WO2023138147A1 - Triptolide prodrug, preparation method therefor and pharmaceutical use thereof - Google Patents
Triptolide prodrug, preparation method therefor and pharmaceutical use thereof Download PDFInfo
- Publication number
- WO2023138147A1 WO2023138147A1 PCT/CN2022/128608 CN2022128608W WO2023138147A1 WO 2023138147 A1 WO2023138147 A1 WO 2023138147A1 CN 2022128608 W CN2022128608 W CN 2022128608W WO 2023138147 A1 WO2023138147 A1 WO 2023138147A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- acid
- triptolide
- cancer
- compound
- hours
- Prior art date
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J73/00—Steroids in which the cyclopenta[a]hydrophenanthrene skeleton has been modified by substitution of one or two carbon atoms by hetero atoms
- C07J73/001—Steroids in which the cyclopenta[a]hydrophenanthrene skeleton has been modified by substitution of one or two carbon atoms by hetero atoms by one hetero atom
- C07J73/003—Steroids in which the cyclopenta[a]hydrophenanthrene skeleton has been modified by substitution of one or two carbon atoms by hetero atoms by one hetero atom by oxygen as hetero atom
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
- A61P11/06—Antiasthmatics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/12—Drugs for disorders of the urinary system of the kidneys
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/18—Antivirals for RNA viruses for HIV
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/02—Antineoplastic agents specific for leukemia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
Definitions
- the invention belongs to the field of medicinal chemistry, and relates to a class of triptolide water-soluble prodrugs or pharmaceutically acceptable salts thereof, pharmaceutical compositions containing these compounds, their preparation methods and medical applications.
- Tripterygium wilfordii extract The main active components of Tripterygium wilfordii extract are diterpenoids, triterpenoids and alkaloids, among which triptolide, a diterpenoid, is one of the main active ingredients of Tripterygium wilfordii, and it is also the main active ingredient of preparations such as tripterygium glycosides and tripterygium.
- triptolide can effectively prevent the rejection caused by transplanted organs after heart, kidney, liver or bone marrow transplantation, and significantly prolong the survival period of animals [Transplantation 2000; 70(3):447-55; Transplant Proc 1999; 31(7):2719-23].
- triptolide regulates the overall transcription level of cells by covalently binding and inhibiting XPB activity in the TFHII transcription complex, so that triptolide has broad-spectrum anti-tumor activity [Angew Chem Int Ed Engl, 2015, 54(6): 1859-1863; Mol Cancer Ther, 2003, 2: 65-72].
- the object of the present invention is to provide a triptolide prodrug or a pharmaceutically acceptable salt thereof whose water solubility is significantly improved and can be rapidly transformed in plasma as represented by general formula (I).
- HA is selected from hydrochloric acid, sulfuric acid, carbonic acid, citric acid, succinic acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid or ferulic acid;
- Liquid phase analysis method mobile phase 0.1% FA-H2O (A) and ACN (B); flow rate: 0.3mL/min; gradient elution degree: 0 ⁇ 2min, 15%B ⁇ 80%B; 2 ⁇ 3min, 80%B ⁇ 80%B; 3 ⁇ 4min, 80%B ⁇ 15%B; 4 ⁇ 5min, 15%B ⁇ 15%B; injection volume: 5 ⁇ L;
- the compound TP-P1 could dose-dependently inhibit the growth of the tumor, and had no effect on the body weight of the mice; the tumor inhibition rate at a dose of 25 ⁇ g/kg/d reached 54.31%, and the dose of 100 ⁇ g/kg/d could completely regress the xenograft tumor, and the tumor inhibition rate reached 100% (Table 4).
- the compound TP-P1 could dose-dependently inhibit the growth of the tumor after continuous intraperitoneal injection for 4 weeks, and had no effect on the body weight of the mice.
- the tumor inhibition rate at a dose of 100 ⁇ g/kg/d reached 93.87%
- the dose of 300 ⁇ g/kg/d and above could completely regress the transplanted tumor, and the tumor inhibition rate reached 100% (Table 5).
- the tumor inhibition rate of the combination group was 78.12% higher than that of the corresponding dose of single drug geritinib group (48.27%), and higher than that of the double dose of single drug geritinib group (71.96%) (Table 6); therefore, the combination of compound TP-P1 and geritinib for the treatment of acute myeloid leukemia has a synergistic effect.
- TP-P1 alleviates the effect of LPS-induced sepsis mice lung inflammation
- TP-P1 can inhibit the release of inflammatory factors such as IL-1 ⁇ , IL-6, TNF- ⁇ , IFN- ⁇ , and in a dose-dependent manner, relieve LPS-induced lung inflammation in septic mice (Table 7).
- mice 8 male SD rats were divided into 2 groups by random grouping. The randomly grouped SD rats were fasted overnight but allowed to drink water freely; 12 hours later, the first group was given the aqueous solution of compound TP-P1, and the dosage was 1.6 mg/kg; the second group was given the olive oil suspension of TP, and the dosage was 1.0 mg/kg; Blood was collected at min, 90min, 2h, 4h, and 6h, transferred to a 1.5mL centrifuge tube pretreated with sodium heparin, and centrifuged (8000rpm/min, 5min, 4°C) to obtain plasma, which was stored in a -80°C refrigerator.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Oncology (AREA)
- Immunology (AREA)
- Virology (AREA)
- Pulmonology (AREA)
- Communicable Diseases (AREA)
- Pain & Pain Management (AREA)
- AIDS & HIV (AREA)
- Tropical Medicine & Parasitology (AREA)
- Molecular Biology (AREA)
- Rheumatology (AREA)
- Hematology (AREA)
- Dermatology (AREA)
- Transplantation (AREA)
- Urology & Nephrology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The present invention relates to the field of medicinal chemistry. Disclosed are a triptolide prodrug, a preparation method therefor and a pharmaceutical use thereof. The preparation steps are: 1, making triptolide react with acyl chloride under the action of DMAP to generate an intermediate II; 2, making the intermediate II react with a carboxylic acid compound under the action of sodium iodide and potassium carbonate to generate a compound III; and 3, carrying out salt formation on the compound III and an acid to obtain a target compound I. The present invention has better water solubility and pharmacokinetic properties, and has a faster transformation speed in an in vivo blood environment compared with Minnelide, a more efficient synthesis method, and a better therapeutic effect on malignant tumors, inflammatory diseases, autoimmune diseases, etc. In addition, the triptolide prodrug has good water solubility, and therefore is effective in oral administration, and can be prepared into an injection for use.
Description
本发明属于药物化学领域，涉及一类雷公藤甲素水溶性前药或其药学上可接受的盐，以及含有这些化合物的药物组合物，它们的制备方法以及医药用途。The invention belongs to the field of medicinal chemistry, and relates to a class of triptolide water-soluble prodrugs or pharmaceutically acceptable salts thereof, pharmaceutical compositions containing these compounds, their preparation methods and medical applications.
雷公藤(Tripterygium wilfordii Hook.f.)为卫矛科(Celastraceae)雷公藤属植物，具有祛风除湿、活血通络、消肿止痛、杀虫解毒等功效，是临床治疗自身免疫性疾病的首选中药。目前已经有多种雷公藤提取物药物如雷公藤多苷片、雷公藤片(999)、雷公藤双层片、雷公藤总萜片等上市应用于类风湿性关节炎、自身免疫性肝炎、肾炎及肾病综合征等多种免疫性和炎症性疾病的治疗。雷公藤提取物的主要活性成分有二萜类、三萜类和生物碱类，其中二萜类的雷公藤甲素是雷公藤的主要活性成分之一，同时也是雷公藤多苷片、雷公藤片等制剂的主要有效成分。Tripterygium wilfordii Hook.f. is a plant of the genus Tripterygium wilfordii Hook.f. in the family Celastraceae. It has the functions of expelling wind and dampness, promoting blood circulation and dredging collaterals, reducing swelling and pain, killing insects and detoxifying, etc. It is the first choice of traditional Chinese medicine for clinical treatment of autoimmune diseases. At present, there are a variety of tripterygium extract drugs such as Tripterygium wilfordii Polyglycoside Tablets, Tripterygium wilfordii Tablets (999), Tripterygium wilfordii Double-layer Tablets, Tripterygium Total Terpene Tablets, etc., which are used in the treatment of various immune and inflammatory diseases such as rheumatoid arthritis, autoimmune hepatitis, nephritis and nephrotic syndrome. The main active components of Tripterygium wilfordii extract are diterpenoids, triterpenoids and alkaloids, among which triptolide, a diterpenoid, is one of the main active ingredients of Tripterygium wilfordii, and it is also the main active ingredient of preparations such as tripterygium glycosides and tripterygium.
作为雷公藤的高活性成分之一，雷公藤甲素具有较强的免疫抑制、抗炎、抗肿瘤等多种药理作用【J Am Chem Soc,1972,94(20):7194-7195；Drugs R D,2003,4(1):1-18；Trends Pharmacol Sci,2019,40(5):327-341】。自1969年以来，中药雷公藤一直被广泛用于治疗类风湿性关节炎，美国风湿病学会一项双盲临床研究同样显示雷公藤可以明显改善类风湿性关节炎症状，有效率达到58％【J Rheumatol 2003，30(3)：465-467】。对于其他自身免疫性以及炎症性疾病，如***性红斑狼疮、银屑病、强直性脊柱炎、哮喘、肾炎、溃疡性结肠炎、肺纤维化等，雷公藤均表现出令人满意的效果【Chin J Mod Appl Pharm，1999,16(2)：10-13；Br J Clin Pharmacol 2012Sep；74(3):424-36；Rheum Dis Clin North Am 2000；Am J Pathol 2001；158(3):997-1004；26(1):29-50】。鉴于雷公藤的免疫抑制作用，许多学者研究发现雷公藤甲素在心脏、肾脏、肝脏或骨髓移植后，可有效阻止移植器官引起的排斥反应，显著延长动物的存活期【Transplantation 2000；70(3):447-55；Transplant Proc 1999；31(7):2719-23】。在癌症领域，雷公藤甲素通过共价结合并抑制TFHII转录复合体中XPB活性从而调控细胞的整体转录水平，使雷公藤甲素具有广谱的抗肿瘤活性【Angew Chem Int Ed Engl,2015,54(6):1859-1863；Mol Cancer Ther,2003,2:65–72】。此外，雷公藤甲素在体外表现出有效的抗HIV作用【J Nat Prod 2000；63(3):357-61】，并且多项临床实现正在进行【NCT03403569；NCT01817283；NCT02002286】。然而，由于其二萜内酯类的结构水溶性较差，限制了其临床应用。因此，可以通过引入水溶性基团，设计合成雷公藤甲素的水溶性前药，在不影响其药效的情况下，提高其水溶性、改善其成药性。As one of the highly active components of Tripterygium wilfordii, triptolide has strong immunosuppressive, anti-inflammatory, anti-tumor and other pharmacological effects [J Am Chem Soc, 1972, 94(20): 7194-7195; Drugs R D, 2003, 4(1): 1-18; Trends Pharmacol Sci, 2019, 40(5): 327-341]. Since 1969, the traditional Chinese medicine Tripterygium wilfordii has been widely used to treat rheumatoid arthritis. A double-blind clinical study by the American College of Rheumatology also showed that Tripterygium wilfordii can significantly improve the symptoms of rheumatoid arthritis, with an effective rate of 58% [J Rheumatol 2003, 30(3): 465-467]. For other autoimmune and inflammatory diseases, such as systemic lupus erythematosus, psoriasis, ankylosing spondylitis, asthma, nephritis, ulcerative colitis, pulmonary fibrosis, etc., Tripterygium wilfordii has shown satisfactory effects [Chin J Mod Appl Pharm, 1999,16(2): 10-13; Br J Clin Pharmacol 2012Sep; 74(3): 424-36; Rheum Dis Clin North Am 2000; Am J Pathol 2001; 158(3):997-1004; 26(1):29-50]. In view of the immunosuppressive effect of tripterygium wilfordii, many scholars have found that triptolide can effectively prevent the rejection caused by transplanted organs after heart, kidney, liver or bone marrow transplantation, and significantly prolong the survival period of animals [Transplantation 2000; 70(3):447-55; Transplant Proc 1999; 31(7):2719-23]. In the field of cancer, triptolide regulates the overall transcription level of cells by covalently binding and inhibiting XPB activity in the TFHII transcription complex, so that triptolide has broad-spectrum anti-tumor activity [Angew Chem Int Ed Engl, 2015, 54(6): 1859-1863; Mol Cancer Ther, 2003, 2: 65-72]. In addition, triptolide has shown effective anti-HIV effects in vitro [J Nat Prod 2000; 63(3):357-61], and multiple clinical implementations are in progress [NCT03403569; NCT01817283; NCT02002286]. However, due to the poor water solubility of its diterpene lactone structure, its clinical application is limited. Therefore, the water-soluble prodrug of triptolide can be designed and synthesized by introducing a water-soluble group, so as to improve its water solubility and improve its druggability without affecting its drug efficacy.
水溶性前药的设计策略主要是在雷公藤甲素的C-14位通过酯键或缩醛连接一些水溶性基团。如PG490-88是在雷公藤甲素的C-14位直接通过酯键引入水溶性的羧酸，是第一个进入临床研究的雷公藤甲素前药【US5663335】。临床试验中虽然大多数患者的毒副作用是可以控制的，但有两个患者出现了致命副作用，其中一名在12mg/kg剂量下死于中性粒细胞减少性败血症，另一名在18mg/kg剂量下死于复杂的临床综合征。后续的药代动力学实验发现，PG490-88在体内无法迅速转化为雷公藤甲素，其不仅在不同物种(包括小鼠、猴子和人)中出现了很大的差异性，在同一批18名志愿者中也表现出了2-3倍的转化差异。究其原因，可能是PG490-88的C-14位空间位阻较大，阻碍了酯酶对酯键的水解，导致PG490-88在体内转化缓慢且不完全。此外，由于个体之间酯酶活性存在差异，也导致药物的安全剂量难以控制。因此，PG490-88的I期临床试验也被迫于2009年终止。The design strategy of water-soluble prodrugs is mainly to connect some water-soluble groups at the C-14 position of triptolide through ester bonds or acetals. For example, PG490-88 introduces a water-soluble carboxylic acid directly through an ester bond at the C-14 position of triptolide, and is the first triptolide prodrug to enter clinical research [US5663335]. In clinical trials, although the toxic side effects of most patients were controllable, two patients had fatal side effects, one of whom died of neutropenic sepsis at a dose of 12 mg/kg, and the other died of complex clinical syndrome at a dose of 18 mg/kg. Subsequent pharmacokinetic experiments found that PG490-88 could not be rapidly converted into triptolide in vivo, which not only showed great differences in different species (including mice, monkeys and humans), but also showed a 2-3 times conversion difference in the same batch of 18 volunteers. The reason may be that the C-14 steric hindrance of PG490-88 hinders the hydrolysis of ester bonds by esterase, resulting in slow and incomplete transformation of PG490-88 in vivo. In addition, due to differences in esterase activity among individuals, it is also difficult to control the safe dosage of drugs. Therefore, the phase I clinical trial of PG490-88 was also forced to terminate in 2009.
为了克服PG490-88体内转化不完全的问题，科研工作者们致力于对PG490-88的C-14位的连接方式进行优化，以避免空间位阻对断键的影响。2010年，Georg等人以甲缩醛作为连接臂引入磷酸基团设计合成了水溶性雷公藤甲素前药Minnelide【WO2010/129918】。由于 磷酸酯键旁的空间位阻较小，Minnelide很容易被磷酸酯酶水解生成羟甲基中间体，中间体羟甲醚的结构很不稳定，会自动水解释放出C14位的羟基。因此，Minnelide在I期临床试验中表现出较好的药代动力学性质，已经进入了II期临床试验。In order to overcome the problem of incomplete transformation of PG490-88 in vivo, researchers have devoted themselves to optimizing the connection method of the C-14 position of PG490-88 to avoid the influence of steric hindrance on bond breaking. In 2010, Georg et al. designed and synthesized the water-soluble triptolide prodrug Minnelide [WO2010/129918] by using methylal as a linker to introduce a phosphate group. Due to the small steric hindrance next to the phosphate bond, Minnelide is easily hydrolyzed by phosphatase to generate a hydroxymethyl intermediate. The structure of the intermediate hydroxymethyl ether is very unstable, and it will be automatically hydrolyzed to release the hydroxyl group at the C14 position. Therefore, Minnelide showed good pharmacokinetic properties in Phase I clinical trials and has entered Phase II clinical trials.
虽然甲缩醛的结构容易水解断裂，但在体外人血浆转化实验中我们发现Minnelide完全转化成雷公藤甲素仍然需要24小时以上，可能与血浆中碱性磷酸酯酶含量不高有关。此外，由于雷公藤甲素原料价格昂贵，Minnelide的合成难度较大、总收率较低(39％)，使其开发成本较高；且由于反应条件较苛刻，不利于规模化制备。因此，开发一种水溶性好、能够快速完全转化、并且合成工艺简单的雷公藤甲素前药具有重要的市场前景。Although the structure of methylal is easily broken by hydrolysis, we found that it still takes more than 24 hours for Minnelide to be completely converted into triptolide in the in vitro human plasma conversion experiment, which may be related to the low content of alkaline phosphatase in plasma. In addition, due to the expensive raw materials of triptolide, the synthesis of Minnelide is difficult and the total yield is low (39%), making its development cost high; and due to the harsh reaction conditions, it is not conducive to large-scale preparation. Therefore, the development of a triptolide prodrug with good water solubility, rapid and complete conversion, and simple synthesis process has an important market prospect.
羧酸酯酶是人和动物体内含量最丰富的水解酶，参与多种内源性和外源性化合物及药物的代谢过程，本发明通过羟基酸酯(一种容易被酯酶水解的结构)的连接方式在雷公藤甲素的C-14位引入水溶性的脂肪性含氮杂环，发现了一类水溶性好、体内转化迅速、并且易于合成(总收率达到61％)的雷公藤甲素水溶性前药。该前药水溶性好，口服有效，在人血浆中可以在1小时内完全转化成雷公藤甲素，因此，其适用于雷公藤甲素有效治疗的各种炎症性疾病、免疫性疾病、血液***恶性肿瘤和实体瘤的治疗。Carboxylesterase is the most abundant hydrolase in humans and animals, and participates in the metabolic process of various endogenous and exogenous compounds and drugs. The present invention introduces a water-soluble aliphatic nitrogen-containing heterocyclic ring at the C-14 position of triptolide through the connection of a hydroxyester (a structure that is easily hydrolyzed by esterases), and discovers a class of triptolide water-soluble prodrugs that have good water solubility, rapid in vivo transformation, and are easy to synthesize (total yield reaches 61%). The prodrug has good water solubility, is effective when taken orally, and can be completely converted into triptolide in human plasma within 1 hour. Therefore, it is suitable for the treatment of various inflammatory diseases, immune diseases, hematological malignancies and solid tumors that triptolide can effectively treat.
发明内容Contents of the invention
发明目的：本发明的目的在于提供了一种通式(I)所示的水溶性显著提高并且在血浆中可以迅速转化的雷公藤甲素前药或其药学上可接受的盐。Object of the invention: The object of the present invention is to provide a triptolide prodrug or a pharmaceutically acceptable salt thereof whose water solubility is significantly improved and can be rapidly transformed in plasma as represented by general formula (I).
技术方案：本发明所述的一类通过易于自降解的羟基酸酯连接臂引入脂肪性含氮杂环的雷公藤甲素前药或其药学上可接受的盐、多晶型物或溶剂化物；所述衍生物的化学结构式如式(I)所示：Technical solution: a class of triptolide prodrugs or their pharmaceutically acceptable salts, polymorphs or solvates that introduce an aliphatic nitrogen-containing heterocycle through a hydroxyester linking arm that is easy to self-degrade according to the present invention; the chemical structural formula of the derivative is shown in formula (I):
其中：in:
n1是1～6；n1 is 1~6;
n2是1～6；n2 is 1~6;
n3是0或1；n3 is 0 or 1;
X为碳、氮或氧原子；X is a carbon, nitrogen or oxygen atom;
HA选自盐酸、硫酸、碳酸、柠檬酸、琥珀酸、酒石酸、磷酸、乳酸、丙酮酸、乙酸、马来酸、甲磺酸、苯磺酸、对甲苯磺酸或阿魏酸；HA is selected from hydrochloric acid, sulfuric acid, carbonic acid, citric acid, succinic acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid or ferulic acid;
本发明优选的典型化合物如下(所述的雷公藤甲素前药选自下列化合物)，但不限于：Preferred typical compounds of the present invention are as follows (the triptolide prodrug is selected from the following compounds), but not limited to:
本发明的另一目的在于提供一种制备该类雷公藤甲素前药或其药学上可接受的盐、多晶型物或溶剂化物的制备方法；其具体制备步骤如下：Another object of the present invention is to provide a method for preparing the triptolide-like prodrug or its pharmaceutically acceptable salt, polymorph or solvate; its specific preparation steps are as follows:
第一步：雷公藤甲素在DMAP的作用下与酰氯反应生成中间体II；The first step: triptolide reacts with acid chloride under the action of DMAP to generate intermediate II;
将雷公藤甲素和DMAP溶于无水二氯甲烷中，在-10～5℃的低温条件下加入酰氯，后将其置于0～30℃的温度下反应6～12小时；再将得到的反应液依次用稀盐酸、饱和碳酸氢钠和盐水洗，无水硫酸钠干燥；抽滤，浓缩滤液，柱层析纯化得到中间体II；Dissolving triptolide and DMAP in anhydrous dichloromethane, adding acid chloride at a low temperature of -10 to 5°C, and reacting at a temperature of 0 to 30°C for 6 to 12 hours; then washing the obtained reaction solution with dilute hydrochloric acid, saturated sodium bicarbonate and brine in sequence, and drying with anhydrous sodium sulfate; suction filtration, concentration of the filtrate, and purification by column chromatography to obtain intermediate II;
第二步：中间体II在碘化钠和碳酸钾的作用下与相应的羧酸化合物反应生成化合物III；Second step: Intermediate II reacts with the corresponding carboxylic acid compound under the action of sodium iodide and potassium carbonate to generate compound III;
将纯化得到的中间体II溶于无水DMF中，后加入碘化钠和羧酸，进行反应0.5～1小时后加入碳酸钾，再将其加热至40～70℃反应3～12小时得反应液；然后将反应液倒入水中，用乙酸乙酯萃取，碳酸氢钠水溶液和食盐水洗涤，干燥，抽滤，浓缩滤液，柱层析纯化得到化合物III；Dissolve the purified intermediate II in anhydrous DMF, add sodium iodide and carboxylic acid, react for 0.5 to 1 hour, add potassium carbonate, then heat it to 40 to 70°C for 3 to 12 hours to obtain a reaction solution; then pour the reaction solution into water, extract with ethyl acetate, wash with aqueous sodium bicarbonate and saline, dry, filter with suction, concentrate the filtrate, and purify by column chromatography to obtain compound III;
第三步：化合物III与相应的酸成盐得到目标化合物I；The third step: Salt formation of compound III with the corresponding acid to obtain the target compound I;
将纯化得到的化合物III溶于乙酸乙酯中，加入无机酸或有机酸，于0～30℃的温度下反应6～12小时，抽滤，干燥得到目标化合物I。Dissolve the purified compound III in ethyl acetate, add inorganic acid or organic acid, react at a temperature of 0-30° C. for 6-12 hours, filter with suction, and dry to obtain the target compound I.
本发明的目的在于提供一种药物组合物，包括所述的雷公藤甲素前药、其药学上可接受的盐、多晶型物或溶剂化物，以及至少一种药学上可接受的载体、添加剂、助剂或赋形剂。The object of the present invention is to provide a pharmaceutical composition, including the triptolide prodrug, its pharmaceutically acceptable salt, polymorph or solvate, and at least one pharmaceutically acceptable carrier, additive, auxiliary agent or vehicle.
本发明的另一目的在于提供所述雷公藤甲素前药或药学上可接受的盐、及其药物组合物在制备抗肿瘤药物中的用途。Another object of the present invention is to provide the use of the triptolide prodrug or pharmaceutically acceptable salt, and its pharmaceutical composition in the preparation of antitumor drugs.
本发明所述的雷公藤甲素前药在人血浆中可以完全转化成雷公藤甲素，因此，本发明提供的雷公藤甲素前药或其药学上可接受的盐，及其药物组合物可以可作为单一治疗剂，或者与其它抗肿瘤药物联合使用，用于雷公藤甲素有效的多种恶性肿瘤的治疗，具体包括急性髓细胞白血病、淋巴瘤、骨髓瘤、肺癌、肝癌、乳腺癌、结直肠癌、卵巢癌、***、胰腺癌、胆管癌、胃癌、***癌、肾癌、食管癌、胶质母细胞瘤以及成神经细胞瘤等恶性肿瘤等多种恶性肿瘤。The triptolide prodrug of the present invention can be completely converted into triptolide in human plasma. Therefore, the triptolide prodrug or its pharmaceutically acceptable salt and pharmaceutical composition thereof provided by the present invention can be used as a single therapeutic agent, or used in combination with other antineoplastic drugs, for the treatment of various malignant tumors for which triptolide is effective, specifically including acute myeloid leukemia, lymphoma, myeloma, lung cancer, liver cancer, breast cancer, colorectal cancer, ovarian cancer, cervical cancer, pancreatic cancer, gallbladder cancer, etc. Tube cancer, gastric cancer, prostate cancer, kidney cancer, esophageal cancer, glioblastoma, neuroblastoma and other malignant tumors and other malignant tumors.
本发明的另一目的在于提供所述雷公藤甲素前药或药学上可接受的盐、及其药物组合物在制备治疗急性髓细胞白血病药物中的用途；体外抗肿瘤活性实验表明，本发明的化合物能够显著抑制急性髓细胞白血病细胞的增殖；整体动物试验表明，本发明化合物对急性髓细胞白血病具有很好的疗效，起效剂量仅为25μg/kg，并且与FLT3抑制剂联合使用具有协同增效作用；因此，本发明的化合物或其药学上可接受的盐，及其药物组合物可作为单一治疗剂，或者与FLT3抑制剂联合使用，用于急性髓细胞白血病的治疗。Another object of the present invention is to provide the triptolide prodrug or pharmaceutically acceptable salt and its pharmaceutical composition in the preparation of drugs for the treatment of acute myeloid leukemia; in vitro anti-tumor activity experiments show that the compound of the present invention can significantly inhibit the proliferation of acute myeloid leukemia cells; whole animal experiments show that the compound of the present invention has a good curative effect on acute myeloid leukemia, the effective dose is only 25 μg/kg, and it has a synergistic effect when used in combination with FLT3 inhibitors; therefore, the compound of the present invention or its pharmaceutically acceptable The salts of and pharmaceutical compositions thereof can be used as a single therapeutic agent or used in combination with FLT3 inhibitors for the treatment of acute myeloid leukemia.
本发明的另一目的在于提供所述雷公藤甲素前药或药学上可接受的盐、及其药物组合物在制备抗炎、免疫抑制及病毒(例如：抗HIV)药物中的用途；本发明所述的雷公藤甲素前药在人血浆中可以完全转化成雷公藤甲素；因此，本发明提供的雷公藤甲素前药或其药学上可接受的盐，及其药物组合物可以用于雷公藤甲素有效的各种适应症的治疗，包括类风湿性关节炎、强直性脊柱炎、***性红斑狼疮、***性血管炎、银屑病、特发性皮炎、炎症性肠病、哮喘、肺纤维化、肾炎、肾病综合征、免疫排斥反应、以及LPS诱导、CAR-T疗法、细菌感染、病毒感染等引起的细胞因子释放综合征。Another object of the present invention is to provide the purposes of the triptolide prodrug or pharmaceutically acceptable salt and its pharmaceutical composition in the preparation of anti-inflammatory, immunosuppressive and viral (for example: anti-HIV) drugs; the triptolide prodrug of the present invention can be completely converted into triptolide in human plasma; therefore, the triptolide prodrug or its pharmaceutically acceptable salt provided by the present invention, and its pharmaceutical composition can be used for the treatment of various indications that triptolide is effective, including rheumatoid arthritis, acute Ankylosing spondylitis, systemic lupus erythematosus, systemic vasculitis, psoriasis, idiopathic dermatitis, inflammatory bowel disease, asthma, pulmonary fibrosis, nephritis, nephrotic syndrome, immune rejection, and cytokine release syndrome caused by LPS induction, CAR-T therapy, bacterial infection, viral infection, etc.
有益效果：本发明与现有技术相比，本发明的特点是：本发明所述的雷公藤甲素前药具有显著的体内抗肿瘤和免疫抑制活性，相比雷公藤甲素具有更好的水溶性和药代动力学性质，相比Minnelide，不仅体内转化更迅速，而且合成更简单，收率较高(61％)，成本更低，具有良好的产业化前景。Beneficial effects: Compared with the prior art, the present invention is characterized in that the triptolide prodrug of the present invention has significant anti-tumor and immunosuppressive activity in vivo, and has better water solubility and pharmacokinetic properties than triptolide, and compared with Minnelide, not only the in vivo transformation is faster, but also the synthesis is simpler, the yield is higher (61%), the cost is lower, and it has good industrialization prospects.
图1是本发明的制备流程图；Fig. 1 is a preparation flow chart of the present invention;
图2是本发明实施例11中TP-P1、TP-P2和TP-P5在大鼠血浆中的体外转化实验的示意图；Figure 2 is a schematic diagram of the in vitro transformation experiment of TP-P1, TP-P2 and TP-P5 in rat plasma in Example 11 of the present invention;
图3是本发明实施例12中TP-P1、TP-P5在人血浆中的体外转化实验的示意图；3 is a schematic diagram of the in vitro transformation experiment of TP-P1 and TP-P5 in human plasma in Example 12 of the present invention;
图4是本发明实施例13中TP-P1、PG490-88Na、Minnelide在大鼠血浆中的体外转化实验的示意图；4 is a schematic diagram of the in vitro transformation experiment of TP-P1, PG490-88Na, and Minnelide in rat plasma in Example 13 of the present invention;
图5是本发明实施例14中TP-P1、PG490-88Na、Minnelide在人血浆中的体外转化实验 的示意图；Figure 5 is a schematic diagram of the in vitro transformation experiment of TP-P1, PG490-88Na, and Minnelide in human plasma in Example 14 of the present invention;
图6是本发明实施例15中不同浓度TP-P1在人血浆中的体外转化实验的示意图。Fig. 6 is a schematic diagram of in vitro conversion experiments of different concentrations of TP-P1 in human plasma in Example 15 of the present invention.
为了更清楚地说明本发明的技术方案，下面结合附图对本发明的技术方案做进一步的详细说明：In order to illustrate the technical solution of the present invention more clearly, the technical solution of the present invention is described in further detail below in conjunction with the accompanying drawings:
实施例1Example 1
化合物TP-P1的合成Synthesis of compound TP-P1
中间体II-1的合成：将雷公藤甲素TP(1.8g,5.0mmol)溶于50mL无水二氯甲烷中，在0℃下加入DMAP(3.05g,25.0mmol)，然后逐滴加入氯乙酰氯(4.0mL,50.00mmol)，滴加完毕后，于25℃反应12小时；反应完毕后，将反应液依次用5％的稀盐酸、饱和碳酸氢钠和饱和氯化钠溶液洗涤，然后使用无水硫酸钠干燥；抽滤，浓缩滤液，柱层析纯化(二氯甲烷:甲醇＝200:1～100:1)得到白色固体1.77g，收率88.8％。
1H NMR(500MHz,MeOD)δ(ppm):5.14(1H,s),4.78-4.85(2H,m),4.31(2H,d,J＝1.0Hz),3.99(1H,d,J＝3.2Hz),3.67(1H,d,J＝2.6Hz),3.53(1H,d,J＝5.7Hz),2.79-2.81(1H,m),2.24-2.32(2H,m),2.07-2.12(1H,m),1.88-1.98(2H,m),1.50-1.54(1H,m),1.31-1.39(1H,m),1.05(3H,s),0.98(3H,d,J＝7.0Hz),0.86(3H,d,J＝6.9Hz).
Synthesis of Intermediate II-1: Dissolve triptolide TP (1.8g, 5.0mmol) in 50mL of anhydrous dichloromethane, add DMAP (3.05g, 25.0mmol) at 0°C, and then add chloroacetyl chloride (4.0mL, 50.00mmol) dropwise. After the addition, react at 25°C for 12 hours; Wash with sodium chloride solution, and then dry with anhydrous sodium sulfate; filter with suction, concentrate the filtrate, and purify by column chromatography (dichloromethane:methanol=200:1～100:1) to obtain 1.77g of white solid with a yield of 88.8%. 1 H NMR(500MHz,MeOD)δ(ppm):5.14(1H,s),4.78-4.85(2H,m),4.31(2H,d,J＝1.0Hz),3.99(1H,d,J＝3.2Hz),3.67(1H,d,J＝2.6Hz),3.53(1H,d,J＝5.7Hz),2.79-2.81(1H,m),2.24-2.32(2H,m),2.07-2.12(1H,m),1.88-1.98(2H,m),1.50-1.54(1H,m),1.31-1.39(1H,m),1.05(3H,s),0.98(3H,d,J＝7.0Hz),0.86(3H,d,J＝6.9Hz).
中间体III-1的合成：将化合物II-1(1.0g,2.3mmol)溶于50mL无水DMF中，加入碘化钠(860mg,4.6mmol)和吗啉-4-基乙酸(670mg,4.6mmol)；室温反应40分钟后，加入碳酸钾(320mg,2.3mmol)，然后加热至50℃反应4小时；反应完毕后，将反应液倒入水中，乙酸乙酯萃取，合并有机层，依次用碳酸氢钠水溶液和饱和氯化钠溶液洗涤，无水硫酸钠干燥；抽滤，浓缩滤液，柱层析纯化(二氯甲烷:甲醇＝100:1～50:1)得到白色固体1.01g，收率80.4％；
1H NMR(500MHz,MeOD)δ(ppm):5.13(1H,s),4.82-4.85(2H,m),4.77-4.79(2H,m),3.99(1H,d,J＝3.2Hz),3.72-3.75(4H,m),3.65(1H,d,J＝2.5Hz),3.51(1H,d,J＝2.5Hz),3.43(2H,s),2.79-2.81(1H,m),2.63-2.67(4H,m),2.23-2.32(2H,m),2.04-2.10(1H,m),1.88-1.98(2H,m),1.49-1.54(1H,m),1.31-1.39(1H,m),1.05(3H,s),0.98(3H,d,J＝10.7Hz),0.86(3H,d,J＝6.9Hz).
Synthesis of Intermediate III-1: Compound II-1 (1.0g, 2.3mmol) was dissolved in 50mL of anhydrous DMF, and sodium iodide (860mg, 4.6mmol) and morpholin-4-ylacetic acid (670mg, 4.6mmol) were added; after 40 minutes of reaction at room temperature, potassium carbonate (320mg, 2.3mmol) was added, and then heated to 50°C for 4 hours; after the reaction was completed, the reaction solution was poured into water, ethyl acetate Extracted, combined organic layers, washed successively with aqueous sodium bicarbonate solution and saturated sodium chloride solution, dried over anhydrous sodium sulfate; filtered with suction, concentrated the filtrate, and purified by column chromatography (dichloromethane:methanol=100:1～50:1) to obtain 1.01 g of a white solid with a yield of 80.4%;
1H NMR(500MHz,MeOD)δ(ppm):5.13(1H,s),4.82-4.85(2H,m),4.77-4.79(2H,m),3.99(1H,d,J=3.2Hz),3.72-3.75(4H,m),3.65(1H,d,J=2.5Hz),3.51(1H, d,J=2.5Hz),3.43(2H,s),2.79-2.81(1H,m),2.63-2.67(4H,m),2.23-2.32(2H,m),2.04-2.10(1H,m),1.88-1.98(2H,m),1.49-1.54(1H,m),1.31-1 .39(1H,m),1.05(3H,s),0.98(3H,d,J=10.7Hz),0.86(3H,d,J=6.9Hz).
合物TP-P1的合成：将化合物III-1(1.0g,1.83mmol)溶于20mL乙酸乙酯中，加入20mL饱和的氯化氢的乙酸乙酯溶液，于室温反应6小时；反应完成后，抽滤，滤饼用乙酸乙酯洗涤，然后真空干燥得到白色固体0.91g,收率85.3％。
1H NMR(500MHz,DMSO-d
6)δ (ppm):10.93(1H,s),5.46(1H,s),4.88-4.92(2H,m),4.77-4.84(2H,m),4.59(1H,m),4.37(2H,s),3.88(1H,d,J＝4.9Hz),3.72-3.85(4H,m),3.56(1H,d,J＝5.5Hz),3.05-3.30(4H,m),2.64-2.74(1H,m),2.15-2.24(2H,m),1.95-1.98(2H,m),1.80-1.88(1H,m),1.36-1.41(1H,m),1.25-1.32(1H,m),0.93(3H,d,J＝6.5Hz),0.90(3H,s),0.78(3H,d,J＝6.6Hz).
13C NMR(126MHz,DMSO-d
6)δ(ppm):173.6,166.6,166.5,162.5,123.7,75.5,75.4(2C),70.8,66.8,62.5,61.8,59.5(2C),58.1,57.4,52.3(2C),35.4,34.6,30.4,29.2,22.5,17.1,16.3,15.9(2C),14.6.LCMS(ESI):m/z[M+H]
+calcd for C
28H
37ClNO
10
+,582.2；found,582.0.
Synthesis of Compound TP-P1: Compound III-1 (1.0g, 1.83mmol) was dissolved in 20mL of ethyl acetate, 20mL of saturated hydrogen chloride in ethyl acetate was added, and reacted at room temperature for 6 hours; after the reaction was completed, suction filtered, the filter cake was washed with ethyl acetate, and then vacuum-dried to obtain 0.91g of a white solid, with a yield of 85.3%. 1 H NMR(500MHz,DMSO-d 6 )δ (ppm):10.93(1H,s),5.46(1H,s),4.88-4.92(2H,m),4.77-4.84(2H,m),4.59(1H,m),4.37(2H,s),3.88(1H,d,J＝4.9Hz),3.72-3.85(4H,m),3.56(1H,d,J＝5.5Hz),3.05-3.30(4H,m),2.64-2.74(1H,m),2.15-2.24(2H,m),1.95-1.98(2H,m),1.80-1.88(1H,m),1.36-1.41(1H,m),1.25-1.32(1H,m),0.93(3H,d,J＝6.5Hz),0.90(3H,s),0.78(3H,d,J＝6.6Hz). 13 C NMR(126MHz,DMSO-d 6 )δ(ppm):173.6,166.6,166.5,162.5,123.7,75.5,75.4(2C),70.8,66.8,62.5,61.8,59.5(2C),58.1,57.4,52.3(2C),35.4,34.6,30.4,29.2,22.5,17.1,16.3,15.9(2C),14.6.LCMS(ESI):m/z[M+H] + calcd for C 28 H 37 ClNO 10 + ,582.2；found,582.0.
实施例2Example 2
化合物TP-P2的合成Synthesis of compound TP-P2
中间体III-2的合成：将化合物II-1(100mg,0.23mmol)溶于10mL无水DMF中，加入碘化钠(86mg,0.46mmol)和4-甲基-1-哌嗪乙酸(73mg,0.46mmol)；于25℃反应40分钟后，加入碳酸钾(32mg,0.23mmol)，然后加热至50℃反应4小时；反应完毕后，将反应液倒入水中，乙酸乙酯萃取，合并有机层，依次用碳酸氢钠水溶液和饱和氯化钠溶液洗涤，无水硫酸钠干燥；抽滤，浓缩滤液，柱层析纯化(二氯甲烷:甲醇＝100:1～40:1)得到白色固体93mg，收率72.3％；
1H NMR(500MHz,MeOD-d
6)δ(ppm):5.12(1H,s),4.81-4.85(2H,m),4.77-4.80(2H,m),3.98(1H,d,J＝3.1Hz),3.65(1H,d,J＝2.8Hz),3.50(1H,d,J＝5.7Hz),3.47(2H,s),2.79-2.83(1H,m),2.66-2.78(8H,m),2.40(3H,s),2.26-2.32(2H,m),2.04-2.10(1H,m),1.89-1.95(2H,m),1.49-1.54(1H,m),1.31-1.39(1H,m),1.05(3H,s),0.97(3H,d,J＝7.0Hz),0.86(3H,d,J＝6.9Hz).
Synthesis of Intermediate III-2: Dissolve compound II-1 (100mg, 0.23mmol) in 10mL of anhydrous DMF, add sodium iodide (86mg, 0.46mmol) and 4-methyl-1-piperazineacetic acid (73mg, 0.46mmol); after reacting at 25°C for 40 minutes, add potassium carbonate (32mg, 0.23mmol), then heat to 50°C for 4 hours; after the reaction, pour the reaction solution into In water, extracted with ethyl acetate, combined the organic layers, washed successively with aqueous sodium bicarbonate solution and saturated sodium chloride solution, dried over anhydrous sodium sulfate; filtered with suction, concentrated the filtrate, and purified by column chromatography (dichloromethane:methanol=100:1～40:1) to obtain 93 mg of white solid, yield 72.3%;
1H NMR (500MHz, MeOD-d
6)δ (ppm): 5.12 (1H, s), 4.81-4.85 (2H, m), 4.77-4.80 (2H, m), 3.98 (1H, d, J = 3.1Hz), 3.65 (1H, d, J = 2.8Hz), 3.50 (1H, d, J = 5.7Hz), 3.47 (2H, s), 2.79-2 .83(1H,m),2.66-2.78(8H,m),2.40(3H,s),2.26-2.32(2H,m),2.04-2.10(1H,m),1.89-1.95(2H,m),1.49-1.54(1H,m),1.31-1.39(1H,m),1.05(3 H,s),0.97(3H,d,J=7.0Hz),0.86(3H,d,J=6.9Hz).
化合物TP-P2的合成：将化合物III-2(100mg,0.18mmol)溶于5mL乙酸乙酯中，加入3mL饱和的氯化氢的乙酸乙酯溶液，室温反应6小时，反应完成后，抽滤，滤饼用乙酸乙酯洗涤，然后真空干燥得到白色固体75mg,收率70.4％；
1H NMR(500MHz,MeOD)δ(ppm):4.83-4.86(2H,m),4.82(2H,s),4.74(1H,s),4.32(1H,d,J＝5.3Hz),3.95(1H,d,J＝5.4Hz),3.84-3.93(2H,m),3.59-3.71(2H,m),3.53(1H,d,J＝6.1Hz),3.37-3.47(2H,m),3.12-3.28(4H,m),2.96(3H,s),2.82-2.85(1H,m),2.27-2.33(2H,m),2.08-2.14(2H,m),1.93-1.99(1H,m),1.56-1.59(1H,m),1.35-1.41(1H,m),1.05(3H,s),1.01(3H,d,J＝6.8Hz),0.88(3H,d,J＝6.9Hz).
13C NMR(126MHz,MeOD)δ(ppm):174.6,168.4,166.9,162.3,124.3,75.4,75.2,70.7,67.0,62.2,60.5,59.3,58.0,56.8,56.0,52.6(2C),48.9(2C),42.1,39.6,35.3,30.3,28.9,22.4,16.5,14.8,14.4,13.2.LCMS(ESI):m/z[M+H]
+calcd for C
29H
40ClN
2O
9
+,595.2；found,595.2.
Synthesis of compound TP-P2: Dissolve compound III-2 (100 mg, 0.18 mmol) in 5 mL of ethyl acetate, add 3 mL of saturated hydrogen chloride in ethyl acetate, react at room temperature for 6 hours, after the reaction is completed, filter with suction, wash the filter cake with ethyl acetate, and dry in vacuo to obtain 75 mg of a white solid, with a yield of 70.4%;
1H NMR(500MHz,MeOD)δ(ppm):4.83-4.86(2H,m),4.82(2H,s),4.74(1H,s),4.32(1H,d,J=5.3Hz),3.95(1H,d,J=5.4Hz),3.84-3.93(2H,m),3.59-3.71(2H, m),3.53(1H,d,J＝6.1Hz),3.37-3.47(2H,m),3.12-3.28(4H,m),2.96(3H,s),2.82-2.85(1H,m),2.27-2.33(2H,m),2.08-2.14(2H,m),1.93-1.99(1 H,m),1.56-1.59(1H,m),1.35-1.41(1H,m),1.05(3H,s),1.01(3H,d,J＝6.8Hz),0.88(3H,d,J＝6.9Hz).
13C NMR(126MHz,MeOD)δ(ppm):174.6,168.4,166.9,162.3,124.3,75.4,75.2,70.7,67.0,62.2,60.5,59.3,58.0,56.8,56.0,52.6(2C),48.9(2C),42.1, 39.6, 35.3, 30.3, 28.9, 22.4, 16.5, 14.8, 14.4, 13.2.LCMS(ESI):m/z[M+H]
+calcd for C
29h
40ClN
2o
9
+, 595.2; found, 595.2.
实施例3Example 3
化合物TP-P3的合成Synthesis of compound TP-P3
化合物TP-P3的合成：将化合物II-1(100mg,0.23mmol)溶于10mL无水DMF中，加入碘化钠(86mg,0.46mmol)和2-哌啶基乙酸(66mg,0.46mmol)；于25℃反应40分钟后，加入碳酸钾(32mg,0.23mmol)，然后加热至50℃反应4小时；反应完毕后，将反应液倒入水中，乙酸乙酯萃取，合并有机层，依次用碳酸氢钠水溶液和饱和氯化钠溶液洗涤，无水硫酸钠干燥；抽滤，浓缩滤液，柱层析纯化(二氯甲烷:甲醇＝100:1～40:1)得到白色固体67mg，收率53.4％；
1H NMR(500MHz,CDCl
3)δ(ppm):5.11(1H,s),4.81-4.85(2H,m),4.65-4.73(2H,m),3.84(1H,d,J＝3.1Hz),3.64(2H,d,J＝4.2Hz),3.56(1H,d,J＝2.8Hz),3.48(1H,d,J＝5.7Hz),2.87-2.98(4H,m),2.32-2.36(1H,m),2.16-2.26(2H,m),1.89-1.95(2H,m),1.76-1.80(4H,m),1.52-1.59(2H,m),1.32-1.38(2H,m),1.06(3H,s),0.97(3H,d,J＝7.0Hz),0.86(3H,d,J＝6.9Hz).
13C NMR(126MHz,CDCl
3)δ(ppm):173.2,168.0,167.0,159.9,125.7,72.4,69.9,63.5,63.1,61.3,60.8,59.4,57.9,55.4,55.0,53.4(2C),40.3,35.7,29.9,28.0,24.6(2C),23.4,23.0,17.5,17.1,16.7,13.8.LCMS(ESI):m/z[M+H]
+calcd for C
29H
38NO
9
+,544.2；found,544.3.
Synthesis of compound TP-P3: Compound II-1 (100mg, 0.23mmol) was dissolved in 10mL of anhydrous DMF, sodium iodide (86mg, 0.46mmol) and 2-piperidinylacetic acid (66mg, 0.46mmol) were added; after reacting at 25°C for 40 minutes, potassium carbonate (32mg, 0.23mmol) was added, and then heated to 50°C for 4 hours; Extract with ethyl acetate, combine the organic layers, wash successively with aqueous sodium bicarbonate solution and saturated sodium chloride solution, and dry over anhydrous sodium sulfate; filter with suction, concentrate the filtrate, and purify by column chromatography (dichloromethane:methanol=100:1～40:1) to obtain 67 mg of white solid, yield 53.4%;
1H NMR (500MHz, CDCl
3)δ (ppm): 5.11 (1H, s), 4.81-4.85 (2H, m), 4.65-4.73 (2H, m), 3.84 (1H, d, J = 3.1Hz), 3.64 (2H, d, J = 4.2Hz), 3.56 (1H, d, J = 2.8Hz), 3.48 (1H, d, J = 5.7Hz) ,2.87-2.98(4H,m),2.32-2.36(1H,m),2.16-2.26(2H,m),1.89-1.95(2H,m),1.76-1.80(4H,m),1.52-1.59(2H,m),1.32-1.38(2H,m),1.06(3H,s) ,0.97(3H,d,J=7.0Hz),0.86(3H,d,J=6.9Hz).
13C NMR (126MHz, CDCl
3( 2C), 23.4, 23.0, 17.5, 17.1, 16.7, 13.8.LCMS(ESI):m/z[M+H]
+calcd for C
29h
38NO
9
+, 544.2; found, 544.3.
实施例4Example 4
化合物TP-P4的合成Synthesis of compound TP-P4
化合物TP-P4的合成：将化合物II-1(100mg,0.23mmol)溶于无水10mL DMF中，加入碘化钠(86mg,0.46mmol)和2-(吡咯烷-1-基)乙酸(59mg,0.46mmol)；于25℃反应40分钟后，加入碳酸钾(32mg,0.23mmol)，然后加热至50℃反应4小时；反应完毕后，将反应液加入水中，乙酸乙酯萃取，合并有机层，依次用碳酸氢钠水溶液和饱和氯化钠溶液洗涤，无水硫酸钠干燥；抽滤，浓缩滤液，柱层析纯化(二氯甲烷:甲醇＝100:1～40:1)得到白色固体64mg，收率52.3％；
1H NMR(500MHz,MeOD)δ(ppm):5.14(1H,m),4.81-4.85(2H,m),3.98-4.03(2H,m),3.73(1H,m),3.66(1H,d,J＝7.3Hz),3.51(1H,d,J＝5.7Hz),3.27(2H,s),3.14-3.18(1H,m),2.79-2.81(1H,m),2.59-2.62(1H,m)2.22-2.32(2H,m),2.02-2.09(4H,m),1.85-1.92(2H,m),1.49-1.53(1H,m),1.35-1.42(4H,m),1.05(3H,s),0.97(3H,d,J＝7.0Hz),0.86(3H,d,J＝6.9Hz).
13C NMR(126MHz,MeOD)δ(ppm):174.6,172.4,162.4,161.9,124.1,71.7,70.6,65.9,63.5,62.7,61.4,59.6,59.5,55.4,54.8,54.3,52.9,40.0,35.4,29.4,28.2,22.8(2C),22.7,16.5(2C),15.7,12.7.LCMS(ESI):m/z[M+H]
+calcd for C
28H
36NO
9
+,530.2；found,530.2.
Synthesis of compound TP-P4: Compound II-1 (100mg, 0.23mmol) was dissolved in anhydrous 10mL DMF, sodium iodide (86mg, 0.46mmol) and 2-(pyrrolidin-1-yl)acetic acid (59mg, 0.46mmol) were added; after reacting at 25°C for 40 minutes, potassium carbonate (32mg, 0.23mmol) was added, and then heated to 50°C for 4 hours; The reaction solution was added to water, extracted with ethyl acetate, the organic layers were combined, washed successively with aqueous sodium bicarbonate solution and saturated sodium chloride solution, and dried over anhydrous sodium sulfate; filtered with suction, concentrated the filtrate, and purified by column chromatography (dichloromethane:methanol=100:1～40:1) to obtain 64 mg of a white solid, with a yield of 52.3%;
1H NMR(500MHz,MeOD)δ(ppm):5.14(1H,m),4.81-4.85(2H,m),3.98-4.03(2H,m),3.73(1H,m),3.66(1H,d,J=7.3Hz),3.51(1H,d,J=5.7Hz),3.27(2H,s),3. 14-3.18(1H,m),2.79-2.81(1H,m),2.59-2.62(1H,m)2.22-2.32(2H,m),2.02-2.09(4H,m),1.85-1.92(2H,m),1.49-1.53(1H,m),1.35-1.42(4H,m ),1.05(3H,s),0.97(3H,d,J=7.0Hz),0.86(3H,d,J=6.9Hz).
13C NMR(126MHz,MeOD)δ(ppm):174.6,172.4,162.4,161.9,124.1,71.7,70.6,65.9,63.5,62.7,61.4,59.6,59.5,55.4,54.8,54.3,52.9,40.0,35.4,29. 4,28.2,22.8(2C),22.7,16.5(2C),15.7,12.7.LCMS(ESI):m/z[M+H]
+calcd for C
28h
36NO
9
+, 530.2; found, 530.2.
实施例5Example 5
化合物TP-P5的合成Synthesis of compound TP-P5
中间体II-5的合成：将雷公藤甲素(180mg,0.50mmol)溶于20mL无水二氯甲烷中，在0℃条件下加入DMAP(305mg,2.50mmol)，然后逐滴加入4-溴丁酰氯(0.56mL,5.00mmol)，滴加完毕后，于25℃反应12小时；反应完毕后，将反应液依次用5％的稀盐酸、饱和碳酸氢钠和饱和氯化钠溶液洗涤，然后使用无水硫酸钠干燥；抽滤，浓缩滤液，柱层析纯化(二氯甲烷:甲醇＝200:1～100:1)得到白色固体193mg，收率75.6％。
1H NMR(500MHz, CDCl
3)δ(ppm):5.11(1H,s),4.65-4.73(2H,m),4.33(1H,t,J＝7.1Hz),3.85(1H,d,J＝2.9Hz),3.52-3.55(2H,m),3.49(1H,d,J＝5.6Hz),2.60-2.72(4H,m),2.51(1H,t,J＝8.1Hz),2.24-2.30(3H,m),1.89-1.98(2H,m),1.57-1.62(1H,m),1.26-1.35(1H,m),1.07(3H,s),0.98(3H,d,J＝6.9Hz),0.87(3H,d,J＝6.9Hz).
Synthesis of Intermediate II-5: Dissolve triptolide (180mg, 0.50mmol) in 20mL of anhydrous dichloromethane, add DMAP (305mg, 2.50mmol) at 0°C, and then add 4-bromobutyryl chloride (0.56mL, 5.00mmol) dropwise. After the addition, react at 25°C for 12 hours; Wash with saturated sodium chloride solution, then dry with anhydrous sodium sulfate; filter with suction, concentrate the filtrate, and purify by column chromatography (dichloromethane:methanol=200:1～100:1) to obtain 193 mg of white solid, yield 75.6%. 1 H NMR(500MHz, CDCl 3 )δ(ppm):5.11(1H,s),4.65-4.73(2H,m),4.33(1H,t,J＝7.1Hz),3.85(1H,d,J＝2.9Hz),3.52-3.55(2H,m),3.49(1H,d,J＝5.6Hz),2.60-2.72(4H,m),2.51(1H,t,J＝8.1Hz),2.24-2.30(3H,m),1.89-1.98(2H,m),1.57-1.62(1H,m),1.26-1.35(1H,m),1.07(3H,s),0.98(3H,d,J＝6.9Hz),0.87(3H,d,J＝6.9Hz).
中间体III-5的合成：将化合物II-5(117mg,0.23mmol)溶于10mL无水DMF中，加入碘化钠(86mg,0.46mmol)和吗啉-4-基乙酸(67mg,0.46mmol)；于25℃反应40分钟后，加入碳酸钾(32mg,0.23mmol)，然后加热至50℃反应4小时；反应完毕后，将反应液倒入水中，乙酸乙酯萃取，合并有机层，依次用碳酸氢钠水溶液和饱和氯化钠溶液洗涤，无水硫酸钠干燥；抽滤，浓缩滤液，柱层析纯化(二氯甲烷:甲醇＝100:1～50:1)得到白色固体83mg，收率63.2％；
1H NMR(500MHz,MeOD)δ(ppm):5.10(1H,s),4.78-4.84(2H,m),4.24(2H,t,J＝6.4Hz),3.98(1H,d,J＝3.2Hz),3.73(4H,t,J＝4.7Hz),3.65(1H,d,J＝2.8Hz),3.50(1H,d,J＝5.7Hz),3.29(2H,s),2.78-2.81(1H,m),2.61(4H,t,J＝4.6Hz),2.46-2.57(2H,m),2.24-2.32(2H,m),2.09-2.12(1H,m),2.01-2.06(2H,m),1.86-1.95(2H,m),1.49-1.54(1H,m),1.31-1.39(1H,m),1.05(3H,s),0.97(3H,d,J＝7.0Hz),0.86(3H,d,J＝6.9Hz).
Synthesis of Intermediate III-5: Compound II-5 (117mg, 0.23mmol) was dissolved in 10mL of anhydrous DMF, sodium iodide (86mg, 0.46mmol) and morpholin-4-ylacetic acid (67mg, 0.46mmol) were added; after reacting at 25°C for 40 minutes, potassium carbonate (32mg, 0.23mmol) was added, and then heated to 50°C for 4 hours; after the reaction was completed, the reaction solution was poured into water, Extracted with ethyl acetate, combined the organic layers, washed successively with aqueous sodium bicarbonate solution and saturated sodium chloride solution, dried over anhydrous sodium sulfate; filtered with suction, concentrated the filtrate, and purified by column chromatography (dichloromethane:methanol=100:1～50:1) to obtain 83 mg of white solid, yield 63.2%;
1H NMR (500MHz, MeOD) δ (ppm): 5.10 (1H, s), 4.78-4.84 (2H, m), 4.24 (2H, t, J = 6.4Hz), 3.98 (1H, d, J = 3.2Hz), 3.73 (4H, t, J = 4.7Hz), 3.65 (1H, d, J = 2.8Hz), 3.50 (1H,d,J=5.7Hz),3.29(2H,s),2.78-2.81(1H,m),2.61(4H,t,J=4.6Hz),2.46-2.57(2H,m),2.24-2.32(2H,m),2.09-2.12(1H,m),2.01-2.06(2H,m), 1.86-1.95(2H,m),1.49-1.54(1H,m),1.31-1.39(1H,m),1.05(3H,s),0.97(3H,d,J=7.0Hz),0.86(3H,d,J=6.9Hz).
化合物TP-P5的合成：将化合物III-5(100mg,0.17mmol)溶解于5mL乙酸乙酯中，加入3mL饱和的氯化氢的乙酸乙酯溶液，于25℃反应6小时；反应完成后，抽滤，滤饼用乙酸乙酯洗涤，然后真空干燥得到白色固体53mg，收率51.3％。mp:239-241℃.
1H NMR(500MHz,MeOD)δ(ppm):4.81-4.85(2H,m),4.73(1H,s),4.37-4.39(2H,m),4.33(1H,m),4.14(2H,s),3.89-4.03(4H,m),3.55(1H,d,J＝4.9Hz),3.34(1H,d,J＝5.5Hz),2.83-2.86(1H,m),2.48-2.58(2H,m),2.28-2.32(2H,m),2.06-2.11(4H,m),1.94-2.04(2H,m),1.53-1.62(1H,m),1.32-1.40(3H,m),1.25-1.28(1H,m),1.05(3H,s),1.01(3H,d,J＝6.0Hz),0.90(3H,d,J＝6.0Hz).
13C NMR(126MHz,MeOD)δ(ppm):174.7,171.9,165.9,162.4,124.2,75.4,74.1,70.7,66.9,65.1,63.7(2C),62.3,59.7,57.9,56.9,55.9,52.6(2C),39.6,35.3,30.3,30.2,29.1,23.5,22.4,16.6,14.8,14.4,13.1.LCMS(ESI):m/z[M+H]+calcd for C
30H
41ClNO
10
+,610.2；found,610.2.
Synthesis of compound TP-P5: Compound III-5 (100 mg, 0.17 mmol) was dissolved in 5 mL of ethyl acetate, 3 mL of saturated hydrogen chloride in ethyl acetate was added, and reacted at 25° C. for 6 hours; after the reaction was completed, suction filtered, the filter cake was washed with ethyl acetate, and then vacuum-dried to obtain 53 mg of a white solid, with a yield of 51.3%. mp:239-241℃. 1 H NMR(500MHz,MeOD)δ(ppm):4.81-4.85(2H,m),4.73(1H,s),4.37-4.39(2H,m),4.33(1H,m),4.14(2H,s),3.89-4.03(4H,m),3.55(1H,d,J＝4.9Hz),3.34(1H,d,J＝5.5Hz),2.83-2.86(1H,m),2.48-2.58(2H,m),2.28-2.32(2H,m),2.06-2.11(4H,m),1.94-2.04(2H,m),1.53-1.62(1H,m),1.32-1.40(3H,m),1.25-1.28(1H,m),1.05(3H,s),1.01(3H,d,J＝6.0Hz),0.90(3H,d,J＝6.0Hz). 13 C NMR(126MHz,MeOD)δ(ppm):174.7,171.9,165.9,162.4,124.2,75.4,74.1,70.7,66.9,65.1,63.7(2C),62.3,59.7,57.9,56.9,55.9,52.6(2C),39.6,35.3,30.3,30.2,29.1,23.5,22.4,16.6,14.8,14.4,13.1.LCMS(ESI):m/z[M+H]+calcd for C 30 H 41 ClNO 10 + ,610.2；found,610.2.
实施例6Example 6
化合物TP-P6的合成Synthesis of compound TP-P6
中间体II-6的合成：将雷公藤甲素(180mg,0.50mmol)溶于20mL无水二氯甲烷中，在0℃下加入DMAP(305mg,2.50mmol)，然后逐滴加入5-溴戊酰氯(0.70mL,5.00mmol)， 滴加完毕后，于25℃反应12小时；反应完毕后，将反应液依次用5％的稀盐酸、饱和碳酸氢钠和饱和氯化钠溶液洗涤，然后使用无水硫酸钠干燥；抽滤，浓缩滤液，柱层析纯化(二氯甲烷:甲醇＝200:1～100:1)得到白色固体205mg，收率78.4％。
1H NMR(500MHz,CDCl
3)δ(ppm):5.10(1H,s),4.65-4.73(2H,m),3.84(1H,d,J＝3.2Hz),3.55(1H,d,J＝3.0Hz),3.47-3.49(1H,m),3.43(2H,t,J＝6.6Hz),2.69-2.72(1H,m),2.50-2.58(1H,m),2.42(2H,t,J＝7,4Hz),2.31-2.36(1H,m),2.11-2.21(2H,m),1.98-2.01(1H,m),1.90-1.96(2H,m),1.78-1.84(2H,m),1.57-1.60(1H,m),1.22-1.29(1H,m),1.07(3H,s),0.97(3H,d,J＝7.0Hz),0.86(3H,d,J＝6.9Hz).
Synthesis of Intermediate II-6: Dissolve triptolide (180mg, 0.50mmol) in 20mL of anhydrous dichloromethane, add DMAP (305mg, 2.50mmol) at 0°C, and then add 5-bromovaleryl chloride (0.70mL, 5.00mmol) dropwise. After the addition, react at 25°C for 12 hours; Wash with saturated sodium chloride solution, and then dry with anhydrous sodium sulfate; filter with suction, concentrate the filtrate, and purify by column chromatography (dichloromethane:methanol=200:1～100:1) to obtain 205 mg of white solid with a yield of 78.4%. 1 H NMR(500MHz,CDCl 3 )δ(ppm):5.10(1H,s),4.65-4.73(2H,m),3.84(1H,d,J＝3.2Hz),3.55(1H,d,J＝3.0Hz),3.47-3.49(1H,m),3.43(2H,t,J＝6.6Hz),2.69-2.72(1H,m),2.50-2.58(1H,m),2.42(2H,t,J＝7,4Hz),2.31-2.36(1H,m),2.11-2.21(2H,m),1.98-2.01(1H,m),1.90-1.96(2H,m),1.78-1.84(2H,m),1.57-1.60(1H,m),1.22-1.29(1H,m),1.07(3H,s),0.97(3H,d,J＝7.0Hz),0.86(3H,d,J＝6.9Hz).
化合物TP-P6的合成：将化合物II-6(120mg,0.23mmol)溶解于10mL无水DMF中，加入碘化钠(86mg,0.46mmol)和吗啉-4-基乙酸(67mg,0.46mmol)；于25℃反应40分钟后，加入碳酸钾(32mg,0.23mmol)，然后加热至50℃反应4小时；反应完毕后，将反应液倒入水中，乙酸乙酯萃取，合并有机层，依次用碳酸氢钠水溶液和饱和氯化钠溶液洗涤，无水硫酸钠干燥；抽滤，浓缩滤液，柱层析纯化(二氯甲烷:甲醇＝100:1～50:1)得到白色固体88mg，收率65.1％；
1H NMR(500MHz,MeOD)δ(ppm):5.10(1H,s),4.78-4.84(2H,m),4.19(2H,t,J＝5.5Hz),3.98(1H,d,J＝3.0Hz),3.73(4H,t,J＝4.5Hz),3.65(1H,d,J＝3.0Hz),3.50(1H,d,J＝5.6Hz),3.29(2H,s),2.78-2.81(1H,m),2.61(4H,t,J＝4.6Hz),2.48-2.54(1H,m),2.39-2.45(1H,m),2.26-2.31(2H,m),2.05-2.11(1H,m),1.85-1.99(2H,m),1.76-1.79(2H,m),1.50-1.54(1H,m),1.31-1.39(3H,m),1.05(3H,s),0.96(3H,d,J＝7.0Hz),0.86(3H,d,J＝6.9Hz).
13C NMR(126MHz,MeOD)δ(ppm):174.6,172.7,170.2,162.4,124.1,71.3,70.6,66.2(2C),64.0,63.5,62.8,61.3,59.7,58.6,55.3,54.8,52.9(2C),40.1,35.4,33.2,29.4,28.3,27.5,22.8,21.3,16.6,16.5,15.7,12.8.LCMS(ESI):m/z[M+H]+calcd for C
31H
42NO
10
+,588.3；found,588.3.
Synthesis of compound TP-P6: Compound II-6 (120mg, 0.23mmol) was dissolved in 10mL of anhydrous DMF, sodium iodide (86mg, 0.46mmol) and morpholin-4-ylacetic acid (67mg, 0.46mmol) were added; after reacting at 25°C for 40 minutes, potassium carbonate (32mg, 0.23mmol) was added, and then heated to 50°C for 4 hours; after the reaction was completed, the reaction solution was poured into water, Extract with ethyl acetate, combine the organic layers, wash successively with aqueous sodium bicarbonate solution and saturated sodium chloride solution, and dry over anhydrous sodium sulfate; filter with suction, concentrate the filtrate, and purify by column chromatography (dichloromethane:methanol=100:1～50:1) to obtain 88 mg of white solid, yield 65.1%;
1H NMR (500MHz, MeOD) δ (ppm): 5.10 (1H, s), 4.78-4.84 (2H, m), 4.19 (2H, t, J = 5.5Hz), 3.98 (1H, d, J = 3.0Hz), 3.73 (4H, t, J = 4.5Hz), 3.65 (1H, d, J = 3.0Hz), 3.50 (1H,d,J=5.6Hz),3.29(2H,s),2.78-2.81(1H,m),2.61(4H,t,J=4.6Hz),2.48-2.54(1H,m),2.39-2.45(1H,m),2.26-2.31(2H,m),2.05-2.11(1H,m), 1.85-1.99(2H,m),1.76-1.79(2H,m),1.50-1.54(1H,m),1.31-1.39(3H,m),1.05(3H,s),0.96(3H,d,J=7.0Hz),0.86(3H,d,J=6.9Hz).
13C NMR (126MHz, MeOD) δ (ppm): 174.6, 172.7, 170.2, 162.4, 124.1, 71.3, 70.6, 66.2 (2C), 64.0, 63.5, 62.8, 61.3, 59.7, 58.6, 55.3, 54.8, 52.9 (2C), 40.1, 35.4, 33.2, 29.4, 28.3, 27.5, 22.8, 21.3, 16.6, 16.5, 15.7, 12.8. LCMS (ESI): m/z[M+H]+calcd for C
31h
42NO
10
+,588.3; found, 588.3.
实施例7Example 7
化合物TP-P7的合成Synthesis of compound TP-P7
中间体II-7的合成：将雷公藤甲素(180mg,0.50mmol)溶于20mL无水二氯甲烷中，在0℃下加入DMAP(305mg,2.50mmol)，然后逐滴加入6-溴己酰氯(0.77mL,5.00mmol)，滴加完毕后，于25℃反应12小时；反应完毕后，将反应液依次用5％的稀盐酸、饱和碳酸氢钠和饱和氯化钠溶液洗涤，然后使用无水硫酸钠干燥；抽滤，浓缩滤液，柱层析纯化(二氯甲烷:甲醇＝200:1～100:1)得到白色固体191mg，收率71.2％。
1H NMR(500MHz,CDCl
3)δ(ppm):5.07(1H,s),4.63-4.71(2H,m),3.82(1H,d,J＝3.2Hz),3.53(1H,d,J＝2.5Hz),3.46(1H,d,J＝6.1Hz),3.40(2H,t,J＝6.8Hz),2.66-2.69(1H,m),2.43-2.49(1H,m),2.36(2H,t,J＝7.4Hz),2.22-2.32(1H,m),2.12-2.19(1H,m),1.84-1.89(2H,m),1.68-1.73(2H,m),1.60-1.65(2H,m),1.55-1.57(1H,m),1.45-1.51(2H,m),1.18-1.24(1H,m),1.04(3H,s),0.94(3H,d,J＝7.0Hz),0.82(3H,d,J＝6.9Hz).
Synthesis of Intermediate II-7: Dissolve triptolide (180mg, 0.50mmol) in 20mL of anhydrous dichloromethane, add DMAP (305mg, 2.50mmol) at 0°C, and then add 6-bromohexanoyl chloride (0.77mL, 5.00mmol) dropwise. After the addition, react at 25°C for 12 hours; Wash with saturated sodium chloride solution, then dry with anhydrous sodium sulfate; filter with suction, concentrate the filtrate, and purify by column chromatography (dichloromethane:methanol=200:1～100:1) to obtain 191 mg of white solid, with a yield of 71.2%. 1 H NMR(500MHz,CDCl 3 )δ(ppm):5.07(1H,s),4.63-4.71(2H,m),3.82(1H,d,J＝3.2Hz),3.53(1H,d,J＝2.5Hz),3.46(1H,d,J＝6.1Hz),3.40(2H,t,J＝6.8Hz),2.66-2.69(1H,m),2.43-2.49(1H,m),2.36(2H,t,J＝7.4Hz),2.22-2.32(1H,m),2.12-2.19(1H,m),1.84-1.89(2H,m),1.68-1.73(2H,m),1.60-1.65(2H,m),1.55-1.57(1H,m),1.45-1.51(2H,m),1.18-1.24(1H,m),1.04(3H,s),0.94(3H,d,J＝7.0Hz),0.82(3H,d,J＝6.9Hz).
化合物TP-P7的合成：将化合物II-7(124mg,0.23mmol)溶于10mL无水DMF中，加入碘化钠(86mg,0.46mmol)和吗啉-4-基乙酸(67mg,0.46mmol)。于25℃反应40分钟后，加入碳酸钾(32mg,0.23mmol)，然后加热至50℃反应4小时；反应完毕后，将反应液倒入水中，乙酸乙酯萃取，合并有机层，依次用碳酸氢钠水溶液和饱和氯化钠溶液洗涤，无水硫酸钠干燥；抽滤，浓缩滤液，柱层析纯化(二氯甲烷:甲醇＝100:1～50:1)得到白色固体83mg，收率60.1％；
1H NMR(500MHz,MeOD)δ(ppm):5.10(1H,s),4.78-4.84(2H,m),4.16(2H,t,J＝6.6Hz),3.98(1H,d,J＝3.0Hz),3.72(4H,t,J＝4.4Hz),3.64(1H,d,J＝3.0Hz),3.48(1H,d,J＝5.6Hz),3.28(2H,s),2.78-2.81(1H,m),2.62(4H,t,J＝4.4Hz),2.45-2.51(1H,m),2.35-2.42(1H,m),2.24-2.32(2H,m),2.05-2.11(1H,m),1.82-1.95(2H,m),1.64-1.79(2H,m),1.45-1.57(2H,m),1.31-1.39(3H,m),1.05(3H,s),0.96(3H,d,J＝7.0Hz),0.86(3H,d,J＝6.9Hz).
13C NMR(126MHz,MeOD)δ(ppm):174.6,172.9,170.1,162.4,124.1,71.3,70.6,66.1(2C),64.3,63.5,62.8,61.3,59.7,58.6,55.3,54.8,52.9(2C),40.1,35.4,33.6,29.4,28.3,28.0,24.9,24.3,22.8,16.6,16.5,15.8,12.9.LCMS(ESI):m/z[M+H]+calcd for C
32H
44NO
10
+,602.3；found,602.3.
Synthesis of compound TP-P7: Compound II-7 (124 mg, 0.23 mmol) was dissolved in 10 mL of anhydrous DMF, and sodium iodide (86 mg, 0.46 mmol) and morpholin-4-ylacetic acid (67 mg, 0.46 mmol) were added.于25℃反应40分钟后，加入碳酸钾(32mg,0.23mmol)，然后加热至50℃反应4小时；反应完毕后，将反应液倒入水中，乙酸乙酯萃取，合并有机层，依次用碳酸氢钠水溶液和饱和氯化钠溶液洗涤，无水硫酸钠干燥；抽滤，浓缩滤液，柱层析纯化(二氯甲烷:甲醇＝100:1～50:1)得到白色固体83mg，收率60.1％； 1 H NMR(500MHz,MeOD)δ(ppm):5.10(1H,s),4.78-4.84(2H,m),4.16(2H,t,J＝6.6Hz),3.98(1H,d,J＝3.0Hz),3.72(4H,t,J＝4.4Hz),3.64(1H,d,J＝3.0Hz),3.48(1H,d,J＝5.6Hz),3.28(2H,s),2.78-2.81(1H,m),2.62(4H,t,J＝4.4Hz),2.45-2.51(1H,m),2.35-2.42(1H,m),2.24-2.32(2H,m),2.05-2.11(1H,m),1.82-1.95(2H,m),1.64-1.79(2H,m),1.45-1.57(2H,m),1.31-1.39(3H,m),1.05(3H,s),0.96(3H,d,J＝7.0Hz),0.86(3H,d,J＝6.9Hz). 13 C NMR(126MHz,MeOD)δ(ppm):174.6,172.9,170.1,162.4,124.1,71.3,70.6,66.1(2C),64.3,63.5,62.8,61.3,59.7,58.6,55.3,54.8,52.9(2C),40.1,35.4,33.6,29.4,28.3,28.0,24.9,24.3,22.8,16.6,16.5,15.8,12.9.LCMS(ESI):m/z[M+H]+calcd for C 32 H 44 NO 10 + ,602.3；found,602.3.
实施例8Example 8
化合物TP-P8的合成Synthesis of compound TP-P8
化合物TP-P8的合成：将化合物II-1(100mg,0.23mmol)溶于10mL无水DMF中，加入碘化钠(86mg,0.46mmol)和3-(4-吗啉基)丙酸(73mg,0.46mmol)；于25℃反应40分钟后，加入碳酸钾(32mg,0.23mmol)，然后加热至50℃反应4小时；反应完毕后，将反应液倒入水中，乙酸乙酯萃取，合并有机层，依次用碳酸氢钠水溶液和饱和氯化钠溶液洗涤，无水硫酸钠干燥；抽滤，浓缩滤液，柱层析纯化(二氯甲烷:甲醇＝100:1～50:1)得到白色固体84mg，收率65.4％；
1H NMR(500MHz,CDCl
3)δ(ppm):5.10(1H,s),4.75-4.82(2H,m),4.65-4.72(2H,m),3.87(4H,t,J＝4.7Hz),3.85(1H,d,J＝3.1Hz),3.56(1H,d,J＝2.7Hz),3.48(1H,d,J＝5.7Hz),3.14(2H,t,J＝7.3Hz),2.90-2.92(4H,m),2.85-2.89(2H,m),2.69-2.72(1H,m),2.32-2.36(1H,m),2.16-2.21(2H,m),1.88-1.95(2H,m),1.55-1.59(1H,m),1.32-1.36(1H,m),1.07(3H,s),0.98(3H,d,J＝7.0Hz),0.86(3H,d,J＝6.9Hz).
13C NMR(126MHz,CDCl
3)δ(ppm):170.5,167.2,165.0,159.8,125.7,72.3,70.0,65.1(2C),63.6,63.1,61.3,60.9,59.4,55.4,55.0,52.6,52.3(2C),40.3,35.7,29.9,29.5,28.1,23.4,17.5,17.1,16.7,13.8.LCMS(ESI):m/z[M+H]+calcd for C
29H
38NO
10
+,560.2；found,560.3.
Synthesis of compound TP-P8: Dissolve compound II-1 (100 mg, 0.23 mmol) in 10 mL of anhydrous DMF, add sodium iodide (86 mg, 0.46 mmol) and 3-(4-morpholinyl) propionic acid (73 mg, 0.46 mmol); after reacting at 25°C for 40 minutes, add potassium carbonate (32 mg, 0.23mmol), then heat to 50°C for 4 hours; Pour into water, extract with ethyl acetate, combine the organic layers, wash successively with aqueous sodium bicarbonate solution and saturated sodium chloride solution, and dry over anhydrous sodium sulfate; filter with suction, concentrate the filtrate, and purify by column chromatography (dichloromethane:methanol=100:1～50:1) to obtain 84 mg of white solid, yield 65.4%;
1H NMR (500MHz, CDCl
3)δ (ppm): 5.10 (1H, s), 4.75-4.82 (2H, m), 4.65-4.72 (2H, m), 3.87 (4H, t, J = 4.7Hz), 3.85 (1H, d, J = 3.1Hz), 3.56 (1H, d, J = 2.7Hz), 3.48 (1H, d, J = 5.7Hz) ,3.14(2H,t,J＝7.3Hz),2.90-2.92(4H,m),2.85-2.89(2H,m),2.69-2.72(1H,m),2.32-2.36(1H,m),2.16-2.21(2H,m),1.88-1.95(2H,m),1.55-1.5 9(1H,m),1.32-1.36(1H,m),1.07(3H,s),0.98(3H,d,J=7.0Hz),0.86(3H,d,J=6.9Hz).
13C NMR (126MHz, CDCl
3)δ (ppm): 170.5, 167.2, 165.0, 159.8, 125.7, 72.3, 70.0, 65.1 (2C), 63.6, 63.1, 61.3, 60.9, 59.4, 55.4, 55.0, 52.6, 52.3 (2C), 40.3, 35.7, 29.9, 2 9.5, 28.1, 23.4, 17.5, 17.1, 16.7, 13.8. LCMS (ESI): m/z[M+H]+calcd for C
29h
38NO
10
+, 560.2; found, 560.3.
实施例9Example 9
化合物的长期稳定性实验Compound long-term stability test
实验方法：将化合物于室温敞口放置90天以上，通过HPLC归一化法测定化合物的纯度(表1)。Experimental method: the compound was left open at room temperature for more than 90 days, and the purity of the compound was determined by the HPLC normalization method (Table 1).
表1 实施例化合物的长期稳定性实验结果The long-term stability test result of table 1 embodiment compound
结果显示：该系列化合物的化学稳定性很好，常温敞口放置90天后，经HPLC-UV和
1H-NMR谱分析，化合物的纯度未见明显降低。
The results showed that the chemical stability of this series of compounds was very good, and the purity of the compounds did not decrease significantly after being left open at room temperature for 90 days by HPLC-UV and 1 H-NMR spectrum analysis.
实施例10Example 10
化合物TP-P1在纯水及酸性水溶液中的稳定性实验Stability experiment of compound TP-P1 in pure water and acidic aqueous solution
实验方法：将化合物TP-P1溶于pH＝7的纯水及pH＝4、pH＝2的酸性水溶液中，采集不同时间点的样品，通过HPLC归一化法测定化合物的纯度(表2)。Experimental method: Compound TP-P1 was dissolved in pure water with pH=7 and acidic aqueous solution with pH=4 and pH=2, samples at different time points were collected, and the purity of the compound was determined by HPLC normalization method (Table 2).
表2 化合物TP-P1在纯水及酸性水溶液中的稳定性实验结果Table 2 Stability test results of compound TP-P1 in pure water and acidic aqueous solution
结果显示：化合物TP-P1在pH7和pH2、pH4的水溶液中6小时内纯度没有明显变化。The results showed that the purity of the compound TP-P1 did not change significantly within 6 hours in the aqueous solution of pH7, pH2, and pH4.
实施例11Example 11
TP-P1、TP-P2、TP-P5在大鼠血浆中的体外转化实验In Vitro Transformation Experiment of TP-P1, TP-P2, TP-P5 in Rat Plasma
考虑到化合物的水溶性，我们选择水溶性较好的成盐化合物TP-P1、TP-P2、TP-P5进行大鼠的体外血浆转化实验。Considering the water solubility of the compounds, we selected salt-forming compounds TP-P1, TP-P2, and TP-P5 with better water solubility for in vitro plasma conversion experiments in rats.
实验方法：取250μL大鼠空白血浆，加入等体积100μg/mL TP-P1、TP-P2、TP-P5水溶液，于恒温振荡器中60r/min、37℃孵育，于1、5、10、15、30、45、60、90min、2、4、6、8、10、12、24h取20μL含药血浆于预冷的60μL甲醇中，涡旋3min，4℃、14000rpm/min离心10min，取上清进行HPLC分析；Experimental method: Take 250 μL rat blank plasma, add an equal volume of 100 μg/mL TP-P1, TP-P2, TP-P5 aqueous solution, incubate in a constant temperature oscillator at 60 r/min, 37 °C, take 20 μL of drug-containing plasma in pre-cooled 60 μL methanol, and vortex 3 min, centrifuge at 4°C, 14000rpm/min for 10min, and take the supernatant for HPLC analysis;
液相分析方法：ACN:0.1％TFA-H
2O＝35:65等度洗脱，流速：0.6mL/min
Liquid phase analysis method: ACN: 0.1% TFA-H 2 O = 35:65 isocratic elution, flow rate: 0.6mL/min
液相分析方法：ACN:0.1％TFA-H
2O＝35:65等度洗脱，流速：0.6mL/min
Liquid phase analysis method: ACN: 0.1% TFA-H 2 O = 35:65 isocratic elution, flow rate: 0.6mL/min
实验结果如图2所述；The experimental results are as shown in Figure 2;
结果显示：TP-P1、TP-P2和TP-P5三个前药在大鼠血浆中30min内都能完全转化生成TP，但在相同的时间下，15min时TP-P1已经转化52.3％，而TP-P2和TP-P5不到50％，因此TP-P1前药转化成TP的效率要稍高于TP-P2和TP-P5。The results showed that the three prodrugs of TP-P1, TP-P2 and TP-P5 could be completely converted into TP within 30 minutes in rat plasma, but at the same time, 52.3% of TP-P1 had been converted in 15 minutes, while TP-P2 and TP-P5 were less than 50%. Therefore, the conversion efficiency of TP-P1 prodrug into TP was slightly higher than that of TP-P2 and TP-P5.
实施例12Example 12
TP-P1、TP-P5在人血浆中的体外转化实验In Vitro Transformation Experiment of TP-P1 and TP-P5 in Human Plasma
鉴于化合物TP-P1和TP-P2都是采用乙醇酸的连接键，而相同时间内TP-P1转化速率优于TP-P5，我们选择TP-P1、TP-P5进行人的体外血浆转化实验。In view of the fact that both compounds TP-P1 and TP-P2 use glycolic acid linkages, and the conversion rate of TP-P1 is better than that of TP-P5 within the same time period, we chose TP-P1 and TP-P5 for human in vitro plasma conversion experiments.
实验方法：取250μL人空白血浆，加入等体积TP-P1或TP-P5水溶液(100μg/mL或2mg/mL)，于恒温振荡器中60r/min、37℃孵育，于5、10、15、30、45、60、90min、2、4、6、8、10、24h取20μL含药血浆于预冷的60μL甲醇中，涡旋3min，4℃、14000rpm/min离心10min，取上清进行HPLC分析。Experimental method: Take 250 μL of human blank plasma, add an equal volume of TP-P1 or TP-P5 aqueous solution (100 μg/mL or 2 mg/mL), incubate in a constant temperature shaker at 60 r/min, 37 °C, take 20 μL of drug-containing plasma in pre-cooled 60 μL methanol, vortex for 3 min, 4 °C , Centrifuge at 14000rpm/min for 10min, and take the supernatant for HPLC analysis.
液相分析方法：ACN:0.1％TFA-H
2O＝35:65等度洗脱，流速：0.6mL/min
Liquid phase analysis method: ACN: 0.1% TFA-H 2 O = 35:65 isocratic elution, flow rate: 0.6mL/min
实验结果如图3所述；The experimental results are as shown in Figure 3;
结果显示：TP-P1在低浓度下完全转化成TP需要45min，而高浓度下60min才完全转化成TP；TP-P5在低浓度下完全转化成TP需要45min，高浓度下90min才完全转化成TP；说明药物浓度对血浆转化存在影响，低浓度下转化速率较快。TP-P5与TP-P1相比，低浓度下完全转化成TP均需要45min，但在30min TP-P1已经转化90％，而TP-P5才不到80％，因此，同等浓度下，在人的血浆中TP-P1转化生成TP的速率快于TP-P5。The results showed that it took 45 minutes for TP-P1 to be completely converted into TP at low concentrations, and 60 minutes at high concentrations; 45 minutes for TP-P5 to be completely converted to TP at low concentrations, and 90 minutes at high concentrations to completely convert to TP; it indicated that drug concentration had an impact on plasma conversion, and the conversion rate was faster at low concentrations. Compared with TP-P1, it takes 45 minutes for TP-P5 to be completely converted into TP at a low concentration, but 90% of TP-P1 has been converted in 30 minutes, while TP-P5 is less than 80%. Therefore, at the same concentration, the conversion rate of TP-P1 into TP in human plasma is faster than that of TP-P5.
实施例13Example 13
TP-P1、PG490-88Na、Minnelide在大鼠血浆中的体外转化实验In Vitro Transformation Experiment of TP-P1, PG490-88Na and Minnelide in Rat Plasma
实验方法：取400μL大鼠空白血浆，加入等体积1μg/mL TP-P1、PG490-88Na、Minnelide水溶液(血药浓度:500ng/mL)，于恒温振荡器中60r/min、37℃孵育，于1、5、10、15、30、45、60、90min、2、4、6、8、10、12、24h取40μL含药血浆于预冷的120μL甲醇(IS＝1ng/mL)中，涡旋3min，4℃、14000rpm/min离心10min，取上清进行UPLC-MS/MS分析。Experimental method: Take 400μL rat blank plasma, add an equal volume of 1μg/mL TP-P1, PG490-88Na, Minnelide aqueous solution (blood concentration: 500ng/mL), incubate in a constant temperature oscillator at 60r/min, 37℃, take 40μL drug-containing plasma at 1, 5, 10, 15, 30, 45, 60, 90min, 2, 4, 6, 8, 10, 12, 24h Vortex for 3 min in pre-cooled 120 μL methanol (IS=1 ng/mL), centrifuge at 14000 rpm/min at 4°C for 10 min, and take the supernatant for UPLC-MS/MS analysis.
液相分析方法：流动相0.1％FA-H2O(A)和ACN(B)；流速：0.3mL/min；梯度洗脱程度：0～2min,15％B～80％B；2～3min,80％B～80％B；3～4min,80％B～15％B；4～5min,15％B～15％B；进样量：5μL；Liquid phase analysis method: mobile phase 0.1% FA-H2O (A) and ACN (B); flow rate: 0.3mL/min; gradient elution degree: 0~2min, 15%B~80%B; 2~3min, 80%B~80%B; 3~4min, 80%B~15%B; 4~5min, 15%B~15%B; injection volume: 5μL;
实验结果如图4所述；The experimental results are as shown in Figure 4;
结果显示：TP-P1在大鼠的血浆中能够较快的转化生成TP，30min内就可以实现TP的完全转化；PG490-88Na在大鼠的血浆中，90min内也可以完全转化成TP；而Minnelide的转化也相对比较慢，需要6h才能实现TP的完全转化，可能是由于大鼠血浆中的磷酸酯酶相对含量较低；因此，在大鼠血浆的条件下，前药TP-P1转化成TP的速率远高于PG490-88Na和Minnelide。The results show that: TP-P1 can be converted into TP quickly in the plasma of rats, and the complete conversion of TP can be realized within 30 minutes; PG490-88Na can also be completely converted into TP within 90 minutes in the plasma of rats; and the conversion of Minnelide is relatively slow, and it takes 6 hours to realize the complete conversion of TP, which may be due to the relatively low content of phosphatase in rat plasma; therefore, under the condition of rat plasma, the conversion rate of prodrug TP-P1 into TP is much higher than that of PG490-88 Na and Minnelide.
实施例14Example 14
TP-P1、PG490-88Na、Minnelide在人血浆中的体外转化实验In Vitro Transformation Experiment of TP-P1, PG490-88Na and Minnelide in Human Plasma
实验方法：取400μL人空白血浆，加入等体积1μg/mL TP-P1、PG490-88Na、Minnelide水溶液(血药浓度:500ng/mL)，于恒温振荡器中60r/min、37℃孵育，于1、5、10、15、30、45、60、90min、2、4、6、8、10、12、24h取40μL含药血浆于预冷的120μL甲醇(IS＝1ng/mL)中，涡旋3min，4℃、14000rpm/min离心10min，取上清进行UPLC-MS/MS分析。Experimental method: Take 400μL human blank plasma, add an equal volume of 1μg/mL TP-P1, PG490-88Na, Minnelide aqueous solution (blood concentration: 500ng/mL), incubate in a constant temperature oscillator at 60r/min, 37°C, take 40μL of drug-containing plasma at 1, 5, 10, 15, 30, 45, 60, 90min, 2, 4, 6, 8, 10, 12, 24h Vortex for 3 min in pre-cooled 120 μL methanol (IS=1 ng/mL), centrifuge at 14000 rpm/min at 4°C for 10 min, and take the supernatant for UPLC-MS/MS analysis.
液相分析方法：流动相0.1％FA-H2O(A)和ACN(B)；流速：0.3mL/min；梯度洗脱程度：0～2min,15％B～80％B；2～3min,80％B～80％B；3～4min,80％B～15％B；4～5min,15％B～15％B；进样量：5μL；Liquid phase analysis method: mobile phase 0.1% FA-H2O (A) and ACN (B); flow rate: 0.3mL/min; gradient elution degree: 0~2min, 15%B~80%B; 2~3min, 80%B~80%B; 3~4min, 80%B~15%B; 4~5min, 15%B~15%B; injection volume: 5μL;
实验结果如图5所述；The experimental results are as shown in Figure 5;
结果显示：TP-P1在人的血浆中也能够较快的转化生成TP，基本1h内可以完全转化；而PG490-88Na在人的血浆中则转化特别缓慢，24h转化也不到15％，远远低于大鼠血浆中的转化速度；Minnelide的转化也相对比较慢，24h转化接近80％，可能也是由于人血浆中的磷酸酯酶相对含量较低。因此，在人血浆的条件下，前药TP-P1转化成TP的速率远高于PG490-88Na和Minnelide。The results show that: TP-P1 can also be converted into TP quickly in human plasma, and can be completely converted within 1 hour; while PG490-88Na is very slow in human plasma, and the conversion rate is less than 15% in 24 hours, which is far lower than the conversion rate in rat plasma; the conversion of Minnelide is also relatively slow, and the conversion in 24 hours is close to 80%, which may also be due to the relative low content of phosphatase in human plasma. Therefore, under the condition of human plasma, the conversion rate of prodrug TP-P1 to TP is much higher than that of PG490-88Na and Minnelide.
实施例15Example 15
不同浓度TP-P1在人血浆中的体外转化实验In Vitro Transformation Experiment of Different Concentrations of TP-P1 in Human Plasma
实验方法：取400μL人空白血浆，加入等体积TP-P1水溶液(10μg/mL、1μg/mL、100ng/ml)，于恒温振荡器中60r/min、37℃孵育，于1、5、10、15、30、45、60、90min、2、4、6、8、10、12、24h取40μL含药血浆于预冷的120μL甲醇(IS＝1ng/mL)中，涡旋3min，4℃、14000rpm/min离心10min，取上清进行UPLC-MS/MS分析；Experimental method: Take 400 μL of human blank plasma, add equal volume of TP-P1 aqueous solution (10 μg/mL, 1 μg/mL, 100 ng/ml), incubate in a constant temperature oscillator at 60 r/min, 37 ° C, take 40 μL of drug-containing plasma in pre-cooled 120 μL methanol ( IS=1ng/mL), vortex for 3min, centrifuge at 14000rpm/min for 10min at 4°C, and take the supernatant for UPLC-MS/MS analysis;
液相分析方法：流动相0.1％FA-H2O(A)和ACN(B)；流速：0.3mL/min；梯度洗脱程度：0～2min,15％B～80％B；2～3min,80％B～80％B；3～4min,80％B～15％B；4～5min,15％B～15％B；进样量：5μL；Liquid phase analysis method: mobile phase 0.1% FA-H2O (A) and ACN (B); flow rate: 0.3mL/min; gradient elution degree: 0~2min, 15%B~80%B; 2~3min, 80%B~80%B; 3~4min, 80%B~15%B; 4~5min, 15%B~15%B; injection volume: 5μL;
实验结果如图6所述；The experimental results are as shown in Figure 6;
结果显示：TP-P1在人的血浆中不同低浓度(50ng/mL、500ng/mL、5000ng/mL)下都能够较快的转化生成TP，1h内基本可以实现完全转化。The results show that: TP-P1 can be converted into TP relatively quickly at different low concentrations (50ng/mL, 500ng/mL, 5000ng/mL) in human plasma, and complete conversion can be basically achieved within 1h.
实施例16Example 16
TP-P1和TP对多种肿瘤细胞株的增殖抑制活性Proliferation inhibitory activity of TP-P1 and TP on various tumor cell lines
表3 TP-P1对多种肿瘤细胞的体外增殖抑制活性Table 3 In vitro proliferation inhibitory activity of TP-P1 on various tumor cells
实验方法：MV-4-11、THP-1、KG-1和HL-60是人源急性髓细胞白血病细胞，PANC-1为人源胰腺癌细胞，U937是人组织细胞淋巴瘤细胞，CAG、ARP-1、H929是人源骨髓瘤细胞，HepG2和Hep3B为人源肝癌细胞，HT-29和HCT-116为人源结肠癌细胞，MDA-MB-231是人源乳腺癌细胞，Hela是人源***细胞，A549是人源肺癌细胞；悬浮生长细胞采用CCK-8方法测定化合物对肿瘤细胞的体外抗增殖活性：用培养基3倍梯度稀释受试化合物至终浓度的两倍，取200μL至2mL EP管中备用；取适量处于对数生长期的细胞重悬于培养基中，等体积加入到含有受试化合物的培养基中，上下颠倒10次混匀，依次加入96孔板中，每孔100μL；于37℃、5％CO
2孵箱培养48h后，每孔加入10μL CCK-8，继续孵育2h；酶标仪读取OD450吸光度值，重复两次实验；采用Graphpad Prism 8软件分析处理数据，求得IC
50；贴壁生长细胞采用MTT方法测定化合物对实体瘤细胞的体外抗增殖活性：胰酶消化处于对数生长期的细胞，计数，取适量细胞重悬于培养液中，每孔100μL加于96孔板中，过夜培养后，每孔加入100μL 3倍梯度稀释的受试化合物或对照的培养基，于37℃、5％CO
2孵箱培养48h后，每孔加入20μL MTT，继续37℃孵育4h，酶标仪读取OD490吸光度值，重复两次实验；采用Graphpad Prism 8软件分析处理数据，求得IC
50值。
Experimental method: MV-4-11, THP-1, KG-1 and HL-60 are human acute myeloid leukemia cells; PANC-1 is human pancreatic cancer cells; U937 is human histiocyte lymphoma cells; CAG, ARP-1, H929 are human myeloma cells; HepG2 and Hep3B are human liver cancer cells; HT-29 and HCT-116 are human colon cancer cells; Human breast cancer cells, Hela is human cervical cancer cells, A549 is human lung cancer cells; CCK-8 method was used to determine the in vitro anti-proliferation activity of the compound on tumor cells: dilute the test compound with a 3-fold gradient in the medium to twice the final concentration, and take 200 μL to 2 mL EP tube for later use; take an appropriate amount of cells in the logarithmic growth phase and resuspend in the medium, add an equal volume to the medium containing the test compound, mix up and down 10 times, and add to a 96-well plate in turn, 100 per well μL; at 37°C, 5% CO
2After 48 hours of incubation in the incubator, add 10 μL of CCK-8 to each well and continue to incubate for 2 hours; read the OD450 absorbance value with a microplate reader, and repeat the experiment twice; use Graphpad Prism 8 software to analyze and process the data to obtain the IC
50; For adherent growth cells, the in vitro anti-proliferation activity of the compound on solid tumor cells was determined by the MTT method: cells in the logarithmic growth phase were digested with trypsin, counted, and an appropriate amount of cells were resuspended in culture medium, and 100 μL per well was added to a 96-well plate.
2After culturing in the incubator for 48 hours, add 20 μL MTT to each well, continue to incubate at 37°C for 4 hours, read the OD490 absorbance value with a microplate reader, and repeat the experiment twice; use Graphpad Prism 8 software to analyze and process the data to obtain the IC
50value.
结果显示：TP-P1及TP对大多数肿瘤细胞具有较强的增殖抑制活性，其中对人源急性髓细胞白血病细胞系THP-1、KG-1、MV-4-11和HL-60的抑制活性最强，对骨髓瘤、淋巴瘤以及其他实体瘤细胞的抑制活性弱于人源急性髓细胞白血病细胞系(表3)。The results showed that TP-P1 and TP had strong proliferation inhibitory activity on most tumor cells, among which the inhibitory activity on human acute myeloid leukemia cell lines THP-1, KG-1, MV-4-11 and HL-60 was the strongest, and the inhibitory activity on myeloma, lymphoma and other solid tumor cells was weaker than that of human acute myeloid leukemia cell lines (Table 3).
实施例17Example 17
TP-P1在MV-4-11裸鼠移植瘤模型上的抗肿瘤效果Antitumor effect of TP-P1 on MV-4-11 nude mouse xenograft tumor model
实验方法：MV-4-11细胞体外培养扩增，取适量处于对数生长期的细胞重悬于无血清IMDM培养基与Matrigel(1:1)混悬液中，无菌条件下制备成5×10
6/100μL细胞悬液，用注射器将100μL细胞悬液接种于雄性Balb/c裸小鼠前左肢腋窝皮下；待肿瘤体积生长至100-200mm
3时，选取肿瘤大小适中的动物随机分组，每组5只；分别给予空白媒介(PBS)、受试化合物TP-P1低剂量(25μg/kg/d)、TP-P1中剂量(50μg/kg/d)、TP-P1高剂量(100μg/kg/d)，每天腹腔注射一次，给药4周；给药期间，每天测量裸小鼠体重和瘤径；实验 结束后颈椎脱臼处死，取瘤称重。
Experimental method: MV-4-11 cells were cultured and expanded in vitro, and an appropriate amount of cells in the logarithmic growth phase were resuspended in serum-free IMDM medium and Matrigel (1:1) suspension, and prepared under sterile conditions into 5×10
6/100μL cell suspension, use a syringe to inoculate 100μL cell suspension subcutaneously in the armpit of the anterior left limb of male Balb/c nude mice; wait until the tumor volume grows to 100-200mm
3At the same time, animals with moderate tumor size were randomly divided into 5 groups. They were given blank vehicle (PBS), low dose of TP-P1 (25 μg/kg/d), medium dose of TP-P1 (50 μg/kg/d), and high dose of TP-P1 (100 μg/kg/d) respectively, and injected intraperitoneally once a day for 4 weeks. During the administration period, the body weight and tumor diameter of nude mice were measured every day;
肿瘤体积(tumor volume，TV)的计算公式为：TV＝1/2×a×b
2，a表示肿瘤长径；b表示肿瘤短径。
The calculation formula of tumor volume (TV) is: TV=1/2×a×b 2 , where a represents the long diameter of the tumor; b represents the short diameter of the tumor.
表4 TP-P1在MV-4-11裸鼠移植瘤模型上的抗肿瘤效果Table 4 Anti-tumor effect of TP-P1 on MV-4-11 nude mouse xenograft model
*,p<0.05；**,p<0.01；***,p<0.001(与溶剂对照比较)。*, p<0.05; **, p<0.01; ***, p<0.001 (compared to solvent control).
结果显示：在MV-4-11裸鼠移植瘤模型上，连续腹腔注射给药4周，化合物TP-P1能够剂量依赖性地抑制肿瘤的生长，并且对小鼠体重没有影响；其中25μg/kg/d剂量下的抑瘤率达到54.31％，100μg/kg/d剂量可使移植瘤完全消退，抑瘤率达到100％(表4)。The results showed that: on the MV-4-11 nude mouse xenograft tumor model, administered intraperitoneally for 4 weeks, the compound TP-P1 could dose-dependently inhibit the growth of the tumor, and had no effect on the body weight of the mice; the tumor inhibition rate at a dose of 25 μg/kg/d reached 54.31%, and the dose of 100 μg/kg/d could completely regress the xenograft tumor, and the tumor inhibition rate reached 100% (Table 4).
实施例18Example 18
TP-P1在THP-1裸鼠移植瘤模型上的抗肿瘤效果Anti-tumor effect of TP-P1 on THP-1 nude mouse xenograft tumor model
实验方法：THP-1细胞体外培养扩增，取适量处于对数生长期的细胞重悬于无血清1640培养基与Matrigel(1:1)混悬液中，无菌条件下制备成5×10
6/100μL细胞悬液，用注射器将100μL细胞悬液接种于雄性Balb/c裸小鼠前左肢腋窝皮下；待肿瘤体积生长至100-200mm
3时，选取肿瘤大小适中的动物随机分组，每组5只；分别给予空白媒介(PBS)、阳性对照药TP(180μg/kg/d)、受试化合物TP-P1(100μg/kg/d)、TP-P1(300μg/kg/d)、TP-P1(600μg/kg/d)，TP-P1(1200μg/kg/d)，每天腹腔注射一次，给药4周；给药期间，每天测量裸小鼠体重和瘤径。实验结束后颈椎脱臼处死，取瘤称重。
实验方法：THP-1细胞体外培养扩增，取适量处于对数生长期的细胞重悬于无血清1640培养基与Matrigel(1:1)混悬液中，无菌条件下制备成5×10 6 /100μL细胞悬液，用注射器将100μL细胞悬液接种于雄性Balb/c裸小鼠前左肢腋窝皮下；待肿瘤体积生长至100-200mm 3时，选取肿瘤大小适中的动物随机分组，每组5只；分别给予空白媒介(PBS)、阳性对照药TP(180μg/kg/d)、受试化合物TP-P1(100μg/kg/d)、TP-P1(300μg/kg/d)、TP-P1(600μg/kg/d)，TP-P1(1200μg/kg/d)，每天腹腔注射一次，给药4周；给药期间，每天测量裸小鼠体重和瘤径。 After the experiment, the animals were killed by cervical dislocation, and the tumors were weighed.
肿瘤体积(tumor volume，TV)的计算公式为：TV＝1/2×a×b
2，a表示肿瘤长径；b表示肿瘤短径。
The calculation formula of tumor volume (TV) is: TV=1/2×a×b 2 , where a represents the long diameter of the tumor; b represents the short diameter of the tumor.
表5 TP-P1在THP-1裸鼠移植瘤模型上的抗肿瘤效果Table 5 Antitumor effect of TP-P1 on THP-1 nude mouse xenograft model
*,p<0.05；**,p<0.01；***,p<0.001(与溶剂对照比较)。*, p<0.05; **, p<0.01; ***, p<0.001 (compared to solvent control).
结果显示：在THP-1裸鼠移植瘤模型上，连续腹腔注射给药4周，化合物TP-P1能够剂量依赖性地抑制肿瘤的生长，并且对小鼠体重没有影响。其中100μg/kg/d剂量下的抑瘤率达到93.87％，300μg/kg/d及以上剂量可使移植瘤完全消退，抑瘤率达到100％(表5)。The results showed that: on the THP-1 nude mouse xenograft tumor model, the compound TP-P1 could dose-dependently inhibit the growth of the tumor after continuous intraperitoneal injection for 4 weeks, and had no effect on the body weight of the mice. Among them, the tumor inhibition rate at a dose of 100 μg/kg/d reached 93.87%, and the dose of 300 μg/kg/d and above could completely regress the transplanted tumor, and the tumor inhibition rate reached 100% (Table 5).
实施例19Example 19
TP-P1与吉瑞替尼联合给药在MV-4-11裸鼠移植瘤模型上的抗肿效果Anti-tumor effect of TP-P1 combined with geritinib on MV-4-11 nude mouse xenograft tumor model
实验方法：MV-4-11细胞体外培养扩增，取适量处于对数生长期的细胞重悬于无血清IMDM培养基与Matrigel(1:1)混悬液中，无菌条件下制备成5×10
6/100μL细胞悬液，用注射器将100μL细胞悬液接种于雄性Balb/c裸小鼠前左肢腋窝皮下；待肿瘤体积生长至100-200mm
3时，选取肿瘤大小适中的动物随机分组，每组6只；分别给予空白媒介(PBS)及羧甲基纤维素钠(CMC-Na)、受试化合物TP-P1剂量组(50μg/kg/d)、吉瑞替尼低剂量(0.5mg/kg/d)、吉瑞替尼高剂量(1mg/kg/d)，TP-P1联用吉瑞替尼低剂量组；每天TP-P1腹腔注射，吉瑞替尼灌胃给药一次，给药3周；给药期间，每天测量裸小鼠体重和瘤径；实验结束后颈椎脱臼处死，取瘤称重。
Experimental method: MV-4-11 cells were cultured and expanded in vitro, and an appropriate amount of cells in the logarithmic growth phase were resuspended in serum-free IMDM medium and Matrigel (1:1) suspension, and prepared under sterile conditions into 5×10
6/100μL cell suspension, use a syringe to inoculate 100μL cell suspension subcutaneously in the armpit of the anterior left limb of male Balb/c nude mice; wait until the tumor volume grows to 100-200mm
3At the same time, the animals with moderate tumor size were randomly divided into groups, 6 animals in each group; they were given blank medium (PBS) and carboxymethylcellulose sodium (CMC-Na), the test compound TP-P1 dose group (50 μg/kg/d), geritinib low dose (0.5 mg/kg/d), geritinib high dose (1 mg/kg/d), TP-P1 combined with geritinib low dose group; TP-P1 intraperitoneal injection every day, geritinib intragastric administration once, The administration was administered for 3 weeks; during the administration period, the body weight and tumor diameter of the nude mice were measured every day; after the experiment, the mice were sacrificed by cervical dislocation, and the tumors were weighed.
肿瘤体积(tumor volume，TV)的计算公式为：TV＝1/2×a×b
2，a表示肿瘤长径；b表示肿瘤短径。
The formula for calculating tumor volume (TV) is: TV=1/2×a×b 2 , where a represents the long diameter of the tumor; b represents the short diameter of the tumor.
表6 TP-P1与吉瑞替尼联合给药在MV-4-11裸鼠移植瘤模型上的抗肿效果Table 6 Anti-tumor effect of TP-P1 combined with geritinib on MV-4-11 nude mouse xenograft model
*,p<0.05；**,p<0.01；***,p<0.001(与溶剂对照比较)。^,p<0.05；^^,p<0.01；^^^,p<0.001(与吉瑞替尼低剂量组比较)。*, p<0.05; **, p<0.01; ***, p<0.001 (compared to solvent control). ^, p<0.05; ^^, p<0.01; ^^^, p<0.001 (compared with low-dose geritinib group).
结果显示：在MV-4-11裸鼠移植瘤模型上，连续给药3周，联合组的抑瘤率78.12％高于对应剂量单药吉瑞替尼组(48.27％)，且高于两倍剂量单药吉瑞替尼组(71.96％)(表6)；因此，化合物TP-P1与吉瑞替尼联合用于急性髓细胞白血病的治疗具有协同增效作用。The results showed that: on the MV-4-11 nude mouse transplanted tumor model, after continuous administration for 3 weeks, the tumor inhibition rate of the combination group was 78.12% higher than that of the corresponding dose of single drug geritinib group (48.27%), and higher than that of the double dose of single drug geritinib group (71.96%) (Table 6); therefore, the combination of compound TP-P1 and geritinib for the treatment of acute myeloid leukemia has a synergistic effect.
实施例20Example 20
TP-P1缓解LPS诱导的脓毒症小鼠肺部炎症的作用The role of TP-P1 in alleviating LPS-induced lung inflammation in septic mice
实验方法：将雌性Balb/c小鼠采用随机分组法分为5组，每组6只，分别为溶剂对照组、模型组、TP-P1低剂量组(500μg/kg/d)、TP-P1中剂量组(1000μg/kg/d)、TP-P1高剂量组(1500μg/kg/d)，各组均在注射LPS前两天进行预防性给药，每天腹腔注射一次，连续给药2天，给药体积为10ml/kg；第3天，分别给予模型组、TP-P1低、中、高各剂量组腹腔注射一次10mg/kg LPS，给药体积为5ml/kg；第4天，无菌摘取小鼠肺组织，通过RT-qPCR技术检测IL-1β、IL-6、TNF-α、IFN-γ等炎性因子的表达情况，各组数据分别与溶剂对照组做归一化处理。Experimental method: Female Balb/c mice were randomly divided into 5 groups, 6 in each group, respectively solvent control group, model group, TP-P1 low-dose group (500 μg/kg/d), TP-P1 medium-dose group (1000 μg/kg/d), TP-P1 high-dose group (1500 μg/kg/d), each group was given preventive administration two days before LPS injection, intraperitoneal injection once a day, continuous administration for 2 days, and the administration volume was 1 0ml/kg; on the third day, the model group, TP-P1 low, medium, and high dose groups were given an intraperitoneal injection of 10 mg/kg LPS, and the administration volume was 5ml/kg; on the fourth day, the lung tissue of the mice was aseptically removed, and the expressions of inflammatory factors such as IL-1β, IL-6, TNF-α, IFN-γ were detected by RT-qPCR technology, and the data of each group were normalized with the solvent control group.
表7 TP-P1缓解LPS诱导的脓毒症小鼠肺部炎症的作用Table 7 TP-P1 alleviates the effect of LPS-induced sepsis mice lung inflammation
*,p<0.05；**,p<0.01；***,p<0.001(与溶剂对照组比较)。^,p<0.05；^^,p<0.01；^^^,p<0.001(给药组与模型组比较)。*, p<0.05; **, p<0.01; ***, p<0.001 (compared with solvent control group). ^, p<0.05; ^^, p<0.01; ^^^, p<0.001 (comparison between administration group and model group).
结果显示：TP-P1能够抑制IL-1β、IL-6、TNF-α、IFN-γ等炎性因子的释放，并且呈剂量依赖性，缓解LPS诱导的脓毒症小鼠肺部炎症(表7)。The results showed that: TP-P1 can inhibit the release of inflammatory factors such as IL-1β, IL-6, TNF-α, IFN-γ, and in a dose-dependent manner, relieve LPS-induced lung inflammation in septic mice (Table 7).
实施例21Example 21
TP-P1的药代动力学性质Pharmacokinetic properties of TP-P1
实验方法：将8只雄性SD大鼠采用随机分组法分为2组，随机分组的SD大鼠禁食过夜但可以自由饮水；12h后，第一组给予化合物TP-P1的水溶液，灌胃给药，给药剂量为1.6mg/kg；第二组给予TP的橄榄油混悬液，灌胃给药，给药剂量为1.0mg/kg；采用眼眶取血法，分别于2min、5min、10min、15min、30min、45min、60min、90min、2h、4h、6h取血，转移至肝素钠预先处理的1.5mL的离心管后离心分离(8000rpm/min，5min，4℃) 得到血浆，并保存于-80℃的冰箱中。Experimental method: 8 male SD rats were divided into 2 groups by random grouping. The randomly grouped SD rats were fasted overnight but allowed to drink water freely; 12 hours later, the first group was given the aqueous solution of compound TP-P1, and the dosage was 1.6 mg/kg; the second group was given the olive oil suspension of TP, and the dosage was 1.0 mg/kg; Blood was collected at min, 90min, 2h, 4h, and 6h, transferred to a 1.5mL centrifuge tube pretreated with sodium heparin, and centrifuged (8000rpm/min, 5min, 4°C) to obtain plasma, which was stored in a -80°C refrigerator.
处理方法：采用1:3甲醇蛋白沉淀法处理血浆样本，涡旋离心后，取上清通过UPLC-MS/MS分析血浆中的TP血药浓度，采用Phoenix 64软件处理数据，得到药代动力学参数。Processing method: Plasma samples were processed by 1:3 methanol protein precipitation method, and after vortex centrifugation, the supernatant was taken to analyze the concentration of TP in plasma by UPLC-MS/MS, and the data were processed by Phoenix 64 software to obtain pharmacokinetic parameters.
表8 TP-P1的药代动力学性质The pharmacokinetic property of table 8 TP-P1
结果显示：TP-P1的AUC
(0-t)、Cmax等药代参数都优于TP，在等摩尔口服给药的情况下TP-P1的吸收明显高于TP(表8)。
The results showed that the pharmacokinetic parameters such as AUC (0-t) and Cmax of TP-P1 were superior to those of TP, and the absorption of TP-P1 was significantly higher than that of TP in the case of equimolar oral administration (Table 8).
以上仅是本发明的优选实施方式，本发明的保护范围并不仅局限于上述实施例，凡属于本发明思路下的技术方案均属于本发明的保护范围。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理前提下的若干改进和润饰，应视为本发明的保护范围。The above are only preferred implementations of the present invention, and the protection scope of the present invention is not limited to the above-mentioned embodiments, and all technical solutions under the idea of the present invention belong to the protection scope of the present invention. It should be pointed out that for those skilled in the art, some improvements and modifications without departing from the principle of the present invention should be regarded as the protection scope of the present invention.
Claims (8)
- 一类雷公藤甲素前药或其药学上可接受的盐、多晶型物或溶剂化物，其特征在于，所述化合物的化学结构式如式(I)所示：A class of triptolide prodrugs or pharmaceutically acceptable salts, polymorphs or solvates thereof, characterized in that the chemical structural formula of the compound is as shown in formula (I):
其中：in:n1是1～6；n1 is 1~6;n2是1～6；n2 is 1~6;n3是0或1；n3 is 0 or 1;X为碳、氮或氧原子；X is a carbon, nitrogen or oxygen atom;HA选自盐酸、硫酸、碳酸、柠檬酸、琥珀酸、酒石酸、磷酸、乳酸、丙酮酸、乙酸、马来酸、甲磺酸、苯磺酸、对甲苯磺酸或阿魏酸。HA is selected from hydrochloric acid, sulfuric acid, carbonic acid, citric acid, succinic acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid or ferulic acid. - 根据权利要求1所述的一类雷公藤甲素前药或其药学上可接受的盐、多晶型物或溶剂化物，其特征在于，A class of triptolide prodrugs or pharmaceutically acceptable salts, polymorphs or solvates thereof according to claim 1, characterized in that,所述的雷公藤甲素前药选自下列化合物：Described triptolide prodrug is selected from following compounds:
- 如权利要求1和2所述的一类雷公藤甲素前药或其药学上可接受的盐、多晶型物或溶剂化物的制备方法，其特征在于，其具体制备步骤如下：The preparation method of a class of triptolide prodrug or its pharmaceutically acceptable salt, polymorph or solvate as claimed in claim 1 and 2, is characterized in that, its specific preparation steps are as follows:
第一步：雷公藤甲素在DMAP的作用下与酰氯反应生成中间体II：The first step: triptolide reacts with acid chloride under the action of DMAP to generate intermediate II:将雷公藤甲素和DMAP溶于无水二氯甲烷中，在-10～5℃的低温条件下加入酰氯，后将其置于0～30℃的温度下反应6～12小时；再将得到的反应液依次用稀盐酸、饱和碳酸氢钠和盐水洗，无水硫酸钠干燥；抽滤，浓缩滤液，柱层析纯化得到中间体II；Dissolving triptolide and DMAP in anhydrous dichloromethane, adding acid chloride at a low temperature of -10 to 5°C, and reacting at a temperature of 0 to 30°C for 6 to 12 hours; then washing the obtained reaction solution with dilute hydrochloric acid, saturated sodium bicarbonate and brine in sequence, and drying with anhydrous sodium sulfate; suction filtration, concentration of the filtrate, and purification by column chromatography to obtain intermediate II;第二步：中间体II在碘化钠和碳酸钾的作用下与羧酸化合物反应生成化合物III：Second step: intermediate II reacts with carboxylic acid compound under the effect of sodium iodide and potassium carbonate to generate compound III:将纯化得到的中间体II溶于无水DMF中，后加入碘化钠和羧酸，进行反应0.5～1小时后加入碳酸钾，再将其加热至40～70℃反应3～12小时得反应液；后将反应液倒入水中，用乙酸乙酯萃取，碳酸氢钠水溶液和食盐水洗涤，干燥，抽滤，浓缩滤液，柱层析纯化得到化合物III；Dissolve the purified intermediate II in anhydrous DMF, then add sodium iodide and carboxylic acid, react for 0.5 to 1 hour, add potassium carbonate, then heat it to 40 to 70°C and react for 3 to 12 hours to obtain a reaction solution; then pour the reaction solution into water, extract with ethyl acetate, wash with aqueous sodium bicarbonate and saline, dry, filter with suction, concentrate the filtrate, and purify by column chromatography to obtain compound III;第三步：化合物III与酸成盐得到目标化合物I：The third step: Salt formation of compound III with acid to obtain target compound I:将纯化得到的化合物III溶于乙酸乙酯中，加入无机酸或有机酸，于0～30℃的温度下反应6～12小时，抽滤，干燥；最终得到目标化合物I。Dissolve the purified compound III in ethyl acetate, add inorganic acid or organic acid, react at a temperature of 0-30° C. for 6-12 hours, filter with suction, and dry; the target compound I is finally obtained. - 一种药物组合物，包括如权利要求1中所述的一类雷公藤甲素前药或其药学上可接受的盐、多晶型物或溶剂化物。A pharmaceutical composition, comprising a class of triptolide prodrugs or pharmaceutically acceptable salts, polymorphs or solvates thereof as described in claim 1.
- 一种如权利要求4所述的药物组合物在制备用于预防/治疗恶性肿瘤、炎症性疾病、 免疫性疾病及病毒感染性疾病的药物。A pharmaceutical composition as claimed in claim 4 is used in the preparation of medicines for preventing/treating malignant tumors, inflammatory diseases, immune diseases and viral infectious diseases.
- 根据权利要求5中所述的恶性肿瘤，其特征在于，所述恶性肿瘤包括急性髓细胞白血病、淋巴瘤、骨髓瘤、肺癌、肝癌、乳腺癌、结直肠癌、卵巢癌、***、胰腺癌、胆管癌、胃癌、***癌、肾癌、食管癌、胶质母细胞瘤及成神经细胞瘤。The malignant tumor according to claim 5, wherein the malignant tumor comprises acute myeloid leukemia, lymphoma, myeloma, lung cancer, liver cancer, breast cancer, colorectal cancer, ovarian cancer, cervical cancer, pancreatic cancer, bile duct cancer, gastric cancer, prostate cancer, kidney cancer, esophageal cancer, glioblastoma and neuroblastoma.
- 根据权利要求5中所述的免疫性疾病和炎症性疾病，其特征在于，所述免疫性疾病和炎症性疾病包括类风湿性关节炎、强直性脊柱炎、***性红斑狼疮、***性血管炎、银屑病、特发性皮炎、炎症性肠病、哮喘、肺纤维化、肾炎、肾病综合征、免疫排斥反应及LPS诱导、CAR-T疗法、细菌感染、病毒感染引起的细胞因子释放综合征。According to the immune disease and inflammatory disease described in claim 5, it is characterized in that, the immune disease and inflammatory disease include rheumatoid arthritis, ankylosing spondylitis, systemic lupus erythematosus, systemic vasculitis, psoriasis, idiopathic dermatitis, inflammatory bowel disease, asthma, pulmonary fibrosis, nephritis, nephrotic syndrome, immune rejection and LPS induction, CAR-T therapy, bacterial infection, cytokine release syndrome caused by viral infection.
- 一种如权利要求4所述的药物组合物与FLT3抑制剂联合用于治疗急性髓细胞白血病的用途。A use of the pharmaceutical composition as claimed in claim 4 in combination with an FLT3 inhibitor for the treatment of acute myeloid leukemia.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210072229.0A CN114478684B (en) | 2022-01-21 | 2022-01-21 | Tripterine prodrug, preparation method and medical application thereof |
| CN202210072229.0 | 2022-01-21 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2023138147A1 true WO2023138147A1 (en) | 2023-07-27 |
Family
ID=81473481
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/CN2022/128608 WO2023138147A1 (en) | 2022-01-21 | 2022-10-31 | Triptolide prodrug, preparation method therefor and pharmaceutical use thereof |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN114478684B (en) |
| WO (1) | WO2023138147A1 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114478684B (en) * | 2022-01-21 | 2023-10-31 | 南京中医药大学 | Tripterine prodrug, preparation method and medical application thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1246121A (en) * | 1996-03-01 | 2000-03-01 | 法玛吉尼西斯公司 | Immunosuppressive compounds and methods |
| WO2008087202A1 (en) * | 2007-01-18 | 2008-07-24 | Pierre Fabre Medicament | Triptolide derivatives for use in the treatment of acute myeloid leukemia |
| CN101235041A (en) * | 2007-10-26 | 2008-08-06 | 北京美迪克斯生物技术有限公司 | Four kinds of tripterygium wilfordii derivative and preparing method of pharmaceutics thereof |
| CN104327152A (en) * | 2014-10-14 | 2015-02-04 | 厦门大学 | Triptolide derivatives and application thereof |
| US20210023238A1 (en) * | 2019-07-23 | 2021-01-28 | City Of Hope | Triptolide antibody conjugates |
| CN114478684A (en) * | 2022-01-21 | 2022-05-13 | 南京中医药大学 | Triptolide prodrug, preparation method and medical application thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP4680589B2 (en) * | 2002-05-31 | 2011-05-11 | ファーマジェネシス， インコーポレイテッド | Triptolide derivatives for the regulation of apoptosis and immunosuppression |
| CN101759763A (en) * | 2009-12-29 | 2010-06-30 | 中山大学 | Triptolide derivative, and preparation method and application thereof |
-
2022
- 2022-01-21 CN CN202210072229.0A patent/CN114478684B/en active Active
- 2022-10-31 WO PCT/CN2022/128608 patent/WO2023138147A1/en unknown
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1246121A (en) * | 1996-03-01 | 2000-03-01 | 法玛吉尼西斯公司 | Immunosuppressive compounds and methods |
| WO2008087202A1 (en) * | 2007-01-18 | 2008-07-24 | Pierre Fabre Medicament | Triptolide derivatives for use in the treatment of acute myeloid leukemia |
| CN101235041A (en) * | 2007-10-26 | 2008-08-06 | 北京美迪克斯生物技术有限公司 | Four kinds of tripterygium wilfordii derivative and preparing method of pharmaceutics thereof |
| CN104327152A (en) * | 2014-10-14 | 2015-02-04 | 厦门大学 | Triptolide derivatives and application thereof |
| US20210023238A1 (en) * | 2019-07-23 | 2021-01-28 | City Of Hope | Triptolide antibody conjugates |
| CN114478684A (en) * | 2022-01-21 | 2022-05-13 | 南京中医药大学 | Triptolide prodrug, preparation method and medical application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114478684A (en) | 2022-05-13 |
| CN114478684B (en) | 2023-10-31 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP5539224B2 (en) | Therapeutic formulations based on asiatic acid and selected salts thereof | |
| JP5792902B2 (en) | Tetracycline compounds substituted with 9-aminomethyl | |
| EP3290408B1 (en) | Carboxylic acid urat1 inhibitor containing diarylmethane structure, preparation method and use thereof | |
| CA2346943A1 (en) | Cyclic tetrapeptide compound and use thereof | |
| HUT70207A (en) | 21-norrapamycin, pharmaceutical compositions containing it and process for preparing them | |
| WO2023138147A1 (en) | Triptolide prodrug, preparation method therefor and pharmaceutical use thereof | |
| CN111647036B (en) | Ocotillol esterified derivatives, preparation method thereof and application thereof in preparing anti-inflammatory drugs | |
| CN115068462A (en) | Application of novel leonurine marine derivative in preparation of medicines for preventing and treating inflammatory diseases or allergy | |
| US9266852B2 (en) | Daidzein derivative, pharmaceutically acceptable salt and preparation method thereof, and pharmaceutical composition containing same | |
| CN108530511B (en) | C19-acylated triptolide derivative | |
| EP2088152B1 (en) | N'-{n-[3-oxo-lupen-28-oyl]-9-aminononanoyl}-3-amino-3-phenylpropeonic acid and the pharmaceutically acceptable derivatives thereof, a method for the production and the use thereof in the form of a medicinal agent | |
| JP2675271B2 (en) | Novel acronisin analog, method for producing the same, and pharmaceutical composition containing the same | |
| JP4004567B2 (en) | Immunosuppressant containing WB2663B substance | |
| WO2017193562A1 (en) | Water soluble rapamycin derivative | |
| WO2021138847A1 (en) | Macrolide compound and its use of treatment chronic respiratory disease | |
| CN115181154B (en) | Pyxinol amide derivative, preparation method thereof and application thereof in pharmaceutical field | |
| JPH064563B2 (en) | Triterpene compound and anticancer agent | |
| CN113105391B (en) | Lappaconitine derivative with analgesic activity and preparation method and application thereof | |
| CN111269222B (en) | Compound and preparation method and application thereof | |
| US5596014A (en) | Hydroxymethyl-polythiophene derivatives | |
| CN112494650A (en) | Application of hematoporphyrin derivative and chemical medicine in liver cancer treatment | |
| CN111303237A (en) | Polyhydroxy-substituted oleanane pentacyclic triterpenoid, preparation method thereof and application thereof in preparing antifungal medicines | |
| WO1990015811A1 (en) | Spicamycin x and its use | |
| JP2001213875A (en) | Derivative of 3,6-dihydro-1,2-dioxin and antitumor agent | |
| JPH10509187A (en) | Production of immunosuppressants using microorganisms |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 22921571 Country of ref document: EP Kind code of ref document: A1 |