WO2020088053A1 - Synthetic route towards teixobactin and analogue thereof - Google Patents

Abstract

Disclosed are a synthetic route towards teixobactin and an analogue thereof, and method for preparing unnatural amino acid. The method comprises: oxidative ring closure processing of a compound of formula (I), wherein R5 is independently Fmoc, Boc, Phth and Cbz, and R6 is independently H, Fmoc, Boc, Phth and Cbz. The initially provided method implements oxidative ring closure reaction for obtaining a high yield of L-allo-Enduracidine (End), an unnatural amino acid, low byproducts and facilitates separation and purification.

Description

Synthetic route of antibiotic teixobactin and its analogues

Priority information

This application requests the priority and rights of the patent application with the patent application number 201811290315.9 filed with the State Intellectual Property Office of China on October 31, 2018, and the full text of which is hereby incorporated by reference.

Technical field

The present invention relates to the field of medicinal chemistry. Specifically, the present invention relates to a method for preparing new antibiotics for the treatment of drug-resistant Gram-positive bacteria and tuberculosis. More specifically, the present invention relates to the synthetic route of antibiotics teixobactin and its analogs.

Background technique

Gram-positive bacterial infections are common and frequently-occurring diseases that endanger human health. In recent years, Gram-positive cocci infections have increased day by day, and the detection rate of methicillin-resistant Staphylococcus aureus (MRSA) has increased. Penicillin-resistant Streptococcus pneumoniae (PRSP) has spread in many countries and regions. The emergence of antibiotic-resistant vancomycin-resistant enterococci (VRE), multi-drug tuberculosis has increased. In order to effectively control these existing antibiotic and antimicrobial drug-resistant bacterial infections, research and development of drugs to treat Gram-positive drug-resistant bacterial infections has become a worldwide concern.

At present, bacteria can be mutated to obtain drug resistance when they are exposed to antibacterial drugs. There are four main mechanisms: ① production of antibiotic enzymes and inactivation of antibiotics; ② variation of target position, no response to drugs; Drug entry; ④Enhance efflux and accelerate the pumping out of the drug into the bacteria. In the "encounter battle" with antibacterial drugs, the surviving bacteria have accumulated a wealth of "combat experience" and become resistant varieties, and then evolved cross-resistance, multi-resistance, pan-resistance, and full resistance "Super bacteria" such as medicine. Not long ago, a joint study by Chinese and foreign researchers whose results were published in the new issue of "The Lancet Infectious Diseases" attracted attention. The study shows that there is a special gene MCR-1, the bacteria carrying this gene show strong resistance to polymyxin, and this resistance can also be quickly transferred to other strains, which means a new The "super bacteria" was found.

In 2015, the detection rate of Escherichia coli in China to the third-generation cephalosporins was 59%, and the detection rate of quinolones was 53.5%. The detection rate of Klebsiella pneumoniae to the third-generation cephalosporin-resistant bacteria was 36.5%, and the detection rate of methicillin-resistant Staphylococcus aureus was 35.8%. The detection rate of these representative drug-resistant bacteria Are at a high level. It should be pointed out that vancomycin-resistant Enterococcus faecium has become a major challenge in developed countries, and the bloodstream infection rate of Enterococcus faecium vancomycin in ICU patients in the US is as high as 80.7%. Globally, the average detection rate of vancomycin resistance in Enterococcus faecium in North America is as high as 66.8%, and in Latin America is 39.4%. Some multi-drug resistant and pan-resistant bacteria have pushed the clinic to a desperate situation, and it is urgent to accelerate the development of new antibacterial drugs.

Carbapenem antibiotics are currently recognized as the "ultimate line of defense" for bacteria, and are often used to treat severe infections caused by multi-drug resistant bacteria. In recent years, the spread of carbapenem-resistant bacteria through plasmids has caused serious bacterial resistance problems, including the recently discovered colistin resistance. Because of their high toxicity, these life-saving drugs are often limited in clinical use. Therefore, there is a need to provide an antibacterial compound with a novel structure. In 2015, an antibiotic research work published in Nature attracted positive reactions worldwide. Professor Kim Lewis of Northeastern University in the United States discovered the first new antibiotic, teixobactin, in the past 30 years through iChip technology, which can kill many deadly pathogens such as MRSA and Mycobacterium tuberculosis. This study not only presented a novel antibiotic structure, but also brought hope to the emergence of new non-resistant antibiotics in the future, and even called teixobactin as a super antibiotic. Teixobactin is an undecapeptide with a certain complexity. The structure contains a cyclic tetrapeptide formed by a lactone bond between the C-terminal 8 threonine and the 11 isoleucine. In addition to four D-type amino acids and one N-methylphenylalanine, the molecule also contains an L-allo-Enduracidine (End) unnatural amino acid. In vitro activity experiments show that teixobactin has a MIC of 0.31 ～ 0.125ug / mL against different Mycobacterium tuberculosis, and can effectively inhibit drug-resistant Mycobacterium tuberculosis; and teixobactin will not cause cytotoxicity, hemolysis, hERG inhibition, genotoxicity, etc .; initial in vitro Pharmacokinetic studies have shown that the half-life is more appropriate.

Therefore, constructing an efficient convergent synthetic route is an important means to study the structure-activity relationship of teixobactin. Among them, the L-allo-Enduracidine (End) unnatural amino acid plays an important role in antibacterial activity, but its structure is complicated, and there is currently no simple synthesis strategy. Furthermore, the total synthesis of teixobactin is more difficult.

Therefore, the siixobactin and its analogues are all simplified, especially the simplification of End's synthetic route.

Summary of the invention

The synthesis difficulties of Teixobactin are mainly the synthesis of the unnatural amino acid L-allo-enduracididine (End) and the construction of a 13-membered ring. Among them, regarding the synthesis of End, there are currently several synthetic routes in the prior art:

1) Rudolph and colleagues obtained L-allo-enduracididine in four steps from the starting materials (shown below). However, the diastereomer of L-allo-enduracididine, L-enduracididine, was also synthesized, and the ratio to L-allo-enduracididine was 1: 6, which caused subsequent separation difficulties.

2) Yuan and his colleagues established a highly stereoselective method for the synthesis of L-allo-enduracididine. The synthetic route uses trans-hydroxyproline as the starting material, and the target product is obtained after ten steps of reaction, with a yield of about 31%, and the diastereomer ratio even exceeds 50: 1 (as shown below). However, this method has a long synthetic route and low yield.

3) Payne and his research team completed the synthesis of L-allo-enduracididine protected by double Cbz (as shown below). This synthesis method uses Boc-L-Asp-OtBu as the starting material, and obtains L-allo-enduracididine protected by Fmoc and double Cbz through a series of reactions. The difficulty of this synthesis is the stereoselective reduction of the carbonyl group to an alcoholic hydroxyl group. This route uses lithium tri-sec-butylborohydride as a reducing agent, and two products (2S, 4R) and (2S, 4S) are obtained, the ratio of which is 5: 1. However, the two products are a pair of diastereomers, and only need to pass Flash column chromatography to obtain a single product.

It can be known from the above method that the related art unnatural amino acid End has a long synthetic route and is difficult to separate and purify. Furthermore, the total synthesis of teixobactin and its analogues has also been constrained from development. It also faces complex synthesis routes, many by-products in the synthesis process, difficulty in separation and purification, low yield, and is not suitable for industrial production.

Based on the discovery and recognition of the above facts and problems, the inventor has proposed a new synthetic route of L-allo-Enduracidine (End) unnatural amino acid. The operation is simple, the product can be obtained quickly and conveniently, and the yield is high. At the same time, based on the proposal of the synthetic route of the above End, the inventor also proposed a new total synthesis strategy of teixobactin and its analogues. The synthesis process has fewer by-products, simple separation and purification, high yield, and is suitable for industrial production.

In the first aspect of the present invention, the present invention proposes a method for preparing unnatural amino acids. According to an embodiment of the present invention, the method includes: subjecting the compound represented by formula (1) to oxidative ring-closure treatment,

Among them, R _{5 is} independently Fmoc, Boc, Phth, and Cbz, and R _{6 is} independently H, Fmoc, Boc, Phth, and Cbz. The inventor proposed for the first time that L-allo-Enduracidine (End) unnatural amino acids can be obtained in one step using an oxidative ring-closing reaction, with high yield, few by-products, and simple separation and purification.

According to an embodiment of the present invention, the above method may further include at least one of the following additional technical features:

According to an embodiment of the present invention, the method further includes deprotecting the oxidation ring-closure treatment product to obtain the compound represented by formula (I),

The deprotection treatment refers to the removal of R ⁵ and R ⁶ substituents.

According to an embodiment of the present invention, the oxidation ring-closure treatment is performed under the condition of an oxidant.

According to an embodiment of the present invention, the oxidizing agent includes at least one selected from elemental iodine, NIS, iodophthalic acid, NBS, Dess-Martin oxidizing agent (Dess-Martin high iodine reagent) and trivalent iodine oxidizing agent.

It should be noted that the amount of the oxidizing agent is not particularly limited as long as it satisfies the oxidation ring-closure reaction of the compound represented by formula (I). According to an embodiment of the present invention, the molar ratio of the compound represented by formula (1) to the oxidant is 1: (1-5).

According to an embodiment of the present invention, the molar ratio of the compound represented by formula (1) to the oxidant is 1: 3.

According to an embodiment of the present invention, the deprotection treatment is performed under acidic conditions. Under acidic conditions, it can simultaneously remove R ₅ and R ₆ groups.

According to an embodiment of the present invention, the acidic conditions are provided by TFA / H ₂ O. Under the conditions of TFA / H ₂ O, the efficiency of removing R ₅ and R ₆ groups is higher.

In the second aspect of the present invention, the present invention provides a method for preparing the compound represented by formula (3). According to an embodiment of the present invention, the method includes: 1) performing an esterification reaction between the oxidized ring-closing product of the compound represented by formula (1) and R ₇ OH, so as to obtain the compound represented by formula (2), 2) combining (2) The compound shown in the above is subjected to a protective treatment to obtain the compound shown in formula (3),

Among them, R ₇ is methyl, ethyl, or propyl. Through the introduction of protective groups, a raw material for the subsequent synthesis of teixobactin and its analogs is obtained. The compound obtained according to the method of the embodiment of the present invention can be further used in the total synthesis of teixobactin and its analogs, which greatly simplifies the total synthesis route and improves the yield.

According to an embodiment of the present invention, the esterification reaction is carried out under acidic conditions. As a result, the esterification efficiency is high and there are few by-products.

According to an embodiment of the present invention, the acidic conditions are provided by formic acid or acetic acid. As a result, the esterification efficiency is higher and there are fewer by-products.

According to an embodiment of the present invention, the upper protection treatment is carried out in the presence of a leaving group with an R ₆ group.

According to an embodiment of the present invention, the leaving group bearing the R ₆ group is Cbz-OSu or Fmoc-Osu. This makes it possible to more effectively connect only the guanidine group to the protecting group R ⁶ .

According to an embodiment of the present invention, R _{5 is} different from R ₆ , and R ₅ is easier to remove than R ₆ . Thus, the ease of removal of the protecting group is used to control the groups required for subsequent attachment at the corresponding sites.

In the third aspect of the present invention, the present invention proposes a compound. According to an embodiment of the present invention, the compound has the structure represented by formula (3). The compound according to the embodiment of the present invention can be further used in the total synthesis of teixobactin and its analogs, which greatly simplifies the route of total synthesis and improves the yield.

In the fourth aspect of the present invention, the present invention proposes a compound. According to an embodiment of the present invention, the compound is obtained by the method described above. The compound according to the embodiment of the present invention can be further used in the total synthesis of teixobactin and its analogs, which greatly simplifies the route of total synthesis and improves the yield.

In the fifth aspect of the present invention, the present invention proposes a method for preparing the compound represented by formula (II) or the salt of the compound represented by formula (II). According to an embodiment of the present invention, the method includes: performing condensation treatment on the aforementioned compound with several amino acids, so as to obtain the compound represented by formula (II). The inventors for the first time proposed the synthetic route of the aforementioned L-allo-Enduracidine (End) unnatural amino acid and its derivatives. The operation is simple, the product can be obtained quickly and conveniently, and the yield is high. In the total synthesis of the product, there are few by-products in the synthesis process, simple separation and purification, high yield, suitable for industrial production.

It should be noted that, according to the different protecting groups of R ⁵ , R ⁶ , and R ⁷ , those skilled in the art can appropriately adjust the removal reagents for removing the above groups to perform the corresponding operations according to the embodiments of the present invention. According to a specific embodiment of the present invention, the R ⁵ is a Fmoc group, the R ⁶ is a Cbz group, and is connected to the guanidine nitrogen, the R ⁷ is a methyl group, the method further includes: 1 ) The above-mentioned compound is subjected to de-Fmoc group treatment; 2) The condensation reaction of the product obtained in step 1) with Alloc-Ala-OH is performed to obtain the compound represented by formula (7); 3) The formula (7) ) The compound represented by the formula (8) is hydrolyzed to obtain the compound represented by formula (8); 4) The compound represented by formula (6) and the compound represented by formula (8) are subjected to condensation reaction to obtain the compound represented by formula (9) Compound; 5) The compound represented by the formula (9) is subjected to the Allyl group and Alloc group removal treatment: 6) The step 5) removal treatment product is subjected to condensation treatment, so as to obtain the compound represented by the formula (10); 7) The compound represented by the formula (10) and the compound represented by the formula (11) are subjected to condensation treatment to obtain the compound represented by the formula (II);

Wherein, R ₁ is hydrogen, C _{1 ~ 15} alkyl group, C _{2 ~ 15} alkylene group, C _{2 ~ 15} alkynyl group, C _6-10 aryl group or an acyl group;

R ₂ is hydrogen, halo, C _{1 ~ 15} alkyl group, C _{2 ~ 15} alkylene group, C _{2 ~ 15} alkynyl group, C _6-10 aryl group or an acyl group, wherein the C _6-10 aryl group may optionally be 1 ～ 5 Rx substitutions;

Rx is halogen, C _1-6 alkyl, halogenated C _1-6 alkyl;

R ₃ is amino, guanidino, urea or formamide;

R ₄ is C _{1 ~ 15} alkyl group, C _{2 ~ 15} alkylene group, C _{2 ~ 15} alkynyl group, C _6-10 aryl group;

X is O, S or NH. The inventor for the first time proposed the above-mentioned new total synthesis strategy of teixobactin and its analogues. The synthesis process has fewer by-products, simple separation and purification, high yield, and is suitable for industrial production.

According to an embodiment of the present invention, the step 7) further includes: 7-1) subjecting the compound represented by the formula (10) to Boc group removal treatment; 7-2) the removal obtained in step 7-1) The compound except for the Boc group undergoes a condensation reaction with the compound of formula (11); 7-3) The condensation product obtained in step 7-2) is subjected to Cbz group, t-Bu group and Boc group removal treatment in order to obtain The compound represented by formula (II).

According to an embodiment of the present invention, the removal treatment of the Boc group in the step 7-1) is performed under the condition of an ethyl acetate hydrochloric acid solution for 15 to 20 minutes. Therefore, the removal efficiency of the Boc group is high, and it does not affect other protecting groups, such as t-Bu and Cbz.

According to an embodiment of the present invention, the concentration of the hydrochloric acid ethyl acetate solution is 2M-3M. Thus, the removal efficiency of the Boc group is higher.

According to an embodiment of the present invention, the condensation reaction in step 7-2) is performed under the conditions of DEPBT / DIEA.

According to an embodiment of the present invention, the molar ratio of formula (10), DEPBT, and DIEA in step 7-2) is 1: 1: 1.

According to an embodiment of the present invention, the removal treatment of the Cbz group, t-Bu group and Boc group in step 7-3) is between trifluoroacetic acid, trifluoromethanesulfonic acid, anisole and It is carried out under a mixed solvent of cresol. Thereby, the removal of the Cbz group, t-Bu group and Boc group can be performed simultaneously, and the removal efficiency is high.

According to an embodiment of the present invention, the volume ratio of the trifluoroacetic acid, trifluoromethanesulfonic acid, anisole, and m-cresol is (70 ± 5) :( 12 ± 5) :( 10 ± 5) :( 8 ± 5). Thus, the removal efficiency is high.

According to an embodiment of the present invention, the volume ratio of trifluoroacetic acid, trifluoromethanesulfonic acid, anisole, and m-cresol is 70: 12: 10: 8. Thus, the removal efficiency is higher.

According to an embodiment of the present invention, the removal of the Fmoc group in the step 1) is carried out under the condition of diethylamine. Thus, only the Fmoc group is removed without affecting the removal of other protective groups such as Cbz group and methyl ester group.

According to an embodiment of the present invention, the condensation reaction in the step 2) is carried out under the condition that the condensation agent is HATU / DIEA or PyAOP, and the solvent is a mixed solvent of dichloromethane and DMF.

According to an embodiment of the present invention, the molar ratio of the compound shown in claim 5 or 6 in the step 2), Alloc-Ala-OH, HATU, and DIEA is 1: 2: 2: 2. Thereby, the condensation efficiency is further improved.

According to an embodiment of the present invention, the hydrolysis reaction in the step 3) is carried out under the condition that the mixed solvent of tetrahydrofuran / water and the base is LiOH. Thus, only the methyl ester group can be hydrolyzed without affecting other protecting groups such as Cbz group and Alloc group.

According to an embodiment of the present invention, the molar ratio of the compound represented by formula (7) to LiOH is 1: 1.5. Thereby, the hydrolysis efficiency is further improved.

According to an embodiment of the present invention, the condensation reaction in the step 4) is carried out under the condition that the condensation agent is DEPBT / DIEA and the solvent is a mixed solvent of THF and DMF.

According to an embodiment of the present invention, the molar ratio of the compound represented by formula (6), the compound represented by formula (8), DEPBT, and DIEA in step 4) is 1: 0.5: 1: 1. Thereby, the condensation efficiency is further improved.

According to an embodiment of the present invention, the removal treatment in step 5) is carried out under acidic conditions provided by tetratriphenylphosphine palladium catalyst and 1,3-dimethylbarbituric acid. Thus, the Allyl group and the Alloc group can be removed at the same time, and the removal efficiency is high.

According to an embodiment of the present invention, the molar ratio of the compound represented by formula (9), tetratriphenylphosphine palladium, and 1,3-dimethylbarbituric acid in step 5) is 0.3: 0.06: 0.6. Thus, the removal efficiency is further improved.

According to an embodiment of the present invention, the condensation reaction in the step 6) is carried out under the condition that the condensation agent is HATU / HOAT / DIEA or PyAop.

According to an embodiment of the present invention, the molar ratio of the compound represented by formula (9), HATU, HOAT, and DIEA in step 6) is 0.3: 1: 1: 2. Thus, the condensation efficiency is high.

According to an embodiment of the present invention, the salt is hydrochloride, trifluoroacetate, acetate, or sulfonate.

According to an embodiment of the present invention, R ₁ is hydrogen, C _{1 ~ 10} alkyl group, C _{2 ~ 10} alkylene group, C _{2 ~ 10} alkynyl group, C _6-8 acyl group or an aryl group;

R ₂ is hydrogen, halo, C _{1 ~ 10} alkyl group, C _{2 ~ 10} alkylene group, C _{2 ~ 10} alkynyl group, C _6-8 acyl or aryl group, wherein the C _6-8 aryl group can be optionally 1 ～ 5 Rx substitutions;

Rx is halogen, C _1-6 alkyl, halogenated C _1-6 alkyl;

R ₄ is C _{1 ~ 10} alkyl group, C _{2 ~ 10} alkylene group, C _{2 ~ 10} alkynyl group, C _6-8 aryl group.

According to an embodiment of the present invention, R ₁ is hydrogen, C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _6-7 aryl or acyl;

R ₂ is hydrogen, halogen, C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _6-7 aryl or acyl, wherein the C _6-7 aryl or C _{5- 6} Heteroaryl groups can be optionally substituted with 1 to 3 Rx;

Rx is halogen, C _1-4 alkyl, halogenated C _1-4 alkyl;

R ₄ is C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl, and C _6-7 aryl.

According to an embodiment of the present invention, R ₁ is hydrogen, methyl, ethyl, n-propyl, isopropyl, butyl, vinyl, propenyl, allyl, ethynyl, propynyl, propargyl, Phenyl, benzyl or acyl;

R ₂ is hydrogen, halogen, methyl, ethyl, n-propyl, isopropyl, butyl, vinyl, propenyl, allyl, ethynyl, propynyl, propargyl, phenyl, benzyl Or acyl, wherein the phenyl or benzyl can be optionally substituted with 1 to 3 Rx;

Rx is halogen, methyl, ethyl, n-propyl, isopropyl, butyl, monofluoromethyl, difluoromethyl, trifluoromethyl;

R ₄ is methyl, ethyl, n-propyl, isopropyl, butyl, vinyl, propenyl, allyl, ethynyl, propynyl, propargyl, phenyl, benzyl.

According to an embodiment of the present invention, the compound represented by formula (6) is obtained by the following steps: esterification, aminolysis, and removal of Fmoc group between the compound represented by formula (5) and Fmoc-L-isoleucine Group reaction to obtain the compound represented by formula (6).

According to an embodiment of the present invention, the esterification reaction is carried out under the condition that the condensing agent is EDCI and the catalyst is DMAP.

According to an embodiment of the present invention, the molar ratio of the compound represented by formula (5), Fmoc-L-isoleucine, EDCI, and DMAP is 9: 10: 10: 1.

According to an embodiment of the present invention, the ammonolysis and Fmoc group removal reactions are carried out under the conditions of a diethylamine solution.

According to an embodiment of the present invention, the concentration of diethylamine is 33%.

According to an embodiment of the present invention, the compound represented by formula (5) is obtained by the following steps: the compound represented by formula (4) is subjected to a condensation reaction with Boc-Ser (tBu) -OH to obtain the formula Compound.

According to an embodiment of the present invention, the condensation reaction is carried out in a mixed solvent of dichloromethane and DMF with HCTU / DIEA as the condensing agent;

According to an embodiment of the present invention, the compound represented by formula (11) is obtained by a solid-phase synthesis method. Thus, the synthesis is simple and efficient.

According to an embodiment of the present invention, the solid phase synthesis method is performed by the following steps:

A) A substitution reaction between 2-Cl resin and Fmoc-L-isoleucine is performed to obtain the compound represented by formula (a), wherein the circle represents the resin;

B) Combine the compound represented by formula (a) with N-Fmoc-N'-trityl-D-glutamine, Fmoc-L-serine, Fmoc-L-isoleucine, Boc-D-4 , 4'-diphenylphenylalanine and N-tert-butoxycarbonyl-N-methyl-D-phenylalanine in the terminal amino acid condensation reaction, so as to obtain the compound represented by formula (b);

C) The compound represented by formula (b) is subjected to a resin-removing reaction to obtain the compound represented by formula (11);

According to an embodiment of the present invention, the 2-Cl resin is subjected to an activation treatment in advance, and the activation treatment is performed in dichloromethane and DMF for 20 minutes.

According to an embodiment of the present invention, after the activation treatment and before the coupling reaction, a solvent extraction treatment is further included.

According to an embodiment of the present invention, the coupling reaction in step 1) is carried out under the condition that DIEA is a condensing agent and DMF is a solvent.

According to an embodiment of the present invention, before each condensation reaction in step 2), the raw materials to be condensed need to be de-Fmoc treated in advance, and the de-Fmoc treatment is performed in a piperidine solution.

According to an embodiment of the present invention, the condensation reaction in step 2) is carried out under the condition that the condensation agent is HATU / DIEA and room temperature.

According to the method of the embodiment of the present invention, the deprotection and upper protection reaction (orthogonal protection strategy) strategies are used to successfully realize the introduction of different reactive protective groups at different reaction sites, and then use the protection of different reactive activities The removal order of the groups introduces different groups at different reaction sites. Furthermore, the method according to the embodiment of the present invention is used to synthesize intermediates having different protecting groups, such as compounds represented by formulas (7), (8), etc. The operation is simple, the product can be conveniently and quickly obtained, and the yield is high, and the above The compound is applied to the total synthesis of teixobactin and its analogues. The synthesis process has few by-products, simple separation and purification, high yield, and is suitable for industrial production.

detailed description

The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are intended to explain the present invention, and should not be construed as limiting the present invention.

It should be noted that the condensation reaction of the present invention can select a conventional condensing agent to perform the condensation reaction. The yield may vary with different condensing agents, but as long as the condensation reaction can be completed, they are all protected by the present invention Within range.

It should be noted that if any step in the overall synthesis route of the present invention can be commercially purchased raw materials, the commercially purchased raw materials can be used for the reaction, or the method proposed in the present invention can be used to synthesize the raw materials for the reaction. The raw materials of the present invention obtained by other methods can be used for the reaction.

It should be noted that the serial number of the present application does not limit the actual synthesis sequence, and those skilled in the art can adjust it according to their needs in order to obtain the desired target product.

Unless otherwise specified, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art to which the present invention belongs. All patents and publications related to the present invention are incorporated by reference in their entirety.

Unless otherwise stated, the following definitions used herein shall apply. For the purposes of the present invention, chemical elements are consistent with the CAS version of the periodic table of elements, and the Handbook of Chemistry and Physics, 75th edition, 1994. In addition, the general principles of organic chemistry can refer to the descriptions in "Organic Chemistry", Thomas Sorrell, University Science, Books, Sausalito: 1999, and "March's Advanced Organic Chemistry" by Michael B. Smith and Jerry March, John Wiley & Sons, New York: 2007 , The entire contents of which are incorporated herein by reference.

Unless otherwise stated or there is a clear conflict in the context, the articles "a", "an" and "said" as used herein are intended to include "at least one" or "one or more". Therefore, the articles used herein refer to one or more than one (ie, at least one) object articles. For example, "a component" refers to one or more components, that is, there may be more than one component that is considered to be employed or used in the implementation of the embodiment.

As described in the present invention, the compounds of the present invention may be optionally substituted with one or more substituents, such as the compounds of the general formula above, or like the specific examples, subclasses, and inclusions of the present invention in the examples. A class of compounds. It should be understood that the term "optionally substituted" can be used interchangeably with the term "substituted or unsubstituted". In general, the term "substituted" means that one or more hydrogen atoms in a given structure are replaced with specific substituents. Unless otherwise indicated, an optional substituent may be substituted at each substitutable position of the group. When more than one position in the given structural formula can be substituted with one or more substituents selected from specific groups, then the substituents can be substituted at the same positions or at different positions.

In addition, it should be noted that, unless it is clearly indicated in other ways, the description methods "each ... independently" and "... independently" and "... independently" can be interchangeable in the present invention. It should be understood in a broad sense. It can mean that the specific options expressed between the same symbols in different groups do not affect each other, or it can mean the specific options expressed between the same symbols in the same group. Do not affect each other.

In each part of this specification, the substituents of the compounds disclosed in the present invention are disclosed according to the type or range of groups. In particular, the present invention includes each independent sub-combination of each member of these group types and ranges. For example, the term "C ₁ - ₆ alkyl" refers particularly to the disclosure independently methyl, ethyl, C ₃ alkyl, C ₄ alkyl, C ₅ alkyl, and C ₆ alkyl.

In various parts of the invention, linking substituents are described. When the structure clearly requires a linking group, the Markush variables listed for the group should be understood as the linking group. For example, if the structure requires a linking group and the Markush group definition for the variable lists "alkyl" or "aryl", it should be understood that the "alkyl" or "aryl" represents the linked group, respectively An alkylene group or an arylene group.

The term "alkyl" or "alkyl group" used in the present invention means a saturated linear or branched monovalent hydrocarbon group containing 1 to 20 carbon atoms, wherein the alkyl group may be optionally Are replaced by one or more substituents described in this invention. Unless otherwise specified, alkyl groups contain 1-20 carbon atoms. In one embodiment, the alkyl group contains 1-15 carbon atoms; in another embodiment, the alkyl group contains 1-6 carbon atoms; in yet another embodiment, the alkyl group contains 1 -4 carbon atoms; in still another embodiment, the alkyl group contains 1-3 carbon atoms.

Examples of alkyl groups include, but are not limited to, methyl (Me, -CH ₃ ), ethyl (Et, -CH ₂ CH ₃ ), n-propyl (n-Pr, -CH ₂ CH ₂ CH ₃ ), Isopropyl (i-Pr, -CH (CH ₃ ) ₂ ), n-butyl (n-Bu, -CH ₂ CH ₂ CH ₂ CH ₃ ), isobutyl (i-Bu, -CH ₂ CH (CH ₃ ) ₂ ), sec-butyl (s-Bu, -CH (CH ₃ ) CH ₂ CH ₃ ), tert-butyl (t-Bu, -C (CH ₃ ) ₃ ), n-pentyl (-CH ₂ CH ₂ CH ₂ CH ₂ CH ₃ ), 2-pentyl (-CH (CH ₃ ) CH ₂ CH ₂ CH ₃ ), 3-pentyl (-CH (CH ₂ CH ₃ ) ₂ ), 2-methyl -2-butyl (-C (CH ₃ ) ₂ CH ₂ CH ₃ ), 3-methyl-2-butyl (-CH (CH ₃ ) CH (CH ₃ ) ₂ ), 3-methyl-1- Butyl (-CH ₂ CH ₂ CH (CH ₃ ) ₂ ), 2-methyl-1-butyl (-CH ₂ CH (CH ₃ ) CH ₂ CH ₃ ), n-hexyl (-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₃ ), 2-hexyl (-CH (CH ₃ ) CH ₂ CH ₂ CH ₂ CH ₃ ), 3-hexyl (-CH (CH ₂ CH ₃ ) (CH ₂ CH ₂ CH ₃ )), 2-methyl-2-pentyl (-C (CH ₃ ) ₂ CH ₂ CH ₂ CH ₃ ), 3-methyl-2-pentyl (-CH (CH ₃ ) CH (CH ₃ ) CH ₂ CH ₃ ), 4-methyl-2-pentyl (-CH (CH ₃ ) CH ₂ CH (CH ₃ ) ₂ ), 3-methyl-3-pentyl (-C (CH ₃ ) (CH ₂ CH ₃ ) _2), 2-methyl-3-pentyl _{(-CH (CH 2 CH 3)} CH (CH 3) 2), 2,3- dimethyl 2-butyl _{(-C (CH 3) 2 CH} (CH 3) 2), 3,3- dimethyl-2-butyl _{(-CH (CH 3) C (} CH 3) 3), n-heptyl Base, n-octyl, etc.

The term "alkylene" means a saturated divalent hydrocarbon group obtained by removing two hydrogen atoms from a saturated linear or branched hydrocarbon group. Unless otherwise specified, the alkylene group contains 1-12 carbon atoms. In one embodiment, the alkylene group contains 1-6 carbon atoms; in another embodiment, the alkylene group contains 1-4 carbon atoms; in yet another embodiment, the alkylene group The group contains 1-3 carbon atoms; also in one embodiment, the alkylene group contains 1-2 carbon atoms. Such examples include methylene (-CH _2- ), ethylene (-CH ₂ CH _2- ), isopropylidene (-CH (CH ₃ ) CH _2- ) and the like.

The term "alkenyl" refers to a straight-chain or branched monovalent hydrocarbon group containing 2-15 carbon atoms, in which there is at least one site of unsaturation, that is, there is a carbon-carbon sp ² double bond, wherein the alkenyl group The group may be optionally substituted with one or more substituents described in the present invention, which includes the positioning of "cis" and "tans", or the positioning of "E" and "Z". In one embodiment, the alkenyl group contains 2-8 carbon atoms; in another embodiment, the alkenyl group contains 2-6 carbon atoms; in yet another embodiment, the alkenyl group contains 2 -4 carbon atoms. Examples of alkenyl groups include, but are not limited to, vinyl (-CH = CH ₂ ), allyl (-CH ₂ CH = CH ₂ ), and the like.

The term "alkynyl" refers to a straight-chain or branched monovalent hydrocarbon group containing 2-15 carbon atoms, in which there is at least one unsaturated site, that is, there is a carbon-carbon sp triple bond, wherein, the alkynyl group It may be optionally substituted with one or more substituents described in the present invention. In one embodiment, the alkynyl group contains 2-8 carbon atoms; in another embodiment, the alkynyl group contains 2-6 carbon atoms; in yet another embodiment, the alkynyl group contains 2 -4 carbon atoms. Examples of alkynyl groups include, but are not limited to, ethynyl (-C≡CH), propargyl (-CH ₂ C≡CH), 1-propynyl (-C≡C-CH ₃ ), etc. .

The term "aryl" refers to monocyclic, bicyclic and tricyclic carbocyclic systems containing 6-14 ring atoms, or 6-12 ring atoms, or 6-10 ring atoms, wherein at least one ring system is aromatic Family, where each ring system contains a ring of 3-7 atoms, and one or more attachment points are connected to the rest of the molecule. The term "aryl" may be used interchangeably with the term "aromatic ring". Examples of aryl groups may include phenyl, naphthyl, and anthracene. The aryl group may be independently optionally substituted with one or more substituents described in the present invention.

As used herein, "pharmaceutically acceptable salts" refer to organic and inorganic salts of the compounds of the present invention. Pharmaceutically acceptable salts are well known in the art, as described in the literature: SMBerge et al., Describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66: 1-19. Salts formed by pharmaceutically acceptable non-toxic acids include, but are not limited to, inorganic acid salts formed by reaction with amino groups include hydrochloride, hydrobromide, phosphate, sulfate, perchlorate, And organic acid salts such as acetate, oxalate, maleate, tartrate, citrate, succinate, malonate, or other methods described in the literature such as ion exchange These salts. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphoric acid Salt, camphorsulfonate, cyclopentylpropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate Salt, gluconate, hemisulfate, enanthate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, Malate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, palmitate, paraben, pectate, persulfate, 3 -Phenylpropionate, picrate, pivalate, propionate, stearate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc. Salts obtained by suitable bases include alkali metal, alkaline earth metal, ammonium and N ⁺ (C _1-4 alkyl) ₄ salts. The present invention also contemplates the quaternary ammonium salt formed by any compound containing N groups. Water-soluble or oil-soluble or dispersed products can be obtained by quaternization. The alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Pharmaceutically acceptable salts further include suitable, non-toxic ammonium, quaternary ammonium salts and amine cations formed by counter-ions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, C _{1 -8} sulfonate and aromatic sulfonate.

Pharmaceutically acceptable acid addition salts can be formed with inorganic and organic acids, such as acetate, aspartate, benzoate, benzenesulfonate, bromide / hydrobromide, bicarbonate / Carbonate, bisulfate / sulfate, camphorsulfonate, chloride / hydrochloride, chlorotheophylline, citrate, ethanedisulfonate, fumarate, glucoheptonate, glucose Urate, glucuronate, hippurate, hydroiodide / iodide, isethionate, lactate, lactobionate, lauryl sulfate, malate, male Salt, malonate, mandelate, mesylate, methyl sulfate, naphthoate, naphthalenesulfonate, nicotinate, nitrate, octadecate, oleate, oxalate Salt, palmitate, parathionate, phosphate / hydrogen phosphate / dihydrogen phosphate, polygalactate, propionate, stearate, succinate, sulfosalicylate, tartrate , Tosylate and trifluoroacetate.

Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like.

Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid , Ethanesulfonic acid, p-toluenesulfonic acid, sulfosalicylic acid, etc.

Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases.

Inorganic bases from which salts can be derived include, for example, ammonium salts and metals from Groups I to XII of the periodic table. In certain embodiments, the salt is derived from sodium, potassium, ammonium, calcium, magnesium, iron, silver, zinc, and copper; particularly suitable salts include ammonium, potassium, sodium, calcium, and magnesium salts.

Organic bases from which salts can be derived include primary, secondary, and tertiary amines, and substituted amines include naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like. Certain organic amines include, for example, isopropylamine, benzathine, cholinate, diethanolamine, diethylamine, lysine, meglumine, piperazine, and tromethamine .

The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound, basic or acidic moieties using conventional chemical methods. In general, such salts can be prepared by reacting the free acid form of these compounds with a stoichiometric amount of a suitable base (such as Na, Ca, Mg, or K hydroxide, carbonate, bicarbonate, etc.), or by These compounds are prepared by reacting the free base forms of these compounds with a stoichiometric amount of a suitable acid. This type of reaction is usually carried out in water or an organic solvent or a mixture of both. Generally, where appropriate, non-aqueous media such as diethyl ether, ethyl acetate, ethanol, isopropanol, or acetonitrile need to be used. In, for example, "Remington's Pharmaceuticals", 20th edition, Mack Publishing Company, Easton, Pa., (1985); and "Handbook of medicinal salts: properties, selection and application (Handbook of Pharmaceuticals Salts: Properties, Selection, and Use) ", Stahl and Wermuth (Wiley-VCH, Weinheim, Germany, 2002) can find a list of other suitable salts.

In addition, the compounds disclosed in the present invention, including their salts, can also be obtained in the form of their hydrates or in the form of solvents containing them (eg, ethanol, DMSO, etc.) for their crystallization. The compounds disclosed in the present invention can form solvates inherently or by design with pharmaceutically acceptable solvents (including water); therefore, the present invention is intended to include both solvated and unsolvated forms.

Any structural formula given by the present invention is also intended to represent the forms in which these compounds are not isotopically enriched and in isotopically enriched forms. Isotope-enriched compounds have the structure depicted by the general formula given in this invention, except that one or more atoms are replaced with atoms having a selected atomic weight or mass number. Exemplary isotopes that can be incorporated into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, such as ² H, ³ H, ¹¹ C, ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁷ O , ¹⁸ O, ¹⁸ F, ³¹ P, ³² P, ³⁵ S, ³⁶ Cl and ¹²⁵ I.

On the other hand, the compounds described in the present invention include isotopically enriched compounds as defined in the present invention, for example, those in which radioactive isotopes such as ³ H, ¹⁴ C, and ¹⁸ F are present, or in which non-radioactive isotopes are present, such as ² H and ¹³ C. Such isotopically enriched compounds can be used in metabolic studies (using ¹⁴ C), reaction kinetic studies (using eg ² H or ³ H), detection or imaging techniques such as positron emission tomography (PET) or including drugs or Single-photon emission computed tomography (SPECT) for the determination of the distribution of substrate tissues may be used in patient radiotherapy. ¹⁸ F enriched compounds are particularly ideal for PET or SPECT studies. Isotope-enriched compounds represented by formula (I) can be prepared by conventional techniques familiar to those skilled in the art or as described in the examples and preparation procedures of the present invention by using appropriate isotope-labeled reagents instead of the unlabeled reagents originally used.

In addition, the substitution of heavier isotopes, especially deuterium (ie, ² H or D), may provide certain therapeutic advantages that are brought about by higher metabolic stability. For example, an increase in half-life in the body or a decrease in dosage requirements or an improvement in the therapeutic index. It should be understood that deuterium in the present invention is regarded as a substituent of the compound of formula (I). Isotope enrichment factors can be used to define the concentration of this type of heavier isotope, especially deuterium. As used herein, the term "isotopic enrichment factor" refers to the ratio between the isotopic abundance and the natural abundance of the specified isotope. If the substituent of the compound of the present invention is designated as deuterium, the compound has at least 3500 for each designated deuterium atom (52.5% deuterium incorporation at each designated deuterium atom), at least 4000 (60% deuterium incorporation), At least 4500 (67.5% deuterium incorporation), at least 5000 (75% deuterium incorporation), at least 5500 (82.5% deuterium incorporation), at least 6000 (90% deuterium incorporation), at least 6333.3 (95% of Deuterium incorporation), at least 6466.7 (97% deuterium incorporation), at least 6600 (99% deuterium incorporation) or at least 6633.3 (99.5% deuterium incorporation) isotope enrichment factor. The pharmaceutically acceptable solvates of the present invention include those in which the crystallization solvent may be isotopically substituted, such as D ₂ O, acetone-d ₆ , DMSO-d ₆ .

The abbreviations involved in the present invention are as follows:

HCTU: 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate

DIEA: N, N-diisopropylethylamine

EDCI carbodiimide

DMAP 4-dimethylaminopyridine

Fmoc-IIe-OH Fmoc-L-Isoleucine

DMF N, N-dimethylformamide

Et2OH Diethylamine

LiOH Lithium hydroxide

DEPBT 3- (diethoxyphosphoryloxy) -1,2,3-benzotriazin-4-one

Pd (PPh3) 4 Palladium tetraphenylphosphine

HATU 2- (7-benzotriazole) -N, N, N ', N'-tetramethylurea hexafluorophosphate

HOAT 1-hydroxy-7-azobenzotriazole

Example 1:

Conditions: a) i) 3equiv. I ₂ or NIS, 4 equiv. NaHCO ₃ .0 ℃, 45min; ii) MeOH / AcOH (9: 1), 30 ℃, 15min, 68% for 2steps; b) 2.5equiv. CbzOSu, 3.5equiv. DIEA, DCM, 30 ° C, 4h, 80%; c) i) 3equiv. LiOH, THF / H ₂ O; ii) Pd (OH) ₂ / C, H ₂ , MeOH.

Synthesis of compound represented by formula 2:

Add the compound (10 mmol) represented by Formula 1 to a round-bottom flask, add 20 mL of acetonitrile, add sodium bicarbonate solid (40 mmol) and stir at zero degree, slowly add elemental iodine (30 mmol) and stir for one hour, then spin dry acetonitrile; Next, add a mixed solution of acetic acid and methanol (acetic acid: methanol / 1: 9). After stirring at room temperature for 15 minutes, spin off the methanol. After washing with sodium bicarbonate aqueous solution, separate it through a silica gel column (dichloromethane: methanol / 20: 1) to obtain the target. Product, yield 71%. 1H NMR (400MHz, DMSO) δ (ppm) 7.90-7.88 (d, J = 7.48Hz, 2H), 7.82-7.80 (d, J = 7.8Hz, 1H), 7.71-7.69 (m, 2H), 7.43 7.31 (m, 8H), 5.21 (s, 2H), 4.36-4.26 (m, 3H), 4.22-4.21 (m, 1H), 3.97-3.92 (t, J = 9.48Hz, 1H), 3.87-3.80 ( m, 1H), 3.62 (s, 3H), 3.51-3.47 (m, 1H), 1.86-1.82 (m, 2H) .13C NMR (400MHz, DMSO) δ172.82,156.07,152.86,151.95,143.74,142.56,140.73 , 139.41,137.41,135.61,128.92,128.48,128.39,128.24,127.86,127.64,127.28,127.08,125.19,124.90,121.38,120.13,120.02,109.76,67.33,65.64,54.18,52.70,51.96,51.53,50.71,46 , 37.35.

Synthesis of the compound shown in Formula 3:

The compound represented by Formula 2 (2 mmol) was added to the round bottom flask, 10 ml of dichloromethane was added, CbzOSu (6 mmol) was added and stirred at room temperature, and N, N-diisopropylethylamine (6 mmol) was slowly added, 30 After stirring at ℃ for 5 hours, the solvent was spin-dried and separated through a normal phase silica gel column (dichloromethane: methanol / 20: 1) to obtain the target product with a yield of 80%. 1H NMR (400MHz, DMSO) δ (ppm) 7.89-7.87 (d, J = 7.52Hz, 2H), 7.84-7.82 (d, J = 7.92Hz, 1H), 7.71-7.69 (d, J = 7.40Hz, 2H), 7.48-7.46 (d, J = 6.56 Hz, 2H), 7.43-7.42 (m, 2H), 7.36-7.29 (m, 10H), 5.15 (s, 2H), 5.05 (s, 2H), 4.36 -4.35 (m, 2H), 4.24-4.23 (m, 2H), 4.00-3.95 (t, J = 9.56Hz, 1H), 3.84-3.81 (m, 1H), 3.62 (s, 3H), 2.12-2.08 (m, 1H), 1.88-1.81 (m, 1H).

Synthesis of compound represented by formula 5:

Place Boc-Ser (tBu) -OH in a round bottom flask, add dichloromethane and N, N-dimethylformamide as a mixed solvent 50ml, HCTU (6-chlorobenzotriazole-1,1,1, 3,3-tetramethylurea hexafluorophosphate) (11 mmol) and DIEA (N, N-diisopropylethylamine) (11 mmol) were added to the reaction solution, and then the compound represented by Formula 4 (10 mmol) was added, After stirring at room temperature for 3 hours, dilute hydrochloric acid was added to quench the reaction. Add 100ml of dichloromethane to dilute the reaction solution, wash with sodium bicarbonate and saturated aqueous solution of sodium chloride respectively, distill off the dichloromethane under reduced pressure, and separate the resulting product using a silica gel column (petroleum ether: ethyl acetate 2: 1). 5. The compound shown. ¹ H NMR (400 MHz, CDCl ₃ ) δ (ppm) 7.23-7.21 (d, J = 8.72 Hz, 1H), 5.92-5.85 (m, 1H), 5.44 (s, 1H), 5.34-5.29 (dd, J ₁ = 1.28 Hz, J ₂ = 17.2 Hz, 1H), 5.24-5.21 (dd, J ₁ = 1.04 Hz, J ₂ = 10.36 Hz, 1H), 4.66-4.64 (d, J = 7.44 Hz, 2H), 4.61 -4.58 (m, 1H), 4.31-4.30 (m, 1H), 4.20 (s, 1H), 3.76-3.75 (m, 1H), 3.45-3.41 (m, 1H), 1.46-1.43 (m, 9H) , 1.21-1.15 (m, 12H). ¹³ C NMR (400MHz, CDCl ₃ ) δ171.22,170.48,155.65,131.58,119.03,118.94,80.19,74.17,68.25,66.16,61.77,57.51,54.91,28.41,27.46,20.02 .

Synthesis of compound represented by formula 6:

Put Fmoc-L-isoleucine (10 mmol) in a round bottom flask, add EDCI (carbodiimide) (10 mmol), DMAP (4-dimethylaminopyridine) (1 mmol) to the reaction solution and stir for 20 min, After further adding the compound represented by Formula 5 (9 mmol) at room temperature and stirring overnight, dilute hydrochloric acid was added to quench the reaction. Add 100ml of dichloromethane to dilute the reaction solution, wash with sodium bicarbonate and saturated aqueous sodium chloride, distill off the dichloromethane under reduced pressure, add the intermediate to the round bottom flask, and add 30ml of 33% diethylamine solution. After the reaction was completed, the resulting product was separated using a silica gel column (petroleum ether: ethyl acetate 2: 1) to obtain compound 6 with a yield of 71%. ¹ H NMR (400 MHz, CDCl ₃ ) δ (ppm) 7.21-7.20 (s, 1H), 5.90-5.82 (m, 1H), 5.50-5.44 (m, 2H), 5.33-5.29 (d, J = 17.24 Hz , 1H), 5.26-5.23 (d, J = 9.36Hz, 1H), 4.86-4.83 (d, J = 10.36Hz, 1H), 4.64-4.52 (m, 2H), 4.24 (s, 1H), 3.80 ( s, 1H), 3.46-3.42 (m, 1H), 3.24-3.23 (d, J = 4.84Hz, 1H), 1.68-1.67 (s, 1H), 1.60 (s, 1H), 1.45 (s, 9H) , 1.29-1.28 (d, J = 6.40 Hz, 3H), 1.20 (s, 1H), 1.19-1.17 (m, 2H), 0.90-0.89 (m, 6H). ¹³ C NMR (400 MHz, CDCl ₃ ) delta 174 .47,171.36,169.05,155.63,131.35,119.29,80.19,74.21,71.29,66.41,61.72,59.68,55.52,54.82,38.73,28.44,27.53,24.18, 17.06,15.80,11.68.

Preparation of compound represented by formula 8

Place compound 3 (1 mmol) in a round-bottom flask, add 10 ml of 33% diethylamine, stir at room temperature for 15 minutes, and spin-dry the solvent; Alloc-Ala-OH (2 mmol), HATU (2 mmol), DIEA (2 mmol) Add to a round bottom flask, add dichloromethane and N, N-dimethylformamide as a mixed solvent 10mL, stir at room temperature for 3 hours, add dilute hydrochloric acid to quench the reaction. Add 100mL of dichloromethane to dilute the reaction solution, wash with sodium bicarbonate and saturated aqueous solution of sodium chloride respectively, distill off the dichloromethane under reduced pressure, and separate the resulting product by silica gel column (petroleum ether: ethyl acetate 1: 1) 7 shows the compound. ¹ H NMR (400 MHz, CDCl ₃ ) δ (ppm) 7.40-7.34 (m, 10H), 5.94-5.87 (m, 1H), 5.73-5.71 (d, J = 8 Hz, 1H), 5.31-5.27 (m, 1H), 5.24-5.17 (m, 5H), 4.83-4.82 (m, 1H), 4.57-4.55 (d, J = 5.32 Hz, 2H), 4.39-4.36 (m, 1H), 4.13-4.10 (m, 1H), 3.74 (s, 3H), 3.47-3.45 (m, 1H), 2.22-2.20 (m, 1H), 1.87-1.89 (m, 1H), 1.43-1.41 (d, J = 7Hz, 3H). ¹³ C NMR (400 MHz, CDCl ₃ ) δ 173.23, 171.73, 155.87, 152.27, 135.75, 134.88, 133.00, 128.87-128.43 (t, 10C), 117.56, 68.77, 68.13, 65.70, 60.52, 53.56, 52.84, 50.65, 50.46, 50.33,37.96,19.07,0.13.

Place compound 7 in a round bottom flask, add a mixed solvent of tetrahydrofuran / water (3: 1), add lithium hydroxide (1.5 mmol), stir at room temperature for 3 minutes, add dilute hydrochloric acid to quench the reaction, add ethyl acetate to extract, The solvent was spin-dried to give compound 8.

Preparation of compound represented by formula 9

Place compound 6 (1 mmol) and compound 8 (0.5 mmol) in a round-bottom flask, add 5 mL of a mixed solvent of tetrahydrofuran and N, N-dimethylformamide, and stir under zero degrees, then mix DEPBT (1 mmol) and DIEA ( 1 mmol) was added to the reaction solution, stirred at room temperature overnight, and diluted hydrochloric acid was added to quench the reaction. Add 100ml of dichloromethane to dilute the reaction solution, wash with sodium bicarbonate and saturated aqueous solution of sodium chloride respectively, distill off the dichloromethane under reduced pressure, and separate the resulting product using a silica gel column (dichloromethane: methanol / 10: 1). The compound shown in 9 has a yield of 58%. ¹ H NMR (400 MHz, CDCl ₃ ) δ (ppm) 7.41-7.38 (m, 4H), 7.33-7.31 (m, 6H), 6.62-6.61 (m, 1H), 5.92-5.86 (m, 2H), 5.48 (s, 1H), 5.31 (s, 1H), 5.27-5.26 (m, 1H), 5.23-5.22 (m, 2H), 5.20-5.15 (m, 3H), 4.69-4.68 (m, 1H), 4.57 (s, 2H), 4.52 (s, 2H), 4.32 (s, 1H), 4.31-4.30 (m, 1H), 4.27-4.23 (m, 1H), 4.20-4.18 (m, 1H), 4.04-4.03 (m, 2H), 3.68-3.66 (m, 1H), 3.61-3.59 (m, 2H), 2.10 (s, 1H), 1.91-1.89 (m, 2H), 1.44 (s, 9H), 1.37-1.35 (m, 3H), 1.26-1.25 ( m, 3H), 1.17 (s, 9H), 0.91-0.90 (m, 6H). 13 C NMR (400MHz, CDCl 3) δ175.92,173.75,173.08,171.28,169.97, 158.19,157.38,152.55,138.02,136.85,134.22,132.90,129.56-128.97 (m, 10C), 118.90,117.60,80.74,74.65,72.79,69.22,68.34,67.25,67.18,66.93,66.90,66.87,66.58,63. , 58.81,56.71,56.62,56.36,54.81,52.31,50.81,38.57,37.83,28.71,27.73,25.93,18.07,17.21,16.27,16.02,11.87.

Preparation of compound represented by formula 10

Place the compound represented by formula 9 (0.3 mmol), tetratriphenylphosphine palladium (0.06 mmol), and 1,3-dimethylbarbituric acid in a round bottom flask, add 5 ml of methylene chloride, and stir at room temperature for 2 hours After the reaction is complete, add a mixed solvent to 100 ml (dichloromethane: N, N-dimethylformamide / 4: 1), HATU (2- (7-benzotriazole) -N, N , N ', N'-tetramethylurea hexafluorophosphate-condensing agent (1mmol), HOAT (1-hydroxy-7-azobenzotriazole) (1mmol), DIEA (2mmol) were added to the reaction solution During stirring at room temperature for 24 hours, washing, dichloromethane was distilled off under reduced pressure, and the resulting product was separated using a silica gel column (dichloromethane: methanol 10: 1) to obtain the compound represented by formula 10. ¹ H NMR (400 MHz, CDCl ₃ ) δ (ppm) 7.40-7.37 (m, 4H), 7.31-7.29 (m, 6H), 5.59-5.57 (m, 1H), 5.28-5.23 (m, 2H), 5.20-5.12 (m, 2H), 4.63 -4.59 (m, 1H), 4.24-4.22 (d, J = 8.12Hz, 1H), 4.14-4.11 (m, 2H), 4.09-4.05 (m, 2H), 3.67-3.61 (m, 3H), 3.31 -3.30 (m, 2H), 2.16 (m, 1H), 2.07 (m, 1H), 1.73 (s, 1H), 1.45 (s, 9H), 4.42-1.40 (m, 3H), 1.32-1.30 (d , J = 6.52 Hz, 3H), 1.26-1.24 (m, 1H), 1.20 (s, 9H), 0.92-0.86 (m, 6H).

Preparation of compound represented by formula 11

(a) (i) piperidine, DMF; (ii) Fmoc-AA-OH, HATU, DIEA, DMF;

(b) TFE: DCM / 1: 4,3h

According to the method of peptide solid-phase synthesis, place 2-Cl resin (0.5 mmol) in a solid-phase synthesis tube, add dichloromethane and N, N-dimethylformamide, activate for 20 min, extract the solvent, and remove Fmoc-L -Isoleucine (5mmol), DIEA (5mmol) was dissolved in DMF and added to the reflection instrument, stirred for 2h, the solvent was removed, Fmoc was removed with 20% piperidine solution for 15min, Fmoc-D-Isoleucine ( 1.5mmol), HATU (1.5mmol), DIEA (3mmol) was added to the reaction vessel, stirred at room temperature for 50min. Next, in the same way, the N-Fmoc-N'-trityl-D-glutamine, Fmoc-L-serine, Fmoc-L-isoleucine and Boc-D-4,4 ' -Diphenylaniline or N-tert-butoxycarbonyl-N-methyl-D-phenylalanine is connected to the last amino acid. Finally, 25wt% trifluoroethanol (diluted with dichloromethane) was added to the reaction tube, stirred at room temperature for 4h, and the solvent was spin-dried to obtain the compound represented by Formula 11.

Preparation of compound represented by formula (II)

Conditions: a) 1.0equiv.compound 10, 3M HCl in dioxane, 15min; b) 1.2equiv.compound 11, 11, 1.2equiv.DEPBT, 1.2equiv.DIEA, THF: DMF / 1: 1, 12h, 60%; c) TFA: TfOH: thioanisole: mcresol (70: 12: 10: 8v / v / v / v).

The compound (0.1 mmol) represented by Formula 10 was placed in a round bottom flask, 5 ml of 2M hydrochloric acid ethyl acetate solution was added, stirred at room temperature for 20 min, diluted with 10 ml of ethyl acetate, washed with saturated aqueous sodium bicarbonate solution, and the organic phase was dried After drying over sodium sulfate, the ethyl acetate solvent was distilled off under reduced pressure to obtain a crude intermediate. Then put compound 11 (0.13mmol) in a round bottom flask, add tetrahydrofuran and N, N-dimethylformamide as a mixed solvent 4ml, put it in an ice water bath, add DEPBT (0.13mmol), DIEA (0.13mmol) After 1 hour of reaction, it was stirred at room temperature overnight. Dilute hydrochloric acid was added to quench the reaction. The reaction solution was diluted with 10 ml of ethyl acetate, washed with a saturated aqueous solution of sodium chloride, and ethyl acetate was distilled off under reduced pressure. The resulting product was separated using a silica gel column (dichloromethane: methanol 10: 1). Finally, the mixed solvent (trifluoroacetic acid: trifluoromethanesulfonic acid: anisole: m-cresol / 70: 12: 10: 8) was added to the round bottom flask, and all the protective groups were removed to obtain the final product 12, which was used The solvent was blown dry with nitrogen, precipitated by adding ice ether, and centrifuged to obtain a crude product, which was separated by reverse-phase HPLC and freeze-dried.

The NMR results of the final compound 12 are shown in Table 1 below.

Table 1:

Among them, the MRI data of compound 12Teixobactin is on the left, and the positive control is on the right. ¹ H-NMR (400 MHz, CD ₃ OD) δ (ppm) From the above data, the structure of the prepared compound is correct.

In the description of this specification, the description referring to the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" means specific features described in conjunction with the embodiment or examples , Structure, material or characteristic is included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. In addition, without contradicting each other, those skilled in the art may combine and combine different embodiments or examples and features of the different embodiments or examples described in this specification.

Although the embodiments of the present invention have been shown and described above, it can be understood that the above-mentioned embodiments are exemplary and cannot be construed as limitations to the present invention, and those of ordinary skill in the art can The embodiments are changed, modified, replaced, and modified.

Claims (10)

1. A method for preparing unnatural amino acids, characterized in that

   The compound represented by formula (1) is subjected to oxidation ring closure treatment,

   

   Among them, R _{5 is} independently Fmoc, Boc, Phth, Cbz,

   R _{6 is} independently H, Fmoc, Boc, Phth, Cbz.
2. The method according to claim 1, further comprising deprotecting the oxidation ring-closure treatment product to obtain the compound represented by formula (I),

   
3. The method according to claim 1 or 2, characterized in that the oxidation ring closure treatment is performed under the condition of an oxidant;

   Optionally, the oxidant includes at least one selected from elemental iodine, NIS, iodophthalic acid, NBS, Dess-Martin oxidant, and trivalent iodine oxidant;

   Optionally, the molar ratio of the compound represented by formula (1) to the oxidant is 1: (1-5), preferably 1: 3;

   Optionally, the deprotection treatment is performed under acidic conditions;

   Optionally, the acidic conditions are provided by TFA / H ₂ O.
4. A method for preparing the compound represented by formula (3), characterized in that

   1) Esterification reaction of the ring-closure product of the compound represented by formula (1) with R ₇ OH to obtain the compound represented by formula (2),

   2) The compound represented by formula (2) is subjected to upper protection treatment to obtain the compound represented by formula (3),

   

   Among them, R ₇ is methyl, ethyl, propyl;

   Optionally, the esterification reaction is carried out under acidic conditions;

   Optionally, the acidic conditions are provided by formic acid or acetic acid;

   Optionally, the upper protection treatment is carried out in the presence of a leaving group with an R ₆ group;

   Optionally, the leaving group bearing the R ₆ group is Cbz-OSu or Fmoc-Osu;

   Optionally, the R _{5 is} different from R ₆ , and R ₅ is easier to remove than R ₆ .
5. A compound characterized by having a structure represented by formula (3).
6. A compound characterized by being obtained by the method of claim 4.
7. A method for preparing a compound represented by formula (II) or a salt of a compound represented by formula (II), characterized in that it includes:

   The compound of claim 5 or 6 is subjected to condensation treatment with several amino acids to obtain the compound represented by formula (II).
8. The method according to claim 7, wherein the R ⁵ is a Fmoc group, the R ⁶ is a Cbz group, and is connected to the guanidine nitrogen, the R ⁷ is a methyl group, the The method further includes:

   1) The compound according to claim 5 or 6 is subjected to de-Fmoc group treatment;

   2) Condensation reaction of the product obtained in step 1) with Alloc-Ala-OH to obtain the compound represented by formula (7);

   3) Hydrolyzing the compound represented by the formula (7) to obtain the compound represented by the formula (8);

   4) Condensation reaction of the compound represented by formula (6) with the compound represented by formula (8) to obtain the compound represented by formula (9);

   5) Allyl group and Alloc group removal treatment of the compound represented by the formula (9):

   6) The product of step 5) is removed and subjected to condensation treatment, so as to obtain the compound represented by formula (10);

   7) The compound represented by the formula (10) and the compound represented by the formula (11) are subjected to condensation treatment to obtain the compound represented by the formula (II);

   

   

   Wherein, R ₁ is hydrogen, C _{1 ~ 15} alkyl group, C _{2 ~ 15} alkylene group, C _{2 ~ 15} alkynyl group, C _6-10 aryl group or an acyl group;

   R ₂ is hydrogen, halo, C _{1 ~ 15} alkyl group, C _{2 ~ 15} alkylene group, C _{2 ~ 15} alkynyl group, C _6-10 aryl group or an acyl group, wherein the C _6-10 aryl group may optionally be 1 ～ 5 Rx substitutions;

   Rx is halogen, C _1-6 alkyl, halogenated C _1-6 alkyl;

   R ₃ is amino, guanidino, urea or formamide;

   R ₄ is C _{1 ~ 15} alkyl group, C _{2 ~ 15} alkylene group, C _{2 ~ 15} alkynyl group, C _6-10 aryl group;

   X is O, S or NH;

   Optionally, the step 7) further includes:

   7-1) The compound represented by the formula (10) is subjected to Boc group removal treatment;

   7-2) The Boc group-removing compound obtained in step 7-1) undergoes a condensation reaction with the compound of formula (11);

   7-3) The condensation product obtained in step 7-2) is subjected to Cbz group, t-Bu group, and Boc group removal treatment to obtain the compound represented by formula (II);

   Optionally, the removal treatment of the Boc group in step 7-1) is carried out under the condition of hydrochloric acid ethyl acetate solution for 15-20 min;

   Optionally, the concentration of the hydrochloric acid ethyl acetate solution is 2M-3M;

   Optionally, the condensation reaction in step 7-2) is carried out under the conditions of DEPBT / DIEA;

   Optionally, in step 7-2), the molar ratio of formula (10), DEPBT, and DIEA is 1: 1: 1;

   Optionally, the removal treatment of the Cbz group, t-Bu group and Boc group in the step 7-3) is performed in trifluoroacetic acid, trifluoromethanesulfonic acid, anisole and m-cresol Carried out under a mixed solvent;

   Optionally, the volume ratio of the trifluoroacetic acid, trifluoromethanesulfonic acid, anisole and m-cresol is (70 ± 5): (12 ± 5): (10 ± 5): (8 ± 5 );

   Preferably, the volume ratio of trifluoroacetic acid, trifluoromethanesulfonic acid, anisole and m-cresol is 70: 12: 10: 8;

   Optionally, the removal of the Fmoc group in step 1) is carried out under the condition of diethylamine;

   Optionally, the condensation reaction in step 2) is carried out under the condition that the condensation agent is HATU / DIEA or PyAOP, and the solvent is a mixed solvent of methylene chloride and DMF;

   Optionally, the molar ratio of the compound shown in claim 5 or 6 in the step 2), Alloc-Ala-OH, HATU, DIEA is 1: 2: 2: 2;

   Optionally, the hydrolysis reaction in step 3) is carried out under the condition that the mixed solvent of tetrahydrofuran / water and the base is LiOH;

   Optionally, the molar ratio of the compound represented by formula (7) to LiOH is 1: 1.5;

   Optionally, the condensation reaction in step 4) is performed under the condition that the condensation agent is DEPBT / DIEA and the solvent is a mixed solvent of THF and DMF;

   Optionally, the molar ratio of the compound represented by formula (6), the compound represented by formula (8), DEPBT, and DIEA in step 4) is 1: 0.5: 1: 1;

   Optionally, the removal treatment in step 5) is carried out under the acidic conditions provided by the catalyst being palladium tetratriphenylphosphine and 1,3-dimethylbarbituric acid;

   Optionally, the molar ratio of the compound represented by formula (9), tetratriphenylphosphine palladium, and 1,3-dimethylbarbituric acid in step 5) is 0.3: 0.06: 0.6;

   Optionally, the condensation reaction in step 6) is carried out under the condition that the condensation agent is HATU / HOAT / DIEA or PyAop;

   Optionally, the molar ratio of the compound represented by formula (9), HATU, HOAT and DIEA in step 6) is 0.3: 1: 1: 2;

   Optionally, the salt is hydrochloride, trifluoroacetate, acetate, sulfonate;

   Optionally, R ₁ is hydrogen, C _{1 ~ 10} alkyl group, C _{2 ~ 10} alkylene group, C _{2 ~ 10} alkynyl group, C _6-8 acyl group or an aryl group;

   R ₂ is hydrogen, halo, C _{1 ~ 10} alkyl group, C _{2 ~ 10} alkylene group, C _{2 ~ 10} alkynyl group, C _6-8 acyl or aryl group, wherein the C _6-8 aryl group can be optionally 1 ～ 5 Rx substitutions;

   Rx is halogen, C _1-6 alkyl, halogenated C _1-6 alkyl;

   R ₄ is C _{1 ~ 10} alkyl group, C _{2 ~ 10} alkylene group, C _{2 ~ 10} alkynyl group, C _6-8 aryl group;

   Optionally, R ₁ is hydrogen, C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _6-7 aryl or acyl;

   R ₂ is hydrogen, halogen, C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _6-7 aryl or acyl, wherein the C _6-7 aryl or C _{5- 6} Heteroaryl groups can be optionally substituted with 1 to 3 Rx;

   Rx is halogen, C _1-4 alkyl, halogenated C _1-4 alkyl;

   R ₄ is C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _6-7 aryl;

   Optionally, R ₁ is hydrogen, methyl, ethyl, n-propyl, isopropyl, butyl, vinyl, propenyl, allyl, ethynyl, propynyl, propargyl, phenyl, Benzyl or acyl;

   R ₂ is hydrogen, halogen, methyl, ethyl, n-propyl, isopropyl, butyl, vinyl, propenyl, allyl, ethynyl, propynyl, propargyl, phenyl, benzyl Or acyl, wherein the phenyl or benzyl group may be optionally substituted with 1 to 3 Rx;

   Rx is halogen, methyl, ethyl, n-propyl, isopropyl, butyl, monofluoromethyl, difluoromethyl, trifluoromethyl;

   R ₄ is methyl, ethyl, n-propyl, isopropyl, butyl, vinyl, propenyl, allyl, ethynyl, propynyl, propargyl, phenyl, benzyl;

   Optionally, the compound represented by formula (6) is obtained by the following steps:

   The compound represented by formula (5) is reacted with Fmoc-L-isoleucine for esterification, aminolysis, and Fmoc group removal to obtain the compound represented by formula (6);

   Optionally, the esterification reaction is carried out under the condition that the condensing agent is EDCI and the catalyst is DMAP;

   Optionally, the molar ratio of the compound represented by formula (5), Fmoc-L-isoleucine, EDCI, and DMAP is 9: 10: 10: 1;

   Optionally, the aminolysis and Fmoc group removal reactions are carried out under the condition of diethylamine solution;

   Optionally, the concentration of the diethylamine is 33%;

   Optionally, the compound represented by formula (5) is obtained by the following steps:

   The compound represented by formula (4) and Boc-Ser (tBu) -OH are subjected to a condensation reaction to obtain the compound represented by formula (5);

   Optionally, the condensation reaction is carried out in a mixed solvent of dichloromethane and DMF with HCTU / DIEA as the condensing agent;

   
9. The method according to claim 8, wherein the compound represented by formula (11) is obtained by a solid phase synthesis method;

   Optionally, the solid phase synthesis method is performed by the following steps:

   A) A substitution reaction between 2-Cl resin and Fmoc-L-isoleucine is performed to obtain the compound represented by formula (a), wherein the circle represents the resin;

   B) Combine the compound represented by formula (a) with N-Fmoc-N'-trityl-D-glutamine, Fmoc-L-serine, Fmoc-L-isoleucine, Boc-D-4 , 4'-diphenylphenylalanine and N-tert-butoxycarbonyl-N-methyl-D-phenylalanine in the terminal amino acid condensation reaction, so as to obtain the compound represented by formula (b);

   C) The compound represented by formula (b) is subjected to a resin-removing reaction to obtain the compound represented by formula (11);

   
10. The method according to claim 9, wherein the 2-Cl resin is subjected to an activation treatment in advance, and the activation treatment is performed in dichloromethane and DMF for 20 min;

    Optionally, after the activation treatment and before the coupling reaction, a solvent extraction treatment is further included;

    Optionally, the coupling reaction in step 1) is carried out under the condition that DIEA is a condensing agent and DMF is a solvent;

    Optionally, before each condensation reaction in step 2), the raw materials to be condensed need to be de-Fmoc treated in advance, and the de-Fmoc treatment is performed in a piperidine solution;

    Optionally, the condensation reaction in step 2) is carried out under the condition that the condensation agent is HATU / DIEA at room temperature.