CN113563415A

CN113563415A - Dipeptide, preparation method and application thereof, metallo-beta-lactamase inhibitor and pharmaceutical composition

Info

Publication number: CN113563415A
Application number: CN202110836429.4A
Authority: CN
Inventors: 何松宏; 张冰; 杨玉凤
Original assignee: Guangzhou Baicao Sangzi Pharmaceutical Technology Co ltd
Current assignee: Guangzhou Baicao Sangzi Pharmaceutical Technology Co ltd
Priority date: 2021-07-23
Filing date: 2021-07-23
Publication date: 2021-10-29
Anticipated expiration: 2041-07-23
Also published as: CN113563415B

Abstract

The invention relates to a dipeptide, a preparation method and application thereof, a metal-beta-lactamase inhibitor and a pharmaceutical composition. The amino group of the dipeptide is connected with a protective group, the dipeptide is synthesized by condensation reaction of the carboxyl group of a first amino acid and the amino group of a second amino acid, the first amino acid is one of alanine, serine, cysteine, phenylalanine, tyrosine, tryptophan and aspartic acid, and the second amino acid is cysteine. The dipeptide has good inhibitory effect on the activity of metallo-beta-lactamase, and can be used as an active ingredient of MBLIs.

Description

Dipeptide, preparation method and application thereof, metallo-beta-lactamase inhibitor and pharmaceutical composition

Technical Field

The invention relates to the technical field of biological medicines, in particular to dipeptide, a preparation method and application thereof, a metal-beta-lactamase inhibitor and a pharmaceutical composition.

Background

The discovery of antibiotics is a very important milestone in recent medical history, and makes a great contribution to the improvement of the survival rate of infected patients. However, the improper use or even abuse of antibiotics has accelerated the development of bacterial resistance and has created significant challenges for human health.

Beta-lactam antibiotics are a generic term for antibiotics that contain a beta-lactam ring within their chemical structure. Beta-lactam antibiotics cause lysis of bacteria by causing damage to cell walls through inhibition of enzymes involved in cell wall synthesis, i.e., Penicillin Binding Proteins (PBPs). The problem of drug resistance of beta-lactam antibiotics, which are one of the most widely used antibiotics in clinical use, has been accompanied by a long history and is becoming serious.

In 1940, the first beta-lactamase enzyme was first reported and was found to hydrolyze the beta-lactam ring, thereby rendering the beta-lactam antibiotic ineffective. With the continuous research and development of beta-lactam antibiotics by various pharmaceutical companies, more and more antibiotics enter the market, so that the selection pressure of bacteria is greatly increased, a unique survival advantage is provided for the bacteria producing beta-lactamase, and almost all clinical gram-negative bacteria are found to express beta-lactamase, wherein the most common bacteria are escherichia coli, klebsiella pneumoniae, pseudomonas aeruginosa and the like. Over 2000 naturally occurring beta-lactamases have been identified, each enzyme having a unique amino acid sequence and hydrolytic properties.

For the classification of beta-lactamases, there are currently two main approaches, namely, the Ambler and the Bush-Jacoby classification. In the more common class of Ambler, beta-lactamases are classified into 4 classes, i.e. A, B, C, D, according to protein sequence and substrate specificity. According to the catalytic property of the active site of beta-lactamase, the beta-lactamase is divided into serine beta-lactamase (SBLs) and metallo-beta-lactamase (MBLs). SBLs include the A, C, D class of enzymes, which utilize serine to hydrolyze the β -lactam ring. MBLs include class B enzymes, which require divalent zinc ions to hydrolyze the beta-lactam ring, but MBLs containing other divalent metals, such as ferrous ions, have also been reported to be active. MBLs are further divided into three subclasses according to amino acid sequence, substrate specificity and the number of metal ions in the active site: b1, B2 and B3. Of these, the B1 and B3 subclasses of MBLs bind two zinc ions at the active site, whereas the B2 subclass of MBLs bind only one zinc ion for maximum catalytic activity and are inhibited when the second metal ion is present at the active center of the B2 subclass. MBLs have been reported to include New Delhi metallo beta-lactamases (NDMs), Verona integrase-encoded Imipenemases (VIMs) and Imipenemases (IMPs), among others.

MBLSs can hydrolyze almost all β -lactam antibiotics except monocyclic β -lactam antibiotics, and are of great harm. Metallo-beta-lactamase inhibitors (MBLIs) can inhibit the hydrolytic activity of MBLs, thereby restoring the sensitivity of bacteria to beta-lactam antibiotics and having great application potential. However, at present, although serine-beta-lactamase inhibitors such as clavulanic acid, sulbactam, tazobactam and abamectin which are already on the market in clinic are widely applied, no MBLIs is available for clinical use so far, and the clinical requirement is not met.

Disclosure of Invention

Based on this, the development of MBLIs is imminent. The present invention provides a dipeptide having MBLs inhibitory activity.

In addition, a preparation method of the dipeptide, application of the dipeptide in preparing a metallo-beta-lactamase inhibitor, the metallo-beta-lactamase inhibitor and a medicament are also provided.

A dipeptide having a protecting group attached to an amino group, the dipeptide being synthesized by a condensation reaction of a carboxyl group of a first amino acid selected from one of alanine, serine, cysteine, phenylalanine, tyrosine, tryptophan and aspartic acid with an amino group of a second amino acid, the second amino acid being cysteine.

The dipeptide has a structure in which an amino group of cysteine of the dipeptide is linked to a carboxyl group of another amino acid to form a thioglycolic acid structure to inhibit the activity of MBLs, and the dipeptide has a shorter distance between the thiol group and the carboxyl group than a dipeptide formed by condensing the carboxyl group of cysteine and the amino group of another amino acid, so that the dipeptide has a better effect of inhibiting MBLs; meanwhile, the free amino group of the dipeptide is subjected to Fmoc protection, so that the polarity of a branch chain outside a thioglycollic acid core structure is reduced, and the inhibitory activity of the dipeptide on MBLs is further improved. In addition, the dipeptide has broad inhibitory effect on MBLs, and has good inhibitory effect on NDM, VIM and IMP.

In one embodiment, the protecting group is selected from one of fluorenylmethyloxycarbonyl, benzyloxycarbonyl and tert-butoxycarbonyl.

In one embodiment, the dipeptide has the structure:

wherein, -R¹Is composed of

-CH₂SH、CH₂OH or-CH₂COOH；-R²is-Fmoc.

A method for producing a dipeptide comprising the steps of:

carrying out condensation reaction on a first amino acid with an amino group connected with a protective group and cysteine to prepare dipeptide; wherein the first amino acid is selected from one of alanine, serine, cysteine, phenylalanine, tyrosine, tryptophan and aspartic acid.

The dipeptide or the dipeptide prepared by the preparation method of the dipeptide is applied to the preparation of a metal-beta-lactamase inhibitor.

A metallo-beta-lactamase inhibitor comprises an active ingredient, wherein the active ingredient comprises the dipeptide or the dipeptide prepared by the preparation method of the dipeptide.

In one embodiment, the metallo-beta-lactamase inhibitor further comprises an adjuvant.

A pharmaceutical composition comprising a non-monocyclic beta-lactam antibiotic and said dipeptide or said dipeptide produced by the process for its preparation.

In one embodiment, the non-monocyclic β -lactam antibiotic is selected from at least one of a penicillin antibiotic, a cephalosporin antibiotic, a cephamycin antibiotic, a thiomycin antibiotic, and a carbapenem antibiotic.

In one embodiment, the non-monocyclic β -lactam antibiotic is selected from at least one of meropenem, ertapenem, and doripenem.

Drawings

FIG. 1 is a graph of the residual activity of Fmoc-F-C, Fmoc-Y-C and Fmoc-W-C on NDM-1 in example 2.

Detailed Description

The present invention will now be described more fully hereinafter for purposes of facilitating an understanding thereof, and may be embodied in many different forms and are not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Furthermore, the terms "first," "second," and the like are used for descriptive purposes only and are not to be construed as indicating or implying relative importance. Herein, "Fmoc" is fluorenylmethyloxycarbonyl.

In one embodiment, the present invention provides a dipeptide, wherein a protecting group is attached to an amino group of the dipeptide, the dipeptide is synthesized by a condensation reaction of a carboxyl group of a first amino acid and an amino group of a second amino acid, the first amino acid is one selected from the group consisting of alanine (a), serine (S), cysteine (C), phenylalanine (F), tyrosine (Y), tryptophan (W), and aspartic acid (D), and the second amino acid is cysteine.

The dipeptide has a structure in which the amino group of cysteine of the dipeptide is linked to the carboxyl group of another amino acid to form a thioglycolic acid structure to inhibit the activity of MBLSs, and the dipeptide has a shorter distance between the thiol group and the carboxyl group than a dipeptide formed by condensing the carboxyl group of cysteine with the amino group of another amino acid, so that the dipeptide has a better effect of inhibiting MBLs; meanwhile, the free amino group of the dipeptide is subjected to Fmoc protection, so that the polarity of a branch chain outside a thioglycollic acid core structure is reduced, and the inhibitory activity of the dipeptide on MBLs is further improved. In addition, the dipeptide has broad inhibitory effect on MBLs, and has good inhibitory effect on NDM, VIM and IMP.

The protecting group is used for protecting the free amino group of the dipeptide obtained by condensation reaction of the first amino acid and cysteine. Optionally, the protecting group is selected from one of fluorenylmethyloxycarbonyl (Fmoc), benzyloxycarbonyl (Cbz) and tert-butyloxy (Boc). It is understood that in other embodiments, the protecting group is not limited to the above, but may be other groups capable of protecting the amino group.

Further, the structure of the dipeptide is:

wherein, -R¹Is composed of

-CH₂SH、CH₂OH or-CH₂COOH；-R²is-Fmoc. That is, the first amino acid of the above dipeptide is phenylalanine, tyrosine or tryptophan, the second amino acid is cysteine, and the protecting group is fluorenylmethyloxycarbonyl. When the first amino acid is phenylalanine, tyrosine or tryptophan, the protecting group not only can reduce the polarity of a branched chain except a thioglycollic acid core structure, but also can increase the stacking effect of aromatic ring pi-pi. Proved by verification, the dipeptide not only has good affinity and high inhibition activity to MBLs, but also has synergistic effect with beta-lactam antibiotics, and can recoverThe antibacterial activity of the compound beta-lactam antibiotics.

In addition, an embodiment of the present invention provides a method for producing the above dipeptide, the method comprising the steps of:

the first amino acid with the amino group connected with the protective group and cysteine are subjected to condensation reaction to prepare the dipeptide.

Optionally, the method for preparing the dipeptide comprises the following steps: carrying out condensation reaction on cysteine and a first amino acid which are fixed on a solid phase carrier to prepare dipeptide connected on the solid phase carrier; and separating the dipeptide from the solid phase carrier to obtain the free dipeptide. The purification of the dipeptide obtained is facilitated by the immobilization of cysteine on a solid support.

The preparation method of the dipeptide is simple, convenient and easy to operate, and is beneficial to industrial production.

Based on the good inhibitory effect of the above dipeptides on the activity of MBLs. The application also provides an application of the dipeptide or the dipeptide prepared by the dipeptide preparation method in preparing MBLIs.

In addition, an embodiment of the present application also provides a metallo-beta-lactamase inhibitor, abbreviated as MBLIs, which comprises an active ingredient comprising the above dipeptide.

Optionally, the MBLIs further comprises an auxiliary material. The auxiliary materials are not particularly limited and may be selected according to actual requirements.

Optionally, the MBLIs is in the form of tablet, capsule or powder. Of course, the formulation of MBLIs is not limited to the above, and other formulations are possible.

The metallo-beta-lactamase inhibitor comprises the dipeptide and has good inhibition effect on the activity of MBLs.

In addition, the application also provides a pharmaceutical composition, which comprises the dipeptide or the dipeptide prepared by the preparation method of the dipeptide, and non-monocyclic beta-lactam antibiotics.

Optionally, the non-monocyclic β -lactam antibiotic is selected from at least one of a penicillin antibiotic, a cephalosporin antibiotic, a cephamycin antibiotic, a thiomycin antibiotic, and a carbapenem antibiotic.

In one embodiment, the penicillin antibiotic is at least one selected from the group consisting of penicillin, ampicillin, and amoxicillin. The cephalosporin antibiotics are selected from at least one of cefalexin, cefuroxime and ceftazidime. The cephamycine antibiotics are at least one of cefoxitin, cefotetan and cefmetazole. The thiomycin antibiotics are selected from thiomycin. The carbapenem antibiotic is at least one selected from Meropenem (Meropaem), Ertapenem (Ertapaem) and Doripenem (Doripaem).

In some embodiments, the non-monocyclic β -lactam antibiotic is a carbapenem antibiotic.

Optionally, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

Optionally, the pharmaceutical composition is a solid preparation, a liquid preparation or a semisolid preparation. In some embodiments, the solid formulation is selected from one of a tablet, a powder, a granule, and a capsule. The liquid preparation is injection. The semisolid preparation is selected from one of ointment and cream. It is understood that the dosage form of the above-mentioned drugs is not limited to the above-mentioned dosage forms, and may be adjusted according to actual needs.

In some embodiments, the pharmaceutical composition comprises MBLIs and a non-monocyclic β -lactam antibiotic as described above.

The pharmaceutical composition comprises the dipeptide and the non-monocyclic beta-lactam antibiotic, the activity of the dipeptide for inhibiting the MBLs is prevented, so that the non-monocyclic beta-lactam antibiotic is not hydrolyzed to be ineffective, and the killing/inhibiting effect of the non-monocyclic beta-lactam antibiotic on MBLs drug-resistant bacteria is effectively recovered. Moreover, the dipeptide has a synergistic effect with the non-monocyclic beta-lactam antibiotics through verification, and the antibacterial activity of the non-monocyclic beta-lactam antibiotics can be enhanced.

In addition, an embodiment of the present application also provides a medicament comprising the above dipeptide or the dipeptide obtained by the above process for producing a dipeptide, and a non-monocyclic β -lactam antibiotic.

In particular, the non-monocyclic β -lactam antibiotics are as described above and will not be described in further detail herein.

The medicament comprises the dipeptide and the non-monocyclic beta-lactam antibiotic, and can effectively recover the killing/inhibiting effect of the non-monocyclic beta-lactam antibiotic on MBLs drug-resistant bacteria.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The following detailed description is given with reference to specific examples. The following examples are not specifically described, and other components except inevitable impurities are not included. Reagents and instruments used in the examples are all conventional in the art and are not specifically described. The experimental procedures, in which specific conditions are not indicated in the examples, were carried out according to conventional conditions, such as those described in the literature, in books, or as recommended by the manufacturer. As used herein, "A-C" refers to a dipeptide formed by the condensation reaction of the carboxyl group of alanine with the amino group of cysteine; "Fmoc-A-C" refers to a dipeptide formed by the condensation reaction of the carboxyl group of alanine with the amino group of cysteine, and the free amino group of the alanine of the dipeptide is Fmoc protected; "S-C" refers to a dipeptide formed by the condensation reaction of the carboxyl group of serine and the amino group of cysteine; Fmoc-S-C is a dipeptide formed by condensation reaction of a carboxyl group of serine and an amino group of cysteine, and the free amino group of the serine of the dipeptide is protected by Fmoc; "Fmoc-C-C" refers to a dipeptide formed by the condensation of two cysteines and the free amino group of the dipeptide is protected by Fmoc; "M-C" is a dipeptide formed by the condensation reaction of the carboxyl group of methionine and the amino group of cysteine; "F-C" refers to a dipeptide formed by the condensation reaction of the carboxyl group of phenylalanine with the amino group of cysteine; "Fmoc-F-C" refers to a dipeptide formed by the condensation reaction of the carboxyl group of phenylalanine and the amino group of cysteine, and the free amino group on the phenylalanine of the dipeptide is protected by Fmoc; "Y-C" refers to a dipeptide formed by the condensation reaction of the carboxyl group of tyrosine and the amino group of cysteine; "Fmoc-Y-C" refers to a dipeptide formed by the condensation reaction of the carboxyl group of tyrosine and the amino group of cysteine, and the free amino group on the tyrosine of the dipeptide is protected by Fmoc; "W-C" refers to a dipeptide formed by the condensation reaction of the carboxyl group of tryptophan and the amino group of cysteine; "Fmoc-W-C" refers to a dipeptide formed by the condensation reaction of the carboxyl group of tryptophan and the amino group of cysteine, and the free amino group of tryptophan of the dipeptide is protected by Fmoc; "D-C" refers to a dipeptide formed by the condensation reaction of the carboxyl group of aspartic acid and the amino group of cysteine; "Fmoc-D-C" refers to a dipeptide formed by the condensation of the carboxyl group of aspartic acid with the amino group of cysteine, and the free amino group of aspartic acid of the dipeptide is Fmoc protected; "N-C" refers to a dipeptide formed by the condensation reaction of the carboxyl group of asparagine and the amino group of cysteine; "H-C" refers to a dipeptide formed by the condensation reaction of the carboxyl group of histidine with the amino group of cysteine; "Fmoc-H-C" refers to a dipeptide formed by the condensation reaction of the carboxyl group of histidine with the amino group of cysteine, and the free amino group of the histidine of the dipeptide is protected by Fmoc; "Fmoc-K-C" refers to a dipeptide formed by the condensation reaction of the carboxyl group of lysine and the amino group of cysteine, and the free amino group of the lysine of the dipeptide is protected by Fmoc; "Fmoc-R-C" refers to a dipeptide formed by the condensation of the carboxyl group of arginine with the amino group of cysteine, and the free amino group of arginine of the dipeptide is Fmoc protected.

Example 1

Synthesis of dipeptides

The dipeptides in table 1 were synthesized according to the following synthetic route:

in the above formula, HBTU is benzotriazole-N, N, N ', N' -tetramethyluronium hexafluorophosphate; "DIEA" is N, N-diisopropylethylamine; "Trt" is trityl; "DMF" is N, N-dimethylformamide; "TFA" is trifluoroacetic acid; "EDT" is 1, 2-ethanedithiol; "Tis" is triisopropylsilane; "deprotection" refers to deprotection, replacing-Fmoc with-H;

refers to a CTC resin, wherein "CTC" is 2-chlorotrityl chloride;

-R¹is selected from-CH₃、-CH₂OH、-CH₂SH、-(CH₂)₂SCH₃、-CH₂COOH、-CH₂CONH₂、-(CH₂)₄NH₂、-(CH₂)₃NHC(＝NH)NH₂、

One of (1);

-R²is-Fmoc or H.

TABLE 1

The specific steps for the synthesis of A-C in Table 1 include:

(1) resin grafting preparation: 10mmol of CTC resin was placed in a reaction tube at room temperature, DMF (15mL per gram of resin) was added and shaken for 30 min. DMF was filtered off by suction, 10mmol of Fmoc-L-Cys (Trt) -OH was added, 20mmol of DIEA was added, and finally an appropriate amount of DMF was added to dissolve it, and the mixture was shaken for 30 min. Blocked with methanol (1mL per gram of resin). DMF was removed and 20% piperidine DMF solution (15mL per gram of resin) was added with shaking for 5min and 20% piperidine DMF solution (15mL per gram of resin) was removed with shaking for 30 min. Then washed twice with DMF (10mL per gram of resin), twice with methanol (10mL per gram of resin) and twice with DMF (10mL per gram of resin).

(2) Condensation: dissolving 30mmol of Fmoc-protected alanine and 30mmol of HBTU in DMF as little as possible, adding into a reaction tube, immediately adding 100mmol of DIEA, and reacting for 30 min. Wash twice with DMF (10mL per gram of resin), twice with methanol (10mL per gram of resin), twice with DCM (10mL per gram of resin), twice with methanol (10mL per gram of resin) and suction dried for 10 min.

(3) Removing Fmoc protecting groups: 20% piperidine DMF solution (15mL per gram of resin) was added and shaken for 30 min. Then washed 5 times with DMF (10mL per gram of resin) and drained for 10 min.

(4) Cutting and purifying: the resin was cleaved with a cleavage solution (TFA 92.5% (v/v), water 2.5% (v/v), EDT 2.5% (v/v), TIS 2.5% (v/v)) added (10mL per gram of resin for 120 min. The lysate is washed with ether for six times and then put into a vacuum pump for pumping to obtain a dipeptide crude product. Purification was then carried out to above 95% using preparative HPLC.

The synthesis of Fmoc-A-C in Table 1 is essentially the same as the synthesis of A-C, except that the third step is not required: and removing the Fmoc protecting group.

The synthesis of S-C in Table 1 is approximately the same as that of A-C, except that alanine is replaced with serine in the second step.

Fmoc-S-C synthesis in Table 1 is essentially the same as S-C synthesis, except that the third step is not required: and removing the Fmoc protecting group.

Fmoc-C-C synthesis in Table 1 is essentially the same as Fmoc-A-C synthesis except that alanine is replaced with cysteine in the second step.

The synthesis of M-C in Table 1 is approximately the same as that of A-C, except that alanine is replaced with methionine in the second step.

The synthesis of F-C in Table 1 is approximately the same as that of A-C, except that alanine is replaced with phenylalanine in the second step.

The synthesis of Fmoc-F-C in Table 1 is essentially the same as the synthesis of F-C, except that the third step is not required: and removing the Fmoc protecting group.

The synthesis of Y-C in Table 1 is approximately the same as the synthesis of A-C, except that alanine is replaced with tyrosine in the second step.

The synthesis of Fmoc-Y-C in Table 1 is essentially the same as the synthesis of Y-C, except that the third step is not required: and removing the Fmoc protecting group.

The synthesis of W-C in Table 1 is approximately the same as the synthesis of A-C, except that alanine is substituted for tryptophan in the second step.

The synthesis of Fmoc-W-C in Table 1 is essentially the same as that of W-C, except that the third step is not required: and removing the Fmoc protecting group.

The synthesis of D-C in Table 1 is approximately the same as that of A-C, except that alanine is replaced with aspartic acid in the second step.

The synthesis of Fmoc-D-C in Table 1 is essentially the same as that of D-C, except that the third step is not required: and removing the Fmoc protecting group.

The synthesis of N-C in Table 1 is approximately the same as the synthesis of A-C, except that alanine is replaced with asparagine in the second step.

The synthesis of H-C in Table 1 is approximately the same as the synthesis of A-C, except that alanine is replaced with histidine in the second step.

The synthesis of Fmoc-H-C in Table 1 is essentially the same as the synthesis of H-C, except that the third step is not required: and removing the Fmoc protecting group.

The synthesis of Fmoc-K-C in Table 1 was identical to that of Fmoc-A-C except that alanine was replaced with lysine in the second step.

The synthesis of Fmoc-R-C in Table 1 was identical to that of Fmoc-A-C except that alanine was replaced with arginine in the second step.

After each dipeptide in table 1 was synthesized, the molecular weight of each dipeptide was measured by the mass spectrometry method, and the results are shown in table 1.

Example 2

Measurement of inhibitory Activity of dipeptide of example 1 on MBLs

(1)K_mDetermination of value

Michaelis constant (K)_m) Is a characteristic constant for characterizing enzyme, shows the affinity degree of protease and substrate, and also shows the catalytic efficiency of enzyme to a certain extent. K_mThe substrate concentration at 1/2, which is numerically equal to the maximum reaction rate of the enzymatic reaction, can be determined by the Mie equation v₀＝V_max[S]/(K_m+[S]) And (4) calculating. The enzyme kinetics experiment was performed in a 96-well plate using Meropenem (Meropenem) as a substrate, a detection wavelength of 300nm, and HEPES buffer (50mM, pH 7.0) supplemented with 1mM ZnSO₄0.01% (v/v) triton X-100 and 0.1. mu.g/mL BSA). Meropenem solution with different volume concentration of 500 mu M is added into a 96-well plate, so that the final concentration of the Meropenem is 0 mu M-500 mu M.Then, 10. mu.L of metallo-beta-lactamase solution (NDM-1, VIM-2 and IMP-7) was added to make the final concentrations 1nM, 2nM and 2.5nM, respectively), and the change in absorbance of meropenem was immediately detected once every 17s for a total of 15 min. The reaction rate of 20% before the reaction was taken for K_mThe results of the experiment are shown in Table 2.

TABLE 2

(2)IC₅₀Determination of value

Median Inhibitory Concentration (IC)₅₀) I.e. the concentration of the desired compound at which the enzymatic activity is half inhibited during the enzymatic reaction. IC (integrated circuit)₅₀Can be used for evaluating the inhibitory activity of the compound on the enzyme, and the smaller the value, the stronger the inhibitory effect of the compound on the enzyme is.

Inhibitor solution with the concentration of 640 MuM is added into the 1 st to 2 nd rows of a 96-well plate, and the inhibitor solution is diluted by twice gradient with HEPES buffer solution with the same volume, so that the final concentration of the inhibitor is 0 MuM to 640 MuM. Followed by addition of 10. mu.L of enzyme solution for preincubation at 25 ℃ for 15min to allow sufficient binding of the inhibitor to the enzyme. After incubation, 20. mu.L of meropenem (final concentration of 250. mu.M) was added to each well and the change in absorbance was rapidly measured, once every 17s for a total of 15 min. The reaction rate of 20% before the reaction was taken for IC₅₀And (4) calculating.

And calculating the inhibition rate of the inhibitor with different concentrations on the metallo-beta-lactamase. Reaction rate V without addition of inhibitor₀The reaction rate of adding different concentrations of inhibitor is V_iResidual activity (%) of the enzyme is 1- (1-V)_i/V₀) X 100%. The concentration of the compound was plotted against the residual activity of the enzyme (the residual activity curve of NDM-1 after addition of Fmoc-F-C, Fmoc-Y-C and Fmoc-W-C is shown in FIG. 1; the abscissa in FIG. 1 is the concentration and the ordinate is the residual activity), and the IC was calculated by fitting the curve using the software GraphPad 8.0₅₀The values and experimental results are shown in table 3.

TABLE 3

Note: C-Y was purchased from Zhejiang ang Ontolaisi Biotech Ltd.

As shown in Table 3, Fmoc-A-C, Fmoc-S-C, Fmoc-C-C, M-C, Fmoc-F-C, Y-C, Fmoc-Y-C, Fmoc-W-C and Fmoc-D-C have excellent inhibitory activities against NDM-1, VIM-2 or IMP-7.

(3)K_iCalculation of values

K_iThe value is the inhibition constant of the compound on the action of the protease, and can indicate the degree of affinity of the compound with the enzyme, and the smaller the value, the stronger the affinity of the compound with the protease. K_iValue is given by formula K_i＝IC₅₀/(1+[S]/K_m) Calculated and obtained, and the results are shown in table 4.

TABLE 4

In Table 4, "-" indicates that no valid data was obtained because of the following formula K_i＝IC₅₀/(1+[S]/K_mCalculate Ki, and IC₅₀Data greater than 500 do not have a definite value, so K_iNor does it have a certain value.

As can be seen from Table 4, Fmoc-A-C, Fmoc-S-C, Fmoc-C-C, M-C, Fmoc-F-C, Y-C, Fmoc-Y-C, Fmoc-W-C and Fmoc-D-C have good affinity for NDM-1, VIM-2 and IMP-7.

The results show that Fmoc-W-C and Fmoc-D-C have good broad-spectrum inhibitory activity on three MBLs. Fmoc-W-C and Fmoc-Y-C have the best inhibitory activity against NDM-1; Fmoc-C-C and Fmoc-D-C have the best inhibitory activity against VIM-2; Fmoc-W-C and Fmoc-S-C have the best inhibitory activity against IMP-7.

Example 3

Evaluation of Effect of dipeptide in example 1 in combination with Meropenem in inhibiting MBLs drug-resistant bacteria

(1) Minimum Inhibitory Concentration (MIC) determination of MBLs drug-resistant strains

The minimum inhibitory concentration of the dipeptide (Fmoc-F-C, Fmoc-Y-C or Fmoc-W-C) combined with meropenem to MBLs drug-resistant strain is determined by a broth microdilution method. E.coli BL21(DE3)/pMAL-c5x-IMP-7, E.coli BL21(DE3)/pET24a-VIM-2 and E.coli BL21(DE3)/pET26b-NDM-1 used for the experiments were purchased from Shanghai Biotech Co., Ltd; coli BAA-2452 (bla)_NDM-1) Coli BAA-2340 (bla)_KPC-2) Purchased from biotechnology limited of baio bowei, beijing.

The antimicrobial activity of the drugs alone was evaluated according to the micro broth dilution method of the american Clinical and Laboratory Standards Institute (CLSI) to determine the MIC of the individual drugs. Diluting the reserved antibacterial drugs or compounds to be tested to 256 mug/mL to be used as working solution, adding 200 mug L of the working solution into a first row of A1-H1 wells of a 96-well plate, respectively adding 100 mug L of LB culture medium into the other wells, sucking 100 mug L of the working solution from the first row to a second row by using an 8-channel micropipette, repeatedly blowing and beating the working solution for a plurality of times to dilute and mix the medicinal solution, sucking 100 mug L of the liquid from the row to the next row, repeating the operation until the last row, sucking 100 mug L of the liquid in the last row, and discarding the liquid after sucking 100 mug L. Three parallel controls were set for each experiment. The 96-well plate was incubated in a 37 ℃ incubator for 24 hours, and the results were observed and MIC values were recorded.

The effect of the meropenem and the dipeptide combined antibacterial effect is evaluated by adopting a chessboard broth microdilution method. Dilution of meropenem: diluting meropenem to 512 mug/mL as a working solution, adding 100 muL of the working solution into a first row of A1-H1 wells of a 96-well plate, respectively adding 50 muL of culture medium into the other wells, sucking 50 muL of the antibacterial drug working solution from the first row to a second row by using an 8-channel micropipette, repeatedly blowing and beating the working solution for several times to dilute and mix the liquid medicine, sucking 50 muL of the liquid from the row to the next row, repeating the operation until the last row, discarding 50 muL of the liquid left in the last row at the moment, and finishing the dilution of the meropenem; ② adding dipeptide: diluting the dipeptide into 512 mu g/mL, 256 mu g/mL, 128 mu g/mL, 64 mu g/mL, 32 mu g/mL, 16 mu g/mL, 8 mu g/mL and 4 mu g/mL, and sucking 50 mu L of liquid medicine from high concentration to low concentration to each row in turn, namely adding 512 mu g/mL liquid medicine to A1-A12 hole, adding 256 mu g/mL liquid medicine to B1-B12 hole, and the like to the last row. 100 μ L of bacterial suspension was added to each well, and three parallel controls were set for each experiment. ATCC25922 was used as a quality control standard, and a sterile well and a drug-free well were provided. The 96-well plate was incubated in a 37 ℃ incubator for 24 hours, and the results were observed and MIC values were recorded. The results of the individual administration and the combination administration at a dipeptide concentration of 128. mu.g/mL are shown in Table 5. The combination therapy with a dipeptide concentration of 128. mu.g/mL means that the dipeptide concentration of 128. mu.g/mL is administered simultaneously with meropenem.

TABLE 5

Note:^ain order to construct the strain of bacteria,^bis a clinical strain

As can be seen from table 5, the MIC of meropenem is higher when meropenem is used alone, and the MIC of meropenem can be reduced to a certain extent when the meropenem is used in combination with different dipeptides, which indicates that the dipeptides have different effects of inhibiting MBLs; and when the Fmoc-F-C or the Fmoc-W-C is combined with meropenem, the MIC value is obviously smaller than that of other dipeptides, and the synergistic effect is better.

(2) Synergistic antimicrobial index (FICI) calculation

The synergistic antibacterial index is used for judging the interaction condition when two medicaments are used in combination, and the numerical value is calculated according to the following equation: FICI ═ FIC_A+FIC_B＝C_A/MIC_A+C_B/MIC_BIf the FICI is less than or equal to 0.5, the two medicines are considered to have synergistic action, if the FICI is more than 0.5 and less than or equal to 1, the two medicines are added, if the FICI is more than 1 and less than or equal to 2, the two medicines are unrelated, and if the FICI is more than or equal to 4, the two medicines are considered to have antagonistic action. The smaller the FICI, the stronger the synergistic effect of the drug. The results of the experiment are shown in Table 6.

TABLE 6

Note:^ain order to construct the strain of bacteria,^bis a clinical strain

The dipeptide of example 1 in combination with meropenem was tested for antibacterial activity against construction strains expressing MBLs or clinically isolated drug-resistant strains by the above experimental method. The results show that when the composition is used with meropenem, Fmoc-F-C and Fmoc-W-C have effective synergistic antibacterial activity on various drug-resistant bacteria expressing metal beta-lactamase and simultaneously have good synergistic inhibition effect on a strain expressing KPC-2, which indicates that Fmoc-F-C and Fmoc-W-C can be used as MBLIs to reverse the drug resistance of carbapenem drug-resistant bacteria to beta-lactam antibiotics and effectively improve the antibacterial activity of meropenem on MBLs drug-resistant bacteria.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, so as to understand the technical solutions of the present invention specifically and in detail, but not to be understood as the limitation of the protection scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. It should be understood that the technical solutions obtained by logical analysis, reasoning or limited experiments based on the technical solutions provided by the present invention are all within the protection scope of the appended claims of the present invention. Therefore, the protection scope of the present invention should be subject to the content of the appended claims, and the description and the drawings can be used for explaining the content of the claims.

Claims

1. A dipeptide having a protecting group attached to an amino group, wherein the dipeptide is synthesized by a condensation reaction of a carboxyl group of a first amino acid selected from one of alanine, serine, cysteine, phenylalanine, tyrosine, tryptophan, and aspartic acid with an amino group of a second amino acid, which is cysteine.

2. The dipeptide of claim 1, wherein the protecting group is selected from one of fluorenylmethyloxycarbonyl, benzyloxycarbonyl and tert-butyloxycarbonyl.

3. The dipeptide of claim 1, wherein the dipeptide has the structure:

wherein, -R¹Is composed of

-CH₂SH、CH₂OH or-CH₂COOH；-R²is-Fmoc.

4. A method for producing a dipeptide, comprising the steps of:

5. Use of a dipeptide according to any of claims 1 to 3 or a dipeptide according to the method of claim 4 for the preparation of a metallo-beta-lactamase inhibitor.

6. A metallo-beta-lactamase inhibitor comprising an active ingredient comprising the dipeptide according to any one of claims 1 to 3 or the dipeptide obtained by the process for producing the dipeptide according to claim 4.

7. The metallo-beta-lactamase inhibitor of claim 6, further comprising an adjuvant.

8. A pharmaceutical composition comprising a non-monocyclic β -lactam antibiotic and a dipeptide according to any of claims 1 to 3 or a dipeptide according to claim 4 obtained by a process for the preparation of a dipeptide.

9. The pharmaceutical composition of claim 8, wherein the non-monocyclic β -lactam antibiotic is selected from at least one of the group consisting of penicillin antibiotics, cephalosporin antibiotics, cephamycins antibiotics, thiomycins antibiotics, and carbapenem antibiotics.

10. The pharmaceutical composition of claim 8, wherein the non-monocyclic β -lactam antibiotic is selected from at least one of meropenem, ertapenem, and doripenem.