Acid-stable phosphoarginine analogs maintaining alpha-amino acid structure

Abstract

The present invention relates to an acid-stable phosphoarginine analog maintaining an alpha-amino acid structure, and provides a novel phosphoarginine analog maintaining an alpha-amino acid structure while being acid-stable, and a synthesis method therefor. The phosphoarginine analogs of the present invention can not only overcome the instability to acids and the absence of an alpha-amino acid structure, which are disadvantages found in previously attempted examples in which N-P bonds are replaced with C-P bonds, but are also shown to be structurally and electronically extremely similar to pArg, and thus are expected to be usefully utilized in a wide range of applications related to phosphorylated arginine.

Description

Acid-stable phosphoarginine analogues that maintain the α-amino acid structure

[0001] The present invention relates to an acid-stable phosphoarginine analogue that maintains an α-amino acid structure.

[0002] This application claims priority to Korean Patent Application No. 10-2024-0093283, filed July 15, 2024, the entire disclosure of which is incorporated herein by reference.

[0003] This application was carried out with the support of the National Research Foundation of Korea under the Ministry of Science and ICT as a research management specialized institution under the project identification number 1711195241 (subproject number 00214461) and the project name [Development and construction of a library of multi-oxygen-substituted cyclohexane natural product structural analogue compounds for discovery of new drug materials].

[0004] This application was supported by the Korea Health Industry Development Institute under the Ministry of Health and Welfare, under the research management specialized institution, project number 2460004262 (subproject number 02173063), project name [Development and application research of new chemical adjuvant based on heterobiological structure].

[0005] This application was supported by the National Research Foundation of Korea under the Ministry of Science and ICT, under the research management specialized institution, Project No. 1711180692 (Subproject No. 2021R1A2C1011196), Project Name [Research on the Control Mechanism of Transcription Factors Regulating Cancer Metastasis].

[0006] This application was supported by the National Research Foundation of Korea under the Ministry of Education, under the research management specialized organization, Project No. 1345365762 (Subproject No. 2021R1A6A1A03044296), Project Name [Chung-Ang University Pharmaceutical Research Institute].

[0007] Protein phosphorylation, which occurs in both prokaryotes and eukaryotes, is a key post-translational modification (PTM) that influences protein function. Both prokaryotes and eukaryotes control the activity of numerous enzymes and receptors through phosphorylation and dephosphorylation of specific amino acid residues, playing a crucial role in cellular regulation.

[0008] Phosphorylation is the process of attaching a phosphate group (PO4) to a polar functional group on an amino acid side chain, catalyzed by kinases in the presence of a phosphate donor, typically ATP. This reversible modification is central to diverse cellular processes, including protein synthesis, degradation, and signal transduction, and is deeply linked to numerous diseases. Therefore, phosphorylation and dephosphorylation have become valuable therapeutic targets, leading to the development of various drugs to treat cancer, neurological disorders, infections, and immune disorders.

[0009] In particular, arginine phosphorylation and dephosphorylation are known to play a crucial role in phosphorylated proteins. Originally identified in histone proteins, arginine phosphorylation has emerged as a crucial regulatory mechanism essential for cellular physiology and survival under specific conditions following the discovery of McsB, a bacterial protein arginine kinase. Interest in protein arginine phosphorylation is growing.

[0010] However, the nitrogen-phosphorus bond in phosphoarginine is known to be unstable and easily cleaved under acidic conditions, a factor that has hindered research into the arginine phosphorylation / dephosphorylation process and the biological role of phosphoarginine. This has highlighted the need to develop stable phosphoarginine (pArg) analogs for a wide range of applications.

[0011] For the study of protein phosphorylation, methods for manipulating the phosphorylation state of the target protein at a specific or non-specific amino acid side chain have mainly been used. However, in the case of phosphorylated arginine, analogs developed to date are known to have omitted the α-amino acid structure, and these have the characteristic that they cannot be included in a specific amino acid sequence.

[0012] Although various studies are being conducted to develop stable arginine analogs, little is known about phosphoarginine analogs that are stable, maintain the function of phosphorylated proteins, and contain an α-amino acid structure.

[0013] An object of the present invention is to provide a phosphoarginine analog represented by the following chemical formula 1:

[0014] [Chemical Formula 1]

[0015]

[0016] The above R1 is each independently one selected from the group consisting of hydrogen, a C1 to C6 alkyl group, a C1 to C6 heteroalkyl group, an aryl group, a benzyl group, and a heteroaryl group,

[0017] The above R2 to R4 are each independently one selected from the group consisting of hydrogen and amine protecting groups, and the amine protecting group is characterized by one selected from the group consisting of Boc (tert-butyloxycarbonyl), Fmoc (9-Fluorenylmethyl carbamates), Cbz (Benzyl carbamates), Alloc (allyloxycarbonyl), Phtalimide, acetyl group (Ac), trityl group (Tr), and sulfonyl group,

[0018] The above R5 is a substituted or unsubstituted hydroxyl group or a substituted or unsubstituted C1 to C6 alkoxy group, and the substitution or unsubstitution is characterized by being substituted or unsubstituted with a C1 to C6 alkyl group, a C1 to C6 heteroalkyl group, an aryl group, or a heteroaryl group.

[0019] n is 0 to 3.

[0020] Another object of the present invention is to provide a method for producing a phosphoarginine analog comprising the following steps:

[0021] (S1) A step of producing an intermediate, methyl thioamide salt, by replacing the nitrile group of diethyl(cyanomethyl)phosphonate with a thioamide group;

[0022] (S2) a step of reacting the methyl thioamide salt with a compound containing an amine protecting group to prepare an intermediate having a protected amine group; and

[0023] (S3) A step of removing at least one from the group consisting of an amine protecting group, an aryl group, and an ethyl group of the intermediate in which the amine group is protected obtained in the step (S2).

[0024] Another object of the present invention is to provide a method for producing a phosphoarginine analog comprising the following steps:

[0025] (Sa) A step of producing an intermediate, cyanomethyl phosphonate, by replacing hydrogen of dibenzyl phosphonate with chlorine;

[0026] (Sb) A step of reacting the above cyanomethyl phosphonate with a compound containing an amine protecting group to produce an intermediate having a protected amine group;

[0027] (Sc) a step of removing at least one of the amine protecting group, aryl group, and ethyl group of the intermediate in which the amine group is protected obtained in the above step (Sb); and

[0028] (Sd) A step of introducing an amine protecting group to the amine group of the compound obtained in the above (Sc) step.

[0029] Another object of the present invention is to provide a kit for preparing a phosphoarginine analog, comprising at least one selected from the group consisting of diethyl(cyanomethyl)phosphonate and dibenzyl phosphonate; and an instruction manual, wherein the instruction manual teaches the preparation method.

[0030]

[0031] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by a person having ordinary skill in the technical field to which the present invention belongs from the description below.

[0032] The present invention provides a phosphoarginine analog represented by the following chemical formula 1:

[0033] [Chemical Formula 1]

[0034]

[0035] The above R1 is each independently one selected from the group consisting of hydrogen, a C1 to C6 alkyl group, a C1 to C6 heteroalkyl group, an aryl group, a benzyl group, and a heteroaryl group,

[0036] The above R2 to R4 are each independently one selected from the group consisting of hydrogen and amine protecting groups, and the amine protecting group is characterized by one selected from the group consisting of Boc (tert-butyloxycarbonyl), Fmoc (9-Fluorenylmethyl carbamates), Cbz (Benzyl carbamates), Alloc (allyloxycarbonyl), Phtalimide, acetyl group (Ac), trityl group (Tr), and sulfonyl group,

[0037] The above R5 is a substituted or unsubstituted hydroxyl group or a substituted or unsubstituted C1 to C6 alkoxy group, and the substitution or unsubstitution is characterized by being substituted or unsubstituted with a C1 to C6 alkyl group, a C1 to C6 heteroalkyl group, an aryl group, or a heteroaryl group.

[0038] n is 0 to 3.

[0039] In one embodiment of the present invention, R1 may be any one selected from the group consisting of hydrogen, a C1 to C6 alkyl group, and a benzyl group, but is not limited thereto.

[0040] In one embodiment of the present invention, the amine protecting group may be any one selected from the group consisting of Boc (tert-butyloxycarbonyl) and Fmoc (9-Fluorenylmethyl carbamates), but is not limited thereto.

[0041] In one embodiment of the present invention, R5 may be any one selected from the group consisting of an unsubstituted hydroxyl group and a C1 to C6 alkoxy group substituted with a C1 to C6 alkyl group, but is not limited thereto.

[0042] In one embodiment of the present invention, the phosphoarginine analogue may include, but is not limited to, one or more selected from the group consisting of compounds comprising:

[0043] , , , , , , , and .

[0044] In one embodiment of the present invention, the phosphoarginine analog may include, but is not limited to, an α-amino acid structure.

[0045] In one embodiment of the present invention, the phosphoarginine analogue may be characterized by, but is not limited to, acid stability.

[0046] In one embodiment of the present invention, the phosphoarginine analogue may include a CP bond (Carbon-Phosphorus bond) and may not include a NP bond (Nitrogen-Phosphorus bond), but is not limited thereto.

[0047] The present invention provides a method for preparing a phosphoarginine analog comprising the following steps:

[0048] (S1) A step of producing an intermediate, methyl thioamide salt, by replacing the nitrile group of diethyl(cyanomethyl)phosphonate with a thioamide group;

[0049] (S2) a step of reacting the methyl thioamide salt with a compound containing an amine protecting group to prepare an intermediate having a protected amine group; and

[0050] (S3) A step of removing at least one from the group consisting of an amine protecting group, an aryl group, and an ethyl group of the intermediate in which the amine group is protected obtained in the step (S2).

[0051] The present invention provides a method for preparing a phosphoarginine analog comprising the following steps:

[0052] (Sa) A step of producing an intermediate, cyanomethyl phosphonate, by replacing hydrogen of dibenzyl phosphonate with chlorine;

[0053] (Sb) A step of reacting the above cyanomethyl phosphonate with a compound containing an amine protecting group to produce an intermediate having a protected amine group;

[0054] (Sc) a step of removing at least one of the amine protecting group, aryl group, and ethyl group of the intermediate in which the amine group is protected obtained in the above step (Sb); and

[0055] (Sd) A step of introducing an amine protecting group to the amine group of the compound obtained in the above (Sc) step.

[0056] The present invention provides a kit for preparing a phosphoarginine analog, comprising at least one selected from the group consisting of diethyl(cyanomethyl)phosphonate and dibenzyl phosphonate; and an instruction manual, wherein the instruction manual teaches the preparation method.

[0057] According to the acid-stable phosphoarginine analogue maintaining the α-amino acid structure, a novel phosphoarginine analogue that is acid-stable while maintaining the α-amino acid structure and a method for synthesizing the same are provided. The phosphoarginine analogue of the present invention not only overcomes the absence of the α-amino acid structure, which is a drawback found in examples in which the NP bond in the past was replaced with the CP bond, but also shows extreme structural and electronic similarity to pArg, and is expected to be usefully utilized in a wide range of applications related to phosphoarginine.

[0058] Figure 1 illustrates a known N-phosphorylated amino acid and its analogs, and a novel pArg analog (compound 8) synthesized according to the method of the present invention.

[0059] Figure 2 shows the synthesis process of a novel pArg analogue (compound 8, etc.) of the present invention.

[0060] Figures 3a and 3b show the compound 10. 1 H and 13 C NMR spectra are shown (600 MHz and 150 MHz, respectively).

[0061] Figures 3c and 3d show the compound 11. 1 H and 13 C NMR spectra are shown (600 MHz and 150 MHz, respectively).

[0062] Figures 3e to 3g are of compound 13. 1 H, 13 C and 31 P NMR spectra are shown (600 MHz, 150 MHz, and 243 MHz, respectively).

[0063] Figures 3h to 3j are of compound 8. 1 H, 13 C and 31 P NMR spectra are shown (600 MHz, 150 MHz, and 243 MHz, respectively).

[0064] Figures 3k to 3m are compounds 24 to 26. 1 This shows the H NMR spectra.

[0065] Figure 4 shows an electrostatic potential map comparing the structural and electronic characteristics of a novel pArg analogue (compound 8) of the present invention with those of pArg.

[0066] Protein phosphorylation is widely known as a crucial post-translational modification (PTM) of proteins. However, research on N-phosphorylated amino acids is extremely limited due to the acid-labile nature of the NP bond. In particular, analogs in which the NP bond is replaced by a CP bond have been applied to some pLys and pHis, but acid-stable pArg analogs that maintain the α-amino acid structure have not been developed.

[0067] Including an α-amino acid structure preserves the basic skeleton that constitutes a protein, and has a decisive influence on resistance to degradation in vivo, enzyme selectivity, transporter recognition, etc. The present invention discloses a method for synthesizing a novel pArg analogue comprising an acid-stable iminoethylphosphonic acid pArg analogue having an α-amino acid structure.

[0068] In general, a total of ten phosphorylated amino acids are known, and they are classified into three groups based on the phosphorylation site: O-phosphorylated amino acids (pSer, pThr, pTyr, pAsp, pGlu), S-phosphorylated amino acids (pCys), and N-phosphorylated amino acids (pLys, 1-pHis, 3-pHis, pArg). Research on phosphorylated amino acids has mainly focused on serine, threonine, and tyrosine. While phosphate monoesters are relatively stable and amenable to acidic analytical methods, phosphate amidates, such as those in pLys, pHis, and pArg, are known to be unstable under similar conditions due to the acid-labile N-P bond, making their study under established experimental conditions complicated.

[0069]

[0070] To address this instability of phosphoamidates, attempts have been made to synthesize stable N-phosphorylated amino acid derivatives under acidic conditions. This was achieved by replacing the labile NP bond with a non-hydrolyzable bond through equivalence (Figure 1). For example, pLys derivatives 1 and 2 were designed by rearranging the NP bond into a CP bond and an OP bond, respectively, while maintaining the length of the side chain and α-amino acid backbone (Figure 1A). Similarly, stable pHis analogs 3 to 5 were prepared by replacing the NP bond with a CP bond through replacement of the imidazole ring with a triazole or pyrazole structure (Figure 1B). For pArg, phosphonate amidine compounds 6 and 7 were developed in which the NP bond was replaced with a CP bond (Figure 1C). However, unlike pLys and pHis derivatives 1–5, the iminoethylphosphonic acid structure of pArg analogs 6 and 7, although functionally mimicking the phosphorylated side chain of native pArg, has a major limitation in that the lack of an α-amino acid group limits their incorporation into peptides and proteins, which limits their application scope. Consequently, rather than incorporating into specific amino acid sequences via amide bond formation at the amine and carboxylic acid groups, they can only be nonspecifically incorporated into lysine residues via glutaraldehyde crosslinking.

[0071] Accordingly, the present invention provides a phosphoarginine analog represented by the following chemical formula 1:

[0072] [Chemical Formula 1]

[0073]

[0074] The above R1 is each independently one selected from the group consisting of hydrogen, a C1 to C6 alkyl group, a C1 to C6 heteroalkyl group, an aryl group, a benzyl group, and a heteroaryl group,

[0075] The above R2 to R4 are each independently one selected from the group consisting of hydrogen and amine protecting groups, and the amine protecting group is characterized by one selected from the group consisting of Boc (tert-butyloxycarbonyl), Fmoc (9-Fluorenylmethyl carbamates), Cbz (Benzyl carbamates), Alloc (allyloxycarbonyl), Phtalimide, acetyl group (Ac), trityl group (Tr), and sulfonyl group,

[0076] The above R5 is a substituted or unsubstituted hydroxyl group or a substituted or unsubstituted C1 to C6 alkoxy group, and the substitution or unsubstitution is characterized by being substituted or unsubstituted with a C1 to C6 alkyl group, a C1 to C6 heteroalkyl group, an aryl group, or a heteroaryl group.

[0077] n is 0 to 3.

[0078] In one embodiment of the present invention, R1 may be any one selected from the group consisting of hydrogen, a C1 to C6 alkyl group, and a benzyl group, but is not limited thereto.

[0079] In addition, the above R2 can be any one of the functional groups described above independently depending on the position.

[0080] It is known that each amino acid linked to the N-terminus of a peptide chain must be protected at the N-terminus and side chain using an appropriate protecting group, such as Boc (acid labile) or Fmoc (base labile), depending on the side chain and the protection strategy used. It is essential to use N-terminal and side chain protecting groups during peptide synthesis to avoid undesirable side effects, such as polymerization due to self-association of activated amino acids. Therefore, in the present invention, the “amine protecting group” may mean a group for protecting an amine, which is a functional group of an amino acid side chain, and may be used interchangeably with any term generally referred to in the art. In one embodiment of the present invention, the amine protecting group may be any one selected from the group consisting of Boc (tert-butyloxycarbonyl) and Fmoc (9-Fluorenylmethyl carbamates), but is not limited thereto.

[0081] In one embodiment of the present invention, R5 may be any one selected from the group consisting of an unsubstituted hydroxyl group and a C1 to C6 alkoxy group substituted with a C1 to C6 alkyl group, but is not limited thereto.

[0082] In one embodiment of the present invention, the substituted or unsubstituted C1 to C6 alkyl group, heteroalkyl group, aryl group, and heteroaryl group may include isomers. For example, the substituted or unsubstituted C1 to C6 alkoxy group may include isomers, and as an example, OBu may include OtBu, which is a tert isomer.

[0083] In one embodiment of the present invention, the phosphoarginine analogue may include, but is not limited to, one or more selected from the group consisting of compounds comprising:

[0084] , , , , , , , and .

[0085] Therefore, in one embodiment of the present invention, the phosphoarginine analogue may include, but is not limited to, one or more selected from the group consisting of compounds consisting of, in order:

[0086] (tert-butyl (S)-2-((tert-butoxycarbonyl)amino)-5-(2-(diethoxyphosphoryl)acetimidamido)pentanoate);

[0087] (S)-2-amino-5-(2-phosphonoacetimidamido)pentanoic acid;

[0088] tert-butyl (S)-5-(2-(bis(benzyloxy)phosphoryl)acetimidamido)-2-((tert-butoxycarbonyl)amino)pentanoate;

[0089] (S)-2-amino-5-(2-(bis(benzyloxy)phosphoryl)acetimidamido)pentanoic acid;

[0090] (S,Z)-5-(N'-(((9H-fluoren-9-yl)methoxy)carbonyl)-2-(bis(benzyloxy)phosphoryl)acetimidamido)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)pentanoic acid;

[0091] tert-butyl (2S)-5-(2-((benzyloxy)(hydroxy)phosphoryl)acetimidamido)-2-((tert-butoxycarbonyl)amino)pentanoate;

[0092] (2S)-2-amino-5-(2-((benzyloxy)(hydroxy)phosphoryl)acetimidamido)pentanoic acid; and

[0093] (2S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(2-((benzyloxy)(hydroxy)phosphoryl)acetimidamido)pentanoic acid.

[0094] In one embodiment of the present invention, the phosphoarginine analog may include, but is not limited to, an α-amino acid moiety.

[0095] In the present invention, “amino acid” refers to an amino group (-NH, which is protonated under biological conditions) 3+ form), carboxyl group (-COO deprotonated under biological conditions - (form), an organic compound with a specific side chain (R group). That is, an amino acid can refer to a molecule that has both an amino group (-NH2) and a carboxyl group (-COOH). The elements present in all amino acids are carbon (C), hydrogen (H), oxygen (O), and nitrogen (N). Additionally, sulfur (S) is present in the side chains of cysteine ​​and methionine, and selenium (Se) is present in the side chain of selenocysteine, a less common amino acid. As of 2020, there are more than 500 naturally occurring amino acids. Some of these are known to constitute the monomeric units of peptides, including proteins, but only 22 α-amino acids appear in the genetic code, 20 of which have unique designated codons, and the remaining two (selenocysteine, present in all eukaryotes, and pyrrolysine, present in some prokaryotes) have specialized coding mechanisms.

[0096] Amino acids can be classified as alpha-amino acids (α-amino acids), beta-amino acids (β-amino acids), gamma-amino acids (γ-amino acids), or delta-amino acids (δ-amino acids) based on the location of their key structural functional groups. Other classification categories relate to polarity, ionizability, and the type of side-chain functional groups (aliphatic, acyclic, aromatic, containing hydroxyl groups, or sulfur). In human muscle and other tissues, amino acid residues in the form of proteins make up the second largest component (water is the largest). In addition to their role as protein residues, amino acids can be involved in various processes, such as neurotransmitter synthesis and biosynthesis.

[0097] In the present invention, “α-amino acid” may refer to an alpha-amino acid (HOOC-CH(R)-NH2) among amino acids having both an amino group (-NH2) and a carboxyl group (-COOH) in which both functional groups are attached to a single carbon. In addition, the α-amino acid may be a zwitterion, but is not limited thereto.

[0098] When synthesizing phosphorylated proteins, the inclusion of an α-amino acid moiety may be essential for their physiological utility. In the present invention, phosphoarginine was developed as a phosphorylated amino acid that exhibits both an α-amino acid moiety and acid stability, using the synthetic method of the present invention. Therefore, in one embodiment of the present invention, the phosphoarginine analog may be characterized by acid stability, but is not limited thereto.

[0099] As described above, phosphoarginine analogs are generally known to have significantly poor acid stability due to the instability of the NP bond. Therefore, research has been conducted in the art to replace the NP bond with a non-hydrolyzable bond, and a method of replacing it with a CP bond has been applied accordingly. However, existing phosphoarginine analogs that have replaced the CP bond have a significant drawback in that they do not include an α-amino acid moiety, which limits their incorporation into peptides and proteins. However, the phosphoarginine analog of the present invention can be characterized by replacing the NP bond with a CP bond, thereby having excellent acid stability while also including an α-amino acid moiety.

[0100] That is, the phosphoarginine analog of the present invention may have an NP bond replaced with a CP bond compared to a conventional phosphoarginine analog or a wild type, and therefore, in one embodiment of the present invention, the phosphoarginine analog may include a CP bond (Carbon-Phosphorus bond) and may not include an NP bond (Nitrogen-Phosphorus bond), but is not limited thereto. In addition, as described above, the phosphoarginine analog of the present invention may be characterized by acid stability.

[0101] In the present invention, “phosphoarginine analogue” may refer to a compound structurally or functionally similar to phosphorylated arginine. In the present invention, it was confirmed that it is structurally and electronically extremely similar to phosphorylated arginine. In addition, the phosphoarginine analogue of the present invention may include a chemically acceptable salt thereof. In one embodiment of the present invention, compound 8 in the form of an HBr salt was synthesized, but this is not limited thereto, and all salts in the form of including HX, 2HX (X = Cl, Br, I, etc.) may be included.

[0102] In one embodiment of the present invention, a phospho-arginine analogue was synthesized through the following method. First, the process began with the preparation of thioamide salt (11) from diethyl (cyanomethyl)phosphonate (9). Specifically, in order to substitute the nitrile group of compound 9 with the thioamide group of compound 10, the reaction was performed by treating with a tetrahydrofuran (THF) solution of H2S and Et3N in pyridine at 50°C for 24 hours. As a result, a yield of 39% was achieved. Thereafter, the successfully synthesized compound 10 was methylated to produce methyl thioamide salt (11) in an excellent yield (90%). For methylation, MeI was treated in acetone at rt for 3 hours.

[0103] For the amino acid backbone, protected ornithine Boc-L-Orn-OtBu(12) was chosen as the starting point because it is suitable for subsequent deprotection. Here, the Boc and tert-butyl ester groups were chosen because they can be efficiently removed under acidic conditions required to remove the ethyl group of the diethyl methylphosphonate group. Therefore, the coupling of compounds 11 and 12 proceeded smoothly in MeOH at rt, 1 h by treating compound 11 with ornithine Boc-L-Orn-OtBu, thereby affording compound 13.

[0104] Compound 8 was then synthesized by removing the ethyl group of the phosphonate using TMSBr (Bromotrimethylsilane) under MeCN, rt, 48 h conditions. In this step, the acid-labile tert-butyl and Boc protecting groups were also removed simultaneously, resulting in the acquisition of compound 8, a pArg analogue of the HBr salt, in 54% yield.

[0105] Accordingly, for compounds 8, 10, 11, and 13 of the present invention, the present invention provides a method for preparing a phosphoarginine analog comprising the following steps:

[0106] (S1) A step of producing an intermediate, methyl thioamide salt, by replacing the nitrile group of diethyl(cyanomethyl)phosphonate with a thioamide group;

[0107] (S2) a step of reacting the methyl thioamide salt with a compound containing an amine protecting group to prepare an intermediate having a protected amine group; and

[0108] (S3) A step of removing at least one from the group consisting of an amine protecting group, an aryl group, and an ethyl group of the intermediate in which the amine group is protected obtained in the step (S2).

[0109] In one embodiment of the present invention, the step (S1) may include, but is not limited to, the following steps:

[0110] (S1-A) A step of reacting diethyl(cyanomethyl)phosphonate by treating it with hydrogen sulfide (H2S) and triethylamine (Et3N); and

[0111] (S1-B) A step of producing a methyl thioamide salt intermediate by methylating the reactant obtained in (S1-A) in acetone.

[0112] In the present invention, the reactants according to step (S1-A) may be represented by the following chemical formula, but are not limited thereto: . Additionally, the methyl thioamide salt intermediate may be represented by the following chemical formula, but is not limited thereto: .

[0113] In one embodiment of the present invention, the step (S1-A) may be performed in pyridine at a temperature of 40°C to 60°C for 12 to 36 hours, but is not limited thereto.

[0114] In the present invention, the step (S1-A) is 40°C to 59°C, 40°C to 58°C, 40°C to 57°C, 40°C to 56°C, 40°C to 55°C, 40°C to 54°C, 40°C to 53°C, 40°C to 52°C, 40°C to 51°C, 40°C to 50°C, 42°C to 60°C, 42°C to 59°C, 42°C to 58°C, 42°C to 57°C, 42°C to 56°C, 42°C to 55°C, 42°C to 54°C, 42°C to 53°C, 42°C to 52°C, 42°C to 51°C, 42°C to 50°C, 44°C to 60°C, 44°C to 59°C, 44℃ to 58℃, 44℃ to 57℃, 44℃ to 56℃, 44℃ to 55℃, 44℃ to 54℃, 44℃ to 53℃, 44℃ to 52℃, 44℃ to 51℃, 44℃ to 50℃, 46℃ to 60℃, 46℃ to 59℃, 46℃ to 58℃, 46℃ to 57℃, 46℃ to 56℃, 46℃ to 55℃, 46℃ to 54℃, 46℃ to 53℃, 46℃ to 52℃, 46℃ to 51℃, 46℃ to 50℃, 48℃ to 60℃, 48℃ to 59℃, 48℃ to 58℃, 48℃ to 57℃, 48℃ to It can be performed at, but is not limited to, 56°C, 48°C to 55°C, 48°C to 54°C, 48°C to 53°C, 48°C to 52°C, 48°C to 51°C, 48°C to 50°C, or 50°C.

[0115] In addition, in the present invention, the (S1-A) step is 12 hours to 34 hours, 12 hours to 30 hours, 12 hours to 28 hours, 12 hours to 26 hours, 12 hours to 24 hours, 16 hours to 36 hours, 16 hours to 34 hours, 16 hours to 30 hours, 16 hours to 28 hours, 16 hours to 26 hours, 16 hours to 24 hours, 18 hours to 36 hours, 18 hours to 34 hours, 18 hours to 30 hours, 18 hours to 28 hours, 18 hours to 26 hours, 18 hours to 24 hours, 20 hours to 36 hours, 20 hours to 34 hours, 20 hours to 30 hours, 20 hours to 28 hours, 20 hours to 26 hours, 20 hours to It can be performed for 24 hours, 22 to 36 hours, 22 to 34 hours, 22 to 30 hours, 22 to 28 hours, 22 to 26 hours, 22 to 24 hours, or 24 hours, but is not limited thereto.

[0116] In one embodiment of the present invention, the reactant in step (S1-B) may be methylated by reacting with methyl iodide (MeI), but is not limited thereto, and may be methylated using any substance that can be generally used for methylation in the art, and methyl iodide may be an example of the substance used. Therefore, it can be clearly inferred that the methylation activity equivalent to that exhibited when methyl iodide is used in the present invention by using other substances that can be used for methylation.

[0117] In addition, the present invention may perform an alkylation process instead of methylation in the step (S1-B). Accordingly, the present invention may include a step (S1-B) of alkylating the reactant obtained in the step (S1-A) to produce a thioamide salt intermediate, instead of the step (S1-B). At this time, the step (S1-B) may be performed by adding iodoalkane, bromoalkane, diazoalkane, alkyl methanesulfonate, alkyl trifluoromethanesulfonate, or the like. The additive material for alkylation is disclosed as an example in the same manner as the additive material for methylation, and any material that can be generally used for alkylation in the art may be used.

[0118] In addition, since the thioamide salt at this time does not contain a methyl group, in this case, the present invention provides a method for producing a phosphoarginine analog comprising the following steps:

[0119] (S1) A step of producing an intermediate, a methyl thioamide salt, by replacing the nitrile group of diethyl(cyanomethyl)phosphonate with a thioamide group;

[0120] (S2) a step of reacting the above thioamide salt with a compound containing an amine protecting group to prepare an intermediate having a protected amine group; and

[0121] (S3) A step of removing at least one from the group consisting of an amine protecting group, an aryl group, and an ethyl group of the intermediate in which the amine group is protected obtained in the step (S2).

[0122] In one embodiment of the present invention, the step (S1-B) may be performed at room temperature for 1 to 6 hours, but is not limited thereto.

[0123] In the present invention, the (S1-B) step may be performed for 1 hour to 5 hours, 1 hour to 4.5 hours, 1 hour to 4 hours, 1 hour to 3.5 hours, 1 hour to 3 hours, 1.5 hours to 6 hours, 1.5 hours to 5 hours, 1.5 hours to 4.5 hours, 1.5 hours to 4 hours, 1.5 hours to 3.5 hours, 1.5 hours to 3 hours, 2 hours to 6 hours, 2 hours to 5 hours, 2 hours to 4.5 hours, 2 hours to 4 hours, 2 hours to 3.5 hours, 2 hours to 3 hours, 2.5 hours to 6 hours, 2.5 hours to 5 hours, 2.5 hours to 4.5 hours, 2.5 hours to 4 hours, 2.5 hours to 3.5 hours, 2.5 hours to 3 hours, or 3 hours. However, it is not limited to these.

[0124] In one embodiment of the present invention, in the step (S2), the amine protecting group may be any one selected from the group consisting of Boc (tert-butyloxycarbonyl), Fmoc (9-Fluorenylmethyl carbamates), Cbz (Benzyl carbamates), Alloc (allyloxycarbonyl), Phtalimide, an acetyl group (Ac), a trityl group (Tr), and a sulfonyl group, but is not limited thereto.

[0125] In the present invention, the term “compound containing an amine protecting group” includes any compound containing a functional group for protecting an amine group, and may have a broad meaning generally understood in the art.

[0126] In the present invention, Boc was used as a compound containing an amine protecting group, and was selected because it is useful for removal in subsequent synthetic steps, but is not limited thereto.

[0127] In one embodiment of the present invention, the compound including the amine protecting group may additionally include a tert-butyl group (tert-buryl), but is not limited thereto.

[0128] In the present invention, the Boc and tert-butyl ester groups were selected because they can be efficiently removed under acidic conditions to remove the ethyl group of the diethyl methylphosphonate group. That is, since the compound containing the amine protecting group of the present invention contains both functional groups (Boc and tert-butyl ester groups), the ethyl group is effectively removed even under acidic conditions, and thus the final result, compound 8 of the present invention, a phosphoarginine analogue, can be obtained with high efficiency.

[0129] In one embodiment of the present invention, the step (S2) may be performed in methanol (MeOH) at room temperature for 0.1 to 6 hours, but is not limited thereto.

[0130] In the present invention, the step (S2) is 0.1 hour to 6 hours, 0.1 hour to 5 hours, 0.1 hour to 4 hours, 0.1 hour to 3 hours, 0.1 hour to 2 hours, 0.1 hour to 1 hour, 0.3 hour to 6 hours, 0.3 hour to 5 hours, 0.3 hour to 4 hours, 0.3 hour to 3 hours, 0.3 hour to 2 hours, 0.3 hour to 1 hour, 0.6 hour to 6 hours, 0.6 hour to 5 hours, 0.6 hour to 4 hours, 0.6 hour to 3 hours, 0.6 hour to 2 hours, 0.6 hour to 1 hour, 0.8 hour to 6 hours, 0.8 hour to 5 hours, 0.8 hour to 4 hours, 0.8 hour to 3 hours, 0.8 hour to 2 hours, It can be performed for 0.8 hours to 1 hour, or 1 hour, but is not limited thereto.

[0131] In one embodiment of the present invention, the step (S2) may be a step for removing the ethyl group of the step (S3) to be performed under acidic conditions, but is not limited thereto.

[0132] The phosphoarginine analogue of the present invention was proven to have excellent stability under acidic conditions even though the NP bond was replaced with a CP bond because it was prepared under highly acidic conditions.

[0133] In one embodiment of the present invention, the step (S3) may be performed by adding any one selected from the group consisting of TMSBr, TMSCl, HBr, HCl, hydrogen, palladium / carbon, and platinum (IV) oxide, but is not limited thereto.

[0134] In this example, the amine protecting group and ethyl group were removed together, but they do not necessarily need to be removed together; they can be removed using separate steps or materials. In this case, any method commonly used in the art can be applied to remove the amine protecting group and ethyl group.

[0135] Additionally, in order to synthesize the compound of the present invention, an aryl group may be substituted for the ethyl group in step (S3). At this time, the elimination reaction condition is hydrogen + (palladium on activated carbon or platinum(IV) oxide), and under the above conditions, the amine protecting group may not be removed.

[0136]

[0137] In one embodiment of the present invention, the step (S3) may be performed in acetonitrile (MeCN) at room temperature for 36 to 60 hours, but is not limited thereto.

[0138] In the present invention, the step (S3) is performed for 36 hours to 56 hours, 36 hours to 54 hours, 36 hours to 52 hours, 36 hours to 50 hours, 36 hours to 48 hours, 40 hours to 60 hours, 40 hours to 56 hours, 40 hours to 54 hours, 40 hours to 52 hours, 40 hours to 50 hours, 40 hours to 48 hours, 42 hours to 60 hours, 42 hours to 56 hours, 42 hours to 54 hours, 42 hours to 52 hours, 42 hours to 50 hours, 42 hours to 48 hours, 44 hours to 60 hours, 44 hours to 56 hours, 44 hours to 54 hours, 44 hours to 52 hours, 44 hours to 50 hours, 44 hours to 48 hours, It can be performed for, but is not limited to, 46 ​​to 60 hours, 47 to 56 hours, 47 to 54 hours, 47 to 52 hours, 47 to 50 hours, 47 to 48 hours, or 48 hours.

[0139] In one embodiment of the present invention, the ethyl group in the step (S3) may be, but is not limited to, an ethyl group of a diethyl methylphosphonate group.

[0140] In one embodiment of the present invention, the yield of the phosphoarginine analogue may be 50% or more relative to the total amount of the intermediate in which the amine group is protected in the above manufacturing method, but is not limited thereto. In one embodiment of the present invention, when the manufacturing method of the present invention was applied, the yield of the final phosphoarginine analogue, Compound 8, was obtained at 54%.

[0141] In addition, for compounds 24 to 26 of the present invention, the present invention provides a method for preparing a phosphoarginine analog comprising the following steps:

[0142] (Sa) A step of producing an intermediate, cyanomethyl phosphonate, by replacing hydrogen of dibenzyl phosphonate with chlorine;

[0143] (Sb) A step of reacting the above cyanomethyl phosphonate with a compound containing an amine protecting group to produce an intermediate having a protected amine group;

[0144] (Sc) a step of removing at least one of the amine protecting group, aryl group, and ethyl group of the intermediate in which the amine group is protected obtained in the above step (Sb); and

[0145] (Sd) A step of introducing an amine protecting group to the amine group of the compound obtained in the above (Sc) step.

[0146] In the present invention, the (Sa) step can be performed in benzene at 0°C, rt, for 0.5 to 3 hours, or in THF at -100 to -50°C for 1 to 3 hours, but is not limited thereto. In addition, in the present invention, the (Sb) step can be performed in t-BuOH or MeOH at rt o / n or for 1 to 3 hours, but is not limited thereto. In the present invention, the (Sc) step can be performed under the conditions of the (S1-B) step, that is, at room temperature, for 1 to 6 hours, but is not limited thereto. Finally, in the present invention, the (Sd) step can be performed under water / dioxane at room temperature for 12 to 36 hours, but is not limited thereto.

[0147] In addition, the present invention provides a kit for preparing a phosphoarginine analog, comprising at least one selected from the group consisting of diethyl(cyanomethyl)phosphonate and dibenzyl phosphonate; and an instruction manual, wherein the instruction manual teaches the preparation method.

[0148] In the present invention, the "kit" refers to a tool that enables the production of diethyl (cyanomethyl) phosphonate of the present invention, or an arginine phosphate analogue including diethyl (cyanomethyl) phosphonate. In addition to the above-mentioned materials, the kit of the present invention may include other components, compositions, solutions, devices, etc. that are typically required for methods of storing and processing them. As a specific example, in this case, each component may be applied once or more without limitation on the number of times, there is no limitation on the order in which each material is applied, and the application of each material may be performed simultaneously or microscopically.

[0149] In the present invention, the kit may include a container; instructions; and the like. The container may serve to package the substance, and may also serve to store and fix the substance. The material of the container may take the form of, for example, a bottle, a tub, a sachet, an envelope, a tube, an ampoule, and the like, and these may be formed partially or wholly from plastic, glass, paper, foil, wax, and the like. The container may be initially equipped with a completely or partially detachable stopper, which may be part of the container or may be attached to the container by mechanical, adhesive, or other means, and may also be equipped with a stopper for allowing access to the contents by means of a syringe needle. The kit may include an outer package, and the outer package may include instructions for the use of the components.

[0150] The present invention also provides the use of the phosphoarginine analogue of the present invention as a building block.

[0151] Compounds used in natural products, pharmaceuticals, and other products are synthesized through a multi-step process. For example, when synthesizing key pharmaceutical raw materials, such as drugs containing phosphorylated proteins, various reaction processes are used, including the process of synthesizing phosphorylated proteins. Compounds synthesized through these reaction processes are extremely difficult and complex, and extensive research is underway to simplify reaction conditions and achieve practicality.

[0152] Thus, the most efficient method for manufacturing highly functionalized complex drugs is the use of building blocks. The phosphoarginine analog of the present invention is designed to function as a building block mimicking phosphoarginine. Because it maintains its α-amino acid structure while exhibiting excellent acid stability, it can be specifically incorporated into peptides and proteins at target sites and selectively facilitate various reactions, making it a useful building block.

[0153] In the present invention, when the term "comprising" is used, it does not exclude other components unless specifically stated otherwise, but rather means that other components can be included. As used throughout the present invention, the terms "step of ~" or "step of ~" do not mean "step for ~."

[0154] Hereinafter, preferred examples are presented to aid in understanding the present invention. However, the following examples are provided solely to facilitate a better understanding of the present invention, and the scope of the present invention is not limited by the following examples.

[0155]

[0156] [Example]

[0157]

[0158] Example 1. Synthesis of pArg analogues

[0159] Example 1-1. Compounds 8, 10, 11, and 13

[0160] Compounds 10, 11, 13, and 8 were synthesized sequentially as pArg having excellent acid stability while maintaining the α-amino acid portion, and the process is as shown in Figure 2.

[0161]

[0162] Preparation of compound 10

[0163] Compound 10 was prepared by modifying the procedure reported in a previous study (H. Ouyang, C. Fu, S. Fu, Z. Ji, Y. Sun, P. Deng, Y. Zhao, Org. Biomol. Chem. 14(2016) 1925–1929). Diethyl (cyanomethyl)phosphonate (compound 9) (2.00 g, 11.29 mmol) was added to a sealed vial equipped with a magnetic bar and a solution of H2S in THF (42.00 mL, 0.80 M, 33.87 mmol). Pyridine (0.46 mL, 5.65 mmol) and Et3N (4.72 mL, 33.87 mmol) were then added at room temperature, and the mixture was stirred at 50°C for 24 h. After cooling to room temperature, the mixture was concentrated under reduced pressure. The resulting residue was purified by flash chromatography on silica gel (hexane / EtOAc, 1:4, v / v) to obtain compound 10 (0.93 g, 39%) as a light brown solid. R f = 0.21(EtOAc only); 1 H NMR (CDCl3, 600 MHz) δ = 8.58 (s, 1H), 8.04 (s, 1H), 4.20-4.10 (m, 4H), 3.42 (d, J = 21.1 Hz, 2H), 1.33 (t, J = 7.1 Hz, 6H); 13 C NMR (CDCl3, 150 MHz) δ = 197.9, 63.4, 63.3, 43.7, 42.9, 16.4, 16.3.

[0164]

[0165] Preparation of compound 11

[0166] Compound 10 (1.45 g, 6.87 mmol) was suspended in acetone (10 mL), and methyl iodide (methyl iodide, 2.20 mL, 35.35 mmol) was added at room temperature. The mixture was stirred at the same temperature for 3 hours. Upon completion of the reaction, a white solid precipitated. The mixture was filtered, rinsed with ether, and dried under vacuum to obtain compound 11 (2.20 g, 90%) as a white solid that is sensitive to air. 1 H NMR (600 mHz, CDCl3) δ = 1H NMR (CDCl3, 600 MHz) δ 4.43 - 4.24 (m, 4H), 3.97 (d, J = 22.5 Hz, 2H), 3.03 (s, 3H), 1.39 (t, J = 7.1 Hz, 3H); 13 C NMR (CDCl3, 150 MHz) δ = 187.9, 65.1, 36.8, 35.9, 18.3, 16.5.

[0167]

[0168] Preparation of compound 13

[0169] Compound 12 (95.2 mg, 0.33 mmol) was suspended in MeOH (1.8 mL), and compound 11 (107.3 mg, 0.33 mmol) was added at room temperature and stirred at the same temperature for 1 h. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel flash chromatography (CHCl3 / MeOH, 30:1) to yield compound 13 (108 mg, 75%) as a dark brown sticky oil. R f = 0.44 (CH2Cl2 / MeOH / AcOH, 7:1:0.01, v / v / v); 1H NMR (CD3OD-d4, 600 mHz) δ = 4.26 - 4.18 (m, 4H), 4.02 - 3.95 (m, 1H), 3.38 - 3.32 (m, 2H), 1.92 - 1.83 (m, 1H), 1.80 - 1.64 (m, 3H), 1.47(s, 9H), 1.45(s, 9H), 1.37(t, J = 7.1 Hz, 6H); 13C NMR (CD3OD-d4, 150 MHz) δ = 173.1, 161.4, 158.1, 82.8, 80.6, 65.0, 64.9, 55.2, 49.8, 43.5, 29.8, 28.7, 28.2, 25.3, 16.7; 31 P NMR (243 MHz, CD3OD-d4) δ = 18.16; HRMS(FAB): C 20 H 40 N3O7P [M+H] + Calculated value 466.2676, measured value 466.2671.

[0170]

[0171] Preparation of compound 8HBr

[0172] Compound 13 (70.0 mg, 0.15 mmol) was suspended in MeCN (2.80 mL), and bromotrimethylsilane (0.20 mL, 1.53 mmol) was added at room temperature, and the mixture was stirred at the same temperature for 2 days. The reaction mixture was concentrated under reduced pressure. The resulting residue was triturated and purified with CH2Cl2 and acetone to obtain compound 8 (27 mg, 54%) as a white solid. R f = 0.33 (MeCN / H2O / CF3CO2H, 1:1.1:0.02); 1 H NMR (600 MHz, DO) δ = 4.02 (t, J = 6.2 Hz, 1H), δ 3.36 (t, J = 6.8 Hz, 2H), δ 2.90 (d, J = 20.2 Hz, 2H), δ 2.08 - 1.92 (m, 2H), δ 1.89 - 1.70(m, 2H); 13 C NMR (DO, 150 MHz) δ = 172.2, 162.8, 52.8, 41.6, 34.5, 33.7, 27.1, 22.7; 31P NMR (243 MHz, DO) δ = 11.44; mp 238.0-240.6℃ (decomposed); HRMS(FAB): C7H 16 N3O5P [M+H] + Calculated value 254.0900, measured value 254.0905.

[0173]

[0174] Example 1-2. Synthesis of compounds 24 to 26

[0175] Compounds 24 to 26 were synthesized as pArg analogues with excellent acid stability while maintaining the α-amino acid moiety.

[0176]

[0177] Preparation of compound 22a

[0178]

[0179] After suspending 1a (0.84 mL, 3.81 mmol) in benzene (1.90 mL), sulfuryl chloride (0.35 mL, 4.19 mmol) was added at 0°C and stirred at room temperature for 30 minutes. The reaction mixture was concentrated under reduced pressure to obtain compound 22a. R f = 0.5 (hexane / EtOAc, 3:1); 1 H NMR (600 MHz, Chloroform-d) δ 7.42 - 7.32 (m, 10H),.27 - 5.12 (m, 4H).

[0180]

[0181] Preparation of compound 23a

[0182]

[0183] After suspending MeCN (0.3 mL, 5.71 mmol) in THF (10 mL), LDA (2.86 mL, 5.71 mmol) was added at -78°C and stirred at the same temperature for 1 hour. Afterwards, compound 22a was suspended in THF (13 mL), added to the reaction flask at -78°C and stirred at the same temperature for 2 hours. Upon completion of the reaction, ammonium chloride was added at room temperature and stirred at the same temperature for 5 minutes. The residue was extracted with ethyl acetate, concentrated under reduced pressure, and purified by silica gel flash chromatography (hexane / EtOAc, 2:1) to yield compound 23a (386 mg, 33%) as a brown, sticky oil. R f = 0.13 (hexane / EtOAc, 2:1); 1 H NMR (600 MHz, Chloroform-d) δ 7.43 - 7.33 (m, 10H), 5.20 - 5.03 (m, 4H), 2.71 (d, J = 21.1 Hz, 2H).

[0184]

[0185] Preparation of compound 22b

[0186]

[0187] Compound 21b (2.50 g, 6.82 mmol) was suspended in tert-butanol (25.0 mL), and Boc2O (2.51 mL, 10.92 mmol) and DMAP (416 mg, 3.41 mmol) were added at room temperature, and the mixture was stirred at the same temperature for 16 h. Upon completion of the reaction, DMAP was removed through a silica filter. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel flash chromatography (hexane / EtOAc, 4:1) to give compound 22b (2.4 g, 83%) as a colorless, sticky oil. R f = 0.5 (hexane / EtOAc, 2:1); 1H NMR (600 MHz, Chloroform-d) δ = 7.35 (d, J = 4.2 Hz, 5H), 4.22 - 4.14 (m, 1H), 3.21 (q, J = 6.5 Hz, 2H), 1.86 - 1.76 (m, 1H), 1.64 - 1.53 (m, 3H), 1.45(s, 9H), 1.43(s, 9H).

[0188]

[0189] Preparation of compound 23b

[0190]

[0191] Compound 22b (2.4 g, 5.68 mmol) was suspended in MeOH (63 mL), and Et₃N (1.58 mL, 11.36 mmol) was added at room temperature. Then, Pd / C (423 mg, 0.397 mmol) was added at room temperature, and the mixture was bubbled with hydrogen gas for 5 min, and stirred at the same temperature for 3 h. Upon completion of the reaction, Pd / C was removed through a Celite filter. The reaction mixture was concentrated under reduced pressure to yield compound 23b (1.60 g, 98%) as a colorless, sticky oil. 1 H NMR (600 MHz, Methanol-d4) δ = 3.94 (dd, J = 8.8, 5.1 Hz, 1H), 2.67 (ddd, J = 7.8, 6.6, 2.5 Hz, 2H), 1.81 - 1.74 (m, 1H), 1.66 - 1.60 (m, 1H), 1.59 - 1.53(m, 2H), 1.47(s, 9H), 1.45(d, J = 0.9 Hz, 9H).

[0192]

[0193] Preparation of compound 24

[0194]

[0195] Compound 23b (1.6 g, 5.55 mmol) was suspended in MeOH (10 mL), and Et₃N (1.16 mL, 8.32 mmol) and CuCl(I) (675 mg, 6.82 mmol) were added at room temperature. Then, compound 23a (2.51 g, 8.32 mmol) was suspended in MeOH (20 mL), added at room temperature, and stirred at 70°C for 24 h. After cooling to room temperature, the mixture was concentrated under reduced pressure, extracted with CH2Cl2, and dried over MgSO4. The resulting residue was purified by flash chromatography on silica gel (CH2Cl2 / MeOH, 10:1) to give compound 24 (860 mg, 25%) as a light yellow solid. R f = 0.2(CH2Cl2 / MeOH, 10:1); 1 H NMR (600 MHz, Methanol-d4) δ = 7.38 - 7.35 (m, 2H), 7.34 - 7.30 (m, 2H), 7.28 - 7.24 (m, 1H), 4.94 (d, J = 7.7 Hz, 2H), 3.98 - 3.88 (m, 1H), 3.22 - 3.12(m, 2H), 2.80(dd, J = 19.9, 8.6 Hz, 1H), 1.82 - 1.73(m, 1H), 1.69 - 1.58(m, 3H), 1.43(s, 9H), 1.42(s, 9H).

[0196]

[0197] Preparation of compound 25

[0198]

[0199] Compound 24 (860 mg, 1.72 mmol) was suspended in 1N HCl in 1,4-dioxane (16 mL) at room temperature and stirred at the same temperature for 18 h. The reaction mixture was concentrated under reduced pressure, and the residue was purified by reverse-phase silica gel chromatography (MeCN / DW, 10:1) to yield compound 25 (150 mg, 23%) as a white solid. R f = 0.22(MeCN / DW, 10:1);1 H NMR (600 MHz, Deuterium Oxide) δ 7.27(d, J = 7.1 Hz, 5H), 4.80(d, J = 8.4 Hz, 2H), 3.84(t, J = 6.4 Hz, 1H), 3.07(t, J = 6.9 Hz, 2H), 2.75(d, J = 20.2 Hz, 2H), 1.85(dtq, J = 39.5, 13.3, 7.8, 6.9 Hz, 2H), 1.69(tp, J = 12.2, 5.9 Hz, 1H), 1.60(ddp, J = 17.2, 11.5, 5.8 Hz, 1H).

[0200]

[0201] Preparation of compound 26

[0202]

[0203] Compound 25 (150 mg, 0.436 mmol) was suspended in water (14 mL), and NaHCO3 was added at room temperature to adjust the pH to 8-9. Then, Fmoc-OSu was suspended in 1,4-dioxane (14 mL), added at room temperature, and stirred at the same temperature for 18 h. The reaction mixture was concentrated under reduced pressure, and the residue was purified by reverse-phase silica gel chromatography (MeCN / DW, 7:1→5:1→3:1) to give compound 26 (73 mg, 30%) as a white solid. R f = 0.5(MeCN / DW, 1:1); 1H NMR (600 MHz, Deuterium Oxide) δ 7.69 (dd, J = 13.0, 7.6 Hz, 2H), 7.51 (dd, J = 26.6, 7.5 Hz, 2H), 7.33 - 7.07 (m, 9H), 4.74 (d, J = 8.2 Hz, 2H), 4.50(s, 1H), 4.34(dd, J = 10.9, 5.1 Hz, 1H), 4.10(t, J = 5.1 Hz, 1H), 3.57(t, J = 6.6 Hz, 1H), 2.85(t, J = 7.4 Hz, 2H), 2.71(s, 1H), 2.68(s, 1H), 1.48 - 1.37(m, 1H), 1.29(dq, J = 14.5, 7.8 Hz, 1H), 1.16(q, J = 7.7 Hz, 2H).

[0204]

[0205] Example 2. NMR analysis of pArg analogs

[0206] NMR analysis was performed on compounds 8, 10, 11, 13, and 24 to 26 prepared in Example 1.

[0207]

[0208] Example 2-1. Compounds 8, 10, 11, and 13

[0209] For compounds 8, 10, 11, and 13 1 H, 13 C, and 31 The results of P NMR spectra analysis were confirmed in the order of compounds 10, 11, 13, and 8, as shown in Figures 3a to 3j.

[0210]

[0211] Accordingly, the stable properties of compound 8 under acidic conditions were clearly demonstrated, especially as compound 8 was obtained in excellent yield after exposure to highly acidic conditions (TMSBr). In addition, compound 8 was designed to act as a building block by mimicking pArg, and considering the structural formula and specific functional groups, it is suggested that compound 8 of the present invention is ideal for specific incorporation into peptides capable of forming amide bonds with both amine and carboxylic acid moieties via appropriate protecting groups.

[0212]

[0213] Example 2-2. Compounds 24 to 26

[0214] For compounds 24 to 26 1 The results of H NMR spectra analysis were confirmed as shown in Figs. 3k to 3m.

[0215]

[0216] Example 3. Confirmation of similarity of pArg analogues to pArg

[0217] The structural and electronic properties of compound 8, a novel pArg analog prepared in Example 1, were compared with those of pArg. Specifically, the electrostatic charge potential Z was used to conduct a preliminary evaluation of the similarity between pArg and compound 8. Specifically, the electrostatic potential map was visualized using Jmol version 16.1.47. The chemical structures of pArg and compound 8 were described using the Jmol / JME 2D Molecular Editor, and the 3D structure was generated accordingly. The electrostatic map was rendered using the 'Molecular Electrostatic Potential' tool, and the range was set from -0.1 to 0.1.

[0218]

[0219] As a result, the structure and electrostatic potential map of pArg and compound 8 of the present invention were confirmed as shown in Fig. 4.

[0220] It is generally known that under physiological conditions, the side chain of pArg exists in a zwitterionic form with a net charge of -1 together with the α-amino acid moiety. In this case, one positive charge is distributed on the three nitrogen atoms of the planar guanidinium moiety, and the two negative charges are localized to the two oxygen anions of the phosphate group. In addition, it has a structural difference in that it contains sp3 hybridized CH2 units instead of the planar imidate found in natural pArg.

[0221]

[0222] Likewise, the side chain of compound 8, a pArg analog of the present invention, was also confirmed to exist in the form of a zwitterion with a net charge of -1 according to the pKa, and to maintain the α-amino acid structure. Accordingly, it was confirmed through Figure 4 that pArg and compound 8 exhibit high electronic and structural similarity.

[0223]

[0224] According to these results, it was confirmed that the present invention can successfully present the synthesis and preliminary characterization of compound 8, a new pArg analogue that is very similar to natural pArg. In particular, the compound of the present invention was found to maintain the α-amino acid structure even though the NP bond was replaced with a CP bond, and despite this minimal structural change, it was confirmed that it could function as an acid-stable analogue of pArg according to the isotropic theory, and also had an excellent level of electronic and structural similarity to pArg. Accordingly, it was proven that the preparation method of the present invention and the pArg analogue prepared therefrom can effectively complement the limitations of existing pArg analogues.

[0225]

[0226] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

[0227] The present invention relates to an acid-stable phosphoarginine analogue that maintains an α-amino acid structure, and provides a novel phosphoarginine analogue that is acid-stable while maintaining an α-amino acid structure, and a method for synthesizing the same. The phosphoarginine analogue of the present invention not only overcomes the shortcomings of acid instability and the absence of an α-amino acid structure found in examples in which the NP bond was replaced with a CP bond in the past attempts, but also shows extreme structural and electronic similarity to pArg, and is expected to be usefully utilized in a wide range of applications related to phosphorylated arginine, and thus its industrial applicability is recognized.

Claims

1. A phosphoarginine analog represented by the following chemical formula 1: [Chemical Formula 1] The above R1 is each independently one selected from the group consisting of hydrogen, a C1 to C6 alkyl group, a C1 to C6 heteroalkyl group, an aryl group, a benzyl group, and a heteroaryl group, The above R2 to R4 are each independently one selected from the group consisting of hydrogen and amine protecting groups, and the amine protecting group is characterized by one selected from the group consisting of Boc (tert-butyloxycarbonyl), Fmoc (9-Fluorenylmethyl carbamates), Cbz (Benzyl carbamates), Alloc (allyloxycarbonyl), Phtalimide, acetyl group (Ac), trityl group (Tr), and sulfonyl group, The above R5 is a substituted or unsubstituted hydroxyl group or a substituted or unsubstituted C1 to C6 alkoxy group, and the substitution or unsubstitution is characterized by being substituted or unsubstituted with a C1 to C6 alkyl group, a C1 to C6 heteroalkyl group, an aryl group, or a heteroaryl group. n is 0 to 3.

2. In paragraph 1, An arginine phosphate analogue, wherein the above R1 is any one selected from the group consisting of hydrogen, a C1 to C6 alkyl group, and a benzyl group.

3. In paragraph 1, An arginine phosphate analogue, wherein the amine protecting group is any one selected from the group consisting of Boc (tert-butyloxycarbonyl) and Fmoc (9-Fluorenylmethyl carbamates).

4. In paragraph 1, An arginine phosphate analogue, wherein the above R5 is any one selected from the group consisting of an unsubstituted hydroxyl group and a C1 to C6 alkoxy group substituted with a C1 to C6 alkyl group.

5. In paragraph 1, The above phosphoarginine analogue comprises at least one selected from the group consisting of compounds consisting of: , , , , , , , and .

6. In paragraph 1, The above phosphoarginine analogue is a phosphoarginine analogue comprising an α-amino acid structure.

7. In paragraph 1, The above phosphoarginine analogue is characterized by acid stability.

8. In paragraph 1, A phosphoarginine analogue, characterized in that the above phosphoarginine analogue contains a CP bond (Carbon-Phosphorus bond) and does not contain a NP bond (Nitrogen-Phosphorus bond).

9. A method for producing a phosphoarginine analog comprising the following steps: (S1) A step of producing an intermediate, methyl thioamide salt, by replacing the nitrile group of diethyl(cyanomethyl)phosphonate with a thioamide group; (S2) a step of reacting the methyl thioamide salt with a compound containing an amine protecting group to prepare an intermediate having a protected amine group; and (S3) A step of removing at least one from the group consisting of an amine protecting group, an aryl group, and an ethyl group of the intermediate in which the amine group is protected obtained in the step (S2).

10. A method for producing a phosphoarginine analog comprising the following steps: (Sa) A step of producing an intermediate, cyanomethyl phosphonate, by replacing hydrogen of dibenzyl phosphonate with chlorine; (Sb) A step of reacting the above cyanomethyl phosphonate with a compound containing an amine protecting group to produce an intermediate having a protected amine group; (Sc) a step of removing at least one of the amine protecting group, aryl group, and ethyl group of the intermediate in which the amine group is protected obtained in the above step (Sb); and (Sd) A step of introducing an amine protecting group to the amine group of the compound obtained in the above (Sc) step.

11. A kit for preparing a phosphoarginine analog, comprising at least one selected from the group consisting of diethyl(cyanomethyl)phosphonate and dibenzyl phosphonate; and instructions. A kit for manufacturing a phosphoarginine analog, wherein the above description teaches a manufacturing method of any one of claims 9 or 10.

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