A Tiancimycin analogue and its preparation method and application

Abstract

The present invention belongs to the field of biopharmaceutical technology and specifically relates to a 6-alkoxy-substituted Tiancimycin analogue, a preparation method thereof, and its anti-tumor application. The structural formula of the Tiancimycin analogue is as follows: #imgabs0# wherein R1 is a C1-C6 alkyl group. The Tiancimycin analogue in the present invention is obtained by alkylating the 6-OH group of Tiancimycin A. The Tiancimycin analogue in the present invention has improved selectivity for normal cells and two tumor cell lines, A549 and KPL-4, and has a larger therapeutic window compared to Tiancimycin A.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a preparation method of a 6-alkoxy substituted Tiancimycin analogue and its anti-tumor application. Background Art

[0002] Enediynes are among the most potent cytotoxic small molecules known to date, with a unique mechanism of action that can inhibit DNA synthesis and replication in tumor cells by inducing single-strand or double-strand breaks. However, their clinical application is significantly limited by their lack of selective killing of tumor cells when used alone.

[0003] Anthraquinone-conjugated enediyne contains an anthraquinone structural unit and a ten-membered cycloenediyne structural unit ( Figure 1 ). Anthraquinone structural units play an important role in their interaction with DNA and biological activity. Anthraquinone-conjugated enediynes have attracted widespread attention due to their simple structure and potential as antibody-drug conjugated warhead molecules. Since the first anthraquinone-conjugated enediyne Dynemicin (DYN) was discovered in 1989, Uncialamycin (UCM, 2) / Tiancimycin (TNMs, 3–5, 9) / Yangpumicin (YPMs, 6–8) and Sealutomicin (10) have been discovered ( Figure 1 Among them, Tiancimycin A is being actively studied and is expected to become a warhead molecule for antibody-drug conjugates. However, although Tiancimycin currently has a strong inhibitory effect on tumor cells, it is also highly toxic to normal cells, thus limiting its application in medicine. Summary of the Invention

[0004] The present invention aims to provide a Tiancimycin analogue and provide the relative toxicity of the compound to tumor cells.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] A Tiancimycin analogue, whose structural formula is as follows:

[0007]

[0008] Wherein, R1 is a C1-C6 alkyl group.

[0009] In one preferred embodiment, R1 is a C1-C3 alkyl group.

[0010] The present invention also claims a method for preparing the Tiancimycin analog, comprising the following steps:

[0011] S1. Dissolve Tiancimycin A, add a basic catalyst and an electrophilic reagent, and stir overnight;

[0012] S2, removing the catalyst and solvent, re-dissolving the obtained solid, and purifying to obtain the Tiancimycin analog;

[0013] The electrophilic reagent is 5-methyl-5H-dibenzo[b,d]thiophen-5-ium trifluoromethanesulfonate, iodomethane or (CH3)2SO4.

[0014] In the present invention, potassium carbonate acts as a base to undergo an acid-base neutralization reaction with a phenolic hydroxyl compound to generate a phenolic hydroxyl anion, which then undergoes electrophilic substitution with an electrophilic reagent to obtain an alkylation product.

[0015] In a preferred embodiment, the electrophilic reagent is 5-methyl-5H-dibenzo[b,d]thiophen-5-ium trifluoromethanesulfonate.

[0016] While 5-methyl-5H-dibenzo[b,d]thiophen-5-ium triflate, iodomethane, and (CH3)2SO4 can all be used as electrophiles to complete the reaction process of the present invention, 6-methyl-5H-dibenzo[b,d]thiophen-5-ium triflate exhibits significantly different reactivity from CH3I and (CH3)2SO4, depending on the different sterically hindered leaving groups. When CH3I and (CH3)2SO4 are used, multiple methyl-substituted products are generated, such as 26-OH, 17-OH, 13-OH, and 6-OH alkylated products. However, when methyl-5H-dibenzo[b,d]thiophen-5-ium triflate is used, only the 6-OH alkylated product is generated, resulting in higher selectivity and higher yield.

[0017] In one preferred embodiment, the molar ratio of Tiancimycin A to the catalyst is 1:(1-3).

[0018] In one preferred embodiment, the molar ratio of Tiancimycin A to the electrophilic reagent is 1:(1-3).

[0019] In one preferred embodiment, Tiancimycin A is dissolved in a solvent.

[0020] In a preferred embodiment, the solvent is one or more of acetonitrile, a mixed solvent of dichloromethane / water = 20 / 1, N,N-dimethylformamide, and ethyl acetate.

[0021] In the present invention, the electrophilic substitution reaction of the phenolic hydroxyl group requires an appropriate base. This type of reaction needs to be carried out in a homogeneous system, so the choice of solvent is very important. Generally speaking, the reaction needs to select a non-hydrogenated solvent and all reactants need to be soluble in the reaction solvent to ensure the reaction proceeds. According to the reaction system of the present invention, the solvent can use one or more of acetonitrile, a mixed solvent of dichloromethane / water = 20 / 1, N,N-dimethylformamide, and ethyl acetate.

[0022] In one preferred embodiment, the catalyst is removed by filtration and the solvent is removed by distillation under reduced pressure.

[0023] In one preferred embodiment, the catalyst is any one of K2CO3, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 1,5-diazabicyclo[4.3.0]-5-nonene (DBN), and cesium carbonate.

[0024] The reaction of the present invention requires a base as a catalyst, but a strong base can cause the oxirane ring of Tiancimycin to open, and then the enediyne pharmacophore undergoes Bergman cyclization and deterioration, thereby losing its efficacy. Therefore, the reaction of the present invention can only use any one of K2CO3, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 1,5-diazabicyclo[4.3.0]-5-nonene (DBN), and cesium carbonate. The base has an appropriate degree of alkalinity and can catalyze the reaction without causing ring opening.

[0025] In a preferred embodiment, the re-dissolving is to re-dissolve the solid with a small amount of acetonitrile.

[0026] In a preferred embodiment, the purification is performed by semi-preparative HPLC.

[0027] In one preferred embodiment, in step S2, the crude product is purified by semi-preparative HPLC on an AQ-C18 column using an isocratic MeCN (50%) / H2O (50%) solvent system to obtain the Tiancimycin analog 3b with a purity greater than 95%.

[0028] In one preferred embodiment, step S2 further includes detection, and the detection includes high-resolution mass spectrometry, nuclear magnetic resonance, and high-performance liquid chromatography analysis.

[0029] The present invention further provides the use of the Tiancimycin analog in the preparation of anti-tumor drugs.

[0030] Preferably, the tumor includes the treatment of squamous cell carcinoma, including squamous cell carcinoma, skin cancer, head and neck cancer, esophageal cancer, lung cancer, cervical cancer, vaginal cancer, penile cancer, malignant lymphoma, brain tumor, thyroid cancer, germ cell tumor, malignant melanoma, glioma, Hodgkin's lymphoma, testicular cancer, prostate cancer, liver cancer, gastric cancer, lung cancer, colon cancer and digestive tract tumors.

[0031] In one preferred embodiment, the tumor includes a lung cancer cell line Jurkat, a breast tumor cell line KPL-4, and an acute T-cell leukemia cell line Jurkat.

[0032] The present invention will be further explained below:

[0033] The present invention provides a Tiancimycin analogue, specifically a new Tiancimycin analogue obtained by alkylating the 6-OH group of Tiancimycin A. It is also found that the Tiancimycin analogue significantly reduces the viability of tumor cells and has the ability to effectively kill various types of tumor cell lines. In addition, the compound Tiancimycin analogue has an IC of 2.5-3.0 for normal cells. 50 The value is 60 times that of KPL-4 and Jurkat cells. This higher selectivity than Tiancimycin and its analogs provides a larger therapeutic window. In in vivo anti-tumor activity evaluation, Tiancimycin analogs showed excellent in vivo anti-tumor activity and minimal systemic toxicity.

[0034] Compared with existing compounds containing anthraquinone-conjugated enediyne structures, the Tiancimycin analogues of the present invention have the following obvious advantages:

[0035] The Tiancimycin analogues of the present invention have improved selectivity for normal cells and two tumor cells A549 and KPL-4, and have a larger therapeutic window compared with Tiancimycin A. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 These are natural anthraquinone-coupled enediynes discovered in the past three decades;

[0037] Figure 2 This is the high-resolution mass spectrum of 3b;

[0038] Figure 3 is the 3b H NMR spectrum;

[0039] Figure 4 is the 3b carbon NMR spectrum;

[0040] Figure 5 The results show that Tiancimycin A and two different doses of 3b inhibit tumor growth;

[0041] Figure 6These are the results of routine blood tests for Tiancimycin A and two different doses of 3b;

[0042] Figure 7 HE staining results of organs of Tiancimycin A and two different doses of 3b 1;

[0043] Figure 8 The results of HE staining of organs of Tiancimycin A and two different doses of 3b are shown in Figure 2. DETAILED DESCRIPTION

[0044] The present invention will be described in detail below with reference to the accompanying drawings and in combination with embodiments. It should be noted that, in the absence of conflict, the embodiments and features of the embodiments of the present invention may be combined with each other.

[0045] Example 1

[0046] Preparation of Tiancimycin Analogues

[0047] Tiancimycin A (4.8 mg, 0.01 mmol) was dissolved in 2 mL of acetonitrile, and K2CO3 (2.8 mg, 0.02 mmol) and 5-methyl-5H-dibenzo[b,d]thiophen-5-ium trifluoromethanesulfonate (7.0 mg, 0.02 mmol) were added and reacted at room temperature overnight. K2CO3 was removed by filtration, the solvent was distilled off under reduced pressure, and the product was redissolved in a small amount of acetonitrile. The crude product was purified by semi-preparative HPLC on an AQ-C18 column using an isocratic MeCN (50%) / H2O (50%) solvent system to obtain the Tiancimycin derivative (4.0 mg, yield = 80%).

[0048] Analysis of compounds:

[0049] The Tiancimycin derivatives were analyzed and HRMS spectra were recorded on a LTQ-ORBITRAP-ETD instrument. NMR spectra were obtained using a Brucker, 500 MHz mass spectrometer. 13 C NMR spectrum, DMSO-d6 (δ = 40.0 ppm), for 1 H NMR reports chemical shifts in ppm in DMSO-d6 (δ = 3.40 ppm). The results are as follows Figure 2-Figure 4 Shown: HRMS (ESI) m / z calcd for C 28 H 22 NO8,[M+H] + =500.1345;Found:500.1335. 1H NMR (500MHz, DMSO-d6) δ13.21(s,1H),9.85(s,1H),8.47(s,1H),8.07(d,J=8.7Hz,1H),7.53(d,J=8.9Hz,1H),6.05(d,J=9 .9Hz,1H),5.97(d,J=9.9Hz,1H),5.16(s,1H),5.03(s,1H),4.31(s,1H),3.97(s,3H),3.82(s,3H),1.31(d,J=6.4Hz,3H). 13 C NMR (125MHz, DMSO-d6) δ186.5,183.2,159.9,149.4,143.2,135.6,130.1,129.5,129.2,127.9,126.0,124 .8,124.4,123.7,117.2,112.8,112.6,101.0,99.5,90.1,87.9,76.6,64.2,63.5,60.9,56.9,43.7,22.4.

[0050] Structural analysis description:

[0051] Tiancimycin derivatives were fully characterized by comprehensive structural analysis. Figure 2-Figure 4 It can be seen that the [M+H] + The molecular ion peak (m / z) is 500.1335, which is consistent with its standard molecular formula C 28 H 21 NO8([C 28 H 21 NO8+H] + , molecular weight is 500.1345) is consistent with the numerical value. 1 H and 13 C nuclear magnetic resonance spectroscopy (NMR), and two-dimensional NMR combined analysis to characterize the structural part of the Tiancimycin derivatives. 1 H and 13 Its structure was further confirmed by C and 2D spectra. Its structure was identified by separation and structural identification (the strong HMBC correlation between H-8 and 6-OCH3 and C-6 in the Tiancimycin derivative supports that the methoxy substitution position is C6).

[0052] The structural formula of the Tiancimycin derivative is determined as follows:

[0053]

[0054] The following is named 3b.

[0055] Example 2

[0056] In vitro antitumor activity test

[0057] Cancer is one of the major health problems facing mankind, and its incidence and mortality rate pose a serious threat to human health. In 2020, there were an estimated 19.29 million confirmed cases of cancer worldwide, of which China had 4.569 million confirmed cases and 3.003 million deaths, both ranking first in the world. Although various treatment strategies including surgery, radiotherapy and drug therapy have developed into more sophisticated and accurate tumor treatment methods, the number of tumor-related deaths has not decreased significantly. In the present disclosure, 3b has the ability to effectively kill multiple types of tumor cell lines.

[0058] The in vitro anti-tumor activity of 3b and Tiancimycin A and its analogs of the present invention on three different types of human tumor cell lines is now listed: including lung cancer cell line A549, breast tumor cell line KPL-4, acute T-cell leukemia cell line Jurkat, and the cytotoxicity of human normal cell line NCM460. NCM460 cells and Jurkat cells were cultured in RPMI 1640 medium, and A549 cells and KPL-4 cells were cultured in DMEM medium. All culture media were supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. All test cells were incubated at 37°C and 5% humidified CO2. Cell viability was determined by CCK-8 method. Briefly, the experimental process was as follows: cells were seeded in 96-well plates (5×10 cells per well). 3 Cells were plated in 4% paraformaldehyde (5% paraformaldehyde) and stabilized for 24 hours. The cells were then treated with 100 μL of culture medium containing various drug concentrations. After 72 hours of incubation, 10 μL of CCK-8 reagent was added to each well and incubated at 37°C for 0.5–2 hours. Cell viability was assessed by measuring absorbance at 450 nm using a microplate reader (Spark 10M Tecan, Grodig, Austria). Fluorescence was observed using an inverted fluorescence microscope (DMIL-LED, Leica Microsystems, Wetzlar, Germany) to determine viability of live cells. 50 Values using PRISM TM Software, version 8.0 (GraphPad software, La Jolla, CA, USA).

[0059]

[0060] Table 1 IC of Tiancimycin A and its analogs against normal cells 50 IC of tumor cells 50 Ratio

[0061]

[0062]

[0063] From the overall data, the cytotoxicity of 3b against the three tumor cell lines listed reached the nanomolar level, indicating that it can effectively kill tumor cells. It is particularly important to note that compared with normal cells NCM460 (IC 50 =6.0 nM) compared with the toxicity of 3b to two tumor cells Jurkat (IC 50 =0.1 nM), KPL-4 (IC 50 =0.1 nM) showed good selectivity, with a 60-fold difference in toxicity. The toxicity of Tiancimycin A against normal cells differed only 4- and 10-fold between that of Jurkat and KPL-4 tumor cells, respectively. Tiancimycin A and its derivatives were significantly more toxic to normal cells than 3b, and their selectivity was also significantly lower than that of 3b. Furthermore, Tiancimycin analogs with ethoxy and propoxy substitutions at the C6 position also showed significant relative toxicity improvements over Tiancimycin A, ranging from 25-50 times.

[0064] Example 3: Evaluation of the anti-breast cancer activity of 3b and Tiancimycin A in vivo

[0065] Experimental Procedure: 6-week-old specific pathogen-free (SPF) female ICR mice weighing 25 ± 2 g were housed in an SPF animal room at an ambient temperature of 26°C, a relative humidity of 50%, and a 12-h light / dark cycle. Blood was collected from the orbital vein of BALB / c nude mice, and fresh serum was prepared for hemolysis analysis. 5 × 10 6 KPL-4 cells (50 μL PBS + 50 μL Matrigel [BD Biocoat]) were inoculated subcutaneously in the right axilla of BALB / c nude mice to establish a subcutaneous tumor-bearing model in nude mice. When the tumor volume of each BALB / c nude mouse reached approximately 100 mm 3 At the same time, nude mice were randomly divided into 4 groups (n=5): blank control group (0.9% normal saline), drug group (tiancimycin A, 0.05 mg / kg), 3b low-dose drug group (3b, 0.05 mg / kg), 3b high-dose drug group (3b, 0.2 mg / kg). The two administrations were separated by seven days.

[0066] The length (a) and width (b) of the tumor were measured every day starting from the time of administration. The tumor volume V was calculated by the following formula: V = 1 / 2ab 2Finally, after 14 days of administration, all BALB / c nude mice were sacrificed, and the solid tumors were removed, weighed, and photographed. The heart, liver, spleen, lungs, and kidneys were separated, and blood was collected. The tissues were then fixed in 10% neutral formaldehyde and paraffin sections were prepared for histological examination (HE staining). The remaining tissues were stored in a -80°C freezer until further use. The blood was then used for inflammatory analysis using a Mindray BC-2800vet three-differential hematology analyzer.

[0067] The experimental process of HE staining is as follows:

[0068] 1. Dewax the paraffin sections to water: place the sections in xylene I for 20 minutes, xylene II for 20 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, and 75% alcohol for 5 minutes, and then wash with tap water.

[0069] 2. Hematoxylin staining: Stain the sections with hematoxylin solution for 3-5 minutes, wash with tap water, differentiate with differentiation solution, wash with tap water, blue with bluing solution, and rinse with running water.

[0070] 3. Eosin staining: Dehydrate the sections in 85% and 95% graded alcohol for 5 minutes each, and then stain them in eosin solution for 5 minutes.

[0071] 4. Dehydration and sealing: Put the slices into anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, anhydrous ethanol III for 5 minutes, xylene I for 5 minutes, and xylene II for 5 minutes to make them transparent, and then seal the slices with neutral gum.

[0072] 5. Microscopic examination, image acquisition and analysis.

[0073] After staining, the nucleus appears blue and the cytoplasm appears red.

[0074] The results showed that the anti-tumor activity was studied by subcutaneous KPL-4 human breast tumor xenograft. The injection dose of 3b was 0.2 or 0.05 mg / kg. The results showed that 0.05 mg / kg Tiancimycin A and two different doses of 3b had different degrees of inhibitory effects on tumor growth, among which 3b (0.2 mg / kg) had the strongest inhibitory effect on tumors, and there was no statistical difference in tumor volume between the 0.05 mg / kg Tiancimycin A and 0.05 mg / kg 3b groups. Tiancimycin A caused a decrease in mouse body weight after administration, however, 3b had almost no effect on mouse body weight at high or low doses, and there was no statistical difference between the two groups and the saline group ( Figure 5Routine organ and blood tests showed that except for slight abnormalities in monocytes (Mono) and neutrophils (Gran), other indicators of all groups were within the normal range. Among them, three groups of mice, including the saline group, TNM A (0.05mg / kg) and 3b (0.2mg / kg), showed slight abnormalities in monocytes (Mono) and neutrophils (Gran), indicating the occurrence of inflammatory response ( Figure 6 In addition, HE staining results of various organs showed that there was no obvious damage to the organs of all mice ( Figure 7 and Figure 8 ).

[0075] 0.05 mg / kg of ethoxy and propoxy substituted Tiancimycin analogs at the C6 position also had a significant inhibitory effect on tumors.

[0076] The above experimental results show that the Tiancimycin analogue has improved selectivity for normal cells and two tumor cells, Jurkat and KPL-4, and has a larger therapeutic window compared with TNMA. Therefore, this compound has more application prospects as an effective anti-tumor compound.

[0077] The contents described in the above embodiments should be understood as these embodiments are only used to more clearly illustrate the present invention, and are not used to limit the scope of the present invention. After reading the present invention, various equivalent modifications to the embodiments made by those skilled in the art fall within the scope defined by the claims attached to the present invention.

Claims

1. A Tiancimycin analogue, characterized in that: Its structural formula is as follows: Wherein, R1 is methyl.

2. The method for preparing a Tiancimycin analogue according to claim 1, characterized in that: The following steps are involved: S1. Dissolve Tiancimycin A, add a basic catalyst and an electrophilic reagent, and stir overnight; S2, removing the catalyst and solvent, re-dissolving the obtained solid, and purifying to obtain the Tiancimycin analog; The electrophilic reagent is 5-methyl-5H-dibenzo[b,d]thiophen-5-ium trifluoromethanesulfonate, iodomethane or (CH3)2SO4.

3. The preparation method according to claim 2, characterized in that The molar ratio of Tiancimycin A to the catalyst is 1: (1-3); the molar ratio of Tiancimycin A to the electrophilic reagent is 1: (1-3).

4. The preparation method according to claim 2, characterized in that Tiancimycin A is dissolved in a solvent.

5. The preparation method according to claim 4, characterized in that The solvent is one or more of acetonitrile, a mixed solvent of dichloromethane / water = 20 / 1, N,N-dimethylformamide, and ethyl acetate.

6. The preparation method according to claim 2, characterized in that The catalyst is any one of K2CO3, 1,8-diazabicyclo[5.4.0]undec-7-ene, 1,5-diazabicyclo[4.3.0]-5-nonene, and cesium carbonate.

7. The preparation method according to claim 2, characterized in that The re-dissolution is to re-dissolve the solid with a small amount of acetonitrile.

8. The preparation method according to claim 2, characterized in that The purification is performed by semi-preparative HPLC; in step S2, the crude product is purified by semi-preparative HPLC on an AQ-C18 column using an isocratic 50% MeCN / 50% H2O solvent system to obtain a Tiancimycin analogue of formula I with a purity greater than 95%.

9. The use of the Tiancimycin analogue in the preparation of anti-tumor drugs according to claim 1, characterized in that: The tumors are breast tumors and acute T-cell leukemia.

Images