Patent Application
20190352511
The present invention relates to lanthanide complexes based on dendrimers. In particular, the subject of the present invention is dendrimeric compounds, grafted to an antenna, capable of complexing with lanthanides.
The methods related to fluorescence (microscopy and macroscopy) have the advantages of being very sensitive, to allow real-time measurements that are little dangerous for biological media (because of the small quantity of imaging agents required subject to using suitable excitation wavelengths) and very accessible (in terms of manufacturing and use costs), experimental time, mobility and degree of specialization of users.
The vast majority of commercial organic fluorophores are highly photosensitive and degrade in the presence of excitation light.
Fluorescent reporters based on semiconductor nanocrystals are another range of optical imaging agent choices. Another limiting factor is the high toxicity of the metals that make up these nanoparticles (cadmium, tellurium and selenium) in the case where the latter dissociate.
The family of lanthanides includes 14 elements with extremely interesting and unique optical properties, characterized by narrow and precise emission bands, ranging from visible to near infrared (>1200 nm). Each lanthanide has distinct and identifiable spectral properties. It is thus possible to identify, on the basis of the same technology, a whole range of different wavelengths simply by choosing the nature of the lanthanide to be incorporated in the molecule. These emission bands are much narrower than organic fluorophores and fluorescent nanoparticles (quantum dots), which allows for better spectral discrimination and multiplexed assays. In addition, the position (in nm) of these emission bands does not vary according to the environment (cell, pH, temperatures, hydrophilic/hydrophobic sites . . . ) which facilitates their detection and minimizes the adaptation of the equipment (unique filter for a given lanthanide).
Despite the strong demand from biology researchers and physicians, to date there are no fluorescent probes compatible with biological applications and operating in lower energy conversion based on lanthanides which are ideally excitable and emitters above 600 nm, corresponding to the biological window. Overall, the current near-infrared probes are of an organic nature, while the commercial probes are few and suffer from limitations such as the tendency towards photobleaching and restricted Stokes displacements.
The object of the present invention is therefore to provide a polymetallic lanthanide dendrimeric complex emitting in the near infrared and capable of being excited in the near infrared.
Another object of the invention is to provide an absorbing and emitting luminescent system in the near infrared and to observe various biological systems without destroying them or interfering with their operation. Another object of the invention is to provide a luminescent system for limiting spurious fluorescence/luminescence signals generated by biological materials (autofluorescence).
Thus, the present invention relates to a complex comprising at least one dendrimer (D) and at least one lanthanide (Ln), in which the dendrimer (D) comprises a unit of formula (I) below:
in which:
said unit of formula (I) being covalently connected to at least one antenna which absorbs at a wavelength ranging from 500 nm to 900 nm.
Luminescent compounds containing lanthanides have, among other advantages, a spectral specificity in the visible and near infrared spectral discrimination by their narrow emission bands specific to the nature of each lanthanide. In order to obtain a good luminescence intensity, it is important to introduce functional groups on the molecule that make it capable of absorbing a large amount of light radiation and to transfer the resulting energy onto the luminescent lanthanide in order to obtain a luminescence radiation by return of the lanthanide to the fundamental state.
Near infrared offers many unique advantages. It makes it possible to dispense with the auto-fluorescence of the tissues, and thus makes it possible to improve the signal-to-noise ratio and thus the detection sensitivity. Moreover, the biological tissues do not, or only slightly, absorb between 640 nm and 1100 nm (window of biological transparency), which allows excitation of the probes in depth and non-invasive biological observation (diagnosis and research).
In addition, the lanthanide complexes are photostable. This photostability property is crucial in allowing the imaging agent to be excited i) over long experimental times and/or ii) during successive experiments and/or iii) by powerful excitation sources (lasers for confocal microscopy, for example). A larger amount of photons may thus be collected without disturbing or damaging the functioning of the biological system to be studied (it is important to remember here that the excitation wavelength (>650 nm) interacts extremely weakly with fluids and tissues. and increases the intensity and quality of the signal collected.
The structure of the dendrimers makes it possible to group on the same molecule a large density of lanthanides and antennas making it possible to increase the quantity of photons emitted per unit volume, which increases the intensity of the signal per molecule and therefore the sensitivity of the detection.
The present invention thus relates to an entity obtained by complexation between a dendrimer (D) as defined above and at least one lanthanide. The complexes thus obtained are luminescent molecules.
The lanthanides are encapsulated within the dendrimer due to the presence of oxygen and nitrogen atoms. The arrangement of the dendrimer branches around the lanthanides partially protects them from direct interactions with the solvent and water molecules in particular.
Preferably, the complexes according to the invention are obtained by placing a dendrimer (D) in contact with a solution of lanthanides. The dendrimer (D) is dissolved, for example, in a solution of DMSO and a solution of lanthanide salt is added to the solution containing the compound (D), in particular in an eight to one ratio. According to one embodiment, the reaction mixture was mixed and the resulting dendrimer-lanthanide conjugate was isolated by dialysis.
According to one embodiment, in the compound (D), the unit of formula (I) is connected via at least one arm to at least one antenna as defined above.
Preferably, of the 16 peripheral nitrogen atoms in the formula (I), at least one is covalently connected to at least one antenna via an arm.
According to the invention, the term “antenna” designates an entity capable of absorbing a large amount of excitation light to transfer the energy corresponding to the lanthanides and/or to emit directly by fluorescence.
Preferably, the antenna is selected from the group consisting of anthraquinones, cyanines, especially cyanines 5, cyanines 5.5 and cyanines 7, aza-BODIPY, perylenediimides, porphyrins, phenothiazine salts and their derivatives.
Among the antennas, it is also possible to use compounds of the “IR dyes” type well known to those skilled in the art. Among these antennas, may be mentioned for example the following compounds:
These antennas are fixed on the dendrimers via chlorine by substitution and thus in the final form there is no chlorine but a C, N, S and O.
According to the invention, the term “arm” refers to an entity for covalently connecting the pattern of formula (I) and the antenna.
According to one embodiment, the antenna is connected to the unit of formula (I) in a covalent manner via at least one arm corresponding to the following formula (II):
-A4-X-[A5-Z]i-((A6)j-Y)k- (II)
in which:
In the formula (II) as defined above, it is through the radical A4 that the arm binds to the dendrimer (in particular the unit of formula (I) above) and it is via Y, or Z when k=0, that the arm binds to the antenna.
According to a preferred embodiment, the dendrimer has the following formula:
C1-{A1-N[A2-N(A3-N(A4-R′)2)2]2}4 (III)
in which:
—NH—C(O)-[A5-Z]i-((A6)j-Y)k-L
—NH—C(O)-[A5-Z]i-((A6)j-Y)k-L′
—NH—C(O)-A5-L″
According to the invention, the term “water-solubilising group” designates a chemical entity making it possible to increase the solubility of the probe in aqueous media.
Among the water-solubilising groups, may be mentioned, for example, phosphates, sulphonates, sugars and PEG chains.
According to the invention, the term “targeting group” refers to a molecule, biological or not, capable of recognizing and/or binding a specific biological site.
Targeting groups include, for example, antibodies, proteins, peptides, carbohydrates, lipids, polysaccharides, fatty acids, amino acids, deoxyribonucleic acids, ribonucleic acids, oligonucleotides, medicaments, and ligands.
Among the water-solubilising groups and the targeting groups, may be mentioned the following groups:
Formula (II) above contains 32 peripheral R′ groups, of which at least one comprises an antenna connected to the dendrimer via an arm. Thus, the dendrimers according to the invention may comprise at least one arm through which at least one antenna is linked.
Thus, as indicated above, at least one of the R′ groups has the formula (1) above.
According to one embodiment, the dendrimer is a generation 4 dendrimer of the following formula (III′):
C1-{A1N[A2-N(A3-N(A4-N[(CH2)2—C(O)—NH—(CH2)2—R′]2)2)2]2}4
R′ being as defined above.
Preferably, in formula (1), k=0.
Preferably, in formula (1), k=0 and i=1.
According to one embodiment, in the formula (II), at least one of the R′ groups corresponds to the following formula (1′):
—NH—C(O)-A5-Z-L
A5 and Z being as defined in formula (II), and
L being as defined above.
According to one embodiment, in the formula (II), all the identical R′ groups correspond to the formula (1′).
Preferably, in the formula (1′) above, A5 is chosen from alkylene radicals, linear or branched, comprising from 1 to 4 carbon atoms.
Preferably, in formula (1′) above, Z is selected from the group consisting of —O—, —NH—, —S—, —C(═O)—O—, —O—C(═O)—, —NH—C(═O)—, —N(Alk)-C(═O)—, —C(═O)—NH— et —C(═O)—N(Alk)- groups, Alk representing an alkyl group having from 1 to 6 carbon atoms.
According to one embodiment, in the formula (II), at least one of the R′ groups corresponds to the following formula (1″):
A5 and L being as defined above.
Preferably, in formula (1″) above, A5 is chosen from alkylene radicals, linear or branched, comprising from 1 to 4 carbon atoms.
According to one embodiment, in the formula (II), all the identical R′ groups correspond to the formula (1″).
According to one embodiment, in formula (II), at least one of the R′ groups corresponds to the following formula (1′″):
—NH—C(O)-A5-O-L
A5 and L being as defined above.
Preferably, in the formula (1′″) above, A5 is chosen from alkylene radicals, linear or branched, comprising from 1 to 4 carbon atoms.
According to one embodiment, in the formula (II), all the identical R′ groups correspond to the formula (1′″).
According to one embodiment, the antenna responds to one of the following formulas:
When the antenna meets the formula
this may contain a metal, chosen in particular from Ag, Al, As, Au, Cd, Co, Cu, Fe, Ir, Mg, Mn, Ni, Os, Pd, Pt, Rh, Ru, Sb, Sn, V, Zn, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu.
Preferably, the dendrimer (D) according to the invention has the following formula:
in which L2 is one of the following antennas:
Preferably, the dendrimer (D) according to the invention has the following formula:
in which R corresponds to the following formula:
Preferably, the dendrimer (D) according to the invention has the following formula:
in which L2 is one of the following antennas:
Preferably, the dendrimer (D) according to the invention has the following formula:
in which:
According to one embodiment, in the complexes according to the invention, the lanthanide is chosen from the group consisting of Yb, Nd, Ho, Tm, Sm, Dy, Eu, Pr and Er.
The present invention also relates to a conjugate comprising a biological molecule and a complex as defined above, wherein said complex is linked to the biological molecule via a linker, said biological molecule being chosen from the group consisting of antibodies, proteins, peptides, carbohydrates, lipids, polysaccharides, fatty acids, amino acids, deoxyribonucleic acids, ribonucleic acids, oligonucleotides, drugs, and ligands.
The present invention also relates to the use of a complex as defined above as a fluorescent chromophore.
According to the invention, the term “fluorescent chromophore” refers to a molecule that can re-emit light after excitation with a quantum yield greater than 10−6 (10−4%).
The complexes according to the invention may especially be used in the field of cell imaging, veterinary imaging, blood tests, biopsies, histological sectional analyzes, high throughput screening assays, bioanalytical assays. plates 96, 396 and 1536 wells or assisted (guided) surgery by imaging.
The present invention also relates to the use of a complex as defined above as a photodynamic therapy (PDT) agent.
This antenna (10) is obtained according to the reaction scheme below:
8.94 ml of para-anisaldehyde (10 g, 73.45 mmol, 1 eq.) and 8.58 ml of acetophenone (8.825 g, 73.45 mmol, 1 eq.) are dissolved in a flask in 150 mL ethanol. The flask is immersed in an ice bath and 5.876 g of sodium hydroxide (149.9 mmol, 2 eq.) dissolved in 50 ml of water are added dropwise. The solution is allowed to warm to room temperature and stirred overnight. The next day, the flask is immersed in an ice bath and cold water is added to the mixture. A yellow precipitate forms and the mixture is sintered and washed with water. The precipitate is then recrystallized from ethanol yielding 11.698 g of chalcone 1 (49.1 mmol, 67%) as white crystals.
1H NMR (250 MHz, Chloroform-d) δ8.06-7.97 (m, 2H), 7.79 (d, J=15.0 Hz, 1H), 7.64-7.59 (m, 2H), 7.58-7.45 (m, 3H), 7.42 (d, J=15.6 Hz, 1H), 6.98-6.91 (m, 2H), 3.86 (s), 3H).
0.546 g (2.29 mmol, 1 eq.) of 0.621 mL of nitromethane (11.46 mmol, 5 eq.) and 1.185 mL of diethylamine (11.46 mmol, 5 eq.) are dissolved in a flask with 150 mL of methanol. The solution is heated to reflux overnight. After the reaction is complete, the solvent is evaporated in vacuo and the residue dissolved in dichloromethane. The solution is washed with a solution of 1M KHSO4 then saturated NaCl. The organic phases are combined, dried over MgSO4, filtered and concentrated in vacuo. The residue is purified by chromatography on silica with petroleum ether/ethyl acetate providing nitrobutanone 2 (0.654 g, 2.18 mmol, 95%) as a pale yellow viscous oil.
1H NMR (250 MHz, Chloroform-d) δ7.96-7.88 (m, 2H), 7.63-7.53 (m, 1H), 7.51-7.41 (m, 2H), 7.25-7.16 (m, 2H), 6.91-6.81 (m, 2H), 4.80 (ddd, J=12.3, 6.7, 0.5 Hz, 1H), 4.65 (ddd, J=12.3, 7.9, 0.4 Hz, 1H), 4.18 (p, J=7.0 Hz, 1H), 3.78 (s, 3H), 3, 42 (dd, J=7.0, 2.1 Hz, 2H).
In a flask are dissolved 3.5 g of nitrobutanone 2 (11.69 mmol, 1 eq.) and 5 eq. of KOH in 100 mL of MeOH/THF (1:2). The solution is stirred at room temperature for one hour and then added dropwise to a solution of concentrated H2SO4 (2 mL/mmol) dissolved in 100 mL of MeOH at 0° C. After the addition is complete, the ice bath is removed and the solution stirred at room temperature for 1 h. The mixture is then poured into an Erlenmeyer flask containing water and ice and the solution is neutralized by adding a 4M sodium hydroxide solution. Once neutralized, the mixture is extracted with dichloromethane and the organic phase is dried over MgSO4, filtered and the solvent evaporated under reduced pressure. The residue is dissolved in 100 mL glacial acetic acid and 5 eq. ammonium acetate are added. The solution is heated at reflux for one hour during which the color of the solution changes from yellow to deep blue. The solution is cooled to room temperature and the acetic acid evaporated under reduced pressure. The black solid is then dissolved in dichloromethane and the solution is washed several times with a saturated solution of sodium bicarbonate and brine. The organic phase is dried over MgSO4, filtered and concentrated under reduced pressure. The residue is then dissolved in a minimum of dichloromethane and the petroleum ether is added slowly until a precipitate forms. The precipitate is filtered on Buchner and washed several times with petroleum ether. Pyrrole 3 is obtained with a yield of 60% (1742 g, 6.99 mmol) in the form of a slightly colored powder.
1H NMR (250 MHz, DMSO-d6) δ11.32 (s, 1H), 7.66 (dt, J=7.7, 1.1 Hz, 2H), 7.57-7.48 (m, 2H), 7.36 (dd, J=8.4, 7.0 Hz, 2H), 7.21 (dd, J=2.8, 1.7 Hz, 1H), 7.20-7.12 (m, 1H), 6.94-6.84 (m, 3H), 3.75 (s, 3H).
0.546 g of pyrrole 3 (2.19 mmol, 1 eq.) are dissolved under argon in a flask in 100 ml of anhydrous dichloromethane. The solution is cooled to −78° C. and 5.48 ml of a solution of 1M BBr3 in dichloromethane (5.48 mmol, 2.5 eq.) are slowly added. The reaction is stirred for 3 h at −78° C. and then overnight at room temperature. The next day, the solution is cooled to −78° C. and MeOH is added to the mixture. The solution is stirred for one hour then it is diluted in dichloromethane and washed with saturated NaCl. The organic phase is dried over MgSO4, filtered and concentrated under reduced pressure. The residue is dissolved in a minimum of dichloromethane and the product is precipitated by slow addition of petroleum ether. The precipitate is filtered on Buchner and washed with petroleum ether yielding 0.495 g (2.10 mmol, 96%) of pyrrole 4 in the form of a slightly purplish white powder.
1H NMR (250 MHz, DMSO-d6) δ11.25 (s, 1H), 9.15 (s, 1H), 7.69-7.61 (m, 2H), 7.43-7.31 (m, 4H), 7.20-7.11 (m, 2H)), 6.81 (dd, J=2.7, 1.7 Hz, 1H), 6.77-6.69 (m, 2H).
1H NMR (250 MHz, acetone-d6) δ10.48 (s, 1H), 8.09 (s, 1H), 7.71-7.65 (m, 2H), 7.49-7.41 (m, 2H), 7.41-7.31 (m, 2H), 7.21-7.14 (m, 2H), 6.85 (dd, J=2.8, 1.7 Hz, 1H). 6.84 -6.80 (m, 2H).
13C NMR (63 MHz, Acetone) δ156.27, 134.16, 133.40, 129.58, 128.76, 126.92, 126.80, 126.55, 124.38, 116.24, 115 , 94, 104.14.
LRMS: calcd: 235.0997, measured [M+H]+: 236.1
IR (cm−1): 3443, 3300, 1600, 1581, 1494, 1244, 1132, 921, 834, 804, 778, 751, 717, 690, 609.
Mp: 209° C.
0.725 g of 4 (3.08 mmol, 1 eq.) in 60 ml of DMF are dissolved in a flask. 1.278 g of K2CO3 (9.24 mmol, 3 eq.), 1.08 ml of methyl chloroacetate (12.33 mmol, 4 eq.) and a catalytic amount of potassium bromide are added to the solution which is stirred at room temperature all night. The solution is then extracted with diethyl ether and washed three times with brine. The aqueous phase is then extracted three times with diethyl ether. The organic phases are combined and dried over MgSO4, filtered and the solvent evaporated under vacuum. The residue is dissolved in a minimum of dichloromethane and petroleum ether is added until a precipitate forms. The precipitate is filtered through Buchner and washed with petroleum ether to give 0.553 g of 5 as an off-white powder.
1H NMR (250 MHz, Chloroform-d) δ8.43 (s, 1H), 7.55-7.46 (m, 4H), 7.44-7.35 (m, 2H), 7.25-7.19 (m, 1H), 7.07 (dd, J=2.7, 1.7 Hz, 1H), 6.97-6.88 (m, 2H), 6.76 (dd, J=2.8, 1.7 Hz, 1H), 4.66 (s, 2H), 3.82 (s, 3H).
13C NMR (63 MHz, CDCl3) δ169.57, 156.10, 133.02, 132.51, 129.56, 128.93, 126.45, 126.38, 126.11, 123.83, 114 , 98, 114.94, 103.86, 65.58, 52.25.
LRMS: Calcd: 307.1208, Measured [M+H]+: 308.4
IR (cm−1): 3430, 2941, 1759, 1600, 1581, 1496, 1438, 1212, 1178, 1131, 1075, 834, 801, 774, 758, 719, 693.
Mp: 165° C.
0.593 g of pyrrole 3 (2.38 mmol, 1 eq.) in 50 ml of ethanol and 0.48 ml of concentrated HCl (0.2 ml/mmol) are dissolved in a flask at room temperature. 0.189 g of sodium nitrite (2.74 mmol, 1.15 eq.) dissolved in water (concentration 0.6 mol/l) are added dropwise. The solution is stirred for 30 min and is then cooled to 0° C. A second portion of concentrated HCl (2.38 mL, 1 mL/mmol) is added. The solution is stirred for one hour then it is dissolved in dichloromethane and washed with brine. The organic phase is dried, filtered and concentrated under reduced pressure. The residue is dissolved in a minimum volume of ethanol and excess aqueous solution of sodium acetate and ice are added and the mixture is stirred for one hour. The solution is then extracted with dichloromethane and washed with brine. The organic phase is dried over MgSO4, filtered and concentrated in vacuo. The residue is then dissolved in a minimum volume of dichloromethane and the product is precipitated by slow addition of petroleum ether. The solid is filtered on Buchner and washed with petroleum ether. 0.503 g of nitrosopyrrole 6 (1.81 mmol, 76%) are obtained in the form of a green powder.
1H NMR (250 MHz, Chloroform-d) δ8.20-8.13 (m, 2H), 7.79 (dd, J=6.9, 3.0 Hz, 2H), 7.51 (dd, J=5.1, 1.8 Hz, 3H), 7.07 (s, 1H), 7.02 (d, J=8.9 Hz, 2H), 3.89 (s, 3H).
20 g of glacial acetic acid (0.112 g of nitrosopyrrole 6 (0.40 mmol, 1 eq.), 0.124 g of pyrrole (0.40 mmol, 1 eq. 0.40 mL of acetic anhydride. The solution is stirred and heated at reflux for one hour during which the color changes to dark blue. The solution is then cooled and the solvent evaporated under reduced pressure. The residue is then dissolved in dichloromethane and the solution is washed with saturated NaHCO3 and saturated NaCl. The organic phase is dried over MgSO4, filtered and concentrated in vacuo. The residue is then dissolved in a minimum of dichloromethane and the petroleum ether is added until a precipitate forms which is filtered on Buchner and washed with petroleum ether. Azadipyrromethene 7 is obtained in the form of a dark blue powder (0.197 g, 0.35 mmol, 87%).
1H NMR (250 MHz, Chloroform-d) δ8.05-7.98 (m, 4H), 7.93 (ddd, J=7.8, 6.4, 1.5 Hz, 4H), 7.58-7.44 (m, 7H), 7.11 (s, 1H), 7.09 (s, 1H), 6.96 (dd, J=8.9, 1.8 Hz, 4H), 4.71 (s, 2H), 3.90 (s, 3H), 3.85 (s, 3H).
IR (cm−1): 3426, 2948, 1758, 1597, 1495, 1211, 1175, 1075, 1002, 902, 833, 801, 772, 759, 745, 718, 692, 637, 605.
Mp: 230° C.
HRMS (ESI): m/z calcd for [C36H30N3O4]: 568.223083, measured 568.222834 (−0.4 ppm)
In a flask under argon are dissolved 0.183 g of azadipyrromethene 7 (0.32 mmol, 1 eq.) and 0.548 ml of DIPEA (3.22 mmol, 10 eq.) in freshly distilled dichloromethane. After stirring for a few min at room temperature, 0.613 ml of distilled BF3.Et2O (4.84 mmol, 15 eq.) are added and the solution is refluxed for two hours. The solution is then cooled to room temperature and the organic phase is washed with brine three times. The aqueous phase is then re-extracted three times with dichloromethane and the combined organic phases are dried over MgSO4, filtered and concentrated in vacuo. The residue is purified by chromatography on silica with a gradient of PE/DCM. Azabodipy 8 is obtained as an iridescent dark blue powder (0.196 g, 0.318 mmol, 99%).
1H NMR (400 MHz, Acetone-d6) δ8.23 (dd, J=8.8, 6.2 Hz, 4H), 8.18-8.09 (m, 4H), 7.58-7.46 (m, 6H), 7.33 (d, J=1.4 Hz, 2H), 7.14 (dd, J=9.0, 7.2 Hz, 4H), 4.88 (s, 2H);), 3.92 (s, 3H), 3.79 (s, 3H).
13C NMR (101 MHz, acetone) δ169.66, 162.39, 160.53, 132.72, 131.94, 131.87, 131.70, 131.62, 130.55, 130.51, 129.36, 125.87, 118.92, 115.83, 115.26, 65.70, 55.88, 52.29.
19F NMR (235 MHz, Acetone-d6) δ−130.38 (dd, J=62.8-31.4 Hz).
HRMS (ESI): m/z calcd for [C36H29BF2N3O4]: 616.221996, measured 616.221217 (−1.3 ppm)
Mp: 179° C.
IR (cm−1): 3288, 2918, 2584, 1758, 1728, 1601, 1504, 1487, 1454, 1388, 1277, 1252, 1228, 1175, 1129, 1100, 1068, 1024, 999, 970, 929, 904, 868, 836, 818, 767, 742, 690, 641, 615.
0.308 g of azabodipy 8 (0.5 mmol, 1 eq.) in a THF/water/H3PO4 mixture (50 ml: 25 ml: 10 ml) is dissolved in a flask. The solution is stirred under reflux for 20 hours until no trace of the ester is visible by TLC. After cooling, the solution is extracted with dichloromethane. The organic phase is washed with brine and then the aqueous phases are re-extracted with dichloromethane until no blue color is observed in the aqueous phase. The organic phase is dried over MgSO4, filtered and concentrated under reduced pressure. The residue may optionally be purified by chromatography with DCM/MeOH if the ester is still present in the crude. Azabodipy 9 is obtained as a dark blue solid (0.294 g, 0.49 mmol, 98%).
1H NMR (250 MHz, Acetone-d6) δ8.27-8.18 (m, 4H), 8.18-8.09 (m, 4H), 7.57-7.48 (m, 6H), 7.32 (d, J=1.8 Hz, 2H), 7.19-7.08 (m, 4H), 4.86 (s, 2H), 3.91 (s, 3H).
HRMS (ESI): m/z calcd for [C35H27BF2N3O4]: 602.206329, measured 602.205753 (1.0 ppm)
In a flask under argon are dissolved 0.080 g of 9 (0.13 mmol, 1 eq.), 0.070 ml of DIPEA (0.40 mmol, 3 eq.) and 0.076 g of HBTU (0.20 mmol, 1.5 eq.) in 5 mL of dichloromethane and 1 mL of distilled acetonitrile. The reaction is stirred for 15 min and then 0.032 g of Boc-ethylene diamine (0.20 mmol, 1.5 eq.) dissolved in 2 mL of anhydrous dichloromethane are added. The reaction is stirred 1:30. Then, the solution is extracted with dichloromethane and washed successively with 1M KHSO4, NaHCO3 sat. and NaCl sat. The organic phases are combined and dried over MgSO4, filtered and concentrated in vacuo. The residue is then purified by chromatography on silica with a PE/EA mixture giving 0.069 g of a deep blue solid with metallic reflections (0.093 mmol, 71%).
1H NMR (250 MHz, Chloroform-d) δ8.10-7.98 (m, 8H), 7.51-7.44 (m, 6H), 7.02 (dd, J=9.0, 8), 1 Hz, 4H), 6.94 (d, J=1.1 Hz, 2H), 4.88 (s, 1H), 4.58 (s, 2H), 3.92 (s, 3H), 3.50 (q, J=5.6 Hz, 2H), 3.35 (dd, J=12.1, 6.0 Hz, 2H), 2.80 (s, 3H), 1.43 (s), 9H).
13C NMR (101 MHz, CDCl3) δ131.13, 131.08, 130.96, 130.79, 129.66, 128.69, 125.36, 117.98, 117.94, 115.03, 114.42, 67.41, 55.65, 38.76, 28.49.
HRMS: [M+H]+ C24H41BF2N5O5 m/z calculated 744.317054, measured 744316900 (0.2 ppm).
This antenna (19) is obtained according to the reaction scheme below:
15 g of paramethoxyacetophenone (0.1 mol, 1 eq.), 10.1 ml of benzaldehyde (0.1 mol, 1 eq.) and 400 mg of NaOH (10 mmol, 0.1 eq.) in methanol. The solution is stirred at reflux overnight. Cold water is then added to the mixture and the precipitate formed is filtered and washed with water. The chalcone 10′ is recrystallized from methanol as white crystals in 85% yield (20.254 g, 85 mmol).
1H NMR (CDCl3, 250 MHz): 8.08-8.01 (tt, 2H), 7.80 (d, J=15.7 Hz, 1H), 7.67-7.61 (m, 2H), 7.54 (d, J=15.7 Hz, 1H), 7.44-7.37 (m, 3H), 7.01-6.94 (tt, 2H), 3.87 (s, 3H).).
This compound is synthesized by applying the procedure described for the preparation of compound 2.
Mass obtained: 3.205 g; 10.71 mmol
Yield 85%.
1H NMR (CDCl3, 400 MHz): 7.88-7.86 (d, 2H), 7.31-7.21 (m, 5H), 6.90-6.88 (d, 2H), 4.80 (ddd, J=12.6 Hz, 6.4 Hz, 1.3 Hz, 1H), 4.65 (ddd, J=12.6 Hz, 8.3 Hz, 1.3 Hz), 4.18. (p, J=7.1Hz, 1H), 3.82 (s, 3H), 3.42-3.29 (m, 2H).
13C NMR (CDCl3, 101 MHz): 195.39, 163.88, 139.39, 130.39, 129.52, 129.06, 127.83, 127.51, 113.93, 79.68, 55.56, 41.22, 39.49.
This compound is synthesized by applying the procedure described for the preparation of compound 3.
Mass obtained: 1.917 g; 7.69 mmol
Yield: 62%
13C NMR (CDCl3, 63 MHz): 158.63, 135.79, 133.27, 128.77, 126.64, 125.79, 125.73, 125.46, 125.31, 115.00, 114.55, 103.14, 55.51.
This compound is synthesized by applying the procedure described for the preparation of compound 4.
Mass obtained: 180 mg, 0.77 mmol
Yield: 89%
Mp: >300° C.
HRMS: [M+H]+: 236.1070.
IR (cm−1): u=3215, 1495, 1249, 1171, 1101, 832, 763, 696
1H NMR (Acetone-d6, 250 MHz): 10.41 (s, 1H), 8.29 (s, 1H), 7.62-7.58 (m, 2H), 7.55-7.51 (m, 2H), 7.33-7.27 (m, 2H), 7.22 (dd, J=2.8 Hz, 1.7 Hz, 1H), 7.15-7.08 (m, 1H), 6.89-6.85 (m, 2H), 6.77 (dd, J=2.8 Hz, 1.8 Hz, 1H).
0.450 g of 13 (1.91 mmol, 1 eq.), 0.794 g of K2CO3 (5.74 mmol, 3 eq.) and 0.69 ml of 1,4-dibromobutane (5 g) are dissolved in a flask at room temperature. 74 mmol, 3 eq.) in DMF. The reaction is stirred vigorously at room temperature overnight. The next day, water is added to the reaction and the mixture is extracted with diethyl ether, the organic phase is washed 3 times with sat. NaCl and the aqueous phases are re-extracted with diethyl ether. The organic phases are combined, dried over MgSO4, filtered and concentrated in vacuo. The residue is dissolved in a minimum of dichloromethane, and the product is precipitated by addition of petroleum ether, filtered through Buchner and washed with petroleum ether giving 0.466 g of 14 (1.26 mmol, 66%) form of a white powder.
Mp: 160-162° C.
HRMS: [M(79Br)+H]+: 371.1813, [M(81Br)+H]+: 373,0814, [M+H]+: 372.0781.
IR (cm−1): u=3395, 1497, 1247, 830, 802, 751, 692.
1H NMR (DMSO-d6, 250 MHz): 11.28 (s, 1H,), 7.61-7.57 (m, 4H), 7.34-7.31 (d, 2H), 7.28-7.25 (dd, 1H), 7.14-7.07 (tt, 1H), 6.95 (d, J=2.8 Hz, 2H), 6.80 (dd, J=2.7 Hz, 1.7 Hz, 1H), 4.02 (t, J=6.1 Hz, 2H), 3.62 (t, J=6.5 Hz, 2H), 2.01-1.94 (m, 2H), 1.88-1.81 (m, 2H).
13C NMR (DMSO-d6, 63 MHz): 156.82, 135.86, 132.27, 128.48, 125.59, 124.88, 124.76, 124.49, 124.33, 115.71, 114.71, 101.94, 66.59, 34.86, 29.11, 27.42.
0.450 g of 14 (1.22 mmol, 1 eq.) and 0.395 g of sodium azide (6.08 mmol, 5 eq.) in DMF are dissolved in a flask. The reaction is stirred at room temperature overnight. The mixture is dissolved in dichloromethane and washed several times with sat. NaCl. The organic phase is dried over MgSO4, filtered and concentrated in vacuo. After dilution with a minimum of dichloromethane, the product is precipitated by the addition of petroleum ether. The solid is filtered on Buchner and 243 mg (0.73 mmol, 60%) is obtained as a white powder.
Mp: 177-180° C.
HRMS: [M+H]+: 333.1707.
IR (cm−1): u=3394, 2948, 2875, 2080, 1496, 1246, 831, 752, 692.
1H NMR (CDCl3, 250 MHz): 8.38 (s, 1H), 7.59-7.55 (m, 2H), 7.45-7.39 (m, 2H), 7.37-7.33 (m, 2H), 7.23-7.17 (m, 1H), 7.10-7.08 (m, 1H), 6.94-6.88 (m, 2H), 6.72 (dd, J=2.6 Hz, 1.5 Hz, 1H), 4.01 (t, J=5.8 Hz, 2H), 3.38 (t, J=6.3 Hz, 2H), 1, 89-1.80 (m, 4H)
13C NMR (CDCl3, 63 MHz): 157.86, 135.76, 133.21, 128.77, 126.57, 125.89, 125.77, 125.41, 125.27, 116.09, 115.09, 103.09, 67.41, 51.34, 26.66, 25.90.
This compound (ocher-golden powder) is synthesized by applying the procedure described for the preparation of compound 6.
Mass obtained: 67 mg, 0.186 mmol
Yield: 62%
Melting point: 125-126° C.
HRMS: [M+H]+: 362.11612.
IR (cm−1): u=3280, 2918, 2850, 2092, 1603, 1360, 1258, 1164, 1038, 828, 768, 695, 668.
1H NMR (CDCl3, 250 MHz): 8.14-8.11 (m, 2H), 7.84 (d, J=8.4 Hz, 2H), 7.46 (dd, J=5.2 Hz), 1.9 Hz, 3H), 7.15 (s, 1H), 6.99 (d, J=8.2 Hz, 2H), 4.06 (t, J=5.8 Hz, 2H), 3.39 (t, J=6.4 Hz, 2H), 1.93-1.78 (m, 4H).
13C NMR (CDCl3, 63 MHz): 163.72, 162.05, 142.84, 131.91, 129.71, 129.57, 129.29, 128.93, 122.19, 115.47, 115.03, 67.67, 51.27, 26.53, 25.82.
This compound (intense blue powder) is synthesized by applying the procedure described for the preparation of compound 7.
Mass obtained: 0.080 g, 0.123 mmol
Yield: 89%.
Mp: 150-152° C.
HRMS: [M+H]+: 651.2717.
IR (cm−1): u=2094, 1759, 1600, 1496, 1241, 1166, 903, 806, 764, 694, 675.
1H NMR (CDCl3, 400 MHz): 8.00 (d, J=7.8 Hz, 4H), 7.92 (d, J=8.3 Hz, 2H), 7.80 (d, J=7), 6 Hz, 2H), 7.48 (t, J=7.5 Hz, 2H), 7.39 (dd, J=12.8 Hz, 6.9 Hz, 4H), 7.17 (s, 1H), 6.99 (d, 3H), 6.93 (d, J=8.4 Hz, 2H), 4.69 (s, 2H), 4.06 (t, J=6.1 Hz, 2H), 3.85 (s, 3H), 3.40 (t, J=6.6 Hz, 2H), 1.91 (dt, J=11.4 Hz, 6.1 Hz, 2H), 1.82 (p, J=6.8 Hz, 2H).
13C NMR (CDCl3, 101 MHz): 169.49, 161.27, 160.76, 157.61, 153.83, 148.27, 145.30, 145.27, 138.66, 133.76, 131.94, 130.36, 129.28, 129.22, 129.00, 128.32, 128.22, 128.15, 125.91, 125.50, 117.43, 115.16, 114.52, 111.16, 67.56, 65.53, 52.45, 51.32, 26.61, 25.87.
This compound (intense green solid of metallic appearance) is synthesized by applying the procedure described for the preparation of compound 8.
Mass obtained: 0.065 g, 0.093 mmol
Yield: 85%
1H NMR (400 MHz, Acetone-d6): δ8.27-8.19 (m, 6H), 8.16-8.11 (m, 2H), 7.60 -7.46 (m, 7H), 7.29 (s, 1H), 7.14-7.08 (m, 4H), 4.87 (s, 2H), 4.24-4.17 (m, 2H), 3.78 (d), J=2.1 Hz, 3H), 3.47 (t, J=6.5 Hz, 2H), 1.98-1.89 (m, 2H), 1.83 (q, J=7.4 Hz, 2H).
13C NMR (101 MHz, Acetone): δ133.16, 131.74, 131.33, 130.48, 130.43, 130.29, 129.60, 129.34, 118.67, 115.76, 115.65, 68.49, 65.69, 52.28, 51.76, 27.13, 26.32.
HRMS: [M+H]+C39H34BF2N6O4 m/z calculated 699.270390; measured 699.270354 (0.1 ppm)
0.061 g of 18 (0.087 mmol, 1 eq.) and 0.024 g of Boc-ON (0.096 g, 1.1 eq.) in anhydrous THF are dissolved under argon in a temperature-controlled flask. To this solution are added 0.096 ml of a 1M solution of trimethylphosphine in toluene (0.096 mmol, 1.1 eq.). The solution is stirred overnight at room temperature. The next day, the solution is checked by TLC and if the starting material is still visible, 1 eq. is added. additional PMe3 and Boc-ON to complete the reaction in one hour. The solution is then washed with sat. NaCl and extracted with dichloromethane. The organic phase is dried over MgSO4, filtered and concentrated in vacuo. The residue is purified by chromatography on silica (PE/EA) and 0.061 g of 19 (0.079 mmol, 91%) are obtained in the form of an iridescent dark green solid.
1H NMR (400 MHz, Acetone-d6) δ8.26-8.17 (m, 6H), 8.15-8.09 (m, 2H), 7.52 (td, J=9.6, 8), 9, 4.8 Hz, 7H), 7.25 (s, 1H), 7.11-7.05 (m, 4H), 6.00 (s, 1H), 4.85 (s, 2H) , 4.16 (dt, J=6.6, 3.4 Hz, 2H), 3.78 (s, 3H), 3.17 (td, J=7.6, 3.8 Hz, 2H), 1.84 (q, J=7.1 Hz, 2H), 1.69 (p, J=7.4 Hz, 2H), 1.41 (s, 9H).
13C NMR (101 MHz, Acetone) δ169.64, 163.11, 160.51, 160.29, 158.35, 156.72, 146.61, 145.53, 144.69, 143.18, 13312, 133.07, 133.04, 131.70, 131.27, 130.44, 130.41, 130.37, 130.26, 129.55, 129.31, 126.86, 124.28 , 120.79, 118.57, 115.71, 115.62, 78.39, 68.77, 65.66, 52.28, 41.83, 40.75, 28.68, 27.43, 27.21, 24.44.
HRMS: [M+H]+C42H41BF2N5O5 calculated 744.317054, measured 744.316900 (0.2 ppm).
Compounds 20a-20b
The mixture 1,7- and 1,6-dibromoperylene-3,4,9,10-tetracarboxylic dianhydride (1.03 g, 1.8 mmol, 1.0 eq.) is solubilized under argon in 20 ml of NMP. To this solution are added hexylamine (0.546 g, 5.4 mmol, 700 μL, 7.3 eq.) and acetic acid (0.432 g, 7.2 mmol, 412 μL, 4.0 eq.). The reaction mixture is stirred at 85° C. under an inert atmosphere for 2 h. After returning to ambient temperature, the reaction mixture is poured into ethanol. The red precipitate is filtered in vacuo and washed several times with ethanol. The desired mixture P1a and P1b is obtained in the form of a red powder which is used directly in the next reaction.
Compound 21
The mixture obtained above (20a, 20b) is solubilized in pyrrolidine. The solution is stirred under an inert atmosphere at 85° C. for 16 h. At the end of the reaction, the solution is cooled and a solution of 1M HCl is added until pH=2. The aqueous phase is extracted three times with dichloromethane. The organic phases are combined, dried over Na2SO4, filtered and evaporated under reduced pressure. The crude reaction product is purified on a silica gel chromatography column (Eluent: Hexane/ethyl acetate (7/3)) to give the 1.7 (P2) isomer in the form of a green powder.
1H NMR (600MHz, CDCl3): 8.37 (s, 2H), 8.31 (d, J=8.0 Hz, 2H), 7.53 (d, J=8.0 Hz, 2H), 4H NMR (CDCl3):?, 21 (dd, J=8.6, 6.6 Hz, 2H), 3.68 (brs, 4H), 2.75 (brs, 4H), 2.04 (brs, 4H), 1.94 (b.p. brs, 4H), 1.75 (m, 4H), 1.46 (m, 4H), 1.37-1.34 (m, 8H), 0.90 (t, J=7.0 Hz, 6H).
13C NMR (151 MHz, CDCl3): 164.05, 146.39, 134.10, 129.82, 126.55, 123.71, 122.03, 121.66, 120.66, 119.00, 117, 98, 52.14, 40.54, 31.65, 28.16, 26.87, 25.80, 22.61, 14.11.
Compound 22
Compound 21 (180 mg, 2.58.10−4 mol, 1.0 eq.) is solubilized in tBuOH (25 mL). To this solution is added the milled KOH (73 mg, 1.29.10−3, 5.0 eq.). The reaction mixture is stirred under an inert atmosphere at 95° C. for 3 h. The progress of the reaction is monitored by TLC. At the end of the reaction, the reaction mixture is cooled and 10 mL of acetic acid are added. The reaction mixture is stirred for a further 1 h, then a solution of 1M HCl (10mL) is added. The reaction mixture is stirred for an additional hour. Dichloromethane is then added and the organic phase is washed three times with water. The organic phase is then dried over Na2SO4, filtered and evaporated under reduced pressure. The crude reaction product is purified on a chromatographic column (eluent: hexane/ethyl acetate (6/4)) to give compound P3 in the form of a green powder.
1H NMR (600MHz, CDCl3): 8.49 (s, 1H), 8.43 (s, 1H), 8.42 (d, J=8.0 Hz, 2H), 8.40 (d, J=8.06 Hz, 2H), 7.69 (d, J=8.01 Hz, 2H), 7.55 (d, J=8.03 Hz, 2H), 4.22 (m, 2H), 3.74 (brs, 4H), 2.83 (brs, 4H), 2.11 (brs, 4H), 2.00 (brs, 4H), 1.76 (m, 2H), 1.45 (m, 2H), 1.36-1.30 (m, 8H), 0.90 (t, J=6.98 Hz, 3H)
Compound 23
Compound 22 (180 mg, 2.58.10−4 mol, 1.0 eq.) is solubilized in 10 mL of NMP and 6-amino-1-hexanol (87 mg, 1.4.10−4 mol, 1.0 eq.) is added. The reaction mixture is stirred under an inert atmosphere at 85° C. for 16 h. At the end of the reaction, the reaction mixture is cooled and dichloromethane is then added. The organic phase is washed three times with water. The organic phase is then dried over Na2SO4, filtered and evaporated under reduced pressure. The crude reaction product is purified on a chromatographic column (Eluent: Hexane/ethyl acetate (7/3)) to give the compound P4 in the form of a green powder.
1H NMR (600 MHz, CDCl3): 8.51 (s, 1H), 8.50 (s, 1H), 8.45 (dd, J=8.1, 1.8Hz, 2H), 7.74 (dd, J=8.0, 6.9 Hz, 2H), 4.23 (m, 4H) 3.77 (brs, 4H), 2.85 (brs, 4H), 2.38 (t, J=8.1 Hz, 2H), 2.10 (brs, 4H), 2.00 (brs, 4H), 1.79-17.6 (m, 4H), 1.57 (m, 4H), 1.49-1.44 (m, 4H), 1.38-1.35 (m, 4H), 0.9 (t, J=7.0 Hz, 3H).
Compound 24
Compound 23 (135 mg, 1.9.10−4 mol, 1.0 eq.) is solubilized in dry dichloromethane (20 mL). To this solution are added triethylamine (256 μL, 1.9 mmol, 10.0 eq.) and 4-toluenesulfonyl chloride (366 mg, 1.9 mmol, 10.0 eq.). The reaction mixture is stirred at ambient temperature under an inert atmosphere for 16 h. At the end of the reaction, water is added and the organic phase is washed three times with water. The organic phase is then dried over Na2SO4, filtered and evaporated under reduced pressure. The crude reaction product is purified on a chromatographic column (Eluent: Hexane/ethyl acetate (8/2)) to give the compound P5 in the form of a green powder.
1H NMR (600 MHz, CDCl3): 8.50 (d, J=6.5 Hz, 2H), 8.44 (dd, J=8.09, 6.9 Hz, 2H), 7.79 (m, 2H), 7.73 (t, J=7.8 Hz, 2H), 7.35 (d, J=8.0 Hz, 2H), 4.22 (dt, J=22.9, 7.6); Hz, 4H), 4.03 (t, J=6.5 Hz, 2H), 3.77 (brs, 4H), 2.85 (brs, 4H), 2.44 (s, 3H), 2.10 (brs, 4H), 1.99 (brs, 4H), 1.77-1.66 (m, 6H), 1.45 (t, J=7.6 Hz, 2H), 1.40-1.33 (m, 8H), 0.90 (t, J=7.0 Hz, 3H).
13C NMR (151 MHz, CDCl3): 164.18, 146.58, 144.76, 134.23, 133.34, 129.99, 129.95, 128.01, 126.73, 126.69, 12.91, 123.84, 122.19, 121.86, 120.82, 119.20, 119.01, 118.21, 118.05, 70.71, 52.30, 40.67, 40.30, 31.77, 28.89, 28.29, 28.05, 26.99, 26.61, 25.93, 25.29, 22.74, 21.77, 14.24.
Compound 25
Compound 24 (68 mg, 78.10−3 mol, 1.0 eq.) is solubilized in 20 mL of DMF. To this solution is added sodium azide (51 mg, 7.8.10−3 mol, 10.0 eq.). The reaction mixture is stirred under an inert atmosphere at 50° C. for 12 h. At the end of the reaction, the reaction mixture is cooled and water is added. The aqueous phase is extracted three times with dichloromethane. The organic phases are pooled, dried over Na2SO4, filtered and evaporated under reduced pressure to yield the desired product P6 without further purification.
1H NMR (600MHz, CDCl3): 8.38 (d, J=2.3Hz, 2H), 8.33 (dd, J=8.0, 6.4Hz, 2H), 7.55 (dd, J=12.3, 8.0 Hz, 2H), 4.22 (q, J=7.4 Hz, 4H), 3.69 (brs, 4H), 3.28 (t, J=7.0 Hz, 2H), 2.76 (brs, 4H), 2.05 (brs, 4H), 1.95 (brs, 4H), 1.84-1.72 (m, 6H), 1.65 (t, J=7.0 Hz, 3H), 1.48 (m, 7H), 1.36 (m, 5H), 0.90 (t, J=7.0 Hz, 3H).
13C NMR (151 MHz, CDCl3): 164.20, 146.56, 134.31, 134.20, 129.96, 126.73, 123.89, 122.18, 122.16, 121.85, 121.29, 120.80, 119.19, 119.02, 118.19, 118.05, 52.29, 51.54, 40.68, 40.39, 31.77, 29.83, 29.65, 28.88, 28.29, 28.13, 27.00, 26.81, 26.63, 25.93, 22.74, 14.24.
Compound 26
Triethylene glycol mono methyl ether (2.0 g, 1.22.10−2 mol, 1.0 eq.) is solubilized in 120 mL of dry CH2Cl2. To this solution are added under argon 4-toluenesulfonyl chloride (4.64 g, 2.43.10−2 mol, 2.0 eq.) and triethylamine (2.68 g, 2.43.10−2 mol, 2.0 g). eq.). The reaction mixture is stirred under an inert atmosphere at ambient temperature for 16 h. At the end of the reaction, water is added to the reaction mixture and the organic phase is washed three times with water, dried over Na2SO4, filtered and evaporated under vacuum. The reaction crude is purified by chromatography column on silica gel (Eluent: CH2Cl2/MeOH (0.5%)) to give the desired product in the form of a colorless oil.
1H NMR (600MHz, CDCl3): 7.79 (d, J=8.3 Hz, 2H), 7.33 (d, J=8.0 Hz, 2H), 4.20-4.10 (m, 2H), 3.72-3.64 (m, 2H), 3.63-3.56 (m, 6H), 3.55-3.49 (m, 2H), 3.36 (s, 3H); , 2.44 (s, 3H).
13C NMR (151 MHz, CDCl3): 144.90, 133.14, 129.93, 121.10, 72.03, 70.87, 70.69, 70.67, 69.35, 68.80, 59.15, 21.76.
Compound 27
Compound 26 (2.87 g, 9.02 mmol, 1.0 eq.) is solubilized in 90 mL of dry DMF. To this solution is added under an inert atmosphere, NaN3 (4.70 g, 72.0 mmol, 8 eq.). The reaction mixture is stirred at 50° C. for 16 h. After cooling to room temperature, water is added to the reaction mixture and the aqueous phase is extracted with CH2Cl2. The organic phase is washed with water, dried over Na2SO4 filtered and evaporated under reduced pressure. The desired product is obtained in the form of an oil without further purification.
1H NMR (600 MHz, CDCl3): 3.69-3.65 (m, 8H), 3.57-3.55 (m, 2H), 3.40-3.39 (m, 5H).
13C NMR (151 MHz, CDCl3): 72.09, 70.86, 70.82, 70.77, 70.19, 59.19, 50.84.
Tetraphenylporphyrin (TPPH, compound 28) was synthesized following the Alder-Rothemund method from a mixture of benzaldehyde and pyrrole in propionic acid, heated at 130° C. for two hours. The crystalline product was isolated by filtration in 6% yield. The low yield is in agreement with the literature and the synthetic route used.
The next step is an electrophilic aromatic substitution, also called aromatic nitration. The latter is controllable regioselectively in the para position of the phenyl, by varying the amount of sodium nitrite and the reaction time in the TFA. In fact, after concentrating TPPH in TFA, the latter was treated with 1.8 equivalents of sodium nitrite for exactly 3 min. The reaction mixture was re-engaged without intermediate purification in order to reduce the nitro group to an amino group in the presence of excess of tin chloride and hydrochloric acid. The mono-amino unsymmetric porphyrin (compound 29) was isolated by column chromatography (silica, DCM/Hexane, 9:1).
5,10,15,20-Tetraphenylporphyrin (TPPH, Compound 28)
Benzaldehyde (5.0 mL, 49.1 mmol, 1.2 eq.) and pyrrole (4.0 mL, 57.6 mmol, 1.0 eq.) were added in 250 mL of propionic acid. The solution was stirred at 130° C. for 2 h. The reaction mixture was then cooled to room temperature and filtered. The purple solid was washed with MeOH and hot water to give the desired product as a purple powder (1.70 g, 2.76 mmol, yield=5%).
1H NMR (600 MHz, CDCl3) δppm=−2.76 (s, 2H, H1); 7.76 (m, 12H, H4 and H5); 8.22 (d, J=7Hz, 8H, H3); 8.85 (s, 8H, H2)
13C NMR (150 MHz, CDCl3) δppm=120.28 (Cq); 126.83 (C4); 127.85 (C5); 134.70 (C3); 142.32 (Cq)
ESI-MS m/z calc. for [C44H30N4]=614.3; found: [M+H+]2+=615.3, [M+2H+]2+=308.1
4′-Amino-5,10,15,20-Tetraphenylporphyrin (Compound 29)
TPPH (906 mg, 1.5 mmol, 1.0 eq.) was dissolved in 70 mL of TFA, then sodium nitrite (181 mg, 2.6 mmol, 1.8 eq.) was added. The solution was stirred at room temperature for exactly 3 min. The reaction mixture was then quenched with 200 mL of water. The aqueous solution was extracted with DCM. The organic layer was washed with saturated aqueous NaHCO3 solution, dried over anhydrous Na2SO4, filtered and evaporated in vacuo to give a purple solid. The latter was dissolved without purification in 50 ml of concentrated HCl. Tin (II) chloride dihydrate (3.88 g, 17.0 mmol, 11.5 eq.) was added to this solution. The mixture was stirred at 65° C. for 2 h. The reaction mixture was quenched with 100 mL of cold water. The aqueous solution was basified to pH 14 by addition of ammonium hydroxide solution and extracted with DCM until colorless. The combined organic layer was dried over anhydrous Na2SO4, filtered and evaporated in vacuo to give a purple solid, which was purified by column chromatography (silica, DCM) to give the desired TPP-NH 2 compound as a powder. purple (443 mg, 7.05.10−4 moles, yield=51%).
1H NMR (600 MHz, CDCl3) δppm=−2.75 (s, 2H, H1); 7.08 (d, J=8.9 Hz, 2H, H7 or H8); 7.78-7.73 (m, 9H, H4 and H5); 8.01 (d, J=8.3 Hz, 2H, H7 or H8); 8.22 (d, J=6Hz, 6H, H3); 8.83 (s, 6H, H2); 8.94 (d, J=4.45 Hz, 2H, H6)
13C NMR (150 MHz, CDCl3) δppm=113.61 (C8 or C7); 126.80 (C4); 127.50 (C5); 134.71 (C3); 135.85 (C7 or C8)
ESI-MS m/z calc. for [C44H31N5]=629.3; found: [M+H+]+=630.3; [M+2H]2+=315.6.
In a flask are dissolved n equivalents of 9 (4 eq./NH2 under argon for a total functionalization i.e. 128 eq. or 0.5 eq./NH2 for hemifunctionalization or 16 eq.), N eq. HBTU and n eq. of DIPEA in 10 mL of dry, degassed DMF. The solution is stirred at room temperature for 15 min. Meanwhile, 1 eq. of G3P-(NH2)32 (typically, 0.100 mL of a 12.44% solution of G3P-(NH2)32 in MeOH, i.e. 1.8 μmol of dendrimer) are dissolved in 10 mL of anhydrous DMF. Next, argon was bubbled in the dendrimer solution for 5 min before adding it to the activated azabodipy solution. The reaction is allowed to stir overnight at room temperature in the dark. The next day, the solution is concentrated under reduced pressure to evaporate the maximum of DMF and DIPEA. The pasty blue residue is then dissolved in high quality DMSO for analysis. and placed in a bag of dialysis membrane (10 kDa) sealed with clips. The dialysis rod is immersed in DMSO p.a. in a 500 ml Erlenmeyer flask and stirred gently in the dark. During dialysis, the dialysis DMSO is changed every hour on the first day, then every half day on the following days. After one week of dialysis, no blue coloration is observed in the dialysis solvent, the functionalized dendrimer solution is recovered inside the flange and the maximum of DMSO evaporated by evaporation under vacuum. The functionalized dendrimer G3P-(Bodipy1)n (n=16,32) is recovered as an intense blue powder in quantitative yield.
In order to conjugate the porphyrin derivative (29) to the PAMAM-G3 dendrimer by amide bonds, the thirty-two monoamide succinic acids on the surface of the dendrimer are activated by a mixture of HATU and DIPEA in DMF. The reaction mixture is stirred at room temperature for 30 min, then the monoamino porphyrin (29) is added. The reaction mixture is stirred for 3 days. The final product is purified by dialysis in DMSO using a MWCO 10kDa cut-off membrane. The product (G3-(TPP)32) is characterized by 1H NMR in DMSO-d6 and makes it possible to demonstrate the complete substitution of the dendrimer by thirty-two porphyrins (
Hexynoic acid (428 mg, 3.8.10−4 mol, 40.0 eq.), hydrochloric N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (592 mg, 3.8.10−4 mol, 40.0 eq.), HOBT (516 mg, 3.8.10−4 mol, 40.0 eq.) and DIPEA are solubilized in 40 mL distilled DMF. The solution is stirred under an inert atmosphere for 30 min. To this solution is added a solution of G3P-(NH2)32 (660 mg, 9.5 10−5 mol, 1.0 eq.). The reaction mixture is stirred under an inert atmosphere at 25° C. for 48 h. At the end of the reaction, the DMF is evaporated under reduced pressure. The crude reaction product is purified by dialysis in DMSO for 24 hours. The solvent is then evaporated under reduced pressure. The compound G3P-(alkyne)32 is obtained in the form of an orange oil.
1H NMR (600MHz, DMSO-d6): 7.97 (brs), 7.87 (brs), 312-3.07 (m), 2.72-2.69 (m), 2.47 (brs), 2.22-2.20 (brs), 2.16-2.12 (m), 1.64 (q).
13C NMR (MHz, DMSO-d6): 172.20, 171.89, 171.64, 84.31, 71.56, 52.18, 49.62, 40.43, 39.24, 36.86, 34.41, 33.06, 24.37, 17.58.
G3P-(alkyne)32 (20 mg, 2.0.10−6 mol, 1.0 eq.) and the chromophore bearing an azide function (compound Perylene) (8.0.10−5 mol, 40 eq.) are solubilized in 1 ml of distilled DMF. The solution is degassed under argon for 20 min. To this solution is then added a solution of CuSO4.5H2O and sodium ascorbate in a mixture of H2O/DMF (1 ml, 1/1, v/v). The reaction mixture is stirred under an inert atmosphere at room temperature for 24 h. At the end of the reaction, the solvents are evaporated under reduced pressure and an EDTA solution is added to the reaction crude. The solution is stirred for 12 h and then dialyzed in water for 24 h. The solvent is then evaporated under reduced pressure and the residue obtained is dialysed in DMSO for 24 h. At the end of the purification, the DMSO is evaporated under reduced pressure and the desired product (G3P-(Perylène)32) is obtained in the form of a powder.
With compound 25 as nitrogen chromophore:
(G3P-(Perylène)32) 1H NMR (600 MHz, CDCl3): 8.47 (s), 8.42 (d), 8.06-7.99 (brs), 7.725 (t), 7.54 (brs), 7.17-7.09 (brs), 4.85 (brs), 4.57 (brs), 4.30 (brs), 4.19-4.14 (brm), 3.47 (brs), 3.29 (brs), 2.34 (brs), 2.24 (brs), 1.89 (brs), 1.69 (brs), 1.40 (brs), 1.31 (brs) 0.85-0.81 (m).
G3P-(Alkyne)32 (20 mg, 2.0 10−6 mol, 1.0 eq.) and the azoture chromophore (1.6.10−5 mol, 8.0 eq.) are solubilized in 1 mL DMF. The solution is degassed under argon for 20 min. A solution of CuSO4.5H2O (7.10-6 mol, 3.5 eq.) and sodium ascorbate (7.10−6 mol, 3.5 eq.) (1 mL, DMF/H2O (1/1, v/v)) is then added. The reaction mixture is stirred under argon at 25° C. for 12 h. At the end of the reaction, the solvents are evaporated under reduced pressure. The reaction crude is triturated in an aqueous solution of EDTA for 5 h and then dialyzed in water (MW 2 kDa). At the end of the dialysis, the solvents are evaporated under vacuum and the compound G3P-(Alkyne)x-(L2)y (x=22-24, y=8-10).
With compound 25 as azoture chromophore,
G3P-(Alkyne)x-(Perylene)y (x=22-24, y=8-10)
1H NMR (600 MHz, CDCl3): 8.16-8.09 (brm), 7.48 (brs), 7.19 (brs), 4.32 (brs), 4.18-4.13 (brs), 3.53 (brs), 3.33 (brs), 2.70 (brs), 2.58 (brs), 2.26 (brs), 1.94 (brs), 1.84 (brs), 1.74 (brs), 1.44 (brm), 1.34 (brs m), 0.89 (brs)
With cyanine as azoture chromophore, G3P-(Alkyne)x-(Cyanine)y (x=22-24, y=8-10)
1H NMR (600 MHz, DSMO-d6): 7.95 (brs), 7.88 (brs), 7.74 (brs), 7.42 (brm), 7.27 (brm), 7.17 (brm). 7.04 (brm), 5.64 (m), 4.51 (brs), 3.93 (brs), 3.77 (brs), 2.75 (brm), 2.68 (brs) , 2.20 (brs), 2.14 (brs), 1.76 (brs), 1.65 (brs), 1.57 (brs).
With compound 18 as nitrogen chromophore
G3P-(Alkyne)x-(Bodipy2)y (x=22-24, y=8-10)
1H NMR (600 MHz, DSMO-d6): 7.95 (brs), 7.89 (brs), 7.55-7.41 (m), 7.12 (brm), 6.92 (brm), 4.93-4.73 (m), 4.37 (brs), 3.51 (brs), 3.39 (brs), 3.08 (brs), 2.75 (brs), 2.67 (brs), 2.21 (brs), 2.15 (brs), 1.76 (brs), 1.65 (t).
The dendrimer G3P-(Alkyne)x-(L2)y (x=22-24, y=8-10) is solubilized in 1 mL of DMF. To this solution is added compound 27, the nitrogenous hydrosolubilizing group (4.0.10−5 mol, 20.0 eq.). The solution is degassed under argon for 20 min. A solution of CuSO4.5H2O (2.0.10−5 mol, 10.0 eq.) and sodium ascorbate (2.0.10−5 mol, 10.0 eq.) (1 mL, DMF/H2O (1/1, v/v)) is then added. The reaction mixture is stirred under argon at 25° C. for 12 h. At the end of the reaction, the solvents are evaporated under reduced pressure. The reaction crude is triturated in an aqueous solution of EDTA for 5 h and then dialyzed in water (MW 2 kDa). At the end of the dialysis, the solvents are evaporated under reduced pressure and the residue is solubilized in DMSO and then dialysed in DMSO (MW 10 kDa). At the end of the dialysis, the DMSO is evaporated under reduced pressure. The desired product G3P-(R2)x-(L2)y (x=22-24, y=8-10) is obtained as a powder.
With compound 25 as azide chromophore and compound 27 as nitrogenous water-solubilising group, G3P-(PEG)x (Perylene)y (x=22-24, y=8-10)
1H NMR (600 MHz, CDCl3): 8.16-80.9 (brm), 7.45 (brs), 7.56 (s), 7.49 (brs), 7.13 (brs), 4.49 (s), 4.33 (brs), 4.19-4.14 (brm), 3.84 (s), 3.61 (s), 3.61 (s), 3.36 (s), 3.32 (brs), 2.71 (s), 2.35 (brs), 2.25 (brs), 1.95 (brs), 1.85 (brs), 1.74 (brs), 1 , 44 (brs), 1.34 (brs).
With compound 18 as azide chromophore and compound 27 as nitrogenous hydrosolubilizing group, G3P-(PEG)x-(Bodipy2)y (x=22-24, y=8-10)
1H NMR (600 MHz, DSMO-d6): 7.96 (brs), 7.86 (brs), 7.81 (s), 7.47-7.36 (brsm), 7.00 (brm), 6.70-6.65 (brm), 5.16 (brs), 4.89 (brs), 4.45 (s), 3.78 (s), 3.78 (s), 3.50 (s), 3.46 (brs), 3.39 (brs), 3.34 (brs), 3.31 (brs), 3.08 (brs), 2.78 (brs), 2.65 (brs), 2.59-2.56 (brm), 2.42 (brs), 2.19 (brs), 2.11 (t), 1.79 (t).
To prepare dendrimer complexes containing the lanthanide cations, a dendrimer solution in DMSO was treated with 8 eq. of lanthanide nitrate for 7 days at room temperature.
Photophysical Properties of Aza-Bodipy-Based Molecules
The absorption spectrum of the Bodipy chromophore (compound 9) measured in a DMSO solution has a broad band of up to 770 nm with a low energy main band at 672 nm (ϵ=2.1.105 M−1 cm−1),
Under excitation centered at 650 nm, corresponding to the maximum absorption of the chromophore, the compound 9 as well as the dendrimers (Ln8-)G3P-(Bodipy1)n (n=16, 32, Ln=Yb3+, Nd3+) show broad, very similar emission bands with a maximum at ˜735 nm from energy levels centered on the aza-Bodipy chromophore (
The absolute quantum yields of the chromophore (compound 9) and dendrimers (Ln8-)G3P-(Bodipy1)n (n=16, 32; Ln=Yb3+, Nd3+) are summarized in Table 1. Significant decreases (6 and 3.4 times) in quantum emission efficiencies from the aza-BODIPY-COOH chromophoric moieties for the functionalized dendrimers, G3P-(aza-BODIPY)32 and G3P-(aza-BODIPY)16, respectively. Such an effect is probably caused by the self-quenching of the chromophores located on the PAMAM G3P dendrimer. The presence of Ln3+ within the branches of G3P-(aza-BODIPY)16 does not affect the quantum efficiency values which remain the same taking into account the experimental error. On the other hand, the Q values are increased by 1.7 and 1.4 times for G3P-(Bodipy1)32 during the encapsulation of the lanthanide cations Yb3+ and Nd3+, respectively. This result can be explained by the formation of different conformations of dendrimers in Ln8-G3P-(Bodipy)32 with reduced interactions between chromophore units thus modulating self-extinction.
aThe values 2σ are indicated in parentheses. Experimental errors: Q, ±10%. Quantum yields (Q) measured under excitation at 650 nm by collecting the emission in the range of 660 to 850 nm.
To test the photostability of the system, the chromophore (compound 9) and the dendrimers (Ln8-)G3P-(Bodipy1)n (n=16, 32; Ln =Yb3+, Nd3+) were continuously illuminated with 670 lumen. nm and the emission signal was monitored at 735 nm. The results (
In order to determine the potential toxicity of the described dendrimers, an Alamar Blue test was performed on the HeLa ovarian carcinoma human cell line (
Confocal microscopy experiments were performed to confirm the intracellular localization of the dendrimers (
Differences in signal intensity in fully or partially functionalized dendrimer cells, with or without encapsulated Ln3+, can be attributed to the importance of cell internalization, which varies with differences in solubility in the cell. water or conformations of macromolecules. However, in all cases, the intracellular signal distribution was observed in the cytoplasm and more specifically in the lysosomes, with a particularly strong signal for the Yb8-G3P-(aza-BODIPY)16 dendrimer. In addition, a formation of several long and fine filipodia, structures derived from F-actin-rich plasma membranes were observed for the various dendrimers studied and the aza-BODIPY-COOH molecules (
It should be noted that under these experimental conditions used, no autofluorescence signal was detected, which again demonstrates the advantages of using probes with excitation and emission wavelengths in the biological imaging window.
Cytometry has been used to quantify cell internalization of dendrimers and to better understand signal intensity differences observed in confocal microscopy. The mechanisms of passive (non-dependent energy) and active (energy dependent) internalization of cells have been studied. Active transport was inhibited with sodium azide (NaN3), while incubation at 4° C. inhibited active and passive transport pathways by increasing the plasma membrane stiffness of HeLa cells (S. Vranic, N. Boggetto, V. Contremoulins, S. Mornet, N. Reinhardt, F. Marano, A. Baeza-Squiban, S. Boland, Deciphering the mechanisms of cellular uptake of nanoparticles by accurate evaluation of internalization for cytometry, Particle and Fiber Toxicology, 10 (2013) 1-16).
The results obtained make it possible to conclude without ambiguity that the partially functionalized dendrimers (Ln8-)G3P-(Bodipy1)16 (Ln=Yb3+, Nd3+) are significantly more internalized by the cells compared to fully functionalized dendrimers (Ln8-)G3P-(Bodipy1)32 (Ln=Yb3+, Nd3+) (
aThe percentages of inhibition of the passive mechanisms are presented as the percentage difference between the internalization inhibited at 4° C. and that inhibited with sodium azide.
Epifluorescence Microscopy with Bodipy-Based Molecules
The detection capacity of the signal emitted in the near infrared by the compounds 9 and the dendrimers (Ln8-) G3P-(Bodipy1)n (n=16, 32; Ln=Yb3+, Nd3+) was confirmed by epifluorescence microscopy after 30 min incubation of HeLa cells with a solution of 1.5 μM of corresponding dendrimer complex. Due to the near-infrared emission bandwidth of the aza-Bodipy chromophore, the signal was collected by epifluorescence with a long pass filter of 785 nm at emission and a 655±40 nm filter at excitation (
The results can again be explained by differences in cell internalization (Table 2) and emission intensities (Table 1) for the different dendrimers studied.
Photophysical Properties of Cyanine-Based Molecules
The dendrimer G3P-(Alkyne)x-(Cyanine)y (x=22-24, y=8-10) and its complexes with Nd and Yb show a broad absorption band centered at 665 nm (
The continuous illumination of G3P-(Alkyne)x-(Cyanine)y (x=22-24, y=8-10) with a light centered at 665 nm makes it possible to estimate the photostability of the dendrimers which appears to be rather weak. (
Epifluorescence Microscopy of Cyanine-Based Molecules
To establish the proof of principle that the emission of this dendrimer can be detected by epifluorescence microscopy after incubation with HeLa cells. A fluorescence signal was observed using a 655±40 nm excitation filter and a 750±50 nm emission filter (
Photophysical Properties of Porphyrin-Based Molecules
Absorption spectra of the Yb8-G3P-(TPP)32 complex were recorded in DMSO and in the DMSO/(Opti-MEM/FCS, 6: 94%) mixture (
The absorption spectrum of Yb8-G3P-(TPP)32 measured in a solution of DMSO exhibited the typical porphyrin absorbance bands: the Soret band centered at 420 nm (ϵ˜5.9·106 M−1·cm−1), the Q IV band centered at 517 nm (ϵ˜16.6·104 M−1·cm−1), the Q III band centered at 553 nm (ϵ˜16.6·104 M−1·cm−1), the Q II band centered at 592 nm (ϵ˜8.5·104 M−1·cm−1), and the IQ band centered at 648 nm (ϵ˜9.1·104 M−·cm−1).
The bands present on the absorption spectrum of Yb8-G3P-(TPP)32 in the DMSO/(Opti-MEM: FCS) mixture are slightly redshifted by 3-11 nm. It should be noted here that in our case we have been limited by the low solubility of Yb8-G3P-(TPP)32 in the cell culture medium and, therefore, light scattering is important.
With excitation centered at 520 nm, Yb8-G3P-(TPP)32 shows a typical porphyrin emission: two bands in the visible region: centered at 664 nm and 717 nm,
The excitation spectrum of Yb8-G3-(TPP)32 shows bands comparable to those observed on the absorption spectrum, thus indicating that the sensitization of Yb3+ occurs by the energy transfer of the tetraphenylporphyrin chromophore to the lanthanide cation,
The quantum yield of the Yb3+ emission in the Yb8-G3-(TPP)32, complex, 37 μM in a DMSO solution could not be determined due to the too low intensity of the emission signal. Nevertheless, luminescence decay curves have been measured and can be deconvolved by bi-exponential functions with individual lifetime values of 68 (1) μs (48 (5)%) and 17.4 (1) μs (52 (5)%).
Epifluorescence Microscopy of Porphyrin-Based Molecules and Potential Activity in Photodynamic Therapy
The ability of the Yb8-G3-(TPP)32 complex to be used as an imaging agent in the near-infrared region has been confirmed by epifluorescence microscopy experiments. Intense signals were detected in the near-infrared excitation at 417 nm (60 nm bandpass filter) in HeLa cells incubated with the Yb8-G3-(TPP)32 complex (
In the case of cells not incubated with Yb8-G3-(TPP)32, we did not observe autofluorescence in the near infrared region (
| Number | Date | Country | Kind |
|---|---|---|---|
| 17 50245 | Jan 2017 | FR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2018/050510 | 1/10/2018 | WO | 00 |
