Patent Application
20140151548
The present invention relates to a matrix used for ionizing a material to be analyzed in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry.
Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is soft ionization mass spectrometry that is widely used to analyze a biological molecule rapidly. The use of MALDI mass spectrometry makes it possible to make a highly precise analysis of, for example, a high-molecular-weight protein, which has not easily been attained by any other ionizing method. Accordingly, this mass spectrometry has been used mainly to make mass spectrometry of biological polymers.
In MALDI mass spectrometry, a mixed crystal of a material to be analyzed and a matrix is prepared, and the crystal is irradiated with a laser beam to ionize the material to be analyzed. The matrix absorbs the light energy of the laser to be ionized, and is simultaneously heated rapidly to be gasified. By the irradiation with the laser, molecules of the sample are not directly gasified. However, these molecules are desorbed together with the matrix molecules surrounding the sample molecules. Subsequently, protons, electrons and others are exchanged between the ionized matrix molecules and sample molecules, so that the material to be analyzed is ionized. As a source for the laser, a nitrogen laser (wavelength: 337 nm) or YAG laser (wavelength: 355 nm) is generally used; thus, as the matrix, a substance having an absorption band in this wavelength region is used.
In recent years, MALDI mass spectrometry has been used also to analyze low-molecular-weight compounds. The spectrometry can attain a rapid analysis and a microanalysis, and can further be applied to molecular imaging. For this reason, the spectrometry has been expected to be applied to metabolome analysis. Whether or not a MALDI mass spectrometry succeeds depends largely on the performance of a matrix therefor. Thus, demands for a matrix suitable for the analysis of low-molecular-weight compounds have been increasing. For example, Patent Document 1 suggests a 1H-tetrazole derivative as a matrix suitable for cationizing low-molecular-weight compounds.
Many biological low-molecular-weight molecules are anionic compounds, such as carboxylic acids, amino acids, and phosphates. Thus, the importance of the development of a matrix suitable for a negative ion mode for detecting anions has been increasing. For example, Non-Patent Document 1 discloses that 9-aminoacridine is suitable as a matrix for MALDI mass spectrometry in a negative ion mode.
Patent Document 1: JP 2010-204050 A
Non-Patent Document 1: “9-Aminoacrydine as a matrix for negative mode matrix-assisted laser desorption/ionization”, Rachal L. Vermillion-Salsbury and David M. Hercules, Rapid Communications in Mass Spectrometry, vol. 16, No. 16, pp. 1,575-1,581, published on Aug. 30, 2002 by John Wiley & Sons Co.
However, the 1H-tetrazole derivative described in Patent Document 1 is a matrix for MALDI mass spectrometry in a positive ion mode. It is unclear whether or not the derivative is applicable to the negative ion mode. 9-Aminoacridine described in Non-Patent Document 1 is currently the most popular as a matrix for MALDI mass spectrometry in a negative ion mode. However, according to the matrix, many compounds are not measurable. Thus, this compound is not necessarily an optimal matrix. As described above, although demands for a matrix suitable for negative-ion-mode MALDI mass spectrometry for low-molecular-weight compounds have been increasing, there has not yet been a matrix having versatility in the present circumstances.
The present invention has been made in light of such problems, and an object thereof is to provide a matrix for MALDI mass spectrometry that has a high ability of ionizing low-molecular-weight compounds, and makes it possible to make measurement in a negative ion mode.
The present invention provides a matrix for MALDI mass spectrometry according to any one of the following items [1] to [4].
[1] A matrix for matrix-assisted laser desorption/ionization mass spectrometry, including:
wherein in the formula (I),
X is a carbon or nitrogen atom,
R1 is a group selected from the group consisting of a hydrogen atom, an alkyl group, an aryl group, a substituted aryl group, an arylalkyl group, a substituted arylalkyl group, a heteroaryl group, and a substituted heteroaryl group provided that a case where each of R1 and R2 is a hydrogen atom is excluded, and
R2 is a group selected from the group consisting of a hydrogen atom, an alkyl group, an alkoxyl group, NR3R4, a halogen atom, a nitro group, an allyl group, an aryl group, a substituted aryl group, a heteroaryl group, and a substituted heteroaryl group,
wherein R3 and R4 are each independently a group selected from the group consisting of a hydrogen atom, an alkyl group, an allyl group, an aryl group, a substituted aryl group, an arylalkyl group, a substituted arylalkyl group, a heteroaryl group, and a substituted heteroaryl group;
in the formula (II),
R5 is a group selected from the group consisting of a hydrogen atom, an alkyl group, an allyl group, an aryl group, a substituted aryl group, an arylalkyl group, a substituted arylalkyl group, a heteroaryl group, and a substituted heteroaryl group, and
R6 is a group selected from the group consisting of a hydrogen atom, an alkyl group, an alkoxyl group, NR3R4, a halogen atom, a nitro group, an allyl group, an aryl group, a substituted aryl group, a heteroaryl group, and a substituted heteroaryl group,
wherein R3 and R4 are each independently a group selected from the group consisting of a hydrogen atom, an alkyl group, an allyl group, an aryl group, a substituted aryl group, an arylalkyl group, a substituted arylalkyl group, a heteroaryl group, and a substituted heteroaryl group; and
in the formula (III),
Z is a carbon or nitrogen atom, and
R7 and R8 are each independently a group selected from the group consisting of a hydrogen atom and an amino group provided that a case where each of R7 and R8 is an amino group is excluded.
[2] The matrix for MALDI mass spectrometry according to item [1], which is a matrix for making measurement in a negative ion mode.
[3] The matrix for MALDI mass spectrometry according to item [1] or [2], wherein a material to be analyzed is an organic compound having a molecular weight of 1000 or less.
[4] The matrix for MALDI mass spectrometry according to any one of items [1] to [3], wherein the compound is one or more selected from the group consisting of compounds each represented by any one of the following formulae (5), (17), (18), (21), (24), (30), (35), (36), and (37) to (42):
According to the the present invention, a novel matrix for MALDI mass spectrometry is provided which has a higher ability of ionizing many low-molecular-weight compounds, in particular, biological low-molecular-weight compounds than 9-aminoacridine and further makes it possible to attain mass spectrometry in a negative ion mode with a high sensitivity. Since the matrix of the invention for MALDI mass spectrometry makes it possible to attain high-sensitivity MALDI mass spectrometry of biological molecules or metabolites thereof, the matrix can be used suitably for analyzing a metabolome, and for others.
A matrix for MALDI mass spectrometry according to an embodiment of the present invention is a compound having a structure represented by the following general formula (I), (II) or (III), or their salts thereof:
In the formula (I),
X is a carbon or nitrogen atom,
R1 is a group selected from the group consisting of a hydrogen atom, an alkyl group, an aryl group, a substituted aryl group, an arylalkyl group, a substituted arylalkyl group, a heteroaryl group, and a substituted heteroaryl group, and
R2 is a group selected from the group consisting of a hydrogen atom, an alkyl group, an alkoxyl group, NR3R4, a halogen atom, a nitro group, an allyl group, an aryl group, a substituted aryl group, a heteroaryl group, and a substituted heteroaryl group,
wherein R3 and R4 are each independently a group selected from the group consisting of a hydrogen atom, an alkyl group, an allyl group, an aryl group, a substituted aryl group, an arylalkyl group, a substituted arylalkyl group, a heteroaryl group, and a substituted heteroaryl group provided that a case where each of R1 and R2 is a hydrogen atom is excluded.
In the formula (II),
R5 is a group selected from the group consisting of a hydrogen atom, an alkyl group, an allyl group, an aryl group, a substituted aryl group, an arylalkyl group, a substituted arylalkyl group, a heteroaryl group, and a substituted heteroaryl group, and
R6 is a group selected from the group consisting of a hydrogen atom, an alkyl group, an alkoxyl group, NR3R4, a halogen atom, a nitro group, an allyl group, an aryl group, a substituted aryl group, a heteroaryl group, and a substituted heteroaryl group,
wherein R3 and R4 are each independently a group selected from the group consisting of a hydrogen atom, an alkyl group, an allyl group, an aryl group, a substituted aryl group, an arylalkyl group, a substituted arylalkyl group, a heteroaryl group, and a substituted heteroaryl group.
In the formula (III),
Z is a carbon or nitrogen atom, and
R7 and R8 are each independently a group selected from the group consisting of a hydrogen atom and an amino group (NH2) provided that a case where each of R7 and R8 is an amino group is excluded.
Specific examples of the alkyl group include linear, branched and cyclic alkyl groups having 1 to 10 carbon atoms. The alkyl groups are preferably methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-butyl, t-butyl, 1-pentyl, cyclopentyl, 1-hexyl, and cyclohexyl groups, more preferably methyl, ethyl, 1-propyl and 2-propyl groups.
Specific examples of the alkoxyl group include alkoxyl groups each having a linear, branched or cyclic alkyl group having 1 to 10 carbon atoms. The alkoxyl groups are preferably methoxy, ethoxy, 1-propyloxy, 2-propyloxy, 1-butyloxy, 2-butyloxy, t-butyloxy, 1-pentyloxy, cyclopentyloxy, 1-hexyloxy, and cyclohexyloxy groups, more preferably methoxy, ethoxy, 1-propyloxy and 2-propyloxy groups.
Specific examples of the aryl group include phenyl, naphthyl, anthranyl, and phenanthryl groups. Specific examples of the heteroaryl group include pyrrolyl, pyridyl, imidazolyl, thiophenyl, quinolyl, and isoquinolyl groups. Specific examples of the substituent on each of the substituted aryl group and the substituted heteroaryl group are the same as described in the case of R2 and R6.
The halogen atom is any of fluorine, chlorine, bromine, and iodine. Preferred are fluorine, chlorine, and bromine.
The matrix for MALDI mass spectrometry is preferably one or more compounds selected from the group consisting of compounds each represented by any one of the following formulae (5), (17), (18), (21), (24), (30), (35), (36), and (37) to (42):
Compounds of the matrix for MALDI mass spectrometry are partially commercially available. Compounds that are not commercially available can be synthesized from the commercially available compounds, respectively, through several steps by any known method.
The thus obtained matrix for MALDI mass spectrometry can be handled in the same way as ordinarily used matrices. For example, a measurement sample for MALDI mass spectrometry can be prepared by dissolving a material to be analyzed and the matrix in any appropriate solvent such as acetonitrile or THF, dropping the resultant solution onto a sample plate, and drying the dropped solution.
The following 37 compounds 2 to 36, 41 and 42 were synthesized. In the chemical formula list shown below, 9-aminoacridine (9-AA) used as a target for comparison is illustrated together.
About compounds 37 (anthracene), 38 (2-aminoantracene), 39 (acridine), and 40 (1-aminoantracene), commercially available products were used.
Synthesis of (9-phenylamino)acridine hydrochloride (4) and (9-phenylamino)acridine (5):
Synthesis of (9-phenylamino)acridine hydrochloride (4):
Reference document: Cope, H. Mutter, R.; Heal, W.; Pascoe, C.; Brown, P.; Pratt, S.; Chen, B. Europian Journal of Medicinal Chemistry, 2006, 41, 1124-1143.
To a mixture of 9-chloroacridine (110 mg, 0.5 mmol), aniline (55.9 mg, 0.6 mmol, 1.2 equivalents), and 2.5 mL of 1-methyl-2-pyrrolidone (NMP), three drops of concentrated hydrochloric acid were added with a Pasteur pipette, and then the resultant was stirred at ambient temperature for 6 hours. Thereafter, thereto was added 20 mL of ethyl acetate, and then the resultant was stirred at ambient temperature for 1 hour. The precipitated crystal was suction-filtered while washed with ethyl acetate. Methanol and ethyl acetate were used to recrystallize the crystal. Yield: 104 mg, 68%.
1H NMR (600 MHz, DMSO-d6) δ: 7.39-7.45 (m, 5H), 7.49-7.53 (m, 2H), 8.01 (dd, J=12, 12 Hz, 2H), 8.10 (d, J=8.8 Hz, 2H), 8.24 (d, J=8.8 Hz, 2H), 13C NMR (100 MHz, DMSO-d6) δ: 113.62, 119.23, 123.74, 124.66, 125.76, 127.51, 129.96, 135.27, 140.09, 140.92, 155.24, MS (ESI) m/z: 271 (M+H)+
Synthesis of (9-phenylamino)acridine (5):
Next, 63.5 mg of the hydrochloride salt and 120 mg of NaOH were added to 1.5 mL of water. The resultant was stirred for 1 hour, and then subjected to extraction with ethyl acetate. The extract was washed with water two times, washed with saturated sodium chloride solution, dried over sodium sulfate, and then concentrated. The resultant crystal was recrystallized with ethyl acetate. Yield: 38.0 mg, 57%; granular yellow crystal; m.p.: 227.2-228.9° C.
Synthesis of 9-(4-bromophenylamino)acridine (10):
To a mixture of 9-chloroacridine (110 mg, 0.5 mmol), 4-bromoaniline (103 mg, 0.6 mmol, 1.2 equivalents), and 2.5 mL of NMP, three drops of concentrated hydrochloric acid were added with a Pasteur pipette, and then the resultant was stirred at ambient temperature for 6 hours. Thereafter, thereto was added 20 mL of ethyl acetate, and then the resultant was stirred at ambient temperature for 1 hour. The precipitated crystal was suction-filtered while washed with ethyl acetate. Methanol and acetonitrile were used to recrystallize the crystal. Yield: 127 mg, 66%; m.p.: 231.4° C.
1H NMR (400 MHz, DMSO-d6) δ: 7.41 (d, J=8.8 Hz, 2H), 7.52 (t, J=8.8 Hz, 2H), 7.71 (d, J=8.8 Hz, 2H), 8.02-8.10 (m, 4H), 8.25 (d, J=8.8 Hz, 2H), 13C NMR (100 MHz, DMSO-d6) δ: 114.10, 114.94, 119.30, 123.98, 125.74, 126.06, 132.63, 135.32, 140.11, 140.71, 154.96; MS (ESI) m/z: 349 (M+H)+
Next, 77 mg of the hydrochloride salt and 120 mg of NaOH were added to 1.5 mL of water. The resultant was stirred for 1 hour, and then subjected to extraction with ethyl acetate. The extract was washed with water two times, washed with saturated sodium chloride solution, dried over sodium sulfate, and then concentrated. The resultant crystal was recrystallized with ethyl acetate. Yield: 36.3 mg, 52%; granular yellow crystal; m.p.: 220.7-221.9° C.
Synthesis of 9-(4-chlorophenylamino)acridine (11):
To a mixture of 9-chloroacridine (110 mg, 0.5 mmol), 4-chloroaniline (76.5 mg, 0.6 mmol, 1.2 equivalents), and 2.5 mL of NMP, three drops of concentrated hydrochloric acid were added with a Pasteur pipette, and then the resultant was stirred at ambient temperature for 6 hours. Thereafter, thereto was added 20 mL of ethyl acetate, and then the resultant was stirred at ambient temperature for 1 hour. The precipitated crystal was suction-filtered while washed with ethyl acetate. Methanol was used to recrystallize the crystal. Yield (hydrochloride salt): 149 mg, 87%.
1H NMR (400 MHz, DMSO) δ:7.46-7.51 (m, 4H), 7.56 (d, J=8.8 Hz, 2H), 8.02 (t, J=8.8 Hz, 2H), 8.17 (d, J=8.8 Hz, 2H), 8.30 (d, J=8.8 Hz, 2H), 13C NMR (100 MHz, DMSO-d6) δ: 114.02, 119.28, 123.96, 125.77, 125.88, 129.72, 131.04, 135.30, 140.12, 155.04, MS (ESI) m/z: 305 (M+H)+
Next, 149 mg of the hydrochloride salt and 240 mg of NaOH were added to 3 mL of water. The resultant was stirred for 1 hour, and then subjected to extraction with ethyl acetate. The extract was washed with water two times, washed with saturated sodium chloride solution, dried over sodium sulfate, and then concentrated. The resultant crystal was recrystallized with acetonitrile. Yield: 56.9 mg, 43%; granular yellow crystal; m.p.: 208.7-209.6° C.
Synthesis of 9-(4-nitrophenylamino)acridine (12):
To a mixture of 9-chloroacridine (110 mg, 0.5 mmol), 4-nitroaniline (82.8 mg, 0.6 mmol, 1.2 equivalents), and 2.5 mL of NMP, three drops of concentrated hydrochloric acid were added with a Pasteur pipette, and then the resultant was stirred at ambient temperature for 6 hours. Thereafter, thereto was added 20 mL of ethyl acetate, and then the resultant was stirred at ambient temperature for 1 hour. The precipitated crystal was suction-filtered while washed with ethyl acetate. Yield (hydrochloride salt): 167 mg, 95%.
1H NMR (400 MHz, DMSO-d6) δ: 7.49 (d, J=8.8 Hz, 2H), 7.60 (t, J=7.2 Hz, 2H), 8.10 (t, J=7.2 Hz, 2H), 8.21-8.33 (m, 6H), MS (ESI) m/z: 316 (M+H)+
Next, 167 mg of the hydrochloride salt and 240 mg of NaOH were added to 3 mL of water. The resultant was stirred for 1 hour, and then subjected to extraction with ethyl acetate. The extract was washed with water two times, washed with saturated sodium chloride solution, dried over sodium sulfate, and then concentrated. The resultant crystal was recrystallized with acetonitrile. Yield: 96.7 mg, 65%; reddish orange needles; m.p.: 218.5-222.6° C.
Synthesis of 9-(4-methoxyphenylamino)acridine (13):
To a mixture of 9-chloroacridine (110 mg, 0.5 mmol), 4-anisidine (73.8 mg, 0.6 mmol, 1.2 equivalents), and 2.5 mL of NMP, three drops of concentrated hydrochloric acid were added with a Pasteur pipette, and then the resultant was stirred at ambient temperature for 6 hours. Thereafter, thereto was added 20 mL of ethyl acetate, and then the resultant was stirred at ambient temperature for 1 hour. The precipitated crystal was suction-filtered while washed with ethyl acetate. Yield (hydrochloride salt): 158 mg, 94%.
1H NMR (400 MHz, DMSO-d6) δ: 3.84 (s, 3H), 7.10 (d, J=8.8 Hz, 2H), 7.42 (t, J=8.8 Hz, 4H), 7.97 (t, J=8.8 Hz, 2H), 8.10 (d, J=8.8 Hz, 2H), 8.24 (d, J=8.8 Hz, 2H), 13C NMR (100 MHz, DMSO-d6) δ: 55.46, 113.15, 115.11, 119.06, 123.49, 125.65, 126.40, 133.21, 135.05, 140.02, 155.31, 158.53, MS (ESI) m/z: 301 (M+H)+
Next, 158 mg of the hydrochloride salt and 240 mg of NaOH were added to 3 mL of water. The resultant was stirred for 1 hour, and then subjected to extraction with ethyl acetate. The extract was washed with water two times, washed with saturated sodium chloride solution, dried over sodium sulfate, and then concentrated. The resultant crystal was recrystallized with water/methanol. Yield: 79.5 mg, 56%; dark red needles.
Synthesis of N-(4-methylphenyl)acridine-9-amine hydrochloride (14) and N-(4-methylphenyl)acridine-9-amine (36):
Synthesis of 9-(4-fluorophenylamino)acridine hydrochloride (14):
To a mixture of 9-chloroacridine (110 mg, 0.5 mmol), 4-fluoroaniline (66.7 mg, 0.6 mmol, 1.2 equivalents), and 2.5 mL of NMP, three drops of concentrated hydrochloric acid were added with a Pasteur pipette, and then the resultant was stirred at ambient temperature for 6 hours. Thereafter, thereto was added 20 mL of ethyl acetate, and then the resultant was stirred at ambient temperature for 1 hour. The precipitated crystal was suction-filtered while washed with ethyl acetate. The crystal was recrystallized with acetonitrile. Yield: 125 mg, 77%; yellow needles.
1H NMR (400 MHz, DMSO-d6) δ: 7.38 (t, J=8.8 Hz, 2H), 7.45 (t, J=7.6 Hz, 2H), 7.51-7.55 (m, 2H), 7.99 (t, J=7.6 Hz, 2H), 8.16 (d, J=8.8 Hz, 2H), 8.26 (d, J=8.8 Hz, 2H), 13C NMR (100 MHz, DMSO-d6) δ: 113.49, 116.73 (d, J=23 Hz), 119.18, 123.73, 125.73, 126.84 (d, J=9.1 Hz), 135.16, 137.25, 140.08, 155.35, 160.73 (d, J=246 Hz); MS (ESI) m/z: 289 (M+H)+
Synthesis of 9-(4-fluorophenylamino)acridine (36):
To 1.5 mL of water were added 64.8 mg of the hydrochloride salt (14) and 120 mg of NaOH. The resultant was stirred for 1 hour, and then subjected to extraction with ethyl acetate. The extract was washed with water two times, washed with saturated sodium chloride solution, dried over sodium sulfate, and then concentrated. The resultant crystal was recrystallized with acetonitrile. Yield: 35.7 mg, 62%; yellow needles; m.p.: 183.5-196.1° C.
Synthesis of 9-(4-methylphenylamino)acridine (15):
To a mixture of 9-chloroacridine (110 mg, 0.5 mmol), 4-toluidine (64.5 mg, 0.6 mmol, 1.2 equivalents), and 2.5 mL of NMP, three drops of concentrated hydrochloric acid were added with a Pasteur pipette, and then the resultant was stirred at ambient temperature for 6 hours. Thereafter, thereto was added 20 mL of ethyl acetate, and then the resultant was stirred at ambient temperature for 1 hour. The precipitated crystal was suction-filtered while washed with ethyl acetate. Yield (hydrochloride salt): 117 mg, 82%.
1H NMR (400 MHz, CDCl3) δ: 2.41 (s, 3H), 7.44 (t, J=7.6 Hz, 4H), 7.97-8.06 (m, 8H), 8.23 (d, J=9.6 Hz, 2H), MS (ESI) m/z: 285 (M+H)+
To 3 mL of water were added 117 mg of the hydrochloride salt and 240 mg of NaOH. The resultant was stirred for 1 hour, and then subjected to extraction with ethyl acetate. The extract was washed with water two times, washed with saturated sodium chloride solution, dried over sodium sulfate, and then concentrated. The resultant crystal was recrystallized with acetonitrile. Yield: 62.5 mg, 54%; orange needles; m.p.: 172.7-174.3° C.
Synthesis of 9-(naphthalene-1-yl-amino)acridine (16):
To a mixture of 9-chloroacridine (110 mg, 0.5 mmol), 1-naphthylamine (85.9 mg, 0.6 mmol, 1.2 equivalents), and 2.5 mL of NMP, three drops of concentrated hydrochloric acid were added with a Pasteur pipette, and then the resultant was stirred at ambient temperature for 6 hours. Thereafter, thereto was added 20 mL of ethyl acetate, and then the resultant was stirred at ambient temperature for 1 hour. The precipitated crystal was suction-filtered while washed with ethyl acetate. Yield (hydrochloride): 110 mg, 69%.
1H NMR (400 MHz, DMSO-d6) δ: 7.35 (t, J=7.6 Hz, 2H), 7.59-7.70 (m, 4H), 7.98 (t, J=7.6 Hz, 2H), 8.07-8.17 (m, 7H), MS (ESI) m/z: 321 (M+H)+
To 3 mL of water were added 110 mg of the hydrochloride salt and 240 mg of NaOH. The resultant was stirred for 1 hour, and then subjected to extraction with ethyl acetate. The extract was washed with water two times, washed with saturated sodium chloride solution, dried over sodium sulfate, and then concentrated.
Synthesis of 9-aminoanthracene (17):
Reference document: Adams, H.; Bawa, R. A.; McMillan, K. G.; Jones, S. Tetrahedron: Asymmetry, 2007, 18, 1003-1012
9-Nitroantracene (446 mg, 2.00 mmol) was added to acetic acid (9.6 g, 160 mmol, 80 equivalents), and the resultant was stirred at 70° C. for 1 hour. Thereto was slowly added a solution obtained by dissolving SnCl2 (1.89 g, 10 mmol, 5 equivalents) in concentrated hydrochloric acid (7.3 g, 200 mmol, 100 equivalents), and the resultant was stirred at 80° C. for 1 hour. The precipitated crystal was then suction-filtered while washed with concentrated hydrochloric acid. Thereafter, the filtrate was added to 30 mL of a 10% NaOH aqueous solution, and the resultant was stirred for 1 hour. The resultant was then suction-filtered while washed with water. The resultant crude crystal was recrystallized with methanol. Yield: 270 mg, 70%; reddish purple needles; m.p.: 137.9-171.2° C.
1H NMR (400 MHz, CDCl3) δ: 4.87 (s, 2H), 7.39-7.46 (m, 4H), 7.88 (s, 1H), 7.88-7.98 (m, 4H), MS (ESI) m/z: 194 (M+H)+
Synthesis of 4-(N-benzyl)amino-7-chloroquinoline (18)
Reference documents: Pellegrini, S.; Grad, J -N.; Bousquet, T.; Pelinski, L. Tetrahedron Lett. 2011, 52, 1742-1744, and
de Souza, M. V. N.; Pais, K. C.; Kaiser, C. R.; Peralta, M. A.; Ferreira, M. de L.; Lourenco, M. C. S. Bioorganic and Medicinal Chemistry, 2009, 17, 1474-1480.
To 25 mL of phenol was added 4,7-dichloroquinoline (1.98 g, 10 mmol), and then the resultant was stirred at 120° C. Thereafter, the temperature thereof was raised to 160° C., and thereto was added benzylamine (1.61 g, 15 mmol, 1.5 equivalents). The resultant was stirred for 12 hours, and then the temperature thereof was returned to ambient temperature. Thereto was added 30 mL of acetone, and the temperature thereof was set to 0° C. The resultant was stirred for 1 hour. The precipitated crystal was then suction-filtered while washed with acetone. The resultant crystal was added to 100 mL of a 10% NaOH aqueous solution. The resultant was stirred for 1 hour, and subjected to extraction with chloroform. The extract was washed with water two times, washed with saturated sodium chloride solution, dried over sodium sulfate, and then concentrated. The crystal was recrystallized with ethyl acetate. Yield: 552 mg, 21%; colorless needles; m.p.: 173.1-174.6° C.
1H NMR (400 MHz, CDCl3) δ: 4.53 (d, J=5.2 Hz, 2H), 5.32 (s, 1H), 6.46 (d, J=6.0 Hz, 1H), 7.34-7.40 (m, 6H), 7.69 (d, J=8.8 Hz, 1H), 7.98 (d, J=2.0 Hz, 1H), 8.53 (d, J=4.8 Hz, 1H), 13C NMR (100 MHz, CDCl3) δ: 47.61, 99.70, 117.13, 120.84, 125.50, 127.59, 127.98, 129.01, 134.94, 137.19, 149.17, 149.41, 152.14, MS (ESI) m/z: 269 (M+H)+
Synthesis of 4-(N-benzylamino)quinoline (19):
Reference document: Masatomo Hamana, Kazuhisa Funakoshi, Yakugaku Zasshi, 1964, 84, 42-47.
4-(N-benzyl)amino-7-chloroquinoline (485 mg, 1.80 mmol) was dissolved in 12 mL of methanol. Thereto was added Pd/C (10%, 25.5 mg, 0.024 mmol, 0.01 equivalents). Hydrogen was added to the resultant while the system was bubbled therewith at ambient temperature under normal pressure. The resultant was stirred for 3 hours. Thereafter, the resultant was filtered through celite, and concentrated. Next, thereto was added 30 mL of a 10% NaOH aqueous solution. The resultant was stirred for 1 hour, and subjected to extraction with chloroform. The extract was washed with water two times, washed with saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The crystal was recrystallized with acetonitrile. Yield: 335 mg, 66%; colorless needles; m.p.: 131.5-132.2° C. (bibliographic data: 113-115° C. (benzene/petroleum benzine)).
1H NMR (400 MHz, CDCl3) δ: 4.54 (d, J=4.8 Hz, 2H), 5.40 (s, 1H), 6.46 (d, J=4.8 Hz, 1H), 7.35-7.45 (m, 6H), 7.64 (t, J=7.6 Hz, 1H), 7.77 (d, J=8.8 Hz, 1H), 8.00 (d, J=8.8 Hz, 1H), 8.55 (d, J=5.2 Hz, 1H), 13C NMR (100 MHz, CDCl3-d6) δ: 47.55, 99.39, 118.73, 119.26, 124.75, 127.54, 127.82, 128.93, 129.03, 130.07, 137.52, 148.46, 149.39, 151.11, MS (ESI) m/z: 235 (M+H)+
Synthesis of 7-chloro-4-(N-phenylamino)quinoline (20):
Reference documents: Chambers, R. A.; Pearson, D. E. J Org. Chem. 1963, 28, 3144-3147, and
Souza, M. Bioorganic and Medicinal Chemistry, 2009, 17, 1474-1480.
To 25 mL of phenol was added 4,7-dichloroquinoline (1.98 g, 10 mmol), and then the resultant was stirred at 120° C. Thereafter, the temperature thereof was raised to 160° C., and thereto was added aniline (1.40 g, 15 mmol, 1.5 equivalents). The resultant was then stirred for 12 hours, and then the temperature thereof was returned to ambient temperature. Thereto was added acetone, and the temperature thereof was set to 0° C. The resultant was stirred for 1 hour. The precipitated crystal was then suction-filtered while washed with acetone. The resultant crystal was added to 100 mL of a 10% NaOH aqueous solution. The resultant was stirred for 1 hour, and subjected to extraction with chloroform. The extract was washed with water two times, washed with saturated sodium chloride solution, dried over sodium sulfate, and then concentrated. The crystal was recrystallized with acetonitrile. Yield: 888 mg, 35%; granular colorless crystal.
1H NMR (400 MHz, DMSO-d6) δ: 6.92 (d, J=6.0 Hz, 1H), 7.17 (t, J=7.6 Hz, 1H), 7.36-7.46 (m, 4H), 7.58 (dd, J=2.0, 7.6 Hz, 1H), 7.90 (d, J=2.0 Hz, 1H), 8.43-8.47 (m, 2H), 9.10 ppm (s, 1H), 13C NMR (100 MHz, DMSO) δ: 101.71, 118.29, 122.64, 124.04, 124.42, 124.88, 127.65, 129.40, 133.85, 140.11, 147.95, 149.58, 151.95, MS (ESI) m/z: 255 (M+H)+
Synthesis of 4-(N-phenylamino)quinoline (21):
Reference documents: Alan R. Katritzky, A. R.; Tian-Bao Huang, T -B.; Voronkov, M. V. J. Org. Chem. 2001, 66, 1043-1045, and
Souza, M. Bioorganic and Medicinal Chemistry, 2009, 17, 1474-1480.
In 30 mL of methanol was dissolved N-phenyl-7-chloroquinoline-4-amine (180 mg, 0.709 mmol). Thereto was added Pd/C (10%, 10 mg, 0.0009 mmol, 0.013 equivalents). Hydrogen was added to the resultant while the system was bubbled therewith at ambient temperature under normal pressure. The resultant was stirred for 3 hours. Thereafter, the resultant was filtered through celite, and concentrated. Next, thereto was added 10 mL of a 10% NaOH aqueous solution. The resultant was stirred for 1 hour, and subjected to extraction with chloroform. The extract was washed with water two times, washed with saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The resultant was recrystallized with acetonitrile. Yield: 57.7 mg, 37%; granular light yellow crystal; m.p.: 194.8-195.6° C. (bibliographic data m.p.: 197-198° C.).
1H NMR (400 MHz, CDCl3) δ: 6.77 (s, 1H), 7.00 (d, J=5.2 Hz, 1H), 7.19, (t, J=6.8 Hz, 1H) 7.31 (d, J=8.8 Hz, 2H), 7.42 (t, J=8.0 Hz, 2H), 7.50 (t, J=8.0 Hz, 1H), 7.69 (t, J=6.8 Hz, 1H), 7.95 (d, J=8.8 Hz, 1H), 8.05 (d, J=8.8 Hz, 1H) 8.58 (d, J=5.2 Hz, 1H), 13C NMR (100 MHz, CDCl3) δ: 102.23, 119.57, 119.71, 122.60, 124.59, 125.32, 129.34, 129.68, 130.19, 139.87, 147.40, 149.13, 150.93, MS (ESI) m/z: 221 (M+H)+
Synthesis of 4-nitro-2-(phenylamino)benzoic acid (22):
Reference documents: Ullmann, F.; Wagner, C. Justus Liebigs Ann. Chem. 1907, 355, 359-371, and
Ramage, R. WO2007/049057 (May 3, 2007).
Potassium carbonate (1.6 g, 0.012 mol, 1.15 equivalents) was added to a mixed solution of aniline (4.66 g, 0.05 mol, 5 equivalents) and 2-chloro-4-nitrobenzoic acid (2.02 g, 0.01 mol, 1 equivalent). The temperature of the resultant was set to 160° C., and copper acetate (91 mg, 0.456 mmol) was added thereto. Thereafter, the resultant was stirred at 180° C. for 10 hours. Thereafter, 30 mL of water was added to the reaction solution. Thereto was added a 6 M hydrochloric acid solution until the pH of the solution was turned to 2. The solution was then stirred for 1 hour. The resultant was crushed in a mortar, and then dried in a desiccator. The resultant was purified through a silica gel column (400 g of silica gel) with the following developing solvent: 1% methanol/chloroform. The resultant was recrystallized with acetonitrile. Yield: 910 mg, 35%; orange needles; m.p.: 232.9-234.0° C. (bibliographic data: 230° C.).
1H NMR (400 MHz, DMSO-d6) δ: 3.33 (broad, 1H), 7.22 (t, J=7.6 Hz, 1H), 7.35 (d, J=7.6 Hz, 2H), 7.43-7.52 (m, 3H), 7.82 (d, J=2 Hz, 1H), 8.12 (d, J=8.8 Hz, 1H), 9.78 (broad, 1H), 13C NMR (100 MHz, DMSO-d6) δ: 107.52, 110.67, 117.04, 122.74, 124.71, 129.79, 133.58, 139.19, 147.84, 150.83, 168.64, MS (ESI) m/z: 257 (M−H)−
Synthesis of 3-nitro-9-chloroacridine (23):
Reference documents: Robert Faure, Jean-Pierre Galzy, Jacques Barbe, Abdel Lhatif Boukir, Emile-Jean Vincent, Gerard Boyer, Jose Elguero, Bull. Soc. Chim. Belges, 1991, 100, 639-646, and
Ramage, R. WO2007/049057 (May 3, 2007).
Phosphorous oxychloride (5.36 g, 35 mmol, 25 equivalents) was added to 4-nitro-2-(phenylamino)benzoic acid (361 mg, 1.4 mmol). The resultant was stirred at 130° C. for 30 minutes. Thereafter, the resultant was cooled to ambient temperature, and then thereto was added a 28% ammonia aqueous solution until the system became basic. The resultant was subjected to extraction with chloroform. The extract was washed with water two times, washed with saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The crystal was recrystallized with ethyl acetate. Yield: 192 mg, 53%; yellow; m.p.: 213.0-214.2° C. (bibliographic data: 213° C.).
1H NMR (400 MHz, DMSO-d6) δ: 7.35 (t, J=8.0 Hz, 1H), 7.61 (d, J=8.8 Hz, 1H), 7.82 (t, J=8.8 Hz, 1H), 7.96 (d, J=9.6 Hz, 1H), 8.26 (d, J=8.8 Hz, 1H), 8.44 (dd, J=2.8, 8.8 Hz, 2H), 13C NMR (100 MHz, DMSO-d6) δ: 113.34, 114.29, 117.72, 120.98, 122.21, 123.35, 126.09, 128.47, 134.42, 140.56, 141.28, 150.06, 176.20, MS (EI) m/z: 258 (M+H)+
Synthesis of 3-nitro-9-aminoacridine (28):
Reference document: Ramage, R. WO2007/049057 (May 3, 2007).
Phenol (419 mg, 4.45 mmol, 10 equivalents) was added to 3-nitro-9-chloroacridine (115 mg, 0.445 mmol). The resultant was stirred at 70° C. for 1 hour. Thereafter, thereto was added 64 mg (0.668 mmol, 1.5 equivalents) of ammonium carbonate. The temperature of the resultant was raised to 120° C., and then the resultant was stirred for 6 hours. The temperature was returned to ambient temperature, and then thereto was added acetone. The temperature was set to 0° C., and the resultant was stirred for 1 hour. Next, thereto was added 15 mL of a 2.5 M NaOH aqueous solution, and the resultant was stirred for 1 hour. The resultant was subjected to extraction with ethyl acetate. The extract was washed with water two times, washed with saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The crystal was recrystallized with methanol. Yield: 68.3 mg, 64%; red; m.p.: 216.2° C. (decomposed).
1H NMR (400 MHz, DMSO-d6) δ: 7.45 (t, J=8.0 Hz, 1H), 7.76 (t, J=8.0 Hz, 1H), 7.91 (d, J=8.8 Hz, 1H), 7.96 (dd, J=3.2, 8.8 Hz, 1H), 8.18 (broad, 2H), 8.46 (d, J=8.8 Hz, 1H) 8.62-8.66 (m, 2H), 13C NMR (100 MHz, DMSO-d6) δ: 113.54, 113.62, 115.25, 123.35, 124.84, 126.07, 129.03, 131.13, 147.39, 148.23, 150.06, 150.71, MS (EI) m/z: 240 (M+H)+
Synthesis of 7-chloro-N-(4-fluorophenylamino)quinoline (25):
Reference document: Motiwala, F. Australian Journal of Chemistry 2007, 60, 369-374
4,7-dichloroquinoline (1.98 g, 10 mmol) was added to 25 mL of phenol. The resultant was stirred at 120° C., and then the temperature thereof was raised to 170° C.
Thereto was added 4-fluoroaniline (1.67 g, 15 mmol, 1.5 equivalents), and then the resultant was stirred for 12 hours. The resultant was then cooled to ambient temperature, and thereto was added 30 mL of acetone. The temperature of the system was set to 0° C., and then the resultant was stirred for 1 hour. The precipitated crystal was suction-filtered while washed with acetone. Next, the filtrate was added to 100 mL of a 10% NaOH aqueous solution, and the resultant was stirred for 1 hour. The resultant was subjected to extraction with chloroform. The extract was washed with water two times, washed with saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The crystal was recrystallized with acetonitrile. Yield: 1.35 g, 50%; granular purple crystal.
1H NMR (400 MHz, DMSO-d6) δ: 6.78 (d, J=5.2 Hz, 1H), 7.28 (t, J=8.8 Hz, 2H), 7.39 (dd, J=5.2, 8.8 Hz, 2H), 7.58 (dd, J=2.0, 9.6 Hz, 1H), 7.90 (d, J=2.0 Hz, 1H), 8.40-8.46 (m, 2H), 9.08 (broad, 1H), 13C NMR (100 MHz, DMSO-d6) δ: 101.24, 116.13 (d, J=22 Hz), 118.04, 124.59 (d, J=59 Hz), 125.27 (d, J=8.2 Hz), 127.66, 133.86, 136.27, 148.39, 148.50, 151.96, 158.89 (d, J=242 Hz), 168.25, MS (ESI) m/z: 273 (M+H)+
Synthesis of N-(4-fluorophenyl)quinoline-4-amine (26):
7-chloro-N-(4-fluorophenyl)quinoline-4-amine (1.29 g, 4.74 mmol) was dissolved in 30 mL of ethyl acetate. Thereto was added Pd/C (10%, 65.6 mg, 0.0616 mmol, 0.013 equivalents). Hydrogen was added to the resultant while the system was bubbled therewith at ambient temperature under normal pressure. The resultant was stirred for 10 hours. Thereafter, the resultant was filtered through celite, and concentrated. Next, thereto was added 100 mL of a 10% NaOH aqueous solution. The resultant was stirred for 1 hour, and subjected to extraction with chloroform. The extract was washed with water two times, washed with saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The crystal was recrystallized with acetonitrile. Yield: 662 mg, 59%; granular colorless crystal.
1H NMR (400 MHz, DMSO-d6) δ: 6.79 (d, J=4.8 Hz, 1H), 7.27 (t, J=8.8 Hz, 2H)7.40 (dd, J=4.8, 8.8 Hz, 2H), 7.53 (t, J=8.0 Hz, 2H), 7.70 (t, J=8.0 Hz, 1H), 7.88 (d, J=8.0 Hz, 1H), 8.37 (d, J=8.8 Hz, 2H), 8.44 (d, J=6.0 Hz, 1H), 8.93 ppm (broad, 1H), 13C NMR (100 MHz, DMSO-d6) δ: 100.94, 115.95, 116.17, 119.48, 121.95, 124.56, 125.00 (d, J=8.2 Hz), 129.17, 136.72, 148.09, 148.84, 150.66, 158.69 (d, J=241 Hz), MS (ESI) m/z: 239 (M+H)+
Synthesis of 4-aminoquinoline (27):
Reference document: Souza, M. Bioorganic and Medicinal Chemistry, 2009, 17, 1474-1480.
4,7-dichloroquinoline (198 mg, 1 mmol) was dissolved in phenol (1.8 mL, 20 mmol, 20 equivalents). The resultant was stirred at 70° C. for 1 hour. Thereto was then added ammonium carbonate (144 mg, 1.5 mmol, 1.5 equivalents), and then the resultant was stirred at 120° C. for 1.5 hours. The resultant was then cooled to ambient temperature, and thereto was added acetone. The temperature of the system was set to 0° C., and then the resultant was stirred for 1 hour. The precipitated crystal was suction-filtered while washed with acetone. Next, the filtrate was added to 10 mL of a 10% NaOH aqueous solution, and the resultant was stirred for 1 hour. The resultant was subjected to extraction with chloroform. The extract was washed with water two times, washed with saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The crystal was purified through a silica gel column (silica gel: 30 g) with the following developing solvent: 5-20% methanol/chloroform. Yield: 103 mg, 58%; dark red.
Next, 4-amino-7-chloroquinoline (103 mg, 0.577 mmol) was dissolved in 10 mL of methanol. Thereto was added Pd/C (10%, 8.16 mg, 0.0008 mmol, 0.013 equivalents). Hydrogen was added to the resultant while the system was bubbled therewith at ambient temperature under normal pressure. The resultant was stirred for 3 hours. Thereafter, the resultant was filtered through celite, and concentrated. Next, thereto was added 10 mL of a 10% NaOH aqueous solution. The resultant was stirred for 1 hour, and subjected to extraction with chloroform. The extract was washed with water two times, washed with saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The crystal was recrystallized with acetonitrile. Yield: 42 mg, 51%; granular colorless crystal.
1H NMR (400 MHz, DMSO-d6) δ: 6.85 (d, J=6.8 Hz, 1H), 7.68 (t, J=6.8 Hz, 1H), 7.93-8.02 (m, 2H), 8.41 (d, J=6.8 Hz, 1H), 8.52 (d, J=8.0 Hz, 1H), 9.14 (broad, 2H), 13C NMR (100 MHz, DMSO-d6) δ: 101.72, 115.95, 119.81, 123.82, 126.14, 133.76, 138.32, 141.34, 158.26, MS (ESI) m/z: 145 (M+H)+
Synthesis of 3-methoxy-9-aminoacridine (29):
Phosphorous oxychloride (8.09 g, 52.8 mmol, 22 equivalents) was added to 4-methoxy-2-(phenylamino)benzoic acid (584 mg, 2.4 mmol). The resultant was stirred at 130° C. for 30 minutes. Thereafter, the resultant was cooled to ambient temperature, and then thereto was added a 28% ammonia aqueous solution until the system became basic. The resultant was subjected to extraction with chloroform. The extract was washed with water two times, washed with saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The resultant crude crystal was recrystallized with methanol. Yield: 391 mg, 67%; light yellow; m.p.: 169.4-169.5° C.
1H NMR (400 MHz, CDCl3) δ: 4.01 (s, 3H), 7.30 (d, J=10 Hz, 1H), 7.43 (s, 1H), 7.58 (t, J=8.8 Hz, 1H), 7.79 (t, J=8.0 Hz, 1H), 8,14 (d, J=8.8 Hz, 1H), 831 (d, J=9.6 Hz, 1H), 8.39 (d, J=8.8 Hz, 1H), 13C NMR (100 MHz, CDCl3) δ: 55.65, 105.24, 120.39, 122.24, 123.13, 124.63, 125.74, 125.80, 128.99, 130.49, 140.88, 149.11, 150.66, 161.50, MS (EI) m/z: 243 (M+H)+
Synthesis of 3-methoxy-9-aminoacridine (31):
Phenol (1.02 g, 10.8 mmol, 10 equivalents) was added to 3-methoxy-9-chloroacridine (262 mg, 1.08 mmol). The resultant was stirred at 70° C. for 1 hour. Thereafter, thereto was added ammonium carbonate (207 mg, 2.16 mmol, 1.5 equivalents). The temperature of the resultant was raised to 120° C., and then the resultant was stirred for 3 hours. The temperature was returned to ambient temperature, and then thereto was added acetone. The temperature was set to 0° C., and the resultant was stirred for 1 hour. Next, thereto was added 15 mL of 2.5 M NaOH, and the resultant was stirred for 1 hour. The resultant was subjected to extraction with ethyl acetate. The extract was washed with water two times, washed with saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The crystal was recrystallized with methanol. Yield: 194 mg, 80%; granular yellow crystal; m.p.: 196.9-199.8° C.
1H NMR (400 MHz, DMSO-d6) δ: 3.92 (s, 3H), 6.99 (d, J=8.8 Hz, 1H), 7.17 (s, 1H), 7.27 (t, J=7.6 Hz, 1H), 7.62 (t, J=8.8 Hz, 1H), 7.69 (broad, 2H), 7.77 (d, J=8.8 Hz, 1H), 8.31-8.37 ppm (m, 2H), 13C NMR (100 MHz, DMSO-d6) δ: 55.17, 105.45, 108.16, 112.78, 115.41, 120.87, 123.27, 124.81, 128.17, 129.77, 149.19, 149.82, 150.86, 160.62 ppm, MS (ESI) m/z: 225 (M+H)+
Synthesis of 3-phenyl-9-chloroacridine (34):
Phosphorous oxychloride (11.5 g, 75 mmol, 25 equivalents) was added to 2-([1,1′-biphenyl]-3-yl-amino)benzoic acid (868 mg, 3 mmol). The temperature of the resultant was set to 130° C., and the resultant was stirred at 130° C. for 30 minutes. Thereafter, the resultant was cooled to ambient temperature, and then thereto was added a 28% ammonia aqueous solution until the system became basic. The resultant was subjected to extraction with chloroform. The extract was washed with water two times, washed with saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The crystal was recrystallized with acetonitrile. Yield: 593 mg, 68%; light yellow needles; m.p.: 104.0-107.9° C.
1H NMR (400 MHz, DMSO-d6) δ: 7.36-7.38 (m, 2H), 7.54 (t, J=6.8 Hz, 2H), 7.57-7.66 (m, 2H), 7.75-7.85 (m, 5H), 7.95 (dd, J=2.0, 8.8 Hz, 1H), 13C NMR (100 MHz, DMSO-d6) δ: 124.70, 125.00, 125.23, 126.82, 126.85, 126.97, 127.02, 127.45, 127.69, 128.37, 129.12, 129.45, 129.55, 129.78, 130.16, 130.61, 130.67, MS (ESI) m/z: 290 (M+H)+
Synthesis of 3-phenyl-9-aminoacridine (35):
Phenol (941 mg, 10 mmol, 10 equivalents) was added to 3-phenyl-9-chloroacridine (290 mg, 1.00 mmol). The resultant was stirred at 70° C. for 1 hour. Thereafter, thereto was added ammonium carbonate (192 mg, 2.00 mmol, 2 equivalents), and then the temperature of the resultant was raised to 120° C. The resultant was then stirred for 3 hours. The temperature was returned to ambient temperature, and then thereto was added acetone. The temperature was set to 0° C., and the resultant was stirred for 1 hour. Next, thereto was added 10 mL of 2.5 M NaOH, and the resultant was stirred for 1 hour and then subjected to extraction with ethyl acetate. The extract was washed with water two times, washed with saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The crystal was recrystallized with acetonitrile. Yield: 198 mg, 73%; granular yellow crystal.
1H NMR (400 MHz, DMSO-d6) δ: 7.53-7.62 (m, 4H), 7.86-8.02 (m, 5H), 8.21 (s, 1H), 8.75 (d, J=8.8 Hz, 1H), 8.83 (d, J=8.8 Hz, 1H), 10.13 (broad, 2H), 13C NMR (100 MHz, DMSO-d6) δ: 110.68, 111.70, 115.44, 118.71, 122.81, 123.77, 124.88, 125.78, 127.24, 129.35, 135.48, 137.89, 139.58, 139.88, 146.44, 157.42, MS (ESI) m/z: 271 (M+H)+
Synthesis of 4-(N-p-fluorobenzyl)amino-7-chloroquinoline (41):
4,7-Dichloroquinoline (1.98 g, 10 mmol) was added to 25 mL of phenol. The resultant was stirred at 120° C., and then the temperature was raised to 160° C. Thereto was then added p-fluorobenzylamine (1.61 g, 15 mmol, 1.5 equivalents). The resultant was stirred for 6 hours, and the temperature was returned to ambient temperature. Thereto was added 30 mL of acetone. The temperature of the system was set to 0° C., and then the resultant was stirred for 1 hour. The precipitated crystal was suction-filtered while washed with acetone. The resultant crystal was added to 100 mL of a 10% NaOH aqueous solution, and the resultant was stirred for 1 hour. The resultant was subjected to extraction with chloroform. The extract was washed with water two times, washed with saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The crystal was recrystallized with acetonitrile. Yield: 1.52 g, 53%; colorless needles; m.p.: 194.9-196.0° C.
IR (KBr) 3217 (NH) cm−1; 1H NMR (400 MHz, DMSO-d6) δ: 4.52 (d, J=5.9 Hz, 2H), 6.34 (d, J=5.9 Hz, 1H), 7.14 (t, J=8.8 Hz), 7.41-7.49 (m, 3H), 7.79 (s, 1H), 8.03 (s, 1H), 8.31-8.33 (m, 2H), 13C NMR (100 MHz, DMSO-d6) δ: 47.79, 99.41, 115.06, 115.28, 117.51, 123.97, 124.30, 127.57, 128.83, 128.92, 133.45, 134.69, 134.71, 149.04, 149.78, 151.79, 159.99, 162.43; MS (EI) m/z: 286 (M+H)+
Synthesis of 4-(N-p-fluorobenzylamino)quinoline (42):
4-(N-9-fluorobenzyl)amino-7-chloroquinoline (41) (287 mg, 1 mmol) was dissolved in 20 mL of methanol. Thereto was added Pd/C (10%, 11 mg, 0.01 mmol, 0.01 equivalents). Hydrogen was added to the resultant while the system was bubbled therewith at ambient temperature under normal pressure. The resultant was stirred for 4 hours. Thereafter, the resultant was filtered through celite, and concentrated. Next, thereto was added 20 mL of a 10% NaOH aqueous solution. The resultant was stirred for 1 hour, and subjected to extraction with chloroform. The extract was washed with water two times, washed with saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The crystal was recrystallized with acetonitrile. Yield: 179 mg, 71%; colorless needles; m.p.: 180.3-181.7° C.
IR (KBr) 3221 (NH) cm−1; 1H NMR (400 MHz, DMSO-d6) δ: 4.53 (d, J=5.8 Hz, 2H), 6.32 (d, J=4.9 Hz, 1H), 7.14 (t, J=8.8Hz), 7.40-7.46 (m, 3H), 7.61 (t, J=8.8 Hz, 1H), 7.78 (d, J=7.8 Hz, 1H), 7.89 (s, 1H), 8.26-8.31 (m, 2H), 13C NMR (100 MHz, DMSO-d6) δ: 44.79, 98.95, 115.03, 115.24, 118.90, 121.57, 124.00, 128.75, 128.83, 129.09, 134.97, 135.01, 148.28, 149.57, 150.54, 159.95, 162.38; MS (EI) m/z: 252 (M+H)+
Six samples were prepared, in each of which plural anionic compounds were mixed with each other (Tables 1 to 6 shown below). Each of these mixtures was subjected to MALDI mass spectrometry in a negative ion mode to evaluate an effect of each of the matrices that was produced on the ability of ionizing each of the anionic compounds and on the peak strength thereof. Each of the mixtures was mixed with the matrix at a ratio selected at will. The mixture was naturally dried on a stainless steel plate for MALDI. This sample was measured using a MALDI mass spectrometer (MALDI-TOF-MS: AXIMA, Performance, manufactured by Shimadzu Corp.).
By contrast,
Table 2 shown below about the composition thereof) in a negative ion mode. Observations are made of respective remarkable peaks of nicotinic acid, adipic acid, quinolinic acid, azelaic acid, and sebacic acid.
By contrast,
About the six anionic compound mixtures in Tables 1 to 6 shown above, various matrices were each used to make MALDI mass spectrometry in a negative ion mode. The results were compared with a result of a case where 9-AA was used as a matrix to make the same measurement, so that the order of some compounds was prepared in such a manner that about the compounds, the respective peak strength ratios between the case of using each of the matrices and that of using 9-AA were successively lined up from the largest value toward the smallest value. The orders obtained from the measurement results about the mixtures shown in Tables 1 and 2 shown above are shown in Tables 7 and 8, respectively. Each number described in each of the tables represents a matrix compound used for the measurement, and refers to the number of one of the matrix compounds in the present specification.
In the case of using 9-AA, which is a matrix according to the prior art, many compounds cannot be detected. By contrast, it has been demonstrated that the matrix compounds created in the invention cause most of the anionic compounds to be efficiently ionized, so that these compounds can be detected in a wide range with a high sensitivity. From these results, it has been understood that the compounds 5, 17, 18, 21, 24, 30, 35 and 36 have a particularly high ionizing ability. It has been verified that, in particular, the compounds 17, 18 and 36 have a remarkable ionizing ability for many materials to be analyzed.
cis-Cinnamic acid, which is a substance acting on plants, and analogues thereof (see Table 9) were subjected to MALDI mass spectrometry in a negative ion mode to evaluate an effect of each of the matrix compounds 37 to 42 that was produced on the ability of ionizing each of the anionic compounds and on the peak strength thereof. Each of the carboxylic acids was mixed with the matrix at a ratio selected at will. Thereafter, the mixture was naturally dried on a stainless steel plate for MALDI. This sample was measured using a MALDI mass spectrometer (MALDI-TOF-MS: AXIMA, Performance, manufactured by Shimadzu Corp.).
In MALDI mass spectrometry measurement of low-molecular-weight biological components, 2,5-dihydroxybenzoic acid (DHB) is frequently used as a matrix. However, it is not said that the compound is high in ionizing ability. Thus, many molecules are not detected therewith. In recent years, it has been shown that when 9-aminoacridine (9-AA) is used as a matrix in negative-ion-mode measurement, various low-molecular-weight biological components can be analyzed with a relatively high sensitivity (see, for example, Non-Patent Document 1). However, according to 9-AA, many compounds still cannot be measured. Thus, it has been desired to develop a higher-performance matrix for metabolome analysis, for which a rapid and high-sensitivity analysis is required. According to the present invention, the detection of low-molecular-weight compounds originating from living bodies, which have not been easily detected in MALDI mass spectrometry, has been successfully achieved by synthesizing 9-aminoanthracene and derivatives thereof, 9-aminoquinoline and derivatives thereof, and 9-aminoacridine derivatives, which show a higher ionizing ability and sensitivity than 9-aminoacridine. Moreover, the selection of a matrix suitable for a biological component as a target makes it possible to avoid the disturbance of peak detection that is based on peaks of ions of the matrix itself. The present invention is particularly useful for the detection or bio-imaging of a specific minor biological component.
Results obtained so far have suggested that an amino group on a condensed polycyclic aromatic ring, or a condensed polycyclic hetero-ring or aromatic ring is desired for a requirement of a matrix. The condensed polycyclic aromatic ring is desirably, for example, anthracene or phenanthrene. The condensed polycyclic hetero-ring is desirably acridine or quinoline. By changing a substituent on the aromatic ring or a substituent on the amino group, the ionizing ability or the sensitivity can be adjusted. The amino group is desirably a primary or secondary amino group. The substituent on the amino group is desirably an allyl, aryl, benzyl or alkyl group. A salt (such as hydrochloride) of such an amine is also usable. The substituent on the condensed aromatic ring that is different from any amino group may be an alkoxyl, amino, aryl, allyl or nitro group. However, the substituent is not limited thereto. Any one of these compounds is commercially available, or can easily be synthesized through several steps from a commercially available material.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2011-152328 | Jul 2011 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2012/067227 | 7/5/2012 | WO | 00 | 2/14/2014 |
