2,7-disubstituted cephalosporin derivatives as beta-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 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 the time period correlates to susceptibility of the pathogenic bacteria to the drug.

31. The in vitro method of claim 30 , further comprising monitoring for acquisition of resistance to the anti-Mycobacterial drug by the pathogenic Mycobacteria by the steps of obtaining a biological sample after a treatment period with the anti-Mycobacterial drug; repeating steps (b) to (e); wherein an increase in fluorescence levels over the time period correlates to resistance to the anti-Mycobacterial drug.

32. 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.

33. The assay system of claim 32 , said assay device comprising a platform having means for receiving an incubation mixture comprising a biological sample of the pathogenic bacteria, a drug effective against the pathogenic bacteria, and the fluorescent, luminescent or color-producing substrate; and means for capturing and concentrating a colored product produced by the β-lactamase activity upon the substrate in fluid connection to the receiving means.