Patent Application
20170327509
The present invention relates to new one-photon or two-photon absorbing fluorophores, a method for preparing the same, and a method for cellular imaging using the same.
Two-photon microscopy (TPM) is an imaging technique of capturing fluorescence through excitation of fluorophores by using two photons having energy equal to half of the energy of a photon used in one-photon microscopy (OPM).
TPM allows excitation of fluorophores by light with energy equal to half of that used in OPM (i.e., with a wavelength that is two times longer), and thus offers the advantages of deeper tissue penetration, and less photodamage and photobleaching of living tissues and cells in bioimaging. Also, TPM is less influenced by autofluorescence generated from intrinsic biomoleucles and is able to provide very high resolution images since excitation occurs only at the focal point (Zipfel, W. R. et al. Nat. Biotechnol. 2003, 21, 1369; Helmchen, F. et al. Nat. Methods, 2005, 2, 932; Willams, W. R. et al. Curr. Opin. Chem. Biol. 2001, 5, 603).
Therefore, efficient two-photon absorbing fluorophores used together with two-photon microscopy to obtain images in vivo are also very important materials in the bioimaging field. An efficient two-photon absorbing fluorophore needs to have a large two-photon absorption cross-section value within a proper biological optical window wavelength region (800 to 1000 nm) to minimize auto fluorescence of living tissue and also needs to ensure photostability, permeability into biological matters, and biocompatibility.
The two-photon absorbing fluorophores satisfying such requirements for bioimaging are limited in number, and generally, D-π-A dipolar dyes that have an electron donor (D) and an electron acceptor (A) in an aromatic ring (π-system) have been widely used. —As a representative example of the dipolar dyes, 1-(6-dimethylaminonaphthalen-2-yl)ethanone (acedan) represented by Formula 19 is used to obtain bright images by two-photon microscopy in living cells and tissues due to high photostability and a quite large two-photon absorption cross-section value (Kim, H. M. et al. Angew. Chem. Int. Ed. 2007. 46, 3460; Kim, H. M. et al. Angew. Chem. Int. Ed. 2008, 47, 5167; Kim, H. M. et al. Angew. Chem. Int. Ed. 2007, 46, 7445).
However, such D-π-A dipolar dyes generate intramolecular charge transfer excited state, thus resulting the fluorescence property highly sensitive towards the environment polarity (polarity of a solvent), and such a property is significantly applied in the detection of a substrate accompanying polarity changes in vivo.
On the other hand, these dipolar dyes have a critical disadvantage of poor fluorescence intensities in aqueous solution, resulting in low fluorescence quantum yield and two-photon absorption cross-section value (MacGregor, R. B. et al. Nature 1986, 319, 70; Hutterer, R. et al. J. Fluoresc. 1998, 8, 365; Gaus, K. et al. Proc. Natl. Acad. Sci. USA 2003, 100, 15554).
That is, such dipolar dyes need to overcome the environmental polarity sensitivity which causes poor fluorescence intensities in aqueous solution.
Therefore, to overcome the above-mentioned problems of the conventional art, the inventors developed new two-photon absorbing fluorophores having high fluorescence quantum yield and a two-photon absorption cross-section value, thereby completing the present invention.
Accordingly, an objective of the present invention is directed to providing compounds represented by Formula 1 or a pharmaceutically acceptable salt thereof.
Another objective of the present invention is directed to providing a method for cellular imaging using the compounds.
Still another objective of the present invention is directed to providing a method for preparing the compound.
However, technical problems to be solved in the present invention are not limited to the above-described problems, and other problems which are not described herein will be fully understood by those of ordinary skill in the art from the following description.
To achieve the above-mentioned objectives of the present invention, the present invention provides compounds represented by Formula 1.
wherein R1 is hydrogen or
R2 is hydrogen,
R3 is hydrogen or
and
R4 and R5 are hydrogen or
linked by a 6-membered ring.
In one embodiment of the present invention, the compound may be one-photon absorbing fluorophores or two-photon absorbing fluorophores.
In addition, the present invention provides a method for cellular imaging using the compounds or a pharmaceutically acceptable salt thereof.
In one embodiment of the present invention, the method may include treating cells with the compounds or a pharmaceutically acceptable salt thereof and measuring fluorescence using a fluorescence microscope.
In another embodiment of the present invention, the fluorescence microscope may be a one-photon fluorescence microscope or a two-photon fluorescence microscope.
Further, the present invention provides a method for preparing a compound of Formula 2, which includes the following steps: 1) synthesizing 4-(6-bromonaphthalene-2-ylamino)cyclohexanol by adding trans-4-aminocyclohexanol and sodium metabisulfite to 6-bromo-2-naphthol; 2) synthesizing 4-(6-bromonaphthalene-2-ylamino)cyclohexyl methanesulfonate by adding triethylamine and methanesulfonylchloride to the 4-(6-bromonaphthalene-2-ylamino)cyclohexanol; 3) synthesizing 7-(6-bromonaphthalene-2-yl)7-azacyclo[2.2.1]heptane by adding dimethylformamide to the 4-(6-bromonaphthalene-2-ylamino)cyclohexyl methanesulfonate; and 4) adding palladium(II)acetate, diphenylphosphinopropane, ethyleneglycolvinylether, and triethylamine to the 7-(6-bromonaphthalene-2-yl)7-azacyclo[2.2.1]heptane.
In addition, the present invention provides a method for preparing a compound of Formula 3, which includes the following steps: 1) synthesizing 6-bromo-N-isopropylnaphthalene-2-amine by adding isopropylamine and sodium metabisulfite to 6-bromo-2-naphthol; and 2) adding palladium(II)acetate, diphenylphosphinopropane, ethyleneglycolvinylether, and triethylamine to the 6-bromo-N-isopropylnaphthalene-2-amine
In addition, the present invention provides a method for preparing a compound of Formula 5, which includes the following steps: 1) synthesizing 4-(6-bromonaphthalene-2-ylamino)cyclohexanol by adding trans-4-aminocyclohexanol and sodium metabisulfite to 6-bromo-2-naphthol; and 2) adding palladium(II)acetate, diphenylphosphinopropane, ethyleneglycolvinylether, and triethylamine to the 4-(6-bromonaphthalene-2-ylamino)cyclohexanol.
In addition, the present invention provides a method for preparing a compound of Formula 9, which includes adding a formaldehyde aqueous solution, sodium cyanoborohydride, and zinc chloride to the compound of Formula 5.
In addition, the present invention provides a method for preparing a compound of Formula 6, which includes the following steps: 1) synthesizing 1-(6-hydroxynaphthalen-2-yl)ethanone by adding palladium(II)acetate, diphenylphosphinopropane, ethyleneglycolvinylether, and triethylamine to 6-bromo-2-naphthol; 2) synthesizing 1-(6-(4-aminocyclohexylamino)naphthalene-2-yl)ethanone by adding trans-1,4-diaminocyclohexane and sodium metabisulfite to the 1-(6-hydroxynaphthalen-2-yl)ethanone; and 3) adding acetic anhydride to the 1-(6-(4-aminocyclohexylamino)naphthalene-2-yl)ethanone.
In addition, the present invention provides a method for preparing a compound of Formula 8, which includes the following steps: 1) synthesizing 4-(6-bromonaphthalene-2-yl)morpholine by adding morpholine and sodium metabisulfite to 6-bromo-2-naphthol; and 2) adding palladium(II)acetate, diphenylphosphinopropane, ethyleneglycolvinylether, and triethylamine to the 4-(6-bromonaphthalene-2-yl)morpholine.
In addition, the present invention provides a method for preparing a compound of Formula 10, which includes adding trans-4-aminocyclohexanol to 6-bromo-2-(2-hydroxyethyl)-1H-benzo[de]isoquinoline-1,3 (2H)-dione.
In addition, the present invention provides a method for preparing a compound of Formula 13, which includes adding sodium metabisulfite and (1S,2S)-2-aminocyclohexanol to 1-(6-hydroxynaphthalen-2-yl)ethanone.
In addition, the present invention provides a method for providing a compound of Formula 14, which includes adding sodium metabisulfite and (1R,2S)-2-aminocyclohexanol to 1-(6-hydroxynaphthalen-2-yl)ethanone.
In addition, the present invention provides a method for preparing a compound of Formula 15, which includes the following steps: 1) synthesizing 1-(6-(((1S,2S)-2-aminocyclohexyl)amino)naphthalene-2-yl)ethanone by adding sodium metabisulfite and (1R,2R)-cyclohexane-1,2-diamine to 1-(6-hydroxynaphthalen-2-yl)ethanone; and 2) adding benzenesulfonyl chloride and triethylamine to the 1-(6-(((1S,2S)-2-aminocyclohexyl)amino)naphthalene-2-yl)ethanone.
In addition, the present invention provides a method for preparing a compound of Formula 16, which includes the steps: 1) synthesizing 1-(6-(((1R,4R)-4-aminocyclohexyl)amino)naphthalene-2-yl)ethanone by adding sodium metabisulfite and (1R,4R)-cyclohexane-1,4-diamine to 1-(6-hydroxynaphthalen-2-yl)ethanone; and 2) adding benzenesulfonyl chloride and triethylamine to the 1-(6-(((1R,4R)-4-aminocyclohexyl)amino)naphthalene-2-yl)ethanone.
In addition, the present invention provides a method for preparing a compound of Formula 17, which includes adding sodium metabisulfite and cyclohexaneamine to 1-(6-hydroxynaphthalen-2-yl)ethanone.
In addition, the present invention provides a method for preparing a compound of Formula 18, which includes adding sodium metabisulfite and pyrrolidine to 1-(6-hydroxynaphthalen-2-yl)ethanone.
Substituents included in a the newly developed compounds of the present invention are expected to be useful for the development of new bright D-π-A fluorophores, and particularly, the introduction of a 4-hydroxycyclohexylamino group as shown in a compound of Formula 5 to different D-π-A fluorophores, is expected to resulting the development of fluorophores having higher fluorescence quantum yield and two-photon absorption cross-section value in aqueous solution.
The present invention is characterized by providing new one-photon absorbing fluorophores and/or two-photon absorbing fluorophores, which is a compound represented by Formula 1 as shown below.
Here, R1 may be hydrogen or
R2 may be hydrogen,
R3 may be hydrogen or
and
R4 and R5 may be hydrogen or
linked by a 6-membered ring, but the present invention is not limited thereto.
Most preferably, Compound 1 may be a substituent represented by Formulas 2, 3, 5, 6, 8, 9, 10, 13, 14, 15, 16, 17, or 18 as shown below.
In one embodiment of the present invention, it was confirmed that compounds of the present invention have two-photon absorption cross-section values higher than those of conventional two-photon absorbing fluorophores and thus are able to provide excellent bright fluorescent images through bioimaging under two-photon microscopy (Experimental Examples 1 to 6).
Therefore, the present invention may provide a method for cellular imaging using the compounds of the present invention.
Herein after, exemplary examples will be provided to help in understanding of the present invention. However, the following examples are merely provided to facilitate understanding of the present invention, and the scope of the present invention is not limited to the following examples.
A general synthetic pathway of Compound 2 is shown in Scheme 1.
Compound 2a, 4-(6-bromonaphthalene-2-ylamino)cyclohexanol, was synthesized by the inventors.
Specifically, water (15 mL) was added to a sealed tube containing starting materials for synthesis such as 6-bromo-2-naphthol (1.5 g, 6.72 mmol, Sigma-aldrich B73406), trans-4-aminocyclohexanol (1.55 g, 13.45 mmol), and sodium metabisulfite (2.56 g, 13.45 mmol), and the tube was closed. The resulting mixture was stirred at 180° C. for 96 hours using a silicone oil container. After the mixture was cooled to room temperature (25° C.), the container was opened to dilute the mixture with ethyl acetate (EtOAc, 300 mL). An organic layer was washed with water (80 mL), a 5% sodium bicarbonate aqueous solution (50 mL), and a saturated saline solution (50 mL) and dehydrated with anhydrous sodium sulfate (Na2SO4, 30 g). The solvent was removed under a reduced pressure condition of 40 mbar, and the resulting product was purified by column chromatography through a silica gel (Merck-silicagel 60, 230-400 mesh; using 5% EtOAc/hexane as a developer), thereby obtaining a brown solid, Compound 2a (600 mg, 38%; 27% 6-bromo-2-naphthol was recovered).
1H NMR (CDCl3, 300 MHz, 298 K, δ): 7.79 (d, J=1.5 Hz, 1H), 7.53-7.38 (m, 1H), 6.84 (dd, J=8.7, 2, 1 Hz, 1H), 6.74 (d, J=2.1 Hz, 1H), 3.76-3.66 (m, 2H), 3.43-3.33 (m, 1H), 2.24-2.19 (m, 2H), 2.08-2.03 (m, 2H), 1.55-1.42 (m, 4H), 1.33-1.19 (m, 4H); 13C NMR (CDCl3, 75 MHz, 298 K, δ): 145.4, 133.9, 129.7, 128.5, 128.3, 127.6, 119.2, 115.1, 104.7, 70.3, 51.3, 34.2, 31.1; IR (KBr, cm−1): 2934, 1625, 1590; HRMS (FAB): m/z calcd for C16H18BrNO [M+] 319.0572, [M++2] 321.0553; found 319.0570 [M+], 321.0558 [M++2]; calcd for C16H19BrNO [MH+] 320.0604, [MH++2] 322.0585; found 320.0607 [MH+], 322.0673 [MH++2]; mp: 139-141° C.
Compound 2b, 4-(6-bromonaphthalen-2-ylamino)cyclohexyl methanesulfonate, was synthesized by the inventors.
Specifically, Compound 2a (474 mg, 1.48 mmol) obtained in Example 1-1 was dissolved in anhydrous dichloromethane (CH2Cl2, 10 mL), and triethylamine (Et3N, 268 μL, 1.93 mmol) obtained through distillation was added thereto. The resulting mixture was cooled to 0° C. using ice, and a solution prepared by dissolving methanesulfonyl chloride (137 μL, 1.78 mmol) in anhydrous dichloromethane (1 mL) was slowly added dropwise for 5 minutes. The resulting mixture was stirred at 0° C. for 30 minutes (the reaction progress was checked by thin-layer chromatography (TLC)), cold water (10 mL) was added to terminate the reaction, and then extraction was performed with ethyl acetate (2×100 mL). An organic layer was washed with water (50 mL) and a saturated saline solution (50 mL) and dehydrated with anhydrous sodium sulfate (10 g). The solvent was removed under a reduced pressure condition of 40 mbar, and the resulting product was purified by column chromatography through a silica gel (Merck-silicagel 60, 230-400 mesh; using 5% hexane/CH2Cl2 as a developer), thereby obtaining a brown solid, Compound 2b (384 mg, 65%).
1H NMR (CDCl3, 300 MHz, 298 K, δ): 7.8 (d, J=1.8 Hz, 1H), 7.54-7.39 (m, 3H), 6.85 (dd, J=8.7, 2.1 Hz, 1H), 4.77-4.68 (m, 1H), 3.48-3.39 (m, 1H), 3.04 (s, 3H), 2.3-2.21 (m, 4H), 1.86-1.72 (m, 2H), 1.48-1.3 (m, 2H).
Compound 2c, 7-(6-bromonaphthalen-2-yl)-7-azabicyclo[2.2.1]heptane, was synthesized by the inventors.
Specifically, Compound 2b (384 mg, 1.48 mmol) obtained through Example 1-2 and anhydrous dimethylformamide (N,N-dimethylformamide, DMF, 20 mL) were added to a oven-dried flask and charged with argon gas. The resulting mixture was stirred at 135° C. for 4 hours using a silicone oil container (the reaction progress was confirmed by TLC). The mixture was cooled to room temperature and diluted with ethyl acetate (300 mL). An organic layer was washed with water (3×50 mL) and a saturated saline solution (50 mL) and dehydrated with anhydrous sodium sulfate (30 g). The solvent was removed under a reduced pressure condition of 40 mbar, and the resulting product was purified by column chromatography through a silica gel (Merck-silicagel 60, 230-400 mesh; using EtOAc/hexane as a developer), thereby obtaining a yellow solid, Compound 2c (228 mg, 89%).
1H NMR (CDCl3, 300 MHz, 298 K, δ): 7.83 (d, J=1.8 Hz, 1H), 7.58 (d, J=9.0 Hz, 1H), 7.5 (d, J=8.7 Hz, 1H), 7.42 (dd, J=8.7, 1.8 Hz, 1H), 7.21 (dd, J=9.0, 2.1 Hz, 1H), 7.07 (d, J=2.1 Hz, 1H), 4.3-4.29 (m, 2H), 1.85-1.82 (m, 4H), 1.49-1.47 (m, 4H); 13C NMR (CDCl3, 75 MHz, 298 K, δ): 146.5, 133.5, 129.6, 129.5, 129.4, 128.2, 120.2, 116.3, 110.7, 58.3, 29; IR (KBr, cm−1): 2945, 1621; HRMS: m/z calcd for C16H16BrN [M+] 301.0466, [M++2] 303.0447; found 301.0462 [M+], 303.0433 [M++2]; calcd for C16H17BrN [MH+]302.0499, [MH++2] 304.0479; found 302.0511 [MH+], 304.0515 [MH++2]; mp: 181-183° C.
Compound 2, 1-(6-(7-azabicyclo[2.2.1]heptan-7-yl)naphthalen-2-yl)ethanone, was synthesized by the inventors.
Specifically, Compound 2c obtained in Example 1-3 (184 mg, 0.61 mmol), palladium(II) acetate (Pd(OAc)2, 6.8 mg, 0.03 mmol), diphenylphosphinopropane (DPPP, 25.2 mg, 0.06 mmol), and ethyleneglycol (1.5 mL) were added to an oven-dried flask with two necks and charged with argon gas. After oxygen present in the mixture was removed by adding the argon gas to the mixture, ethyleneglycol vinyl ether (279 μL, 1.53 mmol) and Et3N (255 μL, 1.83 mmol) obtained by distillation were sequentially added thereto. The resulting mixture was stirred at 145° C. for 5 hours using a silicone oil container. The mixture was cooled to room temperature, and stirred with a 6N hydrochloric acid (HCl) aqueous solution (4 mL) at 60° C. for 4 hours. The mixture was cooled to room temperature, and diluted with ethyl acetate (100 mL). An organic layer was washed with water (50 mL), a 5% sodium bicarbonate aqueous solution (50 mL), and a saturated saline solution (50 mL) and dehydrated with anhydrous sodium sulfate (10 g). The solvent was removed under a reduced pressure condition of 40 mbar, and the resulting product was purified by column chromatography through a silica gel (Merck-silicagel 60, 230-400 mesh; using EtOAc/hexane as a developer), thereby obtaining a yellow solid, Compound 2 (100 mg, 62%). By further purification using recrystallization (using 3% CH2Cl2/hexane as a solvent), a yellow solid, Compound 2 (32 mg, 20%), was obtained.
1H NMR (CDCl3, 300 MHz, 298 K, δ): 8.32 (d, J=1.5 Hz, 1H), 7.93 (dd, J=8.7, 1.8 Hz, 1H), 7.78 (d, J=9.0 Hz, 1H), 7.64 (d, J=8.7 Hz, 1H), 7.24 (dd, J=8.7, 2.1 Hz, 1H), 7.11 (d, J=2.4 Hz, 1H), 4.37-4.34 (m, 2H), 2.67 (s, 3H), 1.87-1.84 (m, 4H), 1.54-1.5 (m, 4H); 13C NMR (CDCl3, 75 MHz, 298 K, δ): 198.0, 148.6, 137.7, 131.9, 131.0, 130.4, 127.0, 126.7, 124.7, 119.8, 110.3, 58.3, 29.0, 26.7; IR (KBr, cm−1): 1670; HRMS: m/z calcd for C18H19NO [M+] 265.1467, C18H20NO [MH+] 266.1545; found 265.1467 [M+], 266.1547 [MH+]; mp: 118-120° C.
A general synthetic pathway of Compound 3 is shown in Scheme 2.
Compound 3a, 6-bromo-N-isopropylnaphthalen-2-amine, was synthesized by the inventors.
Specifically, water (10 mL) was added to a sealed tube containing starting materials for synthesis such as 6-bromo-2-naphthol (1.0 g, 4.50 mmol), isopropyl amine (4 mL, 48.86 mmol), and sodium metabisulfite (1.3 g, 6.80 mmol), and the tube was closed. The resulting mixture was stirred at 180° C. for 48 hours using a silicone oil container. The mixture was cooled to room temperature and diluted with ethyl acetate (300 mL). An organic layer was washed with water (80 mL), a 5% sodium bicarbonate aqueous solution (50 mL), and a saturate saline solution (50 mL) and dehydrated with anhydrous sodium sulfate (30 g). The solvent was removed under a reduced pressure condition of 40 mbar, and the resulting product was purified by column chromatography through a silica gel (Merck-silicagel 60, 230-400 mesh; using 5% EtOAc/hexane as a developer), thereby obtaining a yellow solid, Compound 3a (1.07 g, 68%).
1H NMR (CDCl3, 300 MHz, 298 K, δ): 7.79 (d, J=1.8 Hz, 1H), 7.52 (d, J=9.0 Hz, 1H), 7.48-7.45 (m, 1H), 7.42-7.38 (m, 1H), 6.86-6.82 (m, 1H), 6.74 (d, J=2.1 Hz, 1H), 3.79-3.70 (m, 2H), 1.28 (d, J=6.3 Hz, 6H); 13C NMR (CDCl3, 75 MHz, 298 K, δ): 145.6, 134.0, 129.7, 129.6, 128.4, 128.2, 127.7, 119.3, 44.4, 23.0; IR (KBr, cm−1): 2966, 1627, 1517; mp: 56-58° C.
Compound 3, 1-(6-(isopropylamino)naphthalene-2-yl)ethanone, was synthesized by the inventors.
Specifically, Compound 3a obtained in Example 2-1 (550 mg, 2.1 mmol), Pd(OAc)2 (23 mg, 0.11 mmol), DPPP (86 mg, 0.22 mmol), and ethyleneglycol (3 mL) were added to an oven-dried flask with two necks and charged with argon gas. Oxygen present in the mixture was removed by adding the argon gas to the mixture, and ethyleneglycolvinylether (1.14 mL, 6.2 mmol) and Et3N obtained by distillation (723 μL, 5.2 mmol) were sequentially added thereto. The mixture was stirred at 145° C. for 5 hours using a silicone oil container. The mixture was cooled to room temperature and stirred with a 6N HCl aqueous solution (5 mL) at 60° C. for 4 hours. The mixture was cooled to room temperature and diluted with ethyl acetate (100 mL). An organic layer was washed with water (50 mL), a 5% sodium bicarbonate aqueous solution (50 mL), and a saturated saline solution (50 mL) and dehydrated with anhydrous sodium sulfate (10 g). The solvent was removed under a reduced pressure condition of 40 mbar, and the resulting product was purified by column chromatography through a silica gel (Merck-silicagel 60, 230-400 mesh; using 5% EtOAc/hexane as a developer), thereby obtaining a yellow solid, Compound 3 (322 mg, 68%). By further purification using recrystallization (using 4% CH2Cl2/hexane as a solvent), a yellow solid, Compound 3 (134 mg, 28%), was obtained.
1H NMR (CDCl3, 300 MHz, 298 K, δ): 8.26 (d, J=9.3 Hz, 1H), 7.91 (dd, J=8.7, 2.1 Hz, 1H), 7.71 (d, J=9.0 Hz, 1H), 7.56 (d, J=8.7 Hz, 1H), 6.86 (dd, J=9.0, 2.4 Hz, 1H), 6.76 (d, J=2.1 Hz, 1H), 3.94 (s, 1H), 3.83-3.75 (m, 1H), 2.66 (s, 3H), 1.3 (d, J=6.3 Hz, 6H); 13C NMR (CDCl3, 75 MHz, 298 K, δ): 197.9, 147.6, 138.3, 131.0, 130.8, 130.6, 126.0, 125.8, 124.9, 119.0, 104.0, 44.2, 26.6, 22.9; IR (KBr, cm−1): 1665; HRMS: m/z calcd for C15H17NO [M+] 227.1310, C15H18NO [MH+] 228.1388; found 227.1312 [M+], 228.1390 [M+H+]; mp: 112-114° C.
A general synthetic pathway of Compounds 4 and 7 is shown in Scheme 3.
Compound 4a, 1-(6-hydroxynaphthalen-2-yl)ethanone, was synthesized by the inventors.
Specifically, starting materials for synthesis such as 6-bromo-2-naphthol (2.0 g, 8.97 mmol), Pd(OAc)2 (100 mg, 0.45 mmol), DPPP (370 mg, 0.9 mmol), and ethylene glycol (3 mL) were added to an oven-dried flask with two necks and charged with argon gas. Oxygen present in the mixture was removed by adding the argon gas to the resulting mixture, and ethyleneglycolvinylether (2.41 mL, 27 mmol) and Et3N obtained distillation (3.12 mL, 22.4 mmol) were sequentially added. The mixture was stirred at 145° C. for 4 hours using a silicone oil container. The mixture was cooled to room temperature and stirred with dichloromethane (15 mL) and a 5% HCl aqueous solution (30 mL) at room temperature for 1 hour. The resulting mixture was extracted with dichloromethane (2×30 mL), and an organic layer was washed with water (30 mL) and dehydrated with anhydrous sodium sulfate (6 g). The solvent was removed under a reduced pressure condition of 40 mbar, and the resulting product was purified by column chromatography through a silica gel (Merck-silicagel 60, 230-400 mesh; using CH2Cl2 as a developer), thereby obtaining a solid, Compound 4a (1.33 g, 80%).
1H NMR (CDCl3, 300 MHz, 298 K, δ): 8.41 (1H, s), 7.98 (1H, dd, J=8.7, 1.6 Hz), 7.87 (1H, d, J=8.7 Hz), 7.70 (1H, d, J=8.7 Hz), 7.16 (1H, dd, J=8.7, 1.6 Hz), 5.4 (1H, s), 2.71 (3H, s); mp 172° C.
Compound 4, 1-(6-(methylamino)naphthalen-2-yl)ethanone, was synthesized by the inventors.
Specifically, water (20 mL) was added to a sealed tube containing Compound 4a obtained in Example 3-1 (2.0 g, 10.75 mmol), 50% methyl amine aqueous solution (4 mL, 53.75 mmol), and sodium metabisulfite (3.4 g, 21.5 mmol), and the tube was closed. The resulting mixture was stirred at 145° C. for 48 hours using a silicone oil container. The mixture was cooled to room temperature, and the resulting precipitate was filtered using a filter paper with a pore size of 8 μm and washed with water (10 mL). The filtered precipitate was purified by column chromatography through a silica gel (Merck-silicagel 60, 230-400 mesh; using 5% MeOH/CH2Cl2 as a developer), thereby obtaining a solid, Compound 4 (1.82 g, 85%).
1H NMR (CDCl3, 300 MHz, 298 K, δ): 8.3 (1H, s), 7.91 (1H, dd, J=8.7, 1.6 Hz), 7.70 (1H, d, J=8.7 Hz), 7.62 (1H, d, J=8.7 Hz), 6.89 (1H, dd, J=8.8, 2.2 Hz), 6.77 (1H, s), 4.17 (1H, br. s), 2.97 (3H, s), 2.67 (3H, s); mp 182° C.
Compound 7, 1-(6-(2-hydroxyethylamino)naphthalen-2-yl)ethanone, was synthesized by the inventors.
Specifically, water (15 mL) was added to a sealed tube containing Compound 4a obtained in Example 3-1 (1.0 g, 5.37 mmol), 2-aminoethanol (2-aminoethanol, 1.62 mL, 26.85 mmol), and sodium metabisulfite (2.0 g, 10.74 mmol), and the tube was closed. The resulting mixture was stirred at 145° C. for 48 hours using a silicone oil container. The mixture was cooled to room temperature, and the resulting precipitate was filtered using a filter paper with a pore size of 8 μm and washed with water (10 mL). The precipitated was purified by column chromatography through a silica gel (Merck-silicagel 60, 230-400 mesh; using 2% MeOH/CH2Cl2 as a developer), thereby obtaining a solid, Compound 7 (0.86 g, 70%).
1H NMR (CDCl3, 300 MHz, 298 K, δ): 8.31 (1H, s), 7.91 (1H, dd, J=9.0, 3.0 Hz, s), 7.72 (1H, d, J=9.0 Hz), 7.60 (1H, d, J=9.0 Hz), 6.94 (1H, dd, J=9.0 Hz), 6.84 (1H, s), 4.46 (1H, br. s), 3.94 (2H, t), 3.44 (2H, t), 2.67 (3H, s), 1.66 (1H, br. s); 13C NMR (CDCl3+DMSO-d6, 75 MHz, 298 K, δ): 197.74, 148.56, 138.05, 130.68, 130.63, 130.34, 125.87, 125.82, 124.60, 118.83, 103.45, 60.49, 45.75, 26.39; HRMS: m/z calcd for C14H15NO2 [M+] 229.28; found 229.11 [M+].
A general synthetic pathway of Compounds 5 and 9 is shown in Scheme 4.
Compound 5, 1-(6-(4-hydroxycyclohexylamino)naphthalen-2-yl)ethanone, was synthesized by the inventors.
Specifically, Compound 2a obtained in Example 1-1 (320 mg, 1.0 mmol), Pd(OAc)2 (11.2 mg, 0.055 mmol), DPPP (45.39 mg, 0.11 mmol), and ethylene glycol (2 mL) were added to an oven-dried flask with two necks and charged with argon gas. Oxygen present in the resulting mixture was removed by adding the argon gas to the mixture, and ethyleneglycolvinylether (456 μL, 2.5 mmol) and Et3N (417 μL, 3.0 mmol) obtained by distillation were sequentially added thereto. The mixture was stirred at 145° C. for 5 hours using a silicone oil container. The mixture was cooled to room temperature and stirred with a 6N HCl aqueous solution (2 mL) at 60° C. for 4 hours. The mixture was cooled to room temperature and diluted with ethyl acetate (150 mL). An organic layer was washed with water (50 mL), a 5% sodium bicarbonate aqueous solution (50 mL), and a saturated saline solution (50 mL) and dehydrated with anhydrous sodium sulfate (15 g). The solvent was removed under a reduced pressure condition of 40 mbar, and the resulting product was purified by column chromatography through a silica gel (Merck-silicagel 60, 230-400 mesh; using 50% EtOAc/CH2Cl2 as a developer), thereby obtaining a bright yellow solid, Compound 5 (203 mg, 72%). By further purification using recrystallization (using 7% CH2Cl2/hexane as a solvent), a bright yellow solid, Compound 5 (120 mg, 42%) was obtained.
1H NMR (CD3CN, 300 MHz, 298 K, δ): 8.33 (s, 1H), 7.84 (dd, J=8.7, 1.8 Hz, 1H), 7.73 (d, J=9.0 Hz, 1H), 7.59 (d, J=8.7 Hz, 1H), 6.96 (dd, J=9.0, 2.4 Hz, 1H), 6.83 (d, J=1.8 Hz, 1H), 4.84 (d, J=7.8 Hz, 1H), 3.62-3.5 (s, 1H), 3.46-3.34 (m, 1H), 2.71 (d, J=4.5 Hz, 1H), 2.59 (s, 3H), 2.13-2.09 (m, 4H), 1.46-1.2 (m, 4H); 1H NMR (DMSO-d6, 500 MHz, δ): 8.33 (d, J=1 Hz, 1H), 7.77-7.73 (m, 2H), 7.58 (d, J=8.5 Hz, 1H), 7.02 (dd, J=9.0, 2.0 Hz, 1H), 6.76 (d, J=1.5 Hz, 1H), 6.21 (d, J=7.5 Hz, 1H), 4.57 (d, J=4.5 Hz, 1H), 3.49-3.43 (m, 1H), 3.35-3.3 (m, 1H), 2.58 (s, 3H), 2.03-2 (m, 2H), 1.89-1.86 (m, 2H), 1.38-1.31 (m, 2H), 1.27-1.2 (m, 2H); 13C NMR (CDCl3, DMSO-d6, 125 MHz, 300K, δ): 196.8, 148.3, 138.0, 130.4, 129.4, 125.2, 124.6, 124.0, 119.0, 102.0, 68.4, 50.1, 33.9, 30.1, 26.3; IR (KBr, cm−1): 1669; HRMS: m/z calcd for C18H21NO2 [M+] 283.1572, C18H22NO2 [MH+] 284.1651; found 283.1575 [M+], 284.1648 [MH+]; mp: 186-188° C.
Compound 9, 1-(6-((4-hydroxycyclohexyl)(methyl)amino)naphthalen-2-yl)ethanone, was synthesized by the inventors.
Specifically, Compound 5 obtained in Example 4-1 (50 mg, 0.176 mmol) was dissolved in methanol (5 mL) and stirred in a 37% formaldehyde aqueous solution (43 μL, 0.53 mmol). A solution prepared by dissolving sodium cyanoborohydride (11.1 mg, 0.176 mmol) and zinc chloride (12 mg, 0.088 mmol) in methanol (2 mL) was added to the resulting mixture and stirred at room temperature for 2 hours (the reaction progress was checked by TLC). An 0.1N sodium hydroxide (NaOH) aqueous solution (2 mL) was added to the mixture, methanol was removed under a reduced pressure condition of 40 mbar, and then extraction with ethyl acetate (3×10 mL) was performed. An organic layer was washed with water (10 mL) and a saturated saline solution (10 mL) and dehydrated with anhydrous magnesium sulfate (MgSO4, 3 g). The solvent was removed under a reduced pressure condition of 40 mbar, and the resulting product was purified by column chromatography through a silica gel (Merck-silicagel 60, 230-400 mesh; using 50% EtOAc/hexane as a developer), thereby obtaining a bright yellow solid, Compound 9 (43 mg, 81%). By further purification using recrystallization (using 5% CH2Cl2/hexane as a solvent), a bright yellow solid, Compound 9 (27 mg, 45%) was obtained.
1H NMR (CDCl3, 300 MHz, 298 K, δ): 8.31 (s, 1H), 7.90 (dd, J=8.7, 1.8 Hz, 1H), 7.78 (d, J=9.3 Hz, 1H), 7.61 (d, J=9.0 Hz, 1H), 6.19 (dd, J=9.3, 2.4 Hz, 1H), 6.91 (d, J=2.1 Hz, 1H), 3.87-3.77 (m, 1H), 3.71-3.66 (m, 1H), 2.91 (s, 3H), 2.67 (s, 3H), 2.17-2.02 (m, 2H), 1.88-1.80 (m, 2H), 1.73-1.37 (m, 4H); 13C NMR (CDCl3, 75 MHz, 300 K, δ): 198.0, 150.2, 138.0, 131.1, 131.0, 130.5, 126.4, 124.9, 117.1, 106.3, 70.4, 57.4, 35.1, 31.6, 29.9, 27.9, 26.6; IR (KBr, cm−1): 1672; HRMS: m/z calcd for C19H23NO2 [M+] 297.1729, C19H24NO2 [MH+] 298.1761; found 297.1727 [M+], 297.1766 [MH+]; mp: 192-194° C.
A general synthetic pathway of Compound 6 is shown in Scheme 5.
Compound 6b, 1-(6-(4-aminocyclohexylamino)naphthalen-2-yl)ethanone, was synthesized by the inventors.
Specifically, water (10 mL) was added to a sealed tube containing Compound 4a obtained in Example 3-1 (418 mg, 1.68 mmol), trans-1,4-diaminocyclohexane (383 mg, 3.36 mmol), and sodium metabisulfite (640 mg, 3.36 mmol), and the tube was closed. The resulting mixture was stirred at 180° C. for 72 hours using a silicone oil container. The mixture was cooled to room temperature (25° C.) and filtered using cotton. Following removal of the solvent under a reduced pressure condition of 40 mbar, the filtered liquid was purified by column chromatography through a silica gel (Merck-silicagel 60, 230-400 mesh; using 5% MeOH/CH2Cl2 as a developer), thereby obtaining a brown solid, Compound 6b (355 mg, 56%). By further purification using recrystallization (using 25% MeOH/CH2Cl2 as a solvent), a brown solid, Compound 6b (139 mg, 22%) was obtained.
1H NMR (CD3OD, 300 MHz, 298 K, δ): 8.34 (d, J=1.5 Hz, 1H), 7.82 (dd, J=8.7, 1.8 Hz, 1H), 7.71 (d, J=9.0 Hz, 1H), 7.56 (d, J=8.7 Hz, 1H), 6.97 (dd, J=9.0, 2.4 Hz, 1H), 6.80 (d, J=2.1 Hz, 1H), 3.46-3.41 (m, 1H), 3.08-3.02 (m, 1H), 2.64 (s, 3H), 2.25-2.21 (m, 2H), 2.11-2.07 (m, 2H), 1.53-1.43 (m, 2H), 1.42-1.30 (m, 2H); 13C NMR (CD3OD, 75 MHz, 298 K, δ): 200.6, 149.9, 140.2, 132.2, 132.1, 131.4, 127.1, 126.9, 125.4, 120.3, 104.3, 51.6, 51.2, 32.1, 31.9, 26.5; IR (KBr, cm−1): 3321, 1668, 1550; mp: 198-200° C.
Compound 6, N-(4-(6-acetylnaphthalen-2-ylamino)cyclohexyl)acetamide, was synthesized by the inventors.
Specifically, compound 6b obtained in Example 5-1 (283 mg, 1.0 mmol) was dissolved in anhydrous dichloromethane (50 mL), and a solution prepared by dissolving acetic anhydride (94 μL, 1.0 mmol) in anhydrous dichloromethane (10 mL) was added to the resulting mixture. The mixture was stirred at room temperature for 2 hours, and a saturated ammonium chloride (NH4Cl) aqueous solution (10 mL) was added. An organic layer was washed with water (10 mL) and a saturated saline solution (10 mL) and dried with anhydrous magnesium sulfate (6 g). The solvent was removed under a reduced pressure condition of 40 mbar, the resulting product was purified by column chromatography through a silica gel (Merck-silicagel 60, 230-400 mesh; using 5% MeOH/CH2Cl2 as a developer), thereby obtaining a brown solid, Compound 6 (299 mg, 92%). By further purification using recrystallization (using 5% MeOH/CH2Cl2 as a solvent), a brown solid, Compound 6 (125 mg, 38%), was obtained.
1H NMR (CDCl3, 300 MHz, 298 K, δ): 8.27 (s, 1H), 7.9 (dd, J=8.7, 1.8 Hz, 1H), 7.69 (d, J=8.7 Hz, 1H), 7.57 (d, J=8.7 Hz, 1H), 6.84 (dd, J=8.7, 2.4 Hz, 1H), 6.74 (d, J=1.8 Hz, 1H), 5.36 (d, J=8.1 Hz, 1H), 3.98 (br, 1H), 3.85-3.83 (m, 1H), 3.39 (br, 1H), 2.66 (s, 3H), 2.25-2.23 (m, 2H), 2.11-2.08 (m, 2H), 1.99 (s, 3H), 1.37-1.30 (m, 4H); 13C NMR (CDCl3, 75 MHz, 298 K, δ): 198.0, 169.6, 147.3, 138.2, 131.2, 131.1, 130.6, 126.0, 125.0, 118.9, 104.1, 51.3, 48.2, 32.1, 32.0, 26.6, 23.8; IR (KBr, cm−1): 1653, 1576; HRMS: m/z calcd for C20H24N2O2 [M+] 324.1838, [M+] 325.1869; found 324.1835 [M+], 325.1871 [M+]; mp: above 250° C.
A general synthetic pathway of Compound 8 is shown in Scheme 6.
Compound 8a, 4-(6-bromonaphthalen-2-yl)morpholine, was synthesized by the inventors.
Specifically, water (15 mL) was added to a sealed tube containing starting materials for synthesis such as 6-bromo-2-naphthol (1.5 g, 6.72 mmol), morpholine (morpholine, 2.93 g, 33.60 mmol), and sodium metabisulfite (2.56 g, 13.45 mmol), and the tube was closed. The resulting mixture was stirred at 180° C. for 72 hours using a silicone oil container. The mixture was cooled to room temperature and diluted with ethyl acetate (300 mL) following opening of the tube. An organic layer was washed with water (80 mL), a 5% sodium bicarbonate aqueous solution (50 mL), and a saturated saline solution (50 mL), and dehydrated with anhydrous sodium sulfate (30 g). The solvent was removed under a reduced pressure condition of 40 mbar, and the resulting product was purified by column chromatography through a silica gel (Merck-silicagel 60, 230-400 mesh; using 1% MeOH/CH2Cl2 as a developer), thereby obtaining a brown solid, Compound 8a (1.21 g, 62%).
1H NMR (CDCl3, 300 MHz, 298 K, δ): 7.87 (d, J=1.8 Hz, 1H), 7.64 (d, J=9.3 Hz, 1H), 7.55 (d, J=9.0 Hz, 1H), 7.46 (dd, J=8.7, 2.1 Hz, 1H), 7.24-7.28 (m, 1H), 7.06 (m, 1H), 3.89-3.95 (m, 4H), 3.24-3.30 (m, 4H); 13C NMR (CDCl3, 75 MHz, 298 K, δ): 149.6, 133.2, 129.8, 129.7, 128.6, 128.2, 119.87, 117.1, 110.0, 67.1, 49.7; IR (KBr, cm−1): 1617, 1570; mp: 158-160° C.
Compound 8, 1-(6-morpholinonaphthalen-2-yl)ethanone, was synthesized by the inventors.
Specifically, Compound 8a obtained in Example 6-1 (97 mg, 0.33 mmol), Pd(OAc)2 (3.8 mg, 0.017 mmol), DPPP (13.8 mg, 0.034 mmol), and ethylene glycol (1 mL) were added to an oven-dried flask with two necks and charged with argon gas. Oxygen present in the resulting mixture was removed by adding the argon gas to the mixture, and ethyleneglycolvinylether (183 μL, 1.0 mmol) and Et3N (116 μL, 0.84 mmol) obtained by distillation were sequentially added thereto. The mixture was stirred at 145° C. for 4 hours using a silicone oil container. The mixture was cooled to room temperature and stirred with a 6N HCl aqueous solution (1.5 mL) at 60° C. for 4 hours. The mixture was cooled to room temperature and diluted with ethyl acetate (100 mL). An organic layer was washed with water (3×50 mL) and a saturated saline solution (50 mL) and dehydrated with anhydrous sodium sulfate (10 g). The solvent was removed under a reduced pressure condition of 40 mbar, and the resulting product was purified by column chromatography through a silica gel (Merck-silicagel 60, 230-400 mesh; using 1% MeOH/CH2Cl2 as a developer), thereby obtaining a brown solid, Compound 8 (54 mg, 64%). By further purification using recrystallization (using 1% MeOH/CH2Cl2 as a solvent), a brown solid, Compound 8 (36 mg, 43%), was obtained.
1H NMR (CDCl3, 300 MHz, 298 K, δ): 8.34 (s, 1H), 7.97 (d, J=8.7 Hz, 1H), 7.84 (d, J=9.3 Hz, 1H), 7.69 (d, J=8.7 Hz, 1H), 7.26-7.31 (m, 1H), 7.1 (s, 1H), 3.91 (t, J=4.8 Hz, 1H), 3.32 (t, J=4.6 Hz, 1H), 2.68 (s, 3H); 13C NMR (CDCl3, 75 MHz, 298 K, δ): 198.0, 151.2, 137.4, 132.4, 130.9, 130.2, 127.3, 127.1, 124.9, 119.0, 109.2, 67.0, 49.0, 26.7; IR (KBr, cm−1): 1666; HRMS: m/z calcd for C16H17NO2 [M+] 255.1259, C16H18NO2 [MH+] 256.1292; found 255.1256 [M+], 256.1279 [MH+]; mp: 149-151° C.
A general synthetic pathway of Compound 10 is shown in Scheme 7.
Compound 10, 6-(4-hydroxycyclohexylamino)-2-(2-hydroxyethyl)-1H-benzo[de] isoquinoline-1,3(2H)-dione, was synthesized by the inventors. Specifically, N-methyl pyrrolidone (NMP, 2 mL) was added to a sealed tube containing known starting materials for synthesis such as Compound 13 (S. Ghorbanian, S. et al. J. Chem. Technol. Biotechnol. 75, 1127.; 320 mg, 1.0 mmol) and trans-4-aminocyclohexanol (230 mg, 2.0 mmol), and the tube was closed. The resulting mixture was stirred at 115° C. for 24 hours using a silicone oil container. The mixture was cooled to room temperature and diluted with ethyl acetate (200 mL). An organic layer was washed with water (50 mL) and a saturated saline solution (50 mL) and dehydrated with anhydrous sodium sulfate (20 g). After the solvent was removed under a reduced pressure condition of 40 mbar, hexene (20 mL) was slowly added to the resulting product dissolved in chloroform (2 mL), thereby obtaining a yellow precipitate. The precipitate was filtered using a filter paper with a pore size of 8 μm and washed with water (10 mL) and hexene (10 mL). The precipitate was purified by column chromatography through a silica gel (Merck-silicagel 60, 230-400 mesh; using EtOAc as a developer), thereby obtaining an orange solid, Compound 10 (205 mg, 64%).
1H NMR (DMSO-d6, 300 MHz, 298 K, δ): 8.75 (d, J=8.1 Hz, 1H), 8.42 (d, J=6.9 Hz, 1H), 8.24 (d, J=8.4 Hz, 1H), 7.64 (t, J=7.5 Hz, 1H), 7.23 (d, J=7.8 Hz, 1H), 6.85 (d, J=9.0 Hz, 1H), 4.76 (t, J=6.0 Hz, 1H), 4.63 (d, J=4.2 Hz, 1H), 4.10 (t, J=6.6 Hz, 1H), 3.61-3.57 (m, 3H), 3.61-3.57 (m, 1H), 2.00 (d, J=11.7 Hz, 2H), 1.89 (d, J=11.7 Hz, 2H), 1.35-52 (m, 4H); 13C NMR (DMSO-d6, 125 MHz, 298 K, δ): 164.4, 163.5, 150.3, 034.7, 131.1, 130.1, 129.3, 124.5, 122.4, 120.6, 108.0, 104.7, 68.9, 58.5, 51.5, 41.8, 34.5, 30.2; HRMS: m/z calcd for C20H23N2O4 [MH+] 355.1658; found 355.1659 [MH+]; mp: above 250° C.
A general synthetic pathway of Compound 13 is shown in Scheme 8.
Compound 13, 1-6-(((1S,2S)-2-hydroxycyclohexyl)amino)naphthalen-2-yl)ethanone, was synthesized by the inventors. Specifically, water (15 mL) was added to a sealed tube containing Compound 4a obtained in Example 3-1 (1.25 g, 6.73 mmol), sodium metabisulfite (2.56 g, 13.45 mmol), and (1S,2S)-2-aminocyclohexanol (3.87 g, 33.65 mmol), and the tube was closed. The resulting mixture was stirred at 180° C. for 72 hours using a silicone oil container. After the mixture was cooled to room temperature, the container was opened to dilute the mixture with ethyl acetate (300 mL). An organic layer was washed with water (80 mL), a 5% sodium bicarbonate aqueous solution (50 mL), and a saturated saline solution (50 mL) and dehydrated with anhydrous sodium sulfate (30 g). The solvent was removed under a reduced pressure condition of 40 mbar, and the resulting product was purified by column chromatography through a silica gel (Merck-silicagel 60, 230-400 mesh; using 30% EtOAc/hexane as a developer), thereby obtaining a solid, Compound 13 (1.18 g, 62%).
1H NMR (CDCl3, 300 MHz, 298 K, δ): 8.18 (s, 1H), 7.83 (dd, J=8.7, 1.8 Hz, 1H), 7.58 (d, J=9.0 Hz, 1H), 7.48 (d, J=8.7 Hz, 1H), 6.84 (dd, J=8.7, 2.1 Hz, 1H), 6.72 (d, J=1.8 Hz, 1H), 4.61 (br, s, 1H), 4.13 (br, s, 1H), 3.533.50 (m, 1H), 2.92 (d, J=3.3 Hz, 1H), 2.59 (s, 3H), 1.90-1.86 (m, 1H), 1.781.58 (m, 5H), 1.471.37 (m, 2H); 13C NMR (CDCl3, 75 MHz, 298 K, δ): 198.3, 147.5, 138.1, 130.9, 130.6, 130.5, 125.8, 125.7, 124.6, 119.0, 104.1, 67.7, 54.3, 31.8, 26.7, 26.4, 24.1, 20.0.
A general synthetic pathway of Compound 14 is shown in Scheme 9.
Compound 13, 1-6-(((1 S,2R)-2-hydroxycyclohexyl)amino)naphthalen-2-yl)ethanone, was synthesized by the inventors. Specifically, water (15 mL) was added to a sealed tube containing Compound 4a obtained in Example 3-1 (1.25 g, 6.73 mmol), sodium metabisulfite (2.56 g, 13.45 mmol), and (1S,2R)-2-aminocyclohexanol (3.87 g, 33.65 mmol), and the tube was closed. The mixture was stirred at 180° C. for 72 hours using a silicone oil container. After the mixture was cooled to room temperature, the container was opened to dilute the mixture with ethyl acetate (300 mL). An organic layer was washed with water (80 mL), a 5% sodium bicarbonate aqueous solution (50 mL), and a saturated saline solution (50 mL) and dehydrated with anhydrous sodium sulfate (30 g). The solvent was removed under a reduced pressure condition of 40 mbar, and the resulting product was purified by column chromatography through a silica gel (Merck-silicagel 60, 230-400 mesh; using 30% EtOAc/hexane as a developer), thereby obtaining a solid, Compound 14 (1.18 g, 62%).
1H NMR (CDCl3, 300 MHz, 298 K, δ): 8.18 (s, 1H), 7.83 (dd, J=8.7, 1.8 Hz, 1H), 7.58 (d, J=9.0 Hz, 1H), 7.48 (d, J=8.7 Hz, 1H), 6.84 (dd, J=8.7, 2.1 Hz, 1H), 6.72 (d, J=1.8 Hz, 1H), 4.61 (br, s, 1H), 4.13 (br, s, 1H), 3.533.50 (m, 1H), 2.92 (d, J=3.3 Hz, 1H), 2.59 (s, 3H), 1.90-1.86 (m, 1H), 1.781.58 (m, 5H), 1.471.37 (m, 2H); 13C NMR (CDCl3, 75 MHz, 298 K, δ): 198.3, 147.5, 138.1, 130.9, 130.6, 130.5, 125.8, 125.7, 124.6, 119.0, 104.1, 67.7, 54.3, 31.8, 26.7, 26.4, 24.1, 20.0.
A general synthetic pathway of Compound 15 is shown in Scheme 10.
Compound 15a, 1-(6-(((1S,2S)-2-aminocyclohexyl)amino)naphthalen-2-yl)ethanone, was synthesized by the inventors. Specifically, water (15 mL) was added to a sealed tube containing Compound 4a obtained in Example 3-1 (1.25 g, 6.73 mmol), sodium metabisulfite (2.56 g, 13.45 mmol), and (1R,2R)-cyclohexane-1,2-diamine (1.53 g, 13.46 mmol), and the tube was closed. The resulting mixture was stirred at 180° C. for 72 hours using a silicone oil container. After the mixture was cooled to room temperature, the container was opened to dilute the mixture with ethyl acetate (300 mL). An organic layer was washed with water (80 mL), 5% sodium bicarbonate aqueous solution (50 mL) and a saturated saline solution (50 mL), and dehydrated with anhydrous sodium sulfate (30 g). The solvent was removed under a reduced pressure condition of 40 mbar, and the resulting product was purified by column chromatography through a silica gel (Merck-silicagel 60, 230-400 mesh; using 30% EtOAc/hexane as a developer), thereby obtaining a solid, Compound 14 (1.27 g, 67%).
1H NMR (CDCl3, 300 MHz, 298 K, δ): 8.19 (s, 1H), 7.83 (d, J=8.7 Hz, 1H), 7.59 (d, J=8.7 Hz, 1H), 7.50 (d, J=8.7 Hz, 1H), 6.92 (d, J=8.1 Hz, 1H), 6.80 (s, 1H), 4.38 (br, s, 1H), 3.873.43 (br, 3H), 3.23-3.08 (m, 1H), 2.59 (s, 3H), 2.152.11 (m, 1H), 2.011.94 (m, 1H), 1.721.68 (m, 2H), 1.391.10 (m, 3H), 1.09-0.89 (m, 1H).
Compound 15, N-((2S)-2-((6-acetylnaphthalen-2-yl)amino)cyclohexyl)benzenesulfonamide, was synthesized by the inventors. Specifically, Compound 15a obtained in Example 10-1 (93 mg, 0.33 mmol), benzenesulfonyl chloride (64 mg, 0.36 mmol), and triethylamine (36 mg, 0.36 mmol) were added to a flask and charged with argon gas. The resulting mixture was dissolved in dichloromethane, stirred at room temperature for 3 hours, and then diluted with dichloromethane (100 mL). An organic layer was washed with water (3×50 mL) and a saturated saline solution (50 mL) and dehydrated with anhydrous sodium sulfate (10 g). The solvent was removed under a reduced pressure condition of 40 mbar, and the resulting product was purified by column chromatography through a silica gel (Merck-silicagel 60, 230-400 mesh; using 1% MeOH/CH2Cl2 as a developer), thereby obtaining a solid, Compound 15 (120 mg, 86%).
1H NMR (CDCl3, 300 MHz, 298 K, δ): 8.27 (d, J=1.2 Hz, 1H), 7.937.85 (m, 3H), 7.66 (d, J=8.7 Hz, 1H), 7.607.54 (m, 2H), 7.507.45 (m, 2H), 6.73 (dd, J=8.7, 2.1 Hz, 1H), 6.67 (d, J=2.1 Hz, 1H), 4.87 (d, J=6.6 Hz, 1H), 4.23 (d, J=7.2 Hz, 1H), 3.253.18 (m, 1H), 3.153.08 (m, 1H), 2.66 (s, 3H), 2.33-2.29 (m, 1H), 1.931.89 (m, 1H), 1.751.65 (m, 2H), 1.391.23 (m, 3H), 1.191.12 (m, 1H); 13C NMR (CDCl3, 75 MHz, 298 K, δ): 198.1, 147.4, 140.9, 138.1, 132.9, 131.1, 131.1, 130.6, 129.4, 127.1, 126.2, 126.1, 124.9, 119.1, 104.0, 57.1, 56.7, 33.4, 32.1, 26.6, 24.8, 24.2.
A general synthetic pathway of Compound 16 is shown in Scheme 11.
Compound 15a, 1-(6-(((1S,2S)-2-aminocyclohexyl)amino)naphthalen-2-yl)ethanone, was synthesized by the inventors. Specifically, water (10 mL) was added to a sealed tube containing Compound 4a obtained in Example 3-1 (418 mg, 1.68 mmol), trans-1,4-diaminocyclohexane (383 mg, 3.36 mmol), and sodium metabisulfite (640 mg, 3.36 mmol), and the tube was closed. The mixture was stirred at 180° C. for 72 hours using a silicone oil container. The mixture was cooled to room temperature (25° C.) and filtered using cotton. After the solvent was removed under a reduced pressure condition of 40 mbar, the filtered liquid was purified by column chromatography through a silica gel (Merck-silicagel 60, 230-400 mesh; using 5% MeOH/CH2Cl2 as a developer), thereby obtaining a brown solid, Compound 6b (355 mg, 56%). By further purification using recrystallization (using 25% MeOH/CH2Cl2 as a solvent), a brown solid, Compound 6b (139 mg, 22%) was obtained.
1H NMR (CD3OD, 300 MHz, 298 K, δ): 8.34 (d, J=1.5 Hz, 1H), 7.82 (dd, J=8.7, 1.8 Hz, 1H), 7.71 (d, J=9.0 Hz, 1H), 7.56 (d, J=8.7 Hz, 1H), 6.97 (dd, J=9.0, 2.4 Hz, 1H), 6.80 (d, J=2.1 Hz, 1H), 3.46-3.41 (m, 1H), 3.08-3.02 (m, 1H), 2.64 (s, 3H), 2.25-2.21 (m, 2H), 2.11-2.07 (m, 2H), 1.53-1.43 (m, 2H), 1.42-1.30 (m, 2H); 13C NMR (CD3OD, 75 MHz, 298 K, δ): 200.6, 149.9, 140.2, 132.2, 132.1, 131.4, 127.1, 126.9, 125.4, 120.3, 104.3, 51.6, 51.2, 32.1, 31.9, 26.5; IR (KBr, cm−1): 3321, 1668, 1550; mp: 198-200° C.
Compound 16, N-((1R,4R)-4-((6-acetylnaphthalen-2-yl)amino)cyclohexyl)benzenesulfonamide, was synthesized by the inventors. Specifically, Compound 6b obtained in Example 11-1 (93 mg, 0.33 mmol), benzenesulfonyl chloride (64 mg, 0.36 mmol), and triethylamine (36 mg, 0.36 mmol) were added to a flask and then charged with argon gas, and then the resulting mixture was dissolved with dichloromethane. The mixture was stirred at room temperature for 3 hours and diluted with dichloromethane (100 mL). An organic layer was washed with water (3×50 mL) and a saturated saline solution (50 mL) and dehydrated with anhydrous sodium sulfate (10 g). The solvent was removed under a reduced pressure condition of 40 mbar, and the resulting product was purified by column chromatography through a silica gel (Merck-silicagel 60, 230-400 mesh; using 1% MeOH/CH2Cl2 as a solvent), thereby obtaining a solid, Compound 15 (120 mg, 86%).
1H NMR (CDCl3, 300 MHz, 298 K, δ): 8.26 (d, J=1.2 Hz, 1H), 7.94-7.89 (m, 3H), 7.67 (d, J=8.7 Hz, 1H), 7.607.50 (m, 4H), 6.80 (dd, J=9.0, 2.4 Hz, 1H), 6.69 (d, J=2.1 Hz, 1H), 4.794.77 (m, 1H), 3.89 (br, s, 1H), 3.333.18 (m, 2H), 2.65 (s, 3H), 2.172.13 (m, 2H), 1.971.93 (m, 2H), 1.441.31 (m, 2H), 1.271.14 (m, 2H); 13C NMR (CDCl3, 75 MHz, 298 K, δ): 198.0, 147.1, 141.4, 138.1, 131.2, 131.1, 129.4, 127.1, 126.0, 125.0, 118.8, 104.1, 52.6, 50.7, 32.8, 31.7, 26.6.
A general synthetic pathway of Compound 17 is shown in Scheme 12.
Compound 17, 1-6-(cyclohexylamino)naphthalen-2-yl)ethanone, was synthesized by the inventors. Specifically, water (15 mL) was added to a sealed tube containing Compound 4a obtained in Example 3-1 (1.25 g, 6.73 mmol), sodium metabisulfite (2.56 g, 13.45 mmol), and cyclohexaneamine (3.33 g, 33.65 mmol), and the tube was closed. The mixture was stirred at 180° C. for 72 hours using a silicone oil container. The mixture was cooled to room temperature and diluted with ethyl acetate (300 mL). An organic layer was washed with water (80 mL), a 5% sodium bicarbonate aqueous solution (50 mL), and a saturated saline solution (50 mL) and dehydrated with anhydrous sodium sulfate (30 g). The solvent was removed under a reduced pressure condition of 40 mbar, and the resulting product was purified by column chromatography through a silica gel (Merck-silicagel 60, 230-400 mesh; using 30% EtOAc/hexane as a developer), thereby obtaining a solid, Compound 17 (1.25 g, 70%).
1H NMR (CDCl3, 300 MHz, 298 K, δ): 8.27 (d, J=1.5 Hz, 1H), 7.89 (dd, J=8.7, 1.8 Hz, 1H), 7.69 (d, J=8.7 Hz, 1H), 7.57 (d, J=8.7 Hz, 1H), 6.85 (dd, J=9.0, 2.4 Hz, 1H), 6.76 (d, J=2.1 Hz, 1H), 4.00 (br, s, 1H), 3.463.39 (m, 1H), 2.66 (s, 3H), 2.152.11 (m, 2H), 1.841.78 (m, 2H), 1.841.78 (m, 2H), 1.69-1.61 (m, 1H), 1.481.38 (m, 2H), 1.321.21 (m, 3H); 13C NMR (CDCl3, 75 MHz, 298 K, δ): 197.9, 147.5, 138.3, 131.1, 130.8, 130.6, 126.0, 125.8, 124.9, 118.9, 103.9, 51.6, 33.3, 26.6, 26.0, 25.1.
A general synthetic pathway of Compound 18 is shown in Scheme 13.
Compound 18, 1-6-(pyrrolidin-1-yl)naphthalen-2-yl)ethanone, was synthesized by the inventors. Specifically, water (15 mL) was added to a sealed tube containing Compound 4a obtained in Example 3-1 (1.25 g, 6.73 mmol), sodium metabisulfite (2.56 g, 13.45 mmol), and pyrrolidine (2.59 g, 33.65 mmol), and the tube was closed. The mixture was stirred at 180° C. for 72 hours using a silicone oil container. After the mixture was cooled to room temperature, the container was opened to dilute the mixture with ethyl acetate (300 mL). An organic layer was washed with water (80 mL), a 5% sodium bicarbonate aqueous solution (50 mL), and a saturated saline solution (50 mL) and dehydrated with anhydrous sodium sulfate (30 g). The solvent was removed under a reduced pressure condition of 40 mbar, and the resulting product was purified by column chromatography through a silica gel (Merck-silicagel 60, 230-400 mesh; using 30% EtOAc/hexane as a developer), thereby obtaining a solid, Compound 18 (1.15 g, 72%).
1H NMR (CDCl3, 300 MHz, 298 K, δ): 8.29 (d, J=0.9 Hz, 1H), 7.89 (dd, J=8.7, 1.8 Hz, 1H), 7.74 (d, J=9.0 Hz, 1H), 7.57 (d, J=8.7 Hz, 1H), 6.96 (dd, J=9.0, 2.4 Hz, 1H), 6.69 (d, J=2.1 Hz, 1H), 3.38 (t, J=6.6 Hz, 4H), 2.65 (s, 3H), 2.092.00 (m, 4H); 13C NMR (CDCl3, 75 MHz, 298 K, δ): 197.8, 147.8, 138.1, 131.0, 130.8, 130.2, 125.9, 124.8, 124.7, 116.4, 104.4, 47.8, 26.5, 25.6.
Confirmation of Absorbing Properties of Two-Photon Absorbing Fluorophores
The inventors examined the absorbing properties of two-photon absorbing fluorophores of the present invention, and the results are shown in
Specifically, to confirm the absorbing properties of two-photon absorbing fluorophores, the inventors measured absorbance spectra for Compounds 1 to 9 at the concentration of 10 μM in HEPES buffer (containing 1% DMSO, pH 7.4) and water (containing 1% DMSO), contained in quartz cells with a light path length of 1 cm, and the results are respectively shown in the left and right graphs of
Further, molar extinction coefficients of Compounds 1 to 9 in HEPES buffer (containing 1% DMSO, pH 7.4), water (containing 1% DMSO), ethanol, acetonitrile, dimethylformamide, dichloromethane, and cyclohexane at the maximum absorption wavelength (
Confirmation of Fluorescence Properties of Two-Photon Absorbing Fluorophores
The inventors examined fluorescence properties of two-photon absorbing fluorophores of the present invention, and the results are shown in
Specifically, to confirm the fluorescence properties of two-photon absorbing fluorophores, the inventors measured fluorescence spectra for Compounds 1 to 9 at the concentration of 10 μM in HEPES buffer (containing 1% DMSO, pH 7.4) and water (containing 1% DMSO), contained in quartz cells with a light path length of 1 cm, and the results are respectively shown in the left and right graphs of
Fluorescence spectra for Compounds 1 to 9 at the concentration of 10 μM in ethanol and acetonitrile, and the results are respectively shown in the left and right graphs of
Further, fluorescence intensities for Compounds 1 to 9 at the concentration of 10 μM in HEPES buffer (containing 1% DMSO, pH 7.4) and water (containing 1% DMSO) were compared, and the results are shown in
Further, fluorescence quantum yields of Compounds 1 to 9 in dichloromethane, acetonitrile, and water (containing 1% DMSO) were measured, and the results are shown in
In addition, to compare the fluorescence intensities of the compounds in aqueous solution according to a structural change, fluorescence intensities of Compounds 1, 5, and 13 to 18 at the concentration of 1 μM in water (containing 1% DMSO) were compared, and the results are shown in
Confirmation of the Properties of Fluorescence Due to Two-Photon Excitation of the Two-Photon Absorbing Fluorophores
The inventors examined the properties of fluorescence due to two-photon excitation of the two-photon absorbing fluorophores of the present invention, and the results are shown in
Specifically, to confirm the fluorescence properties of the two-photon absorbing fluorophores under two-photon excitation, the inventors measured fluorescence spectra by two-photon excitation using a titanium:sapphire oscillator (Ti:sapphire oscillator), which was pumped by a frequency-doubled neodimium:yttrium orthovanadate laser (Nd:YVO4 laser; Verdi, Coherent) with an output power of 5.0 W. Output pulse energy was 40 nJ, and repetition rate was 380 kHz.
After quartz cells with a light path length of 1 mm were charged with Compound 1 at the concentration of 10 μM in water (containing 1% DMSO), fluorescence spectra by one-photon (black) and two-photon (red) excitation were measured at 740 nm (a), 760 nm (b), and 780 nm (c), and the results are shown in
For Compound 5 at the concentration of 10 μM in water (containing 1% DMSO), fluorescence spectra by one-photon (black) and two-photon (red) excitation were measured at 740 nm (a), 760 nm (b), and 780 nm (c), and the results are shown in
For Compound 6 at the concentration of 10 μM in water (containing 1% DMSO), fluorescence spectra by one-photon (black) and two-photon (red) excitation were measured at 740 nm (a), 760 nm (b), and 780 nm (c), and the results are shown in
For Compound 7 at the concentration of 10 μM in water (containing 1% DMSO), fluorescence spectra by one-photon (black) and two-photon (red) excitation were measured at 740 nm (a), 760 nm (b) and 780 nm (c), and the results are shown in
In addition, two-photon absorption cross-section values of each of Compounds 1 and 5 to 7 in dichloromethane solution, acetonitrile solution, and water (containing 1% DMSO) were measured, and the results are shown in
Observation of Two-Photon Fluorescence Microscopic Images of NIH3T3 Cells Treated with Compounds 1 and 5
The inventors observed fluorescence changes after NIH3T3 cells were treated with the conventional acedan (Compound 1) and Compound 5 through two-photon microscopy, and the results are shown in
Specifically, NIH3T3 cells were prepared in a 60 mm dish at a density of 2×106 cells/dish. The cells were cultured in Dulbecco's Modified Eagles Medium (DMEM, Hyclone) with 10% fetal bovine serum (Hyclone) and 1% antibiotics (WelGENE) in a 5% CO2-95% air atmosphere at 37° C. For cellular imaging, each cell dish was treated with Compounds 1 and 5 at the concentration of 50 μM, stored under the same conditions described above for 30 minutes, and observed using a two-photon microscope. Before fluorescence measurement, any amount of a compound that did not penetrate into the cells was removed by pipette suction, and a phosphate buffer saline (PBS) buffer solution was added. The two-photon microscope was composed of an upright microscope (BX51, Olympus) and a 20× objective lens (HCX APO, 11507751, NA 1.0, Leica) and used a titanium (Ti):sapphire laser (Chameleon Ultra II, Coherent) with a power of 50 mW at a two-photon excitation wavelength of 760 nm. Referring to
Observation of Two-Photon Fluorescence Microscopic Images of HeLa Cells Treated with Compounds 1, 5, 10, and 12
The inventors observed fluorescence changes after HeLa cells were treated with Compounds 1, 5, 10, and 12 of the present invention through two-photon microscopy, and the results are shown in
Specifically, the HeLa cells were prepared in a 60 mm dish at a density of 2×104 cells/dish. The cells were cultured in a DMEM (Hyclone) with 10% fetal bovine serum (Hyclone) and penicillin-streptomycin (Hyclone) in a 5% CO2-95% air atmosphere at 37° C. For cellular fluorescence imaging, each cell dish was treated with Compounds 1, 5, 10, and 12 at the concentration of 100 μM, stored under the same storage conditions as described above for 30 minutes, and observed using a two-photon microscope. Before a fluorescence measurement, any amount of compound that did not penetrate into the cells was removed by pipette suction, and the cells were washed with a PBS buffer solution three times and fixed with 4% paraformaldehyde for 10 minutes. The two-photon microscope was composed of an upright microscope (BX51, Olympus) and 20× and 40× objective lenses (XLUMPLEN, NA 1.0, Olympus) and used a titanium:sapphire laser (Ti:Sapphire laser; Chameleon Ultra II, Coherent) outputting a laser power of 160 mW at two-photon excitation wavelengths of 740 nm (Compounds 1 and 5), 880 nm (Compounds 1 and 5) and 900 nm (Compounds 10 and 12). Referring to
Observation of Two-Photon Fluorescence Microscopic Images of Mouse Tissues Treated with Compounds 1, 5, 10, and 12
The inventors observed fluorescence changes in mouse tissues treated with Compounds 1, 5, 10, and 12 of the present invention using a two-photon microscope, and the results are shown in
Specifically, a C57BL6 mouse (5-week-old, male, SAMTAKO Co.) was used, and an experiment was performed under a light-protected condition (dark room). The brain, liver and kidney of the mouse were extracted, washed with a PBS buffer solution, and frozen with dry-ice for 5 minutes. The frozen organs were shattered with a hammer and cut to a thickness of 16 μm using a slicer (cryostat machine, Leica, CM3000 model), thereby preparing a tissue slice sample. To fix the organs onto the slicer, an optical cutting temperature (OCT) compound, 10% polyvinyl alcohol, 25% polyethylene glycol, and 85.5% inactive species were used. The tissue slice sample was mounted on a specimen block (Paul Marienfeld GMbH & Co.), the specimen block was immersed in 4% paraformaldehyde for 10 minutes and washed with a PBS buffer solution, and the tissue was fixed again using a mounting solution (Gel Mount, BIOMEDA). The prepared tissue slice sample was immersed in PBS buffer of the concentration of 100 μM Compounds 1, 5, 10, and 12 for 10 minutes, washed with PBS buffer three times, and fixed in 4% paraformaldehyde. The two-photon microscope was composed of an upright microscope (BX51, Olympus) and 20× and 40× objective lenses (XLUMPLEN, NA 1.0, Olympus) and used a Ti:Sapphire laser (Chameleon Ultra II, Coherent) with a power of 120 mW at two-photon excitation wavelengths of 740 nm (Compounds 1 and 5), 880 nm (Compounds 1 and 5) and 900 nm (Compounds 10 and 12). Referring to
It would be understood by those of ordinary skill in the art that the above descriptions of the present invention are exemplary, and the exemplary embodiments disclosed herein can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. Therefore, the exemplary embodiments described above should be interpreted as illustrative and not limited in any aspect.
Since one-photon or two-photon absorbing fluorophores of the present invention have much higher fluorescence quantum yield and two-photon absorption cross-section values in aqueous solution, compared to those of the conventional fluorophores, the new dyes are is expected to be highly promising to use for bioimaging research, especially under two-photon microscopy.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2014-0127353 | Sep 2014 | KR | national |
| 10-2015-0061828 | Apr 2015 | KR | national |
This application claims priority to and the benefit of Korean Patent Application No. 10-2014-0127353, filed on Sep. 24, 2014, Korean Patent Application No. 10-2015-0061828, filed on Apr. 30, 2015 and International Patent Application No. PCT/KR2015/004508, filed on May 6, 2015, the disclosure of which is incorporated herein by reference in its entirety. The present invention was undertaken with the support of Korea Health Technology R&D Project No. HI13C1378 grant funded by the Ministry of Health & Welfare of Korea, Global Research Program No. 2014K1A1A2064569 grant funded by the National Research Foundation (NRF) of Korea.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2015/004508 | 5/6/2015 | WO | 00 |
