Active oxygen activated antibacterial prodrug and application thereof in treatment of bacterial infection

Abstract

The invention provides an active oxygen activated antibacterial prodrug and application thereof in treatment of bacterial infection, the active oxygen activated antibacterial prodrug has a structure as shown in the following formula I, and R1-R5 and X are defined in the specification. The compound disclosed by the invention can be specifically activated into an active molecule with a therapeutic effect at a bacterial infection part in a targeting manner, is high in antibacterial activity, has a bactericidal effect on gram-positive bacteria and gram-negative bacteria, and is low in toxicity, high in biological safety and large in therapeutic window.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to an active oxygen-activated antibacterial prodrug and its application in treating bacterial infections. Background Art

[0002] Bacterial resistance is a major global public health crisis. The emergence of multidrug-resistant bacteria complicates clinical treatment, prolongs treatment time, reduces the number of available medications, significantly lowers treatment success rates, and places a significant burden on society and families. To combat the growing problem of antibiotic resistance, the development of highly effective antibacterial compounds with novel structures is urgently needed.

[0003] Delocalized lipid-soluble cationic compounds have high efficiency and broad-spectrum antibacterial activity and have been widely used for surface and environmental disinfection. However, they are easily electrostatically attracted to plasma proteins, thereby reducing the actual drug concentration reaching the infected lesion, and therefore are greatly limited in the treatment of living bacterial infections. Lipid-soluble cationic compounds can potentially cause cytotoxicity by disrupting the mitochondrial membrane potential of normal cells, so the safe treatment window is relatively small. In response to the above problems, there is an urgent need in this field to develop new prodrug activation strategies to maintain the antibacterial activity of drugs while improving their circulation time and biosafety. Summary of the Invention

[0004] The purpose of the present invention is to provide an active oxygen-activated antibacterial prodrug and its application in treating bacterial infection.

[0005] The first aspect of the present invention provides a compound, or an optical isomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof or a solvate thereof, having a structure shown in Formula I,

[0006]

[0007] In Formula I,

[0008] R1 and R2 are each independently selected from: H and substituted or unsubstituted C1-C 20 Alkyl; or R1, R2, and the nitrogen atom to which they are attached together form an optionally substituted 4-12 membered nitrogen-containing heterocyclic ring;

[0009] R3 and R4 are each independently selected from: H and substituted or unsubstituted C1-C 20 Alkyl; or R3, R4, and the nitrogen atom to which they are attached together form an optionally substituted 4-12 membered nitrogen-containing heterocyclic ring;

[0010] R5 is H, halogen, -CN, -NO2, substituted or unsubstituted C1-C 20 Alkyl, or substituted or unsubstituted C6-C 14 aryl;

[0011] X is O, S, -Si(R6)2- or -C(R6)2-;

[0012] R6 is H, halogen or substituted or unsubstituted C1-C 20 alkyl;

[0013] The term "substituted" refers to being substituted by 1, 2, 3 or 4 substituents selected from the group consisting of: C1-C 20 Alkyl, C6-C 14 Aryl, halogen, optionally substituted by 1, 2, 3 or 4 groups selected from C6-C 14 Aryl and C1-C 20 Alkyl substituents substituted C2-C 20 Alkenyl, C3-C 20 Cycloalkyl, and optionally C1-C 20 Alkyl-substituted mercapto groups.

[0014] The second aspect of the present invention provides a pharmaceutical composition comprising (1) a safe and effective amount of one or more compounds described in the first aspect of the present invention, or their optical isomers, pharmaceutically acceptable salts, hydrates or solvates; and (2) a pharmaceutically acceptable carrier or excipient.

[0015] The third aspect of the present invention provides use of the compound described in the first aspect of the present invention, or its optical isomers, pharmaceutically acceptable salts, hydrates or solvates in the preparation of drugs for treating and / or preventing bacterial infections.

[0016] The fourth aspect of the present invention provides a method for converting the compound of formula I described in the first aspect of the present invention into a compound of formula II, the method comprising the steps of:

[0017]

[0018] The compound of formula I is contacted with active oxygen, wherein the active oxygen is selected from one or more of ·OH, NO, ONOOH and HClO. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Results of the spectral response test of compound CCR1 to biologically active oxygen species. (A) Changes in UV-visible absorbance spectrum with the addition of active oxygen species; (B) Changes in absorbance of compound CCR1 at 556 nm with the amount of active oxygen equivalents; (C) Changes in fluorescence emission spectrum with the addition of active oxygen species (excitation wavelength 556 nm); (D) Changes in fluorescence intensity of compound CCR1 at 580 nm with the amount of active oxygen equivalents; (E) Reaction kinetics of compound CCR1.

[0020] Figure 2This is the result of the selectivity test of compound CCR1 for active substances.

[0021] Figure 3 Figure 2 shows the in vitro antibacterial activity of various compounds against methicillin-resistant Staphylococcus aureus (MRSA). (A) Minimum inhibitory concentration (MIC) (µg / mL) of various compounds against three Gram-positive MRSA strains; (B) Time-dependent absorbance changes of CCR1 in the presence of different concentrations of reactive oxygen species (ROSup); (C) Structural formula of the control compound.

[0022] Figure 4 This is the bactericidal curve of different compounds against MRSA (ATCC43300).

[0023] Figure 5 Acute toxicity studies of various compounds. (A) Lethal dose (LD50) of CCR1 / RD1 in mice; (B) Toxicity study of CCR1 / RD1 in zebrafish; (C) Cytotoxicity study of CCR1 / RD1.

[0024] Figure 6 Figure 3 shows the in vivo antibacterial effects of CCR1 and RD1 on MRSA skin-infected mice. (A) Schematic diagram of the drug administration process; (B) Quantitative analysis of weight changes in mice receiving different treatments on day 8 (n = 4); (C) Images of infected skin wounds in each treatment group; (D) Quantitative analysis of wound area in wound tissues of MRSA-infected mice receiving different treatments on day 8 (n = 6-8); (E) Quantitative analysis of bacterial counts in wound tissues of MRSA-infected mice receiving different treatments on day 8 (n = 6-8); (F) Masson and H&E staining of skin tissues of MRSA-infected mice receiving different treatments.

[0025] Figure 7 Schematic diagram of the activation mechanism of the compound of formula I of the present invention. DETAILED DESCRIPTION

[0026] To facilitate understanding of the features and effects of the present invention by those skilled in the art, the following provides a general description and definition of the terms and expressions used in the specification and claims. Unless otherwise indicated, all technical and scientific terms used herein have the ordinary meanings as understood by those skilled in the art regarding the present invention. In the event of conflict, the definitions in this specification shall prevail.

[0027] The theories or mechanisms described and disclosed herein, whether correct or incorrect, should not limit the scope of the present invention in any way, that is, the present invention can be implemented without being limited by any specific theory or mechanism.

[0028] Herein, “comprising,” “including,” “containing” and similar terms encompass the meanings of “consisting essentially of” and “consisting of,” for example, when “A comprises B and C” is disclosed herein, “A consists essentially of B and C” and “A consists of B and C” should be deemed to have been disclosed herein.

[0029] Throughout this document, all features, such as values, amounts, amounts, and concentrations, specified in numerical ranges or percentage ranges are provided for simplicity and convenience only. Accordingly, the description of numerical ranges or percentage ranges should be considered to encompass and specifically disclose all possible subranges and individual values ​​within those ranges (including integers and fractions).

[0030] In this document, unless otherwise specified, percentage refers to mass percentage and ratio refers to mass ratio.

[0031] Herein, when describing embodiments or examples, it should be understood that they are not intended to limit the present invention to these embodiments or examples. On the contrary, all alternatives, modifications and equivalents of the methods and materials described herein are encompassed within the scope defined by the claims.

[0032] In this document, for the sake of brevity, not all possible combinations of the various technical features in each embodiment or example are described. Therefore, as long as there are no contradictions in the combination of these technical features, the various technical features in each embodiment or example can be combined in any way, and all possible combinations should be considered to be within the scope of this specification.

[0033] As used herein, "alkyl" refers to a straight or branched monovalent saturated hydrocarbon radical having a specified number of carbon atoms, specific alkyl groups are those having 1 to 20 carbon atoms ("C1-C 20 alkyl”), usually containing 1 to 10 carbon atoms (C 1-10 Alkyl), preferably containing 1 to 6 carbon atoms (C 1-6 Alkyl), more preferably containing 1-4 carbon atoms (C 1-4 Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like.

[0034] As used herein, "aryl" or "Ar" refers to an unsaturated aromatic carbocyclic group having a single ring (e.g., phenyl) or multiple fused rings (e.g., naphthyl or anthracenyl), wherein the fused rings may or may not be aromatic. In one variation, aryl contains 6 to 14 ring carbon atoms, preferably C 6-10Aryl. Aryl groups having more than one ring, at least one of which is non-aromatic, may be attached to the parent structure at either an aromatic ring position or a non-aromatic ring position. In one variation, aryl groups having more than one ring, at least one of which is non-aromatic, are attached to the parent structure at an aromatic ring position. Examples of aryl groups include phenyl, naphthyl, phenanthrenyl, anthracenyl, indenyl, azulenyl, biphenyl, biphenylene, and fluorenyl.

[0035] As used herein, "cycloalkyl" or "carbocyclyl" refers to a saturated cyclic hydrocarbon having 3 to 20 ring carbon atoms, comprising one ring such as cyclohexyl or multiple rings such as adamantyl. Cycloalkyl groups comprising more than one ring may be fused, spirocyclic, or bridged, or a combination thereof. Preferred cycloalkyl groups are saturated cyclic hydrocarbons having 3 to 8 ring carbon atoms ("C3-C8 cycloalkyl"). In some embodiments, cycloalkyl groups have 5 to 15 ring carbon atoms ("C5-C8 cycloalkyl"). 15 In some embodiments, a cycloalkyl group has 7 to 12 ring carbon atoms ("C7-C 12 Examples of cycloalkyl groups include adamantyl, decahydronaphthyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

[0036] As used herein, "halo" or "halogen" refers to an element of Group 17 with an atomic number of 9 to 85.

[0037] "Halogen" or "halogen atom" refers to F, Cl, Br, and I. "Halogenated" means substituted with an atom selected from F, Cl, Br, and I. "Halide anion" refers to an anion formed when a halogen atom gains an electron.

[0038] Including Cl — 、

[0039] Br — , I — 、F — .

[0040] As used herein, "mercapto" refers to -SH, and "alkyl-substituted mercapto" refers to mercapto substituted with an alkyl group as defined above.

[0041] As used herein, as a group or as part of another group, the term "alkenyl" means a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms and containing at least one double bond, connected to the remainder of the molecule by a single bond. In some embodiments, an alkenyl group contains 2 to 20 atoms. In some embodiments, an alkenyl group contains 2 to 18 carbon atoms. In some embodiments, an alkenyl group contains 2 to 16 carbon atoms. In some embodiments, an alkenyl group contains 2 to 12 carbon atoms. In some embodiments, an alkenyl group contains 2 to 10 carbon atoms. In some embodiments, an alkenyl group contains 2 to 8 carbon atoms. In some embodiments, an alkenyl group contains 2 to 6 carbon atoms. In some embodiments, an alkenyl group contains 2 to 4 carbon atoms. In some embodiments, an alkenyl group contains 2 to 3 carbon atoms. Non-limiting examples of alkenyl groups include, but are not limited to, ethenyl, propenyl, allyl, butenyl, but-1-enyl, but-2-enyl, pentenyl, pent-1-enyl, pentadienyl, penta-1,4-dienyl, and the like. Unless stated otherwise specifically in the specification, an alkenyl group may be optionally substituted.

[0042] As used herein, "optionally" or "optionally" means that the subsequently described event or circumstance may or may not occur, and the description includes both instances where the event or circumstance occurs and instances where it does not occur. For example, "optionally substituted alkyl" means that the alkyl group is substituted or unsubstituted, and the description includes both substituted and unsubstituted alkyl groups. The "optionally" substituents described in the claims and description of this disclosure are selected from alkyl, alkenyl, alkynyl, alkoxy, halogen, haloalkyl, haloalkenyl, haloalkynyl, cyano, amino, hydroxy, nitro, sulfo, carboxyl, optionally substituted aryl, optionally substituted aryloxy, optionally substituted heteroaryl, optionally substituted cycloalkyl, and optionally substituted heterocyclyl.

[0043] As used herein, the term "substituted", whether or not preceded by the term "optionally" (i.e., equivalent to substituted or unsubstituted), refers to the replacement of one or more hydrogens of a specified group or portion by a "suitable substituent". Herein, the number of substituents may be one or more, i.e., 1, 2, 3, 4, 5, or 6 or more, depending on the group being substituted and the nature of the substituent. For example, when the substituent of an ethyl group is a halogen, the group may be substituted by 1, 2, 3, 4, or 5 substituents, such as trifluoroethyl, pentafluoroethyl, etc., depending on the structure of the substituted group. In some embodiments, the number of substituents is 1, 2, or 3. In some embodiments, the number of substituents is 1 or 2. In some embodiments, the number of substituents is 1. It will be understood that "substituted" or "substituted by..." includes implicit conditions, i.e., such replacement is carried out according to the allowed valence of the substituted atom, and the replacement produces a stable or chemically feasible compound, such as a compound that will not spontaneously transform, such as by rearrangement, cyclization, elimination, etc. Unless otherwise indicated, an "optionally substituted" group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted by more than one substituent selected from a specified group, the substituents may be the same or different at each position. It will be understood by those skilled in the art that the substituents themselves may be substituted if appropriate. Unless specifically indicated as "unsubstituted," chemical moieties referred to herein are understood to include substituted variants. For example, reference to an "alkenyl" group or moiety implicitly includes both unsubstituted alkenyl groups and substituted variants.

[0044] Throughout this application, the “suitable substituents” mentioned above are understood to include, but are not limited to, the alkyl, alkenyl, alkynyl, halogen, haloalkyl, haloalkenyl, haloalkynyl, alkoxy, cyano, amino, hydroxy, carboxyl, sulfo, monoalkylamino, dialkylamino, nitro, aryl, aryloxy, heteroaryl, cycloalkyl (e.g., cycloalkyl, cycloalkenyl, etc.), heterocyclyl, etc. described herein; these groups as substituents, including alkyl, alkenyl, alkynyl, amino, hydroxy, sulfo, carboxyl, alkyl in haloalkyl, alkenyl in haloalkenyl, alkynyl in haloalkynyl, alkoxy, alkyl in monoalkylamino, alkyl in dialkylamino, aryl, aryl in aryloxy, heteroaryl, cycloalkyl and heterocyclyl themselves are also optionally substituted, for example, they may be optionally substituted with one or more groups selected from alkyl, halogen, haloalkyl, alkoxy, hydroxy, amino, monoalkylamino, dialkylamino, nitro, aryl, heteroaryl, cycloalkyl and heterocyclyl.

[0045] As used herein, the terms "alleviate," "prevent," and "preventing" include reducing the likelihood of a disease or condition occurring or worsening in a patient.

[0046] As used herein, the term "treatment" and other similar synonyms include the following meanings:

[0047] (i) preventing a disease or condition from occurring in a mammal, particularly where such mammal is susceptible to the disease or condition but has not yet been diagnosed as having the disease or condition;

[0048] (ii) inhibiting the disease or condition, i.e., curbing its development;

[0049] (iii) alleviate the disease or condition, that is, cause regression of the disease or condition; or

[0050] (iv) Alleviate the symptoms of the disease or condition.

[0051] As used herein, the terms "administer," "administer," "dosing," and the like refer to methods by which a compound or composition can be delivered to the desired site for biological action. These methods include, but are not limited to, oral routes, intraduodenal routes, parenteral injections (including intravenous, subcutaneous, intraperitoneal, intramuscular, intraarterial injection or infusion), topical administration, and rectal administration. Those skilled in the art are familiar with administration techniques that can be used for the compounds and methods described herein, such as those discussed in Goodman and Gilman, The Pharmacological Basis of Therapeutics, current ed.; Pergamon; and Remington's, Pharmaceutical Sciences (current edition), Mack Publishing Co., Easton, Pa. In preferred embodiments, the compounds and compositions discussed herein are administered orally.

[0052] Those skilled in the art will also appreciate that in the methods described herein, the functional groups of the intermediate compounds may need to be protected by appropriate protecting groups. Such functional groups include hydroxyl, amino, sulfhydryl, and carboxylic acid. Suitable hydroxyl protecting groups include trialkylsilyl or diarylalkylsilyl (e.g., tert-butyldimethylsilyl, tert-butyldiphenylsilyl, or trimethylsilyl), tetrahydropyranyl, benzyl, and the like. Suitable amino, amidino, and guanidino protecting groups include tert-butyloxycarbonyl, benzyloxycarbonyl, and the like. Suitable sulfhydryl protecting groups include -C(O)-R" (wherein R" is alkyl, aryl, or aralkyl), p-methoxybenzyl, trityl, and the like. Suitable carboxyl protecting groups include alkyl, aryl, or aralkyl esters.

[0053] Protecting groups can be introduced and removed according to standard techniques known to those skilled in the art and as described herein. The use of protecting groups is described in detail in Greene, TW and PGM Wuts, Protective Groups in OrganiSynthesis, (1999), 4th Ed., Wiley. Protecting groups can also be polymeric resins.

[0054] As used herein, "drug-resistant Staphylococcus aureus (MRSA)" refers to a strain of Staphylococcus aureus that is resistant to all penicillins, including methicillin and other beta-lactamase-resistant penicillins.

[0055] This study designed a class of antibacterial prodrug molecules that undergo charge reversal upon activation by reactive oxygen species (ROS). This approach exploits the inherent ROS burst at sites of bacterial infection to achieve targeted, specific activation of the prodrug. Compared to the active molecule, the prodrug exhibits significantly reduced biotoxicity and improved water solubility, demonstrating robust antibacterial activity in vitro and in skin infection models.

[0056] The present invention provides a compound having a structure shown in Formula I (referred to as Formula I compound), or an optical isomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof.

[0057]

[0058] in,

[0059] R1 and R2 are each independently selected from: H and substituted or unsubstituted C1-C 20 Alkyl; or R1, R2, and the nitrogen atom to which they are attached together form an optionally substituted 4-12 membered nitrogen-containing heterocyclic ring;

[0060] R3 and R4 are each independently selected from: H and substituted or unsubstituted C1-C 20 Alkyl; or R3, R4, and the nitrogen atom to which they are attached together form an optionally substituted 4-12 membered nitrogen-containing heterocyclic ring;

[0061] R5 is H, halogen, -CN, -NO2, substituted or unsubstituted C1-C 20 Alkyl, or substituted or unsubstituted C6-C 14 aryl;

[0062] X is O, S, -Si(R6)2- or -C(R6)2-;

[0063] R6 is H, halogen or substituted or unsubstituted C1-C 20 alkyl;

[0064] The term "substituted" refers to being substituted by 1, 2, 3 or 4 substituents selected from the group consisting of: C1-C 20 Alkyl, C6-C 14 Aryl, halogen, optionally substituted by 1, 2, 3 or 4 groups selected from C6-C 14 Aryl and C1-C 20 Alkyl substituents substituted C2-C 20 Alkenyl, C3-C 20 Cycloalkyl, and optionally C1-C 20 Alkyl-substituted mercapto groups.

[0065] In some embodiments, R1 and R2 are each independently selected from: H and substituted or unsubstituted C1-C 10 alkyl; or R1, R2, and the nitrogen atom to which they are attached together form an optionally substituted 5-10 membered nitrogen-containing heterocyclic ring. Preferably, R1 and R2 are each independently selected from: H and substituted or unsubstituted C1-C6 alkyl; or R1, R2, and the nitrogen atom to which they are attached together form an optionally substituted 6-8 membered nitrogen-containing heterocyclic ring. More preferably, R1 and R2 are each independently selected from: H and substituted or unsubstituted C1-C6 alkyl. In some embodiments, R1 and R2 are each independently C1-C4 alkyl. Preferably, R1 and R2 are optionally substituted by 1, 2, 3 or 4 substituents selected from the group consisting of: C1-C6 alkyl, C6-C 10 Aryl, halogen and C3-C8 cycloalkyl.

[0066] In some embodiments, R3 and R4 are each independently selected from: H and substituted or unsubstituted C1-C 10 alkyl; or R3, R4, and the nitrogen atom to which they are attached together form an optionally substituted 5-10 membered nitrogen-containing heterocycle. Preferably, R3 and R4 are each independently selected from: H and substituted or unsubstituted C1-C6 alkyl; or R3, R4, and the nitrogen atom to which they are attached together form an optionally substituted 6-8 membered nitrogen-containing heterocycle. More preferably, R3 and R4 are each independently selected from: H and substituted or unsubstituted C1-C6 alkyl. In some embodiments, R3 and R4 are each independently C1-C4 alkyl. Preferably, R3 and R4 are optionally substituted by 1, 2, 3 or 4 substituents selected from the group consisting of: C1-C6 alkyl, C6-C 10 Aryl, halogen and C3-C8 cycloalkyl.

[0067] In some embodiments, R5 is H, halogen, -CN, -NO2, optionally substituted by 1, 2, 3 or 4 carbon atoms selected from C3-C 20 Cycloalkyl, C6-C 14 Aryl and halogen substituted C1-C 20 Alkyl, or optionally substituted C6-C 14 Aryl, the C6-C14 Aryl is optionally substituted with 1, 2, 3 or 4 substituents selected from the group consisting of: C1-C 20 Alkyl, C6-C 14 Aryl, halogen, optionally substituted by 1, 2, 3 or 4 groups selected from C6-C 14 Aryl and C1-C 20 Alkyl substituents substituted C2-C 20 alkenyl, and optionally C1-C 20 Alkyl-substituted mercapto groups.

[0068] Preferably, R5 is H, halogen, optionally substituted by 1, 2, 3 or 4 groups selected from C5-C 15 Cycloalkyl, C6-C 10 Aryl and halogen substituted C1-C 10 Alkyl, or optionally substituted C6-C 14 Aryl, the C6-C 14 Aryl is optionally substituted with 1, 2, 3 or 4 substituents selected from the group consisting of: C1-C 10 Alkyl, C6-C 10 Aryl, halogen, optionally substituted by 1, 2, 3 or 4 groups selected from C6-C 10 Aryl and C1-C 10 Alkyl substituents substituted C2-C 10 alkenyl, and optionally C1-C 10 Alkyl-substituted mercapto groups.

[0069] More preferably, R5 is H, optionally substituted by 1, 2, 3 or 4 selected from C7-C 12 Cycloalkyl, C6-C 10 C1-C6 alkyl substituted with aryl and halogen, or optionally substituted C6-C 10 Aryl, the C6-C 10 Aryl is optionally substituted with 1, 2, 3 or 4 substituents selected from the group consisting of C1-C6 alkyl, halogen, optionally substituted with one or two C6-C 10 an aryl-substituted C2-C6 alkenyl group, and a mercapto group optionally substituted by a C1-C6 alkyl group.

[0070] In some embodiments, R5 is H, optionally replaced by 1 or 2 C7-C 12 Cycloalkyl-substituted C1-C6 alkyl, or optionally substituted C6-C 10 Aryl, the C6-C 10 Aryl is optionally substituted with 1, 2, 3 or 4 substituents selected from the group consisting of halogen, optionally substituted with one or two C6-C 10 In some embodiments, R5 is optionally substituted with 1 or 2 C8-C6 alkenyl, C2-C6 alkenyl, and C1-C6 alkyl substituted mercapto.10 Cycloalkyl-substituted C1-C6 alkyl, or optionally substituted C6-C 10 Aryl, the C6-C 10 The aryl group is optionally substituted by 1, 2, 3 or 4 substituents selected from the group consisting of halogen (e.g., fluorine, chlorine), optionally substituted by a C6-C 10 an aryl-substituted C2-C4 alkenyl group, and a mercapto group optionally substituted by a C1-C4 alkyl group.

[0071] In some embodiments, X is O, S, -Si(R6)2- or -C(R6)2-, and R6 is H, halogen, or C1-C 10 Preferably, X is O, -Si(R6)2- or -C(R6)2-, and R6 is H, halogen or C1-C6 alkyl. More preferably, X is O.

[0072] In some embodiments, in Formula I,

[0073] R1 and R2 are each independently selected from: H and optionally substituted by 1, 2, 3 or 4 selected from C6-C 10 C1-C6 alkyl substituted with aryl, halogen and C3-C8 cycloalkyl substituents; or R1, R2, and the nitrogen atom to which they are attached together form a 5-10 membered nitrogen-containing heterocyclic ring, the nitrogen-containing heterocyclic ring is optionally substituted with 1, 2, 3 or 4 substituents selected from C1-C6 alkyl, C6-C 10 aryl, halogen and C3-C8 cycloalkyl substituents;

[0074] R3 and R4 are each independently selected from: H and optionally substituted by 1, 2, 3 or 4 selected from C6-C 10 C1-C6 alkyl substituted with aryl, halogen and C3-C8 cycloalkyl; or R3, R4, and the nitrogen atom to which they are attached together form a 5-10 membered nitrogen-containing heterocyclic ring, the nitrogen-containing heterocyclic ring is optionally substituted with 1, 2, 3 or 4 C1-C6 alkyl, C6-C 10 aryl, halogen and C3-C8 cycloalkyl substituents;

[0075] R5 is H, halogen, optionally substituted by 1, 2, 3 or 4 C3-C 20 Cycloalkyl-substituted C1-C 20 Alkyl, or optionally substituted C6-C 14 Aryl, the C6-C 14 Aryl is optionally substituted with 1, 2, 3 or 4 substituents selected from the group consisting of: C1-C 20 Alkyl, C6-C 14 Aryl, halogen, optionally substituted by 1, 2, 3 or 4 C6-C 14 Aryl-substituted C2-C 20 alkenyl, and optionally C1-C 20Alkyl-substituted mercapto groups;

[0076] X is O, -Si(R6)2- or -C(R6)2-; R6 is H, halogen or C1-C 10 alkyl.

[0077] Preferably, in Formula I,

[0078] R1 and R2 are each independently selected from: H and optionally substituted by 1, 2, 3 or 4 selected from C6-C 10 C1-C6 alkyl substituted with aryl, halogen and C3-C8 cycloalkyl substituents;

[0079] R3 and R4 are each independently selected from: H and optionally substituted by 1, 2, 3 or 4 selected from C6-C 10 C1-C6 alkyl substituted with aryl, halogen and C3-C8 cycloalkyl substituents;

[0080] R5 is optionally replaced by 1 or 2 C8-C 15 Cycloalkyl-substituted C1-C 10 Alkyl, or optionally substituted by 1, 2, 3 or 4 groups selected from halogen, C6-C 14 Aryl-substituted C2-C 10 Alkenyl, and C1-C 10 Alkyl substituted mercapto substituents substituted C6-C 10 aryl;

[0081] X is O.

[0082] Preferably, in Formula I, R1, R2, R3 and R4 are each independently selected from C1-C4 alkyl, R5 is optionally substituted by 1 or 2 C8-C 15 C1-C4 alkyl substituted by cycloalkyl, or optionally substituted by 1, 2, 3 or 4 groups selected from halogen, C6-C 10 C2-C4 alkenyl substituted with aryl, and C6-C4 alkyl substituted with mercapto 10 Aryl, X is O.

[0083] In some embodiments, the compound of formula I is selected from the group consisting of:

[0084]

[0085] The present invention also includes optical isomers, pharmaceutically acceptable salts, hydrates or solvates of the compound of formula I. Herein, "pharmaceutically acceptable salts" refers to a salt that retains the desired biological activity of the parent compound and does not produce any adverse toxicological effects (see, for example, Berge, SM et al., 1977, J.Pharm.Sci.66:1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include salts derived from nontoxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous acid, and salts derived from nontoxic organic acids such as aliphatic monocarboxylic acids and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids. Base addition salts include those derived from alkaline earth metals such as sodium, potassium, magnesium, calcium, and the like, as well as salts derived from non-toxic organic amines such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, and the like.

[0086] As used herein, the term "optical isomer" refers to compounds that have the ability to rotate plane-polarized light and have chiral atoms, and these compounds are generally represented using the conventional R / S configuration. The term "optical isomer" includes enantiomers and diastereomers, as well as compounds that can be distinguished from each other by the designations (D) and (L). Unless otherwise specified, in all compounds of the present invention, each chiral carbon atom (chiral center) can optionally be in the R configuration or the S configuration, or a mixture of the R and S configurations.

[0087] As used herein, "solvate" or "solvate" is a form of a compound in which solvent molecules are combined in certain ratios as an integral part of the crystal structure of the compound.

[0088] As used herein, "hydrate" refers to a complex formed by the coordination of a compound of the present invention with water.

[0089] Unless otherwise specified, the structural formulas described herein are intended to include all isomeric forms (e.g., enantiomers, diastereomers, and geometric isomers (or conformers)): for example, R and S configurations containing asymmetric centers, (Z) and (E) isomers of double bonds, etc. Therefore, single stereochemical isomers of the compounds of the present invention or mixtures of their enantiomers, diastereomers, or geometric isomers (or conformers) are all within the scope of the present invention.

[0090] Because the compounds of the present invention have antibacterial activity and can be specifically activated by reactive oxygen species at sites of bacterial infection in the body to form drug molecules with stronger antibacterial activity, the present invention provides a pharmaceutical composition for treating and / or preventing bacterial infection. The pharmaceutical composition of the present invention comprises (1) a safe and effective amount of one or more compounds of Formula I of the present invention, or an optical isomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof; and (2) a pharmaceutically acceptable carrier or excipient.

[0091] The pharmaceutical composition of the present invention comprises a safe and effective amount of the compound of the present invention and a pharmaceutically acceptable excipient or carrier. The "safe and effective amount" means an amount of the compound sufficient to significantly improve the condition without causing serious side effects.

[0092] The compounds provided herein can be formulated into pharmaceutical compositions using methods available in the art and disclosed herein. Any compound disclosed herein can be provided in an appropriate pharmaceutical composition and administered by an appropriate route of administration. Administration of the compounds described herein to a subject can be systemic or non-systemic, such as topically, intradermally, or intralesionally. In some embodiments, the compound can be administered by topical administration.

[0093] As solid compositions for oral administration, tablets, pills, hard gelatin capsules, powders or granules can be used. In these compositions, the active product is mixed with one or more inert diluents or adjuvants (e.g., sucrose, lactose or starch). These compositions may contain substances other than the diluent, such as lubricants, e.g., magnesium stearate, or coatings for controlled release.

[0094] As liquid compositions for oral administration, pharmaceutically acceptable solutions, suspensions, emulsions, syrups and elixirs containing inert diluents (such as water or liquid paraffin) can be used. These compositions may also contain substances other than diluents, and in some embodiments, contain wetting, sweetening or flavoring products.

[0095] As compositions for topical application, lotions, tinctures, creams, emulsions, gels or ointments may be used. In these compositions, the active product is mixed with one or more inert excipients including water, acetone, ethanol, ethylene glycol, propylene glycol, butane 1,3 diol, isopropyl myristate, isopropyl palmitate, polyethylene glycol, polyethylene glycol-15 hydroxystearate, mineral oil and mixtures thereof.

[0096] "Pharmaceutically acceptable carriers" refer to one or more compatible solid or liquid fillers or gel substances that are suitable for human use and must be of sufficient purity and sufficiently low toxicity. "Compatibility" as used herein refers to the ability of the components of the composition to blend with the compounds of the present invention, and with each other, without significantly reducing the efficacy of the compounds. Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (e.g., sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (e.g., stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (e.g., propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (e.g., Tween), wetting agents (e.g., sodium lauryl sulfate), colorants, flavorings, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0097] Typical pharmaceutical compositions and dosage forms comprise one or more excipients. Suitable excipients are well known to those skilled in the art of pharmacy, and in some embodiments, suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skimmed milk powder, glycerine, propylene glycol, water, ethanol etc. Whether specific excipients are suitable for being incorporated into pharmaceutical compositions or dosage forms depends on multiple factors well known in the art, includes but is not limited to the specific active ingredient in the mode and dosage form of the object being administered to the dosage form. If desired, compositions or single unit dosage form can also contain a small amount of wetting agent or emulsifying agent, or pH buffer.

[0098] In some embodiments, the pharmaceutical compositions of the present invention are administered topically or transdermally. The present invention also provides a transdermal, topical dosage form. Transdermal and topical dosage forms include, but are not limited to, sprays, aerosols, creams, lotions, ointments, gels, solutions, emulsions, suspensions, or other forms known to those skilled in the art. In addition, transdermal dosage forms include "reservoir-type" or "matrix-type" patches, which can be applied to the skin and worn for a specific period of time to allow the desired amount of active ingredient to penetrate.

[0099] Suitable carriers (e.g., excipients and diluents) and other materials that can be used to provide the transdermal and topical dosage forms encompassed herein are well known to those skilled in the pharmaceutical arts and depend on the specific tissue to which a given pharmaceutical composition or dosage form is to be applied. With this fact in mind, typical carriers include, but are not limited to, water, acetone, ethanol, ethylene glycol, propylene glycol, butane 1,3 diol, isopropyl myristate, isopropyl palmitate, mineral oil, and mixtures thereof to form nontoxic and pharmaceutically acceptable lotions, tinctures, creams, emulsions, gels, or ointments. In some embodiments, the materials that can be used as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; (4) astragalus powder; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn starch, etc. Rice oil and soybean oil; (10) glycols such as propylene glycol; (11) polyols such as glycerol, sorbitol, mannitol, polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) phosphate buffer solution; and (21) other non-toxic compatible substances used in pharmaceutical formulations. If desired, moisturizers or humectants may also be added to the pharmaceutical compositions and dosage forms. Examples of such additional ingredients are well known in the art.

[0100] Depending on the specific tissue to be treated, additional components may be used before, in combination with, or after treatment with the provided active ingredient. In some embodiments, a penetration enhancer may be used to assist in delivering the active ingredient to the tissue. Suitable penetration enhancers include, but are not limited to, acetone; various alcohols such as ethanol, oleyl alcohol, and tetrahydrofuran methanol; alkyl sulfoxides such as dimethyl sulfoxide; dimethylacetamide; dimethylformamide; polyethylene glycol; pyrrolidones such as polyvinylpyrrolidone; Kollidon grades (Povidone, Polyvidone); urea; and various water-soluble or insoluble sugar esters such as Tween 80 (Polysorbate 80) and Span 60 (Sorbitol monostearate).

[0101] The pH value of pharmaceutical composition or dosage form can also be regulated, or the pH value of the tissue that pharmaceutical composition or dosage form are applied to, to improve the sending of one or more active components.Similarly, the polarity of solvent carrier, its ionic strength or tension (tonicity) can be regulated to improve sending. Compounds such as stearate can also be added to pharmaceutical composition or dosage form, to advantageously change the hydrophilicity or lipophilicity of one or more active components, thereby improve sending.In this respect, stearate can be used as lipid carrier, emulsifying agent or surfactant of preparation and as sending enhancing or penetration enhancer. Different salts, hydrates or solvates of active component can be used to further regulate the character of resulting composition.

[0102] The compounds of the present invention may be administered alone or in combination with other pharmaceutically acceptable compounds (eg, drugs for treating bacterial infections).

[0103] When administered in combination, the pharmaceutical composition may further comprise one or more (2, 3, 4, or more) other pharmaceutically acceptable compounds (e.g., drugs for treating bacterial infections). One or more (2, 3, 4, or more) of the other pharmaceutically acceptable compounds (e.g., drugs for treating bacterial infections) may be used simultaneously, separately, or sequentially with the compound of the present invention to prevent and / or treat diseases associated with Gram-positive or Gram-negative bacteria infections.

[0104] When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is administered to a mammal (e.g., a human) in need of treatment, wherein the dosage is a pharmaceutically effective dosage. For a 60 kg human, the daily dosage is generally 1 to 2000 mg, preferably 20 to 500 mg. Of course, the specific dosage will also take into account factors such as the route of administration and the patient's health condition, all of which are within the skill of a skilled physician.

[0105] The compounds of the present invention are a class of antibacterial prodrug molecules with charge reversal activated by reactive oxygen species. They utilize the characteristics of reactive oxygen species bursts at bacterial infection sites in the body to achieve targeted specific activation of the prodrug. Compared with active molecules, the compounds of the present invention have significantly reduced biotoxicity and improved water solubility, and exhibit good antibacterial activity in vitro and in skin infection models. Therefore, the present invention provides the use of compounds of Formula I of the present invention, or their optical isomers, pharmaceutically acceptable salts, hydrates, or solvates in the preparation of medicaments for treating and / or preventing bacterial infections. Herein, the bacteria causing bacterial infection may be Gram-positive or Gram-negative bacteria. Preferably, the bacteria are Gram-positive bacteria. In some embodiments, the bacteria are drug-resistant bacteria. Exemplary bacteria include (but are not limited to) Staphylococcus aureus (such as ATCC43300, USA300SF8300, and Newman). In some embodiments, the bacteria are drug-resistant Staphylococcus aureus (MRSA).

[0106] The present invention provides use of the compound of formula I of the present invention, or its optical isomers, pharmaceutically acceptable salts, hydrates or solvates in the preparation of bactericides or antibacterial agents.

[0107] The present invention provides a compound of formula I of the present invention, or its optical isomers, pharmaceutically acceptable salts, hydrates or solvates thereof for use in the preparation of a medicament for treating and / or preventing a disease or symptom caused by a bacterial infection. The disease or condition is caused by infection with Staphylococcus aureus. The disease is selected from the group consisting of skin infection, bacteremia, bloodstream infection, infective endocarditis, breast infection (mastitis), impetigo, toxic epidermal necrolysis, staphylococcal bloodstream infection, neonatal scalded skin syndrome, folliculitis, cellulitis, osteomyelitis and lung infection (pneumonia). The condition is selected from the group consisting of hair root (follicle) infection, blisters, impetigo, abscesses (sores or furuncles), painful empyema, breast abscesses, high fever, shortness of breath, cough, expectoration with blood in sputum, lung abscesses, pus accumulation (called empyema), dyspnea, persistent high fever, heart failure (with dyspnea), chills, fever, bone pain, redness and swelling of the skin and soft tissues and adjacent joint effusion.

[0108] The present invention also provides a method for treating or preventing bacterial infection, comprising administering to a subject in need thereof an effective amount of a compound of formula I, or an optical isomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof, or a pharmaceutical composition containing the compound of formula I or a pharmaceutically acceptable salt thereof.

[0109] The compound of formula I of the present invention can be used as a prodrug molecule, which can be activated by reactive oxygen species to achieve charge reversal. Therefore, the present invention also provides a method for converting the compound of formula I into the compound of formula II, the method comprising the steps of:

[0110]

[0111] The compound of formula I is contacted with active oxygen, which can be one or more selected from OH, NO, ONOOH and HClO. Preferably, the active oxygen is a ROS solution.

[0112] In Formula II, X and R1-R5 are as defined herein in any embodiment.

[0113] The compound of formula I of the present invention can utilize the characteristics of the outbreak of active oxygen in the bacterial infection site in the body to achieve targeted specific activation of the prodrug. The activation mechanism of the compound of formula I is as follows Figure 7 shown.

[0114] The carboxylate of the compound of formula I loses an electron in the presence of active oxygen to become an oxygen free radical, which then undergoes intramolecular charge transfer to leave CO2 and simultaneously generates a trityl free radical. The trityl free radical loses an electron in the presence of oxygen to generate the compound of formula II.

[0115] Advantages of the present invention include:

[0116] (1) The compounds of the present invention can be targeted and specifically activated at the site of bacterial infection to become active molecules with therapeutic effects;

[0117] (2) The compounds of the present invention have high antibacterial activity and have bactericidal effects on Gram-positive bacteria and Gram-negative bacteria.

[0118] (3) The compounds of the present invention have low toxicity, high biosafety, and a wide therapeutic window.

[0119] (4) The compounds of the present invention can be activated as prodrugs by active oxygen to active molecules with charge reversal, and the activation process is highly efficient.

[0120] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0121] Example 1: Preparation of Compound CCR1

[0122] Compound CCR1 was prepared according to the following scheme

[0123]

[0124] Specifically, the synthesis of 3,3'-diethylaminodiphenyl ether (Compound 3) involved placing 3-bromo-N,N-diethylaniline (Compound 1, 2 g, 1 equiv., 8.77 mmol), 3-diethylaminophenol (Compound 2, 2.17 g, 1.5 equiv., 13.15 mmol), cesium carbonate (4.28 g, 1.5 equiv., 13.15 mmol), and cuprous bromide (126 mg, 0.1 equiv., 0.88 mmol) in a 100 ml round-bottom flask. Using 50 ml of anhydrous N,N-dimethylformamide (DMF) as the solvent, the system was purged with argon for 30 minutes to remove oxygen. The reaction was then stirred vigorously at 140°C under an argon balloon for 20 hours. After TLC confirmed the completion of the reaction, DMF was removed by rotary evaporation, and the mixture was extracted three times with dichloromethane and water. The organic phase was dried over anhydrous magnesium sulfate, filtered, and spin-dried to obtain a crude product, which was separated and purified by column chromatography (eluent: petroleum ether:ethyl acetate = 100:1, v / v) to obtain 2.25 g of a colorless, transparent oily liquid with a yield of 82%.

[0125] 1 H NMR (400MHz, CDCl3): δ7.12(t,J=8.3Hz,2H),6.42-6.40(m,4H),6.27(dd,J=8.3Hz,2.0Hz,2H),3.33(q,J=7.0Hz,8H),1.15(t,J=7.0Hz,12H). 13 C NMR(101MHz, CDCl3)δ158.7,149.4,130.0,106.8,105.6,102.7,44.5,12.7.EI-HRMS(m / z):[M+H] + calcd.forC 20 H 28 N2O,312.2202; found 312.2201

[0126] Synthesis of 3,3'-diethylamino-6,6'-dibromodiphenyl ether (Compound 4). 3,3'-diethylaminodiphenyl ether (Compound 3, 2 g, 1 equiv., 6.40 mmol) was added to a 100-ml round-bottom flask containing 50 ml of dichloromethane. A solution of liquid bromine (0.66 ml, 2 equiv., 12.80 mmol) in 20 ml of dichloromethane was placed in a constant pressure dropping funnel and slowly added dropwise to the round-bottom flask under an ice bath. After stirring for one hour, TLC confirmed the completion of the reaction. Saturated sodium bicarbonate solution was added to neutralize the reaction, and the mixture was extracted three times with dichloromethane and water. The organic phase was dried over anhydrous magnesium sulfate, filtered, and spin-dried to yield 3.01 g of a white solid.

[0127] 1H NMR (400MHz, CDCl3): δ7.35(d,J=8.9Hz,2H), 6.33(dd,J=8.9Hz,2.8Hz,2H), 6.16(d,J=2.8Hz,2H), 3.23(q,J=7.0Hz,8H), 1.07(t,J=7.0Hz,12H). 13 C NMR(101MHz, CDCl3)δ154.1,148.5,133.6,108.9,103.5,98.5,44.7,12.5.EI-HRMS(m / z):[M+H] + calcd.for C 20 H 27 Br2N2O,471.0470; found 471.0456

[0128] Synthesis of Compound RD1. 3,3'-Bisdiethylamino-6,6'-dibromodiphenyl ether (Compound 4, 200 mg) was dissolved in anhydrous tetrahydrofuran. 2.5 equivalents of a 2.5M n-butyllithium solution in n-hexane (0.45 mL) were added dropwise at -78°C using a syringe. The mixture was stirred for 15 minutes to prepare the dilithium reagent. Methyl benzoate (100 mg) was dissolved in 10 mL of anhydrous tetrahydrofuran and added dropwise to the dilithium reagent. After reacting for 1 hour, saturated ammonium chloride solution was added to quench the excess lithium reagent. The reaction solution was extracted three times with dichloromethane and water. The organic phase was dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated to obtain a red oily crude product. This was separated and purified using normal phase silica gel column chromatography (eluent: dichloromethane:methanol = 95:5, v / v) to obtain the corresponding compound. 261 mg of Compound RD1 was obtained as a purple-red solid with a yield of 91%.

[0129] 1 H NMR (400MHz, CDCl3): δ7.63-7.62(m,3H),7.38-7.34(m,4H),6.94(dd,J=8.0Hz, 1.6Hz, 2H), 6.87 (d, J = 1.6Hz, 1H), 3.67 (q, J = 7.0Hz, 8H), 1.34 (t, J = 7.0Hz, 12H).

[0130] Preparation of Compound CCR1. First, compound RD1 (100 mg, 0.25 mmol) was added to a 100 mL round-bottom flask and dissolved in 40 mL of tetrahydrofuran. Tetrabutylammonium fluoride (TBAF) (98.16 mg, 0.38 mmol) and trimethylsilyl cyanide (TMSCN) (0.05 mL, 0.50 mmol) were added, and the mixture was heated under reflux with stirring for 2 hours. The reaction was quenched with saturated aqueous ammonium chloride and extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate and the solvent was removed by rotary evaporation to yield an oil. This oil was then dissolved in 20 mL of concentrated HCl and heated under reflux for 8 hours. After the reaction, sodium hydroxide was added to neutralize the concentrated hydrochloric acid, and the mixture was extracted three times with dichloromethane. The organic phase was dried over anhydrous sodium sulfate and the solvent was removed by rotary evaporation. The mixture was separated by column chromatography (DCM:MeOH = 50:1) to yield compound CCR1 (60 mg, 57%) as a light pink solid.

[0131] 1H NMR (400MHz, CDCl3): δ7.33(d,J=7.9Hz,2H),7.26(t,J=7.9Hz,2H),7.19(t,J=7.3Hz,1H),6.80(d,J=8.4Hz ,2H),6.34(d,J=2.7Hz,2H),6.29(dd,J=8.4,2.7Hz,2H),3.31(q,J=7.0Hz,8H),1.14(t,J=7.0Hz,12H).13C NMR (101 MHz, CDCl3): δ 179.37, 152.14, 148.34, 145.49, 131.90, 129.45, 127.95, 126.52, 110.45, 107.84, 97.90, 55.01, 44.43, 12.81. ESI-MS (m / z): [M+H]+ calculated for C28H33N2O3 445.2491; found: 445.2492.

[0132] Example 2: Preparation of Compound CCR2

[0133]

[0134] Compound 3 (1.5 g, 4.80 mmol, 1.0 equiv.) and methyl pyruvate (0.54 g, 5.28 mmol, 1.1 equiv.) were dissolved in 40 mL of dichloroethane. Under an oxygen-free environment, 0.53 mL of titanium tetrachloride (0.91 g, 4.8 mmol, 1.0 equiv.) was added, and the mixture was stirred at 80°C for 12 h. After completion of the reaction, saturated Na2CO3 solution was added to the reaction flask to adjust the pH to neutral. The titanium salt was removed by filtration, and the crude product was extracted, dried, and concentrated to obtain a crude product. Column chromatography (PE:EA = 60:1, v:v) afforded 0.52 g of a purple solid, compound 5, in a 30% yield.

[0135] 1 H NMR (400MHz, CDCl3) δ7.12 (dd, J=8.8, 3.0Hz, 2H), 6.47 (dt, J=8.7, 2.8Hz, 2H), 6.41 (t, J=2.9Hz, 2H), 3 .65(d,J=1.9Hz,3H),3.42(qd,J=7.0,2.1Hz,8H),1.88(d,J=3.3Hz,3H),1.25(td,J=7.1,2.2Hz,12H). 13 C NMR(101MHz, CDCl3)δ176.31,151.16,148.13,127.86,111.11,107.23,98.56,52.66,44.40,44.16,29.17,12.77.ESI-HRMS,m / z,[M] + ,calcd.for C 28 H 33 N2O3 + ,397.2486;found 397.2490.

[0136] Compound 5 (0.32 g, 0.80 mmol, 1.0 equiv.) was placed in a 50 mL round-bottom flask and dissolved in a small amount of tetrahydrofuran. 1 mL of aqueous NaOH (0.32 g, 8.07 mmol, 10.0 equiv.) was added to the flask and refluxed at 70°C for 24 h. After completion of the reaction, the solvent was removed, and the residue was neutralized with 3 M hydrochloric acid to a weak acidic state. The residue was extracted three times with DCM / H₂O. The organic phase was dried over anhydrous Na₂SO₄ and concentrated to obtain the crude product, which was separated by column chromatography (PE:EA = 5:1, v:v) to afford compound CCR2 (101 mg, 33% yield) as a light pink solid.

[0137] 1H NMR (400MHz, CDCl3) δ7.11 (d, J=8.7Hz, 2H), 6.40 (dd, J=8.8, 2.6Hz, 2H), 6.32 (d,J=2.6Hz,2H),3.34(q,J=7.1Hz,8H),1.79(s,3H),1.17(t,J=7.0Hz,12H). 13 C NMR(151MHz, CDCl3)δ151.25,148.32,127.98,110.11,107.25,98.53,44.35,43.85,27.94,12.68.ESI-HRMS,m / z,[M+H] + ,calcd.for C 23 H 31 N2O3 + ,Calculated value, 383.2335; Measured value, 383.2330.

[0138] Example 3: Preparation of Compound CCR3

[0139]

[0140] Compound 3 (1.5 g, 4.80 mmol, 1.0 equiv.) and methyl 2-oxobutanoate (0.61 g, 5.28 mmol, 1.1 equiv.) were dissolved in 40 mL of dichloroethane. Under an oxygen-free environment, 0.53 mL of titanium tetrachloride (0.91 g, 4.8 mmol, 1.0 equiv.) was added to the mixture. The mixture was stirred at 80°C for 12 h. After completion of the reaction, saturated NaCO solution was added to the reaction flask to adjust the pH to neutral using TLC. The salts were removed by filtration, and the residue was extracted three times with DCM / H2O. The organic phase was dried over anhydrous Na2SO4 and concentrated to obtain the crude product. After column chromatography (PE:EA = 60:1, v:v), 0.82 g of a purple solid, compound 6, was obtained in a 42% yield.

[0141] 1 H NMR (400MHz, CDCl3) δ6.97(dd,J=8.8,2.1Hz,2H),6.46–6.39(m,2H),6.35(d,J=2.8Hz,2H),3.61(d,J= 2.1Hz,3H),3.37(q,J=7.2Hz,8H),2.26(q,J=7.4Hz,2H),1.20(t,J=7.1Hz,12H),0.56(t,J=7.3Hz,3H). 13C NMR(101MHz, CDCl3)δ176.34,152.32,148.06,127.69,108.50,107.40,98.38,52.53,49.09,44.36,33.21,12.78,8.43.ESI-HRMS,m / z,[M] + ,calcd.forC 25 H 35 N2O3 + ,411.2642;found 411.2646.

[0142] Compound 6 (0.05 g, 1.22 mmol, 1.0 equiv.) was placed in a 50 mL round-bottom flask and dissolved in a small amount of tetrahydrofuran. 1 mL of aqueous NaOH (0.49 g, 12.18 mmol) was then added to the flask and allowed to react at 70°C for 24 h. After completion of the reaction, as monitored by TLC, the solvent was removed and the residue was neutralized to a weakly acidic state with 3 M hydrochloric acid. The residue was then extracted three times with DCM / H₂O. The organic phase was dried over anhydrous Na₂SO₄ and concentrated to obtain the crude product, which was then separated by column chromatography (PE:EA = 5:1, v:v) to afford compound CCR3 (210 mg, 42% yield) as a light pink solid.

[0143] 1 H NMR(400MHz, CDCl3) δ7.04(d,J=8.7Hz,2H),6.40(dd,J=8.8,2.7Hz,2H),6.32(d,J=2.6Hz,2H ),3.34(q,J=7.1Hz,8H),2.23(q,J=7.3Hz,2H),1.18(t,J=7.0Hz,12H),0.53(t,J=7.3Hz,3H). 13 C NMR(101MHz, CDCl3)δ180.27,152.59,148.42,128.09,107.66,107.61,98.56,49.03,44.52,32.01,12.90,8.63.ESI-HRMS,m / z,[M] + ,calcd.forC 24 H 33 N2O3 + , calculated value 397.2486; measured value 397.2490.

[0144] Example 4: Preparation of Compound CCR4

[0145]

[0146] Methyl(phenyl)sulfane (compound 7, 3.00 g, 24.15 mol, 1.0 equiv.) and aluminum trichloride (3.96 g, 28.99 mmol, 1.20 equiv.) were dissolved in 60 mL of anhydrous dichloromethane. Ethyl oxalyl chloride (compound 8) in anhydrous dichloromethane (3.20 mL) was slowly added dropwise to the dichloromethane solution at 0°C using a constant pressure dropping funnel. The mixture was allowed to react overnight at room temperature. After completion of the reaction, saturated ammonium chloride solution was added at 0°C to quench the unreacted acyl chloride. The crude product was extracted, dried, and concentrated to obtain 3.50 g of a yellow liquid, Compound 9, after separation by column chromatography (PE:EA = 10:1, v:v). The yield was 65%.

[0147] 1 H NMR (400MHz, CDCl3) δ7.93(d,J=1.9Hz,1H),7.91(d,J=1.9Hz,1H),7.30(d,J=1.9Hz, 1H),7.28(d,J=1.8Hz,1H),4.44(q,J=7.1Hz,2H),2.53(s,3H),1.42(t,J=7.2Hz,3H).

[0148] Compound 3 (1.5 g, 4.80 mmol, 1.0 equiv.) and ethyl 2-(4-(methylthio)phenyl)-2-oxoacetate (compound 9, 1.18 g, 5.28 mmol, 1.0 equiv.) were dissolved in 40 mL of dichloroethane. Under an oxygen-free atmosphere, 0.53 mL of titanium tetrachloride (0.91 g, 4.8 mmol, 1.0 equiv.) was added to the mixture, and the mixture was stirred at 80°C for 12 h. After TLC confirmed the completion of the reaction, saturated NaCO solution was added to the reaction flask to adjust the pH to neutral, and the mixture was extracted three times with DCM / H2O. The organic phase was dried over anhydrous Na2SO4 and concentrated to obtain the crude product. After column chromatography (PE:EA = 35:1, v:v), 1.90 g of a yellow solid, compound 10, was obtained in an 80% yield.

[0149] 1 H NMR (400MHz, CDCl3) δ7.24–7.13(m,4H),6.87(d,J=8.5Hz,2H),6.35(d,J=11.3Hz,4H ), 4.20 (q, J = 7.2Hz, 2H), 3.34 (t, J = 7.2Hz, 8H), 2.46 (s, 3H), 1.19 (q, J = 7.5Hz, 15H). 13C NMR (101MHz, CDCl3) δ173.87,152.06,148.14,143.20,136.25,131.46,129.73,125.99 ,110.78,107.22,97.96,61.59,54.66,44.40,15.82,14.21,12.80.ESI-HRMS,m / z,[M] + ,calcd.forC 31 H 39 N2O3S + ,519.2676;found 519.2682.

[0150] Compound 10 (1.40 g, 2.70 mmol, 1.0 equiv.) was placed in a 50 mL round-bottom flask and dissolved in a small amount of tetrahydrofuran. 1 mL of aqueous NaOH (0.36 g, 8.91 mmol, 3.3 equiv.) was added to the flask and refluxed at 70°C for 24 h. After completion of the reaction, the residue was neutralized to a weakly acidic state by TLC, and extracted three times with DCM / H₂O. The organic phase was dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to obtain the crude product. This was then separated by column chromatography (PE:EA = 5:1, v:v) to afford compound CCR4 (700 mg, 53% yield) as a light pink solid.

[0151] 1 H NMR (400MHz, CDCl3) δ7.33–7.26(m,3H),7.20–7.13(m,2H),6.92–6.85(m,2H),6.39–6.28(m, 4H),3.35(q,J=7.1Hz,8H),2.48(d,J=1.7Hz,3H),2.09(d,J=1.6Hz,1H),1.21–1.13(m,12H). 13 C NMR (101MHz, CDCl3) δ178.01,151.94,147.86,143.75,135.59,131.94,131.53,130.29,130.06,126.02 ,125.82,117.79,113.99,111.68,107.30,97.90,54.90,46.13,44.38,15.94,12.75.ESI-HRMS,m / z,[M] + ,calcd.for C 29 H 35 N2O3S + , calculated value 491.2363; measured value 491.2369.

[0152] Example 5: Preparation of Compound CCR5

[0153]

[0154] Fluorobenzene (Compound 11, 2.00 g, 20.81 mol, 1.0 equiv.) and aluminum trichloride (3.41 g, 24.97 mmol, 1.20 equiv.) were dissolved in 60 mL of anhydrous dichloromethane. A solution of ethyl oxalyl chloride (Compound 8) in anhydrous dichloromethane (3.33 g, 24.97 mmol, 1.20 equiv.) was slowly added dropwise to the solution at 0°C via a constant pressure dropping funnel. The mixture was allowed to react overnight at room temperature. After completion of the reaction, saturated ammonium chloride solution was added at 0°C to quench the unreacted acyl chloride. The reaction was then extracted three times with DCM / H₂O. The organic phase was dried over anhydrous Na₂SO₄ and concentrated to yield the crude product. This was separated by column chromatography (PE:EA = 20:1, v:v) to afford 1.80 g of a yellow liquid, Compound 12, in a 45% yield.

[0155] 1 H NMR (400MHz, CDCl3) δ8.08(dd,J=8.8,5.4Hz,2H),7.19(t,J=8.6Hz,2H),4.45(q,J=7.1Hz,2H),1.43(t,J=7.2Hz,3H).

[0156] Compound 3 (1.5 g, 4.80 mmol, 1.0 equiv.) and compound 12 (1.04 g, 5.28 mmol, 1.0 equiv.) were dissolved in 100 mL of dichloroethane. Under an oxygen-free atmosphere, 0.53 mL of titanium tetrachloride (0.91 g, 4.80 mmol, 1.0 equiv.) was added to the mixture, and the mixture was stirred at 80°C for 16 h. After the reaction, saturated NaCO solution was added to the reaction flask to adjust the pH to neutral. The salts were removed by filtration, and the reaction was extracted three times with DCM / H2O. The organic phase was dried over anhydrous Na2SO4 and concentrated to obtain the crude product. Column chromatography (PE:EA = 100:1-60:1, v:v) afforded 1.45 g of a yellow solid, compound 13, in a 65% yield.

[0157] 1H NMR (400MHz, CDCl3) δ7.25–7.19(m,2H),6.94(t,J=8.7Hz,2H),6.82(d,J=8.5Hz,2H),6.34(s,3H) ,6.32(d,J=2.7Hz,1H),4.17(q,J=7.1Hz,2H),3.33(q,J=7.1Hz,8H),1.16(td,J=7.0,3.9Hz,15H). 13 C NMR (101MHz, CDCl3) δ173.83,151.99,148.15,131.24,130.86,130.78,114.71,114.50,110.75,107.20,97.95,61.62,44.35,14.14,12.74. 19 F NMR (565MHz, CDCl3δ-116.88.ESI-HRMS, m / z, [M] + ,calcd.for C 30 H 36 FN2O3 + ,491.2704;found 491.2711.

[0158] Compound 13 (0.90 g, 1.83 mmol, 1.0 equiv.) was placed in a 50 mL round-bottom flask and dissolved in a small amount of tetrahydrofuran. 1 mL of aqueous NaOH (0.73 g, 18.34 mmol, 10.0 equiv.) was added to the flask and stirred at 70°C for 24 h. After TLC confirmed the completion of the reaction, the THF solvent was removed, and the residue was neutralized with 3 M hydrochloric acid to a weak acidic state. The mixture was then extracted three times with DCM / H₂O. The organic phase was dried over anhydrous Na₂SO₄ and concentrated to obtain the crude product, which was then separated by column chromatography (PE:EA = 5:1 to 1:1, v:v) to afford compound CCR5 (360 mg, 43% yield) as a light pink solid.

[0159] 1 H NMR (400MHz, CDCl3) δ7.37–7.29(m,1H),6.97(t,J=8.7Hz,1H),6.80(d,J=8.8Hz,1H),6.40–6.28(m,2H),3.34(q,J=7.0Hz,4H),1.17(t,J=7.0Hz,6H). 13C NMR (101MHz, CDCl3δ179.67,152.01,148.29,131.59,131.10,131.02,114.78,114.57,110.21,107.28,97.88,54.35,44.41,12.73. 19 F NMR(565MHz,CDCl3)δ-75.51.ESI-HRMS,m / z,[M] + ,calcd.forC 28 H 32 FN2O3 + , calculated value 463.2391; measured value 463.2398.

[0160] Example 6: Preparation of Compound CCR6

[0161]

[0162] 1-Bromo-4-phenylvinylbenzene (Compound 14, 2.00 g, 7.72 mmol, 1 equiv.) and diethyl oxalate (Compound 15, 1.10 g, 8.49 mmol, 1.1 equiv.) were dissolved in 50 mL of anhydrous tetrahydrofuran. 3.40 mL of n-butyllithium (0.55 g, 8.49 mmol, 1.1 equiv.) was slowly added dropwise to the tetrahydrofuran solution of 1-bromo-4-phenylvinylbenzene at -78°C under an anhydrous atmosphere and the reaction was maintained at -78°C for 30 min. After TLC confirmed the complete reaction of 1-bromo-4-phenylvinylbenzene, the above solution was slowly added to a THF solution of diethyl oxalate at 0°C and the reaction was continued at room temperature for 2 hours. After TLC confirmed the completion of the reaction, saturated ammonium chloride solution was added to quench the unreacted phenyllithium reagent, and the mixture was extracted three times with DCM / H2O. The organic phase was dried over anhydrous Na2SO4 and concentrated to obtain a crude product, which was separated by column chromatography (PE:EA=5:1-1:1, v:v) to obtain 0.36 g of a yellow solid, compound 16, with a yield of 51%.

[0163] 1 H NMR (400MHz, CDCl3) δ8.02(d,J=8.0Hz,2H),7.63(d,J=8.1Hz,2H),7.56(d,J=7.7Hz,2H),7.40(t,J= 7.6Hz,2H),7.35–7.28(m,1H),7.14(d,J=16.2Hz,1H),4.46(q,J=7.3Hz,2H),1.44(t,J=7.1Hz,3H). 13C NMR (151MHz, CDCl3) δ185.61,163.90,136.45,132.72,131.25,130.67,128.87,128.65,127.15,127.00,126.75,62.33,14.13.ESI-HRMS, m / z,[M] + ,calcd.for C 18 H 17 O3 + ,281.1172;found 281.1132.

[0164] Compound 3 (1.10 g, 3.52 mmol, 1.0 equiv.) and compound 16 (1.01 g, 3.59 mmol, 1.0 equiv.) were dissolved in 50 mL of dichloroethane. Under an oxygen-free environment, 0.39 mL of titanium tetrachloride (0.67 g, 3.52 mmol, 1.0 equiv.) was added to the mixture, and the mixture was stirred at 80°C for 16 h. After completion of the reaction as confirmed by TLC, saturated NaCO solution was added to the reaction flask to adjust the pH to neutral, and the salts were removed by filtration. The residue was extracted three times with DCM / H2O. The organic phase was dried over anhydrous Na2SO4 and concentrated to obtain the crude product. After column chromatography (PE:EA = 80:1-20:1, v:v), 1.13 g of a yellow solid, compound 17, was obtained in a 55% yield.

[0165] 1 H NMR (400MHz, CDCl3) δ7.54–7.46(m,2H),7.44–7.39(m,2H),7.34(t,J=7.6Hz,2H),7.25(dd,J=7.9,2.1Hz,3H),7.07(s,2H),6.87(d,J =8.6Hz,2H),6.35(t,J=2.7Hz,3H),6.31(d,J=2.7Hz,1H),4.19(q,J=7.1Hz,2H),3.33(q,J=7.0Hz,8H),1.17(dt,J=12.8,7.1Hz,15H). 13 C NMR (101MHz, CDCl3) δ152.04,148.14,145.72,137.49,135.34,131.57,129.57,128.73,128.58,128 .41,127.59,126.55,126.05,110.75,107.22,97.93,61.59,44.37,14.20,12.77.ESI-HRMS,m / z,[M] + ,calcd.for C38 H 43 N2O3 + ,575.3268;found575.3276.

[0166] Compound 17 (1.00 g, 1.74 mmol, 1.0 equiv.) was placed in a 50 mL round-bottom flask and dissolved in a small amount of tetrahydrofuran. Then, 1 mL of aqueous NaOH (0.70 g, 17.40 mmol, 10.0 equiv.) was added to the flask and stirred at 70°C for 24 h. After TLC confirmed the reaction was complete, the reaction mixture was neutralized to a weakly acidic state with 3 M hydrochloric acid and extracted three times with DCM / H₂O. The organic phase was dried over anhydrous Na₂SO₄ and concentrated to obtain the crude product, which was separated by column chromatography (PE:EA = 5:1 to 1:1, v:v) to afford compound CCR6 (480 mg, 51% yield) as a light pink solid.

[0167] 1 H NMR(400MHz, CDCl3)δ7.68(t,J=6.2Hz,2H),7.60(q,J=7.3,6.7Hz,2H),7.57–7.48(m,4H),7.44(d,J=10.4Hz,1H) ,7.02(td,J=8.8,2.9Hz,2H),6.51(ddt,J=19.9,11.1,4.5Hz,4H),3.51(p,J=6.7Hz,8H),1.34(q,J=6.9Hz,12H). 13 C NMR (101MHz, CDCl3) δ179.30,152.06,148.25,145.14,137.50,135.44,131.84,129.76,128.72,128. 62,128.42,127.59,126.56,126.07,110.41,107.30,97.87,54.82,44.40,12.77.ESI-HRMS,m / z,[M] + ,calcd.for C 36 H 39 N2O3 + , calculated value 547.2995; measured value 547.2962.

[0168] Example 7: Preparation of compound CCR53

[0169]

[0170] Compound CCR53 was prepared using a method similar to Example 1.

[0171] Preparation of compound RD53. 3,3'-Bisdiethylamino-6,6'-dibromodiphenyl ether (compound 4, 200 mg) was dissolved in anhydrous tetrahydrofuran. 2.5 equivalents of a 2.5M n-butyllithium solution in n-hexane (0.45 mL) were added dropwise at -78°C using a syringe. The mixture was stirred for 15 minutes to prepare the dilithium reagent. Adamantane acetate methyl ester (100 mg) was dissolved in 10 mL of anhydrous tetrahydrofuran and added dropwise to the dilithium reagent. After reacting for 1 hour, saturated ammonium chloride solution was added to quench the excess lithium reagent. The reaction solution was extracted three times with dichloromethane and water. The organic phase was dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated to obtain a red oily crude product. This was separated and purified using normal phase silica gel column chromatography (eluent: dichloromethane:methanol = 95:5, v / v) to obtain compound RD53 as a purple-red solid in an 82% yield.

[0172] 1 H NMR (400MHz, CDCl3): δ7.87(d,J=9.6Hz,2H), 6.96(dd,J=9.5Hz,2H), 6.57(d,J=1.0Hz,2H), 3.53(q,J=7.0Hz,8H),3.05(s,2H),1.81(s,3H),1.51-1.41(m,12H),1.21(t,J=7.1Hz,12H). 13 C NMR (101MHz, CDCl3): δ157.6,156.9,155.0,130.9,114.4,113.3,95.6,45.7,43.5,40.8,37.2,36.1,28.6,12.5.EI-HRMS(m / z):[M] + calcd.for C 32 H 43 N2O,471.3375; found 471.3376

[0173] Preparation of Compound CCR53. First, compound RD53 (100 mg, 0.21 mmol) was added to a 100 mL round-bottom flask and dissolved in 40 mL of tetrahydrofuran. TBAF (98.16 mg, 0.38 mmol) and TMSCN (0.05 mL, 0.50 mmol) were then added and stirred under reflux for 2 hours. The reaction was quenched with saturated aqueous ammonium chloride and extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate and the solvent was removed by rotary evaporation to yield an oil. This oil was then dissolved in 20 mL of concentrated HCl and heated under reflux for 8 hours. After the reaction, sodium hydroxide was added to neutralize the concentrated hydrochloric acid. The product was extracted three times with dichloromethane. The organic phase was dried over anhydrous sodium sulfate and the solvent was removed by rotary evaporation. The product was separated by column chromatography (DCM:MeOH = 50:1) to yield compound CCR53 (60 mg, 58% yield) as a light pink solid.

[0174] 1H NMR (400MHz, CDCl3): δ7.02(d,J=8.7Hz,2H),6.37(dd,J=8.7,2.6Hz,2H),6.29(d,J=2.6Hz,2H),3.34( q,J=7.1Hz,8H),2.16(s,2H),1.65(s,3H),1.49–1.36(m,6H),1.17(t,J=7.1Hz,12H),1.09(s,6H).13C NMR (101 MHz, CDCl3): δ 178.62, 151.50, 148.63, 129.12, 109.03, 107.54, 98.92, 52.67, 47.02, 44.50, 43.74, 36.98, 33.81, 28.88, 12.75. ESI-MS (m / z): [M+H]+ C33H45N2O3 calculated value 517.3430; found value 517.3432.

[0175] Example 8: Responsiveness test of the present compound to active oxygen and selectivity test for active substances

[0176] Compound CCR1 was dissolved in analytical grade DMF to a 10 mM stock solution. Argon was then purged into the bottle, the solution sealed to prevent air ingress, and stored in a -20°C refrigerator. The test solution consisted of a 10 mM PBS buffer solution (pH 7.4) containing 0.2% (volume fraction) DMF. The prodrug CCR1 concentration in the test solution was 5 μM. Aqueous solutions of varying amounts of reactive oxygen species, bioreducing substances, and metal ions were added to the test system, and fluorescence kinetic curves of the reaction were collected. After kinetic stabilization, spectra were collected using a UV-visible spectrophotometer and a fluorescence spectrophotometer.

[0177] The responsiveness of prodrug CCR1 to reactive oxygen species was tested as follows Figure 1 The selectivity test of prodrug CCR1 for active substances such as reactive oxygen species, bioreducing substances, and metal ions is shown in FIG. Figure 2 In the selectivity test, the types and concentrations of active substances added are as follows: Cys: 500μM, GSH: 500μM, Fe 3+ :100μM,K + :10mM,Na + :10mM,Zn 2+ :1mM,O 2- :250μM,H2O2:250μM, 1 O2:10μM, HClO:10μM, OH·:75μM, NO:75μM, ONOO -:10μM.

[0178] Example 9: Minimum Inhibitory Concentration Test

[0179] The minimum inhibitory concentration (MIC) of CCR1 / RD1 against different strains was determined by broth dilution method, referring to the standards of the Clinical and Laboratory Standards Institute (CLSI). The specific procedures are as follows:

[0180] a) streaking Staphylococcus aureus ATCC43300, Staphylococcus aureus USA300 SF8300, and Staphylococcus aureus Newman strains from cryopreservation tubes;

[0181] b) The next day, pick a single colony and culture it in LB medium overnight at 37°C and 220 rpm.

[0182] c) Dilute the overnight culture 1:1000 into fresh LB tube medium, about 2×10 5 CFU / mL;

[0183] d) Add 100 μL of the diluted bacterial solution to a round-bottom 96-well microplate and add an equal volume of fresh LB medium containing different concentrations of CCR1 / RD1.

[0184] Incubate at 37°C for 16-18 hours, observe the growth with the naked eye, and the lowest concentration in the well with no bacterial growth is the MIC.

[0185] The MIC values ​​of the present compound CCR1 and the control compound are shown in Figure 3 (A) The antibacterial prodrug CCR1 was activated into RD1 using ROSup (purchased from Beyotime Biotechnology Co., Ltd.). Figure 3 (B). The structures of the control compounds are shown in Figure 3 (C).

[0186] Example 10: Sterilization curve determination

[0187] The concentration of the ROSup solution used to activate the CCR1 drug was 0.05 μg / mL in normal saline. The specific steps of the time-kill curve determination method are as follows:

[0188] a) Pick a single colony of MRSA (ATCC43300) into 2 mL of LB medium and culture overnight for 16–18 h at 37°C and 220 rpm.

[0189] b) Dilute the overnight bacterial suspension into normal saline at a ratio of 1:100 and aliquot into 2 mL of normal saline solution containing bacteria;

[0190] c) Add compounds to the above solutions to final concentrations of 2.5 μg / mL RD1, 2.5 μg / mL CCR1, 0.05 μg / mL ROSup, and 2.5 μg / mL CCR1 + 0.05 μg / mL ROSup, respectively;

[0191] d) At 0, 1, 4, 8, and 12 h, 200 μL of the solution was taken for plate counting and determination of OD556 absorbance.

[0192] f) After the plates are dried, place them in a 37°C incubator and incubate for 24 hours before counting the number of colonies.

[0193] The time dependence curves of different compounds killing MRSA (ATCC43300) are as follows Figure 4 The pathogen was grown to early exponential phase and challenged with the reagents shown. Data represent the results of three independent experiments, and error bars indicate SD.

[0194] Example 11: Toxicity studies

[0195] 1. Cytotoxicity

[0196] 1. Cell Culture

[0197] RAW264.7 cells were cultured in RPMI-1640, while HeLa and HpG2 cells were cultured in DMEM high-glucose medium (both supplemented with 10% fetal bovine serum and 1× penicillin-streptomycin solution). The cells were placed in a cell culture incubator at 37°C with 5% CO2 to allow for adherent growth. When the cell confluence reached 85%, RAW264.7 cells were washed twice with PBS and then dispersed directly with PBS. HeLa and HpG2 cells were digested with 0.25% Trypsin-EDTA for an appropriate time. The cells were then passaged or evenly plated in 96-well plates according to experimental needs.

[0198] 2. Cytotoxicity Assay

[0199] Take cells in logarithmic growth phase, digest them with 1mL trypsin, pipette to make cell suspension, and count the cell concentration. 4Cells were plated at a density of 100 μL / well and incubated in a cell culture incubator at 37°C with 5% CO2 for 24 hours to allow the cells to adhere. The culture medium was then removed, 100 μL of PBS was added and aspirated (to remove serum), and 100 μL of serum-free culture medium containing CCR1, CCR53, RD1, or RD53 compounds was added to each well. The concentrations of CCR1, CCR53, RD1, or RD53 compounds in each serum-free culture medium were 0, 0.35, 0.78, 1.56, 3.13, 6.25, 12.5, and 25 μg / mL, respectively. Culture was continued for 24 hours. The culture medium was aspirated, 100 μL of PBS was added and aspirated again, and this was repeated (to remove compound residues). The wells containing cells without drug addition served as the control group, and the blank culture medium without cells served as the blank group. Six replicate wells were prepared for each experimental group. Dilute the CCK-8 solution 10-fold in serum-free culture medium. Pipette 100 μL into each well and incubate the plate in a cell culture incubator for 1–4 hours. Measure the absorbance of each well at 450 nm using a microplate reader. Calculate cell viability using the following formula. GraphPad Prism software is then used to estimate the drug concentration that inhibits 50% cell viability (IC50).

[0200] Cell viability (%) = [(As-Ab) / (Ac-Ab)] × 100%

[0201] As: absorbance of the experimental group (containing cells, culture medium, CCK-8 solution, and RD1 or CCR1 or CCR53 or RD53 solution);

[0202] Ac: absorbance of the control group (containing cells, culture medium, and CCK-8 solution, but no drug);

[0203] Ab: absorbance of blank group (containing culture medium and CCK-8 solution, but without cells or drugs).

[0204] The results of the cytotoxicity study of CCR1 / RD1 are as follows Figure 5 (C) shown.

[0205] 2. Acute toxicity in mice

[0206] 1. Experimental Animals

[0207] 60 male and 60 female ICR mice (4-6 weeks old, weight: male 25-30g, female 20-25g) were obtained from Shanghai Shengchang Biotechnology Co., Ltd. The experiment began after 3-5 days of adaptive feeding of experimental animals. The experimental animals were fed with normal basal feed, and males and females were kept separately. At the beginning of the drug administration, the weight difference of each group of mice was controlled to not exceed ±20% of the average body weight. All animal experiment applications for this experiment were approved by the Experimental Animal Ethics and Use Committee of Shanghai Jiao Tong University (Animal Experiment Lot No.: 202101309).

[0208] 2. Preparation of CCR1 / RD1 Compounds

[0209] Weigh an appropriate amount of compound and add melted 5%-10% Solutol HS-15 (50°C water bath). Add 1.5-2 mL of anhydrous ethanol and sonicate until completely dissolved. Then, use a rotary evaporator to dry the alcohol (approximately 5-6 minutes). Add a calculated volume of physiological saline and dissolve to the target concentration, 10 mg / mL for CCR1 and 5 mg / mL for RD1 (prepare immediately for use).

[0210] 3. Acute toxicity study of CCR1 / RD1 compounds in mice

[0211] First, a pilot study was conducted to explore the dose range and determine the absolute lethal dose and maximum tolerated dose (MTD). Mice were fasted overnight (8-12 hours) prior to dosing. After fasting, mice were weighed and the compound formulation was injected via the tail vein. The pilot study employed a stepwise approach, with three mice administered at each step. The starting dose was 150 mg / kg for the CCR1 compound and 50 mg / kg for the RD1 compound. If the animals were still alive, a second group of mice was given a higher dose. If the first group of mice had died or were dying, a second group was given a lower dose. The absolute lethal dose and MTD were determined until all mice receiving the high dose of CCR1 / RD1 died and all mice receiving the low dose survived. Based on the pilot study, CCR1 was administered via the tail vein at doses of 135, 130, 125, 120, 115, and 110 mg / kg, and RD1 was administered via the tail vein at doses of 50, 48, 45, 40, and 25 mg / kg. The present invention adopts a one-time administration method, and the mice are observed individually and regularly within 24 hours after injection, especially within the first 4 hours, and then observed every day for a total of 14 days. The mortality of each group of mice was counted, and the probability unit weighted regression method (Bliss method) was used to calculate the dose (LD50) of the experimental animals using SPSS software. The research results are as follows Figure 5 (A) shown.

[0212] 3. Study on the toxicity of CCR1 and RD1 on zebrafish embryo survival

[0213] 1) At 7 PM the day before the experiment, wild-type AB zebrafish were mated and separated by a partition. The next morning at 9 AM, the partition was removed. After waiting for half an hour, embryos were collected from the bottom of the mating tank and mixed into 10 cm plastic dishes. The dishes were then incubated at 28.5°C. At 6 hpf, developmentally delayed and low-quality embryos were removed.

[0214] At 48 hpf, treat the embryos with 1 mg / mL proteinase for 6 minutes to completely remove the eggshell. After removing the eggshell, wash three times with fresh E3 medium to remove the proteinase on the embryo surface and avoid damage to the embryo due to over-digestion.

[0215] 3) Randomly place 15 embryos per well in a 6-well plate, for a total of 24 groups. Remove excess liquid from the 6-well plate, add 4 mL of fresh E3 medium, and add CCR1 and RD1 at concentrations of 0.00001, 0.00005, 0.0001, 0.0002, 0.00025, 0.0005, 0.00075, 0.001, 0.002, 0.003, 0.01, 0.05, and 0.3 mg / ml, respectively. Two wells containing 0.1% (volume fraction) DMSO and two wells containing E3 medium alone served as controls.

[0216] 4) Observe and record the survival status of each group of zebrafish under a stereomicroscope every day from 1 to 5 days after administration, and remove dead embryos.

[0217] The research results are as follows Figure 5 (B) shown.

[0218] Example 12: Evaluation of Skin Infection Treatment

[0219] 1. Experimental Animals

[0220] Sixteen female BALB / c mice (6-8 weeks old, weighing 18-22 g) were obtained from Shanghai Shengchang Biotechnology Co., Ltd. The animals were acclimated for 3-5 days before the experiment began, and the animals were fed a normal basal diet. At the beginning of modeling, the weight difference between each group of mice was controlled to not exceed ±20% of the mean body weight. All animal experiments in this experiment were approved by the Experimental Animal Ethics and Use Committee of Shanghai Jiao Tong University (Animal Experiment Lot No.: 202101309).

[0221] 2. Establishment of Skin Infection Model

[0222] a) Overnight culture of USA300 SF8300 strain was transferred to TSB medium at a dilution of 1:100 and incubated at 37°C, 220 rpm, for 2 h.

[0223] b) Centrifuge at 5000 rpm for 10 min at 4°C, wash once with PBS, and resuspend to the target OD bacterial solution (OD = 1, ~ 10 9 CFU / mL).

[0224] c) Mice were randomly divided into four groups, with four mice in each group, and anesthetized by intraperitoneal injection of 4% chloral hydrate (0.1 mL / 10 g);

[0225] d) Shave the mouse's hair with a razor, create a 6 mm wound, drip 10 μL of the diluted bacterial solution onto the wound, allow the solution to dry, and then apply a protective film.

[0226] 3. Skin infection treatment and observation

[0227] a) Preparation of CCR1 / RD1 compound: Weigh an appropriate amount of compound and add melted 5%-10% Solutol HS-15 (50°C water bath). Add 1.5-2 mL of anhydrous ethanol and sonicate until completely dissolved. Then, remove the alcohol by rotary evaporation (approximately 5-6 minutes). Add a calculated volume of normal saline to dissolve to the target concentration. The concentration of CCR1 / RD1 / vancomycin is 4 mg / mL (prepare immediately before use).

[0228] b) 2 hours after modeling, 50 μL of the above preparation was injected into the wound under the protective film and protected from light. The specific groups were as follows:

[0229] Grouping preparation Treatment Model Group 10% Solutol + saline Apply to wound (50μL) Positive control group Vancomycin preparation (4 mg / mL) Apply to wound (50μL) CCR1 experimental group CCR1 preparation (4 mg / mL) Apply to wound (50μL) RD1 experimental group RD1 formulation (4 mg / mL) Apply to wound (50μL)

[0230] c) Observe skin condition and take photos every two days and record body weight;

[0231] d) On day 8, the mice were sacrificed, the wound size was recorded, and the skin wound was cut off for subsequent experiments;

[0232] 4. Skin Wound Colony Count

[0233] On day 8, skin was minced and weighed. The appropriate volume of saline (1 mL / 100 mg) and an appropriate amount of abrasive strain were added according to weight, and the skin tissue was broken using a tissue grinder. The tissue resuspension was diluted 1, 10, 100, 1,000, and 10,000-fold with saline, and 5 μL of each was spotted onto an antibiotic-free LB plate. After the plate was air-dried, it was placed in a 37°C incubator and incubated. After 24 hours, colony counts (CFU / mg) were performed.

[0234] 5. Pathological section detection of mouse skin wounds

[0235] On day 8, skin wounds were removed from mice and stored in 4% paraformaldehyde solution. The fixed tissues were dehydrated using graded ethanol and then embedded in paraffin wax. The paraffin-encapsulated tissues were sliced ​​into 4 μm sections using a tissue slicer, dewaxed, stained with hematoxylin and eosin (H&E) and Masson stain, and observed under a microscope.

[0236] The experimental results are as follows Figure 6 As shown, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Claims

1. A compound, or an optical isomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof or a solvate thereof, characterized in that: Having the structure shown in Formula I, In Formula I, R1 and R2 are each independently selected from: H and substituted or unsubstituted C1-C 20 Alkyl; or R1, R2, and the nitrogen atom to which they are attached together form an optionally substituted 4-12 membered nitrogen-containing heterocyclic ring; R3 and R4 are each independently selected from: H and substituted or unsubstituted C1-C 20 Alkyl; or R3, R4, and the nitrogen atom to which they are attached together form an optionally substituted 4-12 membered nitrogen-containing heterocyclic ring; R5 is H, halogen, -CN, -NO2, substituted or unsubstituted C1-C 20 Alkyl, or substituted or unsubstituted C6-C 14 aryl; X is O, S, -Si(R6)2- or -C(R6)2-; R6 is H, halogen or substituted or unsubstituted C1-C 20 alkyl; The term "substituted" refers to being substituted by 1, 2, 3 or 4 substituents selected from the group consisting of: C1-C 20 Alkyl, C6-C 14 Aryl, halogen, optionally substituted by 1, 2, 3 or 4 groups selected from C6-C 14 Aryl and C1-C 20 Alkyl substituents substituted C2-C 20 Alkenyl, C3-C 20 Cycloalkyl, and optionally C1-C 20 Alkyl-substituted mercapto groups.

2. The compound according to claim 1, or its optical isomer, pharmaceutically acceptable salt, hydrate or solvate, wherein: R1 and R2 are each independently selected from: H and substituted or unsubstituted C1-C 10 alkyl; or R1, R2, and the nitrogen atom to which they are attached together form an optionally substituted 5-10 membered nitrogen-containing heterocyclic ring; preferably, R1 and R2 are each independently selected from: H and substituted or unsubstituted C1-C6 alkyl; or R1, R2, and the nitrogen atom to which they are attached together form an optionally substituted 6-8 membered nitrogen-containing heterocyclic ring; more preferably, R1 and R2 are each independently selected from: H and substituted or unsubstituted C1-C6 alkyl; R3 and R4 are each independently selected from: H and substituted or unsubstituted C1-C 10 alkyl; or R3, R4, and the nitrogen atom to which they are attached together form an optionally substituted 5-10 membered nitrogen-containing heterocyclic ring; preferably, R3 and R4 are each independently selected from: H and substituted or unsubstituted C1-C6 alkyl; or R3, R4, and the nitrogen atom to which they are attached together form an optionally substituted 6-8 membered nitrogen-containing heterocyclic ring; more preferably, R3 and R4 are each independently selected from: H and substituted or unsubstituted C1-C6 alkyl; In R1, R2, R3 and R4, the "substituted" refers to being substituted by 1, 2, 3 or 4 substituents selected from the group consisting of C1-C6 alkyl, C6-C 10 Aryl, halogen and C3-C8 cycloalkyl.

3. The compound according to claim 1, or its optical isomer, pharmaceutically acceptable salt, hydrate or solvate, wherein: R5 is H, halogen, -CN, -NO2, optionally substituted by 1, 2, 3 or 4 groups selected from C3-C 20 Cycloalkyl, C6-C 14 Aryl and halogen substituted C1-C 20 Alkyl, or optionally substituted C6-C 14 Aryl, the C6-C 14 Aryl is optionally substituted with 1, 2, 3 or 4 substituents selected from the group consisting of: C1-C 20 Alkyl, C6-C 14 Aryl, halogen, optionally substituted by 1, 2, 3 or 4 groups selected from C6-C 14 Aryl and C1-C 20 Alkyl substituents substituted C2-C 20 alkenyl, and optionally C1-C 20 Alkyl-substituted mercapto groups; Preferably, R5 is H, halogen, optionally substituted by 1, 2, 3 or 4 groups selected from C5-C 15 Cycloalkyl, C6-C 10 Aryl and halogen substituted C1-C 10 Alkyl, or optionally substituted C6-C 14 Aryl, the C6-C 14 Aryl is optionally substituted with 1, 2, 3 or 4 substituents selected from the group consisting of: C1-C 10 Alkyl, C6-C 10 Aryl, halogen, optionally substituted by 1, 2, 3 or 4 groups selected from C6-C 10 Aryl and C1-C 10 Alkyl substituents substituted C2-C 10 alkenyl, and optionally C1-C 10 Alkyl-substituted mercapto groups; More preferably, R5 is H, optionally substituted by 1, 2, 3 or 4 selected from C7-C 12 Cycloalkyl, C6-C 10 C1-C6 alkyl substituted with aryl and halogen, or optionally substituted C6-C 10 Aryl, the C6-C 10 Aryl is optionally substituted with 1, 2, 3 or 4 substituents selected from the group consisting of C1-C6 alkyl, halogen, optionally substituted with one or two C6-C 10 an aryl-substituted C2-C6 alkenyl group, and a mercapto group optionally substituted by a C1-C6 alkyl group.

4. The compound according to claim 1, or its optical isomer, pharmaceutically acceptable salt, hydrate or solvate, wherein: X is O, S, -Si(R6)2- or -C(R6)2-, R6 is H, halogen or C1-C 10 Alkyl; preferably, X is O, -Si(R6)2- or -C(R6)2-, R6 is H, halogen or C1-C6 alkyl; more preferably, X is O.

5. The compound according to claim 1, or its optical isomer, pharmaceutically acceptable salt, hydrate or solvate, wherein: In Formula I, R1 and R2 are each independently selected from: H and optionally substituted by 1, 2, 3 or 4 selected from C6-C 10 C1-C6 alkyl substituted with aryl, halogen and C3-C8 cycloalkyl substituents; or R1, R2, and the nitrogen atom to which they are attached together form a 5-10 membered nitrogen-containing heterocyclic ring, the nitrogen-containing heterocyclic ring is optionally substituted with 1, 2, 3 or 4 substituents selected from C1-C6 alkyl, C6-C 10 aryl, halogen and C3-C8 cycloalkyl substituents; R3 and R4 are each independently selected from: H and optionally substituted by 1, 2, 3 or 4 selected from C6-C 10 C1-C6 alkyl substituted with aryl, halogen and C3-C8 cycloalkyl; or R3, R4, and the nitrogen atom to which they are attached together form a 5-10 membered nitrogen-containing heterocyclic ring, the nitrogen-containing heterocyclic ring is optionally substituted with 1, 2, 3 or 4 C1-C6 alkyl, C6-C 10 aryl, halogen and C3-C8 cycloalkyl substituents; R5 is H, halogen, optionally substituted by 1, 2, 3 or 4 C3-C 20 Cycloalkyl-substituted C1-C 20 Alkyl, or optionally substituted C6-C 14 Aryl, the C6-C 14 Aryl is optionally substituted with 1, 2, 3 or 4 substituents selected from the group consisting of: C1-C 20 Alkyl, C6-C 14 Aryl, halogen, optionally substituted by 1, 2, 3 or 4 C6-C 14 Aryl-substituted C2-C 20 alkenyl, and optionally C1-C 20 Alkyl-substituted mercapto groups; X is O, -Si(R6)2- or -C(R6)2-; R6 is H, halogen or C1-C 10 alkyl; Preferably, in Formula I, R1 and R2 are each independently selected from: H and optionally substituted by 1, 2, 3 or 4 selected from C6-C 10 C1-C6 alkyl substituted with aryl, halogen and C3-C8 cycloalkyl substituents; R3 and R4 are each independently selected from: H and optionally substituted by 1, 2, 3 or 4 selected from C6-C 10 C1-C6 alkyl substituted with aryl, halogen and C3-C8 cycloalkyl substituents; R5 is optionally replaced by 1 or 2 C8-C 15 Cycloalkyl-substituted C1-C 10 Alkyl, or optionally substituted by 1, 2, 3 or 4 groups selected from halogen, C6-C 14 Aryl-substituted C2-C 10 Alkenyl, and C1-C 10 Alkyl substituted mercapto substituents substituted C6-C 10 aryl; X is O; Preferably, in Formula I, R1, R2, R3 and R4 are each independently selected from C1-C4 alkyl, R5 is optionally substituted by 1 or 2 C8-C 15 C1-C4 alkyl substituted by cycloalkyl, or optionally substituted by 1, 2, 3 or 4 groups selected from halogen, C6-C 10 C2-C4 alkenyl substituted with aryl, and C6-C4 alkyl substituted with mercapto 10 Aryl, X is O.

6. The compound according to claim 1, or its optical isomer, pharmaceutically acceptable salt, hydrate or solvate, wherein: The compound is selected from the group consisting of:

7. A pharmaceutical composition, characterized in that The invention comprises (1) a safe and effective amount of one or more compounds according to any one of claims 1 to 6, or optical isomers thereof, pharmaceutically acceptable salts thereof, hydrates thereof or solvates thereof; and (2) a pharmaceutically acceptable carrier or excipient.

8. Use of the compound according to any one of claims 1 to 6, or its optical isomer, pharmaceutically acceptable salt, hydrate or solvate in the preparation of a medicament for treating and / or preventing bacterial infection.

9. The use according to claim 8, characterized in that The bacteria are Gram-positive bacteria or Gram-negative bacteria; preferably, the bacteria are Gram-positive bacteria; preferably, the bacteria are Staphylococcus aureus; preferably, the bacteria are drug-resistant bacteria; preferably, the bacteria are drug-resistant Staphylococcus aureus (MRSA).

10. A method for converting a compound of formula I according to any one of claims 1 to 6 into a compound of formula II, characterized in that: The method comprises the steps of: The compound of formula I is contacted with active oxygen, wherein the active oxygen is selected from one or more of ·OH, NO, ONOOH and HClO.

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