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

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.