NOVEL COMPOUNDS WITH DUAL ACTIVITY
20180296727 ยท 2018-10-18
Assignee
Inventors
Cpc classification
C09D7/70
CHEMISTRY; METALLURGY
A61L2420/04
HUMAN NECESSITIES
International classification
Abstract
Provided are novel compounds and uses thereof in preventing antifouling by unicellular organisms and in attracting cells from multicellular organisms.
Claims
1.-91. (canceled)
92. A compound comprising: at least one antifouling moiety, at least one surface-adsorbing moiety, and at least one amino acid sequence promoting adherence of cells, wherein the at least one antifouling moiety is a fluorine (F) atom or a group comprising at least one fluorine atom; said at least one surface-adsorbing moiety is selected 3,4-dihydroxy-L-phenylalanin (DOPA), a DOPA containing moiety and dopamine; and wherein said at least one amino acid sequence promoting adherence of cells comprises an amino acid sequence selected from RGD; RGDS; KQAGDV; YIGSR; REDV; IKVAV; RNIAEIIKDI; KHIFSDDSSE; VPGIG; FHRRIKA; KRSR; APGL; VRN; NSPVNSKIPKACCVPTELSAI; and AAAAAAAAA.
93. The compound according to claim 92, wherein the at least one antifouling moiety comprises between 1 and 20 fluorine atoms.
94. The compound according to claim 92, wherein the at least one antifouling moiety is a fluorinated carbon group.
95. The compound according to claim 94, wherein the fluorinated carbon group is a substituted or unsubstituted alkyl group, substituted or unsubstituted alkenyl group or substituted or unsubstituted alkynyl group.
96. The compound according to claim 95, wherein the fluorinated carbon group is a C.sub.1-C.sub.20 group comprising between 1 and 20 fluorine atoms or a C.sub.2-C.sub.20 group comprising between 1 and 20 fluorine atoms.
97. The compound according to claim 94, wherein the at least one antifouling moiety is a fluorinated substituted or unsubstituted aryl.
98. The compound according to claim 92, wherein the at least one antifouling moiety comprises one or more fluorinated amino acids.
99. The compound according to claim 98, wherein the fluorinated amino acid is a fluorinated phenylalanine derivative, wherein the fluorine atom substitutes one or more phenyl ring positions.
100. The compound according to claim 99, wherein the fluorinated phenylalanine is selected from o-fluorophenylalanine, m-fluorophenylalanine and p-fluorophenylalanine.
101. The compound according to claim 92, wherein the at least one amino acid sequence promoting adherence of cells is RGD (Arg-Gly-Asp) or RGDS (Arg-Gly-Asp-Ser).
102. The compound according to claim 92, being a compound selected from J-XZ, J-ZX, XZ-J, X-J-Z, ZX-J and Z-J-X; wherein J is the surface binding moiety, X is the antifouling moiety, Z is the amino acid sequence promoting adherence of cells; and each - designates a covalent bond or a linker moiety.
103. The compound according to claim 102, wherein the linker moiety is selected from substituted or unsubstituted carbon chains.
104. The compound according to claim 103, wherein the linker moiety is composed of two or more amino acids.
105. The compound according to claim 92, being a compound of the general Formula (I):
J-XZ(I) wherein J is the surface binding moiety, X is the antifouling moiety, Z is the amino acid sequence promoting adherence of cells, and each - designates a covalent bond or a linker moiety.
106. The compound according to claim 105, being a compound of general Formula (III):
J.sub.n-X.sub.mZ.sub.k(III) wherein each of J, X and Z is as defined in claim 105 and each of n, m and k, independently of the other, is an integer between 1 and 10.
107. The compound according to claim 106, being a compound of Formula (IV):
J-XXZ(IV) wherein each of the two X groups are optionally the same.
108. The compound according to claim 107, being the compound: DOPA-Phe(4F)-Phe(4F)-Arg-Gly-Asp, designated Peptide 1.
109. The compound according to claim 105, being a pentapeptide or a hexapeptide of the general Formula (V) or Formula (VI):
XX-J-Z(V)
ZXX-J(VI) wherein each of J, X and Z are as defined in claim 105.
110. The compound according to claim 105, being a hexapeptide selected from:
J-XX-RGD(VII)
XX-J-RGD(VIII)
RGD-XX-J(IX)
RGDS-XX-J(X) and
XX-J-RGDS(XI).
111. The compound according to claim 110, having the structure: ##STR00006##
112. A formulation comprising a combination of at least two peptides selected from J-X, XZ, J-Z and X-J, wherein J is a surface binding moiety, X is an antifouling moiety, Z is the amino acid sequence promoting adherence of cells, and each - designates a covalent bond or a linker moiety; provided that the combination comprises at least one peptide having moiety J, at least one peptide having moiety X and at least one peptide having moiety Z; wherein the at least one antifouling moiety is a fluorine (F) atom or a group comprising at least one fluorine atom; said at least one surface-adsorbing moiety is selected 3,4-dihydroxy-L-phenylalanin (DOPA), a DOPA containing moiety and dopamine; and wherein said at least one amino acid sequence promoting adherence of cells comprises an amino acid sequence selected from RGD; RGDS; KQAGDV; YIGSR; REDV; IKVAV; RNIAEIIKDI; KHIFSDDSSE; VPGIG; FHRRIKA; KRSR; APGL; VRN; NSPVNSKIPKACCVPTELSAI; and AAAAAAAAA.
113. A method selected from: a method of anchoring or attracting cells onto a surface region of an implantable device or object, said method comprising forming on a surface region of the implantable device or object a film of at least one compound according to claim 92 and implanting said device or object, wherein the film promotes anchoring or attachment of cells thereto; a method of reducing biofouling caused by an organism on an implant post-operatively, said method comprising forming on a surface region of the implant a film of at least one compound according to claim 92 and implanting said implant, wherein the film promotes anchoring or attachment of cells thereto; a method of attracting body cells to and reducing biofouling on an implant, said method comprising forming a film of at least one compound according to claim 92 on a surface region of the implant and implanting said implant; a method for preventing, ameliorating, or treating a disease or disorder related to an implant having been placed in a body cavity or on a tissue, said method comprising the step of forming a film of at least one compound according to claim 92 on a surface region of an implant prior to implantation; a method for prolonging lifetime of an implant intended for implantation in a subject, said method comprising coating a surface region of the implant prior to implantation with a film comprising at least one compound according to claim 92; and a method for stimulating or encouraging bone healing or absorption of an implant after implantation, the method comprising coating a surface region of the implant prior to implantation with at least one compound according to claim 92, thereby inducing bone healing.
114. A medical device or an implant comprising a film of at least one compound according to claim 92.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0131] In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:
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DETAILED DESCRIPTION OF THE INVENTION
[0141] Numerous pentapeptidea and hexapeptides according to the invention have been prepared and studied. For the sake of demonstrating the efficacy and uniqueness of the invention disclosed herein, the following hexapeptides have been prepared:
##STR00005##
[0142] As shown in
[0143] To coat the Ti substrate with a peptide, a bare titanium substrate was dipped in 1 mg\ml peptide solution (Tris buffer). The change of the surface contact angle due to the assembly of the peptide on the substrate was measured.
[0144] As can be seen in
[0145] Using X-ray Photoelectron Spectrometry (XPS) analysis, the presence of fluorine atoms on the surface and the thickness of the peptide layer were characterized. As expected, in the case of a bare titanium, there was no indication of the presence of fluorine, while in all cases of substrate-coated with peptides, the presence of fluorine signal indicated the deposition of the peptides on the surface (
[0146] In order to further examine the film formed, Scanning Electron Microscopy (SEM) was utilized. SEM revealed formation of large spherical assemblies in the case of Peptide 3 and Peptide 4 (
[0147] For further characterization of the coating topography and thickness, Atomic Force Microscopy (AFM) analysis of Ti surfaces coated with the different peptides was performed. AFM analysis clearly showed (
[0148] As mentioned before, Peptide 3 formed big spherical assemblies on the surface. For that reason it was not possible to perform ATR-FTIR analysis as no additional peaks were observed in the case of Peptide 3 modified surface compared to titanium surface (
[0149] To assess the bacterial attachment to the surfaces and the anti-fouling activity of the peptides, the adherence of Escherichia Coli to bare and peptide coated substrates was promoted. Following this, the surfaces incubated overnight in the bacteria medium to enable the biofilm formation. After the incubation, the surfaces were slowly rinsed, swabbed the adhered bacteria and seeded them in order to evaluate the number of live bacteria on the substrates. The plot in
Materials and Methods
[0150] All chemicals, solvents, proteins and bacteria were purchased from commercially available companies and used as supplied unless otherwise stated. Fmoc-DOPA(ac)-COOH was obtained from Novabiochem/EMD chemicals (San-Diego, USA). L and D-4-fluoro phenylalanine, Boc-penta Fluoro phe-COOH were purchased from chem-impex Inc. (Wood Dale, USA). Solvents and TFA were purchased from Bio-lab (Jerusalem, Israel). NMR solvents (CDCl.sub.3 and DMSO-d.sub.6) were supplied by Sigma-Aldrich (Jerusalem, Israel). Piperidine used for deprotection of Fmoc group was obtained from Alfa-Aesar (UK). The proteins BSA, fibrinogen and lysozyme were obtained from Sigma-Aldrich (Jerusalem, Israel), Chem impex INC. (Wood Dale, USA) and Merck (Darmstadt, Germany) respectively. Pseudomonas aeruginosa (ATCC 27853) and Escherichia coli (ATCC 1655) were purchased from ATCC (Virginia, USA). Crystal violet was obtained from Merck (Germany).
[0151] Substrates
[0152] Silicon wafers (100) with a diameter of 2 inches were coated with 50 nm titanium (as measured by quartz crystal) by electron beam evaporation (TFDS-141E, VST) at a rate of 1 ?/sec. In the same manner silicon wafers were coated with 15 nm titanium and then 150 nm of gold. The coated wafers were diced (7100 2 Pro-Vectus, ADT) into 1 cm?1 cm pieces.
[0153] High Performance Liquid Chromatography (HPLC)
[0154] Analytical reversed-phase (RP) HPLC analysis was performed on a Waters Alliance HPLC with UV detection (220 nm and 280 nm) using a XSelect C18 column (3.5 m, 130 ?, 4.6?150 mm). Preparative RP-HPLC was performed on a Waters 150QLC system using a XSelect C18 column (5 m, 130 ?, 30?250 mm). Linear gradients of acetonitrile (with 0.1% TFA) in water (with 0.1% TFA) were used for all systems to elute bound peptides. The flow rates were 1 mL/min (analytical, column heated at 30? C.) and 20 mL/min (preparative).
[0155] Mass Spectrometry (MS)
[0156] Electrospray ionization MS was performed on LCQ Fleet Ion Trap mass spectrometer (Thermo Scientific).
[0157] Surface Modification
[0158] 1 cm?1 cm titanium surfaces were sonicated for 5 minutes in ethanol, washed with TDW and dried under nitrogen. The clean surfaces were dipped in a peptide solution (1 mg/mL in TRIS buffer at pH=8.5, 10 mM concentration and ionic strength of 154 mM with NaCl) and left for incubation overnight at room temperature. Then, the modified substrate were rinsed three times with 1 ml TDW and dried under nitrogen.
[0159] Contact Angle Measurements
[0160] Contact angle measurements were carried out using a Theta Lite optical tensiometer (Attension, Finland).
[0161] ATR-FTIR Analysis
[0162] ATR spectra were recorded using FT-IR (Thermo scientific, Model Nicolet 6700) with GeATR arrangement (Harrick Scientific's VariGATR). For all the surfaces spectra were collected with applied force of 350 N, at 4 cm.sup.?1 resolution with 3000 scans averaged signal and an incident angle of 65?. The transmittance minimal values were determined by the OMNIC analysis program (Nicolet).
[0163] FTIR Analysis
[0164] Infrared spectra were recorded using a Nicolet 6700 FT-IR spectrometer with a deuterated triglycine sulfate (DTGS) detector (Thermo Fisher Scientific, MA, USA). Peptide solution were deposited on a CaF.sub.2 plate and dried by vacuum. The peptide deposits were resuspended with D.sub.2O and subsequently dried to form thin films. The resuspension procedure was repeated twice to ensure maximal hydrogen-to-deuterium exchange. The measurements were taken using a 4 cm.sup.?1 resolution and averaging 1000 scans. The transmittance minimal values were determined by the OMNIC analysis program (Nicolet).
[0165] NMR Analysis
[0166] NMR spectra were obtained at 400.13 MHz (1H) using a Bruker DRX 400 spectrometer. The mass of the peptides was measured using Applied Biosystem Voyager-DE pro MALDI TOF mass spectrometer. The peptides were synthesized by a conventional solution-phase method using a racemization free strategy. The Boc group and Fmoc group were used for N-terminal protection and the C-terminus was protected as a methyl ester. Couplings were mediated by dicyclohexylcarbodiimide/1-hydroxybenzotriazole (DCC/HOBt). The intermediate compounds were characterized by .sup.1H NMR and MALDI-TOF mass spectroscopy and final peptides were fully characterized by .sup.1H NMR, .sup.13C NMR, .sup.19F NMR, MALDI-TOF.
[0167] AFM
[0168] Surfaces were prepared in the same manner as was mentioned before. AFM images were taken in AC mode with Si tip with spring constant 3 N/m in JPK instrument (NanoWizard 3).
[0169] Biofilm Formation
[0170] Escherichia coli were grown in LB medium for 5.5 hours at 37? C. in loosely capped erlenmeyer flask with agitation (120 rpm). The OD.sub.?=600 nm of the culture was measured using UV-1650PC spectrophotometer (SHIMADZU) to confirm absorbance of ?1. 5 uL of the bacteria culture were then seeded onto each substrate (1 cm?1 cm) in 24-well plates and incubated for 75 min at room temperature to allow initial adhesion of the bacteria. Afterward, LB media (2 mL) were added to each well and the plates were incubated at 37? C. overnight.
[0171] Quantification of Live Bacteria on the Substrates
[0172] Each surface was rinsed by shaking the surface in sterilized water three times. A sterile swab was then used to pick up the biofilms attached to the surfaces. The swabs were inserted into eppendorf tubes containing 1 mL of sterilized water and vortexed for 1 min. Each sample was decimally diluted and plated out on dried LB agar (10 g LB and 7.5 g agar per 0.5 L TDW) plate (8 drops of 10 ul for each dilution). Plate counts (CFU mL-1) were carried out after incubation over night at 37? C. The data presents 3 experiments with triplicates for each experiment. The data is given as mean values?standard deviations. Significant differences between group means were analyzed by t-test and confidence levels were set at 95%.
[0173] Crystal Violet Assay
[0174] The substrates were rinsed by shaking the surface in sterilized water three times and stained with 0.2% crystal violet for 30 minutes. The stained samples were washed with running water and left to dry in air. Eventually the bound dye was eluted with 30% acetic acid. Absorbance values were recorded at 590 nm in a microplate reader (Synergy 2, BioTek). All measurements were performed in triplicates and averaged.
[0175] Cell Culture
[0176] Cellular experiments were performed using the Human Embryonic Kidney (HEK) 293T17 cells. HEK293 cells were cultured in P medium supplemented with 10% (v/v) fetal bovine serum (FBS), 50 U/mL penicillin, and 1% (w/v) L-glutamine. Cells were maintained at 37? C., in a humidified atmosphere containing 5% (v/v) CO.sub.2, changing culture medium twice a week. Upon reaching 90% confluence, cells were detached by trypsin and subcultured into a new flask.
[0177] Peptide Synthesis
[0178] The peptides were synthesized manually by solid phase peptide synthesis (SPPS) on 0.25 mmol scale using rink amide resin. The Fmoc protecting group was removed with solution of 20% piperidine in DMF (15 min?2) and then washed with DMF. The amino acids were activated using DIEA/HATU mixture for 3 minutes. amino acids were used in 5-fold excess, except the (4F)Phe and DOPA that was 2-fold excess. The amount of DIEA and HATU was determined according to the amount of the amino acids (2 and 1 molar equiv. for DIEA and HATU, respectively). The amino acids were allowed to couple for 1.5 hours with constant shacking. The cleavage of the peptide from the resin was done after washing the resin with DMF and DCM and drying under vacuum. The peptide was cleaved using a mixes solution of 30 ml TFA/TDW/TIPS (95:2.5:2.5) for 3 hours. The cleavage solution was evaporated to minimum volume by bubbling nitrogen and the crude peptide product was precipitated with diethyl ether. The precipitated crude peptide was centrifuged and the crude peptide was dissolved in minimum volume of 0.1% TFA in TDW and lyophilized before purification with HPLC.
A. Synthesis of Peptide 5
[0179] 1. Boc-L-(4F)Phe-COOH 8a:
[0180] A solution of L-4F-Phe-COOH 1.97 g (10 mmol) in a mixture of dioxane (20 mL), water (20 mL) and 1 M NaOH (10 mL) was stirred and cooled in an ice-water bath. Ditert-butylpyrocarbonate 2.4 g (11 mmol) was added and stirring was continued at room temperature for 6 h. Then the solution was concentrated in vacuum to about 15-20 mL, cooled in an ice water bath, covered with a layer of ethyl acetate (about 30 mL) and a dilute solution of KHSO.sub.4 was added to acidify (pH 2-3). The aqueous phase was extracted with ethyl acetate and this operation was done three times. The ethyl acetate extracts were collected and dried over anhydrous Na.sub.2SO.sub.4 and evaporated in a vacuum. The pure material was obtained as a waxy solid.
[0181] Yield: 2.115 g (7.25 mmol, 72.5%)
[0182] .sup.1H NMR (DMSO-d6, 400 MHz, ?ppm): 12.60 [s, 1H COOH], 7.29-7.25 & 7.11-7.07 [m, 4H, Aromatic protons], 4.10-3.00 [m, 1H, C?H 4F Phe], 3.03-2.77 [m, 2H, C?H 4F Phe], 1.33 [s, 9H, Boc].
[0183] MALDI-TOF (matrix:?-cyano-4-hydroxy cinnamic acid (CHCA)):m/z=[M+H]+ 284.12 (calculated), 284.29 (observed), [M+Na]+ 306.11 (calculated), 306.25 (observed).
[0184] 2. Boc-L-(4F)Phe(2)-L-(4F)Phe(3)-COOMe 9a:
[0185] 500 mg (1.766 mmol) of Boc-L-(4F)Phe-OH was dissolved in 25 mL dry DCM in an ice-water bath. NH.sub.2-L-(4F)Phe-OMe 697.13 mg (3.532 mmol) was isolated from the corresponding methyl ester hydrochloride by neutralization, subsequent extraction with ethyl acetate and solvent evaporation. It was then added to the reaction mixture, followed immediately by 365 mg (1.766 mmol) dicyclohexylcarbodiimide (DCC) and 239 mg (1.766 mmol) of HOBt. The reaction mixture was allowed to come to room temperature and stirred for 48 h. DCM was evaporated and the residue was dissolved in ethyl acetate (60 mL) and dicyclohexyl urea (DCU) was filtered off. The organic layer was washed with 2 M HCl (3?30 mL), brine (2?30 mL), 1 M sodium carbonate (3?30 mL) and brine (2?30 mL) and dried over anhydrous sodium sulfate; and evaporated in a vacuum to yield compound 8a, as a white solid. The product was purified by silica gel (100-200 mesh) using n hexane-ethyl acetate (4:1) as eluent.
[0186] Yield: 616.6 mg (1.334 mmol, 75.5%)
[0187] .sup.1H NMR (CDCl3, 400 MHz, ?ppm): 7.16-7.12 & 6.99-6.90 [m, 8H, Aromatic protons], 6.27-6.25 [d, 1H, NH 4F Phe(3)], 4.93 [b, 1H, NH 4F Phe(2)], 4.77-4.72 [m, 1H, C?H 4F Phe(3)], 4.28-4.27 [m, 1H, C?H 4F Phe(2)], 3.67 [s, 3H, OMe], 3.08-2.98 [m, 4H, C?H 4F Phe(2) and 4F Phe(3)], 1.41 [s, 9H, Boc].
[0188] MALDI-TOF (matrix:?-cyano-4-hydroxy cinnamic acid (CHCA)):m/z=[M+Na]+ 485.18 (calculated), 485.45 (observed), [M+K]+ 501.16 (calculated), 501.32 (observed).
[0189] 3. NH.sub.2-L-(4F)Phe(2)-L-(4F)Phe(3)-COOMe 10a:
[0190] 600 mg (1.298 mmol) compound 8a was dissolved in 16 mL of DCM in an ice bath. Then 4 ml of TFA was added and stirred for 2 h. The progress of reaction was monitored through TLC (Thin layer chromatography). After completion of reaction all the solvents were evaporated in rotary evaporator. The product was dissolved in water, neutralized with NaHCO.sub.3 solution and extracted with ethyl acetate, dried over anhydrous sodium sulphate, evaporated into rotary evaporator to get oily product 10a.
[0191] Yield: 435.3 mg (1.202 mmol, 92.6%)
[0192] .sup.1H NMR (DMSO-d.sub.6, 400 MHz, ?.sub.ppm): 9.06-9.05 [d, 1H, NH 4F Phe(3)], 7.32-7.26 & 7.17-7.04 [m, 8H, Aromatic protons], 4.57-4.51 [m, 1H, C?H 4F Phe(3)], 4.04-3.96 [m, 1H, C?H 4F Phe(2)], 3.61 [s, 3H, OMe], 3.18-2.91 [m, 4H, C?H 4F Phe(2) and 4F Phe(3)].
[0193] MALDI-TOF (matrix:?-cyano-4-hydroxy cinnamic acid (CHCA)):m/z=[M+2H].sup.+ 364.14 (calculated), 364.34 (observed), [M+H.sub.2O].sup.+ 480.15 (calculated), 480.35 (observed).
[0194] 4. Fmoc-L-DOPA(ac)-L-(4F)Phe(2)-L-(4F)Phe(3)-COOMe 11a:
[0195] 430 mg (1.187 mmol) of compound 10a was dissolved in 25 mL dry DCM in an ice-water bath and 652.37 mg (1.42 mmol) of Fmoc-L-DOPA(ac)-COOH was added. Then 245 mg (1.187 mmol) dicyclohexylcarbodiimide (DCC) and 161 mg (1.187 mmol) of HOBt were added to reaction mixture. The reaction mixture was allowed to come to room temperature and stirred for 48 h. DCM was evaporated and the residue was dissolved in ethyl acetate (60 mL) and dicyclohexylurea (DCU) was filtered off. The organic layer was washed with water, extracted, dried over anhydrous sodium sulfate and evaporated in a vacuum to yield compound 11a, as a white solid. The product was purified by silica gel (100-200 mesh) using n hexane-ethyl acetate (4:1) as eluent.
[0196] Yield: 594.8 mg (0.74 mmol, 62.4%).
[0197] .sup.1H NMR (CDCl.sub.3, 400 MHz, ?.sub.ppm): 7.77-7.75, 7.54-7.50, 7.42-7.38, 7.33-7.29 [d & m, 8H, Fmoc aromatic protons], 7.05-6.86 [m, 8H, 4F Phe(2) and 4F Phe(3) aromatic protons], 6.62-6.55 [s & m, 3H, DOPA aromatic protons], 6.50 [b, 1H, NH 4F Phe(2)], 6.19 [b, 1H, NH 4F Phe(3)], 5.17 [b, 1H, NH DOPA], 4.68-4.66 [m, 1H, C?H DOPA], 4.54-4.52 [m, 1H, C?H 4F Phe(2)], 4.47-4.42 [m, 1H, C?H 4F Phe(3)], 4.31 (b, 2H, C?H Fmoc], 4.20-4.17 [m, 1H, C?H Fmoc], 3.65 [s, 3H, OMe], 2.98-2.92 [m, 6H, C?H 4F Phe(2) 4F Phe(3) & DOPA], 1.62 [s, 6H, 2?COCH.sub.3].
[0198] MALDI-TOF (matrix:?-cyano-4-hydroxy cinnamic acid (CHCA)):m/z=[M+H].sup.+ 804.31 (calculated), 804.70 (observed), [M+Na+2H].sup.+ 828.30 (calculated), 828.07 (observed), [M+K+H].sup.+ 843.27 (calculated), 843.60 (observed).
[0199] 5. NH.sub.2-L-DOPA(ac)-L-(4F)Phe(2)-L-(4F)Phe(3)-COOMe 12a:
[0200] 580 mg (0.721 mmol) of compound 11a was treated 15 mL with 20% piperidine solution and stirred for 3 h in room temperature. The completion of reaction was monitored by TLC. Then the solution was lyophilized and purified with column chromatography to get pure sticky compound 12a.
[0201] Yield: 275.6 mg (0.474 mmol, 65.8%)
[0202] .sup.1H NMR (DMSO-d.sub.6, 400 MHz, ?.sub.ppm): 8.53 [b, 1H, NH 4F Phe(2)], 7.96 [b, 1H, NH 4F Phe(3)], 7.24-7.23, 7.10-7.04 [m, 8H, 4F Phe(2) and 4F Phe(3) aromatic protons], 6.69-6.65, 6.55-6.53 [m, 3H, DOPA aromatic protons], 5.56 [m, 1H, C?H DOPA], 4.56 [m, 1H, C?H 4F Phe(2)], 4.47 [m, 1H, 4F Phe(3)], 3.61 [s, 3H, OMe], 3.12-2.73 [m, 6H, C?H 4F Phe(2) 4F Phe(3) & DOPA], 1.61-1.58 [d, 6H, 2?COCH.sub.3].
[0203] MALDI-TOF (matrix:?-cyano-4-hydroxy cinnamic acid (CHCA)):m/z=[M+H].sup.+ 582.23 (calculated), 582.25 (observed), [M+Na].sup.+ 604.22 (calculated), 604.37 (observed), [M+K].sup.+ 620.20 (calculated), 620.19 (observed).
[0204] 6. NH.sub.2-L-DOPA-L(4F)-Phe(2)-L(4F)-Phe(3)-COOMe 5:
[0205] 260 mg (0.447 mmol) of compound 12a, was stirred in 10 mL of 95% TFA in water for 6 h. The progress of the reaction was monitored through TLC. After completion of reaction the solvent was evaporated in rotary evaporator. The product was washed with hexane, cold ether and water three times each to get final Peptide 5.
[0206] Yield: 139.1 mg (0.257 mmol, 57.5%)
[0207] .sup.1H NMR (DMSO-d.sub.6, 500 MHz, ?.sub.ppm): 8.72-8.70 [d, 1H, NH 4F Phe(2)], 8.66-8.64 [d, 1H, NH 4F Phe(3)], 7.88 [b, 2H, OH DOPA], 7.29-7.23, 7.12-7.05 [m, 8H, 4F Phe(2) and 4F Phe(3) aromatic protons], 6.7-6.64, 6.5-6.47 [m, 3H, DOPA aromatic protons], 4.60-4.58 [m, 1H, C?H 4F Phe(2)], 4.53-4.52 [m, 1H, C?H 4F Phe(3)], 3.83 [m, 1H, C?H DOPA], 3.58 [s, 3H, OMe], 3.08-2.75 [m, 6H, C?H 4F Phe(2) 4F Phe(3) & DOPA]. .sup.13C NMR (DMSO-d.sub.6, 125 MHz, ?.sub.ppm): 171.9, 170.1, 168.5, 158.9, 158.54, 145.2, 144.5, 131.5, 125.2, 117.4, 115.5, 115.4, 115.3, 11.2, 114.5, 53.9, 52.3, 47.5, 36.2, 33.8, 25.8, 24.9. .sup.19F NMR (DMSO-d6, 470 MHz, ?.sub.ppm): ?116.42, ?116.71.
[0208] MALDI-TOF (matrix:?-cyano-4-hydroxy cinnamic acid (CHCA)):m/z=[M+H].sup.+ 542.20 (calculated), 542.57 (observed), [M+Na].sup.+ 564.19 (calculated), 564.46 (observed), [M+K].sup.+ 580.16 (calculated), 580.32 (observed).
B. Synthesis of Peptide 6
[0209] 1. Boc-L-(4F)Phe(2)-D-(4F)Phe(3)-COOMe 9b:
[0210] The compound was synthesized with the same procedure as compound 9a.
[0211] .sup.1H NMR (CDCl.sub.3, 400 MHz, ?.sub.ppm): 7.13-7.10 & 6.98-6.91 [m, 8H, Aromatic protons], 6.51 [b, 1H, NH 4F Phe(3)], 4.91-4.89 [d, 1H, NH 4F Phe(2)], 4.82-4.77 [m, 1H, C?H 4F Phe(3)], 4.33 [m, 1H, C?H 4F Phe(1)], 3.68 [s, 3H, OMe], 3.09-2.93 [m, 4H, C?H 4F Phe(2) and 4F Phe(3)], 1.38 [s, 9H, Boc].
[0212] MALDI-TOF (matrix:?-cyano-4-hydroxy cinnamic acid (CHCA)):m/z=[M+2H].sup.+ 464.21 (calculated), 464.15 (observed), [M+Na+2H].sup.+ 586.18 (calculated), 586.37, [M+K+H].sup.+ 502.16 (calculated), 502.25 (observed).
[0213] 2. NH.sub.2-L-(4F)Phe(2)-D-(4F)Phe(3)-COOMe 10b:
[0214] The compound was synthesized with the same procedure as compound 10a.
[0215] .sup.1H NMR (DMSO-d.sub.6, 400 MHz, ?.sub.ppm): 8.34 [d, 1H, NH 4F Phe(3)], 7.23-7.19 & 7.12-7.01 [m, 8H, Aromatic protons], 4.61-4.51 [m, 1H, C?H 4F Phe(3)], 3.62 [s, 3H, OMe], 3.44-3.41 [m, 1H, C?H 4F Phe(2)], 3.03-2.74 [m, 4H, C?H 4F Phe(2) and 4F Phe(3)]. 2.35 (b, 2H, free NH.sub.2].
[0216] MALDI-TOF (matrix:?-cyano-4-hydroxy cinnamic acid (CHCA)):m/z=[M+2H].sup.+ 364.14 (calculated), 364.41 (observed).
[0217] 3. Fmoc-L-DOPA(ac)-L-(4F)Phe(2)-D-(4F)Phe(3)-COOMe 11b:
[0218] The compound was synthesized with the same procedure as compound 11a.
[0219] .sup.1H NMR (DMSO-d.sub.6, 400 MHz, ?.sub.ppm): 8.68-8.55 [d, 1H, NH Phe(2)], 8.15-7.92 [d, 1H, NH 4F Phe(3)], 7.88-7.86, 7.61-6.96 [d & m, 16H, Fmoc aromatic protons, 4F Phe(2) and 4F Phe(3) aromatic protons], 6.75 & 6.64 [s, 3H, DOPA aromatic protons], 5.83 [d, 1H, NH DOPA], 4.62-4.53 [m, 2H, C?H 4F Phe(2) and Phe(3)], 4.14-4.02 [m, 3H, C?H DOPA & C?H Fmoc], 3.63 [s, 3H, OMe], 2.76-2.57 [m, 6H, C?H 4F Phe(2), 4F Phe(3) & DOPA], 1.55 [s, 6H, 2?COCH.sub.3].
[0220] MALDI-TOF (matrix:?-cyano-4-hydroxy cinnamic acid (CHCA)):m/z=[M+Na].sup.+ 826.29 (calculated), 826.15 (observed), [M+K].sup.+ 842.27 (calculated), 841.94 (observed).
[0221] 4. NH.sub.2-L-DOPA(ac)-L-(4F)Phe(2)-D-(4F)Phe(3)-COOMe 12b:
[0222] The compound was synthesized with the same procedure as compound 12a.
[0223] .sup.1H NMR (DMSO-d.sub.6, 400 MHz, ?.sub.ppm): 8.66-8.64 [b, 1H, NH 4F Phe(2)], 7.95 [b, 1H, NH 4F Phe(3)], 7.30-6.80 [m, 8H, 4F Phe(2) and 4F Phe(3) aromatic protons], 6.68-6.64, 6.56-6.53 [m, 3H, DOPA aromatic protons], 5.57-5.55 [m, 1H, C?H DOPA], 4.56 [m, 1H, C?H 4F Phe(2)], 4.47 [m, 1H, 4F Phe(3)], 3.63 [s, 3H, OMe], 3.05-2.67 [m, 6H, C(H 4F Phe(2), 4F Phe(3) & DOPA]. 1.59-1.57 [s, 6H, 2?COCH.sub.3].
[0224] MALDI-TOF (matrix:?-cyano-4-hydroxy cinnamic acid (CHCA)): m/z=[M+Na].sup.+ 604.22 (calculated), 604.06 (observed), [M+K].sup.+ 620.20 (calculated), 619.88 (observed).
[0225] 5. NH.sub.2-L-DOPA-L-(4F)Phe(2)-D-(4F)Phe(3)-COOMe 6:
[0226] The Peptide 6 was synthesized with the same procedure as Peptide 5.
[0227] .sup.1H NMR (DMSO-d.sub.6, 500 MHz, ?.sub.ppm): 8.77-8.75 [d, 1H, NH 4F Phe(2)], 8.66-8.64 [d, 1H, NH 4F Phe(3)], 7.80 [b, 2H, OH DOPA], 7.27-7.24, 7.11-7.00 [m, 8H, 4F Phe(2) and 4F Phe(3) aromatic protons], 6.71-6.60 [m, 3H, DOPA aromatic protons], 5.15 [b, 2H, NH2], 4.62-4.60 [m, 1H, C?H 4F Phe(2)], 4.52-4.49 [m, 1H, C?H 4F Phe(3)], 3.83 [m, 1H, C?H DOPA], 3.65 [s, 3H, OMe], 3.10-2.73 [m, 6H, C?H 4F Phe(2), 4F Phe(3) & DOPA]. .sup.13C NMR (DMSO-d.sub.6, 125 MHz, ?.sub.ppm): 117.43, 170.42, 147.86, 146.63, 143.75, 143.69, 141.30, 135.47, 128.64, 127.75, 127.23, 127.13, 125.054, 121.81, 120.02, 118.04, 109.44, 108.25, 67.20, 53.02, 52.33, 47.09, 37.91, 31.94, 29.71, 25.89.
[0228] .sup.19F NMR (DMSO-d.sub.6, 470 MHz, ?.sub.ppm): ?116.43, ?116.91.
[0229] MALDI-TOF (matrix:?-cyano-4-hydroxy cinnamic acid (CHCA)):m/z=[M+H].sup.+ 542.20 (calculated), 542.65 (observed), [M+Na].sup.+ 564.19 (calculated), 564.55 (observed), [M+K].sup.+ 580.16 (calculated), 580.57 (observed).
C. Synthesis of Peptide 7
[0230] 1. Boc-D-(4F)Phe-COOH 13b:
[0231] The compound 13b was synthesized as compound 13a.
[0232] .sup.1H NMR (DMSO-d.sub.6, 400 MHz, ?.sub.ppm): 12.59 [s, 1H COOH], 7.29-7.26 & 7.12-7.08 [m, 4H, Aromatic protons], 4.10-3.57 [m, 1H, C?H 4F Phe], 3.03-2.77 [m, 2H, C?H 4F Phe], 1.32 [s, 9H, Boc].
[0233] MALDI-TOF (matrix:?-cyano-4-hydroxy cinnamic acid (CHCA)):m/z=[M+H].sup.+ 284.12 (calculated), 284.36 (observed), [M+Na].sup.+ 306.11 (calculated), 306.28 (observed).
[0234] 2. Boc-D-(4F)Phe(2)-L-(4F)Phe(3)-COOMe 9c:
[0235] The compound was synthesized with the same procedure as compound 9a.
[0236] .sup.1H NMR (CDCl.sub.3, 400 MHz, ?.sub.ppm): 7.14-7.09 & 6.99-6.93 [m, 8H, Aromatic protons], 6.50 [b, 1H, NH 4F Phe(3)], 4.88 [b, 1H, NH 4F Phe(2)], 4.82-4.77 [m, 1H, C?H 4F Phe(3)], 4.33 [m, 1H, C?H 4F Phe(2)], 3.68 [s, 3H, OMe], 3.09-2.91 [m, 4H, C?H 4F Phe(2) and 4F Phe(3)], 1.38 [s, 9H, Boc].
[0237] MALDI-TOF (matrix:?-cyano-4-hydroxy cinnamic acid (CHCA)):m/z=[M+Na].sup.+ 485.18 (calculated), 485.88 (observed), [M+K].sup.+ 501.16 (calculated), 501.75 (observed).
[0238] 3. NH.sub.2-D-(4F)Phe(2)-L-(4F)Phe(3)-COOMe 10c:
[0239] The compound was synthesized with the same procedure as compound 10a.
[0240] .sup.1H NMR (DMSO-d.sub.6, 400 MHz, ?.sub.ppm): 8.71-8.67 [d, 1H, NH 4F Phe(3)], 7.25-7.21 & 7.12-7.03 [m, 8H, Aromatic protons], 5.49 [b, 2H, NH.sub.2], 4.56-4.54 [m, 1H, C?H 4F Phe(2)], 3.77-3.70[m, 1H, C?H 4F Phe(3)], 3.64 [s, 3H, OMe] 3.07-2.57 [m, 4H, C?H 4F Phe(2) and 4F Phe(3)].
[0241] MALDI-TOF (matrix:?-cyano-4-hydroxy cinnamic acid (CHCA)):m/z=[M+2H].sup.+ 364.14 (calculated), 364.26 (observed).
[0242] 4. Fmoc-L-DOPA(ac)-D-(4F)Phe(2)-L-(4F)Phe(3)-COOMe 11c:
[0243] The compound was synthesized with the same procedure as compound 11a.
[0244] .sup.1H NMR (CDCl.sub.3, 400 MHz, ?.sub.ppm): 7.79-7.72, 7.51-7.47, 7.42-7.38, 7.33-7.29 [d & m, 8H, Fmoc aromatic protons], 6.94-6.88 [m, 8H, 4F Phe(2) and 4F Phe(3) aromatic protons], 6.76-6.61 [s & m, 3H, DOPA aromatic protons], 6.54 [b, 1H, NH 4F Phe(2)], 6.18 [b, 1H, NH 4F Phe(3)], 5.20 [b, 1H, NH DOPA], 4.76-4.68 [m, 1H, C?H DOPA], 4.67-4.57 [m, 1H, C?H 4F Phe(2)], 4.43-4.35 [m, 1H, C?H 4F Phe(3)],], 4.30-4.21 [m, 1H, C?H Fmoc], 4.19-4.01 (b, 2H, C?H Fmoc], 3.62 [s, 3H, OMe], 3.09-2.75 [m, 6H, C?H 4F Phe(2), 4F Phe(3) & DOPA], 1.63 [s, 6H, 2?COCH.sub.3].
[0245] MALDI-TOF (matrix:?-cyano-4-hydroxy cinnamic acid (CHCA)):m/z=[M+H].sup.+ 804.31 (calculated), 804.74 (observed), [M+Na+H].sup.+ 827.30 (calculated), 827.32 (observed), [M+K+H].sup.+ 843.27 (calculated), 843.62 (observed).
[0246] 5. NH.sub.2-L-DOPA(ac)-D-(4F)Phe(2)-L-(4F)Phe(3)-COOMe 12c:
[0247] The compound was synthesized with the same procedure as compound 12a.
[0248] .sup.1H NMR (DMSO-d.sub.6, 400 MHz, ?.sub.ppm): 8.66-8.64 [b, 1H, NH 4F Phe(2)], 7.95 [b, 1H, NH 4F Phe(3)], 7.29-6.81 [m, 8H, 4F Phe(2) and 4F Phe(3) aromatic protons], 6.68-6.64, 6.54-6.53 [m, 3H, DOPA aromatic protons], 5.57-5.55 [m, 1H, C?H DOPA], 4.60 [m, 1H, C?H 4F Phe(2)], 4.48 [m, 1H, 4F Phe(3)], 3.63 [s, 3H, OMe], 2.88-2.73 [m, 6H, C?H 4F Phe(2), 4F Phe(3) & DOPA]. 1.59-1.56 [s, 6H, 2?COCH.sub.3].
[0249] MALDI-TOF (matrix:?-cyano-4-hydroxy cinnamic acid (CHCA)):m/z=[M+H].sup.+ 582.23 (calculated), 581.93 (observed), [M+Na].sup.+ 604.22 (calculated), 604.01 (observed), [M+K].sup.+ 620.20 (calculated), 619.85 (observed).
[0250] 6. NH.sub.2-L-DOPA-D-(4F)Phe(2)-L-(4F)Phe(3)-COOMe 7:
[0251] The Peptide 7 was synthesized with the same procedure as Peptide 5.
[0252] .sup.1H NMR (DMSO-d.sub.6, 500 MHz, ?.sub.ppm): 8.72-8.71 [d, 1H, NH 4F Phe(2)], 8.65-8.64 [d, 1H, NH 4F Phe(3)], 7.89 [b, 2H, OH DOPA], 7.28-7.23, 7.12-7.06 [m, 8H, 4F Phe(2) and 4F Phe(3) aromatic protons], 6.67-6.64, 6.49-6.47 [m, 3H, DOPA aromatic protons], 4.61-4.50 [m, 1H, C?H 4F Phe(2)& Phe(3)], 3.85-3.80 [m, 1H, C?H DOPA], 3.58 [s, 3H, OMe], 3.05-2.72 [m, 6H, C?H 4F Phe(2), 4F Phe(3) & DOPA]. .sup.13C NMR (DMSO-d.sub.6, 125 MHz, ?.sub.ppm): 171.9, 170.9, 168.6, 162.5, 160.6, 158.5, 158.23, 145.7, 145.1, 133.8, 133.7, 133.5, 131.5, 125.8, 120.7, 117.3, 116.1, 115.5, 115.4, 115.3, 115.2, 54.3, 53.9, 52.4, 46.2, 37.3, 37.0, 36.2, 26.7, 25.3, 24.7.
[0253] .sup.19F NMR (DMSO-d.sub.6, 470 MHz, ?.sub.ppm): ?116.31, ?116.53.
[0254] MALDI-TOF (matrix:?-cyano-4-hydroxy cinnamic acid (CHCA)):m/z=[M+H].sup.+ 542.20 (calculated), 542.51 (observed), [M+Na].sup.+ 564.19 (calculated), 564.53 (observed), [M+K].sup.+ 580.16 (calculated), 580.43 (observed).
D. Synthesis of Peptide 8
[0255] 1. Boc-D-(4F)Phe(2)-D-(4F)Phe(3)-COOMe 9d:
[0256] The compound was synthesized with the same procedure as compound 9a.
[0257] 1H NMR (CDCl3, 400 MHz, ?ppm): 7.18-7.15 & 7.01-6.925 [m, 8H, Aromatic protons], 6.25-6.23 [d, 1H, NH 4F Phe(3)], 4.93 [b, 1H, NH 4F Phe(2)], 4.77-4.76 [m, 1H, C?H 4F Phe(2)], 4.30-4.28 [m, 1H, C?H 4F Phe(3)], 3.7 [s, 3H, OMe], 3.10-3.00 [m, 4H, C?H 4F Phe(2) and 4F Phe(3)], 1.40 [s, 9H, Boc].
[0258] MALDI-TOF (matrix:?-cyano-4-hydroxy cinnamic acid (CHCA)):m/z=[M+Na+H].sup.+ 486.18 (calculated), 485.93 (observed), [M+K+H].sup.+ 502.16 (calculated), 502.00 (observed).
[0259] 2. NH.sub.2-D-(4F)Phe(2)-D-(4F)Phe(3)-COOMe 10d:
[0260] The compound was synthesized with the same procedure as compound 10a.
[0261] .sup.1H NMR (DMSO-d6, 400 MHz, ?ppm): 8.36-8.34 [d, 1H, NH 4F Phe(3)], 8.02 [b, 1H, NH 4F Phe(2)], 7.22-7.17 & 7.11-7.01 [m, 8H, aromatic protons], 4.55-4.50 [m, 1H, C?H 4F Phe(3)], 4.08-3.92 [m, 1H, C?H 4F Phe(2)], 3.60 [s, 3H, OMe], 3.04-2.84 [m, 4H, C]3H 4F Phe(2) and 4F Phe(3)].
[0262] MALDI-TOF (matrix:?-cyano-4-hydroxy cinnamic acid (CHCA)):m/z=[M+2H]+ 364.14 (calculated), 364.29 (observed), [M+Na+H]+ 486.13 (calculated), 486.33 (observed).
[0263] 3. Fmoc-L-DOPA(ac)-D-(4F)Phe(2)-D-(4F)Phe(3)-COOMe 11d:
[0264] The compound was synthesized with the same procedure as compound 11a.
[0265] .sup.1H NMR (CDCl3, 400 MHz, ?ppm): 7.77-7.75, 7.55-7.53, 7.42-7.40 [d & m, 8H, Fmoc aromatic protons], 6.94-6.55 [m, 8H, 4F Phe(2) and 4F Phe(3) aromatic protons], 6.71-6.52 [m, 3H, DOPA aromatic protons], 6.52-6.45 [b, 1H, NH 4F Phe(2)], 6.15 [b, 1H, NH 4F Phe(3)], 5.31 [b, 1H, NH DOPA], 4.73-4.65 [m, 1H, C?H DOPA], 4.64-4.56 [m, C?H 4F Phe(2)], 4.51-4.42 [m, 1H, C?H 4F Phe(3)], 4.24-4.11 [m, 1H, C?H Fmoc], 4.19 (b, 2H, C?H Fmoc], 3.61 [s, 3H, OMe], 3.08-2.72 [m, 6H, C?H 4F Phe(2) 4F Phe(3) & DOPA], 1.62 [s, 6H, 2?COCH3].
[0266] MALDI-TOF (matrix:?-cyano-4-hydroxy cinnamic acid (CHCA)):m/z=[M+Na+2H]+ 828.30 (calculated), 828.03 (observed), [M+K+2H]+ 844.27 (calculated), 844.12 (observed).
[0267] 4. NH.sub.2-L-DOPA(ac)-D-(4F)-Phe(2)-D-(4F)-Phe(3)-COOMe 12d:
[0268] The compound was synthesized with the same procedure as compound 12a.
[0269] .sup.1H NMR (DMSO-d6, 400 MHz, ?ppm): 8.58-8.53 [d, 1H, NH 4F Phe(2)], 8.12 [d, 1H, NH 4F Phe(3)], 7.31-7.09 [m, 8H, 4F Phe(2) and 4F Phe(3) aromatic protons], 6.69-6.68, 6.61-6.60 [m, 3H, DOPA aromatic protons], 5.63-5.61 [m, 1H, C?H DOPA], 4.61 [m, 1H, C?H 4F Phe(2)], 4.52 [m, 1H, 4F Phe(3)], 3.64 [s, 3H, OMe], 3.15-2.65 [m, 6H, C(H 4F Phe(2) 4F Phe(3) & DOPA]. 1.54 [d, 6H, 2?COCH.sub.3].
[0270] MALDI-TOF (matrix:?-cyano-4-hydroxy cinnamic acid (CHCA)):m/z=[M+Na]+ 604.22 (calculated), 604.23 (observed), [M+K]+ 620.20 (calculated), 620.12 (observed).
[0271] 5. NH.sub.2-L-DOPA-D-(4F)Phe(2)-D-(4F)Phe(3)-COOMe 8:
[0272] The Peptide 8 was synthesized with the same procedure as Peptide 5.
[0273] .sup.1H NMR (DMSO-d6, 500 MHz, ?ppm): 8.80-8.77 [d, 1H, NH 4F Phe(2)], 7.95 [b, 2H, OH DOPA], 7.31-7.20, 7.12-7.03 [m, 8H, 4F Phe(2) and 4F Phe(3) aromatic protons], 6.59-6.57, 6.22-6.20 [m, 3H, DOPA aromatic protons], 5.58 [b, 2H, free NH2)], 4.75-4.62 [m, 1H, C?H 4F Phe(2)], 4.51-4.45 [m, 1H, C?H 4F Phe(3)], 3.91-3.82 [m, 1H, C?H DOPA], 3.62 [s, 3H, OMe], 3.08-2.62 [m, 6H, C?H 4F Phe(2), 4F Phe(3) & DOPA]. 13C NMR (DMSO-d6, 125 MHz, ?ppm): 172.01, 171.20, 168.27, 162.77, 158.59, 158.27, 157.09, 145.65, 145.02, 133.58, 133.71, 131.46, 131.45, 131.37, 125.74, 120.65, 117.37, 115.95, 115.63, 115.42, 115.31, 115.09, 54.14, 52.44, 47.97, 33.80, 25.78, 24.92. 19F NMR (DMSO-d6, 470 MHz, ?ppm): ?116.08, ?116.42.
[0274] MALDI-TOF (matrix:?-cyano-4-hydroxy cinnamic acid (CHCA)):m/z=[M+H]+ 542.20 (calculated), 542.85 (observed), [M+Na]+ 564.19 (calculated), 564.55 (observed), [M+K]+ 580.16 (calculated), 580.40 (observed).
E. Synthesis of Peptide 9
[0275] 1. Boc-L-(F5)Phe(2)-L-(F5)Phe(3)-COOMe 9e.
[0276] We have purchased Boc-L-(F5)Phe-COOH. We first deprotected the Boc group by treatment of TFA/DCM, then evaporate all the solvents and esterification of NH2-Phe(F5)-COOH was done by treating with thionyl chloride and methanol. Then the compound 9e was synthesized by coupling of Boc-L-(F5)Phe-COOH with NH2-L-(F5)Phe-COOMe as described for compound 9a.
[0277] .sup.1H NMR (CDCl3, 400 MHz, ?ppm): 6.52 [b, 1H, NH Phe(3)], 4.93 [b, 1H, NH 4F Phe(2)], 4.92-4.85 [m, 1H, C?H Phe(3)], 4.42-4.29 [m, 1H, C?H Phe(2)], 3.81 [s, 3H, OMe], 3.42-2.95 [m, 4H, C?H Phe(2) and Phe(3)], 1.44 [s, 9H, Boc].
[0278] MALDI-TOF (matrix:?-cyano-4-hydroxy cinnamic acid (CHCA)):m/z=[M+Na+H]+ 630.11 (calculated), 630.08 (observed), [M+K+H]+ 646.08 (calculated), 646.13 (observed).
[0279] 2. NH.sub.2-L-(F5)Phe(2)-L-(F5)Phe(3)-COOMe 10e.
[0280] The compound 9e was prepared as described for compound 10a.
[0281] .sup.1H NMR (DMSO-d6, 400 MHz, ?ppm): 8.93-8.90 [d, 1H, NH Phe(3)], 8.40 [b, 1H, free NH2], 4.72-4.70 [m, 1H, C?H Phe(3)], 3.90 [m, 1H, C?H Phe(2)], 3.61 [s, 3H, OMe], 3.17-2.99 [m, 4H, C?H Phe(2) and Phe(3)].
[0282] MALDI-TOF (matrix:?-cyano-4-hydroxy cinnamic acid (CHCA)):m/z=[M+Na+H]+ 530.05 (calculated), 530.16 (observed), [M+K+H]+ 546.03 (calculated), 646.53 (observed).
[0283] 3. Fmoc-DOPA(ac)-L-(F5)Phe(2)-L-(F5)Phe(3)-COOMe 11e.
[0284] The compound 11e was prepared as described for compound 11a.
[0285] .sup.1H NMR (DMSO-d6, 400 MHz, ?ppm): 8.75-8.72 [d, 1H, NH Phe(2)], 8.36-8.34 [b, 1H, NH Phe(3)], 7.88-7.26 [m, 8H, Fmoc aromatic protons], 6.79-6.67 [m, 3H, DOPA aromatic protons], 5.57-5.55 [b, 1H, NH DOPA], 4.66-4.63 [m, 2H, C?H Fmoc], 4.14-4.09 [m, 3H, C?H DOPA, C?H Phe(2), C?H Phe(3)], 3.62 [s, 3H, OMe], 3.05-2.90 [m, 6H, C?H Phe(2), Phe(3) & DOPA], 1.56 [s, 6H, 2?COCH3].
[0286] MALDI-TOF (matrix:?-cyano-4-hydroxy cinnamic acid (CHCA)):m/z=[M+Na]+ 970.21 (calculated), 970.22 (observed), [M+K]+ 986.19 (calculated), 986.04 (observed).
[0287] 4. NH.sub.2-DOPA(ac)-L-(F5)Phe(2)-L-(F5)Phe(3)-COOMe 12e
[0288] The compound 12e was prepared as described for compound 12a.
[0289] 1H NMR (DMSO-d6, 400 MHz, ?ppm): 8.73-8.71 [d, 1H, NH Phe(2)], 6.69-6.55 [m, 3H, DOPA aromatic protons], 5.57-5.55 [d, 1H, NH Phe(3)], 4.64-6.63 [m, 1H, C?H DOPA], 4.54 [m, 1H, C?H Phe(2)], 4.13-4.08 [m, 1H, C?H Phe(3)], 3.61 [s, 3H, OMe], 3.15-2.67 [m, 6H, C?H Phe(2), Phe(3) & DOPA], 1.60 [s, 6H, 2?COCH3].
[0290] MALDI-TOF (matrix:?-cyano-4-hydroxy cinnamic acid (CHCA)):m/z=[M+Na]+ 748.15 (calculated), 748.23 (observed), [M+K]+ 764.12 (calculated), 764.06 (observed).
[0291] 5. NH.sub.2-DOPA-L-(F5)Phe(2)-L-(F5)Phe(3)-COOMe 9
[0292] Peptide 9 was prepared as described for Peptide 5.
[0293] .sup.1H NMR (DMSO-d6, 400 MHz, ?ppm): 9.46 [b, 1H, NH Phe(2)], 9.25 [b, 1H, NH Phe(2)], 8.39 [b, 2H, free NH2), 6.68-6.54 [m, 3H, DOPA aromatic protons], 4.69-4.65 [m, 2H, C?H Phe (1) & Phe(2)], 4.55 [m, 1H, C?H DOPA], 3.61 [s, 3H, OMe], 3.01-2.95 67 [m, 6H, C?H Phe(2) Phe(2) & DOPA]0.13C NMR (DMSO-d6, 100 MHz, ?ppm): 193.6, 158.5, 158.2, 144.3, 140.8, 139.5, 133.7, 129.9, 128.5, 127.8, 124.4, 53.8, 44.2, 33.8, 30.5, 29.4, 22.6, 17.6. 19F (DMSO-d6, 470 MHz, ?ppm): ?141.7, ?142.4, ?157.6, ?163.1, ?163.4.
[0294] MALDI-TOF (matrix:?-cyano-4-hydroxy cinnamic acid (CHCA)):m/z=[M+Na]+ 748.15 (calculated), 748.23 (observed), [M+K]+ 764.12 (calculated), 764.06 (observed).
F. Synthesis of Peptide 7
[0295] 1. Boc-L-DOPA-COOH:
[0296] The compound was synthesized as compound 13a.
[0297] .sup.1H NMR (DMSO-d6, 400 MHz, ?ppm): 9.13 (b, 2H, 2?OH], 7.35-7.33 [d, 1H, NH DOPA], 7.03-6.88[m, 3H, DOPA aromatic protons], 4.45-4.37 [m, 1H, C?H DOPA], 3.22-2.92 [m, 1H, C?H DOPA], 1.75 [s, 9H, OMe].
[0298] MALDI-TOF (matrix:?-cyano-4-hydroxy cinnamic acid (CHCA)):m/z=[M+Na]+ 320.11 (calculated), 320.51 (observed), [M+K]+ 336.08 (calculated), 336.29 (observed).
[0299] 2. Boc-L-DOPA-L-(4F) Phe-COOMe:
[0300] The compound was synthesized as compound 9a.
[0301] 1H NMR (CDCl3, 400 MHz, ?ppm): 7.26-7.24 [d, 1H, NH Phe], 6.90-6.50 [m, 7H, all aromatic protons], 5.24 [b, 1H, NH DOPA], 4.82-4.77 [m, 1H, C?H DOPA], 4.36 [b, 1H, C?H Phe], 3.64 [s, 3H, OMe], 2.99-2.87 [m, 4H, C?H DOPA & Phe], 1.42 [s, 9H, Boc].
[0302] MALDI-TOF (matrix:?-cyano-4-hydroxy cinnamic acid (CHCA)):m/z=[M+Na+H]+ 500.18 (calculated), 500.02 (observed), [M+K+H]+ 516.16 (calculated), 516.24 (observed).
[0303] 3. NH.sub.2-L-DOPA-L-(4F) Phe-COOMe 6:
[0304] This compound was synthesized as described for 10a.
[0305] 1H NMR (DMSO-d6, 400 MHz, ?ppm): 8.92 & 8.81 [s, 2H, 2?OH], 8.02 [b, 2H, free NH2], 7.27-7.09 [m, 4H, aromatic proton Phe], 6.67-6.48 [m, 3H, aromatic protons DOPA], 4.58-4.52 [m, 1H, C?H DOPA], 3.90-3.86 [b, 1H, C?H Phe], 3.61 [s, 3H, OMe], 3.08-2.67 [m, 4H, C?H DOPA & Phe]. 13C NMR (DMSO-d6, 100 MHz, ?ppm): 171.4, 168.7, 162.7, 160.4, 158.5, 145.6, 145.0, 133.3, 133.2, 131.4, 125.6, 120.6, 117.2, 115.9, 115.5, 115.3, 54.1, 53.9, 52.4, 41.0, 36.8, 36.2, 23.6.]. 19F NMR(DMSO-d6, 470 MHz, ?ppm): ?(116.25-116.29).
[0306] MALDI-TOF (matrix:?-cyano-4-hydroxy cinnamic acid (CHCA)):m/z=[M+H] 377.15 (calculated), 377.25 (observed), [M+Na]+ 399.13 (calculated), 399.24 (observed).
[0307] Another two exemplary peptide derivatives have been synthesized using solid or solution phase synthesis. The purity and identify of the peptides was determined using HPLC and MS spectrometer.