CN113185573B - Preparation method and anti-tumor application of conformational locking melittin Anoplin derivative - Google Patents

The invention relates to a conformational locking melittin derivative, which is characterized in that a peptide chain contains a fragment shown in a formula (I), the fragment is positioned at the end part or the middle part of the Anoplin peptide chain, each x is derived from a sequence in the melittin, and a hydrocarbon bracket is formed on the side chain of the Anoplin peptide chain to stabilize the alpha-helical conformation of polypeptide, so that the aim of conformational locking is fulfilled. Pharmacological experiments in vivo and in vitro show that the compound disclosed by the invention has strong inhibition activity on melanoma cells B16F10, has the advantages of high efficiency, stability and the like, and has great potential in the aspect of preparing anti-melanoma medicaments.

一种构象锁定蜂毒肽Anoplin衍生物的制备方法与抗肿瘤 应用Preparation method and anti-tumor properties of a conformation-locked melittin Anoplin derivative application

技术领域Technical field

本发明涉及药物化合物技术领域，具体地说，是关于一种构象锁定蜂毒肽Anoplin衍生物的制备方法与抗肿瘤应用。The present invention relates to the technical field of pharmaceutical compounds. Specifically, it relates to a preparation method and anti-tumor application of a conformation-locked melittin Anoplin derivative.

背景技术Background technique

黑色素瘤是一种恶性程度极高的实体肿瘤，它具有高侵袭性、高转移和预后差的特点，主要涉及皮肤，是由遗传性基因变异和所处环境的风险导致的，最重要的外源性致病因素是暴露在紫外线的照射中。在全球范围，每年新诊断的原发性恶性肿瘤(不包括非黑色素瘤皮肤癌)中有232100例(占比1.7％)是皮肤黑色素瘤，并导致每年有55500例患者死亡(占所有癌症死亡的0.7％)。黑色素瘤一旦扩散，将直接关乎生命安危。近年来，随着免疫和靶向治疗研究的进展，患者预后得到了极大的改善，但是对于不适合靶向和免疫治疗的，或者这两种治疗都失败的晚期患者来说，化疗成了他们最后的治疗手段，而传统化疗药物存在毒性大和耐药的问题，因此发展高效、低毒、持久的新型抗黑色素瘤药物具有重要的意义。Melanoma is a highly malignant solid tumor with the characteristics of high invasiveness, high metastasis and poor prognosis. It mainly involves the skin and is caused by hereditary gene variation and environmental risks. The most important external factors are The primary causative factor is exposure to ultraviolet radiation. Globally, cutaneous melanoma accounts for 232,100 (1.7%) of newly diagnosed primary malignancies (excluding non-melanoma skin cancers) annually and is responsible for 55,500 patient deaths annually (1.7% of all cancer deaths). 0.7%). Once melanoma spreads, it will be directly related to life safety. In recent years, with the progress of immune and targeted therapy research, patient prognosis has been greatly improved. However, for advanced patients who are not suitable for targeted and immunotherapy, or who have failed both treatments, chemotherapy has become the only option. Their last treatment, traditional chemotherapy drugs have problems of high toxicity and drug resistance, so it is of great significance to develop new anti-melanoma drugs that are efficient, low-toxic and durable.

Anoplin是从黄蜂Anoplius samariensis的毒液旋中提取分离得到的一条具有两亲性、α-螺旋构象的超短多肽，其氨基酸序列为：Gly-Leu-Leu-Lys-Arg-Ile-Lys-Thr-Leu-Leu-NH₂，化学结构如下所示：Anoplin is an ultra-short polypeptide with amphipathic and α-helical conformation extracted and isolated from the venom spiral of the wasp Anoplius samariensis. Its amino acid sequence is: Gly-Leu-Leu-Lys-Arg-Ile-Lys-Thr- Leu-Leu-NH ₂ , the chemical structure is as follows:

近年来研究表明，Anoplin具有极其广泛的药理活性，包括抗细菌、抗真菌、抗疟疾以及抗肿瘤等。由于其良好的药理活性，极其简单的结构，且无溶血活性，使得众多学者试图通过结构修饰来研究Anoplin的构效关系和作用机制，继而开发新药来应对恶性肿瘤和多药耐药细菌感染等人类重大疾病。但是，研究也发现Anoplin存在一些缺点限制了它的临床应用，例如活性仍待提高和不耐蛋白酶水解等。In recent years, studies have shown that Anoplin has an extremely wide range of pharmacological activities, including antibacterial, antifungal, antimalarial and antitumor activities. Due to its good pharmacological activity, extremely simple structure, and no hemolytic activity, many scholars have tried to study the structure-activity relationship and mechanism of action of Anoplin through structural modification, and then develop new drugs to deal with malignant tumors and multi-drug-resistant bacterial infections. Major human diseases. However, studies have also found that Anoplin has some shortcomings that limit its clinical application, such as its activity that still needs to be improved and its intolerance to protease hydrolysis.

订书肽(Stapled peptides)是近年来获得广泛关注的一类稳定性强、透膜性好的多肽模拟物，在天然多肽的修饰以及很多胞内蛋白-蛋白相互作用的调控方面都展示了很好的潜力。其中，Verdine等发展的含有α-甲基非天然氨基酸的订书肽策略能够有效地提高多肽的螺旋性、稳定性和细胞通透性，从而成功实现多肽对体外及体内靶标的有效作用。Stapled peptides are a type of peptide mimetics with strong stability and good membrane permeability that have received widespread attention in recent years. They have demonstrated great potential in the modification of natural peptides and the regulation of many intracellular protein-protein interactions. Good potential. Among them, the stapled peptide strategy containing α-methyl unnatural amino acids developed by Verdine et al. can effectively improve the helicity, stability and cell permeability of the peptide, thereby successfully achieving the effective effect of the peptide on targets in vitro and in vivo.

因此，通过订书肽的策略将Anoplin进行构象锁定，增加其α-螺旋程度和耐蛋白酶稳定性，同时考察其体内外对抗黑色素瘤活性的提高。目前还未见类似的报道。Therefore, the conformation of Anoplin was locked through the strategy of stapled peptides to increase its α-helical degree and protease resistance stability, and the improvement of its anti-melanoma activity in vitro and in vivo was also investigated. There have been no similar reports so far.

发明内容Contents of the invention

本发明的目的在于针对Anoplin现有的缺点，提供一类构象锁定Anoplin衍生物。The purpose of the present invention is to provide a class of conformation-locked Anoplin derivatives in view of the existing shortcomings of Anoplin.

本发明的再一的目的是，提供一类构象锁定Anoplin衍生物的制备方法。Another object of the present invention is to provide a method for preparing a type of conformationally locked Anoplin derivatives.

本发明的另一的目的是，提供一类构象锁定Anoplin衍生物的抗肿瘤用途。Another object of the present invention is to provide a class of conformationally locked Anoplin derivatives for anti-tumor use.

为实现上述目的，本发明采取的技术方案是：In order to achieve the above objects, the technical solutions adopted by the present invention are:

一类构象锁定Anoplin的衍生物，所述的构象锁定Anoplin的衍生物结构通式如下：A type of derivatives of conformation-locked Anoplin. The general structural formula of the derivatives of conformation-locked Anoplin is as follows:

其中，n为0～5的整数，m为0～5的整数，各x来源于蜂毒肽Anoplin中连续的氨基酸Among them, n is an integer from 0 to 5, m is an integer from 0 to 5, and each x is derived from the consecutive amino acids in Anoplin.

为实现上述第二个目的，本发明采取的技术方案是：In order to achieve the above second purpose, the technical solution adopted by the present invention is:

一类构象锁定Anoplin衍生物的制备方法，该方法包括下列步骤：A method for preparing a type of conformationally locked Anoplin derivatives, which method includes the following steps:

(a)首先依据固相合成法，以9-芴基甲氧基羰基保护基的天然氨基酸和(2R)-2-N-芴甲氧羰基氨基-2-甲基-6-庚烯酸(Fmoc-S₅-OH)为原料，以Rink Amide MBHA氨基树脂作为载体，O-(1H-6-氯苯并三氮唑-1-基)-1，1，3，3-四甲基脲六氟磷酸酯/N，N-二异丙基乙胺为缩合体系，合成线性多肽。(a) Firstly, according to the solid phase synthesis method, natural amino acids with 9-fluorenylmethoxycarbonyl protecting group and (2R)-2-N-fluorenylmethoxycarbonylamino-2-methyl-6-heptenoic acid ( Fmoc-S ₅ -OH) as raw material, Rink Amide MBHA amino resin as carrier, O-(1H-6-chlorobenzotriazol-1-yl)-1,1,3,3-tetramethylurea Hexafluorophosphate/N, N-diisopropylethylamine is a condensation system to synthesize linear polypeptides.

(b)在树脂上应用Grubbs第一代催化剂对肽段进行烯烃复分解反应，实现α-螺旋结构的锁定，然后将其从树脂上切下，即可得到构象锁定蜂毒肽Anoplin衍生物。(b) Apply Grubbs' first-generation catalyst on the resin to perform an olefin metathesis reaction on the peptide segment to lock the α-helical structure, and then cut it out from the resin to obtain the conformation-locked melittin Anoplin derivative.

为实现上述第三个目的，本发明采取的技术方案是：In order to achieve the above third purpose, the technical solution adopted by the present invention is:

一类构象锁定Anoplin衍生物在制备抗黑色素瘤药物中的应用。Application of a class of conformationally locked Anoplin derivatives in the preparation of anti-melanoma drugs.

所述的黑色素瘤细胞为B16F10细胞。The melanoma cells are B16F10 cells.

本发明的构象锁定Anoplin衍生物是在Anoplin的序列中引入非天然氨基酸，在Anoplin肽段侧链形成碳氢支架以稳定多肽的α-螺旋构象，达到构象锁定的目的。基于订书肽策略的构象锁定Anoplin衍生物首先能够稳定α-螺旋构象，对Anoplin的α-螺旋程度有明显提升。其次，该类构象锁定Anoplin衍生物与Anoplin相比能够在酶稳定性上得到显著改善，进而提高药物的生物利用度。最后，体内外的药理实验均表明该类构象锁定Anoplin衍生物对黑色素瘤细胞B16F10表现出较好的抑制活性，具有高效、稳定等优点，因此可用于制备抗黑色素瘤药物。The conformation-locked Anoplin derivative of the present invention introduces unnatural amino acids into the sequence of Anoplin, and forms a hydrocarbon scaffold in the side chain of the Anoplin peptide segment to stabilize the α-helical conformation of the polypeptide, thereby achieving the purpose of conformation locking. The conformation-locked Anoplin derivatives based on the stapled peptide strategy can first stabilize the α-helical conformation and significantly improve the α-helical degree of Anoplin. Secondly, this type of conformation-locked Anoplin derivatives can significantly improve enzyme stability compared with Anoplin, thereby improving the bioavailability of the drug. Finally, pharmacological experiments in vivo and in vitro have shown that this type of conformationally locked Anoplin derivatives exhibit good inhibitory activity against melanoma cell B16F10 and have the advantages of high efficiency and stability, so they can be used to prepare anti-melanoma drugs.

附图说明Description of the drawings

图1为本发明构象锁定Anoplin衍生物的耐糜蛋白酶稳定性。Figure 1 shows the chymotrypsin-resistant stability of the conformationally locked Anoplin derivative of the present invention.

图2为本发明构象锁定Anoplin衍生物的体内抗肿瘤作用。(A)为构象锁定Anoplin衍生物治疗后，小鼠的实际肿瘤大小；(B)为小鼠体重的变化曲线Figure 2 shows the in vivo anti-tumor effect of the conformation-locked Anoplin derivative of the present invention. (A) is the actual tumor size of mice after treatment with conformationally locked Anoplin derivatives; (B) is the change curve of mouse body weight

具体实施方式Detailed ways

以下结合具体实施例，对本发明作进一步说明。应理解，以下实施例仅用于说明本发明而非用于限定本发明的范围。The present invention will be further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

实施例1本发明构象锁定Anoplin衍生物Ano-1～6的制备Example 1 Preparation of conformationally locked Anoplin derivatives Ano-1 to 6 of the present invention

表1本发明构象锁定Anoplin衍生物Ano-1～6的氨基酸序列Table 1 Amino acid sequences of conformationally locked Anoplin derivatives Ano-1 to 6 of the present invention

表3所示各构象锁定Anoplin衍生物可通过多肽固相合成法制备。Each conformation-locked Anoplin derivative shown in Table 3 can be prepared by solid-phase peptide synthesis.

下面以制备构象锁定Anoplin衍生物Ano-1为例进行具体描述，其余构象锁定Anoplin衍生物的制备操作相同。The preparation of the conformation-locked Anoplin derivative Ano-1 will be described in detail below as an example. The preparation procedures for other conformation-locked Anoplin derivatives are the same.

合成路线：synthetic route:

合成步骤：Synthesis steps:

称取667mg氨基树脂(取代度0.3mmol/g)放入多肽合成管中，于室温加入5mL g二氯甲烷溶胀20rnin，然后用水泵抽干溶剂。向上述合成管中加入5mL N，N-二甲基甲酰胺洗涤树脂后，向合成管中加入7mL 20％哌啶/N，N-二甲基甲酰胺溶液脱去氨基树脂上的Fmoc保护基，于35℃晃动反应10min，抽干反应液。再向合成管中加入7ml 20％哌啶/N，N-二甲基甲酰胺溶液，于35℃晃动反应10min，抽干反应液。重复上述洗涤树脂，即3次N，N-二甲基甲酰胺、3次二氯甲烷、3次N，N-二甲基甲酰胺，用量及方法如上所述。称取Fmoc-Leu-OH(353mg，1mmol)放入10mL离心管中，再向上述离心管中加入O-(1H-6-氯苯并三氮唑-1-基)-1，1，3，3-四甲基脲六氟磷酸酯(372mg，0.9mmol)、N，N-二异丙基乙胺(259mg，2mmol)，加入6mL N，N-二甲基甲酰胺混匀活化三分钟。将活化好的氨基酸加入到固相合成管中，于35℃反应1h后洗涤。然后脱去第一个氨基酸Leu的Fmoc保护基，方法同上。我们用上述方法依次接入Fmoc-Leu-OH，Fmoc-Thr(tBu)-OH，Fmoc-Lys(Boc)-OH，Fmoc-Ile-OH，Fmoc-S₅-OH，Fmoc-Lys(Boc)-OH，Fmoc-Leu-OH，Fmoc-Leu-OH，Fmoc-S₅-OH，脱Fmoc保护。拿到接好线性多肽的树脂之后，用10mL 1，2-二氯乙烷在固相合成管中洗涤树脂，重复三次。加入5mLGrubbs一代催化剂的DCE溶液(10mmol/L)，35℃反应2小时，抽干重复上述反应一次，洗涤树脂，干燥。向多肽合成管中加入15mL切割试剂(95％三氟乙酸，2.5％，水，2.5％三异丙基硅烷)，室温振荡2小时。将切割试剂过滤至50mL离心管中，再用5mL三氟乙酸润洗树脂，用氩气将离心管中的三氟乙酸吹干。然后向该离心管中，加入35mL冰乙醚，振荡沉淀，用3500转/分的离心机离心3min，倾倒出上清溶液，重复上述沉淀过程三次，风干即得粗肽。最后经制备液相纯化后冻干得到构象锁定Anoplin衍生物Ano-1。Weigh 667 mg of amino resin (degree of substitution 0.3 mmol/g) into a peptide synthesis tube, add 5 mL g of methylene chloride at room temperature to swell for 20 min, and then drain the solvent with a water pump. After adding 5mL N,N-dimethylformamide to the above synthesis tube to wash the resin, add 7mL 20% piperidine/N,N-dimethylformamide solution to the synthesis tube to remove the Fmoc protecting group on the amino resin. , shake for 10 minutes at 35°C, and drain the reaction solution. Then add 7 ml of 20% piperidine/N, N-dimethylformamide solution into the synthesis tube, shake and react at 35°C for 10 minutes, and drain the reaction solution. Repeat the above-mentioned washing of the resin, that is, 3 times of N,N-dimethylformamide, 3 times of dichloromethane, and 3 times of N,N-dimethylformamide. The dosage and method are as described above. Weigh Fmoc-Leu-OH (353 mg, 1 mmol) into a 10 mL centrifuge tube, and then add O-(1H-6-chlorobenzotriazol-1-yl)-1,1,3 to the above centrifuge tube. , 3-tetramethylurea hexafluorophosphate (372mg, 0.9mmol), N,N-diisopropylethylamine (259mg, 2mmol), add 6mL N,N-dimethylformamide, mix and activate for three minutes . Add the activated amino acid into the solid-phase synthesis tube, react at 35°C for 1 hour, and then wash. Then remove the Fmoc protecting group of the first amino acid Leu with the same method as above. We use the above method to access Fmoc-Leu-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ile-OH, Fmoc-S ₅ -OH, Fmoc-Lys(Boc) -OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-S ₅ -OH, deprotected by Fmoc. After getting the resin connected to the linear peptide, wash the resin in the solid-phase synthesis tube with 10 mL of 1,2-dichloroethane, repeat three times. Add 5 mL of DCE solution (10 mmol/L) of Grubbs first-generation catalyst, react at 35°C for 2 hours, drain and repeat the above reaction once, wash the resin, and dry. Add 15 mL of cutting reagent (95% trifluoroacetic acid, 2.5% water, 2.5% triisopropylsilane) to the peptide synthesis tube, and shake at room temperature for 2 hours. Filter the cutting reagent into a 50 mL centrifuge tube, rinse the resin with 5 mL of trifluoroacetic acid, and blow dry the trifluoroacetic acid in the centrifuge tube with argon. Then add 35 mL of glacial ether to the centrifuge tube, oscillate and precipitate, centrifuge at 3500 rpm for 3 min, pour out the supernatant solution, repeat the above precipitation process three times, and air-dry to obtain the crude peptide. Finally, after preparative liquid phase purification and freeze-drying, the conformation-locked Anoplin derivative Ano-1 was obtained.

实施例中所用试剂均为市售分析纯，制备液相所用溶剂为色谱纯。The reagents used in the examples are all commercially available analytical grade, and the solvents used to prepare the liquid phase are chromatographically pure.

实施例2本发明构象锁定Anoplin衍生物Ano-1～6的高分辨质谱和α-螺旋程度数据Example 2 High-resolution mass spectrometry and α-helix degree data of the conformation-locked Anoplin derivatives Ano-1 to 6 of the present invention

本发明构象锁定Anoplin衍生物Ano-1～6的高分辨质谱和α-螺旋程度数据见表2。The high-resolution mass spectra and α-helix degree data of the conformationally locked Anoplin derivatives Ano-1 to 6 of the present invention are shown in Table 2.

表2本发明构象锁定Anoplin衍生物Ano-1～6的高分辨质谱和α-螺旋程度数据表Table 2 High-resolution mass spectra and α-helix degree data table of the conformationally locked Anoplin derivatives Ano-1 to 6 of the present invention

由上表可见，本发明化合物均增加了Anoplin的α-螺旋程度。It can be seen from the above table that the compounds of the present invention all increase the degree of α-helix of Anoplin.

实施例3本发明构象锁定Anoplin衍生物体外抗肿瘤实验Example 3 In vitro anti-tumor experiment of conformationally locked Anoplin derivatives of the present invention

3.1实验方法：采用CCK-8细胞增殖毒性检测试剂盒测试构象锁定Anoplin衍生物对黑色素瘤细胞B16F10的抗增殖活性。在96孔板中配置200μL的B16F10细胞悬液，将培养板在37℃，5％CO₂培养箱预培养24小时。向培养板加入10μL不同浓度的构象锁定Anoplin衍生物，并在培养箱孵育24小时。向每孔加入10μL CCK-8溶液。将培养板在培养箱内孵育2小时后用酶标仪测定96孔板在450nm处的吸光度。按如下公式计算药物对B16F10细胞的抑制率：抑制率＝[(Ac-As)]/[(Ac-Ab)]×100％，As：实验孔(含有细胞的培养基、CCK-8、待测药物)，Ac：对照孔(含有细胞的培养基、CCK-8、没有待测药物)，Ab：空白孔(不含细胞和待测药物的培养基、CCK-8)。3.1 Experimental method: CCK-8 cell proliferation toxicity detection kit was used to test the anti-proliferative activity of conformation-locked Anoplin derivatives on melanoma cells B16F10. Prepare 200 μL of B16F10 cell suspension in a 96-well plate, and pre-culture the culture plate in a 37°C, 5% _CO2 incubator for 24 hours. Add 10 μL of different concentrations of conformationally locked Anoplin derivatives to the culture plate and incubate in the incubator for 24 hours. Add 10 μL of CCK-8 solution to each well. After incubating the culture plate in the incubator for 2 hours, use a microplate reader to measure the absorbance of the 96-well plate at 450 nm. Calculate the inhibitory rate of the drug on B16F10 cells according to the following formula: Inhibition rate = [(Ac-As)]/[(Ac-Ab)] × 100%, As: experimental well (culture medium containing cells, CCK-8, to be test drug), Ac: control well (medium containing cells, CCK-8, and no drug to be tested), Ab: blank well (medium containing cells and drug to be tested, CCK-8).

3.2实验结果3.2 Experimental results

体外抗肿瘤实验见表3In vitro anti-tumor experiments are shown in Table 3

表3本发明的化合物Ano-1～6体外抗肿瘤实验结果Table 3 In vitro anti-tumor experimental results of compounds Ano-1 to 6 of the present invention

由上表可见，本发明化合物均对B16F10细胞有较强的抑制活性，极高地提升了Anoplin的抗肿瘤活性。It can be seen from the above table that the compounds of the present invention all have strong inhibitory activity on B16F10 cells, which greatly improves the anti-tumor activity of Anoplin.

实施例4本发明构象锁定Anoplin衍生物的耐糜蛋白酶稳定性Example 4 The chymotrypsin resistance stability of the conformationally locked Anoplin derivative of the present invention

优选上述实验α-螺旋程度和体外抗肿瘤活性均较高的Ano-3进行耐糜蛋白酶稳定性的考察。称取适量的药物配制成浓度为1mM的肽储存液。称取一定量的糜蛋白酶溶于含有2mM CaCl₂的磷酸盐缓冲液(50mM，pH 7.4)至糜蛋白酶的浓度为0.5ng/μL。在2mL离心管中分别加入1000μL含糜蛋白酶的磷酸盐缓冲液和100μL的药物储存液进行酶消化反应。分别取药物各个时间点的100μL反应液加入20μL 1M的盐酸淬灭糜蛋白酶活性。最后使用高效液相色谱仪分析不同时间点多肽的百分残余量并作图。Ano-3, which has higher α-helix degree and in vitro anti-tumor activity in the above experiment, is preferred for investigation of chymotrypsin resistance stability. Weigh an appropriate amount of drug to prepare a peptide stock solution with a concentration of 1mM. Weigh a certain amount of chymotrypsin and dissolve it in phosphate buffer (50mM, pH 7.4) containing 2mM _CaCl2 until the concentration of chymotrypsin is 0.5ng/μL. Add 1000 μL of phosphate buffer containing chymotrypsin and 100 μL of drug storage solution to a 2 mL centrifuge tube for enzymatic digestion reaction. Take 100 μL of the reaction solution at each time point of the drug and add 20 μL of 1M hydrochloric acid to quench the chymotrypsin activity. Finally, a high-performance liquid chromatograph was used to analyze the percentage residual amount of the polypeptide at different time points and draw a graph.

图1为多肽对糜蛋白酶稳定性结果，直链肽Anoplin迅速降解，半衰期为11.35分钟，而经过构象锁定能够显著提高Ano-3的稳定性，8小时后依然没有降解。Figure 1 shows the stability results of polypeptides against chymotrypsin. The linear peptide Anoplin is rapidly degraded with a half-life of 11.35 minutes. However, conformational locking can significantly improve the stability of Ano-3, and it is still not degraded after 8 hours.

实施例5本发明构象锁定Anoplin衍生物的体内抗肿瘤作用Example 5 In vivo anti-tumor effect of conformationally locked Anoplin derivatives of the present invention

5.1模型建立5.1 Model establishment

雌性C57小鼠，右腹股沟皮下接种B16F10细胞(5×10⁵个)，约5天形成肿瘤。Female C57 mice were subcutaneously inoculated with B16F10 cells (5 × 10 ⁵ cells) in the right groin, and tumors formed in about 5 days.

5.2评价构象锁定Anoplin衍生物的体内抗肿瘤作用5.2 Evaluation of the in vivo anti-tumor effect of conformationally locked Anoplin derivatives

实验分组：①空白组；②Anoplin组；③Ano-3组。给药方式：采用瘤内注射的方式，每只1mg药物，水溶液体积50μL，连续给药3天。评价指标为：①以瘤体积2000mm³为终点，解剖出小鼠肿瘤，比较各组肿瘤大小；②跟踪记录实验过程中小鼠一般健康状况，饮食、饮水和体重变化，比较副作用。Experimental groups: ① Blank group; ② Anoplin group; ③ Ano-3 group. Method of administration: Intratumoral injection, 1 mg of drug per animal, aqueous solution volume 50 μL, administered continuously for 3 days. The evaluation indicators are: ① Taking the tumor volume of ^2000mm3 as the end point, dissect the mouse tumors and compare the tumor sizes of each group; ② Track and record the general health status of the mice during the experiment, changes in diet, drinking water and weight, and compare side effects.

图2A为构象锁定Anoplin衍生物Ano-3治疗后，C57小鼠皮下黑色素瘤的实际肿瘤。与空白组和Anoplin组相比，Ano-3能较好抑制肿瘤大小。图2B为C57小鼠实际体重曲线，无明显变化。Figure 2A shows actual tumors of subcutaneous melanoma in C57 mice after treatment with the conformationally locked Anoplin derivative Ano-3. Compared with the blank group and Anoplin group, Ano-3 can better inhibit tumor size. Figure 2B shows the actual body weight curve of C57 mice, with no obvious change.

以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员，在不脱离本发明方法的前提下，还可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。The above are only the preferred embodiments of the present invention. It should be pointed out that those of ordinary skill in the art can also make several improvements and supplements without departing from the method of the present invention. These improvements and supplements should also be regarded as It is the protection scope of the present invention.