Discovery of β-Carboline Derivatives as a Highly Potent Cardioprotectant against Myocardial Ischemia-Reperfusion Injury

Cardioprotectant against Myocardial Ischemia-Reperfusion Injury

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Discovery of β‑Carboline Derivatives as a Highly Potent

∥

Hong Zhang, Rong-Hong Zhang, Xiang-Ming Liao, Dan Yang, Yu-Chan Wang, Yong-Long Zhao,

Guo-Bo Xu, Chun-Hua Liu, Yong-Jun Li, Shang-Gao Liao,* and Meng Zhou *

Cite This: J. Med. Chem. 2021, 64, 9166 −9181

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ABSTRACT: Timely myocardial reperfusion salvages ischemic

myocardium from infarction, whereas reperfusion itself induces

cardiomyocyte death, which is called myocardial ischemia/

reperfusion (MI/R) injury. Herein, β-carboline derivative 17c

was designed and synthesized with obvious myocardial protective

activity for the ﬁrst time. Pretreatment of 17c eﬀectively protected

the cardiomyocyte H9c2 cells from H O -induced lactate

dehydrogenase leakage and restored the endogenous antioxidants,

superoxide dismutase (SOD) and glutathione peroxidase (GSH-

Px). Besides, 17c eﬀectively protected the mitochondria through

decreasing the reactive oxygen species overproduction and

enhancing the mitochondrial membrane potential. As a result,

7c signiﬁcantly reduced the necrosis of cardiomyocytes in H O -

2 2

induced oxidative stress, which was more potent than polydatin. In MI/R injury rats, 17c pretreatment obviously increased the levels

of SOD and GSH-Px and inhibited the apoptosis of cardiomyocytes. Through this way, the size of myocardial infarction was

signiﬁcantly reduced after MI/R injury in vivo, better than that of polydatin, suggesting that 17c is a promising cardioprotectant for

the prevention of MI/R injury.

INTRODUCTION

dismutase (SOD) and glutathione (GSH), was proven to

■

16,17

ameliorate MI/R injury and reduce infarct size.

Develop-

Acute myocardial infarction is a widespread public health

problem with high morbidity and mortality.

restoration of the blood ﬂow to the heart muscle after

myocardial ischemia is the most eﬀective therapeutic strategy

to attenuate myocardial ischemic injury. Nevertheless, reperfu-

sion itself is considered to cause additional cellular injury,

ment of drugs with a capacity to reduce ROS overproduction is

therefore an eﬀective strategy for the prevention and treatment

of MI/R injury.

Timely

β-Carboline (1, Figure 1A) is a well-known alkaloid with a

common tricyclic pyrido-[3,4-b]indole ring and was originally

isolated from the seeds of Peganum harmala L. which is

including contractile dysfunction and cellular damage, and

18,19

traditionally used to treat digestive cancer and malaria.

Pharmacological research revealed that β-carboline derivatives

ultimately leads to arrhythmia, contractile dysfunction, and

heart failure. This pathological process is called myocardial

exhibited a wide range of biological eﬀects, such as anticancer,

ischemia/reperfusion (MI/R) injury. Although the patho-

genesis of MI/R injury is not completely understood so far,

numerous reports demonstrated that reactive oxygen species

antivirus, antibacterial, anti-inﬂammatory, and antithrom-

botic activities. The β-carboline derivative harmine was

reported to possess marked anti-inﬂammatory activity through

suppressing the levels of tumor necrosis factor-alpha,

interleukin-6, and nitric oxide in lipopolysaccharide-stimulated

RAW264.7 cells. In addition, its reduction product harmaline

displayed a strong antiplatelet activity and was promising in

(

ROS), calcium overload, neutrophil inﬁltration, and myocar-

dial necrosis and apoptosis are all involved in the occurrence of

9−12

MI/R injury.

MI/R injury induces an imbalance between

antioxidants and toxic-free radicals, which increases the

susceptibility of tissues to oxidative damage via lipid, protein,

and DNA oxidation, leading to the disfunction of myocardial

tissue. Thus, ROS overproduction is considered as a key event in

Received: March 3, 2021

Published: June 16, 2021

MI/R injury. The overproduction of ROS during MI/R injury

overwhelms the intrinsic antioxidant and causes the death of the

cardiomyocyte, which is believed to be a signiﬁcant cellular

mechanism responsible for MI/R injury. Decreasing the ROS

level by antioxidants or scavengers, such as superoxide

2021 American Chemical Society

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J. Med. Chem. 2021, 64, 9166−9181

Figure 1. Design diagram of β-carboline derivatives with potential myocardial protective eﬀect. (A) Structures of β-carboline (1) and its analogues

harmine and harmaline with anti-inﬂammatory and antiplatelet eﬀects, respectively. (B) Cardiomyocyte protective compound 2MAC3C derived from

α-carboline 2. (C) Speculated β-carboline derivative (3) with probable myocardial protective activity. (D) Reverse synthesis analysis of β-carboline.

(E) Optimization strategies on 1, 3, 9 positions of β-carboline 4 with the aim of ﬁnding potential cardioprotectant candidates.

Scheme 1. Synthesis of Target Compounds

Reagents and conditions: (a) TFA, DCM, rt, 4 h; (b) KMnO , DMF, 0°C-rt, 3.5 h; (c) KOH, THF/H O (2:1), 68 °C, 8 h; (d) HBTU, DIEA,

THF, rt, 10 h; (e) m-CPBA, DCM, 0 °C, 12 h; (f) acetic anhydride, 110 °C, 2 h; (g) NaOH, THF/H O (1:1), 0.5 h; (h) toluene, 70 °C, 8 h for

acetone/H O (1:1), rt, 0.5 h; (l) SnCl ·2H O, EA, reﬂux, 3 h

5a and 15b; Et N, 4-diaminopyridine, DCM, rt, 12 h for 15c and 15d; (i) KOH, dry THF, rt, 4 h; (j) NaOH, DMF, 70 °C, 4 h; (k) Na CO ,

the ﬁght against stroke or coronary artery diseases. Further

research demonstrated that harmine and harmaline could

induce vasorelaxation in rat aorta by promoting the release of

nitrous oxide and inhibiting the contractions of the vascular

carboline derivatives in the treatment of cardiovascular diseases

like hypertension, myocardial infarction, and atherosclerosis.

In previous research, we found that α-carboline derivatives

(2) displayed obvious cardiomyocyte protective eﬀect against

7,28

smooth muscles,

indicating the potential application of β-

Na S O -induced hypoxia/reoxygenation injury in H9c2 cells.

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J. Med. Chem. 2021, 64, 9166−9181

Journal of Medicinal Chemistry

Article

Table 1. Protective Eﬀects of β-Carboline Derivatives against H O -Induced Oxidative Stress

viabilities of H9c2 cardiomyocyte (%)

Compd.

2.5 μM

5 μM

10 μM

20 μM

40 μM

53.3 ± 1.6

56.6 ± 2.1

60.9 ± 1.3

64.2 ± 1.5

76.1 ± 1.2***

79.3 ± 1.0***

81.4 ± 1.3***

63.5 ± 1.3

52.5 ± 0.8

59.6 ± 2.1

71.0 ± 3.2*

64.2 ± 2.2

64.2 ± 2.1

59.0 ± 1.1

63.5 ± 2.2

66.1 ± 1.9*

66.6 ± 4.9

67.1 ± 2.4

70.5 ± 3.6*

60.2 ± 9.1

69.4 ± 1.1***

69.7 ± 2.4**

67.8 ± 0.6**

71.3 ± 2.4**

70.6 ± 2.9*

65.8 ± 3.2

93.0 ± 5.3**

65.6 ± 4.1

62.2 ± 2.6

59.8 ± 1.3

52.6 ± 1.7

53.3 ± 1.4

56.3 ± 1.8

61.7 ± 1.4

75.0 ± 1.0***

80.5 ± 0.4***

76.8 ± 2.2***

60.1 ± 0.3

50.6 ± 0.4

58.7 ± 1.2

66.2 ± 3.2

65.4 ± 1.8

66.3 ± 3.6*

58.1 ± 2.6

59.6 ± 4.6

65.8 ± 3.3

61.0 ± 6.7

70.0 ± 2.0**

74.4 ± 3.8*

55.3 ± 3.3

72.5 ± 2.9**

76.2 ± 1.7***

70.7 ± 1.8**

74.9 ± 3.8*

73.0 ± 6.6

63.4 ± 4.7

83.5 ± 4.7**

62.6 ± 6.1

61.6 ± 4.1

53.8 ± 1.8

53.2 ± 2.1

58.6 ± 1.3

61.6 ± 1.0

72.3 ± 0.2***

79.2 ± 0.5***

73.1 ± 1.4***

58.5 ± 0.1

51.7 ± 0.3

60.3 ± 0.5

68.2 ± 4.9

63.7 ± 1.7

65.1 ± 3.2

59.2 ± 3.2

56.3 ± 4.7

71.5 ± 3.3*

60.9 ± 6.7

65.4 ± 3.6

73.6 ± 3.8*

56.0 ± 4.3

77.8 ± 1.6***

80.2 ± 1.1***

65.0 ± 2.1

79.8 ± 4.6*

72.7 ± 7.0

69.6 ± 4.9

79.8 ± 5.2*

60.6 ± 5.9

66.1 ± 2.1*

48.5 ± 2.0

52.6 ± 2.0

59.3 ± 1.4

64.2 ± 1.1*

70.8 ± 0.2***

81.5 ± 0.5***

67.4 ± 0.8**

54.4 ± 0.2

46.5 ± 0.1

60.9 ± 0.6

63.2 ± 3.2

63.1 ± 1.9

70.3 ± 3.2*

61.2 ± 3.5

56.0 ± 5.2

75.7 ± 3.0**

59.9 ± 6.6

66.5 ± 3.2

77.4 ± 3.1**

55.4 ± 2.3

86.4 ± 1.3***

89.4 ± 2.2***

62.9 ± 2.2

83.1 ± 4.6*

74.5 ± 6.3*

71.4 ± 4.8*

81.8 ± 5.2*

57.8 ± 4.3

44.6 ± 2.1

50.6 ± 2.5

64.9 ± 1.0**

65.9 ± 1.0**

63.1 ± 0.6*

90.4 ± 0.4***

65.4 ± 0.5**

52.3 ± 0.2

47.6 ± 0.1

67.7 ± 3.0*

64.6 ± 4.3

65.7 ± 4.1

68.2 ± 3.2*

62.8 ± 1.2**

53.3 ± 3.0

60.3 ± 2.1

56.4 ± 5.7

68.2 ± 3.5*

80.7 ± 1.8***

57.2 ± 8.6

93.2 ± 1.0***

95.8 ± 2.1***

48.9 ± 2.3

86.1 ± 3.6**

70.3 ± 6.3

77.1 ± 4.9*

90.6 ± 6.4**

54.8 ± 4.6

Pol

H O only

69.8 ± 2.0**

72.7 ± 1.3**

Positive control. *p < 0.05, **p < 0.01, ***p < 0.001 vs H O -only group.

Through optimizations on α-carbolines, derivative 2MAC3C

Figure 1B) was obtained and further encapsulated by

carboline. Acetaldehyde and propionaldehyde were both

selected as raw materials to react with L-tryptophan methyl

ester (5) through the Pictet−Spengler reaction to provide

various alkyl substituents at C1. For acetaldehyde, the reaction

gave two tetrahydro-β-carboline epimers 6a and 6b, both of

which were then transformed to β-carboline 7 through the

oxidative dehydrogenation reaction under the oxidant KMnO₄.

The methyl ester of 7 was then hydrolyzed (8), and glycine, L-

glutamate, and 4-methoxyaninine were introduced to C3

through a dehydration reaction to give compounds 9a, 9b,

and 9c, respectively, in the presence of the condensing agent O-

(1H-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexa-

ﬂuorophosphate (HBTU) and base N,N-diisopropylethylamine

(

amphiphilic chitosan micelles, which displayed improved

solubility and protective activity against Na S O -induced

hypoxia/reoxygenation injury. Based on our preliminary

studies and the reported cardioprotective potential of β-

carboline derivatives, we speculated that optimizations on β-

carboline probably give potent cardioprotectant candidates

(

Figure 1C).

Although there are numerous natural products derived from

β-carboline, only limited synthetic strategies of β-carboline have

been reported. The Pictet−Spengler reaction of L-tryptophan

and aldehyde followed by a dehydrogenation reaction is the

33−35

most widely used route to access β-carboline (Figure 1D).

Considering the synthetic route of β-carboline, modiﬁcations on

, 3, 9 positions were performed to acquire β-carboline

(

DIEA).

In the case of propionaldehyde, the generated tetrahydro-β-

carboline epimers 10a and 10b were both dehydrated to give the

intermediate 11. To perform structural modiﬁcations on C-1, N-

derivatives in a short time (Figure 1E). Interestingly, a series

of β-carboline analogues were synthesized through this strategy,

which displayed signiﬁcant cardiomyocyte protective eﬀects

of 11 was oxidized by 3-chloroperoxybenzoic acid (mCPBA)

to aﬀord compound 12, the reaction of which with acetic

anhydride gave acetate 13 . Further hydrolysis of 13 yielded 14

against H O -induced oxidative stress. In addition, compound

7c markedly decreased the infarct size induced by MI/R injury

(

crystal structure, see Figure S1 ), esteriﬁcation of which with

in vivo, indicating a strong cardioprotective activity.

succinic anhydride, glutaric anhydride, benzoic acid, and o-

phthalic anhydride provided β-carbolines 15a, 15b, 15c, and

15d, respectively. In addition, diﬀerent benzyl groups (i.e.,

benzyl, 4-ﬂuorobenzyl, 4-chlorobenzyl, 4-methoxybenzyl, and 2-

nitrobenzyl) were introduced to N-9 via their bromides under

base condition to give 16a, 17a, 18a, 19a, and 20a. The methyl

RESULTS AND DISCUSSION

■

Synthetic Chemistry. The synthetic route is depicted in

Scheme 1, and the Pictet−Spengler reaction followed by a

dehydrogenation reaction was used to aﬀord the core of β-

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J. Med. Chem. 2021, 64, 9166−9181

Figure 2. Protective eﬀects of 17c on the necrosis of H9c2 cells after H O (0.3 mM) injury. (A) Eﬀect of 17c on the release of LDH at the end of

incubation with H O . (B) Representative ﬂuorescent photomicrographs of Hoechst 33258 staining. Blue: cell nuclei stained by Hoechst 33258 dye.

(C) Representative ﬁgures of H9c2 cells stained with FDA (green)/PI (red). (D) Percentage of dead cells through FDA/PI staining. (E) Contour

plots of Annexin-V/PI, showing necrotic cells in Q1 (Annexin-V/PI −/+; upper left) and apoptotic cells in Q2 (Annexin-V/PI +/+; upper right)

among H9c2 cells. (F) Percentages of viable, apoptotic, and necrotic cells calculated. Scar bar = 100 μm and refers to all panels. ***p < 0.001 vs control

###

&&&

group; p < 0.01, p < 0.01, p < 0.001 vs H O only group; p < 0.05, p < 0.01,

p < 0.001 vs Pol group.

esters 16a, 17a, 18a, and 19a were further hydrolyzed to provide

their corresponding acids 16b, 17b, 18b, and 19b, respectively.

To improve the water solubility, 17b was transformed to its

sodium salt 17c with a saturated Na CO aqueous solution. The

against the embryonic rat heart-derived H9c2 cell line at the

concentration of 40 μM, except compounds 9a, 15b, and 15c (p

0.05). Thus, biological evaluations were carried out with

concentrations lower than 80 μM.

It is reported that ROS is overproduced during MI/R injury,

nitro group of 20a was further reduced by SnCl to give 20b

carrying an amino counterpart.

and excessive accumulation of ROS would cause oxidative stress

Protective Eﬀects against H O -Induced Oxidative

and even cell death. H O is a major component of ROS, and

Stress. Prior to the investigation of cardiomyocyte protective

eﬀect, the cytotoxicity of all β-carboline derivatives (6a−20b)

was analyzed by the MTT assay (Table S1). No signiﬁcant

toxicity was observed for most of the synthetic compounds

H O -induced oxidative stress in H9c2 cells is extensively used

7,38

in oxidative stress studies.

Thus, the cardiomyocyte

protective activity of the title compounds was then evaluated

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J. Med. Chem. 2021, 64, 9166−9181

Figure 3. Mitochondrial protective eﬀect of 17c against H O (0.3 mM)-induced oxidative stress in H9c2 cells. (A) Detection of intracellular ROS

(green ﬂuorescence) by DCFH-DA staining. (B) Histogram analysis of ROS ﬂuorescence intensity. (C) Analysis of mitochondrial membrane

potential (ΔΨ ) by using the JC-1 ﬂuorescent probe. ΔΨ (green ﬂuorescence) decreased signiﬁcantly in the oxidative damage group, while high

ΔΨ (orange ﬂuorescence) was retained in the 17c pretreatment group. (D) Histogram analysis of MMP ﬂuorescence intensity. Scar bar = 100 μm

###

&&&

and refers to all panels. *p < 0.05, ***p < 0.001 vs control group; p < 0.05, p < 0.001 vs H O only group; p < 0.05, p < 0.01,

group.

p < 0.001 vs Pol

by using H O -induced oxidative stress in H9c2 cells, and

Interestingly, introduction of benzyl groups to the N-9

position of β-carboline (16a, 18a, and 19a) obviously enhanced

the cardiomyocyte protective eﬀect, and hydrolyzing C-3 methyl

ester to its carboxy counterpart (as in 16a to 16b, 17a to 17b,

18a to 18b, and 19a to 19b) improved the hydrophilicity and

remarkably increased the activity, which was consistent with the

above optimization results on the C3 position. Meanwhile, all

39−41

polydatin (Pol) was used for comparison.

The cytotoxicity of H O at di ﬀ erent concentrations against

H9c2 cells was ﬁrst assessed (Figure S2), and the concentration

(

0.3 mM) with the cell viability of 61.0 ± 0.6% at 0.3 mM was

used in the subsequent experiments. Further protective eﬀect

evaluations showed that pretreatment of Pol at concentrations

of 10, 20, and 40 μM for 24 h signiﬁcantly alleviated H O -

these carboxylic acids were more potent than Pol against H O

injury at the same concentrations. Among them, pretreatment of

H9c2 cells with 17b signiﬁcantly enhanced the protective eﬀect

in a dose-dependent manner, with cell viabilities of 69.4% (p <

induced cell injury (p < 0.05, Table 1). As we speculated earlier,

the oxidative injury of cardiomyocytes was obviously improved

with pretreating of the substituted β-carbolines. The preliminary

results showed that optimizations on C-1, C-3, and N-9 of β-

carboline gave the products (9a, 9b, 9c, 15c, 16b, 17b, 18b, 19a,

.001), 72.5 (p < 0.01), 77.8% (p < 0.001), 86.4% (p < 0.001),

and 93.2% (p < 0.001) at the concentrations of 2.5, 5, 10, 20, and

0 μM, respectively. Moreover, sodium salt 17c was more active

than its parent compound 17b, with cell viabilities of 69.7% (p <

.01), 76.2% (p < 0.001), 80.2% (p < 0.001), 89.4% (p < 0.001),

19b, and 20a) with better cardiomyocyte protective activities

than the intermediates (7, 8, and 11−14), and no protective

eﬀect was observed for tetrahydro-β-carbolines (6a, 6b, 10a, and

and 95.8% (p < 0.001) at 2.5, 5, 10, 20, and 40 μM, respectively.

Representative photographs of the cultured H9c2 cells exposed

to 0.3 mM H O with or without 17c pretreatment are shown in

10b). Specially, introduction of hydrophilic amino acids to the

carboxyl group at C3 (9a and 9b) was desired for improving the

protective activity, indicating that improving the hydrophilicity

at the C3 position was beneﬁcial to activity. By contrast,

increasing the hydrophilic level of C1 of the β-carbolines (15a,

Figure S3 . Compared with the control group, the loss of cell

volume and cell shrinkage were observed in the H O treatment

group, which was the morphological hallmark of cell death,

15b, and 15d) reduced the eﬀect. However, the β-carboline with

whereas pretreatment of 17c obviously ameliorated the injury.

Furthermore, no cytotoxicity of 17c was observed even at 80 μM

(Table S1 ). Except 17c, 20a with an o-nitrobenzyl group at the

N-9 position also showed remarkable protective eﬀect, but no

concentration dependence was observed, and this eﬀect

a hydrophobic benzene ring at C1 (15c) displayed acceptable

activity at 2.5, 10, and 20 μM (p < 0.05), suggesting that the

hydrophobicity of C1 played an important role in enhancing the

protective eﬀect.

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Figure 4. Eﬀects of 17c (10, 20, 40, and 80 μM) on the activities of MDA (A), SOD (B), and GSH-Px (C). Cardiomyocytes were lysed, and MDA,

###

SOD, and GSH-Px levels were measured. ***p < 0.001 vs control group; p < 0.05, p < 0.01, p < 0.001 vs H O only group; p < 0.05, p < 0.01,

&&&

p < 0.001 vs Pol group.

disappeared when the nitro group was reduced to an amino one

20b), indicating that maintaining the hydrophilicity of the N-9

Mitochondria Protective Eﬀect of 17c against H O₂

Injury. During the process of MI/R, with the recovery of

oxygen, the electron transport chain was re-energized and the

(

benzyl group was necessary for activity. Therefore, 17c was

chosen for further cardioprotective investigations (Table 1).

Inhibitory Eﬀect of 17c against H O -Induced Cell

mitochondrial membrane potential (ΔΨ ) was re-established.

Excessive ROS was abruptly generated, which contributed to the

opening of mitochondrial membrane permeability transition

Necrosis. Since obvious cell death was observed in the above

experiment after exposure to H O₂(Figure S3), lactate

pore and Ca overload. Along with the rapid normalization of

pH, the dissipation in ΔΨ occurred, leading to swelling of

dehydrogenase (LDH) leakage assay was performed to assess

the membrane integrity of H9c2 cells to evaluate the protective

eﬀect of 17c. Compared with the control group, the LDH release

was markedly elevated in the H O group (p < 0.001, Figure

44−46

mitochondria and, ﬁnally, cardiomyocyte death.

Thus, the

overproduction of ROS and decreasing of mitochondrial ΔΨ

play a pivotal role in the mitochondrial dysfunction during the

MI/R injury, and these two factors were then investigated.

Intracellular ROS was commonly estimated using the oxidation-

sensitive dye 2′,7′-dichloroﬂuorescin-diacetate (DCFH-DA),

which was oxidized to give the ﬂuorescent product 2′,7′-

A), whereas pretreatment of the cells with 17c signiﬁcantly

reduced the release of LDH (p < 0.01). In particular, the level of

LDH was almost restored to normal with 17c pretreatment at 40

or 80 μM (p < 0.001), much lower than that achieved with 80

μM Pol (p < 0.001). Hoechst 33,258 staining showed that the

nuclei of H9c2 cells in the control group were stained in blue and

were uniform in shape (Figure 2B). Nuclear pyknosis and

karyorrhexis were observed after the exposure to H O , which

dichloroﬂuorescein (DCF), and JC-1, a reliable ﬂuorescent

probe, was applied to detect the mitochondrial ΔΨ .

Compared with the control group, the ﬂuorescence (green)

intensity of the H O group was signiﬁcantly enhanced (p <

0.05, Figure 3A,B), indicating the overproduction of ROS in

mitochondria. However, the ﬂuorescence intensity was

decreased in a dose-dependent manner when the cells were

pretreated with 17c (p < 0.05), and the eﬀect of 17c at 10 μM

was more obvious than that of Pol at 80 μM (p < 0.05),

suggesting the potential of 17c in scavenging excessive ROS in

cardiomyocytes. Similarly, low mitochondrial membrane

potential (green) was observed in the H O -only group (Figure

were the morphological hallmark of necrosis. However, the

cell death of cardiomyocytes was alleviated through pretreat-

ment with diﬀerent concentrations of 17c, and the eﬀect was

ampliﬁed with the increase of concentration.

In addition, double staining with ﬂuorescein diacetate

(

FDA)/propidium iodide (PI) was further used to analyze the

dead cells caused by H O (Figure 2C). No dead cell with PI

staining (red) was detected in the control group, whereas quite a

number of dead cells were observed in the H O group.

C). By contrast, 17c pretreatment signiﬁcantly enhanced the

mitochondrial ΔΨ (orange ﬂuorescence increased) with

Nevertheless, compound 17c obviously increased the vital cells

that took up the ﬂuorogen FDA (green) and decreased the dead

cells stained by PI. Further percentage analysis revealed that 5

μM 17c markedly reduced the necrosis cells when compared

concentrations increasing (p < 0.001, Figure 3D), which was

superior to Pol at concentrations higher than 10 μM (p < 0.05).

These results manifested that 17c eﬀectively protected the

mitochondria of H9c2 cells against H O -induced intracellular

with the H O group (p < 0.001, Figure 2D), and the eﬀect was

ROS production.

better than 80 μM Pol (p < 0.05). Flow cytometry analysis

Figure 2E) showed that the percentage of necrosis (Q1, 70%)

of H9c2 cells was greatly enhanced after exposure to H O (p <

Eﬀect of 17c in Improving Endogenous Antioxidant

Capacity. Maintenance of adequate antioxidant levels is crucial

to prevent MI/R injury. Thus, to better understand the

protective activity of 17c against ROS induced by H O ,

(

.001, Figure 2F), whereas pretreatment of 10, 20, 40, and 80

μM of 17c signiﬁcantly alleviated the necrosis rate to 48.9% (p <

detailed investigations were carried out on the innate

antioxidant status of H9c2 cells. The major enzymatic innate

.001), 44.5% (p < 0.001), 13.6% (p < 0.001), and 3.53% (p <

.001), respectively, better than that of Pol (51.2%, p < 0.05).

antioxidants including malondialdehyde (MDA), SOD, and

These results suggested that 17c eﬀectively improved the

glutathione peroxidase (GSH-Px) were evaluated to deter-

mine the endogenous antioxidant capacity of cardiomyocytes

necrosis of H9c2 cells induced by H O .

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Article

Figure 5. Protective eﬀect of 17c on MI/R injury in rats. (A) Experimental protocol for investigating the eﬀect of 17c (25, 50, and 100 mg/kg) on the

left ventricular function during acute MI/R injury. (B) Representative TTC-stained slides of the myocardium sections from each group. (C)

Percentage of myocardial infarct to the total myocardium. (D) Representative photographs of H&E staining heart sections of each group. The yellow

arrows represent inﬂammatory cell inﬁltration, and the black arrows represent disrupted myoﬁbrillar structure. Scar bar = 100 μm and refers to all

&&&

panels. ***p < 0.001 vs sham group; p < 0.001 vs MI/R group;

p < 0.001 vs Pol group. n = 12 per group. Bars represent mean ± SEM.

(

Figure 4). Compared with that in the control group, the MDA

with 17c remarkably improved the SOD activity in a dose-

dependent manner (p < 0.05). By contrast, no signiﬁcant eﬀect

was observed for Pol. For GSH-Px, the content was severely

decreased after H O injury (p < 0.001, Figure 4C). Whereas

pretreatment of H9c2 cells with 17c signiﬁcantly increased the

content of GSH-Px even at 5 μM (p < 0.01), 10 μM 17c

exhibited better activity than 80 μM of Pol (p < 0.01). The

above data showed that 17c eﬀectively enhanced the

activity in the H O group increased about 4-fold (p < 0.001,

Figure 4A). However, the level of MDA was almost restored to

normal in the 17c pretreatment group (20 μM, p < 0.001), which

was better than that of 80 μM Pol (p < 0.05). As a major

antioxidant enzyme, SOD was then measured in diﬀerent

groups. After exposing to H O , the SOD activity was

signiﬁcantly decreased (p < 0.001, Figure 4B), but pretreatment

172

J. Med. Chem. 2021, 64, 9166−9181

Figure 6. Eﬀects of 17c (25, 50 and 100 mg/kg) on the activities of LDH (A), MDA (B), SOD (C), and GSH-Px (D). Blood was collected at 24 h of

###

reperfusion, and LDH, MDA, SOD, and GSH-Px levels were measured. ***p < 0.001 vs sham group; p < 0.01 and p < 0.001 vs MI/R group; p <

.05 and p < 0.01 vs Pol group. n = 5 per group.

Figure 7. (A) Eﬀects of 17c (25, 50 and 100 mg/kg) on the expression of apoptotic-related proteins (Bax, Bcl-2, and caspase3). Histogram analysis of

Bax/GAPDH (B), Bcl-2/GAPDH (C), and caspase3/GAPDH (D). ***p < 0.001 vs sham group; p < 0.001 vs MI/R group; p < 0.05, p < 0.01,

and

p < 0.001 vs Pol group. n = 5 per group.

endogenous antioxidant capacity of cardiomyocytes against

H O -induced oxidative stress.

segment elevation was markedly ameliorated after reperfusion in

the rats in the 17c pretreatment group.

Protective Eﬀect of 17c in Rat MI/R Injury. To investigate

the eﬀect against MI/R injury in vivo, 17c was administered (i.

g.) to the animals for 7 days. During the experiment, no

statistical diﬀerence in the body weight was observed between

the animals administrated with 17c (25, 50, and 100 mg/kg) and

saline (Figure S4), indicating the low toxicity of 17c. Through

transient ischemia occlusion of the left anterior descending

coronary artery (LAD) ligation of rat for 1 h followed by 24 h

reperfusion, the MI/R injury model was established in vivo

The infarct size was evaluated by using the 2,3,5-

triphenyltetrazolium chloride (TTC) staining assay (Figure

B). Compared with the model group (32.71%), groups

pretreated with 17c exhibited eﬀectively reduced myocardial

infarction sizes (16.11, 10.96, and 9.88%, respectively, for 25, 50,

and 100 mg/kg) (p < 0.001, Figure 5C), which were better than

that of Pol (p < 0.001). In addition, hematoxylin & eosin (H&E)

staining was performed to appraise the cardiac pathological

changes (Figure 5D). In the MI/R group, the myocardial ﬁbers

were disordered, and obvious infarction, inﬂammatory inﬁltra-

tion, and myocardial death were observed. Nevertheless, 17c-

pretreated groups showed less severe myocardial damage, as

evidenced by the relieving edema, leukocyte inﬁltration, and

myocardial death. Moreover, the myocardial protective activity

was enhanced with the increasing doses.

(

Figure 5A). Electrocardiograph (ECG) was monitored

throughout the whole procedure (Figure S5). Results showed

that the ST-segment under the second lead in the MI/R group

was obviously higher than that in the sham group (>0.2 mV),

whereas a signiﬁcant ST-segment resolution was observed after

reperfusion for 2 h, indicating the successful reperfusion of the

ischemic region. Compared with the MI/R group, the ST-

173

J. Med. Chem. 2021, 64, 9166−9181

Journal of Medicinal Chemistry

Article

Furthermore, the cardiac biomarker LDH in serum was

analyzed at 24 h postreperfusion injury, which is a sensitive index

ROS contents induced by MI/R injury and potently inhibited

myocardial apoptosis and thus eﬀectively alleviated the infarct

size. Overall, these results demonstrated the potential of 17c as a

novel cardioprotective candidate. However, the target of 17c

and the mechanism of action of this compound series remain to

be further studied. According to our results, 17c might inhibit

the mitochondrial ROS production and regulate the metabolism

of mitochondria during the process of MI/R injury, and the

study of the detailed biological eﬀect of 17c on mitochondria

will be carried out in the next work of our laboratory.

of the myocardial zymogram. In comparison with that of the

sham group, the serum LDH level was signiﬁcantly increased

after MI/R injury (p < 0.001, Figure 6A). In accordance with the

above observations, 17c markedly reduced the MI/R-induced

LDH release in a dose-dependent manner (p < 0.001). Since

ROS overproduction induced during MI/R injury would lead to

lipid, protein, and DNA oxidation, the activities of MDA, SOD,

and GSH-Px were then investigated. Similarly, a signiﬁcant

elevation of MDA (p < 0.001, Figure 6B) and decreased

activities of SOD and GSH-Px (p < 0.001 Figure 6C,D) were

observed in the MI/R group compared with the sham group.

However, pretreatment of 17c dramatically enhanced the

activities of these enzymatic antioxidant defense systems in a

dose-dependent manner. Speciﬁcally, pretreatment with 50 and

EXPERIMENTAL SECTION

■

General Methods and Materials. All analytical grade reagents

and chemicals were purchased from commercial suppliers and used as

received. BCA protein concentration detection kit was purchased from

Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Anti-B-

cell lymphoma 2 (Bcl-2) antibody, anti-Bcl-2 associated X protein

00 mg of 17c signiﬁcantly decreased the MDA level by almost a

half (p < 0.001, Figure 6B), better than that in the Pol group (p <

.01). Meanwhile, 17c pretreatment increased the activity of

(

Bax) antibody, cysteine protease-3 antibody (caspase3), and

glyceraldehyde-3-phosphate dehydrogenase antibody (GAPDH) were

purchased from ProteinTech (WuHan, China).

SOD to more than 1.2-fold (p < 0.001, Figure 6C) and the level

of GSH-Px to more than1.3-fold (p < 0.001, Figure 6D) at the

concentrations of 50 and 100 mg/kg, which were both better

than Pol (p < 0.05). All these data demonstrated that 17c

pretreatment signiﬁcantly decreased the myocardial ROS

contents induced by MI/R injury and thus alleviated the infarct

size.

H NMR and C NMR spectra were recorded on a JEOL

spectrometer (400 MHz) using CDCl , dimethyl sulfoxide (DMSO-

d ), or CD OD as solvents with the internal standard of

tetramethylsilane. High-resolution mass spectrum (HRMS) was

recorded on a Bruker UHPLC-MicroQ-TOF-II spectrometer. All

ﬁnal compounds were puriﬁed to >95% purity as determined by HPLC

(

Dionex Ultimate 3000, USA) analysis using the following methods.

Purity analysis of ﬁnal compounds was performed through an ACE

Excel-5-C₁₈(particle size = 5 μm, pore size = 4.6 nm, dimensions = 250

mm) column. The injection volume was 5 μL with a gradient of 5 to

Antiapoptotic Activity of 17c during MI/R Injury. Cell

damages induced by MI/R injury lead to apoptosis and necrosis,

and these two types of cell death contribute diﬀerentially to MI/

R injury. Previous reports revealed that short-term myocardial

ischemia mainly led to apoptosis, whereas prolonged periods of

5% methanol/water or acetonitrile/water. The ﬂow rate was 1.0 mL/

min, and each analysis lasted for 30 min. The retention time (R_T_,_H_P_L_C

)

of each sample was displayed in the analytical data of the respective

compounds. All melting points were obtained on a WRS-2 micro-

computer melting point apparatus.

Synthesis of (1S,3S)-Methyl 1-Methyl-2,3,4,9-tetrahydro-1H-

pyrido[3,4-b]indole-3-carboxylate (6a) and (1R,3S)-Methyl 1-

Methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxy-

late (6b). To a stirred solution of aldehyde (0.40 g, 9.16 mmol) and L-

tryptophan methyl ester 5 (1.00 g, 4.58 mmol) in dichloromethane

(DCM, 50 mL) was added triﬂuoroacetic acid (TFA, 1.57 g, 11.45

mmol); the mixture was stirred at room temperature for 3.5 h

myocardial ischemia increased the rate of necrosis. In addition,

apoptosis plays an important role in lethal reperfusion injury as

indicated by numerous studies in various animal models.

Therefore, the apoptosis of cardiomyocytes was then inves-

tigated, and the expression levels of key apoptosis-related

proteins (Bax, Bcl-2, and caspase3) were evaluated by Western

6,57

blot.

the expression of Bcl-2 and caspase3 and increased the Bax level

Figure 7A). However, these MI/R-related alterations were

Results showed that MI/R injury obviously impaired

[

monitored by thin-layer chromatography (TLC)]. Then, the mixture

(

was evaporated to dryness in vacuum, 60 mL of water was added, and

the mixture was extracted with DCM (3 × 20 mL). The combined

organic layers were dried over anhydrous Na SO and evaporated in

alleviated by 17c pretreatment. Quantitative analysis revealed

that the Bax level was signiﬁcantly decreased in each dose group

(p < 0.001, Figure 7B), and 25 mg/kg of 17c was more eﬀective

vacuo, and the obtained mixture was separated by silica gel column

than 200 mg/kg of Pol (p < 0.01). By contrast, the expressions of

Bcl-2 and caspase3 were signiﬁcantly restored in 50 and 100 mg/

kg 17c groups (p < 0.001, Figure 7C,D), better than that of Pol

chromatography (DCM/MeOH = 90:1) to give 6a and 6b with yields

of 51.2 and 37.1%, respectively. 6a: mp 161.0−174.0 °C; H NMR (400

MHz, CDCl ): δ 8.01 (s, 1H), 7.47 (d, J = 7.2 Hz, 1H), 7.27 (t, J = 7.6

(

p < 0.05). Therefore, the results suggested that 17c

Hz, 1H), 7.18−7.05 (m, 2H), 4.27−4.22 (m, 1H), 3.86−3.79 (m, 4H),

preconditioning potently inhibited reperfusion-induced apop-

tosis.

3.16−3.10 (m, 1H), 2.83 (m, 1H), 2.01 (s, 1H), 1.48 (d, J = 6.8 Hz,

H). C NMR (100 MHz, CDCl ): δ 174.31, 136.35, 135.95, 127.15,

21.83, 119.58, 118.18, 110.89, 106.63, 52.60, 52.31, 45.82, 25.15,

1.70, 14.30. ESI-HRMS (m/z): calcd C H N O for [M − H] ,

43.1128; found, 243.1146. R

−

CONCLUSIONS

■

= 18.34 min. 6b: mp 62.0−65.0 °C;

T,HPLC

In summary, a series of β-carboline derivatives were designed

and synthesized with obvious myocardial protective activities for

the ﬁrst time. Among them, compound 17c signiﬁcantly reduced

H O -induced oxidative stress in vitro and alleviated LAD

H NMR (400 MHz, CDCl ): δ 7.85 (s, 1H), 7.47 (d, J = 7.6 Hz, 1H),

7.26 (t, J = 7.6 Hz, 1H), 7.16−7.07 (m, 2H), 4.38 (q, J = 6.4 Hz, 1H),

3.99 (t, J = 5.6 Hz, 1H), 3.74 (s, 3H), 3.12 (dd, J = 15.6, 5.2 Hz, 1H),

2.98 (dd, J = 15.6, 7.2 Hz, 1H), 2.27 (s, 1H), 1.44 (d, J = 6.8 Hz, 3H).

C NMR (100 MHz, CDCl ): δ 174.12, 136.19, 135.79, 126.99,

ligation-induced MI/R injury in vivo. Pretreatment of H9c2 cells

with 17c improved the H O -induced LDH leakage and

restored the endogenous antioxidant capacity and thus

eﬀectively protected the mitochondrial by decreasing the

overproduction of ROS and restoring the mitochondrial

membrane potential ΔΨ . As a result, the death of H9c2

cardiomyocytes induced by H O was signiﬁcantly decreased by

21.66, 119.41, 118.02, 110.73, 106.47, 52.47, 52.18, 45.69, 25.03,

1.58. ESI-HRMS (m/z): calcd C H N O for [M + H] , 245.01;

found, 245.01. R_T_,_H_P_L_C= 12.81 min. The same procedure was also

followed for the synthesis of 10a and 10b.

Synthesis of Methyl 1-Methyl-9H-pyrido[3,4-b]indole-3-

carboxylate (7). Compound 6a (1.00 g, 4.17 mmol) was dissolved

in dimethyl formamide (DMF, 25 mL), and KMnO (1.32 g, 8.34

17c. More attractively, 17c markedly reduced the myocardial

mmol) was slowly added at 0 °C. The reaction was stirred at 0 °C for 1 h

174

J. Med. Chem. 2021, 64, 9166−9181

Journal of Medicinal Chemistry

Article

and brought to room temperature. After 2.5 h, the mixture was ﬁltered.

7.28 (m, 1H), 7.17−7.08 (m, 2H), 4.14 (dd, J = 8.0, 4.4 Hz, 1H), 3.99−

3.96 (m, 1H), 3.74 (s, 3H), 3.14−3.09 (m, 1H), 3.02−2.96 (m 1H),

50 mL of water was added to the ﬁltrate, and the mixture was extracted

with ethyl acetate (EA, 3 × 20 mL). The combined organic solvent was

1.85−1.69 (m, 2H), 1.06 (t, J = 7.6 Hz, 3H). C NMR (100 MHz,

dried over anhydrous Na SO and evaporated in vacuo, and the residue

CDCl ): δ 176.46, 135.95, 135.59, 127.15, 121.76, 119.51, 118.14,

was puriﬁed by silica gel column chromatography (DCM/MeOH =

110.85, 107.08, 51.70, 52.28, 51.76, 28.52, 25.12, 10.77. ESI-HRMS

−

40:1) to aﬀord 7 with a yield of 88.9%. mp 244.0−254.0 °C; H NMR

(m/z): calcd C H N O for [M − H] , 257.1284; found, 257.1319.

(

400 MHz, CDCl ): δ H NMR (400 MHz): δ 9.59 (s, 1H), 8.79 (s,

T,HPLC

= 19.83 min.

H), 8.16 (d, J = 8.0 Hz, 1H), 7.60−7.53 (m, 2H), 7.36−7.32 (m, 1H),

Methyl(1S,3S)-1-ethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]-

.01 (s, 3H), 2.81 (s, 3H). C NMR (100 MHz, CDCl ): δ 166.89,

indole-3-carboxylate (10b). White solid, 37.5% yield. mp 79.0−96.0

42.12, 140.71, 137.03, 136.53, 128.87, 128.30, 122.23, 122.00, 121.06,

°C; H NMR (400 MHz, CDCl ): δ 7.99 (s, 1H), 7.48 (d, J = 7.6 Hz,

16.63, 112.17, 52.70, 20.32. ESI-HRMS (m/z): calcd C H N O for

1H), 7.29 (t, J = 7.6 Hz, 1H), 7.16−7.08 (m, 2H), 4.14 (m, 1H),3.81 (s,

3H), 3.79 (dd, J = 7.2, 4 Hz, 1H), 3.13 (dq, J = 15.2, 2 Hz, 1H), 2.85−

2.79 (m, 1H), 2.02−1.95 (m, 1H), 1.77−1.68 (m, 1H), 1.03 (d, J = 7.2

−

[

M − H] , 239.0815; found, 239.0743. R

= 17.65 min. The same

T,HPLC

procedure was also followed for the synthesis of 11.

Synthesis of 1-Methyl-9H-pyrido[3,4-b]indole-3-carboxylic

Hz, 3H). C NMR (100 MHz, CDCl ): δ 174.31, 136.35, 135.95,

Acid (8). Compound 7 (1.0 g, 4.17 mmol) and KOH (1.17 g, 20.83

127.15, 121.83, 119.58, 118.18, 110.89, 106.63, 52.60, 52.31, 45.82,

mmol) were dissolved in tetrahydrofuran (THF)/H O (2:1, 30 mL)

25.15, 21.70, 14.30. ESI-HRMS (m/z): calcd C H N O for [M −

15 18

= 19.55 min.

−

and stirred at 66 °C for 8 h (monitored by TLC). Then, the mixture was

moved to room temperature, adjusted to pH 5−6 with HCl (1 M), and

extracted with EA (3 × 20 mL). The combined organic layers were

dried over anhydrous Na SO and evaporated in vacuo, and the mixture

H] , 257.1284; found, 257.1319. R

T,HPLC

Methyl 1-Ethyl-9H-pyrido[3,4-b]indole-3-carboxylate (11).

White solid, 86.8% yield. mp 218.0−223.0 °C; H NMR (400 MHz,

CDCl ): δ 9.97 (s, 1H), 8.80 (s, 1H), 8.16 (d, J = 8.0 Hz, 1H), 7.61−

was puriﬁed by silica gel column chromatography (DCM/MeOH =

7.53 (m, 2H), 7.33 (td, J = 8.0, 1.2 Hz, 1H), 4.00 (s, 3H), 3.08 (q, J = 8.0

0:1) to give a white solid (8) with a yield of 78.2%. mp 251.0−267.0

Hz, 2H), 1.25 (t, J = 7.6 Hz, 3H). C NMR (100 MHz, CDCl ): δ

(

C; H NMR (400 MHz, DMSO-d ): δ 9.62 (s, 1H), 7.01 (s, 1H), 6.68

d, J = 6.4 Hz, 1H), 6.13 (d, J = 6.8 Hz, 1H), 6.07 (t, J = 6.4 Hz, 1H),

.84 (t, J = 6.4 Hz, 1H), 2.66 (br s, 1H), 2.26 (s, 3H). C NMR (100

167.09, 147.36, 140.67, 136.80, 135.75, 128.67, 128.43, 122.04, 121.80,

120.77, 116.62, 112.14, 52.64, 27.54, 13.01. ESI-HRMS (m/z): calcd

−

for [M − H] , 253.0971; found, 253.0887. RT,HPLC

MHz, DMSO-d ): δ 167.34, 142.27, 141.33, 137.00, 136.65, 128.93,

19.19 min.

27.59, 122.69, 121.86, 120.65, 116.10, 112.82, 20.80. ESI-HRMS (m/

Synthesis of 1-Ethyl-3-(methoxycarbonyl)-9H-pyrido[3,4-b]-

indole 2-Oxide (12). Compound 11 (50 mg, 197 μmol) was dissolved

in DCM (15 mL) and stirred at 0 °C. To the solution was added

mCPBA (102 mg, 590 μmol) in batches, and the mixture was stirred at

0 °C for 10 min and then reacted at room temperature for 49 h

(monitored by TLC). The reaction was terminated by excess sodium

sulﬁte and washed with saturated sodium chloride solution (2 × 10

mL). The organic layer was evaporated in vacuo, and the mixture was

puriﬁed by silica gel column chromatography (DCM/MeOH = 50:1)

−

z): calcd C H N O for [M − H] , 225.0658; found, 225.0676.

= 7.99 min.

T,HPLC

Synthesis of Ethyl(1-methyl-9H-pyrido[3,4-b]indole-3-

carbonyl)glycinate (9a). To a solution of compound 7 (60 mg,

77 μmol) in THF (10 mL) was added HBTU (101 mg, 266 μmol) and

DIEA (27 mg, 212 μmol), and the solution was left stirring at room

temperature for 10 min. Then, glycine ethyl ester (27 mg, 266 μmol)

was added, and the reaction was continued at room temperature for 10

h (monitored by TLC). The resulting mixture was spin-dried and

puriﬁed by column chromatography (DCM/MeOH = 20:1) to give a

to aﬀord a white solid (12) with a yield of 77.1%. H NMR (400 MHz,

OD): δ 8.13 (s, 1H), 7.92 (d, J = 8.0 Hz, 1H), 7.46−7.41 (m, 2H),

white solid (9a) with a yield of 85.1%. mp 156.0−159.0 °C; H NMR

7.20−7.16 (m, 1H), 3.98 (s, 3H), 3.20 (q, J = 7.6 Hz, 2H), 1.31 (t, J =

7.2 Hz, 3H). C NMR (100 MHz, CD

(400 MHz, CDCl ): δ 9.32 (s, 1H), 8.60 (s, 1H), 8.07 (s, 1H), 7.97 (d, J

OD): δ 162.95, 142.28, 139.93,

7.6 Hz, 1H), 7.55 (td, J = 8, 0.8 Hz, 1H), 7.50 (d, J = 8Hz, 1H), 7.31

135.72, 133.05, 128.29, 121.15, 121.04, 120.91, 120.81, 115.53, 111.61,

(

td, J = 7.6, 0.8Hz, 1H), 5.28 (s, 1H), 4.37−4.31 (m, 4H), 2.73 (s, 3H),

52.12, 19.66, 9.61. ESI-HRMS (m/z): calcd C15

H N O for [M + H] ,

14 2 3

.37 (t, J = 7.2 Hz, 3H). C NMR (100 MHz, CDCl ): δ 171.11,

271.1077; found, 271.1187. RT,HPLC = 13.99 min.

65.91, 149.91, 140.45, 140.10, 135.72, 128.45, 128.29, 122.15, 121.90,

Synthesis of Methyl(R)-1-(1-acetoxyethyl)-9H-pyrido[3,4-b]-

indole-3-carboxylate (13). Compound 12 (41 mg, 152 μmol) was

dissolved in acetic anhydride (10 mL) and stirred at 110 °C for 2 h

20.54, 112.36, 112.06, 61.70, 41.40, 20.21, 14.24. ESI-HRMS (m/z):

−

calcd C H N O for [M − H] , 310.1186; found, 310.1054. R

T,HPLC

(

monitored by TLC). To the mixture was added 40 mL of water and

9.59 min. The same procedure was also followed for the synthesis of

b and 9c.

adjusted to pH 6−7 with NaHCO . Then, the solution was extracted

with EA (3 × 15 mL), and the combined organic layers were dried over

Dimethyl(1-methyl-9H-pyrido[3,4-b]indole-3-carbonyl)-L-

glutamate (9b). White solid, 89.5% yield. mp 156.0−159.0 °C; H

NMR (400 MHz, CDCl ): δ 8.85 (br s, 1H), 8.66 (br s, 1H), 8.40 (s,

anhydrous Na SO and evaporated in vacuo. The residue was subjected

2 4

to silica gel column chromatography (PE/EA = 3:1) to aﬀord a white

H), 8.00 (d, J = 8.0 Hz, 1H), 7.56−7.49 (m, 2H), 7.31−7.27 (m, 1H),

solid (13) with a yield of 57.2%. H NMR (400 MHz, CDCl ): δ 8.21

.93−4.88 (m, 1H), 3.82 (s, 3H), 3.65 (s, 3H), 2.79 (s, 3H), 2.63−2.48

(s, 1H), 7.74 (s, 1H), 7.20 (d, J = 6.0 Hz, 1H), 6.76−6.72 (m, 2H),

6.56−6.53 (m, 1H), 5.90 (t, J = 5.6 Hz, 1H), 3.90 (s, 3H), 2.38 (s, 3H),

(

m, 2H), 2.46−2.38 (m, 1H), 2.26−2.17 (m, 1H). C NMR (100

MHz, CDCl ): δ 173.20, 173.03, 140.51, 140.31, 135.99, 128.60,

2.14 (d, J = 5.6 Hz, 3H). C NMR (100 MHz, CDCl ): δ 171.24,

22.36, 122.05, 112.77, 112.69, 111.98, 52.62, 51.97, 51.83, 30.51,

166.73, 142.28, 140.65, 137.21, 135.20, 130.23, 129.19, 121.91, 121.82,

−

7.74, 20.22. ESI-HRMS (m/z): calcd C H N O for [M − H] ,

121.15, 117.97, 112.23, 71.88, 52.81, 21.42, 18.89. ESI-HRMS (m/z):

82.1397; found, 382.1256. R

= 20.07 min.

calcd C17

for [M + H] , 313.1182; found, 313.1218. RT,HPLC

T,HPLC

N-(4-Methoxyphenyl)-1-methyl-9H-pyrido[3,4-b]indole-3-

9.96 min.

carboxamide (9c). White solid, 86.7% yield. mp 156.0−159.0 °C; H

NMR (400 MHz, DMSO-d ): δ 12.04 (s, 1H), 10.37 (s, 1H), 8.79 (s,

Synthesis of Methyl(R)-1-(1-hydroxyethyl)-9H-pyrido[3,4-

b]indole-3-carboxylate (14). To a solution of 13 (50 mg, 160

H), 8.38 (d, J = 8.0 Hz, 1H), 7.83 (d, J = 8.8 H, 2H), 7.66 (d, J = 8.0

μmol) in THF/H O (1:1, 15 mL) was added NaOH (13 mg, 320

Hz, 1H), 7.60 (dt, J = 6.8, 1.2 H, 1H), 7.30 (dt, J = 7.6, 0.8 H, 1H), 6.96

μmol) and stirred at room temperature for 30 min. Then, the reaction

was adjusted to pH 5−6 with HCl (1 M) and extracted with EA (3 × 15

(

d, J = 8.8 Hz, 2H), 3.76 (s, 3H), 2.91 (s, 3H). C NMR (100 MHz,

DMSO-d ): δ 174.96, 163.45, 156.00, 141.51, 141.46, 139.33, 136.59,

mL). The combined organic layers were dried over anhydrous Na SO

2 4

32.41, 128.92, 128.20, 122.71, 121.96, 121.86, 120.57, 114.46, 112.78,

and evaporated in vacuo, and the mixture was puriﬁed by silica gel

column chromatography (PE/EA = 2:1) to give a white solid (14) with

−

5.76, 20.91. ESI-HRMS (m/z): calcd C H N O for [M − H] ,

30.1237; found, 330.1098. R

= 23.87 min.

a yield of 80.0%. mp 168.7−171.7 °C; [α]

46.32 (c 1.9, MeOH); H

T,HPLC

Methyl(1R,3S)-1-ethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]-

NMR (400 MHz, DMSO-d ): δ 11.65 (s, 1H), 8.79 (s, 1H), 8.32 (d, J =

indole-3-carboxylate (10a). White solid, 52.4% yield. mp 56.0−71.0

7.2 Hz, 1H), 7.73 (d, J = 8.4 Hz, 1H), 7.53 (td, J = 7.2, 0.8 Hz 1H),

7.26−7.22 (m, 1H), 5.86 (d, J = 4.4 Hz, 1H), 5.23−5.16 (m, 1H), 3.86

°C; H NMR (400 MHz, CDCl ): δ 7.86 (s, 1H), 7.30−7.27 (m, 1H),

175

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Journal of Medicinal Chemistry

Article

(

s, 3H), 1.54 (d, J = 6.8 Hz, 3H). ¹³C NMR (100 MHz, DMSO-d ): δ

66.62, 149.31, 141.49, 135.55, 134.43, 128.89, 128.84, 122.28, 121.23,

stirred at room temperature for 30 min; then, benzyl bromide (404 mg,

2.36 mmol) was added, and the mixture was reacted for 3 h (monitored

by TLC). The solution was diluted with water (15 mL), adjusted to pH

7−8 with 1 M HCl, extracted with EA (3 × 20 mL), and washed with

brine (15 mL). The organic liquid was dried over anhydrous Na SO ,

20.52, 117.21, 113.40, 70.39, 52.45, 23.16. ESI-HRMS (m/z): calcd

−

C H N O for [M − H] , 269.0920; found, 269.0778. R

T,HPLC

7.60 min.

Synthesis of (R)-4-(1-(3-(Methoxycarbonyl)-9H-pyrido[3,4-

b]indol-1-yl)ethoxy)-4-oxobutanoic Acid (15a). To a solution of

4 (100 mg, 370 μmol) in toluene (15 mL) was added succinic

concentrated under reduced pressure, and puriﬁed by silica gel column

chromatography (PE/EA = 8:1) to give a white solid (16a) with a yield

of 89.0%. mp138.6−140.4 °C; H NMR (400 MHz, CDCl ): δ 8.83 (s,

anhydride (111 mg, 1.11 mmol) and stirred at 70 °C for 8 h. The

reaction was monitored by TLC. After cooling to room temperature, 20

mL of water was added, and the solution was extracted with EA (3 × 20

mL). The extract was dried with anhydrous Na SO for 30 min, ﬁltered,

and evaporated in vacuo, and the crude product was puriﬁed by silica gel

H), 8.23 (d, J = 7.6 Hz, 1H), 7.57 (t, J = 8.4 Hz, 1H), 7.42−7.35 (m,

H), 7.26−7.25 (m, 2H), 6.95 (d, J = 6.8 Hz, 2H), 5.81 (s, 2H), 4.05 (s,

H), 3.23 (q, J = 7.6 Hz, 2H), 1.37 (t, J = 7.6 Hz, 3H). C NMR (100

MHz, CDCl ): δ 152.90, 137.08, 133.32, 129.23, 129.20, 128.58,

23.17, 122.72, 122.58, 121.63, 119.73, 116.87, 116.75, 116.26, 112.43,

column chromatography (DCM/MeOH = 10:1) to aﬀord a white solid

07,73, 61.62, 58.25, 42.64, 31.15. ESI-HRMS (m/z): calcd

(

15a) with a yield of 75.1%. mp 164.7−168.4 °C; H NMR (400 MHz,

−

C H N O for [M + HCOOH − H] , 383.1495; found, 389.1457.

DMSO-d ): δ 12.13 (s, 1H), 12.07 (s, 1H), 8.90 (s, 1H), 8.42 (d, J = 7.6

= 27.82 min. The same procedure was also followed for the

T,HPLC

Hz, 1H), 7.72 (d, J = 8 Hz, 1H), 7.63 (t, J = 7.2 Hz, 1H), 7.33 (t, J = 7.6

Hz, 1H), 6.28 (q, J = 6.4 Hz,1H), 3.92 (s, 3H), 2.45 (t, J = 7.2 Hz, 2H),

synthesis of 17a, 18a, 19a, and 20a.

Synthesis of 9-Benzyl-1-ethyl-9H-pyrido[3,4-b]indole-3-car-

boxylic Acid (16b). To a solution of 16a (100 mg, 291 μmol) in DMF

(15 mL) was added NaOH (35 mg, 872 μmol) and stirred at 70 °C for 3

h. Then, the mixture was diluted with water (10 mL), adjusted to pH

5−6 with 1 M of HCl, extracted with EA (3 × 20 mL), and washed with

brine (15 mL). The organic liquid was dried over anhydrous Na SO ,

.24 (d, J = 6.4 Hz, 2H), 1.71 (d, J = 6.4 Hz, 3H). C NMR (100 MHz,

DMSO-d ): δ 173.82, 172.27, 166.40, 166.38, 143.65, 141.68, 136.39,

34.52, 129.31, 121.46, 117.74, 113.14, 71.69, 52.47, 29.48, 29.28,

−

9.42. ESI-HRMS (m/z): calcd C H N O for [M − H] , 369.1081;

found, 369.1140. R

= 12.95 min. The same procedure was also

T,HPLC

followed for the synthesis of 15b.

R)-5-(1-(3-(Methoxycarbonyl)-9H-pyrido[3,4-b]indol-1-yl)-

ethoxy)-5-oxopentanoic Acid (15b). White solid, 78.6% yield. mp

concentrated under reduced pressure, and puriﬁed by silica gel column

(

chromatography (DCM/MeOH = 8:1) to aﬀord a white solid with a

yield of 90%. mp184.3−185.1; H NMR (400 MHz, DMSO-d ): δ 8.82

55.7−158.8 °C; H NMR (400 MHz, DMSO-d ): δ 11.87 (s, 1H),

(s, 1H), 8.42 (d, J = 8.0 Hz, 1H), 7.68 (d, J = 8.4 Hz, 1H), 7.57 (t, J = 8.0

Hz, 1H), 7.32 (t, J = 8.4 Hz, 1H), 7.25−7.17 (m, 3H), 6.88 (d, J = 7.6

Hz, 2H), 5.91 (s, 2H), 3.11 (q, J = 7.6 Hz, 2H), 1.20 (t, J = 7.6 Hz, 3H).

.81 (s, 1H), 8.33 (d, J = 7.6 Hz, 1H), 7.67 (d, J = 8.0 Hz, 1H), 7.57 (t, J

7.6 Hz, 1H), 7.28 (t, J = 7.2 Hz, 1H), 6.25 (q, J = 6.4 Hz, 1H), 3.87 (s,

H), 2.41 (t, J = 7.6 Hz, 2H), 2.21 (t, J = 7.6 Hz, 4H), 1.67 (d, J = 6.8

C NMR (100 MHz, DMSO-d ): δ 167.17, 146.40, 142.64, 138.68,

Hz, 3H). C NMR (100 MHz, DMSO-d ): δ 174.49, 174.44, 172.67,

37.52, 136.16, 129.52, 129.46, 129.42, 127.84, 125.82, 125.51, 121.49,

66.42, 143.80, 141.65, 136.45, 134.50, 129.23, 122.46, 121.49, 117.68,

21.34, 116.04, 111.43, 48.26, 28.26, 13.88. ESI-HRMS (m/z): calcd

13.10, 71.25, 52.45, 33.35, 33.18, 20.49, 19.63. ESI-HRMS (m/z):

−

C H N O for [M − H] , 329.1284; found, 329.1335. R

−

21 18

T,HPLC

calcd C H N O for [M − H] , 383.1277; found, 383.1066. R

T,HPLC

7.53 min. The same procedure was also followed for the synthesis of

4.26 min.

Synthesis of Methyl(R)-1-(1-(benzoyloxy)ethyl)-9H-pyrido-

3,4-b]indole-3-carboxylate (15c). Compound 14 (100 mg, 370

μmol) and triethylamine (225 mg, 2.22 mmol) were dissolved in DCM

20 mL) and stirred for 5 min at room temperature. To the solution was

added benzoic anhydride (251 mg, 1.11 mmol) and 4-diaminopyridine

7b, 18b, and 19b.

Methyl 1-Ethyl-9-(4-ﬂuorobenzyl)-9H-pyrido[3,4-b]indole-

-carboxylate (17a). White solid, 22.0% yield. mp 202.0−217.0 °C;

[

H NMR (400 MHz, CDCl ): δ 8.83 (s, 1H), 8.24 (dt, J = 7.6, 0.8 Hz,

(

H), 7.61−7.57 (m, 1H), 7.41−7.37 (m, 2H), 6.99−6.91 (m, 4H), 5.79

(s, 2H), 4.06 (s, 3H), 3.24 (q, J = 7.6 Hz, 2H), 1.38 (t, J = 7.6 Hz, 3H).

(

13.56 mg, 1.11 mmol) and reacted for 12 h at room temperature

monitored by TLC). Then, 20 mL of water was added and the mixture

C NMR (100 MHz, CDCl ): δ 163.51, 161.06, 147.00, 142.33,

36.33, 132.90, 132.87, 129.94, 129.19, 127.14, 127.06, 121.89, 121.86,

extracted with EA (3 × 20 mL). The organic liquid was dried with

anhydrous Na SO and evaporated in vacuo, and the crude product was

21.30, 116.38, 116.29, 116.07, 110.34, 52.89, 48.00, 28.96, 14.75. ESI-

−

HRMS (m/z): calcd C H FN O for [M − H] , 361.1346; found,

361.1355. R

puriﬁed by silica gel column chromatography (PE/EA = 8:1) to give a

22 19

= 26.52 min.

white solid (15c) with a yield of 80.1%. mp 205.1−209.6; H NMR

T,HPLC

-Ethyl-9-(4-ﬂuorobenzyl)-9H-pyrido[3,4-b]indole-3-car-

(400 MHz, DMSO-d ): δ 12.19 (s, 1H), 8.94 (s, 1H), 8.43 (d, J = 8.0

boxylic Acid (17b). White solid, 63.5% yield. mp 160.0−175.0 °C; H

Hz, 1H), 8.06 (d, J = 7.2 Hz, 2H), 7.73 (d, J = 8.4 Hz, 1H), 7.39−7.62

NMR (400 MHz, CDCl ): δ 8.83 (s, 1H), 8.22 (d, J = 7.6 Hz, 1H), 7.61

(m, 2H), 7.54 (t, J = 8.0 Hz, 2H), 7.34 (t, J = 7.6 Hz, 1H), 6.55 (q, J =

(t, J = 7.6 Hz, 1H), 7.41−7.38 (m, 2H), 7.00−6.91 (m, 4H), 5.80 (s,

.4 Hz, 1H), 3.91 (s, 3H), 1.89 (d, J = 6.4Hz, 3H). C NMR (100

H), 3.20 (q, J = 7.2 Hz, 2H), 1.39 (t, J = 7.2 Hz, 3H). C NMR (100

MHz, DMSO-d ): δ 166.36, 165.91, 143.55, 141.71, 136.51, 134.79,

MHz, CDCl ): δ 165.39, 163.64, 161.18, 145.09, 142.66, 136.95,

33.90, 130.35, 130.00, 129.86, 129.35, 129.26, 129.07, 122.67, 122.38,

135.40, 132.74, 130.94, 129.69, 127.13, 122.10, 121.71, 116.44, 116.22,

21.52, 118.00, 117.89, 113.13, 72.25, 52.48, 19.33. ESI-HRMS (m/z):

−

114.35, 110.39, 48.21, 27.88, 13.10. ESI-HRMS (m/z): calcd

calcd C H N O for [M − H] , 373.1182; found, 373.1140. R

T,HPLC

−

C H FN O for [M − H] , 347.1190; found, 347.1072. R

T,HPLC

2.95 min. The same procedure was also followed for the synthesis of

21 17

7.74 min.

5d.

Sodium 1-Ethyl-9-(4-ﬂuorobenzyl)-9H-pyrido[3,4-b]indole-

-carboxylate (17c). To a solution of 17b (200 mg, 574.10 μmol)

(

R)-2-((1-(3-(Methoxycarbonyl)-9H-pyrido[3,4-b]indol-1-yl)-

ethoxy)carbonyl)benzoic Acid (15d). White solid, 72.71% yield.

mp 102.6−105.2 °C; H NMR (400 MHz, DMSO-d ): δ 12.75 (s, 1H),

in acetone/H O (1:1, 10 mL) was slowly added a saturated aqueous

solution of Na CO (30.42 mg, 287.05 μmol) and stirred at room

.84 (s, 1H), 8.32 (d, J = 8.0 Hz, 1H), 7.73 (d, J = 7.6 Hz, 2H), 7.54 (t, J

temperature for 30 min. The resultant solid was collected by ﬁltration

7.2 Hz, 2H), 7.50−7.37 (m, 2H), 7.25 (t, J = 7.6 Hz, 1H), 6.43 (q, J =

and washed with PE to give a white solid with a yield of 70.5%. H NMR

.4 Hz, 1H), 3.89 (s, 3H), 1.82 (d, J = 6.4 Hz, 3H). C NMR (100

(

400 MHz, DMSO-d ): δ 8.69 (s, 1H), 8.24 (d, J = 7.6 Hz, 1H), 7.57−

MHz, DMSO-d ): δ 169.81, 168.98, 166.49, 143.47, 142.03, 137.38,

.49 (m, 2H), 7.24 (t, J = 6.8 Hz, 1H), 7.03−6.99 (m, 2H), 6.92−6.91

35.95, 134.98, 132.98, 130.67, 129.29, 129.07, 128.01, 125.90, 122.32,

(

m, 2H), 5.78 (s, 2H), 3.09 (q, J = 7.2 Hz, 2H), 1.14 (t, J = 7.2 Hz, 3H).

21.36, 120.71, 117.92, 113.43, 72.73, 52.45, 18.73. ESI-HRMS (m/z):

−

C NMR (100 MHz, DMSO-d ): δ 169.45, 163.06, 160.64, 146.07,

calcd C H N O for [M − H] , 417.0871; found, 417.0894. R

T,HPLC

1.76 min.

145.64, 142.67, 114.23 134.89, 134.89, 130.05, 128.88, 127.97, 121.99,

121.89, 116.03, 115.81, 114.38, 110.00, 47.56, 28.57, 14.91. ESI-HRMS

Synthesis of Methyl 9-Benzyl-1-ethyl-9H-pyrido[3,4-b]-

indole-3-carboxylate (16a). To a solution of 11 (200 mg, 787

μmol) in DMF (15 mL) was added KOH (110 mg, 1.97 mmol) and

(m/z): calcd C H FN NaO for [M + H] , 371.1166; found,

371.1166. R_T_,_H_P_L_C= 18.59 min.

176

J. Med. Chem. 2021, 64, 9166−9181

Journal of Medicinal Chemistry

Article

Methyl 9-(4-Chlorobenzyl)-1-ethyl-9H-pyrido[3,4-b]indole-

solution was changed after 24 h on the second day. When the cells

reached 80% fusion, they were digested and passaged with trypsin.

Cytotoxicity Assay. The cytotoxicity of the title compounds was

tested through MTT analysis on H9c2 cells. Cells were seeded in 96-

well plates at a density of 10,000 cells/well. At the end of 24 h, the cells

-carboxylate (18a). White solid, 57.1% yield. mp113.4−117.0 °C;

H NMR (400 MHz, CDCl ): δ 8.83 (s, 1H), 8.23 (d, J = 7.2 Hz, 1H),

(

.58 (t, J = 8.4 Hz, 1H), 7.40−7.36 (m, 2H), 7.27−7.23 (m, 2H), 6.90

d, J = 8.4 Hz, 2H), 5.78 (s, 2H), 4.05 (s, 3H), 3.21 (q, J = 7.6 Hz, 2H),

−1

.38 (t, J = 7.6 Hz, 3H). C NMR (100 MHz, CDCl ): δ 166.80,

were treated with test compounds (2.5, 5, 10, 20, 40, and 80 μmol·L )

46.94, 142.21, 137.47, 136.37, 135.73, 133.69, 129.89, 129.39, 129.16,

26.86, 121.93, 121.86, 121.31, 116.37, 110.28, 52.88, 48.08, 29.05,

and incubated at 37 °C for 24 h. MTT (Sigma) was added to each well

with a ﬁnal concentration of 0.5 mg/mL and incubated for 4 h. Then,

the medium was decanted, and 150 μL of DMSO was added to each

well. The absorbance was obtained by a microplate reader (B20-TEK

ELX800UV, USA) at 570 nm, and all measurements were conducted

three times under the same condition. IC50 values were calculated by

Graphpad Prism 5.

4.72. ESI-HRMS (m/z): calcd C H ClN O for [M + H] ,

79.1207; found, 379.1334. R_T_,_H_P_L_C= 28.26 min.

-(4-Chlorobenzyl)-1-ethyl-9H-pyrido[3,4-b]indole-3-car-

boxylic Acid (18b). White solid, 30.3% yield. mp 183.6−186.8 °C; H

NMR (400 MHz, DMSO-d ): δ 8.87 (s, 1H), 8.47 (d, J = 7.6 Hz, 1H),

H O -Induced Oxidative Stress. H9c2 cells were seeded in 96-

.73 (d, J = 8.4 Hz, 1H), 7.63 (t, J = 8.4 Hz, 1H), 7.39−7.34 (m, 3H),

well plates and were cultured for 24 h. The cells were incubated with

.95 (d, J = 8.4 Hz, 2H), 5.96 (s, 2H), 3.14 (q, J = 7.6 Hz, 2H), 1.26 (t, J

100 μL of complete culture medium containing 0, 2.5, 5, 10, 20, and 40

7.2 Hz, 3H)· C NMR (100 MHz, DMSO-d ): δ 167.16, 146.34,

−

μmol·L of test samples for 24 h, and Pol was used for comparison. 50

37.75, 137.65, 136.04, 132.38, 129.59, 129.53, 129.41, 129.41, 127.82,

22.57, 121.54, 121.44, 116.09, 111.39, 100.00, 47.70, 28.24, 13.86.

R_T_,_H_P_L_C= 17.63 min.

μL of H O (1.5 mM) was added to each well with a ﬁnal concentration

of 0.3 mM and incubated for another 0.5 h. Then, the viability was

tested using the MTT assay.

Methyl 1-Ethyl-9-(4-methoxybenzyl)-9H-pyrido[3,4-b]-

Hoechst 33258 Staining. The cell death of H9c2 cardiomyocytes

indole-3-carboxylate (19a). White solid, 78.2% yield. mp 101.2−

was evaluated by Hoechst 33258 staining. H9c2 cells (4 × 10 ) were

04.1 °C; H NMR (400 MHz, CDCl ): δ 7.71 (s, 1H), 7.20 (d, J = 6.4

^c^u^l^t^u^r^e^dⁱⁿ²⁴^-^w^e^l^l^p^l^a^t^e^s^a^t³⁷^°^Cⁱⁿ⁵^%^C^O2 for 24 h. After

Hz, 1H), 6.68 (t, J = 6.0 Hz, 1H), 6.57 (d, J = 6.8 Hz, 1H), 6.52 (t, J =

.0 Hz, 1H), 6.15 (d, J = 6.8 Hz, 2H), 6.07 (d, J = 6.8 Hz, 2H), 5.22 (s,

preincubating with 17c (5, 10, 20, 40, and 80 μM) and Pol (80 μM) for

4 h, the cells were exposed to H O (0.3 mM) for 0.5 h. Then, after

H), 3.89 (s, 3H), 3.61 (s, 3H), 3.25 (q, J = 6.0 Hz, 2H), 1.76 (t, J = 6.0

2 2

removal of the medium and washing with phosphate-buﬀered saline

PBS), the cells were stained with 10 μg/mL Hoechst 33258 (10 mg/

Hz, 3H). C NMR (100 MHz, CDCl ): δ 166.90, 159.11, 147.09,

(

42.36, 137.21, 136.45, 129.64, 129.19, 128.96, 126.64, 121.80, 121.66,

21.03, 116.29, 114.48, 114.48, 110.50, 55.30, 52.79, 48.02, 29.10,

4.71. ESI-HRMS (m/z): calcd C H N O for [M + HCOOH −

mL, Sigma, St. Louis, MO, USA) and incubated in the dark at room

temperature for 10 min. The cells were observed and imaged with a

ﬂuorescence microscope (Nikon ECLIPSE TE2000-U, Japan).

−

H] , 419.1601; found, 419.1630. R

= 26.31 min.

T,HPLC

Live/Dead Cell Analysis. H9c2 cells (4 × 10 ) were cultured in a

-Ethyl-9-(4-methoxybenzyl)-9H-pyrido[3,4-b]indole-3-car-

24-well plate at 37 °C in 5% CO for 24 h. The cells were washed with

boxylic Acid (19b). White solid, 86.5% yield. mp 266.6−269.3 °C. H

PBS and incubated with a medium containing 17c (5, 10, 20, 40, and 80

μM) or Pol (80 μM) for 24 h and treated with 0.3 mM H O for

NMR (400 MHz, DMSO-d ): δ 8.82 (s, 1H), 8.41 (d, J = 7.6 Hz, 1H),

.66 (d, J = 8 Hz, 1H), 7.56 (t, J = 7.6 Hz, 1H), 7.31 (t, J = 7.2 Hz,

another 0.5 h. Then, after removal of the medium and washing with

PBS, the cells were stained with the solution containing 100 μL of FDA

H),6.81−6,77 (m, 4H), 5.82 (s, 2H), 3,62 (s, 3H), 3.14 (q, J = 6.8 Hz,

H), 1.24 (t, J = 7.6 Hz, 3H). C NMR (100 MHz, DMSO-d ): δ

(

0.02 mg/mL, Sigma, St. Louis, MO, USA) and 30 μL of PI (0.02 mg/

67.21, 158.94, 146.38, 142.59, 137.46, 136.14, 130.41, 129.47, 127.07,

mL, Sigma, St. Louis, MO, USA) and incubated in the dark at room

temperature for 10 min. Afterward, the cells were observed and imaged

with a ﬂuorescence microscope.

22.49, 121.51, 121.28, 116.07, 114.77, 111.49, 100.00, 55.51, 47.71,

−

8.34, 13.91. ESI-HRMS (m/z): calcd C H N O for [M − H] ,

59.1390; found, 359.1462. R

= 10.71 min.

T,HPLC

Cell Death Assay. The death of H9c2 cells was evaluated through

Methyl 1-Ethyl-9-(2-nitrobenzyl)-9H-pyrido[3,4-b]indole-3-

ﬂow cytometric analysis using a FACScan ﬂow cytometer (BECKMAN

carboxylate (20a). White solid, 31.8% yield. mp 216.0−220.7 °C;

CytomicsTM FC 500, USA). H9c2 cells (1 × 10 ) were cultured in a 60

H NMR (400 MHz, CDCl ): δ 8.80 (s, 1H), 8.24 (dd, J = 8.0 Hz, 2H),

mm culture dish at 37 °C overnight and incubated in triplicate with 17c

(

.55 (t, J = 8.0 Hz, 1H), 7.45−7.29 (m, 4H), 6.38 (d, J = 8.0 Hz, 1H),

(

10, 20, 40, and 80 μM) or Pol (80 μM) for 24 h. Then, the cells were

.18 (s, 2H), 4.02 (s, 3H), 3.01 (q, J = 7.6 Hz, 2H), 1.32 (t, J = 7.2 Hz,

exposed to H O (0.3 mM). After 0.5 h, the medium was removed and

_H₎_.₁3C NMR (100 MHz, CDCl ): δ 166.74, 146.98, 146.78, 142.02,

the cells were washed with PBS; the suspension cells were centrifuged at

000 rpm for 5 min. The adherent cells were digested with trypsin

37.84, 136.28, 134.77, 133.55, 130.02, 129.33, 128.91, 127.7, 125.96,

21.95, 121.57, 116.33, 110.03, 52.84, 46.85, 28.95, 14.60. ESI-HRMS

without ethylenediaminetetraaceticacid and washed with PBS. Then, all

cells were resuspended in 500 μL of binding buﬀer, and 5 μL of Annexin

V-FITC and 5 μL of PI (Keygen Biotech, China) working solution were

successively added to 500 μL of cell suspension and incubated for 15

and 5 min in total dark, respectively. Subsequently, ﬂow cytometry was

performed using FACScan ﬂow cytometry. Data were analyzed by

FlowJo10 software.

−

m/z): calcd C H N O for [M − H] , 388.1291; found, 388.1332.

T,HPLC

= 26.20 min.

Synthesis of Methyl 9-(2-Aminobenzyl)-1-ethyl-9H-pyrido-

3,4-b]indole-3-carboxylate (20b). To a solution of 20a (100 mg,

57 μmol) in EA (30 mL) was added SnCl ·2H O (174 mg, 771 μmol)

[

and heated to reﬂux for 3 h (monitored by TLC). The mixture was

diluted with water (30 mL), adjusted to pH 9 with Na CO , and

Measurement of Intracellular ROS Production. DCFH-DA

ﬂuorescent labeling was used to detect intracellular ROS accumulation

extracted with water saturated n-BuOH (3 × 15 mL). The organic

liquid was concentrated under reduced pressure and puriﬁed by silica

in H9c2 cells. The cells (4 × 10 ) were cultured in a 24-well plate at 37

gel column chromatography (DCM/MeOH = 8:1) to give a white solid

C overnight and incubated in triplicate with 17c (5, 10, 20, 40, and 80

(

20b) with a yield of 81.6%. mp 146.0−150.7 °C; H NMR (400 MHz,

μM) or Pol (80 μM) for 24 h and treated with H O (0.3 mM) for 0.5 h.

DMSO-d ): δ 8.82 (s, 1H), 8.22 (d, J = 8.0 Hz, 1H), 7.56 (t, J = 7.6 Hz,

Then, the cell supernatants were removed and continually incubated

with 10 mM DCFH-DA (Nanjing Jiancheng Bioengineering Institute,

China) at 37 °C for 20 min. The medium was removed, the cells were

washed with PBS twice, and images were acquired using a ﬂuorescence

microscope. The formation of ﬂuorescent DCF with excitation

wavelength of 488 nm and emission wavelength of 525 nm was

quantiﬁed by a microplate reader (BIO-TEK ELX800UV, USA).

(

H), 7.38−7.34 (m, 2H), 7.07 (t, J = 7.2 Hz, 1H), 6.79 (d, J = 7.6 Hz,

H), 6.51 (t, J = 7.6 Hz, 1H), 6.15 (d, J = 7.6 Hz, 1H), 5.60 (s, 2H), 4.03

s, 3H), 3.13 (q, J = 7.2 Hz, 2H), 1.35 (t, J = 7.2 Hz, 3H). ¹³C NMR

100 MHz, CDCl ): δ 166.92, 147.28, 142.56, 142.45, 137.32, 136.54,

29.79, 129.07, 128.55, 125.52, 121.80, 121.74, 121.72, 121.14, 119.73,

16.53, 116.32, 116.38, 52.83, 45.01, 28.72, 1.90.

Cell Lines and Culture Conditions. H9c2 cells were cultured with

Measurement of Mitochondrial Membrane Potential (ΔΨ ).

a DMEM high glucose medium containing 10% fetal bovine serum

HyClone, GE Healthcare, Australia) at 37 °C and 5% CO , and the

5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl-carbocyanine

(

iodide (JC-1) (Sigma-Aldrich, St. Louis, USA) was used to detect the

177

J. Med. Chem. 2021, 64, 9166−9181

Journal of Medicinal Chemistry

The Supporting Information is available free of charge at

Article

mitochondrial membrane potential ΔΨ . J-aggregate (the predom-

white. The infarct area and the percentage of infarct area in the

ventricular heart area were calculated by ImageJ software.

inant form at high ΔΨ ) emits orange ﬂuorescence, and JC-1 monomer

(

the predominant form at low ΔΨ ) emits green ﬂuorescence. In short,

Histopathological Analysis of Heart. Heart tissues were ﬁxed in

4% paraformaldehyde solution for 24 h and embedded in paraﬃn to

make a paraﬃn section of 4 μm, which was dewaxed with xylene and

stained with hematoxylin & eosin. Gradient ethanol dehydration,

preparation, and pathological changes of the myocardial tissue were

observed by an optical microscope (×400).

after the above treatment, H9c2 cells were washed twice and incubated

with JC-1 with a ﬁnal concentration of 2 μM at 37 °C for 30 min in the

dark following the manufacturer’s instructions. Images were captured

using a ﬂuorescence microscope, and the ﬂuorescence intensity was

calculated by ImageJ software. The ﬂuorescence intensity was measured

with a ﬂuorescence microscope. The green excitation wavelength and

emission wavelength were 485 and 530 nm, respectively. The orange

excitation wavelength and emission wavelength were 530 and 580 nm,

respectively.

Western Blotting Method to Analyze Apoptosis-Related

Proteins. The heart tissue of the rat left ventricle was homogenized

with commercial RIPA (Beyotime). The protein supernatant was

obtained by centrifugation at a low temperature for 20 min after

cleavage on ice for 1 h. The protein content was determined by a BCA

kit. Equal amounts of 30 μg per lane of protein were placed on Tris/HCl

gel, and the protein was separated by sodium dodecyl sulfate

polyacrylamide gel electrophoresis. After the electrophoresis, the

samples were transferred to the polyvinylidene diﬂuoride (Milibo,

Co. Ltd., USA) membrane and blocked with 5% skimmed milk powder

for 2 h. Diluted primary antibodies Bcl-2 (1:3000), Bax (1:10000),

caspase-3 (l:2000), and GAPDH (1:2000) were incubated overnight at

4 °C followed by a horseradish peroxidase-labeled goat anti-rabbit IgG

secondary antibody (ProteinTech, China) for 2 h at room temperature.

Immunoreactivity was developed with diaminobenzidine solution,

scanned, and recorded by a gel imaging system (G:BDX Chemi XL1.4,

Genome Co., Ltd.).

Measurement of the Activities of LDH, MDA, SOD, and GSH-

Px. H9c2 cells were cultured in 6-well plates with 1 × 10 cells/well and

treated with the test compounds and H O like the above experiments

in vitro. The LDH activity in the culture medium was tested by the LDH

assay kit (Nanjing Jiancheng Bioengineering Institute, China)

according to the manufacturer’s protocol. Then, the cells were collected

to measure the MDA, SOD, and GSH-Px (Nanjing Jiancheng

Bioengineering Institute, China) activities with the corresponding

detection kits according to the manufacturer’s instructions. For the in

vivo assay, the whole blood of rats was centrifuged at a speed of 10,000

rpm in a centrifuge containing heparin (10 mg/mL) for 10 min, and the

supernatant was taken to obtain the plasma. After the treatment, the

plasma was collected to measure the LDH, MDA, SOD, and GSH-Px

(

Nanjing Jiancheng Bioengineering Institute, China) activities with the

Statistical Analysis. The experimental results are collected from

three independent experiments and expressed as mean ± SEM. Data

were tested for normal distribution (Kolmogorov−Smirnoﬀ test) and

subsequently analyzed with one-way analysis of variance and t-test using

GraphPad Prism 8.0, and p < 0.05 indicates that the diﬀerence is

statistically signiﬁcant. Histograms were drawn with GraphPad Prism

corresponding detection kits according to the manufacturer’s

instructions.

Animals. A total of 72 speciﬁc pathogen-free male Sprague-Dawley

(

SD) rats, aged 7−8 weeks and weighing 220−230 g, were obtained

from the Laboratory Animal Center of Guizhou Medical University,

and the study was approved by the Animal Ethics Committee of

Guizhou Medical University (no. 1603117). Rats were maintained in

ventilated cages at a standard 12 h:12 h LD cycle for 7 days before the

experiment.

8.0 (La Jolla, California, USA).

ASSOCIATED CONTENT

sı Supporting Information

■

Preparation of the Rat MI/R Injury Model. Rats were randomly

divided into 6 groups with 12 rats in each group: sham, MI/R, Pol (200

mg/kg) + MI/R, low dose (17c, 25 mg/kg) + MI/R, medium dose

https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00384.

(

17c, 50 mg/kg) + MI/R, and high dose (17c, 100 mg/kg) + MI/R

Molecular formula strings (CSV )

groups. All test samples were administered (i. g.) to the animals in each

group for 7 days, and the same volume of normal saline was

administered to the sham group. The body weight of each animal

was recorded every day. Two hours after administration on the seventh

day, the rats were anesthetized by intraperitoneal injection of 500 mg/

kg uratan, and the trachea was intubated and connected to a ventilator

Cytotoxicity of β-carboline derivatives; X-ray crystal

structure of 14; toxicity of H O against H9c2 cells; cell

morphology of H9c2 cells pretreatment with 17c; toxicity

of 17c in vivo; representative ECG traces; and H and C

NMR spectral data for compounds 10a−18b, 21, and

(

Tai Meng, HX-300s-S, China). After opening of the chest along the left

2a−24d (PDF )

edge of the sternum and exposure of the heart, a 6−0 silk thread was

threaded through the live knot at 1−2 mm from the left anterior

descending branch of the coronary artery. Then, the heart was sent back

to the thoracic cavity and closed. After 1 h of ischemia, the living knot

was opened, and the heart was allowed 24 h of reperfusion. The sham

operation group experienced the same protocol without vascular

occlusion. The left anterior descending branch of the coronary artery

was ligated in the MI/R group, positive group, and each dose group.

During the entire surgical process, the body temperature of the animal

was maintained at 37 °C using a heating blanket. After the blood supply,

the heart was quickly removed, and the apical tissue was removed and

rinsed with normal saline.

AUTHOR INFORMATION

Corresponding Authors

■

Shang-Gao Liao − School of Pharmacy, Guizhou Medical

University, Guian New District, Guizhou 550025, P. R. China;

Email: lshangg@163.com

Meng Zhou − State Key Laboratory of Functions and

Applications of Medicinal Plants, Engineering Research Center

for the Development and Application of Ethnic Medicine and

TCM (Ministry of Education), Guizhou Medical University,

Guiyang 550004, P. R. China; School of Pharmacy, Guizhou

Medical University, Guian New District, Guizhou 550025, P.

R. China; orcid.org/0000-0003-4353-0776;

Email: gmu_mengzhou@163.com

ECG Monitoring. After constructing the MI/R model, the rats in

the sham group, model group, administration group, and positive

control group (at least three in each group) were connected to ECG

(

Chengdu Instrument Factory, Chengdu, China), and the changes of

the ST segment were calculated as lead II electrocardiograph.

Determination of Myocardial Infarct Size. After the blood

supply, the heart was quickly removed. The apical tissue was removed,

rinsed with normal saline, and absorbed by a ﬁlter paper. Then, the

heart was cut into ﬁve equal pieces at a line parallel to the coronary

sulcus and below the ligation line and weighed. All slices were incubated

in 1% TTC (Sigma-Aldrich, USA) in pH 7.4 buﬀer at 37 °C in the dark

for 15 min, and the normal tissue was dark red and the infarct site was

Authors

Hong Zhang − State Key Laboratory of Functions and

Applications of Medicinal Plants, Engineering Research Center

for the Development and Application of Ethnic Medicine and

TCM (Ministry of Education), Guizhou Medical University,

Guiyang 550004, P. R. China; School of Pharmacy, Guizhou

178

J. Med. Chem. 2021, 64, 9166−9181

Journal of Medicinal Chemistry

S.; Santos, H. A.; Shahbazi, M. A. Combination Therapy of Killing

Article

Medical University, Guian New District, Guizhou 550025, P.

R. China

tetramethyluronium hexaﬂuorophosphate; HE, hematoxylin−

eosin; LAD, left anterior descending coronary artery; LDH,

lactate dehydrogenase; MDA, malondialdehyde; MI/R, my-

ocardial ischemia/reperfusion; mPTP, membrane permeability

transition pore; MTT, 2-(4,5-dimethyl-2-thiazolyl)-3,5-diphen-

yl-2H-tetrazol-3-ium bromide; Pol, polydatin; PI, propidium

iodide; ROS, reactive oxygen species; SOD, superoxide

dismutase; TTC, 2,3,5-triphenyltetrazolium chloride

Rong-Hong Zhang − State Key Laboratory of Functions and

Applications of Medicinal Plants, Engineering Research Center

for the Development and Application of Ethnic Medicine and

TCM (Ministry of Education) and Center for Tissue

Engineering and Stem Cell Research, Key Laboratory of

Regenerative Medicine of Guizhou Province, Guizhou Medical

University, Guiyang 550004, P. R. China

Xiang-Ming Liao − School of Pharmacy, Guizhou Medical

University, Guian New District, Guizhou 550025, P. R. China

Dan Yang − School of Pharmacy, Guizhou Medical University,

Guian New District, Guizhou 550025, P. R. China

Yu-Chan Wang − School of Pharmacy, Guizhou Medical

University, Guian New District, Guizhou 550025, P. R. China

Yong-Long Zhao − School of Pharmacy, Guizhou Medical

University, Guian New District, Guizhou 550025, P. R. China

Guo-Bo Xu − School of Pharmacy, Guizhou Medical University,

Guian New District, Guizhou 550025, P. R. China

Chun-Hua Liu − State Key Laboratory of Functions and

Applications of Medicinal Plants, Engineering Research Center

for the Development and Application of Ethnic Medicine and

TCM (Ministry of Education), Guizhou Medical University,

Guiyang 550004, P. R. China; School of Pharmacy, Guizhou

Medical University, Guian New District, Guizhou 550025, P.

R. China

Yong-Jun Li − State Key Laboratory of Functions and

Applications of Medicinal Plants, Engineering Research Center

for the Development and Application of Ethnic Medicine and

TCM (Ministry of Education), Guizhou Medical University,

Guiyang 550004, P. R. China; School of Pharmacy, Guizhou

Medical University, Guian New District, Guizhou 550025, P.

R. China

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∥

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ACKNOWLEDGMENTS

■

This work was supported by the National Natural Science

Foundation of China (81660570, U1812403-05), the Guizhou

Medical University 2018 Academic New Seedling Training and

Innovation Exploration Project ([2017]5718, [2018]5779-13,

[

2018]5779-75), the Key Research Project of Science and

Technology Department of Guizhou Province (Qiankehejichu

2020]1Z008, [2020]1Z068), the Special Foundation of the

Central Government to Guide Local Science and Technology

[2019]4008), and the Guizhou Science and Technology

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hyde 3-phosphate dehydrogenase; GSH, glutathione; GSH-Px,

glutathione peroxidase; H2DCFDA, 2′,7′-dichlorodihydroﬂuor-

escein diacetate; HBTU, O-(1H-benzotriazol-1-yl)-N,N,N′,N′-

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