Beta-carboline alkaloid compounds and uses thereof

Abstract

The application provides a beta-carboline alkaloid compound and application thereof. The beta-carboline alkaloid compound has a structure shown in formula I. The compound 1 and the compound 2 provided by the application have a significant inhibitory effect on myocardial fibroblast proliferation and migration, and can be applied to the preparation of drugs for treating cardiovascular diseases such as myocardial fibrosis, myocardial infarction, myocardial hypertrophy and heart failure.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a β-carboline alkaloid compound and its applications. Background Technology

[0002] Myocardial fibrosis (MF) is a major pathological process in cardiovascular diseases (myocardial infarction, myocardial hypertrophy, and heart failure, etc.), characterized by the over-activation, proliferation, and migration of cardiac fibroblasts (CFs) (Circ.Res.2023,133,237-251). As a disease that impairs cardiac function, there are currently no effective drugs approved by the Federal Drug Administration (FDA) for myocardial fibrosis (Signal Transduct.Target.Ther.2022,7,206). The drugs pirfenidone and nintedanib, used to treat idiopathic pulmonary fibrosis, have significant drawbacks when used to treat myocardial fibrosis. The mechanism of action of pirfenidone is unclear; in a phase 2 clinical trial in heart failure patients, it moderately reduced myocardial fibrosis but failed to improve cardiac function (Nat.Med.2021,27,1477-1482). Nintedanib has potential cardiotoxicity and is therefore unsuitable for treating patients with underlying chronic heart disease (Acta Oncol. 2009, 48, 964-970). Angiotensin-converting enzyme inhibitors (ACEIs) such as captopril can alleviate ventricular dilation and remodeling (Heart. Fail. Rev., 2022, 27, 1119-1136) and reduce myocardial hypertrophy and fibrosis (Am. J. Hypertens. 2009, 22, 228-234), but also have side effects such as hypotension, renal failure, proteinuria, and paroxysmal cough (Br. J. Pharmacol. 2010, 160, 1273-1292). Therefore, there is a lack of effective clinical treatments for myocardial fibrosis, and there is an urgent need to develop drugs to treat it.

[0003] β-carboline alkaloids are a large class of natural or chemically synthesized alkaloids with a pyrido[3,4-b]indole skeleton. These alkaloids not only possess complex and diverse structures but also exhibit a variety of significant biological activities, such as antitumor, antiviral, anti-inflammatory, antibacterial, anti-Alzheimer's disease, anticonvulsant, anti-anxiety, and reduction of myocardial ischemia-reperfusion injury (Eur. J. Med. Chem. 2018, 157, 622-656; Eur. J. Med. Chem. 2021, 216, 113321; J. Med. Chem. 2021, 64, 9166-9181), making them an important source for drug development. Currently, nine drugs containing the β-carboline alkaloid skeleton have been marketed (Eur.Urol. 2014,65,587–596; CNSDrugs 2014,28,29–43; Biochem.Biophys.Res.Commun. 2015,462,402–408; Drugs 2020,80,1345–1353).

[0004] Given the high drug-likeness of the β-carboline alkaloid structure, the development of anti-myocardial fibrosis drugs with the β-carboline alkaloid structure is of great clinical significance. Summary of the Invention

[0005] To address the challenge of the lack of effective clinical treatments for myocardial fibrosis, the present invention aims to provide a β-carboline alkaloid compound and its applications.

[0006] According to one aspect of the present invention, a β-carbamoline alkaloid compound is provided having the structure shown in Formula I:

[0007]

[0008] Compound 1 (1-acetyl-7-fluoro-N-phenethyl-9H-pyrido[3,4-b]indole-3-carboxamide), as shown in Formula I, has no reported structure or biological activity; compound 2 (1-acetyl-9-methyl-N-phenethyl-9H-pyrido[3,4-b]indole-3-carboxamide), has no reported biological activity.

[0009] Compound 1 and compound 2, as shown in Formula I, were synthesized by the following method:

[0010]

[0011] Using L-tryptophan or 6-FL-tryptophan as a starting material, the corresponding tryptophan methyl ester hydrochloride was quantitatively generated by reacting with thionyl chloride in methanol solvent. Subsequently, it was reacted with acetone aldehyde via a Pictet-Spengeler reaction to generate β-carboline methyl ester intermediate 3a / b.

[0012] 3a is hydrolyzed under alkaline conditions to generate the corresponding carboxylic acid compound 4, which is then condensed with phenylethylamine to generate compound 1.

[0013] 3b first reacts with the methylating agent iodomethane to generate N-methylated product 5, then undergoes hydrolysis with lithium hydroxide to generate the corresponding carboxylic acid compound 6, and finally condenses with phenylethylamine to generate compound 2.

[0014] According to a second aspect of the invention, pharmaceutical use of said compounds 1 and 2 is provided, specifically, the use of the above-described β-carboline alkaloid compounds (compounds 1 and 2 and their pharmaceutically acceptable salts) in the preparation of drugs for inhibiting myocardial fibrosis is provided.

[0015] Furthermore, the β-carboline alkaloid compound serves as a lead compound for the preparation of drugs that inhibit myocardial fibrosis.

[0016] According to a third aspect of the invention, the use of the above-described β-carboline alkaloid compounds (compound 1 and compound 2 and their pharmaceutically acceptable salts) in the preparation of a medicament for treating cardiovascular diseases is provided.

[0017] Furthermore, the β-carboline alkaloid compound serves as a lead compound for the preparation of drugs for treating cardiovascular diseases.

[0018] Furthermore, the cardiovascular disease includes one or more of myocardial infarction, myocardial hypertrophy, and heart failure.

[0019] Compared with the prior art, the present invention has at least one of the following beneficial effects:

[0020] Compounds 1 and 2 provided by this invention were tested for their inhibitory activity on the proliferation and migration of myocardial fibroblasts. The results showed that compounds 1 and 2 had significant inhibitory effects on the proliferation and migration of myocardial fibroblasts, which were stronger than those of the positive control drug captopril. Therefore, compounds 1 and 2 provided by this invention can be used as drugs or lead compounds for the development of treatments for cardiovascular diseases such as myocardial fibrosis, myocardial infarction, myocardial hypertrophy, and heart failure. Attached Figure Description

[0021] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings:

[0022] Figure 1 The results of Compound 1 and Compound 2 of Formula I in Example 3 of this invention inhibiting the proliferation of cardiomyocytes;

[0023] Figure 2 The results of Compound 1 and Compound 2 of Formula I in Example 3 of this invention inhibiting the migration of myocardial fibroblasts are shown. Detailed Implementation

[0024] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0025] The chemical reagents and solvents used in the embodiments of this invention are all known products and were obtained by purchasing commercially available products.

[0026] The following are abbreviations used in embodiments of the present invention:

[0027] DMF: N,N-dimethylformamide; DMAP: 4-dimethylaminopyridine; HATU: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate; TLC: thin-layer chromatography.

[0028] Example 1

[0029] This embodiment provides a method for preparing compound 1, as follows:

[0030] 0.25 g (1.0 eq.) of 6-FL-tryptophan was added to 10 mL of methanol solution containing 191 μL (2.3 eq.) of thionyl chloride. The reaction solution was stirred and refluxed at 70 °C for 16 h. The reaction was confirmed to be complete by TLC. The solvent was recovered under reduced pressure to obtain 0.30 g of tryptophan methyl ester hydrochloride (yield 98%), which was directly used in the next step of the reaction.

[0031] In 10 mL of methanol solution containing 0.30 g (1.0 eq.) of tryptophan methyl ester hydrochloride, iodine (0.28 g, 1.0 eq.), p-toluenesulfonic acid monohydrate (0.21 g, 1.0 eq.), and 40% acetone aldehyde (1.00 g, 5.0 eq.) were added sequentially. The reaction mixture was stirred and refluxed at 70 °C for 3 h, and the reaction was confirmed to be complete by TLC. The solvent was recovered under reduced pressure, and the residue was dispersed in 50 mL of NaHCO3 (0.18 g, 2.0 eq.) aqueous solution and stirred at room temperature for 0.5 h. Subsequently, Na2SO3 (0.14 g, 1.0 eq.) was added, and the mixture was stirred at 0 °C for 1 h. The mixture was filtered to obtain 0.14 g of yellow solid 3a (yield 44%), which was used directly in the next reaction.

[0032] 0.14 g (1.0 eq.) of yellow solid 3a was dissolved in 10 mL of tetrahydrofuran, and then 0.023 g (1.0 eq., 5 mL of water) of LiOH aqueous solution was added. The resulting reaction solution was stirred at 40 °C for 2 h, and the pH was adjusted to 2-3 with 1 M HCl. The mixture was filtered to give compound 4 (0.087 g, 65% yield). This compound was used directly in the next reaction.

[0033] The 1H NMR, 1C NMR, and mass spectrometry data of compound 4 are as follows: 1 H NMR (400MHz, DMSO-d6): δ12.98(s,1H),12.31(s,1H),9.15(s,1H),8.50(dd,J=8.8 ,5.6Hz,1H),7.55(dd,J=9.6,2.4Hz,1H),7.21(td,J=9.6,2.4Hz,1H),2.84(s,3H); 13 C NMR (100MHz, DMSO-d6): δ201.2,166.2,163.0(J CF =242.0Hz), 143.2(J) CF =14.0Hz), 136.9, 135.6 (J) CF =1.0Hz), 135.0, 131.1, 124.2 (J) CF =11.0Hz),120.9,117.1,109.5(J CF =25.0Hz), 99.6(J) CF =27.0Hz),25.8; HRESIMS:calcd for C 14 H8FN2O3[MH] - :271.0519,found:271.0518.

[0034] Compound 4 (0.085 g, 1.0 eq.), DMAP (0.0057 g, 0.15 eq.), and HATU (0.182 g, 1.5 eq.) were dissolved in 20 mL of DMF. Triethylamine (65 μL, 1.5 eq.) and phenylethylamine (60 μL, 1.5 eq.) were added. The reaction mixture was stirred at room temperature for 2 h, followed by extraction with ethyl acetate (20 mL × 3). The organic phase was washed successively with potassium carbonate aqueous solution (0.108 g, 2.5 eq., 30 mL water) and 10% citric acid aqueous solution. The organic phases were combined, the solvent was recovered under reduced pressure, and the mixture was purified by silica gel column chromatography, eluting with petroleum ether / ethyl acetate in a 3:1 ratio, to give a pale yellow solid, which was compound 1 (0.070 g, yield 60%).

[0035] The proton, carbon, and mass spectrometry data of compound 1 are as follows: 1H NMR (400MHz, CDCl3): δ10.42(s,1H),9.03(s,1H),8.15(dd,J=8.8,5.2Hz,1H),8.06(t,J=6.0Hz,1H),7.34( m,3H),7.28(m,2H),7.12(td,J=8.8,2.4Hz,1H),3.86(q,J=6.8Hz,2H),3.01(t,J=6.8Hz,2H),2.75(s,3H); 13 C NMR (100MHz, CDCl3): δ202.4,164.5,164.2(J CF =246.0Hz), 142.5 (J) CF =13.0Hz),139.9,139.1,136.9(J CF =1.0Hz),133.6,132.3,129.1,129.1,128.9,128.9,126.8,123.8(J CF =11.0Hz), 118.1, 117.6 (J) CF =1.0Hz), 110.5(J) CF =25.0Hz), 99.2(J CF =26.0Hz),40.6,36.0,25.8; HRESIMS:calcd for C 22 H 19 FN3O2[M+H] + :376.1461,found:376.1461; calcd for C 22 H 18 FN3O2Na[M+Na] + :398.1281,found:398.1279.

[0036] Example 2

[0037] This embodiment provides a method for preparing compound 2, as follows:

[0038] Compound 3b was prepared according to the method in Example 1a above, starting with 0.38 g of L-tryptophan, yielding 0.25 g of compound 3b, with a yield of 50%.

[0039] 0.25 g of compound 3b (1.0 eq.) was dissolved in 10 mL of anhydrous DMF and stirred. Sodium hydride (60%, 0.041 g, 1.1 eq.) and methyl iodide (174 μL, 3.0 eq.) were added separately at 0 °C. After the addition was complete, the ice bath was removed, and the mixture was stirred at room temperature for 0.5 h. 20 mL of ice water was added, and the mixture was extracted with ethyl acetate (10 mL × 3). The organic phases were combined, the solvent was recovered under reduced pressure, and the mixture was purified by silica gel column chromatography, eluting with petroleum ether / ethyl acetate 5:1, to give a pale yellow solid compound 5 (0.209 g, yield 79%).

[0040] Compound 5 was hydrolyzed with lithium hydroxide according to the steps in Example 1 above to obtain compound 6, which was then condensed with phenylethylamine to obtain compound 2.

[0041] The proton, carbon, and mass spectrometry data of compound 6 (0.175 g, 88% yield) are as follows: 1 H NMR (400MHz, DMSO-d6): δ12.96(s,1H),9.12(s,1H),8.50(d,J=8.4Hz,1H),7.82(d,J =8.4Hz,1H),7.73(t,J=8.4Hz,1H),7.41(t,J=8.4Hz,1H),3.91(s,3H),2.90(s,3H); 13 C NMR (100MHz, DMSO-d6): δ200.8,166.1,143.2,139.5,135.8,135.0,131.5,129.6,122.1,121.1,120.3,119.6,111.0,33.6,28.2; HRESIMS:calcd for C 15 H 11 N2O3[MH] - :267.0770,found:267.0766.

[0042] The proton, carbon, and mass spectrometry data of compound 2 (0.155 g, yield 64%) are as follows: 1 H NMR (400MHz, CDCl3): δ9.04(s,1H),8.22(d,J=8.0Hz,1H),8.01(t,J=6.0Hz,1H),7.67(t,J=8.4Hz,1H),7.53(d, J=8.4Hz,1H),7.35(m,4H),7.28(m,1H),3.96(s,3H),3.85(q,J=6.8Hz,2H),3.01(t,J=6.8Hz,2H),2.77(s,3H); 13C NMR (100MHz, CDCl3): δ200.6,164.6,144.0,139.2,138.2,137.6,136.2,133.3,129.7,129.1,129. 1,128.9,128.9,126.8,121.9,121.4,121.3,117.2,110.5,40.6,36.1,34.3,28.3; HRESIMS:calcd for C 23 H 22 N3O2[M+H] + :372.1712,found:372.1710; calcd for C 23 H 21 N3O2Na[M+Na] + :394.1531,found:394.1528.

[0043] Example 3

[0044] This embodiment provides an anti-myocardial fibrosis activity test for the above-mentioned compounds 1 and 2.

[0045] (1) Isolation of primary myocardial fibroblasts

[0046] Primary cardiomyocytes (CFs) were isolated according to the methods described in the literature (Circ.Res.2023,133,237–251; Transduct.Target.Ther.2024,9,45).

[0047] Specifically, Sprague-Dawley neonatal rats (1-2 days after birth) were selected, and their ventricles were removed after euthanasia. The ventricles were digested in Hank's balanced salt solution (HBSS) containing 0.1% trypsin (Invitrogen, USA) and 0.05% type II collagenase (Worthington, USA). After centrifugation, the supernatant was discarded, and the cells were gently dispersed and cultured in culture dishes for 2 hours. Then, the supernatant (containing cardiomyocytes) in the culture dishes was removed, and the medium was replaced with fresh DMEM (Dulbecco's Modified Eagle Medium) containing 10% fetal bovine serum, and cultured for another 48 hours. Subsequently, the CFs were digested and passaged.

[0048] (2) Cell pretreatment

[0049] Compound 1, Compound 2, and the positive control captopril were prepared in advance to a concentration of 10 mM. CF cells were removed from the incubator, digested, collected, and reseeded. After cell attachment, the diluted compounds (diluted to 10 μM with serum-containing culture medium) were added to the corresponding wells.

[0050] (3) Cell viability detection

[0051] Cell viability was measured using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT). Cells were loaded at 3 × 10⁻⁶ cells / year. 3 The cells were seeded at a concentration of [number] per well into 96-well plates and incubated at 37°C for 24 h. Then, 20 μM isoproterenol (ISO) and a dilution of the compound were added, and the plates were incubated for another 48 h. Next, MTT solution (5 mg / mL, dissolved in PBS) was added, and the plates were incubated at 37°C for 4 h. Then, 150 μL of dimethyl sulfoxide (DMSO) was added. After the MTT dissolved, the OD value at 490 nm was measured using a microplate reader. Data were analyzed using GraphPad Prism 8.0 software.

[0052] (4) Cell scratch test

[0053] Cells were seeded into six-well plates. When the cell confluence reached approximately 90%, a straight line was drawn on the bottom of the plate using the tip of a 200 μL pipette, aligned with a ruler. After rinsing the cells with PBS, DMEM culture medium was added for further culture. Cell migration was photographed at 0, 24, and 48 hours after the scratch.

[0054] (5) Statistical Analysis

[0055] All data were analyzed using GraphPad Prism 8.0 software and are expressed as mean ± standard deviation. One-way ANOVA was used, and Dunnett's test was used for multiple comparisons.

[0056] Figure 1 This invention presents the results of the inhibition of cardiac fibroblast proliferation by compounds 1 and 2 of Formula I in Example 3. The CON group consisted of untreated cardiac fibroblasts; the ISO group consisted of cardiac fibroblasts treated with isoproterenol (ISO), and the same applies below. Compounds 1 and 2 (10 μM) and captopril (Cap, 10 μM) were incubated with isoproterenol (ISO, 20 μM) for 48 h. Cell viability was detected by the MTT assay. # P<0.05 (compared to the CON group), ***P<0.001 (compared to the ISO group), indicating that compounds 1 and 2 in the embodiments of the present invention significantly inhibited the proliferation of myocardial fibroblasts.

[0057] Figure 2The results of Compound 1 and Compound 2 of Formula I in Example 3 of this invention inhibiting the migration of cardiomyocytes are shown. Compounds 1 and 2 (10 μM), captopril (Cap, 10 μM), and isoproterenol (ISO, 20 μM) were incubated with cells for 48 h. Cell migration was analyzed by photographing at 0, 24, and 48 h after scratching (×200, bar = 500 μm). ## P<0.01 (compared to the CON group), *P<0.05, **P<0.01 (compared to the ISO group). Cell scratch assays showed that compounds 1 and 2 significantly inhibited the migration of cardiomyocytes.

[0058] Based on the above experimental results, compounds 1 and 2 have significant inhibitory effects on the proliferation and migration of myocardial fibroblasts, and can be used to prepare drugs or lead compounds for anti-myocardial fibrosis and cardiovascular diseases such as myocardial infarction, myocardial hypertrophy, and heart failure.

[0059] Specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention. The above preferred features can be used in any combination without conflict.

Claims

1. A β-carboline alkaloid compound, characterized in that, It has the structure shown in Equation I:

2. The use of the β-carboline alkaloid compound of claim 1 in the preparation of a drug for inhibiting myocardial fibrosis.

3. The application according to claim 2, characterized in that, The β-carboline alkaloid compound is used as a lead compound for the preparation of drugs that inhibit myocardial fibrosis.

4. The use of the β-carboline alkaloid compound of claim 1 in the preparation of a drug for treating cardiovascular diseases.

5. The application according to claim 4, characterized in that, The β-carboline alkaloid compound is used as a lead compound for the preparation of drugs for treating cardiovascular diseases.

6. The application according to claim 4, characterized in that, The cardiovascular diseases mentioned include one or more of myocardial infarction, myocardial hypertrophy, and heart failure.

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