QUINOLINE-3-CARBOXAMIDE COMPOUNDS AND THEIR USE IN DIAGNOSIS

Abstract

The present application provides quinoline-3-carboxamide compounds covalently linked to a label for use in the diagnosis of an inflammatory disease at local site. The above mentioned compounds can be used to detect or image accumulation of S100A9 in the body of a subject at sites of inflammation, using in vivo non-invasive molecular imaging techniques for the detection of said compounds. Accordingly, labeled quinoline-3-carboxamide compounds can be applied to evaluate the risk of a subject of developing an inflammatory disease and to follow the progress of the disease.

Claims

1. A compound in which a substituted or unsubstituted quinoline-3-carboxamide is covalently linked to a label, wherein the compound is not: ##STR00056##

2. The compound according to claim 1 having formula (I) ##STR00057## wherein R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are each independently selected from the group consisting of H, optionally substituted linear or branched C1-C6 alkyl, optionally substituted linear or branched C2-C6-alkenyl, optionally substituted linear or branched C2-C6-alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, halogen, hydroxyl, amino, cyano, and optionally substituted linear or branched C1-C6 alkoxy; or each of R.sup.1 and R.sup.2, R.sup.2 and R.sup.3, and/or R.sup.3 and R.sup.4, together with the atoms to which they are attached, form an optionally substituted 4- to 8-membered carbocyclic or heterocyclic ring; R.sup.5 is selected from the group consisting of H, optionally substituted linear or branched C1-C6 alkyl, optionally substituted linear or branched C2-C6-alkenyl, optionally substituted linear or branched C2-C6-alkynyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted cycloalkyl; R.sup.6 is H or an optionally substituted group selected from the group consisting of aryl, heteroaryl, alkyl, cycloalkyl, alkenyl and alkynyl; R.sup.7 is an optional linker; X is O or S; and R.sup.8 is a label; or a salt, isomer, or tautomer thereof.

3. The compound according to claim 2, wherein R.sup.3 and R.sup.4 are each H.

4. The compound according to claim 2 or 3, wherein R.sup.1 and R.sup.2 are each independently selected from the group consisting of H, Cl, —CH.sub.3, —CH.sub.2CH.sub.3, —CH.sub.2CH.sub.2CH.sub.3, and —CH(CH.sub.3).sub.2.

5. The compound according to claim 4, wherein R.sup.1 is —CH.sub.2CH.sub.3 and R.sup.2 is H.

6. The compound according to claim 2 or 3, wherein R.sup.1 and R.sup.2, together with the atoms to which they are attached, form an optionally substituted 5- or 6-membered aryl or heteroaryl ring.

7. The compound according to claim 6, wherein the optionally substituted 5 or 6 membered aryl or heteroaryl ring is selected from the group consisting of: ##STR00058##

8. The compound according to any one of claims 2 to 6, wherein R.sup.5 is an optionally substituted C1-C6 alkyl.

9. The compound according to claim 8, wherein R.sup.5 is an optionally substituted C1-C3 alkyl.

10. The compound according to claim 9, wherein R.sup.5 is —CH.sub.3, —CH.sub.2CH.sub.3, —CH.sub.2CH.sub.2CH.sub.3, or —CH(CH.sub.3).sub.2.

11. The compound according to claim 10, wherein R.sup.5 is —CH.sub.3.

12. The compound according to any one of claims 2 to 11, wherein X is O.

13. The compound according to any one of claims 2 to 12, wherein R.sup.6 is an optionally substituted group selected from the group consisting of aryl, heteroaryl, and alkyl.

14. The compound according to claim 13, wherein R.sup.6 is —CH.sub.3 or phenyl.

15. The compound according to claim 2, wherein R.sup.3 and R.sup.4 are each H; R.sup.1 and R.sup.2 are each independently selected from the group consisting of H, Cl, —CH.sub.3, —CH.sub.2CH.sub.3, —CH.sub.2CH.sub.2CH.sub.3, and —CH(CH.sub.3).sub.2; R.sup.5 is —CH.sub.3; X is O; and R.sup.6 is —CH.sub.3 or phenyl, preferably phenyl.

16. The compound of claim 15, wherein R.sup.1 is —CH.sub.2CH.sub.3 or —CH(CH.sub.3).sub.2, and wherein R.sup.2 is H.

17. The compound of claim 16, wherein R.sup.1 is —CH.sub.2CH.sub.3.

18. The compound of claim 2, wherein R.sup.1 and R.sup.2, together with the atoms to which they are attached, form a 5 or 6 membered aryl or heteroaryl ring; R.sup.3 and R.sup.4 are each H; R.sup.5 is —CH.sub.3; X is O; and R.sup.6 is —CH.sub.3 or phenyl, preferably phenyl.

19. The compound of claim 18, wherein the 5 or 6 membered aryl or heteroaryl ring is selected from the group consisting of: ##STR00059##

20. The compound according to any one of claims 2 to 19, wherein R.sup.7 is a linker comprising ##STR00060## wherein n is an integer from 0 to 20.

21. The compound according to claim 20, wherein n is from 1 to 10.

22. The compound according to claim 21, wherein n is from 1 to 5.

23. The compound according to claim 22, wherein n is 3.

24. The compound according to any one of claims 2-23, wherein R.sup.8 is a metal binding group for a metal selected from .sup.99mTc, .sup.186Re, .sup.188Re, .sup.111In, .sup.67Ga, .sup.68Ga, .sup.64 Cu and/or .sup.89Zr.

25. The compound of claim 24, wherein the metal binding group coordinates said metal together with an additional binding group.

26. The compound according to any one of claims 1 to 25, wherein the label is any one of a single photon emission tomography (SPECT) label, a positron emission tomography (PET) label, an optical imaging label, a magnetic resonance imaging (MRI) label, an ultrasound label or a photoacoustic imaging label.

27. The compound according to claim 26, wherein the label comprises a group selected from the group consisting of .sup.18F, .sup.68Ga, .sup.123I, .sup.124I, .sup.125I, .sup.99mTc, .sup.111In, .sup.67Ga, .sup.64Cu, .sup.11C, .sup.89Zr, fluorescent dyes and absorbers.

28. The compound according to claim 26 or 27, wherein the label comprises a PET label selected from the group consisting of: ##STR00061##

29. The compound according to claim 1 or 2 having formula (III) ##STR00062## or a salt, hydrate, isomer or tautomer thereof.

30. The compound according to claim 26 or 27, wherein the label comprises a SPECT label selected from the group consisting of: ##STR00063## wherein each R.sup.9 is independently selected from the group consisting of —CH.sub.3, —CH.sub.2COOH, ##STR00064## preferably wherein each R.sup.9 is the same, more preferably wherein R.sup.9 is methyl.

31. The compound according to claim 1 or 2 having formula (IV) ##STR00065## or a salt, hydrate, isomer or tautomer thereof.

32. The compound according to claim 1 or 2 having formula (V) ##STR00066## or a salt, hydrate, isomer or tautomer thereof.

33. The compound according to claim 26 or 27, wherein the label comprises a photoacoustic imaging label.

34. The compound according to claim 33, wherein the photoacoustic imaging label is a phthalocyanine, a naphthalocyanine, or a polymethine dye.

35. The compound according to claim 34, wherein the photoacoustic imaging label is a polymethine dye.

36. The compound according to claim 33, wherein the label comprises a photoacoustic imaging label which is an absorber selected from the group consisting of: ##STR00067## ##STR00068## wherein each R.sup.10 is independently selected from the group consisting of sulphonic acids, ammonium salts, and thioethers, preferably wherein each R.sup.10 is the same; wherein o is an integer from 0 to 20, preferably from 1 to 10, more preferably from 1 to 5, most preferably o is 3; and wherein M.sup.2+ is Fe, Cu, Ni, or V(=O).

37. The compound according to claim 26 or 27, wherein the label comprises an optical imaging label.

38. The compound according to claim 37, wherein the label is a dye.

39. The compound according to claim 38, wherein the dye is selected from the group consisting of fluorescein isothiocyanate (FITC), 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl indotricarbocyanine iodide (DiR), a coumarin dye, a rhodamine dye, a carbopyronin dye, an oxazine dye, a fluorescein dye, a cyanine dye, a boron-dipyrromethene (BODIPY) dye, a squaraine dye, and a squaraine rotaxane dye.

40. The compound according to claim 37, wherein the optical imaging label is a fluorophore.

41. The compound according to claim 40, wherein the label is a polymethine dye.

42. The compound according to claim 41, wherein the polymethine dye is a cyanine dye.

43. The compound according to claim 42, wherein the cyanine dye is cyanine 3, cyanine 3.5, cyanine 5, cyanine 5.5, or cyanine 7.

44. The compound according to claim 43, wherein the cyanine dye is cyanine 5.5.

45. The compound according to claim 43, wherein the cyanine dye is cyanine 7.

46. The compound according to claim 1 or 2 having formula (II) ##STR00069## or a salt, isomer, or tautomer thereof.

47. The compound according to claim 1 or 2 having formula (VIII) ##STR00070## or a salt, isomer, or tautomer thereof.

48. The compound according to any one of claims 1-47, or the compound ##STR00071## wherein the compound is a diagnostic compound.

49. A diagnositic composition comprising a compound according to any one of claims 1 to 48 and a pharmaceutically or diagnostically acceptable excipient.

50. The compound according to any one of claims 1 to 48 for use in a method of diagnosis.

51. The compound for use according to claim 50, wherein the diagnosis is diagnosis of an inflammatory disease in a subject.

52. The compound for use according to claim 51, wherein the inflammatory disease is associated with phagocyte and/or epithelial cell activation in said subject.

53. The compound for use according to claim 52, wherein the inflammatory disease is further associated with an overexpression and accumulation of S100A9 in said subject.

54. The compound for use according to any one of claims 50 to 53, wherein the inflammatory disease comprises dermatitis, atherosclerosis, psoriasis, autoimmune diseases, arthritis, allergies, cardiovascular processes, local and systemic infections, neuroinflammatory diseases, acute lung injury (ALI) and tumors.

55. The compound for use according to any one of claims 50 to 54, wherein the method is an in vivo non-invasive molecular imaging method.

56. The compound for use according to claim 55, wherein the method is any one of single photon emission tomography (SPECT), positron emission tomography (PET), optical imaging, magnetic resonance imaging (MRI), ultrasound or photoacoustic imaging.

57. The compound for use according to any one of claims 50 to 56, wherein the method of diagnosis is a method of an early stage diagnosis.

58. The compound for use according to any one of claims 50 to 57, wherein the inflammatory disease is at local site.

59. The compound for use according to any one of claims 50 to 58, wherein said subject is a mammal.

60. The compound for use according to claim 59, wherein said mammal is mouse, rat, guinea pig, rabbit, cat, dog, monkey, horse, or human.

61. The compound for use according to any one of claims 50 to 60, wherein the method comprises administering said compound to the subject.

62. The compound for use according to any one of claims 50 to 61, wherein said administration is carried out orally, parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, by intranasal instillation, by implantation, by intracavitary or intravesical instillation, intraocularly, intraarterially, intralesionally, transdermally, or by application to mucous membranes.

63. A method of diagnosing an inflammatory disease in a subject, comprising: a) administering to said subject a compound according to any one of claims 1 to 48, b) detecting the administered compound using an in vivo non-invasive molecular imaging technique, thereby collecting imaging data, c) comparing the imaging data received in step b) to reference imaging data.

64. A non-invasive method of detecting or imaging accumulation of S100A9 in the body of a subject to whom a compound of any one of claims 1 to 48 has been pre-delivered, comprising: a) detecting the administered compound using an in vivo non-invasive molecular imaging technique, thereby collecting imaging data, b) comparing the imaging data received in step a) to reference imaging data.

65. The method according to claim 63 or 64, wherein a significantly increased signal in the imaging data from the subject as compared to reference imaging data indicates the presence of an inflammatory disease in said subject.

66. The method according to claim 63 or 64, wherein no difference in the signal in the imaging data from the subject as compared to reference imaging data indicates no presence of an inflammatory disease in said subject.

67. The method according to any one of claims 63 to 66, wherein the inflammatory disease is associated with phagocyte and/or epithelial cell activation in said subject.

68. The method according to claim 67, wherein the inflammatory disease is further associated with an overexpression and accumulation of S100A9 in said subject.

69. The method according to any one of claims 63 to 68, wherein the inflammatory disease comprises dermatitis, atherosclerosis, psoriasis, autoimmune diseases, arthritis, allergies, cardiovascular processes, local and systemic infections, neuroinflammatory diseases, acute lung injury (ALI) and tumors.

70. The method according to any one of claims 63 to 69, wherein said in vivo non-invasive molecular imaging method is any one of single photon emission tomography (SPECT), positron emission tomography (PET), optical imaging, magnetic resonance imaging (MRI), ultrasound or photoacoustic imaging.

71. The method according to any one of claims 63 to 70, wherein the method of diagnosing is a method of an early stage diagnosing.

72. The method according to any one of claims 63 to 71, wherein the inflammatory disease is at local site.

73. The method according to any one of claims 63 to 72, wherein said subject is a mammal.

74. The method according to claim 73, wherein said mammal is mouse, rat, guinea pig, rabbit, cat, dog, monkey, horse, or human.

75. Use of a compound according to any one of claims 1 to 48 for the preparation of a diagnostic composition for diagnosing an inflammatory disease associated with phagocyte and/or epithelial cell activation in a subject.

76. A method for evaluating whether a subject may be at risk of developing an inflammatory disease associated with phagocyte and/or epithelial cell activation, the method comprising: a) administering to said subject a compound according to any one of claims 1 to 48, b) detecting the administered compound using an in vivo non-invasive molecular imaging technique, thereby collecting imaging data, c) comparing the imaging data received in step b) to reference imaging data.

77. The method according to claim 76, wherein a significantly increased signal in the imaging data from the subject as compared to reference imaging data indicates that said subject is at higher risk of developing an inflammatory disease associated with phagocyte and/or epithelial cell activation.

78. The method according to claim 76, wherein a signal in the imaging data at a normal level as compared to reference imaging data indicates that said subject is at lower risk of developing an inflammatory disease associated with phagocyte and/or epithelial cell activation.

79. A method of monitoring or evaluating the progression of an inflammatory disease associated with phagocyte and/or epithelial cell activation in a patient, the method comprising: a) administering to said subject a compound according to any one of claims 1 to 48, b) detecting the administered compound using an in vivo non-invasive molecular imaging technique, thereby collecting imaging data, c) comparing the imaging data received in step b) to reference imaging data obtained from said patient at an earlier date, wherein the result of the comparison of c) provides an evaluation of the progression of the inflammatory disease associated with phagocyte and/or epithelial cell activation in said patient.

80. The method according to claim 79, wherein a significantly increased signal in the imaging data from the subject as compared to reference imaging data obtained from said patient at an earlier date indicates a progression of the inflammatory disease associated with phagocyte and/or epithelial cell activation in said patient.

81. The method according to claim 79, wherein no change or decrease in the signal in the imaging data from the subject as compared to reference imaging data obtained from said patient at an earlier date indicates no progression or a regression of the inflammatory disease associated with phagocyte and/or epithelial cell activation in said patient.

82. A method of imaging an inflammatory disease in a subject, comprising: a) administering to said subject a compound according to any one of claims 1 to 48, b) detecting the administered compound using an in vivo non-invasive molecular imaging method, thereby collecting imaging data.

83. An in vitro method of diagnosing an inflammatory disease in a subject to whom a compound according to any one of claims 1 to 48 has been pre-delivered, comprising: a) analyzing a sample taken from said subject, b) detecting said pre-delivered compound using a non-invasive molecular imaging method, thereby collecting imaging data, c) comparing the imaging data received in step b) to reference imaging data.

84. The method according to claim 83, wherein an increased signal in the imaging data from the subject as compared to reference imaging data indicates the presence of an inflammatory disease in said subject.

85. The method according to claim 83, wherein no difference in the imaging signal in the imaging data from the subject as compared to reference imaging data indicates no presence of an inflammatory disease in said subject.

86. A process for the preparation of a compound of the formula (I) or its salts, isomers, tautomers or solvates thereof, as claimed in one or more of claims 2-48, comprising reacting a compound of the formula (VI) with a compound of the formula (VII) to give a compound of the formula I, ##STR00072## wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8 and X are defined as in claims 2-48.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0139] FIG. 1: Overview of quinoline-3-carboxamide compound variations.

[0140] FIG. 2: Examples of labels for use in molecular imaging applications.

[0141] FIG. 3: Overview of the synthesis of 3-quinoline carboxylic acid 8 as an intermediate for the preparation of quinoline-3-carboxamide compounds covalently linked to a label.

[0142] FIG. 4: Overview of the synthesis of the cyanine 5.5-conjugated quinoline-3-carboxamide (Cy5.5-CES271) as a S100A9 ligand for fluorescence-based optical imaging.

[0143] FIG. 5: ELISA-based blocking study showing binding of S100A9 protein to TLR4/MD2 in the presence or absence of quinoline-3-carboxamide compounds covalently linked to a label, in this case Cy5.5-CES271.

[0144] FIG. 6: Accumulation of Cy5.5-CES271 in a mouse model of contact dermatitis (ICD). (A) FRI images showing accumulation of a quinoline-3-carboxamide compound covalently linked to a label (Cy5.5-CES271) in a mouse model of contact dermatitis of the left ear in WT (left) and S100A9 deficient (right) mice. (B) FRI images and S100A9 staining showing accumulation of a quinoline-3-carboxamide compound covalently linked to a label (Cy5.5-CES271) in a mouse model of contact dermatitis.

[0145] FIG. 7: Accumulation of Cy5.5-CES271 in a mouse model of myocardial infarction. One day post occlusion of the LAD in a C57BI/6 mouse: FRI of the explanted heart (longitudinal mid-infarction cut) two hours after i.v. injection of 2 nmol Cy5.5-CES271. The tracer accumulates in the myocardial infarction (MI) in concordance to the presence of S100A9 shown by histological staining. Furthermore, absence of S100A9 in the remote myocardium (RM) is accompanied by the lack of Cy5.5-CES271.

[0146] FIG. 8: Accumulation of Cy5.5-CES271 in a mouse model of atherosclerosis in a HypoE mouse, 10 days on HFC diet. FRI of the explanted aorta (2h p.i.) shows accumulation of the Cy5.5-CES271 (yellow) in plaque lesions (white patches, bright light). Systematic histological analysis of the aorta shows high levels of S100A9 in high uptake areas of Cy5.5-CES271 (A) and absence/very low levels of S100A9 in low uptake areas (B) of the tracer.

[0147] FIG. 9: Overview of the synthesis of 5-ethyl-4-hydroxy-1-methyl-N-{1-(1-methyl-1H-imidazol-2-yl)-2-[(1-methyl-1H-imidazol-2-yl)methyl]-5,8,11-trioxa-2-azatridecan-13-yl-2-oxo-N-phenyl-1,2-dihydroquinoline-3-carboxamide and the corresponding Re-complex fac-[Re(CO).sub.3(5-ethyl-4-hydroxy-1-methyl-N-{1-(1-methyl-1H-imidazol-2-yl)-2-[(1-methyl-1H-imidazol-2-yl)methyl]-5,8,11-trioxa-2-azatridecan-13-yl}-2-oxo-N-phenyl-1,2-dihydroquinoline-3-carboxamide)].sup.+.

[0148] FIG. 10: Overview of the synthesis of fac-[.sup.99mTc(CO).sub.3(5-ethyl-4-hydroxy-1-methyl-N-(1-(1-methyl-1H-imidazol-2-yl)-2-[(1-methyl-1H-imidazol-2-yl)methyl]-5,8,11-trioxa-2-azatridecan-13-yl}-2-oxo-N-phenyl-1,2-dihydroquinoline-3-carboxamide)].sup.+ (.sup.99mTc-FEB054) as a S100A9 ligand for single photon emission tomography (SPECT).

[0149] FIG. 11: Characterisation of fac-[.sup.99mTc(CO).sub.3(5-ethyl-4-hydroxy-1-methyl-N-{1-(1-methyl-1H-imidazol-2-yl)-2-[(1-methyl-1H-imidazol-2-yl)methyl]-5,8,11-trioxa-2-azatridecan-13-yl}-2-oxo-N-phenyl-1,2-dihydroquinoline-3-carboxamide)].sup.+ (.sup.99mTc-FEB054). [0150] (A): Quality control via Co-injection. Chromatogram of the free ligand and the corresponding Re-complex (red line) and of .sup.99mTc-FEB054 (blue line) performed by gradient-HPLC using a Knauer system with a K-500 pump, a K-501 pump, a K-2000 UV detector, a Nal(TI) Scintibloc 51 SP51 γ-detector and a reversed phase C.sub.18 column (ACE-126-2510, 10 mm×250 mm). Eluent A: water (0.1% TFA). Eluent B: Methanol (0.1% TFA). Gradient from 70% A to 0% A over 30 minutes, holding for 8 minutes and back to 70% A over 5 minutes at a flow rate of 5.5 ml min.sup.−1, detection at λ=254 nm. [0151] (B): Mass analysis for product identification (.sup.99mTc-FEB054) on trace level: ESI-HR-MS System: Thermo Fisher Scientific (Bremen, Deutschland), Exactive; scan range: m/z 98-2000; resolution: Ultra High (100000@1Hz); AGC target: balanced; maximum inject time: 1000 ms. Parameter: Sheath gas flow rate: 40; Aux gas flow rate: 5; Sweep gas flow rate: 0; Spray voltage in |kV|: 3.8; Capillary temp in ° C: 300; Capillary voltage in V: 50 (pos), −87.5 (neg); Tube lens voltage in V: 90 (pos), −130 (neg); Skimmer voltage: 22 (pos), −30 (neg).

[0152] FIG. 12: Overview of the synthesis of fac-[.sup.99mTc(bathophenanthrolinedisulfonic acid)(CO).sub.3(5-ethyl-4-hydroxy-1-methyl-2-oxo-N-phenyl-N-[1-(pyridin-3-yl)-2,5,8,11-tetraoxatridecan-13-yl]-1,2-dihydroquinoline-3-carboxamide)].sup.− (.sup.99mTc-FEB105) as a S100A9 ligand for single photon emission tomography (SPECT).

[0153] FIG. 13: Characterisation of fac-[.sup.99mTc(bathophenanthrolinedisulfonic acid)(CO).sub.3(5-ethyl-4-hydroxy-1-methyl-2-oxo-N-phenyl-N-(pyridin-3-yl)-2,5,8,11-tetraoxatridecan-13-yl]-1,2-dihydroquinoline-3-carboxamide)].sup.− (99mTc-FEB105). Chromatogram of the UV and gamma channel. The UV channel shows the signal given by the co ligand with a retention time around 8 minutes. At a retention time of 14.8 minutes, the signal given by Precursor can be observed. In the gamma channel, three peaks with retention times between 11 and 12 minutes are caused by.sup.99mTc-FEB105.

[0154] FIG. 14: Overview of the synthesis of 1-[4-(5-ethyl-4-hydroxy-N,1-dimethyl-2-oxo-1,2-dihydroquinoline-3-carboxamido)phenyl]-4-fluoro-1-oxobutane-2-sulfonic acid as a S100A9 ligand for positron emission tomography (PET).

[0155] FIG. 15: Binding of Cy5.5-CES271 to human and murine S100A9. Estimation of the binding constant of Cy5.5-CES271 to human (A) and murine (B) S100A9. The constant was calculated using the one site saturation regression model. Each dot represents the mean value of four independent experiments.

[0156] FIG. 16: Biodistribution of Cy5.5-CES271 in healthy Balb/c mice. The tracer was intravenously injected at a dose of 2 nmol per mouse. The tracer accumulation was measured 1 and 3 h post injection (n=5 for each time point Data shows a decrease of tracer concentration in the blood between 1 h and 3 h post injection, while in the same time interval the tracer concentration in the urine increases significantly, reflecting renal tracer elimination. Kidney, liver and lung present with higher tracer concentrations at 3 h post injection, which can be partly related to tracer metabolization. In other tissues analysed (heart, spleen, muscle, brain) tracer concentrations are at the same level when comparing 1 h and 3 h post injection time points.

[0157] FIG. 17: Human serum blood stability of [.sup.99mTc]FEB054. In human blood serum stability tests, no decomposition was observed over a period of 120 min. Blood serum stability was tested in freshly prepared human blood serum at 37° C. Samples were taken after 10, 20, 30, 60, 90 and 120 minutes and analysed using a gradient HPLC system. 5 MBq of [.sup.99mTc]FEB054 in 20 μL PBS buffer was added to 200 μL of a freshly prepared human blood serum sample and incubated at 37° C. After 10, 20, 30, 60, 90 and 120 minutes, 20 μL were separated and diluted with 50 μL dichloromethane and 50 μL methanol. After centrifugation, 10 μL of the solution were analyzed via gradient-HPLC using a Knauer system with two Smartline 1000 pumps, Smartline UV detector 2500 (Herbert Knauer GmbH), a GabiStar γ-detector (Raytest lsotopenmessgeräte GmbH) and a reversed phase C.sub.18 column (Nucleosil 100-5 C-18 column 4.6 mm×250 mm). Eluent A: water (0.1% TFA). Eluent B: Methanol (0.1% TFA).Gradient from 70% A to 0% A over 15 minutes, holding for minutes and back to 70% A over 5 minutes at a flow rate of 5.5 ml min.sup.−1, detection at λ=254 nm.

[0158] FIG. 18: Biodistribution (A) and elimination (B) of [.sup.99mTc]FEB054 in healthy Balb/c mice. The tracer was intravenously injected at a dose of 62 MBq per mouse. The tracer accumulation and elimination was measured 0-90 min post injection. In vivo biodistribution experiments show a good tracer availability in the blood in the first 10 minutes and predominant hepato-biliary elimination within 20 min resulting in intensive accumulation of the tracer in the liver and intestines.

[0159] FIG. 19: Accumulation of [.sup.99mTc]FEB054 in the inflamed ear and muscle in a mouse model of contact dermatitis (ICD). The tracer was intravenously in a mouse model of contact dermatitis of the left ear in WT mice (=inflamed, right hand side) at a dose of 66 MBq per mouse and measured 0-60 min post injection. The given image example 45 minutes post injection reveals a higher tracer uptake in the inflamed ear as compared to the healthy control ear. Quantitative image analysis shows increasing uptake ratios of tracer in the inflamed ear versus blood and muscle, respectively.

[0160] FIG. 20: Accumulation of [.sup.99mTc]FEB054 in a mouse model of collagen induced arthritis. The tracer was intravenously injected at a dose of 55 MBq per mouse. The upper image shows a SPECT/CT-scan of a healthy control mouse, the lower image a SPECT/CT scan of an arthritis mouse. The tracer distribution was measured in vivo 60 min post injection using SPECT/CT. The volume rendered images (CT: grey-white color scale, [.sup.99mTc]FEB054-SPECT: red color scale) of two representative mice show tracer accumulation in the inflamed joints of the hind limbs in the arthritis mouse (red arrows) in clear contrast to the hind limbs of a healthy control mouse (green arrows). Besides this mouse model specific finding, SPECT images show intensive tracer accumulation in liver, intestines, kidneys and the urinary bladder.

[0161] FIG. 21: FRI images of Cy5.5-CES271 and Cy7-CES271 in a mouse model of contact dermatitis (ICD). (A) Representative Fluorescence Reflectance images of a Cy7-CES271 and Cy5.5-aS100A9 coinjected animal. The left panel shows the signal recorded in the Cy7 channel, representing the Cy7-CES271 accumulation. The right panel shows the

[0162] Cy5.5-aS100A9 accumulation. (B) Comparison of the Total Radiant Efficiency in inflamed and control ears reveals highly significant correlation between the Cy7-CES271 and Cy5.5-aS100A9 signal (R.sup.2=0.96; n=8; p<0.001).

[0163] FIG. 22: Accumulation of Cy5.5-CES271 in a mouse model of acute lung inflammation (ALI). (A) At different time points after the intranasal application of either 10 pg or 50 pg LPS the local and systemic levels of S100A8/S100A9 are compared to control animals. Exemplary paraffin sections of inflamed and healthy lung tissue, taken at 8 h after LPS (10 pg) application were stained for S100A9-expression. Histological data of control (upper image) and lung inflammation (lower image) confirmed local expression of these proteins. (B) The comparison of the tracer accumulation in explanted mice lungs 3 h and 6 h after intranasal application of 50 pg LPS and parallel tracer injection is displayed. 50 pg LPS treated mice injected with glycine saturated Cy5.5 served as perfusion controls. (C) S100A8/S100A9 serum levels of LPS treated and control animals are correlated with the measured Mean Fluorescence Intensity over the lungs (R.sup.2=0.69; n=19; p<0.001).

EXAMPLES

[0164] The following examples illustrate the invention. These examples should not be construed as to limit the scope of this invention. The examples are included for purposes of illustration and the present invention is limited only by the claims. The person of skill in the art will understand that other synthetic routes and variations to the quinoline-3-carboxamide compound, linker, and/or label described below are possible and within the scope of the present disclosure.

Experimental Details for the Synthesis of Cy5.5-CES271

Diethyl 2-(4-nitro-3-oxo-3H-isobenzofuran-1-yl idene)malonate

[0165] ##STR00031##

[0166] To diethyl malonate (152 mL, 160 g, 1 mol), acetic anhydride (480 mL, 519 g, 5.1 mol) and triethylamine (137 mL, 99.2 g, 0.98 mol) was added at 35° C. under slight cooling (temperature should be maintained between 35 and 40° C.) 3-nitrophthalic acid anhydride (193.1 g, 1 mol). After stirring for 2 h at 40° C. and cooling to room temperature, the mixture was poured onto crushed ice (1.4 kg) and hydrochloric acid (32% w/v; 186 mL). After stirring for 30 min, the solvent was decanted and the residue suspended with 360 mL acetone. The precipitate was isolated by suction, washed with cold acetone and dried in vacuo. Yield: 293.2 g (0.87 mol; 87%)

[0167] mp.: 169° C. (acetone)

[0168] .sup.1H NMR (600 MHz, chloroform-d)

[0169] δ [ppm]=9.01 (dd, .sup.3J.sub.H,H=8.1 Hz, .sup.4J.sub.H,H=0.8 Hz, 1H, 2-CH), 8.11 (dd, .sup.3J.sub.H,H=7.9 Hz, .sup.4J.sub.H,H=0.8 Hz, 1H, 4-CH), 7.98 (m, 1H, 3-CH), 4.40, 4.38 (q, .sup.3J.sub.H,H=7.1 Hz, 4H, 11-CH, 11′-CH), 1.37, 1.36 (t, .sup.3J.sub.H,H7.1 Hz, 6H, 12-CH, 12′-CH).

[0170] .sup.13C NMR (151 MHz, chloroform-d)

[0171] δ [ppm]=162.8, 162.3 (s, C-10, C-10′), 158.5 (s, C-8), 152.5 (s, C-7), 146.9 (s, C-1), 138.2 (s, C-5), 136.5 (d, C-3), 131.7 (d, C-2), 127.6 (d, C-4), 118.5 (s, C-6), 112.0 (s, C-9), 62.7 (t, C-11, C-11′), 14.1, 14.1 (q, C-12, C-12′).

[0172] MS (ESI.sup.+): m/z=358.0536; calculated [C.sub.15H.sub.13NO.sub.8]Na.sup.+([M+Na]).sup.+): 358.0533. 390.0797; calculated [C.sub.15H.sub.13NO.sub.8]Na.sup.+ MeOH ([M+Na+MeOH].sup.+): 390.0796. 693.1176; calculated [C.sub.30H.sub.26N.sub.2O.sub.16].sub.2Na.sup.+ ([2M+Na]).sup.+): 693.1175. 725.1435; calculated [C.sub.30H.sub.26N.sub.2O.sub.16].sub.2Na.sup.+.Math.MeOH ([2M+Na+MeOH].sup.+): 693.1175. 757.1693; calculated [C.sub.30H.sub.26N.sub.2O.sub.16].sub.2Na.sup.+.Math.2MeOH ([2M+Na+2MeOH].sup.+): 757.1699.

2-Acetyl-6-nitrobenzoic acid

[0173] ##STR00032##

[0174] Hydrochloric acid (32% w/v; 351 mL), water (38 mL) and toluene (7.5 mL) were charged in a round bottom flask and diethyl 2-(4-nitro-3-oxo-3H-isobenzofuran-1-ylidene)malonate (147 g, 438 mmol) was added. The mixture was stirred and warmed to 95° C. over 2 h. At 70° C. evolution of carbon dioxide started. Stirring was continued for 20 h at 95° C., and the mixture then cooled to 10° C. The solid product was filtered off, washed with water and dried at 65° C. in vacuo. Yield: 84.5 g (404mmo1; 92%).

[0175] mp.: 169 ° C. (toluene)

[0176] .sup.1H NMR (400 MHz, NaOD, 0.1M in deuterium oxide)

[0177] δ [ppm]=8.26 (dd, .sup.3J.sub.H,H=8.3, .sup.4J.sub.H,H=1.1 Hz, 1H, 3-CH), 8.17 (dd, .sup.3J.sub.H,H=7.8, .sup.4J.sub.H,H=1.1 Hz, 1H, 5-CH), 7.64 (dd, .sup.3J.sub.H,H=8.3, 7.8 Hz, 1H, 4-CH).

[0178] .sup.13C NMR (101 MHz, NaOD, 0.1M in deuterium oxide)

[0179] δ [ppm]=202.5 (s, C-7), 173.0 (s, C-9), 145.3 (s, C-2), 135.9 (s, C-6), 135.0 (d, C-5), 134.5 (s, C-1), 128.6 (d, C-4), 127.9 (d, C-3), 27.9 (m, C-8).

[0180] MS (ESI.sup.+): m/z=232.0220; calculated [C.sub.10H.sub.9NO.sub.3]Na.sup.+ ([M+Na].sup.+): 232.0216. 245.0038; calculated [C.sub.10H.sub.8NNaO.sub.3]2Na.sup.+ ([M−H +2Na].sup.+): 254.0036.

2-amino-6-ethylbenzoic acid

[0181] ##STR00033##

[0182] Sodium hydroxide (1M, 50 mL) and water (50 mL) were charged in a steel tube for high pressure hydrogenations and 2-acetyl-6-nitrobenzoic acid (10 g, 48 mmol) was added (resulting pH: 12.3). After adding Platinum(IV)oxide (200 mg) the reaction vessel was flushed with hydrogen three times and the mixture was stirred at 90° C. for 3 h at 20 bar (H.sub.2). Then Raney-Nickel suspension (3.6 g) was added and after further stirring (3 h, 110° C., 20 bar H.sub.2) the resulting mixture was filtrated through Celite®. The filtrate was adjusted to pH 3.5 with concentrated hydrochloric acid and extracted with ethyl acetate (3×200 mL). After removing the solvent and drying in vacuo a white solid was obtained. Yield: 6.1 g (37 mmol; 77%).

[0183] mp.: 105° C. (ethyl acetate)

[0184] .sup.1H NMR (400 MHz, DMSO-d.sub.6)

[0185] δ [ppm]=8.04 (s, 2H, NH.sub.2), 7.02 (dd, .sup.3J.sub.H,H=8.2, 7.5 Hz, 1H, 4-CH), 6.59 (dd, .sup.3J.sub.H,H=8.2, .sup.4J.sub.H,H=1.2 Hz, 1H, 3-CH), 6.42 (dd, .sup.3J.sub.H,H=7.5Hz, .sup.4J.sub.H,H=1.2 Hz, 1H, 5-CH), 2.71 (q, .sup.3J.sub.H,H=7.6 Hz, 2H, 7-CH), 1.12 (t, .sup.3J.sub.H,H=7.6 Hz, 3H, 8-CH).

[0186] .sup.13C NMR (101 MHz, DMSO-d.sub.6)

[0187] δ [ppm]=170.5 (s, C-9), 148.8 (s, C-2), 144.6 (s, C-6), 131.2 (d, C-4), 117.2 (d, C-5), 114.7 (s, C-1), 114.0 (d, C-3), 27.9 (t, C-7), 16.3 (q, C-8).

[0188] MS (ESI): m/z=166.0864; calculated [C.sub.9H.sub.11NO.sub.2]H.sup.+ ([M+H].sup.+): 166.0864. 186.0689; calculated [C.sub.6H.sub.11NO.sub.2]Na.sup.+ ([M+Na].sup.+): 188.0682.

5-Ethyl-1H-benzo[d][1,3]oxazine-2,4-dione

[0189] ##STR00034##

[0190] A solution of phosgene in toluene (20%, 14.7 mL, 28 mmol) was added dropwise to a slurry of 2-amino-6-ethylbenzoic acid (3.7 g, 22.4 mmol) in absolute THF (20 mL) keeping the temperature below 20° C. (ice cooling). After the mixture was stirred for 1h at room temperature, the reaction mixture was poured onto ice water (110 mL) and the resulting precipitate was collected, washed with water, and dried in vacuo to yield the isatoic anhydride: yield 3.73 g (87%).

[0191] mp.: 204° C. (H.sub.2O).

[0192] .sup.1H NMR (400 MHz, DMSO-d.sub.6)

[0193] δ [ppm]=11.59 (s, 1H, 9-NH), 7.54 (dd, .sup.3J.sub.H,H=8.2, 7.6 Hz, 1H, 4-CH), 7.00 (dd, .sup.3J.sub.H,H=7.6 Hz, .sup.4J.sub.H,H=1.1 Hz, 1H, 5-CH), 6.95 (dd, .sup.3J.sub.H,H=8.2, .sup.4J.sub.H,H=1.2 Hz, 1H, 3-CH), 2.97 (q, .sup.3J.sub.H,H=7.4 Hz, 2H, 7-CH.sub.2), 1.11 (t, .sup.3J.sub.H,H=7.4 Hz, 3H, 8-CH.sub.3).

[0194] .sup.13C NMR (101 MHz, DMSO-d.sub.6)

[0195] δ [ppm]=158.5 (s, C-11), 148.5 (s,C-6), 147.1 (s, C-10), 142.7 (s, C-2), 136.1 (d, C-4), 124.5 (d, C-5), 113.5 (d, C-3), 107.8 (s, C-1), 27.1 (t, C-7), 15.0 (q, C-8).

[0196] MS (ESI.sup.+): m/z=192.0655; calculated [C.sub.10H.sub.9NO.sub.3]H.sup.+ ([M+H .sup.+): 192.0655. 214.0475; calculated [C.sub.10H.sub.9NO.sub.3]Na.sup.+ ([M+Na].sup.+): 214.0475. 405.1056; calculated [C.sub.20H.sub.18N.sub.2O.sub.6].sub.2Na.sup.+ ([2M+Na].sup.+): 405.1057.

5-Ethyl-4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid

[0197] ##STR00035##

[0198] The isatoic anhydride (10 g, 52.3 mmol) was dissolved in DMF (100 mL) and cooled on an ice bath, and sodium hydride (95%, 1.58 g, 62.8 mmol) followed by methyl iodide (4.2 mL, 9.6 g, 68 mmol) was added at a rate to keep the temperature below 5° C. After stirring at room temperature overnight, excess methyl iodide was removed by evacuating for 30 min at approximately 30 mbar. Sodium hydride (95%, 1.58 g, 62.8 mmol) followed by diethylmalonate (9.5 mL, 10.06 g, 62.8 mmol) was added, and the mixture was heated at 85° C. for 2 h, then cooled, and quenched with water (500 mL). The aqueous solution was acidified with 1 M HCl, and the resulting precipitate was collected by filtration, washed with water, and dried to afford a light brown solid. Yield: 7.54 g (27.4 mmol; 52%).

[0199] mp.: 73° C. (H.sub.2O).

[0200] .sup.1H NMR (400 MHz, chloroform-d)

[0201] δ [ppm]=14.98 (s, 1H, 7-OH), 7.47 (dd, .sup.3J.sub.H,H=8.6, 7.5 Hz, 1H, 3-CH), 7.12 (dd, .sup.3J.sub.H,H=8.6 Hz, .sup.4J.sub.H,H=1.1 Hz, 1H, 2-CH), 6.98 (dd, .sup.3J.sub.H,H=7.5 Hz, .sup.4J.sub.H,H=1.1 Hz, 1H, 4-CH), 4.46 (q, .sup.3J.sub.H,H=7.1 Hz, 2H, 14-CH.sub.2), 3.58 (s, 3H, 13-CH.sub.3), 3.18 (q, .sup.3J.sub.H,H=7.4 Hz, 2H, 11-CH.sub.2), 1.44 (t, .sup.3J.sub.H,H=7.1 Hz, 3H, 15-CH.sub.3), 1.23 (t, .sup.3J.sub.H,H=7.4 Hz, 3H, 12-CH.sub.3).

[0202] .sup.13C NMR (101 MHz, chloroform-d)

[0203] δ [ppm]=174.5 (s, C-7), 173.5 (s, C-10), 159.2 (s, C-9), 147.0 (s, C-5), 142.9 (s, C-1), 133.6 (d, C-3), 124.7 (d, C-4), 113.3 (s, C-6), 112.6 (d, C-2), 97.5 (s, C-8), 62.3 (t, C-14), 30.3 (t, C-11), 29.9 (q, C-13), 16.4 (q, C-12), 14.2 (q, C-15).

[0204] MS (ESI.sup.+): m/z=276.1234; calculated [C.sub.15H.sub.17NO.sub.4]H.sup.+ ([M+H].sup.+): 276.1230. 298.1049; calculated [C.sub.15H.sub.17NO.sub.4]Na.sup.+ ([M+Na].sup.+): 298.1050. 573.2207; calculated [C.sub.30H.sub.34N.sub.2O.sub.8].sub.2Na.sup.+ ([2M+Na].sup.+): 573.2207.

5-ethyl-4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid

[0205] ##STR00036##

[0206] The ethyl ester (7.15g, 26 mmol ) was heated at 60° C. for six hours in a mixture of hydrochloric acid and acetic acid (80 mL, 2.8M HCl in AcOH). Afterwards, the reaction mixture was poured on iso-Propanol (200 mL). The formed precipitate washed with iso-Propanol (10 mL) and the product was obtained as a white solid. Yield: 3.18 g (12.9 mmol; 50%).

[0207] mp.: decomposition at 220° C. (iso-Propanol).

[0208] 1H NMR (400 MHz, chloroform-d)

[0209] δ [ppm]=16.12 (s, 1H, OH), 15.60 (s, 1H, OH), 7.66 (dd, 3JH-H=8.6, 7.5 Hz, 1H, 3-CH), 7.33 (dd, 3JH-H=8.7 Hz, 4JH-H=1.1 Hz, 1H, 2-CH), 7.20 (dd, 3JH-H=7.5, 4JH-H=1.1, 1H, 4-CH), 3.72 (s, 3H, 13-CH3), 3.24 (q, 3JH-H =7.4 Hz, 2H, 11-CH2), 1.26 (t, 3JH-H=7.4 Hz, 3H, 12-CH3).

[0210] 13C NMR (101 MHz, chloroform-d)

[0211] δ [ppm]=174.6 (s, C-10) , 174.4 (s, C-7), 164.4 (s, C-9), 147.8 (s, C-5), 141.4 (s, C-1), 134,5 (d, C-3), 126.4 (d, C-4), 114.3 (s, C-6), 113.4 (d, C-2), 94,7 (s, C-8), 30.4 (q, C-13), 30.0 (t, C-11), 16.3 (q, C-12).

[0212] MS (ESI+): m/z=248.0917; calculated [C13H13NO4]H+ ([M+H]+): 248.0917. 270.0735; calculated [C13H13NO4]Na+ ([M+Na]+): 270.0737. 517.1581; calculated [C26H26N2O8]2Na+ ([2M+Na]+): 517.1490.

N-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)aniline

[0213] ##STR00037##

[0214] Subsequently the azido-PEG-bromide (7.34 g, 26 mmol) and aniline (4.75 mL, 52 mmol) were emulsified in water (30 mL) and heated to reflux overnight. After cooling to room temperature water (100 mL) was added and the aqueous emulsion was extracted with ethyl acetate (100 mL) four times. The organic phases were dried over magnesium sulphate and concentrated to dryness. The resulting oil was chromatographed on a silica gel column (cyclohexane/EtOAc 2/1 to 1/1) to give a yellow oil. Yield: 3.41 g (11.6 mmol, 45%).

[0215] .sup.1H NMR (400 MHz, chloroform-d)

[0216] δ [ppm]=7.20-7.14 (m, 2H, 2-CH, 2′-CH), 6.73-6.68 (m, 1H, 1-CH), 6.64-6.60 (m, 2H, 3-CH, 3′-CH), 3.71-3.62 (m, 12H, 6-CH.sub.2 bis 11-CH.sub.2), 3.35 (t, .sup.3J.sub.H,H=5.1 Hz, 2H, 12-CH.sub.2), 2.29 (t, .sup.3J.sub.H,H=5.1 Hz, 2H, 5-CH.sub.2).

[0217] .sup.13C NMR (101 MHz, chloroform-d)

[0218] δ [ppm]=148.2 (s, C-4), 129.1 (d, C-2, C-2′), 117.3 (d, C-1), 112.9 (d, C-3, C-3′), 70.6, 70.5, 70.5, 70.2, 69.9 (t, C-7 bis C-11), 69.5 (t, C-6), 50.5 (t, C-12), 43.4 (t, C-5).

[0219] MS (ESI.sup.+): m/z=295.1769; calculated [C.sub.14H.sub.22N.sub.4O.sub.3]H.sup.+ ([M+H].sup.+): 295.1765. 317.1585; calculated [C.sub.14H.sub.22N.sub.4O.sub.3]Na.sup.+ ([M+Na].sup.+): 317.1584.

N-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-5-ethyl-4-hydroxy-1-methyl-2-oxo-N-phenyl-1,2-dihydroquinoline-3-carboxamide

[0220] ##STR00038##

[0221] The 1,2-dihydroquinoline-3-carboxylic acid (1 g, 4.05 mmol) was dissolved under argon atmosphere in dichloro methane (10 mL) and triethylamine (2.1 mL, 1.54 g, 15.2 mmol) and the N-PEG-aniline (1.43 g, 4.86 mmol) were added. At 0° C. (ice bath) thionyl chloride (0.38 mL, 0.63 g, 5.3 mmol), dissolved in dichloromethane (0.6 mL), was added dropwise within 30 minutes. The reaction mixture was stirred for 4 h at 0° C. and then overnight at room temperature. After removing the solvent in vacuo the resulting oil was chromatographed on a silica gel column (EtOAc/MeOH 9/1 to 6/1) to give a yellow sticky oil. Yield: 1.73 g (3.3 mmol, 82%).

[0222] .sup.1H NMR (400 MHz, chloroform-d)

[0223] δ [ppm]=7.42-7.37 (m, 1H, 3-CH), 7.23-7.10 (m, 5H, 15-CH bis 17-CH), 7.03-7.00 (m, 1H, 2-CH), 7.00-6.96 (m, 1H, 4-CH), 3.71-3.58 (m, 12H, 19-CH.sub.2 bis 24-CH.sub.2), 3.62 (s, 3H, 13-CH.sub.3), 3.36-3.32 (m, 2H, 25-CH.sub.2), 3.29-3.24 (m, 2H, 18-CH.sub.2), 3.20 (q, .sup.3J.sub.H,H=7.4 Hz, 2H, 11-CH.sub.2), 1.25 (t, .sup.3J.sub.H,H=7.4 Hz, 3H, .sup.12-CH.sub.3).

[0224] .sup.13C NMR (101 MHz, chloroform-d)

[0225] δ [ppm]=174.5 (s, n.z.), 170.1 (s, n.z.), 167.7 (s, n.z.), 148.2 (s, C-14), 145.8 (s, C-5), 142.0 (s, C-1), 131.9 (d, C-3), 129.2, 128.5, 126.3 (d, 15-C bis 17-C), 124.4 (d, C-4), 113.6 (s, C-6), 112.4 (d, C-2), 103.9 (s, C-8), 70.6, 70.6, 70.4, 70.0, 69.6, 67.8 (t, 19-C bis 24-C), 50.6 (t, C-25), 43.5 (t, C-18), 30.0 (t, C-11), 29.6 (q, C-13), 16.7 (q, C-12).

[0226] MS (ESI.sup.+): m/z=524.2507; calculated [C.sub.27H.sub.33N.sub.5O.sub.6]H.sup.+ ([M+H].sup.+): 524.2504. 546.2326; calculated [C.sub.27H.sub.33N.sub.5O.sub.6]Na.sup.+ ([M+Na].sup.+): 546.2323.

N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-5-ethyl-4-hydroxy-1-methyl-2-oxo-N-phenyl-1,2-dihydroquinoline-3-carboxamide

[0227] ##STR00039##

[0228] N-(2-{2-[2-(2-Azidoethoxy)ethoxy]ethoxy}ethyl)-5-ethyl-4-hydroxy-1-methyl-2-oxo-N-phenyl-1,2-dihydrochinolin-3-carboxamide (1.128 g, 2.15 mmol, 1.0 eq.) was dissolved under argon in tetrahydrofuran (15 mL) and a few mg of Pd/C were added. The argon atmosphere was changed to hydrogen (balloon) and the reaction mixture was stirred at room temperature for 16 h. After filtration on Celite® and evaporation of the solvent the desired product was obtained as a light brown solid.

[0229] yield: 840 mg (1.69 mmol, 79%).

[0230] mp.: 53 ° C. (THF)

[0231] .sup.1H NMR (400 MHz, methanol-d.sub.4)

[0232] δ [ppm]=7.42-7.39, 7.15-7.10 (m, 5H, 15-CH bis 17-CH), 7.26-7.21 (m, 1H, 3-CH), 7.10-7.05 (m, 1H, 2-CH), 6.82-6.79 (m, 1H, 4-CH), 3.91-3.86, 3.77-3.74, 3.68-3.52 (m, 12H, 19-CH.sub.2 bis 24-CH.sub.2), 3.52-3.46 (m, 2H, 11-CH.sub.2), 3.40 (s, 3H, 13-CH.sub.3), 3.29-3.22 (m, 2H, 18-CH.sub.2), 3.21-3.18 (m, 2H, 25-CH.sub.2), 1.14 (t, .sup.3J.sub.H,H=7.4 Hz, 3H, 12-CH.sub.3).

[0233] .sup.13CNMR (101 MHz, methanol-d.sub.4)

[0234] δ [ppm]=174.5 (s, C-7), 174.2 (s, C-10), 162.8 (s, C-9), 147.2 (s, C-5), 143.0 (s, C-1), 142.8 (s, C-14), 130.4 (d, C-3), 129.2, 128.3, 128.2, (d, C-15 bis C-17), 124.4 (d, C-4), 121.7 (s, C-6), 113.5 (d, C-2), 109.2 (s, C-8), 71.3, 71.0 , 71.0, 70.9, 70.7, 68.8, 68.1 (t, C-19 bis C-24), 44.7 (t, C-18), 40.7 (t, C-25), 30.3 (t, C-11), 29.8 (q, C-13), 17.8 (t, C-12).

[0235] MS (ESI.sup.+): m/z=498.2598; calculated [C.sub.27H.sub.35N.sub.3O.sub.6]H.sup.+ ([M+H].sup.+): 498.2599. 520.2420; calculated [C.sub.27H.sub.35N.sub.3O.sub.6]Na.sup.+ ([M+Na].sup.+): 520.2418. 995.5115; calculated [C.sub.54H.sub.70N.sub.6O.sub.12].sub.2H.sup.+ ([2M+H].sup.+): 995.5124. 1017.4920; calculated [C.sub.54H.sub.70N.sub.6O.sub.12].sub.2Na.sup.+ ([2M+Na].sup.+): 1017.4944.

3-ethyl-2((1E,3E,5E)-5-(3-(1-(5-ethyl-4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinolin-3-yl)-1,15-dioxo-2-phenyl-5,8,11-trioxa-2,14-diazanonadecan-19-yl)-1,1-dimethyl-6,8-disulfonato-1H-benzo[e]indol -2(3H)-ylidene)penta-1,3-dien-1-yl)-1,1-dimethyl-1H-benzo[e]indol-3-ium-6,8-disulfonate (Cy5.5-CES271)

[0236] ##STR00040##

[0237] The amino-functionalized precursor (3.0 mg, 4.2 pmol) was dissolved in 400 μL dry dimethylformamide provided with 10 μL triethylamine. To this solution, Cy5.5-NHS ester (GE) (1 mg, 0.9 μmol) was added. The reaction mixture was vortexed for 16 h at room temperature in the dark. Purification of Cy5.5-CES271 was performed by gradient-HPLC using a Knauer system with two K-1800 pumps, an S-2500 UV detector and a RP-HPLC Nucleosil 100-5 C18 column (250 mm×4.6 mm). Eluent A: water (0.1% TFA). Eluent B: Acetonitrile (0.1% TFA). Gradient from 95% A to 40% A over 19 minutes, holding for 5 minutes and back to 95% in one minute at a flow rate of 5.5 ml/min, detection at λ=254 nm. The appropriate fractions (t.sub.R=16.5 min) were collected, lyophilized, redissolved in 1 mL water and finally stored at −20° C. The average content of Cy5.5-CES271 was 0.45±0.02 μmol/ml (≈50%) as determined by fluorometer measurements with λ.sub.abs=678 nm and ε.sub.678=250000 M.sup.−1 cm−1.

[0238] MS (ES.sup.−): m/e=464.1 (100%), 464.5, 464.8 [M].sup.3−; 696.7, 697.2, 697.7 [M+H].sup.2−.

Experimental Details for the Synthesis of fac-].sup.99mTc(CO).sub.3(5-ethyl-4-hydroxy-1-methyl-N-{1-(1-methyl-1H-imidazol-2-yl)-2-[1-methyl-1H-imidazol-2-yl) methyl]-5, 8, 11-trioxa-2-azatridecan-13-yl}2-oxo-N-phenyl-1,2-dihydroquinoline-3-carboxamide)].sup.+ (.sup.99mTc-FEB054)

2-{2-[2-(2-Azidoethoxy)ethoxy]ethoxyl-N,N-bis[(1-methyl-1H-imidazole-2-yl)methyl]ethylazide

[0239] ##STR00041##

[0240] 1-Methyl-1H-imidazole-2-carboxaldehyde (1.32 g, 12.0 mmol, 2.6 eq.) was added to a stirred solution of 2-{2-[2-(2-azidoethoxy)ethoxy]ethoxy}ethan-1-amine (1.00 g, 4.6 mmol, 1.0 eq.) in 1,2-dichloroethane (50 mL) under an Argon atmosphere and was heated for 30 minutes at 75° C. Afterwards, the solution was cooled to 0° C., Sodium triacetoxyborohydride (3.22 g, 15.2 mmol, 3.3 eq.) was added, and the resulting mixture was allowed to stir at room temperature overnight. Upon completion, a saturated solution of NaHCO.sub.3 (20 mL) was added and the reaction mixture extracted with ethyl acetate (2×50 mL). The combined organic layers were washed with brine, dried over MgSO.sub.4 and concentrated under reduced pressure. The residue was purified by column chromatography over silica gel (ethyl acetate/methanol) to give the product as a yellow oil. Yield: 1.37 g (3.4 mmol, 74%).

[0241] .sup.1H NMR (400 MHz, chloroform-d)

[0242] δ [ppm]=6.73 (d, .sup.3J.sub.H,H=1.3 Hz, 2H, 2-CH), 6.67 (d, .sup.3J.sub.H,H=1.3 Hz, 2H, 1-CH), 3.60 (s, 4H, 5-CH.sub.2), 3.46-3,41 and 3.33-3.30 (m, 12H, 7-CH.sub.2 to 12-CH.sub.2), 3.32 (s, 6H, 3-CH.sub.3), 3.18 (t, .sup.3J.sub.H,H=5.0 Hz, 2H, 13-CH.sub.2), 2.60 (t, .sup.3J.sub.H,H=5.0 Hz, 2H, 6-CH.sub.2).

[0243] .sup.13C NMR (101 MHz, chloroform-d)

[0244] δ [ppm]=145.2 (s, C-4), 126.5 (s, C-2), 121.2 (s, C-1), 70.4, 70.3, 70.3, 69.8, 69.7, 69.7 (s, C-7 to C-12), 52.7 (s, C-6), 50.3 (s, C-13 and C-5), 32.1 (s, C-3).

[0245] MS (ESI.sup.+): m/z=407.2517; calculated for [C18H30N8O3]H+ ([M+H]+): 407.2514. 429.2331; calculated for [C18H30N8O3]Na+ ([M+Na]+): 429.2333.

2-{2-(2-Azidoethoxy)ethoxy]ethoxyl}-N,N-bis[(1-methyl-1H-imidazole-2-yl)methyl]ethylamine

[0246] ##STR00042##

[0247] 2-{2-[2-(2-Azidoethoxy)ethoxy]ethoxyl}-N,N-bis[(1-methyl-1H-imidazole-2-yl)methyl]ethylazide (1.12 g, 2.8 mmol, 1.0 eq.) was dissolved in tetrahydrofuran (10 mL), was treated with palladium on activated charcoal and stirred under a hydrogen atmosphere over night at room temperature. The reaction mixture was filtered over Celite® and concentrated under reduced pressure to give the product as a yellow oil. Yield: 1.02 g (2.7 mmol, 96%).

[0248] .sup.1H NMR (400 MHz, chloroform-d)

[0249] δ [ppm]=6.81 (d, .sup.3J.sub.H,H=1.2 Hz, 2H, 2-CH), 6.73 (d, .sup.3J.sub.H,H=1.3 Hz, 2H, 1-CH), 3.68 (s, 4H, 5-CH.sub.2), 3.53-3.48 and 3.43-3.38 (m, 12H, 7-CH.sub.2 to 12-CH.sub.2), 3.40 (s, 6H, 3-CH.sub.3), 2.77 (t, .sup.3J.sub.H,H=5.2 Hz, 2H, 13-CH.sub.2), 2.70 (t, .sup.3J.sub.H,H=5.2 Hz, 2H, 6-CH.sub.2).

[0250] .sup.13C NMR (101 MHz, chloroform-d)

[0251] δ [ppm]=145.5 (s, C-4), 127.0 (s, C-2), 121.4 (s, C-1), 72.8, 70.5, 70.4, 70.2, 69.9, 69.9 (s, C-7 bis C-12), 52.9 (s, C-6), 50.5 (s, C-5), 41.4 (s, C-13), 32.3 (s, C-3).

[0252] MS (ESI.sup.+): m/z=381.2607; calculated for [C18H32N6O3]H+ ([M+H]+): 381.2609. 403.2425; calculated for [C18H32N6O3]Na+ ([M+Na]+): 403.2428.

N-{1-(1-methyl-1H-imidazol-2-yl)-2-[(1-methyl-1H-imidazol-2-yl]methyl]-5,8,11-trioxa-2-azatridecan-13-yl}aniline

[0253] ##STR00043##

[0254] 2-{2-[2-(2-Azidoethoxy)ethoxy]ethoxy}-N,N-bis[(1-methyl-1H-imidazole-2-yl)methyl]ethylamine (139 mg, 0.37 mmol, 1.2 eq.) was dissolved in 2-propanol (5 mL), followed by the addition of copper(I) iodide (6 mg, 0.03 mmol, 0.1 eq.), K.sub.3PO.sub.4 (132 mg, 0.62 mmol, 2.0 eq.), ethylene glycol (35 μL, 0.62 mmol, 2.0 eq.) and lodobenzene (35 μL, 0.31 mmol, 1.0 eq.). The reaction vessel was flushed with argon and stirred at 80° C. for 23 hours. Water (10 mL) was added, the reaction mixture extracted with ethyl acetate (2×15 mL) and the solvent of the combined organic phases removed under reduced pressure. The crude product was purified by column chromatography over silica gel (chloroform/methanol) to give the product as a yellow oil. Yield: 84 mg (0.18 mmol, 58%).

[0255] .sup.1H NMR (400 MHz, methanol-d.sub.4)

[0256] δ [ppm]=7.10 d(d, .sup.3J.sub.H,H=8.7 Hz, 7.3 Hz, 2H, 16-CH, 16′-CH), 6.98 (d, .sup.3J.sub.H,H=1.3 Hz, 2H, 2-CH, 2′-CH), 6.83 (d, .sup.3J.sub.H,H=1.3 Hz, 2H, 1-CH, 1′-CH), 6.66-6.60 (m, 3H, 17-CH, 15-CH, 15′-CH), 3.71 (s, 4H, 5-CH.sub.2, 5′-CH.sub.2), 3.64-3.55 and 3.46-3.40 (m, 12H, 7-CH.sub.2 to 12-CH.sub.2), 3.51 (s, 6H, 3-CH.sub.3, 3′-CH.sub.3), 3.22 (t, .sup.3J.sub.H,H=5.5 Hz, 2H, 13-CH.sub.2), 2.66 (t, .sup.3J.sub.H,H=5.3 Hz, 2H, 6-CH.sub.2).

[0257] .sup.13C NMR (101 MHz, chloroform-d)

[0258] δ [ppm]=150.0 (s, C-14), 146.4 (s, C-4, C-4′), 130.1 (d, C-16, C-16′), 126.9 (d, C-1, C-1′), 123.4 (d, C-2, C-2′), 118.3 (d, C-17), 114.2 (d, C-15, C-15′), 71.6, 71.6, 71.4, 71.1, 70.7, 70.6 (t, C-7 bis C-12), 54.2 (t, C-6), 51.6 (t, C-5), 44.7 (t, c-13), 33.1 (q, C-3, C-3′).

[0259] MS (ESI.sup.+): m/z=457.2921; calculated for [C.sub.24H.sub.36N.sub.6O.sub.3]H.sup.+ ([M+H].sup.+): 457.2933. 479.2743; calculated for [C.sub.24H.sub.36N.sub.6O.sub.3]Na.sup.+ ([M+Na].sup.+): 479.2752.

5-ethyl-4-hydroxy-1-methyl-N-{1-(1-methyl-1H-imidazol-2-yl)-2-[(1-methyl-1H-imidazol-2-yl)methyl]-5,8,11-trioxa-2-azatridecan-13-yl}-1-2-oxo-N-phenyl-1,2-dihydroquinoline-3-carboxamide

[0260] ##STR00044##

[0261] 5-ethyl-4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid (319 mg, 1.29 mmol, 1.0 eq.) was dissolved in tetrahydrofuran (5 mL), cooled to 0° C. under an argon atmosphere and triethylamine (679 μL, 4.90 mmol, 3.8 eq.) was added dropwise. The resulting solution was treated with N-{1-(1-methyl-1H-imidazol-2-yl)-2-[(1-methyl-1H-imidazol-2-yl)methyl]-5,8,11-trioxa-2-azatridecan-13-yl}aniline (578 mg, 1.29 mmol, 1.0 eq.) in tetrahydrofuran (2 mL) followed by the drop wise addition of thionyl chloride (112 μL, 1.55 mmol, 1.2 eq.) in tetrahydrofuran (488 μL) over 15 minutes at 0° C. After 20 h at room temperature the reaction mixture was washed with saturated NaHCO.sub.3 solution, extracted with chloroform (2×10 mL) and the solvent of the combined organic layers was removed under reduced pressure. After column chromatography over silica gel (ethyl acetate/methanol), the product was obtained as a brown solid. Yield: 600 mg (0.88 mmol, 68%).

[0262] mp.: 73° C. (methanol).

[0263] .sup.1H NMR (400 MHz, acetone-d.sub.6)

[0264] δ [ppm]=7.42 (t, .sup.3J.sub.H,H=8.0 Hz, 1H, 3-CH), 7.38-7.34 and 7.21-7.09 (m, 5H, 15CH to 17-CH), 7.21-7.26 (m, 1H, 2-CH), 7.01 (d, .sup.3J.sub.H,H=1.3 Hz, 2H, 29-CH, 29′-CH), 7.01-6.96 (m, 1H, 4-CH), 6.87 (d, .sup.3J.sub.H,H=1.3 Hz, 2H, 28-CH, 28′-CH), 3.83 (s, 4H, 26-CH.sub.2), 3.57 (s, 3H, 30-CH.sub.3), 3.56-3.41 (m, 14H, 18-CH.sub.2 to 24-CH.sub.2), 3.37 (s, 3H, 13-CH.sub.3), 3.26 (q, .sup.3J.sub.H,H=7.2 Hz, 2H, 11-CH.sub.2), 2.74 (t, .sup.3J.sub.H,H=5.2 Hz, 2H, 25-CH.sub.2), 1.22 (t, .sup.3J.sub.H,H=7.2 Hz, 1H, 12-CH.sub.3).

[0265] .sup.13C NMR (101 MHz, acetone-d.sub.6)

[0266] δ [ppm]=174.2 (NA), 168.9 (NA), 159.7 (NA), 146.2 (s, C-27, C-27), 145.8 (s, C-5), 144.0 (s, C-14), 142.7 (s, C-1), 131.9 (d, C-3), 129.0, 128.0, 127.6 (d, C-15 to 17C), 127.0 (d, C-28, C-28′), 125.1 (d, C-4), 122.5 (d, C-29, C-29′), 115.2 (s, C-8), 113.5 (d, C-2), 71.3, 71.2, 71.2, 70.9, 70.7, 70.6, 68.8 (t, C-18 to C-24), 53.6 (t, C-25), 51.4 (t, C-26), 32.8 (q, C-30), 30.7 (t, C-11), 29.5 (q, C-13), 17.6 (q, C-12).

[0267] C-6 could not be identified.

[0268] MS (ESI.sup.+): m/z=686.3660; calculated for [C.sub.37H.sub.47N.sub.7O.sub.6]H.sup.+ ([M+H].sup.+): 686.3661. 708.3480; calculated for [C.sub.37H.sub.47N.sub.7O.sub.6]Na.sup.+ ([M+Na].sup.+): 708.3480. 747.2795; calculated for [C.sub.37H.sub.46N.sub.7O.sub.6]Na.sup.+ ([M+Cu−H].sup.+): 747.2800.

fac-[Re(CO).SUB.3.(5-ethyl-4-hydroxy-1-methyl-N-{1-(1-methyl-1H-imidazol-2-yl)-2-[(1-methyl-1H-imidazol-2-yl)methyl]-5,8,11-trioxa-2-azatridecan-13-yl}-2-oxo-N-phenyl-1,2-dihydroquinoline-3-carboxamide).SUP.+

[0269] ##STR00045##

[0270] [ReBr.sub.3(CO).sub.3][NEt.sub.4].sub.2 (56 mg, 0.07 mmol, 1.0 eq.) was dissolved in Water (1 mL), added to a solution of 5-ethyl-4-hydroxy-1-methyl-N-{1-(1-methyl-1H-imidazol-2-yl)-2-[(1-methyl-1H-imidazol-2-yl)methyl]-5,8,11-trioxa-2-azatridecan-13-yl}-2-oxo-N-phenyl-1,2-dihydroquinoline-3-carboxamide (50 mg, 0.07 mmol, 1 eq.) in methanol (4 mL) and stirred over night at room temperature. The solvent was removed under reduced pressure and the product purified by column chromatography over C-18 reversed phase silica gel (water/methanol) to give a brown solid. Yield: 41 mg (0.04 mmol, 60%).

[0271] mp.: 115 (methanol)

[0272] MS (ESI.sup.+): m/z=956.2972; calculated for [C.sub.40H.sub.47N.sub.7O.sub.9Re].sup.+ ([M].sup.+): 956.2989.

[0273] HPLC: (Nucleosil 100-5 C18 (4×250 mm))

[0274] t.sub.Ret: 17.65 min

fac-[.SUP.99m.Tc(CO).SUB.3.(5-ethyl-4-hydroxy-1-methyl-N-{1-(1-methyl-1H-imidazol-2-yl)-2-[(1-methyl-1H-imidazol-2-yl)methyl]-5,8,11-trioxa-2-azatridecan-13-yl}-2-oxo-N-phenyl-1,2-dihydroquinoline-3-carboxamide)].SUP.+ ([^{99m.Tc]FEB054)}

[0275] ##STR00046##

[0276] To a mixture of K.sub.2[H.sub.3BCO.sub.2] (4.5 mg, 33 μmol), KNa-tartrate*4 H.sub.2O (7 mg, 25 μmol) and Na.sub.2B.sub.4O.sub.7*10 H.sub.2O (7 mg, 18 μmol) under argon atmosphere was added freshly eluated .sup.99mTcO.sub.4 (3950 MBq) in isotonic NaCl solution (1 mL). The reaction mixture was heated 20 minutes at 110° C. yielding fac-[.sup.99mTc(CO).sub.3(OH.sub.2).sub.3].sup.+. Then 5-ethyl-4-hydroxy-1-methyl-N-{1-(1-methyl-1H-imidazol-2-yl)-2-[(1-methyl-1H-imidazol-2-yl)methyl]-5,8,11-trioxa-2-azatridecan-13-yl}-2-oxo-N-phenyl-1,2-dihydroquinoline-3-carboxamide (1 mg, 1.4 μmol) in isotonic NaCl solution (200 μL) was added and the mixture heated for 15 minutes at 50° C. Purification of fac-[.sup.99mTc(CO).sub.3(5-ethyl-4-hydroxy-1-methyl-N-{1-(1-methyl-1H-imidazol-2-yl)-2-[(1-methyl-1H-imidazol-2-yl)methyl]-5,8,11-trioxa-2-azatridecan-13-yl}-2-oxo-N-phenyl-1,2-dihydroquinoline-3-carboxamide)].sup.+ was performed by gradient-HPLC using a Knauer system with a K-500 pump, a K-501 pump, a K-2000 UV detector, a Nal(TI) Scintibloc 51 SP51 γ-detector and a reversed phase C.sub.18 column (ACE-126-2510, 10 mm×250 mm). Eluent A: water (0.1% TFA). Eluent B: methanol (0.1% TFA).Gradient from 70% A to 0% A over 30 minutes, holding for 8 minutes and back to 70% A over 5 minutes at a flow rate of 5.5 ml min.sup.−1, detection at λ=254 nm. After collection of the product fraction, the solvent was removed under reduced pressure. The resulting residue was resolved in ethanol (10 μL) and a solution of Tween® 80 (190 μL, 1.6% in isotonic NaCl solution). Quality control of the solution was performed with co injection of the faci-[Re(CO).sub.3(5-ethyl-4-hydroxy-1-methyl-N-{1-(1-methyl-1H-imidazol-2-yl)-2-[(1-methyl-1H-imidazol-2-yl)methyl]-5,8,11-trioxa-2-azatridecan-3-yl}-2-oxo-N-phenyl-1,2-dihydroquinoline-3-carboxamide)].sup.+ (0.1 mg, 0.1 μmol) in methanol (15 μL) by gradient-HPLC using a Knauer system with two Smartline 1000 pumps, Smartline UV detector 2500 (Herbert Knauer GmbH), a GabiStar γ-detector (Raytest lsotopenmessgerate GmbH) and a reversed phase C.sub.18 column (Nucleosil 100-5 C-18 column 4.6 mm×250 mm). Eluent A: water (0.1% TFA). Eluent B: methanol (0.1% TFA).Gradient from 70% A to 0% A over 15 minutes, holding for minutes and back to 70% A over 5 minutes at a flow rate of 5.5 ml min.sup.−1, detection at λ=254 nm.

Experimental Details for the Synthesis of fac-[.sup.99mTc(bathophenanthrolinedisulfonic acid)(CO).sub.3(5-ethyl-4-hydroxy-1-methyl-2-oxo-N-phenyl-N-[1-(pyridin-3-yl)-2, 5, 8, 11-tetraoxatridecan-13-yl]1, 2-dihydroquinoline-3-carboxamide) [.sup.99mTc-FEB105)

3-(13-azido-2,5,8,11-tetraoxatridecyl)pyridine

[0277] ##STR00047##

[0278] Under an argon atmosphere, 2-{2-[2-(2-azidoethoxy)ethoxy]ethoxy}ethan-1-ol (4.47 g, 20.4 mmol, 1.2 eq) was dissolved in DMF (80 mL), cooled to 0° C. and stepwise treated with NaH (95%, 945 mg, 37.4 mmol, 2.2 eq.). After 30 minutes a solution of 3-(bromomethyl)pyridine hydrobromide (4.30 g, 17.0 mmol, 1.0 eq.) in DMF (60 mL) was added dropwise at 0° C. After three hours, the solvent was removed under reduced pressure, the residue redissolved in water (50 mL) and extracted with DCM (3×50 mL). The combined organic layers were washed with brine, dried over MgSO.sub.4 and concentrated under reduced pressure. The residue was purified by column chromatography over silica gel (chloroform/methanol) to give the product as a yellow oil. Yield: 2.94 g (9.4 mmol, 55%).

[0279] .sup.1H NMR (400 MHz, methanol-d.sub.4)

[0280] δ [ppm]=8.56-8.53 (m, 1H, 5-CH), 8.48-8.45 (m, 1H, 1-CH), 7.88-7.84 (m, 1H, 3-CH), 7.45-7.41 (m, 1H, 2-CH), 4.62 (s, 2H, 6-CH2), 3.70-3.62 (m, 14H, 7-CH2 to 13-CH2), 3.35 (t, 3JH,H=5.0 Hz, 2H, 14-CH2).

[0281] .sup.13C NMR (101 MHz, methanol-d.sub.4)

[0282] δ [ppm]=149.4 (d, C-5), 149.2 (d, C-1), 137.7 (d, C-3), 136.3 (s, C-4), 125.1 (d, C-2), 71.6, 71.6, 71.6, 71.6, 71.5, 71.1, 71.1 (t, C-7 to C-12), 71.3 (t, C-6), 51.8 (t, C-14).

[0283] MS (ESI.sup.+): m/z=311.1712; calculated for [C.sub.14H.sub.22N.sub.4O.sub.4]H.sup.+ ([M+H].sup.+): 311.1714. 333.1532; calculated for [C.sub.14H.sub.22N.sub.4O.sub.4]Na.sup.+ ([M+Na].sup.+): 333.1533.

1-(pyridin-3-yl)-2,5,8,11-tetraoxatridecan-13-amine

[0284] ##STR00048##

[0285] 3-(13-azido-2,5,8,11-tetraoxatridecyl)pyridine (1.23 g, 4.15 mmol, 1.0 eq.) was dissolved in THF (20 mL) and PPh.sub.3 (3.27 g, 12.45 mmol, 3.0 eq.) was added. After gas evolution stopped, H.sub.2O (5 mL) was added and the reaction mixture stirred at 40° C. for 16 hours. Afterwards, the solvent was removed under reduced pressure and the residue was purified by column chromatography over silica gel (ethyl acetate/methanol) to give the product as a yellow oil. Yield: 845 mg (3.0 mmol, 71%).

[0286] .sup.1H NMR (400 MHz, methanol-d.sub.4)

[0287] δ [ppm]=8.55-8.53 (m, 1H, 5-CH), 8.48-8.45 (m, 1H, 1-CH), 7.88-7.84 (m, 1H, 3-CH), 7.45-7.41 (m, 1H, 2-CH), 4.62 (s, 2H, 6-CH.sub.2), 3.70-3.59 (m, 12H, 7-CH.sub.2 to 12-CH.sub.2), 3.50 (t, .sup.3J.sub.H,H=5.3 Hz, 2H, 13-CH), 2.75 (t, .sup.3J.sub.H,H=5.3 Hz, 2H, 14-CH.sub.2).

[0288] .sup.13C NMR (101 MHz, methanol-d.sub.4)

[0289] γ [ppm]=149.4 (d, C-5), 149.3 (d, C-1), 137.7 (d, C-3), 136.3 (s, C-4), 125.1 (d, C-2), 73.5(t, C-13), 71.6, 71.6, 71.6, 71.3, 71.3, 71.1 (t, C-7 to C-12), 71.3 (t, C-6), 42.1 (t, C-14).

[0290] MS (ESI.sup.+): m/z=285.1808; calculated for [C.sub.14H.sub.22N.sub.2O.sub.4]H.sup.+ ([M+H].sup.+): 285.1809. 307.1628; calculated for [C.sub.14H.sub.22N.sub.2O.sub.4]Na.sup.+ ([M+Na].sup.+): 307.1628.

N-phenyl-1-(pyridin-3-yl)-2,5,8,11-tetraoxatridecan-13-amine

[0291] ##STR00049##

[0292] To a solution of 1-(pyridin-3-yl)-2,5,8,11-tetraoxatridecan-13-amine (400 mg, 1.41 mmol, 1.1 eq.) in i-propanol (10 mL), iodobenzene (143 μL, 1.28 mmol, 1.0 eq.), Cul (48 mg, 0.26 mmol, 0.2 eq.), K.sub.3PO.sub.4 (543 mg, 2.56 mmol, 2.0 eq.) and ethylene glycol (143 μml, 2.56 mmol, 2.0 eq.) were added and the reaction mixture was stirred at 80° C. for 40 hours. Afterwards, the reaction mixture was filtered over Celite®, the solvent was removed under reduced pressure and the residue was purified by column chromatography over silica gel (cyclohexane/ethyl acetate) to give the product as a yellow oil. Yield: 155 mg (0.43 mmol, 34%).

[0293] .sup.1H NMR (400 MHz, chloroform-d)

[0294] δ [ppm]=8.58-8.55 (m, 1H, 5-CH), 8.53-8.50 (m, 1H, 1-CH), 7.69-7.65 (m, 1H, 3-CH), 7.27-7.23 (m, 1H, 2-CH), 7.16-7.12 (m, 2H, 17-CH, 17′-CH), 6.70-6.65 (m, 1H, 18-CH), 6.62-6.59 (m, 2H, 16-CH, 16′-CH), 4.56 (s, 2H, 6-CH.sub.2), 3.68-3.63 (m, 14H, 7-CH.sub.2 to 13-CH.sub.2), 3.27 (t, .sup.3J.sub.H,H=5.3 Hz, 2H, 14-CH).

[0295] .sup.13C NMR (101 MHz, chloroform -d)

[0296] δ [ppm]=149.2 (d, C-5), 149.1 (d, C-1), 148.3 (s, C-15), 135.5 (d, C-3), 133.8 (s, C-4), 129.2 (d, C-17, C-17′), 123.5 (d, C-2), 117.5 (d, C-18), 113.1 (s, C-16, C-16′), 70.8 (t, C-6), 70.8, 70.7, 70.7, 70.6, 70.4, 69.9, 69.7, 69.7 (t, C-7 to C-13), 43.6 (t, C-14).

[0297] MS (ESI.sup.+): m/z=361.2120; calculated for [C.sub.20H.sub.28N.sub.2O.sub.4]H.sup.+ ([M+H].sup.+): 361.2122. 383.1936; calculated for [C.sub.20H.sub.28N.sub.2O.sub.4]Na.sup.+ ([M+Na].sup.+): 383.1941.

5-ethyl-4-hydroxy-1-methyl-2-oxo-N-phenyl-N-[1-(pyridin-3-yl)-2,5,8,11-tetraoxatridecan-13-yl]-1,2-dihydroquinoline-3-carboxamide

[0298] ##STR00050##

[0299] 5-ethyl-4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid (81 mg, 0.33 mmol, 1.0 eq.) was dissolved in tetrahydrofuran (4 mL), cooled to 0° C. under an argon atmosphere and triethylamine (173 μL, 1.25 mmol, 3.8 eq.) was added dropwise. The resulting solution was treated with N-phenyl-1-(pyridin-3-yl)-2,5,8,11-tetraoxatridecan-13-amine (118 mg, 0.33 mmol, 1.0 eq.) in tetrahydrofuran (2 mL) followed by the dropwise addition of thionyl chloride (29 μL, 0.40 mmol, 1.2 eq.) in tetrahydrofuran (471 μL) over 15 minutes at 0° C. After 20 h at room temperature the reaction mixture was washed with saturated NaHCO.sub.3 solution, extracted with chloroform (2×10 mL) and the solvent of the combined organic layers was removed under reduced pressure. After column chromatography over silica gel (ethyl acetate/methanol), the product was obtained as a brown solid. Yield: 40 mg (0.07 mmol, 21%).

[0300] .sup.1H NMR (600 MHz, methanol-d.sub.4)

[0301] δ [ppm]=8.52-8.50 (m, 1H, 5-CH), 8.44-8.41 (m, 1H, 1-CH), 7.83-7.81 (m, 1H, 3-CH), 7.44-7.40, 7.25-7.22 and 7.02-6.99 (m, 3H, 24-CH to 26-CH), 7.35-7.31 and 7.21-7.11 (m, 5H, 16-CH to 18-CH), 4.58 (s, 2H, 6-CH.sub.2), 3.69-3.59 (m, 14H, 7-CH.sub.2 to 13-CH.sub.2), 3.48-3.45 (m, 3H, 29-CH.sub.3), 3.25-3.22 (m, 2H, 14-CH.sub.2), 3.20-3.15 (m, 2H, 30-CH.sub.2), 1.19-1.15 (m, 3H, .sup.31-CH.sub.3).

[0302] .sup.13C NMR (151 MHz, methanol-d.sub.4)

[0303] δ [ppm]=147.9 (d, C-5), 147.9 (d, C-1), 136.2 (d, C-3), 131.0, 124.8 and 112.5 (d, C-24 to C-26), 128.2, 127.5, 126.8 (d, C-16 to C-18), 123.7 (d, C-2), 145.0 (s, C-23), 141.1 (s, C-27), 134.8 (s, C-4), 125.2 (s, C-22), 112.9 (s, C-20), 69.7 (t, C-6), 70.2, 70.1, 70.1, 70.0, 69.9, 69.7, 67.4 (t, C-7 to C-13), 43.3 (t, C-14), 29.8 (C-30), 28.9 (q, C-29), 16.1 (q, C-31).

[0304] MS (ESI.sup.+): m/z=590.2854; calculated for [C.sub.33H.sub.39N.sub.3O.sub.7]H.sup.+ ([M+H].sup.+): 590.2861. 612.2670; calculated for [C.sub.20H.sub.28N.sub.2O.sub.4]Na.sup.+ ([M+Na].sup.+): 612.2680.

[0305] ACE-126-2510, 10 mm×250 mm

[0306] HPLC: (ACE-126-2510, (10 mm×250 mm)), Eluent A: water (0.1% TFA). Eluent B: methanol (0.1% TFA).Gradient from 70% A to 0% A over 30 minutes, holding for 8 minutes and back to 70% A over 5 minutes at a flow rate of 5.5 ml min.sup.−1, detection at λ=254 nm.

[0307] t.sub.Ret: 23.68 min

fac-[.SUP.99m.Tc(bathophenanthrolinedisulfonic acid)(CO).SUB.3.(5-ethyl-4-hydroxy-1-methyl-2-oxo-N-phenyl-N-[1-(pyridin-3-yl)-2,5,8,11-tetraoxatridecan-13-yl]-1,2-dihydroquinoline-3-carboxamide)].SUP.− (^{99m.Tc-FEB105)}

[0308] ##STR00051##

[0309] To a mixture of K.sub.2[H.sub.3BCO.sub.2] (4.5 mg, 33 μmol), KNa-tartrate*4 H.sub.2O (7 mg, 25 μmol) and Na.sub.2B.sub.4O.sub.7*10 H.sub.2O (7 mg, 18 μmol) under argon atmosphere was added freshly eluated .sup.99mTcO.sub.4 in isotonic NaCl solution (1 mL). The reaction mixture was heated 20 minutes at 110° C. yielding fac-[.sup.99mTc(CO).sub.3(OH.sub.2).sub.3].sup.+. Then 5-ethyl-4-hydroxy-1-methyl-2-oxo-N-phenyl-N-[1-(pyridin-3-yl)-2,5,8,11-tetraoxatridecan-13-yl]-1,2-dihydroquinoline-3-carboxamide (0.5 mg, 0.8 μmol) in isotonic NaCl solution (150 μL) and bathophenanthrolinedisulfonic acid disodium salt hydrate (0.7 mg, 1.3 μmol) in isotonic NaCI solution (150 μL) were added and the mixture heated for 15 minutes at 50° C. Analysis was performed by gradient-HPLC using a Knauer system with two Smartline 1000 pumps, Smartline UV detector 2500 (Herbert Knauer GmbH), a GabiStar γ-detector (Raytest lsotopenmessgeräte GmbH) and a reversed phase C.sub.18 column (Nucleosil 100-5 C-18 column 4.6 mm×250 mm). Eluent A: water (0.1% TFA). Eluent B: methanol (0.1% TFA).Gradient from 70% A to 0% A over 25 minutes, holding for 5 minutes and back to 70% A over 5 minutes at a flow rate of 1 ml min.sup.−1, detection at λ=254 nm.

Experimental Details for the synthesis of 1-]14-(5-ethyl-4-hydroxy-N,1-dimethyl-2-oxo-1,2-dihydroquinoline-3-carboxamido)phenyl4-fluoro-1-oxobutane-2-sulfonic acid

Methyl 4-[(tert-butoxycarbonyl)(methyl)amino]benzoate

[0310] ##STR00052##

[0311] Methyl 4-(methylamino)benzoate (1.0 g, 6.1 mmol, 1.0 eq.) was dissolved in THF (40 mL). Then NEt.sub.3 (3.9 mL, 18.2 mmol, 3.0 eq.) and di-tert-butyl dicarbonate (1.2 mL, 9.1 mmol, 1.5 eq.) were added and the reaction mixture was refluxed for 16 hours. The solvent was removed under reduced pressure and the residue was purified by column chromatography over silica gel (cyclohexane/ethyl acetate) to give the product as a colorless oil. Yield: 780 mg (2.9 mmol, 49%).

[0312] .sup.1H NMR (400 MHz, chloroform-d)

[0313] δ [ppm]=7.99 (m, 2H, 2-CH/6-CH), 7.32 (m, 2H, 3-CH/5-CH), 3.90 (s, 3H, 8-CH3), 3.29 (s, 3H, 9-CH3), 1.46 (s, 9H, 12-CH3 to 14-CH3).

[0314] .sup.13C NMR (101 MHz, chloroform-d)

[0315] δ [ppm]=166.8 (C-7), 154.3 (C-10), 148.1 (C-4), 130.1 (C-2/C-6), 126.4 (C-1), 124.5 (C-3/C-5), 81.2 (C-11), 52.2 (C-8), 37.0 (C-9), 28.4 (C-12 to C-14).

[0316] MS (ESI.sup.+): m/z=288.1214; calculated for [C.sub.14H.sub.19NO.sub.4]Na.sup.+ ([M+Na].sup.+): 288.1206.

tert-Butyl [4-(2,2-dioxido-1,2-oxathiolane-3-carbonyl)phenyl](methyl)carbamate

[0317] ##STR00053##

[0318] In a flame dried schlenk flask under argon atmosphere, 1,3-propane sultone (123 mg, 1.01 mmol, 1.35 eq.) was dissolved in dry THF (15 mL) and cooled to −78° C. Over 30 minutes, n-buthyl lithium (0.63 mL, 1.6 M in hexane, 1.01 mmol, 1.35 eq.) was added dropwise. After 1 hour, methyl 4-[(tert-butoxycarbonyl)(methyl)amino]benzoate (200 mg, 0.754 mmol, 1.0 eq.) in THF (10 mL) was added dropwise at −78° C. After two hours at −78° C., acetic acid (500 μL) was added and the reaction mixture was allowed to warm to room temperature. The reaction mixture was washed with water (50 mL) and extracted with dichloromethane (3×100 mL). The combined organic fractions were dried over MgSO.sub.4, the solvent was removed under reduced pressure and the residue was purified by column chromatography over silica gel (cyclohexane/ethyl acetate) to give the product as a colorless oil. Yield: 110 mg (0.31 mmol, 41%).

[0319] .sup.1H NMR (400 MHz, chloroform-d)

[0320] δ [ppm]=8.05 (m, 2H, 2-CH/6-CH), 7.47 (m, 2H, 3-CH/5-CH), 5.08 (dd, .sup.3J.sub.H,H=8.9, 5.7 Hz, 1H, 8-CH), 4.65 (ddd, .sup.3J.sub.H,H=8.9, 7.4, 6.1 Hz, 1H, 10-CHa), 4.55 (ddd, .sup.3J.sub.H,H=8.9, 7.4, 6.1 Hz, 1H, 10-CHb), 3.33 (s, 3H,11-CH3), 3.30 (dddd, .sup.3J.sub.H,H32 13.2, 7.4, 6.1, 5.7 Hz, 1H, 9-CHa), 2.72 ((dddd, .sup.3J.sub.H,H32 13.2, 8.9, 7.4, 6.1 Hz,1H, 9-CHb), 1.50 (s, 9H, 14-CH3 to 16-CH3).

[0321] .sup.13C NMR (101 MHz, chloroform-d)

[0322] δ [ppm]=186.2 (C-7), 154.0 (C-12), 149.8 (C-4), 131.1 (C-1), 129.9 (C-2/C-6), 124.5 (C-3/C-5), 81.8 (C-13), 68.3 (C-10), 59.8 (C-8), 36.8 (C-11), 28.4 (C-14 bis C-16), 27.0 (C-9).

[0323] MS (ESI.sup.+): m/z=378.0985; calculated for [C.sub.16H.sub.21NO.sub.6S]Na.sup.+ ([M+Na].sup.+): 378.0982. 733.2051; calculated for [C.sub.16H.sub.21NO.sub.6S].sub.2Na.sup.+ ([2M+Na].sup.+): 733.2071.

(2,2-dioxido-1,2-oxathiolan-3-yl)(4-(methylamino)phenyl)methanone

[0324] ##STR00054##

[0325] tert-butyl [4-(2,2-dioxido-1,2-oxathiolane-3-carbonyl)phenyl](methyl)carbamate (305 mg, 0.86 mmol, 1.0 eq.) was cooled to 0° C. and HCl (2 mL, 4 M in dioxane, 8.0 mmol, 9.3 eq.) was added dropwise. After 30 minutes, a saturated solution of NaHCO.sub.3 (2 mL) was added. Water (10 mL) was added and the reaction mixture was extracted with ethyl acetate (3×50 mL). The combined organic fractions were dried over MgSO.sub.4, and the solvent was removed under reduced pressure. The product was obtained as a brownish solid without further purification. Yield: 180 mg (0.71 mmol, 82%).

[0326] MS (ESI.sup.+): m/z=256.0636; calculated for [C.sub.11H.sub.13NO.sub.4S]Na.sup.+ ([M+Na].sup.+): 256.0638. 278.0458; calculated for [C.sub.11H.sub.13NO.sub.4S]Na.sup.+ ([M+Na].sup.+): 278.0457.

N-[4-(2,2-dioxido-1,2-oxathiolane-3-carbonyl)phenyl]-5-ethyl-4-hydroxy-N,1-dimethyl-2-oxo-1,2-dihydroquinoline-3-carboxamide

[0327] ##STR00055##

[0328] 5-ethyl-4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid (190 mg, 0.77 mmol 1.1 eq.) and NEt.sub.3 (368 μL, 2.65 mmol, 3.8 eq.) were dissolved in dry THF (5 mL) under an argon atmosphere and cooled to 0° C. Over a period of five minutes SOCl.sub.2 (66 μL, 0.91 mmol, 1.3 eq.) in dry THF (454 μL) was added dropwise and stirred for 30 minutes. Afterwards, (2,2-dioxido-1,2-oxathiolan-3-yl)(4-(methylamino)phenyl)methanone (178 mg, 0.70 mmol, 1.0 eq.) in dry THF (5 mL) was added and the reaction mixture was allowed to warm to room temperature. After 90 minutes, a saturated solution of NaHCO.sub.3 (1 mL) and water (20 mL) were added and the reaction mixture was extracted with ethyl acetate (3×50 mL). The combined organic fractions were dried over MgSO.sub.4, and the solvent was removed under reduced pressure.

Binding of S100A9 protein TLR-4/MD2

[0329] Cy5.5-CES271 was successfully synthesized as described in the previous example. Studies were then conducted to verify that Cy5.5-CES271 maintained strong binding affinity for S100A9. In this regard, a specific ELISA (Enyme-linked Immunosorbent Assay) was developed to analyse S100A9 binding to TLR4. In this newly developed assay, the binding of S100A9 to TLR4 can be measured.

[0330] Briefly, TLR4/MD2 (3146-TM-050/CF, R&D Systems) was coupled to the wells of a 96-well plate and served as capturing molecule. After blocking of the unspecific binding sites by PBS/5% skim milk powder, plates were washed three times. S100A9 protein was added at a concentration of 2 μg/ml in the presence or absence of 100μM CES271-Cy5.5 and incubated for two hours at room temperature. Unbound S100 protein was removed by washing the plates for three times, followed by the addition of a primary anti-S100A9-antibody (1 μg/ml, polyclonal, rabbit). After a washing step, the secondary anti-rabbit-IgG-antibody coupled to HRP (1 μg/ml, Cell Signalling) was added. TMB was used as substrate for HRP to quantify binding efficiency by absorbance readings at 450 nm in an ELISA reader (Anthos Mikrosysteme).

[0331] The addition of novel non-peptidic S100A9 ligand (i.e. Cy5.5-CES271) was shown to markedly block binding of S100A9 to TLR4, as indicated by a decrease of signal given by the TLR4-S100A9 ELISA. The results, which are shown in FIG. 5, confirm that Cy5.5-CES271 binds to S100A9. This blocking study proves target specificity of Cy5.5-CES271, and shows that binding of S100A9 to TLR4/MD2 (which produces high absorption) can be efficiently blocked in the presence of Cy5.5-CES271 (resulting in low absorption).

Binding of Cv5.5-CES271 to Human and Murine S100A9

[0332] The binding constant of Cy5.5-CES271 to murine and human S100A9 was determined by fluorimetric measurements. 0,3788 μM S100A9 (5 μg ml.sup.−1 of homodimer S100A9) solved in 50 μl PBS was coated to the bottom of a 96-well plate and served as capturing molecule. For each S100A9 coated well a control well was used with 50 μl PBS alone. After a washing step, unspecific binding sites were blocked by PBS/5° A, skim milk powder. Cy5.5-CES271 was added at increasing concentrations. After 1 h incubation at 4° C., the supernatants were removed and the fluorescence intensity was measured with a fluorimeter. Non-linear regression analysis was performed with a one site saturation model, to calculate the binding constant of Cy5.5-CES271 to either murine or human S100A9. The K.sub.d-values of 2.66 μM (murine) and 2.06 μM (human) confirm that Cy5.5-CES271 is eligible for imaging purposes (FIG. 15). A strong binding affinity of the tracer to S100A9 could be observed that is not affected by the attachment of Cy5.5 to CES271.

Mice

[0333] Balb/c mice (Harlan Laboratories) and S100A9-deficient mice (S100A9.sup.−/−), backcrossed from C57BL/6 to Balb/c background (F10 generation) were used at the age of 10- to 14 weeks, sex and age matched for each set of experiments and housed under specific pathogen-free conditions.

Biodistribution of Cy5.5-CES271

[0334] The biodistribution of Cy5.5-CES271 and [.sup.99mTc]FEB054 injected to healthy Balb/c mice was analysed by the measurement of fluorescence intensity in various organs. FIG. 16 shows the tracer accumulation 1 and 3 h after injection. The tracer was injected at a dose of 2 nmol per mouse. We could observe a good tissue availability of Cy5.5-CES271 and an elimination that was mainly driven by renal excretion, as indicated by the high renal uptake and the increasing concentrations of the tracer in the urinary bladder urine in comparison to the relatively low hepatic uptake. This kinetics, that is different to previously published antibody based tracer anti-S100A9-Cy5.5.sup.7 kinetics favors Cy5.5-CES271 for imaging of organs neighboring the liver like the lung or the heart.

Human Serum Blood Stability of [.sup.99mTc]FEB054

[0335] Blood serum stability was tested in freshly prepared human blood serum at 37° C. (FIG. 17). Samples were taken after 10, 20, 30, 60, 90 and 120 minutes and analysed using a gradient HPLC system. 5 MBq of [.sup.99mTc]FEB054 in 20 μl PBS buffer was added to 200 μl of a freshly prepared human blood serum sample and incubated at 37° C. After 10, 20, 30, 60, 90 and 120 minutes, 20 μl were separated and diluted with 50 μl dichloromethane and 50 μl methanol. After centrifugation, 10 μl of the solution were analysed via gradient-HPLC using a Knauer system with two Smartline1000 pumps, Smartline UV-detector 2500 (Herbert Knauer GmbH), a GabiStar γ-detector (Raytest lsotopenmessgeräte GmbH) and a reverse phase C.sub.15 column 4.6 mm×250 mm). Eluent A: water (0.1% TFA). Eluent B: methanol (0.1% TFA). Gradient from 70% A to 0% A over 15 minutes, holding four minutes and bad to 70% A over 5 minutes at a flow rate of 5.5 ml min.sup.−1, detection at λ=254 nm.

Biodistribution of ].sup.99mTc]FEB054

[0336] Mice were injected and scanned under isoflurane/oxygen inhalation anaesthesia (1.4-1.8% isoflurane, 0.5 I O.sub.2/min). All animals were injected with a target dose of 2.5 MBq/g body weight) and a total injection volume ≦150 μl. For the image acquisition we used a NanoSPECT/CT-Plus preclinical camera (Mediso Medical Imaging Systems; Hungary), a 4-head gamma camera equipped with multi-pinhole collimators and a cone beam CT imaging system. In the shown studies we employed 9 pinholes/head with a diameter of 1.0 mm and a field of view of 30 mm×16 mm. 10 projections/scan and 60 seconds/projection resulting in a minimum of 100 kcounts/projection (in order to achieve high quality images with high statistics) were acquired. At the end of the SPECT measurement a CT scan was performed (same FOV as the SPECT scan) (55 kVp; 180 projections/rotation; 500 ms exposure time and 1 mm pitch with constant statistics). Data analysis was performed by CT based definitions of regions-of-interest and quantitative mesurements are expressed as kBq/mL.

In vivo Near-Infrared Optical Imaging (FRI)

[0337] In vivo fluorescence reflectance imaging was performed with an IVIS Spectrum small-animal imaging system (Xenogen). Images were acquired and analyzed using Living Image 4.X software (Xenogen). For the measurements the Cy5.5® filter set was used. Identical excitation/emission settings were used for all experiments. Fluorescence emission was measured by Fluorescence emission radiance per incident excitation irradiance (p/sec/cm2/sr/μW/cm2).

Irritant Contact Dermatitis (ICD)

[0338] ICD was induced by the application of 20 μl 1% croton oil in olive oil-acetone (1:4) to the ventral surface of the left ear of mice for 24 h (n=5 per group), whereas the right ear served as a control. FRI was performed 3 h after tracer application, corresponding to 27 h after croton oil treatment. The dermatitis mouse model showed significant accumulation of a quinoline-3-carboxamide compound covalently linked to a label at sites of inflammation. As shown in the results of imaging experiments depicted in FIG. 6, Cy5.5-CES271 showed significant accumulation in the inflamed ear of a dermatitis mouse model, which was not observed in S100A9.sup.−/− deficient mouse model. FIG. 6 shows preliminary FRI images of Cy5.5-CES271 in a mouse model of contact dermatitis of the left ear (WT vs. S100A9 deficient mice). Moreover, FRI images of Cy5.5-CES271 and Cy7-CES271 comparing the Total Radiant Efficiency in inflamed and control ears reveals highly significant correlation between the Cy7-CES271 and Cy5.5-aS100A9 signal in a mouse model of contact dermatitis (ICD), (FIG. 21). The comparison of Cy7-CES271 to the well characterized aS100A9-Cy5.5 was performed by parallel injection of the tracers. As we could not compare two tracers linked with the identical dye Cy5.5, CES271 was labeled with Cy7. This dye has distinct excitation and emission wavelengths and allows for a direct comparison of Cy7-CES271 to aS100A9-Cy5.5 by parallel injection of the tracers. FIG. 21 shows two images of the identical mouse recorded in the Cy5.5 (antibody) and Cy7 channel. In the direct comparison of Cy7-CES271 and aS100A9-Cy5.5 we observed an excellent correlation between the signal recorded from each tracer (FIG. 21 B; R.sup.2=0.96; n=8; p<0.001). This shows, that the binding of CES271 to S100A9 has the same dependency on local S100A9 expression as the S100A9 specific antibody based tracer aS100A9-Cy5.5. In the Cy7 channel the quantum yield is much lower compared to the Cy5.5 channel, which means that the recorded signal with Cy5.5-CES271 is higher than compared to Cy5.5-CES271 (right image; FIG. 21). The difference was qualified by a direct comparison CES271-Cy5.5 with its Cy7 derivate. Any significant differences between the relative accumulation of these tracers in the target tissue (n=6; p=0.33; data not shown) could not be observed while the absolute radiant efficiency obtained with Cy7-CES271 was lower than the one obtained with -Cy5.5-CES271 by a ratio of 0.199±0.013 (n=6; p<0.001; data not shown). In addition, it could be observed that the tracers have different kinetics which means that at the ideal imaging time points and their specific signal differ, which means that they have different ideal time point for imaging (data not shown). CES271-Cy5.5 was synthesized according to our published procedure (Faust et al. 2015). Cy5.5-NHS ester and Cy7-NHS ester was purchased from GE Healthcare at the highest purity grade available. Antibodies and antibody labeling (from Nat Com). Rabbit-derived antibodies addressing S100A9 were purified via protein G-sepharose and labelled with the fluorochrome Cy5.5 according to the manufacturer's instructions (GE Healthcare), as described previously. Briefly, 5 mg of the antibody was dialysed towards 100 mM Na.sub.2CO.sub.3 buffer, pH 8.0 and a 20-fold excess of the fluorochrome was added for 90 min at RT. The resulting tracer was purified from unbound dye using size exclusion chromatography (PD10 column). The labelling efficacy (dye/antibody ratio) was determined on the basis of ultraviolet-spectra of the purified dye-antibody compound using PBS as a reference buffer. Typically, the labelling resulted in 2.5-3.0 fluorochrome molecules per antibody, irrespective of the precursors.

[0339] Additionally, hybrid nuclear imaging (SPECT) and computed tomography (CT) was performed in the mouse model of contact dermatitis of the left ear. FIG. 19 shows a significant uptake of the 99mTc-labeled quinoline-3-carboxamide compound in the inflamed ear in contrast to the healthy control ear. Quantitative image analysis of the dynamic imaging 0-60 minutes post injection shows increasing uptake ratios of tracer in the inflamed ear versus blood and muscle, respectively. This improve in image contrast is of utmost importance for imaging based diagnostics.

[0340] These results demonstrate that tracer-tagged quinoline-3-carboxamide compounds provided herein show significant accumulation at sites of inflammation and/or inflammatory active diseases.

Collagen Induced Arthritis

[0341] Induction and imaging of CIA: Arthritis was induced in DBA/1j mice by injection of bovine collagen type II. Bovine collagen type II (bCII, MD Biosciences) was dissolved in 0.05 M acetic acid at a concentration of 2 mg/ml. DBA/1j mice were injected subcutaneously at the tail base with 100 μg bCII emulsified in CFA (Difco) and boostered on day 21 with 100 μg bCII in IFA at the same location. Mice were regularly inspected from day 14 after disease induction and scored for swelling, erythema and deformation of each joint three times a week. Imaging was performed after arthritis was clinically detectable in the majority of treated animals at indicated time points after tracer application of either Cy5.5-CES271 or [99mTc]FEB054.

[0342] SPECT/CT imaging of inflamed joints was recorded 1 h after iv injection of [99mTc]FEB054 (55 MBq of [99mTc]FEB054 per mouse). Individual feet of the mice were analysed separately according to the histological score. S100A9-expression as depicted by SPECT was in excellent correlation with clinical scoring, clearly discriminating clinically mild from severe joint inflammation with high SNR (FIG. 20). Even single affected joints could be clearly identified.

Myocardial Infarction

[0343] Myocardial infarction was induced by opening the chest of a mouse and transient ligation of the left coronary artery for 60 minutes. The ischemic event triggers acute inflammatory response to the tissue injury in the first hours/days followed by a remodeling process (involving less intense inflammatory processes) after about one week. FRI was performed one day after the surgical intervention to assess the intense acute inflammatory response and the images in FIG. 7 were acquired two hours after injection of 2 nmol Cy5.5-CES-271. FRI experiments in mouse models of myocardial infarction and atherosclerosis show significant accumulation of quinoline-3-carboxamide compound covalently linked to a label at sites of inflammation. FIG. 7 shows the accumulation of Cy5.5-CES271 at one day post occlusion of the LAD in a C57BI/6 mouse. FRI of the explanted heart (longitudinal mid-infarction cut) two hours after i.v. injection of 2 nmol Cy5.5-CES271 is shown. These results demonstrate that the tracer accumulates in the myocardial infarction (MI) in accordance with the presence of S100A9 as shown by histological staining. The absence of S100A9 in the remote myocardium (RM) is accompanied by a lack of Cy5.5-CES271 accumulation.

Atherosclerosis, Histology and Immunohistochemistry

[0344] Atherosclerosis is an inflammatory disease of the vessel wall. SR-BI.sup.−/−/apoE.sup.R61 h/h- mice (HypoE) are known to develop atherosclerotic lesions especially when set on a high fat and high choleresterol diet (HFC). Typical predeliction site is the aortic arch. 12-14 week old mice were set on HFC diet for 10 days. At day 10 of HFC diet Cy5.5-CES271 was injected i.v., the mice were sacrificed two hours p.i., the aortic arches were explanted, and measured ex-vivo by FRI.

[0345] The hearts and aorta were fixed overnight in 4% paraformaldehyde and embedded in paraffin. Sections measuring 4 μm in thickness were analyzed. For immunohistochemistry, paraffin skin sections (4 μm) were dewaxed, blocked with 10% fetal bovine serum, and incubated with rabbit anti-S100A9 or MAC-3 antibodies for 1 h at room temperature followed by a goat-anti-rabbit biotinylated antibody and 3,3′ diaminobenzidine (DAB).

[0346] FRI imaging of the explanted aorta (2 h p.i.) shows accumulation of Cy5.5-CES271 (yellow) in plaque lesions (white patches, bright light), FIG. 8. Systematic histological analysis of the aorta shows high levels of S100A9 in high uptake areas of Cy5.5-CES271 (A) and absence/very low levels of S100A9 in low uptake areas (B) of the tracer.

Acute Lung Injury (ALI)

[0347] LPS-induced lung inflammation was elicited via intranasal application of 50 pg or 10 pg of LPS. LPS from Escherichia coli 055:B5 was obtained from Sigma-Aldrich. Directly after the administration of LPS 2nmol of tracer were injected intravenously. Glycine saturated Cy5.5 served as a perfusion control for CES271-Cy5.5, while Cy5.5-labelled rabbit IgG without relevant specificity served as a control for aS100A9-Cy5.5. Mice were either assigned to fluorescence mediated tomography (FMT) to fluorescence reflectance imaging (FRI). For FRI imaging, the mice were sacrificed at the indicated time points. Afterwards, BALF was obtained and the lungs were fixed with 1% low melt aggarose.

[0348] 2D FRI lung imaging was performed using the Bruker FX Pro Imaging Station (Bruker Corporation). Excitation light was set to 630 nm using an appropriate bandpass filter. Emission at 700 nm was recorded using a filtre-equipped high-sensitivity (4-million-pixel) cooled charge-coupled device camera. Acquisition time was 5 s for each image.

[0349] Mice were intravenously injected with Cy5.5-labelled CES271 (CES271-Cy5.5). The animals underwent optical imaging at various time points after injection. In our blocking experiments each mouse received 2 pmol CES271 1h before 2 nmol of CES271-Cy5.5 were injected. Control mice received the same volume PBS. Imaging was performed at identical time points. 2D FRI lung imaging was performed using the Bruker FX Pro Imaging Station (Bruker Corporation). Excitation light was set to 630 nm using an appropriate bandpass filter.

[0350] Emission at 700 nm was recorded using a filtre-equipped high-sensitivity (4-million-pixel) cooled charge-coupled device camera. Acquisition time was 5 s for each image. Immunohistochemistry of ear sections (cryo) was performed as described earlier using purified rabbit anti-sera against murine S100A9 (Petersen et al. 2013). Briefly, after inhibition of endogenous peroxidase activity in frozen tissue sections Fc receptors were blocked by incubating in PBS/1% BSA including 50% normal goat serum (NGS). Slides were immunostained in a two-step procedure of incubation of primary antibody or isotype control followed by a horseradish peroxidase-conjugated secondary antibody using AEC as chromogen. Images were acquired by using an upright microscope (Axioskop, Zeiss). Statistical analysis (from Nat Com). Results are presented throughout as mean values±standard deviation (s.d.). P-values are given in the figure legends and values of p>0.05 were considered not to be significant. Statistical analyses were performed by parametric tests.

[0351] Cy5.5-CES271 enables imaging of LPS-induced lung inflammation with high sensitivity. Acute lung inflammation is frequently accompanied by extraordinary high levels of S100A8/A9 in BALF and serum. To evaluate whether visualizing S100A9-expression with CES271-Cy5.5 could facilitate early diagnosis of acute lung injury we tested a model of acute LPS-induced lung injury in mice in a pronounced, high dose and a milder, low dose setting. Systemic and local S100A8/A9 expression levels showed a dose and time dependent increase (FIG. 22A). The concentration of S100A8/S100A9 was especially high in the bronchoalveolar fluid (BALF) early after LPS application. Expression changes were confirmed by immunohistochemical analysis (FIG. 22A). Limited access of OI to the deep tissue target region on the one hand and continuous movement of the target region on the other hand are known challenges for imaging in this model. Therefore ex vivo analysis of the explanted lungs of affected animals was performed. At 3 and 6 hours after parallel LPS-/Cy5.5-CES271-application specific tracer accumulation could clearly be delineated. Unaffected lungs of animals that received LPS free saline as a control (FIG. 22B; n=3; p<0.01) did not show a similar tracer accumulation. As a control for possible early perfusion changes or tissue swelling in LPS treated animals, which could by themselves cause an increase in the recorded signal, we introduced an additional control. Glycine saturated Cy5.5 was injected into either LPS or saline treated mice. The recorded signal was comparable to the Cy5.5-CES271 control signal and significantly lower than Cy5.5-CES271 signal in LPS treated mice (FIG. 22B; n=3; p<0.01). This shows that Cy5.5-CES271 enables the detection of ALI early on in the disease course. S100A8/S100A9 serum levels were then measured by ELISA and correlated with the mean fluorescence intensity over the explanted lungs. We observed a good correlation between the systemic S100A8/S100A9 levels and the local inflammatory activity represented by the tracer uptake (FIG. 22C, R.sup.2=0.69; n=19; p<0.001).

[0352] A very interesting finding regarding the disease model was that the perfusion control did not show any significant differences in the tracer uptake between LPS treated and saline treated animals, which means that perfusion changes could be a late event in the development of an acute lung injury. This could be an explanation why to date perfusion based contrast agents fail to visualize ALI early on. Accordingly, these results aim to provide a new developmental option for the early diagnosis of ALI in the clinical setting.

[0353] The new tracer Cy5.5-CES271 represents the first approach with the potential of easy and quick translation of inflammation imaging in a broad range of clinical settings. It is based on a proven S100A9-binding Q-compound (Faust et al. 2015) that has already passed phase III trials. The linked indocyanine green derivate has separately found its way into clinical use as an optical imaging perfusion marker. Despite the limitations of this perfusion based contrast agent, the introduction of an optical scanner for visualization of the disease activity in rheumatoid arthritis proves that optical imaging is finding its way into clinical practice. In fact, Cy5.5-CES271 could readily be used for optical imaging of epithelial lesions. It enables monitoring of cutaneous inflammation and could find general use in the growing field of clinical fluorescence endoscopy. Monitoring of inflammatory bowel disease patients could be a first application, because this disease shows a burst of local expression of S100A8/S100A9 (calprotectin) early on and calprotectins proven prognostic potential makes it an attractive target in this setting.

[0354] The proven biocompatibility of its basic elements makes it presumable that Cy5.5-CES271also displays good biocompatibility and could pave the way for quick and successful introduction into clinical inflammation imaging. Compared to antibody based tracers Cy5.5-CES271 has much faster kinetics. This is especially important after radiolabelling for potential PET or SPECT usage of the tracer, which allows for inflammation monitoring in the whole body of the patient. For this purpose, a faster clearance of the radioactive tracer from the blood would reduce the effective dose that the patient receives under imaging purposes. In addition, antibodies are mainly cleared via the liver while our tracer is excreted via the Ren and adds an alternative route of elimination for patients that are compromised in either of the eliminating organs.

[0355] It will be clear that the invention may be practiced otherwise than as particularly described in the foregoing description and examples. Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, are within the scope of the appended claims.

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