WO2015196944A1

WO2015196944A1 - Gnrh analog-cytotoxic molecule conjugate and preparation method and use thereof

Info

Publication number: WO2015196944A1
Application number: PCT/CN2015/081781
Authority: WO
Inventors: 刘克良; 周宁; 马永涛; 王晨宏; 冯思良
Original assignee: Institute of Pharmacology and Toxicology of AMMS
Current assignee: Institute of Pharmacology and Toxicology of AMMS
Priority date: 2014-06-26
Filing date: 2015-06-18
Publication date: 2015-12-30
Anticipated expiration: 2016-12-26
Also published as: CN105198966A; CN105198966B

Abstract

Provided are a GnRH analog-cytotoxic molecule conjugate as shown by the following formula (I), and a preparation method and a use thereof in the preparation of drugs for treating tumours. The cytotoxic molecule is a taxol derivative or a docetaxel derivative. The conjugate can maintain the binding activity with GnRH receptors, and has a relatively good plasma stability and the growth inhibition activity on tumour cells. (X)Aaa¹Aaa²-Aaa³-Ser-Aaa⁵-Aaa⁶(Y)-Leu-Aaa⁸-Pro-Aaa¹⁰-NH₂

Description

GnRH analogue-cytotoxic molecule conjugate, preparation method and use thereof

Technical field

本发明属于医药、化学领域，具体涉及GnRH类似物-细胞毒分子缀合物、其制备方法及其在制备预防和/或治疗肿瘤的药物中的用途。The invention belongs to the field of medicine and chemistry, and particularly relates to a GnRH analogue-cytotoxic molecule conjugate, a preparation method thereof and use thereof for preparing a medicament for preventing and/or treating a tumor.

Background technique

促性腺激素释放激素(GnRH)又称促黄体素释放激素(LHRH)，是下丘脑以脉冲形式分泌的十肽激素(Glp–His–Trp–Ser–Tyr–Gly–Leu–Arg–Pro–Gly·NH₂)，是调节生殖系统的关键分子。由下丘脑的神经元产生，通过门静脉到达垂体。在垂体部位，GnRH活化了其受体GPCR,一个七重跨膜G-蛋白偶联受体，进而促进LH(黄体生成素)和FSH(卵泡刺激素)的合成和分泌。最近的研究显示，GnRH在下丘脑和垂体外的其他器官中也有作用。在子宫，卵巢，输卵管，胎盘，乳腺，前列腺，外周血液单核细胞，结肠，直肠和肾的肿瘤细胞中，以及淋巴瘤和黑色素瘤中都发现了GnRH受体。甚至消化系统中也发现了GnRH的受体。另外，GnRH受体在正常组织中分布有限，这使得将GnRH受体作为抗肿瘤药物靶向给药的受体和用GnRH类似物作为靶向基团成为可能。Gonadotropin-releasing hormone (GnRH), also known as luteinizing hormone releasing hormone (LHRH), is a decapeptide hormone secreted by the hypothalamus in a pulsed form (Glp–His–Trp–Ser–Tyr–Gly–Leu–Arg–Pro–Gly) · NH ₂ ), a key molecule that regulates the reproductive system. It is produced by neurons in the hypothalamus and reaches the pituitary through the portal vein. In the pituitary, GnRH activates its receptor GPCR, a seven-fold transmembrane G-protein coupled receptor, which in turn promotes the synthesis and secretion of LH (luteinizing hormone) and FSH (follicle stimulating hormone). Recent studies have shown that GnRH also plays a role in the hypothalamus and other organs outside the pituitary. GnRH receptors are found in uterus, ovaries, fallopian tubes, placenta, breast, prostate, peripheral blood mononuclear cells, colon, rectal and renal tumor cells, as well as in lymphoma and melanoma. Even the receptor for GnRH is found in the digestive system. In addition, the GnRH receptor has a limited distribution in normal tissues, which makes it possible to use the GnRH receptor as a receptor for targeted administration of antitumor drugs and to use GnRH analogs as targeting groups.

以GnRH类似物为靶向基团，通过一个可降解的连接臂如：二硫键、酯键、腙键等，与细胞毒分子如：阿霉素、柔红霉素、紫杉醇、多西他赛、甲氨蝶呤、喜树碱等直接缀合，以受体介导的方式进入肿瘤细胞，进而在肿瘤细胞内部特异性的微环境下(酸性环境以及特异性酶的高表达)，释放出游离的细胞毒分子，最终抑制肿瘤的生长。Using a GnRH analogue as a targeting group, through a degradable linker such as a disulfide bond, an ester bond, a hydrazone bond, etc., with cytotoxic molecules such as: doxorubicin, daunorubicin, paclitaxel, docetaxel Race, methotrexate, camptothecin, etc. are directly conjugated, enter the tumor cells in a receptor-mediated manner, and then release in the specific microenvironment inside the tumor cells (acidic environment and high expression of specific enzymes) Free cytotoxic molecules are released, ultimately inhibiting tumor growth.

目前，关于该类化合物的研究获得了长足的进展，个别化合物已进入临床研究阶段，但仍存在稳定性不高的缺点，容易过早的释放出细胞毒分子，进而产生毒副作用。At present, the research on this kind of compound has made great progress. Individual compounds have entered the stage of clinical research, but there are still shortcomings of low stability, and it is easy to release cytotoxic molecules prematurely, and then produce toxic and side effects.

为了解决上述问题，尚需要研究新的GnRH类似物-细胞毒分子缀合物，在保持较高的肿瘤细胞生长抑制活性、较强的受体结合活性的同时，提高缀合物的稳定性。In order to solve the above problems, it is necessary to study a novel GnRH analogue-cytotoxic molecule conjugate, which maintains high tumor cell growth inhibitory activity and strong receptor binding activity. At the same time, improve the stability of the conjugate.

发明内容Summary of the invention

本发明的第一方面涉及式(I)所示的化合物、其立体异构体、溶剂合物或其无生理毒性的盐，A first aspect of the invention relates to a compound of the formula (I), a stereoisomer thereof, a solvate thereof or a salt thereof which is not physiologically toxic,

(X)Aaa¹-Aaa²-Aaa³-Ser-Aaa⁵-Aaa⁶(Y)-Leu-Aaa⁸-Pro-Aaa¹⁰-NH₂I(X)Aaa ¹ -Aaa ² -Aaa ³ -Ser-Aaa ⁵ -Aaa ⁶ (Y)-Leu-Aaa ⁸ -Pro-Aaa ¹⁰ -NH ₂ I

其中，among them,

Aaa¹为L或D型的Glp或Nal；Aaa ¹ is L or D type Glp or Nal;

Aaa²为L或D型的His或Cpa；Aaa ² is an L or D type His or Cpa;

Aaa³为L或D型的Trp或Pal；Aaa ³ is L or D type Trp or Pal;

Aaa⁵为L或D型的Tyr或Aph(L-Hor)；Aaa ⁵ is the L or D-Tyr or Aph (L-Hor);

Aaa⁶为L或D型的Lys或Aph；Aaa ⁶ is Lys or Aph of L or D type;

Aaa⁸为L或D型的Arg或ILys；Aaa ⁸ is L or D type Arg or ILys;

Aaa¹⁰为L或D型的Gly或Ala；Aaa ¹⁰ is L or D type Gly or Ala;

X选自H，Ac,-a-b,-Xaa¹-Xaa²-a-b；X is selected from the group consisting of H, Ac, -ab, -Xaa ¹ -Xaa ² -ab;

Y选自H，Cbm,-a-b,-Xaa¹-Xaa²-a-b；Y is selected from the group consisting of H, Cbm, -ab, -Xaa ¹ -Xaa ² -ab;

其中Xaa¹、Xaa²各自独立地为亲水性氨基酸；Wherein Xaa ¹ and Xaa ² are each independently a hydrophilic amino acid;

其中a为

其中m＝1-6，例如为1-3，例如为2；Where a is

Wherein m=1-6, for example 1-3, for example 2;

a通过酰胺键与肽链连接(a的羧基与肽链上的氨基形成酰胺键)。a is linked to the peptide chain via an amide bond (the carboxyl group of a forms an amide bond with the amino group on the peptide chain).

b为小分子修饰的紫杉醇或多西紫杉醇，修饰位点位于其2’位羟基。在本发明的实施方案中，所述小分子为如下所示的C1或C2，其中n＝1-10(例如为1、2、3、4、5、6、7、8、9、10；优选为1、3、5、10)，C1或C2通过酯键与紫杉醇或多西紫杉醇连接(C1的羧基与紫杉醇或多西紫杉醇的2’位羟基成酯，碳酸酯C2与紫杉醇或多西紫杉醇的2’位羟基通过酯交换反应成酯)。 b is a small molecule modified paclitaxel or docetaxel, and the modification site is located at its 2' hydroxyl group. In an embodiment of the invention, the small molecule is C1 or C2 as shown below, wherein n = 1-10 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; Preferably, 1, 3, 5, 10), C1 or C2 is linked to paclitaxel or docetaxel via an ester bond (the carboxyl group of C1 is esterified with the 2' hydroxyl group of paclitaxel or docetaxel, carbonate C2 with paclitaxel or docetaxel The 2' hydroxyl group of paclitaxel is converted to an ester by transesterification.

在本发明的实施方案中，所述小分子修饰的紫杉醇或多西紫杉醇b为以下所示的b1、b2、b3或b4，其中n＝1-10(例如为1、2、3、4、5、6、7、8、9、10；优选为1、3、5、10)。In an embodiment of the invention, the small molecule modified paclitaxel or docetaxel b is b1, b2, b3 or b4 shown below, wherein n = 1-10 (eg 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; preferably 1, 3, 5, 10).

在本发明的实施方案中，所述小分子修饰的紫杉醇b通过二硫键(如b2，b4的二硫键与a上的巯基进行交换反应形成二硫键)或硫醚键(如b1，b3上的马来酰亚胺与a上的巯基进行亲核取代形成硫醚键)与多肽链上连接的a偶联。In an embodiment of the present invention, the small molecule-modified paclitaxel b forms a disulfide bond by a disulfide bond (eg, a disulfide bond of b2, b4 is exchanged with a sulfhydryl group on a) or a thioether bond (eg, b1, The maleimide on b3 is nucleophilically substituted with a thiol group on a to form a thioether bond) coupled to a attached to the polypeptide chain.

根据本发明第一方面任一项的化合物、其立体异构体、溶剂合物或其无生理毒性的盐，其中所述的水溶性氨基酸的种类为本领域所公知，例如选自Arg、Gln、Glu、Ser、Asp、Asn、Thr等。A compound according to any one of the first aspects of the invention, a stereoisomer thereof, a solvate thereof or a non-physiologically toxic salt thereof, wherein the type of the water-soluble amino acid is known in the art For example, it is selected from the group consisting of Arg, Gln, Glu, Ser, Asp, Asn, Thr, and the like.

在本发明的实施方案中，所述-Xaa¹-Xaa²例如选自L或D型的以下肽片段，-Arg-Gln，-Gln-Arg，-Arg-Arg，-Gln-Gln,-Arg-Ser,-Arg-Asp。In an embodiment of the present invention, the -Xaa ¹ -Xaa ² for example, a peptide fragment is selected from L or D form, -Arg-Gln, -Gln-Arg , -Arg-Arg, -Gln-Gln, -Arg -Ser, -Arg-Asp.

在本发明中，所述-Xaa¹-Xaa²通过酰胺键与a连接。In the present invention, the -Xaa ¹ -Xaa ² is bonded to a through an amide bond.

在本发明中，所述a与b通过硫醚键或二硫键连接。In the present invention, the a and b are linked by a thioether bond or a disulfide bond.

在本发明中，-Xaa¹-Xaa²通过与Aaa¹的α氨基或Aaa⁶的侧链氨基形成酰胺键与多肽链相连。In the present invention, -Xaa ¹ -Xaa ² is bonded to the polypeptide chain by forming an amide bond with an α-amino group of Aaa ¹ or a side chain amino group of Aaa ⁶ .

在本发明中，-Xaa¹-Xaa²中的Xaa¹位于C端。In the present invention, Xaa ¹ in -Xaa ¹ -Xaa ² is located at the C-terminus.

在本发明中，X通过取代Aaa¹的α氨基上的H与Aaa¹相连。In the present invention, X is bonded to Aaa ¹ by substituting H on the α-amino group of Aaa ¹ .

在本发明中，Y通过取代Aaa⁶的侧链氨基上的H与Aaa⁶相连。In the present invention, Y is bonded to Aaa ⁶ by substituting H on the side chain amino group of Aaa ⁶ .

根据本发明第一方面任一项的化合物、其立体异构体、溶剂合物或其无生理毒性的盐，其选自以下化合物：A compound according to any one of the first aspects of the invention, a stereoisomer thereof, a solvate thereof or a non-physiologically toxic salt thereof, which is selected from the group consisting of:

1、(R1)Glp-His-Trp-Ser-Tyr-D-Lys(R2)-Leu-Arg-Pro-Gly-NH₂；1. (R1)Glp-His-Trp-Ser-Tyr-D-Lys(R2)-Leu-Arg-Pro-Gly-NH ₂ ;

2、(R1)Glp-His-Trp-Ser-Tyr-D-Lys(R3)-Leu-Arg-Pro-Gly-NH₂；2. (R1)Glp-His-Trp-Ser-Tyr-D-Lys(R3)-Leu-Arg-Pro-Gly-NH ₂ ;

3、(R1)Glp-His-Trp-Ser-Tyr-D-Lys(R4)-Leu-Arg-Pro-Gly-NH₂；3. (R1)Glp-His-Trp-Ser-Tyr-D-Lys(R4)-Leu-Arg-Pro-Gly-NH ₂ ;

4、(R1)Glp-His-Trp-Ser-Tyr-D-Lys(R5)-Leu-Arg-Pro-Gly-NH₂；4. (R1)Glp-His-Trp-Ser-Tyr-D-Lys(R5)-Leu-Arg-Pro-Gly-NH ₂ ;

5、(R1)Glp-His-Trp-Ser-Tyr-D-Lys(R6)-Leu-Arg-Pro-Gly-NH₂；5. (R1)Glp-His-Trp-Ser-Tyr-D-Lys(R6)-Leu-Arg-Pro-Gly-NH ₂ ;

6、(R1)Glp-His-Trp-Ser-Tyr-D-Lys(R7)-Leu-Arg-Pro-Gly-NH₂；6. (R1)Glp-His-Trp-Ser-Tyr-D-Lys(R7)-Leu-Arg-Pro-Gly-NH ₂ ;

7、(R1)Glp-His-Trp-Ser-Tyr-D-Lys(R8)-Leu-Arg-Pro-Gly-NH₂；7. (R1)Glp-His-Trp-Ser-Tyr-D-Lys(R8)-Leu-Arg-Pro-Gly-NH ₂ ;

8、(R1)Glp-His-Trp-Ser-Tyr-D-Lys(R9)-Leu-Arg-Pro-Gly-NH₂；8. (R1)Glp-His-Trp-Ser-Tyr-D-Lys(R9)-Leu-Arg-Pro-Gly-NH ₂ ;

9、(R10)D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(R2)-Leu-ILys-Pro-D-Ala-NH₂；9, (R10) D-Nal -D-Cpa-D-Pal-Ser-Aph (L-Hor) -D-Aph (R2) -Leu-ILys-Pro-D-Ala-NH 2;

10、(R10)D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(R3)-Leu-ILys-Pro-D-Ala-NH₂；10, (R10) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R3)-Leu-ILys-Pro-D-Ala-NH ₂ ;

11、(R10)D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(R4)-Leu-ILys-Pro-D-Ala-NH₂；11. (R10) D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(R4)-Leu-ILys-Pro-D-Ala-NH ₂ ;

12、(R10)D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(R5)-Leu-ILys-Pro-D-Ala-NH₂；12. (R10) D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(R5)-Leu-ILys-Pro-D-Ala-NH ₂ ;

13、(R10)D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(R6)-Leu-ILys-Pro-D-Ala-NH₂；13. (R10) D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(R6)-Leu-ILys-Pro-D-Ala-NH ₂ ;

14、(R10)D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(R7)-Leu-ILys-Pro-D-Ala-NH₂；14. (R10) D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(R7)-Leu-ILys-Pro-D-Ala-NH ₂ ;

15、(R10)D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(R8)-Leu-ILys-Pro-D-Ala-NH₂；15. (R10) D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(R8)-Leu-ILys-Pro-D-Ala-NH ₂ ;

16、(R10)D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(R9)-Leu-ILys-Pro-D-Ala-NH₂； 16. (R10) D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(R9)-Leu-ILys-Pro-D-Ala-NH ₂ ;

17、(R2)D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(R11)-Leu-ILys-Pro-D-Ala-NH₂；17. (R2)D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(R11)-Leu-ILys-Pro-D-Ala-NH ₂ ;

18、(R3)D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(R11)-Leu-ILys-Pro-D-Ala-NH₂；18, (R3) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R11)-Leu-ILys-Pro-D-Ala-NH ₂ ;

19、(R4)D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(R11)-Leu-ILys-Pro-D-Ala-NH₂；19. (R4) D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(R11)-Leu-ILys-Pro-D-Ala-NH ₂ ;

20、(R5)D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(R11)-Leu-ILys-Pro-D-Ala-NH₂；20, (R5) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R11)-Leu-ILys-Pro-D-Ala-NH ₂ ;

21、(R6)D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(R11)-Leu-ILys-Pro-D-Ala-NH₂；21, (R6) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R11)-Leu-ILys-Pro-D-Ala-NH ₂ ;

22、(R7)D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(R11)-Leu-ILys-Pro-D-Ala-NH₂；22. (R7) D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(R11)-Leu-ILys-Pro-D-Ala-NH ₂ ;

23、(R8)D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(R11)-Leu-ILys-Pro-D-Ala-NH₂；23, (R8) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R11)-Leu-ILys-Pro-D-Ala-NH ₂ ;

24、(R9)D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(R11)-Leu-ILys-Pro-D-Ala-NH₂；24, (R9) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R11)-Leu-ILys-Pro-D-Ala-NH ₂ ;

25、(R5)D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(R5)-Leu-ILys-Pro-D-Ala-NH₂；25, (R5) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R5)-Leu-ILys-Pro-D-Ala-NH ₂ ;

26、(R6)D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(R6)-Leu-ILys-Pro-D-Ala-NH₂；26, (R6) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R6)-Leu-ILys-Pro-D-Ala-NH ₂ ;

27、(7)D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(R7)-Leu-ILys-Pro-D-Ala-NH₂；27, (7) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R7)-Leu-ILys-Pro-D-Ala-NH ₂ ;

28、(R8)D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(R8)-Leu-ILys-Pro-D-Ala-NH₂；28, (R8) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R8)-Leu-ILys-Pro-D-Ala-NH ₂ ;

29、(R9)D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(R9)-Leu-ILys-Pro-D-Ala-NH₂；29, (R9) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R9)-Leu-ILys-Pro-D-Ala-NH ₂ ;

30、(R1)Glp-His-Trp-Ser-Tyr-D-Lys(R12)-Leu-Arg-Pro-Gly-NH₂；30, (R1) Glp-His-Trp-Ser-Tyr-D-Lys (R12)-Leu-Arg-Pro-Gly-NH ₂ ;

31、(R1)Glp-His-Trp-Ser-Tyr-D-Lys(R13)-Leu-Arg-Pro-Gly-NH₂；31, (R1) Glp-His-Trp-Ser-Tyr-D-Lys (R13)-Leu-Arg-Pro-Gly-NH ₂ ;

32、(R1)Glp-His-Trp-Ser-Tyr-D-Lys(R14)-Leu-Arg-Pro-Gly-NH₂；32, (R1) Glp-His-Trp-Ser-Tyr-D-Lys (R14)-Leu-Arg-Pro-Gly-NH ₂ ;

33、(R1)Glp-His-Trp-Ser-Tyr-D-Lys(R15)-Leu-Arg-Pro-Gly-NH₂；33, (R1) Glp-His-Trp-Ser-Tyr-D-Lys (R15)-Leu-Arg-Pro-Gly-NH ₂ ;

34、(R1)Glp-His-Trp-Ser-Tyr-D-Lys(R16)-Leu-Arg-Pro-Gly-NH₂；34, (R1) Glp-His-Trp-Ser-Tyr-D-Lys (R16)-Leu-Arg-Pro-Gly-NH ₂ ;

35、(R1)Glp-His-Trp-Ser-Tyr-D-Lys(R17)-Leu-Arg-Pro-Gly-NH₂；35, (R1) Glp-His-Trp-Ser-Tyr-D-Lys (R17)-Leu-Arg-Pro-Gly-NH ₂ ;

36、(R1)Glp-His-Trp-Ser-Tyr-D-Lys(R18)-Leu-Arg-Pro-Gly-NH₂；36, (R1) Glp-His-Trp-Ser-Tyr-D-Lys (R18)-Leu-Arg-Pro-Gly-NH ₂ ;

37、(R1)Glp-His-Trp-Ser-Tyr-D-Lys(R19)-Leu-Arg-Pro-Gly-NH₂；37, (R1) Glp-His-Trp-Ser-Tyr-D-Lys (R19)-Leu-Arg-Pro-Gly-NH ₂ ;

38、(R10)D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(R12)-Leu-ILys-Pro-D-Ala-NH₂；38, (R10) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R12)-Leu-ILys-Pro-D-Ala-NH ₂ ;

39、(R10)D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(R13)-Leu-ILys-Pro-D-Ala-NH₂；39, (R10) D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(R13)-Leu-ILys-Pro-D-Ala-NH ₂ ;

40、(R10)D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(R14)-Leu-ILys-Pro-D-Ala-NH₂；40, (R10) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R14)-Leu-ILys-Pro-D-Ala-NH ₂ ;

41、(R10)D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(R15)-Leu-ILys-Pro-D-Ala-NH₂；41, (R10) D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(R15)-Leu-ILys-Pro-D-Ala-NH ₂ ;

42、(R10)D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(R16)-Leu-ILys-Pro-D-Ala-NH₂；42. (R10)D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(R16)-Leu-ILys-Pro-D-Ala-NH ₂ ;

43、(R10)D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(R17)-Leu-ILys-Pro-D-Ala-NH₂；43. (R10)D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(R17)-Leu-ILys-Pro-D-Ala-NH ₂ ;

44、(R10)D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(R18)-Leu-ILys-Pro-D-Ala-NH₂；44, (R10) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R18)-Leu-ILys-Pro-D-Ala-NH ₂ ;

45、(R10)D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(R19)-Leu-ILys-Pro-D-Ala-NH₂； 45, (R10) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R19)-Leu-ILys-Pro-D-Ala-NH ₂ ;

46、(R12)D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(R11)-Leu-ILys-Pro-D-Ala-NH₂；46, (R12) D-Nal -D-Cpa-D-Pal-Ser-Aph (L-Hor) -D-Aph (R11) -Leu-ILys-Pro-D-Ala-NH 2;

47、(R13)D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(R11)-Leu-ILys-Pro-D-Ala-NH₂；47, (R13) D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(R11)-Leu-ILys-Pro-D-Ala-NH ₂ ;

48、(R14)D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(R11)-Leu-ILys-Pro-D-Ala-NH₂；48, (R14) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R11)-Leu-ILys-Pro-D-Ala-NH ₂ ;

49、(R15)D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(R11)-Leu-ILys-Pro-D-Ala-NH₂；49, (R15) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R11)-Leu-ILys-Pro-D-Ala-NH ₂ ;

50、(R16)D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(R11)-Leu-ILys-Pro-D-Ala-NH₂；50, (R16) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R11)-Leu-ILys-Pro-D-Ala-NH ₂ ;

51、(R17)D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(R11)-Leu-ILys-Pro-D-Ala-NH₂；51, (R17) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R11)-Leu-ILys-Pro-D-Ala-NH ₂ ;

52、(R18)D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(R11)-Leu-ILys-Pro-D-Ala-NH₂；52, (R18) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R11)-Leu-ILys-Pro-D-Ala-NH ₂ ;

53、(R19)D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(R11)-Leu-ILys-Pro-D-Ala-NH₂；53. (R19) D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(R11)-Leu-ILys-Pro-D-Ala-NH ₂ ;

54、(R15)D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(R15)-Leu-ILys-Pro-D-Ala-NH₂；54. (R15)D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(R15)-Leu-ILys-Pro-D-Ala-NH ₂ ;

55、(R16)D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(R16)-Leu-ILys-Pro-D-Ala-NH₂；55, (R16) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R16)-Leu-ILys-Pro-D-Ala-NH ₂ ;

56、(R17)D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(R17)-Leu-ILys-Pro-D-Ala-NH₂；56, (R17) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R17)-Leu-ILys-Pro-D-Ala-NH ₂ ;

57、(R18)D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(R18)-Leu-ILys-Pro-D-Ala-NH₂；57, (R18) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R18)-Leu-ILys-Pro-D-Ala-NH ₂ ;

58、(R19)D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(R19)-Leu-ILys-Pro-D-Ala-NH₂；58. (R19) D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(R19)-Leu-ILys-Pro-D-Ala-NH ₂ ;

其中R1-R19对应修饰基团见表1。Wherein R1-R19 corresponds to a modifying group as shown in Table 1.

表1Table 1

在本发明中，上述1-58的编号即为该化合物编号，可代表该化合物。In the present invention, the above 1-58 number is the compound number, which may represent the compound.

在本发明中，如未特别指明，则氨基酸为L型氨基酸。In the present invention, the amino acid is an L-form amino acid unless otherwise specified.

本发明第二方面涉及药物组合物，其含有至少一种本发明第一方面任一项的化合物、其立体异构体、溶剂合物或其无生理毒性的盐，以及可药用载体或赋形剂。A second aspect of the invention relates to a pharmaceutical composition comprising at least one compound according to any one of the first aspects of the invention, a stereoisomer thereof, a solvate thereof or a salt thereof which is not physiologically toxic, and a pharmaceutically acceptable carrier or Shape agent.

本发明的第三方面涉及本发明第一方面任一项的化合物、其立体异构体、溶剂合物或其无生理毒性的盐的制备方法，其包括以下步骤：A third aspect of the invention relates to a process for the preparation of a compound according to any one of the first aspects of the invention, a stereoisomer thereof, a solvate thereof or a salt thereof which is not physiologically toxic, comprising the steps of:

(1)6位氨基酸修饰：按照固相合成Boc、Fmoc交叉保护策略从C端向N端合成肽序列至6位氨基酸，20-50％哌啶/DMF溶液脱除6位氨基酸侧链氨基上的Fmoc保护基，加入修饰单元(对甲苄基保护的巯基丙酸)、DIC、HOBT，反应4个小时后茚三酮检测，反应完全后，继续依次完成氨基酸的缩合，肽序列完成后，无水HF裂解肽树脂，10-30％醋酸水溶液溶解，冷冻干燥后得粗肽，中压色谱纯化得纯品；(1) 6-amino acid modification: According to the solid phase synthesis Boc, Fmoc cross-protection strategy, the peptide sequence was synthesized from the C-terminus to the N-terminus to the 6-position amino acid, and the 20-50% piperidine/DMF solution was removed from the 6-position amino acid side chain amino group. The Fmoc protecting group is added with a modifying unit (p-methylbenzyl-protected mercaptopropionic acid), DIC, HOBT, and the ninhydrin is detected after 4 hours of reaction. After the reaction is completed, the amino acid condensation is sequentially completed, and after the peptide sequence is completed, Anhydrous HF-cleaved peptide resin, dissolved in 10-30% aqueous acetic acid solution, lyophilized to obtain a crude peptide, and purified by medium pressure chromatography to obtain a pure product;

(2)N端修饰(包括同时修饰N端和6位氨基酸)：按照固相合成Boc、Fmoc交叉保护策略从C端向N端合成肽序列至6位氨基酸或N端，20-50％哌啶/DMF溶液脱除6位氨基酸侧链氨基上的Fmoc保护基，加入修饰单元(对甲苄基保护的巯基丙酸)、DIC、HOBT，反应4个小时后茚三酮检测，反应完全后，继续依次完成氨基酸的缩合，直至肽序列所有氨基酸及修饰单元完成缩合，最后用无水HF裂解肽树脂，10-30％醋酸水溶液溶解，冷冻干燥后得粗肽，中压色谱纯化得纯品；(2) N-terminal modification (including simultaneous modification of N-terminal and 6-position amino acids): synthesis of peptide sequence from C-terminus to N-terminus to 6-position amino acid or N-terminus according to solid phase synthesis Boc and Fmoc cross-protection strategy, 20-50% piperidine The acyl/DMF solution was used to remove the Fmoc protecting group on the amino acid side chain amino acid of the 6-position amino acid, and the modified unit (p-methylbenzyl-protected mercaptopropionic acid), DIC, HOBT was added. After the reaction for 4 hours, the ninhydrin was detected. After the reaction was completed, the reaction was completed. Continue to complete the condensation of amino acids in sequence until all amino acids and modified units of the peptide sequence are condensed. Finally, the peptide resin is lysed with anhydrous HF, dissolved in 10-30% aqueous acetic acid solution, and lyophilized to obtain crude peptide, which is purified by medium pressure chromatography. ;

(3)肽序列以硫醚键与小分子修饰的紫杉醇的偶联：将纯化后的肽序列与小分子修饰的紫杉醇在50％的乙腈/水溶液中，室温搅拌反应,反应完全后，中压制备色谱纯化得纯品；(3) Coupling of the peptide sequence with a small molecule modified paclitaxel: the purified peptide sequence and the small molecule modified paclitaxel are stirred in a 50% acetonitrile/water solution at room temperature, and after the reaction is completed, the medium is pressed. Prepared by chromatography to obtain a pure product;

(4)肽序列以二硫键与小分子修饰的紫杉醇的偶联：将纯化后的肽序列与小分子修饰的紫杉醇在DMSO中，室温搅拌反应,反应完全后，中压制备色谱纯化得纯品。(4) Coupling of the peptide sequence with a small molecule modified paclitaxel: the purified peptide sequence and the small molecule modified paclitaxel in DMSO, stirring at room temperature, the reaction is complete After that, it was purified by medium pressure preparative chromatography to obtain a pure product.

本发明的第四方面涉及本发明第一方面任一项的化合物、其立体异构体或其无生理毒性的盐在制备用于预防和/或治疗肿瘤的药物中的用途。A fourth aspect of the invention relates to the use of a compound according to any one of the first aspects of the invention, a stereoisomer thereof or a non-physiologically toxic salt thereof for the manufacture of a medicament for the prevention and/or treatment of a tumor.

根据本发明第四方面任一项所述的用途，其中所述肿瘤包括但不限于，皮肤基底细胞癌，胆道癌；膀胱癌；骨癌；脑和CNS癌；乳腺癌；宫颈癌；绒毛膜癌；结肠和直肠癌；结缔组织癌；消化系统的癌症；子宫内膜的癌症；食道癌；眼癌；头部和颈部的癌症；胃癌；上皮细胞内的赘生物；肾癌；喉癌；白血病；肝癌；肺癌(例如小细胞和非小细胞)；淋巴瘤包括霍奇金的和非霍奇金的淋巴瘤；黑素瘤；骨髓瘤；成神经细胞瘤；口腔癌(例如，唇，舌，口腔和咽)；卵巢癌；胰腺癌；前列腺癌；成视网膜细胞瘤；横纹肌肉瘤；直肠癌；呼吸系统的癌；肉瘤；皮肤癌；睾丸癌；甲状腺癌；子宫癌；泌尿系统的癌症以及其它的癌和肉瘤。Use according to any of the fourth aspects of the invention, wherein the tumor comprises, but is not limited to, cutaneous basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and CNS cancer; breast cancer; cervical cancer; chorion Cancer; colon and rectal cancer; connective tissue cancer; cancer of the digestive system; cancer of the endometrium; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer; neoplasms in epithelial cells; Leukemia; liver cancer; lung cancer (eg small cells and non-small cells); lymphoma including Hodgkin's and non-Hodgkin's lymphoma; melanoma; myeloma; neuroblastoma; oral cancer (eg, lip , tongue, mouth and pharynx; ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; respiratory cancer; sarcoma; skin cancer; testicular cancer; thyroid cancer; uterine cancer; urinary system Cancer and other cancers and sarcomas.

本发明第五方面涉及预防和/或治疗肿瘤的方法，所述方法包括给有需要的受试者预防和/或治疗有效量的本发明第一方面任一项的化合物、其立体异构体或其无生理毒性的盐的步骤。A fifth aspect of the invention relates to a method of preventing and/or treating a tumor comprising administering to a subject in need thereof a prophylactically and/or therapeutically effective amount of a compound according to any one of the first aspects of the invention, a stereoisomer thereof Or a step of its physiologically toxic salt.

根据本发明第五方面任一项所述的方法，其中所述肿瘤包括但不限于，皮肤基底细胞癌，胆道癌；膀胱癌；骨癌；脑和CNS癌；乳腺癌；宫颈癌；绒毛膜癌；结肠和直肠癌；结缔组织癌；消化系统的癌症；子宫内膜的癌症；食道癌；眼癌；头部和颈部的癌症；胃癌；上皮细胞内的赘生物；肾癌；喉癌；白血病；肝癌；肺癌(例如小细胞和非小细胞)；淋巴瘤包括霍奇金的和非霍奇金的淋巴瘤；黑素瘤；骨髓瘤；成神经细胞瘤；口腔癌(例如，唇，舌，口腔和咽)；卵巢癌；胰腺癌；前列腺癌；成视网膜细胞瘤；横纹肌肉瘤；直肠癌；呼吸系统的癌；肉瘤；皮肤癌；睾丸癌；甲状腺癌；子宫癌；泌尿系统的癌症以及其它的癌和肉瘤。The method according to any of the fifth aspects of the invention, wherein the tumor comprises, but is not limited to, cutaneous basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and CNS cancer; breast cancer; cervical cancer; chorion Cancer; colon and rectal cancer; connective tissue cancer; cancer of the digestive system; cancer of the endometrium; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer; neoplasms in epithelial cells; Leukemia; liver cancer; lung cancer (eg small cells and non-small cells); lymphoma including Hodgkin's and non-Hodgkin's lymphoma; melanoma; myeloma; neuroblastoma; oral cancer (eg, lip , tongue, mouth and pharynx; ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; respiratory cancer; sarcoma; skin cancer; testicular cancer; thyroid cancer; uterine cancer; urinary system Cancer and other cancers and sarcomas.

根据本发明第五方面任一项所述的方法，其中所述受试者为哺乳动物，例如牛科动物、马科动物、羊科动物、猪科动物、犬科动物、猫科动物、啮齿科动物、灵长类动物；其中，优选的受试者为人。The method according to any of the fifth aspects of the invention, wherein the subject is a mammal, such as a bovine, an equine, a sheep, a porcine, a canine, a feline Animals, rodents, primates; wherein the preferred subject is a human.

本发明第六方面涉及本发明第一方面任一项的化合物、其立体异构体或其无生理毒性的盐，其用于预防和/或治疗肿瘤。A sixth aspect of the invention relates to a compound according to any one of the first aspects of the invention, a stereoisomer thereof or a non-physiologically toxic salt thereof for use in the prevention and/or treatment of a tumor.

根据本发明第六方面任一项所述的组合物、其立体异构体或其无生理毒性的盐，其中所述肿瘤包括但不限于，皮肤基底细胞癌，胆道癌；膀胱癌；骨癌；脑和CNS癌；乳腺癌；宫颈癌；绒毛膜癌；结肠和直肠癌；结缔组织癌；消化系统的癌症；子宫内膜的癌症；食道癌；眼癌；头部和颈部的癌症；胃癌；上皮细胞内的赘生物；肾癌；喉癌；白血病；肝癌；肺癌(例如小细胞和非小细胞)；淋巴瘤包括霍奇金的和非霍奇金的淋巴瘤；黑素瘤；骨髓瘤；成神经细胞瘤；口腔癌(例如，唇，舌，口腔和咽)；卵巢癌；胰腺癌；前列腺癌；成视网膜细胞瘤；横纹肌肉瘤；直肠癌；呼吸系统的癌；肉瘤；皮肤癌；睾丸癌；甲状腺癌；子宫癌；泌尿系统的癌症以及其它的癌和肉瘤。The composition according to any one of the sixth aspects of the present invention, or a stereoisomer thereof, or a physiologically toxic salt thereof, wherein the tumor includes, but is not limited to, cutaneous basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer ; brain and CNS cancer; breast cancer; cervical cancer; choriocarcinoma; colon and rectal cancer; connective tissue cancer; cancer of the digestive system; cancer of the endometrium; esophageal cancer; eye cancer; cancer of the head and neck; Gastric cancer; neoplasms in epithelial cells; kidney cancer; laryngeal cancer; leukemia; liver cancer; lung cancer (eg, small cells and non-small cells); lymphoma including Hodgkin's and non-Hodgkin's lymphoma; melanoma; Myeloma; neuroblastoma; oral cancer (eg, lips, tongue, mouth and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; respiratory cancer; sarcoma; Cancer; testicular cancer; thyroid cancer; uterine cancer; cancer of the urinary system and other cancers and sarcomas.

本发明第七方面涉及本发明第一方面任一项的化合物、其立体异构体或其无生理毒性的盐用于制备试剂的用途，所述试剂用于杀伤和/或杀死肿瘤细胞。A seventh aspect of the invention relates to the use of a compound according to any one of the first aspects of the invention, a stereoisomer thereof or a non-physiologically toxic salt thereof for the preparation of a reagent for killing and/or killing tumor cells.

在本发明的一个实施方案中，所述试剂用于体外方法中。In one embodiment of the invention, the agent is for use in an in vitro method.

在本发明的一个实施方案中，所述试剂用于体内方法中。In one embodiment of the invention, the agent is for use in an in vivo method.

在本发明的一个实施方案中，其中所述肿瘤细胞为肿瘤细胞系，或者来自受试者的肿瘤细胞。In one embodiment of the invention, wherein the tumor cell is a tumor cell line, or a tumor cell from a subject.

根据本发明第七方面任一项所述的用途，其中所述肿瘤细胞包括但不限于，皮肤基底癌细胞，胆道癌细胞；膀胱癌细胞；骨癌细胞；脑和CNS癌细胞；乳腺癌细胞；宫颈癌细胞；绒毛膜癌细胞；结肠和直肠癌细胞；结缔组织癌细胞；消化系统的癌细胞；子宫内膜的癌细胞；食道癌细胞；眼癌细胞；头部和颈部的癌细胞；胃癌细胞；肾癌细胞；喉癌细胞；白血病细胞；肝癌细胞；肺癌细胞(例如小细胞和非小细胞)；淋巴瘤细胞包括霍奇金的和非霍奇金的淋巴瘤细胞；黑素瘤细胞；骨髓瘤细胞；成神经细胞瘤细胞；口腔癌细胞(例如，唇，舌，口腔和咽癌细胞)；卵巢癌细胞；胰腺癌细胞；前列腺癌细胞；成视网膜瘤细胞；横纹肌肉瘤细胞；直肠癌细胞；呼吸系统的癌细胞；肉瘤细胞；皮肤癌细胞；睾丸癌细胞；甲状腺癌细胞；子宫癌细胞；泌尿系统的癌细胞以及其它的癌细胞和肉瘤细胞。The use according to any of the seventh aspects of the invention, wherein the tumor cells include, but are not limited to, skin basal cancer cells, biliary cancer cells; bladder cancer cells; bone cancer cells; brain and CNS cancer cells; Cervical cancer cells; choriocarcinoma cells; colon and rectal cancer cells; connective tissue cancer cells; cancer cells of the digestive system; cancer cells of the endometrium; esophageal cancer cells; Gastric cancer cells; renal cancer cells; laryngeal cancer cells; leukemia cells; liver cancer cells; lung cancer cells (eg, small cells and non-small cells); lymphoma cells including Hodgkin's and non-Hodgkin's lymphoma cells; Tumor cell Tumor cells; neuroblastoma cells; oral cancer cells (eg, lips, tongue, mouth and pharyngeal cancer cells); ovarian cancer cells; pancreatic cancer cells; prostate cancer cells; retinal tumor cells; rhabdomyosarcoma cells; Cancer cells of the respiratory system; sarcoma cells; skin cancer cells; testicular cancer cells; thyroid cancer cells; uterine cancer cells; cancer cells of the urinary system and other cancer cells and sarcoma cells.

本发明第八方面涉及杀伤和/或杀死肿瘤细胞的方法，所述方法包括给所述细胞施用有效量的本发明第一方面任一项的化合物、其立体异构体或其无生理毒性的盐。An eighth aspect of the invention relates to a method of killing and/or killing a tumor cell, the method comprising administering to the cell an effective amount of a compound according to any one of the first aspects of the invention, a stereoisomer thereof or no physiological toxicity thereof Salt.

在本发明的一个实施方案中，所述方法在体外进行。In one embodiment of the invention, the method is carried out in vitro.

在本发明的一个实施方案中，所述方法在体内进行。In one embodiment of the invention, the method is carried out in vivo.

根据本发明第八方面任一项所述的用途，其中所述肿瘤细胞包括但不限于，皮肤基底癌细胞，胆道癌细胞；膀胱癌细胞；骨癌细胞；脑和CNS癌细胞；乳腺癌细胞；宫颈癌细胞；绒毛膜癌细胞；结肠和直肠癌细胞；结缔组织癌细胞；消化系统的癌细胞；子宫内膜的癌细胞；食道癌细胞；眼癌细胞；头部和颈部的癌细胞；胃癌细胞；肾癌细胞；喉癌细胞；白血病细胞；肝癌细胞；肺癌细胞(例如小细胞和非小细胞)；淋巴瘤细胞包括霍奇金的和非霍奇金的淋巴瘤细胞；黑素瘤细胞；骨髓瘤细胞；成神经细胞瘤细胞；口腔癌细胞(例如，唇，舌，口腔和咽癌细胞)；卵巢癌细胞；胰腺癌细胞；前列腺癌细胞；成视网膜瘤细胞；横纹肌肉瘤细胞；直肠癌细胞；呼吸系统的癌细胞；肉瘤细胞；皮肤癌细胞；睾丸癌细胞；甲状腺癌细胞；子宫癌细胞；泌尿系统的癌细胞以及其它的癌细胞和肉瘤细胞。The use according to any of the eighth aspects of the present invention, wherein the tumor cells include, but are not limited to, skin basal cancer cells, biliary cancer cells; bladder cancer cells; bone cancer cells; brain and CNS cancer cells; Cervical cancer cells; choriocarcinoma cells; colon and rectal cancer cells; connective tissue cancer cells; cancer cells of the digestive system; cancer cells of the endometrium; esophageal cancer cells; Gastric cancer cells; renal cancer cells; laryngeal cancer cells; leukemia cells; liver cancer cells; lung cancer cells (eg, small cells and non-small cells); lymphoma cells including Hodgkin's and non-Hodgkin's lymphoma cells; Tumor cells; myeloma cells; neuroblastoma cells; oral cancer cells (eg, lips, tongue, mouth and pharyngeal cancer cells); ovarian cancer cells; pancreatic cancer cells; prostate cancer cells; retinal tumor cells; rhabdomyosarcoma cells Rectal cancer cells; cancer cells of the respiratory system; sarcoma cells; skin cancer cells; testicular cancer cells; thyroid cancer cells; uterine cancer cells; cancer of the urinary system Cells and other cancer cells and sarcoma cells.

本发明第九方面涉及本发明第一方面任一项的化合物、其立体异构体或其无生理毒性的盐，其用于杀伤和/或杀死肿瘤细胞。A ninth aspect of the invention relates to a compound according to any one of the first aspects of the invention, a stereoisomer thereof or a non-physiologically toxic salt thereof for use in killing and/or killing tumor cells.

在本发明的一个实施方案中，其用于体外方法中。 In one embodiment of the invention, it is used in an in vitro method.

在本发明的一个实施方案中，其用于体内方法中。In one embodiment of the invention, it is used in an in vivo method.

根据本发明第九方面任一项所述的化合物、其立体异构体或其无生理毒性的盐，其中所述肿瘤细胞包括但不限于，皮肤基底癌细胞，胆道癌细胞；膀胱癌细胞；骨癌细胞；脑和CNS癌细胞；乳腺癌细胞；宫颈癌细胞；绒毛膜癌细胞；结肠和直肠癌细胞；结缔组织癌细胞；消化系统的癌细胞；子宫内膜的癌细胞；食道癌细胞；眼癌细胞；头部和颈部的癌细胞；胃癌细胞；肾癌细胞；喉癌细胞；白血病细胞；肝癌细胞；肺癌细胞(例如小细胞和非小细胞)；淋巴瘤细胞包括霍奇金的和非霍奇金的淋巴瘤细胞；黑素瘤细胞；骨髓瘤细胞；成神经细胞瘤细胞；口腔癌细胞(例如，唇，舌，口腔和咽癌细胞)；卵巢癌细胞；胰腺癌细胞；前列腺癌细胞；成视网膜瘤细胞；横纹肌肉瘤细胞；直肠癌细胞；呼吸系统的癌细胞；肉瘤细胞；皮肤癌细胞；睾丸癌细胞；甲状腺癌细胞；子宫癌细胞；泌尿系统的癌细胞以及其它的癌细胞和肉瘤细胞。The compound according to any one of the ninth aspects of the present invention, the stereoisomer thereof or the non-physiologically toxic salt thereof, wherein the tumor cells include, but are not limited to, skin basal cancer cells, biliary cancer cells; bladder cancer cells; Bone cancer cells; brain and CNS cancer cells; breast cancer cells; cervical cancer cells; choriocarcinoma cells; colon and rectal cancer cells; connective tissue cancer cells; cancer cells of the digestive system; cancer cells of the endometrium; esophageal cancer cells ; eye cancer cells; cancer cells in the head and neck; gastric cancer cells; renal cancer cells; laryngeal cancer cells; leukemia cells; liver cancer cells; lung cancer cells (eg, small cells and non-small cells); lymphoma cells including Hodgkin And non-Hodgkin's lymphoma cells; melanoma cells; myeloma cells; neuroblastoma cells; oral cancer cells (eg, lips, tongue, mouth and pharyngeal cancer cells); ovarian cancer cells; pancreatic cancer cells Prostate cancer cells; retinal neoplasia cells; rhabdomyosarcoma cells; rectal cancer cells; cancer cells of the respiratory system; sarcoma cells; skin cancer cells; testicular cancer cells; Cancer cells; uterine cancer cells; cancer cells of the urinary system and other cancer cells and sarcoma cells.

本发明还涉及一种用于杀伤和/或杀死肿瘤细胞的试剂盒，所述的试剂盒包括本发明第一方面任一项的化合物、其立体异构体或其无生理毒性的盐，且，任选地还包括使用说明。The invention also relates to a kit for killing and/or killing tumor cells, the kit comprising a compound according to any one of the first aspects of the invention, a stereoisomer thereof or a non-physiologically toxic salt thereof, And, optionally, instructions for use are also included.

在本发明中，所述肿瘤包括但不限于，皮肤基底细胞癌，胆道癌；膀胱癌；骨癌；脑和CNS癌；乳腺癌；宫颈癌；绒毛膜癌；结肠和直肠癌；结缔组织癌；消化系统的癌症；子宫内膜的癌症；食道癌；眼癌；头部和颈部的癌症；胃癌；上皮细胞内的赘生物；肾癌；喉癌；白血病；肝癌；肺癌(例如小细胞和非小细胞)；淋巴瘤包括霍奇金的和非霍奇金的淋巴瘤；黑素瘤；骨髓瘤；成神经细胞瘤；口腔癌(例如，唇，舌，口腔和咽)；卵巢癌；胰腺癌；前列腺癌；成视网膜细胞瘤；横纹肌肉瘤；直肠癌；呼吸系统的癌；肉瘤；皮肤癌；睾丸癌；甲状腺癌；子宫癌；泌尿系统的癌症以及其它的癌和肉瘤。 In the present invention, the tumor includes, but is not limited to, cutaneous basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and CNS cancer; breast cancer; cervical cancer; choriocarcinoma; colon and rectal cancer; connective tissue cancer ; cancer of the digestive system; cancer of the endometrium; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer; neoplasms in epithelial cells; kidney cancer; laryngeal cancer; leukemia; liver cancer; And non-small cells; lymphomas include Hodgkin's and non-Hodgkin's lymphoma; melanoma; myeloma; neuroblastoma; oral cancer (eg, lips, tongue, mouth and pharynx); ovarian cancer Pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; respiratory cancer; sarcoma; skin cancer; testicular cancer; thyroid cancer; uterine cancer; urinary cancer and other cancers and sarcomas.

发明的有益效果Advantageous effects of the invention

本发明人经研究发现本发明第一方面所示化合物，其保持了良好的GnRH受体结合活性，且具有较强的肿瘤细胞生长抑制活性；而且具有一定的肿瘤细胞选择性，同时稳定性得到了大幅度的提高，有利于降低药物由于过早的释放抗癌因子而产生的毒副作用。另外，本发明的化合物分别保持了其配体的GnRH受体激动和拮抗活性，对于性激素依赖的肿瘤(如：前列腺癌、子宫内膜癌、乳腺癌等)可能起到协同治疗的作用。The present inventors have found that the compound of the first aspect of the present invention maintains a good GnRH receptor binding activity and has strong tumor cell growth inhibitory activity; and has certain tumor cell selectivity and stability. A substantial increase is beneficial to reduce the side effects of drugs caused by premature release of anti-cancer factors. In addition, the compounds of the present invention retain the GnRH receptor agonistic and antagonistic activities of their ligands, respectively, and may play a synergistic therapeutic role for sex hormone-dependent tumors (e.g., prostate cancer, endometrial cancer, breast cancer, etc.).

DRAWINGS

图1缀合物1和4的血浆稳定性Figure 1 Plasma stability of conjugates 1 and 4

图2缀合物9-11和缀合物17-19的血浆稳定性Figure 2 Plasma stability of conjugate 9-11 and conjugate 17-19

图3缀合物12、16、20、24和29的血浆稳定性Figure 3 Plasma stability of conjugates 12, 16, 20, 24 and 29

图4紫杉醇(PTX)的受体饱和实验图Figure 4: Receptor saturation experiment of paclitaxel (PTX)

图5缀合物4的受体饱和实验图Figure 5: Receptor saturation experiment diagram of conjugate 4

图6缀合物9的受体饱和实验图Figure 6 Receptor saturation experiment diagram of conjugate 9

图7缀合物20的受体饱和实验图Figure 7 is a graph of acceptor saturation of conjugate 20

detailed description

下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。The embodiments of the present invention will be described in detail below with reference to the accompanying drawings, however, the following examples are intended to illustrate the invention and are not intended to limit the scope of the invention. Those who do not specify the specific conditions in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are conventional products that can be obtained commercially.

在本发明中使用的缩写具有下面的含义：The abbreviations used in the present invention have the following meanings:

Ac–乙酰基Ac-acetyl

Ala-丙氨酸Ala-alanine

Arg-精氨酸 Arg-arginine

Aph-4-氨基苯丙氨酸Aph-4-aminophenylalanine

Boc-叔丁氧羰基Boc-tert-butoxycarbonyl

Cbm-氨基甲酰基Cbm-carbamoyl

Cpa-4-氯苯丙氨酸Cpa-4-chlorophenylalanine

DIC-二异丙基碳二亚胺DIC-diisopropylcarbodiimide

DMF-N,N-二甲基甲酰胺DMF-N, N-dimethylformamide

Fmoc-芴甲氧羰基Fmoc-芴methoxycarbonyl

Gly-甘氨酸Gly-glycine

Gln-谷氨酰胺Gln-glutamine

Glp-焦谷氨酸Glp-pyroglutamic acid

HF-氢氟酸HF-hydrofluoric acid

HOBt-1-羟基苯并三唑HOBt-1-hydroxybenzotriazole

L-Hor-L-4，5-二氢乳清酸L-Hor-L-4,5-dihydroorotate

Leu-亮氨酸Leu-leucine

Lys-赖氨酸Lys-lysine

ILys-异丙基赖氨酸ILys-isopropyl lysine

MBHA-苯基氨甲基树脂MBHA-phenylaminomethyl resin

Nal-萘丙氨酸Nal-naphthylalanine

Pal-3-吡啶丙氨酸Pal-3-pyridine alanine

Pro-脯氨酸Pro-proline

Ser-丝氨酸Ser-serine

Trp-色氨酸Trp-tryptophan

His-组氨酸His-histidine

Tyr-酪氨酸Tyr-tyrosine

Glu-谷氨酸Glu-glutamic acid

Asp-天冬氨酸Asp-aspartate

Asn-天冬酰胺Asn-asparagine

Thr-苏氨酸 Thr-threonine

除非另有说明，在本申请中使用的术语具有以下含义:Terms used in this application have the following meanings unless otherwise stated:

本发明中，所有氨基酸构型除注明为D-型外，均为L-型；In the present invention, all amino acid configurations are L-form except for the D-type;

侧链氨基指氨基酸上α氨基之外的氨基；A side chain amino group means an amino group other than the alpha amino group of the amino acid;

茚三酮指茚三酮指示试剂：Ninhydrin refers to ninhydrin indicator reagent:

(a)茚三酮/乙醇(5g/100mL)；(a) ninhydrin/ethanol (5 g/100 mL);

(b)苯酚/乙醇(4g/mL)；(b) phenol/ethanol (4 g/mL);

(c)KCN/吡啶溶液(2mL×0.01mol/L/100mL)。(c) KCN/pyridine solution (2 mL x 0.01 mol/L/100 mL).

本发明还涉及含有作为活性成分的有效剂量的至少一种式(I)和/或其立体异构体或其无生理毒性的盐以及常规药物赋形剂或辅剂的药物组合物。The invention further relates to a pharmaceutical composition comprising as an active ingredient an effective amount of at least one of the formula (I) and/or its stereoisomers or its non-physiologically toxic salts, as well as conventional pharmaceutical excipients or adjuvants.

本发明所用术语“常规药物赋形剂或辅剂”包括任一种或所有溶剂，分散介质，包衣，抗菌剂或抗真菌剂，等渗及缓释试剂，以及类似的生理配伍制剂，以适合静脉注射，肌肉注射，皮下注射，或其它给药方式，如口服给药。根据给药的方式，可将活性化合物包衣以保护化合物免受酸或其它自然条件的影响而失活。The term "conventional pharmaceutical excipient or adjuvant" as used herein includes any or all solvents, dispersion media, coatings, antibacterial or antifungal agents, isotonic and sustained release agents, and similar physiologically compatible formulations, Suitable for intravenous, intramuscular, subcutaneous, or other modes of administration, such as oral administration. Depending on the mode of administration, the active compound may be coated to protect the compound from the effects of acid or other natural conditions.

本发明所用术语“无生理毒性的盐”是指可保留母体化合物预期生理活性而不会产生任何意料之外毒副作用的盐或者它们的组合物。例如：盐酸盐，氢溴酸盐，硫酸盐，磷酸盐，硝酸盐，以及醋酸盐，草酸盐，酒石酸盐，琥珀酸盐，苹果酸盐，苯甲酸盐，双羟萘酸盐，海藻酸盐，甲磺酸盐，萘磺酸盐等。根据盐中含有的阳离子又可为：钾盐，锂盐，锌盐，铜盐，钡盐，铋盐，钙盐等无机盐，还可为诸如三烷基铵盐等有机盐。The term "physiologically toxic salt" as used in the present invention refers to a salt or a combination thereof which retains the expected physiological activity of the parent compound without causing any unexpected toxic side effects. For example: hydrochloride, hydrobromide, sulfate, phosphate, nitrate, and acetate, oxalate, tartrate, succinate, malate, benzoate, pamoate , alginate, methanesulfonate, naphthalenesulfonate, and the like. The cation according to the salt may be an inorganic salt such as a potassium salt, a lithium salt, a zinc salt, a copper salt, a cerium salt, a cerium salt or a calcium salt, and may also be an organic salt such as a trialkylammonium salt.

本发明式(I)及其立体异构体、溶剂合物、无生理毒性盐或含有它的药物组合物可以以已知的任何方式给药，如口服、肌肉、皮下等，给药剂型例如片剂、胶囊、口含片、咀嚼片、酏剂、混悬剂、透皮剂、微囊包埋剂、埋植剂、糖浆剂等。可以是普通制剂、缓释制剂、控释制剂及各种微粒给药系统。为了将单位给药剂型制成片剂，可以广泛使用本领域公知的各种生物可降解的或生物相容载体。关于载体的例子，如盐水基及各种缓冲水溶液、乙醇或其它多元醇、脂质体、聚乳酸、乙酸乙烯酯、聚酐、聚羟乙酸、胶原、聚原酸酯等。The formula (I) of the present invention and its stereoisomers, solvates, non-physiological salts or pharmaceutical compositions containing the same may be administered in any manner known per se, such as oral, intramuscular, subcutaneous, etc., for example, for administration of a dosage form, for example Tablets, capsules, buccal tablets, chewable tablets, tinctures, suspensions, transdermal agents, microcapsules, implants, syrups, and the like. It may be a general preparation, a sustained release preparation, a controlled release preparation, and various microparticle delivery systems. In order to form a unit dosage form into tablets, various biodegradable or biocompatible carriers well known in the art can be widely used. About the carrier Examples are, for example, saline-based and various buffered aqueous solutions, ethanol or other polyols, liposomes, polylactic acid, vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and the like.

本发明式(I)所示的化合物、其立体异构体、溶剂合物或其无生理毒性的盐或含有它的药物组合物的给药剂量取决于许多因素，例如所要预防或治疗疾病的性质和严重程度，患者或动物的性别、年龄、体重，敏感性及个体反应，所用的具体化合物，给药途径，给药次数以及所希望达到的治疗效果等。上述剂量可以单一剂量形式或分成几个，例如二、三、四个剂量形式给药。单个最大剂量一般不超过30mg/Kg体重，例如0.001-30mg/Kg，优选0.01-5mg/Kg，较佳剂量范围为0.5-2mg/Kg体重。但是，在某些情况下，也可能使用30mg/Kg体重以上或者0.001mg/Kg以下的单个剂量。The dose of the compound of the formula (I), a stereoisomer thereof, a solvate thereof or a non-physiologically acceptable salt thereof or a pharmaceutical composition containing the same according to the present invention depends on many factors such as the disease to be prevented or treated. Nature and severity, the sex, age, weight, sensitivity and individual response of the patient or animal, the particular compound employed, the route of administration, the number of doses, and the desired therapeutic effect. The above dosages may be administered in a single dosage form or divided into several, for example two, three or four dosage forms. The single maximum dose will generally not exceed 30 mg/kg body weight, such as 0.001-30 mg/Kg, preferably 0.01-5 mg/Kg, with a preferred dosage range of 0.5-2 mg/Kg body weight. However, in some cases, it is also possible to use a single dose of 30 mg/kg or more or 0.001 mg/kg or less.

实施例所用固相合成载体MBHA树脂为天津南开合成责任有限公司产品；DIC、HOBT以及Boc-保护或Fmoc-保护的天然氨基酸为上海吉尔生化公司以及成都凯泰新技术有限责任公司产品，TFA、DIEA为ACROS公司产品，Boc-或Fmoc-保护的非天然氨基酸除说明外均由本实验室合成提供。The solid phase synthesis carrier MBHA resin used in the examples is Tianjin Nankai Synthetic Co., Ltd.; DIC, HOBT and Boc-protected or Fmoc-protected natural amino acids are products of Shanghai Jill Biochemical Co., Ltd. and Chengdu Kaitai New Technology Co., Ltd., TFA, DIEA is a product of ACROS, and Boc- or Fmoc-protected non-natural amino acids are provided by our laboratory, except for instructions.

实施例1：肽序列的合成Example 1: Synthesis of peptide sequences

以0.5g MBHA树脂(0.54mmol)为固相载体，DIC/HOBt为缩合剂，根据化合物的氨基酸序列，按照Boc、Fmoc交叉保护策略依次连接好所有氨基酸及修饰单元，将上述肽树脂放入HF切割仪的反应器中，加入1.0mL苯甲醚，0.5mL乙二硫醇，装好后将HF切割仪的体系抽成真空，用液氮冷却反应器，转入约10mL液态HF，于0℃反应1h。用油泵抽走HF，取下反应器，加入冷冻无水乙醚沉淀出固体，再将混悬液转移至砂芯漏斗中。用少量冷却的无水乙醚洗涤三次，再用10％的乙酸水溶液冲洗至树脂不再相互粘附，收集洗涤液，冷冻干燥后得白色絮状固体。0.5g MBHA resin (0.54mmol) was used as the solid phase carrier, and DIC/HOBt was used as the condensing agent. According to the amino acid sequence of the compound, all amino acids and modifying units were sequentially connected according to the Boc and Fmoc cross-protection strategy, and the above peptide resin was placed in HF. In the reactor of the cutter, 1.0 mL of anisole and 0.5 mL of ethanedithiol were added. After the installation, the system of the HF cutter was evacuated, and the reactor was cooled with liquid nitrogen, and transferred to about 10 mL of liquid HF at 0. °C reaction for 1h. The HF was pumped off with an oil pump, the reactor was removed, solidified by adding anhydrous diethyl ether, and the suspension was transferred to a sand core funnel. It was washed three times with a small amount of cooled anhydrous diethyl ether, and then washed with a 10% aqueous solution of acetic acid until the resin no longer adhered to each other, and the washing liquid was collected, and lyophilized to obtain a white flocculent solid.

以该方法合成了8条肽序列，具体结构如下： Eight peptide sequences were synthesized by this method, and the specific structure is as follows:

实施例2：硫醚键连接的GnRH类似物-紫杉醇缀合物的合成Example 2: Synthesis of a thioether-linked GnRH analogue-paclitaxel conjugate

称取等摩尔量的肽序列与紫杉醇衍生物b1或b3在50％的乙腈/水溶液中，室温搅拌反应,反应完全后，中压制备色谱纯化得纯品。An equimolar amount of the peptide sequence was weighed and the paclitaxel derivative b1 or b3 was stirred in a 50% acetonitrile/water solution at room temperature, and after completion of the reaction, the medium was purified by medium pressure preparative chromatography to obtain a pure product.

以该方法合成了缀合物：1、2、3、5、6、7、9、10、11、13、14、15、17、18、19、21、22、23、26、27、28、30、31、32、34、35、36、38、39、40、42、43、44、46、47、48、50、51、52、55、56、57。The conjugates were synthesized in this way: 1, 2, 3, 5, 6, 7, 9, 10, 11, 13, 14, 15, 17, 18, 19, 21, 22, 23, 26, 27, 28 30, 31, 32, 34, 35, 36, 38, 39, 40, 42, 43, 44, 46, 47, 48, 50, 51, 52, 55, 56, 57.

实施例3：二硫键连接的GnRH类似物-紫杉醇缀合物的合成 Example 3: Synthesis of a disulfide-linked GnRH analogue-paclitaxel conjugate

称取等摩尔量的肽序列与紫杉醇衍生物b2或b4在DMSO中，室温搅拌反应,反应完全后，中压制备色谱纯化得纯品。The equimolar amount of the peptide sequence was weighed out with the paclitaxel derivative b2 or b4 in DMSO, and the reaction was stirred at room temperature. After the reaction was completed, the pure product was purified by medium pressure preparative chromatography.

以该方法合成了缀合物：4、8、12、16、20、24、25、29、33、37、41、45、49、53、54、58。The conjugates were synthesized in this way: 4, 8, 12, 16, 20, 24, 25, 29, 33, 37, 41, 45, 49, 53, 54, 58.

实施例2和实施例3的质谱表征数据见表2。The mass spectrometric characterization data of Example 2 and Example 3 is shown in Table 2.

表2 GnRH类似物-紫杉醇缀合物的Maldi-Tof-MS表征Table 2 Maldi-Tof-MS Characterization of GnRH Analog-Paclitaxel Conjugates

实施例4：肿瘤细胞生长抑制活性及GnRH受体结合活性Example 4: Tumor cell growth inhibitory activity and GnRH receptor binding activity

肿瘤细胞生长抑制活性测定方法：取对数生长期的细胞(人乳腺癌细胞MCF-7以及人结直肠癌细胞HT-29)消化、接种于96孔板，培养基稀释至细胞悬液密度约3×10⁴个/mL,每孔100uL接种，于细胞培养箱中孵育24h。Tumor cell growth inhibitory activity assay method: cells in logarithmic growth phase (human breast cancer cell MCF-7 and human colorectal cancer cell line HT-29) were digested, inoculated into a 96-well plate, and the medium was diluted to a cell suspension density. 3×10 ⁴ cells/mL, inoculated at 100 uL per well, and incubated in a cell culture incubator for 24 h.

分别称取紫杉醇及缀合物，以DMSO溶解，制成10^-3mol/L的母液，加培养基逐级稀释成2000nmol/L,400nmol/L,80nmol/L,16nmol/L,3.2nmol/L,0.64nmol/L等浓度的培养基溶液。取出在培养箱中孵育24h的96孔板，吸除培养基，加入含有不同浓度药物的培养基溶液，每孔100μL,每个浓度点重复5个孔，然后放入培养箱中孵育48h，之后除去药物的培养基溶液，PBS洗涤，再每孔加入20％的MTS/PBS溶液100μL，培养箱中孵育1h后，通过多功能酶标仪测定培养板各孔在490nm下的吸光度，计算各浓度抑制率及各化合物的半数抑制浓度(IC₅₀值)。Paclitaxel and conjugate were weighed separately and dissolved in DMSO to prepare 10 ^-3 mol/L mother liquor. The medium was diluted to 2000 nmol/L, 400 nmol/L, 80 nmol/L, 16 nmol/L, 3.2 nmol/ L, a medium solution of a concentration of 0.64 nmol/L. Remove the 96-well plate incubated in the incubator for 24 h, aspirate the medium, add a medium solution containing different concentrations of the drug, 100 μL per well, repeat 5 wells per concentration point, and then incubate in the incubator for 48 h, then The medium solution of the drug was removed, washed with PBS, and then 100 μL of 20% MTS/PBS solution was added to each well, and after incubating for 1 hour in the incubator, the absorbance of each well of the culture plate at 490 nm was measured by a multi-function microplate reader, and each concentration was calculated. Inhibition rate and half-inhibitory concentration (IC ₅₀ value) of each compound.

按照上述方法，测定结果见表3。According to the above method, the measurement results are shown in Table 3.

GnRH受体结合活性测定方法：GnRH receptor binding activity assay method:

本实验通过运用胞内钙离子荧光检测技术，以稳定表达GnRHr的CHO-K1/Gα15细胞为受试对象，在37℃/5％CO₂培养箱中培养，当细胞汇合度达到90％时，进行消化处理，将收集到的细胞悬液，以20,000个细胞每孔的密度接种到384微孔板，然后放入37℃/5％CO₂培养箱中继续培养至少18h，加入染料，每孔20ul，随后，将10ul配置好的化合物溶液加到细胞板内，然后将细胞板放到37℃/5％CO₂培养箱孵育1个小时，最后于室温平衡15min。然后通过FLIPR检测，对于每个检测孔而言，以1-20s的平均荧光强度值作为基线，21到120s的最大荧光强度值减去基线值即为相对荧光强度值。In this experiment, CHO-K1/Gα15 cells stably expressing GnRHr were cultured by intracellular calcium ion fluorescence detection technique, and cultured in a 37° C./5% CO ₂ incubator. When the cell confluence reached 90%, Digestion treatment, the collected cell suspension was inoculated into a 384-well plate at a density of 20,000 cells per well, and then placed in a 37 ° C / 5% CO ₂ incubator for at least 18 h, adding dye, each well 20 ul, then, 10 ul of the configured compound solution was added to the cell plate, and then the cell plate was incubated in a 37 ° C / 5% CO ₂ incubator for 1 hour, and finally equilibrated at room temperature for 15 min. Then, by FLIPR detection, for each detection well, the average fluorescence intensity value of 1-20 s is used as a baseline, and the maximum fluorescence intensity value of 21 to 120 s minus the baseline value is the relative fluorescence intensity value.

％激动率＝{1-(ΔRFU_化合物-ΔRFU_背景)/(ΔRFU_{参照激动剂}–ΔRFU_背景)}×100% agonism = {1 - (ΔRFU _{compound -} ΔRFU _background ) / (ΔRFU _{reference agonist -} ΔRFU _background )} × 100

％抑制率＝{1-(ΔRFU_化合物-ΔRFU_背景)/(ΔRFU_{参照拮抗剂}–ΔRFU_背景)}×100% inhibition rate = {1 - (ΔRFU _{compound -} ΔRFU _background ) / (ΔRFU _{reference antagonist -} ΔRFU _background )} × 100

通过S曲线拟合计算出其EC₅₀或IC₅₀，测定结果如表3所示。The EC ₅₀ or IC _{50 was} calculated by S curve fitting, and the measurement results are shown in Table 3.

表3table 3

a：GnRH激动活性,参照Leuprorelin：EC₅₀＝0.8nmol/L；a: GnRH agonistic activity, refer to Leuprorelin: EC ₅₀ = 0.8 nmol / L;

b：GnRH拮抗活性,参照Degarelix：IC₅₀＝0.2nmol/L；b: GnRH antagonistic activity, refer to Degarelix: IC ₅₀ = 0.2 nmol / L;

c：对照组紫杉醇：IC₅₀＝9.53±0.63nmol/L；c: paclitaxel in the control group: IC ₅₀ = 9.53 ± 0.63 nmol / L;

d：对照组紫杉醇：IC₅₀＝10.3±2.2nmol/L。d: Control group paclitaxel: IC ₅₀ = 10.3 ± 2.2 nmol / L.

肿瘤细胞生长抑制活性结果表明，缀合物在两种细胞(人乳腺癌细胞(MCF-7)以及人结直肠癌细胞(HT-29))中均表现出了良好的肿瘤细胞生长抑制活性，个别化合物甚至与紫杉醇活性相当。Tumor cell growth inhibitory activity results showed that the conjugate showed good tumor cell growth inhibitory activity in both cells (human breast cancer cells (MCF-7) and human colorectal cancer cells (HT-29)). Individual compounds are even comparable to paclitaxel.

缀合物的受体结合活性结果显示，缀合物均保持了与GnRH受体较高的结合活性，说明缀合物具有通过GnRH受体介导进入肿瘤细胞的潜力。The receptor binding activity of the conjugate showed that the conjugates maintained a higher binding activity to the GnRH receptor, indicating that the conjugate has the potential to mediate into the tumor cells via the GnRH receptor.

实施例5：缀合物血浆稳定性Example 5: Conjugate plasma stability

血浆稳定性测定方法：将待测样品DMSO溶液(12.5mg/mL)10μL，加入人血浆2mL，涡旋均匀，37℃恒温振荡器中孵育，不同时间点取样200μL，三倍体积乙腈终止反应；高速离心(12000r/min)10min，取上清液进行HPLC分析，由峰面积计算其原型保留量。Plasma stability determination method: 10 μL of DMSO solution (12.5 mg/mL) to be tested, added to human plasma 2 mL, vortexed evenly, incubate in a 37 ° C constant temperature oscillator, sample 200 μL at different time points, and terminate the reaction with three volumes of acetonitrile; High-speed centrifugation (12000 r / min) for 10 min, the supernatant was taken for HPLC analysis, and the prototype retention was calculated from the peak area.

按照上述方法，测定结果见图1-3。According to the above method, the measurement results are shown in Figures 1-3.

血浆稳定性测定结果显示：缀合物在血浆中均表现出了较高的稳定性，大部分缀合物在血浆中孵育24h以后原型保留在50％以上，相较于AN-152(临床III期)在人血浆中半衰期为2h，有了明显的提高(Nagy A,Plonowski A,Schally AV.Stability of cytotoxic luteinizing hormone-releasing hormone conjugate(AN-152)containing doxorubicin 14-O-hemiglutarate in mouse and human serum in vitro:implications for the design of preclinical studies.Proc.Natl.Acad.Sci.U.S.A.2000；97:829–834.)。个别缀合物如9、10原型保留百分比高达90％以上，血浆稳定性获得了极大的改善。另外，选取肽序列N端进行修饰的缀合物(9、10、11)与选取6位进行修饰的缀合物(17、18、19)相比，血浆稳定性没有显著性差异；以硫醚键作为连接臂，相比二硫键连接，缀合物的稳定性明显提高。The results of plasma stability assay showed that the conjugate showed high stability in plasma. Most of the conjugates retained more than 50% of the conjugate after incubation for 24 hours in plasma, compared with AN-152 (Clinical III). Period) has a half-life in human plasma of 2 h, with a significant improvement (Nagy A, Plonowski A, Schally AV. Stability of cytotoxic luteinizing hormone-releasing hormone conjugate (AN-152) containing doxorubicin 14-O-hemiglutarate in mouse and human serum in vitro: impurities for the design of preclinical studies. Proc. Natl. Acad. Sci. U.S.A. 2000; 97:829-834.). Individual conjugates such as 9, 10 prototype retention percentages of up to 90% or more, plasma stability has been greatly improved. In addition, there was no significant difference in plasma stability between the conjugates modified with the N-terminus of the peptide sequence (9, 10, 11) and the conjugate modified with the 6 positions (17, 18, 19); The ether bond acts as a linker and the stability of the conjugate is significantly improved compared to the disulfide bond.

实施例6：缀合物的细胞选择性Example 6: Cellular selectivity of conjugates

通过细胞受体饱和实验间接考察缀合物的细胞选择性，实验方法：以GnRH受体阳性的MCF-7细胞为受试对象，并将其分成两组，其中一组加入不同浓度的Degarelix的培养基溶液预处理，另一组加入空白培养基，在37℃5％CO₂恒温培养箱中孵育1小时后，吸除培养基，然后加入紫杉醇或缀合物的药物溶液，考察其在两组细胞的生长抑制活性的差异。The cell selectivity of the conjugate was investigated indirectly by cell receptor saturation assay. The experimental method: GnRH receptor-positive MCF-7 cells were used as subjects, and they were divided into two groups, one of which was added with different concentrations of Degarelix. The medium solution was pretreated, and the other group was added to the blank medium, and after incubating for 1 hour in a 37 ° C 5% CO ₂ incubator, the medium was aspirated, and then the drug solution of paclitaxel or conjugate was added to examine it in two The difference in growth inhibitory activity of the group of cells.

细胞受体饱和实验结果显示(如图4-图7)：经不同浓度的Degarelix预饱和处理的，其GnRH受体被不同程度的屏蔽，紫杉醇(PTX)对GnRH受体预饱和的MCF-7细胞和未经预饱和处理的MCF-7细胞的生长抑制活性没有表现出差异；而对于缀合物，GnRH受体预饱和的MCF-7细胞的存活率升高。显现了缀合物通过GnRH受体介导进入细胞的途径，奠定了缀合物的细胞选择性的基础。The results of cell receptor saturation experiments (Figure 4-7): GnRH receptors were screened to different extents by different concentrations of Degarelix pre-saturation, and paclitaxel (PTX) pre-saturated MCF-7 to GnRH receptors. The growth inhibitory activity of the cells and the MCF-7 cells which were not pre-saturated did not show a difference; whereas for the conjugate, the survival rate of the GnRH receptor-pre-saturated MCF-7 cells was increased. The pathway by which the conjugates mediated into the cell via the GnRH receptor is revealed, laying the foundation for cell selectivity of the conjugate.

尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。 Although specific embodiments of the invention have been described in detail, those skilled in the art will understand. Various modifications and alterations of the details are possible in light of the teachings of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

a compound represented by the formula (I), a stereoisomer thereof, a solvate thereof or a salt thereof which is not physiologically toxic,

(X)Aaa ¹ -Aaa ² -Aaa ³ -Ser-Aaa ⁵ -Aaa ⁶ (Y)-Leu-Aaa ⁸ -Pro-Aaa ¹⁰ -NH ₂

I

among them,

Aaa ¹ is L or D type Glp or Nal;

Aaa ² is an L or D type His or Cpa;

Aaa ³ is L or D type Trp or Pal;

Aaa ⁵ is Ty or Aph (L-Hor) of L or D type;

Aaa ⁶ is Lys or Aph of L or D type;

Aaa ⁸ is L or D type Arg or ILys;

Aaa ¹⁰ is L or D type Gly or Ala;

X is selected from the group consisting of H, Ac, -ab, -Xaa ¹ -Xaa ² -ab;

Y is selected from the group consisting of H, Cbm, -ab, -Xaa ¹ -Xaa ² -ab;

Wherein Xaa ¹ and Xaa ² are each independently a hydrophilic amino acid;

Where a is

Where m=1-6;

b is a small molecule modified paclitaxel or docetaxel, the modification site is located at its 2' hydroxyl group;

The small molecule is C1 or C2 as shown below, wherein n = 1-10, and the small molecule is linked to the 2' hydroxyl group through an ester bond.

A compound according to claim 1, a stereoisomer, a solvate thereof or a non-physiologically acceptable salt thereof, wherein the small molecule modified paclitaxel or docetaxel is as shown below B1, b2, b3 or b4, where n=1-10:

A compound according to claim 1 or 2, a stereoisomer thereof, a solvate thereof or a salt thereof which is not physiologically toxic, wherein said b is b1 or b2, wherein n = 1-10;

The compound according to claim 1 or 2, a stereoisomer thereof, a solvate thereof or a non-physiologically toxic salt thereof, wherein the hydrophilic amino acid is selected from the group consisting of Arg, Gln, Glu, Ser, Asp, Asn and Thr.

The compound according to claim 1 or 2, a stereoisomer thereof, a solvate thereof or a physiologically toxic salt thereof, wherein the -Xaa ¹ -Xaa ^{2 is} selected from the following peptide fragment of L or D type, -Arg-Gln, -Gln-Arg, -Arg-Arg, -Gln-Gln.

A compound according to any one of claims 1 to 5, a stereoisomer thereof, a solvate thereof or a non-physiologically toxic salt thereof, which is selected from the group consisting of the following compounds:

1. (R1) Glp-His-Trp-Ser-Tyr-D-Lys (R2)-Leu-Arg-Pro-Gly-NH ₂ ;

2. (R1) Glp-His-Trp-Ser-Tyr-D-Lys (R3)-Leu-Arg-Pro-Gly-NH ₂ ;

3, (R1) Glp-His-Trp-Ser-Tyr-D-Lys (R4)-Leu-Arg-Pro-Gly-NH ₂ ;

4. (R1) Glp-His-Trp-Ser-Tyr-D-Lys (R5)-Leu-Arg-Pro-Gly-NH ₂ ;

5. (R1) Glp-His-Trp-Ser-Tyr-D-Lys (R6)-Leu-Arg-Pro-Gly-NH ₂ ;

6. (R1) Glp-His-Trp-Ser-Tyr-D-Lys (R7)-Leu-Arg-Pro-Gly-NH ₂ ;

7. (R1) Glp-His-Trp-Ser-Tyr-D-Lys (R8)-Leu-Arg-Pro-Gly-NH ₂ ;

8. (R1) Glp-His-Trp-Ser-Tyr-D-Lys (R9)-Leu-Arg-Pro-Gly-NH ₂ ;

9. (R10) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R2)-Leu-ILys-Pro-D-Ala-NH ₂ ;

10, (R10) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R3)-Leu-ILys-Pro-D-Ala-NH ₂ ;

11. (R10) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R4)-Leu-ILys-Pro-D-Ala-NH ₂ ;

12. (R10) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R5)-Leu-ILys-Pro-D-Ala-NH ₂ ;

13. (R10) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R6)-Leu-ILys-Pro-D-Ala-NH ₂ ;

14, (R10) D-Nal -D-Cpa-D-Pal-Ser-Aph (L-Hor) -D-Aph (R7) -Leu-ILys-Pro-D-Ala-NH 2;

15. (R10) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R8)-Leu-ILys-Pro-D-Ala-NH ₂ ;

16. (R10) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R9)-Leu-ILys-Pro-D-Ala-NH ₂ ;

17. (R2) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R11)-Leu-ILys-Pro-D-Ala-NH ₂ ;

18, (R3) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R11)-Leu-ILys-Pro-D-Ala-NH ₂ ;

19, (R4) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R11)-Leu-ILys-Pro-D-Ala-NH ₂ ;

20, (R5) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R11)-Leu-ILys-Pro-D-Ala-NH ₂ ;

21, (R6) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R11)-Leu-ILys-Pro-D-Ala-NH ₂ ;

22, (R7) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R11)-Leu-ILys-Pro-D-Ala-NH ₂ ;

23, (R8) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R11)-Leu-ILys-Pro-D-Ala-NH ₂ ;

24, (R9) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R11)-Leu-ILys-Pro-D-Ala-NH ₂ ;

25, (R5) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R5)-Leu-ILys-Pro-D-Ala-NH ₂ ;

26, (R6) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R6)-Leu-ILys-Pro-D-Ala-NH ₂ ;

27, (R7) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R7)-Leu-ILys-Pro-D-Ala-NH ₂ ;

28, (R8) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R8)-Leu-ILys-Pro-D-Ala-NH ₂ ;

29, (R9) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R9)-Leu-ILys-Pro-D-Ala-NH ₂ ;

30, (R1) Glp-His-Trp-Ser-Tyr-D-Lys (R12)-Leu-Arg-Pro-Gly-NH ₂ ;

31, (R1) Glp-His-Trp-Ser-Tyr-D-Lys (R13)-Leu-Arg-Pro-Gly-NH ₂ ;

32, (R1) Glp-His-Trp-Ser-Tyr-D-Lys (R14)-Leu-Arg-Pro-Gly-NH ₂ ;

33, (R1) Glp-His-Trp-Ser-Tyr-D-Lys (R15)-Leu-Arg-Pro-Gly-NH ₂ ;

34, (R1) Glp-His-Trp-Ser-Tyr-D-Lys (R16)-Leu-Arg-Pro-Gly-NH ₂ ;

35, (R1) Glp-His-Trp-Ser-Tyr-D-Lys (R17)-Leu-Arg-Pro-Gly-NH ₂ ;

36, (R1) Glp-His-Trp-Ser-Tyr-D-Lys (R18)-Leu-Arg-Pro-Gly-NH ₂ ;

37, (R1) Glp-His-Trp-Ser-Tyr-D-Lys (R19)-Leu-Arg-Pro-Gly-NH ₂ ;

38, (R10) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R12)-Leu-ILys-Pro-D-Ala-NH ₂ ;

39, (R10) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R13)-Leu-ILys-Pro-D-Ala-NH ₂ ;

40, (R10) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R14)-Leu-ILys-Pro-D-Ala-NH ₂ ;

41, (R10) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R15)-Leu-ILys-Pro-D-Ala-NH ₂ ;

42. (R10) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R16)-Leu-ILys-Pro-D-Ala-NH ₂ ;

43. (R10) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R17)-Leu-ILys-Pro-D-Ala-NH ₂ ;

44, (R10) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R18)-Leu-ILys-Pro-D-Ala-NH ₂ ;

45, (R10) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R19)-Leu-ILys-Pro-D-Ala-NH ₂ ;

46, (R12) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R11)-Leu-ILys-Pro-D-Ala-NH ₂ ;

47, (R13) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R11)-Leu-ILys-Pro-D-Ala-NH ₂ ;

48, (R14) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R11)-Leu-ILys-Pro-D-Ala-NH ₂ ;

49, (R15) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R11)-Leu-ILys-Pro-D-Ala-NH ₂ ;

50, (R16) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R11)-Leu-ILys-Pro-D-Ala-NH ₂ ;

51, (R17) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R11)-Leu-ILys-Pro-D-Ala-NH ₂ ;

52, (R18) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R11)-Leu-ILys-Pro-D-Ala-NH ₂ ;

53. (R19) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R11)-Leu-ILys-Pro-D-Ala-NH ₂ ;

54, (R15) D-Nal -D-Cpa-D-Pal-Ser-Aph (L-Hor) -D-Aph (R15) -Leu-ILys-Pro-D-Ala-NH 2;

55, (R16) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R16)-Leu-ILys-Pro-D-Ala-NH ₂ ;

56, (R17) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R17)-Leu-ILys-Pro-D-Ala-NH ₂ ;

57, (R18) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R18)-Leu-ILys-Pro-D-Ala-NH ₂ ;

58. (R19) D-Nal-D-Cpa-D-Pal-Ser-Aph (L-Hor)-D-Aph (R19)-Leu-ILys-Pro-D-Ala-NH ₂ ;

Wherein R1-R19 corresponds to a modifying group as follows:

A pharmaceutical composition comprising at least one compound, a stereoisomer, a solvate thereof or a non-physiologically acceptable salt thereof, according to any one of claims 1 to 6, and a pharmaceutically acceptable carrier or excipient.

A process for the preparation of a compound according to any one of claims 1 to 6, a stereoisomer thereof, a solvate thereof or a salt thereof which is not physiologically toxic, comprising the steps of:

(1) 6-amino acid modification: According to the solid phase synthesis Boc, Fmoc cross-protection strategy, the peptide sequence was synthesized from the C-terminus to the N-terminus to the 6-position amino acid, and the 20-50% piperidine/DMF solution was removed from the 6-position amino acid side chain amino group. The Fmoc protecting group is added with a modifying unit (p-methylbenzyl-protected mercaptopropionic acid), DIC, HOBT, and the ninhydrin is detected after 4 hours of reaction. After the reaction is completed, the amino acid condensation is sequentially completed, and after the peptide sequence is completed, Anhydrous HF-cleaved peptide resin, dissolved in 10-30% aqueous acetic acid solution, lyophilized to obtain a crude peptide, and purified by medium pressure chromatography to obtain a pure product;

(2) N-terminal modification (including simultaneous modification of N-terminal and 6-position amino acids): synthesis of peptide sequence from C-terminus to N-terminus to 6-position amino acid or N-terminus according to solid phase synthesis Boc and Fmoc cross-protection strategy, 20-50% piperidine The acyl/DMF solution was used to remove the Fmoc protecting group on the amino acid side chain amino acid of the 6-position amino acid, and the modified unit (p-methylbenzyl-protected mercaptopropionic acid), DIC, HOBT was added. After the reaction for 4 hours, the ninhydrin was detected. After the reaction was completed, the reaction was completed. Continue to complete the condensation of amino acids in sequence until all amino acids and modified units of the peptide sequence are condensed. Finally, the peptide resin is lysed with anhydrous HF, dissolved in 10-30% aqueous acetic acid solution, and lyophilized to obtain crude peptide, which is purified by medium pressure chromatography. ;

(3) Coupling of the peptide sequence with a small molecule modified paclitaxel: the purified peptide sequence and the small molecule modified paclitaxel are stirred in a 50% acetonitrile/water solution at room temperature, and after the reaction is completed, the medium is pressed. Prepared by chromatography to obtain a pure product;

(4) Coupling of the peptide sequence with the disulfide bond and the small molecule modified paclitaxel: the purified peptide sequence and the small molecule modified paclitaxel are stirred in DMSO at room temperature, and the reaction is completed, and the medium pressure preparative chromatography is purified to be pure. Product.

Use of a compound according to any one of claims 1 to 6, a stereoisomer thereof, a solvate thereof or a salt thereof which is not physiologically toxic for the preparation of a medicament for the prevention and/or treatment of a tumor.

The use according to claim 9, wherein the tumor is selected from the group consisting of cutaneous basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and CNS cancer; breast cancer; cervical cancer; choriocarcinoma; colon and rectal cancer; connective tissue cancer; Cancer of the digestive system; cancer of the endometrium; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer; neoplasms in epithelial cells; kidney cancer; laryngeal cancer; leukemia; liver cancer; Non-small cells; lymphomas include Hodgkin's and non-Hodgkin's lymphoma; melanoma; myeloma; neuroblastoma; oral cancer (eg, lips, tongue, mouth and pharynx); ovarian cancer; Pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; respiratory cancer; sarcoma; skin cancer; testicular cancer; thyroid cancer; uterine cancer; urinary cancer and other cancers and sarcomas.

A method of preventing and/or treating a tumor, the method comprising administering to a subject in need thereof an effective amount of a compound according to any one of claims 1 to 6, a stereoisomer thereof, a solution A pharmaceutical composition or a salt thereof that is not physiologically toxic.

The method of claim 11 wherein said tumor is selected from the group consisting of cutaneous basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and CNS cancer; breast cancer; cervical cancer; choriocarcinoma; colon and rectal cancer; connective tissue cancer; Cancer of the digestive system; cancer of the endometrium; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer; neoplasms in epithelial cells; kidney cancer; laryngeal cancer; leukemia; liver cancer; Non-small cells; lymphomas include Hodgkin's and non-Hodgkin's lymphoma; melanoma; myeloma; neuroblastoma; oral cancer (eg, lips, tongue, mouth and pharynx); ovarian cancer; Pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; respiratory cancer; sarcoma; skin cancer; testicular cancer; thyroid cancer; uterine cancer; urinary cancer and other cancers and sarcomas.

Use of a compound according to any one of claims 1 to 6, a stereoisomer thereof, a solvate thereof or a non-physiologically toxic salt thereof for the preparation of a reagent for killing and/or killing tumor cells.

The use of claim 13 for use in an in vitro or in vivo method.

The use according to claim 13 or 14, wherein the tumor cell is a tumor cell line or a tumor cell derived from a subject, preferably, the tumor cell includes, but is not limited to, a cutaneous basal cancer cell, a biliary cancer cell; a bladder cancer Cells; bone cancer cells; brain and CNS cancer cells; breast cancer cells; cervical cancer cells; choriocarcinoma cells; colon and rectal cancer cells; connective tissue cancer cells; cancer cells of the digestive system; cancer cells of the endometrium; Cancer cells; eye cancer cells; cancer cells in the head and neck; gastric cancer cells; renal cancer cells; laryngeal cancer cells; leukemia cells; liver cancer cells; lung cancer cells (eg, small cells and non-small cells); lymphoma cells including Huo Qijin and non-Hodgkin's lymphoma cells; melanoma cells; myeloma cells; neuroblastoma cells; oral cancer cells (eg, lips, tongue, mouth and pharyngeal cancer cells); ovarian cancer cells; Cancer cells; prostate cancer cells; retinal tumor cells; rhabdomyosarcoma cells; rectal cancer cells; cancer cells of the respiratory system; sarcoma cells; skin cancer cells; testicular cancer Cells; thyroid cancer cells; uterine cancer cells; cancer cells of the urinary system and other cancer cells and sarcoma cells.

A method of killing and/or killing a tumor cell, the method comprising administering to the cell an effective amount of a compound according to any one of claims 1 to 6, a stereoisomer thereof or a salt thereof which is not physiologically toxic.

The method of claim 16 which is carried out in vitro or in vivo.

The method of claim 16 or 17, wherein the tumor cell is a tumor cell line, or a tumor cell from a subject, preferably, the tumor cell includes, but is not limited to, a cutaneous basal cancer cell, a biliary cancer cell; a bladder cancer Cells; bone cancer cells; brain and CNS cancer cells; breast cancer cells; cervical cancer cells; choriocarcinoma cells; colon and rectal cancer cells; connective tissue cancer cells; cancer cells of the digestive system; cancer cells of the endometrium; Cancer cells; eye cancer cells; cancer cells in the head and neck; gastric cancer cells; renal cancer cells; laryngeal cancer cells; leukemia cells; liver cancer cells; lung cancer cells (eg, small cells and non-small cells); lymphoma cells including Huo Qijin and non-Hodgkin's lymphoma cells; melanoma cells; myeloma cells; neuroblastoma cells; oral cancer cells (eg, lips, tongue, mouth and pharyngeal cancer cells); ovarian cancer cells; Cancer cells; prostate cancer cells; retinal tumor cells; rhabdomyosarcoma cells; rectal cancer cells; cancer cells of the respiratory system; sarcoma cells; skin cancer cells; testicular cancer Cells; thyroid cancer; uterine cancer; cancer of the urinary system as well as other cancer and sarcoma cells.

A compound according to any one of claims 1 to 6, a stereoisomer thereof or a non-physiologically toxic salt thereof for killing and/or killing tumor cells.

A compound according to claim 19, a stereoisomer thereof or a physiologically toxic salt thereof, for use in an in vitro or in vivo method.

The compound according to claim 19 or 20, a stereoisomer thereof or a physiologically toxic salt thereof, wherein the tumor cell is a tumor cell line or a tumor cell derived from a subject, preferably, the tumor cell includes but not Limited to, skin basal cancer cells, biliary cancer cells; bladder cancer cells; bone cancer cells; brain and CNS cancer cells; breast cancer cells; cervical cancer cells; chorionic cancer cells; colon and rectal cancer cells; connective tissue cancer cells; Systemic cancer cells; endometrial cancer cells; esophageal cancer cells; eye cancer cells; cancer cells in the head and neck; gastric cancer cells; renal cancer cells; laryngeal cancer cells; leukemia cells; liver cancer cells; Small cells and non-small cells; lymphoma cells include Hodgkin's and non-Hodgkin's lymphoma cells; melanoma cells; myeloma cells; neuroblastoma cells; oral cancer cells (eg, lips, tongue) , oral and pharyngeal cancer cells; ovarian cancer cells; pancreatic cancer cells; prostate cancer cells; retinal tumor cells; rhabdomyosarcoma cells; rectal cancer cells; Cells; sarcoma cells; skin cancer; testicular cancer; thyroid cancer; uterine cancer; cancer of the urinary system as well as other cancer and sarcoma cells.

A kit for killing and/or killing a tumor cell, the kit comprising the compound according to any one of claims 1 to 6, a stereoisomer thereof or a salt thereof which is not physiologically toxic, and optionally The ground also includes instructions for use.