COMPOUNDS AND METHODS FOR AMINE OXIDASE IMAGING
20230104758 · 2023-04-06
Inventors
Cpc classification
C12Y104/03006
CHEMISTRY; METALLURGY
G01N21/6428
PHYSICS
International classification
Abstract
The invention pertains to a functionalizable and enzyme-activatable fluorescent probe and methods for monitoring the activity of amine oxidases. Amine oxidases catalyze the oxidative deamination, e.g. of the ε-amine of a lysine to an aldehyde which in turn can form covalent bonds with neighboring side chains, e.g. in the context of collagen cross-linking. Amine oxidase activity can be correlated with collagen-associated diseases including pulmonary and hepatic fibrosis, cardiomyopathy and tumor metastasis.
Claims
1. A compound according to Formula I ##STR00053## wherein a and b are independently an integer from 0 to 10; A is a structure selected from the group consisting of ##STR00054## R.sup.3 and R.sup.4 are independently selected from the group consisting of NHR.sup.9′, —OR.sup.9′, SR.sup.9′, ##STR00055## linear or branched, substituted or non-substituted (C.sub.1-10)alkyl, (C.sub.2-10)alkenyl and (C.sub.2-10)alkynyl, or substituted or non-substituted carbocycle selected from the group consisting of (C.sub.3-10)carbocycle; substituted or non-substituted triphenylphosphine connected via the phosphorous; N-maleimidyl; and halogens, or one of R.sup.3 or R.sup.4 is a proteinogenic amino acid, a non-proteinogenic amino acid or a peptide, and the other of R.sup.3 or R.sup.4 is a halogen; L is absent or a linker; X, Y and Z are independently selected from the group consisting of O, N and S; R.sup.9′ is selected from the group consisting of hydrogen; linear or branched, substituted or non-substituted, optionally sulfonated, (C.sub.1-10)alkyl, (C.sub.2-10)alkenyl, or (C.sub.2-10)alkynyl; linear or branched, substituted or non-substituted (C.sub.1-20)alkyl ether, (C.sub.2-10)alkenyl ether, (C.sub.2-10)alkynyl ether, or (C.sub.4-10)carbocyclic ether; substituted or non-substituted carbocycle selected from the group consisting of (C.sub.3-10)carbocycle; substituted or non-substituted triphenylphosphine connected via the phosphorous; substituted or non-substituted (C.sub.3-6)heterocycle and (C.sub.7-C.sub.10)carbo- or hetero-bicycle having 1 to 3 heteroatoms each independently selected from N, O and S; a proteinogenic amino acid or non-proteinogenic amino acid; a peptide or a peptide comprising 1 to 2000 amino acids; a collagen peptide, fibronectin peptide, fibrillin peptide, elastin peptide, or an RGD (arginylglycylaspartic acid) peptide; and an antibody; R.sup.9 is selected from residues defined for R.sup.9′ and is further selected from the group consisting of azide, N-maleimidyl, —NH.sub.2, —OH and —SH.sub.2; R.sup.9″ is selected from the group consisting of halogen, substituted or non-substituted triphenylphosphine connected via the phosphorous, azide, —SR.sup.10, ##STR00056## R.sup.10 is selected from the group consisting of linear or branched, substituted or non-substituted (C.sub.1-10)alkyl, (C.sub.2-10)alkenyl, or (C.sub.2-10)alkynyl; and linear or branched, substituted or non-substituted (C.sub.1-20)alkyl ether, (C.sub.2-10)alkenyl ether, (C.sub.2-10)alkynyl ether, or (C.sub.4-10)carbocyclic ether; R.sup.5 is selected from the group consisting of hydrogen or —OH; and halogens; R.sup.6 and R.sup.8 are independently selected from the group consisting of hydrogen, —OH, and halogen; and R.sup.7 is each independently selected from the group consisting of hydrogen; linear or branched, substituted or non-substituted (C.sub.1-10)alkyl, (C.sub.2-10) alkenyl, or (C.sub.2-10)alkynyl; linear or branched, substituted or non-substituted (C.sub.1-20)alkyl ether, (C.sub.2-10)alkenyl ether, (C.sub.2-10)alkynyl ether, or (C.sub.4-10)carbocyclic ether; —N.sub.2 forming an azide with the nitrogen atom of A; and tert-Butyloxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (Fmoc), benzyloxycarbonyl(Cbz), acetyl (Ac), trifluoroacetic acid (TFA), phthalimide, benzyl (Bn), triphenylmethyl (Tr), benzylidene, or para-toluenesulfonyl (Ts); and salts and solvates thereof.
2. The compound according to claim 1, wherein a and b are independently an integer from 0 to 3; A is a structure selected from the group consisting of ##STR00057## R.sup.3 and R.sup.4 are independently selected from the group consisting of —NHR.sup.9′, —OR.sup.9′, SR.sup.9′, ##STR00058## linear or branched, substituted or non-substituted (C.sub.1-10)alkyl; triphenylphosphine connected via the phosphorous; N-maleimidyl; and halogen or one of R.sup.3 or R.sup.4 is a proteinogenic amino acid, a non-proteinogenic amino acid or a peptide, and the other of R.sup.3 or R.sup.4 is a halogen; L is absent or a linker selected from the group consisting of linear or branched, substituted or non-substituted (C.sub.1-10)alkyl; ##STR00059## wherein c and d are independently selected from 1, 2, 3, 4, and 5; and linear or branched, substituted or non-substituted (C.sub.1-20)alkyl ether; X, Y and Z are independently selected from the group consisting of O, N and S; R.sup.9′ is selected from the group consisting of hydrogen; linear or branched, substituted or non-substituted, optionally sulfonated, (C.sub.1-10)alkyl, (C.sub.2-10)alkenyl, or (C.sub.2-10)alkynyl; linear or branched, substituted or non-substituted (C.sub.1-20)alkyl ether; triphenylphosphine connected via the phosphorous; a proteinogenic amino acid or a non-proteinogenic amino acid; a peptide or a peptide comprising 1 to 2000 amino acids; a collagen peptide, fibronectin peptide, fibrillin peptide, elastin peptide, or an RGD (arginylglycylaspartic acid) peptide; and an antibody; R.sup.9 is selected from residues defined for R.sup.9′ and is further selected from the group consisting of azide, N-maleimidyl, —NH.sub.2, —OH and —SH.sub.2; R.sup.9″ is selected from the group consisting of azide, —SR.sup.10, ##STR00060## R.sup.10 is selected from the group consisting of linear or branched, substituted or non-substituted (C.sub.1-10)alkyl; and linear or branched, substituted or non-substituted (C.sub.1-20)alkyl ether; R.sup.5 is selected from the group consisting of hydrogen or —OH; and halogen; R.sup.6 and R.sup.8 are independently selected from the group consisting of hydrogen and halogen; and R.sup.7 is each independently selected from the group consisting of hydrogen; linear or branched, substituted or non-substituted (C.sub.1-10)alkyl; linear or branched, substituted or non-substituted (C.sub.1-20)alkyl ether; —N.sub.2 forming an azide with the nitrogen atom of A; and Boc, Fmoc, Cbz, Ac, TFA, phthalimide, Bn, Tr, benzylidene, or Ts;
3. The compound according to claim 1, wherein a is an integer from 0 to 3; b is 0; A is a structure selected from the group consisting of ##STR00061## R.sup.3 is ##STR00062## R.sup.4 is hydrogen, linear or branched, substituted or non-substituted (C.sub.1-10)alkyl; L is absent or a linker selected from the group consisting of linear or branched, substituted or non-substituted (C.sub.1-10)alkyl; ##STR00063## wherein c and d are independently selected from 1, 2, 3, 4, and 5; and linear or branched, substituted or non-substituted (C.sub.1-10)alkyl ether; X is selected from the group consisting of O, N and S; Z is O; R.sup.9 is selected from the group consisting of hydrogen or N-maleimidyl; linear or branched, substituted or non-substituted (C.sub.1-10)alkyl, (C.sub.2-10)alkenyl, or (C.sub.2-10)alkynyl; linear or branched, substituted or non-substituted (C.sub.1-20)alkyl ether; triphenylphosphine connected via the phosphorous: a proteinogenic amino acid, a non-proteinogenic amino acid, lysine, proline, glycine, 4-hydroxyproline, 4-aminoproline, or 4-aminooxyproline; a peptide or a peptide comprising 1 to 2000 amino acids; a collagen peptide, fibronectin peptide, fibrillin peptide, elastin peptide, or an RGD (arginylglycylaspartic acid) peptide; an antibody an anti-collagen, anti-elastin, anti-fibronectin, or anti-fibrillin antibody; and azide, —NH.sub.2, —OH, or —SH.sub.2; R.sup.9″ is selected from the group consisting of azide, —SR.sup.10, ##STR00064## R.sup.10 is selected from the group consisting of linear or branched, substituted or non-substituted (C.sub.1-10)alkyl, (C.sub.2-10)alkenyl, and (C.sub.2-10) )alkynyl; R.sup.5 is selected from the group consisting of hydrogen or —OH; and halogen; R.sup.6 and R.sup.8 are independently selected from the group consisting of hydrogen and halogen; and R.sup.7 is each independently selected from the group consisting of hydrogen; linear or branched, substituted or non-substituted (C.sub.1-10)alkyl; —N.sub.2 forming an azide with the nitrogen atom of A; and Boc, Fmoc, or Cbz.
4. The compound according to claim 1, wherein the compound is a compound according to Formula II ##STR00065## wherein a is 1 or 2; b is 0; A is a structure selected from the group consisting of ##STR00066## R.sup.3 is ##STR00067## R.sup.4 is hydrogen or linear or branched, substituted or non-substituted (C.sub.1-10)alkyl; L is absent, ##STR00068## wherein c and d are independently selected from 1, 2, 3, 4, and 5; X is selected from the group consisting of O and N; Z is O; R.sup.9 is selected from the group consisting of hydrogen or N-maleimidyl; linear or branched, substituted or non-substituted (C.sub.1-10)alkyl; triphenylphosphine connected via the phosphorous; a proteinogenic amino acid, a non-proteinogenic amino acid, lysine, proline, glycine, 4-hydroxyproline, 4-aminoproline, or 4-aminooxyproline; a collagen peptide, fibronectin peptide, fibrillin peptide, elastin peptide, or an RGD (arginylglycylaspartic acid) peptide; and azide, —NH.sub.2, —OH and —SH.sub.2; R.sup.9″ is selected from the group consisting of azide, ##STR00069## and R.sup.7 is each independently selected from the group consisting of hydrogen; and linear or branched, substituted or non-substituted (C.sub.1-10)alkyl.
5. (canceled)
6. (canceled)
7. (canceled)
8. (canceled)
9. (canceled)
10. (canceled)
11. A method for the detection of amine oxidase activity comprising the following steps: (a) providing a compound according to claim 1; (b) providing and contacting a cell, tissue, or body fluid with the compound of step (a) under conditions which allow for amine oxidase activity; (c) measuring fluorescence in the cell, tissue or body fluid, and (d) determining the activity and/or location of the amine oxidase based on the fluorescence measured in step (c).
12. A method for the diagnosis of an amine oxidase-associated disease and/or a collagen- or elastin-associated disease in a patient or sample of a patient in need thereof, comprising the following steps: (a) providing a compound according to claim 1; (b) contacting the patient or the sample of the patient in need thereof with an effective amount of the compound of step (a), wherein the effective amount is effective for detecting an amine oxidase activity; (c) measuring fluorescence in the patient or sample of the patient; and (d) determining activity and/or location of the amine oxidase based on the fluorescence measured in step (c).
13. The method according to claim 12, wherein the amine oxidase is selected from the group consisting of a lysyl oxidase (LOX), lysyl oxidase homolog 1 (LOXL1), lysyl oxidase homolog 2 (LOXL2), lysyl oxidase homolog 3 (LOXL3), lysyl oxidase homolog 4 (LOXL4), a primary-amine oxidase, a diamine oxidase, and a monoamine oxidase.
14. The method according to claim 12, wherein the disease is selected from the group consisting offibrosis, optionally pulmonary and hepatic fibrosis, cardiomyopathy, occipital horn syndrome (OHS), Menkes' syndrome, myocardial ischaemia, atherosclerosis, scleroderma, keloid disorder, liver cirrhosis, Alzheimer's and non-Alzheimer's dementia, Wilson's disease, primary biliary cirrhosis, chronic venous insufficiency, pseudoexfoliation syndrome, glaucoma, pelvic organ prolapse, endometriosis, intracranial aneurysms, heart failure, and cancer including tumor metastasis.
15. The method of claim 14, wherein the cancer is selected from the group consisting of colorectal cancer, bladder cancer, pancreatic cancer, breast cancer, head and neck squamous-cell carcinoma (HNSCC), laryngeal cancer, lung cancer, gastric cancer, prostate cancer, esophageal squamous cell cancer, endometrial cancer, testicular seminoma cancer, hepatocellular cancer, renal clear cell cancer, and basal and squamous skin cell carcinoma.
16. The method according to claim 13, wherein the amine oxidase is amine oxidase copper containing 1 (AOC1), amine oxidase copper containing 1 (AOC2), amine oxidase copper containing 1 (AOC3), monoamine oxidase A (MAOA), or monoamine oxidase (MAOAB).
17. The method according to claim 13, wherein the amine oxidase is a lysyl oxidase that has at least 80% sequence identity with at least one of SEQ ID NOs: 1 to 3.
18. The method according to claim 13, wherein the amine oxidase is a monoamine oxidase that has at least 80% sequence identity with at least one of SEQ ID NOs: 4 to 7.
19. The method according to claim 11, wherein the amine oxidase is selected from the group consisting of a lysyl oxidase (LOX), lysyl oxidase homolog 1 (LOXL1), lysyl oxidase homolog 2 (LOXL2), lysyl oxidase homolog 3 (LOXL3), lysyl oxidase homolog 4 (LOXL4), a primary-amine oxidase, a diamine oxidase, and a monoamine oxidase.
20. The method according to claim 19, wherein the amine oxidase is amine oxidase copper containing 1 (AOC1), amine oxidase copper containing 1 (AOC2), amine oxidase copper containing 1 (AOC3), monoamine oxidase A (MAOA), or monoamine oxidase (MAOAB).
21. The method according to claim 19, wherein the amine oxidase is a lysyl oxidase that has at least 80% sequence identity with at least one of SEQ ID NOs: 1 to 3.
22. The method according to claim 19, wherein the amine oxidase is a monoamine oxidase that has at least 80% sequence identity with at least one of SEQ ID NOs: 4 to 7.
23. The compound according to claim 1, wherein the linear or branched, substituted or non-substituted (C.sub.1-10)alkyl for at least one of R.sup.3, R.sup.4, R.sup.7, R.sup.9′ and/or R.sup.10 is selected from methyl, ethyl, and propyl.
24. The compound according to claim 1, wherein the (C.sub.3-10)carbocycle for at least one of R.sup.3, R.sup.4 and/or R.sup.9′ is an aromatic (C.sub.6)carbocycle.
25. The compound according to claim 1, wherein R.sup.3 and/or R.sup.4 are an aromatic (C.sub.6)carbocycle if a and/or b are 0.
26. The compound according to claim 1, wherein for at least one of R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.8 and/or R.sup.9″ the halogen is selected from F, Cl, Br, and I.
27. The compound according to claim 1, wherein if one of R.sup.3 or R.sup.4 is a proteinogenic amino acid, a non-proteinogenic amino acid, or a peptide, the other of R.sup.3 or R.sup.4 is a halogen.
28. The compound according to claim 1, wherein the linker is selected from the group consisting of: linear or branched, substituted or non-substituted (C.sub.1-10)alkyl, (C.sub.2-10)alkenyl, or (C.sub.2-10)alkynyl; a (C.sub.1-10) alkyl comprising one or more amide functionalities in the alkyl chain; ##STR00070## wherein c and d are independently selected from 1, 2, 3, 4, and 5; and linear or branched, substituted or non-substituted (C.sub.1-20)alkyl ether, (C.sub.2-10)alkenyl ether, (C.sub.2-10)alkynyl ether, and (C.sub.4-10)carbocyclic ether.
29. The compound according to claim 1, wherein Z is selected from the group consisting of O and N.
30. The compound according to claim 1, wherein for at least one of R.sup.7, R.sup.9 and/or R.sup.10, the (C.sub.1-20)alkyl ether is a polyethylene glycol (PEG) chain or a PEG chain with 1 to 10 ethylene oxide entities.
31. The compound according to claim 1, wherein for R.sup.9′, the (C.sub.3-10)carbocycle is a carbocycle substituted by a substituent selected from the group consisting of Cl, F, Br, substituted or non-substituted methyl, —(CF.sub.3), ethyl, propyl and cyclopropyl.
32. The compound according to claim 1, wherein for R.sup.9′, the (C.sub.7-C.sub.10)heterobicycle is a substituted or non-substituted (C.sub.7)heterobicycle having 2 heteroatoms selected from N and S.
33. The compound according to claim 1, wherein for R.sup.9′, the proteinogenic amino acid or non-proteinogenic amino acid is an aminooxy or hydrazide derivative of the proteinogenic or non-proteinogenic amino acid.
34. The compound according to claim 1, wherein for R.sup.9′, the proteinogenic amino acid or non-proteinogenic amino acid is selected from the group consisting of lysine, proline, glycine, 4-hydroxyproline, 4-aminoproline, and 4-aminooxyproline.
35. The compound according to claim 1, wherein for R.sup.9′, the peptide comprises 1 to 2000 amino acids.
36. The compound according to claim 1, wherein for R.sup.9′, the peptide comprises 1 to 10, [proline]-[4-hydroxyproline]-[glycine] units.
37. The compound according to claim 1, wherein for R.sup.9′, the antibody is an anti-collagen, anti-elastin, anti-fibronectin or anti-fibrillin antibody.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0194] The following Figures and Examples serve to illustrate the invention and are not intended to limit the scope of the invention as described in the appended claims. The term “Pacific Blue” or “PB”, as used herein, corresponds to 3-acetoxy-6,8-difluoro-7-hydroxycumarin.
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DETAILED DESCRIPTION OF THE INVENTION
Example 1: General Experimental Aspects
[0239] Materials and reagents were of highest commercially available grade and used without further purification. They were purchased from Acros Organics (Switzerland), Sigma Aldrich (Switzerland), Fischer (Switzerland), Bachem (Switzerland), Chem-Impex (USA), and TCI (Germany). Water used for peptide preparation and purification was nanopure with resistivity of 18.2 MΩ*cm, prepared by a Sartorius Arium611VF (Switzerland) water purification system or bi-distilled, water was purchased from AppliChem Panreac (Switzerland). For small molecule synthesis, reactions were monitored by thin layer chromatography using Merck Millipore (Switzerland) silica gel 60 F254 glass-backed plates. Visualization of compounds was achieved by UV-Vis or via staining with KMnO.sub.4. Flash chromatography and plug filtrations were performed using silica gel (60 Å pore size, and 230-400 mesh particle size, Sigma Aldrich (Switzerland)). Solvents for extraction and chromatography were of technical quality and distilled before usage. .sup.1H and .sup.13C NMR spectra were recorded on a Bruker DRX 400, a Bruker AV III 400 (400 MHz/100 MHz), or a Bruker AV III 600 (600 MHz/150 MHz). All spectra were recorded at 25° C., unless stated otherwise. Chemical shifts (5) are reported in parts per million (ppm) relative to the signal of tetramethylsilane (TMS) or residual solvent. The signals were assigned with COSY, HSQC, HMBC, and NOESY spectra. Solid phase peptide synthesis (SPPS) was performed on Rink amide ChemMatrix resin from Biotage (Sweden), and automated peptide synthesis on a Syro I peptide synthesizer (MultiSynTech GmbH, Witten Germany). High-resolution mass spectrometry (HRMS) was performed by the Molecular and Biomolecular Analysis (MoBiAs) service of the D-CHAB at ETH Zurich using a Bruker Daltons maXis equipped with an ESI (electrospray ionization) source and a Q-TOF ion analyzer, or a Bruker Daltonics SOLARIX equipped with a MALDI (matrix-assisted laser desorption/ionization)/ESI source and a Q-TOF ion analyzer. α-Cyano-4-hydroxycinnamic acid (CHCA) was used as MALDI-MS matrix. Analytical reversed-phase high-performance liquid chromatography (RP HPLC) was performed on a Dionex UHPLC, Ultimate 3000 (Thermo Fisher Scientific, Waltham/USA). Preparative RP HPLC purification were carried out on a Dionex UHPLC, Ultimate 3000 (Thermo Fisher Scientific, Waltham/USA). Circular dichroism (CD) spectra were recorded on a Chirascan plus spectrometer (Applied Photophysics Ltd, Leatherhead/UK) with a Nitropack nitrogen generator (Parker Balston, Haverhill/USA) and a temperature controller TC 125 (Quantum Northwest). The solutions were measured in a quartz cell with a path length of 1.0 mm (Hellma 110-QS). Peptides were dried by lyophilization on a Christ Alpha 2-4 LD plus (Kuhnner A G, Birsfelden/CH) lyophilizer. Absorption and emission spectroscopy. UV-visible spectra were obtained with a Cary-500 Scan spectrophotometer. The spectra were measured in quartz cuvettes (ThorLabs, CV10Q3500, Newton, N.J. USA). Samples were irradiated with an LED transilluminator (Roithner Lasertechnik, LED405-06V, Vienna, Austria) emitting at a wavelength as stated with an incident intensity of ca. 2 mW cm.sup.−2, measured with a power-meter (ThorLabs, PM100D, Newton, N.J. USA) equipped with a Si photodiode detector (ThorLabs, S120VC, Newton, N.J. USA). Fluorescence spectroscopy was measured in a Fluorolog 3 fluorimeter (Horiba Jobin-Yvon, Germany) fluorimeter with a cuvette sample changer for quartz cuvettes (ThorLabs, CV10Q3500F-E, Newton, N.J. USA). All measurements were conducted at 25° C. in 50 mM PBS pH 7.4 buffer solution under red light for ambient illumination to avoid photoactivation. Quantum yields were determined in PBS and as applicable correlated with known reference for Pacific Blue (Φ.sub.F=0.884 meas. versus Φ.sub.F=0.89). Ex vivo Fluorescence measurements were taken using a Tecan Spark 10M Multi-Mode Microplate Reader (Tecan, Mannedorf, Switzerland) at ambient temperature with a filter for excitation/emission (e.g. 405/460 nm respectively) unless stated otherwise, and values are expressed in relative fluorescent units (RFUs) for each experiment. All experiments were performed in triplicate. Graphical presentation and statistical analysis were performed using Graphpad Prism 7 software, (GraphPad Software, Inc., San Diego, USA.) with statistical significance determined as p<0.05 by ordinary one-way ANOVA with multiple comparisons.
Example 2: General and Exemplary Experimental Protocol for Determining Amine-Oxidase Activity
[0240] Fluorescence experiments for determining amine oxidase activity of a given enzyme are conducted using a fluorescence plate reader and a standard 96-well black polystyrene plate with a clear flat bottom. To the plate 150 μL of the following solutions are added to individual wells, e.g. with at least three replicates: buffer (e.g. 25 mM at pH 7.5), a solution in buffer of the amine-oxidase reactive probe, e.g. Compound 2, (e.g. at 100 μM), a buffered solution of the sample containing an amine-oxidase at the desired concentration(s) (e.g. 40 μg/mL), and a buffered solution of the sample containing amine-oxidases with addition of the probe, e.g. Compound 2, (e.g. at 100 μM). The plate is incubated in the dark at 37° C. for a period of time between 15 min and 24 hours before measurements are taken. Fluorescence is measured by exciting the unmasked probe at the absorbance maximum (e.g. 360 nm) or at 405 nm, and measuring the fluorescence at the emission maximum of the probe, for example at 460 nm. Enzyme activity is observed as fluorescence measured relative to the untreated controls, for example as expressed in relative fluorescent units (RFUs). The probe activation, and thus amine oxidase activity can be displayed in a diagram (see
Example 3: Synthesis of 3-Carboxymethyl-6,8-difluoro-7-hydroxycoumarin (“Pacific Blue” Ester)
[0241] ##STR00033##
[0242] Pacific Blue (ester) was prepared as previously reported in Chang, D.; Kim, K. T.; Lindberg, E.; Winssinger, N., Bioconj. Chem. 2018, 29, 158-163 via Pechmann condensation of 2,4-difluororesorcinol with dimethyl acetylsuccinate. .sup.1H NMR (400 MHz, DMF-d.sub.7) δ 7.61 (dd, J=11.8, 2.2 Hz, 1H), 3.79 (s, 2H), 3.71 (s, 3H), 2.48 (s, 3H).
Example 3a: Synthesis of 3-Carboxymethyl-6,8-dichloro-7-hydroxycoumarin
[0243] ##STR00034##
[0244] 2,4-dichlororesorcinol (1.68 g, 9.4 mmol) was dissolved in dimethyl acetylsuccinate (1.77 g, 9.5 mmol) by trituration. Concentrated H.sub.2SO.sub.4 (1.5 mL) was added, and the solution was stirred at r.t. for 24 h. The resulting viscous liquid was homogenized with the addition of methanol (1-2 mL) and then poured over crushed ice (100 mL). The precipitate was collected by filtration, and recrystallized from methanol/water to yield 3-carboxymethyl-6,8-dichloro-7-hydroxycoumarin as a purple crystalline solid. .sup.1H NMR (400 MHz, CD.sub.3CN) δ 7.71 (s, 1H), 3.66 (s, 5H), 2.35 (s, 3H). .sup.13C NMR (101 MHz, CD.sub.3CN) δ 171.54, 161.19, 152.23, 149.77, 149.10, 125.08, 118.93, 115.46, 109.76, 52.75, 33.51, 15.88. HRMS (ESI): m/z calcd. for C.sub.13H.sub.10Cl.sub.2NaO.sub.5: 338.9797 [M+Na].sup.+; found: 338.9795.
Example 4: 3-methylacetate-6,8-difluoro-7-(4-((tert-butoxycarbonyl)amino)propoxy)-coumarin
[0245] ##STR00035##
[0246] Pacific Blue (5.4 mmol, 1.56 g) and 3-(tert-butoxycarbonylamino)-propyl bromide (11 mmol, 2.6 g) were dissolved in 18 mL of anhydrous DMF. C.sub.5CO.sub.3 (8.1 mmol, 2.64 g) was added, and the resulting solution was heated to 60° C. for 2 hours. The solution was cooled to room temperature, and the reaction was quenched with 200 mL of an aqueous ammonium chloride solution, extracted with EtOAc (3×50 mL), dried over Na.sub.2SO.sub.4(s), and concentrated under reduced pressure. The crude mixture was purified by chromatography on silica gel (1-5% gradient of MeOH in dichloromethane), and concentrated under reduced pressure to produce 3-methylacetate-6,8-difluoro-7-(4-((tert-butoxycarbonyl)amino)propoxy)-coumarin (“Boc-PB-LOX-OMe”) as a clear oil (2.3 g, 96%). .sup.1H NMR (400 MHz, CD.sub.3CN) δ 7.27 (dd, J=12.0, 2.2 Hz, 1H), 5.38 (s, 1H), 4.25 (t, J=6.2 Hz, 2H), 3.60 (s, 5H), 3.16 (q, J=6.6 Hz, 2H), 2.26 (s, 3H), 1.84 (p, J=6.4 Hz, 2H), 1.32 (s, 9H). .sup.13C NMR (101 MHz, CD.sub.3CN) δ 171.31, 160.66, 156.92, 152.40 (dd, J=243.1, 4.6 Hz), 149.54 (t, J=2.8 Hz), 143.70 (dd, J=249.3, 6.3 Hz), 139.43 (dd, J=10.3, 2.5 Hz), 138.78 (dd, J=16.0, 10.9 Hz), 120.63 (d, J=0.8 Hz), 116.21 (dd, J=9.2, 1.3 Hz), 107.68 (dd, J=22.9, 3.6 Hz), 79.12, 73.76, 52.76, 37.83, 33.62, 31.21, 28.60, 15.96. .sup.19F NMR, F-H decoupled (377 MHz, CD.sub.3CN) δ −133.72 (d, J=5.4 Hz), −150.18 (d, J=5.4 Hz). .sup.19F NMR (377 MHz, CD.sub.3CN) δ −133.72 (dd, J=11.9, 5.4 Hz), −150.18 (d, J=5.4 Hz). HRMS (ESI): m/z calcd. for C.sub.21H.sub.25F.sub.2NNaO.sub.7: 464.1491 [M+Na].sup.+; found: 464.1494.
Example 4a: Synthesis of 3-methylacetate-6,8-dichloro-7-(4-((tert-butoxycarbonyl)amino)propoxy)-coumarin
[0247] ##STR00036##
[0248] 3-Carboxymethyl-6,8-dichloro-7-hydroxycoumarin (0.71 mmol, 227 mg) and 3-(tert-butoxycarbonylamino)-propyl bromide (1.45 mmol, 347 mg) were dissolved in 2.4 mL of anhydrous DMF. C.sub.sCO.sub.3 (1.07 mmol, 349 mg) was added, and the resulting solution was stirred and heated to 60° C. for 2 hours. The solution was cooled to room temperature, and the reaction was quenched with 100 mL of an aqueous ammonium chloride solution, extracted with EtOAc (3×50 mL), dried over Na.sub.2SO.sub.4(s), and concentrated under reduced pressure. The crude mixture was purified by chromatography on silica gel (1-5% gradient of MeOH in dichloromethane), and concentrated under reduced pressure to produce 3-methylacetate-6,8-dichloro-7-(4-((tert-butoxycarbonyl)amino)propoxy)-coumarin as a white solid (175 mg, 52%). .sup.1H NMR (400 MHz, CD.sub.3CN) δ 7.70 (s, 1H), 5.44 (s, 1H), 4.09 (t, J=6.2 Hz, 2H), 3.66 (s, 2H), 3.66 (s, 3H), 3.28 (q, J=6.6 Hz, 2H), 2.34 (s, 3H), 2.01-1.94 (m, 2H), 1.40 (s, 9H). .sup.13C NMR (101 MHz, CD.sub.3CN) δ 171.27, 160.85, 156.93, 154.33, 149.34, 148.90, 125.42, 125.26, 120.79, 118.99, 117.72, 73.13, 52.78, 38.20, 33.62, 33.50, 31.19, 28.63, 15.99. HRMS (ESI): m/z calcd. for C.sub.21H.sub.25Cl.sub.2NNaO.sub.7: 496.0900 [M+Na].sup.+; found: 496.0901.
Example 4b: Synthesis of 3-methylacetate-6,8-dichloro-7-(4-ammoniumpropoxy)-coumarin Chloride
[0249] ##STR00037##
[0250] 3-methylacetate-6,8-dichloro-7-(4-((tert-butoxycarbonyl)amino)propoxy)-coumarin (0.042 mmol, 20 mg) was dissolved in 250 μL of 4N HCl in dioxane at ambient temperature and stirred for 1 hour. Solvent was then removed from the resulting slurry by evaporation with a gentle stream of compressed air, and the white solid was dried overnight under high vacuum to produce the HCl salt 3-methylacetate-6,8-dichloro-7-(4-ammoniumpropoxy)-coumarin chloride as a white solid (17 mg, quant.). .sup.1H NMR (400 MHz, Deuterium Oxide) δ 7.77 (s, 1H), 4.26 (t, J=5.7 Hz, 2H), 3.81 (s, 2H), 3.77 (s, 3H), 3.39 (t, J=7.5 Hz, 2H), 2.39 (s, 3H), 2.26 (dt, J=12.7, 6.1 Hz, 2H). .sup.13C NMR (126 MHz, D.sub.2O) δ 173.01, 162.07, 152.47, 151.06, 146.91, 124.69, 124.52, 118.74, 117.94, 116.36, 71.80, 52.88, 37.41, 32.71, 27.32, 15.10.
Example 5: 3-carboxy-6,8-difluoro-7-(4-((tert-butoxycarbonyl)amino)propoxy)-coumarin (Compound 1)
[0251] ##STR00038##
[0252] Boc-PB-LOX-OMe (5.2 mmol, 2.3 g) was dissolved in 8 mL of 1:1 THF:MeOH, and NaOH (15.6 mmol, 624 mg) was added as an aqueous solution in 1 mL of H.sub.2O, and the resulting solution was heated to 50° C. for 5 hours. Upon completion as observed by thin layer chromatography, (R.sub.f=0.1 for 5% MeOH in DCM), the solution was cooled to room temperature, and the reaction was carefully acidified to pH 2 with 1M HCl, and immediately extracted with EtOAc (3×50 mL), dried over Na.sub.2SO.sub.4(s), and concentrated under reduced pressure to give Compound 1 as a white solid (2.2 g, 99%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 12.53 (s, 1H), 7.66 (d, J=12.5 Hz, 1H), 6.89 (t, J=5.7 Hz, 1H), 4.29 (t, J=6.3 Hz, 2H), 3.63 (s, 2H), 3.11 (q, J=6.5 Hz, 2H), 2.38 (s, 3H), 1.84 (p, J=6.6 Hz, 2H), 1.37 (s, 9H). .sup.13C NMR (101 MHz, DMSO) δ 171.06, 159.32, 155.61, 152.13, 152.08, 149.71, 149.67, 148.34, 143.36, 143.29, 140.88, 140.82, 137.90, 137.88, 137.80, 137.78, 137.32, 137.21, 137.16, 137.05, 119.92, 115.16, 115.07, 107.40, 107.18, 77.53, 72.55, 36.41, 32.94, 29.97, 28.21, 15.39. .sup.19F NMR, F-H decoupled (376 MHz, DMSO-d.sub.6) δ −132.45 (d, J=5.0 Hz), −149.31 (d, J=5.1 Hz). .sup.19F NMR (376 MHz, DMSO-d.sub.6) δ −132.45 (dd, J=11.9, 4.9 Hz), −149.31 (d, J=4.7 Hz). HRMS (ESI): m/z calcd. for C.sub.20H.sub.23F.sub.2NNaO.sub.7: 450.1335 [M+Na].sup.+; found: 450.1340.
Example 5a: Synthesis of MRA_3102
[0253] ##STR00039##
[0254] MRA_3102. 2 (0.68 mmol, 300 mg) was dissolved in 3.4 mL of THF, and N-hydroxysuccinimide (0.68 mmol, 78 mg) was added. The resulting solution was cooled in an ice batch, and DCC was added (0.68 mmol, 140.2 mg). The reaction mixture was stirred on ice for 30 minutes, and then allowed to come warm to ambient temperature overnight. DCU was filtered, and the solution was concentrated by rotary evaporation. The crude residue was resuspended in EtOAc, and filtered, and again concentrated by rotary evaporation, and this process was repeated 2× until no more precipitate could be observed in solution. The crude NHS-ester was then concentrated under reduced pressure to produce MRA_3102 as a white solid (353 mg, 99%). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.17 (dd, J=11.2, 2.2 Hz, 1H), 4.80 (s, 1H), 4.35 (t, J=6.0 Hz, 2H), 4.08 (s, 2H), 3.36 (q, J=6.2 Hz, 2H), 2.83 (s, 4H), 2.41 (s, 3H), 2.00 (p, J=6.4 Hz, 2H), 1.44 (s, 9H). .sup.13C NMR (101 MHz, CDCl.sub.3) δ 168.81, 165.57, 159.57, 156.18, 151.73 (dd, J=246.0, 4.2 Hz), 149.64 (t, J=2.7 Hz), 143.15 (dd, J=253.2, 5.7 Hz), 138.97 (dd, J=10.3, 2.6 Hz), 138.70 (dd, J=15.6, 10.7 Hz), 119.85 (d, J=13.4 Hz), 117.63, 114.98 (d, J=8.8 Hz), 106.25 (dd, J=22.4, 3.7 Hz), 77.36, 73.09, 37.66, 30.41, 30.08, 28.52, 25.70, 16.06. .sup.19F NMR, F-H decoupled (376 MHz, CDCl.sub.3) δ −131.48 (d, J=5.8 Hz), −146.94 (d, J=5.8 Hz). .sup.19F NMR (376 MHz, CDCl.sub.3) δ −131.48 (dd, J=10.4, 5.5 Hz), −146.94 (d, J=4.8 Hz).
Example 6: 3-carboxy-6,8-difluoro-7-(4-((tert-butoxycarbonyl)amino)propoxy)-coumarin (Compound 2)
[0255] ##STR00040##
[0256] Compound 1 (0.047 mmol, 20 mg) was dissolved in 250 μL of 4N HCl in dioxane at ambient temperature and stirred for 1 hour. Solvent was then removed from the resulting slurry by evaporation with a gentle stream of compressed air, and the white solid was dried overnight under high vacuum to produce the HCl salt of Compound 2 as a white solid (17 mg, quant.). .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.13 (s, 3H), 7.67 (dd, J=12.1, 2.1 Hz, 1H), 4.37 (t, J=6.1 Hz, 2H), 3.62 (s, 2H), 2.97 (t, J=7.5 Hz, 2H), 2.36 (s, 3H), 2.05 (p, J=6.4 Hz, 2H). .sup.13C NMR (101 MHz, DMSO-d.sub.6) δ 171.14, 159.38, 150.92 (dd, J=242.9, 4.2 Hz), 148.43 (t, J=2.5 Hz), 142.14 (dd, J=249.1, 6.2 Hz), 137.87 (dd, J=10.1, 2.3 Hz), 136.95 (dd, J=16.3, 11.0 Hz), 120.14, 115.41 (d, J=9.3 Hz), 107.44 (dd, J=22.5, 3.4 Hz), 71.92, 66.43, 35.84, 33.06, 27.65, 15.51. .sup.19F NMR, F-H decoupled (376 MHz, DMSO-d.sub.6) δ −132.51 (d, J=4.9 Hz), −149.30 (d, J=4.9 Hz). .sup.19F NMR (376 MHz, DMSO-d.sub.6) δ −132.51 (dd, J=12.0, 4.9 Hz), −149.30 (d, J=5.1 Hz). HRMS (ESI): m/z calcd. for C.sub.15H.sub.16F.sub.2NO.sub.5: 328.0991 [M+H].sup.+; found: 328.0989.
Example 7: 3-methylacetate-6,8-difluoro-7-(4-((tert-butoxycarbonyl)amino)butoxy)-coumarin (MRA 3068)
[0257] ##STR00041##
[0258] 3-Carboxymethyl-6,8-difluoro-7-hydroxycoumarin (Pacific Blue) (1 mmol, 284 mg) and 3-(tert-butoxycarbonylamino)-butyl bromide (2 mmol, 504 mg) were dissolved in 2 mL of anhydrous DMF. C.sub.sCO.sub.3 (1.5 mmol, 489 mg) was added, and the resulting solution was heated to 60° C. for 2 hours. The solution was cooled to room temperature, and the reaction was quenched with 100 mL of an aqueous ammonium chloride solution, extracted with EtOAc (3×50 mL), dried over Na.sub.2SO.sub.4(s), and concentrated under reduced pressure. The crude mixture was purified by chromatography on silica gel (1-5% gradient of MeOH in dichloromethane), and concentrated under reduced pressure to produce MRA_3068 as a white solid (341 mg, 75%). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.13 (dd, J=11.4, 2.3 Hz, 1H), 4.60 (s, 1H), 4.29 (tt, J=6.2, 1.0 Hz, 2H), 3.73 (s, 2H), 3.71 (s, 3H), 3.19 (s, 2H), 2.34 (s, 3H), 1.86-1.78 (m, 2H), 1.73-1.65 (m, 2H), 1.43 (s, 9H). .sup.13C NMR (101 MHz, CDCl.sub.3) δ 170.35, 160.05, 156.13, 151.71 (dd, J=245.5, 4.5 Hz), 148.03 (t, J=2.7 Hz), 143.14 (dd, J=253.0, 6.0 Hz), 138.76 (dd, J=10.5, 2.5 Hz), 138.33 (dd, J=15.7, 10.7 Hz), 119.76, 115.20 (dd, J=8.8, 1.0 Hz), 106.03 (dd, J=22.4, 3.7 Hz), 74.73 (t, J=3.5 Hz), 52.54, 33.01, 28.53, 27.34, 26.54, 15.81. .sup.19F NMR, F-H decoupled (376 MHz, CDCl.sub.3) δ −131.81 (d, J=5.6 Hz), −147.25 (d, J=5.8 Hz). .sup.19F NMR (376 MHz, CDCl.sub.3) δ −131.81 (dd, J=11.4, 5.7 Hz), −147.25 (dd, J=5.7, 2.0 Hz). HRMS (ESI): m/z calcd. for C.sub.22H.sub.31F.sub.2N.sub.2O.sub.7: 473.2094 [M+NH.sub.4].sup.+; found: 473.2087.
Example 8: 3-Carboxy-6,8-difluoro-7-(4-((tert-butoxycarbonyl)amino)butoxy)-coumarin (MRA_3069)
[0259] ##STR00042##
[0260] MRA_3068 (0.5 mmol, 241 mg) was dissolved in 1.3 mL of 1:1 THF:MeOH, and NaOH (1.6 mmol, 64 mg) was added as an aqueous solution in 0.6 mL of H.sub.2O, and the resulting solution was heated to 50° C. for 5 hours. Upon consumption of starting material as observed by thin layer chromatography, (R.sub.f=0.1 for 5% MeOH in DCM), the solution was cooled to room temperature, and the reaction was carefully acidified to pH 2 with 1M HCl, and immediately extracted with EtOAc (3×50 mL), dried over Na.sub.2SO.sub.4(s), and concentrated under reduced pressure to produce MRA_3069 as a clear oil (231 mg, 97%). .sup.1H NMR (400 MHz, Methanol-d.sub.4) δ 7.40 (dd, J=11.8, 2.2 Hz, 1H), 4.30 (t, J=6.2 Hz, 2H), 3.70 (s, 2H), 3.11 (t, J=6.8 Hz, 2H), 2.38 (s, 3H), 1.85-1.76 (m, 3H), 1.71-1.63 (m, 3H), 1.42 (s, 9H). .sup.13C NMR (101 MHz, Methanol-d.sub.4) δ 173.51, 161.70, 158.51, 153.00 (dd, J=244.3, 4.4 Hz), 150.12 (t, J=2.7 Hz), 144.08 (dd, J=250.5, 6.2 Hz), 139.63 (dd, J=10.3, 2.5 Hz), 139.28 (dd, J=16.0, 10.9 Hz), 121.06, 116.66 (d, J=9.1 Hz), 107.66 (dd, J=22.9, 3.6 Hz), 79.85, 75.86 (t, J=3.4 Hz), 40.86, 33.62, 28.77, 28.34, 27.25, 15.68. .sup.19F NMR, F-H decoupled (376 MHz, Methanol-d.sub.4) δ −133.41 (d, J=5.5 Hz), −150.56 (d, J=5.5 Hz). .sup.19F NMR (377 MHz, Methanol-d.sub.4) δ −133.41 (dd, J=11.8, 5.4 Hz), −150.55 (d, J=5.4 Hz). HRMS (ESI): m/z calcd. for C.sub.21H.sub.23F.sub.2NNaO.sub.7: 462.1346 [M+Na-2H].sup.−; found: 462.1356.
Example 9: Ex Vivo Experiments—General Aspects
[0261] Skin tissue used for enzymatic analysis of LOX activity was harvested (1 cm×1 cm) and placed in 2 ml of ice-cold PBS. Skin tissue was homogenized using an Ultra-TURRAX homogenizer (KA®-Werke GmbH & CO. KG, Staufen, Germany) and samples were spun at 2000×g to pellet out tissue debris. The supernatant was further purified using a 0.2 m filter. Tissue homogenates were then evaluated with regard to total in solution protein retrieval using a nanospec absorbance measurement standardized to BSA at 1 mg/mL (Pierce™, Thermo Fisher Scientific Inc, Waltham, USA). Homogenates were diluted to appropriate concentrations (e.g. 1 mg/ml total protein) with 25 mM PIPES, 0.5% Triton X-100 (Sigma Aldrich, St Louis, Mass., USA) and pre-incubated with the probe (10 μM or 100 μM) at 37° C. in the dark for the designated period of time prior to fluorescence measurements. The resultant fluorescence was then measured using a Tecan Spark 10M Multi-Mode Microplate Reader (Tecan, Männedorf, Switzerland) at ambient temperature with a filter for excitation/emission (e.g. 405/460 nm respectively) as defined for the experiment. Activity is observed as fluorescence measured, and is expressed in relative fluorescent units (RFUs). Graphical presentation and statistical analysis were carried out using Graphpad Prism 7 software, (GraphPad Software, Inc., San Diego, USA.) with statistical significance determined as p<0.05.
Example 10: General Protocols for the Synthesis of Collagen Model Peptides 3, 4 and 5
[0262] Protocol A—General procedure for swelling: Before automated peptide synthesis, the resin was swelled in CH.sub.2Cl.sub.2 for 15 min while shaking. Then the resin was drained and washed with DMF (3×6 mL) and drained again. Protocol B—General protocol for automated peptide synthesis: For automated peptide synthesis, a Syro I peptide synthesizer (Biotage, Sweden) was used. Couplings were performed either with the appropriate Fmoc-protected amino acid or the trimer Fmoc-Pro-Hyp-Gly-OH. After swelling the resin in DMF on the synthesizer, i-Pr.sub.2NEt (9 equiv. as a 3 M solution in NMP (N-methyl-2-pyrrolidone)), HATU (3 equiv., 0.5 M in DMF) and the Fmoc-amino acid/Fmoc-tripeptide (3 equiv., 0.5 M in DMF) were added to the resin. The mixture was allowed to react in intervals of 1 min. agitation and 5 min. rests for 30 min. (2×) and was then washed with DMF (5×). Fmoc-deprotection was carried out by addition of a solution of 40% (v/v) piperidine in DMF and reaction for 1 min. This step was repeated 4 times. The resin was then washed with DMF (5×). Tripeptide couplings and Fmoc-deprotections were repeated until the desired peptides were obtained. For the automated synthesis of CMPs no acylation (capping) was performed. Protocol C—On resin N-terminal functionalization with Compound 1: Functionalization was performed manually at room temperature on the solid support-bound peptide. Compound 1 (2.0 equiv.), HATU (1.9 equiv.) and i-Pr.sub.2NEt (4 equiv.) were dissolved in DMF (1-2 mL). After pre-activation for 5 min, the coupling mixture was added to the resin and agitated for 1-2 hrs. The resin was washed with CH.sub.2Cl.sub.2 (3×), DMF (3×), CH.sub.2Cl.sub.2 (3×), and petroleum ether (2×). The reaction was monitored by the qualitative color tests on bead or by LC-MS after test cleavage (see Protocol E). Protocol D—Cleavage from the resin: The resin was shaken for 1 h in a mixture of TFA/(i-Pr.sub.2).sub.3Si—H/H.sub.2O (92.5:2.5:2.5), and washed with pure TFA (2×). The peptide in solution was collected by filtration in a conical flask. Addition of ice-cold Et.sub.2O afforded the peptide as a white precipitate. The solid was isolated by centrifugation followed by decantation. The solid was suspended in Et.sub.2O, sonicated, centrifuged again and the supernatant was decanted. The residual white solid was dissolved in water/CH.sub.3CN, frozen, and lyophilized to obtain a white foam. Protocol E—Purification and analysis by RP HPLC: CH.sub.3CN (A) and H.sub.2O containing 1% CH.sub.3CN and 0.1% TFA (B) were used as eluents. For semi-preparative HPLC a flow rate of 6 mL/min, for analytical HPLC a flow rate of 1 mL/min and for LC-MS a flow rate of 0.5 mL/min was used. After purification by semi-preparative HPLC all collected fractions were analyzed by analytical HPLC or LC-MS and only pure fractions were combined. Amine containing CMPs were desalted with a VariPure cartridge prior to lyophilizing. Preparative Columns: Reprosil Gold 120 C18, 150×10 mm. Analytical Columns: Phenomenex, Jupiter 5 μm, 300 Å, 250×4.6 mm. LC-MS: Reprosil Gold C18 5 μm, 125×3 mm. Protocol F— Gel permeation chromatography: Nanopure H.sub.2O was used as eluent. A flow rate of 0.1 mL/min. was used at room temperature.
Example 11: Synthesis of 1-[ProHypGly].SUB.3.-AopProGly-[ProHypGly].SUB.3.—NH.SUB.2 .(Compound 3)
[0263] ##STR00043##
[0264] Compound 3 was synthesized on Rink amide ChemMatrix resin (.sup.˜0.5 mmol/g). The resin was swelled according to protocol A. Fmoc-γ-Aminoxyproline(Aop)-OH, Fmoc-ProHypGly-OH, Fmoc-Pro-OH, and Fmoc-Gly-OH, and Fmoc-Ahx-OH, were coupled according to protocol B, and Compound 1 was coupled to the resin using the manual protocol C. Compound 3 was cleaved from the solid support according to protocol D and purified according to protocol E using a gradient of 92% B to 72% B over 20 min, tR=14.0 min. After desalting and lyophilization, Compound 3 was obtained as a white foam that was stored at −20° C. in the dark. Analytical reverse-phase HPLC: 91% to 60% B over 20 min, tR=8.99 min; Purity determined by analytical HPLC using UV detection at 214 nm: >97%. HRMS (MALDI): m/z calcd. for [C.sub.105H.sub.148F.sub.2N.sub.25O.sub.33].sup.+: 2325.0634; found: 2325.0668 [M+H].sup.+.
Example 12: Synthesis of 1-Ahx-[ProHypGly].SUB.7.—NH.SUB.2 .(Compound 4)
[0265] ##STR00044##
[0266] Compound 4 was synthesized on Rink amide ChemMatrix resin (.sup.˜0.5 mmol/g). The resin was swelled according to protocol A. Fmoc-ProHypGly-OH and Fmoc-Ahx-OH were coupled according to protocol B, and Compound 1 was coupled to the resin using protocol C. Compound 4 was cleaved from the solid support according to protocol D and purified according to protocol E using a gradient of 92% B to 60% B over 20 min, t.sub.R=15.1 min. After desalting and lyophilization, Compound 4 was obtained as a white foam that was stored at −20° C. in the dark. Analytical reverse-phase HPLC: 91% to 45% B over 20 min, t.sub.R=7.47 min; Purity determined by analytical HPLC using UV detection at 214 nm: >97%. HRMS (MALDI): m/z calcd. for [C.sub.105H.sub.147F.sub.2N.sub.24O.sub.33].sup.+: 2310.0525; found: 2310.0537 [M+H].sup.+.
Example 13a: Synthesis of 1-Ahx-[ProProGly].SUB.7.—NH.SUB.2 .(Compound 5)
[0267] ##STR00045##
[0268] Compound 5 was synthesized on Rink amide ChemMatrix resin (.sup.˜0.5 mmol/g). The resin was swelled according to protocol A. Fmoc-ProProGly-OH and Fmoc-Ahx-OH were coupled according to protocol B, and Compound 1 was coupled to the resin using protocol C. Compound 5 was cleaved from the solid support according to protocol D and purified according to protocol E using a gradient of 92% B to 62% B over 20 min, t.sub.R=14.8 min. After desalting and lyophilization, Compound 5 was obtained as a white foam that was stored at −20° C. in the dark. Analytical reverse-phase HPLC: 91% B to 60% B over 20 min, t.sub.R=13.7 min. Purity determined by analytical HPLC using UV detection at 214 nm: >99%. HRMS (MALDI): m/z for [C.sub.105H.sub.147F.sub.2N.sub.24O.sub.26].sup.+: 2198.0881; found: 2198.0909 [M+H].sup.+.
Example 13b: Synthesis of 1-Ahx-[ProProGly].SUB.7.—NH.SUB.2 .(Compound 6)
[0269] ##STR00046##
[0270] Compound 6 was synthesized on Rink amide ChemMatrix resin (.sup.˜0.5 mmol/g). The resin was swollen according to protocol A. Fmoc-(4S)Aminoxyproline(Boc)-OH and Fmoc-Ahx-OH were coupled according to protocol B, and Compound 1 was coupled to the resin using the manual protocol C. The peptide was cleaved from the solid support according to protocol D and purified according to protocol E using a gradient of 98% B to 50% B over 20 min, t.sub.R=10.1 min. After desalting and lyophilization, Compound 6 was obtained as a white foam that was stored at −20° C. in the dark. Analytical reverse-phase HPLC: 98% B to 40% B over 20 min, t.sub.R=8.1 min. Purity determined by analytical HPLC using UV detection at 214 nm: >99%.
Example 14: CD Spectra of Compounds 3, 4, and 5
[0271] CD spectra (see
Example 15: General Procedure for Determination of T.SUB.m.-Values
[0272] The thermal denaturation experiments were performed with 0.2 mM solutions of Compounds 3, 4 and 5 that were equilibrated at 5° C. for at least 24 hrs in 50 mM PBS buffer (pH=7.4). The samples were heated with a heating rate of 1° C./100 s while monitoring the molar ellipticity at 224 nm. The experiment was repeated for each sample at least 3 times. The data obtained from the thermal denaturation experiments were fit to an all-or-none transition in which three single strands combine to a triple helix as previously reported by J. Engel, H. T. Chen, D. J. Prokop, H. Klump, Biopolymers 1997, 16, 601-622 and S. Frank, R. A. Kammerer, D. Mechling, T. Schulthess, R. Landwehr, J. Bann, Y. Guo, A. Lustig, H. P. Bächinger, J. Engel, J. Mol. Biol. 2001, 308, 1081-1089. The fit was performed using Micromath Scientist 3.0 with H=−500000 and Tm=40 as initial values. The model used is shown below: [0273] //Model two state; IndVars: TEMP; DepVars: F, CD, K; Params: H, DEU, REFU, DEN, REFN, Tm; R=8.31; K=EXP(H/(R*(TEMP+273.15))*((TEMP+273.15)/(Tm+273.15)−1)-In(0.75*0.0002{circumflex over ( )}2)); P=1/(3*K*(0.0002{circumflex over ( )}2)); U=(−P/2+(P{circumflex over ( )} 2/4+P{circumflex over ( )} 3/27){circumflex over ( )}(½)){circumflex over ( )}(⅓); V=−(P/2+(P{circumflex over ( )} 2/4+P{circumflex over ( )} 3/27){circumflex over ( )}(½)){circumflex over ( )}(⅓); F=U+V+1; CDU=REFU+DEU*(TEMP+273.15); CDN=REFN+DEN*(TEMP+273.15); CD=F*(CDN−CDU)+CDU.
[0274] The thermal denaturation curves of Compounds 3, 4 and 5 are shown in
Example 16: General Procedure for In Vivo Assays
[0275] Animals: Mice were housed and fed according to Swiss guidelines and all animal experiments were approved by the local veterinary authorities (Kantonales Veterinäramt Zürich). All mice used for these experiments are on a C57BL/6 background. Administration of Compounds 3, 4 and 5 into skin: Peptide solutions at 100 μM were brought to room temperature for 30 min. Male or female mice at 8 weeks of age were anesthetized by intraperitoneal injection of ketamine/xylazine (100 mg ketamine/5-10 mg xylazine per kg body weight), skin shaved and an injection of 50 μL was made intradermally into the back skin. Mice were housed for an additional five days and then skin harvested for histological analysis.
[0276] This example demonstrates the successful design and synthesis of a representative activity-based fluorescent compound according to the present invention which is capable of real-time quantification of lysyl oxidase activity in fibrotic conditions. The activation of the compound by enzymes was confirmed in ex vivo tissue homogenate models (see above), and demonstrated in vivo via conjugation to peptides that specifically and precisely target collagen and elastin undergoing real-time cross-linking and remodeling in the extracellular matrix. Additionally, the compounds according to the present invention with the addition of a conjugated peptide show utility for analysis of enzyme-mediated tissue remodeling in a relevant model of fibrogenesis.
Example 17: Synthesis of (5-((3-((Boc)amino)propyl)amino)-5-oxopentyl)triphenyl-phosphonium bromide
[0277] ##STR00047##
[0278] (4-Carboxybutyl)triphenyl-phosphonium bromide (2.26 mmol, 1 g) and 1,1′-carbonyldimidazole (2.26 mmol, 366 mg) were dissolved in anhydrous DMF (15 mL) and stirred for 30 min at room temperature. N-Boc-1,3-propanediamine (2.26 mmol, 393 mg) was added to the suspension, and stirred overnight. The crude mixture was concentrated by rotary evaporation, and purified by silica gel chromatography eluting with a gradient of 2-10% methanol in dichloromethane to yield (5-((3-((Boc)amino)propyl)amino)-5-oxopentyl)triphenylphosphonium bromide as a white foam (860 mg, 73%). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.47 (t, J=6.0 Hz, 1H), 7.85-7.65 (m, 15H), 5.54 (t, J=5.7 Hz, 1H), 3.75-3.58 (m, 2H), 3.20 (q, J=6.2 Hz, 2H), 3.00 (q, J=6.2 Hz, 2H), 2.59 (t, J=6.8 Hz, 2H), 1.93 (q, J=7.0, 6.6 Hz, 2H), 1.68-1.55 (m, 4H), 1.39 (s, 9H). .sup.13C NMR (101 MHz, CDCl.sub.3) δ 173.30, 156.29, 135.23 (d, J=3.1 Hz), 133.77 (d, J=10.0 Hz), 130.60 (d, J=12.6 Hz), 118.12 (d, J=86.1 Hz), 78.62, 50.70, 36.53 (d, J=138.0 Hz), 34.17, 29.49, 28.59, 26.21 (d, J=17.0 Hz), 22.45 (d, J=50.7 Hz), 21.09 (d, J=4.2 Hz). .sup.31P NMR (162 MHz, CDCl.sub.3) δ 24.67. HRMS (ESI): m/z calcd. for C.sub.31H.sub.40N.sub.2O.sub.3P: 519.2771 [M].sup.+; found: 519.2764.
Example 17a: Synthesis of (5-((3-ammoniopropyl)amino)-5-oxopentyl)triphenyl-phosphonium chloride
[0279] ##STR00048##
[0280] (4-Carboxybutyl)triphenyl-phosphonium bromide (2.26 mmol, 1 g) and 1,1′-carbonyldimidazole (2.26 mmol, 366 mg) were dissolved in anhydrous DMF (15 mL) and stirred for 30 min at room temperature. N-Boc-1,3-propanediamine (2.26 mmol, 393 mg) was added to the suspension, and stirred overnight. The crude mixture was concentrated by rotary evaporation, and purified by silica gel chromatography eluting with a gradient of 2-10% methanol in dichloromethane to yield (5-((3-ammoniopropyl)amino)-5-oxopentyl)triphenylphosphonium chloride as a white solid (860 mg, 73%). .sup.1H NMR (400 MHz, MeOD) δ 7.92-7.72 (m, 15H), 3.50-3.41 (m, 2H), 3.22 (t, J=6.7 Hz, 2H), 2.91 (t, J=7.4 Hz, 2H), 2.29 (t, J=7.4 Hz, 2H), 1.89-1.77 (m, 4H), 1.76-1.64 (m, 2H). .sup.13C NMR (101 MHz, MeOD) δ 175.94, 136.31 (d, J=3.1 Hz), 134.85 (d, J=10.0 Hz), 131.55 (d, J=12.6 Hz), 119.86 (d, J=86.5 Hz), 38.27, 36.92, 35.74, 28.67, 27.63 (d, J=17.4 Hz), 23.12 (d, J=4.1 Hz), 22.55 (d, J=51.6 Hz). .sup.31P NMR (162 MHz, MeOD) δ 23.74. HRMS (ESI): m/z calcd. for C.sub.26H.sub.32N.sub.2OP: 419.2247 [M].sup.+; found: 419.2241.
Example 17b: Synthesis of MRA_3103
[0281] ##STR00049##
[0282] (5-((3-ammoniopropyl)amino)-5-oxopentyl)triphenylphosphonium chloride (0.21 mmol, 89 mg) was dissolved in anhydrous DMF (1.4 mL) and cooled to 0° C. with stirring. N,N-Diisopropylethylamine (0.424 mmol, 74 μL) was added, followed by MRA_3102 (0.19 mmol, 100 mg), and the solution was allowed to warm to room temperature and stir overnight. The crude mixture was concentrated by rotary evaporation, and purified by RP-HPLC with a gradient of 90-10% acetonitrile in water with 0.1% TFA. Fractions containing product as determined by HPLC-MS were combined, flash frozen, and lyophilized to yield MRA_3103 as a white solid (14 mg, 9%). .sup.1H NMR (600 MHz, CD.sub.3CN) δ 7.88-7.82 (m, 3H), 7.72-7.65 (m, 12H), 7.37 (dd, J=12.0, 2.2 Hz, 1H), 6.94 (t, J=5.9 Hz, 1H), 6.77 (t, J=5.6 Hz, 1H), 5.46 (s, 1H), 4.30 (t, J=6.2 Hz, 2H), 3.52 (s, 2H), 3.24-3.16 (m, 4H), 3.06 (dq, J=12.9, 6.3 Hz, 4H), 2.35 (s, 3H), 2.13 (t, J=7.1 Hz, 2H), 1.89 (p, J=6.5 Hz, 2H), 1.72 (p, J=7.2 Hz, 2H), 1.60 (dq, J=15.8, 7.7 Hz, 2H), 1.49 (p, J=6.6 Hz, 2H), 1.38 (s, 9H). .sup.19F NMR (F-H decoupled) (376 MHz, CD.sub.3CN) δ −133.94 (d, J=5.2 Hz), −150.46 (d, J=6.0 Hz). .sup.19F NMR (376 MHz, CD.sub.3CN) δ −133.94 (dd, J=12.7, 5.8 Hz), −150.46 (d, J=6.3 Hz). .sup.31P NMR (162 MHz, CD.sub.3CN) δ 23.50. .sup.13C NMR (151 MHz, CD.sub.3CN) δ 173.51, 170.28, 161.20, 157.08, 152.47 (dd, J=242.9, 4.5 Hz), 149.91, 143.78 (dd, J=248.9, 6.2 Hz), 139.49 (dd, J=10.1, 1.9 Hz), 138.66 (dd, J=16.1, 11.0 Hz), 136.11 (d, J=3.1 Hz), 134.61 (d, J=10.0 Hz), 131.26 (d, J=12.6 Hz), 121.60, 119.56, 118.99, 116.69 (d, J=9.2 Hz), 107.71 (dd, J=22.7, 3.5 Hz), 79.27, 73.85, 37.85, 37.21, 36.93, 35.68, 35.49, 31.20, 30.07, 28.60, 27.05 (d, J=17.3 Hz), 22.44 (d, J=4.1 Hz), 22.40 (d, J=51.8 Hz), 16.10. HRMS (ESI): m/z calcd. for C.sub.46H.sub.53F.sub.2N.sub.3O.sub.7P: 828.3584 [M].sup.+; found: 828.3582.
Example 18: General Protocols for the Synthesis of Mitochondrial-Targeting Peptides
[0283] Protocol A—General Procedure for Swelling
[0284] Before automated peptide synthesis, the resin was swelled in CH.sub.2Cl.sub.2 for 15 min while shaking. Then the resin was drained and washed with DMF (3×6 mL) and drained again.
[0285] Protocol B—General Protocol for Automated Peptide Synthesis
[0286] For automated peptide synthesis, a Syro I peptide synthesizer (Biotage, Sweden) was used. Couplings were performed either with the appropriate Fmoc-protected amino acid or the trimer Fmoc-Pro-Hyp-Gly-OH. After swelling the resin in DMF on the synthesizer, i-Pr.sub.2NEt (9 equiv. as a 3 M solution in NMP (N-methyl-2-pyrrolidone)), HATU (3 equiv., 0.5 M in DMF) and the Fmoc-amino acid/Fmoc-tripeptide (3 equiv., 0.5 M in DMF) were added to the resin. The mixture was allowed to react in intervals of 1 min. agitation and 5 min. rests for 30 min. (2×) and was then washed with DMF (5×). Fmoc-deprotection was carried out by addition of a solution of 40% (v/v) piperidine in DMF and reaction for 1 min. This step was repeated 4 times. The resin was then washed with DMF (5×). Tripeptide couplings and Fmoc-deprotections were repeated until the desired peptides were obtained. For the automated synthesis of CMPs no acylation (capping) was performed.
[0287] Protocol C—on Resin N-Terminal Functionalization with Compound 1
[0288] Functionalization was performed manually at room temperature on the solid support-bound peptide. Compound 1 (2.0 equiv.), HATU (1.9 equiv.) and i-Pr.sub.2NEt (4 equiv.) were dissolved in DMF (1-2 mL). After pre-activation for 5 min, the coupling mixture was added to the resin and agitated for 1-2 hrs. The resin was washed with CH.sub.2Cl.sub.2 (3×), DMF (3×), CH.sub.2Cl.sub.2 (3×), and petroleum ether (2×). The reaction was monitored by the qualitative color tests on bead or by LC-MS after test cleavage (see Protocol E).
[0289] Protocol D—Cleavage from the Resin
[0290] The resin was shaken for 1 h in a mixture of TFA/(i-Pr.sub.2).sub.3Si—H/H.sub.2O (92.5:2.5:2.5), and washed with pure TFA (2×). The peptide in solution was collected by filtration in a conical flask. Addition of ice-cold Et.sub.2O afforded the peptide as a white precipitate. The solid was isolated by centrifugation followed by decantation. The solid was suspended in Et.sub.2O, sonicated, centrifuged again and the supernatant was decanted. The residual white solid was dissolved in water/CH.sub.3CN, frozen, and lyophilized to obtain a white foam.
[0291] Protocol E—Purification and Analysis by RP HPLC
[0292] For semi-preparative HPLC, H.sub.2O containing 1% CH.sub.3CN and 0.1% TFA (A) and CH.sub.3CN (B) were used as eluents. For semi-preparative HPLC a flow rate of 6 mL/min, for analytical HPLC a flow rate of 1 mL/min and for LC-MS a flow rate of 0.5 mL/min was used. After purification by semi-preparative HPLC all collected fractions were analyzed by analytical HPLC or LC-MS and only pure fractions were combined. For analytical HPLC, CH.sub.3CN (A) and H.sub.2O containing 1% CH.sub.3CN and 0.1% TFA (B) were used as eluents. Amine containing CMPs were desalted with a VariPure cartridge prior to lyophilizing.
[0293] Preparative Columns: Reprosil Gold 120 C18, 150×10 mm. Analytical Columns: Phenomenex, Jupiter 5 μm, 300 Å, 250×4.6 mm. LC-MS: Reprosil Gold C18, 125×3 mm.
Example 18a: Synthesis of 1-Ahx-[Char].SUB.3.—NH.SUB.2
[0294] ##STR00050##
[0295] The peptide was synthesized on Rink amide resin (.sup.˜0.5 mmol/g). The resin was swelled according to protocol A. Fmoc-D-Arginine(Boc)-OH, Fmoc-Cha-OH, and Fmoc-Ahx-OH, were coupled according to protocol B, and Compound 1 was coupled to the resin using the manual protocol C. The peptide was cleaved from the solid support according to protocol D and purified according to protocol E using a gradient of 15% B to 40% B over 20 min, t.sub.R=11.4 min. After desalting and lyophilization, the peptide was obtained as a white foam that was stored at −20° C. in the dark. Analytical reverse-phase HPLC: 90% to 10% B over 20 min, t.sub.R=8.3 min; Purity determined by analytical HPLC using UV detection at 214 nm: >99%. HRMS (MALDI): m/z calcd. for [C.sub.66H.sub.110F.sub.2N.sub.18O.sub.11].sup.2+: 684.4279; found: 684.4269 [M+H].sup.2+.
Example 18b: Synthesis of MRA_3069-Ahx-(ChaR).SUB.3.—NH.SUB.2
[0296] ##STR00051##
[0297] The peptide was synthesized on Rink amide resin (.sup.˜0.5 mmol/g). The resin was swelled according to protocol A. Fmoc-D-Arginine(Boc)-OH, Fmoc-Cha-OH, and Fmoc-Ahx-OH, were coupled according to protocol B, and MRA_3069 was coupled to the resin using the manual protocol C. The peptide was cleaved from the solid support according to protocol D and purified according to protocol E using a gradient of 15% to 48% B over 20 min, t.sub.R=15.0 min. After desalting and lyophilization, the peptide was obtained as a white foam that was stored at −20° C. in the dark. Analytical reverse-phase HPLC: 10% to 90% B over 20 min, t.sub.R=8.7 min; Purity determined by analytical HPLC using UV detection at 214 nm: >95%. HRMS (MALDI): m/z calcd. for [C.sub.66H.sub.110F.sub.2N.sub.18O.sub.11].sup.2+: 691.4358; found: 691.4343 [M+H].sup.2+
Example 18c: Synthesis of PB-Ahx-(ChaR).SUB.3.—NH.SUB.2
[0298] ##STR00052##
[0299] The peptide was synthesized on Rink amide resin (.sup.˜0.5 mmol/g). The resin was swelled according to protocol A. Fmoc-D-Arginine(Boc)-OH, Fmoc-Cha-OH, and Fmoc-Ahx-OH, were coupled according to protocol B. Pacific Blue (PB) NHS was coupled to the resin directly using the manual protocol C, but without the addition of HATU. The peptide was cleaved from the solid support according to protocol D and purified according to protocol E using a gradient of 15% B to 60% B over 20 min, t.sub.R=13.9 min. After desalting and lyophilization, the peptide was obtained as a white foam that was stored at −20° C. in the dark. Analytical reverse-phase HPLC: 10% to 90% B over 20 min, t.sub.R=9.56 min; Purity determined by analytical HPLC using UV detection at 214 nm: >99%. HRMS (MALDI): m/z calcd. for [C.sub.63H.sub.103F.sub.2N.sub.17O.sub.11].sup.2+: 655.899; found: 655.8979 [M+H].sup.2+.
Example 19: Cellular Assays for the Determination of Monamine Oxidase Activity from MAO A/MAO B
[0300] Cell culture: MCF-7 and SY5Y cells were obtained from the Health Protection Agency (www.HPA.org.uk) or the American Type Culture Collection. The cells were grown in a humidified 5% CO.sub.2 atmosphere at 37° C. using Kaighn's Modification of Ham's F-12 medium (F-12KTM) supplemented with L-glutamine (4 mM), penicillin (100 U/mL penicillin), streptomycin (100 g/mL), and 10% fetal calf serum (FCS) superior (standardized). Culture medium DMEM high glucose, F-12KTM, L-glutamine (200 mM), penicillin (10.000 U/mL), streptomycin (10 mg/mL), and trypan blue solution were purchased from Sigma, Invitrogen, ATCC or BioConcept. Trypsin-EDTA (0.05%/0.02%) in Ca2+- and Mg2+-deficient phosphate buffered saline (PBS) (1:250) was purchased from Amimed. PBS (pH 7.4) was purchased from Invitrogen. FCS superior was bought fromOxoid AG and Biochrom AG. Cell culture flasks as well as serological pipettes were purchased from BD Biosciences and Sarstedt. Ethylenediaminetetraacetic acid (EDTA) was purchased from Sigma-Aldrich. Hoechst 33342 was purchased from Invitrogen. MitoTracker Red mitochondrial staining dye and live cell imaging solution were purchased from ThermoFisher.
[0301] Confocal Microscopy: Fluorescence images of cells were collected using a Nikon Eclipse T1 microscope equipped with a Yokogawa spinning-disk confocal scanner unit CSU-W1-T2, two sCMOS cameras (Orca Flash 4.0 V2) and a LUDL BioPrecision2 stage with piezo focus. Emission in the blue channel was filtered with a 450/50 bandpass filter, emission in the green channel was filtered with a 525/50 bandpass filter and emission in the far-red channel with a 700/75 bandpass filter. Fluorescence images were obtained using an oil-immersion objective with a magnification of 100×1.49 CFI Apo TIRF. The microscope was operated using VisiVIEW (Metamorph).
[0302] Prior to the measurement, cells were seeded at a density of 10′000 cells/well in ibidi 8-well plates and were grown for 1 day in DMEM (10% FCS) at 37° C. Afterwards cells were washed with PBS and fresh DMEM was added. Compounds were added to the cells as stock solutions in PBS to reach the final 10 mM concentration. The cells were incubated for two hours at 37° C., washed with PBS and incubated with MitoTracker Red based on the manufacturers recommended procedure for 5 min at 37° C. Cells were washed with PBS and the live cell imaging solution (200 μL) was added. The live cells were then immediately examined on a confocal microscope (Vistron Spinning Disk). For detection of the coumarin-activation a laser line 405 nm and for MitoTracker a laser line of 561 nm was used.
Example 20: Biochemical and Histological Analysis of the Skin Treated with Compounds 3 to 6
[0303] Skin tissue used for histology was harvested and immediately embedded and frozen in tissue freezing Medium® (Leica Biosystems, Wetzlar, Germany). Tissue sections (7 μM) were fixed using ice-cold acetone for 10 min at −20° C. and stained with propidium iodide to visualize nuclei. Images were taken using a 20× objective. For immunofluorescent analysis of collagen, antibodies against collagen I (Southern Biotech, Birmingham, Ala., USA; 1310-01) or collagen III (Abcam, Cambridge, UK, ab7778) were used. Fixed tissue sections were incubated with 1% bovine serum albumin (BSA) in PBS for 30 min followed by collagen I (1:400 dilution in 1% BSA) or collagen III (1:800 dilution in 1% BSA) antibodies for 60 min at room temperature. After washing (3×5 min PBS), secondary antibodies (anti-rabbit AF488 (711-547-003) or anti-goat AF488 (705-545-147), Jackson ImmunoResearch) were incubated on the tissues for 30 min at room temperature (both at 1:400 dilution in 1% BSA) and nuclei visualized using propidium iodide. All images were taken using a 20× objective.
Example 21: Biochemical and Histological Analysis of Tissue Sections Treated with Compounds 3 to 6
[0304] Skin tissue used for histology was harvested and immediately embedded and frozen in tissue freezing Medium® (Leica Biosystems, Wetzlar, Germany). Tissue sections (7 uM) were treated by the addition of 50 μL of each compound at 100 μM in PBS (Compounds 3, 4, 5, or 6) by pipette to completely cover the surface of the section, incubated at 37° C. for 4 hours in the dark, and washed 2× with PBS. Sections were fixed using ice-cold acetone for 10 min at −20° C. and stained with propidium iodide to visualize nuclei. Images were taken using a 20× objective.
[0305] For immunofluorescent analysis of collagen, antibodies against collagen I (Southern Biotech, 1310-01) or collagen III (Abcam, ab7778) were used. Fixed tissue sections were incubated with collagen I or collagen III antibodies for 60 min at room temperature. After washing, secondary antibodies (anti-rabbit AF488 (711-547-003) or anti-goat AF488 (705-545-147), Jackson ImmunoResearch) were incubated on the tissues for 30 min at room temperature and nuclei visualized using propidium iodide. All images were taken using a 20× objective.