CN113912669A

CN113912669A - Design, synthesis and application of antibacterial compound based on ring conformation

Info

Publication number: CN113912669A
Application number: CN202110716523.6A
Authority: CN
Inventors: 许正双; 李旺; 李洋
Original assignee: Peking University Shenzhen Graduate School
Current assignee: Peking University Shenzhen Graduate School
Priority date: 2021-06-28
Filing date: 2021-06-28
Publication date: 2022-01-11

Abstract

The invention provides a derivative of Pyridomycin, the macrocyclic derivative of which retains the skeleton structure of the 12-membered heterocycle in the natural product and the 3-hydroxy-2-picolinic acid structure at the N6 position: (1) The C2 position The unsaturated double bond outside the ring is replaced by a cyclic amino acid, and the cyclic amino acid can regulate the conformation of the macrocycle; specifically, the size of the ring and the chiral configuration of the alpha-position of the carboxyl group are used to control the conformation of the macrocycle; (2) Pyridomycin molecule is used to control the conformation of the macrocycle. The 6-7-position ortho-diamine structural feature retains the two activity-related structures of 3-hydroxy-2-picolinic acid and 3-pyridine in Pyridomycin. By adjusting the dihedral angle of the ortho-diamine, the small molecule has the unique conformation of Pyridomycin. The structural compound has better activity and application prospect.

Description

Design, synthesis and application of antibacterial compound based on ring conformation

Technical Field

The invention specifically relates to the design, synthesis and application of antibacterial compounds based on ring conformation, and relates to a synthesis method.

Background introduction

Tuberculosis is an infectious disease caused by infection of mycobacterium tuberculosis, and about 1000 million people suffer from tuberculosis in the world in 2019 according to a worldwide tuberculosis report of 2020 recently issued by the World Health Organization (WHO). Tuberculosis affects men and women and people of all age groups, and China still belongs to a country with serious tuberculosis infection. The proportion of multidrug-resistant or rifampicin tuberculosis among the number of tuberculosis cases remains stable worldwide. From the data, the tuberculosis situation is still severe, but for the infection caused by multi-drug resistant tubercle bacillus, the effect of the traditional treatment method is gradually weakened, and the discovery and the development of new drugs and methods for treating drug resistant tuberculosis are urgent.

Pyridomycin was first isolated from streptomycin strain 6706 in 1953 by Umezawa, H. Such natural products have specific activity against mycobacteria but no activity against other bacteria. Experiments show that the isoniazid resistant clinical isolate of mycobacterium tuberculosis carrying katG mutation can be killed. The research shows that the action target of Pyridomycin is InhA. In InhA, it can occupy multiple NHAD binding sites of InhA active center, thus inhibiting the combination of NHAD and InhA, preventing the formation of mycoderm and making it have the ability to resist tubercle bacillus. Therefore, the research on Pyridomycin and derivatives thereof has profound significance for developing new drugs against drug-resistant tubercle bacillus.

The structural formula of Pyridomycin is as follows:

total synthesis of Pyridomycin was reported by Mitsuhiro in 1989. In 2012, k. -h.altmann reported synthetic routes and activities for Pyridomycin derivatives, and found that exocyclic unsaturated double bond structure was not an essential group for its anti-mycobacterium tuberculosis activity. Subsequently, the group studied the structure-activity relationship of Pyridomycin by synthesizing derivatives of different structures, and found that the larger the C2 substituent, the higher the anti-tubercle bacillus activity, and that the compound has a reduced anti-tubercle bacillus activity when the C2 substituent is a polar group. The chirality of C2 influences the antitubercular activity of the compounds. The N-6 substituted 3-hydroxypicolinic acid is an essential group, and the deletion or the change of the hydroxyl substitution position can cause the activity of the compound against mycobacterium tuberculosis to be weakened or even disappear.

Disclosure of Invention

The document shows that the cycloolefine in the structure of Pyridomycin is not necessary for the anti-tubercle bacillus activity and is a main site designed for derivatives. The invention designs Pyridomycin derivatives and small molecules by taking the C2 position as a derivative site to synthesize and research the activity of the Pyridomycin derivatives and the small molecules. The invention specifically comprises the following steps:

a derivative of Pyridomycin, which has the structure:

. The macrocyclic derivatives are: .

The macrocyclic derivative reserves the skeleton structure of a 12-membered heterocyclic ring and the 3-hydroxy-2-picolinic acid structure at the N6 position in a natural product, wherein R, R³Respectively hydrogen, C1-C4 alkyl, fluorine, chlorine, amino, hydroxyl, nitro, sulfonic group, aryl, substituted aryl, heteroaryl and substituted heteroaryl. R¹，R²Respectively hydrogen, C1-C4 alkyl. R⁴Hydrogen, C1-C4 alkyl, fluoro, chloro, amino, hydroxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl. X is CH₂Oxygen or nitrogen. n is 1-2. Aryl refers to aromatic structures of benzene and naphthalene. Heteroaryl refers to aromatic heterocyclic structures such as oxazole, thiazole, pyridine, pyrimidine, quinoline, and the like.

Further:

(1) substitution of the exocyclic unsaturated double bond at C2 with a cyclic amino acid that can modulate the conformation of the macrocycle. In particular, the conformation of the macrocycle is controlled by the size of the cycle and the alpha-position chiral configuration of the carboxyl.

(2) By utilizing the structural characteristics of ortho-diamine at the 6-7 position of a Pyridomycin molecule, two activity related structures of 3-hydroxy-2-picolinic acid and 3-pyridine in Pyridomycin are kept, and the micromolecule has the unique conformation of Pyridomycin by adjusting the dihedral angle of ortho-diamine.

Here, small molecule means: . Wherein R, R³Respectively hydrogen, C1-C4 alkyl, fluorine, chlorine, amino, hydroxyl, nitro, sulfonic group, aryl, substituted aryl, heteroaryl and substituted heteroaryl. R⁵，R⁶， R⁷，R⁸Respectively hydrogen, C1-C4 alkyl, combined into a ring or an aryl structure. R⁶Is oxygen and forms a double bond with the carbon atom to which it is attached to form a carbonyl group. R⁵And R⁶Can be cyclized into a five-membered ring or a six-membered ring structure. R⁶And R⁷Can be cyclized into a five-membered or six-membered saturated ring structure, or can form a benzene ring and a substituted benzene ring together with the connected atoms. R⁷And R⁸Can be cyclized into a five-membered ring or a six-membered ring structure. R⁹Hydrogen, C1-C4 alkyl, fluoro, chloro, amino, hydroxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl. R⁹And R⁵Can be cyclized into a five-membered ring or a six-membered ring structure. R⁹Form a double bond with the carbon atom to which it is attached to form a carbonyl group. m is 1-4.

Furthermore, the specific structure of D-proline (D-Pro-8), L-proline (L-Pro-8) and D-pipecolic acid (D-Pip-8) is as follows:

D-Pro-8 and L-Pro-8 correspond to Pyridomycin in place of its functional group at position 1-2, with proline controlling the conformation of the macrocycle. D-Pip-8 and L-Pip-8 correspond to Pyridomycin in place of its functional group at position 1-2, with pipecolic acid controlling the conformation of the macrocycle. All molecules retain the original substituted pyridine ring and retain their ability to bind to the target protein.

Further, the small molecule retains a simplified conformational form of the small molecule with two terminal pyridine binding sites. The structure of o-diamine at the 6-7 position of a Pyridomycin molecule is simplified into structures such as phenyl (S-1, S-3, S-5), cyclohexanediamine (S-2, S-4), piperidine (S-6, S-7) and the like, and the two pyridines of Pyridomycin are in a form of combined conformation by simulating macrocyclic conformation through adjusting the dihedral angle of the o-diamine. The structure of the o-diamine can also be replaced by the structural forms of cyclopentediamine, proline and the like.

The phenyl is N, N' -pyridine derivative-o-phenylenediamine micromolecules, and the configuration of the phenyl comprises: the phenyl first structure (S-1), the phenyl second structure (S-3) and the phenyl third structure (S-5) are sequentially as follows:

the cyclohexanediamine is N, N' -pyridine derivative-cyclohexanediamine micromolecules, and the configuration of the cyclohexanediamine comprises: the first structure (S-2) of the cyclohexanediamine and the second structure (S-4) of the cyclohexanediamine have the following specific structures in sequence:

the piperidine is N, N' -pyridine derivative-piperidine acid micromolecule, and the configuration of the piperidine comprises: the piperidine first structure (S-6) and the piperidine second structure (S-7) have the following specific structures:

furthermore, the 1-2 atom of pyridomycin is replaced by five-membered cyclic amine (D/L-proline), which is D-proline and/or L-proline. To control the macrocyclic conformation. The derivatives are designated as D-Pro-8 and L-Pro-8. The synthesis process comprises the following steps: and carrying out amidation reaction on the compound D/L-Pro-1 and the compound L-Tre-1 under EDCI condition to obtain the compound D/L-Pro-2. The compound D/L-Pro-1 is N-Fmoc-D-Pro-OH, and the compound L-Tre-1 is N-Boc-L-Thr-OBn. And then removing Fmoc protective groups from the compound D/L-Pro-2 under the conditions of diethylamine and dichloromethane to obtain a compound D/L-Pro-3, and then carrying out amidation reaction with pyridine unnatural amino acid triethylamine salt P2 to obtain a compound D/L-Pro-4. And then carrying out palladium-carbon hydrogenation reaction on the compound D/L-Pro-4 to obtain a ring-closing precursor, and then carrying out one-step cyclization under the conditions of HATU, DIPEA and DMF to obtain the compound D/L-Pro-6. Finally, the Boc protecting group on the compound D/L-Pro-6 is removed by TFA, and then amidation reaction is carried out with 3-hydroxy-2-picolinic acid under the conditions of HATU and DIPEA, thereby completing the synthesis of the compound D/L-Pro-8.

Further, the 1-2 atom of pyridomycin is replaced with a six-membered cyclic amine (D/L-pipecolic acid) to control the macrocyclic conformation. The hexatomic cyclic amine is D-pipecolic acid and/or L-pipecolic acid. The derivatives are marked as D-Pip-8 and L-Pip-8. The synthesis process comprises the following steps: and carrying out peptide-connecting reaction on the compound D/L-Pip-1{ N-Fmoc-D/L-Pro-OH } and the compound L-Tre-1{ N-Boc-L-Thr-OBn } under the EDCI condition to obtain the compound D/L-Pip-2. The compound D/L-Pip-1 is N-Fmoc-D/L-Pro-OH. The compound L-Tre-1 is N-Boc-L-Thr-OBn. And then removing Fmoc protective groups from the compound D/L-Pip-2 under the conditions of diethylamine and dichloromethane to obtain a compound D/L-Pip-3, and carrying out amidation reaction on the compound D/L-Pip-3 and pyridine unnatural amino acid triethylamine salt P2 to obtain a compound D/L-Pip-4. And then carrying out palladium-carbon hydrogenation reaction on the compound D/L-Pip-4, and then carrying out one-step cyclization reaction under the conditions of HATU and DIPEA to obtain the compound D/L-Pip-6. And finally, removing the Boc protecting group on the compound D/L-Pip-6 by TFA, and then reacting with 3-hydroxy-2-picolinic acid under the conditions of HATU and DIPEA to obtain the compound D/L-Pip-8.

Furthermore, the synthesis process of the N, N' -pyridine derivative-o-phenylenediamine micromolecules S-1, S-3 and S-5 comprises the following steps: the chemical structure of the compound S-1-1 (please specifically introduce the chemical structure of S-1-1, such as the existing product which can be directly given the trade name or the common name) and tert-butyloxycarbonyl o-phenylenediamine are subjected to amidation reaction to obtain the compound S-1-2, and then the tert-butyloxycarbonyl protecting group is removed by TFA. Protecting the hydroxyl of the 3-hydroxyl-2-picolinic acid by methoxy methyl ether, then carrying out amidation reaction with a compound S-2-3, and removing the MOM protecting group to obtain a target product S-1.

Furthermore, the synthesis process of the N, N' -pyridine derivative-cyclohexanediamine micromolecules S-2 and S-4 comprises the following steps:¹H NMR (300MHz,CDCl₃)δ11.81(s,1H),8.96(d,J＝2.3Hz,1H),8.67(dd,J＝4.9, 1.7Hz,1H),8.21(d,J＝7.4Hz,1H),8.07–7.97(m,2H),7.37–7.29(m, 2H),7.28–7.25(m,1H),7.23(dd,J＝6.9,4.0Hz,1H),4.12–3.87(m,2H), 2.36(dt,J＝12.5,2.8Hz,1H),2.14(dt,J＝12.5,2.6Hz,1H),1.95–1.77 (m,2H),1.68–1.27(m,4H).HRMS(ESI)calcd for:C₁₈H₂₁N₄O₃+[M+H]+341.16082, found 341.16165；C₁₈H₂₀N₄NaO₃ ⁺[M+Na]⁺363.14276；found 363.14346。

furthermore, the synthesis method of the N, N' -pyridine derivative-pipecolic acid small molecular compound S-6 comprises the following steps: dissolving the compound S-4-1 in dry dichloromethane under the protection of nitrogen, cooling to 0 ℃, adding EDCI, 3-aminomethyl pyridine and DMAP, recovering the room temperature, stirring, and monitoring by thin-layer chromatography. After the reaction is completed, water is added for quenching, and DCM is used for extracting organic phase, and water and saturated NaHCO are used for next time₃Solution washing, saturated NaCl solution washing, anhydrous Na₂SO₄And (5) drying. Filtering, concentrating under reduced pressure, and separating by normal phase silica gel column chromatography to obtain compound S-6-2.

The compound S-6-2 is dissolved in dichloromethane, added with diethylamine at room temperature for reaction at room temperature, and monitored by thin layer chromatography. After the reaction is completed, the crude product of the compound S-6-3 is obtained by spin drying and is directly used for the next step. The crude compound S-6-3 was dissolved in dry DMF and MOM protected sodium salt of 3-hydroxy-2-pyridinecarboxylic acid was added and cooled. HATU and DIPEA were added sequentially, stirred overnight at room temperature, and monitored by LC-MS. Adding water to the system for quenching, extracting by ethyl acetate, and using saturated NaHCO to the organic phase₃Washing with solution, water, saturated NaCl solution, anhydrous Na₂SO₄And (5) drying. After filtration, concentration under reduced pressure, the residue is dissolved in THF, diluted hydrochloric acid is added, stirring is carried out overnight at room temperature, and monitoring is carried out by thin-layer chromatography. Diluting with ethyl acetate, separating organic phase, extracting water phase with ethyl acetate to remove ester-soluble impurities, retaining water phase, adding methanol, cooling, adjusting pH with NaOH solution to alkaline, extracting reaction system with ethyl acetate, extracting organic phase with saturated NaHCO₃Washing with solution, water, saturated NaCl solution, anhydrous Na₂SO₄And (5) drying. Filtering, concentrating under reduced pressure, and separating by normal phase silica gel column chromatography to obtain compound S-6.

Furthermore, the macrocyclic conformation of the pyridomycin natural product is simulated, five-membered ring structures or six-membered ring structures such as proline, pipecolic acid and the like are used for controlling the macrocyclic conformation, and two pyridine ring derivative structures on side chains are reserved. And further using six-membered or five-membered cyclic o-diamine such as phenylenediamine, cyclohexanediamine or pipecolic acid to simulate a 6-7-position o-diamine structure of the pyridomycin molecule, regulating dihedral angle through the change of the o-diamine substitution form, simulating the conformation of a natural product, and adjusting the binding capacity of two pyridine derivative functional groups on side chains and the target protein.

The molecules, or reasonable salt forms and combination forms thereof, can have potential activities of resisting microorganisms, bacteria, fungi and the like, and particularly have certain resistance to tubercle bacillus. Further may have other potential biological activities.

Advantageous technical effects

The technical effect of the invention was evaluated in an activity test:

all compounds to be screened were dissolved in 100% dimethyl sulfoxide (DMSO) and stored at a concentration of 2 mg/mL. Ribavirin sodium salt (Resazurin sodium salt) powder was dissolved in distilled water at a concentration of 0.01% (w/v) and stored at 4 degrees (spent a week) by sterile filtration. All conventional chemicals were purchased commercially without further purification.

M.smegmatis, m.aurum, m.absccess, m.avium, BCG were cultured at 37 degrees in Middlebrook 7H9 medium containing 10% (v/v) ADC (oleic acid, albumin, glucose, catalase), and agar 80 containing 0.05% Tween.

The MIC was determined in a reinazolin reduction microplate assay (REMA) (see experimental procedures of anitirob. ingredients chemither.2002, 46,2720). Compounds (diluted in gradients) were added to the diluted bacterial suspension in 96-well plates (100 μ L), the plates were sealed with PCR membranes and incubated (7 days, 37 ℃). Bacterial viability was determined using a redox indicator (0.025% w/v, 16h) and by fluorescence (Ex: 560nm, Em: 590nm) using a Tecan Infinite M200 plate reader. The Minimum Inhibitory Concentration (MIC) was determined as the lowest concentration of compound with background levels of rinsable sodium fluorescence (equivalent to no bacterial control). Experiments were performed at least twice and the average values were reported.

Active form

Remarking: MIC (ug/mL) < 10; , + represents 10< MIC < 50; + represents 50< MIC;

as can be seen from the above table, the product of the present invention achieved the desired purpose and was excellent in activity.

As shown in figure 1, the macrocyclic derivative retains the skeleton structure of a 12-membered heterocyclic ring and the 3-hydroxy-2-picolinic acid structure at the N6 position in a natural product, and replaces the exocyclic unsaturated double bond at the C2 position with D, L-proline and D, L-pipecolic acid. The small molecule reserves two activity related structures of 3-hydroxy-2-picolinic acid and 3-pyridine in Pyridomycin.

Drawings

FIG. 1 is a summary diagram of the structural formulas of the derivatives and the small molecules of the present invention.

FIG. 2 is a flow chart of the synthetic process of D-Pro-8 (synthesis of non-natural amino acid fragments) which is a D-proline derivative.

FIG. 3 is a flow chart of the synthesis process of D-proline derivatives.

FIG. 4 is a flow chart of a synthetic process of L-proline derivatives.

FIG. 5 is a flow chart of the synthesis process of the D-pipecolic acid derivative.

FIG. 6 is a flow chart of a synthetic process for L-pipecolic acid derivatives.

FIG. 7 is a flow chart of the synthesis process of compound S-1.

FIG. 8 is a flow chart of the synthesis process of compound S-2.

FIG. 9 is a flow chart of the synthesis process of compound S-3.

FIG. 10 is a flow chart of the synthesis process of compound S-4.

FIG. 11 is a flow chart of the synthesis process of compound S-5.

FIG. 12 is a flow chart of the synthesis process of compound S-6.

FIG. 13 is a flow chart of the synthesis process for compound S-7.

Detailed Description

Technical features of the present invention will now be described in detail with reference to the accompanying drawings.

Referring to fig. 1, a derivative of Pyridomycin, which has the structure:

. The macrocyclic derivatives are: .

The macrocyclic derivative reserves the skeleton structure of a 12-membered heterocyclic ring and the 3-hydroxy-2-picolinic acid structure at the N6 position in a natural product, wherein R, R³Respectively hydrogen, C1-C4 alkyl, fluorine, chlorine, amino, hydroxyl, nitro, sulfonic group, aryl, substituted aryl, heteroaryl and substituted heteroaryl. R¹，R²Respectively hydrogen, C1-C4 alkyl. R⁴Hydrogen, C1-C4 alkyl, fluoro, chloro, amino, hydroxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl. X is CH₂Oxygen or nitrogen. n is 1-2. Aryl refers to aromatic structures of benzene and naphthalene. Heteroaryl refers to aromatic heterocyclic structures such as oxazole, thiazole, pyridine, pyrimidine, quinoline, and the like. (1) Substitution of the exocyclic unsaturated double bond at C2 with a cyclic amino acid that can modulate the conformation of the macrocycle. In particular, the conformation of the macrocycle is controlled by the size of the cycle and the alpha-position chiral configuration of the carboxyl. (2) By utilizing the structural characteristics of ortho-diamine at the 6-7 position of a Pyridomycin molecule, two activity related structures of 3-hydroxy-2-picolinic acid and 3-pyridine in Pyridomycin are kept, and the micromolecule has the unique conformation of Pyridomycin by adjusting the dihedral angle of ortho-diamine.

The first embodiment is as follows: d-proline derivative D-Pro-8

The synthesis of non-natural amino acid fragments is shown in FIG. 2. The synthesis route reported by Mitsuhiro topic group is referred to and improved, for example, the solvent is changed from pyridine to dichloromethane during the methane sulfonation, triethylamine is added as an acid-binding agent, the synthesis of the compound 2-4 can be completed in a short time, the yield and the diastereoisomer ratio are unchanged, and the operation is simpler. In addition, sodium chlorite and sodium dihydrogen phosphate are used to replace bromine as oxidizing condition to obtain compound 2-6, and the ester exchange reaction is finally carried out in triethylamine methanol to obtain compound P2.

As shown in figure 3, amidation reaction is carried out on a compound D-Pro-1 and a compound L-Tre-1 under EDCI condition to obtain a compound D-Pro-2, then Fmoc protective groups of the compound D-Pro-2 are removed under diethylamine and dichloromethane conditions to obtain a compound D-Pro-3, and then amidation reaction is carried out on the compound D-Pro-3 and pyridine unnatural amino acid triethylamine salt P2 to obtain a compound D-Pro-4. And then carrying out palladium-carbon hydrogenation reaction on the compound D-Pro-4 to obtain a ring-closing precursor, and then carrying out one-step cyclization under the conditions of HATU, DIPEA and DMF to obtain the compound D-Pro-6. Finally, the Boc protecting group on the compound D-Pro-6 is removed by TFA, and then amidation reaction is carried out with 3-hydroxy-2-pyridinecarboxylic acid under the conditions of HATU and DIPEA to complete the synthesis of the compound D-Pro-8.

(1) Synthesis of Compound D-Pro-2

Protection by nitrogenNext, compound D-Pro-1(1g, 1eq.) was dissolved in 15mL of dichloromethane, cooled to 0 ℃, EDCI (1.14g, 2eq.) and compound L-Tre-1(0.92g, 1eq.) were added, 4-dimethylaminopyridine (DMAP, 0.18g, 0.5eq.) was added, the ice bath was removed, the reaction was returned to room temperature for 2h, and monitored by thin layer chromatography. After the reaction was complete, 10mL of water was added to the system and quenched, the organic phase was separated, the aqueous phase was extracted with dichloromethane (20 mL. times.3), the organic phases were combined and separately saturated NaHCO₃Solution (20mL), water (20mL), saturated NaCl solution (20mL) washed with anhydrous Na₂SO₄And (5) drying. After filtration, the mixture was concentrated under reduced pressure and subjected to normal phase silica gel column chromatography to give compound D-Pro-2(1.8 g, 96%).¹H NMR(400MHz,CDCl₃)δ7.76(d,J＝7.4Hz,2H),7.61(ddd,J＝ 20.9,14.8,7.4Hz,2H),7.35(dp,J＝20.0,7.5Hz,9H),5.50(ddt,J＝21.1, 7.9,5.3Hz,1H),5.13(d,J＝15.6Hz,2H),4.64–4.04(m,5H),3.61(td, J＝8.5,6.9,3.7Hz,1H),3.48(dtd,J＝10.4,7.3,3.5Hz,1H),2.18–1.98 (m,1H),1.83(ddt,J＝28.2,14.8,8.8Hz,3H),1.49(s,3H),1.45(s,6H), 1.36(d,J＝6.5Hz,2H),1.25(d,J＝6.4Hz,1H).HRMS(ESI)calcd for C₃₆H₄₀N₂NaO₈ ⁺[M+Na]⁺651.26769；found 651.26782.

(2) Synthesis of Compound D-Pro-4

Compound D-Pro-2(0.6g, 1eq.) was dissolved in 5mL of dichloromethane and 5mL of DEA was added at room temperature. The reaction was carried out at room temperature for 1.5h, and methylene chloride and DEA were directly removed by concentration under reduced pressure, and methylene chloride (3 mL. times.3) was added to conduct concentration under reduced pressure to obtain a crude product (0.60g) of Compound D-Pro-3, which was used in the next step without purification. Triethylamine salt P2(0.25g, 1eq.) of the unnatural pyridine amino acid was dissolved in 4mL dichloromethane, cooled to 0 ℃, HATU (0.54g, 2eq.) was added and stirred for 5min, the crude product of compound D-Pro-3 (0.30g, 0.7eq.) was dissolved in 4mL dichloromethane and added to the reaction system, stirred for 5min at 0 ℃, DIPEA (0.63mL, 5eq.) was added, stirred for 30min at 0 ℃, and monitored by thin layer chromatography. After completion of the reaction, 4mL of water was added to the system to quench, the organic phase was separated, the aqueous phase was extracted with ethyl acetate (10 mL. times.3), the organic phases were combined, washed with water (10mL), saturated NaCl solution (10mL) and anhydrous Na₂SO₄Drying, filtering, and concentrating under reduced pressureThen, the residue was subjected to normal-phase silica gel column chromatography to recover 0.29 g of unreacted D-Pro-2 and obtain compound D-Pro-4(0.15g, 47% yield in two steps).¹H NMR(400MHz, CDCl₃)δ8.56(d,J＝2.2Hz,1H),8.54–8.49(m,1H),7.63(dt,J＝7.9,2.0 Hz,1H),7.35(d,J＝3.3Hz,5H),7.27–7.22(m,1H),5.51(td,J＝6.5,2.5 Hz,1H),5.12(d,J＝2.5Hz,2H),4.49(dd,J＝9.8,2.5Hz,1H),4.42(dd, J＝8.6,4.2Hz,1H),3.88(dt,J＝9.7,6.4Hz,1H),3.73(dt,J＝7.3,3.1Hz, 1H),3.54(dt,J＝9.7,7.2Hz,1H),3.37(ddd,J＝8.5,6.4,2.2Hz,1H),3.17 (dd,J＝14.0,8.3Hz,1H),3.13–3.05(m,1H),3.02(dd,J＝7.9,6.6Hz, 1H),2.17–2.03(m,1H),1.92(p,J＝6.3Hz,2H),1.79–1.75(m,1H),1.47 (s,9H),1.32(d,J＝6.5Hz,3H),1.06(d,J＝6.8Hz,3H).HRMS(ESI)calcd for C₃₂H₄₃N₆O₈ ⁺[M+H]⁺639.31369；found 639.31384；C₃₂H₄₂N₆NaO₈ ⁺[M+Na]⁺661.29563； found 661.29553.

(3) Synthesis of Compound D-Pro-6

Dissolving the compound D-Pro-4(0.1g, 1eq.) in 8mL of methanol, purging with a vacuum water pump under reduced pressure, replacing the residual air in the solvent with nitrogen, adding a palladium-carbon catalyst (10%, 67.30mg, 0.4eq.), purging with a vacuum water pump under reduced pressure, replacing the nitrogen in the system with hydrogen, stirring at room temperature for 3h under a hydrogen balloon condition, and monitoring by thin layer chromatography. After the reaction was completed, the reaction system was directly filtered with celite to remove the palladium on carbon catalyst, and the solvent was removed by concentration under reduced pressure to obtain a crude product of compound D-Pro-5, which was used directly in the next step, and after removing a small amount of residual water by azeotropic distillation with dry toluene (10mL × 3), 156mL of dry DMF was added, and the mixture was cooled to 0 ℃, HATU (0.6g, 10eq.), stirred at 0 ℃ for 5min, DIPEA (0.42mL, 15eq.) was added, and stirred at room temperature slowly overnight and monitored by thin layer chromatography. After completion of the reaction, DMF was removed by concentration under reduced pressure, and the mixture was dissolved in 10mL each of ethyl acetate and water, and the organic phase was separated and the aqueous phase was extracted with ethyl acetate (10 mL. times.5). The organic phases were combined and the organic phase was washed with water (10mL) and saturated NaHCO, respectively₃Solution (10mL), saturated NaCl solution (10mL) wash, anhydrous Na₂SO₄And (5) drying. Filtering, concentrating under reduced pressureThen, the resulting mixture was subjected to normal phase silica gel column chromatography to give compound D-Pro-6(20mg, 26%).¹H NMR(400MHz, CDCl₃)δ8.57(d,J＝2.2Hz,1H),8.49(dd,J＝4.9,1.7Hz,1H),7.66(dt, J＝7.9,2.0Hz,1H),7.26–7.21(m,1H),5.28(d,J＝11.1Hz,1H),4.54(dd, J＝8.5,3.5Hz,1H),4.26(s,1H),3.92(d,J＝7.7Hz,1H),3.58(ddd,J＝ 9.9,7.5,5.4Hz,1H),3.52(s,1H),3.49–3.39(m,1H),3.06–2.94(m,2H), 2.50–2.34(m,1H),2.22(d,J＝14.8Hz,1H),2.12(dh,J＝13.0,5.9Hz, 2H),2.00(d,J＝7.6Hz,1H),1.46(d,J＝4.6Hz,9H),1.34(d,J＝7.3Hz, 3H),1.29(d,J＝6.5Hz,3H).HRMS(ESI)calcd for C₂₅H₃₇N₄O₇ ⁺[M+H]⁺505.26568,found 505.26575.

(4) Synthesis of D-Pro-8

Compound D-Pro-6(6mg, 1eq.) was dissolved in 0.5mL of dichloromethane, added with 0.5mL of TFA, and reacted at room temperature for 2h, monitored by thin layer chromatography. After the reaction was completed, dichloromethane and TFA were removed by concentration under reduced pressure, dichloromethane (2mL × 3) was added to remove residual TFA by concentration under reduced pressure, toluene (2mL × 3) was added, and residual water in the system was removed by azeotropic distillation under reduced pressure to obtain a crude product of the compound D-Pro-7, which was directly used in the next step, the crude product of the compound L-Pro-7 was dissolved in 0.5mL of dry acetonitrile, 3-hydroxy-2-pyridinecarboxylic acid (8.1mg, 5eq.), cooled to 0 ℃, stirred for 5min, HATU (44.4mg, 10eq.), DIPEA (0.04mL, 15eq.), slowly warmed to room temperature for reaction overnight, and monitored by LC-MS. After completion of the reaction, 1mL of water was added to the system for quenching, extraction was performed with ethyl acetate (2 mL. times.5), and the organic phases were combined and successively saturated NaHCO was used₃Solution (2mL), water (2mL), saturated NaCl solution (2mL) washed with anhydrous Na₂SO₄And (5) drying. After filtration, the reaction mixture was concentrated under reduced pressure and subjected to normal-phase silica gel column chromatography to give compound D-Pro-8(1.6mg, 25%).¹H NMR(500MHz,Methanol-d4)δ8.40(d,J＝2.2 Hz,1H),8.18(dd,J＝4.9,1.6Hz,1H),8.17–8.07(m,1H),7.70(dt,J＝ 7.8,2.0Hz,1H),7.48(dd,J＝8.5,4.3Hz,1H),7.38(dd,J＝8.5,1.4Hz, 1H),7.19(dd,J＝7.8,4.9Hz,1H),5.15(dd,J＝6.5,5.2Hz,1H),4.72(d, J＝5.1Hz,1H),4.25(dd,J＝9.0,5.2Hz,1H),3.81–3.65(m,2H),3.60(tt, J＝10.2,6.9Hz,1H),2.99(d,J＝7.6Hz,1H),2.82–2.69(m,1H),2.33– 2.18(m,1H),2.12(dd,J＝13.3,7.7Hz,1H),2.09–1.92(m,2H),1.37(dd, J＝11.6,6.8Hz,3H),1.29(s,3H).¹³C NMR(150MHz,Methanol-d4)δ178.46, 172.66,169.75,169.65,150.96,147.91,147.90,139.27,139.24,136.35,136.33, 130.49,124.90,124.88,78.06,72.31,61.51,57.87,55.59,48.39,39.76,37.24, 30.80,29.62,26.03,16.01.HRMS(ESI)calcd for C₂₆H₃₂N₅O₇ ⁺[M+H]⁺526.22962,found 526.22974.

Example two: synthesis of L-proline derivatives

Referring to example one, as shown in the figure (FIG. 4), firstly compound L-Pro-1 is subjected to amidation reaction with compound L-Tre-1 under EDCI condition to obtain compound L-Pro-2, then compound L-Pro-2 is subjected to Fmoc protection group removal under diethylamine and dichloromethane conditions to obtain compound L-Pro-3, and then amidation reaction with pyridine unnatural amino acid triethylamine salt P2 to obtain compound L-Pro-4. And then carrying out palladium-carbon hydrogenation reaction on the compound L-Pro-4 to obtain a ring-closing precursor, and then carrying out one-step cyclization under the conditions of HATU and DIPEA to obtain the compound L-Pro-6. Finally, the Boc protecting group on the compound L-Pro-6 is removed by TFA, and then the compound L-Pro-8 is synthesized with 3-hydroxy-2-pyridinecarboxylic acid under the conditions of HATU and DIPEA.

(1) Synthesis of Compound L-Pro-2

Compound L-Pro-2(1.7g, 91%).¹H NMR(400MHz,CDCl₃)δ7.76(dd,J＝7.6, 4.0Hz,2H),7.66–7.48(m,2H),7.45–7.23(m,9H),5.54–5.36(m,1H), 5.25–4.92(m,2H),4.57–4.42(m,1H),4.43–4.30(m,2H),4.25(dq,J ＝18.7,6.2,5.0Hz,2H),3.70–3.56(m,1H),3.51(dt,J＝10.4,7.1Hz,1H), 2.19(dtd,J＝18.1,8.2,3.8Hz,1H),2.02–1.78(m,3H),1.42(d,J＝2.2 Hz,9H),1.30(d,J＝6.4Hz,2H),1.19(d,J＝6.4Hz,1H).HRMS(ESI)calcd for C₃₆H₄₀N₂NaO₈+[M+Na]⁺651.26769；found 651.26782.

(2) Synthesis of Compound L-Pro-4

Compound L-Pro-4(0.17g, 56% yield over two steps).¹H NMR(500MHz,Methanol-d4)δ8.51 (d,J＝2.1Hz,1H),8.43(dd,J＝4.9,1.6Hz,1H),7.84(dt,J＝7.9,1.9Hz, 1H),7.47–7.28(m,6H),5.38(tt,J＝6.5,3.3Hz,1H),5.25–5.06(m,2H), 4.48–4.33(m,2H),3.83–3.71(m,2H),3.65(dt,J＝9.8,6.5Hz,1H),3.58 (ddd,J＝8.8,6.0,1.8Hz,1H),3.17(dd,J＝14.0,8.8Hz,1H),3.12–2.94 (m,2H),2.16(dt,J＝12.6,8.0Hz,1H),2.01–1.91(m,2H),1.84(dt,J＝ 13.4,5.6Hz,1H),1.45(s,9H),1.22(d,J＝6.4Hz,3H),1.06(d,J＝6.9Hz, 3H).HRMS(ESI)calcd for C₃₂H₄₃N₆O₈ ⁺[M+H]⁺639.31369；found 639.31384；C₃₂H₄₂N₆O₈Na⁺ [M+Na]⁺661.29563；found 661.29553.

(3) Synthesis of Compound L-Pro-6

Compound L-Pro-6(25mg, 31.6%).¹H NMR(400MHz,Methanol-d4)：δ8.45(d, J＝2.2Hz,1H),8.38(dd,J＝4.9,1.6Hz,1H),7.78(dt,J＝7.8,1.9Hz,1H), 7.38(dd,J＝7.9,4.9Hz,1H),5.59(dt,J＝9.7,4.7Hz,1H),4.36(ddd,J ＝13.0,9.4,5.2Hz,3H),3.76–3.40(m,3H),3.07–2.83(m,2H),2.62(dq, J＝9.9,6.5Hz,1H),2.20(dddd,J＝28.9,15.0,7.8,2.9Hz,2H),2.11–2.00 (m,1H),1.92(qd,J＝6.6,2.7Hz,1H),1.44(s,9H),1.29(d,J＝6.2Hz,3H), 1.06(d,J＝6.5Hz,3H).HRMS(ESI)calcd for C₂₅H₃₇N₄O₇ ⁺[M+H]⁺505.26568,found 505.26575.

(4) Synthesis of Compound L-Pro-8

Compound L-Pro-8(1mg, 16%).¹H NMR(500MHz,Methanol-d4)δ8.46(d,J＝2.2 Hz,1H),8.37(dd,J＝4.9,1.6Hz,1H),8.22–8.13(m,1H),7.79(dt,J＝ 7.9,2.0Hz,1H),7.48(dd,J＝8.5,4.4Hz,1H),7.37(ddd,J＝8.4,3.5,1.1 Hz,2H),5.73(qd,J＝6.4,3.4Hz,1H),4.45(dd,J＝8.5,3.1Hz,1H),4.39 (dd,J＝9.4,6.1Hz,1H),3.79–3.64(m,2H),3.60(d,J＝9.9Hz,1H),3.49 (dt,J＝11.7,7.6Hz,1H),3.23(q,J＝7.4Hz,1H),3.07–2.91(m,2H),2.71 (dq,J＝9.9,6.6Hz,1H),2.39–2.25(m,1H),2.25–2.12(m,1H),2.07(ddt, J＝12.9,6.6,3.5Hz,1H),1.94(ddt,J＝15.2,7.1,3.5Hz,1H),1.33(d,J ＝6.4Hz,3H),1.12(d,J＝6.6Hz,3H).¹³C NMR(150MHz,Methanol-d4)δ175.27, 172.84,168.98,168.69,149.26,146.73,137.46,135.32,135.12,129.04,128.99, 125.45,123.85,123.80,74.85,73.53,59.34,54.90,51.38,46.29,41.17,34.42, 30.50,22.73,15.18,13.49.HRMS(ESI)calcd for C₂₆H₃₂N₅O₇ ⁺[M+H]⁺526.22962,found 526.22974。

Example three: synthesis of D-pipecolic acid derivatives

As shown in figure 5, a compound D-Pip-1 and a compound L-Tre-1 are subjected to a peptide-joining reaction under an EDCI condition to obtain a compound D-Pip-2, then the compound D-Pip-2 is subjected to Fmoc protection group removal under the conditions of diethylamine and dichloromethane to obtain a compound D-Pip-3, and then the compound D-Pip-3 and pyridine unnatural amino acid triethylamine salt P2 are subjected to an amidation reaction to obtain a compound D-Pip-4. And then carrying out palladium-carbon hydrogenation reaction on the compound D-Pip-4, and then carrying out one-step cyclization reaction under the conditions of HATU and DIPEA to obtain the compound D-Pip-6. And finally, removing the Boc protecting group on the compound D-Pip-6 by TFA, and then reacting with 3-hydroxy-2-picolinic acid under the conditions of HATU and DIPEA to obtain the compound D-Pip-8. (1) Synthesis of Compound D-Pip-2

Compound D-Pip-2(1.48g, 90%).¹HNMR(400MHz,CDCl₃)δ7.77(d,J＝7.6Hz, 2H),7.61(t,J＝6.7Hz,1H),7.53(dd,J＝7.5,4.6Hz,1H),7.49–7.25(m, 9H),5.49(d,J＝6.5Hz,1H),5.34–5.03(m,3H),4.94–4.65(m,1H),4.43 (ddd,J＝31.7,14.3,9.4Hz,3H),4.26(dt,J＝29.9,7.1Hz,1H),4.14–3.94 (m,1H),3.15–2.85(m,1H),2.10(t,J＝15.5Hz,1H),1.71–1.58(m,4H), 1.47(s,3H),1.40(s,6H),1.32(d,J＝6.4Hz,3H).HRMS(ESI)calcd for C₃₇H₄₂N₂NaO₈ ⁺ [M+Na]⁺665.28334,found 665.28339。

(2) Synthesis of Compound D-Pip-4

Compound D-Pip-4(0.15g, 46% yield over two steps).¹H NMR(500MHz,CDCl₃)δ8.61–8.46 (m,2H),7.64(d,J＝8.1Hz,1H),7.36(q,J＝4.7,3.9Hz,5H),7.25(d,J ＝4.7Hz,1H),5.52–5.47(m,1H),5.28(d,J＝5.8Hz,1H),5.15(q,J＝12.3 Hz,2H),4.55(dd,J＝9.8,2.7Hz,1H),4.27(d,J＝5.5Hz,1H),3.92(d,J ＝13.6Hz,1H),3.79(s,1H),3.41(td,J＝7.4,2.6Hz,1H),3.27–3.17(m, 1H),3.14–3.08(m,2H),2.19(d,J＝14.1Hz,1H),1.77–1.62(m,5H),1.47 (s,9H),1.32(d,J＝6.4Hz,3H),1.10(d,J＝6.9Hz,3H).HRMS(ESI)calcd for C₃₃H₄₅N₆O₈ ⁺[M+H]⁺653.32934,found 653.33130。

(3) Synthesis of Compound D-Pip-6

Compound D-Pip-6(24mg, 31.5%).¹H NMR(400MHz,Methanol-d4)δ8.46(s, 1H),8.36(d,J＝4.9Hz,1H),7.73(d,J＝7.8Hz,1H),7.32(dd,J＝7.8,5.0 Hz,1H),5.26(d,J＝5.7Hz,1H),5.17–5.01(m,1H),4.30(d,J＝6.0Hz, 1H),4.07(t,J＝7.4Hz,1H),3.93–3.76(m,1H),3.64(s,1H),3.00(dd, J＝13.5,7.4Hz,1H),2.90(dt,J＝15.0,8.4Hz,2H),2.79(q,J＝7.3Hz, 1H),2.29(d,J＝13.9Hz,1H),1.81–1.50(m,4H),1.46(s,10H),1.33(d, J＝7.2Hz,3H),1.26(d,J＝6.4Hz,3H).HRMS(ESI)calcd for C₂₆H₃₉N₄O₇ ⁺[M+H]⁺ 519.28133,found 519.28149.

(4) Synthesis of Compound D-Pip-8

Compound D-Pip-8(0.6mg, 16%).¹H NMR(400MHz,Methanol-d4)δ8.39(d,J ＝2.2Hz,1H),8.14(dd,J＝4.4,1.4Hz,1H),8.04(dd,J＝4.9,1.6Hz,1H), 7.67(dt,J＝7.9,1.9Hz,1H),7.50(dd,J＝8.5,4.3Hz,1H),7.39(dd,J＝ 8.5,1.4Hz,1H),7.07(dd,J＝7.8,4.9Hz,1H),5.33(d,J＝5.7Hz,1H),5.24 (t,J＝6.4Hz,1H),4.79(d,J＝6.4Hz,1H),4.17(ddd,J＝8.6,6.3,2.2Hz, 1H),3.88(d,J＝13.5Hz,1H),3.75(t,J＝1.6Hz,1H),3.02–2.82(m,4H), 2.32(d,J＝13.8Hz,1H),1.84–1.66(m,2H),1.67–1.41(m,3H),1.38(d, J＝7.3Hz,3H),1.28(s,3H).¹³C NMR(151MHz,MeOD)δ179.09,168.97,168.30, 167.97,157.73,149.74,146.32,139.72,138.02,134.81,130.54,129.14,125.91, 123.24,76.14,68.60,56.40,52.80,51.57,43.62,35.75,35.59,25.00,24.62, 20.19,17.08,12.96.HRMS(ESI)calcd for C₂₇H₃₄N₅O₇ ⁺[M+H]⁺540.24527,found 540.24512。

Example four: synthesis of L-pipecolic acid derivatives

As shown in figure 6, the compound L-Pip-1 and the compound L-Tre-1 are subjected to a peptide-joining reaction under the EDCI condition to obtain a compound L-Pip-2, then the compound L-Pip-2 is subjected to Fmoc protection group removal under the conditions of diethylamine and dichloromethane to obtain a compound L-Pip-3, and then the compound L-Pip-3 and pyridine unnatural amino acid triethylamine salt P2 are subjected to an amidation reaction to obtain a compound L-Pip-4. And then carrying out palladium-carbon hydrogenation reaction on the compound L-Pip-4 to obtain a ring-closing precursor, and then carrying out one-step cyclization reaction under the conditions of HATU and DIPEA to obtain the compound L-Pip-6. Finally, the Boc protecting group on the compound L-Pip-6 is removed by TFA, and then the compound L-Pip-8 is synthesized with 3-hydroxy-2-picolinic acid under the conditions of HATU and DIPEA.

(1) Synthesis of Compound L-Pip-2

Compound L-Pip-2(1.5g, 91%).¹H NMR(500MHz,CDCl₃)δ7.77(d,J＝7.1Hz, 2H),7.57(ddd,J＝44.2,17.2,7.5Hz,2H),7.41(td,J＝7.4,2.9Hz,2H), 7.37–7.27(m,7H),5.49(dt,J＝12.9,4.6Hz,1H),5.29–5.01(m,3H), 4.96–4.74(m,1H),4.51(dq,J＝10.8,7.7,5.6Hz,1H),4.46–4.34(m, 1H),4.34–4.18(m,1H),4.05(td,J＝15.8,13.8,7.6Hz,1H),3.11–2.72 (m,1H),2.25–2.11(m,1H),1.65(dd,J＝13.3,6.6Hz,3H),1.40(s,7H), 1.37(s,3H),1.31(d,J＝6.4Hz,2H),1.20(d,J＝6.8Hz,1H).HRMS(ESI) calcd for C₃₇H₄₂N₂NaO₈ ⁺[M+Na]⁺665.28334,found 665.28339。

(2) Synthesis of Compound L-Pip-4

Compound L-Pip-4(0.16g, 49% yield over two steps).¹H NMR(400MHz,Methanol-d₄)δ8.52 (d,J＝2.2Hz,1H),8.43(dd,J＝4.9,1.6Hz,1H),7.85(dt,J＝7.9,1.9Hz, 1H),7.49–7.27(m,6H),5.58–5.24(m,2H),5.24–5.03(m,2H),4.40(d, J＝3.5Hz,1H),3.92(dd,J＝42.0,10.8Hz,1H),3.76(dd,J＝8.4,1.8Hz, 1H),3.71–3.45(m,1H),3.27–2.90(m,4H),2.26(dd,J＝31.8,13.7Hz, 1H),1.71–1.47(m,5H),1.46(d,J＝15.0Hz,9H),1.33(d,J＝6.3Hz,1H), 1.26(d,J＝6.4Hz,2H),1.05(d,J＝6.9Hz,3H).HRMS(ESI)calcd for C₃₃H₄₅N₆O₈ ⁺ [M+H]⁺653.32934,found 653.33130。

(3) Synthesis of Compound L-Pip-6

Compound L-Pip-6(25mg, 28%).¹H NMR(400MHz,Methanol-d4)δ8.44(d,J ＝2.2Hz,1H),8.37(dd,J＝5.0,1.6Hz,1H),7.77(d,J＝7.9Hz,1H),7.37 (ddd,J＝7.9,4.9,0.9Hz,1H),5.48(dd,J＝6.4,3.6Hz,1H),4.46(dd,J ＝6.9,3.4Hz,1H),4.38(d,J＝3.5Hz,1H),4.36–4.21(m,2H),3.53(d, J＝9.4Hz,1H),3.24–3.07(m,2H),3.06–2.85(m,2H),2.78(dq,J＝9.6, 6.6Hz,1H),2.05–1.68(m,3H),1.68–1.51(m,2H),1.43(s,9H),1.29(d, J＝6.3Hz,3H),1.04(d,J＝6.6Hz,3H).HRMS(ESI)calcd for C₂₆H₃₉N₄O₇ ⁺[M+H]⁺ 519.28133,found 519.28149。

(4) Synthesis of Compound L-Pip-8

Compound L-Pip-8(1mg, 24%).¹H NMR(400MHz,Methanol-d₄)δ8.45(d,J＝ 2.2Hz,1H),8.36(dd,J＝4.9,1.6Hz,1H),8.14(dd,J＝4.4,1.4Hz,1H), 7.77(dt,J＝8.0,1.9Hz,1H),7.47(dd,J＝8.5,4.4Hz,1H),7.43–7.26 (m,2H),5.65–5.56(m,1H),4.61–4.52(m,2H),4.40–4.25(m,2H),3.58 (d,J＝9.4Hz,1H),3.20(td,J＝13.0,4.2Hz,1H),3.04–2.79(m,4H),2.00 –1.80(m,3H),1.62(q,J＝4.9Hz,2H),1.32(d,J＝6.4Hz,3H),1.09(d, J＝6.6Hz,3H).¹³C NMR(150MHz,MeOD)δ177.79,171.59,168.93,168.74,149.26, 149.23,146.73,146.49,137.77,137.49,135.10,129.04,123.79,123.56,75.14, 73.37,55.59,55.01,51.48,38.97,38.70,34.61,25.63,23.13,17.97,15.21, 13.49.HRMS(ESI)calcd for C₂₇H₃₄N₅O₇ ⁺[M+H]⁺540.24527,found 540.24512。

Example five: synthesis of Small molecule S-1

As shown in the figure (figure 7), amidation reaction is carried out on the compound S-1-1 and tert-butyloxycarbonyl o-phenylenediamine to obtain a compound S-1-2, and then the tert-butyloxycarbonyl protecting group is removed by TFA. Protecting the hydroxyl of the 3-hydroxyl-2-picolinic acid by methoxy methyl ether, then carrying out amidation reaction with a compound S-2-3, and removing the MOM protecting group to obtain a target product S-1.

Dissolving 0.2g of compound S-1-1 in 8mL of dry DMF under the protection of nitrogen, cooling to 0 ℃, adding EDCI (0.62g, 2eq.) sequentially, tert-butoxy o-phenylenediamine (0.35g, 1eq.) and DMAP (0.02g, 0.1eq.) sequentially, recovering to room temperature, reacting for 5h, and obtaining a thin layerAnd (5) carrying out chromatographic monitoring. To the system was added 8mL ethyl acetate, 10mL saturated NaHCO₃The solution was quenched, the organic phase was separated, the aqueous phase was extracted with ethyl acetate (10 mL. times.3), the organic phase was in turn extracted with water (10mL), saturated NaHCO₃Solution (10mL) wash, saturated NaCl solution (10mL) wash, anhydrous Na₂SO₄And (5) drying. After filtration, the reaction mixture was concentrated under reduced pressure and subjected to normal-phase silica gel column chromatography to obtain compound S-1-2(0.51g, 98%).

0.32g of the compound S-1-2 was dissolved in 5mL of dichloromethane, and a 2M ethanol solution of hydrogen chloride (5mL, 10eq.) was added at room temperature, followed by reaction overnight at room temperature and monitoring by thin layer chromatography. The ethanol solution was concentrated under reduced pressure to remove excess hydrogen chloride, methylene chloride (5 mL. times.3) was added, the mixture was concentrated under reduced pressure to remove the residual acid, and toluene (5 mL. times.3) was added to remove the residual water from the system by azeotropic distillation under reduced pressure. 0.2g of a crude product of the compound S-1-3 was obtained.

0.2g of the crude product of compound S-1-3 was dissolved in 4.3mL of dry DMF and MOM protected sodium salt of 3-hydroxy-2-pyridinecarboxylic acid (0.28g, 1eq.) was added and cooled to 0 ℃. HATU (1.66g, 5eq.) and DIPEA (1.55mL, 10eq.) were added sequentially, stirred overnight at room temperature and monitored by LC-MS. The system was quenched by addition of 5mL of water, extracted with ethyl acetate (20 mL. times.3), and the organic phase was quenched with saturated NaHCO₃Solution (10mL), water (10mL), saturated NaCl solution (10mL) washed with anhydrous Na₂SO₄And (5) drying. After filtration and concentration under reduced pressure, the residue is dissolved in 5mL of THF, 5mL of 2M dilute hydrochloric acid are added, stirring is carried out overnight at room temperature, and monitoring is carried out by thin-layer chromatography. Diluting with 10mL ethyl acetate, separating organic phase, extracting the aqueous phase with ethyl acetate (10mL × 3) to remove ester-soluble impurities, retaining the aqueous phase, adding 5mL methanol, cooling to 0 deg.C, adjusting pH with 10% NaOH solution to alkaline, extracting the reaction system with ethyl acetate (10mL × 5), extracting the organic phase with saturated NaHCO₃Solution (10mL), water (10mL), saturated NaCl solution (10mL) washed with anhydrous Na₂SO₄And (5) drying. After filtration, the mixture was concentrated under reduced pressure and subjected to normal-phase silica gel column chromatography to give compound S-1(0.09g, 27% in the four steps).¹H NMR(400MHz,DMSO-d₆)δ12.03–11.78(m,1H),10.57(d, J＝16.3Hz,2H),9.16(d,J＝2.3Hz,1H),8.77(dd,J＝4.9,1.7Hz,1H),8.33 (dt,J＝8.0,2.0Hz,1H),8.11(d,J＝4.3Hz,1H),7.92(d,J＝7.9Hz,1H), 7.62–7.49(m,3H),7.45(dd,J＝8.5,1.4Hz,1H),7.34(dtd,J＝24.0,7.6, 1.6Hz,2H).HRMS(ESI)calcd for:C₁₈H₁₅N₄O₃ ⁺[M+H]⁺335.11387,found 335.11386。

Example six: synthesis of small molecule S-2

Referring to FIG. 8, and to example five, Compound S-2(28mg, 25% yield over four steps) was obtained.¹H NMR(300 MHz,CDCl₃)δ11.81(s,1H),8.96(d,J＝2.3Hz,1H),8.67(dd,J＝4.9,1.7 Hz,1H),8.21(d,J＝7.4Hz,1H),8.07–7.97(m,2H),7.37–7.29(m,2H), 7.28–7.25(m,1H),7.23(dd,J＝6.9,4.0Hz,1H),4.12–3.87(m,2H),2.36 (dt,J＝12.5,2.8Hz,1H),2.14(dt,J＝12.5,2.6Hz,1H),1.95–1.77(m, 2H),1.68–1.27(m,4H).HRMS(ESI)calcd for:C₁₈H₂₁N₄O₃+[M+H]+341.16082, found 341.16165；C₁₈H₂₀N₄NaO₃ ⁺[M+Na]⁺363.14276；found 363.14346。

Example seven: synthesis of small molecule S-3

See FIG. 9 and example five to yield compound S-3(0.07g, 20% overall yield over four steps).¹H NMR(500MHz,DMSO-d6)δ11.91(s,1H),10.39(s,1H),10.22(s,1H),8.55(d, J＝2.2Hz,1H),8.41(dd,J＝4.8,1.6Hz,1H),8.14(d,J＝4.4Hz,1H),7.82 (d,J＝7.8Hz,1H),7.74(dt,J＝8.0,2.0Hz,1H),7.59(dd,J＝8.5,4.3Hz, 1H),7.47(dd,J＝8.5,1.3Hz,1H),7.38(dd,J＝7.6,1.8Hz,1H),7.28(dtd, J＝22.0,7.6,5.3Hz,3H),3.77(s,2H).HRMS(ESI)calcd for:C₁₉H₁₇N₄O₃ ⁺[M+H]⁺ 349.12952,found 349.12958；C₁₉H₁₆N₄NaO₃ ⁺[M+Na]⁺371.11146；found 371.11148。

Example eight: synthesis of small molecule S-4

Referring to FIG. 10, and referring to example five, Compound S-2(20mg, 27% yield over four steps) was obtained.¹H NMR (500MHz,Methanol-d₄)δ8.31(d,J＝2.2Hz,1H),8.14(dd,J＝4.9,1.6Hz, 1H),7.98(dd,J＝4.4,1.3Hz,1H),7.53(dt,J＝8.0,1.8Hz,1H),7.39(dd, J＝8.5,4.3Hz,1H),7.29(dd,J＝8.5,1.3Hz,1H),6.97(dd,J＝7.9,4.9 Hz,1H),3.84(dtd,J＝27.9,10.7,3.8Hz,2H),3.53–3.36(m,2H),2.11– 1.92(m,2H),1.90–1.67(m,2H),1.58–1.29(m,4H).HRMS(ESI)calcd for: C₁₉H₂₃N₄O₃ ⁺[M+H]⁺355.17647,found 355.17740；C₁₉H₂₂N₄NaO₃ ⁺[M+Na]⁺377.15841；found 377.15930。

Example nine: synthesis of Compound S-5

Referring to FIG. 11, and referring to example five, Compound S-5(0.050g, four-step overall yield 30%) was obtained.¹H NMR(400 MHz,Methanol-d₄)δ8.60(d,J＝2.2Hz,1H),8.39(dd,J＝4.9,1.6Hz,1H), 8.20(dd,J＝4.4,1.4Hz,1H),7.90(dt,J＝8.0,2.0Hz,1H),7.49(dd,J＝ 8.5,4.3Hz,1H),7.43(dd,J＝7.8,1.5Hz,1H),7.41–7.35(m,2H),7.08 (td,J＝7.8,1.6Hz,1H),6.76(td,J＝7.6,1.3Hz,1H),6.69(dd,J＝8.2, 1.3Hz,1H),4.45(s,2H).¹³C NMR(100MHz,Methanol-d₄)δ168.17,158.13,148.02, 147.11,142.41,139.64,136.47,136.09,131.62,128.93,127.34,126.05,125.82, 123.84,123.09,117.68,113.00,44.76.HRMS(ESI)calcd for:C₁₈H₁₇N₄O₂ ⁺[M+H]⁺ 321.13460,found 321.13458。

Example ten: synthesis of Compound S-6

Referring to fig. 12, compound S-4-1(0.5g, 1eq.) was dissolved in 7mL of dry dichloromethane under nitrogen, cooled to 0 ℃, EDCI (0.50g,2eq.), 3-aminomethylpyridine (0.15g,1eq.), DMAP (0.01 g,0.1eq.) added, and stirred for 5h at room temperature for tlc monitoring. After the reaction was completed, water was added to quench, and DCM was used to extract the organic phase, followed by water (5mL) and saturated NaHCO₃Solution (5mL) wash, saturated NaCl solution (5mL) wash, anhydrous Na₂SO₄And (5) drying. After filtration, the reaction mixture was concentrated under reduced pressure and subjected to normal-phase silica gel column chromatography to obtain compound S-6-2(0.58g, 92%).

0.2g of the compound S-6-2 was dissolved in 2mL of dichloromethane, and diethylamine (2mL) was added at room temperature to react for 2 hours at room temperature, followed by thin layer chromatography. After the reaction is completedSpin-dry to give the crude compound S-6-3, which was used directly in the next step. The crude compound S-6-3 was dissolved in dry DMF (3mL), MOM protected sodium salt of 3-hydroxy-2-pyridinecarboxylic acid (0.0.19g, 1eq.) was added, and cooled to 0 ℃. HATU (1.14g, 5eq.) and DIPEA (1.06 mL, 10eq.) were added sequentially, stirred overnight at room temperature and monitored by LC-MS. The system was quenched by addition of 3mL of water, extracted with ethyl acetate (10 mL. times.3), and the organic phase was quenched with saturated NaHCO₃Solution (5mL), water (5mL), saturated NaCl solution (5mL) washed with anhydrous Na₂SO₄And (5) drying. After filtration, concentration under reduced pressure and dissolution of the residue in 5mL of THF, addition of 5mL of 2M diluted hydrochloric acid, stirring overnight at room temperature, monitoring by thin layer chromatography. Diluting with 10mL ethyl acetate, separating organic phase, extracting the aqueous phase with ethyl acetate (10mL × 3) to remove ester-soluble impurities, retaining the aqueous phase, adding 5mL methanol, cooling to 0 deg.C, adjusting pH with 10% NaOH solution to alkaline, extracting the reaction system with ethyl acetate (10mL × 5), extracting the organic phase with saturated NaHCO₃Solution (10mL), water (10mL), saturated NaCl solution (10mL) washed with anhydrous Na₂SO₄And (5) drying. After filtration, the mixture was concentrated under reduced pressure and subjected to normal phase silica gel column chromatography to give compound S-6(31mg, 20% yield in four steps).¹H NMR(400MHz,CDCl₃)δ7.70(d,J＝7.8Hz,2H),7.41–7.27(m,4H),7.23– 7.13(m,1H),4.14(dd,J＝11.9,5.2Hz,1H),3.95(dd,J＝14.9,6.8Hz,1H), 3.55(dd,J＝14.9,6.0Hz,1H),2.87(dd,J＝12.5,5.3Hz,1H),2.76(dd,J ＝10.7,3.5Hz,1H),2.39–2.10(m,2H),2.08–1.95(m,1H),1.90–1.75 (m,3H).HRMS(ESI)calcd for:C₁₈H₁₉N₄O₃ ^-[M-H]^-339.14626,found 39.14597。

Example eleven: synthesis of Compound S-7

Referring to FIG. 13, compound S-8(50mg, 25% yield over four steps) was obtained according to example five.¹H NMR(500 MHz,Methanol-d₄)δ8.53–8.28(m,2H),8.22–7.95(m,1H),7.78(dd,J＝26.5,7.9Hz,1H),7.50–7.24(m,3H),5.28(d,J＝5.4Hz,1H),3.58(ddt, J＝49.5,13.3,6.8Hz,2H),2.95(tdq,J＝19.5,12.9,6.5,5.7Hz,3H),2.43 –2.29(m,1H),1.64(ddt,J＝16.8,13.5,4.4Hz,2H),1.49(tt,J＝12.0, 3.7Hz,2H),1.43–1.16(m,2H).HRMS(ESI)calcd for:C₁₉H₂₃N₄O₃ ⁺[M+H]⁺355.17647, found 355.17749,C₁₉H₂₂N₄NaO₃ ⁺[M+Na]⁺377.15841,found 377.15936。

Claims

1. A derivative of Pyridomycin, characterized in that: (1) replacing the exocyclic unsaturated double bond at position C2 with a cyclic amino acid that can modulate the conformation of the macrocycle; specifically, the conformation of the macrocycle is controlled by the size of the macrocycle and the alpha-position chiral configuration of carboxyl; (2) by utilizing the structural characteristics of ortho-diamine at the 6-7 position of a Pyridomycin molecule, two activity related structures of 3-hydroxy-2-picolinic acid and 3-pyridine in Pyridomycin are kept, and the micromolecule has the unique conformation of Pyridomycin by adjusting the dihedral angle of ortho-diamine.

2. The derivative of Pyridomycin according to claim 1, wherein: the cyclic amino acid is D-proline (D-Pro-8), L-proline (L-Pro-8), D-pipecolic acid (D-Pip-8) and/or L-pipecolic acid (L-Pip-8), and the specific structures are as follows in sequence:

D-Pro-8 and L-Pro-8 correspond to Pyridomycin replacing the functional group at position 1-2, proline controlling the conformation of the macrocycle; D-Pip-8 and L-Pip-8 correspond to Pyridomycin in place of its functional group at position 1-2, with pipecolic acid controlling the conformation of the macrocycle; all molecules retain the original substituted pyridine ring and retain their ability to bind to the target protein.

3. The derivative of Pyridomycin according to claim 1, wherein: the small molecule retains the small molecule simplified conformation form of pyridine binding sites at two ends; the ortho-diamine structure at the 6-7 position of the Pyridomycin molecule is simplified into a phenyl, cyclohexanediamine and/or piperidine structure, and the macrocyclic conformation is simulated by adjusting the dihedral angle of the ortho-diamine, so that the two pyridines of Pyridomycin conform to the form of the bound conformation. The structure of the o-diamine can also be replaced by the structural forms of cyclopentediamine, proline and the like;

the phenyl is N, N' -pyridine derivative-o-phenylenediamine micromolecules, and the configuration of the phenyl comprises: phenyl first structure (S-1), phenyl second structure (S-3), phenyl third structure (S-5), the concrete structure is in proper order:

4. the derivative of Pyridomycin according to claim 1, wherein: substituting 1-2 site atoms of pyridomycin with five-membered cyclic amine, wherein the five-membered cyclic amine is D-proline and/or L-proline; to control the macrocyclic conformation; the derivatives are marked as D-Pro-8 and L-Pro-8;

the synthesis process of the five-membered cyclic amine comprises the following steps: carrying out amidation reaction on the compound D/L-Pro-1 and the compound L-Tre-1 under the EDCI condition to obtain a compound D/L-Pro-2; the compound D/L-Pro-1 is N-Fmoc-D-Pro-OH, and the compound L-Tre-1 is N-Boc-L-Thr-OBn; removing Fmoc protecting groups from the compound D/L-Pro-2 under the conditions of diethylamine and dichloromethane to obtain a compound D/L-Pro-3, and then carrying out amidation reaction with pyridine unnatural amino acid triethylamine salt P2 to obtain a compound D/L-Pro-4; then, carrying out palladium-carbon hydrogenation reaction on the compound D/L-Pro-4 to obtain a ring-closing precursor, and then carrying out one-step cyclization under the conditions of HATU, DIPEA and DMF to obtain a compound D/L-Pro-6; finally, the Boc protecting group on the compound D/L-Pro-6 is removed by TFA, and then amidation reaction is carried out with 3-hydroxy-2-picolinic acid under the conditions of HATU and DIPEA, thereby completing the synthesis of the compound D/L-Pro-8.

5. The derivative of Pyridomycin according to claim 1, wherein: replacing the 1-2 atom of pyridomycin with a six-membered cyclic amine to control the macrocyclic conformation; the hexatomic cyclic amine is D-pipecolic acid and/or L-pipecolic acid; the derivatives are marked as D-Pip-8 and L-Pip-8;

the synthesis process of the hexabasic cyclic amine comprises the following steps: carrying out peptide-joining reaction on a compound D/L-Pip-1{ N-Fmoc-D/L-Pro-OH } and a compound L-Tre-1{ N-Boc-L-Thr-OBn } under the EDCI condition to obtain a compound D/L-Pip-2; the compound D/L-Pip-1 is N-Fmoc-D/L-Pro-OH; the compound L-Tre-1 is N-Boc-L-Thr-OBn; then removing Fmoc protective groups from the compound D/L-Pip-2 under the conditions of diethylamine and dichloromethane to obtain a compound D/L-Pip-3, and carrying out amidation reaction on the compound D/L-Pip-3 and pyridine unnatural amino acid triethylamine salt P2 to obtain a compound D/L-Pip-4; then, carrying out palladium-carbon hydrogenation reaction on the compound D/L-Pip-4, and then carrying out one-step cyclization reaction under the conditions of HATU and DIPEA to obtain a compound D/L-Pip-6; and finally, removing the Boc protecting group on the compound D/L-Pip-6 by TFA, and then reacting with 3-hydroxy-2-picolinic acid under the conditions of HATU and DIPEA to obtain the compound D/L-Pip-8.

6. The derivative of Pyridomycin according to claim 3, wherein: the synthesis process of the N, N' -pyridine derivative-o-phenylenediamine micromolecules S-1, S-3 and S-5 comprises the following steps: subjecting compound S-1-1{ please specifically introduce the chemical structure of S-1-1, such as the existing product with trade name or common name } to amidation reaction with t-butyloxycarbonyl o-phenylenediamine to obtain compound S-1-2, and then removing the t-butyloxycarbonyl protecting group with TFA; protecting the hydroxyl of the 3-hydroxyl-2-picolinic acid by methoxy methyl ether, then carrying out amidation reaction with a compound S-2-3, and removing the MOM protecting group to obtain a target product S-1.

7. The derivative of Pyridomycin according to claim 3, wherein: the synthesis method of the N, N' -pyridine derivative-pipecolic acid micromolecule compound S-6 comprises the following steps: dissolving a compound S-4-1 in dry dichloromethane under the protection of nitrogen, cooling to 0 ℃, adding EDCI, 3-aminomethyl pyridine and DMAP, recovering the room temperature, stirring, and monitoring by thin-layer chromatography; after the reaction is completed, water is added for quenching, and DCM is used for extracting organic phase, and water and saturated NaHCO are used for next time₃Solution washing, saturated NaCl solution washing, anhydrous Na₂SO₄Drying; filtering, concentrating under reduced pressure, and separating with normal phase silica gel column chromatography to obtain compound S-6-2;

8. The derivative of Pyridomycin according to claim 1, wherein: the large ring conformation of the pyridomycin natural product is simulated, the five-membered ring structure or the six-membered ring structure of proline, pipecolic acid and the like is utilized to control the large ring conformation, and the two pyridine ring derivative structures of the side chains are reserved. And further using six-membered or five-membered cyclic o-diamine such as phenylenediamine, cyclohexanediamine or pipecolic acid to simulate a 6-7-position o-diamine structure of the pyridomycin molecule, regulating dihedral angle through the change of the o-diamine substitution form, simulating the conformation of a natural product, and adjusting the binding capacity of two pyridine derivative functional groups on side chains and the target protein.

9. The derivative of Pyridomycin according to claim 1, wherein: the derivative molecules or reasonable salt forms and combination forms thereof can have potential activities of resisting microorganisms, bacteria, fungi and the like, and particularly have certain resistance to tubercle bacillus; further may have other potential biological activities.