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. 2023 Feb 7;86(2):317–329. doi: 10.1021/acs.jnatprod.2c00924

Synthesis of Quinoline and Dihydroquinoline Embelin Derivatives as Cardioprotective Agents

Pedro Martín-Acosta †, Irene Cuadrado ‡, Laura González-Cofrade ‡, Roberto Pestano †, Sonsoles Hortelano §,*, Beatriz de las Heras ‡,*, Ana Estévez-Braun †,*
PMCID: PMC9972480  PMID: 36749898

Abstract

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A set of new dihydroquinoline embelin derivatives was obtained from the reaction of the natural benzoquinone embelin (1) with anilines and aromatic aldehydes in the presence of AgOTf. The synthesis of these compounds involves the formation of a Knoevenagel adduct, followed by nucleophilic addition of aniline and subsequent electrocyclic ring closure. The scope of the reaction regarding the aldehydes and anilines was determined. Quinoline derivatives were also obtained from the corresponding dihydroquinolines under oxidation with DDQ. The cardioprotective activity of the synthesized compounds was screened using a doxorubicin-induced cardiotoxicity model in H9c2 cardiomyocytes. Some structure–activity relationships were outlined, and the best activities were achieved with quinoline-embelin derivatives having a 4-nitrophenyl group attached at the pyridine ring. The obtained results indicated that embelin derivatives 4i, 6a, 6d, 6k, and 6m could have potential as cardioprotective agents, as they attenuated a DOX-induced cardiotoxicity effect acting on oxidative stress and apoptosis.

Quinolines and dihydroquinolines are important biologically active nitrogen-containing heterocycles found widespread in nature.1 These molecules present a wide range of applications, particularly in industry as sensors,2 agrochemicals,3 or luminescent materials,4 and in medicinal chemistry, where they play an important role in drug discovery. Many molecules that contain quinoline structures as a core structure or fused to other bioactive relevant scaffolds exhibit antimalarial,5,6 antibacterial,7 anticancer,8 anti-inflammatory,9 and antioxidant activity.10 Some representative examples are the antimalarial quinine,11 megistoquinones I and II with antibacterial activity,12 ciprofloxacin used for the treatment of respiratory and urinary infections,13 and lenvatinib, a tyrosine kinase inhibitor used as an effective antitumor agent.14 Another example of special interest is 4-azapodophyllotoxin, a potent inhibitor of tubulin polymerization,15 synthesized to overcome some therapeutic limitations of podophyllotoxins such as water solubility, metabolic inactivation, or toxicity (Figure 1).16−18

Figure 1.

Figure 1

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Representative bioactive compounds with quinoline and dihydroquinoline structures.

Quinolines can be obtained through diverse methodologies such as the Skraup,19 Doebner–von Miller,20 Friedleander,21 Gould–Jacobs,22 Pfitzinger,23 and Combes syntheses.24 However, efficient access to compounds that have quinoline-type structures fused to other biologically relevant scaffolds continues to represent a challenge from an academic and industrial perspective. In this sense, the three-component reaction of aromatic amines, aldehydes, and 1,3-dicarbonyl compounds represents an efficient method for the synthesis of these potentially bioactive heterocycles.25

Only a few methodologies are reported to access molecules with a quinone core fused to quinoline or dihydroquinoline heterocycles, all of which are limited to 2-hydroxy-1,4-naphthoquinone as starting material (Scheme 1). Shunjun et al. reported the first synthesis of this type of compounds fused to p-naphthoquinone fragments employing ionic liquids,26 while Singh et al. reported the first synthesis of quinoline derivatives incorporating an o-naphthoquinone moiety.27 Aiming to obtain 4-azapodophyllotoxin analogs, Wu’s group optimized a synthesis by condensation of 3,4-methylenedioxyaniline, aldehydes, and 2-hydroxy-1,4-naphthoquinone in the presence of l-proline.28 Pélinski et al. synthesized this type of compounds from 2-aminobenzyl alcohols and 2-hydroxy-1,4-naphthoquinone.29

Scheme 1. Synthetic Approaches toward Quinoline and Dihydroquinoline Fused to Naphthoquinones.

Scheme 1

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Embelin (1) is a natural benzoquinone isolated from Oxalis erythrorhiza.30 This compound and some derivatives have shown activity against multiple targets such as CK2,31,32 XIAP,33 α-glucosidase,34 B-RAF,35 or PKC.36 It acts as an inhibitor of neuroserpin polymerization37 and as a GPR84 agonist,38 modulates Akt/β-catenin39 and p38 MAPK,40 and blocks the NF-κβ signaling pathway.41,42 This plethora of protein targets and pathway modulations have attracted the interest of medicinal chemists.43−45

Our research group improved selectivity and potency of this natural product by increasing its complexity and structural diversification, using a domino Knoevenagel–Michael addition–intramolecular cyclization sequence developed by us.46,47 Aiming to extend the chemical diversity around nitrogen-containing heterocycles fused to this natural benzoquinone, we decided to carry out the synthesis of embelin derivatives fused to dihydroquinoline and quinoline structures.

Previous studies have reported the protective effects of embelin against myocardial injury.48−50 However, embelin has not been tested so far for its cardioprotective effects against anthracycline-induced toxicity. In this work we investigated whether cardiotoxicity induced by doxorubicin could be attenuated by embelin derivatives.

Results and Discussion

Chemistry

Initially, we carried out the reaction from embelin (1), 4-nitrobenzaldehyde (2a), and aniline (3a) in CHCl3, under reflux with pTSA (20 mol %) as a catalyst, obtaining product 4a in 39% yield (Table 1, entry 1). Next, we tried the use of microwave irradiation to improve the yields and reduce the reaction time. When the reaction mixture was irradiated at 120 °C for 1 h, the dihydropyridine adduct (4a) was obtained (41%) together with arylaminoembelin (5a) (14%), resulting in the direct nucleophilic attack of aniline (entry 2). Additionally, other reaction conditions were explored. Thus, an excess of aldehyde and aniline (1.5 equiv) led to an improvement in the yield of the process, obtaining 4a in a 63% yield together with 5a (18%) (entry 3). Other types of catalysts were used such as EDDA, Et3N, TFA, and Lewis acids (InCl3, FeCl3, BiCl3, AgOTf). Different solvents, reaction times, and temperatures were also evaluated. The results obtained are shown in Table 1.

Table 1. Improvement of Reaction Conditions with Embelin (1), 4-Nitrobenzaldehyde (2), and Aniline (3).

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          Yield (%)
Entry 1/2a/3 Catalyst Reaction conditions Time 4a 5a
1 1.0/1.0/1.0 20% pTSA CHCl3, reflux 48 h 39  
2 1.0/1.0/1.0 20% pTSA CHCl3, MW, 120 °C 1 h 41 14
3 1.0/1.5/1.5 20% pTSA CHCl3, MW, 120 °C 1 h 63 18
4 1.0/1.5/1.5 20% pTSA CHCl3, MW, 150 °C 30 min 49 25
5 1.0/1.5/1.5 20% pTSA DCE, MW, 120 °C 1 h 33 18
6 1.0/1.5/1.5 20% pTSA EtOH, MW, 120 °C 30 min 56 31
7 1.0/1.5/1.5 20% pTSA EtOH, MW, 150 °C 20 min 66 20
8 1.0/1.5/1.5 20% TFA EtOH, MW, 150 °C 15 min 43 27
9 1.0/1.5/1.5 20% EDDA DCE, MW, 120 °C 15 min    
10 1.0/1.5/1.5 20% Et3N EtOH, MW, 150 °C 15 min 28  
11 1.0/1.5/1.5 20% InCl3 EtOH, MW, 150 °C 15 min 57 24
12 1.0/1.5/1.5 20% FeCl3 EtOH, MW, 150 °C 15 min 31 14
13 1.0/1.5/1.5 20% BiCl3 EtOH, MW, 150 °C 15 min 43 21
14 1.0/1.5/1.5 20% AgOTf EtOH, MW, 150 °C 15 min 80 13
15 1.0/1.5/1.5 10% AgOTf EtOH, MW, 150 °C 15 min 64 11
16 1.0/1.5/1.5 20% AgOTf H2O, MW, 150 °C 15 min 46 6
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Use of polar and more environmentally friendly solvents, such as EtOH instead of CHCl3, as well as higher reaction temperatures (150 °C, entry 7), afforded product 4a in a 66% yield in 20 min. Different Lewis acids such as InCl3, FeCl3, BiCl3, and AgOTf were evaluated, obtaining the best yields with AgOTf (20% mol) as catalyst in EtOH after 15 min of reaction at 150 °C (entry 14). Under these reaction conditions, 4a was obtained in 80% yield together with 5a (13%). At this point, and since the arylamino embelin sideproduct could be of interest for their biological potential, the reaction conditions shown in entry 14 were selected.

The improved reaction conditions were used to evaluate the scope of the reaction with respect to the aldehydes and aromatic amines. The use of substituted aromatic aldehydes with electron-withdrawing groups such 4-nitro (4a, 80%) or 4-methyl ester (4g, 81%) led to the best yields. Moderate to good yields (62–74%) were obtained when introducing halogens in the aromatic ring (4c–4f). Good results were also obtained with the use of heteroaromatic aldehydes such as 3-thiophenecarboxaldehyde (4j, 83%). On the contrary, the introduction of electron-donating groups like 3,4-dimethyl or dimethoxy substituents (4i and 4j) decreased the yields (52% and 56%, respectively). The formation of the corresponding products was not observed when aliphatic aldehydes were used.

We also evaluated the scope with different substituted aromatic and heteroaromatic amines, finding, as expected, a behavior opposite to that observed with the aldehydes. The use of anilines substituted with electron-donating groups such as 4-methoxyaniline (3b) (entry 11) and 3,5-dimethylaniline (3c) (entry 12) led to the corresponding dihydropyridines 4k and 4l in 80% and 92% yield, respectively. A moderate yield (67%) was obtained with 4-bromoaniline (3d) (entry 13), while the use of aromatic anilines substituted with an electron-withdrawing group (3-nitroaniline) (3e) did not afford the corresponding dihydropyridin derivative, and only the 3-nitrophenylembeline derivative (5d) was quantitatively formed.

Table 2. Scope of the Aldehydes and Anilines.

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Entry R1 R2 Yield (%)
1 4-NO2-Ph H 4a (80%) + 5a (13%)
2 Ph H 4b (70%) + 5a (17%)
3 4-Cl-Ph H 4c (74%) + 5a (11%)
4 4-Br-Ph H 4d (62%) + 5a (15%)
5 4-F-Ph H 4e (72%) + 5a (10%)
6 3-F-Ph H 4f (72%) + 5a (12%)
7 4-COOCH3 H 4g (81%) + 5a (14%)
8 3-thienyl H 4h (83%) + 5a (10%)
9 3,4-(CH3)2-Ph H 4i (52%) + 5a (21%)
10 3,4-(OCH3)2-Ph H 4j (56%) + 5a (25%)
11 4-NO2-Ph 4-OCH3 4k (80%) + 5b (14%)
12 4-NO2-Ph 3,5-(CH3)2 4l (92%)
13 4-NO2-Ph 4-Br 4m (67%) + 5c (29%)
14 4-NO2-Ph 3-NO2 4n (0%) + 5d (100%)
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Regarding the reaction mechanism, different mechanistic proposals are found in the literature for similar types of transformations. Some of them involve either the direct nucleophilic attack of the carbon next to the amino group51−53 or the nucleophilic attack of the amino group to a previously generated Michael adduct and subsequent electrocyclic ring closure.27,54 Recently, Sun et al. demonstrated the formation of dimeric species between the aldehyde and the corresponding 1,3-dicarbonyl compounds as reaction intermediates.55 This dimers evolve through the nucleophilic attack of the aromatic amine, subsequent hydrolysis, and electrocyclic ring closure.55

The structure of product 4a was corroborated by the key correlations observed in the HMBC spectrum and the NOE effects observed in selective NOE experiments (Figure 2).

Figure 2.

Figure 2

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Key HMBC and ROESY correlations for compound 4a.

Quinoline derivatives were obtained from the dehydrogenation of the corresponding dihydroquinoline derivatives. A wide range of oxidizing reagents was used (MnO2, NBS, Br2, p-chloroanil, and DDQ), of which DDQ turned out to be the most suitable not only for the short reaction times but also for the easy isolation of the corresponding reaction products. All the dihydroquinoline derivatives previously synthesized (4a–4m) were treated with DDQ, affording the corresponding quinoline derivatives (6a–6m) in moderate to good yields (55–88%). Results are depicted in Table 3.

Table 3. Synthesis of Pyridine Embelin Derivatives.

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Cardioprotective Effects

Doxorubicin, which possesses an anthracycline core, is frequently used to treat various types of cancers, including hematological neoplasia and solid malignancies. However, its cardiac side effects, leading to cardiomyopathy and congestive heart failure, have limited the clinical use of this potent anticancer drug.56 Therefore, more effective therapeutic agents are needed considering previous reports of embelin against myocardial injury,48−50 and since so far protective effects of embelin and embelin derivatives against anthracycline-induced cardiotoxicity have not been tested,57 in the present study, we aimed to determine whether cardiotoxicity induced by chemotherapy could be attenuated by embelin derivatives when administrated in conjunction with chemotherapeutical agents.

The potential cytotoxicity of embelin (1) together with derivatives (4a–4m, 5a–5d, 6a–6m) was tested in cardiomyocytes and macrophages in order to ensure that compounds lack toxicity also in immune cells. Cell viability was determined by the MTT assay.58 Most of the compounds exhibited cytotoxicity when incubated with cells for 24 h, as shown in Table 4. Four dihydroquinoline derivatives (4b, 4f, 4i, and 4m) and six quinoline compounds (6a, 6d, 6e, 6i, 6k, and 6m) maintained higher viable cell rates in both types of cells. IC50 values of these derivatives were higher than 40 μM and were selected for further analysis.

Table 4. Effects of Embelin (1) and Nitrogenated Embelin Derivatives (4a–4m, 5a–5d, 6a–6m) on Cell Viabilities in J774 Macrophages and H9c2 Cardiomyocytesa.

  Cell viability (%)
  J774 macrophages
 H9c2 cardiomyocytes
Compound 10 μM 20 μM 10 μM 20 μM
1 21.3 ± 2.7e 15.3 ± 0.8e 36.9 ± 5.8e 8.3 ± 0.1e
4a 50.0 ± 5.6e 43.7 ± 2.0e 94.8 ± 3.5 89.4 ± 0.6b
4b 99.7 ± 1.1 98.7 ± 1.2 98.1 ± 6.8 95.8 ± 7.7
4c 49.1 ± 3.4e 39.5 ± 3.6e 98.9 ± 3.4 93.9 ± 2.3
4d 38.5 ± 5.9e 32.9 ± 3.4e 99.5 ± 2.7 98.9 ± 6.1
4e 57.3 ± 3.8e 44.6 ± 3.3e 93.4 ± 7.2 83.7 ± 1.5e
4f 99.5 ± 1.3 97.8 ± 0.6 88.7 ± 5.3b 81.1 ± 2.9e
4g 31.2 ± 5.3e 29.6 ± 3.9e 72.6 ± 1.1e 62.1 ± 1.3e
4h 84.9 ± 2.6e 51.4 ± 2.9e 92.5 ± 6.6 75.8 ± 2.3e
4i 96.2 ± 6.8 72.6 ± 1.7e 84.3 ± 6.8e 82.2 ± 6.2e
4j 89.1 ± 5.5d 36.9 ± 3.8e 68.9 ± 3.6e 60.6 ± 4.9e
4k 55.6 ± 6.7e 37.8 ± 4.8e 99.7 ± 1.1 90.7 ± 1.5
4l 46.7 ± 6.6e 33.1 ± 4.5e 98.2 ± 6.6 88.3 ± 6.1b
4m 94.7 ± 4.2 77.2 ± 4.9e 96.5 ± 5.5 96.2 ± 4.8
5a 56.9 ± 4.0e 56.2 ± 2.0e 99.0 ± 7.1 83.0 ± 7.0e
5b 68.4 ± 3.8e 48.3 ± 1.7e 89.3 ± 9.2b 87.4 ± 6.7c
5c 56.9 ± 5.5e 39.4 ± 5.3e 61.9 ± 2.7e 56.1 ± 3.4e
5d 65.5 ± 1.9e 50.8 ± 7.0e 67.8 ± 2.1e 57.5 ± 1.4e
6a 96.3 ± 2.1 76.9 ± 2.8e 97.6 ± 7.0 87.8 ± 6.9c
6b 81.8 ± 4.7e 63.9 ± 6.6e 99.7 ± 2.1 68.1 ± 4.6e
6c 83.4 ± 0.5e 55.8 ± 5.7e 95.0 ± 8.4 74.3 ± 4.8e
6d 82.1 ± 3.7e 74.5 ± 3.9e 99.4 ± 0.2 99.3 ± 2.6
6e 93.1 ± 4.5 76.6 ± 5.9e 96.7 ± 8.1 93.9 ± 0.9
6f 79.3 ± 4.6e 62.2 ± 4.3e 98.3 ± 4.9 84.5 ± 4.3d
6g 78.4 ± 5.7e 42.2 ± 0.3e 88.7 ± 10.4b 70.6 ± 3.0e
6h 73.5 ± 2.8e 56.7 ± 3.3e 75.3 ± 5.0e 71.1 ± 2.8e
6i 93.3 ± 4.3 75.1 ± 2.9e 99.8 ± 3.0 92.6 ± 5.4
6j 85.2 ± 3.7e 64.0 ± 2.6e 85.5 ± 6.2d 68.4 ± 8.6e
6k 81.9 ± 4.6e 76.3 ± 5.3e 99.2 ± 0.8 94.4 ± 6.3
6l 82.3 ± 0.3e 63.8 ± 4.8e 64.8 ± 6.7e 55.9 ± 9.4e
6m 99.6 ± 4.6 86.4 ± 8.7e 89.8 ± 7.6b 85.9 ± 8.2d
control 100 100 100 100
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a

Results are expressed as mean ± SD. n = 3.

b

p < 0.05.

c

p < 0.01.

d

p < 0.001.

e

p < 0.0001 vs control (untreatred) cells.

To evaluate the cardioprotective potential of selected compounds, H9c2 cardiomyocytes were exposed to compounds (20 μM) and doxorubicin (1 μM) for 24 h. A significant loss in cell viability was observed in DOX-treated cells versus untreated cells (IC50 = 0.83 μM). Co-treatment with DOX and embelin derivatives significantly attenuated DOX-induced cardiotoxicity. The most prominent cardioprotective effects were observed with compounds 4i, 6a, 6d, 6e, 6k, and 6m, as they significantly increased cell viability reduced by DOX treatment over 80% (81.6 ± 3.7%, 87.5 ± 4.3%, 83.4 ± 5.5%, 81.4 ± 5.9%, 84.3 ± 1.3%, and 91.2 ± 5.9%, respectively) (Figure 3). Derivatives with a quinoline core were more active than the corresponding dihydroquinoline (i.e., 4m vs 6m). Regarding the type of substituent at the aromatic ring attached at the pyridine core, the best activities were achieved with electron-withdrawing substituents in the para position such as nitro, fluoro, or bromo. The presence of a bromo at the aromatic ring fused to the pyridine led to an improved activity (i.e., 6a vs 6m).

Figure 3.

Figure 3

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Protective effects of embelin derivatives on DOX-induced cardiotoxicity in H9c2 cells. H9c2 cells were co-treated with DOX (1 μM) and compounds 4b, 4f, 4i, 4m, 6a, 6d, 6e, 6i, 6k, and 6m (20 μM) for 24 h. Cell viability was measured by MTT assay. Data are expressed as the mean ± SD (n = 3). *p < 0.05, ***p < 0.001, and ****p < 0.0001 vs DOX-treated cells and ####p < 0.0001 vs untreated cells.

Next, cytotoxicity of the selected compounds was also tested in MCF-7 breast cancer cells to disconfirm that protective effects might antagonize the antitumor effects of doxorubicin. As shown in Figure 4, all compounds, except derivative 6e, did not affect the antitumor activity of DOX.

Figure 4.

Figure 4

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Effects of derivatives 4i, 6a, 6d, 6e, 6k, and 6m on the chemotherapeutic activity of DOX in MCF-7 human breast tumor cells. Cells were co-treated with selected compounds at 20 μM and DOX (1 μM) for 24 h. Cell viability was measured by MTT assay. Results are reported as mean of cell viability ± SD (n = 3). ****p < 0.0001 vs DOX-treated cells.

Further investigations were focused on the molecular mechanisms of the cardioprotective properties of derivatives 4i, 6a, 6d, 6k, and 6m. Multiple pathways have been proposed for the cardiotoxic effects of DOX. Among the reported mechanisms, DOX-induced cardiotoxicity is said to be driven by increased oxidative stress and aggravated apoptosis.59

Oxidative stress is produced by an imbalance between radical oxygen species (ROS) formation and endogenous antioxidant activation in reaction to cell injury, leading to myocardial toxicity. Accordingly, DOX-induced ROS production in the H9c2 cells was quantified with the DCFH-DA fluorogenic dye. DOX significantly increased the levels of ROS compared to untreated cells (Figure 5). No effects on ROS production were observed when compounds were incubated alone with cells. Co-treatment with DOX plus embelin derivatives 4i, 6a, 6d, 6k, and 6m significantly inhibited ROS production, reducing Dox-induced oxidative stress. The inhibitory effects of embelin derivatives were similar to those induced by N-acetyl-cysteine (NAC), a well-known antioxidant.

Figure 5.

Figure 5

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Derivatives 4i, 6a, 6d, 6k, and 6m reduced ROS levels produced by DOX in H9c2 cells. Levels of ROS in H9c2 cells were measured fluorometrically after treatment with DOX (1 μM) alone or co-treatment with N-acetyl cysteine (NAC, 1 mM) or selected derivatives (20 μM) for 24 h, using the 2′,7′-dichlorfluorescein-diacetate (DCFH-DA) assay. Values are expressed as mean ± SD with respect to DOX-treated cells (n = 3). ****p < 0.0001 vs DOX-treated cells.

The phosphatidylinositol-3-kinase (PI3K/Akt) and mitogen-activated protein kinase (MAPK) signaling pathways play important roles in regulating cell survival. Oxidative stress is involved in the activation of MAPK signaling pathways that leads to DOX-induced cardiomyocyte apoptosis, via phosphorylation of extracellular signal-regulated kinase (ERK).60 In contrast, activation of PI3K/Akt has been described to be an important mechanism for cardioprotection.61 In order to further understand the cardioprotective mechanism of these five derivatives, we investigated the expression of survival and apoptosis-related proteins (mainly Bcl-2 family genes) following DOX treatment in H9c2 cells.

As shown in Figure 6, all active derivatives suppressed ERK phosphorylation induced by DOX, as determined by Western blot analysis. Moreover, co-treatment with DOX and embelin derivatives resulted in a significant activation of Akt. Finally, our results from Western blot also revealed that in the presence of embelin derivatives pro-apoptotic Bax protein expression was decreased while the expression of anti-apoptotic protein Bcl-2 was increased. Collectively, these results indicate that cell damage induced by DOX was reversed after treatment with embelin derivatives, supporting the cardioprotective effects of these compounds.

Figure 6.

Figure 6

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Analysis of survival and apoptotic protein expression in H9c2 cells incubated with derivatives in combination with DOX. Western blot analysis of pAkt, pERK, Bcl2, and Bax protein expression in cells following exposure to DOX (1 μM) alone or in combination with derivatives (20 μM). β-Actin was immunoblotted as a loading control. Densitometric analysis of the relative expression of pERK, pAkt, Bcl2, and Bax. Data are presented as mean ± SD (n = 3). *p < 0.05, **p < 0.01 vs DOX-treated cells.

In conclusion, a set of dihydroquinoline embelin derivatives was synthesized by reaction between embeline (1), aromatic aldehydes, and anilines. With improved reaction conditions in hand, the substrate scope of the reaction with respect to aldehyde and aniline was evaluated. Dihydroquinolines treated with DDQ gave the corresponding quinoline derivatives in high yields. Due to previous reports of embelin against myocardial injury, the cardioprotective effects of the synthesized compounds were evaluated. The results indicated that the presence of a quinoline moiety substituted by electron-withdrawing groups seems to play an important role for the activity. Five embelin derivatives, 4i, 6a, 6d, 6k, and 6m, could attenuate DOX-induced cardiotoxicity without reducing DOX’s chemotherapeutic effect. Our findings also indicate that these derivatives reduce oxidative stress induced by DOX and may have a potential benefit in preventing cardiotoxicity by doxorubicin.

Experimental Section

General Experimental Procedures

IR spectra were obtained using a Cary 630 FTIR spectrometer. UV spectra were obtained in absolute EtOH on a Thermo Scientific Genesys 180 spectrophotometer. NMR spectra were recorded on a Bruker Avance 500 or Bruker Avance 600 in CDCl3 or DMSO at 500 or 600 MHz for 1H NMR and 125 or 150 MHz for 13C NMR. Chemical shifts are given in (δ) parts per million, and coupling constants (J) in hertz (Hz). 1H and 13C spectra were referenced using the solvent signal as internal standard. Melting points were taken on a capillary melting point apparatus and are uncorrected. EIMS and HREIMS were recorded on a Micromass Autospec spectrometer. HREIMS were recorded using a high-resolution magnetic trisector (EBE) mass analyzer. Analytical thin-layer chromatography plates (Polygram-Sil G/UV254) were used. Preparative thin-layer chromatography was carried out with Analtech silica gel GF plates (20 × 20 cm, 1000 μm) using appropriate mixtures of EtOAc and hexanes. All solvents and reagents were purified by standard techniques reported62 or used as supplied from commercial sources. All compounds were named using the ACD40 Name-Pro program, which is based on IUPAC rules. The embelin (1) used in the reactions was obtained from Oxalis erythrorhiza Gillies ex Hook. & Arn. following the procedure described in ref (30).

General Procedure for the Synthesis of 9,10-Dihydroacridine-1,4-dione Derivatives

In a MW vial equipped with a magnetic stir bar, 20 mg of embelin (0.068 mmol), 1.5 equiv of aldehyde, 1.5 equiv of aniline, and 20 mol % of AgOTf as catalyst were dissolved in 2 mL of EtOH. The MW tube was sealed, and the reaction mixture irradiated at 150 °C for 15 min. The solvent was removed under reduced pressure, and the reaction mixtures were purified by preparative-TLC.

2-Hydroxy-9-(4-nitrophenyl)-3-undecyl-9,10-dihydroacridine-1,4-dione (4a)

The reaction mixture purified by preparative TLC with 20% n-Hex/AcOEt yielded 27.4 mg (80%) of 4a as an amorphous violet solid and 3.2 mg of 5a (13%) as a red solid. Mp 164–166 °C; UV (EtOH) λmax 290, 340 nm; IR (film) νmax 3317, 2963, 1572, 1513, 1442, 1223, 1081, 798 cm–1; 1H NMR (600 MHz, DMSO-d6) δ 0.84 (t, J = 7.1 Hz, 3H), 1.22 (bs, 16H), 1.37 (m, 2H), 2.29 (t, J = 7.6 Hz, 2H), 5.46 (s, 1H), 6.98 (td, J = 0.9, 7.5 Hz, 1H), 7.18 (m, 2H), 7.48 (d, J = 8.3 Hz, 1H), 7.52 (d, J = 8.8 Hz, 2H), 8.12 (d, J = 8.8 Hz, 2H), 10.16 (s, 1H); 13C NMR (125 MHz, CDCl3) δ 14.1 (CH3), 22.5 (CH2), 22.7 (CH2), 28.1 (CH2), 29.3 (CH2), 29.4 (CH2), 29.5 (CH2), 29.6 (CH2 × 2), 29.7 (CH2), 31.9 (CH2), 40.7 (CH), 105.6 (C), 115.1 (C), 116.6 (C), 117.3 (CH), 123.9 (CH × 2), 125.9 (CH), 128.6 (CH), 128.8 (CH × 2), 130.5 (CH), 133.9 (C), 139.3 (C), 146.7 (C), 152.9 (C), 153.9 (C), 178.4 (C), 181.7 (C); EIMS m/z (%) 502 ([M+], 97), 380 (100), 361 (31), 240 (12); HRMS 502.2644 (calcd for C30H34N2O5 [M+] 502.2468).

2-Hydroxy-5-(phenylamino)-3-undecylcyclohexa-2,5-diene-1,4-dione (5a)

1H NMR (500 MHz, CDCl3) δ 0.88 (t, J = 7.1 Hz, 3H), 1.26 (bs, 16H), 1.48 (m, 2H), 2.45 (t, J = 7.8 Hz, 2H), 6.00 (s, 1H), 7.24 (m, 3H), 7.42 (t, J = 7.6 Hz, 2H), 7.96 (bs, 1H); 13C NMR (125 MHz, CDCl3) δ 14.1 (CH3), 22.7 (CH2), 22.8 (CH2), 28.1 (CH2), 29.3 (CH2), 29.4 (CH2), 29.5 (CH2 × 2), 29.6 (CH2), 29.7 (CH2), 31.9 (CH2), 94.5 (CH), 116.3 (C), 122.8 (CH × 2), 126.3 (CH), 129.7 (CH × 2), 136.9 (C), 146.0 (C), 154.3 (C), 180.4 (C), 182.9 (C); EIMS m/z (%) 369 ([M+], 100), 229 (33), 228 (46), 200 (14); HREIMS 369.2298 (calcd for C23H31NO3 [M+] 369.2304).

2-Hydroxy-9-phenyl-3-undecyl-9,10-dihydroacridine-1,4-dione (4b)

The reaction mixture purified by PTLC with 20% n-Hex/AcOEt yielded 22.1 mg (70%) of 4b as an amorphous violet solid (mp 161–162 °C) and 4.2 mg of 5a (17%) as a red solid. UV (EtOH) λmax 288, 322 nm; IR (film) νmax 3310, 2926, 2363, 1599, 1442, 1222, 798 cm–1; 1H NMR (500 MHz, CDCl3) δ 0.87 (t, J = 7.0 Hz, 3H), 1.25 (bs, 16H), 1.45 (m, 2H), 2.39 (t, J = 8.3 Hz, 2H), 5.33 (s, 1H), 6.98 (d, J = 8.0 Hz, 2H), 7.02 (t, J = 7.4 Hz, 1H), 7.11 (d, J = 7.6 Hz, 1H), 7.17 (m, 2H), 7.25 (m, 4H), 7.83 (bs, 1H); 13C NMR (125 MHz, CDCl3) δ 14.1 (CH3), 22.5 (CH2), 22.7 (CH2), 28.1 (CH2), 29.3 (CH2), 29.4 (CH2), 29.5 (CH2), 29.6 (CH2 × 2), 29.7 (CH2), 31.9 (CH2), 40.6 (CH), 107.0 (C), 115.9 (C), 116.9 (CH), 125.3 (C), 125.5 (CH), 126.7 (CH), 127.8 (CH × 4), 128.6 (CH), 130.9 (CH), 133.9 (C), 139.1 (C), 146.4 (C), 154.0 (C), 178.5 (C), 182.2 (C); EIMS m/z (%) 475 ([M+], 90), 381 (69), 380 (100), 316 (35), 240 (16); HREIMS 475.2612 (calcd for C30H34NO3F [M+] 475.2617).

2-Hydroxy-9-(4-chlorophenyl)-3-undecyl-9,10-dihydroacridine-1,4-dione (4c)

The reaction mixture purified by PTLC with 20% n-Hex/AcOEt yielded 24.8 mg (74%) of 4c as an amorphous violet solid and 2.8 mg of 5a (11%) as a red solid. Mp 139–141 °C; UV (EtOH) λmax 287, 380 nm; IR (film) νmax 3317, 3239, 2963, 2918, 2363, 1643, 1572, 1223, 798 cm–1; 1H NMR (500 MHz, CDCl3) δ 0.87 (t, J = 7.0 Hz, 3H), 1.25 (bs, 16H), 1.44 (m, 2H), 2.40 (t, J = 7.7 Hz, 2H), 5.30 (s, 1H), 5.31 (bs, 1H), 5.25 (s, 1H), 6.99 (d, J = 8.1 Hz, 1H), 7.03 (t, J = 7.6 Hz, 1H), 7.08 (d, J = 6.9 Hz, 2H), 7.17 (d, J = 8.5 Hz, 2H), 7.20 (m, 3H), 7.84 (bs, 1H); 13C NMR (125 MHz, CDCl3) δ 14.1 (CH3), 22.4 (CH2), 22.7 (CH2), 28.1 (CH2), 29.3 (CH2), 29.4 (CH2), 29.5 (CH2), 29.6 (CH2 × 2), 29.7 (CH2), 31.9 (CH2), 40.1 (CH), 106.5 (C), 116.1 (C), 116.9 (CH), 124.8 (C), 125.7 (CH), 128.1 (CH), 128.7 (CH × 2), 129.2 (CH × 2), 130.6 (CH), 132.6 (C), 133.9 (C), 139.1 (C), 144.9 (C), 153.9 (C), 178.5 (C), 182.0 (C); EIMS m/z (%) 591 ([M+], 64), 380 (100), 349 (22), 238 (20); HREIMS 493.2211 (calcd for C30H34NO337Cl [M+] 493.2198), 491.2207 (calcd for C30H34NO335Cl [M+] 491.2227).

2-Hydroxy-9-(4-bromophenyl)-3-undecyl-9,10-dihydroacridine-1,4-dione (4d)

The reaction mixture purified by PTLC with 20% n-Hex/AcOEt yielded 22.6 mg (62%) of 4d as an amorphous violet solid and 3.7 mg of 5a (15%) as a red solid. Mp 139–140 °C; UV (EtOH) λmax 290, 295, 325, 339 nm; IR (film) νmax 3317, 3239, 2963, 2918, 2851, 2363, 1643, 1573, 1442, 1222, 1080, 798 cm–1; 1H NMR (600 MHz, DMSO-d6) δ 0.84 (t, J = 6.9 Hz, 3H), 1.22 (bs, 16H), 1.37 (m, 2H), 2.30 (t, J = 7.8 Hz, 2H), 5.27 (s, 1H), 6.98 (t, J = 7.2 Hz, 1H), 7.16 (m, 2H), 7.18 (d, J = 8.4 Hz, 2H), 7.42 (d, J = 8.4 Hz, 2H), 7.45 (d, J = 8.1 Hz, 2H), 10.09 (s, 1H); 13C NMR (125 MHz, CDCl3) δ 13.92 (CH3), 21.83 (CH2), 22.05 (CH2), 27.54 (CH2), 28.68 (CH2), 28.79 (CH2), 28.89 (CH2 × 2), 28.98 (CH2), 29.00 (CH2), 31.25 (CH2), 39.22 (CH), 105.81 (C), 116.00 (C), 117.78 (CH), 119.29 (C), 124.48 (C), 124.50 (CH), 127.54 (CH), 129.48 (CH × 2), 129.71 (CH), 131.22 (CH × 2), 134.83 (C), 139.35 (C), 146.73 (C), 155.61 (C), 178.35 (C), 182.05 (C); EIMS m/z (%) 535 ([M+], 49), 393 (11), 380 (31), 380 (100); HRMS 537.1682 (calcd for C30H34NO381Br [M+] 537.1702), 535.1725 (calcd for C30H34NO379Br [M+] 535.1722).

2-Hydroxy-9-(4-fluorophenyl)-3-undecyl-9,10-dihydroacridine-1,4-dione (4e)

The reaction mixture purified by PTLC with 20% n-Hex/AcOEt yielded 23.2 mg (72%) of 4e as an amorphous violet solid and 1.6 mg of 5a (10%) as a red solid. Mp 140–142 °C; UV (EtOH) λmax 289, 294, 340 nm; IR (film) νmax 3317, 3239, 2963, 2918, 2851, 2363, 1643, 1573, 1442, 1222, 798 cm–1; 1H NMR (500 MHz, CDCl3) δ 0.87 (t, J = 7.0 Hz, 3H), 1.24 (bs, 16H), 1.44 (m, 2H), 2.39 (t, J = 7.7 Hz, 2H), 5.32 (s, 1H), 6.92 (t, J = 8.8 Hz, 2H), 6.99 (d, J = 7.8 Hz, 1H), 7.03 (t, J = 6.7 Hz, 1H), 7.09 (d, J = 7.8 Hz, 1H), 7.20 (m, 3H), 7.85 (bs, 1H); 13C NMR (125 MHz, CDCl3) δ 14.1 (CH3), 22.5 (CH2), 22.7 (CH2), 28.1 (CH2), 29.3 (CH2), 29.4 (CH2), 29.5 (CH2), 29.6 (CH2 × 2), 29.7 (CH2), 31.9 (CH2), 39.9 (CH), 106.8 (C), 115.4 (CH × 2, JC–F = 21.5 Hz), 116.0 (C), 116.9 (CH), 125.1 (C), 125.6 (CH), 127.9 (CH), 129.4 (CH × 2, JC–F = 7.5 Hz), 130.5 (CH), 133.9 (C), 139.0 (C), 154.0 (C), 160.6 (C), 162.5 (C), 178.5 (C), 182.1 (C); EIMS m/z (%) 475 ([M+], 89), 381 (30), 380 (100), 334 (23); HREIMS 475.2513 (calcd for C30H34NO3F [M+] 475.2523).

2-Hydroxy-9-(3-fluorophenyl)-3-undecyl-9,10-dihydroacridine-1,4-dione (4f)

The reaction mixture purified by PTLC with 20% n-Hex/AcOEt yielded 23.1 mg (72%) of 4f as an amorphous violet solid and 3.0 mg of 5a (12%) as a red solid. Mp 240–242 °C; UV (EtOH) λmax 288, 294, 337, 392 nm; IR (film) νmax 3317, 3239, 2963, 2918, 2851, 2363, 1643, 1572, 1513, 1442, 1223, 1080, 798 cm–1; 1H NMR (500 MHz, CDCl3) δ 0.87 (t, J = 6.9 Hz, 3H), 1.24 (bs, 16H), 1.45 (m, 2H), 2.40 (t, J = 7.9 Hz, 2H), 5.34 (s, 1H), 6.85 (td, J = 1.7, 8.2 Hz, 1H), 6.91 (dt, J = 2.1, 9.8 Hz, 1H), 7.00 (d, J = 7.9 Hz, 2H), 7.05 (m, 2H), 7.11 (d, J = 7.1 Hz, 1H), 7.21 (m, 2H), 7.85 (bs, 1H); 13C NMR (125 MHz, CDCl3) δ 14.1 (CH3), 22.5 (CH2), 22.7 (CH2), 28.1 (CH2), 29.3 (CH2), 29.4 (CH2), 29.5 (CH2), 29.6 (CH2 × 2), 29.7 (CH2), 31.9 (CH2), 40.4 (CH), 106.4 (C), 113.7 (CH, JC–F = 21.2 Hz), 114.9 (CH, JC–F = 21.7 Hz), 116.1 (C), 117.0 (CH), 123.4 (CH, JC–F = 2.6 Hz), 124.6 (C), 125.7 (CH), 128.1 (CH), 129.9 (CH, JC–F = 8.1 Hz), 130.4 (CH), 133.9 (C), 139.2 (C), 148.7 (C, JC–F = 6.3 Hz), 153.9 (C), 163.1 (C, JC–F = 245.6 Hz), 178.4 (C), 182.0 (C); EIMS m/z (%) 475 ([M+], 60), 381 (23), 380 (100), 334 (23), 240 (11); HREIMS 475.2519 (calcd for C30H34NO3F [M+] 475.2523).

Methyl 4-(2-hydroxy-1,4-dioxo-3-undecyl-1,4,9,10-tetrahydroacridin-9-yl)benzoate (4g)

The reaction mixture purified by PTLC with 20% n-Hex/AcOEt yielded 28.2 mg (81%) of 4g as an amorphous violet solid and 3.6 mg of 5a (14%) as a red solid. Mp 146–148 °C; UV (EtOH) λmax 286, 293, 340, 378 nm; IR (film) νmax 3317, 3239, 2963, 2918, 2851, 2363, 1644, 1573, 1513, 1442, 1223, 1081, 798 cm–1; 1H NMR (500 MHz, CDCl3) δ 0.87 (t, J = 7.1 Hz, 3H), 1.25 (bs, 16H), 1.45 (m, 2H), 2.40 (t, J = 7.8 Hz, 2H), 3.86 (s, 3H), 5.39 (s, 1H), 7.00 (d, J = 8.1 Hz, 1H), 7.04 (dd, J = 0.8, 7.5 Hz, 1H), 7.08 (d, J = 6.7 Hz, 1H), 7.20 (td, J = 1.3, 8.0 Hz, 1H), 7.32 (d, J = 8.3 Hz, 2H), 7.88 (bs, 1H), 7.92 (d, J = 8.2 Hz, 2H); 13C NMR (125 MHz, CDCl3) δ 14.1 (CH3), 22.5 (CH2), 22.7 (CH2), 28.1 (CH2), 29.3 (CH2), 29.4 (CH2), 29.5 (CH2), 29.6 (CH2 × 2), 29.7 (CH2), 31.9 (CH2), 40.7 (CH), 52.1 (CH3), 106.2 (C), 116.1 (C), 117.1 (CH), 122.8 (C), 124.5 (C), 125.7 (CH), 127.9 (CH × 2), 128.2 (CH), 130.0 (CH × 2), 130.5 (CH), 133.9 (C), 139.2 (C), 151.1 (C), 153.9 (C), 166.8 (C), 178.4 (C), 181.9 (C); EIMS m/z (%) 515 ([M+], 90), 381 (39), 380 (100), 374 (22), 240 (10); HREIMS 515.2672 (calcd for C32H37NO5 [M+] 515.2672).

2-Hydroxy-9-(thiophen-3-yl)-3-undecyl-9,10-dihydroacridine-1,4-dione (4h)

The reaction mixture purified by PTLC with 20% n-Hex/AcOEt yielded 26.1 mg (83%) of 4h as an amorphous violet solid and 2.6 mg of 5a (10%) as a red solid. Mp 190–192 °C; UV (EtOH) λmax 290, 295, 360 nm; IR (film) νmax 3317, 3239, 2963, 2918, 2851, 2363, 1644, 1573, 1442, 1223, 1080, 798 cm–1; 1H NMR (500 MHz, CDCl3) δ 0.87 (t, J = 7.1 Hz, 3H), 1.25 (bs, 16H), 1.44 (m, 2H), 2.39 (t, J = 7.7 Hz, 2H), 5.47 (s, 1H), 6.90 (m, 2H), 6.99 (d, J = 7.8 Hz, 1H), 7.08 (t, J = 7.4 Hz, 1H), 7.20 (m, 3H), 7.88 (bs, 1H); 13C NMR (125 MHz, CDCl3) δ 14.1 (CH3), 22.5 (CH2), 22.7 (CH2), 28.1 (CH2), 29.3 (CH2), 29.4 (CH2), 29.5 (CH2), 29.6 (CH2 × 2), 29.7 (CH2), 31.9 (CH2), 35.5 (CH), 106.4 (C), 115.9 (C), 116.8 (CH), 121.2 (CH), 124.6 (C), 125.5 (CH), 125.8 (CH), 127.3 (CH), 127.9 (CH), 130.3 (CH), 134.2 (C), 139.2 (C), 146.3 (C), 154.0 (C), 178.5 (C), 182.2 (C); EIMS m/z (%) 463 ([M+], 100), 381 (23), 380 (84), 322 (43), 238 (29); HREIMS 463.2162 (calcd for C28H33NO3S [M+] 463.2181).

2-Hydroxy-9-(3,4-dimethylphenyl)-3-undecyl-9,10-dihydroacridine-1,4-dione (4i)

The reaction mixture purified by PTLC with 20% n-Hex/AcOEt yielded 17.3 mg (52%) of 4i as an amorphous violet solid and 5.2 mg of 5a (21%) as a red solid. Mp 106–108 °C; UV (EtOH) λmax 286, 294, 342, 365 nm; IR (film) νmax 3317, 3250, 2922, 2855, 1643, 1595, 1446, 1223, 1084, 767 cm–1; 1H NMR (500 MHz, CDCl3) δ 0.87 (t, J = 7.2 Hz, 3H), 1.25 (bs, 16H), 1.44 (m, 2H), 2.16 (s, 3H), 2.19 (s, 3H), 2.39 (t, J = 7.8 Hz, 2H), 5.25 (s, 1H), 6.99 (m, 5H), 7.11 (d, J = 7.3 Hz, 1H), 7.16 (dt, J = 1.3, 7.5 Hz, 1H), 7.78 (s, 1H); 13C NMR (125 MHz, CDCl3) δ 14.1 (CH3), 19.4 (CH3), 19.9 (CH3), 22.5 (CH2), 22.6 (CH2), 28.1 (CH2), 29.3 (CH2), 29.4 (CH2), 29.5 (CH2), 29.6 (CH2 × 2), 29.7 (CH2), 31.9 (CH2), 40.2 (CH), 107.1 (C), 115.8 (C), 116.8 (CH), 120.9 (C), 125.2 (CH), 125.5 (CH), 125.6 (C), 127.7 (CH), 129.0 (CH), 130.4 (CH), 133.9 (C), 135.1 (C), 136.8 (C), 139.1 (C), 144.2 (C), 154.1 (C), 178.5 (C), 182.3 (C); EIMS m/z (%) 485 ([M+], 81), 380 (100), 344 (12), 238 (12); HREIMS 485.2926 (calcd for C32H39O3N [M+] 485.2930).

2-Hydroxy-9-(3,4-dimethoxyphenyl)-3-undecyl-9,10-dihydroacridine-1,4-dione (4j)

The reaction mixture purified by PTLC with 20% n-Hex/AcOEt yielded 13.8 mg (56%) of 4j as an amorphous violet solid and 6.4 mg of 5a (25%) as a red solid. Mp 152–154 °C; UV (EtOH) λmax 291, 297, 350 nm; IR (film) νmax 3317, 3239, 2963, 2918, 2851, 2363, 1643, 1573, 1513, 1442, 1222, 1081, 798 cm–1; 1H NMR (500 MHz, CDCl3) δ 0.87 (t, J = 7.1 Hz, 3H), 1.25 (bs, 16H), 1.45 (m, 2H), 2.40 (t, J = 8.0 Hz, 2H), 3.80 (s, 3H), 3.82 (s, 3H), 5.28 (s, 1H), 6.70 (dd, J = 2.0, 8.3 Hz, 1H), 6.82 (d, J = 1.9 Hz, 1H), 7.04 (td, J = 1.1, 7.5 Hz, 1H), 7.13 (d, J = 7.2 Hz, 1H), 7.19 (td, J = 1.4, 7.6 Hz, 1H), 7.81 (bs, 1H); 13C NMR (150 MHz, CDCl3) δ 14.1 (CH3), 22.4 (CH2), 22.7 (CH2), 28.1 (CH2), 29.3 (CH2), 29.4 (CH2), 29.5 (CH2), 29.6 (CH2 × 2), 29.7 (CH2), 31.9 (CH2), 40.1 (CH), 55.8 (CH3), 55.9 (CH3), 107.1 (C), 111.1 (CH), 111.3 (CH), 115.9 (C), 116.8 (CH), 119.9 (CH), 125.4 (C), 125.5 (CH), 127.8 (CH), 130.4 (CH), 133.9 (C), 138.9 (C), 139.4 (C), 147.8 (C), 148.9 (C), 154.0 (C), 178.6 (C), 182.3 (C); EIMS m/z (%) 517 ([M+], 61), 381 (26), 380 (100), 238 (19); HREIMS 517.2839 (calcd for C32H39NO5 [M+] 517.2828).

2-Hydroxy-7-methoxy-9-(4-nitrophenyl)-3-undecyl-9,10-dihydroacridine-1,4-dione (4k)

The reaction mixture purified by PTLC with 20% n-Hex/AcOEt yielded 24.2 mg (80%) of 4k as an amorphous violet solid and 3.7 mg of 5b (14%) as a red solid. Mp 153–155 °C; UV (EtOH) λmax 290, 297, 380 nm; IR (film) νmax 3317, 3239, 2963, 2918, 2851, 2363, 1644, 1573, 1513, 1442, 1222, 1080, 798 cm–1; 1H NMR (500 MHz, CDCl3) δ 0.87 (t, J = 7.0 Hz, 3H), 1.24 (bs, 16H), 1.44 (m, 2H), 2.39 (t, J = 7.9 Hz, 2H), 3.72 (s, 3H), 5.44 (s, 1H), 6.56 (d, J = 2.7 Hz, 1H), 6.80 (dd, J = 2.6, 8.7 Hz, 1H), 7.00 (d, J = 8.7 Hz, 1H), 7.41 (d, J = 8.7 Hz, 2H), 7.93 (bs, 1H), 8.12 (d, J = 8.8 Hz, 2H); 13C NMR (125 MHz, CDCl3) δ 14.1 (CH3), 22.5 (CH2), 22.7 (CH2), 28.0 (CH2), 29.3 (CH2), 29.4 (CH2), 29.5 (CH2), 29.6 (CH2 × 2), 29.7 (CH2), 31.9 (CH2), 40.1 (CH), 55.6 (CH3), 103.9 (C), 114.4 (CH), 115.4 (CH), 116.1 (C), 118.6 (CH), 123.9 (CH × 2), 125.3 (C), 127.3 (C), 128.8 (CH × 2), 139.1 (C), 146.6 (C), 152.8 (C), 154.3 (C), 157.8 (C), 177.7 (C), 181.7 (C); EIMS m/z (%) 532 ([M+], 86), 463 (18), 410 (100), 307 (64); HREIMS 532.2574 (calcd for C31H36N2O6 [M+] 532.2573).

2-Hydroxy-5-((4-methoxyphenyl)amino)-3-undecylcyclohexa-2,5-diene-1,4-dione (5b)

1H NMR (500 MHz, CDCl3) δ 0.88 (t, J = 6.9 Hz, 3H), 1.26 (bs, 16H), 1.48 (m, 2H), 2.44 (t, J = 7.9 Hz, 2H), 3.83 (s, 3H), 5.3 (s, 1H), 6.94 (d, J = 8.8 Hz, 2H), 7.16 (d, J = 9.0 Hz, 2H), 7.87 (bs, 1H); 13C NMR (125 MHz, CDCl3) δ 14.1 (CH3), 22.7 (CH2), 22.8 (CH2), 28.1 (CH2), 29.4 (CH2), 29.5 (CH2), 29.6 (CH2 × 2), 29.7 (CH2), 29.8 (CH2), 31.9 (CH2), 55.6 (CH3), 93.6 (CH), 114.9 (CH × 2), 116.1 (C), 124.8 (CH × 2), 129.5 (C), 146.9 (C), 154.6 (C), 158.1 (C), 179.9 (C), 182.9 (C); EIMS m/z (%) 399 ([M+], 100), 259 (27), 258 (51), 230 (10); HREIMS 399.2401 (calcd for C24H33NO4 [M+] 399.2410).

2-Hydroxy-6,8-dimethyl-9-(4-nitrophenyl)-3-undecyl-9,10-dihydroacridine-1,4-dione (4l)

The reaction mixture purified by PTLC with 20% n-Hex/AcOEt yielded 33.4 mg (92%) of 4l as an amorphous violet solid. Mp 260–262 °C; UV (EtOH) λmax 289, 301, 360 nm; IR (film) νmax 3317, 3239, 2963, 2919, 2851, 2363, 1644, 1573, 1513, 1442, 1222, 1080, 798 cm–1; 1H NMR (500 MHz, CDCl3) δ 0.87 (t, J = 7.0 Hz, 3H), 1.23 (bs, 16H), 1.42 (m, 2H), 2.06 (s, 3H), 2.30 (s, 3H), 2.36 (t, J = 8.0 Hz, 2H), 5.47 (s, 1H), 6.75 (s, 1H), 6.77 (s, 1H), 7.38 (d, J = 8.9 Hz, 2H), 7.84 (bs, 1H), 8.09 (d, J = 8.7 Hz, 2H); 13C NMR (125 MHz, CDCl3) δ 14.1 (CH3), 19.2 (CH3), 20.9 (CH3), 22.5 (CH2), 22.7 (CH2), 28.1 (CH2), 29.3 (CH2), 29.4 (CH2), 29.5 (CH2), 29.6 (CH2 × 2), 29.7 (CH2), 31.9 (CH2), 37.7 (CH), 105.9 (C), 115.6 (CH), 116.1 (C), 119.2 (C), 123.8 (CH × 2), 129.1 (CH), 129.3 (CH × 2), 134.5 (C), 137.9 (C), 138.5 (C), 139.1 (C), 146.4 (C), 151.5 (C), 154.1 (C), 178.0 (C), 181.7 (C); EIMS m/z (%) 530 ([M+], 75), 408 (100), 389 (28), 380 (15), 268 (14); HREIMS 530.2769 (calcd. for C32H38N2O5 [M+] 530.2781).

7-Bromo-2-hydroxy-9-(4-nitrophenyl)-3-undecyl-9,10-dihydroacridine-1,4-dione (4m)

The reaction mixture purified by PTLC with 20% n-Hex/AcOEt yielded 23.4 mg (67%) of 4m as an amorphous violet solid and 8.8 mg of 5c (29%) as a red solid. Mp 236–237 °C; UV (EtOH) λmax 290, 296, 375 nm; IR (film) νmax 3317, 3239, 2963, 2918, 2851, 2363, 1644, 1573, 1513, 1442, 1222, 1080, 798 cm–1; 1H NMR (500 MHz, CDCl3) 0.87 (t, J = 7.2 Hz, 3H), 1.24 (bs, 16H), 1.44 (m, 2H), 2.40 (t, J = 7.7 Hz, 2H), 5.41 (s, 1H), 6.93 (d, J = 8.9 Hz, 1H), 7.17 (d, J = 1.8 Hz, 1H), 7.35 (dd, J = 2.0, 8.5 Hz, 1H), 7.40 (d, J = 8.6 Hz, 2H), 7.89 (bs, 1H), 8.14 (d, J = 8.7 Hz, 2H); 13C NMR (125 MHz, CDCl3) 14.1 (CH3), 22.5 (CH2), 22.7 (CH2), 28.1 (CH2), 29.3 (CH2), 29.4 (CH2), 29.5 (CH2), 29.6 (CH2 × 2), 29.7 (CH2), 31.9 (CH2), 40.5 (CH), 105.7 (C), 116.7 (C), 118.3 (C), 118.7 (CH), 124.1 (CH × 2), 125.6 (C), 128.9 (CH × 2), 131.7 (CH), 133.0 (C), 133.2 (CH), 138.9 (C), 146.9 (C), 152.2 (C), 153.8 (C), 178.7 (C), 181.5 (C); EIMS m/z (%) 580 ([M+], 100), 473 (62), 458 (70), 346 (35), 168 (75); HREIMS 580.1595 (calcd for C30H33N2O579Br[M+] 580.1573), 582.1529 (calcd for C30H33N2O581Br[M+] 582.1552).

5-((4-Bromophenyl)amino)-2-hydroxy-3-undecylcyclohexa-2,5-diene-1,4-dione (5c)

1H NMR (500 MHz, CDCl3) δ 0.88 (t, J = 7.1 Hz, 3H), 1.26 (bs, 16H), 1.48 (m, 2H), 2.45 (t, J = 7.8 Hz, 2H), 5.96 (s, 1H), 7.13 (d, J = 8.6 Hz, 2H), 7.54 (d, J = 8.6 Hz, 2H), 7.89 (bs, 1H); 13C NMR (125 MHz, CDCl3) δ 14.1 (CH3), 22.7 (CH2), 22.8 (CH2), 28.1 (CH2), 29.4 (CH2), 29.5 (CH2), 29.6 (CH2 × 3), 29.7 (CH2), 31.9 (CH2), 95.0 (CH), 116.6 (C), 119.3 (C), 124.2 (CH × 2), 132.9 (CH × 2), 136.1 (C), 145.6 (C), 154.2 (C), 180.5 (C), 182.7 (C); EIMS m/z (%) 448 ([M+], 100), 308 (37), 307 (46), 277 (13); HREIMS 447.1428 (calcd for C23H30NO379Br [M+] 447.1409), 449.1388 (calcd for C23H30NO381Br [M+] 449.1389).

2-Hydroxy-5-((3-nitrophenyl)amino)-3-undecylcyclohexa-2,5-diene-1,4-dione (5d)

A 20 mg amount of embelin (0.068 mmol), 14.1 mg of 3-nitroaniline (0.1 mmol), and 3.5 mg of AgOTf (20 mol %) were dissolved in 2 mL of EtOH. The MW tube was sealed, and the reaction mixture was irradiated at 150 °C for 15 min. The reaction product was filtered and washed with n-hex to yield 28.2 mg (100%) of 5d as an amorphous red solid. 1H NMR (500 MHz, CDCl3) δ 0.88 (t, J = 7.1 Hz, 3H), 1.26 (bs, 16H), 1.48 (m, 2H), 2.47 (t, J = 7.8 Hz, 2H), 6.08 (s, 1H), 7.57 (dd, J = 1.1, 8.0 Hz, 1H), 7.63 (t, J = 8.0 Hz, 1H), 7.78 (s, 1H), 8.01 (s, 1H), 8.09 (ddd, J = 1.1, 2.1, 8.0 Hz, 1H), 8.12 (t, J = 2.0 Hz, 1H); 13C NMR (150 MHz, CDCl3) δ 14.1 (CH3), 22.7 (CH2), 22.8 (CH2), 28.1 (CH2), 29.4 (CH2), 29.5 (CH2), 29.6 (CH2 × 3), 29.7 (CH2), 31.9 (CH2), 96.1 (CH), 117.0 (C), 117.1 (CH), 120.5 (CH), 128.0 (CH), 130.7 (CH), 138.5 (C), 144.9 (C), 149.1 (C), 153.8 (C), 180.9 (C), 182.5 (C); EIMS m/z (%) 414 ([M+], 100), 274 (32), 273 (40), 261 (14); HREIMS 414.2170 (calcd for C23H30N2O5 [M+] 414.2155).

General Procedure for the Synthesis of Acridine-1,4-dione Derivatives

One equivalent of DDQ was added to the corresponding 9,10-dihydroacridine-1,4-dione derivative dissolved in 3 mL of CH2Cl2 at room temperature. The reaction mixture was stirred until the disappearance of the starting material. The solution turned from violet to brown-orange. The reaction mixture was washed with saturated NaHCO3 and extracted with CH2Cl2 to obtain the corresponding acridine-1,4-diones.

2-Hydroxy-9-(4-nitrophenyl)-3-undecylacridine-1,4-dione (6a)

Yield: 14.4 mg (72%) of compound 6a, as an orange oil; UV (EtOH) λmax 291, 297, 344 nm; IR (film) νmax 3373, 2989, 2307, 1703, 1155, 1073, 827 cm–1; 1H NMR (500 MHz, CDCl3): 0.87 (t, J = 7.2 Hz, 3H), 1.25 (bs, 16H), 1.39 (m, 2H), 2.71 (t, J = 7.7 Hz, 2H), 7.41 (d, J = 8.9 Hz, 1H), 7.46 (d, J = 8.7 Hz, 2H), 7.64 (t, J = 7.5 Hz, 2H), 7.95 (d, J = 7.7 Hz, 2H), 8.46 (d, J = 8.5 Hz, 2H), 8.51 (d, J = 8.4 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 14.1 (CH3), 22.7 (CH2), 23.8 (CH2), 28.1 (CH2), 29.3 (CH2), 29.4 (CH2), 29.5 (CH2), 29.6 (CH2), 29.7 (CH2 × 2), 31.9 (CH2), 114.7 (C), 123.9 (CH × 2), 127.3 (CH), 127.6 (C), 127.9 (C), 129.2 (CH × 2), 130.1 (CH), 132.0 (CH), 133.5 (CH), 143.1 (C), 147.6 (C), 147.9 (C), 149.2 (C), 149.4 (C), 154.3 (C), 180.4 (C), 182.2 (C); EIMS m/z (%) 500 ([M+], 100), 472 (10), 372 (21), 359 (26); HREIMS 500.2337 (calcd for C30H32N2O5 [M+] 500.2311).

2-Hydroxy-9-phenyl-3-undecylacridine-1,4-dione (6b)

Yield: 14.7 mg (81%) of compound 6b, as an orange oil; UV (EtOH) λmax 290, 294 nm; IR (film) νmax 3239, 2963, 2918, 2851, 2363, 1662, 1572, 1513, 1442, 1222, 1080, 798 cm–1; 1H NMR (500 MHz, CDCl3) δ 0.87 (t, J = 7.3 Hz, 3H), 1.25 (bs, 16H), 1.39 (m, 2H), 2.70 (t, J = 7.5 Hz, 2H), 7.25 (m, 2H), 7.57 (m, 5H), 7.69 (m, 1H), 6.43 (t, J = 8.2 Hz, 1H), 8.47 (d, J = 8.5 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 14.1 (CH3), 22.7 (CH2), 23.7 (CH2), 28.1 (CH2), 29.3 (CH2), 29.5 (CH2), 29.6 (CH2 × 2), 29.7 (CH2 × 2), 31.9 (CH2), 119.9 (C), 126.7 (C), 127.9 (CH × 2), 128.3 (CH), 128.4 (CH), 128.5 (CH × 2), 128.9 (C), 129.5 (CH), 131.7 (CH), 133.1 (CH), 136.0 (C), 147.8 (C), 149.3 (C), 152.2 (C), 154.4 (C), 180.5 (C), 182.6 (C); EIMS m/z (%) 455 ([M+], 100), 427 (10), 328 (32), 286 (34); HREIMS 455.2445 (calcd for C30H33NO3 [M+] 455.2460).

2-Hydroxy-9-(4-chlorophenyl)-3-undecylacridine-1,4-dione (6c)

Yield: 9.2 mg (94%) of compound 6c, as an orange oil; UV (EtOH) λmax 290, 294, 344 nm; IR (film) νmax 3317, 3239, 2963, 2918, 2851, 2363, 1662, 1573, 1513, 1442, 1223, 1081, 798 cm–1; 1H NMR (500 MHz, CDCl3) δ 0.87 (t, J = 7.1 Hz, 3H), 1.25 (bs, 16H), 1.38 (m, 2H), 2.70 (t, J = 7.4 Hz, 2H), 7.20 (d, J = 7.9 Hz, 2H), 7.53 (d, J = 8.5 Hz, 1H), 7.56 (d, J = 8.1 Hz, 2H), 7.61 (t, J = 7.4 Hz, 1H), 7.92 (t, J = 7.2 Hz, 1H), 8.48 (d, J = 8.5 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 14.1 (CH3), 22.7 (CH2), 23.7 (CH2), 28.1 (CH2), 29.3 (CH2), 29.4 (CH2), 29.5 (CH2), 29.6 (CH2 × 2), 29.7 (CH2), 31.9 (CH2), 116.9 (C), 119.9 (C), 127.0 (C), 127.9 (CH), 128.9 (CH × 2), 129.4 (CH × 2), 129.7 (CH), 131.8 (CH), 133.2 (CH), 134.4 (C), 134.7 (C), 147.7 (C), 149.3 (C), 150.8 (C), 154.4 (C), 180.5 (C), 182.9 (C); EIMS m/z (%) 589 ([M+], 100), 380 (19), 351 (23), 319 (25); HREIMS 489.2055 (calcd for C30H32NO335Cl [M+] 489.2071), 491.2024 (calcd for C30H32NO337Cl [M+] 491.2041).

2-Hydroxy-9-(4-bromophenyl)-3-undecylacridine-1,4-dione (6d)

Yield: 12.1 mg (61%) of compound 6d, as an orange oil; UV (EtOH) λmax 291, 299, 342 nm; IR (film) νmax 3373, 2989, 2307, 1703, 1662, 1551, 1372, 1155, 827 cm–1; 1H NMR (500 MHz, CDCl3) δ 0.87 (t, J = 7.1 Hz, 3H), 1.25 (bs, 16H), 1.59 (m, 2H), 2.70 (t, J = 7.2 Hz, 2H), 7.14 (d, J = 7.9 Hz, 2H), 7.53 (d, J = 8.0 Hz, 1H), 7.62 (t, J = 7.1 Hz, 1H), 7.72 (d, J = 7.7 Hz, 2H), 7.92 (t, J = 7.7 Hz, 1H), 8.47 (d, J = 7.4 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 14.1 (CH3), 22.7 (CH2), 23.7 (CH2), 28.1 (CH2), 29.3 (CH2), 29.4 (CH2), 29.5 (CH2), 29.6 (CH2 × 2), 29.7 (CH2), 31.9 (CH2), 119.8 (C), 122.8 (C), 127.0 (C), 127.9 (CH), 128.6 (C), 129.6 (CH × 2), 129.7 (CH), 131.8 (CH × 2), 132.9 (CH), 133.2 (CH), 134.9 (C), 147.7 (C), 149.3 (C), 150.8 (C), 154.4 (C), 180.5 (C), 182.5 (C); EIMS m/z (%) 535 ([M+], 100), 533 (96), 407 (26), 394 (32), 365 (29); HREIMS 535.1563 (calcd for C30H32NO381Br [M+] 535.1545), 533.1578 (calcd for C30H32NO379Br [M+] 533.1566).

2-Hydroxy-9-(4-fluorophenyl)-3-undecylacridine-1,4-dione (6e)

Yield: 15.4 mg (78%) of compound 6e, as an orange oil; UV (EtOH) λmax 290, 296, 344, 390 nm; IR (film) νmax 3056, 2986, 2683, 2303, 1423.8, 1267.3, 894.6 cm–1; 1H NMR (500 MHz, CDCl3) δ 0.87 (t, J = 6.9 Hz, 3H), 1.25 (bs, 16H), 1.39 (m, 2H), 2.70 (t, J = 7.2 Hz, 2H), 7.23 (m, 2H), 7.29 (m, 2H), 7.54 (d, J = 8.4 Hz, 1H), 7.61 (t, J = 7.1 Hz, 1H), 7.91 (t, J = 7.1 Hz, 1H), 8.47 (d, J = 8.4 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 14.1 (CH3), 22.7 (CH2), 23.7 (CH2), 28.1 (CH2), 29.3 (CH2), 29.4 (CH2), 29.5 (CH2), 29.6 (CH2), 29.7 (CH2 × 2), 31.9 (CH2), 32.6 (CH), 115.8 (CH × 2, JC–F = 21.9 Hz), 120.0 (C), 126.9 (C), 127.9 (CH), 128.9 (C), 129.6 (CH), 129.8 (CH × 2, JC–F = 8.1 Hz), 131.8 (CH), 133.2 (CH), 147.8 (C), 149.3 (C), 151.2 (C), 154.4 (C), 162.8 (C, JC–F = 248.8 Hz), 180.5 (C), 182.5 (C); EIMS m/z (%) 473 ([M+], 100), 346 (23), 335 (27), 304 (28); HREIMS 473.2381 (calcd for C30H32NO3F [M+] 473.2366).

2-Hydroxy-9-(3-fluorophenyl)-3-undecylacridine-1,4-dione (6f)

Yield: 16.4 mg (91%) of compound 6f, as an orange oil; UV (EtOH) λmax 290, 294, 344, 395 nm; IR (film) νmax 3056, 2985, 2684, 2304, 1662, 1423, 1267, 894 cm–1; 1H NMR (500 MHz, CDCl3) δ 0.87 (t, J = 7.0 Hz, 3H), 1.25 (bs, 16H), 1.39 (m, 2H), 2.70 (t, J = 7.6 Hz, 2H), 6.99 (d, J = 8.7 Hz, 1H), 7.03 (d, J = 7.3 Hz, 1H), 7.29 (m, 1H), 6.40 (s, 1H), 7.55 (m, 2H), 7.62 (t, J = 7.3 Hz, 1H), 7.92 (m, 1H), 8.48 (d, J = 8.5 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 14.1 (CH3), 22.7 (CH2), 23.7 (CH2), 28.1 (CH2), 29.3 (CH2), 29.4 (CH2), 29.5 (CH2), 29.6 (CH2), 29.7 (CH2 × 2), 31.9 (CH2), 115.3 (CH, JC–F = 22.3 Hz), 115.5 (CH, JC–F = 20.9 Hz), 119.8 (C), 123.7 (CH, JC–F = 2.9 Hz), 127.0 (C), 127.9 (CH), 128.5 (C), 129.7 (CH), 130.3 (CH, JC–F = 8.3 Hz), 131.8 (CH), 133.3 (CH), 138.1 (C, JC–F = 7.9 Hz), 147.7 (C), 149.3 (C), 150.3 (C), 154.4 (C), 162.9 (C, JC–F = 250.5 Hz), 180.3 (C), 182.5 (C); EIMS m/z (%) 473 ([M+], 100), 345 (39), 335 (46), 304 (20); HREIMS 473.2351 (calcd for C30H32NO3F [M+] 473.2366).

Methyl 4-(2-hydroxy-1,4-dioxo-3-undecyl-1,4-dihydroacridin-9-yl)benzoate (6g)

Yield: 14.0 mg (78%) of compound 6g, as an orange oil; UV (EtOH) λmax 292, 297, 344 nm; IR (film) νmax 3056, 2985, 2303, 1662, 1424, 894 cm–1; 1H NMR (500 MHz, CDCl3) δ 0.87 (t, J = 7.1 Hz, 3H), 1.25 (bs, 16H), 1.38 (m, 2H), 2.70 (t, J = 7.6 Hz, 2H), 4.00 (s, 3H), 7.35 (d, J = 7.6 Hz, 2H), 7.47 (d, J = 8.3 Hz, 1H), 7.60 (t, J = 7.3 Hz, 1H), 7.92 (d, J = 7.3 Hz, 1H), 8.27 (d, J = 7.8 Hz, 2H), 8.49 (d, J = 8.1 Hz, 1H); 13C NMR (150 MHz, CDCl3) δ 14.1 (CH3), 22.7 (CH2), 23.7 (CH2), 28.1 (CH2), 29.3 (CH2), 29.5 (CH2), 29.6 (CH2), 29.6 (CH2), 29.7 (CH2 × 2), 31.9 (CH2), 52.4 (CH3), 119.7 (C), 122.8 (C), 127.1 (C), 127.8 (CH), 128.1 (CH × 2), 129.7 (CH), 129.8 (CH × 2), 130.3 (C), 131.8 (CH), 133.3 (CH), 140.9 (C), 147.7 (C), 149.3 (C), 150.9 (C), 154.4 (C), 166.6 (C), 180.4 (C), 182.4 (C); EIMS m/z (%) 513 ([M+], 100), 386 (20), 369 (40), 344 (16), 228 (18); HREIMS 513.2520 (calcd for C32H35NO5 [M+] 513.2515).

2-Hydroxy-9-(thiophen-3-yl)-3-undecylacridine-1,4-dione (6h)

Yield: 14.6 mg (73%) of compound 6h, as an orange oil; UV (EtOH) λmax 291, 296, 345, 390; IR (film) νmax 3056, 2985, 2304, 1666, 1550, 1423, 1267, 894 cm–1; 1H NMR (500 MHz, CDCl3) δ 0.87 (t, J = 7.0 Hz, 3H), 1.25 (bs, 16H), 1.39 (m, 2H), 2.70 (t, J = 7.7 Hz, 2H), 7.07 (d, J = 4.6 Hz, 1H), 7.25 (dd, J = 1.1, 2.9 Hz, 1H), 7.62 (m, 2H), 7.70 (d, J = 8.3 Hz, 1H), 7.91 (t, J = 7.2 Hz, 1H), 8.46 (d, J = 8.5 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 14.1 (CH3), 22.7 (CH2), 23.7 (CH2), 28.1 (CH2), 29.3 (CH2), 29.4 (CH2), 29.5 (CH2), 29.6 (CH2), 29.7 (CH2 × 2), 31.9 (CH2), 120.5 (C), 123.7 (CH), 126.1 (CH), 126.7 (C), 128.1 (CH), 128.4 (CH), 129.3 (C), 129.6 (CH), 131.7 (CH), 133.4 (CH), 135.1 (C), 147.8 (C), 147.9 (C), 149.3 (C), 154.5 (C), 180.4 (C), 182.6 (C); EIMS m/z (%) 461 ([M+], 100), 334 (26), 323 (25), 292 (35); HREIMS 461.2015 (calcd for C28H31NO3S [M+] 461.2025).

2-Hydroxy-9-(3,4-dimethylphenyl)-3-undecylacridine-1,4-dione (6i)

Yield: 17.6 mg (88%) of compound 6i, as an orange oil; UV (EtOH) λmax 288, 325, 340 nm; IR (film) νmax 3056, 2985, 2929, 2855, 2303, 1662, 1551, 1424, 1267, 895 cm–1; 1H NMR (500 MHz, CDCl3) δ 0.87 (t, J = 7.2 Hz, 3H), 1.25 (bs, 16H), 1.39 (m, 2H), 2.36 (s, 3H), 2.43 (s, 3H), 2.70 (t, J = 7.7 Hz, 2H), 6.98 (d, J = 7.1 Hz, 1H), 7.01 (s, 1H), 7.35 (d, J = 7.3 Hz, 1H), 7.58 (m, 2H), 7.89 (t, J = 7.9 Hz, 1H), 8.46 (d, J = 8.1 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 14.1 (CH3), 19.8 (CH3), 19.9 (CH3), 22.7 (CH2), 23.7 (CH2), 28.1 (CH2), 29.3 (CH2), 29.4 (CH2), 29.5 (CH2), 29.6 (CH2), 29.7 (CH2 × 2), 31.9 (CH2), 119.9 (C), 125.3 (CH), 126.6 (C), 128.5 (CH), 128.9 (CH), 129.2 (C), 129.3 (CH), 129.7 (CH), 131.6 (CH), 132.9 (CH), 133.4 (C), 136.7 (C), 136.9 (C), 147.8 (C), 149.2 (C), 152.8 (C), 154.5 (C), 180.6 (C), 182.7 (C); EIMS m/z (%) 483 ([M+], 100), 356 (27), 343 (22), 314 (30); HREIMS 483.2757 (calcd for C32H37NO3 [M+] 483.2773).

2-Hydroxy-9-(3,4-dimethoxyphenyl)-3-undecylacridine-1,4-dione (6j)

Yield: 9.6 mg (81%) of compound 6j, as an orange oil; UV (EtOH) λmax 291, 297, 342 nm; IR (film) νmax 3056, 2986, 2303, 1662, 1551, 1423, 1267, 895 cm–1; 1H NMR (500 MHz, CDCl3) δ 0.87 (t, J = 7.3 Hz, 3H), 1.25 (bs, 16H), 1.40 (m, 2H), 2.70 (t, J = 7.7 Hz, 2H), 3.86 (s, 3H), 4.02 (s, 3H), 6.75 (s, 1H), 6.80 (d, J = 8.3 Hz, 1H), 7.08 (d, J = 8.2 Hz, 1H), 7.62 (m, 2H), 7.90 (t, J = 6.8 Hz, 1H), 8.47 (d, J = 8.6 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 14.1 (CH3), 22.7 (CH2), 23.7 (CH2), 28.1 (CH2), 29.3 (CH2), 29.4 (CH2), 29.5 (CH2), 29.6 (CH2), 29.7 (CH2 × 2), 31.9 (CH2), 55.9 (CH3), 56.0 (CH3), 110.9 (CH), 111.4 (CH), 120.0 (C), 120.4 (CH), 126.6 (C), 128.2 (C), 128.4 (CH), 129.3 (C), 129.5 (CH), 131.7 (CH), 133.0 (CH), 147.9 (C), 149.1 (C), 149.2 (C), 149.3 (C), 152.2 (C) 154.5 (C), 180.5 (C), 182.7 (C); EIMS m/z (%) 515 ([M+], 100), 397 (35), 369 (37), 229 (36); HREIMS 515.2686 (calcd for C32H37NO5 [M+] 515.2672).

2-Hydroxy-7-methoxy-9-(4-nitrophenyl)-3-undecylacridine-1,4-dione (6k)

Yield: 16.7 mg (83%) of compound 6k, as an orange oil; UV (EtOH) λmax 290, 295 nm; IR (film) νmax 3056, 2926, 2855, 2307, 1662, 1520, 1267, 857 cm–1; 1H NMR (500 MHz, CDCl3) δ 0.86 (t, J = 7.1 Hz, 3H), 1.24 (bs, 16H), 1.38 (m, 2H), 2.68 (t, J = 7.7 Hz, 2H), 3.72 (s, 3H), 6.54 (s, 1H), 7.45 (d, J = 8.2 Hz, 2H), 7.57 (dd, J = 2.6, 9.2 Hz, 1H), 8.38 (d, J = 7.1 Hz, 1H), 8.46 (d, J = 8.5 Hz, 2H); 13C NMR (125 MHz, CDCl3) δ 14.1 (CH3), 22.7 (CH2), 23.7 (CH2), 28.1 (CH2), 29.3 (CH2), 29.4 (CH2), 29.5 (CH2), 29.6 (CH2), 29.7 (CH2 × 2), 31.9 (CH2), 55.7 (CH3), 104.8 (CH), 119.9 (C), 124.0 (CH × 2), 126.1 (CH), 127.8 (C), 128.8 (C), 129.1 (CH × 2), 129.5 (C), 133.5 (CH), 143.6 (C), 145.3 (C), 147.9 (C), 153.9 (C), 160.4 (C), 160.9 (C), 180.7 (C), 182.4 (C); EIMS m/z (%) 530 ([M+], 100), 399 (50), 390 (38), 258 (40), 221 (42); HREIMS 530.2426 (calcd for C31H34N2O6 [M+] 530.2417).

2-Hydroxy-6,8-dimethyl-9-(4-nitrophenyl)-3-undecylacridine-1,4-dione (6l)

Yield: 17.5 mg (71%) of compound 6l, as an orange oil; UV (EtOH) λmax 289, 293, 350 nm; IR (film) νmax 3056, 2986, 2863, 2304, 1662, 1551, 1424, 1267, 895 cm–1; 1H NMR (500 MHz, CDCl3) δ 0.87 (t, J = 7.1 Hz, 3H), 1.24 (bs, 16H), 1.37 (m, 2H), 1.88 (s, 3H), 2.54 (s, 3H), 2.64 (t, J = 7.5 Hz, 2H), 7.26 (s, 1H), 7.52 (d, J = 8.1 Hz, 2H), 8.14 (s, 1H), 8.39 (d, J = 8.2 Hz, 2H); 13C NMR (125 MHz, CDCl3) δ 14.1 (CH3), 21.7 (CH3), 22.7 (CH2), 23.6 (CH2), 24.9 (CH3), 28.0 (CH2), 29.3 (CH2), 29.4 (CH2), 29.5 (CH2), 29.6 (CH2), 29.7 (CH2 × 2), 31.9 (CH2), 119.7 (C), 123.5 (CH × 2), 124.5 (C), 126.4 (C), 129.1 (C), 129.2 (CH × 2), 130.6 (CH), 136.6 (CH), 137.6 (C), 144.5 (C), 146.8 (C), 147.9 (C), 149.5 (C), 151.0 (C), 154.4 (C), 180.5 (C), 182.1 (C); EIMS m/z (%) 528 ([M+], 100), 483 (6), 388 (21), 359 (16); HRIMS 528.2648 (calcd for C32H36N2O5 [M+] 528.2624).

7-Bromo-2-hydroxy-9-(4-nitrophenyl)-3-undecylacridine-1,4-dione (6m)

Yield: 5.4 mg (72%) of compound 6m, as an orange oil; UV (EtOH) λmax 291, 297, 350 nm; IR (film) νmax 3054, 2989, 2926, 2855, 2307, 1662, 1520, 1423, 1267, 895 cm–1; 1H NMR (500 MHz, CDCl3) 0.87 (t, J = 7.1 Hz, 3H), 1.25 (bs, 16H), 1.39 (m, 2H), 2.71 (t, J = 7.7 Hz, 2H), 7.44 (d, J = 8.6 Hz, 2H), 7.51 (d, J = 1.9 Hz, 1H), 8.01 (dd, J = 2.1, 8.9 Hz, 1H), 8.37 (d, J = 8.9 Hz, 1H), 8.48 (d, J = 8.5 Hz, 2H); 13C NMR (125 MHz, CDCl3) 14.1 (CH3), 22.7 (CH2), 23.8 (CH2), 28.1 (CH2), 29.4 (CH2), 29.4 (CH2), 29.5 (CH2), 29.6 (CH2), 29.7 (CH2 × 2), 31.9 (CH2), 116.7 (C), 120.3 (C), 124.1 (CH × 2), 125.1 (C), 127.9 (C), 129.1 (CH × 2), 129.3 (CH), 133.4 (CH), 137.1 (CH), 142.2 (C), 147.7 (C), 147.9 (C), 148.1 (C), 148.2 (C), 154.3 (C), 180.2 (C), 181.9 (C); EIMS m/z (%) 580 ([M+], 100), 578 (90), 528 (12), 439 (41); HREIMS 580.1388 (calcd for C30H31NO581Br [M+] 580.1396), 578.1396 (calcd for C30H31NO579Br [M+] 578.1416).

Biological Assays

Cell Culture

J774A.1 murine macrophage cells, H9c2 embryonic rat heart-derived cells, and MCF-7 breast cancer cells were purchased from American Type Cell Culture (ATCC, Manassas, VA, USA). Cells were cultured in Dulbecco’s modified Eagle medium with 10% fetal bovine serum and 1% penicillin/streptomycin (Lonza) and were maintained at 37 °C in a humidified incubator containing 5% CO2.

Cell Viability Assay

Cell viability was assessed by conducting an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye] assay. Briefly, J 774A.1, H9c2, and MCF7 cells were seeded in 96-well plates for 24 h. Then, cells were treated with DOX in the absence or presence of compounds. MTT (Sigma) reagent was added to the medium for 1 h at 37 °C. Then, the formazan was dissolved in dimethyl sulfoxide (DMSO, 100 μL). Absorbance was measured at 540 nm with a microplate reader (BMG Labtech).

Assessment of Cardioprotective Activity

Compounds were tested in a DOX-induced H9c2 cardiomyocytes model. H9c2 cells treated with DOX (1 μM) served as a positive control. To determine the potential protective effects, cells were co- treated with compounds at 20 and 1 μM of DOX (Sigma) for 24 h. After that, the MTT cell viability assay was performed.

Measurement of Reactive Oxygen Species

Intracellular ROS levels in H9c2 cells were evaluated by monitoring the oxidation of 2′,7′-dichlorfluorescein-diacetate (DCFH-DA) (Sigma) to fluorescent dichlorofluorescein. Cells were treated with DOX (1 μM) in the absence or presence of compounds or NAC as ROS inhibitor (1 mM) (Sigma). Then, cells were incubated with 10 μM DCFH-DA at 37 °C in the dark for 30 min. ROS level was fluorimetrically evaluated at 485/550 nm during 24 h.

Western Blot

Cell lysates were prepared as described previously63 and later subjected to sulfate-polyacrylamide (SDS-PAGE) electrophoresis. The gels were transferred onto a Hybond-PVDF membrane and, after blocking, were incubated with anti-pAkt, anti-Akt, anti-pERK1/2, anti-ERK1/2, anti-Bax, and anti-Bcl-2. β-Actin (Sigma) were used as a loading control. After further incubating with horseradish peroxidase (HRP)-conjugated secondary antibodies for 2 h, the specific proteins bands were developed by an ECL detection system (Amersham).

Statistical Analysis

Statistical analyses were carried out using GraphPad Prism (version 9). Data are presented as means ± standard deviation (SD) from at least three experiments, and a one-way ANOVA was performed. p < 0.05 was considered statistically significant.

Acknowledgments

In memory of Professor Maritza Grande. We gratefully acknowledge the financial support from the Spanish MICIU RTI2018-094356-B-C21 to A.E.B., I.C., and B.H. and Agencia Canaria de Investigación, Innovación y Sociedad de la Información Pro ID 2021010037 to A.E.B. These projects are also cofunded by the European Regional Development Fund (FEDER). We thank Dr. A. Tapia and G. Feresin for providing the natural embeline. We are grateful to Instituto de Salud Carlos III for financial support to S.H.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jnatprod.2c00924.

  • Spectra of compounds (PDF)

Author Contributions

P.M.-A., I.C., and R.P. have contributed equally to this work.

The authors declare no competing financial interest.

Supplementary Material

np2c00924_si_001.pdf (6.8MB, pdf)

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Supplementary Materials

np2c00924_si_001.pdf (6.8MB, pdf)

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