Identification of a Hydrogen-Sulfide-Releasing Isochroman-4-One Hybrid as a Cardioprotective Candidate for the Treatment of Cardiac Hypertrophy

Cardiac pathological hypertrophy is associated with undesirable epigenetic changes and causes maladaptive cardiac remodeling and heart failure, leading to high mortality rates. Specific drugs for the treatment of cardiac hypertrophy are still in urgent need. In the present study, a hydrogen-sulfide-releasing hybrid 13-E was designed and synthesized by appending p-hydroxythiobenzamide (TBZ), an H2S-releasing donor, to an analog of our previously discovered cardioprotective natural product XJP, 7,8-dihydroxy-3-methyl-isochromanone-4. This hybrid 13-E exhibited excellent H2S-generating ability and low cellular toxicity. The 13-E protected against cardiomyocyte hypertrophy In Vitro and reduced the induction of Anp and Bnp. More importantly, 13-E could reduce TAC-induced cardiac hypertrophy In Vivo, alleviate cardiac interstitial fibrosis and restore cardiac function. Unbiased transcriptomic analysis showed that 13-E regulated the AMPK signaling pathway and influenced fatty acid metabolic processes, which may be attributed to its cardioprotective activities.


Introduction
Cardiac hypertrophy is usually characterized by an increase in cardiomyocyte size and the thickening of ventricular walls. Cardiac hypertrophy is commonly classified as physiological, when it is associated with normal cardiac function, or as pathological, when it is associated with cardiac dysfunction [1]. Pathological hypertrophy is a risk factor which is induced by factors such as prolonged and abnormal hemodynamic stress, including hypertension, myocardial infarction, etc. [2]. Pathological hypertrophy is associated with fibrosis, capillary rarefaction, increased production of pro-inflammatory cytokines, cellular dysfunction and undesirable epigenetic changes, leading to maladaptive cardiac remodeling and heart failure [3]. Until now, although many cardioprotective drugs have been found to alleviate hypertrophy, such as diuretics, angiotensin converting enzyme inhibitors (ACEI), angiotensin II receptor blockers and beta-blockers, mortality rate remains high, and specific drugs for the treatment of cardiac hypertrophy are still in urgent need [4].
The discovery of active molecules from natural products is an important strategy to develop novel cardioprotective drugs. Many bioactive derivatives from nature, including polyphenolic compounds, peptides, oligosaccharides, vitamins and unsaturated fatty acids, possess protective effects on cardiovascular diseases [5,6]. 7,8-dihydroxy-3-methylisochromanone-4 (XJP, Figure 1) is a structurally unique natural polyphenolic compound, isolated from the banana (Musasapientum L.) peel by our group previously. Our biological In the present study, a more potent and stable analog of XJP, by protecting the phenolic hydroxyl group, was selected as the parent natural product [21], and p-hydroxythiobenzamide (TBZ) was used as the H2S-releasing moiety, as it is a widely used H2S-releasing compound characterized with controllable and slow-releasing properties [22]. The target hybrid 13-E was firstly designed and synthesized by linking the TBZ group to XJP by a flexible alkyl link, and its effects towards hypertrophy induced by transverse aortic constriction (TAC) were explored. The protective effects of 13-E were tested using primary cultured cardiomyocytes in addition to a well-established animal model of cardiac hypertrophy. The results showed that 13-E possesses good cardioprotective activities and attenuates hypertrophy potently, which deserves further investigations.

Synthesis and Characterization of Hybrid 13-E
The synthesis and structure identification of XJP has been reported by our group previously [23]. Briefly, the commercially available benzaldehyde 1 was reduced to alcohol 2 with sodium borohydride in an almost quantitative yield. The subsequent reaction of alcohol 2 with N-methoxy-N-methyl-2-bromopropanamide afforded Weberamide 3 in a 95% yield. Isochroman-4-one 5 was obtained by the cyclization of 3 with t-BuLi as a base, following the deprotection of benzyl in the presence of Pd-C/H2. Finally, compound 5 engaged in nucleophile substitution with 1,6-dibromohexane to afford compound 6, which then reacted with 4-cyanophenol to produce intermediate 7. The reaction of NaHS and intermediate 7 with appropriate amounts of MgCl2·6H2O in the solvent of DMF gave the target compound 13-E in a 60% yield (Scheme 1). The structure of compound 13-E was identified unambiguously with 1 H-NMR, 13 C-NMR and HR-MS (please see Supporting Information). Hydrogen sulfide (H 2 S) is an endogenous gas signaling molecule, which was first believed to be a toxic byproduct of metabolic processes until Kimura demonstrated H 2 S as an endogenous neuromodulator and discovered its physiological role in regulating smooth muscle relaxation [12]. H 2 S is now regarded as the third gasotransmitter besides nitric oxide (NO) and carbon monoxide (CO) [13]. Mounting evidence has indicated the wide range of physiological and pathological activities of H 2 S in cardiovascular systems endowed with antioxidant, anti-inflammatory, pro-autophagic and cardioprotective properties [14]. Although H 2 S plays an important role in cardiac pathology and physiology, it is not suitable for direct use in clinical treatment due to uncontrollable doses and high toxicity. The use of H 2 S donors represents an exciting and intriguing strategy to be pursued for the treatment of cardiovascular disease [15]. In recent years, H 2 S-releasing compounds, such as GYY4137, have been developed and show promising cardioprotective activities. For example, Xie et al. found that GYY4137 can alleviate atherosclerosis through increasing the expression of HO-1, and Meng et al. found that GYY4137 protects the heart from hypertrophy [16,17]. Our group also developed novel allyl thioesters as potential cardioprotective agents by releasing H 2 S [18].
In recent years, several H 2 S-releasing natural product hybrids have been developed by using the combination principle, in which a H 2 S-releasing moiety was linked to a natural product, to improve activity or reduce side effects. These hybrids may exhibit greater activities than their respective parent natural product, but with less toxicity, representing a superior design strategy for developing H 2 S donors [19,20]. Given the pharmacological effects of XJP, including its antihypertensive and cardioprotective activities, it is closely related to the etiology of cardiac hypertrophy, and there has been extensively reported cardioprotective activity of H 2 S for cardiac hypertrophy. Our design principle proposes a method to fuse the important pharmacophore of XJP and H 2 S donors into a new molecule to further improve its properties and to enhance its activity. (Figure 1).
In the present study, a more potent and stable analog of XJP, by protecting the phenolic hydroxyl group, was selected as the parent natural product [21], and phydroxythiobenzamide (TBZ) was used as the H 2 S-releasing moiety, as it is a widely used H 2 S-releasing compound characterized with controllable and slow-releasing properties [22]. The target hybrid 13-E was firstly designed and synthesized by linking the TBZ group to XJP by a flexible alkyl link, and its effects towards hypertrophy induced by transverse aortic constriction (TAC) were explored. The protective effects of 13-E were tested using primary cultured cardiomyocytes in addition to a well-established animal model of cardiac hypertrophy. The results showed that 13-E possesses good cardioprotective activities and attenuates hypertrophy potently, which deserves further investigations.

Synthesis and Characterization of Hybrid 13-E
The synthesis and structure identification of XJP has been reported by our group previously [23]. Briefly, the commercially available benzaldehyde 1 was reduced to alcohol 2 with sodium borohydride in an almost quantitative yield. The subsequent reaction of alcohol 2 with N-methoxy-N-methyl-2-bromopropanamide afforded Weberamide 3 in a 95% yield. Isochroman-4-one 5 was obtained by the cyclization of 3 with t-BuLi as a base, following the deprotection of benzyl in the presence of Pd-C/H 2 . Finally, compound 5 engaged in nucleophile substitution with 1,6-dibromohexane to afford compound 6, which then reacted with 4-cyanophenol to produce intermediate 7.

H2S-Releasing Capability and Safety of 13-E
We first tested the H2S-releasing capability of 13-E by using the methylene blue (MB) method. As shown in Figure 2A, 13-E exhibited excellent H2S-generating ability and produced H2S rapidly with a peak time of around 5-10 min, whereas the negative control Lcysteine did not release H2S. It is interesting that 13-E released H2S smoothly for a period of time with a maximum of 31.9 μM of H2S at around 1 h and only showed a slight downward trend since 80 min, which is consistent with the slow-releasing process of H2S in vivo. The effects of compound 13-E on cell viability were then evaluated to investigate the

H 2 S-Releasing Capability and Safety of 13-E
We first tested the H 2 S-releasing capability of 13-E by using the methylene blue (MB) method. As shown in Figure 2A, 13-E exhibited excellent H 2 S-generating ability and produced H 2 S rapidly with a peak time of around 5-10 min, whereas the negative control L-cysteine did not release H 2 S. It is interesting that 13-E released H 2 S smoothly for a period of time with a maximum of 31.9 µM of H 2 S at around 1 h and only showed a slight downward trend since 80 min, which is consistent with the slow-releasing process of H 2 S In Vivo. The effects of compound 13-E on cell viability were then evaluated to investigate the safety of 13-E, and adriamycin (AMD) was used as a positive control. As expected, 13-E at a concentration of 100 µM had no effects on cell viability, indicating the safety of this compound ( Figure 2B).

H2S-Releasing Capability and Safety of 13-E
We first tested the H2S-releasing capability of 13-E by using the methylene blue ( method. As shown in Figure 2A, 13-E exhibited excellent H2S-generating ability and duced H2S rapidly with a peak time of around 5-10 min, whereas the negative contr cysteine did not release H2S. It is interesting that 13-E released H2S smoothly for a pe of time with a maximum of 31.9 μM of H2S at around 1 h and only showed a slight do ward trend since 80 min, which is consistent with the slow-releasing process of H vivo. The effects of compound 13-E on cell viability were then evaluated to investigate safety of 13-E, and adriamycin (AMD) was used as a positive control. As expected, at a concentration of 100 μM had no effects on cell viability, indicating the safety of compound ( Figure 2B).

13-E Protects against Cardiomyocyte Hypertrophy In Vitro
We further determined the impact of 13-E on the development of cardiac hypertrophy In Vitro. In order to determine whether the anti-hypertrophic efficiency of 13-E was better than the XJP or combination of the XJP with TBZ, we isolated neonatal rat ventricular cardiomyocytes (

13-E Protects against Cardiomyocyte Hypertrophy In Vitro
We further determined the impact of 13-E on the development of cardiac hyper phy in vitro. In order to determine whether the anti-hypertrophic efficiency of 13-E w better than the XJP or combination of the XJP with TBZ, we isolated neonatal rat vent ular cardiomyocytes (NRVCs) and incubated the cells with XJP, TBZ, XJP+TBZ and 1 and detected the induction of Anp and Bnp after phenylephrine (PE) treatment. The res show that the level of XJP (10 μM), TBZ (10 μM) or XJP (10 μM)+TBZ (10 μM) on Anp Bnp after PE treatment had no statistical difference. In contrast, 13-E significantly redu the induction of Anp and Bnp by PE, suggesting an antihypertrophic role of 13-E ( Fig  3).

13-E Restores Cardiac Function
To further demonstrate the cardioprotective effects of 13-E in vivo, sham or TAC m were subjected to the vehicle or 20 mg/kg 13-E (i.p.) for 4 weeks. There was no differe in the heart beats (BMP) of mice between the sham and TAC groups ( Figure S1). The ec cardiographic results show that TAC groups distinctly decreased the functional para ters of the left ventricular ejection fraction (LVEF, %) and left ventricular fractional sh ening (LVFS, %) as compared to the sham group, and these effects could be alleviated

13-E Restores Cardiac Function
To further demonstrate the cardioprotective effects of 13-E In Vivo, sham or TAC mice were subjected to the vehicle or 20 mg/kg 13-E (i.p.) for 4 weeks. There was no difference in the heart beats (BMP) of mice between the sham and TAC groups ( Figure S1). The echocardiographic results show that TAC groups distinctly decreased the functional parameters of the left ventricular ejection fraction (LVEF, %) and left ventricular fractional shortening (LVFS, %) as compared to the sham group, and these effects could be alleviated by 13-E ( Figure 4). Additionally, both the left ventricular end-diastolic volume (LV vol; d, µL) and left ventricular end-systolic volume (LV vol; s, µL) were remarkably increased in vehicle mice subjected to TAC, which were reversed in 13-E-treated mice ( Figure S2A,B). These results manifest that 13-E may contribute to cardiac functions.

13-E Reduces TAC-Induced Cardiac Hypertrophy
The TAC model displayed the development of left ventricular hypertrophy and wall thickening and the progressive development of left ventricular systolic and diastolic dysfunction. Hematoxylin and Eosin (H and E) staining was further performed to evaluate the histological features of cardiac hypertrophy. As shown in Figure 5A, 13-E treatment restrained the increase in heart size caused by TAC surgery. Furthermore, TAC-induced hypertrophy manifested the increase in ratios of heart weight to body weight (HW/BW, mg/g) or tibia length (HW/TL, mg/mm) compared to mice in the sham group. This consequence was availably repressed by 13-E administration ( Figure 5B,C). The cardiac-hypertrophy-related parameters, such as the left ventricular posterior wall dimension (LVPW, mm), the left ventricular internal dimension (LVID, mm) and the interventricular septum thickness (IVS, mm), were increased in the TAC group and were later suppressed by 13-

13-E Reduces TAC-Induced Cardiac Hypertrophy
The TAC model displayed the development of left ventricular hypertrophy and wall thickening and the progressive development of left ventricular systolic and diastolic dysfunction. Hematoxylin and Eosin (H and E) staining was further performed to evaluate the histological features of cardiac hypertrophy. As shown in Figure 5A, 13-E treatment restrained the increase in heart size caused by TAC surgery. Furthermore, TAC-induced hypertrophy manifested the increase in ratios of heart weight to body weight (HW/BW, mg/g) or tibia length (HW/TL, mg/mm) compared to mice in the sham group. This consequence was availably repressed by 13-E administration ( Figure 5B,C). The cardiac-hypertrophyrelated parameters, such as the left ventricular posterior wall dimension (LVPW, mm), the left ventricular internal dimension (LVID, mm) and the interventricular septum thickness (IVS, mm), were increased in the TAC group and were later suppressed by 13-E administration ( Figure 5D,E; Figure S2C-F). These results demonstrate that 13-E is capable of preserving left ventricular functions and delaying the progression of cardiac hypertrophy.

13-E Alleviates Cardiac Interstitial Fibrosis
Interstitial fibrosis is a distinguishing feature of cardiac hypertrophy; as a consequence, we analyzed the extent of myocardial fibrosis in the mice. Masson trichrome staining was used to evaluate collagen deposition. The collagen fibers stain blue, the muscles

13-E Alleviates Cardiac Interstitial Fibrosis
Interstitial fibrosis is a distinguishing feature of cardiac hypertrophy; as a consequence, we analyzed the extent of myocardial fibrosis in the mice. Masson trichrome staining was used to evaluate collagen deposition. The collagen fibers stain blue, the muscles stain red and the nuclei stain black. As expected, TAC surgery resulted in cardiac hypertrophy, reflected in the deposition of myocardial interstitial collagen. The administration of 13-E significantly decreased the fibrosis fraction in TAC mice compared to the vehicle group (n = 3 for each group) ( Figure 6). These results indicate that 13-E plays a vital role in preventing TAC-induced cardiac fibrosis. In summary, 13-E exhibits an obvious cardioprotective effect in In Vivo pressure overload-induced cardiac hypertrophy murine models by reducing myocardial interstitial fibrosis.
Molecules 2022, 27, x FOR PEER REVIEW 8 of 13-E significantly decreased the fibrosis fraction in TAC mice compared to the ve group (n = 3 for each group) ( Figure 6). These results indicate that 13-E plays a vita in preventing TAC-induced cardiac fibrosis. In summary, 13-E exhibits an obvious c oprotective effect in in vivo pressure overload-induced cardiac hypertrophy murine els by reducing myocardial interstitial fibrosis.

13-E Regulates the AMPK Signaling Pathway and Influences Fatty Acid Metabolic Processes
In order to identify candidate genes and pathways that mediate the 13-E-ind protection against cardiac hypertrophy, we performed an unbiased transcriptomic a sis on the left ventricular tissue of mice 4 weeks following the TAC and 13-E treatm Using RNA sequencing (RNA-seq) analysis, we identified 764 differentially expr genes (p adjust < 0.05), in which 356/764 (47%) were downregulated by TAC, whereas 408/764 (53%) were upregulated (Table S1). We then performed Kyoto Encycloped Genes and Genomes (KEGG) pathway analysis, and the results revealed that severa logical processes were altered by the 13-E treatment, including the AMPK signaling way, the PPAR signaling pathway and the insulin resistance and glucagon signaling way ( Figure 7A). The majority of the genes were clustered in the AMPK pathway, w is an interesting observation that we speculated may reflect a regulation of cellula tabolism induced by 13-E. Gene Ontology (GO) enrichment analysis confirmed this ulation that the majority of the genes were clustered in fatty acid metabolic processes ure 7B).
We then verified the expression of several genes in fatty acid metabolic proc using Q-PCR ( Figure 7C-F). Pparg encodes peroxisome proliferator-activated rec gamma (PPARγ), which plays key roles in the storage and mobilization of lipids a glucose metabolism [24]. The level of Pparg reduced significantly in TAC mice, and treatment restored its level. cAMP responsive element-binding protein 3-like 1 (CREB is a member of the CREB3 family of transcription factors. They regulate the expressi

13-E Regulates the AMPK Signaling Pathway and Influences Fatty Acid Metabolic Processes
In order to identify candidate genes and pathways that mediate the 13-E-induced protection against cardiac hypertrophy, we performed an unbiased transcriptomic analysis on the left ventricular tissue of mice 4 weeks following the TAC and 13-E treatments. Using RNA sequencing (RNA-seq) analysis, we identified 764 differentially expressed genes (p adjust < 0.05), in which 356/764 (47%) were downregulated by TAC, whereas only 408/764 (53%) were upregulated (Table S1). We then performed Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and the results revealed that several biological processes were altered by the 13-E treatment, including the AMPK signaling pathway, the PPAR signaling pathway and the insulin resistance and glucagon signaling pathway ( Figure 7A). The majority of the genes were clustered in the AMPK pathway, which is an interesting observation that we speculated may reflect a regulation of cellular metabolism induced by 13-E. Gene Ontology (GO) enrichment analysis confirmed this speculation that the majority of the genes were clustered in fatty acid metabolic processes ( Figure 7B). and 13-E treatment reduced its level. Accumulating evidence indicates that Angiopoietinlike 4 (ANGPTL4) is associated with the risk of atherosclerosis and type 2 diabetes [26]. SLC27A2 is a member of solute carrier family 27, and it enables fatty acid transmembrane transporters [27]. We found that the level of Angptl4 and slc27a2 decreased significantly in the heart tissue of TAC mice, and 13-E treatment reduced the elevations.

Discussion and Conclusions
Cardiac hypertrophy is a major health problem worldwide, and it is a complex process driven by simultaneous changes in hemodynamics, such as hypertension, characterized by increased heart mass. Pathological cardiac hypertrophy is a key risk factor for heart failure, with systolic and diastolic dysfunction and impaired cardiac function. Clinical drugs that can be used to treat cardiac hypertrophy are limited, largely because of the complex etiology and multiple risk factors during the development of cardiac hypertrophy. Therefore, a multi-target therapeutic strategy may be a better choice for the treatment of cardiac hypertrophy [28]. Polyphenolic isochroman-4-one natural product XJP showed multiple cardioprotective effects, including anti-hypertension, anti-atherosclerosis and anti-inflammation, in our previous work. The current study was performed to determine whether the hydrogen-sulfide-releasing isochroman-4-one hybrid has any therapeutic potential in alleviating cardiac hypertrophy.
Results from this work indicate that hydrogen-sulfide-releasing isochroman-4-one hybrid 13-E prevents PE-induced cardiac hypertrophy in isolated neonatal rat ventricular cardiomyocytes effectively, whereas the components of 13-E, XJP and H2S-releasing donor TBZ only attenuate PE-induced cardiac hypertrophy slightly. These results confirm that compound 13-E exhibits its cardioprotective effects as a whole. Importantly, the development of cardiac hypertrophy induced by TAC can be efficiently alleviated by compound We then verified the expression of several genes in fatty acid metabolic processes using Q-PCR ( Figure 7C-F). Pparg encodes peroxisome proliferator-activated receptor gamma (PPARγ), which plays key roles in the storage and mobilization of lipids and in glucose metabolism [24]. The level of Pparg reduced significantly in TAC mice, and 13-E treatment restored its level. cAMP responsive element-binding protein 3-like 1 (CREB3L1) is a member of the CREB3 family of transcription factors. They regulate the expression of a large variety of genes and play roles in ER stress and lipid metabolism [25]. The level of Creb3l1 increased significantly in TAC mice, which may indicate that enhanced ER stress and 13-E treatment reduced its level. Accumulating evidence indicates that Angiopoietinlike 4 (ANGPTL4) is associated with the risk of atherosclerosis and type 2 diabetes [26]. SLC27A2 is a member of solute carrier family 27, and it enables fatty acid transmembrane transporters [27]. We found that the level of Angptl4 and slc27a2 decreased significantly in the heart tissue of TAC mice, and 13-E treatment reduced the elevations.

Discussion and Conclusions
Cardiac hypertrophy is a major health problem worldwide, and it is a complex process driven by simultaneous changes in hemodynamics, such as hypertension, characterized by increased heart mass. Pathological cardiac hypertrophy is a key risk factor for heart failure, with systolic and diastolic dysfunction and impaired cardiac function. Clinical drugs that can be used to treat cardiac hypertrophy are limited, largely because of the complex etiology and multiple risk factors during the development of cardiac hypertrophy. Therefore, a multi-target therapeutic strategy may be a better choice for the treatment of cardiac hypertrophy [28]. Polyphenolic isochroman-4-one natural product XJP showed multiple cardioprotective effects, including anti-hypertension, anti-atherosclerosis and anti-inflammation, in our previous work. The current study was performed to determine whether the hydrogen-sulfide-releasing isochroman-4-one hybrid has any therapeutic potential in alleviating cardiac hypertrophy.
Results from this work indicate that hydrogen-sulfide-releasing isochroman-4-one hybrid 13-E prevents PE-induced cardiac hypertrophy in isolated neonatal rat ventricular cardiomyocytes effectively, whereas the components of 13-E, XJP and H 2 S-releasing donor TBZ only attenuate PE-induced cardiac hypertrophy slightly. These results confirm that compound 13-E exhibits its cardioprotective effects as a whole. Importantly, the development of cardiac hypertrophy induced by TAC can be efficiently alleviated by compound 13-E In Vivo. In previous work, XJP has been shown to specifically reduce LPS-accelerated vascular inflammation as a novel ACEI [7]. ACEIs have been reported to be effective in reducing left ventricular mass in hypertension and heart failure [29]. These studies are in accordance with what was observed in this study, i.e., that 13-E can efficiently reduce HW/BW and reduce the thickness of the left ventricular posterior wall, LVID and IVS. However, according to our previous reports, XJP can reduce blood pressure effectively in spontaneously hypertensive rats. These anti-hypertrophic effects seem to be derived from a direct protective effect on cardiomyocytes, rather than as a consequence of reductions in blood pressure, as compound 13-E can alleviate hypertrophy in cultured cardiomyocytes. Interstitial fibrosis is an important marker of cardiac dysfunction and a predictor for the poor prognosis of cardiac hypertrophy. In animals treated with compound 13-E, a reduction in interstitial fibrosis determined by Masson staining was observed, indicating that compound 13-E can improve cardiac compliance. As a result, cardiac functions including ejection fraction and fractional shortening were improved in compound 13-E-treated animals.
ATP synthesis and catabolism are dynamic processes in the maintenance of cellular homeostasis, especially in cardiomyocytes with high energy remanding. It is well-known that cellular energy depletion can activate AMPK by increasing the ratio of AMP/ATP. In normal hearts, fatty acid oxidation is the main source of energy for cardiomyocyte constriction. However, during cardiac hypertrophy, fatty acid oxidation is impaired, and there is an imbalance between energy production and consumption, which can incite many signaling pathways associated with cellular energetic metabolism. In fact, H 2 S has been reported to activate AMPK during cardiac dysfunction, and interestingly, we also found that 13-E mainly influences genes associated with fatty acid oxidation and the AMPK pathway, indicating that the protection effects of 13-E on cardiac hypertrophy may be mediated by regulating cellular energetic metabolism.
The combination principle is commonly used to design H 2 S-releasing drugs, as these hybrids often possess superior pharmacodynamic and pharmacokinetic characteristics. The current study demonstrates that hybrid 13-E effectively reduces cardiac hypertrophy and fibrosis and improves cardiac function in TAC-induced cardiac hypertrophy. Interestingly, the co-administration of purified XJP combined with TBZ was also found to alleviate hypertrophy; however, this drug combination is less efficient than hybrid 13-E, suggesting the independence of compound 13-E. Together with the cardioprotective effects of XJP and H 2 S, compound 13-E can be developed to be a promising drug candidate for the treatment of cardiac hypertrophy in the future.

Chemistry
1 H NMR and 13 C NMR spectra were recorded on a Bruker AV-300 NMR, the deuterated solvents were CDCl 3 and DMSO-d 6 and the mass spectra were obtained on an Agilent 1100-LC-MSD-Traps/SL. All reagents and solvents were commercially available and were used without further purification. Silicagel 60 H (200-300 mesh), manufactured by Qingdao Haiyang Chemical Group Co., Ltd. (Qingdao, China), was used for general chromatography.

6-(Benzyloxy)-7-methoxy-3-methylisochroman-4-one (4)
Compound 1 (5.0 g, 1 eq) was added to the appropriate amount of methanol solution, and NaBH 4 (0.4 g, 1 eq) was added several times. After stirring for 30 min at room temperature, the reaction solution became clear. After the reaction was completed, the reaction solution was concentrated under reduced pressure, and a small amount of water was slowly added. The white solid was separated out, and about 6.2 g of compound 2 was obtained by a brewer funnel and was used directly without further purification.
Compound 2 (6.0 g, 1 eq) was dissolved in DMF, N-methoxy-N-methyl-2-bromopropanamide (2.3 mL, 1.5 eq), and a catalytic amount of NaH was added. The reaction was stirred at R.T. for 30 min. After the reaction was completed, the reaction solution was transferred to the separation funnel, and ethyl acetate was extracted three times. Then, the organic layer was combined, was washed with saturated NaCl solution, was dried to obtain about 5.0 g of compound 3 and was used directly without further purification.
Compound 3 (5.0 g, 1 eq) was placed in a three-neck flask and was dissolved in anhydrous THF in a nitrogen-protected atmosphere. Under −78 • C, the tert-butyl lithium solution (25 mL, 2.2 eq) was injected into the reaction bottle and was stirred for about 15 min. After the reaction was completed, the ammonium chloride solution was added to quench the reaction. The excess THF was first spun out, and then it was extracted with water and ethyl acetate three times. About 2.5 g of white solid compound 4 was obtained through column purification (petroleum ether: ethyl acetate 10:1). 1 (5) Compound 4 was dissolved in methanol, and 10% Pd/C was added. Hydrogen was completely replaced, and the reaction was carried out at room temperature overnight. Pd/C was removed by filtration, and the solvent was dried and passed through the column (petroleum ether: ethyl acetate 2:1) to obtain about 1.0 g of white solid compound 5. 1  4.1.3. 6-((6-Bromohexyl)oxy)-7-methoxy-3-methylisochroman-4-one (6) K 2 CO 3 (276 mg, 2.0 eq) was added to a solution of compound 5 (100 mg, 1 eq) in anhydrous acetone, and the mixture was refluxed for 30 min. Then, 1, 6-dibromohexane (210 µL, 3 eq) was added and the mixture and was refluxed for 2 h. After being filtrated and concentrated under reduced pressure, followed by being purified by flash column chromatography with n-hexane/ethyl acetate (8:1, v/v) as an eluent, compound 6 was afforded as a white solid in a yield of 85%. 1 (7) 4-Cyanophenol (40 mg, 1.2 eq) was dissolved in anhydrous acetonitrile, and K 2 CO 3 (130 mg, 3 eq) was added. After refluxing for 30 min in a nitrogen-protected atmosphere, compound 6 (100 mg, 1 eq) was added. The whole reaction system continued to reflux for 6 h. After being filtrated and concentrated under reduced pressure, the filtrate was diluted with ethyl acetate, was washed with water and brine and was dried with anhydrous Na 2 SO 4 . Purified by flash column chromatography, compound 7 (about 100 mg) was afforded as a white solid in a yield of 76%. 1

H 2 S Release Experiment
Sodium phosphate buffer was used to prepare the stock solution of Na 2 S (20 mM) in a 100 mL volumetric flask. Aliquots of Na 2 S stock solution were transferred into a 50 mL volumetric flask to obtain standard solutions of 5, 10, 20, 40, 60, 80, 100 and 150 mM, respectively. An amount of 1 mL of each standard solution was added to react with the MB cocktail (200 mL of 30 mM FeCl 3 in 1.2 M HCl, 200 mL of 20 mM N, N-dimethyl-1,4phenylenediaminesulfate in 7.2 M HCl and 100 mL of 1% w/v of Zn(OAc) 2 in H 2 O) at room temperature for 20 min in a triplicate manner. The mixture was measured at 670 nm in a UV-Vis spectrophotometer, and then the Na 2 S calibration curve was obtained. In order to promote the compounds to release H 2 S, L-cysteine was used as an accelerator. All compounds were dissolved in THF solution (40 mM) and were added into a phosphate buffer in the presence of L-cysteine (1 mM). Then, 2 mL of mixture was transferred to a colorimetric cuvette containing MB + cocktail in the designated time. After incubation for 20 min, the absorbance of each compound was analyzed by a UV-Vis spectrophotometer at 670 nm. The H 2 S concentration of each derivative was calculated through a standard curve.

Determination of Cytotoxicity
Cytotoxicity was evaluated with the colorimetric MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] assay. CHO cells were seeded at 5 × 10 4 cells/well in 96-well plates. After 24 h, the medium was removed and replaced with the tested compounds at different concentrations for 24 h at 37 • C. After the replacement of the tested compounds with 80 µL of medium and 20 µL of MTT in PBS (0.5 mg/mL, final concentration), the cells were incubated for another 4 h. After the removal of MTT, the formazan crystals were dissolved in DMSO. The amount of formazan was measured (570 nm). Cell viability was expressed as the percentage of control cells and was calculated using the formula Ft/Fnt × 100, where Ft is the absorbance of the treated neurons after subtracting the absorbance of the zero-day control, and Fnt is the absorbance of the untreated neurons after subtracting the absorbance of the zero-day control.

Animals
All animal care and experiments were approved by the Committee on Animal Care and Use of Nanjing Medical University and were conducted in compliance with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health. All mice were housed in a controlled environment, with regulations of temperature (22 ± 1 • C) and humidity (55%), a 12:12 h dark-light cycle and being fed with a standard chow diet. A total of 32 male C57BL/6 mice at the age of 8 weeks were randomly divided into four groups: vehicle + sham (n = 8), 13-E + sham (n = 8), vehicle + TAC (n = 8) and 13-E + TAC (n = 8). All TAC group mice were subjected to minimally invasive TAC surgery.
Briefly, mice were anesthetized with 5% isoflurane, were maintained with 2.5% isoflurane during surgery and were secured to an operating table. The hair on the neck and chest was removed using a depilatory agent, and then the surgery area was disinfected with alcohol. The chest was opened, and the aortic arch was isolated by blunt dissection through an intercostal incision. The transverse aorta was constricted by a 7-0 silk suture ligature tied firmly against a 27-gauge needle, and then the needle was subsequently removed after ligation. The sham mice were subjected to the same surgical procedure without constriction of the aorta. The incision was then closed. After surgery, the mice were allowed to fully recover on a heating pad and were housed in standard housing conditions.

Neonatal Rat Cardiomyocyte Isolation, Culture and Treatment
Cardiomyocytes were isolated from neonatal rats of 1 to 4 days old. After disinfecting the chest with 75% ethanol, the sternums were cut, and the hearts were removed. The residual blood clots and atria were removed in Dulbecco's Modified Eagle Medium (DMEM), and the ventricular tissues were minced by dissecting scissors. The ventricular tissues were digested by mixed enzyme solution (0.25% Trypsin-EDTA) in a 37 • C water bath for 5 min. The supernatant was collected and neutralized with DMEM medium containing 20% fetal bovine serum (FBS). Repeated digestions were performed 7-9 times until cells were isolated completely. The collected cell suspension was centrifuged at 2000 rpm for 10 min. The sediments were resuspended in DMEM supplemented with 10% FBS. The cells were then plated with different culture dishes according to the specific experimental requirements at 37 • C in the presence of 5% CO 2 in a humidified incubator. After culturing in a serum-free medium for 6-8 h, the primary cardiomyocytes were incubated for 48 h with 50 µmol/L of phenylephrine (PE) to induce cardiomyocyte hypertrophy In Vitro, and phosphate buffer saline (PBS) was used as a control under the conditions of 37 • C, 5% CO 2 and 95% O 2 .

Echocardiography
Cardiac functions were assessed by echocardiography with Vevo 770 after TAC or sham surgery for 4 weeks. Briefly, mice were anesthetized with 2% isoflurane and were adjusted to maintain heart rates in the range of 415-460/min during echocardiogram acquisition. Transthoracic echocardiography of the left ventricle was performed to measure LV wall thickness, LV chamber size, LV function and LV mass. The parameters of cardiac function that were collected include: LVEF, LVFS, left ventricular volume (LV), LVPW, LVID and IVS.

Histological Staining
After surgery, the heart tissues were fixed with 4% polyformaldehyde and were finally embedded into paraffin. The heart-embedded paraffin blocks were cut into 5 µm sections using a microtome and were mounted on slides. To evaluate cardiac histological changes and fibrosis, the paraffin-embedded heart sections were dewaxed, rehydrated and subjected to H and E staining and Masson's trichrome. Images were taken by a light microscope. The images were analyzed by using the ImageJ analysis system. Supplementary Materials: The spectra of 1 H-NMR and 13 C-NMR for the target compound are available online: https://www.mdpi.com/article/10.3390/molecules27134114/s1, Figure S1: 8 weeks old mice were treated with Vehicle or 13-E, which were performed with Sham or transverse aortic constriction (TAC) operation for 4 weeks (n = 8 for each group). Heart rates were detected for each group. The results were presented as mean ± SEM. ns: no significant; Figure S2