Use of azacycloheptene bisindole derivatives for the preparation of medicaments for the treatment of leukemia

Abstract

The application discloses an application of an azacycloheptene bisindole derivative in preparation of a leukemia treatment drug and belongs to the technical field of drugs. The azacycloheptene bisindole derivative has a structural general formula (I). In the formula (I), R1 is H, -NO2, -OCH3, I or -CH3, R2 is H or -CH3, and R3 is H or -OCH3. The azacycloheptene bisindole derivative has anti-leukemia activity on human erythroleukemia cell strain HEL and human chronic myelogenous leukemia cell strain K562.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of a azircyclic heptaene bisindole derivative in the preparation of drugs for the treatment of leukemia. Background Technology

[0002] Leukemia is a heterogeneous group of hematologic malignancies that threaten human life, characterized by the accumulation of immature hematopoietic progenitor cells in the bone marrow and peripheral blood. Leukemia patients have low survival rates and an increasing incidence rate; relapse remains a common occurrence after treatment, with a poor prognosis. Primary and acquired drug resistance, as well as complex genetic alterations, are the main reasons affecting the efficacy of leukemia treatment. The development of leukemia treatment has been slow; treatment regimens have not changed substantially in the past few decades. Hematopoietic stem cell transplantation combined with chemotherapy is currently the only effective method to cure leukemia; however, some patients often relapse or lack a treatment response after treatment. Currently, traditional demethylating agents (cytarabine, decitabine, etc.) still cannot meet clinical needs as anti-leukemia drugs. Therefore, there is an urgent need to find safer and more effective chemotherapeutic drugs.

[0003] Malignant proliferation of blood cells is directly related to mutations and abnormalities in internal signaling, such as the overexpression of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase / phosphokinase B (PI3K / AKT) signaling pathways in leukemia cells, which affects tumor proliferation and metastasis. Drugs that interfere with these abnormal signaling pathways can exert effective anti-leukemic activity. Among them, the PI3K-AKT pathway plays a key role in regulating cellular functions such as cell growth, proliferation, apoptosis, differentiation, and metabolism. The PI3K-AKT signaling pathway is activated in 50% of leukemia patients, and its activation can serve as a marker of poor prognosis in leukemia. PI3K positively regulates phosphorylated AKT (p-AKT), and activated p-AKT is located in the cytosol and nucleus, regulating various downstream proteins. Forkhead box O (FoxO), as a downstream transcriptional regulator of AKT, can directly participate in the regulation of cell differentiation, growth, survival, metabolism, stress and other pathways. Among them, FoxO3a can regulate related downstream genes that affect the proliferation and survival of AML cells through nucleocytoplasmic shuttle, such as cyclin-dependent factor 1A (CDKN1A), cyclin-dependent factor 2B (CDKN2B), cyclin-dependent kinases (CDKs).

[0004] Iheyamines A is a non-monoterpenoid bisindole alkaloid obtained by isolation and purification from Polycitorella sp. It possesses a unique cycloheptanyl-2,2-bisindole skeleton [Iheyamines, new cytotoxic bisindole pigments from a colonial ascidian, Polycitorella sp. Tetrahedron Letters, 1999; Direct, four-step synthetic pathway to iheyamine A and several analogues. Organic & Biomolecular Chemistry, 2022.]. Structurally, this type of alkaloid has a cycloheptanyl[3,2-b:4,5-b']bisindole skeleton, which consists of two six-membered rings, two five-membered rings, and one seven-membered ring. These azahepten bisindole derivatives have biological activity against some tumor cells, such as U20S (human osteosarcoma cells), A549 (human lung cancer cells), and HT29 (human colon cancer cells), but their efficacy and mechanism of action in leukemia have not been reported. Summary of the Invention

[0005] The purpose of this invention is to overcome the above-mentioned shortcomings and provide the application of a nitrogen-containing heptacyclic bisindole derivative in the preparation of leukemia treatment drugs.

[0006] This invention uses a azircyclic heptaene bisindole derivative as the active ingredient and prepares it into a leukemia treatment drug in any dosage form according to conventional preparation methods.

[0007] This invention uses methyl[5,6,7,12-tetrahydro-4bH-azepino[3,2-b:4,5-b']diindole-4b-carboxylate, an intermediate product in the synthesis of azahepten bisindole alkaloids, as the parent core. Through modification, azahepten bisindole derivative is obtained, with the general structural formula (I):

[0008] (I)

[0009] In formula (I), R1 is H, -NO2, -OCH3, I, -CH3, R2 is H, -CH3, and R3 is H, -OCH3.

[0010]

[0011] The specific compounds of the above-mentioned general formula azahepten bisindole derivatives are:

[0012]

[0013] Compared with existing technologies, this invention has significant beneficial effects. As can be seen from the above technical solutions, experiments have confirmed that the aziridine heptadecene bisindole derivative of this invention has anti-leukemic activity against human erythroleukemia cell line HEL and human chronic myeloid leukemia cell line K562 (see Table 4). In human erythroleukemia cell line HEL, human promyelocytic leukemia cell line HL-60, and human monocytic leukemia cell line THP-1, the two compounds L-38 and L-42 with the best activity were tested and found to have significant inhibitory effects on the proliferation of all the above leukemia cell lines (see Table 6). Among them, compound L-42 inhibited the growth and proliferation of leukemia cells in a concentration- and time-dependent manner (see Table 6). Figure 1 Flow cytometry analysis showed that L-42 induces apoptosis in leukemia cells (see...). Figure 2-4 After 24 h of L-42 treatment, the expression of apoptosis-related proteins cleaved-caspase 3 and cleaved-PARP in THP-1 cells was upregulated. Simultaneously, the expression of pro-apoptotic proteins Bcl-2-associated X protein (Bax) and Bcl-2-associated death promoter (Bad) was upregulated, while the expression of the anti-apoptotic protein Bcl-2 was downregulated (see...). Figure 5 L-42 can induce G1 phase arrest in HEL and THP-1 cells and S phase arrest in HL-60 cells (see [link to relevant documentation]). Figure 6-8 Transcriptome sequencing data analysis showed that L-42 led to a significant enrichment of differentially expressed genes in the PI3K-AKT signaling pathway in THP-1 cells (see Transcriptome Sequencing Data). Figure 9-12 Immunoblotting experiments showed that L-42 can downregulate the expression of phosphorylated proteins p-PI3K (p85), p-AKT, and p-FOXO3, inhibit the PI3K / AKT protein kinase signaling pathway, and regulate the expression of genes related to cell growth and cycle, such as CDKN1A, CDKN2B, CDK1, CDK4, and CDK6 (see [link to study]). Figure 13 After establishing a leukemia model in BALB / c mice by infecting them with Fried mouse leukemia virus (F-MuLV), it was found that L42 could effectively delay the progression of leukemia in mice (see...). Figure 14A-G). The above results indicate that the azacyclic heptaene bisindole derivative can induce apoptosis and cell cycle arrest in leukemia cells. Its mechanism of action is related to the inhibition of the PI3K / AKT protein kinase signaling pathway. At the same time, it delayed the progression of leukemia in mice in animal experiments, indicating that this type of compound can be used for the treatment of leukemia. Attached Figure Description

[0014] Figure 1 The concentration- and time-dependent relationship between L-42 and the inhibition of leukemia cell line growth and proliferation is shown in the figure.

[0015] Figure 2 Image of L-42-induced apoptosis in HEL leukemia cells detected by flow cytometry;

[0016] Figure 3 Image for flow cytometry detection of L-42-induced apoptosis in THP-1 leukemia cells;

[0017] Figure 4 Image for flow cytometry detection of L-42-induced apoptosis in HL-60 leukemia cells;

[0018] Figure 5 Figure showing the changes in the expression levels of proteins related to L-42-induced apoptosis in THP-1 cells as detected by Western blotting assay.

[0019] Figure 6 To detect G1 phase arrest in L-42-induced HEL leukemia cells using flow cytometry;

[0020] Figure 7 To detect G1 phase arrest in L-42-induced leukemia THP-1 cells using flow cytometry;

[0021] Figure 8 To detect S-cycle arrest in L-42-induced leukemia cells HL-60 by flow cytometry;

[0022] Figure 9 Rich maps of differentially expressed genes related to cell signal transduction were obtained using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database.

[0023] Figure 10 Heatmap showing differential gene expression changes in the PI3K / AKT pathway;

[0024] Figure 11 This is a protein-protein interaction diagram of differentially expressed genes in the PI3K / AKT pathway.

[0025] Figure 12A graph showing the changes in the expression levels of related proteins in the PI3K / AKT pathway induced by L-42 in an immunoblotting assay.

[0026] Figure 13 This diagram illustrates the changes in expression levels of downstream genes in the PI3K / ATK pathway as detected by real-time quantitative PCR.

[0027] Figure 14A A graph showing the establishment of a mouse model of leukemia and the timing of drug administration;

[0028] Figure 14B A graph showing the change in spleen size in mice carrying leukemia after drug administration;

[0029] Figure 14C A statistical graph showing the spleen size of mice with leukemia after drug administration;

[0030] Figure 14D Statistical graph showing changes in hematocrit in mice carrying leukemia after drug administration;

[0031] Figure 14E A statistical graph showing the change in body weight in mice carrying leukemia after drug administration;

[0032] Figure 14F HE staining of spleen in mice carrying leukemia after drug administration;

[0033] Figure 14G A statistical chart showing the survival time of mice carrying leukemia after administration of the drug. Detailed Implementation

[0034] Example:

[0035] The method for preparing the azahepten bisindole derivative of the present invention includes the following steps:

[0036] (1) Synthesis of chlorinated derivative 5a

[0037] Using methyl indole-3-carboxylate (1.00 g, 5.70 mmol) as a starting material, and t-BuOH : H2O = 100:1 as a solvent, a THF solution of 2.5 equivalents of N-chlorosuccinimide NCS (2.50 g, 14.3 mmol) was added dropwise to a round-bottom flask at 0 °C. The reaction was carried out at room temperature for about 30 minutes to obtain chlorinated product 5a (1.30 g).

[0038] (2) Synthesis of 3-methyl((2-(1H-indol-3-yl)ethyl)amine)-2-oxoindol-3-carboxylic acid ester 6a:

[0039]

[0040] Compound 5a (1.00 g, 4.40 mmol) and tryptophan (700 mg, 4.40 mmol) were dissolved in 20 mL MeCN, and DIPEA (2.30 mL, 13.3 mmol) was slowly added at 0°C, with stirring at room temperature for 30 minutes. The mixture was concentrated under reduced pressure. The crude product was purified by rapid column chromatography (DCM / MeOH = 300:1-150:1) to give product 6a (735 mg).

[0041] (3) Carboxylic acid ester cyclization products L37-L4

[0042]

[0043] General synthetic steps for the cyclic-closed product: Compound 6a (280 mg, 0.8 mmol) and phosphorus pentoxide (561 mg, 4.0 mmol) were dissolved in 3 mL of sulfolane. The mixture was heated to 80°C under argon protection for 8 hours. After the reaction was complete, the reaction mixture was cooled to room temperature and alkalized to pH 7-8 with saturated sodium bicarbonate aqueous solution. The mixture was filtered to give a yellow solid, and the aqueous layer was extracted with ethyl acetate (200 mL × 5). The organic layer was washed with water (200 mL × 5), the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give an oil. The oil was mixed with the above yellow solid and purified by rapid column chromatography (PE / EA = 6:1) to give the product.

[0044] Spectral data of compounds L37-L44:

[0045]

[0046] L-37: Methyl3-nitro-5,6,7,12-tetrahydro-4bH-azepino[3,2-b:4,5-b']diindole-4b-carboxylate (7g) 33 mg, yield 11%, golden yellow solid, IR (KBr)3420, 3006, 2950, ​​1735, 1616, 1560, 1520, 1455, 1330, 1225, 1149, 748 cm −1 . 1HNMR (600 MHz, DMSO-d6) δ 11.71 (s, 1H), 7.80 (d, J=1.6 Hz, 1H), 7.77 (dd, J=8.1, 1.7 Hz, 1H), 7.60 (d, J=8.0 Hz, 1H), 7.39 (d, J=8.2 Hz, 1H), 7.33 (d, J=8.0 Hz, 1H), 7.19-7.26 (m, 1H), 7.04 (t, J=7.1 Hz, 1H), 4.10 (t, J=4.9 Hz,1H), 3.54 (s, 3H), 3.31 (br. s., 1H), 3.05-3.17 (m, 2H), 2.86-2.97 (m, 1H); 13 C NMR (150 MHz, DMSO-d6) δ 174.2, 170.7, 154.6, 140.2, 138.8, 137.7, 131.7,129.4, 127.2, 124.7, 122.0, 120.9, 119.7, 119.3, 112.0, 90.1, 78.3, 53.1,44.0, 25.6. HRMS (ESI) m / z calcd for C 20 H 16 O4N4Na [M + Na] + : 399.1064, found:399.1058.

[0047]

[0048] L-38: Methyl5,6,7,12-tetrahydro-4bH-azepino[3,2-b:4,5-b']diindole-4b-carboxylate (7a) 106 mg, yield 40%, pale yellow solid, IR (KBr) 3347, 3059,2951, 1733, 1615, 1565, 1455, 1326, 1225, 1006, 749 cm −1 . 1H NMR (600 MHz,DMSO-d6) δ 11.69 (s, 1H), 7.59 (d, J=8.0 Hz, 1H), 7.46-7.53 (m, 2H), 7.36-7.45 (m, 2H), 7.22 (t, J=8.0 Hz, 2H), 7.04 (t, J=7.5 Hz, 1H), 3.96 (br. s.,1H), 3.51 (s, 3H), 3.35-3.42 (m, 1H), 3.06-3.16 (m, 2H), 2.89-2.99 (m, 1H); 13 C NMR (150 MHz, DMSO-d6) δ 174.1, 171.2, 154.9, 137.9, 137.6, 130.2,129.8, 127.4, 125.7, 124.5, 123.2, 120.4, 120.0, 119.7, 119.3, 112.0, 78.3,53.0, 44.3, 25.7. HRMS (ESI) m / z calcd for C 20 H 17 O2N3Na [M + Na] + : 354.1213,found: 354.1204.

[0049]

[0050] L-39: Methyl2',3'-dihydrospiro[indole-3,4'-pyrido[3,2-b]indole]-9b'(1'H)-carboxylate (8a) 50 mg, yield 19%, yellow solid, IR (KBr) 3392, 3060,2950, 1733, 1652, 1558, 1457, 1318, 1234, 1093, 747 cm −1 . 1H NMR (600 MHz,DMSO-d6) δ 10.71 (s, 1H), 7.48 (d, J=7.5 Hz, 1H), 7.43 (t, J=3.8 Hz, 2H),7.39 (d, J=8.0 Hz, 1H), 7.30 (t, J=7.6 Hz, 1H), 7.11 (t, J=7.5 Hz, 1H), 6.99(t, J=7.5 Hz, 1H), 6.84 (d, J=8.0 Hz, 1H), 6.77 (t, J=7.5 Hz, 1H), 4.09 (dd,J=11.4, 3.5 Hz, 1H), 3.68 - 3.73 (m, 1H), 3.67 (s, 3H), 3.07 (dd, J =16.9,2.9 Hz, 1H), 2.85 (ddd, J=16.8, 13.8, 3.2 Hz, 1H); 13 C NMR (150 MHz, DMSO-d6)δ 169.3, 168.5, 155.5, 135.1), 133.3, 127.7, 126.8, 123.1, 122.7, 121.8,118.8, 118.6, 117.9, 111.3, 111.1, 68.1, 64.9, 53.2, 48.7, 22.8. HRMS (ESI)m / z calcd for C 20 H 18 O2N3 [M + H] + : 332.1394, found: 332.1386.

[0051]

[0052] L-40: Methyl3-methoxy-5,6,7,12-tetrahydro-4bH-azepino[3,2-b:4,5-b']diindole-4b-carboxylate (7) 153 mg, yield 53%, yellow solid, IR (KBr) 3446,2949, 2856, 1732, 1615, 1568, 1474, 1337, 1283, 1025, 741 cm −1 . 1H NMR (600MHz, DMSO-d6) δ 11.61 (s, 1H), 7.56 (d, J=8.0 Hz, 1H), 7.42 (d, J=8.4 Hz,1H), 7.37 (d, J=8.2 Hz, 1H), 7.20 (t, J=7.5 Hz, 1H), 7.10 (d, J=2.5 Hz, 1H),7.03 (t, J=7.5 Hz, 1H), 6.95 (dd, J=8.4, 2.5 Hz, 1H), 3.99 (br. s., 1H), 3.79(s, 3H), 3.52 (s, 3H), 3.30 (d, J=9.3 Hz, 1H), 3.15 (br. s., 1H), 3.08 (ddd,J=16.5, 8.7, 3.7 Hz, 1H), 2.94 (ddd, J=16.5, 7.1, 3.5 Hz, 1H); 13 C NMR (150MHz, DMSO-d6) δ 172.0, 171.2, 157.7, 148.2, 139.5, 137.3, 129.9, 127.3,124.0, 120.4, 119.4, 119.2, 119.0, 114.5, 111.8, 109.8, 78.3, 55.6, 52.9,44.5, 25.7. HRMS (ESI) m / z calcd for C 21 H 19 O3N3Na [M + Na] + : 384.1318, found:384.1310.

[0053]

[0054] L-41: Methyl9-methoxy-5,6,7,12-tetrahydro-4bH-azepino[3,2-b:4,5-b']diindole-4b-carboxylate (7h) 147 mg, yield 51%, pale yellow solid, IR (KBr)3337, 2950, 2832, 1720, 1622, 1563, 1454, 1218, 1043, 753 cm −1 . 1H NMR (600MHz, DMSO-d6) δ 11.55 (s, 1H), 7.49 (d, J=7.5 Hz, 1H), 7.35-7.46 (m, 1H),7.29 (d, J=8.7 Hz, 1H), 7.21 (t, J=7.5 Hz, 1H), 7.06 (d, J=2.0 Hz, 1H), 6.88(dd, J=8.9, 2.4 Hz, 1H), 3.93 (br. s., 1H), 3.78 (s, 3H), 3.51 (s, 3H), 3.35(br. s., 1H), 3.04-3.18 (m, 2H), 2.86-2.96 (m, 1H); 13 C NMR (150 MHz, DMSO-d6)δ 173.9, 171.2, 154.9, 153.5, 137.9, 132.9, 130.2, 130.0, 127.5, 125.4,123.0, 119.9, 119.8, 115.5, 112.8 , 100.3, 78.1, 55.4, 52.9, 44.2, 25.6. HRMS(ESI) m / z calcd for C 21 H 20 O3N3 [M + H] + : 362.1499, found: 362.1487.

[0055]

[0056] L-42: Methyl3-iodo-5,6,7,12-tetrahydro-4bH-azepino[3,2-b:4,5-b']diindole-4b-carboxylate (7f) 135 mg, yield 37%, yellow solid, IR (KBr) 3420,2948, 2867, 1723, 1606, 1560, 1507, 1448, 1335, 1233, 1110, 745 cm −1 . 1H NMR(600 MHz, DMSO-d6) δ 11.71 (s, 1H), 7.80 (d, J=1.6 Hz, 1H), 7.77 (dd, J=8.0,1.63 Hz, 1H), 7.60 (d, J=8.0 Hz, 1H), 7.39 (d, J=8.4 Hz, 1H), 7.33 (d, J=8.0Hz, 1H), 7.16-7.27 (m, 1H), 7.04 (t, J=7.5 Hz, 1H), 4.10 (br. s., 1H), 3.54(s, 3H), 3.25-3.33 (m, 1H), 3.05-3.17 (m, 2H), 2.86-2.98 (m, 1H); 13 C NMR (150MHz, DMSO-d6) δ 174.2, 170.7, 154.6, 140.2, 138.8, 137.7, 131.7, 129.4,127.2, 124.7, 122.0, 120.9, 119.7, 119.3, 112.0, 90.1, 78.3, 53.1, 44.0,25.6. HRMS (ESI) m / z calcd for C 20 H 17 O2N3I [M + H] + : 458.0360, found: 458.0358.

[0057]

[0058] L-43: Methyl3-methyl-5,6,7,12-tetrahydro-4bH-azepino[3,2-b:4,5-b']diindole-4b-carboxylate (7b) 143 mg, yield 52%, pale yellow solid, IR (KBr)3420, 3033, 2948, 2886, 1732, 1615, 1567, 1473, 1336, 1229, 1181, 743 cm −1 . 1HNMR (600 MHz, DMSO-d6) δ 11.65 (s, 1H), 7.58 (d, J=8.0 Hz, 1H), 7.38 (dd, J=8.0, 2.7 Hz, 2H), 7.31 (s, 1H), 7.16-7.25 (m, 2H), 6.96-7.07 (m, 1H), 3.89(br. s., 1H), 3.51 (s, 3H), 3.31-3.34 (m, 1H), 3.04-3.19 (m, 2 H), 2.92 (ddd,J=16.1, 7.6, 3.5 Hz, 1H), 2.34 (s, 3H); 13 C NMR (150 MHz, DMSO-d6) δ 173.1,171.2, 152.7, 138.0, 137.5, 135.0, 130.4, 129.9, 127.3, 124.2, 123.7, 119.8,119.6, 119.5, 119.1, 111.9, 78.1, 52.8, 44.3, 25.6, 21.0. HRMS (ESI) m / zcalcd for C 21 H 20 O2N3 [M + H] + : 362.1499, found: 362.1487.

[0059]

[0060] L-44: Methyl1,3-dimethyl-5,6,7,12-tetrahydro-4bH-azepino[3,2-b:4,5-b']diindole-4b-carboxylate (7c) 152 mg, yield 53%, yellow solid, IR (KBr)3419, 2951, 2856, 1731, 1609, 1561, 1457, 1337, 1230, 1016, 740 cm −1 . 1H NMR(600 MHz, DMSO-d6) δ 11.48 (s, 1H), 7.57 (d, J=8.0 Hz, 1H), 7.42 (d, J=8.2Hz, 1H), 7.20 (t, J=8.0 Hz, 1H), 7.11 (s, 1H), 7.00-7.06 (m, 2H), 3.82 (br.s., 1H), 3.50 (s, 3H), 3.30 (d, J=9.6 Hz, 1H), 3.03-3.19 (m, 1 H), 2.93 (ddd,J=16.4, 7.3, 3.4 Hz, 1H), 2.44-2.55 (m, 3H), 2.30 (s, 3H); 13 C NMR (150 MHz, DMSO-d6) δ 172.0, 171.3, 151.1, 137.9, 137.4, 134.9, 131.7, 130.0, 129.0,127.3, 124.1, 121.0, 119.5, 119.4, 119.1, 112.0, 78.3, 52.8, 44.5, 25.7,21.0, 16.5. HRMS (ESI) m / z calcd for C 22 H 21 O2N3Na [M + Na] + : 382.1526, found:382.1518.

[0061] (4) Synthesis of azircyclic heptaene bisindole alkaloids (Iheyamines A):

[0062] Further hydrolysis of the cyclic compound L-38, followed by decarboxylation-dehydrogenation, yielded core L-31 of iheyamine A in 70% yield. Surprisingly, L-31 could also be obtained from the helical cyclic compound L-39 under the same hydrolysis conditions, with a yield of 46%.

[0063] Table 1. Screening of decarboxylation-dehydrogenation conditions

[0064]

[0065] b Note: Reaction with 7a in 4 mL solvent.

[0066] Spectral data of L-31:

[0067]

[0068] L-31: 12H-Azepino [3,2-b:4,5-b'] diindole (1a) 26 mg, yield 70%, purple solid, IR (KBr) 3254, 2925, 1616, 1558, 1506, 1457, 1338, 1241, 1154,745 cm −1 . 1 H NMR (600 MHz, CDCl3) δ 9.11 (d, J=5.5 Hz, 1H), 8.57-8.64 (m,2H), 8.43 (d, J=7.6 Hz, 1H), 7.70-7.78 (m, 1H), 7.67 (s, 1H), 7.60 (d, J=7.5Hz, 1H), 7.46-7.56 (m, 3H); 13 C NMR (150 MHz, CDCl3) δ 151.1, 146.5, 146.2,145.4, 140.5, 136.9, 135.1, 130.6, 129.9, 127.3, 126.6, 122.7, 122.6, 121.9,121.9, 121.2, 117.3, 115.0. HRMS (ESI) m / z calcd for C 18 H 12 N3 [M + H] + :270.1026, found: 270.1020.

[0069] Experimental Example 1: Cellular Experiment of the Aza-Heptene Bisindole Derivative of the Present Invention

[0070] Reagents and materials:

[0071] Table 2. Main Reagents

[0072]

[0073] Table 3. Main Materials

[0074]

[0075] 1.1 Experimental Methods

[0076] 1.1.1 Cell Culture:

[0077] Cells were cultured in DMEM or 1640 medium containing 10% fetal bovine serum and placed in a cell culture incubator at 37°C with 5% CO2. Cell growth and quantity were observed under a microscope. The medium was changed and the cells were passaged every 2-3 days. Depending on the cell growth status and experimental needs, cells that showed logarithmic growth were used for subsequent operations.

[0078] 1.1.2 Cytotoxicity detection:

[0079] (1) Compound screening: Cells were screened at 10 × 10⁻⁶ m². 4 Cells were seeded at a concentration of 90 μL / well in 96-well plates and incubated overnight at 37°C. The compounds were diluted with DMSO to obtain 20 mM azacyclohepten bisindole derivatives (L31 and L37-L44) and corresponding concentrations of positive control stock solutions. These were prepared with serum-free DMEM to a 10× concentration and added to each well at 10 μL / well. Cells were incubated at 37°C for 72 h, with 4-5 replicates per group. After incubation, 5 mg / mL MTT reagent was added to each well at 10 μL / well, and incubated for 4 h. The suspended cells were centrifuged at 1500 rpm for 15 min, the supernatant was discarded, and 150 μL of DMSO was added. The absorbance was measured at OD570 using a multi-plate reader. The IC50 was calculated. 50 Values ​​are used to select effective compounds;

[0080] (2) Compound activity detection: Cells were incubated at 6-8 × 10⁻⁸ cm⁻¹. 4 90 μL of cells at a concentration of 10 mM / mL were seeded into each well of a 96-well plate and incubated overnight at 37°C. A 20 mM solution of azacyclohepten bisindole derivative and a corresponding concentration of positive control drug stock solution was prepared using serum-free DMEM to a concentration of 10×, and 10 μL / well was added to each well. The plates were incubated at 37°C for 72 h, with 4-5 replicates per group. After incubation, 10 μL / well of 5 mg / mL MTT reagent was added to each well and incubated for 4 h. Without centrifugation, 100 μL of a triplet solution (50g SDS, 25mL isobutanol, 50mL hydrochloric acid, and brought to a final volume of 500mL) was added overnight. The absorbance was measured at OD570 using a microplate reader. The IC50 was calculated. 50 IC50 values ​​were compared between different leukemia cell lines. 50 Value; the experiment was repeated three times, and the IC50 values ​​of the compound and different positive control drugs were compared. 50 value.

[0081] 1.1.3 Cell growth curve detection:

[0082] Collect cells in the logarithmic growth phase, pipette the cells to transfer the cell suspension to EP tubes, spin at 800 rpm for 3 min, discard the supernatant, add 1640 medium containing 5% FBS, resuspend appropriately with a pipette, count the cells, and adjust the cell density to 6 × 10⁶ cells / mL. 4 90 μL of culture medium and cell sap were seeded into 96-well plates, and 100 µL of sterile PBS was added to the edge wells around the perimeter. The plates were then placed in a cell culture incubator. A 20 mM stock solution was prepared, and 10× of the corresponding drug concentration was prepared using FBS-free 1640 medium, according to the number of replicates for the drug treatment group and the control group (5-8 replicates for both groups). 10 μL of MTT solution was added to one well of the plate. After 4 h, 100 μL of triplet solution was added to each well using a multipipeline, and the plate was returned to the cell culture incubator. After 12 h, the 96-well plate containing MTT was removed, and the absorbance was measured at OD570 using a microplate reader. This operation was repeated at 24 h, 48 h, and 72 h. The growth curve was plotted using GraphPad software with time on the x-axis and absorbance on the y-axis. The entire process was repeated three times.

[0083] 1.1.4 Flow cytometry detection:

[0084] (1) Apoptosis detection:

[0085] Collect cells in good growth condition, add 1640 medium with 5% FBS, resuspend appropriately with a pipette, count the cells, and adjust the cell density to 10 × 10⁶ cells / year. 4 Cells were seeded at a concentration of 1 / mL and seeded into 6-well plates, with a volume of 2 mL per well. After the cells stabilized, different concentrations of L-42 were added, and the plates were incubated in a cell culture incubator. Cells were collected at 24 h and 48 h, respectively. The cells were resuspended in 1 mL of 1×PBS solution, and the supernatant was discarded. This process was repeated once. Cells were resuspended in 40 μL of 1×Binding Buffer. 2 µL of PI and FITC were added to each sample tube, and the cells were stained at room temperature in the dark for 15 min. The volume was then increased to 500 μL by adding 1×Binding Buffer to each tube, and the mixture was pipetted and analyzed by flow cytometry.

[0086] (2) Cell cycle detection:

[0087] Collect cells in good growth condition, add 1640 medium with 5% FBS, resuspend appropriately with a pipette, count the cells, and adjust the cell density to 10 × 10⁶ cells / year. 4After the cells stabilized, different concentrations of L-42 were added and the cells were cultured in a cell culture incubator. Cells were collected at 24 h and 48 h, resuspended in 1 mL of 1×PBS, and the supernatant was discarded; this process was repeated once. Then, 500 μL of pre-chilled 70% ethanol was added to each sample EP tube, and the cells were resuspended and fixed overnight at 4°C. Cells were gently pipetted with 1 mL of 1×PBS, centrifuged, and then 500 μL of PI staining solution (5 µg / mL) was added to each sample tube. The tubes were incubated at room temperature in the dark for 15 min. After staining, the dye was removed, and the cells were washed twice with pre-chilled 1×PBS. Then, 500 µL of pre-chilled 1×PBS was added to each tube, and the cells were resuspended using a pipette for flow cytometry analysis.

[0088] 1.1.5 Statistical Analysis Experimental data were statistically analyzed using t-test and analysis of variance (ANOVE). Experimental data are expressed as mean ± standard deviation. P < 0.05 is indicated by *, P < 0.01 by **, P < 0.001 by ***, and P < 0.0001 by ****.

[0089] 1.2 Experimental Data

[0090] 1.2.1 Azacyclic heptacyclic bisindole derivatives possess antileukemic activity

[0091] A series of azacyclic heptacyclic bisindole derivatives were designed and synthesized. Their IC50 activity at 72 h was determined using the MTT assay for human erythroleukemia cells (HEL) and human chronic myeloid leukemia cells (K562). 50 The activity of the compounds was evaluated using an IC50 value (see 1.1.2(1)). Compounds with this structure exhibited antileukemic activity (see Table 4), with L-38 and L-42 showing significant inhibitory activity, and L-38 showing an IC50 value of [value missing] in HEL cells. 50 The value was 2.60 ± 0.05 µM, and the IC50 in K562 cells was [value missing]. 50 The value was 1.43 ± 0.01 µM; the IC50 value of L-42 in HEL cells was 1.43 ± 0.01 µM. 50 The value was 2.58 ± 0.05 µM, and the IC50 in K562 cells was... 50 The value was 2.74 ± 0.52 µM (see Table 4). Compared with the similarly structured azacyclic heptadecene bisindole alkaloids L-31 and L-39 (Table 5), these derivatives also exhibited stronger anti-leukemic activity. These results indicate that azacyclic heptadecene bisindole derivatives with the [5,6,7,12-tetrahydro-4bH-acoxane[3,2-b:4,5-b']bisindole-4b carboxylate as the parent nucleus possess anti-leukemic activity.

[0092] Table 4. Inhibitory activity of the aza-hepten bisindole derivative of the present invention in leukemia cells.

[0093]

[0094] Table 5. Inhibitory activity of compounds with similar structures in leukemia cells.

[0095]

[0096] 1.2.2 The azacyclic heptacyclic bisindole derivative L-42 inhibits the growth and proliferation of leukemia cells.

[0097] The anti-leukemia activity of the relatively active derivatives L-38 and L42 was tested. After treating leukemia cell lines (HEL, THP-1, and HL-60) with L-38 for 72 hours, its IC50 value was [not specified]. 50 The values ​​were 0.608±0.173 µM, 0.584±0.152 µM, and 0.380±0.075 µM, respectively; after treating leukemia cell lines (HEL, THP-1, and HL-60) with L-42 for 72 h, their IC50 values ​​were... 50 The values ​​were 0.466±0.099 µM, 0.475±0.084 µM, and 0.356±0.023 µM, respectively (see Table 6) (Note: This section is consistent with IC in 1.2.1). 50 The difference in values ​​was mainly due to the different number of cells plated and the different detection methods in this part of the experiment, see 1.1.2(2)). Compared with the positive drugs etoposide, doxorubicin and cytarabine, it showed effective anti-leukemia activity. However, after L-42 treatment of normal hepatocytes, the IC50 value was significantly lower than that of normal hepatocytes. 50 The value was 2.594 ± 0.27 µM, which was higher than the IC50 value in leukemia cell lines. 50 The values ​​indicate that it has good selectivity for leukemia cells (see Table 6). After treatment with L-42 for 24 h, 48 h, and 72 h, at concentrations of 0.5, 1, and 2 µM, L-42 inhibited the growth and proliferation of leukemia cell lines in a concentration- and time-dependent manner. Figure 1 ).

[0098] Table 6. Inhibitory activity of L-38 and L-42 in leukemia cells

[0099]

[0100] 1.2.3 L-42-induced apoptosis in leukemia cells

[0101] After 24 and 48 hours of treatment with L-42, flow cytometry analysis showed that L-42 induced apoptosis in leukemia cells HEL, THP-1, and HL-60. Figure 2-4 In HEL cells () Figure 2 When L-42 was treated at concentrations of 0.5, 1, and 2 µM, the apoptosis rates at 24 h were 8.28±0.24%, 17.23±1.24% (p<0.05), and 30.77±5.05% (p<0.05), respectively, and the apoptosis rates at 48 h were 10.59±2.52%, 13.74±1.85%, and 82.52±6.85% (p<0.05), respectively; in THP-1 cells ( Figure 3 At concentrations of 0.5, 1, and 2 µM, the apoptosis rates at 24 h were 8.38±1.64%, 26.75±4.17% (p<0.05), and 66.03±2.66% (p<0.05), respectively, and the apoptosis rates at 48 h were 26.50±0.22%, 34.06±5.5% (p<0.05), and 79.19±8.92% (p<0.05), respectively; in HL-60 cells ( Figure 4 At concentrations of 0.5, 1, and 2 µM, the apoptosis rates of L-42 at 48 h were 13.57 ± 0.55% (p < 0.05), 26.27 ± 3.09% (p < 0.05), and 27.44 ± 3.33% (p < 0.05), respectively, and 16.00 ± 4.12%, 57.48 ± 15.12% (p < 0.05), and 96.54 ± 1.14% (p < 0.05), respectively, in a concentration- and time-dependent manner. Western blotting further confirmed that L-42 induced apoptosis. After 24 h of L-42 treatment, the relative expression levels (RD) of apoptosis-related proteins cleaved-caspase 3 and cleaved-PARP in THP-1 cells were upregulated, while the relative expression levels (RD) of pro-apoptotic proteins Bax and Bad were upregulated, and the relative expression level (RD) of anti-apoptotic protein Bcl-2 was downregulated. Figure 5 ).

[0102] 1.2.4 L-42 induces cell cycle arrest in leukemia cells

[0103] After 24 hours of L42 treatment, flow cytometry analysis revealed that L42 induced cell cycle arrest in leukemia cells HEL, THP-1, and HL-60. Figure 6-8In HEL and THP-1 cells, treatment with L-42 at concentrations of 0.25, 0.5, and 1 µM arrested cell cycle in the G1 phase. After 24 h of treatment with different concentrations of L-42, the percentages of cells in the G1 phase in HEL cells were 44.03 ± 1.02% (p < 0.05), 50.66 ± 1.68% (p < 0.05), and 54.54 ± 1.79% (p < 0.05), respectively, which were significantly higher than the 37.32 ± 1.02% in the blank control group. Figure 6 In THP-1 cells, the proportions of cells in G1 phase were 49.9±4.34%, 52.65±3.88% (p<0.05), and 53.74±4.07% (p<0.05), respectively, which were significantly higher than the blank control group (42.83±3.68%). Figure 7 In HL-60 cells, L-42 arrests the cell cycle in the S phase. After treatment with different concentrations of L-42 for 24 h, the percentages of cells in the S phase in HL-60 cells were 36.60±2.09%, 39.8±2.05% (p<0.05), and 44.47±2.98% (p<0.05), respectively, which were significantly higher than the 34.51±2.19% in the blank control group. Figure 8 L-42 induces cell cycle arrest in leukemia cells in a concentration-dependent manner.

[0104] 1.3 Experimental Results

[0105] 1) Azahepten bisindole derivatives with [5,6,7,12-tetrahydro-4bH-acephenin[3,2-b:4,5-b']diindole-4bcarboxylic acid methyl ester as the parent nucleus have anti-leukemic activity.

[0106] 2) The azahepten bisindole derivative L-42 inhibits the growth and proliferation of leukemia cells;

[0107] 3) L-42 induces apoptosis in leukemia cells;

[0108] 4) L-42 causes cell cycle arrest in leukemia cells.

[0109] Experimental Example 2: Molecular Experiment of the Aza-Heptene Bisindole Derivative of the Present Invention

[0110] 2.1 Experimental Methods

[0111] 2.1.1 Gene sequencing and data analysis:

[0112] THP-1 cells treated with L-42 were used for gene sequencing at BGI Genomics. Differentially expressed genes (DEGs) were identified from the gene sequencing data and used for KEGG pathway analysis, heatmap generation, and protein-protein interaction analysis.

[0113] 2.1.2 Immunoblot:

[0114] (1) Detection of protein concentration and protein denaturation

[0115] The collected transfected cells were washed once with PBS, centrifuged at 2500 rpm for 5 min, and the supernatant was discarded, while the cell pellet was retained. Protein lysis buffer and PMSF (100:1) mixture were added according to the number of cells collected, and the mixture was placed on ice for 30 min for lysis. After centrifugation at 12000 rpm for 15 min at 4°C, the supernatant was collected.

[0116] (2) BCA protein concentration assay kit for quantitative protein assay

[0117] Prepare BCA working solution (BCA to Cu volume ratio of 50:1) according to the number of protein samples to be tested, mix thoroughly, and place on ice. Add standard protein (BCA standard to PBS volume ratio of 1:10) to the standard wells at intervals of 0, 2, 4, 6, 8, 12, 16, and 20. Pipette 1 µL of protein sample to the wells of the protein samples to be tested, then add 20 µL to each well. Finally, pipette 200 µL of BCA working solution to each well. Incubate at 37°C for 15 min. Detect the absorbance at 562 nm using a microplate reader for different protein concentrations, plot a standard curve, and calculate the protein concentration of the sample to be tested. Mix the obtained protein with 5× protein loading buffer at a ratio of 4:1 and incubate at 95°C for 5 minutes for denaturation.

[0118] (3) Gel preparation for gel running and transfer: Prepare 10% separating gel and 5% stacking gel according to the molecular weight of the target protein, and apply comb teeth.

[0119] Electrophoresis: Assemble the electrophoresis apparatus and load the protein according to the determined sample volume; perform SDS-polyacrylamide gel electrophoresis (100 V for 120 min).

[0120] Transfer: After electrophoresis, the membrane was assembled into a "sandwich" in the order of "filter paper-gel-PDVF membrane-filter paper" and transferred (300 mA 60 min).

[0121] Blocking: After the transfer is complete, remove the membrane and place it in 3% BSA blocking solution for 60 min.

[0122] Primary antibody incubation: Incubate overnight at 4°C with the corresponding primary antibody solution, and wash 3 times with TBST.

[0123] Secondary antibody incubation: Incubate with fluorescent anti-rabbit secondary antibody solution (1:30000) at room temperature in the dark for 2 h, then wash 3 times with TBST.

[0124] Imprint scanning: The imprint membrane is placed in a dual-color infrared fluorescence scanning imager for scanning analysis.

[0125] 2.1.3 Real-time quantitative PCR (RT-qPCR):

[0126] (1) Extraction of total RNA from cells

[0127] Collect logarithmically growing cells into enzyme-free centrifuge tubes, resuspend and wash with 1×PBS, centrifuge, discard the supernatant, and retain the cell pellet. Add 1 mL of Trizol to each tube to resuspend the cells and lyse on ice for 5 min. Then add chloroform at a ratio of Trizol to chloroform of 5:1, gently shake until thoroughly mixed, and stand on ice for 5 min. Centrifuge at 12,000 rpm for 15 min at 4 °C. After centrifugation, transfer the colorless aqueous supernatant to a new enzyme-free EP tube, add an equal volume of isopropanol, gently shake to mix, and freeze the pellet at -80 °C for 2 h. After thawing, centrifuge at 12,000 rpm for 15 min at 4 °C, discard the supernatant, wash with 1 mL of anhydrous ethanol, centrifuge at 12,000 rpm for 15 min at 4 °C, discard the supernatant, and air dry.

[0128] (2) Measurement of RNA concentration

[0129] Add a certain amount of DEPC water to the obtained RNA precipitate, vortex until the precipitate is fully dissolved, detect the RNA concentration using Nanopore, and store in a -80℃ freezer for later use.

[0130] (3) Preparation of cDNA (reverse transcription of RNA)

[0131] Remove the obtained RNA from the -80°C freezer and thaw it on ice, then vortex to mix. Prepare cDNA according to the reverse transcription kit instructions. The total volume is 20 µL, prepared as shown in the table below:

[0132] Table 7.1. Preparation of the reaction solution

[0133] React at 42℃ for 2 min, then add 4 µL of 5× buffer, 4 µL of DEPC water, 1 µL of reverse transcriptase, and 1 µL of primer to each tube. Mix thoroughly and react at 37℃ for 15 min, then react at 85℃ for 15 s. The resulting product is cDNA.

[0134] Table 7.2. SYBR Green I dye method for real-time fluorescent PCR reaction system

[0135] After thorough mixing, add 10 µL to each PCR tube, repeat 3 times for each sample, cap the tubes, and place them in a real-time quantitative PCR instrument for detection.

[0136] The setup procedure is as follows:

[0137] Table 7.3. Real-time quantitative PCR

[0138]

[0139] 2.2 Experimental Data

[0140] 2.2.1 L-42 regulates the PI3K / AKT protein kinase signaling pathway in leukemia cells

[0141] Following L-42 treatment, RNA transcriptome sequencing analysis revealed that differentially expressed genes related to cell signal transduction were enriched via the PI3K / AKT protein kinase pathway through KEGG. Figure 9 The differential gene upregulation changes in the PI3K / AKT pathway are shown in the heatmap. Figure 10 This pathway involves protein-protein interactions between differentially expressed genes and pathways such as the cell cycle. Figure 11 As shown. Immunoblotting further confirmed that the expression of phosphorylated PI3K (p-PI3K, p85) and phosphorylated PI3K (p-AKT) in the PI3K / AKT pathway was downregulated under the influence of L-42. Figure 12 The downstream protein phosphorylation of FoxO3A (p-FoxO3A) is downregulated in the cytoplasm, while FoxO3 is upregulated in the nucleus. Figure 12 FoxO3, as a transcriptional regulator, can regulate the expression of genes related to cell growth and cell cycle in the cell nucleus, thereby affecting cell growth and proliferation. The expression changes of CDKN1A, CDKN2B, CDK1, CDK4, and CDK6, downstream genes of the PI3K / ATK pathway, were detected by RT-qPCR. Figure 13 Among them, CDKN1A and CDKN2B were upregulated (p<0.05), while CDK1, CDK4, and CDK6 were downregulated (p<0.05), and the expression changes of these genes were consistent with the sequencing results. Figure 10 This result indicates that L-42 inhibits the growth and proliferation of leukemia cells by interfering with the PI3K / AKT signaling pathway.

[0142] 2.3 Experimental Results

[0143] L-42 inhibits the growth and proliferation of leukemia cells by interfering with the PI3K / AKT signaling pathway.

[0144] Experimental Example 3: Animal Experiments of the Aza-Heptene Biindole Derivative of the Present Invention

[0145] 3.1 Experimental Methods

[0146] 3.1.1 Laboratory animals

[0147] The in vivo experiments on mice were conducted in accordance with the relevant "Regulations on the Management of Experimental Animals" issued by the State and the Key Laboratory of Natural Product Chemistry of Guizhou Medical University, using SPF-grade Balb / c mice.

[0148] 3.1.2 Animal Model Construction

[0149] Spleen-to-hematocrit ratio in mice: Newborn Balb / c mice within 48 hours of birth were intraperitoneally injected with Fried's mouse leukemia virus (F-MuLV) solution (100 µL / mouse). At least three female and three male mice were used in each experiment. They were intraperitoneally injected with (0.5% DMSO), L-42 (6 mg / kg), and the positive control etoposide (6 mg / kg), administered every other day for two weeks. Five weeks later, the spleens of the leukemia mice were weighed using an electronic scale. Blood was then collected from the mouse heart using a micro-capillary system containing erythrocytes and centrifuged at 3000 rpm for 15 min. At this point, the capillaries separated into two layers: the upper layer was serum, and the lower layer was erythrocytes. The lengths of both layers were measured with a ruler, and the hematocrit ratio was calculated. Hematocrit ratio = erythrocytes / (serum + erythrocytes).

[0150] Hematoxylin-eosin staining (HE staining): After tissue sampling, fixation with formalin, trimming, dehydration, and clearing, place the cleared tissue block in molten paraffin. Once the paraffin has completely penetrated the tissue block, embed it and allow it to cool and solidify. Fix the embedded paraffin block onto a microtome and cut it into thin sections. Before staining, remove the paraffin from the sections with xylene, then pass through high-concentration to low-concentration alcohol, and finally into distilled water for staining. Alternatively, stain with hematoxylin and eosin, dehydrate the stained sections with alcohol, and then clear them with xylene. Apply resin to the cleared sections and seal with a coverslip.

[0151] Mouse survival curves: At least three female and three male mice were used in each experiment. They were injected intraperitoneally with (0.5% DMSO), L-42 (6 mg / kg), and the positive control etoposide (6 mg / kg), administered every other day for two weeks. The survival time of leukemia mice was recorded in the blank control group.

[0152] 3.2 Experimental Data

[0153] 3.2.1 The azacyclic heptacyclic bisindole derivative L-42 inhibits the progression of leukemia in mice.

[0154] A mouse model of leukemia was established by injecting F-MuLV virus into newborn mice. Five weeks after birth, mice were given medication, including a blank control group, a positive control group (etoposide, 6 mg / kg), and an L-42 group (6 mg / kg). Medication was administered for two weeks, once every two days. Figure 14A The mice were weighed every four days. Mice that died in the blank control group were euthanized, and their spleens were photographed and weighed. Blood samples were collected to measure and analyze the hematocrit. Results showed that the spleens of mice in the L-42 group were significantly smaller, consistent with the normal group. Figure 14B -C), the hematocrit of mice treated with L-42 was also improved compared with the blank control. Figure 14D The mouse body weight did not change significantly after administration. Figure 14E After HE staining of mouse spleens, the spleen tissue structure of the L-42 group (drug-treated group) was not infiltrated by leukemia cells, and was closer to that of the normal control group (Normal), indicating that L-42 improved the clearing and infiltration of leukemia cells into the spleen tissue. Figure 14F Meanwhile, a leukemia mouse model was also used to statistically analyze the effect of L-42 on mouse survival time. The time to death of mice was statistically analyzed, and the results showed that L-42 effectively prolonged the survival time of mice. Figure 14G Therefore, the aziridine heptaene bisindole derivative L-42 can inhibit the progression of leukemia in mice.

[0155] 3.3 Experimental Results

[0156] The aziridine heptaene bisindole derivative L-42 can inhibit the progression of leukemia in mice.

[0157] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, equivalent changes, and alterations made to the above embodiments without departing from the technical essence of the present invention shall still fall within the scope of the present invention.

Claims

1. The application of an azahepten bisindole derivative in the preparation of leukemia treatment drugs, wherein the azahepten bisindole derivative has the general structural formula (I): （I） In formula (I), R1 is H, -NO2, -OCH3, I, -CH3, R2 is H, -CH3, and R3 is H, -OCH3.

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