IN VITRO METHOD FOR DETECTION OF INFECTIONS CAUSED BY PSEUDOMONAS AERUGINOSA

Abstract

In vitro method for detection of infections caused by Pseudomonas aeruginosa. The present invention relates to compounds of general Formula (I) and to their use as haptens. Moreover, the present invention also refers to conjugates comprising the haptens of the invention and to their use for obtaining antibodies. Finally, the invention also relates to an in vitro method for the detection of infections caused by Pseudomonas aeruginosa by means of the identification and/or quantification of the main signaling molecules from the pqs quorum sensing system.

##STR00001##

Claims

1. A compound characterized by the Formula I, ##STR00009## wherein: R.sub.1 is selected among H or OH; R.sub.2 is selected among C3-C15 alkyl, C3-C15 alkenyl or C3-C15 alkynyl; R.sub.3 is (CH.sub.2).sub.m—R.sub.5; m is a whole number between 1 and 6; R.sub.4 is selected among H or OH; and R.sub.5 is selected among COOH, SH, NH.sub.2, OH or PEG.

2. A compound, according to claim 1, selected from the group consisting of: ##STR00010##

3. Use of a compound, according to any of the claim 1 or 2, or any combination thereof, as a hapten.

4. Conjugate comprising at least a hapten according to any of the claim 1 or 2, in combination with a second component which confers antigenicity to the conjugate, characterized in that the R.sub.3 of Formula I forms a covalent bond with the second component.

5. Conjugate, according to claim 4, wherein the second component is a carrier protein, or a fragment thereof, selected from the group comprising: horseshoe crab hemocyanin (HCH), bovine serum albumine (BSA) or keyhole limpet hemocyanin (KLH).

6. Method for producing a conjugate, according to any of the claim 4 or 5, which comprises creating a covalent bond, directly or through a cross-linking agent, between the carrier protein and at least one hapten according to any of the claim 1 or 2, wherein the covalent bond is formed between the carrier protein and the R.sub.3 of the hapten.

7. Use of the conjugate according to any of the claim 4 or 5 for producing antibodies.

8. Antibody characterized in that it specifically recognizes a conjugate as defined in claim 4 or 5, or antiserum comprising said antibody.

9. In vitro method for detecting and/or quantifying at least a quinolone selected from the group: 2-heptyl-4-quinolone (HHQ), 2-heptyl-3-hydroxy-4-quinolone (PQS), and/or 2-heptyl-4-quinolone N-oxide (HQNO), in a biological sample, which comprises the use of an antibody or antiserum as defined in claim 8.

10. In vitro method, according to claim 9, wherein the detection and/or quantification is carried out by an immunochemical technique, preferably the immunochemical technique is an ELISA.

11. In vitro method, according to any of the claim 9 or 10, which comprises: a) immobilizing a conjugate defined in any of claim 4 or 5 on a solid support, b) eliminating the non-immobilized conjugate, c) adding the sample to be analysed and a first antibody defined in claim 9 in the solid support of section a) and incubating, d) eliminating the first antibody not bound to the conjugate, e) adding a second antibody conjugated with a detectable labelling agent, said second antibody recognizing the first antibody and incubating, f) eliminating the second antibody not bound to the first antibody, and g) detecting and/or quantifying the complex obtained according to section e) with a composition containing a chromogenic, fluorogenic and/or chemiluminescent indicator substrate.

12. In vitro method, according to any of claims 9 to 11, wherein the sample is obtained from a subject who may have an infection caused by Pseudomonas aeruginosa.

13. In vitro method, according to any of claims 9 to 12, wherein the sample is selected from the group comprising: sputum, bronchoaspirate, bronchoalveolar lavage, blood, serum and/or plasma.

14. Kit for the detection and/or quantification a quinolone selected from the group: 2-heptyl-4-quinolone (HHQ), 2-heptyl-3-hydroxy-4-quinolone (PQS), and/or 2-heptyl-4-quinolone N-oxide (HQNO), characterized in that it comprises at least one antibody defined in claim 8, or a conjugate defined in any of claim 4 or 5, or any combination thereof.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0051] FIG. 1. Concentration of quinolones measured in culture broth samples at 8 hours of growth from bacterial isolates classified as acute (1-5) or chronic (7-11) according to clinically diagnosed patients.

[0052] The following examples serve to illustrate the invention and should not be considered, in any case, as limiting of the scope thereof.

EXAMPLES

Example 1. Chemistry

General Procedures and Equipment

[0053] The chemicals used in the synthesis of the haptens were obtained from Aldrich Chemical Co. (Milwaukee, Wis., USA), Sigma Chemical Co. (St. Louis, Mo., USA) or Acros Organics B.V.B.A. (Morris Plains, N.J., USA). Thin-layer chromatography (TLC) was performed on 0.25 mm, pre-coated silica gel 60 F254 aluminium sheets (Merck, Darmstadt, Germany). .sup.1H and .sup.13C NMR spectra were obtained with a Varian Mercury-400 spectrometer (400 MHz .sup.1H and 101 MHz for .sup.13C). Liquid chromatography/electrospray ionization/mass spectrometry (LC/ESI/MS) was performed in a Waters (Milford, Mass., USA) model composed by an Acquity UPLC system directly interfaced to a Micromass LCT Premier XE MS system equipped with an ESI LockSpray source for monitoring positive and negative ions. Data were processed with MassLynx (V 4.1) software (Waters).

Example 1.1. Preparation of Intermediates

Intermediates II

4-(2-((tert-butyldimethylsilyl)oxy)ethyl)aniline (1)

[0054] Imidazol (4.7 g, 0.07 mol) was added to a mixture of 2-(4-aminophenyl)ethanol (8.0 g, 0.05 mol) and TBSCl (10.5 g, 0.07 mol) in DMF (115 mL). The reaction was stirred at room temperature for 4 h. Afterwards, water and ethyl acetate were added to the reaction and the mixture was extracted with ethyl acetate 3 times. The combined organic layers were washed with water, brine, dried over MgSO.sub.4 and evaporated under reduced pressure. Crude product was purified by silica flash chromatography using as eluent AcOEt/Hexane 9:1. Pure aniline 1 was obtained 11.3 g, yield 90% as orange oil.

[0055] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 6.99 (d, J=8.5 Hz, 2H), 6.62 (d, J=8.5 Hz, 2H), 3.74 (t, J=7.3 Hz, 2H), 2.72 (t, J=7.3 Hz, 2H), 0.88 (s, 9H), 0.00 (s, 6H). .sup.13C NMR (101 MHz, CDCl.sub.3) δ 144.64, 130.05, 129.25, 115.28, 65.12, 38.95, 26.11, 18.52, −5.20. HRMS: m/z (ES+) for C.sub.14H.sub.26NOSi [(M+H).sup.+] calculated 252.1784 found 252.1788 (+1.6 ppm).

Methyl 3-(4-aminophenyl)propanoate (2)

[0056] Thionyl Chloride (0.75 ml, 0.01 mol) was added to a stirred ice cooled MeOH solution (3 ml). The mixture was left stirring 10 min and 3-(4-aminophenyl) propanoic acid was slowly added (0.5 g, 0,003 mol). The mixture was stirred 16 h under inert atmosphere at room temperature. After completion, the mixture was evaporated under reduced pressure and NaHCO.sub.3 std. solution was added. The aqueous phase was extracted 3 times using ethyl acetate. Organic layers were combined, washed with brine, dried over Na.sub.2SO.sub.4 and evaporated under reduced pressure. The crude product was washed with hexane and evaporated to obtain aniline 2 (0.525 g, 0.0029 mol, yield 97%) as a single product.

[0057] .sup.1H NMR (400 MHz, CD.sub.3OD) δ 6.94 (d, J=8.3 Hz, 2H), 6.66 (d, J=8.3 Hz, 2H), 3.62 (s, 3H), 2.78 (t, J=7.6 Hz, 2H), 2.55 (t, J=7.6 Hz, 2H). .sup.13C NMR (101 MHz, CD.sub.3OD) δ 175.37, 146.69, 131.64, 129.88, 116.93, 51.96, 37.12, 31.26. HRMS: m/z (ES+) for C.sub.10H.sub.13NO.sub.2 [(M+H).sup.+] calculated 180.1025 found 180.1028 (+1.7 ppm).

Intermediate III

5-(1-hydroxyoctylidene)-2,2-dimethyl-1,3-dioxane-4,6-dione (3)

[0058] Meldrum's acid (10 g, 0.069 mol) was dissolved in anhydrous DCM (127.6 mL) and cooled to 0° C. under N.sub.2 atmosphere. After cooling, pyridine (11.18 mL, 0.14 mol) was added followed by dropwise addition of octanoyl chloride (12.414 g, 0.076 mol). The reaction mixture was stirred 1 h at 0° C. then it was left stirring at RT. Reaction progress was monitored by TLC until completion. Reaction mixture was washed with 5% HCl solution. The organic layer was then washed with distilled water before being dried over anhydrous MgSO.sub.4, filtered and concentrated under reduced pressure to yield compound 3 as an orange oil (18.59 g, quantitative yield) which was used in the next step without further purification.

[0059] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 3.06 (t, J=7.8 Hz, 2H), 1.73 (s, 6H), 1.69 (m, 2H), 1.45-1.20 (m, 8H), 0.87 (t, J=6.8 Hz, 3H). .sup.13C NMR (101 MHz, CDCl.sub.3) δ 198.47, 170.72, 160.33, 104.89, 91.37, 35.89, 31.75, 29.46, 29.03, 26.94, 26.29, 22.72, 14.19. HRMS: m/z (ES−) for C.sub.14H.sub.21O.sub.5 [(M−H).sup.−] calculated 269.1389 found 269.1388 (−0.4 ppm).

Intermediate IV

Methyl 3-oxodecanoate (4)

[0060] 5-(1-hydroxyoctylidene)-2,2-dimethyl-1,3-dioxane-4,6-dione 3 (18.5 g, 0.068 mol) was dissolved in MeOH (84 mL) and heated at reflux for 5 h. The reaction was allowed to cool, and the solvent was removed under reduced pressure yielding the crude product as an orange oil. Purification was performed using silica flash chromatography Hex/Et.sub.2O 8:2. Compound β-ketoester 4 was obtained as a yellow oil (8.85 g, 0.044 mol, yield 76%).

[0061] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 3.72 (s, 3H, O—CH.sub.3), 3.43 (s, 2H, OC—CH.sub.2—CO), 2.51 (t, J=7.4 Hz, 2H, CH.sub.2), 1.57 (quin., 2H, CH.sub.2), 1.34-1.17 (m, 8H, 4×CH.sub.2), 0.86 (t, J=6.7 Hz, 3H, CH.sub.3). .sup.13C NMR (101 MHz, CDCl.sub.3) δ 202.94, 167.80, 52.41, 49.12, 43.19, 31.75, 29.12, 29.07, 23.58, 22.70, 14.16. HRMS: m/z (ES−) for C.sub.11H.sub.20O.sub.3 [(M−H).sup.−] calculated 199.1334 found 119.1331 (−2.5 ppm)

Intermediates V

Methyl 3-((4-(3-(tert-butyldimethylsilyl)propyl)phenyl)amino)dec-2-enoate (5)

[0062] To a solution of compound 4 (7.0 g, 35 mmol) in dry hexane (100 mL) was added aniline 1 (9.7 g, 38 mmol) and p-toluene sulfonic acid (0.12 g, 0.7 mmol). The reaction mixture was heated at reflux under a N.sub.2 atmosphere for 16 h. It was allowed to cool down and the reaction mixture was evaporated under reduced pressure, obtaining the crude product 5 (14.5 g, 34 mmol, yield 97%) as an pale orange oil. The product was used in the next step without further purification.

[0063] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 10.22 (s, 1H), 7.16 (d, J=8.3 Hz, 2H), 7.00 (d, J=8.3 Hz, 2H), 4.70 (s, 1H), 3.80 (t, J=6.8 Hz, 2H), 3.68 (s, 3H), 2.79 (t, J=6.8 Hz, 2H), 2.25 (t, J=7.7 Hz, 2H), 1.48-1.35 (m, 2H), 1.38-1.09 (m, 8H), 0.85-0.91 (m, 12H), −0.05 (s, 6H). .sup.13C NMR (101 MHz, CDCl.sub.3) δ 171.19, 164.30, 137.34, 136.82, 129.99, 125.31, 84.09, 64.46, 50.37, 39.11, 32.33, 31.74, 29.21, 28.98, 28.16, 26.05, 22.72, 18.47, 14.19, −5.28. HRMS: m/z (ES+) for C.sub.25H.sub.44NO.sub.3Si [(M+H).sup.+] calculated 434.3090 found 434.3087 (−0.7 ppm).

Methyl 3-((4-(3-methoxy-3-oxopropyl)phenyl)amino)dec-2-enoate (6)

[0064] To a solution of compound 4 (1.0 g, 5 mmol) in dry toluene (15 mL) was added aniline 2 (0.98 g, 5.5 mmol) and p-toluene sulfonic acid (0.017 g, 0.1 mmol). The reaction mixture was heated at 85° C. under a N.sub.2 atmosphere for 16 h. It was allowed to cool down and the reaction mixture was evaporated under reduced pressure, yielding the crude product as a pale orange oil. The crude product 6 (1.947 g) was used in the next step without further purification (purity by NMR about 70%).

[0065] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 10.22 (s, 1H), δ 7.15 (d, J=8.3 Hz, 2H), 7.00 (d, J=8.3 Hz, 2H), 4.70 (s, 1H), 3.68 (s, 3H), 3.67 (s, 3H), 2.94 (t, J=7.8 Hz, 2H), 2.63 (t, J=7.8 Hz, 2H), 2.25 (t, J=8.0 Hz, 2H), 1.40 (quin., 2H), 1.33-1.13 (m, 8H), 0.84 (t, J=6.9 Hz, 3H). .sup.13C NMR (101 MHz, CDCl.sub.3) δ 173.38, 171.16, 164.09, 129.07, 128.94, 125.43, 120.49, 84.38, 51.79, 50.39, 35.74, 32.32, 31.70, 30.48, 29.17, 29.14, 28.94, 22.69, 14.17. HRMS: m/z (ES+) for C.sub.21H.sub.31NO.sub.4 [(M+H).sup.+] calcd 362.2331 found 362.2346 (+4.1 ppm).

Intermediates VI

6-(2-((tert-butyldimethylsilyl)oxy)ethyl)-2-heptylquinolin-4(1H)-one (7)

[0066] Compound κ (13.2 g, 30.4 mmol) was added dropwise to refluxing diphenyl ether (250 ml) at 270° C. and maintained for 2 h. Once the reaction cooled to RT, a silica preparative column was used in order to eliminate the diphenyl ether, using hexane as eluent. The crude product was re-absorbed in the same silica using DCM and evaporating under reduced pressure. Afterwards, the product was purified by flash column chromatography using a concentration gradient of eluent from Hexane/AcOEt 4:6 to 3:7. Quinolone 7 was obtained as off-white solid (6.23 g, 15.5 mmol, yield 51%).

[0067] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 11.99 (s, 1H), 8.18 (d, J=1.9 Hz, 1H), 7.65 (d, J=8.5 Hz, 1H), 7.47 (dd, J=8.5, 1.9 Hz, 1H), 6.20 (s, 1H), 3.82 (t, J=7.0 Hz, 2H), 2.91 (t, J=7.0 Hz, 2H), 2.67 (t, J=7.8 Hz, 2H), 1.76-1.64 (m, 2H), 1.38-1.09 (m, 8H), 0.90-0.74 (m, 12H), −0.04 (s, 6H, H17 and H18). .sup.13C NMR (101 MHz, CDCl.sub.3) δ 178.89, 154.88, 139.34, 134.92, 133.57, 125.05, 124.93, 118.40, 108.04, 64.56, 39.43, 34.50, 31.80, 29.33, 29.22, 29.13, 26.05, 22.71, 18.43, 14.16, −5.24. HRMS: m/z (ES−) for C.sub.24H.sub.38NO.sub.2Si [(M−H).sup.−] calculated 400.2672 found 400.2678 (+1.5 ppm).

Methyl 3-(2-heptyl-4-oxo-1,4-dihydroquinolin-6-yl)propanoate (8)

[0068] Following an analogous procedure to that described for compound 7, but using ester 6, compound 8 was obtained as a pale-brown solid after purification by flash chromatography column using DCM with 2% MeOH (0.927 g, 2.8 mmol, yield 75%).

[0069] 1H NMR (400 MHz, CDCl.sub.3) δ 11.64 (s, 1H), δ 8.18 (d, J=2.0 Hz, 1H), 7.62 (d, J=8.5 Hz, 1H), 7.45 (dd, J=8.5, 2.0 Hz, 1H), 6.20 (s, 1H), 3.64 (s, 3H), 3.04 (t, J=7.8 Hz, 2H), 2.71-2.62 (m, 4H), 1.76-1.61 (m, 2H), 1.34-1.13 (m, 8H), 0.81 (t, J=6.8 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 178.78, 173.28, 154.74, 139.23, 136.16, 132.70, 125.13, 124.26, 118.65, 108.27, 51.79, 35.73, 34.50, 31.79, 30.73, 29.28, 29.13, 29.11, 22.70, 14.15. HRMS: m/z (ES−) for C20H26NO3 [(M−H)−] calculated 328.1913 found 328.1904 (−2.8 ppm).

Example 1.2. Preparation of Haptens I

Haptens 1a. Preparation of 2-heptyl-6-(2-mercaptoethyl)quinolin-4(1H)-one (I-1)

i) 6-(2-bromoethyl)-2-heptylquinolin-4(1H)-one (9)

[0070] To a solution of quinolone 7 (0.15 g, 0.37 mmol) in 5 mL of anhydrous DCM, a solution of boron tribromide in DCM 1M was slowly added under inert atmosphere. The mixture was left under refluxe at 45-50° C. overnight. The reaction mixture was evaporated under reduced pressure. H.sub.2O was added and extracted 3 times with AcOEt. Combined organic layers were washed with NaHCO3std, brine, dried over MgSO4 and evaporated under vacuum. The crude product was purified by silica column chromatography using DCM with 2% MeOH as eluent. It was obtained pure compound 9 (0.13 g, 0.36 mmol, yield 96%).

[0071] .sup.1H NMR (400 MHz, CD.sub.3OD) δ 8.07 (d, J=2.0 Hz, 1H), 7.59 (dd, J=8.6, 2.0 Hz, 1H), 7.54 (d, J=8.5 Hz, 1H), 6.21 (s, 1H), 3.67 (t, J=7.2 Hz, 2H), 3.27 (t, J=7.2 Hz, 2H), 2.69 (t, J=7.8 Hz, 2H), 1.74 (p, J=7.4 Hz, 2H), 1.45-1.25 (m, 8H), 0.89 (t, J=6.7 Hz, 3H). .sup.13C NMR (101 MHz, CD.sub.3OD) δ 180.40, 156.96, 140.49, 136.57, 134.32, 125.52, 125.45, 119.31, 108.83, 39.78, 34.97, 33.68, 32.85, 30.18, 30.17, 30.08, 23.65, 14.38. HRMS: m/z (ES−) for C.sub.18H.sub.23BrNO [(M−H).sup.−] calculated 348.0963 found 348.0963 (+0.0 ppm).

ii) S-(2-(2-heptyl-4-oxo-1,4-dihydroquinolin-6-yl)ethyl) ethanethioate (10)

[0072] A solution of bromoderivative 9 (70 mg, 0.20 mmol) and potassium thioacetate (22.8 mg, 0.20 mmol) in 2.0 mL of anhydrous DMF was stirred during 1 h. The reaction was diluted with AcOEt and washed 3 times with H2O, brine, dried over Na.sub.2SO.sub.4 and evaporated under reduced pressure to obtain pure compound 10 (69 mg, 0.20 mmol, quantitative yield), used in the next step without further purification.

[0073] .sup.1H NMR (400 MHz, CD.sub.3OD) δ 8.05 (d, J=2.0 Hz, 1H), 7.59 (dd, J=8.6, 2.0 Hz, 1H), 7.53 (d, J=8.5 Hz, 1H), 6.21 (s, 1H), 3.16 (t, J=7.1 Hz, 2H), 2.98 (t, J=7.1 Hz, 2H), 2.70 (t, J=7.6 Hz, 2H), 2.30 (s, 3H), 1.75 (p, J=7.5 Hz, 2H), 1.45-1.26 (m, 8H), 0.89 (t, J=6.9 Hz, 3H). .sup.13C NMR (101 MHz, CD.sub.3OD) δ 197.12, 180.40, 156.87, 140.34, 137.50, 134.32, 125.41, 125.24, 119.31, 108.79, 36.49, 34.97, 32.85, 31.25, 30.51, 30.18, 30.08, 23.65, 14.38. HRMS: m/z (ES−) for C.sub.20H.sub.26NO.sub.2S [(M−H).sup.−] calculated 344.1684 found 344.1682 (−0.6 ppm).

iii) 2-Heptyl-6-(2-mercaptoethyl)quinolin-4(1H)-one (I-1)

[0074] Protected thiol 10 (69 mg, 0.2 mmol) and KOH (11.2 mg, 0.2 mmol) were dissolved under inert atmosphere in 1 ml of anhydrous and degassed MeOH. The mixture was stirred during 1 h at RT. The reaction was acidified until pH 6-7 with degassed HCl 1M, diluted with water and extracted 3 times with AcOEt, washed with brine, dried over Na.sub.2SO.sub.4 and evaporated under reduced pressure to obtain the crude product as a pale yellow solid. Eventually, the crude product was purified by crystallization using hexane/AcOEt to obtain compound I-1 (49 mg, 0.16 mmol, yield 81%).

[0075] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 12.17 (s, 1H), 8.17 (d, J=2.0 Hz, 1H), 7.74 (d, J=8.5 Hz, 1H), 7.44 (dd, J=8.5, 2.0 Hz, 1H), 6.26 (s, 1H), 3.00 (t, J=7.3 Hz, 2H), 2.79 (q, J=7.5 Hz, 2H), 2.71 (t, J=7.8 Hz, 2H), 1.72 (p, J=7.6 Hz, 2H), 1.37-1.12 (m, 8H), 0.80 (t, J=6.8 Hz, 3H).

[0076] .sup.13C NMR (101 MHz, CDCl.sub.3) δ 178.54, 155.37, 139.44, 135.62, 133.00, 124.87, 124.70, 118.89, 108.08, 39.94, 34.49, 31.79, 29.30, 29.23, 29.12, 26.09, 22.71, 14.18. HRMS: m/z (ES−) for C.sub.18H.sub.24NOS [(M−H).sup.−] calculated 302.1579 found 302.1575 (−1.0 ppm).

Haptens 1b. Preparation of 3-(2-heptyl-3-hydroxy-4-oxo-1,4-dihydroquinolin-6-yl)propanoic acid (I-2)

i) 3-(3-Formyl-2-heptyl-4-oxo-1,4-dihydroquinolin-6-yl)propanoic acid (11)

[0077] Ester 8 (700 mg, 2.1 mmol), hexamine (601.7 mg, 4.3 mmol) and p-TsOH.Math.H2O (453.6 mg, 2.4 mmol, 1.1 equiv) were dissolved in glacial acetic acid (54 ml). The mixture was heated at reflux for 3 h under a nitrogen atmosphere. After cooling, 6 M HCl (13 ml) was added and heating was continued at 115° C. for 1 h. The mixture was allowed to cool, diluted with water, and extracted with ethyl acetate. The combined organic fractions were washed with brine, dried over MgSO4, and concentrated under reduced pressure. Ethanol was used to recover some precipitated product during filtration, evaporated under vacuo and mixed with crude product for purification. The crude was purified by column chromatography on silica flash chromatography using DCM with 6% MeOH and 0.5% glacial acetic acid to obtain acid 11 as an off-white solid (480 mg, 1.4 mmol, yield 66%).

[0078] 1H NMR (400 MHz, DMSO-d6) δ 12.13 (s, 1H), 10.38 (s, 1H), 7.97 (d, J=2.0 Hz, 1H), 7.62 (dd, J=8.4, 2.0 Hz, 1H), 7.51 (d, J=8.4 Hz, 1H), 3.03 (t, J=7.6 Hz, 2H), 2.94 (t, J=7.4 Hz, 2H), 2.58 (t, J=7.4 Hz, 2H), 1.60 (quin., 2H), 1.41-1.21 (m, 8H), 0.86 (t, J=6.7 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) δ 190.81, 177.98, 173.58, 159.61, 137.98, 137.59, 133.64, 126.12, 123.84, 118.74, 113.23, 48.59, 35.12, 31.54, 31.12, 29.98, 28.98, 28.85, 28.34, 22.04, 13.92. HRMS: m/z (ES−) for C20H24NO4 [(M−H)−] calculated 344.1705 found 344.1708 (+0.9 ppm).

ii) 3-(2-Heptyl-3-hydroxy-4-oxo-1,4-dihydroquinolin-6-yl)propanoic acid (I-2)

[0079] Aqueous hydrogen peroxide (1.05 M, 1.0 ml, 1.0 mmol) and aqueous sodium hydroxide (1.08 M, 1.78 ml, 1.9 mmol) were added to a solution of acid 11 (0.300 g, 0.9 mmol) in ethanol (4.3 ml) under nitrogen atmosphere. The mixture was stirred overnight at room temperature. After completion, the reaction mixture was evaporated under reduced pressure. The crude was purified by flash column chromatography using DCM with 4% MeOH and 0.5% glacial acetic acid. Quinolone 1-2 was obtained (135 mg, 0.4 mmol, yield 47%).

[0080] 1H NMR (400 MHz, DMSO-d6) δ 11.38 (s, 1H), δ 7.90 (d, J=1.6 Hz, 1H), 7.45 (d, J=8.6 Hz, 1H), 7.43 (dd, J=8.6, 1.6 Hz, 1H), 2.91 (t, J=7.5 Hz, 2H), 2.71 (t, J=7.5 Hz, 2H), 2.57 (t, J=7.5 Hz, 2H), 1.64 (quin., 2H), 1.37-1.18 (m, 8H), 0.84 (t, J=6.6 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) δ 173.69, 168.59, 137.76, 135.95, 135.25, 134.14, 130.86, 122.96, 122.07, 117.86, 35.33, 31.19, 30.04, 28.77, 28.47, 28.12, 27.82, 22.06, 13.93. HRMS: m/z (ES−) for C19H25NO4 [(M−H)−] calculated 330.1705 found 330.1699 (−1.8 ppm).

Haptens 1c. Preparation of 6-(2-carboxyethyl)-2-heptyl-4-hydroxyquinoline N-oxide (I-3)

i) Methyl 3-(4-((tert-butoxycarbonyl)oxy)-2-heptylquinolin-6-yl)propanoate (12)

[0081] Ester 8 (129 mg, 0.39 mmol) was dissolved in anhydrous THF (7 ml). Boc.sub.2O (94 mg, 0.43 mmol) and a catalytic quantity of 4-(dimethylamino)pyridine (DMAP) (12 mg, 0.1 mmol) were added. Then, the mixture was heated at 60° C. under nitrogen atmosphere during 1 h 30. The mixture was left to cool to RT and concentrated under reduced pressure. The crude was purified by silica flash chromatography using AcOEt/Hex 8:2 as eluent, obtaining Boc protected compound 12 (148 mg, 0.34 mmol, yield 88%).

[0082] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.97 (d, J=8.6 Hz, 1H), 7.76 (d, J=2.0 Hz, 1H), 7.55 (dd, J=8.6, 2.0 Hz, 1H), 7.25 (s, 1H), 3.68 (s, 3H), 3.13 (t, J=7.8 Hz, 2H), 2.94 (t, J=7.8 Hz, 2H), 2.72 (t, J=7.8 Hz, 2H), 1.85-1.73 (m, 2H), 1.62 (s, 9H), 1.46-1.22 (m, 8H), 0.87 (t, J=6.8 Hz, 3H). .sup.13C NMR (101 MHz, CDCl.sub.3) δ 173.25, 163.68, 154.20, 150.66, 148.62, 138.51, 131.15, 129.14, 120.75, 119.74, 112.28, 84.72, 51.84, 39.64, 35.71, 31.90, 31.21, 30.07, 29.64, 29.30, 27.83, 22.77, 14.22. HRMS: m/z (ES+) for C.sub.25H.sub.36NO.sub.5 [(M+H).sup.+] calculated 430.2593 found 430.2579 (−3.3 ppm).

ii) 4-((tert-butoxycarbonyl)oxy)-2-heptyl-6-(3-methoxy-3-oxopropyl)quinoline N-oxide (13)

[0083] Compound 12 (138 mg, 0.32 mmol) was dissolved in anhydrous DCM (4 ml) and cooled at 4° C. Afterwards, mCPBA (83 mg, 0.48 mmol) was added and the mixture was stirred at 4° C. under nitrogen atmosphere during 4 h. Once the starting material was consumed, more DCM was added, and the solution was washed 3 times with NaHCO.sub.3std. The organic phase was concentrated under reduced pressure and it was obtained, without further purification, pure N-oxide 13 (132 mg, 0.30 mmol, yield 92%) as a yellow oil.

[0084] 1H NMR (400 MHz, CDCl.sub.3) δ 8.70 (d, J=9.0 Hz, 1H), 7.77 (d, J=1.9 Hz, 1H), 7.63 (dd, J=9.0, 1.9 Hz, 1H), 7.31 (s, 1H), 3.66 (s, 3H), 3.13 (m, 4H), 2.72 (t, J=7.8 Hz, 2H), 1.88-1.75 (m, 2H), 1.61 (s, 9H), 1.49-1.22 (m, 8H), 0.87 (t, J=6.8 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 172.93, 150.56, 149.57, 144.13, 141.33, 140.92, 132.08, 122.97, 120.69, 120.59, 113.61, 85.22, 51.90, 35.36, 31.87, 31.81, 30.95, 29.69, 29.19, 27.80, 26.24, 22.76, 14.20. HRMS: m/z (ES+) for C25H36NO6 [(M+H)+] calculated 446.2542 found 446.2539 (−0.7 ppm).

iii) 6-(2-Carboxyethyl)-2-heptyl-4-hydroxyquinoline N-oxide (I-3)

[0085] N-oxide 13 (125 mg, 0.28 mmol) was dissolved in a degassed solution of KOH 5 M in EtOH (2.5 ml). The mixture was stirred at RT under nitrogen atmosphere during 1 h. Afterwards, H.sub.2O was added, and the mixture was left stirring 30 min. The mixture was acidified with HCl cc until pH=1-2 when a white solid precipitated. It was filtered and dried to obtain the crude product. Quinolone N-oxide 1-3 (72 mg, 0.22 mmol, yield 77%) was obtained after crystallization in EtOH/H2O 4:1.

[0086] 1H NMR (400 MHz, CD3OD) δ 8.11 (d, J=2.0 Hz, 1H), 8.04 (d, J=8.8 Hz, 1H), 7.73 (dd, J=8.8, 2.0 Hz, 1H), 6.35 (s, 1H), 3.09 (t, J=7.6 Hz, 2H), 2.92 (t, J=7.6 Hz, 2H), 2.70 (t, J=7.6 Hz, 2H), 1.83-1.73 (m, 2H), 1.53-1.24 (m, 8H), 0.91 (t, J=6.9 Hz, 3H). 13C NMR (101 MHz, CD3OD) δ 176.27, 155.85, 140.65, 139.67, 134.59, 125.20, 124.52, 117.17, 107.29, 36.34, 32.88, 32.48, 31.56, 30.43, 30.11, 28.82, 23.69, 14.39. HRMS: m/z (ES−) for C19H2404 [(M−H)−] calculated 330.1705 found 330.1710 (+1.5 ppm).

Example 2. Immunochemistry

General Procedures and Equipment

[0087] The reagents used were obtained from Aldrich Chemical Co. (Milwaukee, Wis., USA) and from Sigma Chemical Co. (St. Louis, Mo., USA). Purification of conjugates was carried out in ÄKTA Prime Plus using 2 HiTrap desalting columns both from GE Healthcare (Chicago, Ill., USA) or either by dialysis using Spectra/Por membranes from Spectrumlabs (Piraeus, Greece, EU) with molecular weight cut-off of 12-14 kDa. The matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS) was a Bruker autoflex III Smartbeam spectrometer (Billerica, Mass.). The pH and the conductivity of all buffers and solutions were measured with a pH-meter pH 540 GLP and a conductimeter LF 340, respectively (WTW, Weilheim, Germany). Polystyrene microtiter plates were purchased from Nunc (Maxisorp, Roskilde, Denmark). Dilution plates were purchased from Nirco (Barbera del Valles, Spain). Washing steps were performed on a Biotek ELx465 (Biotek Inc.). Absorbances were read on a SpectramaxPlus (Molecular Devices, Sunnyvale, Calif., USA) at a single wavelength mode (450 nm). The competitive curves were analysed with a four-parameter logistic equation using the software GraphPad Prism 7.0 (GraphPad Software Inc., San Diego, Calif., USA).

Buffers

[0088] Unless otherwise indicated, phosphate buffer saline (PBS) corresponds to 10 mM phosphate buffer in 0.8% saline solution (pH 7.5). Coating buffer is a 50 mM bicarbonate-carbonate buffer (pH 9.6). PBST is PBS with 0.05% Tween 20 (pH 7.5). Citrate buffer corresponds to 40 mM sodium citrate (pH 5.5).

[0089] The substrate solution contains 0.01% of 3,3′,5,5′-tetramethylbenzidine (TMB) and 0,004% H.sub.2O.sub.2 prepared in citrate buffer. Borate buffer is 0.2 M sodium borate/boric acid (pH 8.7). All buffers were prepared using ultra-pure Milli-Q® water with a resistivity between 16-18 MΩ cm.

Analysis of Hapten Density

[0090] Hapten densities of protein conjugates were estimated by means of MALDI-TOF-MS comparing molecular weight of natural proteins with that of conjugates. MALDI experiments were conducted by deposition of 2 μL of the matrix solution (10 mg mL.sup.−1 of sinapinic acid in MeCN/H.sub.2O 70:30, 0.1% HCOOH) in the MALDI plate and after drying, 2 μL of the purified sample diluted ½ with MeCN 0.2% HCOOH are added and allowed to dry. Finally, 2 μL of the matrix solution are added over the mixture mentioned above and after drying the resulting spot analyzed by MALDI-TOF. Hapten densities were calculated through the equation: [MW(conjugate)−MW(native protein)]/[MW(hapten)−MW(lost atoms)].

Example 2.1. Immunogens and Coating Antigens Preparation

Conjugation of I-1 Hapten

[0091] The conjugation procedure was carried out in parallel over 25 mg of HCH (Horseshoe Crab Hemocyanin) or 25 mg of BSA (Bovine Serum Albumine). Each protein was dissolved in 4.5 ml of Borax/Borate buffer pH=8.7. Afterwards 12.5 mg (44 μmol) of succinimidyl iodoacetate (SIA) were dissolved in 1 mL of anhydrous DMF. Over each protein solution, dropwise additions (5×100 μL) of SIA were performed. The reactions were stirred 3.5 h at RT and left overnight at 4° C. without agitation to obtain iodoacetate-BSA and iodoacetate-HCH solutions, which were purified by AKTA using 2 HiTrap desalting column and eluting with Borax/Borate buffer.

[0092] 8.0 mg (26 μmol) of I-1 hapten were dissolved in 1.7 mL of anhydrous DMF. Afterwards, 850 μL of I-1 solution were added dropwise to each activated protein solution and the mixtures were stirred 4 h at RT and left overnight at 4° C. without agitation. Finally, each immunoreactive agent obtained was purified by dialysis in PBS 0.5 mM (5×5 L) and Milli-Q water (1×5 L) and lyophilized.

Conjugates Obtained:

[0093] 2-((2-(2-heptyl-4-oxo-1,4-dihydroquinolin-6-yl)ethyl)thio)acetamide-HCH (1-4) [0094] 2-((2-(2-heptyl-4-oxo-1,4-dihydroquinolin-6-yl)ethyl)thio)acetamide-BSA (1-5)

Conjugation of I-2 and I-3 Haptens

[0095] The conjugation procedure was carried out in parallel over a solution of 5 mg of KLH (Keyhole Limpet Hemocyanin) or a solution of 5 mg of BSA (Bovine Serum Albumine) in PBS 10 mM.

[0096] A solution of 2.62 μL (11 μmop of tri-n-butylamine and 1.56 μL (12 μmop isopropyl cloroformiate was added to a solution of the corresponding hapten, 1-2 or 1-3 to activate the carboxylic acid, (10 μmop dissolved in 400 μL of anhydrous DMF. The mixture was left stirring 15 min at 4° C. and 30 min at RT. Then 200 μL of the reaction mixture were added over each protein solution and the mixture was left 2 h stirring at RT and left overnight at 4° C. without agitation. Each immunoreactive agent obtained was purified by dialysis in PBS 0.5 mM (5×5 L) and Milli-Q water (1×5 L) and lyophilized.

Conjugates Obtained:

[0097] 3-(2-heptyl-3-hydroxy-4-oxo-1,4-dihydroquinolin-6-yl)propanamide-KLH (I-6) [0098] 3-(2-heptyl-3-hydroxy-4-oxo-1,4-dihydroquinolin-6-yl)propanamide-BSA (I-7) [0099] 3-(2-heptyl-1-hydroxy-4-oxo-1,4-dihydroquinolin-6-yl)propanamide-KLH (I-8) [0100] 3-(2-heptyl-1-hydroxy-4-oxo-1,4-dihydroquinolin-6-yl)propanamide-BSA (I-9)

Conjugate Density

[0101]

TABLE-US-00002 TABLE 2 Quantity of bioconjugates produced and hapten density of BSA conjugates calculated from MALDI-TOF analysis. HCH or KLH conjugates cannot be analyzed by MALDI-TOF, therefore the degree of conjugation was evaluated by comparison with a paralel conjugation protocol with BSA conjugates. Quantity (mg) Yield (%) N° of residues SIA-BSA — — 22 I-4 18.4   74   — I-5 18.73  75   13 I-6  5.29 105   — I-7  5.95 119   17 I-8  6.68 133   — I-9  3.38  67.6 21

Example 2.2. Antibody Production

[0102] Antibodies were obtained by immunizing female New Zealand white rabbits with the corresponding immunogen, namely 1-4, 1-6 or 1-8.

[0103] The protocol used for the production of antibodies was conducted in accordance with the institutional guidelines under a license from the local government (DAAM 7463) and approved by the Institutional Animal Care and Use Committee at the CID-CSIC.

[0104] The antisera (As) obtained by immunizing the animals were named as:

TABLE-US-00003 Immunogen Antisera Coating antigen I-4 As382, As383 and As384 I-5 I-6 As385, As386 and As387 I-7 I-8 As388, As389 and As390 I-9

[0105] The antibody titer was assessed during the immunization process through non-competitive indirect ELISA. Microtiter plates were coated with a fixed concentration of the homologous competitor conjugate (1 mg mL.sup.−1) and the avidity of the produced antibodies was measured by preparing serial dilutions of the corresponding As. The animals were exsanguinated after 6 immunizations, and the final blood was collected in vacutainer tubes provided with a serum separation gel. Antisera were obtained by centrifugation at 4° C. for 10 min at 10 000 rpm, then stored at −80° C. in the presence of preservative 0.02% sodium azide.

Example 2.3. Non-Competitive Indirect 2D ELISA

[0106] Non-competitive indirect ELISA were carried out to establish the concentrations of a homologous coating antigen (CA) and As dilutions used in competitive assays. For this purpose, concentrations of BSA conjugates (I-5, I-7, I-9) ranging from 5 μg/ml to 5 ng/ml and As dilutions from 1/1000 to 1/1024000 were assessed. The experimental procedure is fully detailed in the next section. However, in this type of assay no analyte is present, therefore the total volume of As dilution added per well is 100 μL.

[0107] Competitive assay conditions were selected at 70% signal saturation giving approximately 1-1.2 units of absorbance.

Example 2.4. Competitive Indirect ELISA

[0108] The competitive assay was carried out in a 96 well Maxisorp flat-bottom plates, coated using 100 μL of a BSA conjugate solution in coating buffer pH=9.6. Then, plates were covered with adhesive plate sealer and incubated overnight at 4° C. The day after, plates were washed with PBST (4×300 μL) using the platewasher Biotek ELx405 HT. Sequentially, 50 μL of the corresponding sample solution, containing the analyte HHQ, PQS or HQNO (2 μM to 0.13 nM) or MH medium were added, followed by 50 μL addition of a fixed As dilution and left without agitation 30 min at RT. After another washing step, a 1/6000 dilution of goat AntiRabbit IgG-HRP in PBST was added and incubated 30 min at RT. After a final washing, 100 μL of a substrate solution was added and left 30 min at RT in the dark. Once the time was consumed, the enzymatic reaction was stopped by adding 50 μl of H.sub.2SO.sub.4 4M solution and the absorbances read at 450 nm. Absorbance data were plotted and analyzed using GraphPad software. The standard calibration curve was fitted to a four-parameter equation according to the following formula: y=B+(A−B)/[1−(x/C).sup.D], where A is the maximum absorbance, B is the minimum absorbance, C is the concentration producing 50% of the maximal absorbance, and D is the slope at the inflection point of the sigmoid curve. Unless otherwise indicated, the data presented correspond to the average of at least two well replicates.

TABLE-US-00004 TABLE 3 Parameters of competitive ELISA assays for detection of A) HHQ, B) PQS and C) HQNO. The data shown correspond to the average of 3 different days suing at least 2 well/replicates per concentration A) PBST MH diluted 1/5 As382 dil. 1/32000 1/64000 I-7 (μg/mL) 0.313 0.156 A.sub.min 0.029 ± 0.005 0.012 ± 0.003 A.sub.max 1.187 ± 0.082 1.165 ± 0.081 Slope −0.874 ± 0.114   −0.704 ± 0.015   IC.sub.50 4.593 ± 0.287 2.851 ± 0.296 Dynamic     0.894 ± 0.214 to     0.448 ± 0.081 to Range 22.797 ± 3.691  21.605 ± 4.736  LOD 0.341 ± 0.132 0.167 ± 0.051 R.sup.2 0.995 ± 0.003 0.998 ± 0.002 PBS + 0.01% B) Tween + 0.1 mM EDTA MH diluted 1/10 As385 dil. 1/48000 1/64000 I-5 (μg/mL) 0.039 0.039 A.sub.min 0.030 ± 0.006 0.019 ± 0.006 A.sub.max 1.445 ± 0.035 1.107 ± 0.025 Slope −0.723 ± 0.006   −0.734 ± 0.110   IC.sub.50 3.872 ± 0.529 6.051 ± 0.155 Dynamic     0.528 ± 0.043 to     0.973 ± 0.250 to Range 24.365 ± 3.182  36.157 ± 6.197  LOD 0.169 ± 0.012 0.362 ± 0.137 R.sup.2 0.997 ± 0.002 0.995 ± 0.003 C) PBS at pH = 6.5 MH diluted 1/5 As389 dil. 1    1/16000 I-5 (μg/mL) 0.250 0.250 A.sub.min 0.091 ± 0.036 0.091 ± 0.020 A.sub.max 1.083 ± 0.059 0.854 ± 0.009 Slope −0.754 ± 0.037   −0.720 ± 0.064   IC.sub.50 4.204 ± 0.858 2.708 ± 0.035 Dynamic     0.723 ± 0.181 to     0.413 ± 0.102 to Range 26.707 ± 0.958  17.044 ± 1.076  LOD 0.266 ± 0.087 0.147 ± 0.053 R.sup.2 0.987 ± 0.013 0.990 ± 0.004

Example 2.5. ELISA Evaluation

Physicochemical Parameters Optimization

[0109] Performance of the assays was evaluated through the modification of different physicochemical parameters in the competition step. The assessed parameters were: competence time, incubation time, pH, ionic strength, presence of a surfactant (% Tween 20), solubility with addition of organic solvents or cation complexation by EDTA.

TABLE-US-00005 TABLE 4 Physicochemical parameters selected after optimization. The parameters improving the features of the assay were assessed separately and in conjunction. Assay in buffer HHQ PQS HQNO As dilution 1/32000 1/32000 1/16000 (As382) (As385) (As389) [Competitor] (μg/mL) 0.313 0.039 0.250 pH 7.5 7.5 6.5 Conductivity (mS/cm) 15 15 15 Tween 20 (%) 0.05 0.01 0.05 Competition time (min) 30 30 30 Preincubation time (min) 0 0 0 Organic solvent (%) 0 0 0

Specificity or Cross Reactivity Studies

[0110] Regarding specificity studies, it was followed the experimental procedure for indirect competitive ELISA described above. It was assessed the avidity of As versus other quinolone effector molecules of the pqs system from P. aeruginosa. Each assay was run using HHQ, PQS and HQNO as analyte. Cross reactivity was calculated through the equation: CR (%)=IC.sub.50(Cross reactant)/IC.sub.50(Analyte)×100.

TABLE-US-00006 TABLE 5 Percentages of Cross Reactivity (CR) calculated for HHQ, PQS and HQNO using the corresponding As under the conditions of the three developed assays. As382 (HHQ) As385 (PQS) As389 (HQNO) Analyte IC50 (nM) C.R. (%) IC50 (nM) C.R. (%) IC50 (nM) C.R. (%) HHQ 2.67 100.00 27.19 13.31 26.35 36.24 PQS 37.27 7.16 3.62 100.00 176.60 5.41 HQNO 85.97 3.11 236.20 1.53 9.55 100.00

Example 2.6. Clinical Isolates

Matrix Effect Study

[0111] Culture broth Mueller-Hinton was diluted 1:2, 1:5, 1:10, 1:20 and used to run the standard calibration curve. Subsequently, the dilution providing the best ELISA parameters was selected and the conditions of CA and As dilution adjusted.

Accuracy Studies

[0112] Blind spiked samples using diluted MH culture broth were prepared, measured and interpolated in the standard curve mentioned above. The experiment was repeated three different days and the final accuracy results are expressed as mean of all replicates.

Reproducibility Study

[0113] The assay was run three different days and three times within the same day.

Growth Curves

[0114] Clinical Pseudomonas aeruginosa isolates coming from patients diagnosed with acute or chronic respiratory airways infection were grown in MH culture broth 8 h at 37° C. In addition, a reference P. aeruginosa strain (PAO) was also grown under the same conditions. Then, aliquots were taken, centrifuged and analysed using the developed immunochemical assays. The results enclosed in FIG. 1 suggest a clear difference in the QS production profile between both types of isolates. Therefore, it would be possible to differentiate and stratify patients depending on the pqs quorum sensing system molecular footprint.

[0115] It has been demonstrated the potential of the immunochemical tools presented here for differentiation of both types of infections. The three studied quinolones are promising biomarkers for diagnostic of infections caused by P. aeruginosa. Moreover, the study of QS could provide much more information about type of infection or disease state.