Synthesis and in vivo screening of isosteviol derivatives as new cardioprotective agents

European Journal of Medicinal Chemistry 219 (2021) 1 13396

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European Journal of Medicinal Chemistry

journal homepage: h ttp://www.elsevier.com/locate/ejmech

Synthesis and in vivo screening of isosteviol derivatives as new

cardioprotective agents

Hanyuan Zhang ¹, Bo Liu ¹, Geng Xu, Chao Xu, E. Ou, Jiansong Liu, XiaoOu Sun^**, Yu Zhao^*

School of Biomedical and Pharmaceutical Sciences, Guangdong University of Technology, Guangzhou, 510006, China

a r t i c l e i n f o

a b s t r a c t

Article history:

Isosteviol, an ent-beyerane diterpenoid, has been repeatedly reported to possess potent cardioprotective

activity. With the aim of discovering new cardioprotective derivatives from isosteviol, 47 compounds,

including 40 new ones, were synthesized and evaluated in vivo using the easy-handling and efﬁcient

zebraﬁsh model. The structure-activity relationship of this type of compounds was thus discussed. Of

these compounds, new derivative 15d exhibited the most pronounced efﬁcacy in vivo. Our results

indicated that 15d could effectively prevent the doxorubicin-induced morphological distortions and

cardiac dysfunction in zebraﬁsh. Its cardioprotective activity is much better than that of isosteviol, and

Levosimendan in zebraﬁsh model. The molecular mechanism underlying in H9c2 cells indicated that 15d

protected cardiomyocyte death and damage through inhibiting the reactive oxygen species over-

production, restoring the mitochondrial membrane potential and maintaining morphology of mito-

chondrial. Thus, 15d merits further development as a potential cardioprotective clinical trial candidate.

The present study is a successful example to combine synthesis, structure-activity relationship study and

in vivo screening to effectively discover new cardioprotective agents from isosteviol.

Received 10 February 2021

Received in revised form

17 March 2021

Accepted 18 March 2021

Available online 28 March 2021

Keywords:

Isosteviol

Cardioprotective activity

Zebraﬁsh

In vivo screening

Structure-activity relationship (SAR)

1. Introduction

be unabated.

Natural products, the vital source of pharmaceutical agents,

Cardiovascular disease (CVDs) is the leading cause of death and

disability globally. It accounts for around 17.9 million death, 31% of

all death worldwide, each year [1]. People with CVDs or who are at

high cardiovascular risk need early detection and treatment. Car-

dioprotective drugs, which can preserve the metabolism, structure

and function of the heart and vasculature and limit their damage

either in primary or secondary prevention, are extremely important

in the treatment of patients with CVDs or at risk for CVDs. Although

several groups of cardioprotective drugs including statins, angio-

tensin converting enzyme inhibitors (ACEIs), beta-blockers (BBs),

and blockers or receptors for angiotensin II type one (ARBs) have

been proven to be effective in achieving the hard endpoints, the

mortality reduction with treatment of these drugs does not exceed

30% and the residual cardiovascular risks are still rather high [2].

Therefore, discovery of novel cardioprotective agents continues to

have long been used to treat CVDs [3]. Stevioside (1, Fig. 1), a

diterpenoid triglycoside, is a natural sweetener. Stevioside (1) is the

principal constituent of the leaves of Stevia rebaudiana, which is a

perennial herb shrub, originated from Brazil and now cultivated

worldwide for producing natural sweeter [4]. Acid hydrolysis of the

glycoside of 1 following by a Wagner-Meerwein rearrangement

generated isosteviol (2, Fig. 1), an ent-beyerane diterpenoid, in a

rather high yield [5]. Both 1 and 2 exhibit broad spectrum phar-

macological effects including antidiabetic, anticancer, anti-

inﬂammatory, anti-hyperglycaemic, anti-hypertensive, anti-diar-

rheal, diuretic and immunomodulator activity, and cardiovascular

protection effects [4,6,7]. The cardioprotective effect of 2 and its

sodium form have been widely evaluated in various in vivo models

[4]. For example, isosteviol (2) was reported to reduce the damage

due to a rat heart ischaemia-reperfusion (IR) injury at 0.5, 1.0 and

2.0 mg/kg [8]. And it was also reported to signiﬁcantly attenuate the

myocardium IR injury induced by IR in the isolated guinea pig heart

[9], and attenuated the IR-induced prolongation of the QTc interval

and inhibited I_kr[10].

* Corresponding author.

** Corresponding author.

E-mail addresses: xiaoousun@gdut.edu.cn (X. Sun), yuzhao@gdut.edu.cn

(Y. Zhao).

Because of their potent pharmacological activities, novel natural

skeleton, good synthetic accessibility, and signiﬁcant abundance in

1

H.Y.Z. and B. L. are co-ﬁrst authors.

https://doi.org/10.1016/j.ejmech.2021.113396

H. Zhang, B. Liu, G. Xu et al.

European Journal of Medicinal Chemistry 219 (2021) 113396

further evaluation of their cardioprotective effect in the zebraﬁsh

model. Finally, 15d with the most pronounced cardioprotective

effect was identiﬁed. And its molecular mechanism underlying in

H9c2 cells was also investigated.

2. Chemistry

Our initial efforts were focused on the modiﬁcation of C-19

carboxylic acid and cyclopentanone in the ring D of 2. Compounds

3e10 were thus synthesized (Scheme 1). Isosteviol (2), the starting

material, was obtained via the acid hydrolysis of 1 with HBr

following the reported method [5]. Esteriﬁcation of 19-COOH of 2

with benzyl bromide (BnBr) or 4-methoxybenzyl chloride (PMBCl)

and K₂CO₃in acetone afforded 3 and 4, respectively. Methyl ester-

iﬁcation of 19-COOH of 2 with methyl iodide (CH₃I) and tetrabu-

tylammonium ﬂuoride (TBAF) produced 5. Conversion of 19-COOH

of 2 into amine via the Curtius rearrangement with diphenyl

phosphoryl azide (DPPA) and Et₃N in t-BuOH yielded 6. Hydroxyl-

ation of 2 via aldol/Cannizzaro reaction with excessive para-

Fig. 1. The structure of stevioside (1) and isosteviol (2).

the plant, extensive modiﬁcations on the 1 and 2 for new de-

rivatives with anticancer activity were accomplished by several

research groups [1 1e23]. However, research on the development of

new cardioprotective derivatives of 2 is rather limited. Only one

paper reported the synthesis of six triphenylphosphonium-

conjugated isosteviol derivatives and their cardioprotective ef-

fects [24]. And another paper reported the cardioprotective effect of

one derivative of 2 in H9c2 cells [25].

In the past two decades, the zebraﬁsh has become increasingly

popular animal model which enable efﬁcient large-scale in vivo

compounds screening in the early phase of drug discovery [26]. As

compared with the cell-based in vitro system, in vivo screening

provides several advantages, such as the ability to evaluate the

compounds in an intact animal and discover compounds with the

ability to be effective in the complex context of the disease interest.

Zebraﬁsh screening have discovered several compounds which

have been advanced in preclinical and clinical trials [27]. The car-

diovascular physiology between human and zebraﬁsh is highly

conserved at anatomical, cellar and membrane-biology levels.

Several cardiovascular drugs were found to exhibit identical effects

on zebraﬁsh physiology [27,28]. Zebraﬁsh as a tool for cardiovas-

cular drug screen combined the advantages of in vitro screening,

such as easy handling, cost-effectiveness, and living organism

evaluation which can easily access important cardiovascular pa-

rameters including heartbeats, ventricular systolic pump function,

and so on [26,29].

Doxorubicin (DOX), a type of anthracycline antibiotic, is widely

used to treat many types of cancers [30]. However, DOX was found

to have serious cardiotoxicity which led to acute inﬂammation,

irreversible degenerative cardiomyopathy, and congestive heart

failure in humans and laboratory animals [31]. The detailed studies

and characterization of the DOX-treated animals such as mice,

zebraﬁsh, indicated that their DOX-induced cardiomyopathy is

comparable to that in humans and thus is an excellent model for

the study of cardiomyopathy progression and cardiovascular pro-

tective drug screening [32e34].

With the aim of discovering new cardioprotective agents from 2,

we initiated the study to synthesize and in vivo assay the de-

rivatives using the easy-handling DOX-treated zebraﬁsh model.

Firstly, modiﬁcation of the functional groups presented in the

structure of 2, C-19 carboxylic acid and cyclopentanone in the ring

D, were performed to explore their structure-activity relationship

(SAR). Subsequently, based on the constructed SAR, a diversity-

oriented modiﬁcation of 2 was conducted. Forty-seven com-

pounds thus synthesized were ﬁrstly evaluated for their ability to

protect zebraﬁsh against DOX-induced mortality. Subsequently,

ﬁve derivatives with potent protective activity were selected for

formaldehyde and NaOH in ethanol produced the 16

a-hydroxy-

15 -hydroxymethyl derivative 7 in high yield [35,36]. The stereo-

b

chemistry of 7, determined with 2D NMR spectroscopy (Fig. S1,

Supporting Information), is consistent with literature [35,36]. The

reaction of 7 with p-methyl phenylsulfonyl chloride (TsCl) in pyri-

dine gave 8. Oxidation of the 16-OH of 8 generated the 16-keto 9

which was further converted to the 16-keto-15-ene 10 via an

elimination reaction.

The synthetic routes employed to modify 16

hydroxymethyl moiety of 7 was depicted in Scheme 2. Selective

acylation of the 15 -hydroxymethyl of 7 with acetic anhydride

a-hydroxy-15b-

b

(Ac₂O), cyclohexanecarboxylic acid, nicotinic acid, isonicotinic acid,

benzoic acid and its analogs, 3,5-dimethoxycinnamic acid, 4-

methoxyphenylacetic acid and 3,4,5-trimethoxyphenylacetic acid

gave 11ae11n, respectively. Further oxidizing 16-OH of 11ae11n

produced their 16-keto derivatives 12ae12n, respectively.

Starting from 7, modiﬁcation of 19-COOH and 16a-hydroxy-15b-

hydroxymethyl moiety was carried out following the routes in

Scheme 3. Conversion of 19-COOH of 7 to the primary NH₂group

via Curtius rearrangement produced 13. Esteriﬁcation of 15b-

hydroxymethyl of 13 with nicotinic acid, 4-methoxybenzoic acid,

2,3-dimethoxybenzoic acid, 2,3,4-trimethoxybenzoic acid, and

3,4,5-trimethoxybenzoic acid gave 14ae14e, respectively. Finally,

the corresponding 16-keto products 15ae15e were obtained from

14aꢀ14e via oxidation reaction with PDC reagents.

3. Results and discussion

3.1. Preliminary screening of 2e15 in DOX-treated zebraﬁsh

embryos and SAR analysis

All synthesized derivatives, including 40 new (4, 11ae11n,

12ae12n, 13, 14ae14e and 15ae15e) and 7 known compounds (3,

5e10), accompanying with the starting material 2, were subjected

to an easy-handling and efﬁcient zebraﬁsh assay system for their

cardioprotective effect evaluation. Levosimendan (LSD), the drug

used in the treatment of heart failure and for cardioprotection, was

used as the positive control [37,38]. Firstly, the survival of the DOX-

treated zebraﬁsh embryos in the absence or presence of each test

compound was measured. The compounds with potent protective

activity, indicating by their ability to protect zebraﬁsh against the

DOX-induced mortality, were then selected for further evaluation,

including dose-response study, toxicity assay and cardioprotective

effect assay.

As shown in Fig. 2, the embryos were treated with DOX (model

group), or DOX with each of test compounds (2e10, 11ae11n,

2

H. Zhang, B. Liu, G. Xu et al.

European Journal of Medicinal Chemistry 219 (2021) 113396

Scheme 1. Reagents and conditions: (a) BnBr for 3 or PMBCl for 4, K₂CO₃, acetone, 50 ^ꢁC; (b) CH₃I, TBAF, THF; (c) DPPA, Et₃N, t-BuOH, reﬂux; (d) NaOH, (HCHO)n, EtOH, reﬂux; (e)

TsCl, pyridine, DMAP, reﬂux; (f) PDC, DMF; (g) Pyridine, DMAP, reﬂux.

Scheme 2. Reagents and conditions (a) Ac₂O, Pyridine, DMAP for 11a; (b) acid, DMAP, EDCI, DCM for 11be11n; (c) PDC, DMF.

12ae12n, 13, 14ae14e, 15ae15e, and LSD) at 5, 15, and 40

mM,

52% to 57%. While treatment of 2 at 15 and 40 mM, respectively, has

respectively. In comparison with the control group (100% survival

rate), the survival rate of the model group dramatically dropped to

no evident effect on the survival rate. Among the 47 derivatives, 37

ones, at each concentration or a certain concentration, reduced the

DOX-induced mortality. In contrast, the other 10 derivatives

including 10, 11n, 12a, 12b, 12e, 12g, 12l, 12n, 13, and 15b, at each

concentration, further increased the DOX-induced mortality.

52%. When treated with LSD, the positive drug, at 5, 15, and 40

the survival rate has dose-dependently risen to 58%, 69% and 74%,

respectively. Isosteviol (2), at 5 M, increased the survival rate from

mM,

m

3

H. Zhang, B. Liu, G. Xu et al.

European Journal of Medicinal Chemistry 219 (2021) 113396

Scheme 3. Reagents and conditions: (a) DPPA, Et₃N, ^tBuOH, reﬂux; (b) Acid, DMAP, EDCI, DCM; (c) PDC, DMF.

Based on the result (Fig. 2), SAR was deduced (Fig. 3). Firstly, we

explored the effect of modiﬁcation of C-19 on the activity. Con-

version the 19-COOH of 2 to its esters (3e5), led to some positive

their appropriate 16-keto derivatives (12ae12r). Among the

various ester groups presented at the 15b-methyl-O-acyl moiety,

the ethyl group (11a, 12a), and cyclohexane (11b, 12b), which could

not decrease or even increased the DOX-induced mortality, are not

conducive for the good activity. While mono-, di-, and tri-methoxy

substituted benzyl group (11g, 11i, 12h, 12i, 12j, and 12k), and

nicotinoyl group (12c) beneﬁt the activity.

effect on the survival rate (around 60%, with 3 at 5

mM, 4 at 15 mM,

and 5 at 40 M, respectively). Replacement of the 19-COOH with

m

the primary amine group (6) signiﬁcantly reduced the DOX-

induced mortality. The survival rate of zebraﬁsh embryos treated

with 6 (85% at 5

m

M) is much higher than 2 (57% at 5

m

M) and LSD

Next, we further synthesized another series of derivatives via

simultaneously replacing the 19-COOH with amine and acylating

the mono-, di-, and tri-methoxy substituted benzoic acid and iso-

(74% at 40 M). These results suggested that 19-COOH of 2 can be

m

modiﬁed for good protective activity. The activity of 19-COOH

modiﬁed derivatives was ranked as amine derivate (6) > ester de-

rivatives (3, 4, and 5) > isosteviol (2).

Next, we explored how the modiﬁcation of cyclopentanone in

the ring D affected the protective activity. Hydroxylation of 2 (7)

and further modiﬁcation of the 16

moiety of 7 via esteriﬁcation and oxidation (8 and 9) led to

improved activity. Derivatives 7, 8, and 9, at 5, 15, 40 M, risen the

nicotinic acid to 15b-hydroxymethyl of 7. Derivatives 13, 14ae14e

and 15ae15e were thus obtained. Hydroxylation of 6 led to 13,

which greatly increased the DOX-induced mortality. Acylation of 13

with various acids led to derivatives 14ae14e with moderate ac-

tivity. Further oxidization of the 16-OH of 14ae14e gave 15ae15e,

among which, 15d and 15e, the most potent ones, at 40 mM, were

found to signiﬁcantly increase the survival rate to around 98% and

a-hydroxy-15b-hydroxymethyl

m

survival rate from 52% to 60%e67%. In contrast, derivative 10 with

the exo-methylene cyclopentanone moiety in the structure signif-

icantly increased the DOX-induced mortality in a dose-dependent

87%, respectively.

3.2. Dose-response and toxicity evaluation

manner. The survival rate treated with 10 at 5, 15, 40 mM is 50%,

38%, 26%, respectively. It has been repeatedly reported that exo-

methylene cyclopentanone moiety is an important pharmaco-

phore for generating signiﬁcant anticancer derivatives of isosteviol

(2) [4]. However, in the present study, exo-methylene cyclo-

pentanone moiety is detrimental.

Based on the above results, derivatives 6, 12j, 12k, 15d, and 15e,

which showed signiﬁcant activity and possesses different structural

features, accompanying with starting material 2 and positive drug

LSD, were selected for further study. Firstly, the dose-response of

these compounds was explored. As shown in Fig. 4, all derivatives

can dose-dependently improve the survival rate at a range of

concentrations. The optimum concentration of 2, 6, 12j, 12k, 15d,

Derivative 7 contained two hydroxyl groups which are readily

available for further modiﬁcation. The above results also suggested

that good activity can be obtained by altering the 16

hydroxymethyl moiety. Therefore, we further performed the

diversity-oriented modiﬁcation on 16 -hydroxy-15 -hydrox-

ymethyl to produce 28 derivatives containing 16 -hydroxy-15

methyl-O-acyl unit (11ae11n) or 16-keto-15 -methyl-O-acyl frag-

a-hydroxy-15b-

15e and LSD are 10, 0.8, 70, 60, 40, 60, and 50 mM, respectively

(Fig. 4). Furthermore, to test whether these compounds will cause

any toxicity, the embryos were treated with the tested compounds

alone at different concentrations. The results demonstrated that

none of the tested compounds caused mortality in zebraﬁsh even at

a much higher concentration than their optimum concentration

(Fig. 4).

a

b

a

b-

b

ment (12ae12n). The preliminary screening results indicated that

most derivatives (21 out of 28) showed protective activity. Seven

derivatives (11g, 11i, 11m, 12h, 12i, 12j, and 12k), at the test con-

centrations, increased the survival rate from 52% to more than 70%.

3.3. Cardioprotective effects of derivatives on DOX-induced

cardiomyopathy in zebraﬁsh embryos

Among them, 12j containing 16-keto-15

trimethyl benzoyl unit and 12k with 16-keto-15

b

-methyl-O-2,3,4-

-methyl-O-3,4,5-

trimethyl benzyl moiety in the structure are most potent ones,

which signiﬁcantly risen the survival rate to 77%e82% (12j) and

68%e80% (12k) at 5,15, and 40 mM, respectively.

In this series of compounds, the hydroxyl group or the ketone

group presented at C-16 is not determinant for the activity, because

both positive and negative effect on the survival rate can be

Subsequently, we further assayed the cardioprotective effect of

2, 6, 12j, 12k, 15d, 15e, and LSD using the transgenic zebraﬁsh

Tg(cmlc2:GFP), which speciﬁcally expressed the green ﬂuorescent

protein (GFP) in the myocardium. Four cardiovascular parameters,

including heartbeat, fractional shortening, stroke volume, and

cardiac output, were measured to evaluate the cardiac function. As

shown in Fig. 5, the zebraﬁsh treated with DOX alone (model

observed when converting 16a-hydroxy derivatives (11ae11r) to

4

Fig. 2. The ability of 2e15 to protect zebraﬁsh against the DOX-induced mortality. The 24 h post-fertilization (hpf) embryos were treated with 100 mM DOX (model), or 100 mM DOX with each test compound at 5, 15, and 40 mM. The

survival of zebraﬁsh was assessed at 96 hpf. The survival rate of DOX treated zebraﬁsh in the presence of compound 2e10 (A), compound 11a-11n (B), compound 12a-12n (C), compound 13, 14a-14e and 15a-15e (D). Data are presented

as mean S.D. (n ¼ 4).

H. Zhang, B. Liu, G. Xu et al.

European Journal of Medicinal Chemistry 219 (2021) 113396

Fig. 3. The SAR analysis, diversity-oriented modiﬁcation and the structure of the derivatives selected for further study.

group) exhibited severe cardiac edema and its heart was distorted

into an elongated shape with compact ventricle and atrium. Sig-

niﬁcant reductions in the fractional shortening, stroke volume, and

cardiac output, and moderate decrease in heart rate were observed.

These cardiovascular parameters and the morphological distortions

clearly indicated the abnormal ventricular ﬁlling and systolic

dysfunction. Cotreatment of DOX and the test compound (2, 6, 12j,

12k, 15d, 15e, and LSD) at their optimum concentration greatly

reduced the cardiac edema, maintained the normal heart shape,

and improved the cardiac performance impaired by DOX. Among

all the tested compounds, 15d exhibited the most signiﬁcant efﬁ-

ciency. The cardioprotective activity was ranked as 15d > 15e > LSD

>6 > 12j > 2 > 12k.

important role in cardiac and vascular abnormalities in various

cardiovascular diseases [39]. Therefore, we then explored if 15d can

affect the DOX-induced ROS overaccumulation. As shown in Fig. 8,

comparing with the control group, the higher ﬂuorescence in-

tensity of H9c2 cells treated with DOX was observed, which indi-

cated that DOX induced ROS overproduction. Co-treatment of 15d

and DOX signiﬁcantly decreased the ﬂuorescence intensity in dose-

dependent manner. These results clearly indicated that 15d can

reduce the overproduction of ROS thus protecting the cell from

oxygen stress damage. Mitochondrial plays a pivotal role in car-

dioprotection and is regarded as the promising target for the new

cardioprotective agents [40]. Mitochondrial membrane potential

(Djm) is commonly used as the indication of the mitochondrial

Furthermore, the cardioprotective effect of 15d was evaluated at

status. A loss of Djm represented the mitochondrial dysfunction.

Subsequently, we, therefore, examined the inﬂuence of 15d on the

Djm of H9c2 cells treated with DOX. JC-1, the membrane sensitive

dye, was used to stain cells to detect the changes of the Djm. Our

result showed that when treated with DOX, the Djm of the cells

was markedly reduced. Cotreatment of 15d dose-dependently

recovered the Djm (Fig. 9). The morphology changes of mito-

chondrial were also explored. Mitochondria treated with DOX

became fragmental and the length of mitochondrial appeared

short. Cotreatment of 15d can ameliorate such morphology changes

(Fig. 9). Taken together, these results indicated that 15d could

effectively attenuate the DOX-induced oxidative stress, restore the

Djm, maintain the mitochondrial morphology and thus protect the

cell from damage.

20, 40, and 60

dependently protected the heart and improve cardiac function.

While 15d at 60 M was unable to rescue the heart so effectively as

it did at 40 M, which conﬁrmed our dose-response result that 15d

exerted its best efﬁcacy at 40 M (Fig. 6).

mM. Our results indicated that 15d (20e40 mM) dose-

m

3.4. Derivative 15d decreased the elevated mRNA level of the

cardiomyopathy biomarkers cTnT and ANP in DOX-treated zebraﬁsh

embryos

Cardiac troponin T (cTnT) and atrial natriuretic peptide (ANP)

are two speciﬁc diagnostic biomarkers for cardiomyopathy disease.

To further conﬁrm the cardioprotective effects of 15d at the gene

level, we assayed the mRNA level of cTnT and ANP of zebraﬁsh by

qPCR. As shown in Fig. 7, DOX treatment led to a signiﬁcant increase

in mRNA level of CTnT and ANP. Derivative 15d markedly decreased

the cTnT and ANP level in a dose-dependent manner. These results

presented strong molecular evidence for the cardioprotective po-

tency of 15d.

4. Conclusion

With an aim of discovering new derivatives with promising

cardioprotective activity from isosteviol (2), a diversity-oriented

modiﬁcation based on the structural feature of 2 was performed.

Forty-seven derivatives, including 40 new ones, were thus syn-

thesized. The cardiovascular protective activity of the derivatives

was evaluated in vivo, with the employment of an easy-handing

and effective zebraﬁsh model. The SAR study of isosteviol de-

rivatives was discussed. Among the 47 derivatives synthesized, 15d

exhibited the most pronounced efﬁciency in vivo. The new deriv-

ative 15d could effectively reduce the cardiac edema, maintain the

normal heart shape and prevent the cardiac dysfunction impaired

by DOX in zebraﬁsh. And its cardioprotective effect is superior to

that of 2 and LSD. The molecular mechanism underlying in DOX-

treated H9c2 cells indicated that 15d protected cardiomyocyte

death and damage via inhibiting the ROS overaccumulation and

restoring the Djm and morphology. Therefore, 15d merits further

3.5. Derivative 15d inhibited reactive oxygen species

overaccumulation, restored mitochondrial membrane potential, and

maintained the mitochondrial morphology in H9c2 cells

After identifying 15d as the most potent derivative, we further

investigated its molecular mechanisms underlying in H9c2 cells.

Firstly, the protective effect of 15d on DOX-induced cytotoxicity in

H9c2 cells was examined. The viability of H9c2 cells treated with

DOX (10

mM) was markedly decreased by around 50%. Derivative

15d (0.5e12.5

m

M) dose-dependently protected the cells from

damage (Fig. 8). Oxidative stress, resulting from the over-

accumulation of the reactive oxygen species (ROS), plays an

6

H. Zhang, B. Liu, G. Xu et al.

European Journal of Medicinal Chemistry 219 (2021) 113396

Fig. 4. Dose-response and toxicity assay of 2, 6, 12j, 12k, 15d, 15e and LSD. The 24 hpf embryos were treated with 100

m

M DOX with or without each test compound or solely treated

with each test compound (toxicity assay). Survival of the zebraﬁsh was examined at 96 hpf. Data are presented as mean S.D. (n ¼ 4). ^###P < 0.001 vs. the control group, *P < 0.05,

**P < 0.01, and ***P < 0.001 vs. the DOX treated only group.

development as a potential cardioprotective clinical trial candidate.

To the best of our knowledge, this is the ﬁrst example to combine

synthesis, SAR study, and in vivo screening to discover new car-

dioprotective derivatives from isosteviol.

were not recrystallized. All ﬁnal compounds were puriﬁed to ꢂ95%

as determined by HPLC, conducted on Waters Alliance e2695 series

system with a Grace Alltima 2.1 mm ꢃ 150 mm HP C18 5

mm col-

umn. ¹H NMR and ¹³C NMR spectra were measured on a Varian

Unity Inova 400 MHz NMR spectrometer with Me₄Si (TMS) as an

internal standard. High-resolution mass spectra were obtained on a

Bruker Maxis II ETD with ESI interface.

5. Experiment section

5.1. Chemistry

5.1.1. General procedures for the synthesis of compounds 3 and 4

Isosteviol (2) (1 eq) and K₂CO₃(1.5 eq) were dissolved in

acetone. The mixture was stirred at 50 ^ꢁC for 30 min. Then BnBr (1.2

eq for synthesizing 3) or PMBCl (1.2 eq, for synthesizing 4) was

added. The reaction was stirred at 50 ^ꢁC for 2 h. The solution was

then diluted with water and extracted with EtOAc for 3 times. The

combined organic layer was washed with brine, dried over Na₂SO₄,

All reagents and solvents were purchased from Energy Chemical

or other commercial source and used without further puriﬁcation.

Thin-layer chromatography (TLC) was performed on 0.20mm silica

gel 60 F-254 plates (Qingdao Ocean Chemical Factory, Shandong,

China). Silica gel chromatography was carried out on Biotage

CombiFlash Rf ﬂash chromatography system. Solid compounds

7

H. Zhang, B. Liu, G. Xu et al.

European Journal of Medicinal Chemistry 219 (2021) 113396

Fig. 5. The cardioprotective effects of 2, 6, 12j, 12k, 15d, 15e, and LSD on DOX-induced cardiomyopathy in zebraﬁsh Tg(cmlc2:GFP). Tg(cmlc2:GFP) zebraﬁsh embryos were treated

with DOX (80 M) in the absence or presence of each of test compound at their optimum concentration at 24 hpf. Four cardiovascular parameters including heartbeat, stroke

m

volume, cardiac output and fractional shortening, were measured at 72 hpf to evaluate the cardiac function. (A) Light microscopy image of the Tg(cmlc2:GFP) zebraﬁsh. (B)

Representative ﬂuorescent microscopy image of atrium and ventricle. (C) Graph of heart rate (D) Graph of fraction shortening (E) graph of stroke volume (F) Graph of cardiac output.

Data are presented as mean S.D. (n ¼ 4). ^##P < 0.01 and ^####P < 0.0001 vs. the control group, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 vs. the DOX treated only

group.

and concentrated in vacuo to give the crude product, which was

chromatographed using a silica gel column to yield the pure

product (3 and 4).

starting from 100 mg of 2; white solid; mp: 100.4e100.9 ^ꢁC;

25

[

a

]

ꢀ59 (c 1.6, CH₃OH); ¹H NMR (CDCl₃, 400 MHz)

d 7.42e7.27

D

(5H, overlap, 2,3,4,5,6-Ph), 5.10 (2H, m, CH₂-Ph), 2.55 (1H, dd,

J ¼ 3.8,18.7 Hz, H-15), 2.21 (1H, d, J ¼ 13.3 Hz, H-3), 1.21 (3H, s, H-

18), 0.97 (3H, s, H-17), 0.61 (3H, s, H-20), 1.90e0.80 (18H, m, CH,

5.1.1.1. Benzyl ent-16-oxobeyeran-19-oate (3). Yield: 117 mg (91%),

8

H. Zhang, B. Liu, G. Xu et al.

European Journal of Medicinal Chemistry 219 (2021) 113396

Fig. 6. The cardioprotective effects of 15d at 20, 40 and 60

m

M, on DOX-induced cardiomyopathy in zebraﬁsh Tg(cmlc2:GFP). (A) Light microscopy image of the Tg(cmlc2:GFP)

zebraﬁsh. (B) Representative ﬂuorescent microscopy image of atrium and ventricle. (C) Graph of heart rate (D) Graph of fraction shortening. (E) Graph of stroke volume. (F) Graph of

cardiac output. Data are presented as mean S.D. (n ¼ 4). ^####P < 0.0001 vs. the control group, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 vs. the DOX treated only

group.

176.99, 135.99, 128.51, 128.51, 128.40, 128.40, 128.17, 66.12, 57.18,

54.66, 54.28, 48.69, 48.37, 43.90, 41.50, 39.80, 39.43, 38.01, 37.96,

37.31, 28.96, 21.73, 20.31, 19.89, 18.97, 13.32; HRMS (ESI, m/z) calcd

for C₂₇H₃₆O₃Na, 431.2562 [M þ Na^þ]; found, 431.2560.

5.1.1.2. 4-Methoxybenzyl ent-16-oxobeyeran-19-oate (4). Yield:

109 mg (79%), starting from 100 mg of 2; white solid; mp

25

79.2e80.7 ^ꢁC; [

d

a

]

_Dꢀ57 (c 1.6, CH₂Cl₂); ¹H NMR (CDCl₃, 400 MHz)

7.28 (2H, m, 2,6-Ph), 6.88 (2H, m, 3,5-Ph), 5.01 (2H, m, CH₂-Ph),

3.82 (3H, s,OCH₃), 2.56 (1H, dd, J ¼ 3.8, 18.7, Hz, H-15), 2.19 (1H, d,

J ¼ 13.3 Hz, H-3), 1.19 (3H, s, H-18), 0.97 (3H, s, H-17), 0.60 (3H, s, H-

20),1.90e0.80 (18H, m, CH, CH₂in ent-beyerane skeleton); ¹³C NMR

Fig. 7. Relative mRNA level of the cardiomyopathy biomarkers ANP and cTnT in control

zebraﬁsh embryos and DOX-treated embryos with or without 15d. Data are presented

as mean S.D. (n ¼ 4). ##P < 0.01 vs. the control group, **P < 0.01 and ***P < 0.001

vs. the DOX treated only group.

(CDCl₃, 100 MHz)

d 222.63, 177.10, 159.53, 130.17, 130.17, 128.13,

113.86, 113.86, 65.90, 57.20, 55.29, 54.67, 54.29, 48.72, 48.40, 43.88,

41.51, 39.82, 39.45, 38.03, 37.96, 37.33, 28.95, 21.72, 20.32, 19.87,

18.97, 13.35; HRMS (ESI, m/z) calcd for C₂₈H₃₈O₄Na, 461.2668

[M þ Na^þ]; found, 461.2661.

CH₂in ent-beyerane skeleton); ¹³C NMR (CDCl₃, 100 MHz)

d 222.46,

Fig. 8. Derivative 15d inhibited the DOX-induced cytotoxicity and ROS overaccumulation in H9c2 Cells. (A) The protective effect 15d on cell viability. (B) Confocal images of ROS

(Scale bar: 100

m

m). (C) Graph of ROS level. Data are presented as mean S.D. (n ¼ 3). ^##P < 0.01 vs. the control group, *P < 0.05, and **P < 0.01 vs. the DOX treated only group.

9

H. Zhang, B. Liu, G. Xu et al.

European Journal of Medicinal Chemistry 219 (2021) 113396

Fig. 9. The effect of 15d on mitochondrial membrane potential and mitochondrial morphology in H9c2 Cells treated with DOX. (A) Confocal images of JC-1stained mitochondrial

potential (Scale bar: 50 mm). (B) Graph of the red to green (R/G) ﬂuorescence intensities. (C) Confocal images of JC-1stained mitochondrial morphology (Scale bar: 50 mm). (D) Graph

of the length of the mitochondrial. Data are presented as mean S.D. (n ¼ 3). ^##P < 0.01 vs. the control group, *P < 0.05, and **P < 0.01 vs. the DOX treated only group.

5.1.2. Synthesis of methyl ent-16-oxobeyeran-19-oate (5)

5.1.4. Synthesis of ent-16a-hydroxy-15b-hydroxymethylbeyeran-

Isosteviol (2) (100 mg, 0.3 mmol), CH₃I (0.04 mL, 0.6 mmol) and

TBAF (0.9 mL, 1 M in THF, 0.9 mmol) were dissolved in anhydrous

THF (2 mL). The mixture was stirred at room temperature until all

starting material was consumed. The solution mixture was then

diluted with EtOAc, washed with brine, dried over anhydrous

Na₂SO₄and concentrated to dryness under reduced pressure. The

19-oic acid (7)

Isosteviol (2) (50 mg, 0.2 mmol), NaOH (31.4 mg, 0.8 mmol) and

paraformaldehyde (37.8 mg, 1.3 mmol) were dissolved in anhy-

drous EtOH (2 mL). The mixture was stirred at 80 ^ꢁC until all

starting material was consumed. The mixture was diluted with

EtOAc, neutralized with HCl (1N). The solution was washed with

brine, dried over anhydrous Na₂SO₄, and concentrated. The residue

residue was chromatographed using a silica gel column to give

25

78 mg of 5 (75%), white solid; mp 215.9e216.8 ^ꢁC; [

a

]

_Dꢀ46 (c 0.6,

was chromatographed using a silica gel column to give 32mg of 7

CH₂Cl₂); ¹H NMR (CDCl₃, 400 MHz)

d

3.64 (3H, s, COCH₃), 2.63 (1H,

(58%), white solid; mp 199.6e200.8 ^ꢁC; [

a]

_Dꢀ31 (c 0.8, CH₃OH);

25

dd, J ¼ 3.8, 18.6 Hz, H-15), 2.18 (1H, d, J ¼ 13.5 Hz, H-3), 1.19 (3H, s,

1H NMR (CD₃OD, 400 MHz)

d

3.83 (1H, dd, J ¼ 5.2, 10.6, Hz, H-1⁰a),

H-18), 0.98 (3H, s, H-17), 0.69 (3H, s, H-20), 1.95e0.80 (18H, m, CH,

3.56 (1H, d, J ¼ 4.7 Hz, H-16), 3.48 (1H, dd, J ¼ 8.5, 10.6 Hz, H-1⁰b),

2.10 (1H, m, H-3), 2.02 (1H, m, H-15), 1.17 (3H, s, H-18), 0.90 (3H, s,

H-17), 0.88 (3H, s, H-20), 1.85e0.80 (17H, m, CH, CH₂in ent-

CH₂in ent-beyerane skeleton); ¹³C NMR (CDCl₃, 100 MHz)

d 222.54,

177.86, 57.08, 54.75, 54.33, 51.27, 48.72, 48.48, 43.79, 41.50, 39.83,

39.46, 37.96, 37.96, 37.33, 28.86, 21.72, 20.34, 19.87, 18.96, 13.18;

HRMS (ESI, m/z) calcd for C₂₁H₃₃O₃, 333.2429 [M þ H^þ]; found,

333.2428.

beyerane skeleton); ¹³C NMR (CD₃OD, 100 MHz)

d 180.40, 84.62,

63.02, 57.97, 57.06, 54.19, 49.84, 42.56, 40.47, 39.62, 38.15, 37.82,

34.69, 33.25, 29.36, 28.22, 24.15, 22.08, 19.19, 18.72, 12.47; HRMS

(ESI, m/z) calcd for C₂₁H₃₄O₄Na, 373.2355 [M þ Na^þ]; found,

373.2357.

5.1.3. Synthesis of ent-4-amino-19-norbeyeran-16-one (6)

5.1.5. Synthesis of ent-16a-hydroxy-15b-(tosyloxy)methylbeyeran-

Isosteviol (2) (250 mg, 0.8 mmol), DPPA (0.2 mL, 0.9 mmol) and

Et₃N (0.25 mL, 1.8 mmol) were dissolved in anhydrous tert-butanol

(6 mL). The mixture was heated to reﬂux and stirred for 7 h. After

concentration under vacuum, the residue was chromatographed

19-oic acid (8)

Compound 7 (100 mg, 0.3 mmol) and TsCl (114.4 mg, 0.6 mmol)

were dissolved in anhydrous pyridine (2 mL). The mixture was

heated to reﬂux and stirred for 5 h. The reaction mixture was then

diluted with EtOAc, neutralized with HCl (1N). The solution was

washed with saturated brine, dried over anhydrous Na₂SO₄, and

concentrated. The residue was chromatographed using a silica gel

using a silica gel column to give 205 mg of 6 (90%), white solid; mp

25

122.5e123.9 ^ꢁC; [

a

]

ꢀ70 (c 1.0, CH₃OH); ¹H NMR (CDCl₃,

D

400 MHz)

d

2.73 (1H, dd, J ¼ 3.8, 18.7 Hz, H-15), 1.38 (3H, s, H-18),

1.01 (3H, s, H-17), 0.98 (3H, s, H-20), 1.90e0.80 (19H, m, CH, CH₂in

column to give 46 mg of 8 (32%), white solid; mp 106.6e107.2 ^ꢁC;

ent-beyerane skeleton); ¹³C NMR (CDCl₃, 100 MHz)

d

222.20, 59.08,

[

a

]

_Dꢀ55 (c 1.1, CH₃OH); ¹H NMR (CDCl₃, 400 MHz)

d 7.81 (2H, d,

25

55.11, 54.76, 54.49, 48.86, 48.70, 41.54, 40.34, 39.18, 38.52, 37.49,

37.11, 31.88, 19.87, 19.87, 19.83, 17.96, 14.06; HRMS (ESI, m/z) calcd

for C₁₉H₃₂NO, 290.2484 [M þ H^þ]; found, 290.2472.

J ¼ 8.3 Hz, 2,6-Ph), 7.36 (2H, d, J ¼ 8.3 Hz, 3,5-Ph), 4.32 (1H, dd,

J ¼ 4.7, 9.5 Hz, H-1⁰a), 3.96 (1H, t, J ¼ 9.2 Hz, H-1⁰b), 3.39 (1H, d,

J ¼ 4.5 Hz, H-16), 2.45 (3H, s, CH₃-Ph), 2.15e2.06 (2H, overlap, H-15,

10

H. Zhang, B. Liu, G. Xu et al.

European Journal of Medicinal Chemistry 219 (2021) 113396

H-3), 1.20 (3H, s, H-18), 0.85 (3H, s, H-17), 0.72 (3H, s, H-20);

5.1.9. General procedures for the synthesis of 11be11n

1.90e0.80 (17H, m, CH, CH₂in ent-beyerane skeleton); ¹³C NMR

Compound (7, 1 eq), EDCI (5 eq), DMAP (2 eq) and appropriate

acid (1.5 eq) were dissolved in anhydrous DCM. The mixture was

stirred at room temperature until all starting material was

consumed. The solution was then diluted with DCM, washed brine,

dried over anhydrous Na₂SO₄, and concentrated in vacuum. The

residue was chromatographed using a silica gel column to give the

pure products (11be11n).

(CDCl₃, 100 MHz)

d 183.41, 144.99, 132.79, 129.97, 128.01, 84.17,

77.23, 72.40, 57.41, 56.84, 53.86, 47.93, 43.46, 43.02, 40.85, 39.32,

38.35, 37.66, 34.53, 32.94, 28.73, 24.87, 21.84, 21.65, 19.55, 18.70,

13.85, 12.71; HRMS (ESI, m/z) calcd for C₂₈H₄₀O₆SNa, 527.2444

[M þ Na^þ]; found, 527.2433.

5.1.6. Synthesis of ent-16-oxo-5b-(tosyloxy)methylbeyeran-19-oic

acid (9)

5.1.9.1. Ent-15

b-((cyclohexanecarbonyl)oxy)methyl-16a-hydrox-

ybeyeran-19-oic acid (11b). Yield: 92 mg (35%), starting from

To a solution of 8 (100 mg, 0.2 mmol) in anhydrous DMF (2 mL)

was added PDC (225.7 mg, 0.6 mmol). The reaction was stirred at

room temperature for 24 h. The mixture was then ﬁltered and

concentrated. The residue was chromatographed using a silica gel

25

200 mg of 7; white solid; mp 91.1e91.5 ^ꢁC; [

CH₃OH); ¹H NMR (CDCl₃, 400 MHz)

a

]

ꢀ57 (c 1.0,

D

d

4.21 (1H, dd, J ¼ 4.8, 10.8 Hz,

H-1⁰a), 4.00 (1H, m, H-1⁰b), 3.52 (1H, d, J ¼ 4.6 Hz, H-16), 2.33 (1H,

m, COCH), 2.24e2.11 (2H, overlap, H-15, H-3), 1.25 (3H, s, H-18),

0.93 (3H, s, H-17), 0.87 (3H, s, H-20), 1.90e0.80 (27H, m, CH, CH₂in

ent-beyerane skeleton or carbon chain); ¹³C NMR (CDCl₃, 100 MHz)

column to yield 65 mg of 9 (65%), white solid; mp 163.5e164.8 ^ꢁC;

25

[

a

]

_Dꢀ31 (c 0.7, CH₃OH); ¹H NMR (CDCl₃, 400 MHz)

d 7.73 (2H, d,

J ¼ 8.3 Hz, 2,6-Ph), 7.35 (2H, d, J ¼ 8.3 Hz, 3,5-Ph), 4.26 (2H, m, H-1⁰),

2.51 (1H, m, H-15), 2.45 (3H, s, CH₃-Ph), 2.17 (1H, d, J ¼ 13.4 Hz, H-

3), 1.25 (3H, s, H-18), 0.91 (3H, s, H-17), 0.70 (3H, s, H-20);

1.90e0.80 (17H, m, CH, CH₂in ent-beyerane skeleton); ¹³C NMR

d

176.23, 173.13, 85.69, 66.21, 57.46, 57.18, 54.13, 47.50, 45.25, 43.28,

42.76, 40.93, 39.40, 38.32, 38.01, 34.85, 33.14, 30.21, 29.22, 29.09,

28.13, 25.74, 25.48, 25.42, 25.07, 19.61, 18.76, 13.98; HRMS (ESI, m/z)

calcd for C₂₈H₄₄O₅Na, 483.3087 [M þ Na^þ]; found, 483.3091.

(CDCl₃, 100 MHz)

d 220.59, 182.68, 144.99, 132.26, 129.87, 129.87,

128.09, 128.09, 67.63, 57.02, 56.88, 52.86, 51.24, 48.07, 43.59, 40.52,

39.57, 38.40, 37.68, 37.12, 35.00, 28.91, 21.70, 21.44, 19.60, 19.53,

18.75, 13.21; HRMS (ESI, m/z) calcd for C₂₈H₃₈O₆SNa, 525.2287

[M þ Na^þ]; found, 525.2280.

5.1.9.2. Ent-16a-hydroxy-15b-(nicotinoyloxy)methylbeyeran-19-oic

acid (11c). Yield: 87 mg (33%), starting from 200 mg of 7; white

25

solid; mp 113.5e114.0 ^ꢁC; [

(CDCl₃, 400 MHz)

a]

ꢀ55 (c 1.4, CH₂Cl₂); ¹H NMR

D

d

9.21 (1H, s, 2-pyrimidine), 8.75 (1H, d,

J ¼ 4.9 Hz, 4-pyrimidine), 8.30 (1H, d, J ¼ 8.0 Hz, 6-pyrimidine), 7.38

(1H, dd, J ¼ 4.9, 8.0 Hz, 5-pyrimidine), 4.52 (1H, dd, J ¼ 4.9, 10.9 Hz,

H-1⁰a), 4.27 (1H, m, H-1⁰b), 3.64 (1H, d, J ¼ 4.8 Hz, H-16), 2.34 (1H,

m, H-15), 1.27 (3H, s, H-18), 0.94 (3H, s, H-17), 0.92 (3H, s, H-20);

2.25e0.80 (18H, m, CH, CH₂in ent-beyerane skeleton); 13C NMR

5.1.7. Synthesis of ent-15-methylene-16-oxobeyeran-19-oic acid

(10)

To a solution of 9 (100 mg, 0.2 mmol) in anhydrous pyridine

(2 mL) was added DMAP (36.7 mg, 0.3mmol). The reaction was

heated to reﬂux and stirred for 3 h. The mixture was then diluted

with EtOAc, neutralized with HCl (1N), and washed with saturated

brine. The solution was dried over anhydrous Na₂SO₄, and

concentrated in vacuo to give the crude product, which was chro-

(CDCl₃, 100 MHz)

d 173.22, 165.52, 153.38, 150.88, 137.28, 126.13,

123.51, 85.40, 67.34, 57.46, 57.16, 54.19, 47.41, 45.30, 42.81, 41.06,

39.35, 38.35, 37.99, 34.93, 33.13, 28.11, 25.06, 22.00, 19.59, 18.80,

14.04; HRMS (ESI, m/z) calcd for C₂₇H₃₈NO₅, 456.2750 [M þ H^þ];

found, 456.2740.

matographed using a silica gel column to produce 30 mg of 10

25

(46%), white solid; mp 177.7e178.3 ^ꢁC; [

a]

_Dꢀ101 (c 1.4, CH₂Cl₂);

¹H NMR (CDCl₃, 400 MHz)

d

6.07 (1H, s, CH₂¼ ), 5.49 (1H, s, CH₂¼ ),

5.1.9.3. Ent-16a-hydroxy-15b-(isonicotinoyloxy)methylbeyeran-19-

oic acid (11d). Yield: 84 mg (32%), starting from 200 mg of 7; white

2.18 (1H, d, J ¼ 12.9 Hz, H-3), 1.28 (3H, s, H-18), 1.02 (3H, s, H-17),

0.69 (3H, s, H-20), 1.90e0.80 (17H, m, CH, CH₂in ent-beyerane

25

solid; mp 139.2e140.4 ^ꢁC; [

(CDCl₃, 400 MHz)

a]

ꢀ44 (c 1.6, CH₃OH); ¹H NMR

D

skeleton); ¹³C NMR (CDCl₃, 100 MHz)

d 210.83, 183.18, 154.40,

d

8.74 (2H, d, J ¼ 5.8 Hz, 3,5-pyrimidine), 7.85 (2H,

116.17, 56.96, 56.63, 53.47, 46.80, 43.78, 40.41, 38.81, 38.13, 37.87,

37.85, 29.71, 29.10, 21.76, 21.04, 20.12, 18.91, 12.41; HRMS (ESI, m/z)

calcd for C₂₁H₃₀O₃Na, 353.2093 [M þ Na^þ]; found, 353.2087.

d, J ¼ 5.8 Hz, 2,6-pyrimidine), 4.50 (1H, dd, J ¼ 5.4, 11.0 Hz, H-1⁰a),

4.25 (1H, dd, J ¼ 8.7, 11.0 Hz, H-1⁰b), 3.58 (1H, d, J ¼ 4.7 Hz, H-16),

2.35 (1H, m, H-15), 2.17 (1H, d, J ¼ 13.2 Hz, H-3), 1.27 (3H, s, H-18),

0.97e0.91 (6H, overlap, H-17, H-20), 2.00e0.85 (17H, m, CH, CH₂in

ent-beyerane skeleton); ¹³C NMR (CDCl₃, 100 MHz)

d 173.11, 165.41,

5.1.8. Synthesis of ent-15b -acetoxymethyl-16a-hydroxybeyeran-

19-oic acid (11a)

150.61, 137.35, 122.99, 85.56, 67.73, 57.43, 57.19, 54.14, 53.44, 47.34,

45.29, 42.77, 41.02, 39.34, 38.33, 37.98, 34.89, 33.07, 29.71, 28.20,

25.02, 21.98, 19.54, 18.84, 14.09; HRMS (ESI, m/z) calcd for

To the solution of 7 (200 mg, 0.6 mmol) in anhydrous pyridine

(5 mL) was added Ac₂O (0.07 mL, 0.7 mmol) and DMAP (73.3 mg,

0.6 mmol). The reaction was stirred at room temperature until all

starting material was consumed. The solution was then diluted

with EtOAc, neutralized with HCl (1N), and washed with brine. The

solution was dried over Na₂SO₄and concentrated. The residue was

C

₂₇H₃₈NO₅, 456.2750 [M þ H^þ]; found, 456.2738.

5.1.9.4. Ent-15b-(benzoyloxy)methyl-16a-hydroxybeyeran-19-oic

acid (11e). Yield: 108 mg (42%), starting from 200 mg of 7; white

25

chromatographed using a silica gel column to give 78 mg of 11a

solid; mp 96.7e96.9 ^ꢁC; [

400 MHz)

a

]

_Dꢀ54 (c 1.1, CH₃OH); ¹H NMR (CDCl₃,

25

(35%), white solid; mp 157.0e158.4 ^ꢁC; [

a]

ꢀ87 (c 0.6, CH₃OH);

d

8.03 (2H, d, J ¼ 7.4 Hz, 2,6-Ph), 7.56 (1H, t, J ¼ 7.4 Hz, 4-

D

¹H NMR (CDCl₃, 400 MHz)

d

4.22 (1H, dd, J ¼ 6.2, 10.8 Hz, H-1⁰a),

Ph), 7.43 (2H, t, J ¼ 7.4 Hz, 3,5-Ph), 4.45 (1H, dd, J ¼ 4.9, 10.9 Hz, H-

1⁰a), 4.22 (1H, m, H-1⁰b), 3.63 (1H, d, J ¼ 4.7 Hz, H-16), 2.32 (1H, m,

H-15), 2.17 (1H, d, J ¼ 13.0 Hz, H-3), 1.27 (3H, s, H-18), 0.96e0.90

(6H, overlap, H-17, H-20); 2.0e0.80 (17H, m, CH, CH₂in ent-

4.05 (1H, dd, J ¼ 8.9, 10.8 Hz, H-1⁰b), 3.45 (1H, d, J ¼ 4.6 Hz, H-16),

2.20 (1H, m, H-15), 2.16e2.06 (4H, overlap, H-3, COCH₃), 1.21 (3H, s,

H-18), 0.91 (3H, s, H-17), 0.84 (3H, s, H-20), 1.85e0.80 (17H, m, CH,

CH₂in ent-beyerane skeleton); ¹³C NMR (CDCl₃, 100 MHz)

d

183.67,

beyerane skeleton); ¹³C NMR (CDCl₃, 150 MHz)

d 173.21, 166.64,

171.56, 85.27, 66.32, 57.48, 56.97, 54.01, 47.36, 43.56, 42.87, 40.81,

39.46, 38.36, 37.72, 34.72, 33.22, 29.70, 28.80, 24.98, 21.91, 19.49,

18.77, 12.93; HRMS (ESI, m/z) calcd for C₂₃H₃₆O₅Na, 415.2461

[M þ Na^þ]; found, 415.2455.

148.84, 125.57, 124.28, 122.98, 116.55, 86.48, 67.95, 61.66, 57.60,

57.21, 56.22, 54.01, 47.82, 45.30, 42.72, 41.02, 39.48, 38.33, 34.99,

33.12, 28.15, 25.12, 22.71, 22.18, 18.78, 13.93; HRMS (ESI, m/z) calcd

for C₂₈H₃₇O₅, 453.2641 [M-H^þ]; found, 453.2650.

11

H. Zhang, B. Liu, G. Xu et al.

European Journal of Medicinal Chemistry 219 (2021) 113396

5.1.9.5. Ent-15

b

-((4-ﬂuorobenzoyl)oxy)methyl-16

a

-hydroxybeyeran-

s, OCH₃), 3.49 (1H, d, J ¼ 4.5 Hz, H-16), 3.21 (1H, s, 16-OH), 2.23 (1H,

m, H-15), 2.12 (1H, d, J ¼ 13.7 Hz, H-3), 1.21 (3H, s, H-18), 0.89 (3H, s,

H-17), 0.83 (3H, s, H-20), 1.85e0.75 (17H, m, CH, CH₂in ent-

19-oic acid (11f). Yield: 95 mg (35%), starting from 200 mg of 7;

25

white solid; mp 108.1e109.1 ^ꢁC; [

(CDCl₃, 400 MHz)

a]

_Dꢀ59 (c 1.4, CH₃OH); ¹H NMR

d

8.05 (2H, m, 2,6-Ph), 7.10 (2H, m, 3,5-Ph), 4.44

beyerane skeleton); ¹³C NMR (CDCl₃, 100 MHz)

d 172.13, 165.07,

(1H, m, H-1⁰a), 4.20 (1H, m, H-1⁰b), 3.59 (1H, d, J ¼ 4.7 Hz, H-16),

2.32 (1H, m, H-15), 2.17 (1H, d, J ¼ 13.4 Hz, H-3), 1.27 (3H, s, H-18),

0.96e0.90 (6H, overlap, H-17, H-20), 1.90e0.80 (17H, m, CH, CH₂in

156.57, 153.08, 141.89, 126.68, 116.36, 106.47, 85.73, 66.72, 60.99,

60.04, 56.58, 56.18, 55.11, 53.03, 46.88, 44.28, 41.61, 39.94, 38.44,

37.31, 33.95, 32.11, 28.68, 27.15, 24.11, 21.16, 18.67, 17.77, 12.92;

HRMS (ESI, m/z) calcd for C₃₁H₄₄O₈Na, 567.2934 [M þ Na^þ]; found,

567.2909.

ent-beyerane skeleton); ¹³C NMR (CDCl₃, 100 MHz)

d 173.14, 165.90,

132.23, 132.14, 126.38, 126.35, 115.78, 115.56, 85.71, 67.03, 57.47,

57.21, 54.18, 47.53, 45.29, 42.77, 40.98, 39.37, 38.34, 38.01, 34.90,

33.10, 28.17, 25.05, 22.03, 19.60, 18.81, 14.06; HRMS (ESI, m/z) calcd

for C₂₈H₃₇FO₅Na, 495.2523 [M þ Na^þ]; found, 495.2539.

5.1.9.10. Ent-16

a-hydroxy-15b-((3,4,5-trimethoxybenzoyl)oxy)meth-

ylbeyeran-19-oic acid (11k). Yield: 97 mg (31%), starting from

25

200 mg of 7; white solid; mp 114.4e115.8 ^ꢁC; [

CH₃OH); ¹H NMR (CDCl₃, 400 MHz)

7.32 (2H, s, 2,6-Ph), 4.43 (1H,

a]

ꢀ35 (c 1.0,

D

5.1.9.6. Ent-16

a

-hydroxy-15

b

-((4-methoxybenzoyl)oxy)methyl-

d

beyeran-19-oic acid (11g). Yield: 75 mg (27%), starting from 200 mg

dd, J ¼ 4.3, 10.9 Hz, H-1⁰a), 4.27 (1H, dd, J ¼ 8.8, 10.9 Hz, H-1⁰b), 3.90

(3H, s, OCH₃), 3.90 (6H, s, 2 ꢃ OCH₃), 3.69 (1H, d, J ¼ 4.7 Hz, H-16),

2.35 (1H, m, H-15), 2.17 (1H, d, J ¼ 13.4 Hz, H-3), 1.28 (3H, s, H-18),

0.94 (3H, s, H-17), 0.92 (3H, s, H-20), 1.90e0.80 (17H, m, CH, CH₂in

25

of 7; white solid; mp 201.7e202.5 ^ꢁC; [

NMR (CDCl₃, 400 MHz)

a]

_Dꢀ53 (c 0.3, CH₃OH); ¹H

d

7.98 (2H, d, J ¼ 8.9 Hz, 3,5-Ph), 6.91 (2H, d,

J ¼ 8.9 Hz, 2,6-Ph), 4.40 (1H, dd, J ¼ 5.0, 10.9 Hz, H-1⁰a), 4.18 (1H, dd,

J ¼ 8.9, 10.9 Hz, H-1⁰b), 3.86 (3H, s, OCH₃), 3.61 (1H, d, J ¼ 4.7 Hz, H-

16), 2.34 (1H, m, H-15), 2.17 (1H, d, J ¼ 13.5 Hz, H-3), 1.26 (3H, s, H-

18), 0.96e0.91 (6H, overlap, H-17, H-20), 1.80e0.80 (17H, m, CH,

ent-beyerane skeleton); ¹³C NMR (CDCl₃, 100 MHz)

d 173.18, 166.43,

153.02, 153.02, 142.28, 125.21, 106.83, 106.83, 85.36, 66.66, 60.94,

57.48, 57.18, 56.28, 56.28, 54.31, 47.47, 45.25, 42.80, 41.04, 39.36,

34.93, 33.16, 31.45, 30.20, 28.13, 25.12, 22.11, 19.65, 18.73, 14.04;

HRMS (ESI, m/z) calcd for C₃₁H₄₄O₈K, 583.2673 [M þ K^þ]; found,

583.2665.

CH₂in ent-beyerane skeleton);¹³C NMR (CDCl₃, 100 MHz)

d 173.13,

166.59, 163.42, 131.64, 131.64, 122.54, 113.77, 113.77, 85.63, 66.57,

57.51, 57.19, 55.44, 54.21, 47.63, 45.30, 42.80, 40.95, 39.40, 38.34,

38.03, 34.91, 33.16, 28.11, 25.09, 22.07, 19.63, 18.80, 14.07; HRMS

(ESI, m/z) calcd for C₂₉H₄₀O₆Na, 507.2723 [M þ Na^þ]; found,

507.2711.

5.1.9.11. Ent-15

16 -hydroxybeyeran-19-oic acid (11l). Yield: 120 mg (39%), starting

from 200 mg of 7; white solid; mp 127.3e128.5 ^ꢁC; [

1.9, CH₂Cl₂); ¹H NMR (CDCl₃, 400 MHz)

b-(((E)-3-(3,5-dimethoxyphenyl)acryloyl)oxy)methyl-

a

25

a

]

ꢀ56 (c

D

5.1.9.7. Ent-15

b

-((2,3-dimethoxybenzoyl)oxy)methyl-16

a

-hydrox-

d

7.61 (1H, d, J ¼ 16.0 Hz,

ybeyeran-19-oic acid (11h). Yield: 102 mg (35%), starting from

CH₂¼ ), 6.68 (2H, d, J ¼ 2.3 Hz, 2,6-Ph), 6.49 (1H, t, J ¼ 2.3 Hz, 4-Ph),

6.44 (1H, d, J ¼ 16.0 Hz, CH₂¼ ), 4.33 (1H, dd, J ¼ 5.0,10.8 Hz, H-1⁰a),

4.14 (1H, m, H-1⁰b), 3.81 (6H, s, 2 ꢃ OCH₃), 3.56 (1H, d, J ¼ 4.6 Hz, H-

16), 2.26 (1H, m, H-15), 2.16 (1H, d, J ¼ 13.1 Hz, H-3), 1.26 (3H, s, H-

18), 0.93 (3H, s, H-17), 0.89 (3H, s, H-20), 1.85e0.80 (17H, m, CH,

25

200 mg of 7; white solid; mp 112.0e113.5 ^ꢁC; [

CH₃OH); ¹H NMR (CDCl₃, 400 MHz)

a]

ꢀ52 (c 1.1,

D

d

7.41 (1H, dd, J ¼ 1.8, 7.6 Hz, 6-

Ph), 7.18e7.00 (2H, overlap, 3,4-Ph), 4.49 (1H, dd, J ¼ 4.5,10.3 Hz, H-

1⁰a), 4.20 (1H, dd, J ¼ 10.3, 12.1 Hz, H-1⁰b), 3.86 (6H, s, 2 ꢃ OCH₃),

3.57 (1H, d, J ¼ 4.4 Hz, H-16), 2.30 (1H, m, H-15), 2.21 (1H, d,

J ¼ 3.5 Hz, H-3), 1.28 (3H, s, H-18), 0.95 (3H, s, H-17), 0.90 (3H, s, H-

20),1.90e0.80 (17H, m, CH, CH₂in ent-beyerane skeleton); ¹³C NMR

CH₂in ent-beyerane skeleton); ¹³C NMR (CDCl₃, 100 MHz)

d 173.14,

167.16, 161.02, 161.02, 145.18, 136.27, 118.40, 106.07, 106.07, 102.82,

85.76, 66.72, 57.45, 57.17, 55.47, 55.47, 54.08, 47.44, 45.28, 42.81,

40.99, 39.38, 38.32, 34.88, 33.16, 29.72, 28.08, 25.04, 22.05, 19.62,

18.77, 13.94; HRMS (ESI, m/z) calcd for C₃₂H₄₄O₇Na, 563.2985

[M þ Na^þ]; found, 563.2996.

(CDCl₃, 150 MHz)

d 173.16, 166.84, 133.03, 130.14, 129.93, 129.59,

129.59, 128.51, 128.51, 85.64, 66.90, 57.48, 57.18, 54.19, 47.55, 45.28,

42.80, 41.00, 39.39, 38.34, 38.01, 34.91, 33.14, 28.11, 25.07, 24.88,

22.07, 19.62, 18.78, 14.04; HRMS (ESI, m/z) calcd for C₃₀H₄₂O₇Na,

537.2829 [M þ Na^þ]; found, 537.2843.

5.1.9.12. Ent-16

a-hydroxy-15b-(2-(4-methoxyphenyl)acetoxy)meth-

ylbeyeran-19-oic acid (11m). Yield: 84 mg (30%), starting from

25

5.1.9.8. Ent-15

b

-((3,4-dimethoxybenzoyl)oxy)methyl-16

a

-hydrox-

200 mg of 7; white solid; mp 75.0e76.4 ^ꢁC; [

CH₃OH); ¹H NMR (CDCl₃, 400 MHz)

a]

ꢀ60 (c 0.9,

D

ybeyeran-19-oic acid (11i). Yield: 116 mg (40%), starting from

d

7.18 (2H, d, J ¼ 8.6 Hz, 2,6-Ph),

25

200 mg of 7; white solid; mp 169.3e170.8 ^ꢁC; [

CH₃OH); ¹H NMR (CDCl₃, 400 MHz)

a

]

ꢀ65 (c 1.0,

6.85 (2H, d, J ¼ 8.6 Hz, 3,5-Ph), 4.21 (1H, dd, J ¼ 5.1, 10.7 Hz, H-1⁰a),

3.96 (1H, t, J ¼ 10.2 Hz, H-1⁰b), 3.78 (3H, s, OCH₃), 3.59 (2H, s,

COCH₂), 3.33 (1H, d, J ¼ 4.6 Hz, H-16), 2.20e2.08 (2H, overlap, H-15,

H-3), 1.23 (3H, s, H-18), 0.87 (3H, s, H-17), 0.86 (3H, s, H-20),

1.85e0.80 (17H, m, CH, CH₂in ent-beyerane skeleton); ¹³C NMR

D

d

7.68 (1H, d, J ¼ 8.4 Hz, 6-Ph),

7.55 (1H, s, 2-Ph), 6.89 (1H, d, J ¼ 8.4 Hz, 5-Ph), 4.41 (1H, dd, J ¼ 4.7,

10.9 Hz, H-1⁰a), 4.22 (1H, dd, J ¼ 8.6, 10.9 Hz, H-1⁰b), 3.93 (3H, s,

OCH₃), 3.92 (3H, s, OCH₃), 3.64 (1H, d, J ¼ 4.6 Hz, H-16), 2.32 (1H, m,

H-15), 2.17 (1H, d, J ¼ 13.5 Hz, H-3), 1.27 (3H, s, H-18), 0.96e0.90

(6H, overlap, H-17, H-20), 1.90e0.80 (17H, m, CH, CH₂in ent-

(CDCl₃, 100 MHz)

d 173.22, 172.09, 158.76, 130.30, 130.30, 126.20,

114.15, 114.15, 85.49, 66.88, 57.41, 57.15, 55.31, 53.95, 47.39, 45.24,

42.70, 40.85, 40.71, 39.34, 38.29, 37.98, 34.88, 34.78, 33.10, 28.09,

24.94, 22.02, 19.58, 13.92; HRMS (ESI, m/z) calcd for C₃₀H₄₃O₆,

499.3059 [M þ H^þ]; found, 499.3080.

beyerane skeleton); ¹³C NMR (CDCl₃, 100 MHz)

d 173.15, 166.63,

153.06, 148.69, 123.64, 122.65, 112.01, 110.44, 85.47, 66.56, 57.51,

57.19, 56.04, 56.04, 54.26, 47.59, 45.29, 42.79, 40.97, 39.39, 38.34,

38.01, 34.92, 33.17, 28.12, 25.11, 22.08, 19.64, 18.79, 14.06; HRMS

(ESI, m/z) calcd for C₃₀H₄₂O₇K, 553.2568 [M þ K^þ]; found,

553.2562.

5.1.9.13. Ent-16

a-hydroxy-15b-(2-(3,4,5-trimethoxyphenyl)acetoxy)

methylbeyeran-19-oic acid (11n). Yield: 109 mg (34%), starting from

25

200 mg of 7; white solid; mp 174.0e175.4 ^ꢁC; [

CH₂Cl₂); ¹H NMR (CDCl₃, 400 MHz)

6.51 (2H, s, 2,6-Ph), 4.25 (1H,

a]

ꢀ51 (c 2.6,

D

5.1.9.9. Ent-16

a

-hydroxy-15

b

-((2,3,4-trimethoxybenzoyl)oxy)meth-

d

ylbeyeran-19-oic acid (11j). Yield: 70 mg (23%), starting from

dd, J ¼ 5.1,10.8 Hz, H-1⁰a), 3.99 (1H, t, J ¼ 10.0 Hz, H-1⁰b), 3.85 (6H, s,

2 ꢃ OCH₃), 3.82 (3H, s, OCH₃), 3.59 (2H, s, COCH₂), 3.37 (1H, d,

J ¼ 4.7 Hz, H-16), 2.23e2.07 (2H, overlap, H-15, H-3), 1.24 (3H, s, H-

18), 0.90e0.81 (6H, overlap, H-17, H-20), 1.85e0.80 (17H, m, CH,

25

200 mg of 7; white solid; mp 96.6e97.2 ^ꢁC; [

a

]

ꢀ54 (c 2.1,

D

CH₃OH); ¹H NMR (CDCl₃, 400 MHz)

d

7.62 (1H, d, J ¼ 9.0 Hz, 6-Ph),

6.68 (1H, d, J ¼ 9.0 Hz, 5-Ph), 4.40 (1H, dd, J ¼ 4.6, 10.2 Hz, H-1⁰a),

4.09 (1H, m, H-1⁰b), 3.84 (3H, s, OCH₃), 3.82 (3H, s, OCH₃), 3.77 (3H,

CH₂in ent-beyerane skeleton); ¹³C NMR (CDCl₃, 100 MHz)

d 173.20,

12

H. Zhang, B. Liu, G. Xu et al.

European Journal of Medicinal Chemistry 219 (2021) 113396

171.69, 153.34, 137.19, 129.63, 106.33, 106.33, 85.39, 66.87, 60.87,

57.37, 57.14, 56.16, 56.16, 53.94, 47.45, 45.24, 42.70, 41.85, 40.82,

39.30, 38.29, 37.96, 34.78, 33.02, 31.45, 28.15, 24.93, 22.01, 19.55,

18.74, 13.94; HRMS (ESI, m/z) calcd for C₃₂H₄₅O₈, 557.3115 [M-H^þ];

found, 557.3111.

5.1.10.5. Ent-15

b

-(benzoyloxy)methyl-16-oxobeyeranbeyeran-19-oic

acid (12e). Yield: 54 mg (58%), starting from 93 mg of 11e; white

25

solid; mp 98.0e98.9 ^ꢁC; [

400 MHz)

a

]

_Dꢀ79 (c 1.7, CH₃OH); ¹H NMR (CDCl₃,

d

7.97 (2H, d, J ¼ 7.7 Hz, 2,6-Ph), 7.54 (1H, t, J ¼ 7.7 Hz, 4-

Ph), 7.42 (2H, t, J ¼ 7.7 Hz, 3,5-Ph), 4.60 (1H, dd, J ¼ 4.8, 11.4 Hz, H-

1⁰a), 4.49 (1H, dd, J ¼ 3.3, 11.4 Hz, H-1⁰b), 2.73 (1H, m, H-15), 2.21

(1H, d, J ¼ 13.1 Hz, H-3), 1.30 (3H, s, H-18), 1.04 (3H, s, H-17), 0.90

(3H, s, H-20), 2.0e0.80 (17H, m, CH, CH₂in ent-beyerane skeleton);

5.1.10. General procedures for the synthesis of 12ae12n

Products 12ae12n were synthesized from 11ae11n following

the same procedure described for preparation of 9.

¹³C NMR (CDCl₃, 100 MHz)

d 221.32, 172.87, 166.27, 133.08, 129.61,

128.47, 129.61, 128.47, 62.57, 57.18, 56.96, 53.22, 50.93, 48.24, 45.31,

40.63, 39.40, 38.45, 37.94, 37.17, 35.44, 28.18, 22.70, 21.60, 19.93,

19.73, 18.81, 14.29; HRMS (ESI, m/z) calcd for C₂₈H₃₆O₅Na, 475.2461

[M þ Na^þ]; found, 475.2465.

5.1.10.1. Ent-15b -acetoxymethyl-16-oxobeyeran-19-oic acid 12a.

Yield: 47 mg (47%), starting from 100 mg of 11a; white solid; mp

25

159.6e160.3 ^ꢁC; [

400 MHz)

a

]

ꢀ30 (c 1.4, CH₂Cl₂); ¹H NMR (CDCl₃,

D

d

4.30 (2H, m, H-1⁰), 2.66 (1H, m, H-15), 2.16 (1H, d,

5 .1.10 . 6 . E n t - 15

b - ( ( 4 - ﬂ u o r o b e n z o yl ) o x y ) m e t h yl - 16 -

J ¼ 13.3 Hz, H-3), 2.03 (3H, s, COCH₃), 1.25 (3H, s, H-18), 0.97 (3H, s,

oxobeyeranbeyeran-19-oic acid (12f). Yield: 102 mg (63%), starting

25

from 162 mg of 11f; white solid; mp 205.4e206.2 ^ꢁC; [

1.1, CH₂Cl₂); ¹H NMR (CDCl₃, 400 MHz)

8.00 (2H, m, 2,6-Ph), 7.09

a

]

_Dꢀ90 (c

H-17), 0.78 (3H, s, H-20), 1.90e0.80 (17H, m, CH, CH₂in ent-

beyerane skeleton); ¹³C NMR (CDCl₃, 100 MHz)

d 221.74, 183.53,

d

(2H, m, 3,5-Ph), 4.56 (1H, dd, J ¼ 5.5, 11.4 Hz, H-1⁰a), 4.46 (1H, dd,

J ¼ 3.6, 11.4 Hz, H-1⁰b), 2.76 (1H, m, H-15), 2.20 (1H, d, J ¼ 13.4 Hz,

H-3), 1.30 (3H, s, H-18), 1.02 (3H, s, H-17), 0.91 (3H, s, H-20),

1.90e0.80 (17H, m, CH, CH₂in ent-beyerane skeleton); ¹³C NMR

170.67, 62.28, 56.93, 53.00, 51.21, 48.11, 43.59, 40.65, 39.51, 38.45,

35.47, 31.52, 30.15, 29.71, 28.92, 21.46, 20.85, 19.79, 19.60, 18.75,

13.17; HRMS (ESI, m/z) calcd for C₂₃H₃₄O₅Na, 413.2304 [M þ Na^þ];

found, 413.2301.

(CDCl₃, 100 MHz)

d 221.13, 172.88, 165.34, 132.26, 132.26, 132.17,

115.78, 115.78, 115.56, 62.71, 57.21, 56.89, 53.21, 51.04, 48.21, 45.32,

40.65, 39.36, 38.44, 37.92, 37.11, 35.49, 28.27, 21.58, 19.96, 19.69,

18.84, 14.32; HRMS (ESI, m/z) calcd for C₂₈H₃₅FO₅Na, 493.2367

[M þ Na^þ]; found, 493.2342.

5.1.10.2. Ent-15b-((cyclohexanecarbonyl)oxy)methyl-16-oxobeyeran-

19-oic acid (12b). Yield: 46 mg (45%), starting from 103 mg of 11b;

25

white solid; mp 145.3e146.6 ^ꢁC; [

a]

_Dꢀ68 (c 2.4, CH₂Cl₂); ¹H NMR

(CDCl₃, 400 MHz)

d

4.34 (1H, dd, J ¼ 4.5, 11.4 Hz, H-1⁰a), 4.23 (1H,

dd, J ¼ 3.2, 11.4 Hz, H-1⁰b), 2.58 (1H, m, H-15), 2.32e2.13 (2H,

overlap, COCH, H-3), 1.26 (3H, s, H-18), 0.98 (3H, s, H-17), 0.82 (3H,

s, H-20), 1.90e0.80 (27H, m, CH, CH₂in ent-beyerane skeleton or

5.1.10.7. Ent-15

b-((4-methoxybenzoyl)oxy)methyl-16-

oxobeyeranbeyeran-19-oic acid (12g). Yield: 41 mg (41%), starting

25

from 100 mg of 11g; white solid; mp 202.2e202.9 ^ꢁC; [

1.5, CH₂Cl₂); ¹H NMR (CDCl₃, 400 MHz)

a

]

_Dꢀ80 (c

carbon chain); ¹³C NMR (CDCl₃, 100 MHz)

d 221.47, 175.54, 172.85,

d

7.92 (2H, d, J ¼ 8.9 Hz, 3,5-

61.81, 57.19, 56.98, 53.09, 50.70, 48.14, 45.26, 43.14, 40.55, 39.42,

38.40, 37.93, 37.13, 35.33, 29.71, 28.99, 28.91, 28.16, 25.73, 25.43,

25.37, 21.57, 19.79, 18.74, 14.19; HRMS (ESI, m/z) calcd for C₂₈H₄₃O₅,

459.3110 [M þ H^þ]; found, 459.3090.

Ph), 6.89 (2H, d, J ¼ 8.9 Hz, 2,6-Ph), 4.54 (1H, dd, J ¼ 5.1, 11.5 Hz, H-

1⁰a), 4.46 (1H, dd, J ¼ 3.3, 11.5 Hz, H-1⁰b), 3.84 (3H, s, OCH₃), 2.73

(1H, m, H-15), 2.20 (1H, d, J ¼ 13.4 Hz, H-3), 1.26 (3H, s, H-18), 1.03

(3H, s, H-17), 0.90 (3H, s, H-20), 1.80e0.80 (17H, m, CH, CH₂in ent-

beyerane skeleton); ¹³C NMR (CDCl₃, 100 MHz)

d 221.42, 172.81,

5.1.10.3. Ent-15b-(nicotinoyloxy)methyl-16-oxobeyeranbeyeran-19-

166.01, 163.46, 131.68, 131.68, 122.18, 113.74, 113.74, 62.32, 57.22,

56.96, 55.42, 53.23, 51.12, 48.20, 45.31, 40.65, 39.41, 38.44, 35.45,

31.45, 30.21, 28.24, 21.61, 19.95, 19.72, 18.83, 14.33; HRMS (ESI, m/z)

calcd for C₂₉H₃₈O₆Na, 505.2566 [M þ Na^þ]; found, 505.2575.

oic acid (12c). Yield: 68 mg (66%), starting from 106 mg of 11c;

25

white solid; mp 108.9e109.6 ^ꢁC; [

(CDCl₃, 400 MHz)

a

]

_Dꢀ40 (c 1.1, CH₃OH); ¹H NMR

d

9.15 (1H, s, 2-pyrimidine), 8.77 (1H, d,

J ¼ 3.7 Hz, 4-pyrimidine), 8.27 (1H, m, 6-pyrimidine), 7.39 (1H, dd,

J ¼ 4.8, 8.0 Hz, 5-pyrimidine), 4.66 (1H, dd, J ¼ 4.9, 11.5 Hz, H-1⁰a),

4.51 (1H, dd, J ¼ 3.6, 11.5 Hz, H-1⁰b), 2.75 (1H, m, H-15), 2.21 (1H, d,

J ¼ 12.8 Hz, H-3), 1.26 (3H, s, H-18), 1.03 (3H, s, H-17), 0.90 (3H, s, H-

20), 2.00e0.80 (17H, m, CH, CH₂in ent-beyerane skeleton); ¹³C

5.1.10.8. Ent-15

b-((2,3-dimethoxybenzoyl)oxy)methyl-16-

oxobeyeranbeyeran-19-oic acid (12h). Yield: 29 mg (30%), starting

25

from 29 mg of 11h; white solid; mp 117.3e118.6 ^ꢁC; [

2.2, CH₃OH); ¹H NMR (CDCl₃, 400 MHz)

a

]

ꢀ67 (c

D

d

7.25 (1H, d, J ¼ 2.0 Hz, 6-

NMR (CDCl₃, 100 MHz)

d 221.01, 172.91, 164.93, 153.53, 150.75,

Ph), 7.15e6.98 (2H, overlap, 3,4-Ph), 4.58 (1H, dd, J ¼ 5.6, 11.5 Hz, H-

1⁰a), 4.50 (1H, dd, J ¼ 3.4,11.5 Hz, H-1⁰b), 3.87 (6H, s, 2 ꢃ OCH₃), 2.74

(1H, m, H-15), 2.17 (1H, d, J ¼ 13.2 Hz, H-3), 1.24 (3H, s, H-18), 0.98

(3H, s, H-17), 0.82 (3H, s, H-20), 2.00e0.80 (17H, m, CH, CH₂in ent-

137.27, 125.76, 123.51, 62.95, 57.13, 56.86, 53.23, 50.72, 48.28, 45.31,

40.60, 39.34, 38.44, 37.08, 35.45, 31.45, 28.19, 21.57, 19.96, 19.70,

18.80, 14.28; HRMS (ESI, m/z) calcd for C₂₇H₃₅NO₅Na, 476.2413

[M þ Na^þ]; found, 476.2382.

beyerane skeleton); ¹³C NMR (CDCl₃, 100 MHz)

d 221.42, 172.94,

165.44, 153.48, 149.40, 125.70, 123.76, 122.23, 115.95, 62.47, 61.67,

57.20, 57.02, 56.10, 52.88, 50.66, 48.21, 45.28, 40.59, 39.46, 38.43,

37.98, 37.22, 35.34, 28.15, 21.64, 19.80, 19.73, 18.73, 14.23; HRMS

(ESI, m/z) calcd for C₃₀H₃₉O₇, 511.2696 [M-H^þ]; found, 511.2647.

5.1.10.4. Ent-15b-(isonicotinoyloxy)methyl-16-oxobeyeranbeyeran-

19-oic acid (12d). Yield: 16 mg (35%), starting from 46 mg of 11d;

25

white solid; mp 72.3e72.9 ^ꢁC; [

(CDCl₃, 400 MHz)

a]

ꢀ56 (c 2.0, CH₃OH); ¹H NMR

D

d

8.76 (2H, d, J ¼ 5.5 Hz, 3,5-pyrimidine), 7.81 (2H,

d, J ¼ 5.5 Hz, 2,6-pyrimidine), 4.61 (1H, dd, J ¼ 5.6, 11.5 Hz, H-1⁰a),

4.50 (1H, dd, J ¼ 3.8, 11.5 Hz, H-1⁰b), 2.79 (1H, m, H-15), 2.20 (1H, d,

J ¼ 13.6 Hz, H-3), 1.26 (3H, s, H-18), 1.03 (3H, s, H-17), 0.92 (3H, s, H-

20), 2.00e0.85 (17H, m, CH, CH₂in ent-beyerane skeleton); ¹³C

5.1.10.9. Ent-15

b-((3,4-dimethoxybenzoyl)oxy)methyl-16-

oxobeyeranbeyeran-19-oic acid (12i). Yield: 60 mg (58%), starting

25

from 104 mg of 11i; white solid; mp 105.3e106.0 ^ꢁC; [

0.7, CH₃OH); ¹H NMR (CDCl₃, 400 MHz)

a]

_Dꢀ72 (c

d

7.61 (1H, dd, J ¼ 2.0,

NMR (CDCl₃, 150 MHz)

d

220.82, 172.93, 164.88, 150.69, 150.69,

8.5 Hz, 6-Ph), 7.47 (1H, d, J ¼ 2.0 Hz, 2-Ph), 6.88 (1H, d, J ¼ 8.5 Hz, 5-

Ph), 4.57 (1H, dd, J ¼ 4.8, 11.4 Hz, H-1⁰a), 4.50 (1H, dd, J ¼ 3.3,

11.4 Hz, H-1⁰b), 3.92 (3H, s, OCH₃), 3.90 (3H, s, OCH₃), 2.72 (1H, m,

H-15), 2.20 (1H, d, J ¼ 13.4 Hz, H-3), 1.26 (3H, s, H-18), 1.04 (3H, s, H-

17), 0.89 (3H, s, H-20), 1.90e0.80 (17H, m, CH, CH₂in ent-beyerane

136.97, 124.00, 122.91, 63.34, 57.15, 56.81, 53.18, 50.84, 48.26, 45.32,

40.64, 39.31, 38.45, 37.05, 35.52, 31.45, 30.21, 28.23, 21.55, 19.99,

18.84, 14.33; HRMS (ESI, m/z) calcd for C₂₇H₃₅NO₅K, 492.2152

[M þ K^þ]; found, 492.2167.

13

H. Zhang, B. Liu, G. Xu et al.

European Journal of Medicinal Chemistry 219 (2021) 113396

skeleton); ¹³C NMR (CDCl₃, 150 MHz)

d

221.36, 172.86, 166.01,

5.1.10.14. Ent-16-oxo-15b-(2-(3,4,5-trimethoxyphenyl)acetoxy)meth-

153.10, 148.66, 122.28, 111.92, 110.37, 62.40, 57.17, 56.99, 56.02,

56.02, 53.24, 50.97, 48.20, 45.31, 39.43, 38.45, 37.95, 37.16, 35.43,

31.45, 30.20, 28.15, 21.61, 19.96, 19.73, 18.78, 14.28; HRMS (ESI, m/z)

calcd for C₃₀H₄₀O₇K, 551.2411 [M þ K^þ]; found, 551.2438.

ylbeyeran-19-oic acid (12n). Yield: 51 mg (50%), starting from

25

102 mg of 11n; white solid; mp 85.1e86.0 ^ꢁC; [

CH₃OH);¹H NMR (CDCl₃, 400 MHz)

6.44 (2H, s, 2,6-Ph), 4.38 (1H,

a]

ꢀ56 (c 1.9,

D

d

dd, J ¼ 4.8, 11.4 Hz, H-1⁰a), 4.25 (1H, dd, J ¼ 3.2, 11.4 Hz, H-1⁰b), 3.84

(6H, s, 2 ꢃ OCH₃), 3.82 (3H, s, OCH₃), 3.60e3.43 (2H, m, COCH₂),

2.56 (1H, m, H-15), 2.16 (1H, d, J ¼ 13.6 Hz, H-3), 1.27 (3H, s, H-18),

0.86 (3H, s, H-17), 0.80 (3H, s, H-20), 1.85e0.80 (17H, m, CH, CH₂in

5.1.10.10. Ent-16-oxo-15b-((2,3,4-trimethoxybenzoyl)oxy)methyl-

beyeran-19-oic acid (12j). Yield: 49 mg (70%), starting from 70 mg

25

of 11j; white solid; mp 96.7e97.6 ^ꢁC; [

NMR (CDCl₃, 400 MHz)

a]

_Dꢀ54 (c 1.1, CH₂Cl₂); ¹H

ent-beyerane skeleton); ¹³C NMR (CDCl₃,100 MHz)

d 221.35,172.89,

d

7.53 (1H, d, J ¼ 9.0 Hz, 6-Ph), 6.68 (1H, d,

170.99, 153.29, 153.29, 137.07, 129.31, 106.35, 106.35, 62.50, 60.84,

57.10, 56.85, 56.11, 56.11, 52.62, 50.66, 48.00, 45.27, 41.79, 40.43,

39.33, 38.38, 37.91, 37.00, 35.26, 28.15, 21.50, 19.63, 19.59, 18.75,

14.20; HRMS (ESI, m/z) calcd for C₃₂H₄₄O₈K,595.2673 [M þ K^þ];

found, 595.2688.

J ¼ 9.0 Hz, 5-Ph), 4.52 (1H, dd, J ¼ 4.7, 11.4 Hz, H-1⁰a), 4.46 (1H, dd,

J ¼ 3.6, 11.4 Hz, H-1⁰b), 3.90 (3H, s, OCH₃), 3.89 (3H, s, OCH₃), 3.86

(3H, s, OCH₃), 2.69 (1H, m, H-15), 2.20 (1H, d, J ¼ 13.4 Hz, H-3), 1.25

(3H, s, H-18), 1.01 (3H, s, H-17), 0.88 (3H, s, H-20), 2.00e0.75 (17H,

m, CH, CH₂in ent-beyerane skeleton); ¹³C NMR (CDCl₃, 100 MHz)

d

221.44, 172.88, 164.65, 157.22, 155.03, 142.90, 126.91, 117.40,

5.1.11. Synthesis of ent-4-amino-15

norbeyeran-16 -ol (13)

b-hydroxymethyl-19-

106.83, 62.15, 61.87, 61.05, 57.19, 57.01, 56.05, 52.90, 50.83, 48.21,

45.30, 40.61, 39.46, 38.44, 37.99, 37.22, 35.39, 28.16, 21.64, 19.82,

19.73, 18.75, 14.26; HRMS (ESI, m/z) calcd for C₃₁H₄₂O₈K, 581.2517

[M þ K^þ]; found, 581.2511.

a

Compound 13 was synthesized from 7 following the same

procedure described for preparation of 6. Yield: 34 mg (37%),

starting from 100 mg of 7; white solid; mp 180.2e181.6 ^ꢁC;

25

[

a

]

_Dꢀ49 (c 1.6, CH₃OH); ¹H NMR (CDCl₃, 400 MHz)

d 3.97 (1H, dd,

5.1.10.11. Ent-16-oxo-15

b

-((3,4,5-trimethoxybenzoyl)oxy)methyl-

J ¼ 5.1, 9.8 Hz, H-1⁰a), 3.62 (1H, d, J ¼ 4.9 Hz, H-16), 3.52 (1H, t,

J ¼ 10.2 Hz, H-1⁰b), 2.12 (1H, m, H-15), 1.34 (3H, s, H-18), 1.07 (3H, s,

H-17), 0.94 (3H, s, H-20); 2.0e0.80 (18H, m, CH, CH₂in ent-

beyeran-19-oic acid (12k). Yield: 75 mg (58%), starting from 130 mg

25

of 11k; white solid; mp 109.9e110.8 ^ꢁC; [

¹H NMR (CDCl₃, 400 MHz)

7.22 (2H, s, 2,6-Ph), 4.60 (1H, dd,

a]

_Dꢀ50 (c 1.0, CH₃OH);

d

beyerane skeleton); ¹³C NMR (CDCl₃, 100 MHz)

d 84.07, 64.88,

J ¼ 4.4, 11.4 Hz, H-1⁰a), 4.53 (1H, dd, J ¼ 3.4, 11.4 Hz, H-1⁰b),

3.78e3.95 (9H, overlap, 3 ꢃ OCH₃), 2.71 (1H, d, J ¼ 4.1 Hz, H-15),

2.20 (1H, d, J ¼ 13.2 Hz, H-3), 1.30 (3H, s, H-18), 1.05 (3H, s, H-17),

0.87 (3H, s, H-20), 1.90e0.80 (17H, m, CH, CH₂in ent-beyerane

59.14, 57.55, 55.45, 55.15, 50.35, 47.80, 42.03, 41.58, 40.85, 40.14,

38.34, 33.85, 32.97, 31.87, 24.95, 19.16, 17.88, 13.76; HRMS (ESI, m/z)

calcd for C₂₀H₃₆NO₂, 322.2746 [M þ H^þ]; found, 322.2725.

skeleton); ¹³C NMR (CDCl₃, 100 MHz)

d

221.26, 172.89, 165.84,

5.1.12. General procedures for the synthesis of 14ae14e

Products 14a-14e were synthesized from 13 following the same

procedure described for preparation of 11a-11n.

152.97, 152.97, 124.78, 107.64, 106.79, 106.79, 60.94, 57.18, 57.01,

56.63, 56.25, 53.25, 50.76, 48.21, 48.18, 45.28, 43.48, 40.65, 38.45,

37.15, 35.42, 28.12, 21.59, 19.98, 19.75, 18.76, 14.26, 13.33; HRMS

(ESI, m/z) calcd for C₃₁H₄₂O₈Na, 565.2778 [M þ Na^þ]; found,

565.2752.

5.1.12.1. Ent-4-amino-16a-hydroxy-19-norbeyeran-15b-methyl nic-

otinate (14a). Yield: 113 mg (43%), starting from 200 mg of 13;

25

white solid; mp 76.9e77.3 ^ꢁC; [

(CDCl₃, 400 MHz)

a

]

ꢀ45 (c 1.6, CH₃OH); ¹H NMR

D

5.1.10.12. Ent-15

b

-(((E)-3-(3,5-dimethoxyphenyl)acryloyl)oxy)

d

9.25 (1H, s, 2-pyrimidine), 8.77 (1H, dd, J ¼ 1.8,

methyl-16-oxobeyeran-19-oic acid (12l). Yield: 53 mg (53%), start-

4.9 Hz, 4-pyrimidine), 8.32 (1H, m, 6-pyrimidine), 7.41 (1H, dd,

J ¼ 4.9, 7.9 Hz, 5-pyrimidine), 4.59 (1H, dd, J ¼ 6.1, 11.0 Hz, H-1⁰a),

4.29 (1H, dd, J ¼ 8.2, 11.0 Hz, H-1⁰b), 3.59 (1H, d, J ¼ 5.0 Hz, H-16),

2.47 (1H, m, H-15), 1.33 (3H, s, H-18), 1.11 (3H, s, H-17), 0.94 (3H, s,

H-20), 1.95e0.80 (18H, m, CH, CH₂in ent-beyerane skeleton); ¹³C

25

ing from 100 mg of 11l; white solid; mp 149.3e150.0 ^ꢁC; [

(c 1.1, CH₂Cl₂); 1H NMR (CDCl₃, 400 MHz)

a

]

_Dꢀ99

d

7.57 (1H, d, J ¼ 16.0 Hz,

CH2 ¼ ), 6.66 (2H, d, J ¼ 2.3 Hz, 2,6-Ph), 6.49 (1H, t, J ¼ 2.3 Hz, 4-Ph),

6.34 (1H, d, J ¼ 16.0 Hz, CH2 ¼ ), 4.45 (2H, m, H-1⁰), 3.80 (6H, s,

2 ꢃ OCH3), 2.71 (1H, m, H-15), 2.15 (1H, d, J ¼ 13.4 Hz, H-3), 1.24

(3H, s, H-18), 1.01 (3H, s, H-17), 0.81 (3H, s, H-20), 1.85e0.80 (17H,

m, CH, CH₂in ent-beyerane skeleton); 13C NMR (CDCl₃, 150 MHz)

NMR (CDCl₃, 100 MHz)

d 165.56, 153.48, 150.94, 137.08, 126.13,

123.48, 85.32, 67.20, 59.08, 57.45, 54.98, 54.44, 47.44, 42.53, 41.68,

40.93, 38.34, 37.68, 33.73, 32.96, 31.84, 24.95, 20.38, 19.12, 17.87,

13.76; HRMS (ESI, m/z) calcd for C₂₆H₃₉N₂O₃, 427.2960 [M þ H^þ];

found, 427.2951.

d

221.78, 182.02, 166.46, 161.01, 161.01, 145.16, 136.21, 118.22, 106.10,

106.10, 102.53, 62.43, 56.98, 56.98, 55.43, 55.43, 53.05, 51.24, 48.18,

43.52, 40.76, 39.55, 38.46, 37.69, 37.22, 35.51, 28.84, 21.55, 19.90,

19.64, 18.76, 13.26; HRMS (ESI, m/z) calcd for C₃₂H₄₃O₇, 539.3009

[M þ H^þ]; found, 539.3019.

5.1.12.2. Ent-4-amino-16

a-hydroxy-19-norbeyeran-15b-methyl 4-

methoxybenzoate (14b). Yield: 93 mg (33%), starting from 200 mg

25

of 13; white solid; mp 173.4e175.2 ^ꢁC; [

¹H NMR (CDCl₃, 400 MHz)

a

]

ꢀ54 (c 0.4, CH₃OH);

D

5.1.10.13. Ent-15

b

-(2-(4-methoxyphenyl)acetoxy)methyl-16-

d

7.95 (2H, d, J ¼ 8.2 Hz, 3,5-Ph), 6.87 (2H,

oxobeyeran-19-oic acid (12m). Yield: 54 mg (56%), starting from

d, J ¼ 8.2 Hz, 2,6-Ph), 4.43 (1H, dd, J ¼ 6.5, 11.0 Hz, H-1⁰a), 4.16 (1H,

dd, J ¼ 7.7, 11.0 Hz, H-1⁰b), 3.79 (3H, s, OCH₃), 3.48 (1H, d, J ¼ 5.0 Hz,

H-16), 2.37 (1H, m, H-15), 1.25 (3H, s, H-18), 1.05 (3H, s, H-17), 0.86

(3H, s, H-20),1.95e0.80 (18H, m, CH, CH₂in ent-beyerane skeleton);

25

96 mg of 11m; white solid; mp 87.1e87.9 ^ꢁC; [

CH₃OH); ¹H NMR (CDCl₃, 400 MHz)

a

]

ꢀ84 (c 1.3,

D

d

7.14 (2H, d, J ¼ 8.6 Hz, 2,6-Ph),

6.84 (2H, d, J ¼ 8.6 Hz, 3,5-Ph), 4.34 (1H, dd, J ¼ 4.9, 11.4 Hz, H-1⁰a),

4.24 (1H, dd, J ¼ 3.2, 11.4 Hz, H-1⁰b), 3.78 (3H, s, OCH₃), 3.52 (2H, d,

J ¼ 3.5 Hz, COCH₂), 2.55 (1H, m, H-15), 2.16 (1H, d, J ¼ 13.5 Hz, H-3),

1.27 (3H, s, H-18), 0.88 (3H, s, H-17), 0.80 (3H, s, H-20); 1.85e0.80

(17H, m, CH, CH₂in ent-beyerane skeleton); ¹³C NMR (CDCl₃,

¹³C NMR (CDCl₃, 100 MHz)

d 165.60, 162.45, 130.58, 130.58, 121.51,

112.78, 112.78, 84.41, 65.41, 58.07, 56.48, 54.43, 53.94, 53.46, 46.69,

41.49, 40.78, 39.77, 37.36, 36.67, 32.70, 31.96, 30.84, 23.95, 19.38,

18.13, 16.88, 12.75; HRMS (ESI, m/z) calcd for C₂₈H₄₂NO₄, 456.3114

[M þ H^þ]; found, 456.3079.

100 MHz)

d 221.45, 172.90, 171.40, 158.74, 130.45, 130.45, 125.81,

114.07, 114.07, 62.38, 57.14, 56.91, 55.32, 52.65, 50.74, 48.01, 45.26,

40.56, 40.44, 39.38, 38.37, 37.94, 37.07, 35.23, 28.14, 21.53, 19.66,

19.63, 18.74, 14.17; HRMS (ESI, m/z) calcd for C₃₀H₄₀O₆Na, 519.2723

[M þ Na^þ]; found, 519.2747.

5.1.12.3. Ent-4-amino-16

a-hydroxy-19-norbeyeran-15b-methyl 2,3-

dimethoxybenzoate (14c). Yield: 110 mg (36%), starting from

25

200 mg of 13; white solid; mp 164.4e165.2 ^ꢁC; [

a

]

ꢀ67 (c 0.7,

D

14

H. Zhang, B. Liu, G. Xu et al.

European Journal of Medicinal Chemistry 219 (2021) 113396

CH₃OH); ¹H NMR (CDCl₃, 400 MHz)

d

7.40 (1H, dd, J ¼ 1.8, 7.7 Hz, 6-

dd, J ¼ 2.9, 11.5 Hz, H-1⁰b), 3.85 (3H, s, OCH₃), 2.92 (1H, m, H-15),

1.96 (1H, m, H-3), 1.34 (3H, s, H-18), 1.09 (3H, s, H-17), 1.01 (3H, s, H-

20),1.95e0.80 (17H, m, CH, CH₂in ent-beyerane skeleton); ¹³C NMR

Ph), 7.12 (2H, m, 3,4-Ph), 4.63 (1H, dd, J ¼ 5.1, 10.4 Hz, H-1⁰a), 4.13

(1H, t, J ¼ 10.9 Hz, H-1⁰b), 3.91 (3H, s, OCH₃), 3.87 (3H, s, OCH₃), 3.60

(1H, m, H-16), 3.08 (1H, s, 16-OH), 2.35 (1H, m, H-15), 1.86 (1H, dd,

J ¼ 5.2,13.8 Hz, H-3),1.37 (3H, s, H-18),1.03 (3H, s, H-17), 0.96 (3H, s,

(CDCl₃, 100 MHz)

d 221.28, 166.10, 163.55, 131.59, 131.59, 122.12,

113.84, 113.84, 62.70, 58.98, 56.78, 55.45, 55.11, 53.36, 51.85, 48.11,

41.53, 40.55, 38.34, 37.80, 36.93, 34.44, 31.83, 19.97, 19.93, 19.24,

17.90, 14.16; HRMS (ESI, m/z) calcd for C₂₈H₄₀NO₄, 454.2957

[M þ H^þ]; found, 454.2945.

H-20), 1.95e0.80 (17H, m, CH, CH₂in ent-beyerane skeleton);¹³

C

NMR (CDCl₃, 100 MHz)

d 166.85, 154.52, 153.52, 125.70, 124.37,

122.70, 116.49, 86.34, 67.83, 61.68, 57.92, 56.22, 56.08, 55.86, 54.12,

47.77, 42.53, 41.00, 38.69, 37.68, 35.70, 34.22, 32.90, 26.95, 25.04,

19.70, 19.14, 17.43, 14.29; HRMS (ESI, m/z) calcd for C₂₉H₄₃NO₅Na,

508.3039 [M þ Na^þ]; found, 508.3063.

5.1.13.3. Ent-4-amino-16-oxo-19-norbeyeran-15

b-methyl 2,3-

dimethoxybenzoate (15c). Yield: 53 mg (53%), starting from

25

100 mg of 14c; white solid; mp 152.3e153.2 ^ꢁC; [

CH₃OH); ¹H NMR (CDCl₃, 400 MHz)

d 7.28 (1H, m, 6-Ph), 7.13e7.05

a]

ꢀ89 (c 0.8,

D

5.1.12.4. Ent-4-amino-16

a

-hydroxy-19-norbeyeran-15

b

-methyl

2,3,4-trimethoxybenzoate (14d). Yield: 79 mg (55%), starting from

(2H, overlap, 3,4-Ph), 4.55 (1H, dd, J ¼ 6.9, 11.5 Hz, H-1⁰a), 4.44 (1H,

dd, J ¼ 3.4, 11.5 Hz, H-1⁰b), 3.89 (3H, s, OCH₃), 3.88 (3H, s, OCH₃),

2.82 (1H, m, H-15), 1.34 (3H, s, H-18), 0.99 (3H, s, H-17), 0.99 (3H, s,

H-20), 2.00e0.75 (18H, m, CH, CH₂in ent-beyerane skeleton); ¹³C

25

90 mg of 13; white solid; mp 51.7e52.9 ^ꢁC; [

CH₃OH);¹H NMR (CDCl₃, 400 MHz)

a

]

ꢀ70 (c 0.4,

D

d

7.71 (1H, d, J ¼ 8.9 Hz, 2-Ph),

6.76 (1H, d, J ¼ 8.9 Hz, 3-Ph), 4.56 (1H, dd, J ¼ 5.0, 10.3 Hz, H-1⁰a),

4.18 (1H, dd, J ¼ 10.3, 11.9 Hz, H-1⁰b), 3.94 (3H, s, OCH₃), 3.91 (3H, s,

OCH₃), 3.85 (3H, s, OCH₃), 3.58 (1H, d, J ¼ 4.7 Hz, H-16), 2.40 (1H, m,

H-15), 1.87 (1H, m, H-3), 1.35 (3H, s, H-18), 1.09 (3H, s, H-17), 0.97

(3H, s, H-20),1.95e0.80 (17H, m, CH, CH₂in ent-beyerane skeleton);

NMR (CDCl₃, 100 MHz)

d 221.04, 165.68, 154.55, 153.65, 125.53,

123.91, 122.04, 116.02, 62.74, 61.74, 57.10, 56.09, 56.00, 53.44, 52.90,

51.47, 48.14, 40.52, 38.69, 37.79, 36.91, 35.66, 34.84, 26.95, 19.81,

19.19, 19.05, 17.44, 14.66; HRMS (ESI, m/z) calcd for C₂₉H₄₂NO₅,

484.3063[M þ H^þ]; found,484.3082.

¹³C NMR (CDCl₃, 100 MHz)

d 166.24, 157.59, 154.08, 142.90, 127.64,

117.48, 107.49, 86.61, 67.69, 62.00, 61.07, 59.07, 57.53, 56.13, 55.05,

54.35, 47.91, 42.40, 41.72, 40.92, 38.36, 37.68, 33.78, 32.98, 31.88,

25.08, 20.48, 19.22, 17.87, 13.64; HRMS (ESI, m/z) calcd for

5.1.13.4. Ent-4-amino-16-oxo-19-norbeyeran-15

b-methyl 2,3,4-

trimethoxybenzoate (15d). Yield: 57 mg (57%), starting from

25

C

₃₀H₄₆NO₆, 516.3325 [M þ H^þ]; found, 516.3315.

100 mg of 14d; white solid; mp 109.2e109.9 ^ꢁC; [

CH₃OH); ¹H NMR (CDCl₃, 400 MHz)

a]

ꢀ70 (c 1.0,

D

d

7.55 (1H, d, J ¼ 8.9 Hz, 6-Ph),

5.1.12.5. Ent-4-amino-16

a

-hydroxy-19-norbeyeran-15

b

-methyl

6.71 (1H, d, J ¼ 8.9 Hz, 5-Ph), 4.52 (1H, dd, J ¼ 6.7, 11.5 Hz, H-1⁰a),

4.43 (1H, dd, J ¼ 3.3, 11.5 Hz, H-1⁰b), 3.92 (3H, s, OCH₃), 3.89 (3H, s,

OCH₃), 3.87 (3H, s, OCH₃), 2.88 (1H, m, H-15), 1.26 (3H, s, H-18), 1.06

(3H, s, H-17), 1.00 (3H, s, H-20), 2.00e0.75 (18H, m, CH, CH₂in ent-

3,4,5-trimethoxybenzoate (14e). Yield: 61 mg (38%), starting from

25

100 mg of 13; white solid; mp 75.4e76.6 ^ꢁC; [

CH₃OH); ¹H NMR (CDCl₃, 400 MHz)

7.33 (2H, s, 2,6-Ph), 4.55 (1H,

a]

ꢀ24 (c 1.7,

D

d

dd, J ¼ 5.5, 10.9 Hz, H-1⁰a), 4.26 (1H, dd, J ¼ 8.3, 10.9 Hz, H-1⁰b), 3.91

(6H, s, 2 ꢃ OCH₃), 3.90 (3H, s, OCH₃), 3.64 (1H, d, J ¼ 5.0 Hz, H-16),

2.44 (1H,m, H-15), 1.34 (3H, s, H-18), 1.10 (3H, s, H-17), 0.94 (3H, s,

H-20), 1.95e0.80 (18H, m, CH, CH₂in ent-beyerane skeleton); ¹³C

beyerane skeleton); ¹³C NMR (CDCl₃, 100 MHz)

d 221.35, 164.71,

157.31, 155.10, 142.96, 126.72, 117.28, 106.92, 62.46, 61.88, 61.04,

58.97, 56.82, 56.06, 55.11, 53.15, 51.63, 48.11, 41.60, 40.51, 38.35,

37.79, 36.97, 34.41, 31.85, 29.71,19.84,19.24,17.89,14.12; HRMS (ESI,

m/z) calcd for C₃₀H₄₄NO₆, 514.3168 [M þ H^þ]; found, 514.3148.

NMR (CDCl₃, 100 MHz)

d 166.45, 153.03, 153.03, 142.31, 125.20,

106.81, 106.81, 85.38, 66.69, 60.93, 59.08, 57.55, 56.25, 56.25, 55.04,

54.55, 47.59, 42.57, 41.73, 40.92, 38.38, 37.69, 33.75, 32.98, 31.86,

25.01, 20.38, 19.18, 17.88, 13.78; HRMS (ESI, m/z) calcd for

5.1.13.5. Ent-4-amino-16-oxo-19-norbeyeran-15

b-methyl 3,4,5-

trimethoxybenzoate (15e). Yield: 65 mg (54%), starting from

25

C

₃₀H₄₆NO₆, 516.3325 [M þ H^þ]; found, 516.3319.

120 mg of 14e; white solid; mp 64.0e64.8 ^ꢁC; [

CH₃OH); ¹H NMR (CDCl₃, 400 MHz)

a

]

ꢀ66 (c 1.1,

D

d

7.24 (2H, s, 2,6-Ph), 4.62 (1H,

5.1.13. General procedures for the synthesis of 15ae15e

Products 15ae15e were synthesized from 14ae14e following

the same procedure described for preparation of 12ae12n.

m, H-1⁰a), 4.50 (1H, m, H-1⁰b), 3.89 (9H, overlap, 3 ꢃ OCH₃), 2.86

(1H, m, H-15),1.35 (3H, s, H-18),1.05 (3H, s, H-17),1.03 (3H, s, H-20),

2.0e0.80 (18H, m, CH, CH₂in ent-beyerane skeleton); ¹³C NMR

(CDCl₃, 100 MHz)

d 221.20, 165.92, 153.00, 153.00, 142.37, 124.73,

5.1.13.1. Ent-4-amino-16-oxo-19-norbeyeran-15

(15a). Yield: 45 mg (57%), starting from 80 mg of 14a; white solid;

b

-methyl nicotinate

106.75, 106.75, 62.91, 60.93, 59.00, 56.94, 56.19, 56.19, 55.09, 53.44,

51.39, 48.14, 41.73, 40.52, 38.38, 37.80, 36.94, 34.36, 31.81, 19.95,

19.95, 19.27, 17.85, 14.09; HRMS (ESI, m/z) calcd for C₃₀H₄₄NO₆,

514.3168 [M þ H^þ]; found, 514.3158.

25

mp 124.8e125.6 ^ꢁC; [

400 MHz)

a

]

ꢀ48 (c 0.9, CH₃OH); ¹H NMR (CDCl₃,

D

d

9.18 (1H, s, 2-pyrimidine), 8.78 (1H, d, J ¼ 4.9 Hz, 4-

pyrimidine), 8.26 (1H, d, J ¼ 8.0 Hz, 6-pyrimidine), 7.42 (1H, dd,

J ¼ 4.9, 8.0 Hz, 5-pyrimidine), 4.67 (1H, dd, J ¼ 6.6, 11.6 Hz, H-1⁰a),

4.50 (1H, dd, J ¼ 3.3, 11.6 Hz, H-1⁰b), 2.91 (1H, m, H-15), 1.96 (1H, d,

J ¼ 13.8 Hz, H-3), 1.35 (3H, s, H-18), 1.07 (3H, s, H-17), 1.02 (3H, s, H-

20),1.95e0.80 (17H, m, CH, CH₂in ent-beyerane skeleton); ¹³C NMR

5.2. Zebraﬁsh embryo collection and drug treatment

The maintenance of wild-type zebraﬁsh and transgenic zebra-

ﬁsh Tg(cmlc2:GFP) were performed following standard procedures.

And the fertilized zebraﬁsh embryos were obtained as previously

described [32,33,41]. Zebraﬁsh experiments were complied with

the local and national animal use protocols. In brief, zebraﬁsh

(3e12 months old), kept at aquaculture system with light cycle,

were fed with brine shrimp twice a day. The fertilized zebraﬁsh

embryos were produced from pairwise breeding and selected by

microscopic examination.

(CDCl₃, 100 MHz)

d 220.92, 165.03, 153.65, 150.83, 137.00, 125.69,

123.54, 63.28, 58.97, 56.74, 55.12, 53.39, 51.39, 48.19, 41.43, 40.48,

38.31, 37.79, 36.88, 34.40, 31.81, 19.93, 19.93, 19.23, 17.86, 14.14;

HRMS (ESI, m/z) calcd for C₂₆H₃₇N₂O₃, 425.2804 [M þ H^þ]; found,

425.2797.

5.1.13.2. Ent-4-amino-16-oxo-19-norbeyeran-15

b-methyl 4-

methoxybenzoate (15b). Yield: 35 mg (52%), starting from 68 mg

25

of 14b; white solid; mp 118.1e119.5 ^ꢁC; [

a]

_Dꢀ48 (c 2.7, CH₃OH);

5.2.1. Survival of the DOX-treated zebraﬁsh embryos in the absence

or presence of test compound

¹H NMR (CDCl₃, 400 MHz)

d

7.95 (2H, d, J ¼ 8.7 Hz, 3,5-Ph), 6.93 (2H,

d, J ¼ 8.7 Hz, 2,6-Ph), 4.56 (1H, dd, J ¼ 7.1, 11.5 Hz, H-1⁰a), 4.42 (1H,

A previously described assay was used [33]. Brieﬂy, the wild-

15

H. Zhang, B. Liu, G. Xu et al.

European Journal of Medicinal Chemistry 219 (2021) 113396

type zebraﬁsh of 24 hpf were distributed into a 24-well plate (20

The H9c2cells were observed using a confocal microscope (Carl

embryos with 1 mL egg water per well), and were treated with DOX

Zeiss), and the ratio of red-to-green ﬂuorescence intensities were

(100

mM) with or without each of the test compound (5, 15, 40

mM)

analyzed to measure the Dj.

for another 48 h. The survival of zebraﬁsh was assessed at 96 hpf

using inverted ﬂuorescence microscope (ZEISS, Germany). The

zebraﬁsh, which lack of motion on touch, or lost contractility in

both atrium and ventricle, were regarded as dead ones.

Author contributions

Y.Z. designed the overall study. H.Y.Z. designed and synthesis the

compounds. H.Y.Z. performed biological experiments in zebraﬁsh.

G.X., C.X., E.O. and J.S.L participated in synthesis and biological

evaluation in zebraﬁsh. X.O.S. supervised the biological experi-

ments in H9c2 cell. X.O.S and B.L performed the biological experi-

ments in H9c2 cells. Y.Z. and H.Y.Z. wrote the manuscript. Y. Z.

supervised the overall study.

5.2.3. Cardioprotective effect evaluation

The transgenic zebraﬁsh Tg(cmlc2:GFP) embryos of 24 hpf were

transferred into 24-well plates (20 embryos per well) and were

treated with DOX (80 mM) with or without the test compound for

another 48 h. The parameters including heartbeat, fractional

shortening, stroke volume, and cardiac output, were examined and

calculated to evaluate the cardiac functions following the methods

previously reported [41].

Declaration of interest statement

The authors declare that they have no known competing

ﬁnancial interests or personal relationships that could have

appeared to inﬂuence the work reported in this paper.

5.2.4. Quantitative polymerase chain reaction

Twenty embryos from each treatment were subjected to RNA

extraction using a standard TRIzol protocol (Generay Biotech). RNA

concentration and quality were manus measured using Nanodrop

2000 S spectrophotometer (Thermo Scientiﬁc). Total RNA was

ampliﬁed by qRT-PCR using a two-step RT-PCR kit (Vazyme,

Nanjing, China). The qRT-PCR analyses were repeated at least three

Declaration of competing interest

The authors declare that they have no known competing

ﬁnancial interests or personal relationships that could have

appeared to inﬂuence the work reported in this paper.

times. Primers for qPCR are listed as following:

b-actin L, CCT ACT

AAT ACA CAG CCA TGG ATG A; -actin R, GTC CCA TGC CAA CCA TCA

b

Acknowledgements

C; cTnT-L, GTC TGC ACT TCG GCG GTT ACA; cTnT-R, AGG TAA AAT

CTA TAT TGT TCA GTG AAA TCT AAC CG; ANP-L, ATG GCC GGG GGA

CTA ATT CT; ANP-R, AGA GTT GCA ACC GAG GGT GC.

This study was supported by National Science and Technology

Major Projects for “Major New Drugs Innovation and Development”

(NO. 2019ZX09301120) and the Science and Technology Planning

Project of Guangzhou (201904010232). We thank Dr. Wen Tan

(School of Biomedical and Pharmaceutical Sciences, Guangdong

University of Technology) for excellent assistance during our

research.

5.3. Cell viability assay

The H9c2 cell were cultured in Dulbecco’s modiﬁed Eagle’s

medium (DMEM) containing 10% fetal bovine serum, 100 U/mL

penicillin and 100 mg/mL streptomycin, in a humidiﬁed incubator

with 5% CO₂at 37 ^ꢁC. The cells were used until they reached 70%

conﬂuence. The Cell Counting Kit-8 (CCK8) assays (Beyotime) were

carried out to evaluate the Cell viability. Brieﬂy, H9c2 cells were

incubated in a 96-well plate with medium containing CCK-8

(0.5 mg/mL) for 4 h, after the treatment of DOX with or without

the test compound for 48 h. The optical density was measured at

450 nm by Spectra Max M5 plate reader.

Appendix A. Supplementary data

Supplementary data to this article can be found online at

https://doi.org/10.1016/j.ejmech.2021.113396.

References

[1] https://www.who.int/health-topics/cardiovascular-diseases#tab¼tab_1.

(Accessed 7 February 2021).

5.4. Assessment of intracellular reactive oxygen species (ROS)

[2] F. Simko, M. Adamcova, What prevents cardioprotective drugs from reaching

the market? Expet Rev. Clin. Pharmacol. 11 (2018) 463e465.

[3] A. Shaito, D.T.B. Thuan, H.T. Phu, T.H.D. Nguyen, H. Hasan, S. Halabi,

S. Abdelhady, G.K. Nasrallah, A.H. Eid, G. Pintus, Herbal medicine for cardio-

vascular diseases: ef ﬁ cacy, mechanisms, and safety, Front. Pharmacol. 11

(2020) 422.

[4] M. Wang, H. Li, F. Xu, X. Gao, J. Li, S. Xu, D. Zhang, X. Wu, J. Xu, H. Hua, D. Li,

Diterpenoid lead stevioside and its hydrolysis products steviol and isosteviol:

biological activity and structural modi ﬁ cation, Eur. J. Med. Chem. 156 (2018)

885e906.

The intracellular reactive oxygen species were assayed using the

using DCFH-DA (Sigma-Aldrich). Brieﬂy, H9c2 cells, seeded in 24-

well plate were treated with or without the test compound for

48 h. DCFH-DA diluted with DMEM to ﬁnal concentration of

5 mmol/L was then added to H9c2cells. The mixture was incubated

for 40 min at 37 ^ꢁC. The dye was then removed, and the wells were

washed three times with phosphate buffered saline (PBS). The

ﬂuorescence intensity was then measured with confocal micro-

scopy (Carl Zeiss) to determine the reactive oxygen species level.

[5] E. Mosettig, U. Beglinger, F. Dolder, H. Lichti, P. Quitt, J.A. Waters, The absolute

con ﬁ guration of steviol and isosteviol, J. Am. Chem. Soc. 85 (1963)

2305e2309.

[6] G. Brahmachari, L.C. Mandal, R. Roy, S. Mondal, A.K. Brahmachari, Stevioside

overview, Arch. Pharm. (Weinheim) 344 (2011) 5e19.

5.5. Measurement of mitochondrial membrane potential (Djm)

[7] N. Moons, W. De Borggraeve, W. Dehaen, Stevioside and steviol as starting

materials in organic synthesis, Curr. Org. Chem. 16 (2012) 1986e1995.

[8] D. Xu, Y. Li, J. Wang, A.K. Davey, S. Zhang, A.M. Evans, The cardioprotective

effect of isosteviol on rats with heart ischemia-reperfusion injury, Life Sci. 80

(2007) 269e274.

[9] D. Xu, S. Zhang, D. Foster JR., J. Wang, The effects of isosteviol against

myocardium injury induced by ischaemia-reperfusion in the isolated Guinea

pig heart, Clin. Exp. Pharmacol. Physiol. 34 (2007) 488e493.

[10] C. Yin, Y. Chen, H. Wu, D. Xu, W. Tan, Attenuation of ischemia/reperfusion-

induced inhibition of the rapid component of delayed recti ﬁ er potassium

Mitochondrial membrane potential was measured using the

cationic JC-1 (Sigma-Aldrich) dye. Brieﬂy, H9c2 cells, seeded in

confocal petri dishes were treated with or without the test com-

pound for 48 h. The JC-1 working solution, prepared to a ﬁnal

concentration of 1 mg/mL, was added to H9c2 cells with 500 mL per

well. The mixture was incubated at 37 ^ꢁC for 20 min. The dye was

then removed, and the wells were washed three times with PBS.

16

H. Zhang, B. Liu, G. Xu et al.

European Journal of Medicinal Chemistry 219 (2021) 113396

current by Isosteviol through scavenging reactive oxygen species, Bba-Bio-

membr. 1859 (2017) 2447e2453.

when cells are exposed to hypoxia-reoxygenation, J. Cell Mol. Med. 24 (2020)

10924e10934.

[11] L.H. Lin, L.W. Lee, S.Y. Sheu, P.Y. Lin, Study on the stevioside analogues of

steviolbioside, steviol, and isosteviol 19-alkyl amide dimers: synthesis and

cytotoxic and antibacterial activity, Chem. Pharm. Bull. (Tokyo) 52 (2004)

1117e1122.

[26] A. Pott, W. Rottbauer, S. Just, Streamlining drug discovery assays for cardio-

vascular disease using zebra ﬁ sh, Expet Opin. Drug Discov. 15 (2020) 27e37.

[27] C.A. MacRae, R.T. Peterson, Zebra ﬁ sh as tools for drug discovery, Nat. Rev.

Drug 14 (2015) 721e731.

[12] Y. Wu, G.-F. Dai, J.-H. Yang, Y.-X. Zhang, Y. Zhu, J.-C. Tao, Stereoselective

synthesis of 15- and 16-substituted isosteviol derivatives and their cytotoxic

activities, Bioorg. Med. Chem. Lett. 19 (2009) 1818e1821.

[13] M. Zou, S.-S. Yu, K. Wang, D.-Y. Zhang, X.-M. Wu, W.-Y. Hua, Glycosylation of

ent-kaurene derivatives and an evaluation of their cytotoxic activities, Chin. J.

Nat. Med. 11 (2013) 289e295.

[14] A. Malki, A. El-Sharkawy, M. El Syaed, S. Bergmeier, Antitumor activities of the

novel isosteviol derivative 10C against liver cancer, Anticancer Res. 37 (2017)

1591e1601.

[15] M. Ukiya, S. Sawada, T. Kikuchi, Y. Kushi, M. Fukatsu, T. Akihisa, Cytotoxic and

apoptosis-inducing activities of steviol and isosteviol derivatives against hu-

man cancer cell lines, Chem. Biodivers. 10 (2013) 177e188.

[16] R.N. Khaybullin, M. Zhang, J. Fu, X. Liang, T. Li, A.R. Katritzky, P. Okunieff, X. Qi,

Design and synthesis of isosteviol triazole conjugates for cancer therapy,

Molecules 19 (2014) 18676e18689.

[17] T.-t. Wang, Y. Liu, L. Chen, Synthesis and cytotoxic activity of nitric oxide-

releasing isosteviol derivatives, Bioorg. Med. Chem. Lett. 24 (2014)

2202e2205.

[28] A. Asnani, R.T. Peterson, The zebra ﬁ sh as a tool to identify novel therapies for

human cardiovascular disease, Dis. Model Mech. 7 (2014) 763e767.

[29] A. Kithcart, C.A. MacRae, Using zebra ﬁ sh for high-throughput screening of

novel cardiovascular drugs, JACC Basic Transl. Sci. 2 (2017) 1e12.

[30] S. Roychoudhury, A. Kumar, D. Bhatkar, N.K. Sharma, Molecular avenues in

targeted doxorubicin cancer therapy, Future Oncol. 16 (2020) 687e700.

[31] K. Renu, G.A. V, B.T. P, S. Arunachalam, Molecular mechanism of doxorubicin-

induced cardiomyopathy - an update, Eur. J. Pharmacol. 818 (2018) 241e253.

[32] C.-C. Huang, A. Monte, J.M. Cook, M.S. Kabir, K.P. Peterson, Zebra ﬁ sh heart

failure models for the evaluation of chemical probes and drugs, Assay Drug

Dev. Technol. 11 (2013) 561e572.

[33] C.C. Huang, P.C. Chen, C.W. Huang, J. Yu, Aristolochic acid Induces heart failure

in zebra ﬁ sh embryos that is mediated by in ﬂ ammation, Toxicol. Sci. 100

(2007) 486e494.

[34] X. Fang, H. Wang, D. Han, E. Xie, X. Yang, J. Wei, S. Gu, F. Gao, N. Zhu, X. Yin,

Q. Cheng, P. Zhang, W. Dai, J. Chen, F. Yang, H.T. Yang, A. Linkermann, W. Gu,

J. Min, F. Wang, Ferroptosis as a target for protection against cardiomyopathy,

Proc. Natl. Acad. Sci. U. S. A. 116 (2019) 2672e2680.

[18] A. Malki, R. Laha, S.C. Bergmeier, Synthesis and cytotoxic activity of MOM-

ether analogs of isosteviol, Bioorg. Med. Chem. Lett. 24 (2014) 1184e1187.

[19] C.J. Liu, T. Zhang, S.L. Yu, X.J. Dai, Y. Wu, J.C. Tao, Synthesis, cytotoxic activity,

and 2D- and 3D-QSAR studies of 19-carboxyl-modi ﬁ ed novel isosteviol de-

rivatives as potential anticancer agents, Chem. Biol. Drug Des. 89 (2017)

870e887.

[20] C.J. Liu, Y.P. Liu, S.L. Yu, X.J. Dai, T. Zhang, J.C. Tao, Syntheses, cytotoxic activity

evaluation and HQSAR study of 1,2,3-triazole-linked isosteviol derivatives as

potential anticancer agents, Bioorg. Med. Chem. Lett. 26 (2016) 5455e5461.

[21] T. Zhang, L.-H. Lu, H. Liu, J.-W. Wang, R.-X. Wang, Y.-X. Zhang, J.-C. Tao, D-ring

modi ﬁ ed novel isosteviol derivatives: design, synthesis and cytotoxic activity

evaluation, Bioorg. Med. Chem. Lett. 22 (2012) 5827e5832.

[22] S.-L. Zhu, Y. Wu, C.-J. Liu, C.-Y. Wei, J.-C. Tao, H.-M. Liu, Design and stereo-

selective synthesis of novel isosteviol-fused pyrazolines and pyrazoles as

potential anticancer agents, Eur. J. Med. Chem. 65 (2013) 70e82.

[23] C.J. Liu, S.L. Yu, Y.P. Liu, X.J. Dai, Y. Wu, R.J. Li, J.C. Tao, Synthesis, cytotoxic

activity evaluation and HQSAR study of novel isosteviol derivatives as po-

tential anticancer agents, Eur. J. Med. Chem. 115 (2016) 26e40.

[24] L. Testai, I. Strobykina, V.V. Semenov, M. Semenova, E.D. Pozzo, A. Martelli,

V. Citi, C. Martini, M.C. Breschi, V.E. Kataev, V. Calderone, Mitochondriotropic

and cardioprotective effects of triphenylphosphonium-conjugated derivatives

of the diterpenoid isosteviol, Int. J. Mol. Sci. 18 (2017).

[35] J.C. Tao, G.Q. Tian, Y.B. Zhang, Y.Q. Fu, G.F. Dai, Y. Wu, Synthesis and bioactivity

of isosteviol derivatives: a facile method for preparation of ent-16

15 -hydroxymethylbeyeran-19-oic acid, Chin. Chem. Lett. 16 (2005)

1441e1444.

a -hydroxy-

b

[36] Y. Wu, J.H. Yang, G.F. Dai, C.J. Liu, G.Q. Tian, W.Y. Ma, J.C. Tao, Stereoselective

synthesis of bioactive isosteviol derivatives as alpha-glucosidase inhibitors,

Bioorg. Med. Chem. 17 (2009) 1464e1473.

[37] A. Pathak, M. Lebrin, A. Vaccaro, J.M. Senard, F. Despas, Pharmacology of

levosimendan: inotropic, vasodilatory and cardioprotective effects, J. Clin.

Pharm. Therapeut. 38 (2013) 341e349.

[38] L. De Luca, W.S. Colucci, M.S. Nieminen, B.M. Massie, M. Gheorghiade, Evi-

dence-based use of levosimendan in different clinical settings, Eur. Heart J.

(2006) 1908e1920.

[39] M.N. Sack, F.Y. Fyhrquist, O.J. Saijonmaa, V. Fuster, J.C. Kovacic, Basic biology of

oxidative stress and the cardiovascular system: part 1 of a 3-part series, J. Am.

Coll. 70 (2017) 196e211.

[40] F. Forini, G. Nicolini, G. Iervasi, Mitochondria as key targets of cardioprotection

in cardiac ischemic disease: role of thyroid hormone triiodothyronine, Int. J.

Mol. Sci. 16 (2015) 6312e6336.

[41] F. Tang, X. Zhou, L. Wang, L. Shan, C. Li, H. Zhou, S.M.-Y. Lee, M.P.-M. Hoi,

A novel compound DT-010 protects against doxorubicin-induced cardiotox-

icity in zebra ﬁ sh and H9c2 cells by inhibiting reactive oxygen species-

mediated apoptotic and autophagic pathways, Eur. J. Pharmacol. 820 (2018)

86e96.

[25] K.S.M. Abdul, J. Rayadurgam, N. Faiz, A. Jovanovic, W. Tan, Cardioprotection by

isosteviol derivate JC105: a unique drug property to activate ERK1/2 only

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