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

Patent metadata

Field	Value
Publication number	US-9834681-B2
Application number	US-201514613214-A
Country	US
Kind code	B2
Filing date	Feb 3, 2015
Priority date	Feb 4, 2014
Publication date	Dec 5, 2017
Grant date	Dec 5, 2017

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 selected from the group consisting of:
(a) C6-C10 aryl, and
(b) C3-C7 heteroaryl;

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;
R a , R b , R c , and R d are independently selected from the group consisting of hydrogen, halogen, nitro, C1-C3 alkyl, and C1-C3 alkyl substituted with one or more halogens; 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. A compound of claim 1 having the formula:

or an ester or a salt thereof.

5. A compound of claim 1 having the formula:

or an ester or a salt thereof.

6. A compound of claim 1 having the formula:

or an ester or a salt thereof.

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

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

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

10. 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.

11. The compound of claim 1 , wherein Z is a fluorescent moiety.

12. The compound of claim 1 , wherein Z is a fluorescent phenolic dye moiety.

13. The compound of claim 1 , wherein Z is selected from the group consisting of a courmarin moiety, a xanthene moiety, a resorufin moiety, a cyanine moiety, a difluoroboradiazaindacene moiety, a bimane moiety, an acridine moiety, an isoindole moiety, a dansyl moiety, an aminophthalic hydrazide moiety, an aminophthalimide moiety, an aminonaphthalimide moiety, a quinine moiety, a dicyanovinyl moiety, a tricyanovinyl moiety, an indolaniline moiety, an indamine moiety, and derivatives thereof.

14. The compound of claim 1 , wherein Z is a xanthene moiety selected from the group consisting of a fluorescein moiety, a rhodol moiety, a rhodamine moiety, and derivatives thereof.

15. The compound of claim 1 , wherein Z is

16. The compound of claim 1 , wherein Z is

wherein R′ is hydrogen or aryl.

17. The compound of claim 16 , wherein R′ is phenyl or substituted phenyl.

18. The compound of claim 1 , wherein Z is

19. The compound of claim 1 , wherein Z is a luciferin moiety.

20. 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.

21. 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.

22. The method of claim 21 , 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.

23. The method of claim 21 , wherein the sample comprises a pathogenic bacterial species selected from Bacteroides, Clostridium, Streptococcus, Staphylococcus, Pseudomonas, Haemophilus, Legionella, Mycobacterium, Escherichia, Salmonella, Shigella , or Listeria.

24. The method of claim 21 , wherein measuring an optical signal comprises measuring fluorescence emission intensity.

25. The method of claim 21 , wherein measuring an optical signal comprises measuring absorbance intensity.

26. The method of claim 21 , wherein measuring an optical signal comprises measuring luminescence emission intensity.

27. The method of claim 21 , wherein measuring an optical signal comprises observing a color change.

28. 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.

29. The assay method of claim 28 , further comprising monitoring for acquisition of resistance to the drug by the pathogenic bacteria by the steps of obtaining a biological sample after a treatment period with the drug; and repeating steps (b) to (e); wherein an increase in signal intensity levels over the time period correlates to resistance to the drug.

30. 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 t

Assignees

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

Inventors

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

Classifications

• C07D501/52

  with the 7-amino radical acylated by an araliphatic carboxylic acid · 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

• C07D501/62

  Compounds further condensed with a carbocyclic ring or ring system · 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