tosyl derivative 11a in 32% yield.

¹H NMR (300 MHz, CDCl₃) δ 7.59–7.52 (m, 2H, CH‐2‴, CH‐6‴), 7.40–7.34 (m, 2H, CH‐2″, CH‐6″), 7.30–7.16 (m, 12H, 2xPh, CH‐3″’, CH‐5″’), 7.10–6.98 (m, 2H, CH‐3″, CH‐5″), 6.75–6.63 (m, 3H, CH‐5, CH‐7, CH‐8), 4.98–4.90 (m, 3H, CH₂‐3^′, OCH₂Ph‐p‐F), 4.29 (t, J = 7.8 Hz, 1H, CH‐9^′), 3.74 (d, J = 7.8 Hz, 2H, CH₂‐8^′), 2.90–2.79 (m, 2H, CH₂‐7^′), 2.73 (t, J = 6.7 Hz, 2H, CH₂−4), 2.39 (s, 3H, CH₃Ph), 2.12–1.98 (m, 2H, CH₂‐2^′), 1.80–1.49 (m, 6H, CH₂‐3, CH₂‐1^′, CH₂‐5^′), 1.46 (s, 3H, CH₃‐4^′), 1.34–1.24 (m, 5H, CH₃−2, CH₂‐6^′). ¹³C NMR (75 MHz, CDCl₃) δ 162.4 (d, J _CF = 245.9 Hz, C‐4″), 151.9 (C‐6), 148.2 (C‐8a), 143.0 (C‐4‴), 141.8 (2C, C‐Ph), 136.9 (C‐1‴), 133.9 (C‐4^′), 133.2 (C‐1″), 129.6 (2C, CH‐3″’, CH‐5″’), 129.4 (d, 2C, J _CF = 8.3 Hz, CH‐2″, CH‐6″), 128.6, 128.2, and 126.7 (10C, CH‐Ph), 127.3 (2C, CH‐2‴, CH‐6‴), 124.7 (CH‐3^′), 121.7 (C‐4a), 117.8 (CH‐5), 115.4 (d, 2C, J _CF = 21.8 Hz, CH‐3″, CH‐5″), 115.2 (CH‐7), 114.4 (CH‐8), 75.6 (C‐2), 70.0 (OCH₂Ph‐p‐F), 52.7 (CH₂‐8^′), 50.4 (CH‐9^′), 48.4 (CH₂‐7^′), 39.3 (CH₂‐1^′), 36.5 (CH₂‐5^′), 30.9 (CH₂‐3), 25.7 (CH₂‐6^′), 24.2 (CH₃‐2), 22.4 (CH₂‐4), 22.1 (CH₂‐2^′), 21.5 (CH₃Ph), 15.7 (CH₃‐4^′). HRMS(ESI) m/z calcd for C₄₆H₅₁FNO₄S [M+H]⁺ 732.3517, found: 732.3518.

4.15. Synthesis of Quinolines

4.15.1. General Procedure for Synthesis of Quinoline Amines (17a, 17b, 18a, 18b)

A mixture of aldehyde 15 or 16 and the corresponding amine (2,2 diphethylamine or 2‐chloro‐3‐(trifluoromethyl)‐benzylamine) in dry dichloroethane was stirred for 15 min at room temperature under N₂. Next, NaBH(OAc)₃ was added in the presence of acetic acid, and stirred at room temperature for an additional 1 h. Reaction mixture was diluted with EtOAc (15 mL) and washed with water (3 × 10 mL), brine (3 × 10 mL), dried over anhydrous Na₂SO₄, and evaporated to dryness. The residue was subjected to silica gel column chromatography (CH₂Cl₂/MeOH, 95:5) to afford quinoline derivatives 17a, 17b, 18a, or 18b.

4.15.2. 6‐(Benzyloxy)‐3‐[N‐(2,2‐diphenylethylethanamine)]‐2‐methylquinoline (17a)

Reagent and conditions: Aldehyde 15 (130 mg, 0.45 mmol), 2,2 diphethylamine (0.75 mmol), dry dichloroethane (2 mL), NaBH(OAc)₃ (0.98 mmol), acetic acid (one drop). The quinoline amine 17a was afforded in a 55% yield as a pale yellow oil. ¹H NMR (300 MHz, CDCl₃) δ 7.94 (d, J = 9.2 Hz, 1H, CH‐8), 7.66 (s, 1H, CH‐4), 7.51–7.37 (m, 6H, CH‐7, OCH₂Ph), 7.32–7.16 (m, 10H, 2xPh), 7.05 (d, J = 2.8 Hz, 1H, CH‐5), 5.20 (s, 2H, OCH₂Ph), 4.25 (t, J = 7.7 Hz, 1H, CH‐4^′), 3.34 (d, J = 7.7 Hz, 2H, CH₂‐3^′), 3.03–2.87 (m, 4H, CH₂‐1^′, CH₂‐2^′), 2.68 (s, 3H, CH₃−2). ¹³C NMR (75 MHz, CDCl₃) δ 156.3 (C‐6), 155.7 (C‐2), 142.6 (C‐8a), 142.5 (2C, C‐Ph), 136.6 (C‐1″), 134.1 (CH‐4), 131.7 (C‐3), 129.7 (CH‐8), 128.6, 127.8, and 127.5 (5C, CH‐Ph), 128.6, 127.9, and 126.6 (10C, 2xPh), 128.1 (C‐4a), 121.6 (CH‐7), 106.1 (CH‐5), 70.2 (OCH₂Ph), 54.3 (CH₂‐3^′), 50.9 (CH‐4^′), 49.2 (CH₂‐2^′), 33.1 (CH₂‐1^′), 22.9 (CH₃−2). HRMS(ESI) m/z calcd for C₃₃H₃₃N₂O [M+H]⁺ 473.2587, found: 473.2566.

4.15.3. 6‐(Benzyloxy)‐3‐[N‐(2‴‐chloro‐3‴‐(trifluoromethyl)benzylethanamine)]‐2‐methylquinoline (17b)

Reagent and conditions: Aldehyde 15 (130 mg, 0.45 mmol), 2‐chloro‐3‐(trifluoromethyl)‐benzylamine (0.75 mmol), dry dichloroethane (2 mL), NaBH(OAc)₃ (0.98 mmol), acetic acid (one drop). The quinoline amine 17b was afforded in a 55% yield as a pale yellow oil. ¹H NMR (300 MHz, CDCl₃) δ 7.93 (d, J = 9.4 Hz, 1H, CH‐8), 7.79 (s, 1H, CH‐4), 7.69–7.57 (m, 3H, CH‐4‴, CH‐5‴, CH‐6‴), 7.51–7.27 (m, 6H, CH‐7, OCH₂Ph), 7.07 (d, J = 2.8 Hz, 1H, CH‐5), 5.13 (s, 2H, OCH₂Ph), 4.07 (s, 2H, CH₂‐3^′), 3.10–2.94 (m, 4H, CH₂‐1^′, CH₂‐2^′), 2.69 (s, 3H, CH₃−2). ¹³C NMR (75 MHz, CDCl₃) δ 156.5 (C‐6), 155.6 (C‐2), 142.3 (C‐8a), 137.0 (C‐1‴), 136.5 (C‐1″), 134.7 (CH‐4), 133.5 (CH‐6‴), 132.0 (C‐2‴), 131.3 (C‐3), 129.5 (CH‐8), 128.6 (C‐3‴), 128.6, 128.1, and 127.5 (5C, CH‐Ph), 128.0 (C‐4a), 126.7 (2C, CH‐4‴, CH‐5‴), 122.9 (q, J _CF = 274.3 Hz, CF₃), 122.0 (CH‐7), 106.0 (CH‐5), 70.2 (OCH₂Ph), 50.4 (CH₂‐3^′), 48.4 (CH₂‐2^′), 32.7 (CH₂‐1^′), 22.7 (CH₃−2). HRMS(ESI) m/z calcd for C₂₇H₂₅ClF₃N₂O [M+H]⁺ 485.1591, found: 485.1583.

4.15.4. 6‐(Benzyloxy)‐2‐[N‐(2,2‐diphenylethylpropanamine)]quinoline (18a)

Reagent and conditions: Aldehyde 16 (130 mg, 0.45 mmol), 2,2 diphethylamine (0.75 mmol), dry dichloroethane (2 mL), NaBH(OAc)₃ (0.98 mmol), acetic acid (one drop). The quinoline amine 18a was afforded in a 92% yield as a pale yellow oil. ¹H NMR (300 MHz, CDCl₃) δ 7.92 (d, J = 8.4 Hz, 1H, CH‐4), 7.58 (d, J = 9.2 Hz, 1H, CH‐8), 7.57–7.30 (m, 6H, CH‐7, OCH₂Ph), 7.31–7.01 (m, 11H, 2xPh, CH‐3), 7.11 (d, J = 2.8 Hz, 1H, CH‐5), 5.17 (s, 2H, OCH₂Ph), 4.54 (t, J = 7.7 Hz, 1H, CH‐5^′), 3.47 (d, J = 7.7 Hz, 2H, CH₂‐4^′), 2.97 (t, J = 7.0 Hz, 2H, CH₂‐1^′), 2.89 (t, J = 6.7 Hz, 2H, CH₂‐3^′), 2.12 (m, 2H, CH₂‐2^′). ¹³C NMR (75 MHz, CDCl₃) δ 158.5 (C‐6), 156.5 (C‐2), 143.3 (C‐8a), 141.5 (2C, C‐Ph), 136.5 (C‐1″), 135.7 (CH‐4), 129.8 (CH‐8), 128.8, 127.9, and 127.0 (10C, 2xPh), 128.6, 128.1, and 127.5 (5C, CH‐Ph), 128.1 (C‐4a), 122.5 (CH‐7), 121.6 (CH‐3), 106.5 (CH‐5), 70.2 (OCH₂Ph), 53.2 (CH₂‐4^′), 49.2 (CH‐5^′), 48.5 (CH₂‐3^′), 35.8 (CH₂‐1^′), 26.9 (CH₂‐2^′). HRMS(ESI) m/z calcd for C₃₃H₃₃N₂O [M+H]⁺ 473.2587, found: 473.2570.

4.15.5. 6‐(Benzyloxy)‐2‐[N‐(2‴‐chloro‐3‴‐(trifluoromethyl)benzylpropanamine)]quinoline (18b)

Reagent and conditions: Aldehyde 16 (130 mg, 0.45 mmol), 2‐chloro‐3‐(trifluoromethyl)‐benzylamine (0.75 mmol), dry dichloroethane (2 mL), NaBH(OAc)₃ (0.98 mmol), acetic acid (one drop). The quinoline amine 18b was afforded in a 72% yield as a pale yellow oil. ¹H NMR (300 MHz, CDCl₃) δ 8.13–8.08 (m, 1H, CH‐6‴), 7.99 (d, J = 8.5 Hz, 1H, CH‐4), 7.69–7.64 (m, 1H, CH‐8), 7.50–7.33 (m, 8H, CH‐7, CH‐4‴, CH‐5‴, OCH₂Ph), 7.25–7.22 (m, 1H, CH‐3), 7.13 (d, J = 2.8 Hz, 1H, CH‐5), 5.16 (s, 2H, OCH₂Ph), 4.21 (s, 2H, CH₂‐4^′), 3.15 (t, J = 6.7 Hz, 2H, CH₂‐1^′), 3.05 (t, J = 6.5 Hz, 2H, CH₂‐3^′), 2.28–2.16 (m, 2H, CH₂‐2^′). ¹³C NMR (75 MHz, CDCl₃) δ 158.6 (C‐2), 156.6 (C‐6), 142.9 (C‐8a), 137.0 (C‐1‴), 136.4 (C‐1″), 136.1 (CH‐4), 135.0 (CH‐6‴), 132.1 (C‐2‴), 129.9 (2C, CH‐8, C‐3‴), 128.7, 128.2, and 127.5 (5C, CH‐Ph), 127.6 (C‐4a), 127.2 (2C, CH‐4‴, CH‐5‴), 122.7 (CH‐7), 121.8 (CH‐3) 106.7 (CH‐5), 70.3 (OCH₂Ph), 49.0 (CH₂‐4^′), 48.9 (CH₂‐3^′), 36.8 (CH₂‐1^′), 26.8 (CH₂‐2^′). HRMS(ESI) m/z calcd for C₂₇H₂₅ClF₃N₂O [M+H]⁺ 485.1602, found: 485.1593.

4.15.6. 6‐(Benzyloxy)‐2‐[N‐(2‴‐chloro‐3‴‐(trifluoromethyl)benzyl)‐N‐(p‐methyl‐benzenesulfonamide)]‐2‐propylquinoline (19b)

A mixture of quinoline amine 18b (140 mg, 0.29 mmol) and p‐toluensulfonyl chloride (55.3 mg, 0.29 mmol) in dry CH₂Cl₂ and triethylamine (0.1 mL) was stirred at 0ºC in a cooling ice bath for 2 h under N₂. Next, the organic layer was washed with water (3 × 10 mL), dried over anhydrous Na₂SO₄, and evaporated to dryness. The residue was subjected to silica gel column chromatography (Hexane/EtOAc, 80:20) to give 19b in 62% yield. ¹H NMR (300 MHz, CDCl₃) δ 7.85–7.75 (m, 2H, CH‐4, CH‐6‴), 7.69–7.61 (m, 3H, CH‐8, CH‐2‴, CH‐6‴), 7.50–7.25 (m, 12H, CH‐3, CH‐5, CH‐7, CH‐4‴, CH‐5‴, CH‐3‴, CH‐5‴, OCH₂Ph), 5.22 (s, 2H, OCH₂Ph), 4.48 (s, 2H, CH₂‐4^′), 3.35–3.22 (m, 4H, CH₂‐1^′, CH₂‐3^′), 2.43 (s, 3H, CH₃Ph), 2.09–1.98 (m, 2H, CH₂‐2^′). ¹³C NMR (75 MHz, CDCl₃) δ 158.6 (C‐2), 156.6 (C‐6), 143.0 (2C, C‐8a, C‐4t″), 137.1 (2C, C‐1‴, C‐1‴), 136.3 (C‐1″), 136.1 (CH‐4), 135.0 (CH‐6‴), 132.2 (C‐2‴), 129.9 (2C, CH‐8, C‐3‴), 128.9 (2C, CH‐3‴, CH‐5‴), 128.7, 128.2 and 127.5 (5C, CH‐Ph), 127.5 (C‐4a), 127.2 (2C, CH‐2‴, CH‐6‴), 126.8 (2C, CH‐4‴, CH‐5‴), 121.9 (2C, CH‐3, CH‐7) 106.9 (CH‐5), 70.6 (OCH₂Ph), 50.4 (CH₂‐4^′), 49.3 (CH₂‐3^′), 31.3 (CH₂‐1^′), 28.8 (CH₂‐2^′), 21.5 (CH₃Ph). HRMS(ESI) m/z calcd for C₃₄H₃₁ClF₃N₂O₃S [M+H]⁺ 639.1691, found: 639.1682.

4.16. Cytotoxicity Bioassays

Human cancer cell lines were purchased from American Type Culture Collection (ATCC, Manassas, Virginia, USA). Synthesized benzopyrans and quinolines were assayed against the human cancer cell lines, including lung A549 (RRID:CVCL_A549; CCL‐185 ATCC), melanoma A2058 (RRID:CVCL_1059; CRL_3601 ATCC), hepatoma HepG2 (RRID:CVCL_0027; HB‐8065 ATCC), breast MCF‐7 (RRID:CVCL_0031; HTB‐22 ATCC), and pancreas Mia PaCa‐2 (RRID:CVCL_0428; CRL‐1420 ATCC) in an MTT test as 10‐point curves with 1:2 dilutions starting at 40 µM in triplicate. Cells were seeded at 4.000 cells/well in a 384‐well plate (Corning 3701) for 24 h and compounds were added with an Echo 550 (Beckman Coulter) that enables the transference of ultralow sample volumes (nanolitres), and cells were incubated for 72 h [40]. Methylmethanesulfonate (MMS, Sigma–Aldrich, 2 mM) was used as the positive control, and DMSO 0.5% as the negative control. After addition of 0.5 mg/mL MMT dye (thiazolyl blue tetrazoliumbromide, ACROS Organics), cells were incubated for 2–3 h and supernatant was removed. Resulting formazan crystals were finally dissolved by means of 50 µL DMSO (100%) and absorbance at 570 nm was read with Envision Multimode Plate Reader (Revvity) and results were analyzed using Genedata Screener software (Genedata, Inc., Basel, Switzerland). Results are shown in percentage of activity normalized to negative (DMSO) and positive (MMS) controls considered as the scale reference set to 0 and −100, respectively. After normalization, the half maximal effective dose (ED₅₀ in µM) for each compound was determined by fitting the normalized activity data to a dose–response curve using the Hill equation. Outliers were excluded if they fell outside the range defined by the mean ± three standard deviations. Normalized data are presented as mean ± standard error of the mean (SEM) activity values plotted against concentration, along with dose–response curves for each compound and cell line (Figures 3 and 4). ED₅₀ values are summarized in Table 1 (benzopyrans) and Table 2 (quinolines) and expressed with 95% confidence interval (CI) per triplicate. ED₅₀ fits results and provides the interval in which the true ED₅₀ value resides with 95% confidence. The lower and upper (+/−) 95% confidence limits (CL) of this interval are calculated on the log scale as: CL = Log AC₅₀ ± margin of error (MOE). The MOE is defined as: SE Log ED₅₀ x T (0.975, f), where SE Log ED₅₀ is the standard error of the Log ED₅₀ estimate, T (1 − α/2, f) is the inverse cumulative t‐distribution with a significance level α of 5% (or a 95% confidence level) and degrees of freedom f.

Supporting Information

Additional supporting information can be found online in the Supporting Information Section. General instrumentation, synthesis of benzopyran and quinoline compounds, and cytotoxicity bioassays are detailed in the Supporting Information file.

Funding

This study was supported by Instituto de Salud Carlos III (Grant PI21/02045), and Conselleria de Cultura, Educación y Ciencia, Generalitat Valenciana (Grant AICO/2021/081).

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supplementary Material

Álvaro Bernabeu‐Sanchis, Mackenzie Thomas A., Ramos Maria C., García Ainhoa, Villarroel‐Vicentea Carlos, Vila Laura, Tormo José R., Cortes Diego, Cabedo Nuria, ChemMedChem 2026, 21, e202500904. 10.1002/cmdc.202500904