Compounds and methods for liquid phase synthesis

Abstract

The present disclosure includes hydrophilic linker compounds of Formula 1, and describes methods of their use in liquid phase synthesis. In Formula 1, "m" is 0-20, "n" is 1-50, and "Z" is a linker compound as described herein.

Description

[0001] This application is submitted together with a sequence list in ST.26 XML format. The sequence list is provided as a file titled "Sequence List X22492" created on August 22, 2022, and is 92 kilobytes in size. The sequence list information in ST.26 XML format is incorporated herein by reference in its entirety.

[0002] background

[0003] The chemical synthesis of peptide and amino acid sequences is accomplished through an iterative approach, in which amino acids are sequentially coupled to each other. This approach involves the following repetitive process: deprotecting the C- or N-terminus of the grown peptide chain, coupling the protected amino acid to it, and then deprotecting the newly coupled amino acid, thus preparing it for the next coupling in the sequence. This iterative process (in which the amino acid coupling and deprotection steps are repeated) amplifies a major challenge in chemical synthesis—the challenge of separating the desired synthetic product from other reaction components (solvents, unreacted starting materials and reagents, and undesirable reaction byproducts).

[0004] Solid-phase peptide synthesis (“SPPS”) is the most commonly used method and system for synthesizing peptides and amino acid sequences. SPPS involves coupling an activated amino acid to a solid support. This solid support is typically a functionalized (e.g., with an NH2 group) polymer resin bead. The next amino acid (which typically has its NH2 terminus protected by an Fmoc, Boc, or other protecting group) reacts with the resin such that the functionalized group on the resin reacts with and binds to the activated COOH group of the terminal amino acid. In this way, the terminal amino acid is covalently linked to the resin.

[0005] Then, in the next step, the NH2 terminus of the terminal amino acid is deprotected, exposing its NH2 group for the next reaction. Thus, a new amino acid is introduced. The NH2 terminus of the new amino acid is protected with a protecting group (e.g., Fmoc, Boc, or another protecting group). Therefore, when this new amino acid is added, the activated ester from the new amino acid reacts with the newly deprotected NH2 group of the terminal amino acid, thereby coupling the two amino acids together. Once this new amino acid is coupled, it also has a protected NH2 group, which can then be deprotected and react with the next amino acid. By repeatedly performing this iterative process, the entire amino acid sequence can be constructed. Once the entire sequence is constructed, it can be uncoupled (cleaved) and deprotected from the resin, thereby producing the amino acid sequence. (It should be noted that the side chains of various amino acids (R1, R2, etc.) added by this method can be orthogonally protected with groups such as BOC, tert-butyl, or triphenylmethyl to prevent these side chains from reacting during amino acid synthesis. Those skilled in the art will understand how to construct, protect, and subsequently deprotect these side chains or other groups during synthesis.)

[0006] In each step of the SPPS method, the grown peptide remains attached to a solid support, which is separated from other reactants by phase separation. The solid support facilitates separation by filtering the desired product while it is attached to the solid support. SPPS is commercially available and remains the standard in peptide synthesis. However, it is expensive, time-consuming, and generates high levels of process waste due to the need for extensive resin washing. Each added amino acid must be deprotected and coupled, which is difficult and often results in the use of large amounts of solvent. Multiple solvent washes are typically required after each reaction to remove residual reagents from the resin. Cycle times in manufacturing facilities can be approximately one amino acid coupling and deprotection cycle per day. Further disadvantageously, many of these solvents are environmentally unfriendly. Furthermore, phase separation in SPPS presents challenges in achieving high product purity. Because the grown peptide is not in the same phase as other reactants, the reaction kinetics are slower than in a liquid phase, and minimizing undesirable side reactions such as aggregation while maximizing conversion to the desired product can be challenging. Monitoring and optimizing the reaction of heterogeneous reaction mixtures can be challenging, especially when using analytical methods that require the analyte to be dissolved in a homogeneous liquid stream, such as high-performance liquid chromatography (“HPLC”).

[0007] In contrast to SPPS, liquid-phase peptide synthesis (“LPPS”) refers to methods for preparing peptides under homogeneous reaction conditions. This can include synthetic methods involving a soluble polymer support moiety on which peptides can be prepared in iterative deprotection and coupling processes similar to those used in SPPS. By allowing the deprotection and coupling reactions to occur in a homogeneous solution phase, LPPS overcomes some of the difficulties involved in SPPS. Typically, LPPS can be actually more efficient than SPPS due to the need for fewer solvents, starting materials, and reagents. Furthermore, liquid-phase reaction kinetics can be faster compared to reactions occurring at phase boundaries. This can be beneficial for reaction optimization attempts, such as allowing the deprotection step to occur in a shorter time or under milder reaction conditions to minimize racemization of amino acid residues in the grown peptide. LPPS also allows for direct monitoring of the reaction, for example, by coupling HPLC with mass spectrometry (“LCMS”), where products linked to the soluble polymer support can be detected and quantified more easily than in similar SPPS methods.

[0008] Despite the advantages of LPPS over SPP, successful implementation of an LPPS strategy can be challenging. The problem of effectively separating the desired product from other reaction components and undesirable byproducts remains. To address this, numerous strategies have been employed to enable the separation of peptide products from the liquid-phase reaction mixture. Hydrophobic soluble linker systems have been developed as label-assisted LPPS supports (see, for example, Takahashi, D. et al. (2017) Angewandte Chemie International Edition 129:7911-7915 and U.S. Patent Application Publication No. US 2018 / 0215782). Using these linker systems, peptides can be elongated at the linker, and byproducts can then be removed by precipitation or by an aqueous post-treatment of extraction. However, several key limitations have been identified, such as peptide length, where solubility becomes a major issue as the peptide chain elongates. Furthermore, purity challenges may exist, as aqueous washing may have limited effectiveness in removing reagents and byproducts. These components can interfere with downstream synthetic steps and lead to undesirable additions and deletions. In addition, high residual water content in the organic layer may have a negative impact on peptide coupling, which may require the addition of a dehydration step.

[0009] Compared to hydrophobic linker systems, hydrophilic linker systems offer key advantages. In these systems, the linker is characterized by a hydrophilic “label,” which allows reaction byproducts to be removed by simple extraction with more environmentally friendly organic solvents. Polyethylene glycol (PEG) has been reported as a hydrophilic support for liquid-phase peptide synthesis (see, e.g., Fischer, PM; Zheleva, DI (2002) Journal of Peptide Science 8: 529-542). However, the PEG derivatives used in these applications are often polydisperse and lack a fixed molecular weight. Variable PEG chain lengths can introduce complexity in the analysis and purification of high molecular weight PEG conjugates. Therefore, finding a new hydrophilic linker system with fixed molecular weight LPPS could be an improvement that would address these shortcomings, particularly in the commercial manufacture of peptides. Further progress would be made if such a system enabled liquid-phase peptide synthesis of long peptides (15-chain segments and above). In fact, this embodiment specifically provides a hydrophilic linker system for label-assisted LPPS and its method of use for commercial-scale peptide synthesis. This article discloses such methods and systems.

[0010] Overview

[0011] This embodiment provides compounds of fixed molecular weight that can be used as hydrophilic linker constructs for liquid-phase organic synthesis, such as LPPS. The compounds of this disclosure are characterized by repeating heterobifunctional PEG-like units linked to the linker, on which peptides or other molecules can be constructed via coupling (e.g., amino acid coupling) and deprotection steps. In particular, the compounds of this disclosure can be used to construct peptides or other molecules via repeating synthetic steps (e.g., amino acid coupling and deprotection steps).

[0012] One embodiment of this disclosure comprises a hydrophilic connector compound of formula 1:

[0013]

[0014] Where "m" is 0-20, "n" is 1-50, and "Z" is a linker group. "Z" is a functional group that can form a covalent bond with an optional protected compound (e.g., an amino acid), thereby allowing iterative deprotection and coupling steps to one or more optional protected compounds (e.g., amino acids or peptides), and the resulting product, such as a polypeptide product, can then be released from the "Z" group through chemical transformation.

[0015] Another embodiment of this disclosure comprises a compound of formula 1, wherein "m" is 0, 1, 2, or 3, and "n" is 1-50. Another embodiment of this disclosure comprises a compound of formula 1, wherein "m" is 0, 1, 2, or 3, and "n" is 1-10. Another embodiment of this disclosure comprises a compound of formula 1, wherein "m" is 1, and "n" is 2-10.

[0016] Another embodiment of this disclosure comprises a compound of formula 1, wherein "Z" is selected from:

[0017] And “m” and “n” are as defined above.

[0018] The hydrophilic linker disclosed herein is used for the synthesis of peptides and other compounds, wherein an activated ester of a compound such as an amino acid or peptide fragment is first coupled to a free alcohol-OH or amine-NH2 of the linker of Formula 1. Subsequently, the compound, for example a peptide, is grown by continuously coupling individual molecular components, such as activated esters of amino acids or peptide fragments, after intermediate deprotection. The final compound (e.g., a peptide) is cleaved from the linker under acidic conditions. Invention Details

[0020] This embodiment provides hydrophilic linker compounds for use in liquid-phase synthesis systems, such as LPPS systems with fixed molecular weights. When used with LPPS, the disclosed compounds enable liquid-phase peptide synthesis methods for long peptides (15-units and above). Specifically, this embodiment provides hydrophilic linker systems for liquid-phase synthesis (e.g., LPPS) and methods for using them to synthesize molecules or peptides on a commercial scale.

[0021] As described above, this hydrophilic connector compound can be a compound of Formula 1 summarized above. Specific preferred examples include:

[0022] Compounds of Formula 1a:

[0023]

[0024] Compounds of Formula 1b:

[0025]

[0026] Compounds of Formula 1c:

[0027]

[0028] Compounds of formula 1d:

[0029]

[0030] Compounds of formula 1e:

[0031]

[0032] Compounds of Formula 1f:

[0033]

[0034] Compound of formula 1g:

[0035]

[0036] Compounds of formula 1h:

[0037]

[0038] Compounds of Formula 1i:

[0039] Compounds of Formula 1j:

[0040]

[0041] Compounds of formula 1k:

[0042]

[0043] Compounds of Formula 1l:

[0044]

[0045] Compounds of formula 1m:

[0046]

[0047] Compounds of formula 1n:

[0048]

[0049] Compounds of formula 1o:

[0050]

[0051] Liquid-phase synthesis using the hydrophilic linker compounds described herein can be used to assemble compounds linked by amide bonds, i.e., by the condensation of a carbonyl group on one molecule with an amino group on another. Peptide synthesis is a clear application of the hydrophilic linker molecules and methods described herein, as peptide bonds are generated by the condensation reaction of a carboxyl group on one amino acid with an amino group on another. Therefore, compounds containing amide bonds can be assembled by iterative coupling and deprotection reactions to assemble molecules that must be released from the support used during synthesis upon completion. Specifically, molecules having amino groups protected by Fmoc groups with available carboxylic acid groups can be coupled to hydrophilic linker molecules as described herein. The resulting molecule can then be deprotected by removing the Fmoc groups, and the unprotected amino group of the resulting molecule can be coupled to a available carboxylic acid group on another molecule having a available amino group. Examples 21-24 illustrate the liquid-phase synthesis of non-peptide molecules using the hydrophilic linker molecules described herein.

[0052] Peptide preparation via SPPS and LPPS involves iterative coupling and deprotection reactions to elongate the peptide, which must be released from the support used during synthesis upon completion. Furthermore, the starting materials of the amino acids or peptide fragments used for synthesis typically have side-chain protecting groups, which help ensure selectivity during the coupling step. The side-chain protecting groups are chosen such that they are stable to the conditions used during the deprotection step in peptide elongation. For example, FMOC groups can be used to protect the amino groups in amino acid starting materials and are readily removed with secondary amine bases. Conversely, BOC and triphenylmethyl (triphenylmethyl) protecting groups are stable under the basic conditions typically used to remove FMOC groups during peptide elongation and can be removed with strong organic acids upon completion. Some peptide synthesis linkers can be cleaved under the same conditions used for amino acid side-chain deprotection. This is called a “hard” cleavage method—the peptide is simultaneously deprotected and cleaved from the resin upon completion of elongation. Complex synthetic strategies can be achieved through careful selection of linker chemistry, which allows cleavage to occur under conditions orthogonal to the side-chain deprotection. This is called a “soft” cleavage method—the peptide cleaves from the resin while some or all of the side-chain protecting groups remain intact.

[0053] The hydrophilic linker disclosed herein can be used in synthetic methods utilizing both "hard" and "soft" cleavage methods. Using a "soft" cleavage synthesis strategy can, for example, enable the synthesized peptides to be used as starting materials in the synthesis of more complex peptides based on hybrid fragments. Furthermore, a hybrid SPPS / LPPS method can be implemented as part of a confluent peptide synthesis strategy using the hydrophilic linker described herein. Peptide fragments can be constructed using SPPS, cleaved from their solid support, isolated, and optionally purified, and then assembled by coupling them to peptides linked to the hydrophilic linker using LPPS. This confluent hybrid SPPS / LPPS strategy can be more practical and efficient at scale-up compared to purely SPPS-based methods.

[0054] Fragment-based confluent peptide synthesis strategies can also be implemented using LPPS with hydrophilic linkers as described herein. In this case, peptide fragments are constructed using LPPS, cleaved from the hydrophilic linker support, isolated, and optionally purified, and then assembled by coupling them to peptides linked to hydrophilic linkers using LPPS.

[0055] The hydrophilic linker compounds disclosed herein can also be used as part of a linker system to facilitate membrane-enhanced peptide synthesis (MEPS). Synthetic strategies built around MEPS employ membrane-based separation (or percolation) of the growing peptide from other reactants. Practical implementation of MEPS in LPPS strategies is facilitated by using systems that allow such separation in the same organic solvent in which the reaction takes place, such as organic solvent nanofiltration (OSN). This membrane-based separation technique achieves separation through the size difference between the growing peptide and other reactants. For this purpose, “nanostar” hub structures can be used as LPPS supports, increasing the molecular size of the growing peptide while remaining compact and readily synthesizable (see, for example, Yeo, J. et al. (2021) Angewandte Chemie International Edition 60: 7786-7795). Aromatic hub structures can serve as central connection points to which peptide synthesis linkers can be attached. These hub structures can also serve as additional UV chromophores for reaction monitoring, for example, by UHPLC-MS (ultra-high performance liquid chromatography-mass spectrometry). The nanostar hub structure increases the mass difference between the grown synthetic peptides and other reactants, thereby improving filtration efficiency.

[0056] The hydrophilic linker compounds disclosed herein can be used as part of a MEPS-based strategy. In particular, the hydrophilic linker compounds disclosed herein can be linked into nanostar hubs. Scheme 1 shows the synthesis of a previously disclosed nanostar structure characterized by linking a Rink- or Wang-type linker to a polyethylene glycol chain of a central benzene ring (Yeo, 2021).

[0057] Option 1

[0058]

[0059] Similar to nanostar structure 2 in scheme 1, the compounds of formula 1 described herein can also be connected to nanostar hubs to obtain compounds of, for example, formula 2:

[0060]

[0061] Where “Z” is

[0062]

[0063] “m” is 0, 1, 2, or 3; “n” is 1-10; and “p” is 2 or 3. Specifically, nanostar compounds of formula 2 can be prepared, where “Z” is…

[0064]

[0065] “m” is 1; “n” is 2, 4, 6, 8 or 10; and “p” is 2 or 3.

[0066] This disclosure also relates to "branched" hydrophilic linker compounds, wherein the linker is characterized by two or more hydrophilic functional groups linked to a peptide linker group. Branched hydrophilic linker systems may comprise compounds of Formula 3:

[0067]

[0068] Wherein “Z” represents a functional group that can form a covalent bond with an optionally protected amino acid, which can thus undergo repeated deprotection and coupling steps to one or more optionally protected amino acids or peptides, and the resulting polypeptide product can then be released from the “Z” group by chemical transformation; “m” is 0, 1, 2, or 3; “n” is 1-10; and “p” is 2 or 3. Specifically, branched compounds of formulas 3a, 3b, and 3c can be prepared:

[0069]

[0070] Where “k” is 1, “m” is 1, and “q” is 1; and “l”, “n” and “t” are each independently 2, 4, 6, 8 or 10.

[0071] Significant progress has been made in recent years in technologies enabling large-scale implementation of flow chemistry processes. In flow chemistry, reagents and reactants are typically pumped together through tubes or pipes into a continuously flowing mixture. Significant advantages can be achieved when implementing flow chemistry strategies into chemical manufacturing processes compared to conventional batch processes. Flow chemistry strategies facilitate the control of reaction parameters such as pressure, temperature, and reaction time. For example, by passing the reaction mixture through a tube, the mixture is exposed to the high surface area of ​​the tube, which increases the heat flux into or out of the reaction, allowing for rapid heating or cooling. Pressurized flow reactors can be used, allowing heating above the boiling point at atmospheric pressure and increasing reaction rates. Conventional batch processes can become complex when scaled up due to mixing and heat transfer rates, while flow chemistry makes it easier to maintain a high degree of control over these parameters. Furthermore, by conducting the reaction in a moving flow, only small amounts of high-energy intermediates are generated at any given time during the process, thus reducing the associated safety risks.

[0072] Flow chemistry principles have been applied to SPPS and liquid-phase synthesis systems, such as LPPS. In SPPS, packed-bed flow systems have been studied on both large and small scales, and such systems are well-suited for automation. In LPPS, immobilized reagents and microreactors have been used for small-scale preparation of peptide fragments (see, for example, Baxendale, IR; et al. (2006) Chemical Communications 4835-4837; and Fuse, S.; et al. (2014) Angewandte Chemie International Edition 53: 851-855), and continuous stirred tank reactor (CSTR) technology has been applied to the large-scale preparation of dipeptide and tripeptide products (see, for example, Jolley, KE; et al. (2017) Organic Process Research and Development 21: 1557-1565).

[0073] The hydrophilic linker compounds disclosed herein are particularly useful for realizing liquid-phase flow chemistry processes, such as LPPS. The rapid reaction kinetics of coupling and deprotection reactions on growth molecules (e.g., peptides) coupled to hydrophilic linkers in solution are an advantageous feature of flow chemistry implementation. The problem remains in solution-phase flow chemistry involving the separation of desired reaction products from undesirable byproducts and unreacted starting materials. The preparation of molecules, such as peptides, using the hydrophilic linker compounds disclosed herein occurs in solution; however, the separation of the desired product occurs during phase separation, allowing for the use of continuous liquid-liquid separation (e.g., using a mixer-sedimentation unit or a continuous flow centrifuge).

[0074] In conventional solid-phase peptide synthesis, such as in washing, coupling, and deprotection steps, large quantities of toxic solvents, such as dimethylformamide, N-methyl-2-pyrrolidine, dimethylacetamide, and dichloromethane, are used, posing challenges to industrial hygiene and environmental protection. Therefore, there is strong interest in developing more environmentally friendly (i.e., “more environmentally friendly”) alternative solvents for peptide synthesis. The methods described herein can utilize such more environmentally friendly solvents. Examples of more environmentally friendly washing solvents include ethyl acetate, isopropyl acetate, MTBE (methyl tert-butyl ether), and CPME (cyclopentyl methyl ether). The coupling reactions described herein can also be carried out in more environmentally friendly solvents such as DMSO.

[0075] As used herein, the term "amino acid" refers to an organic compound containing both carboxylic acid (-CO2H) and amine (-NH2) functional groups. Amino acids can be proteogenic (i.e., incorporated into proteins by biological synthesis during translation), such as glycine, L-alanine, and L-phenylalanine; or non-proteogenic, such as 3-aminoisobutyric acid and 8-amino-3,6-dioxanoic acid.

[0076] As used herein, the term "hydrophilic linker" refers to a chemical part of a molecule, such as a polypeptide, that can be constructed thereon by coupling (e.g., amino acid coupling) and deprotection steps, and is characterized by one or more functional groups that have a high affinity for water.

[0077] As used in this article, the term "flow chemistry" refers to chemical reactions occurring in a continuously flowing stream.

[0078] As used herein, the term “nanostar” refers to a linker structure concept for the synthesis of biopolymers (e.g., peptides), in which a core organic chemical structure serves as a central connection point (or “hub”) for two or more linkers on which biopolymer chains can be constructed. Nanostar structures for peptide construction have been described (see, for example, Yeo, J.; et al. (2021) Angewandte Chemie International Edition 60: 7786-7795).

[0079] As used herein, the term "peptide" or "polypeptide" refers to a polymeric chain of amino acids. These amino acids can be natural or synthetic, including modified amino acids. As used herein, the terms "peptide" and "polypeptide" are used interchangeably.

[0080] The following abbreviations are defined as follows: “AEEA” refers to 2-(2-(2-aminoethoxy)ethoxy)acetyl; “Aib” refers to 2-aminoisobutyric acid; “Boc” refers to tert-butoxycarbonyl; “CAD” refers to electrospray ionization mass spectrometry; “DCM” refers to dichloromethane; “DEPBT” refers to 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazine-4(3H)-one; “DIC” refers to diisopropylcarbodiimide; “DIEA” refers to diisopropylethylamine; “DMF” refers to N,N-dimethylformamide; “DMSO” refers to dimethyl sulfoxide; “DVB” refers to divinylbenzene; “EDC” refers to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; “ESMS” refers to electrospray ionization mass spectrometry; “Fmoc” refers to fluorenylmethoxycarbonyl; “Fmoc-Suberol” refers to 5-Fmoc-amino-2-carboxymethoxy- 10,11-Dihydro-5H-dibenzo[a,d]cycloheptene; "HMPA" refers to 4-(hydroxymethyl)phenoxyacetic acid; "HMPB" refers to 4-(4-hydroxymethyl-3-methoxyphenoxy)butyric acid; "LCMS" refers to liquid chromatography-mass spectrometry; "LPPS" refers to liquid phase peptide synthesis; "MTBE" refers to methyl tert-butyl ether; "oxyma" refers to ethyl cyanohydroxyiminoacetic acid ester; "PEG" refers to polyethylene glycol; "P" refers to... "yBop" refers to (benzotriazol-1-yloxy)tripyrrolidinephosphonium hexafluorophosphate; "PyOxim" refers to [(E)-(1-cyano-2-ethoxy-2-oxoethylidene)amino]oxy-tripyrrolidine-1-ylphosphonium hexafluorophosphate; "SPPS" refers to solid-phase peptide synthesis; "tBu" refers to tert-butyl; "TFA" refers to trifluoroacetic acid; "Trt" refers to triphenylmethyl; and "UPLC" refers to ultra-high performance liquid chromatography.

[0081] Option 2.

[0082]

[0083] Scheme 2 shows the preparation of hydrophilic linker compound 8, wherein “X” represents a functional group with a chemically unstable -OH or -NH2 group that can (respectively) form an ester or amide bond with an optional protected amino acid, thereby undergoing repeated deprotection and coupling steps to one or more optional protected amino acids or peptides, and the resulting polypeptide product can then be released from the “X” group by chemical transformation.

[0084] In Scheme 1, compound 8 is prepared by solid-phase synthesis using a Fmoc protecting group strategy. This synthesis can be performed partially or entirely in an automated peptide synthesizer. In step 1, the Fmoc-Sieber amide resin (1) is deprotected with piperidine, and then coupled with the Fmoc-protected intermediate 2 in step 2 using amide coupling conditions (e.g., Oxyma and DIC) to obtain intermediate 3. An iterative cycle of steps 3 (deprotection with piperidine) and 4 (amide coupling with the Fmoc-protected intermediate 2 using amide coupling conditions such as Pyoxim and an organic base) yields intermediate 4, repeating the cycle “n” minus one to achieve coupling of “n” monomer units together. In step 5, intermediate 4 is deprotected with piperidine, and then coupled with intermediate 5 or 6 in step 6 using an amide (e.g., with Pyoxim and an organic base) to obtain intermediate 7. If intermediate 7 has a Fmoc-protected nitrogen, it is deprotected with piperidine in step 7. Finally, the hydrophilic connector compound 8 was cleaved from the Sieber resin under acidic conditions (e.g., using TFA).

[0085] Option 3.

[0086]

[0087] Scheme 3 shows the use of a hydrophilic linker compound 9 to extend the polymer chain of an amino acid. The hydrophilic linker compound 9 carries a nitrogen atom on which the polymer chain of the amino acid can be constructed, and then cleaved from the linker under acidic conditions. In step 1, an Fmoc-protected amino acid 10 is coupled with the hydrophilic linker compound 9 in a polar aprotic organic solvent such as DMF or DMSO using amide coupling conditions (e.g., Pyoxim and an organic base) to give a first coupling intermediate 11. Upon completion of the reaction, a less polar aprotic solvent, such as MTBE, is added, which causes the coupled intermediate 11 to precipitate from the reaction mixture. The precipitate is separated from the bulk reaction mixture (e.g., by centrifugation and decantation of the supernatant) and optionally washed again by treating the precipitate with a solvent insoluble therein (e.g., MTBE), followed by separation of the precipitate (e.g., by centrifugation and decantation of the supernatant). In this way, intermediate 11 is separated from the reaction mixture and from a large amount of reaction solvent, unreacted starting materials, and reaction waste. In step 2, intermediate 11 is deprotected using piperidine and subjected to precipitation / product separation / optional washing. Then, in step 3, the next protected amino acid (12) is coupled using amide coupling conditions (e.g., Pyoxim and an organic base), followed by precipitation / product separation / optional washing to obtain intermediate 13. At this point, intermediate 13 can be subjected to further chemical transformations (e.g., as outlined in Scheme 6). If the terminal nitrogen protecting group is -Fmoc and polymerization chain elongation continues, steps 2 and 3 are iteratively repeated sequentially with the protected amino acid (e.g., 14) to obtain intermediate 15.

[0088] Option 4.

[0089]

[0090] Scheme 4 shows the use of an oxygen-containing hydrophilic linker compound 16 to extend the polymeric chain of an amino acid, on which the polymeric chain of the amino acid can be constructed, and then cleaved from the linker under acidic conditions. The steps of this method are similar to those outlined in Scheme 3, except that step 1 is an esterification step (carried out using reagents such as PyBOP / organic base or DIC / DMAP). Repeated deprotection and coupling steps with the protected amino acid (steps 2 and 3, respectively) as outlined in Scheme 3 yield polymeric compound 17.

[0091] Option 5.

[0092]

[0093] Scheme 5 shows three pathways for cleaving elongated amino acid polymers from oxygen-linked linkers. When cleaved from these linkers, the amino acid polymers have a free carboxylic acid (-CO2H) at their C-terminus.

[0094] In the first approach, in step 1b, compound 17 undergoes “soft” cleavage under acidic conditions (e.g., a DCM solution of 2-5% TFA), causing the linker to hydrolyze from the amino acid polymer to yield a carboxylic acid group at the C-terminus, and leaving an N-terminal protecting group in compound 18 (and possibly present in R...). 1 R 2 R 3 Other protecting groups (e.g., pyridine). Neutralize with a base such as pyridine, followed by an aqueous post-treatment to separate most of the hydrophilic linker from 18. Then, using an intact N-terminal protecting group, 18 is coupled to another amine, for example, as part of a fragment-based peptide synthesis strategy.

[0095] In the second approach, in step 1a, the N-terminal protecting group is removed under suitable conditions (using piperidine in the case of -Fmoc protection) to obtain 19. In step 2b, the amino acid polymer can be purified leaving behind any remaining intact R group. 1 R 2 R 3 Under conditions such as using a DCM solution of 2-5% TFA, hydrolysis from the linker yields 20, or complete deprotection of the acid-unstable protecting group can be achieved under “hard” cleavage conditions using a mixture of, for example, TFA, triisopropylsilane, 1,2-ethylenedithiol and water (85:5:5:5 v / v ratio).

[0096] In the third pathway, intermediate 19 is coupled with carboxylic acid 21 in step 2a under amide coupling conditions (e.g., DEPBT and an organic base, or 21 can be reacted with an organic base as a succinimide ester) to give 22. In step 3, compound 23 is cleaved from the hydrophilic linker using the “hard” or “soft” cleavage described above.

[0097] Option 6.

[0098]

[0099] Scheme 6 shows two pathways for cleaving elongated amino acid polymers from nitrogen-linked linkers. When cleaved from these linkers, the amino acid polymers have a primary amide (-CONH2) at their C-terminus.

[0100] In the first approach, the N-terminal protecting group on intermediate 24 is removed in step 1a (piperidine is used if the protecting group is -Fmoc), yielding 25, which is then cleaved from the hydrophilic linker under acidic conditions using "soft" cleavage conditions (e.g., a DCM solution of 2-5% TFA), leaving R 1 R 2 R 3Complete protecting groups, or use "hard" cleavage conditions (e.g., 85:5:5:5 TFA, triisopropylsilane, 1,2-ethylenedithiol, and water) to remove acid-labile R groups. 1 R 2 R 3 With the same protecting group, we obtain 26. If the N-terminal protecting group of intermediate 24 is an acid-labile protecting group, such as -Boc, then steps 1a and 2b can be carried out in a single tank under “hard” pyrolysis conditions.

[0101] In the second approach, in step 2a, 25 is coupled to carboxylic acid 27 under amide coupling conditions (e.g., DEPBT and an organic base, or 27 may be reacted using an organic base as a succinimide ester) to obtain 28. In step 3, compound 29 is cleaved from the hydrophilic linker using the “hard” or “soft” cleavage described above. Example

[0102] The following examples further illustrate various embodiments of this disclosure and represent typical synthesis of the compounds disclosed. The reagents and starting materials are readily available or can be readily synthesized by those skilled in the art. It should be understood that these examples are illustrated by way of example and not limitation, and various modifications can be made by those skilled in the art.

[0103] exist LC-MS was performed on an HP1200 liquid chromatography system. Chromatographic conditions - Column: Waters CSH TM C 18 150 × 2.1 mm, 1.7 μm; gradient used: 5-95% solvent B: solvent A, run for 20 to 30 minutes; flow rate: 0.5 mL / min; column temperature: 40-50 °C; solvent A: 0.2% aqueous solution of TFA; solvent B: acetonitrile. Electrospray ionization mass spectrometry (ESMS) was performed on a quadrupole mass spectrometer with a mass-selective detector connected to the chromatographic system.

[0104] Example 1

[0105] Preparation of 2-(4-(amino(2,4-dimethoxyphenyl)methyl)phenoxy)-N-(53-amino-8,17,26,35,44,53-hexaoxo-3,6,12,15,21,24,30,33,39,42,48,51-dodecoxa-9,18,27,36,45-pentazatrialkyl)acetamide [Rink-(AEEA)6-NH2]

[0106]

[0107] The title compound was prepared by solid-phase synthesis using the Fmoc strategy on a Symphony X automated peptide synthesizer (Protein Technologies Inc.) starting with Fmoc-Sieber amide resin (0.8% substituted, 1% DVB styrene, 100-200 mesh).

[0108] Coupling of AEEA units on Sieber resin: Preparation of (AEEA)6 on Fmoc-Sieber amide resin was carried out in 9 batches at a scale of 0.5 mmol (total 4.5 mol). For each batch, the resin was swollen by washing twice with DMF (10 L) for 10 min each time. Then, the deprotection and coupling cycles were performed as follows: the resin was washed with DMF (9 mL, 2 min), deprotected with a 20% piperidine DMF solution (7 mL, 5 min, then 9 mL, 25 min), washed with DMF (9 mL, 1 min, repeated 6 times), coupled with Fmoc-AEEA-OH [0.375 M in DMF (4 mL, 1.5 mmol, 3 equivalents), Oxyma (0.750 M, 2 mL, 3 equivalents), DIC (0.660 M, 2.5 mL, 3.3 equivalents)] mixed under nitrogen bubbling for 1 h 45 min, and finally the reaction vessel was drained and washed with DMF (9 mL, 30 s, repeated 3 times). This deprotection and coupling cycle was repeated a total of six times to obtain Fmoc-(AEEA)6 on the Sieber resin. After the final DMF wash, the resin was washed with DCM (10 mL, 1 min, repeated 5 times). It was then dried in a nitrogen stream for 4 h. For each batch prepared in this manner, the average yield of Fmoc-(AEEA)6 on Sieber resin was 1.156 g.

[0109] To couple the Rink group to (AEEA)6 on Sieber resin: A portion (1.1 g, 0.5 mmol) of Fmoc-(AEEA)6 on Sieber resin was swollen with DMF (10 mL, 20 min, repeated 3 times), deprotected with piperidine in 20% DMF (10 mL, 20 min, repeated 3 times), and then washed with DMF (10 mL, 2 min, repeated 5 times). The Fmoc-Rink connector (p-[a-[1-(9H-fluorene-9-yl)-methoxyformamido]-2,4-dimethoxybenzyl]-phenoxyacetic acid, 0.81 g, 1.5 mmol, 3.0 equivalent), PyOxim (0.79 g, 1.5 mmol, 3.0 equivalent), and DIEA (0.52 mL, 0.39 mg, 3.0 mmol, 6.0 equivalent) in a solution of DMF (9 mL) were added to the reaction vessel and mixed by bubbling with nitrogen for 2 h. The reaction vessel was drained and washed with DMF (10 mL, 2 min, repeated 5 times), followed by deprotection with a 20% piperidine DMF solution (10 mL, 20 min, repeated 3 times). The resin was washed with DMF (10 mL, 2 min, repeated 5 times). After the final DMF wash, the resin was washed with DCM (10 mL, 2 min, repeated 5 times) and dried in a nitrogen stream for 4 h to obtain 1.21 g Rink-(AEEA)6 on Sieber resin.

[0110] Rink-(AEEA)6-NH2 cleavage from Sieber resin: Rink-(AEEA)6 on Sieber resin was mixed with 12 mL of 5% TFA in DCM solution for 30 min, filtered, and washed with another DCM solution. The filtrate was neutralized with DIEA and concentrated under reduced pressure. The resulting oily substance was dissolved in DMSO (2 mL), MTBE (30 mL) was added, and the mixture was centrifuged at 3000 rpm for 3 min. The supernatant was decanted, fresh MTBE (30 mL) was added, and the mixture was centrifuged again at 3000 rpm for 3 min. The supernatant was decanted again to obtain the title compound as an oily precipitate. ESMS m / z 1188.5 (M+H + ).

[0111] Example 2

[0112] Preparation of 2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[4-[Amino-(2,4-dimethoxyphenyl)methyl)phenoxy]phenoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]acetyl]acetamide[Rink-(AEEA)8-NH2]

[0113]

[0114] The title compound was prepared by solid-phase synthesis, using essentially the method described in Example 1, by coupling eight AEEA units to a solid support and then to a Fmoc-Rink linker. After coupling the Fmoc-Rink linker to (AEEA)8 on Sieber resin, Fmoc-Rink-(AEEA)8-NH2 was cleaved from the resin using a 2% TFA solution in DCM (5 volumes, 20 min, repeated 5 times). The combined filtrate was neutralized with DIEA, and the solution was evaporated. DMF (2 volumes) was added, followed by MTBE to initiate phase separation. The mixture was centrifuged, and the supernatant was removed to give Fmoc-Rink-(AEEA)8-NH2, an oily substance. The final Fmoc groups were removed with a 30% piperidine solution in DMF, followed by MTBE to initiate phase separation. The supernatant was removed to give the title compound, an oily substance. ESMS m / z 739.5 (M+2H) + / 2).

[0115] Example 3

[0116] 2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[4-[Amino-(2,4-dimethoxyphenyl)methyl)phenoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]acetamide[Rink-(AEEA] 10 Preparation of [-NH2]

[0117]

[0118] The title compound was prepared by solid-phase synthesis, using essentially the method described in Example 1, to couple 10 AEEA units onto a solid support, followed by coupling at a Fmoc-Rink linker. ESMS m / z 1768.8 (M+H) + ).

[0119] Example 4

[0120] Preparation of 2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[4-[Amino-(2,4-dimethoxyphenyl)methyl)]phenoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]acetamide[Rink-(AEEA)4-NH2]

[0121]

[0122] The title compound was prepared by solid-phase synthesis, using essentially the method described in Example 1, to couple four AEEA units to a solid support, and then to a Fmoc-Rink linker. ESMS m / z 897.4 [(M+H + ].

[0123] Example 5

[0124] Preparation of 2-[2-[2-[2-[2-[2-[4-[amino-(2,4-dimethoxyphenyl)methyl)]phenoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetamide [Rink-(AEEA)2-NH2]

[0125]

[0126] The title compound was prepared by solid-phase synthesis, using essentially the method described in Example 1, to couple two AEEA units to a solid support, and then to a Fmoc-Rink linker. ESMS m / z 607.3 [(M+H + ].

[0127] Example 6

[0128] N-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-amino-2-oxo-ethoxy)ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy] [HMPB-(AEEA)]-4-[4-(hydroxymethyl)-3-methoxy-phenoxy]butyramide 10 Preparation of [-NH2]

[0129]

[0130] Ten AEEA units were coupled to Sieber resin using essentially the method described in Example 1. After the last AEEA unit was coupled to the resin, it was washed with DCM and dried in a nitrogen stream for 4 h. A portion of the resin (0.50 mmol) was deprotected with piperidine in 20% DMF, essentially as described in Example 1, and then the resin was resuspended in DCM (11.4 mL) with TFA (0.6 mL). The suspension was mixed at 25 °C for 30 min, then filtered and washed with DCM. The filtrate was neutralized with DIEA and concentrated under reduced pressure. The resulting oily substance was dissolved in DMSO (2 mL) and then MTBE (30 mL) was added. The mixture was centrifuged, the supernatant was removed, and (AEEA) was obtained in a centrifuge tube.10 -NH2 is an oily substance.

[0131] To those containing (AEEA) 10 Add a solution of DEPBT (150 mg, 0.50 mmol), HMPB (120 mg, 0.50 mmol), and DIEA (0.174 mL, 129 mg, 1.0 mmol) in DMSO (1.5 mL) to a centrifuge tube containing -NH2, allow the solution to stand for 5 min, and then add it to a test tube. Place the resulting mixture on a shaker and mix for 2 h, then add MTBE (20 mL) to induce phase separation. Centrifuge the mixture (2500 rpm, 3 min) and discard the supernatant. Add fresh MTBE (20 mL) to a test tube, centrifuge (2500 rpm, 3 min), discard the supernatant, and obtain the title compound as an oil. ESMS m / z 1712.70 (M+Na-1H).

[0132] Example 7

[0133] Preparation of N-(17-amino-8,17-dioxo-3,6,12,15-tetraoxa-9-azaheptadecyl)-4-(4-(hydroxymethyl)-3-methoxyphenoxy)butyramide [HMPB-(AEEA)2-NH2]

[0134]

[0135] The title compound was prepared essentially as described in Example 6, except that HMPB was coupled with (AEEA)₂-NH₂ to give HMPB-(AEEA)₂-NH₂. ESMS m / z 552.3 (M+Na) + ).

[0136] Example 8

[0137] Preparation of N-(35-amino-8,17,26,35-tetraoxo-3,6,12,15,21,24,30,33-octaoxa-9,18,27-triazapentadecyl)-4-(4-(hydroxymethyl)-3-methoxyphenoxy)butyramide [HMPB-(AEEA)4-NH2]

[0138]

[0139] The title compound was prepared essentially as described in Example 6, except that HMPB was coupled with (AEEA)₄-NH₂ to give HMPB-(AEEA)₄-NH₂. ESMS m / z 842.4 (M+Na)+ ).

[0140] Example 9

[0141] Preparation of N-(53-aza-8,17,26,35,44,53-hexaoxo-3,6,12,15,21,24,30,33,39,42,48,51-dodecoxa-9,18,27,36,45-pentaazatrialkyl(pentaazatripentacontyl))-4-(4-(hydroxymethyl)-3-methoxyphenoxy)butyramide [HMPB-(AEEA)6-NH2]

[0142]

[0143] The title compound was prepared essentially as described in Example 6, except that HMPB was coupled with (AEEA)6-NH2 to give HMPB-(AEEA)6-NH2. ESMS m / z 1132.5 (M+Na) + ).

[0144] Example 102: Preparation of ((5-amino-10,11-dihydro-5H-dibenzo[a,d][7]annulen-3-yl)oxy)-N-(53-amino-8,17,26,35,44,53-hexaoxo-3,6,12,15,21,24,30,33,39,42,48,51-dodecoxa-9,18,27,36,45-pentazatrialkyl)acetamide [Ramage-(AEEA)6-NH2]

[0145]

[0146] Ramage groups were coupled to (AEEA)6 on Sieber resin: A portion (986.8 mg, 0.5 mmol) of Fmoc-(AEEA)6 on Sieber resin was swollen with DMF (10 mL, 20 min, repeated 3 times), deprotected with 20% piperidine in DMF (10 mL, 20 min, repeated 3 times), and then washed with DMF (10 mL, 2 min, repeated 5 times). A solution of Fmoc-octanediol (0.76 g, 1.5 mmol, 3.0 equivalent) in DMF:DMSO (4:1, 5 mL) was added to the reaction vessel, followed by the addition of Oxyma (0.750 M in DMF, 2 mL, 1.5 mmol, 3.0 equivalent) and DIC (0.660 M in DMF, 2.5 mL, 1.65 mmol, 3.3 equivalent), and mixed by bubbling with nitrogen for 4 h. The reaction vessel was drained and washed with DMF (10 mL, 2 min, repeated 5 times), followed by deprotection with 20% piperidine in DMF (10 mL, 20 min, repeated 3 times). The resin was washed with DMF (10 mL, 2 min, repeated 5 times). After the final DMF wash, the resin was washed with DCM (10 mL, 2 min, repeated 5 times) and dried in a nitrogen stream for 4 h to obtain 1.21 g of Rink-(AEEA)6 on Sieber resin.

[0147] Cycling of Ramage-(AEEA)6-NH2 from Sieber resin: Ramage-(AEEA)6-NH2 was cleaved from Sieber resin essentially as described in Example 1, except that 2% TFA in DCM was used and the reaction was stirred for 10 min instead of 30 min prior to further processing. ESMS m / z 1153.55 (M+H).

[0148] Example 11

[0149] 2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[(11-amino-6,11-dihydro-5H-dibenzo[1,2-e:1',2'-f][7]annulen-2-yl)oxy]acetyl]amino] [Ethoxy] [Ethoxy] [Acetyl] [Amino] [Ethoxy] [Ethoxy] [Acetyl] [Amino] [Ethoxy] [Ethoxy] [Acetyl] [Amino] [Ethoxy] [Ethoxy] [Acetyl] [Amino] [Ethoxy] [Ethoxy] [Acetyl] [Amino] [Ethoxy] [Ethoxy] [Acetyl] [Amino] [Ethoxy] [Ethoxy] [Acetyl] [Amino] [Ethoxy] [Ethoxy] [Acetyl] [Amino] [Ethoxy] [Ethoxy] [Acetyl] [Ramage-(AEEA)] 10 Preparation of [-NH2]

[0150]

[0151] Fmoc-octanediol was coupled to (AEEA) on Sieber resin via solid-phase synthesis using the method described in Example 10. 10 Preparation of the title compound. ESMS m / z 1733.8 (M+H).

[0152] Example 12

[0153] Preparation of 2-((5-amino-10,11-dihydro-5H-dibenzo[a,d][7]annulen-3-yl)oxy)-N-(35-amino-8,17,26,35-tetraoxo-3,6,12,15,21,24,30,33-octaoxa-9,18,27-triazapentadecyl)acetamide [Ramage-(AEEA)4-NH2]

[0154]

[0155] The title compound was prepared by solid-phase synthesis using the method described in Example 10, by coupling Fmoc-octanediol to (AEEA)4 on a Sieber resin. ESMS m / z 863.4 (M+H).

[0156] Example 13

[0157] Preparation of 2-((5-amino-10,11-dihydro-5H-dibenzo[a,d][7]annulen-3-yl)oxy)-N-(17-amino-8,17-dioxo-3,6,12,15-tetraoxa-9-azahepta-hepta-yl)acetamide [Ramage-(AEEA)2-NH2]

[0158]

[0159] The title compound was prepared by solid-phase synthesis using the method described in Example 10, by coupling Fmoc-octanediol to (AEEA)2 on a Sieber resin. ESMS m / z 595.3 (M+Na).

[0160] Example 14

[0161] Preparation of N-(2-(2-(2-amino-2-oxoethoxy)ethoxy)ethyl)-2-(2-(2-(2-(4-(hydroxymethyl)phenoxy)acetamido)ethoxy)ethoxy)acetamide [HMPA-(AEEA)2-NH2]

[0162]

[0163] To couple the HMPA group to (AEEA)2 on Sieber resin: Partially swell the Fmoc-(AEEA)2 (845 mg, 0.5 mmol) on the Sieber resin with DMF (10 mL, 20 min, repeated 3 times), deprotect with 20% piperidine in DMF (10 mL, 20 min, repeated 3 times), and then wash with DMF (10 mL, 2 min, repeated 5 times). A solution of HMPA (91 mg, 0.5 mmol, 1.0 equivalent), DIEA (0.174 mL, 1.0 mmol, 2.0 equivalent), and DEPBT (150 mg, 0.5 mmol, 1.0 equivalent) in DMF (10 mL) was added to the reaction vessel and mixed by bubbling with nitrogen for 2 h. The reaction vessel was drained, washed with DMF (10 mL, 2 min, repeated 5 times), then washed with DCM (10 mL, 2 min, repeated 5 times), and dried in a nitrogen stream for 4 h to obtain 908.6 mg of HMPA-(AEEA)2 on Sieber resin.

[0164] Cleavage of HMPA-(AEEA)2-NH2 from Sieber resin: HMPA-(AEEA)2-NH2 was cleaved from Sieber resin essentially as described in Example 1 to give the title compound. ESMS m / z 472.2 (M+H + ).

[0165] Example 15

[0166] 2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[4-(hydroxymethyl)phenoxy]acetyl]amino]eth ... 10 Preparation of [-NH2]

[0167]

[0168] The title compound was prepared by solid-phase synthesis using the method described in Example 14, wherein HMPA was reacted with (AEEA) on Sieber resin. 10 Coupling. ESMS m / z 1632.7(M+H).

[0169] Example 16 - Liquid-phase peptide synthesis and "hard" cleavage from linkers using Rink-(AEEA)6-NH2

[0170] The 19-chain peptide of SEQ ID NO: 1 was prepared using liquid-phase peptide synthesis as follows.

[0171]

[0172] (SEQ ID NO: 1)

[0173] Elongation of the amino acid chain on Rink-(AEEA)6-NH2: Fmoc-Ser(tBu)-OH (0.5 mmol), PyOxim (0.5 mmol), and DIEA (1 mmol) were dissolved in DMF (1 mL). The solution was mixed for 1 min, and then the Rink linker-(AEEA)6-NH2 (prepared in Example 1, 0.16 mmol) was added. The reaction solution was mixed for 10 min, and then MTBE (5 mL) was added. The mixture was centrifuged at 3250 rpm, and the supernatant was discarded. The remaining substance was mixed with 30% piperidine in DMF (1 mL) for 5 min. After mixing for 5 min, MTBE (5 mL) was added. The mixture was centrifuged, and the supernatant was discarded. LCMS confirmed that no product was present in the supernatant containing MTBE at each centrifugation step.

[0174] The amino acid coupling and deprotection steps were repeated, coupling the Fmoc-protected amino acids (side chain -OH and -CO2H groups protected with tBu) in the order from C-terminus to N-terminus given in SEQ ID NO: 1. When the peptide construct is 15 amino acids or less, DMF and DMSO are interchangeable as reaction solvents for the amino acid coupling and deprotection steps. When the peptide length is greater than 15 amino acids, DMSO is preferred as the reaction solvent. Upon completion of the coupling of the final alanine residue in SEQ ID NO: 1, the final Fmoc group was removed using the above-described 30% piperidine / DMF deprotection reaction conditions. The product was lyophilized to give the peptide of SEQ ID NO: 1 (0.612 mg, with residual solvent). ESMS m / z 1697.7 (M+2H) + / 2), 1132.0(M+3H + / 3). The crude separation weights support the LCMS data, indicating that there was no product loss in the supernatant during the phase separation step using MTBE during liquid-phase synthesis.

[0175] Peptide cleavage from the "hard" linker: To remove the soluble linker and protecting group, the peptide of SEQ ID NO: 1 was stirred at 25°C for 2 h in a solution containing TFA:triisopropylsilane:1,2-ethylenedithiol:water (85:5:5:5 v / v ratio) to obtain a peptide with the following sequence: AFIEYLLEGGPSSGAPPPS-NH2 (SEQ ID NO: 2).

[0176] The peptide of SEQ ID NO: 2 was precipitated with MTBE (10:1 MTBE to the reaction volume), centrifuged as described above, and then vacuum dried. High-resolution MS m / z measured 944.4783 (charge state +2, neutral mass 1886.9426), theoretical neutral mass 1886.9414.

[0177] Example 17 - Using Ramage (AEEA) 10- Liquid-phase peptide synthesis of NH2 and "soft" cleavage from linkers

[0178] Ramage-(AEEA) 10 Preparation of SEQ ID NO: 3 on -NH2: Using essentially the method described in Example 16, liquid-phase peptide synthesis was initiated by coupling Fmoc-Ser(tBu)-OH to 3.0 mmol of Ramage-(AEEA). 10-NH, then Fmoc deprotection was performed to prepare the peptide of SEQ ID NO: 3. The remaining Fmoc-protected amino acids (side chain -OH groups protected with tBu) were coupled / deprotected in the order from C-terminus to N-terminus given in SEQ ID NO: 3. Pro(7) and Pro(8) were incorporated as a dimer (Fmoc-Pro-Pro-OH). PyBOP can be used interchangeably with Pyoxim. ESMS m / z 913.1 (M+3H + / 3).

[0179]

[0180] (SEQ ID NO: 3)

[0181] Soft cleavage of the peptide from the linker: 10 volumes of a 2% TFA DCM solution were added to the peptide of SEQ ID NO: 3, and the reaction mixture was incubated at room temperature for 30 min. The reaction mixture was neutralized with 1 equivalent of pyridine and concentrated under vacuum. The starting material was observed in the product, and 20 volumes of a 2% TFA DCM solution were added to it. The mixture was incubated at room temperature for 30 min, neutralized with pyridine, and concentrated under vacuum again. 50 volumes of a 5% TFA DCM solution were added to the residue. After 40 min, the solution was neutralized with pyridine and concentrated under reduced pressure to obtain the crude peptide of SEQ ID NO: 4. ESMS m / z 1042.50 (M+Na+).

[0182] GPS(tBu)-S(tBu)-GAPPPS(tBu)-NH2(SEQ ID NO: 4)

[0183] Example 18 - Liquid-phase peptide synthesis using Rink-(AEEA)2-NH2

[0184]

[0185] (SEQ ID NO: 5)

[0186] Essentially as described in Example 16, the peptide of SEQ ID NO: 5 was prepared using Rink-(AEEA)2-NH2 as a support in liquid-phase peptide synthesis. MTBE was added to the final Fmoc deprotection reaction mixture, and the mixture was then centrifuged. The supernatant was discarded, yielding the peptide of SEQ ID NO: 5 as an oily sediment. ESMS m / z 1631.8 (M+Na) + ), 1609.8(M+H + ), 805.5(M+2H + / 2).

[0187] Example 19 - Using HMPA-(AEEA) 10 Liquid-phase peptide synthesis of -NH2 and “soft” cleavage from the linker were performed to prepare the peptide of SEQ ID NO: 6 using the following liquid-phase peptide synthesis.

[0188]

[0189] (SEQ ID NO: 6)

[0190] HMPA-(AEEA) 10 Elongation of the amino acid chain on -NH2: PyBOP (0.418 g, 0.803 mmol) and DIEA (0.207 g, 1.60 mmol) were added to a solution of Fmoc-Gly-OH (0.2388 g, 0.8032 mmol) in DMSO (2 mL). After mixing for 1 min, MPA-(AEEA) was added. 10 Mix in -NH2 (0.4372 g, 0.2678 mmol) for 90 min, then add MTBE (40 mL). Centrifuge the mixture at 3000 rpm for 3 min at room temperature and discard the supernatant. Repeat this washing three times to obtain the oily layer at the bottom. Repeat the coupling step again, then mix the resulting oily substance with 10% piperidine in DMF (2 mL) for 15 min. Add MTBE (20 mL) and centrifuge in a similar manner as described above to obtain the mixture. Repeat the 10% piperidine / DMF deprotection operation again to obtain the oily layer at the bottom.

[0191] The remaining Fmoc-protected amino acids (glutamine side chain -CONH2 group protected with triphenylmethyl and tryptophan side chain -NH group protected with -Boc) were coupled / deprotected as described above, in the order from C-terminus to N-terminus as shown in SEQ ID NO: 6. The subsequent coupling reaction system was stirred for 45 min instead of 90 min. After stirring the final amino acid (Fmoc-Phe-OH) coupling reaction system for 45 min, isopropyl acetate was added to the mixture, followed by centrifugation and washing with MTBE as described above to obtain the bottom oily layer, which is the title compound. ES / MS m / z 1556.10 (M+2H+ / 2).

[0192] Soft cleavage of peptides from the linker: Essentially as described in Example 17, the peptide of SEQ ID NO: 6 was subjected to soft cleavage by incubation twice for 30 min with a DCM solution of 3% TFA to obtain the peptide of SEQ ID NO: 7. ES / MS m / z 1519.60 (M+Na+).

[0193] Fmoc-FVQ(Trt)-W(Boc)-LIAG-OH(SEQ ID NO:7)

[0194] Example 20 - Using HMPB-(AEEA) 10 Liquid-phase peptide synthesis of -NH2 and "soft" cleavage from the linker

[0195] The peptide of SEQ ID NO: 8 was prepared using liquid-phase peptide synthesis as follows.

[0196]

[0197] (SEQ ID NO: 8)

[0198] HMPB-(AEEA) 10 Elongation of the amino acid chain on -NH2: Essentially as described in Example 16, the Fmoc-protected amino acid (glutamine side chain -CONH2 group protected with triphenylmethyl, tryptophan side chain -NH group protected with -Boc) was coupled, and then the peptide of SEQ ID NO: 8 was prepared by deprotection in the manner described in Example 16, following the order from C-terminus to N-terminus as given in SEQ ID NO: 8, to obtain the peptide of SEQ ID NO: 8. ESMS m / z ESMS m / z 1585.70 (M+2H + / 2).

[0199] Soft cleavage of the peptide of SEQ ID NO: 7: The peptide of SEQ ID NO: 8 was dissolved in 3% TFA in DCM solution (20 volumes) and incubated at room temperature for 30 min. The reaction mixture was neutralized with 1 equivalent of pyridine and then washed with water. The organic layer was concentrated under vacuum to obtain the peptide of SEQ ID NO: 7. ESMS m / z 1497.7 (M+H+).

[0200] Example 21

[0201] 20-[[(1S)-4-[2-[2-[2-[2-[2-[2-[4-[4-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-amino-2-oxo-ethoxy)ethoxy]ethylamino]-2-oxo-ethoxy] ... [Ethylamino]-2-oxo-ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethylamino]-4-oxo-butoxy]-2-methoxy-phenyl]methoxy]-2-oxo-ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethylamino]-1-tert-butoxycarbonyl-4-oxo-butyl]amino]-20-oxo-eicosanoate tert-butyl ester [fatty acid side chain -HMPB-(AEEA)] 10 Preparation of [-NH2]

[0202]

[0203] Method 1 [Coupling sequence - (AEEA)2, γ-Glu, fatty acids]: Add DIC (1.33 mL, 8.469 mmol) to a solution of Fmoc-(AEEA)2-OH (4.493 g, 8.469 mmol) and DMAP (51.7 mg, 0.423 mmol) in DMSO (11 mL), and let stand for 5 min. Then add HMPB-(AEEA) to this solution. 10 -NH2 (2.823 mmol), washed with DMSO (3 mL). The reaction solution was mixed for 2 h, then MTBE (45 mL) was added. The mixture was centrifuged at 3000 rpm for 2 min. The supernatant was decanted, fresh MTBE (45 mL) was added, and the mixture was centrifuged at 3000 rpm for 2 min. The supernatant was discarded, EtOAc (45 mL) was added, and the mixture was centrifuged at 3000 rpm for 2 min, then the supernatant was decanted to obtain an oily precipitate. 30% piperidine / DMF (8 mL) was added to the oily precipitate, mixed for 15 min, then MTBE (45 mL) was added. The mixture was centrifuged at 3000 rpm for 2 min. The supernatant was discarded, fresh MTBE (45 mL) was added, and the mixture was centrifuged at 3000 rpm for 2 min again. The supernatant was decanted again to obtain an oily precipitate.

[0204] Add DIEA (0.983 mL, 5.64 mmol) to a solution of Fmoc-Glu-OtBu (1.201 g, 2.823 mmol) and PyBOP (1.469 g, 2.823 mmol) in DMSO (9 mL). Let the solution stand for 5 min, then add it to the oily precipitate and mix for 30 min. Add MTBE (45 mL) and centrifuge the mixture at 3000 rpm for 2 min. Discard the supernatant, add fresh MTBE (45 mL), and centrifuge the mixture at 3000 rpm for 2 min. Discard the supernatant, add EtOAc (45 mL), and centrifuge the mixture at 3000 rpm for 2 min. Decant the supernatant again to obtain an oily precipitate. Add 30% piperidine / DMF (8 mL) to the oily precipitate, mix for 15 min, then add MTBE (45 mL). Centrifuge the mixture at 3000 rpm for 2 min. Discard the supernatant, add fresh MTBE (45 mL), and then centrifuge the mixture again at 3000 rpm for 2 min. Decant the supernatant again to obtain an oily precipitate.

[0205] DIEA (0.983 mL, 5.64 mmol, 2.0 equivalent) was added to a solution of 20-tert-butoxy-20-oxo-eicosanoic acid (1.13 g, 2.83 mmol, 1.0 equivalent) and DEPBT (844.7 mg, 2.823 mmol, 1.0 equivalent) in DMSO:toluene (9:1, 10 mL). The solution was allowed to stand for 5 min, then added to the oily precipitate and mixed for 30 min. MTBE (45 mL) was added, and the mixture was centrifuged at 3000 rpm for 2 min. The supernatant was decanted, and fresh MTBE (45 mL) was added. The mixture was centrifuged again at 3000 rpm for 2 min, and the supernatant was decanted again. EtOAc (45 mL) was added, and the mixture was centrifuged again at 3000 rpm for 2 min. The supernatant was discarded, yielding the title compound as an oily precipitate. ESMS m / z 1274.10 (M+2H) + / 2).

[0206] Method 2 [Coupling sequence - (AEEA)2, γ-Glu-fatty acid succinimide ester]: As described above, Fmoc-(AEEA)2-OH is coupled with HMPB-(AEEA) in the same scale. 10 -NH2 coupling. Add 30% piperidine / DMF (3 ml) solution to (AEEA)2-HMPB-(AEEA) 10Mix the MTBE (total volume 40 mL) in the NH2 oil for 15 min, then add the MTBE to the reactants and centrifuge the mixture (3000 rpm × 2 min). Decant the MTBE and add 2 mL of DMSO to the oil to dissolve it. Add fresh MTBE (total volume 40 mL), centrifuge the mixture again, and decant the MTBE layer. Dissolve O5-(2,5-dioxopyrrolidone-1-yl)(2S)-2-[(20-tert-butoxy-20-oxo-eicosanoyl)amino]glutaric acid O1-tert-butyl ester (5.76 g, 8.46 mmol) in a mixture of DMSO:toluene (9:1 ratio, 5.4 mL DMSO + 0.6 mL toluene). Add DIEA (3 mL, 16.92 mmol) and allow the resulting solution to stand for 5 min for pre-activation. Add the mixture to a centrifuge tube containing AEEA2-HMPB-(AEEA). 10 Add 2.82 mmol of -NH2 for 30 min, then add MTBE (total volume 40 mL) and centrifuge the mixture (3000 rpm × 2 min). Remove the MTBE layer, add fresh MTBE (40 mL total volume), and centrifuge the mixture again. Discard the supernatant to obtain the title compound as an oil. ESMS m / z 1273.70 (M+2H+ / 2).

[0207] Example 22 2-[2-[2-[2-[2-[(4S)-5-tert-butoxy-4-[(20-tert-butoxy-20-oxo=eicosanoyl)amino]-5-oxo-pentanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetic acid [fatty acid side chain] was prepared by soft pyrolysis.

[0208]

[0209] The fatty acid side chain -HMPB-(AEEA) 10-NH2 (prepared in Example 21, 2500 mg) was added to a 2% TFA / toluene solution (10 volumes, 25 mL). The mixture was mixed for 10 min, then centrifuged at 3000 rpm for 5 min, and the supernatant was collected and neutralized with pyridine (equimolar with TFA). MTBE (25 mL) was added to the remaining oily precipitate, and the mixture was centrifuged at 3000 rpm for 5 min, and the supernatant was collected. Fresh MTBE (25 mL) was added to the oily precipitate, and the mixture was centrifuged again at 3000 rpm for 5 min, and the supernatant was collected again to obtain the oily precipitate. The lysis and washing were repeated more than twice. The organic layer was washed with a saturated NaCl aqueous solution and water, and then the combined organic layers were concentrated under reduced pressure to obtain the title compound. ESMS m / z 874 (M+H+). UPLC-CAD [Column: Waters CSH] was used. TM C 18 150 × 2.1 mm, 1.7 μm; column temperature: 50 °C; gradient -30 to 90% solvent B: solvent A, run for 21 min; flow rate -0.5 mL / min; solvent A: 0.2% TFA aqueous solution; solvent B: acetonitrile; detector: photodiode array UV, CAD] Analyze the crude product. The crude product showed a purity of 61.71%.

[0210] Example 23

[0211] 20-[[(1S)-4-[2-[2-[2-[2-[2-[2-[4-[4-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-amino-2-oxo-ethoxy)ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethyl Preparation of tert-butyl eicosanoate [fatty acid side chain -HMPB-(AEEA)6-NH2]

[0212]

[0213] Add DIC (0.47 mL, 3 mmol) to a solution of Fmoc-AEEA-OH (1156 mg, 3 mmol) and DMAP (18.4 mg, 0.149 mmol) in DMSO (4 mL). Let the resulting solution stand for 5 minutes to activate it, then add HMPB-(AEEA)6-NH2 (1 mmol). Place the reaction mixture on a shaker and mix for 2 hours. Add MTBE (40 mL total volume) to induce oil formation, and centrifuge the mixture (3000 rpm × 2 min). Discard the MTBE layer and add fresh MTBE (40 mL total volume). Centrifuge the mixture again and discard the MTBE. Add 30% piperidine / DMF (3 mL) to the resulting oil, mix for 15 min, then add MTBE (40 mL total volume) and centrifuge the mixture (3000 rpm × 2 min). Decant the MTBE layer, add DMSO (2 mL) to the oily substance to dissolve it, and add fresh MTBE (40 mL total volume). Centrifuge the mixture again and decant the MTBE layer. Dissolve a solution of Fmoc-AEEA-OH (1156 mg, 3 mmol) and PyOxim (1598.1 mg, 3 mmol) in DMSO (4 mL). Add DIEA (1 mL, 6 mmol) and let the resulting solution stand for 5 min for pre-activation. Add the mixture to AEEA-HMPB-(AEEA)6-NH2 (1 mmol) in a centrifuge tube, mix for 30 min, remove Fmoc with 30% piperidine / DMF (3 mL) as described above, and wash with MTBE. O5-(2,5-dioxopyrrolidone-1-yl)(2S)-2-[(20-tert-butoxy-20-oxo-eicosanoyl)amino]glutaric acid O1-tert-butyl ester (2040 mg, 3 mmol) was dissolved in a mixture of DMSO and toluene (9:1 ratio, 2.7 mL DMSO + 0.3 mL toluene). DIEA (1 mL, 6 mmol) was added, and the resulting solution was allowed to stand for 5 min for pre-activation. This mixture was then added to a centrifuge tube containing AEEA2-HMPB-(AEEA)6-NH2 (1 mmol) for 30 min, followed by repeated MTBE washing to induce oil formation. MTBE was discarded, yielding the title compound as an oily precipitate. ESMS m / z 1966.3 (M+H+).

[0214] Example 24

[0215] 20-[[(1S)-4-[2-[2-[2-[2-[2-[2-[4-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-amino-2-oxo-ethoxy)ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethoxy] [HMPA-(AEEA)]-2-oxo-ethoxy]-2-oxo-ethoxy]-2-oxo-ethoxy]-2-oxo-ethoxy]-2-oxo-ethoxy]-2-oxo-ethoxy]-2-oxo-ethoxy]-2-oxo-ethoxy]-2-oxo-ethoxy]-2-oxo-ethoxy]-2-oxo-ethoxy]-2-oxo-ethoxy]-1-tert-butoxycarbonyl-4-oxo-butyl]amino]-20-oxo-eicosane-tert-butyrate [fatty acid side chain -HMPA-(AEEA)] 10 Preparation of [-NH2]

[0216]

[0217] Add DIC (0.235 mL, 1.50 mmol) to a solution of Fmoc-(AEEA)-OH (578 mg, 1.50 mmol) and DMAP (9.2 mg, 0.075 mmol) in DMSO (4 mL). Let the solution stand for 5 min, then add HMPA-(AEEA). 10Mix in 0.500 mmol of -NH2 for 2 h. Add 14 mL of MTBE and centrifuge the mixture at 3000 rpm for 2 min. Discard the supernatant, add 14 mL of fresh MTBE, and centrifuge the mixture again at 3000 rpm for 2 min. Discard the supernatant and perform a second coupling cycle with Fmoc-(AEEA)-OH as described above. After 2 h, add 14 mL of MTBE and centrifuge the mixture at 3000 rpm for 2 min. Add 14 mL of fresh MTBE and centrifuge again at 3000 rpm for 2 min. Discard the supernatant to obtain an oily precipitate. Add 30% piperidine / DMF (3 mL) to the oily precipitate, mix for 15 min, add 14 mL of MTBE, centrifuge the mixture at 3000 rpm for 2 min, discard the supernatant, dissolve the oily precipitate in 1 mL of DMSO, and add 14 mL of fresh MTBE. The mixture was centrifuged again at 3000 rpm for 2 min, and then the supernatant was decanted to obtain an oily precipitate.

[0218] Add DIEA (0.523 mL, 3.0 mmol) to a solution of Fmoc-AEEA-OH (586 mg, 1.5 mmol) and PyOxim (791 mg, 1.5 mmol) in DMSO (4 mL). Let the solution stand for 5 min, then add it to the oily sediment as described above and mix for 30 min. Add MTBE (14 mL) and centrifuge the mixture at 3000 rpm for 2 min. Discard the supernatant, add fresh MTBE (14 mL), and centrifuge the mixture again at 3000 rpm for 2 min. Discard the supernatant to obtain the oily sediment. Add 30% piperidine / DMF (3 mL) to the oily sediment and mix for 15 min. Add MTBE (14 mL), centrifuge the mixture at 3000 rpm for 2 min, and then discard the supernatant. Add fresh MTBE (14 mL), and centrifuge the mixture again at 3000 rpm for 2 min. Discard the supernatant to obtain (AEEA)2-HMPA-(AEEA). 10 -NH2 is an oily sediment.

[0219] Fmoc-Glu-O / Bu is coupled to (AEEA)2-HMPA-(AEEA) in a manner similar to the second coupling of Fmoc-AEEA-OH. 10 The γ-NH2 group was then subjected to MTBE, centrifuged, and then deprotected with piperidine in 30% DMF. MTBE was then added, and the mixture was centrifuged again to obtain Fmoc-γGlu-(AEEA)2-HMPA-(AEEA). 10 -NH2 is an oily sediment.

[0220] Add DIEA (0.523 mL, 3.0 mmol, 6.0 equivalence) to a solution of 20-(tert-butoxy)-20-oxoeicosanoic acid (600 mg, 1.5 mmol, 3.0 equivalence) and PyBOP (781 mg, 1.5 mmol, 3.0 equivalence) in DMSO (4 mL). Let the solution stand for 5 min, during which time the activated ester precipitates out, then add toluene (8 mL), and let stand for another 5 min. Add this solution to Fmoc-γGlu-(AEEA)2-HMPA-(AEEA). 10 In -NH2, mix for 1 hour (complete dissolution of the reaction mixture was not achieved). Add MTBE (14 mL) and centrifuge the mixture at 3000 rpm for 2 min. Decant the supernatant, add fresh MTBE (14 mL), and centrifuge the mixture again at 3000 rpm for 2 min. Decant the supernatant to obtain the title compound as an oil. ESMS m / z 2488.2 (M+H) + ); 1245.5(M+2H + / 2).

[0221] Example 25 - Using HMPB-(AEEA) 10 Liquid-phase peptide synthesis of -NH2 and "soft" cleavage from the linker

[0222]

[0223] (SEQ ID NO: 9)

[0224] HMPB-(AEEA) 10 Elongation of the amino acid chain on -NH2: A mixture of Fmoc-Ala-OH (1.9 g, 6 mmol) and DMAP (36.8 mg, 0.301 mmol) was dissolved in 4 mL of DMSO. DIC (0.93 mL, 6 mmol) was added to the mixture, and it was allowed to stand for 5 min for pre-activation. This mixture was then added to HMPB-(AEEA)2-NH2, and the mixture was shaken at room temperature for 2 h. MTBE was added to bring the volume of the mixture to 40 mL, inducing a phase transition with the coupling product Fmoc-Ala-HMPB-(AEEA)2-NH2, precipitating an oily substance. The mixture was centrifuged (3000 rpm × 2 min), and the MTBE supernatant was decanted. MTBE was added again to the oily precipitate to bring the volume to 40 mL, the mixture was centrifuged, and the MTBE supernatant was decanted. The coupling operation was repeated twice using Fmoc-Ala-OH to achieve complete coupling.

[0225] A 30% piperidine solution in DMF (3 mL) was added to the resulting oily precipitate, and the mixture was shaken for 15 min. Then, MTBE was added to bring the volume to 40 mL, causing the oily precipitate to form. The mixture was centrifuged (3000 rpm × 2 min), and the supernatant was decanted. DMSO (1 mL) was added to the oily precipitate. MTBE was added to bring the volume to 40 mL, and the mixture was centrifuged again. The supernatant was decanted to obtain the oily precipitate.

[0226] The amino acid coupling and deprotection steps were basically repeated as described in Example 16. First, (2S)-6-[[2-[2-[2-[2-[2-[2-[2-[(4S)-5-tert-butoxy-4-[(20-tert-butoxy-20-oxo-eicosanoyl)amino]-5-oxo-pentanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]-2-(9H-fluorene-9-ylmethoxycarbonylamino)hexanoic acid was coupled using DEPBT instead of PyOxim in the order from C-terminus to N-terminus as obtained in SEQ ID NO: 9. Then, the remaining Fmoc-protected amino acids (the aspartic side chain -CO2H group protected by tBu, the glutamine side chain -NH2 protected by triphenylmethyl and the lysine -NH2 protected by -Boc) were coupled using DEPBT instead of PyOxim in the order from C-terminus to N-terminus as obtained in SEQ ID NO: 9. Each coupling operation was mixed for 60 min to obtain the peptide of SEQ ID NO: 9. ESMS m / z 1961.08(M+2H + / 2).

[0227] Soft cleavage to obtain the peptide of SEQ ID NO: 10:

[0228] The peptide of SEQ ID NO: 9 (0.38 mmol, 1.5 g) was incubated in a 2% TFA solution in DCM (15 mL) for 30 min at room temperature. Pyridine (1 equivalent) was then added to neutralize the reaction mixture. The reaction mixture was washed with water, and the organic layer was concentrated under vacuum. The residue was washed with EtOAc and then dissolved in DCM to give the peptide of SEQ ID NO: 10. ESMS m / z 1125 (M+2H+ / 2).

[0229] Using UPLC-MS [column: Waters CSH] TM C 18 150×2.1mm, 1.7mm; column temperature: 50℃; gradient -30-90% solvent B: solvent A, run time 21 min; flow rate: 0.5 mL / min; solvent A: 0.2% TFA aqueous solution; solvent B: acetonitrile; detector: photodiode array UV, ESMS] analyzed the crude product. The crude peptide showed a purity of 71.31%.

[0230]

[0231] (SEQ ID NO: 10)

[0232] Example 26 - Using HMPB-(AEEA) 10 Liquid-phase peptide synthesis of -NH2

[0233] The peptide of SEQ ID NO: 11 was prepared using liquid-phase peptide synthesis as follows.

[0234]

[0235] (SEQ ID NO: 11)

[0236] A mixture of Fmoc-Leu-OH (4.2 g, 12 mmol) and DMAP (73.6 mg, 0.602 mmol) was dissolved in DMSO (5 mL). DIC (1.58 g, 1.96 mL, 12 mmol) was added, and the mixture was allowed to stand for 5 min for pre-activation, if required. This mixture was then added to HMPB-(AEEA). 10 In -NH2 (4 mmol), shake at room temperature for 2 h. Add MTBE to make the mixture volume 40 mL, inducing a phase transition with the coupling product Fmoc-Leu-HMPB-(AEEA)2-NH2, precipitating an oily substance. Centrifuge the mixture (3000 rpm × 3 min), and decant the MTBE supernatant. Add MTBE again to the oily precipitate to make the volume 40 mL, centrifuge the mixture, and decant the MTBE supernatant. Repeat the coupling operation twice with Fmoc-Leu-OH to achieve complete coupling.

[0237] Add 3 mL of a 30% piperidine solution in DMF to the resulting oily precipitate and shake the mixture for 15 min. Then add MTBE to bring the volume to 40 mL, causing the oily precipitate to form. Centrifuge the mixture (3000 rpm × 3 min), decant the supernatant, and add 1 mL of DMSO to the oily precipitate. Add MTBE to bring the volume to 40 mL, centrifuge the mixture again, decant the supernatant, and obtain the oily precipitate.

[0238] The subsequent amino acid coupling and deprotection steps were basically repeated as described in Example 16, so that the Fmoc-protected amino acids (the -OH and -CO2H side chains protected with tBu) were coupled in the order from the C-terminus to the N-terminus as given in SEQ ID NO: 11, to obtain the peptide of SEQ ID NO: 11. ESMS m / z 1893 (M+2H+ / 2), 1262 (M+3H+ / 3).

[0239] Example 27 - Liquid-phase synthesis of tetramerized peptides and "soft" cleavage from HMPB-based linkers

[0240]

[0241] (SEQ ID NO: 12)

[0242] HMPB-(AEEA) 10 Preparation of tetramerized peptide on -NH2: The peptide of SEQ ID NO: 12 was prepared basically as described in Example 16, but with the following changes: the first amino acid (Fmoc-Gly-OH) was reacted with HMPB-(AEEA) as follows. 10 -NH2 coupling: Fmoc-Gly-OH, DIC and DMAP (3:3:0.15 molar ratio) were dissolved in DMSO, mixed for 1 min, and then added to HMPB-(AEEA). 10 -NH2. After 2 hours, MTBE (5 mL) was added to initiate phase separation. The mixture was centrifuged at 3250 rpm, and the supernatant was decanted. A second coupling cycle was performed using Fmoc-Gly-OH as described above. After 2 hours, MTBE (5 mL) was added to initiate phase separation. The mixture was centrifuged at 3250 rpm, and the supernatant was decanted. The Fmoc group was removed using 30% piperidine / DMF, and then subsequent coupling and deprotection were performed substantially as described in Example 16 to obtain the peptide of SEQ ID NO: 12. ESMS m / z 2360.1 (M+Na) + ), 2338.1(M+H + ), 1169.6 (M+2H) + / 2).

[0243] Tetrimester peptide from HMPB-(AEEA) 10 Soft pyrolysis at the -NH2 connector

[0244] Two methods were used for "soft" cleavage of the peptide SEQ ID NO: 12 to obtain the protected tetramerized peptide SEQ ID NO: 13:

[0245] Boc-Y(tBu)-Aib-E(tBu)-G-OH (SEQ ID NO: 13).

[0246] Method 1: Add 5% TFA / DCM solution (10 volumes) to the solution of the peptide in SEQ ID NO: 12. After 30 min, neutralize the solution with pyridine and wash twice with 10% NaCl solution. Dry the organic layer with Na2SO4 and concentrate under reduced pressure. Dissolve the residue in a small amount of DMF and dilute with water (3 volumes). Extract the mixture three times with MTBE, and concentrate the combined organic layers under reduced pressure to obtain the peptide in SEQ ID NO: 13. ESMS m / z 687.4 (M+Na+), 665.4 (M+H+).

[0247] Method 2: Add 2% TFA / toluene solution (10 volumes) to the peptide of SEQ ID NO: 12. Mix the mixture for 10 minutes, then centrifuge at 3000 rpm for 5 minutes. Collect the supernatant and neutralize with pyridine (equimolar with TFA). Add MTBE (3 mL) to the remaining oily sediment and centrifuge the mixture at 3000 rpm for 5 minutes. Collect the supernatant, add fresh MTBE (3 mL) to the oily sediment, and centrifuge the mixture again at 3000 rpm for 5 minutes. Collect the supernatant again to obtain the oily sediment. Repeat the lysis and washing process on the oily sediment at least twice. Wash with saturated NaCl aqueous solution and water, then concentrate the combined organic layers under reduced pressure to obtain the peptide of SEQ ID NO: 13.

[0248]

[0249] (SEQ ID NO: 14)

[0250] Preparation of tetramerized peptide on HMPB-(AEEA)4-NH2: The peptide of SEQ ID NO: 14 was prepared essentially as described above using HMPB-(AEEA)4-NH2. ESMS m / z 1488.7 (M+Na) + ).

[0251] Soft cleavage of the tetramerized peptide at the HMPB-(AEEA)4-NH2 linker: 3% TFA / DCM solution (20 volumes) was added to a solution of the peptide in SEQ ID NO: 14. After 30 min, the solution was neutralized with pyridine and washed twice with water. The organic layer was dried over MgSO4 and concentrated under reduced pressure to obtain the peptide in SEQ ID NO: 13. ESMS m / z 687.4 (M+Na+), 665.4 (M+H+).

[0252]

[0253] (SEQ ID NO: 15)

[0254] Preparation of tetramerized peptide on HMPB-(AEEA)2-NH2: The peptide of SEQ ID NO: 15 was prepared essentially as described above using HMPB-(AEEA)2-NH2. ESMS m / z 1198.5 (M+Na) + ).

[0255]

[0256] (SEQ ID NO: 16)

[0257] Preparation of tetramerized peptide on HMPB-(AEEA)6-NH2: The peptide of SEQ ID NO: 16 was prepared essentially as described above using HMPB-(AEEA)6-NH2. ESMS m / z 1778.8 (M+Na) + ).

[0258] Example 28 - Liquid-phase synthesis of tetramerized peptides and "soft" cleavage from HMPA-based linkers

[0259]

[0260] (SEQ ID NO: 17)

[0261] HMPA-(AEEA) 10 Preparation of tetramerized peptides on -NH2: DIC (60 mg, 0.470891 mmol) and 2,4,6-trimethylpyridine (0.125 mL, 0.945 mmol) were added to a solution of Fmoc-Gly-OH (140 mg, 0.470891 mmol) in DMF (1 mL). HMPA-(AEEA) was then added to this solution. 10 In -NH2 (256 mg, 0.15679 mmol), shaken at room temperature for 2 h. Add MTBE (20 mL) and centrifuge the mixture (5000 rpm × 3 min). Decant the supernatant, washing three times with the extracted MTBE, separating the bottom oily layer from the supernatant by each decanting. Repeat the above coupling and washing process three times to drive the reaction to completion. Mix the deposited oily layer with 25% piperidine in DMF (2 mL) for 20 min. Wash with MTBE (20 mL) and centrifuge three times in a similar manner as described above to obtain the bottom oily layer.

[0262] DIEA (74 μL, 0.424 mmol) was added to a solution of Fmoc-Glu(OtBu)-OH (90.74 mg, 0.2111 mmol) and PyOxim (112.5 mg, 0.2111 mmol) in DMSO (750 μL) and acetonitrile (750 μL), and the solution was mixed for 2 min. This solution was added to the above oily sediment (0.180 g, 0.107 mmol), and the mixture was mixed at room temperature for 30 min, followed by the addition of MTBE (20 mL). The mixture was centrifuged (3000 rpm × 3 min), and the supernatant was decanted. This extraction was washed twice, and the supernatant was decanted, thus separating the bottom oily layer each time. 10% piperidine / DMF (2 mL) was added to the oily sediment, and the mixture was mixed for 15 min. The mixture was washed with MTBE (20 mL), and centrifuged twice in a similar manner as described above to obtain the bottom oily layer.

[0263] DIPEA (55.05 μL, 0.316 mmol) was added to a solution of Fmoc-Aib-OH (68.4 mg, 0.210 mmol) and PyOxim (110.8 mg, 0.2080 mmol) in DMSO (750 μL). The solution was mixed for 2 min and then added to the upper oily layer (0.197 g, 0.105 mmol). Mixing was allowed at room temperature for 30 min, followed by the addition of MTBE (20 mL), resulting in an oily precipitation. The mixture was centrifuged (3000 rpm × 3 min), and the supernatant was decanted. This extraction and washing was performed twice, separating the bottom oily layer from the supernatant by decanting each time. 10% piperidine / DMF (2 mL) was added to the oily precipitate, and the mixture was mixed for 15 min. Similarly, the mixture was washed with MTBE (20 mL) and centrifuged twice as described above to obtain the bottom oily layer.

[0264] DIEA (91 μL, 0.316 mmol) was added to a solution of Boc-Tyr(tBu)-OH (106.4 mg, 0.3153 mmol) and PyOxim (166.3 mg, 0.3153 mmol) in DMSO (750 μL) and acetonitrile (750 μL). The solution was mixed for 2 min and then added to the upper oily layer (201.0 mg, 0.1026 mmol). The mixture was allowed to mix at room temperature for 30 min, followed by the addition of MTBE (20 mL), resulting in an oily precipitation. The mixture was centrifuged (3000 rpm × 3 min). The extraction and washing were performed twice, separating the bottom oily layer from the supernatant by decanting each time. This coupling and washing process was repeated four times to drive the reaction to completion, yielding the peptide of SEQ ID NO: 17, as an oily substance. ESMS m / z 2279.10(M+H+); 1140.20(M+2H+ / 2).

[0265] Tetrimester peptides from HMPA-(AEEA) 10 Soft cleavage at the -NH2 linker: 1% TFA (in DCM (1.22 mL)) was added to the peptide of SEQ ID NO: 17 (102.3 mg, 0.04488 mmol), and the mixture was stirred at room temperature for 1 h. Pyridine (10.6 mg, 0.134 mmol) was added to the resulting solution. The mixture was washed with water (4.5 mL), and the organic layer was collected, concentrated, and the peptide of SEQ ID NO: 13 was obtained as an oil; LCMS showed a conversion of 33% from the starting material to the product. ESMS m / z 665.40 (M+H+); 687.30 (M+Na+).

[0266] HMPA-(AEEA) 10 Preparation of tetramerized peptides on -NH2:

[0267]

[0268] (SEQ ID NO: 18)

[0269] EDC (249.7 mg, 1.609 mmol) and ethyl cyanoglyoxylate-2-oxime (272.4 mg, 1.898 mmol) were added to a solution of Fmoc-Gly-OH (522.5 mg, 1.757 mmol) in DMSO (2.5 mL). This solution was then added to HMPA-(AEEA)2 (0.4143 g, 0.8787 mmol), and the mixture was stirred at room temperature for 2 h. Subsequently, MTBE (30 mL) was added to precipitate an oily substance. The mixture was centrifuged (3000 rpm × 3 min), and the supernatant was decanted. This extraction and washing was performed three times, separating the bottom oily layer from the supernatant by decanting each time. The above coupling and washing process was repeated three times to drive the reaction to completion. The deposited oily layer was mixed with 10% piperidine in DMF (2 mL) for 20 min, washed with MTBE (20 mL), and centrifuged in a similar manner as described above. The subsequent Fmoc removal step is performed again to obtain the bottom oily layer.

[0270] DIEA (0.516 g, 4.01 mmol) was added to a solution of Fmoc-Glu(OtBu)-OH (0.842 g, 1.980 mmol) and PyOxim (1.055 g, 1.980 mmol) in DMSO (3 mL) and acetonitrile (1 mL), and the solution was mixed for 1 min. This solution was added to the above oily precipitate (0.504 g, 0.954 mmol), and the mixture was mixed at room temperature for 45 min, followed by the addition of MTBE (40 mL) to precipitate the oil. The mixture was centrifuged (3000 rpm × 3 min), and the supernatant was decanted. This extraction and washing was performed twice, with the supernatant decanted each time to separate the oily layer from the bottom layer. The coupling and washing steps were repeated again. 10% piperidine / DMF (2 mL) was added to the oily precipitate, and the mixture was mixed for 15 min. The mixture was washed in a similar manner as described above to obtain the bottom oily layer.

[0271] DIEA (782 μL, 4.48 mmol) was added to a solution of Fmoc-Aib-OH (970 mg, 2.98 mmol) and PyOxim (1.57 g, 2.92 mmol) in DMSO (3 mL) and acetonitrile (1 mL), and the solution was mixed for 1 min. This solution was then added to the above oily deposit (1.001 g, 1.402 mmol), and the mixture was mixed at room temperature for 45 min. The mixture was then washed with MTBE and centrifuged as described above. 10% piperidine / DMF (2 mL) was added to the oily deposit, and the mixture was mixed for 15 min. The mixture was washed with MTBE and centrifuged as described above. The subsequent Fmoc removal steps were repeated again to obtain the bottom oily layer.

[0272] DIEA (1.13 mL, 6.48 mmol) was added to a solution of Boc-Tyr(tBu)-OH (1.63 g, 4.83 mmol) and PyOxim (2.55 g, 4.74 mmol) in DMSO (3 mL) and acetonitrile (1 mL), and the solution was mixed for 1 min. This solution was then added to the above oily precipitate (1.289 g, 1.613 mmol), mixed at room temperature for 60 min, washed with MTBE, and centrifuged as described above. The coupling and MTBE washing steps were repeated to obtain the peptide of SEQ ID NO: 18, as an oily substance. ESMS m / z 1118.50 (M+H+); 1140.50 (M+Na+).

[0273] Soft cleavage of the tetrameric peptide at the HMPA-(AEEA)2-NH2 linker: A mixture of 1,1,1,3,3,3-hexafluoro-2-propanol (200 μL, 1.91 mmol) in DCM (0.8 mL) was added to the peptide of SEQ ID NO: 18 (50 mg, 0.045 mmol), and the resulting mixture was stirred at room temperature for 20 min. ACN (2 mL) was added, and the reaction mixture was concentrated under vacuum. The CAN addition and concentration steps were repeated twice. The above conditions were then subjected twice more (stirring in 1,1,1,3,3,3-hexafluoro-2-propanol and DCM for 20 min, followed by CAN addition and concentration) to give the peptide of SEQ ID NO: 13 as an oil; LCMS showed a conversion of 8% from starting material to product. ESMS m / z 664.40 (M+).

[0274] Example 29 - Preparation of peptide of SEQ ID NO: 22 by liquid phase based fragment using Rink linker-(AEEA)6-NH2

[0275] This article describes the synthesis of the peptide SEQ ID NO: 22 via a liquid-phase fragment-based method.

[0276] Preparation of the peptide of SEQ ID NO: 19 on Rink-(AEEA)6-NH2: Using essentially the method described in Example 16, liquid-phase peptide synthesis was initiated by coupling Fmoc-Ser(tBu)-OH onto 0.05 mmol Rink-(AEEA)6-NH2, followed by Fmoc deprotection to prepare the peptide of SEQ ID NO: 19. The remaining Fmoc-protected amino acids (side chain -OH groups protected with tBu) were coupled / deprotected in the order from C-terminus to N-terminus given in SEQ ID NO: 19.

[0277]

[0278] (SEQ ID NO: 19)

[0279] ESMS m / z 1095.7 (M+2H) + / 2), 731.0(M+3H + / 3).

[0280] Coupling of the peptide of SEQ ID NO: 7 to the peptide of SEQ ID NO: 19: Using the coupling method substantially as described in Example 16, DMSO was used as the reaction solvent to couple the peptide of SEQ ID NO: 7 to the peptide of SEQ ID NO: 19, yielding the peptide of SEQ ID NO: 20. The reaction system was sampled after 1 minute, and LCMS analysis showed that the reaction was complete.

[0281]

[0282] (SEQ ID NO: 20)

[0283] ESMS m / z 1835.90(M+2H + / 2), 1224.20(M+3H + / 3). Using the deprotection method substantially as described in Example 16, DMSO was used as the reaction solvent to remove the N-terminal Fmoc group from the peptide of SEQ ID NO: 20, yielding the peptide of SEQ ID NO: 21:

[0284]

[0285] (SEQ ID NO: 21)

[0286] ESMS m / z 1724.9 (M+2H) + / 2), 1150.20(M+3H + / 3).

[0287] Coupling of the peptide of SEQ ID NO: 10 to the peptide of SEQ ID NO: 21: Using the coupling method substantially as described in Example 16, the peptide of SEQ ID NO: 10 (0.06 mmol) was coupled to the peptide of SEQ ID NO: 21 using DMSO as the reaction solvent, and then the Fmoc group was removed using piperidine in 30% DMSO using the deprotection method substantially as described in Example 16, to obtain the peptide of SEQ ID NO: 22.

[0288]

[0289] (SEQ ID NO: 22)

[0290] ESMS m / z = 1820.1(M+3H) + / 3), 1365.3(M+4H + / 4).

[0291] Example 30

[0292] Use Rink-(AEEA) 10 -NH2, Preparation of the peptide of SEQ ID NO: 23 using a fragment-based method

[0293]

[0294] (SEQ ID NO: 23)

[0295] Add PyBOP (0.1781 g, 0.3422 mmol) and DIEA (54.186 μL, 0.311 mmol) to a solution of Fmoc-PPPS(tBu)-OH (SEQ ID NO: 24, 0.2291 g, 0.3395 mmol) in DMSO (2 mL). Mix for 1 min, then add to Rink-(AEEA). 10 Mix in -NH2 (0.500 g, 0.338 mmol) for 60 min, then add MTBE (12 mL) to obtain an oily precipitate. Centrifuge the mixture at room temperature (3000 rpm × 3 min) and decant the supernatant. Repeat the washing process again. Wash the oily precipitate with isopropyl acetate (2 times, 10 mL each time) in a similar manner. Repeat the coupling process more than twice. Mix the oily layer with piperidine (2 mL) in 20% DMF for 20 min, and wash with MTBE (12 mL) and isopropyl acetate in a similar manner to obtain an oily precipitate.

[0296] Add PyBOP (0.180 g, 0.346 mmol) and DIEA (60.34 μL, 0.346 mmol) to a solution of Fmoc-S(tBu)-S(tBu)-GA-OH (SEQ ID NO: 25, 0.2262 g, 0.3454 mmol) in DMSO (2 mL). Mix the solution for 1 min, then add it to the oily precipitate (0.5073 g, 0.2303 mmol) from the previous step and mix for 60 min. Add MTBE (12 mL), precipitate the oily precipitate, centrifuge the mixture (3000 rpm × 3 min), and decant the supernatant. Wash the oily precipitate twice with MTBE and isopropyl acetate as described above. Repeat the coupling reaction at least twice. The oily layer was mixed with 20% piperidine (2 mL) in DMF for 20 min, precipitated with MTBE (12 mL) and isopropyl acetate in a similar manner, washed / centrifuged to obtain an oily deposit.

[0297] Basically, following a similar approach to the coupling and deprotection described above, the following substances were sequentially coupled to the oily sediments separated in the above steps:

[0298] 1.Fmoc-AGGP-OH (SEQ ID NO: 26),

[0299] 2.Fmoc-Q(Trt)-W(Boc)-LI-OH (SEQ ID NO: 27),

[0300] 3.

[0301] (SEQ ID NO: 28)

[0302] 4.Fmoc-KIAQ(Trt)-OH(SEQ ID NO: 29)

[0303] 5.Fmoc-I-Aib-LD(tBu)-OH (SEQ ID NO: 30)

[0304] 6.Fmoc-S(tBu)-D(tBu)-Y(tBu)-S(tBu)-OH (SEQ ID NO: 31)

[0305] After coupling with the peptide of SEQ ID NO: 31, a piperidine / DMF deprotection step was performed, followed by washing / centrifugation with MTBE / isopropyl acetate to obtain the peptide of SEQ ID NO: 23, which was an oily substance. ESMS m / z 1756.50 (M+4H+ / 4).

[0306] sequence list

[0307] SEQ ID NO:1

[0308]

[0309] SEQ ID NO:2

[0310] AFIEYLLEGGPSSGAPPPS-NH2

[0311] SEQ ID NO:3

[0312]

[0313] SEQ ID NO:4

[0314] G-P-S(tBu)-S(tBu)-G-A-P-P-P-S(tBu)-NH2

[0315] SEQ ID NO:5

[0316]

[0317] SEQ ID NO:6

[0318]

[0319] SEQ ID NO:7

[0320] Fmoc-F-V-Q(Trt)-W(Boc)-L-I-A-G-OH

[0321] SEQ ID NO:8

[0322]

[0323] SEQ ID NO:9

[0324]

[0325] SEQ ID NO:10

[0326]

[0327] SEQ ID NO:11

[0328]

[0329] SEQ ID NO:12

[0330]

[0331] SEQ ID NO:13

[0332] Boc-Y(tBu)-Aib-E(tBu)-G-OH

[0333] SEQ ID NO:14

[0334]

[0335] SEQ ID NO:15

[0336]

[0337] SEQ ID NO:16

[0338]

[0339] SEQ ID NO:17

[0340]

[0341] SEQ ID NO:18

[0342]

[0343] SEQ ID NO:19

[0344]

[0345] SEQ ID NO:20

[0346]

[0347] SEQ ID NO:21

[0348]

[0349] SEQ ID NO:22

[0350]

[0351] SEQ ID NO:23

[0352]

[0353] SEQ ID NO:24

[0354] Fmoc-P-P-P-S(tBu)-OH

[0355] SEQ ID NO:25

[0356] Fmoc-S(tBu)-S(tBu)-G-A-OH

[0357] SEQ ID NO:26

[0358] Fmoc-A-G-G-P-OH

[0359] SEQ ID NO:27

[0360] Fmoc-Q(Trt)-W(Boc)-L-I-OH

[0361] SEQ ID NO:28

[0362]

[0363] SEQ ID NO:29

[0364] Fmoc-K-I-A-Q(Trt)-OH

[0365] SEQ ID NO:30

[0366] Fmoc-I-Aib-L-D(tBu)-OH

[0367] SEQ ID NO:31

[0368] Fmoc-S(tBu)-D(tBu)-Y(tBu)-S(tBu)-OH

[0369] SEQ ID NO:32

[0370]

Claims

1. The following compound: Where "m" is 0, 1, 2, or 3, "n" is 1-10, and "Z" is the linker compound selected from: , , , , and .

2. The compound of claim 1, wherein the compound is 。 3. The compound of claim 1, wherein the compound is 。 4. The compound of claim 1, wherein the compound is 。 5. The compound of claim 1, wherein the compound is 。 6. The compound of claim 1, wherein the compound is 。 7. The compound of any one of claims 1-6, wherein "m" is 1 and "n" is 2, 3, 4, 5, 6, 7, 8, 9 or 10.

8. The compound of any one of claims 1-6, wherein the compound is used for liquid-phase synthesis.

9. The compound of claim 8, wherein the liquid-phase synthesis is liquid-phase peptide synthesis (LPPS).

10. The compound of any one of claims 1-6, wherein the compound is hydrophilic.

11. Peptide of SEQ ID NO:

1.

12. A method for preparing the peptide of SEQ ID NO: 2, comprising preparing the peptide of SEQ ID NO: 1 using liquid-phase peptide synthesis, and then treating the prepared peptide of SEQ ID NO: 1 with acid to obtain the peptide of SEQ ID NO:

2.

13. Peptide of SEQ ID NO:

3.

14. A method for preparing the peptide of SEQ ID NO: 4, comprising preparing the peptide of SEQ ID NO: 3 using liquid-phase peptide synthesis, and then treating the prepared peptide of SEQ ID NO: 3 with acid to obtain the peptide of SEQ ID NO:

4.

15. Peptide of SEQ ID NO:

5.

16. Peptide of SEQ ID NO:

6.

17. A method for preparing the peptide of SEQ ID NO: 7, comprising preparing the peptide of SEQ ID NO: 6 using liquid-phase peptide synthesis, and then treating the prepared peptide of SEQ ID NO: 6 with acid to obtain the peptide of SEQ ID NO:

7.

18. Peptide of SEQ ID NO:

8.

19. Peptide of SEQ ID NO:

9.

20. A method for preparing the peptide of SEQ ID NO: 10, comprising preparing the peptide of SEQ ID NO: 9 using liquid-phase peptide synthesis, and then treating the prepared peptide of SEQ ID NO: 9 with acid to obtain the peptide of SEQ ID NO:

10.

21. Peptide of SEQ ID NO:

11.

22. Peptide of SEQ ID NO:

12.

23. Peptide of SEQ ID NO:

14.

24. Peptide of SEQ ID NO:

15.

25. Peptide of SEQ ID NO:

16.

26. Peptide of SEQ ID NO:

17.

27. Peptide of SEQ ID NO:

18.

28. A method for preparing the peptide of SEQ ID NO: 13, comprising preparing the peptide of SEQ ID NO: 11, 12, 14, 15, 16, 17 or 18 using liquid-phase synthesis, and then treating the prepared peptide of SEQ ID NO: 11, 12, 14, 15, 16, 17 or 18 with acid to obtain the peptide of SEQ ID NO:

13.

29. Peptide of SEQ ID NO:

19.

30. Peptide of SEQ ID NO:

20.

31. Peptide of SEQ ID NO:

21.

32. Peptide of SEQ ID NO:

22.

33. Peptide of SEQ ID NO:

23.

34. Methods for preparing compounds of the following formula: The preparation of the compound includes using liquid-phase synthesis, wherein the liquid-phase synthesis comprises: a. Couple Fmoc-AEEA-OH to the linker compound of any one of claims 1-14, and deprotect the resulting product using a base; b. Couple the second Fmoc-AEEA-OH to the product from step a. and deprotect the resulting product using a base; c. Make Fmoc-Glu-O t Bu is coupled to the product of step b, and the resulting product is deprotected using a base; d. Make 20-( tert-butoxy )-20-oxoeicosanoic acid is coupled to the product of step c; and e. Remove the binder compound from the product from step d. using acid.

35. Methods for preparing compounds of the following formula: The preparation of the compound includes using liquid-phase synthesis, wherein the liquid-phase synthesis comprises: a. Couple Fmoc-(AEEA)2-OH to the linker compound of any one of claims 1-10, and deprotect the resulting product using a base; b. Make Fmoc-Glu-O t Bu is coupled to the product of step a. and the resulting product is deprotected using a base; c. Make 20-( tert-butoxy )-20-oxoeicosanoic acid is coupled to the product obtained in step b; and d. Remove the connector compound from the product from step c. using acid.

36. Methods for preparing compounds of the following formula: The preparation of the compound includes using liquid-phase synthesis, wherein the liquid-phase synthesis comprises: a. Couple Fmoc-(AEEA)2-OH to the linker compound of any one of claims 1-10, and deprotect the resulting product using a base; b. To make O5-(2,5-dioxopyrrolidone-1-yl)(2S)-2-[(20- tert-butoxy -20-oxo-eicosanoyl)amino]glutaric acid O1-tert-butyl ester is coupled to the product obtained in step a; and c. Remove the connector compound from the product from step b. using acid.

37. A compound, which is 。 38. A compound, which is 。 39. A compound, which is 。 40. A method for preparing the peptide of SEQ ID NO: 32, comprising preparing part or all of the peptide sequence by liquid-phase peptide synthesis using a compound of any one of claims 1-10.

41. A method for preparing the peptide of SEQ ID NO: 20, comprising coupling the peptide of SEQ ID NO: 19 with the peptide of SEQ ID NO:

7.

42. A method for preparing the peptide of SEQ ID NO: 22, comprising coupling the peptide of SEQ ID NO: 21 with the peptide of SEQ ID NO:

10.

43. A method for preparing the peptide of SEQ ID NO: 32, comprising the following steps: a. The preparation of the peptide of SEQ ID NO: 19 using the compound of any one of claims 1-10 via liquid-phase peptide synthesis; b. Couple the peptide of SEQ ID NO: 19 to the peptide of SEQ ID NO: 7 to generate the peptide of SEQ ID NO: 20; c. Deprotect the peptide of SEQ ID NO: 20 to generate the peptide of SEQ ID NO: 21; d. To couple the peptide of SEQ ID NO: 10 to the peptide of SEQ ID NO: 21; and e. Deprotect the peptide from step d. to generate the peptide of SEQ ID NO:

22.

44. The method of claim 43, further comprising: f. Elongating the peptide from step e. of SEQ ID NO: 22 by coupling a single amino acid, a peptide fragment, or a mixture thereof; g. Peptides obtained by acid treatment; and h. Deprotect the obtained peptide.

45. A method for preparing the peptide of SEQ ID NO: 32, comprising the following steps: a. To couple the peptide of SEQ ID NO: 24 with the compound of any one of claims 1-10, and to deprotect the resulting peptide; b. Couple the peptide from step a. to the peptide of SEQ ID NO: 25, and deprotect the resulting peptide; c. Couple the peptide from step b. to the peptide of SEQ ID NO: 26, and deprotect the resulting peptide; d. Couple the peptide from step c. to the peptide of SEQ ID NO: 27, and deprotect the resulting peptide; e. Couple the peptide from step d. to the peptide of SEQ ID NO: 28, and deprotect the resulting peptide; f. Couple the peptide from step e. to the peptide of SEQ ID NO: 29, and deprotect the resulting peptide; g. Couple the peptide from step f. to the peptide of SEQ ID NO: 30, and deprotect the resulting peptide; and h. Couple the peptide from step g. to the peptide of SEQ ID NO: 31, and deprotect the resulting peptide.

Images