2,7-disubstituted cephalosporin derivatives as β-lactamase substrates and methods for their use for the diagnosis of tuberculosis

Patent metadata

Field	Value
Publication number	US-10344165-B2
Application number	US-201715831266-A
Country	US
Kind code	B2
Filing date	Dec 4, 2017
Priority date	Feb 4, 2014
Publication date	Jul 9, 2019
Grant date	Jul 9, 2019

Abstract

β-Lactamase substrates and methods for using the substrates to detect β-lactamase and to diagnose tuberculosis.

First claim

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A compound having the formula:

or an ester or a salt thereof,
wherein
A is C6-C10 aryl;
R 1 is selected from the group consisting of:
(a) hydrogen,
(b) methoxy, and
(c) ethoxy;

R 2 is selected from the group consisting of hydrogen, C1-C3 alkyl, C1-C3 alkyl substituted with one or more halogens, and substituted piperazine;
R 3 and R 4 are independently selected from the group consisting of hydrogen, methyl, ethyl, propyl, halomethyl, haloethyl, halopropyl, or R 3 and R 4 taken together with the carbon atom to which they are attached form a cyclopropyl or a cyclobutyl ring, provided that R 3 and R 4 are not both hydrogen;
n is 0 or 1; and
Z is a moiety that provides a fluorescent, luminescent, or colorimetric signal when released from the compound.

2. A compound of claim 1 having the formula:

or an ester or a salt thereof.

3. A compound of claim 1 having the formula:

or an ester or a salt thereof.

4. The compound of claim 1 , wherein A is phenyl.

5. The compound of claim 1 , wherein R 1 is methoxy.

6. The compound of claim 1 , wherein R 2 is hydrogen.

7. The compound of claim 1 , wherein R 3 and R 4 taken together with the carbon atom to which they are attached form a cyclopropyl ring.

8. The compound of claim 1 , wherein Z is a moiety that provides a fluorescent, luminescent, or colorimetric signal when released from the compound.

9. The compound of claim 1 , wherein Z is

10. The compound of claim 1 , wherein Z is

wherein R′ is hydrogen or aryl.

11. The compound of claim 1 , wherein Z is

12. A method for detecting β-lactamase in a sample, comprising:
(a) contacting a sample with a compound of claim 1 ; and
(b) measuring an optical signal generated from contacting the sample with the compound.

13. A method for diagnosing tuberculosis, comprising:
(a) contacting a sample with a compound of claim 1 ; and
(b) measuring an optical signal generated from contacting the sample with the compound.

14. The method of claim 13 , wherein the sample is sputum, pleural fluid, spinal fluid, blood, urine, saliva, stool, tissue biopsies, tissue homogenates, directly in live animals or human patients, or a sample obtained by swabbing an area of interest on a subject.

15. The method of claim 13 , wherein the sample comprises a pathogenic bacterial species selected from Bacteroides, Clostridium, Streptococcus, Staphylococcus, Pseudomonas, Haemophilus, Legionella, Mycobacterium, Escherichia, Salmonella, Shigella , or Listeria.

16. The method of claim 13 , wherein measuring an optical signal comprises measuring fluorescence emission intensity.

17. An assay method for determining drug susceptibility of pathogenic bacteria in a subject infected by said pathogenic bacteria, comprising:
(a) obtaining a biological sample from the subject;
(b) contacting said biological sample with a drug effective against the pathogenic bacteria;
(c) contacting said biological sample with a substrate for a β-lactamase of the pathogenic bacteria, wherein the substrate is a compound of claim 1 ;
(d) delivering an excitation wavelength to the biological sample; and
(e) measuring levels of a signal intensity at an emission wavelength produced by the product of the β-lactamase action on the substrate in the biological sample over a period of time; wherein no increase or a decrease in signal intensity levels over the time period correlates to susceptibility of the pathogenic bacteria to the drug.

18. An in vitro method for determining drug susceptibility of a pathogenic Mycobacteria in a subject infected by the same, comprising the steps of:
(a) obtaining a biological sample from the subject;
(b) contacting said biological sample with an anti-mycobacterial drug;
(c) contacting said biological sample with a fluorogenic substrate for Mycobacterial β-lactamase, wherein the substrate is a compound of claim 1 ;
(d) delivering an excitation wavelength to the biological sample; and
(e) measuring levels of fluorescence at an emission wavelength produced by a fluorescent product of the β-lactamase action on the substrate in the biological sample over a period of time; wherein no increase or a decrease in fluorescence over the time period correlates to susceptibility of the pathogenic bacteria to the drug.

19. An assay system for monitoring drug susceptibility of pathogenic bacteria, comprising:
(a) one or more color-producing substrates for a β-lactamase of the pathogenic bacteria, wherein the substrate is a compound of claim 1 ;
(b) an assay device for visibly detecting a product of β-lactamase activity on the substrate; and
(c) a reader configured to quantify visible signals emitted by the detected product.

Assignees

• Texas A & M Univ Sys
• Univ Leland Stanford Junior

Inventors

• Rao Jianghong
• Xie Hexin
• Cheng Yunfeng
• Cirillo Jeffrey D

Classifications

• G01N2800/52

  Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis · CPC title

• C12Q1/18

  Testing for antimicrobial activity of a material · CPC title

• C12Q1/04

  Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor {(C12Q1/6897 takes precedence)} · CPC title

• C09B11/24 Primary

  Phthaleins containing amino groups {; Phthalanes; Fluoranes; Phthalides; Rhodamine dyes; Phthaleins having heterocyclic aryl rings; Lactone or lactame forms of triarylmethane dyes} · CPC title

• C07D501/62

  Compounds further condensed with a carbocyclic ring or ring system · CPC title