本申请要求于2023年4月18日提交中国专利局、申请号为202310411593.X、发明名称为“一种长效坎普他汀化合物”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。This application claims the priority of the Chinese patent application filed with the China Patent Office on April 18, 2023, with application number 202310411593.X and invention name “A Long-Acting Compstatin Compound”, the entire contents of which are incorporated by reference into this application.
本发明涉及一种长效的坎普他汀化合物，该长效化合物可用于预防和治疗干眼症、阵发性睡眠性血红蛋白尿症(PNH)、冷凝集素病(CAD)和C3肾小球肾炎。The present invention relates to a long-acting compstatin compound, which can be used for preventing and treating dry eye, paroxysmal nocturnal hemoglobinuria (PNH), cold agglutinin disease (CAD) and C3 glomerulonephritis.
坎普他汀(Compstatin)是一种十三环肽，与补体C3结合抑制补体激活，坎普他汀与天然C3结合，抑制其被C3转化酶剪切。其结合过程是一种多步骤反应，倾向于与C3b和C3c结合。坎普他汀在体内能完全抑制肝素/鱼精蛋白诱导的补体激活，而对心率或全身性血管、中心静脉和肺动脉压没有副作用。坎普他汀是一种安全有效的补体抑制剂，在临床上具有抑制补体激活的潜力。Compstatin is a 13-cyclic peptide that binds to complement C3 to inhibit complement activation. Compstatin binds to natural C3 and inhibits its cleavage by C3 convertase. Its binding process is a multi-step reaction, preferring to bind to C3b and C3c. Compstatin can completely inhibit heparin/protamine-induced complement activation in vivo without side effects on heart rate or systemic vascular, central venous and pulmonary artery pressure. Compstatin is a safe and effective complement inhibitor with the potential to inhibit complement activation in clinical practice.
由于坎普他汀消除半衰期静脉注射为30分钟，患者需要每天给药，患者顺应性差。本发明的目的就是为患者提供一种长效的坎普他汀化合物，减少给药频率，提高患者用药顺应性。Since the elimination half-life of compstatin after intravenous injection is 30 minutes, patients need to take the drug every day, which leads to poor patient compliance. The purpose of the present invention is to provide patients with a long-acting compstatin compound, reduce the frequency of administration, and improve patient compliance.
发明内容Summary of the invention
本发明提供了一种长效的坎普他汀化合物，该长效化合物可用于干眼症、阵发性睡眠性血红蛋白尿症(PNH)、冷凝集素病(CAD)和C3肾小球肾炎的治疗。The present invention provides a long-acting compstatin compound, which can be used for treating dry eye, paroxysmal nocturnal hemoglobinuria (PNH), cold agglutinin disease (CAD) and C3 glomerulonephritis.
为实现上述目的，本发明首先提供了一种结构式Ⅰ所示的化合物，该化合物所成的可药用的盐、溶剂化物、螯合物或非共价复合物，基于该化合物基础上的药物前体，或上述形式的任意混合物。To achieve the above objectives, the present invention first provides a compound shown in structural formula I, a pharmaceutically acceptable salt, solvate, chelate or non-covalent complex of the compound, a drug precursor based on the compound, or any mixture of the above forms.
R1-Ile-Cys-Val-AA1-Gln-Asp-Trp-Gly-AA2-His-Arg-Cys-Thr-AA3-AA4(R2)-AA5(2-12二硫键)R1-Ile-Cys-Val-AA1-Gln-Asp-Trp-Gly-AA2-His-Arg-Cys-Thr-AA3-AA4(R2)-AA5(2-12 disulfide bond)
结构式ⅠStructural formula Ⅰ
结构式Ⅰ中的AA1为Trp，或为Trp(1-Me)，或为Trp(1-甲酰基)，或为Val；AA1 in the structural formula I is Trp, or Trp(1-Me), or Trp(1-formyl), or Val;
结构式Ⅰ中的AA2为Ala，或为His；AA2 in structural formula I is Ala, or His;
结构式Ⅰ中的AA3为(PEGm1(CH2)m2CO)m3-，或为不存在；AA3 in structural formula I is (PEGm1 (CH2 )m2 CO)m3 -, or is absent;
其中：m1为1至10的整数；Wherein: m1 is an integer from 1 to 10;
m2为1至5的整数；m2 is an integer from 1 to 5;
m3为1至5的整数；m3 is an integer from 1 to 5;
结构式Ⅰ中的AA4为D型或L型的Lys，或为D型或L型的Dap，或为D型或L型的Dab，或为D型或L型的Orn，或为D型或L型的Dah，或为D型或L型的Dao，或为(Lys)6，或为不存在；AA4 in the structural formula I is D- or L-form Lys, or D- or L-form Dap, or D- or L-form Dab, or D- or L-form Orn, or D- or L-form Dah, or D- or L-form Dao, or (Lys)6, or is absent;
结构式Ⅰ中的AA5为NH2，或为OH；AA5 in structural formula I is NH2 or OH;
结构式Ⅰ中的R1为Ac,或为穿模肽，所述的穿模肽选自以下的穿模肽；In the structural formula I, R1 is Ac, or is a penetrating peptide, and the penetrating peptide is selected from the following penetrating peptides;
Tat(49-57)(SEQ ID NO：1)：RKKRRQRRR，Tat(49-57)(SEQ ID NO：1)：RKKRRQRRR，
VP-22(SEQ ID NO：2)：DAATARGRGRSAASRPTERPRAPARSASRPRRPVD，VP-22 (SEQ ID NO: 2):DAATARGRGRSAASRPTERPRAPARSASRPRRPVD,
ANTP-1(SEQ ID NO：3)：RQIKIWFQNRRMKWKK，ANTP-1 (SEQ ID NO: 3): RQIKIWFQNRRMKWKK,
ANTP-2(SEQ ID NO：4)：RWIKIWFQNRRMKWKK，ANTP-2 (SEQ ID NO: 4): RWIKIWFQNRRMKWKK,
ANTP-3(SEQ ID NO：5)：RQIKIWFWNRRMKWKK，ANTP-3 (SEQ ID NO: 5): RQIKIWFWNRRMKWKK,
ANTP-4(SEQ ID NO：5)：RQIKIWFQWRRMKWKK，ANTP-4 (SEQ ID NO: 5): RQIKIWFQWRRMKWKK,
ANTP-5(SEQ ID NO：6)：RWIKIWFWNRRMKWKK，ANTP-5 (SEQ ID NO: 6): RWIKIWFWNRRMKWKK,
ANTP-6(SEQ ID NO：7)：RWIKIWFQWRRMKWKK，ANTP-6 (SEQ ID NO: 7): RWIKIWFQWRRMKWKK,
ANTP-7(SEQ ID NO：8)：RQIKIWFWWRRMKWKK，ANTP-7 (SEQ ID NO: 8): RQIKIWFWWRRMKWKK,
ANTP-8(SEQ ID NO：9)：RWIKIWFWWRRMKWKK，ANTP-8 (SEQ ID NO: 9): RWIKIWFWWRRMKWKK,
Transportan(SEQ ID NO：10)：GWTLNSAGYLLGKINLKALAALAKKIL(SEQ ID NO：11)，Transportan (SEQ ID NO: 10): GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 11),
PEP-1(SEQ ID NO：12)：KETWWETWWTEWSQPKKKRKV，PEP-1 (SEQ ID NO: 12):KETWWETWWTEWSQPKKKRKV,
MPG(SEQ ID NO：13)：GALFLGFLGAAGSTMGAWSQPKKKRKV，MPG (SEQ ID NO: 13): GALFLGFLGAAGSTMGAWSQPKKKRKV,
MAP(SEQ ID NO：14)：KLALKLALKALKAALKLA，MAP (SEQ ID NO: 14): KLALKLALKALKAALKLA,
KALA(SEQ ID NO：15)：WEAKLAKALAKALAKHLAKALAKALKACEA，KALA (SEQ ID NO: 15): WEAKLAKALAKALAKHLAKALAKALKACEA,
ppTG20(SEQ ID NO：16)：GLFRALLRLLRSLWRLLLRA，ppTG20 (SEQ ID NO: 16): GLFRALLRLLRSLWRLLLRA,
Stearyl-R8(SEQ ID NO：17)：Stearyl-RRRRRRRR，或Stearyl-R8 (SEQ ID NO: 17): Stearyl-RRRRRRRR, or
Arg9(SEQ ID NO：18)：RRRRRRRRR；Arg9 (SEQ ID NO: 18): RRRRRRRRR;
结构式Ⅰ中的R2为HO2C(CH2)n1CO-(AA6)n2-(PEGn3(CH2)n4CO)n5-；或为HO2C(CH2)n1CO-(AA6)n2-(AA7)n6-，或为不存在；In the structural formula I, R2 is HO2 C(CH2 )n1 CO-(AA6)n2 -(PEGn3 (CH2 )n4 CO)n5 -; or HO2 C(CH2 )n1 CO-(AA6)n2 -(AA7)n6 -, or is absent;
其中：n1为10至20的整数；Wherein: n1 is an integer from 10 to 20;
n2为1至5的整数；n2 is an integer from 1 to 5;
n3为1至30的整数；n3 is an integer from 1 to 30;
n4为1至5的整数；n4 is an integer from 1 to 5;
n5为0，或为1至10的整数；n5 is 0, or an integer from 1 to 10;
n6为0，或为1至10的整数；n6 is 0, or an integer from 1 to 10;
AA6为γGlu，或为εLys，或为β-Ala，或为γ-氨基丁酸，或为5-Ava；AA6 is γGlu, or εLys, or β-Ala, or γ-aminobutyric acid, or 5-Ava;
AA7为Gly，或为Ser，或为Thr，或为Asp，或为Glu，或为Aad，或为Lys，或为Orn，或为Dab，或为Dap，或为Arg，或为Harg。AA7 is Gly, or Ser, or Thr, or Asp, or Glu, or Aad, or Lys, or Orn, or Dab, or Dap, or Arg, or Harg.
本发明还提供了包括根据本发明化合物的药物组合物，以及提供了本发明化合物的药物组合物用于制备治疗疾病的药物用途。The present invention also provides a pharmaceutical composition comprising the compound according to the present invention, and provides use of the pharmaceutical composition of the compound according to the present invention in preparing a medicament for treating a disease.
作为优选，所述药物组合物在预防和治疗干眼症、阵发性睡眠性血红蛋白尿症(PNH)、冷凝集素病(CAD)和C3肾小球肾炎中的应用。Preferably, the pharmaceutical composition is used in the prevention and treatment of dry eye, paroxysmal nocturnal hemoglobinuria (PNH), cold agglutinin disease (CAD) and C3 glomerulonephritis.
本发明所涉及到的更多内容在以下有详细描述，或者有些也可以在本发明的实施例中体会。More contents involved in the present invention are described in detail below, or some of them can also be experienced in the embodiments of the present invention.
除非另有所指，本文中所用来表示不同成分的数量、反应条件，在任意情况下都可解读为“大致的”、“大约的”意思。相应的，除有明确的特指外，在下述以及权利要求中所引用的数字参数都是大致的参数，在各自的实验条件下由于标准误差的不同，有可能会得到不同的数字参数。Unless otherwise indicated, the quantities of different components and reaction conditions used herein may be interpreted as "roughly" or "approximately" in any case. Accordingly, unless otherwise specified, the numerical parameters cited below and in the claims are approximate parameters, and different numerical parameters may be obtained under respective experimental conditions due to different standard errors.
本文中，当一个化合物的化学结构式和化学名称有分歧或疑义时，以化学结构式确切定义此化合物。本文所描述的化合物有可能含有一个或多个手性中心，和/或者双键以及诸如此类的结构，也可能存在立体异构体，包括双键的异构体(比如几何异构体)、旋光对映异构体或者非对映异构体。相应的，在本文描述范围内的任意化学结构，无论是部分或整体结构中含有上述类似结构，都包括了此化合物的所有可能的对映异构体和非对映异构体，其中也包括了单纯的任一种立体异构体(如单纯的几何异构体、单纯的对映异构体或者单纯的非对映异构体)以及这些异构体的任意一种混合物。这些消旋异构体和立体异构体的混合物由本领域技术人员利用不停的分离技术或手性分子合成的方法也可进一步被拆分成其组成成分的对映异构体或立体异构体。In this article, when there is a disagreement or ambiguity between the chemical formula and the chemical name of a compound, the chemical formula is used to accurately define the compound. The compounds described herein may contain one or more chiral centers, and/or double bonds and structures such as these, and may also exist as stereoisomers, including double bond isomers (such as geometric isomers), optical enantiomers or diastereomers. Accordingly, any chemical structure within the scope of the description herein, whether it contains the above-mentioned similar structures in part or in its entirety, includes all possible enantiomers and diastereomers of the compound, including any single stereoisomer (such as a single geometric isomer, a single enantiomer or a single diastereomer) and any mixture of these isomers. These racemic isomers and mixtures of stereoisomers can also be further separated into their constituent enantiomers or stereoisomers by those skilled in the art using continuous separation techniques or methods of chiral molecule synthesis.
结构式Ⅰ的化合物包含了，但并不仅限于，这些化合物的光学异构体、消旋体和/或其他的混合物。上述情况下，其中单一的对映异构体或非对映异构体，如有旋光的异构体，可以用不对称合成的方法或消旋体拆分的方法获得。消旋体的拆分可用不同的方法实现，如常规的用助拆分的试剂重结晶，或用色谱方法。另外，结构式Ⅰ的化合物也包含了带双键的顺式和/或反式的异构体。The compounds of formula I include, but are not limited to, optical isomers, racemates and/or other mixtures of these compounds. In the above cases, single enantiomers or diastereomers, such as optically active isomers, can be obtained by asymmetric synthesis or racemate resolution. The resolution of the racemate can be achieved by different methods, such as conventional recrystallization with a resolving agent, or by chromatographic methods. In addition, the compounds of formula I also include cis and/or trans isomers with double bonds.
本发明所述化合物包含但不限于，结构式Ⅰ所示化合物以及他们所有的在药学上可用的不同形式。这些化合物的药学上可用的不同形式包括各种可药用的盐、溶剂化物、螯合物、非共价的复合物、基于上述物质基础上的药物前体和上述这些形式的任意混合物。The compounds of the present invention include, but are not limited to, compounds of formula I and all of their pharmaceutically acceptable forms. The pharmaceutically acceptable forms of these compounds include various pharmaceutically acceptable salts, solvates, chelates, non-covalent complexes, prodrugs based on the above substances, and any mixtures of the above forms.
本发明提供的结构式Ⅰ所示的化合物性质稳定，具有预防和治疗干眼症、阵发性睡眠性血红蛋白尿症(PNH)、冷凝集素病(CAD)和C3肾小球肾炎的活性。The compound represented by structural formula I provided by the present invention has stable properties and has the activity of preventing and treating dry eye, paroxysmal nocturnal hemoglobinuria (PNH), cold agglutinin disease (CAD) and C3 glomerulonephritis.
本发明公开了一种长效的坎普他汀化合物，本领域技术人员可以借鉴本文内容，适当改进相关参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的的化合物和制备方法进行改动或适当变更与组合，来实现和应用本发明技术。The present invention discloses a long-acting compstatin compound. Those skilled in the art can refer to the content of this article and appropriately improve the relevant parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in The method of the present invention has been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the compounds and preparation methods described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.
本发明中涉及的英文缩写所对应的中文名称见下表所示：The Chinese names corresponding to the English abbreviations involved in the present invention are shown in the following table:
表1
Table 1
实施例1化合物的制备Example 1 Preparation of Compounds
制备方法，包括：起始树脂为Rink Amide MBHA树脂，用固相多肽合成法制备肽树脂，肽树脂再经酸解得到粗品，最后粗品经过纯化得到纯品；其中固相多肽合成法制备肽树脂的步骤为在载体树脂上通过固相偶联合成法依次接入序列中相对应的保护氨基酸或片段，制备得到肽树脂。The preparation method comprises: the starting resin is Rink Amide MBHA resin, the peptide resin is prepared by solid phase peptide synthesis method, the peptide resin is then acid-hydrolyzed to obtain a crude product, and finally the crude product is purified to obtain a pure product; wherein the step of preparing the peptide resin by solid phase peptide synthesis method is to sequentially access the corresponding protected amino acids or fragments in the sequence on the carrier resin by solid phase coupling synthesis method to prepare the peptide resin.
上述制备方法中，所述的Fmoc-保护氨基酸或保护氨基酸片段的用量为所投料树脂总摩尔数的1.2～6倍；优选为2.5～3.5倍。In the above preparation method, the amount of the Fmoc-protected amino acid or protected amino acid fragment is 1.2 to 6 times the total molar number of the resin fed, preferably 2.5 to 3.5 times.
上述制备方法中，所述的载体树脂取代值为0.2～1.0mmol/g树脂，优选的取代值为0.3～0.5mmol/g树脂。In the above preparation method, the substitution value of the carrier resin is 0.2-1.0 mmol/g resin, and the preferred substitution value is 0.3-0.5 mmol/g resin.
作为本发明优选的方案，所述固相偶联合成法为：前一步反应得到的保护氨基酸-树脂脱去Fmoc保护基后再与下一个保护氨基酸偶联反应。所述的去Fmoc保护的脱保护时间为10～60分钟，优选的为15～25分钟。所述的偶联反应时间为60～300分钟，优选的为100～140分钟。As a preferred embodiment of the present invention, the solid phase coupling synthesis method is: the protected amino acid-resin obtained in the previous step is deprotected from the Fmoc protecting group and then coupled with the next protected amino acid. The deprotection time of the Fmoc deprotection is 10 to 60 minutes, preferably 15 to 25 minutes. The coupling reaction time is 60 to 300 minutes, preferably 100 to 140 minutes.
所述的偶联反应需添加缩合试剂，缩合试剂选自DIC(N,N-二异丙基碳二亚胺)、N,N-二环己基碳二亚胺，六氟磷酸苯并三唑-1-基-氧基三吡咯烷基磷、2-(7-氮杂-1H-苯并三氮唑-1-基)-1,1,3,3-四甲基脲六氟磷酸酯、苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸盐或O-苯并三氮唑-N,N,N',N'-四甲基脲四氟硼酸酯中的一种；优选的为N,N-二异丙基碳二亚胺。所述缩合试剂的摩尔用量为氨基树脂中氨基总摩尔数的1.2～6倍，优选为2.5～3.5倍。The coupling reaction requires the addition of a condensation reagent, which is selected from DIC (N,N-diisopropylcarbodiimide), N,N-dicyclohexylcarbodiimide, benzotriazole-1-yl-oxytripyrrolidinophosphine hexafluorophosphate, 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate or O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate; preferably N,N-diisopropylcarbodiimide. The molar amount of the condensation reagent is 1.2 to 6 times the total molar number of amino groups in the amino resin, preferably 2.5 to 3.5 times.
所述的偶联反应需添加活化试剂，活化试剂选自1-羟基苯并三唑或N-羟基-7-氮杂苯并三氮唑，优选的为1-羟基苯并三唑。活化试剂的用量为氨基树脂中氨基总摩尔数的1.2～6倍，优选的为2.5～3.5倍。The coupling reaction requires the addition of an activation reagent, which is selected from 1-hydroxybenzotriazole or N-hydroxy-7-azabenzotriazole, preferably 1-hydroxybenzotriazole. The amount of the activation reagent is 1.2 to 6 times the total molar number of amino groups in the amino resin, preferably 2.5 to 3.5 times.
作为本发明优选的方案，所述的脱去Fmoc保护的试剂为PIP/DMF(哌啶/N,N-二甲基甲酰胺)混合溶液，混合溶液中含哌啶为10～30％(V)。去Fmoc保护试剂的用量为每克氨基树脂5～15mL，优选的为每克氨基树脂8～12mL。As a preferred solution of the present invention, the Fmoc-removing reagent is a PIP/DMF (piperidine/N,N-dimethylformamide) mixed solution, wherein the piperidine content in the mixed solution is 10-30% (V). The amount of the Fmoc-removing reagent is 5-15 mL per gram of amino resin, preferably 8-12 mL per gram of amino resin.
优选的，肽树脂经酸解同时脱去树脂及侧链保护基得到粗品：Preferably, the peptide resin is subjected to acid hydrolysis to simultaneously remove the resin and the side chain protecting groups to obtain a crude product:
进一步优选的，所述肽树脂酸解时采用的酸解剂为三氟醋酸(TFA)、1,2-乙二硫醇(EDT)和水的混合溶剂，混合溶剂的体积配比为：TFA为80～95％，EDT为1～10％，余量为水。Further preferably, the acid hydrolysis agent used in the acid hydrolysis of the peptide resin is a mixed solvent of trifluoroacetic acid (TFA), 1,2-ethanedithiol (EDT) and water, and the volume ratio of the mixed solvent is: TFA is 80-95%, EDT is 1-10%, and the balance is water.
更进一步优选的，混合溶剂的体积配比为：TFA为89～91％、EDT为4～6％，余量为水。最优的，混合溶剂的体积配比为：TFA为90％、EDT为5％，余量为水。More preferably, the volume ratio of the mixed solvent is: 89-91% TFA, 4-6% EDT, and the balance is water. Optimally, the volume ratio of the mixed solvent is: 90% TFA, 5% EDT, and the balance is water.
所述酸解剂用量为每克肽树脂需要4～15mL酸解剂；优选的，每克肽树脂需要7～10mL酸解剂。The amount of the acid hydrolysis agent is 4 to 15 mL per gram of peptide resin; preferably, 7 to 10 mL per gram of peptide resin.
使用酸解剂裂解的时间为室温条件下1～6小时，优选的为3～4小时。The time for lysis using an acid hydrolysis agent is 1 to 6 hours at room temperature, preferably 3 to 4 hours.
进一步的，粗品经高效液相色谱纯化、冻干得到纯品。Furthermore, the crude product was purified by high performance liquid chromatography and freeze-dried to obtain a pure product.
1、肽树脂的合成1. Synthesis of peptide resin
使用Rink Amide BHHA树脂为载体树脂，通过去Fmoc保护和偶联反应，依次接入序列中对应的保护氨基酸，制得肽树脂。Rink Amide BHHA resin was used as a carrier resin, and the corresponding protected amino acids in the sequence were sequentially connected through Fmoc protection removal and coupling reaction to prepare a peptide resin.
(1)接入主链第1个保护氨基酸(1) Insertion of the first protected amino acid in the main chain
取3mmol第1个保护氨基酸和3mmol HOBt，用适量DMF溶解；另取3mmol DIC，搅拌下慢慢加入至保护氨基酸DMF溶液中，于室温环境中搅拌反应30分钟，得到活化后的保护氨基酸溶液，备用。Take 3 mmol of the first protected amino acid and 3 mmol of HOBt, dissolve them in an appropriate amount of DMF; take another 3 mmol of DIC, slowly add it into the protected amino acid DMF solution with stirring, and stir the reaction at room temperature for 30 minutes to obtain the activated protected amino acid solution for use.
取1mmol的Rink Amide MBHA树脂(取代值约0.4mmol/g)，采用20％PIP/DMF溶液去保护25分钟，洗涤过滤得到去Fmoc的树脂。Take 1 mmol of Rink Amide MBHA resin (substitution value is about 0.4 mmol/g), use 20% PIP/DMF solution to deprotect for 25 minutes, wash and filter to obtain the de-Fmoc resin.
将活化后的第1个保护氨基酸溶液加入到已去Fmoc的树脂中，偶联反应60～300分钟，过滤洗涤，得含1个保护氨基酸的树脂。The activated first protected amino acid solution is added to the Fmoc-free resin, and the coupling reaction is carried out for 60 to 300 minutes. The resin containing one protected amino acid is filtered and washed to obtain.
(2)接入主链保护氨基酸(2) Insertion of main chain protective amino acids
采用上述接入主链第1个保护氨基酸同样方法，依次接入上述对应的保护氨基酸，如需要乙酰化就进行最后乙酰化反应，得含主链肽树脂。The same method as the first protected amino acid in the main chain is used to sequentially insert the corresponding protected amino acids. If acetylation is required, the final acetylation reaction is performed to obtain a main chain peptide resin.
(3)如有侧链接入，接入的方法如下：(3) If there is a side chain access, the access method is as follows:
A、接入侧链第1个保护氨基酸A. Insert the first protected amino acid in the side chain
取3mmol侧链第1个保护氨基酸和3mmol HOBt，用适量DMF溶解；另取3mmol DIC，搅拌下慢慢加入至保护氨基酸DMF溶液中，于室温环境中搅拌反应30分钟，得到活化后的保护氨基酸溶液。Take 3 mmol of the first protected amino acid in the side chain and 3 mmol of HOBt, dissolve them in an appropriate amount of DMF; take another 3 mmol of DIC, slowly add it into the protected amino acid DMF solution with stirring, and stir the reaction at room temperature for 30 minutes to obtain the activated protected amino acid solution.
取2.5mmol四三苯基膦钯和25mmol苯硅烷，用适量二氯甲烷溶解，去保护4小时，过滤洗涤，得到去Alloc的树脂备用。Take 2.5 mmol of tetrakistriphenylphosphine palladium and 25 mmol of phenylsilane, dissolve them in an appropriate amount of dichloromethane, deprotect for 4 hours, filter and wash, and obtain the de-Allocated resin for use.
将加入活化后的侧链第1个保护氨基酸液加入到已去Alloc的树脂，偶联反应60～300分钟，过滤洗涤，得含侧链第1个保护氨基酸的树脂。The activated first side chain protected amino acid solution is added to the de-Allocated resin, and the coupling reaction is carried out for 60 to 300 minutes. The resin containing the first side chain protected amino acid is filtered and washed to obtain a resin.
B、接入侧链保护氨基酸B. Insertion of side chain protected amino acids
采用上述接入主链第1个保护氨基酸同样方法，依次接入侧链对应的保护氨基酸和单保护脂肪酸，得到肽树脂。The same method as described above for inserting the first protected amino acid in the main chain was used to sequentially insert the corresponding protected amino acid and the mono-protected fatty acid in the side chain to obtain a peptide resin.
2、粗品的制备2. Preparation of crude product
取上述肽树脂，加入体积比为TFA︰水︰EDT＝95︰5︰5的裂解试剂(裂解试剂10mL/克树脂)，搅拌均匀，室温搅拌反应3小时，反应混合物使用砂芯漏斗过滤，收集滤液，树脂再用少量TFA洗涤3次，合并滤液后减压浓缩，加入无水乙醚沉淀，再用无水乙醚洗沉淀3次，抽干得类白色粉末。Take the above peptide resin, add a cleavage reagent with a volume ratio of TFA: water: EDT = 95:5:5 (cleavage reagent 10 mL/g resin), stir evenly, and react at room temperature for 3 hours. The reaction mixture is filtered using a sand core funnel, and the filtrate is collected. The resin is washed 3 times with a small amount of TFA, and the filtrate is combined and concentrated under reduced pressure. Anhydrous ether is added to precipitate, and the precipitate is washed 3 times with anhydrous ether and dried to obtain an off-white powder.
所得类白色粉末用20％醋酸水溶液溶解，搅拌下滴加碘/乙醇饱和溶液至完全环化，35～40℃减压浓缩，得粗品浓缩溶液。The obtained off-white powder was dissolved in a 20% acetic acid aqueous solution, and a saturated iodine/ethanol solution was added dropwise under stirring until complete cyclization, and the mixture was concentrated under reduced pressure at 35-40° C. to obtain a concentrated solution of a crude product.
3、纯品的制备3. Preparation of pure product
取上述粗品浓缩溶液，用0.45μm混合微孔滤膜过滤，纯化备用；Take the above crude concentrated solution, filter it with a 0.45 μm mixed microporous filter membrane, and purify it for later use;
采用高效液相色谱法进行纯化，纯化用色谱填料为10μm的反相C18，流动相系统为0.1％TFA/水溶液-0.1％TFA/乙腈溶液，30mm*250mm的色谱柱流速为20mL/min，采用梯度系统洗脱，循环进样纯化，取粗品溶液上样于色谱柱中，启动流动相洗脱，收集主峰蒸去乙腈后，得纯化中间体浓缩液。The product was purified by high performance liquid chromatography, with a 10 μm reverse phase C18 chromatographic filler, a 0.1% TFA/water solution-0.1% TFA/acetonitrile solution as the mobile phase system, a 30 mm*250 mm chromatographic column with a flow rate of 20 mL/min, and a gradient system for elution and cyclic injection purification. The crude product solution was loaded onto the chromatographic column, the mobile phase elution was started, the main peak was collected and the acetonitrile was evaporated to obtain a purified intermediate concentrate.
纯化中间体浓缩液用0.45μm滤膜滤过备用，采用高效液相色谱法进行换盐，流动相系统为1％醋酸/水溶液-乙腈，纯化用色谱填料为10μm的反相C18，30mm*250mm的色谱柱流速为20mL/min(可根据不同规格的色谱柱，调整相应的流速)；采用梯度洗脱，循环上样方法，上样于色谱柱中，启动流动相洗脱，采集图谱，观测吸收度的变化，收集换盐主峰并用分析液相检测纯度，合并换盐主峰溶液，减压浓缩，得到纯品醋酸水溶液，冷冻干燥，得纯品。The purified intermediate concentrate is filtered through a 0.45 μm filter membrane for standby use, and the salt is exchanged by high performance liquid chromatography, the mobile phase system is 1% acetic acid/water solution-acetonitrile, the chromatographic filler for purification is 10 μm reverse phase C18, and the flow rate of the 30 mm*250 mm chromatographic column is 20 mL/min (the corresponding flow rate can be adjusted according to the chromatographic columns of different specifications); the gradient elution and the circular loading method are adopted, the sample is loaded into the chromatographic column, the mobile phase elution is started, the spectrum is collected, the change of the absorbance is observed, the main peak of the salt exchange is collected and the purity is detected by the analytical liquid phase, the main peak solution of the salt exchange is combined, and it is concentrated under reduced pressure to obtain a pure acetic acid aqueous solution, and freeze-dried to obtain a pure product.
用上述方法制备了以下化合物：The following compounds were prepared using the above method:
表2
Table 2
实施例2生物活性的测定Example 2 Determination of biological activity
1、测定方法1. Determination method
用BSA包被96-孔平板，加入人血浆、鸡卵白蛋白(OVA)、多克隆抗体-OVA抗体、试验药物并温育，随后加入抗-人类C3HRP-缀和抗体，温育后加入底物，检测450nm吸收度，计算得到化合物的IC50。A 96-well plate was coated with BSA, and human plasma, chicken ovalbumin (OVA), polyclonal antibody-OVA antibody, and test drugs were added and incubated, followed by the addition of anti-human C3HRP-conjugated antibody. After incubation, the substrate was added, the absorbance at 450 nm was detected, and the IC50 of the compound was calculated.
2、测定结果2. Measurement results
测定结果见下表：The measurement results are shown in the following table:
表3
Table 3
实施例3初步药代特性的测定Example 3 Determination of preliminary pharmacokinetic properties
选择活性最好的化合物进行初步药代特性的测定，试验动物为食蟹猴，雄性食蟹猴2只，皮下给药，剂量为0.1mg/kg，分别于药前(0h)、以及给药后1h、2h、3h、4h、8h、12h、18h、24h、48h、96h、144h、168h静脉取血，离心分离血浆样本，用液质联用法分别测定血浆样本中相应化合物的血药浓度，化合物皮下(SC)给药半衰期见下表：The compound with the best activity was selected for preliminary pharmacokinetic properties determination. The experimental animals were cynomolgus monkeys. Two male cynomolgus monkeys were administered subcutaneously at a dose of 0.1 mg/kg. Blood was collected from the vein before drug administration (0h) and 1h, 2h, 3h, 4h, 8h, 12h, 18h, 24h, 48h, 96h, 144h, and 168h after administration. The plasma samples were separated by centrifugation and the blood drug concentrations of the corresponding compounds in the plasma samples were determined by liquid chromatography-mass spectrometry. The half-life of the compound after subcutaneous (SC) administration is shown in the following table:
表4
Table 4
最后应说明的是：以上各实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述各实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.
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| CN202310411593.XACN118812653A (en) | 2023-04-18 | 2023-04-18 | A long-acting compstatin compound |
| CN202310411593.X | 2023-04-18 |
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| PCT/CN2024/088262PendingWO2024217443A1 (en) | 2023-04-18 | 2024-04-17 | Long-acting compstatin compound |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103687867A (en)* | 2011-05-11 | 2014-03-26 | 阿佩利斯制药公司 | Cell-responsive, long-acting or targeted compstatin analogs and uses thereof |
| CN105051057A (en)* | 2012-11-15 | 2015-11-11 | 阿佩利斯制药公司 | Cell-reactive, long-acting, or targeted compstatin analogs and related compositions and methods |
| US20160215022A1 (en)* | 2013-03-15 | 2016-07-28 | Apellis Pharmaceuticals, Inc. | Cell-penetrating compstatin analogs and uses thereof |
| US20170173107A1 (en)* | 2014-03-17 | 2017-06-22 | The Trustees Of The University Of Pennsylvania | Compstatin Analogs With Improved Potency and Pharmacokinetic Properties |
| US20180057538A1 (en)* | 2016-08-26 | 2018-03-01 | The Regents Of The University Of California | Potent and highly soluble pegylated compstatin peptides |
| WO2022013374A1 (en)* | 2020-07-16 | 2022-01-20 | Zp Spv 3 K/S | Inhibitors of complement factor c3 and their medical uses |
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103687867A (en)* | 2011-05-11 | 2014-03-26 | 阿佩利斯制药公司 | Cell-responsive, long-acting or targeted compstatin analogs and uses thereof |
| CN105051057A (en)* | 2012-11-15 | 2015-11-11 | 阿佩利斯制药公司 | Cell-reactive, long-acting, or targeted compstatin analogs and related compositions and methods |
| US20160215022A1 (en)* | 2013-03-15 | 2016-07-28 | Apellis Pharmaceuticals, Inc. | Cell-penetrating compstatin analogs and uses thereof |
| US20170173107A1 (en)* | 2014-03-17 | 2017-06-22 | The Trustees Of The University Of Pennsylvania | Compstatin Analogs With Improved Potency and Pharmacokinetic Properties |
| US20180057538A1 (en)* | 2016-08-26 | 2018-03-01 | The Regents Of The University Of California | Potent and highly soluble pegylated compstatin peptides |
| WO2022013374A1 (en)* | 2020-07-16 | 2022-01-20 | Zp Spv 3 K/S | Inhibitors of complement factor c3 and their medical uses |
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| CN118812653A (en) | 2024-10-22 |
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