Labdane conjugates protect cardiomyocytes from doxorubicin‐induced cardiotoxicity

The cardiovascular side effects associated with doxorubicin (DOX), a wide spectrum anticancer drug, have limited its clinical application. Therefore, to explore novel strategies with cardioprotective effects, a series of new labdane conjugates were prepared (6a–6c and 8a–8d) from the natural diterpene labdanodiol (1). These hybrid compounds contain anti‐inflammatory privileged structures such as naphthalimide, naphthoquinone, and furanonaphthoquinone. Biological activity of these conjugates against DOX‐induced cardiotoxicity was tested in vitro and the potential molecular mechanisms of protective effects were explored in H9c2 cardiomyocytes. Three compounds 6c, 8a, and 8b significantly improved cardiomyocyte survival, via inhibition of reactive oxygen species‐mediated mitogen‐activated protein kinase signaling pathways (extracellular signal‐regulated kinase and c‐Jun N‐terminal kinase) and autophagy mediated by Akt activation. Some structure–activity relationships were outlined, and the best activity was achieved with the labdane–furonaphthoquinone conjugate 8a having an N‐cyclohexyl substituent. The findings of this study pave the way for further investigations to obtain more compounds with potential cardioprotective activity.

cardiomyocyte apoptosis, and autophagy dysregulation (Prathumsap et al., 2020;Yu et al., 2018). Several stress-responsive signaling pathways, such as the mitogen-activated protein kinases (MAPKs), c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK), regulate inflammation, apoptosis, and autophagy, contributing to DOX-induced cell death. This drug also mediates damage at various steps of cardiac energy metabolism, affecting the sensitivity of cardiac cells to apoptosis and leading to defects in the AMP-activated protein kinase (AMPK) signaling pathway (Octavia et al., 2012).
In the search for new therapeutic strategies for cardioprotection, diterpenes and their derivatives constitute a good starting point since a number of diterpenes have shown protective activities. Among them, kirenol showed cardioprotection through its antiapoptotic and prosurvival effects (Alzahrani et al., 2021). Moreover, other kaurane and labdane diterpenes also exhibited cardioprotective effects against ischemic myocardial injury (Cuadrado-Berrocal et al., 2015;Gao et al., 2021;Marco, 2020). The basic skeletal structure of labdane diterpenes presents two parts: a fused decalin system (C-1-C-10) and a branched six-carbon side chain (C-11-C-16) at C-9.
The remaining four carbons (C-17-C-20) are methyl groups attached at C-8, C-4, and C-10 of the decalin system, respectively. In the present work, from the natural labdanediol (1) (Amaro-Luis & Adrian, 1982) and through its side chain, a novel series of labdane hybrid compounds containing naphthalimide or naphthoquinone were prepared using as linker a triazole or a furan ring (6a-6c and 8a-8d). Moreover, the protective effects of these compounds were evaluated in a DOX-induced cardiotoxicity model.
Results showed that three derivatives 6c, 8a, and 8b, exhibited cardioprotective effects by increasing cell viability in a dose-response manner through regulation of oxidative stress and autophagy. Conjugate 8a was identified as an applicable agent to prevent DOX-induced cardiotoxicity.

| General experimental procedures
Nuclear magnetic resonance (NMR) spectra were recorded in CDCl 3 at 400, 500, or 600 MHz for 1 H NMR and 100, 125, or 150 MHz for 13 C NMR. Chemical shifts (δ) are given in parts per million, and coupling constants (J) in hertz (Hz). 1 H and 13 C spectra were referenced using the solvent signal as an internal standard. High-resolution electron ionization mass spectrometry (HREIMS) was recorded using a highresolution magnetic trisector mass analyzer. Analytical thin-layer chromatography plates used were Polygram-Sil G/UV254. Preparative thin-layer chromatography was carried out with Analtech silica gel GF plates (20 × 20 cm 2 , 1000 μm) using appropriate mixtures of ethyl acetate and hexanes. All solvents and reagents were purified by standard techniques reported (Perrin & Amarego, 1988) or used as supplied from commercial sources. The labdanediol (1) used as starting material was obtained from Oxylobus glanduliferus (Asteraceae) following the procedure described in (Amaro-Luis & Adrian, 1982).
Characterization of labdanediol (1) and synthetic procedures and characterization data for synthesized compounds 2-4, 7, and 9-10 are included in the Supporting information.
2.1.1 | General procedure for the synthesis of labdane conjugates 6a-6c A solution of the labdane azide (4) (1 equiv) and the corresponding alkyne (2 equiv) in 3 ml of CH 2 Cl 2 (dichloromethane [DCM]) was added to a mixture of CuSO 4 ·5H 2 O (4 mol%) and sodium ascorbate (12 mol%) in 3 ml of water. The reaction mixture was stirred vigorously at room temperature until the disappearance of the alkyne. Then, it was extracted with DCM (3 × 15 ml), the organic phases were collected, dried over anhydrous MgSO 4 , filtered, and the solvent was eliminated under reduced pressure. The residue was purified by preparative thin-layer chromatography (TLC) with hexanes:EtOAc (7:3) as eluent.
The reaction mixture was heated under reflux until the disappearance of the starting material. Then, it was cooled to room temperature and the toluene was removed under reduced pressure. The residue was purified by preparative -TLC using hexanes:EtOAc (7:3).

| Cell culture
H9c2 embryonic rat heart-derived cells and MCF-7 breast cancer cells were obtained from the American Type Culture Collection (ATCC). The cells were cultured in Dulbecco's modified Eagle's medium (Sigma), containing 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin, at 37°C in a humidified incubator containing 5% CO 2 .

| Cell viability
To evaluate the cytotoxicity of DOX and tested compounds, cell viability assays were performed using the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) methods. Briefly, H9c2 and MCF7 cells were seeded at a density of 5×10 4 cells/well and 1×10 4 cells/well, respectively, in 96-well plates for 24 h. Then, cells were treated with DOX (Sigma) in the absence or presence of compounds. MTT (Sigma; 5 mg/ml for H9c2 or 2 mg/ml for MCF7) reagent was added to the medium for 1 h at 37°C. Then, the formazan was dissolved in dimethyl sulfoxide (100 μl). Absorbance was measured at 540 nm with a microplate reader (BMG Labtech). LDH was determined in cell supernatants by measuring the conversion of pyruvate to lactate, and analyzed spectrophotometrically at 490 nm, using CytoTox96 ® kit (Promega).

| Cardioprotective activity
The cardioprotective effects of compounds were tested in a DOXinduced H9c2 cardiomyocytes model. H9c2 cells were treated with compounds at 20 μM and exposed to 1 μM of DOX for 24 h to cause impairment and decreases in cell viability. After that, cell viability assays were performed to determine the potential protection of the compounds. Western blot: Cell lysates were extracted as previously reported (Cuadrado et al., 2011) and later subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis. The gels were transferred onto a Hybond-PVDF membrane and, after blocking, were incubated with anti-pAkt, anti-Akt, anti-pAMPK, anti-AMPK, p62 (Santa Cruz), anti-pERK1/ 2, anti-ERK1/2, anti-pJNK, and anti-JNK (Cell Signaling). Glyceraldehyde 3-phosphate dehydrogenase (Abcam) and β-actin (Sigma) were used as a loading control. After further incubation with horseradish peroxidase-conjugated secondary antibodies for 2 h, the specific protein bands were developed by the ECL detection system (Amersham).

| Statistical analysis
All values have been expressed as mean ± SD. The statistical significance was tested using Sigmaplot 11.0 software (Systat Software). Data were analyzed by one-way analysis of variance. A p value <.05 was recognized as a significant difference. The estimated half-maximal inhibitory concentration (IC 50 ) value of the cytotoxic effect of DOX was calculated using GraphPad Prism 8.0 (GraphPad Software) (nonlinear regression).
The introduction of these privileged structures was carried out following two different approaches, namely, a copper(I)-catalyzed Huisgen 1,3-dipolar cycloaddition reaction (Rostovtsev et al., 2002) and Regarding the first approach, it implies the preparation of a labdanoyl azide intermediate (4) as is shown in Scheme 1. Thus, the treatment of labdanediol (1) with BBr 3 in DCM for 24 h afforded compound 2 in 83% yield. The terminal hydroxyl group was converted quantitatively into the corresponding mesyl derivative (3) by treatment with methanesulfonyl chloride in DCM, and finally, the labdanoyl azide 4 was obtained when 3 was reacted with sodium azide in dimethylformamide.
The preparation of labdane-furanonaphthoquinone conjugates was achieved via a MCR from 2-hydroxy-1,4-naphthoquinone, the corresponding isonitrile, and the labdanoyl aldehyde (7) In Table 2 appears the structures and yields obtained when the reaction was carried out using different isocyanides. The best yields were achieved with 2-morpholinoethyl (8b) and benzyl (8c) isocyanides.

| Biological evaluation
To explore the cardioprotective activity of the synthesized compounds, a DOX-induced cardiotoxicity model in H9c2 rat cardiomyocytes was used. DOX induced a dose-dependent reduction of cardiomyocyte viability with an IC 50 = 0.83 µM after 24 h of treatment ( Figure 1a). Thus, based on the IC 50 value, a concentration of 1 µM of DOX was selected for further analysis. Next, the potential | 89 cytotoxicity of the starting diterpenes 1, 2 and the labdane conjugates (6a-6c, 8a-8d) was evaluated. The tested compounds did not show any cytotoxicity at a concentration level of 20 µM, with the exception of compound 1 (Figure 1b).
8b showed high cardioprotective potential, which was manifested by a significant increase in the percentage of the viable cells in the presence of DOX (78.06 ± 4.42%, 82.72 ± 3.11%, and 80.20 ± 2.87%, respectively) (Figure 2a). Cytotoxicity of active conjugates 6c, 8a, and 8b was tested in the range 5-50 µM, with IC 50 values of 73.5, 60.7, and 80.6 µM, respectively (Figure 2b). Treatment with labdane conjugates attenuated DOX-induced injury in a dose-dependent manner up to 20 µM (Figure 2c). This concentration was selected for further experiments. Furthermore, cotreatment with conjugates 6c, 8a, and 8b in the DOX cardiotoxicity model induced a significant decrease in LDH release, a marker of cellular damage (Figure 2d). The results obtained confirmed the protective effects of these three compounds, which were selected for further testing.
An important issue for the design of new cardioprotective compounds is that the reduction of DOX unwanted side effects should not compromise its antitumor potential. To further investigate whether labdane conjugates preserved the antitumor effect of DOX, MCF-7 breast cancer cells were treated with compounds alone or in combination with DOX. Results shown in Figure 3 demonstrated that the three tested diterpene conjugates 6c, 8a, and 8b did not interfere with the DOX anticancer activities in MCF-7 cells.
From the obtained results, some structure-activity relationships were outlined. Among the labdane conjugates with a triazol linker (6a-6c), compound 6c having a naphthalimide moiety was the only one that showed cardioprotective activity. Regarding the labdane-furonaphthoquinone conjugates (8a-8d), the nature of the moiety attached to the nitrogen seems to play an important role in the activity, since 8a with a cyclohexyl substituent and 8b with an ethylmorpholine group showed cardioprotective effects, while conjugates with a benzyl group (8c) or a tert-butyl (8d) resulted less active. These data suggest that this part of the molecule could interact with specific residues of the target and the resultant interactions cause the corresponding modulation of the activity.
Further investigations were focused on the molecular mechanisms of the cardioprotective properties of the selected compounds.
Various signaling pathways involved in autophagy regulation, including Akt/AMPK/m-TOR signaling, have been described to be critical mediators of DOX-induced cardiotoxicity (Chen et al., 2011;Dirks-Naylor, 2013;Timm & Tyler, 2020). The addition of selected compounds, in particular, conjugate 8a, protected cardiomyocytes, as deduced by the increase in Akt phosphorylation and impaired activation of AMPK. Moreover, treatment with conjugates reversed the increased expression of the autophagy marker p62 induced by DOX ( Figure 5). These results indicate that autophagy could be potentially restored with the compounds.
To deepen the structural determinants responsible for the cardioprotective effect two new derivatives, furonaphthoquinones 9 and 10 were prepared as simplified fragments of the active labdane-furonaphthoquinone conjugate 8a (Figure 6).
The effects of these derivatives on H9c2 cell viability were tested ( Figure 7). The results obtained showed that compounds 9 and 10 did F I G U R E 5 Bioactive conjugates 6c, 8a, and 8b regulated DOX-induced activation of autophagy through Akt/AMPK. Immunoblot analysis of Akt, AMPK, and p62 in H9c2 cells following exposure to DOX (1 µM) alone or in combination with conjugates (20 µM). β-Actin and GAPDH were used as a loading control. A representative experiment of three performed is shown. DOX, doxorubicin, GAPDH, glyceraldehyde 3-phosphate dehydrogenase. **p < .01 versus DOX-treated cells.
F I G U R E 6 Structures of compounds 9 and 10 as simplified fragments of conjugate 8a not exert any cardioprotective effects against DOX-induced toxicity in H9c2 cells. This indicates that the presence of both the diterpene unit and the furanophthoquinone moiety is relevant for cardioprotective effects, as the highest protection is achieved with compound 8a.
Targeted molecular pathways involved in the cardioprotective effects of active conjugates are summarized in Figure 8.
Molecular mechanisms involved in the protective effects were investigated. Active compounds exhibited a significant inhibition of oxidative stress and MAPK signaling. Among them, the labdane-furonaphthoquinone conjugate 8a showed the best potential for cardioprotection via its additional ability to regulate upstream Akt/ AMPK/mTOR signaling autophagic pathway in cardiomyocytes. To the best of our knowledge, this is the first ever report to show the cardioprotective potential of labdane diterpenes, in particular conjugates containing the furonaphthoquinone moiety, against DOX-induced cardiotoxicity. Thus, combined administration of labdane conjugates with DOX could improve the use of this chemotherapeutic agent, providing a safer strategy for cardioprotection. F I G U R E 7 Evaluation of simplified derivatives 9 and 10, and conjugate 8a on DOX-induced cardiotoxicity. H9c2 cell viability was measured by MTT after incubation for 24 h with compounds (20 µM) in the absence or presence of DOX (1 µM). Results are reported as the mean of cell viability ± SD (n = 3). DOX, doxorubicin; MTT, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide ### p < .001 versus untreated cells and ***p < .001 versus DOX-treated cells.
F I G U R E 8 Schematic representation for the underlying mechanisms of cardioprotection by active labdane conjugates against DOX-induced toxicity. DOX, doxorubicin; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; ROS, reactive oxygen species. Created with BioRender.com.