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US-12624382-B2 - Beta-lactam compounds and methods of use thereof

US12624382B2US 12624382 B2US12624382 B2US 12624382B2US-12624382-B2

Abstract

Beta-lactam compounds to detect carbapenemases or microbial carbapenem resistance are disclosed. The compounds contain a chemical probe. Upon hydrolysis by carbapenemases, the compounds undergo intramolecular rearrangement and release the chemical probe. Detection of the released chemical probe indicates the presence of carbapenemases and the presence of microbial carbapenem resistance.

Inventors

  • Dan Yang
  • Chi-Wang Ma

Assignees

  • THE UNIVERSITY OF HONG KONG

Dates

Publication Date
20260512
Application Date
20190919

Claims (20)

  1. 1 . A compound to detect carbapenemases or microbial carbapenemase resistance, wherein the compound have the structure of CP-A-D or salts thereof, wherein CP is an azabicyclo moiety composed of a beta-lactam ring and an unsaturated five-membered hetero-ring having a C—C double bond; A is a conjugated system attached to the unsaturated five-membered hetero-ring at a carbon atom of the double bond and is at meta-position relative to the nitrogen atom; and D is connected to A via a methylene bridge and comprises a chemical probe, wherein the beta-lactam ring of the compound can be hydrolyzed by one or more carbapenemases, thereby triggering intramolecular rearrangement to release D from the compounds, wherein D further comprises a self-immolative linker interposed between the methylene bridge and the rest of D, wherein D comprises a luminescence probe, and the luminescence probe is selected from the group consisting of resorufin, fluorescein, Tokyo Green, coumarin, and luciferin, wherein the self-immolative linker is spontaneously separated from the rest of D following the release of D from the compound, wherein the self-immolative linker is 4-methanediyl-2-methoxyphenoxy, and wherein the phenoxy oxygen of the self-immolative linker is connected to the methylene bridge and the methanediyl group of the self-immolative linker is connected to the rest of D.
  2. 2 . A compound to detect carbapenemases or microbial carbapenemase resistance, wherein the compound has the structure of Formulas Ia, Ib, Ic, Id or Ie, or salts thereof, (a) wherein A is a divalent group selected from —(CR 4 ═CR 5 ) m —, —(C≡C) n —, optionally substituted arylenes, optionally substituted heteroarylenes, and covalent adducts thereof, wherein the covalent adducts are conjugated systems and wherein m and n are positive integers; (b) wherein D is connected to A via a methylene bridge and comprises a chemical probe, wherein D comprises a luminescence probe, and the luminescence probe is selected from the group consisting of resorufin, fluorescein, Tokyo Green, coumarin, and luciferin; (c) wherein the beta-lactam ring of the compound can be hydrolyzed by one or more carbapenemases, thereby triggering intramolecular rearrangement to release D from the compound; (d) wherein R 1 , R 2 , R 4 , and R 5 are independently: a hydrogen atom, a halogen atom, a sulfonic acid, an azide group, a cyanate group, an isocyanate group, a nitrate group, a nitrile group, an isonitrile group, a nitrosooxy group, a nitroso group, a nitro group, an aldehyde group, an acyl halide group, a carboxylic acid group, a carboxylate group, an optionally substituted alkyl group, an optionally substituted heteroalkyl group, an optionally substituted alkenyl group, an optionally substituted heteroalkenyl group, an optionally substituted alkynyl group, an optionally substituted heteroalkynyl group, an optionally substituted aryl group, an optionally substituted heteroaryl group; a hydroxyl group optionally containing one substituent at the hydroxyl oxygen, wherein the substituent is an optionally substituted alkyl group, an optionally substituted heteroalkyl group, an optionally substituted alkenyl group, an optionally substituted heteroalkenyl group, an optionally substituted alkynyl group, an optionally substituted heteroalkynyl group, an optionally substituted aryl group, or an optionally substituted heteroaryl group; a thiol group optionally containing one substituent at the thiol sulfur, wherein the substituent is an optionally substituted alkyl group, an optionally substituted heteroalkyl group, an optionally substituted alkenyl group, an optionally substituted heteroalkenyl group, an optionally substituted alkynyl group, an optionally substituted heteroalkynyl group, an optionally substituted aryl group, or an optionally substituted heteroaryl group; a sulfonyl group containing an optionally substituted alkyl group, an optionally substituted heteroalkyl group, an optionally substituted alkenyl group, an optionally substituted heteroalkenyl group, an optionally substituted alkynyl group, an optionally substituted heteroalkynyl group, an optionally substituted aryl group, or an optionally substituted heteroaryl group; an amino group optionally containing one or two substituents at the amino nitrogen, wherein the substituents are optionally substituted alkyl groups, optionally substituted heteroalkyl groups, optionally substituted alkenyl groups, optionally substituted heteroalkenyl groups, optionally substituted alkynyl groups, optionally substituted heteroalkynyl groups, optionally substituted aryl groups, optionally substituted heteroaryl groups, or combinations thereof; an amide group optionally containing one or two substituents at the amide nitrogen, wherein the substituents are optionally substituted alkyl groups, optionally substituted heteroalkyl groups, optionally substituted alkenyl groups, optionally substituted heteroalkenyl groups, optionally substituted alkynyl groups, optionally substituted heteroalkynyl groups, optionally substituted aryl groups, optionally substituted heteroaryl groups, or combinations thereof; an azo group containing an optionally substituted alkyl group, an optionally substituted heteroalkyl group, an optionally substituted alkenyl group, an optionally substituted heteroalkenyl group, an optionally substituted alkynyl group, an optionally substituted heteroalkynyl group, an optionally substituted aryl group, or an optionally substituted heteroaryl group; an acyl group containing an optionally substituted alkyl group, an optionally substituted heteroalkyl group, an optionally substituted alkenyl group, an optionally substituted heteroalkenyl group, an optionally substituted alkynyl group, an optionally substituted heteroalkynyl group, an optionally substituted aryl group, or an optionally substituted heteroaryl group; an ester group containing an optionally substituted alkyl group, an optionally substituted heteroalkyl group, an optionally substituted alkenyl group, an optionally substituted heteroalkenyl group, an optionally substituted alkynyl group, an optionally substituted heteroalkynyl group, an optionally substituted aryl group, or an optionally substituted heteroaryl group; a carbonate ester group containing an optionally substituted alkyl group, an optionally substituted heteroalkyl group, an optionally substituted alkenyl group, an optionally substituted heteroalkenyl group, an optionally substituted alkynyl group, an optionally substituted heteroalkynyl group, an optionally substituted aryl group, or an optionally substituted heteroaryl group; an ether group containing an optionally substituted alkyl group, an optionally substituted heteroalkyl group, an optionally substituted alkenyl group, an optionally substituted heteroalkenyl group, an optionally substituted alkynyl group, an optionally substituted heteroalkynyl group, an optionally substituted aryl group, or an optionally substituted heteroaryl group; an aminooxy group optionally containing one or two substituents at the amino nitrogen, wherein the substituents are optionally substituted alkyl groups, optionally substituted heteroalkyl groups, optionally substituted alkenyl groups, optionally substituted heteroalkenyl groups, optionally substituted alkynyl groups, optionally substituted heteroalkynyl groups, optionally substituted aryl groups, optionally substituted heteroaryl groups, or combinations thereof; or a hydroxyamino group optionally containing one or two substituents, wherein the substituents are optionally substituted alkyl groups, optionally substituted heteroalkyl groups, optionally substituted alkenyl groups, optionally substituted heteroalkenyl groups, optionally substituted alkynyl groups, optionally substituted heteroalkynyl groups, optionally substituted aryl groups, optionally substituted heteroaryl groups, or combinations thereof; (e) wherein R 3 is: a carboxylic acid or carboxylate; an ester group containing an optionally substituted alkyl group, an optionally substituted heteroalkyl group, an optionally substituted alkenyl group, an optionally substituted heteroalkenyl group, an optionally substituted alkynyl group, an optionally substituted heteroalkynyl group, an optionally substituted aryl group, or an optionally substituted heteroaryl group; an amide group optionally containing one or two substituents at the amide nitrogen, wherein the substituents are optionally substituted alkyl groups, optionally substituted heteroalkyl groups, optionally substituted alkenyl groups, optionally substituted heteroalkenyl groups, optionally substituted alkynyl groups, optionally substituted heteroalkynyl groups, optionally substituted aryl groups, optionally substituted heteroaryl groups, or combinations thereof; or a hydroxamate group optionally containing one or two substituents, wherein the substituents are optionally substituted alkyl groups, optionally substituted heteroalkyl groups, optionally substituted alkenyl groups, optionally substituted heteroalkenyl groups, optionally substituted alkynyl groups, optionally substituted heteroalkynyl groups, optionally substituted aryl groups, optionally substituted heteroaryl groups, or combinations thereof; (f) wherein R 1 , R 2 , R 3 , R 4 , and R 5 , and their substituents are optionally and independently substituted with one or more chemical groups, wherein each chemical group is independently: a halogen atom, an alkyl group, a heteroalkyl group, an alkenyl group, a heteroalkenyl group, an alkynyl group, a heteroalkynyl group, an aryl group, a heteroaryl group, —OH, —SH, —NH 2 , —N 3 , —OCN, —NCO, —ONO 2 , —CN, —NC, —ONO, —CONH 2 , —NO, —NO 2 , —ONH 2 , —SCN, —SNCS, —CF 3 , —CH 2 CF 3 , —CH 2 Cl, —CHCl 2 , —CH 2 NH 2 , —NHCOH, —CHO, —OOCl, —COF, —COBr, —COOH, —SO 3 H, —CH 2 SO 2 CH 3 , —PO 3 H 2 , —OPO 3 H 2 , —P(═O)(OR G1 )(OR G2 ), —OP(═O)(OR G1 )(OR G2 ), —BR G1 (OR G2 ), —B(OR G1 )(OR G2 ), or -GR G1 in which -G is —O—, —S—, —NR G2 —, —C(═O)—, —S(═O)—, —SO 2 —, —C(═O)O—, —C(═O)NR G2 —, —OC(═O)—, —NR G2 C(═O)—, —OC(═O)O—, —OC(═O)NR G2 —, —NR G2 C(═O)O—, —NR G2 C(═O)NR G3 —, —C(═S)—, —C(═S)S—, —SC(═S)—, —SC(═S)S—, —C(═NR G2 )—, —C(═NR G2 )O—, —C(═NR G2 )NR G3 —, —OC(═NR G2 )—, —NR G2 C(═NR G3 )—, —NR G2 SO 2 —, —C(═NR G2 )NR G3 —, —OC(═NR G2 )—, —NR G2 C(═NR G3 )—, —NR G2 SO 2 —, —NR G2 SO 2 NR G3 —, —NR G2 C(═S)—, —SC(═S)NR G2 —, —NR G2 C(═S)S—, —NR G2 C(═S)NR G3 —, —SC(═NR G2 )—, —C(═S)NR G2 —, —OC(═S)NR G2 —, —NR G2 C(═S)O—, —SC(═O)NR G2 —, —NR G2 C(═O)S—, —C(═O)S—, —SC(═O)—, —SC(═O)S—, —C(═S)O—, —OC(═S)—, —OC(═S)O—, —SO 2 NR G2 —, —BR G2 —, or —PR G2 —, wherein each occurrence of R G1 , R G2 , and R G3 is, independently, a hydrogen atom, a halogen atom, an alkyl group, a heteroalkyl group, an alkenyl group, a heteroalkenyl group, an alkynyl group, a heteroalkynyl group, an aryl group, or a heteroaryl group.
  3. 3 . The compound of claim 1 , wherein the luminescence probe remains non-luminescent or luminescence-quenched prior to carbapenemase-catalyzed hydrolysis of the compound and becomes luminescent or luminescence-enhanced after being released from the compound.
  4. 4 . The compound of claim 1 , wherein the beta-lactam ring of the compound can be hydrolyzed by one or more carbapenemases independently selected from the group consisting of Class A carbapenemases, Class B carbapenemases, Class C carbapenemases, and Class D carbapenemases.
  5. 5 . A method to detect carbapenemases or microbial carbapenem resistance, comprising: (a) contacting a sample containing one or more populations of bacteria with the compound of claim 1 ; (b) detecting the release of D from the compound, wherein detection of the release of D indicates the presence of carbapenemases, wherein the presence of carbapenemases indicates the presence of microbial carbapenem resistance.
  6. 6 . The method of claim 5 , wherein D comprises a luminescence probe that remains non-luminescent or luminescence-quenched prior to carbapenemase-catalyzed hydrolysis of the compound and becomes luminescent or luminescence-enhanced after being released from the compound.
  7. 7 . The method of claim 6 , wherein the release of D is detected by detecting the luminescence signal of the luminescence probe, wherein the luminescence signal of the luminescence probe reaches between about 80 and about 100% of its maximum value within about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, or about 30 minutes, following the contact of the sample with the compound.
  8. 8 . The method of claim 7 , wherein the luminescence signal of the luminescence probe, detected at about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, or about 30 minutes following the contact of the sample with the compound, is positively correlated with the total concentration of carbapenemases, the total population of bacteria with carbapenem resistance, or both.
  9. 9 . The method of claim 5 , wherein the one or more populations of bacteria comprise enterobacteriaceae.
  10. 10 . The method of claim 9 , wherein the enterobacteriaceae comprise Escherichia coli, Enterobacter aerogenes, Enterobacter cloacae, Klebsiella pneumoniae, Klebsiella oxytoca , or combinations thereof.
  11. 11 . The method of claim 5 , wherein the sample comprises a human or non-human animal bodily fluid, a human or non-human animal tissue, or both.
  12. 12 . The method of claim 5 , further comprising, before step (a), lysing the sample to release carbapenemases from the bacteria.
  13. 13 . The method of claim 5 , further comprising, before, during, or after step (a), contacting the sample with one or more additional compounds that can trigger colorimetric change, luminescence change, or both, of the chemical probe of D after D is released from the compound.
  14. 14 . The method of claim 13 , wherein the one or more additional compounds comprise an enzyme.
  15. 15 . The method of claim 14 , wherein the enzyme is a peroxidase, a luciferase, or a beta-galactosidase.
  16. 16 . A method to test the efficacy of a carbapenemase inhibitor, comprising: (a) contacting a solution or suspension comprising an isolated carbapenemase, a bacterial cell lysate, one or more populations of bacteria, or combinations thereof, with the compound of claim 1 in the absence of the carbapenemase inhibitor and, separately, in the presence of the carbapenemase inhibitor; (b) detecting the release of D from the compound, wherein the magnitude of the difference in the release of D detected in the absence of the carbapenemase inhibitor and in the presence of the carbapenemase inhibitor indicates the efficacy of the carbapenemase inhibitor.
  17. 17 . The method of claim 16 , wherein the compound and the carbapenemase inhibitor are simultaneously added to the solution or suspension.
  18. 18 . The method of claim 17 , wherein the compound and the carbapenemase inhibitor are mixed together before being added to the solution or suspension.
  19. 19 . The method of claim 16 , wherein the compound is added after the addition of the carbapenemase inhibitor to the solution or suspension.
  20. 20 . The method of claim 16 , wherein the carbapenemase inhibitor is added after the addition of the compound to the solution or suspension.

Description

FIELD OF THE INVENTION The invention is generally directed to beta-lactam compounds that are useful in detecting microbial antibiotic resistance, more particularly to beta-lactam compounds that are useful in detecting carbapenemases or microbial carbapenem resistance. BACKGROUND OF THE INVENTION Beta-lactam antibiotics, such as the penam series, have been widely used for treating different kinds of bacterial infections. They contain a beta-lactam ring in their molecular structures and work by inhibiting cell wall biosynthesis in bacterial organisms. However, misuse of beta-lactam antibiotics in human and livestock has led to serious antibiotic resistance. In particular, beta-lactamases are enzymes produced by bacteria that cause antibiotic resistance via hydrolyzing the antibiotics' beta-lactam ring, thereby deactivating their antibacterial properties. In recent decades, the carbapenem series of beta-lactam antibiotics were developed and known to be one of the last resort antibiotics. However, bacterial carbapenemases are capable of hydrolyzing them. To mitigate outbreaks of antibiotic-resistant bacteria, clinicians are advised to prescribe antibiotics only when necessary. They are also advised to start the treatment with narrow spectrum and first-line antibiotics unless patients are not responding to medication. Unfortunately, patients infected with antibiotic-resistant bacteria usually do not exhibit distinctive symptoms. Oftentimes, clinicians have to make rapid decisions on the prescription of antibiotics without knowing whether antibiotic resistance is present. Prescribing carbapenem antibiotics to patients infected with bacteria that have already developed resistance against such antibiotics is ineffective and may even lead to life threatening conditions such as sepsis (Hampton, JAMA, 2016, 315, p. 19; Shorr et al., Critical Care Medicine, 2011, 39, p. 46). The existing methods for identifying carbapenemases or microbial carbapenem resistance, such as agar plate diffusion, are time-consuming and may delay proper treatment. Although compounds with carbapenem-like structures have been designed and synthesized as chromogenic or fluorescence probes for carbapenemases or microbial carbapenem resistance, they frequently suffer from slow kinetics and narrow carbapenemase specificity (Mao et al., ChemBioChem, 2018, doi: 10.1002/cbic.201800126 (epub. ahead of print); Xie et al., CN Patent Application No. 106279178; Mao et al., Angewandte Chemie International Edition, 2017, 56, p. 4468; Xie et al., CN Patent Application No. 106811192; Pfaendler et al., U.S. Pat. No. 9,296,752). There is an urgent need to develop a rapid test for detecting antibiotic resistance, especially carbapenem resistance. There is also a tremendous demand to develop a method for quickly evaluating the efficacy of carbapenem inhibitors for drug development. SUMMARY OF THE INVENTION Therefore, it is an object of the present invention to provide compounds to detect carbapenemases or microbial carbapenem resistance. It is another object of the present invention to provide methods of making such compounds. It is another object of the present invention to provide methods for detecting carbapenemases or microbial carbapenem resistance. It is another object of the present invention to provide methods for testing the efficacy of carbapenemase inhibitors. It is yet another object of the present invention to develop kits for detecting carbapenemases or microbial carbapenem resistance. Throughout the description and claims of this specification, the word “comprise” and variations of the word, such as “comprising” and “comprises,” mean “including but not limited to,” and are not intended to exclude, for example, other additives, components, integers or steps. Any discussion of documents, acts, materials, articles or the like which has been included in the present specification is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each claim of this application. Disclosed are compounds and methods for detecting carbapenemases or microbial carbapenem resistance. Generally, the compound of the present invention has the structure of CP-A-D, wherein CP is an azabicyclo moiety composed of a beta-lactam ring and an unsaturated five-membered hetero-ring having a C—C double bond; A is a conjugated system attached to the unsaturated five-membered hetero-ring at a carbon atom of the double bond and is at meta-position relative to the nitrogen atom; and D is connected to A via a methylene bridge and comprises a chemical probe, wherein the beta-lactam ring of the compound can be hydrolyzed by one or more carbapenemases, thereby triggering intramolecular rearrangement to release D from the compounds. For example, in some forms, the disclosed compounds have the structure of Formulas Ia, Ib, Ic, Id or