THIOCHROMENE TYPE COMPOUND AND USE THEREOF

Abstract

This invention relates to a photocleavable mass tag and the use thereof, and particularly to a thiochromene-type compound useful for MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight) mass spectrometry or matrix-less LDI-TOF (Laser Desorption/Ionization Time-Of-Flight) mass spectrometry and the use thereof.

Claims

1. A compound represented by Formula I below: ##STR00030## In Formula I, R.sub.1 is a C1-12 alkyl group, a C6-60 aryl group, or a C2-60 heteroaryl group containing at least one heteroatom selected from among N, S and O, having a reactive group at a terminal thereof, the reactive group being any one of a N-hydroxysuccinimide ester group, a N-hydroxysulfosuccinimide ester group, a pentafluorophenyl ester group, a 4-sulfo-2,3,5,6-tetrafluorophenyl ester group, a nitrophenyl ester group, a 2,4,5-trichlorophenyl ester group, a phthalimido ester group, a N-hydroxy-5-norbornen-endo-2,3-dicarboimide ester group, and a maleimide group, R.sub.2 and R.sub.3 are independently hydrogen, a C1-12 alkyl, a C6-60 aryl, a C1-12 alkoxy, a C1-12 alkylamino, a C1-12 alkylthio or a fused ring, the fused ring being any one selected from among pyrrole, thiophene, indole, furan, imidazole, triazole, diazole, and pyrimidine, and Ar is benzene or a heteroaromatic ring selected from among pyrrole, thiophene, indole, furan, imidazole, triazole, diazole, and pyrimidine).

2. A compound represented by Formula II below: ##STR00031## in Formula II, R.sub.1 is a C1-12 alkyl group, a C6-60 aryl group, or a C2-60 heteroaryl group containing at least one heteroatom selected from among N, S and O, having a reactive group at a terminal thereof, the reactive group being any one of a N-hydroxysuccinimide ester group, a N-hydroxysulfosuccinimide ester group, a pentafluorophenyl ester group, a 4-sulfo-2,3,5,6-tetrafluorophenyl ester group, a nitrophenyl ester group, a 2,4,5-trichlorophenyl ester group, a phthalimido ester group, a N-hydroxy-5-norbornen-endo-2,3-dicarboimide ester group, and a maleimide group, and R.sub.2 and R.sub.3 are independently hydrogen, a C1-12 alkyl, a C6-60 aryl, a C1-12 alkoxy, a C1-12 alkylamino, a C1-12 alkylthio or a fused ring, the fused ring being any one selected from among pyrrole, thiophene, indole, furan, imidazole, triazole, diazole, and pyrimidine).

3. A compound represented by Formula III below: ##STR00032## in Formula III, R.sub.2 and R.sub.3 are independently hydrogen, a C1-12 alkyl, a C6-60 aryl, a C1-12 alkoxy, a C1-12 alkylamino, a C1-12 alkylthio or a fused ring, C(O)R.sub.4 is N-hydroxysuccinimidyl, N-hydroxy sulfosuccinimidyl, pentafluorophenyloxy, 4-sulfo-2,3,5,6-tetrafluorophenyloxy, nitrophenyloxy, 2,4,5-trichlorophenyloxy, phthalimidoyloxy, N-hydroxy-5-norbomen-endo-2,3-dicarboimidyloxy, or maleimide, and n is an integer of 1-12, the fused ring being any one selected from among pyrrole, thiophene, indole, furan, imidazole, triazole, diazole, and pyrimidine).

4. A compound represented by Formula IV below: ##STR00033## in Formula IV, R.sub.1 is a C1-12 alkyl group, a C6-60 aryl group, or a C2-60 heteroaryl group containing at least one heteroatom selected from among N, S and O, having a reactive group at a terminal thereof, the reactive group being any one of a N-hydroxysuccinimide ester group, a N-hydroxysulfosuccinimide ester group, a pentafluorophenyl ester group, a 4-sulfo-2,3,5,6-tetrafluorophenyl ester group, a nitrophenyl ester group, a 2,4,5-trichlorophenyl ester group, a phthalimido ester group, a N-hydroxy-5-norbornen-endo-2,3-dicarboimide ester group, and a maleimide group, and R.sub.2 and R.sub.3 are independently hydrogen, a C1-12 alkyl, a C6-60 aryl, a C1-12 alkoxy, a C1-12 alkylamino, a C1-12 alkylthio or a fused ring, the fused ring being any one selected from among pyrrole, thiophene, indole, furan, imidazole, triazole, diazole, and pyrimidine).

5. A compound represented by Formula V below: ##STR00034## in Formula V, A is a C1-12 alkyl group, a C6-60 aryl group, or a C2-60 heteroaryl group containing at least one heteroatom selected from among N, S and O, having a reactive group at a terminal thereof, the reactive group being any one of a N-hydroxysuccinimide ester group, a N-hydroxysulfosuccinimide ester group, a pentafluorophenyl ester group, a 4-sulfo-2,3,5,6-tetrafluorophenyl ester group, a nitrophenyl ester group, a 2,4,5-trichlorophenyl ester group, a phthalimido ester group, a N-hydroxy-5-norbornen-endo-2,3-dicarboimide ester group, and a maleimide group, R.sub.2 and R.sub.3 are independently hydrogen, a C1-12 alkyl, a C6-60 aryl, a C1-12 alkoxy, a C1-12 alkylamino, a C1-12 alkylthio or a fused ring, the fused ring being any one selected from among pyrrole, thiophene, indole, furan, imidazole, triazole, diazole, and pyrimidine, and Ar is benzene or a heteroaromatic ring selected from among pyrrole, thiophene, indole, furan, imidazole, triazole, diazole, and pyrimidine).

6. The compound of any one of claims 1 to 5, which is used for MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight) mass spectrometry or matrix-less LDI-TOF (Laser Desorption/Ionization Time-Of-Flight) mass spectrometry.

7. A conjugate compound of a biomaterial and a compound represented by Formula Ia below: ##STR00035## in Formula Ia, A is a C1-12 alkyl group, a C6-60 aryl group, or a C2-60 heteroaryl group containing at least one heteroatom selected from among N, S and O, having a reactive group at a terminal thereof, the reactive group being any one of a N-hydroxysuccinimide ester group, a N-hydroxysulfosuccinimide ester group, a pentafluorophenyl ester group, a 4-sulfo-2,3,5,6-tetrafluorophenyl ester group, a nitrophenyl ester group, a 2,4,5-trichlorophenyl ester group, a phthalimido ester group, a N-hydroxy-5-norbornen-endo-2,3-dicarboimide ester group, and a maleimide group, R.sub.2 and R.sub.3 are independently hydrogen, a C1-12 alkyl, a C6-60 aryl, a C1-12 alkoxy, a C1-12 alkylamino, a C1-12 alkylthio or a fused ring, the fused ring being any one selected from among pyrrole, thiophene, indole, furan, imidazole, triazole, diazole, and pyrimidine, and Ar is benzene or a heteroaromatic ring selected from among pyrrole, thiophene, indole, imidazole, triazole, and pyrimidine) wherein the biomaterial is an antigen, an antibody, a biomarker, a peptide, a nucleic acid, or a glycan.

8. The conjugate compound of claim 7, wherein the antibody is a monoclonal antibody.

9. The conjugate compound of claim 7 or 8, which is used for MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight) mass spectrometry or matrix-less LDI-TOF (Laser Desorption/Ionization Time-Of-Flight) mass spectrometry.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0038] FIG. 1 shows a conventional ferrocene-based mass tag;

[0039] FIG. 2 shows the mass spectrometry spectrum results of ferrocene-based novel mass tags having different masses under matrix-less laser desorption/ionization conditions;

[0040] FIG. 3 shows the concept of a photocleavable mass tag;

[0041] FIG. 4 shows the scheme of a 2-alkylthio-2H-thiochromene derivative-based photocleavable mass tag;

[0042] FIG. 5 shows the UV-Vis absorption spectrum results of compounds 1, 2 and 3a;

[0043] FIG. 6 shows the mass spectrometry spectrum results of compound 3a obtained through matrix-less LDI-TOF MS;

[0044] FIG. 7 shows the matrix-less LDI MS spectrum results of mass tags 3a-3d;

[0045] FIG. 8 shows the comparison results of matrix-less LDI MS sensitivity of compound 3a, which is the mass tag of the present invention, and a conventionally known trityl-SPh derivative;

[0046] FIG. 9 shows the comparison results of matrix-less LDI MS sensitivity of compound 3a, which is the mass tag of the present invention, and a ferrocene-based mass tag disclosed in the related art; and

[0047] FIG. 10 shows the test data for detection limit of the mass tag 3d of the present invention upon matrix-less LDI MS.

DETAILED DESCRIPTION OF THE INVENTION

Example 1: Synthesis of Cleavable Mass Tag (I)

[0048] Mass tags 3a-3d were synthesized from thiochroman-4-one and phenyl hydrazine in accordance with the synthesis route in Synthesis Method 1.

(A) Synthesis of 6,11-dihydrothiochromeno[4,3-b]indole (4)

[0049] ##STR00007##

[0050] Thiochroman-4-one (11.4 g, 69.2 mmol) and phenyl hydrazine (7.49 g, 6.82 mL, 69.2 mmol) were dissolved in ethanol (100 mL) in a reactor, rapidly added with trimethylsilyl chloride (7.52 g, 8.79 mL, 69.2 mmol), and the reactor was then capped. The resulting reaction mixture was heated to reflux for 4 hr and then cooled. A saturated sodium bicarbonate aqueous solution was added until the solution became sufficiently basic, followed by dilution with ethyl acetate (150 mL). The organic layer was separated and the remaining water layer was extracted three times with ethyl acetate (250 mL3). The combined organic layer was washed with a saturated sodium chloride aqueous solution, added with anhydrous sodium sulfate and filtered under reduced pressure. The filtered solvent was completely removed under reduced pressure, and the remaining solid was dispersed in diethyl ether and ethyl acetate at 8:1 (v/v) and then filtered, thus yielding compound 4 as a yellow liquid. (11.8 g, 49.7 mmol, 72%): mp=162-165 C.; R.sub.f=0.33 (EtOAc:hexane=1:4); .sup.1H NMR (400 MHz, DMSO-d.sub.6): 11.58 (s, 1H), 7.71 (d, J=7.2 Hz, 1H), 7.53 (d, J=8.0 Hz, 1H), 7.39 (d, J=8.0 Hz, 1H), 7.34 (d, J=7.6 Hz, 1H), 7.24 (dd, J=8.0, 7.2 Hz, 1H), 7.13-7.17 (m, 2H), 7.04 (dd, J=7.2, 7.0 Hz, 1H), 4.28 (s, 2H); .sup.13C NMR (100 MHz, DMSO-d.sub.6): 136.7, 132.4, 131.9, 127.5, 127.4, 127.2, 125.9, 125.8, 122.9, 122.4, 119.4, 118.5, 111.4, 105.9, 22.7; IR (ZnSe-ATR) 3336 (w), 1957 (w), 1915 (w), 1871 (w), 1785 (w), 1453 (w), 1440 (w), 1416 (w), 1312 (w), 1276 (w), 1177 (w), 1006 (w), 1036 (w), 918 (w), 864 (w), 761 (m), 733 (vs), 670 (w) cm.sup.1; Anal. Calcd for C.sub.15H.sub.11NS: C, 75.92; H, 4.67; N, 5.90; S, 13.51. Found: C, 75.92; H, 4.62; N, 5.91; S, 13.63.

(B) Synthesis of 11-alkyl-6,11-dihydrothiochromeno[4,3-b]indole (5a-5d)

[0051] ##STR00008##

[0052] In a nitrogen atmosphere, 6,11-dihydrothiochromeno[4,3-b]indole (4, 1.50 g, 6.32 mmol, 1.0 equiv) and sodium hydride (60%, dispersion in mineral oil, 0.556 g, 12.6 mmol, 2.0 equiv) were mixed together and anhydrous dimethylformamide (10 mL) was slowly added thereto at 0 C. The resulting reaction mixture was stirred at 0 C. for 30 min, rapidly added with alkyl halide (12.6 mmol, 2.0 equiv) in a nitrogen atmosphere, stirred at room temperature for 2 hr, and then sequentially added with water (10 mL) and ethyl acetate (30 mL). The organic layer was separated and the remaining water layer was extracted three times with ethyl acetate (40 mL3). The combined organic layer was washed with a saturated sodium chloride aqueous solution, added with anhydrous sodium sulfate and filtered under reduced pressure. The solvent of the filtrate was completely removed under reduced pressure, followed by purification through column chromatography (hexane:ethyl acetate=15:1), thereby yielding compound 5 as a yellow liquid.

##STR00009##

[0053] 11-Methyl-6,11-dihydrothiochromeno[4,3-b]indole (5a): 90%; mp=94-97 C; R.sub.f=0.32 (EtOAc:hexane=1:15); .sup.1HNMR (400 MHz, DMSO-d.sub.6): 7.83 (d, J=7.6 Hz, 1H), 7.62 (d, J=8.0 Hz, 1H), 7.50-7.54 (m, 2H), 7.33 (dd, J=7.6, 6.8 Hz, 1H), 7.21-7.25 (m, 2H), 7.11 (d, J=7.6, 7.2 Hz, 1H), 4.15 (s, 2H), 3.95 (s, 3H); .sup.13C NMR (100 MHz, DMSO-d.sub.6): 138.5, 134.5, 133.7, 128.6, 127.8, 127.1, 126.2, 125.0, 124.1, 122.5, 119.8, 118.7, 110.3, 109.7, 32.8, 22.8; IR (ZnSe-ATR) 2887 (w), 1908 (w), 1875 (w), 1834 (w), 1470 (w), 1426 (w), 1360 (w), 1275 (w), 1235 (w), 1219 (w), 1121 (w), 1164 (w), 1082 (w), 1046 (w), 943 (w), 821 (w), 737 (vs), 714 (m), 675 (w) cm.sup.1; Anal. Calcd for C.sub.16H.sub.13NS: C, 76.46; H, 5.21; N, 5.57; S, 12.76. Found: C, 76.49; H, 5.22; N, 5.43; S, 12.64.

##STR00010##

[0054] 11-Ethyl-6,11-dihydrothiochromeno[4,3-b]indole (5b): 70%; mp=122-126 C; R.sub.f=0.36 (EtOAc:hexane=1:15); .sup.1H NMR (400 MHz, DMSO-d.sub.6): 7.68 (d, J=7.6 Hz, 1H), 7.63 (d, J=8.4 Hz, 1H), 7.51-7.56 (m, 2H), 7.35 (dd, J=7.6, 7.2 Hz, 1H), 7.21-7.26 (m, 2H), 7.12 (dd, J=7.6, 7.2 Hz, 1H), 4.40 (q, J=7.0 Hz, 2H), 4.13 (s, 2H), 1.39 (t, J=7.0 Hz, 3H); .sup.13C NMR (100 MHz, DMSO-d.sub.6): 137.7, 133.9, 133.7, 128.7, 127.7, 127.0, 126.4, 124.4, 124.3, 122.6, 119.8, 118.8, 118.7, 110.2, 22.7, 15.5, 15.4; IR (ZnSe-ATR) 2970 (w), 1936 (w), 1847 (w), 1477 (w), 1457 (w), 1363 (w), 1340 (w), 1284 (w), 1208 (w), 1162 (w), 1131 (w), 1086 (w), 1040 (w), 1102 (w), 784 (w), 746 (vs), 712 (w), 670 (w) cm.sup.1; Anal. Calcd for C.sub.17H.sub.15NS: C, 76.94; H, 5.70; N, 5.28; S, 12.08. Found: C, 76.88; H, 5.71; N, 5.29; S, 12.07.

##STR00011##

[0055] 11-Propyl-6,11-dihydrothiochromeno[4,3-b]indole (5c): 94%; mp=75-80 C.; R.sub.f=0.40 (EtOAc:hexane=1:15); .sup.1H NMR (400 MHz, DMSO-d.sub.6): 7.69 (d, J=8.0 Hz, 1H), 7.62 (d, J=7.6 Hz, 1H), 7.56 (d, J=8.4 Hz, 1H), 7.51 (d, J=7.6 Hz, 1H), 7.35 (dd, J=7.6, 7.2 Hz, 1H), 7.20-7.25 (m, 2H), 7.11 (dd, J=8.0, 7.2 Hz, 1H), 4.34 (t, J=7.2 Hz, 2H), 4.13 (s, 2H), 1.73 (tq, J=7.2, 7.2 Hz, 2H), 0.82 (t, J=7.2 Hz, 3H); .sup.13C NMR (100 MHz, DMSO-d.sub.6): 138.2, 133.9, 133.8, 128.8, 128.0, 127.1, 126.4, 124.32, 124.26, 122.6, 119.8, 118.8, 110.6, 110.4, 46.1, 23.3, 22.7, 11.0; IR (ZnSe-ATR) 3056 (w), 2970 (w), 1475 (w), 1460 (w), 1414 (w), 1348 (w), 1204 (w), 1158 (w), 1039 (w), 1012 (w), 754 (m), 738 (vs), 669 (w) cm.sup.1; Anal. Calcd for C.sub.18H.sub.17NS: C, 77.38; H, 6.13; N, 5.01; S, 11.47. Found: C, 77.38; H, 6.17; N, 4.95; S, 11.35.

##STR00012##

[0056] 11-Butyl-6,11-dihydrothiochromeno[4,3-b]indole (5d): 98%; mp=86-90 C; R.sub.f=0.47 (EtOAc:hexane=1:15); .sup.1H NMR (400 MHz, DMSO-d.sub.6): 7.71 (d, J=7.6 Hz, 1H), 7.62 (d, J=8.0 Hz, 1H), 7.55 (d, J=8.4 Hz, 2H), 7.52 (d, J=8.0 Hz, 1H), 7.34 (dd, J=7.6, 7.2 Hz, 1H), 7.20-7.25 (m, 2H), 7.11 (dd, J=7.6, 7.2 Hz, 1H), 4.38 (t, J=7.6 Hz, 2H), 4.12 (s, 2H), 1.68 (tt, J=7.6, 7.6 Hz, 2H), 1.25 (tq, J=7.6, 7.2 Hz, 2H), 0.85 (t, J=7.2 Hz, 3H); .sup.13C NMR (100 MHz, DMSO-d.sub.6): 138.1, 134.0, 133.8, 128.7, 127.9, 127.0, 126.3, 124.4, 124.3, 122.5, 119.8, 118.8, 110.6, 110.5, 44.3, 31.9, 22.7, 19.4, 13.5; IR (ZnSe-ATR) 2962 (w), 2921 (w), 1469 (w), 1454 (w), 1360 (w), 1347 (w), 1196 (w), 1120 (w), 1042 (w), 819 (w), 757 (m), 743 (vs), 681 (w) cm.sup.1; Anal. Calcd for C.sub.19H.sub.19NS: C, 77.77; H, 6.53; N, 4.77; S, 10.93. Found: C, 77.74; H, 6.55; N, 4.81; S, 10.98.

(C) Synthesis of 11-alkyl-6,11-dihydrothiochromeno[4,3-b]indole 5-oxide (6a-6d)

[0057] ##STR00013##

[0058] 11-Alkyl-6,11-dihydrothiochromeno[4,3-b]indole (5a-5d, 1.0 equiv) was dissolved in anhydrous dichloromethane, after which m-chloroperbenzoic acid (69%, 1.1 equiv) was added thereto at 0 C. The resulting reaction mixture was stirred at 0 C for 1 hr and the reaction was terminated with the addition of a 20% sodium thiosulfate aqueous solution. The organic layer was separated and the remaining water layer was extracted three times with ethyl acetate. The combined organic layer was washed with a saturated sodium chloride aqueous solution, added with anhydrous sodium sulfate and filtered under reduced pressure. Thereafter, purification was conducted through the method described below.

##STR00014##

[0059] 11-Methyl-6,11-dihydrothiochromeno[4,3-b]indole 5-oxide (6a): The remaining solid was dispersed in diethyl ether:ethyl acetate at 8:1 (v/v) and then filtered, thus obtaining compound 6a as a yellow liquid. (76%): mp=180-183 C.; R.sub.f=0.29 (EtOAc:hexane=1:1); .sup.1H NMR (400 MHz, DMSO-d.sub.6): 7.89 (d, J=7.6 Hz, 1H), 7.83 (d, J=7.2 Hz, 1H), 7.68-7.71 (m, 2H), 7.58-7.61 (m, 2H), 7.27 (dd, J=7.6, 7.2 Hz, 1H), 7.15 (dd, J=7.6, 7.2 Hz, 1H), 4.55 (d, J=14.2 Hz, 1H), 4.47 (d, J=14.2 Hz, 1H), 4.03 (s, 3H); .sup.13C NMR (100 MHz, DMSO-d.sub.6): 140.3, 138.6, 133.1, 131.5, 128.4, 127.0, 126.4, 125.9, 125.1, 123.2, 120.2, 119.1, 110.6, 100.7, 44.2, 32.3; IR (ZnSe-ATR) 2962 (w), 1581 (w), 1524 (w), 1468 (w), 1423 (w), 1356 (w), 1367 (w), 1261 (w), 1227 (w), 1123 (w), 1070 (m), 1048 (s), 1035 (s), 1025 (m), 1016 (m), 817 (w), 759 (vs), 667 (w) cm.sup.1; HRMS (m/z): [M+Na].sup.+ calcd for C.sub.16H.sub.13NNaOS, 290.0610; found, 290.0610.

##STR00015##

[0060] 11-Ethyl-6,11-dihydrothiochromeno[4,3-b]indole 5-oxide (6b): The remaining solid was dispersed in diethyl ether:ethyl acetate at 8:1 (v/v) and then filtered, thus obtaining compound 6b as a yellow liquid. (79%): mp=210-213 C; R.sub.f=0.33 (EtOAc:hexane=1:1); .sup.1H NMR (400 MHz, DMSO-d.sub.6): 7.83 (d, J=7.6 Hz, 1H), 7.70-7.75 (m, 3H), 7.59-7.62 (m, 2H), 7.27 (dd, J=7.6, 7.2 Hz, 1H), 7.15 (dd, J=7.6, 7.2 Hz, 1H), 4.43-4.57 (m, 4H), 1.41 (t, J=7.0 Hz, 3H); .sup.13C NMR (100 MHz, DMSO-d.sub.6): 140.6, 137.8, 132.3, 131.7, 128.5, 127.0, 126.4, 126.2, 124.4, 123.2, 120.3, 119.2, 110.6, 101.1, 44.2, 15.32, 15.31; IR (ZnSe-ATR) 2974 (w), 1585 (w), 1459 (w), 1339 (w), 1159 (w), 1133 (w), 1075 (w), 1024 (m), 831 (w), 768 (w), 750 (vs), 737 (m), 700 (w), 668 (w) cm.sup.1; HRMS (m/z): [M+Na].sup.+ calcd for C.sub.17H.sub.15NNaOS, 304.0767; found, 304.0768.

##STR00016##

[0061] 11-Propyl-6,11-dihydrothiochromeno[4,3-b]indole 5-oxide (6c): The filtrate was purified through column chromatography (hexane:ethyl acetate=1:1), thus obtaining compound 6c as a yellow liquid. (80%): mp=128-132 C; R.sub.f=0.27 (EtOAc:hexane=1:1); .sup.1H NMR (400 MHz, DMSO-d.sub.6): 7.82 (d, J=8.0 Hz, 1H), 7.69-7.77 (m, 3H), 7.58-7.64 (m, 2H), 7.26 (dd, J=7.6, 7.2 Hz, 1H), 7.15 (dd, J=7.6, 7.2 Hz, 1H), 4.56 (d, J=14.0 Hz, 1H), 4.42-4.46 (m, 3H), 1.77 (tq, J=7.4, 7.0 Hz, 2H), 0.84 (t, J=7.4 Hz, 3H); .sup.13C NMR (100 MHz, DMSO-d.sub.6): 140.6, 138.3, 132.6, 131.6, 128.5, 126.9, 126.6, 126.1, 124.4, 123.2, 120.3, 119.2, 110.9, 101.2, 45.8, 44.1, 23.2, 11.0; IR (ZnSe-ATR) 2958 (w), 1581 (w), 1481 (w), 1455 (w), 1410 (w), 1362 (w), 1209 (w), 1077 (m), 1056 (m), 1036 (m), 1024 (m), 902 (w), 763 (s), 753 (vs), 730 (m) cm.sup.1; HRMS (m/z): [M+Na].sup.+ calcd for C.sub.18H.sub.17NNaOS, 318.0923; found, 318.0925.

##STR00017##

[0062] 11-Butyl-6,11-dihydrothiochromeno[4,3-b]indole 5-oxide (6d): Compound 6d was obtained as a yellow liquid through purification using column chromatography (hexane:ethyl acetate=1:1). (65%): mp=64-68 C; R.sub.f=0.33 (EtOAc:hexane=1:1); .sup.1H NMR (400 MHz, DMSO-d.sub.6): 7.82 (d, J=7.2 Hz, 1H), 7.77 (d, J=7.6 Hz, 1H), 7.69-7.71 (m, 2H), 7.58-7.62 (m, 2H), 7.26 (dd, J=7.8, 7.6 Hz, 1H), 7.15 (dd, J=7.8, 7.6 Hz, 1H), 4.41-4.57 (m, 4H), 1.72 (tt, J=7.2, 7.2 Hz, 2H), 1.27 (tq, J=7.2, 7.2 Hz, 2H), 0.85 (t, J=7.2 Hz, 3H)); .sup.13C NMR (100 MHz, DMSO-d.sub.6): 140.7, 138.2, 132.6, 131.6, 128.5, 126.8, 126.6, 126.1, 124.3, 123.2, 120.3, 119.2, 110.9, 101.2, 44.2, 44.1, 31.8, 19.4, 13.5; IR (ZnSe-ATR) 2950 (w), 1585 (w), 1482 (w), 1456 (w), 1432 (w), 1349 (w), 1266 (w), 1131 (w), 1079 (m), 1052 (m), 1027 (m), 737 (s), 668 (w) cm.sup.1; HRMS (m/z): [M+Na].sup.+ calcd for C.sub.19H.sub.19NNaOS, 332.1080; found, 332.1080.

(D) Synthesis of thiochromeno[4,3-b]indol-5-ium 2,2,2-trifluoroacetate (7a-7d) from Compound 6 (Method A)

[0063] ##STR00018##

[0064] 11-Alkyl-6,11-dihydrothiochromeno[4,3-b]indole 5-oxide (6a-6d, 1.0 equiv) was dispersed in acetonitrile in a nitrogen atmosphere, after which trifluoroacetic anhydride (3.0 equiv) was slowly added thereto at 0 C. The resulting reaction mixture was stirred at room temperature for 5 min, and the remaining reactant and the solvent were dried and removed under reduced pressure. The remaining solid was dispersed in anhydrous diethyl ether, cooled to 0 C., washed with cold diethyl ether, and filtered, thus obtaining sulfonium salts 7a-7d as deep yellow solids. These solids were used for the subsequent reaction without additional purification.

(E) Synthesis of thiochromeno[4,3-b]indol-5-ium 2,2,2-trifluoroacetate (7a-7d) from Compound 5 (Method B)

[0065] ##STR00019##

[0066] In a nitrogen atmosphere, 11-alkyl-6,11-dihydrothiochromeno[4,3-b]indole (5a-5d, 1.0 equiv) and bis(trifluoroacetoxy)iodobenzene (1.1 equiv) were mixed, after which anhydrous acetonitrile was added thereto at 0 C. The resulting reaction mixture was stirred at room temperature for 5 min, and the remaining reactant and the solvent were removed under reduced pressure. The remaining solid was dispersed in anhydrous diethyl ether, cooled to 0 C., washed with cold diethyl ether, and filtered, thus obtaining sulfonium salts 7a-7d as deep yellow solids. These solids were used for the subsequent reaction without additional purification.

(F) Synthesis of 2,5-dioxopyrrolidin-1-yl 3-((11-alkyl-6,11-dihydrothiochromeno[4,3-b]indol-6-yl)thio)propanoate (8a-8d)

[0067] ##STR00020##

[0068] Each of sulfonium salts (7a-7d, 1.0 equiv) was dissolved in acetonitrile, after which 3-mercaptopropionic acid (1.0 equiv) and sodium carbonate (1.0 equiv) were sequentially added thereto. The resulting reaction mixture was stirred at room temperature (for about 20 min) until it was colorless, and was then diluted with ethyl acetate. Water was added until the remaining solid was completely dissolved, after which the organic layer was separated and the remaining water layer was extracted three times with ethyl acetate. The combined organic layer was washed with a saturated ammonium chloride aqueous solution and a saturated sodium chloride aqueous solution in sequence, added with anhydrous sodium sulfate and filtered under reduced pressure, after which the solvent of the filtrate was completely removed under reduced pressure, thus obtaining compounds 8a-8d as yellow solids. These solids were used for the subsequent reaction without additional purification.

##STR00021##

[0069] Each of carboxylic acid compounds 8a-8d (1.0 equiv) and N-hydroxysuccinimide (1.1 equiv) were dissolved in anhydrous dichloromethane in a nitrogen atmosphere. The resulting reaction mixture was added with a solution of N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (3.0 equiv) separately dissolved in anhydrous dichloromethane. The reaction mixture was stirred at room temperature for 4 hr and then diluted with dichloromethane. The resulting solution was washed two times with water, added with anhydrous sodium sulfate and filtered under reduced pressure. The solvent of the filtrate was completely removed under reduced pressure and then purified through column chromatography (hexane:ethyl acetate=1:1), thus obtaining compounds 3a-3d as white solids.

##STR00022##

[0070] 2,5-Dioxopyrrolidin-1-yl 3-((11-methyl-6,11-dihydrothiochromeno[4,3-b]indol-6-yl)thio)propanoate (3a): 31% (yield synthesized from compound 6a using Methods A1 and B); mp=90-96 C. dec; R.sub.f=0.52 (EtOAc:hexane=1:1); .sup.1H NMR (400 MHz, DMSO-d.sub.6): 7.98 (d, J=8.0 Hz, 1H), 7.71 (d, J=8.0 Hz, 1H), 7.59 (d, J=7.6 Hz, 1H), 7.56 (d, J=8.4 Hz, 1H), 7.42 (dd, J=8.0, 7.2 Hz, 1H), 7.33 (dd, J=7.6, 7.2 Hz, 1H), 7.27 (dd, J=8.4, 7.2 Hz, 1H), 7.15 (dd, J=7.6, 7.2 Hz, 1H), 6.23 (s, 1H), 4.00 (s, 3H), 3.10-3.24 (m, 2H), 2.96-3.03 (m, 1H), 2.75-2.82 (m, 5H); .sup.13C NMR (100 MHz, DMSO-d.sub.6): d 170.2, 168.0, 138.2, 134.7, 130.5, 129.7, 127.7, 126.8, 126.7, 125.1, 123.0, 122.7, 120.2, 118.7, 110.7, 110.4, 42.9, 33.0, 31.5, 25.7, 25.5; IR (ZnSe-ATR) 1811 (w), 1782 (w), 1732 (s), 1470 (w), 1426 (w), 1361 (w), 1201 (m), 1064 (m), 824 (w), 743 (s), 668 (w) cm.sup.1; Anal. Calcd for C.sub.23H.sub.20N.sub.2O.sub.4S.sub.2: C, 61.04; H, 4.45; N, 6.19; S, 14.17. Found: C, 61.14; H, 4.48; N, 6.10 S, 14.04.

##STR00023##

[0071] 2,5-Dioxopyrrolidin-1-yl 3-((11-ethyl-6,11-dihydrothiochromeno[4,3-b]indol-6-yl)thio)propanoate (3b): 30% (yield synthesized from compound 6b using Methods A1 and B); mp=82-87 C. dec; R.sub.f=0.32 (EtOAc:hexane=1:1); .sup.1H NMR (400 MHz, DMSO-d.sub.6): 7.81 (d, J=8.0 Hz, 1H), 7.71 (d, J=8.0 Hz, 1H), 7.60 (d, J=7.2 Hz, 1H), 7.58 (d, J=8.0 Hz, 1H), 7.44 (dd, J=7.6, 7.6 Hz, 1H), 7.33 (dd, J=7.6, 7.6 Hz, 1H), 7.27 (dd, J=7.6, 7.6 Hz, 1H), 7.16 (dd, J=7.6, 7.2 Hz, 1H), 6.21 (s, 1H), 4.44 (q, J=7.0 Hz, 2H), 3.08-3.23 (m, 2H), 2.96-3.03 (m, 1H), 2.75-2.82 (m, 5H), 1.42 (t, J=7.0 Hz, 3H); .sup.13C NMR (100 MHz, DMSO-d.sub.6): 170.1, 167.9, 137.5, 133.9, 130.6, 129.9, 127.6, 126.9, 126.7, 124.4, 123.1, 123.0, 120.3, 118.8, 111.2, 110.4, 42.8, 31.5, 25.7, 25.5, 15.4; IR (ZnSe-ATR) 1810 (w), 1781 (w), 1733 (s), 1459 (w), 1426 (w), 1345 (w), 1203 (m), 1066 (m), 1044 (m), 991 (w), 813 (w), 745 (vs) cm.sup.1; Anal. Calcd for C.sub.24H.sub.22N.sub.2O.sub.4S.sub.2: C, 61.78; H, 4.75; N, 6.00; S, 13.74. Found: C, 61.83; H, 4.79; N, 5.97 S, 13.91.

##STR00024##

[0072] 2,5-Dioxopyrrolidin-1-yl 3-((11-propyl-6,11-dihydrothiochromeno[4,3-b]indol-6-yl)thio)propanoate (3c): 25% (yield synthesized from compound 6c using Methods A1 and B); 32% (yield synthesized from compound 5c using Methods A2 and B); mp=77-82 C. dec; R.sub.f=0.36 (EtOAc:hexane=1:1); .sup.1H NMR (400 MHz, DMSO-d.sub.6): 7.83 (d, J=8.0 Hz, 1H), 7.70 (d, J=8.0 Hz, 1H), 7.59 (d, J=7.6 Hz, 2H), 7.43 (dd, J=7.6, 7.6 Hz, 1H), 7.32 (dd, J=7.6, 7.6 Hz, 1H), 7.26 (dd, J=8.0, 7.2 Hz, 1H), 7.15 (dd, J=8.0, 7.2 Hz, 1H), 6.21 (s, 1H), 4.31-4.46 (m, 2H), 3.08-3.21 (m, 2H), 2.96-3.03 (m, 1H), 2.75-2.82 (m, 5H), 1.67-1.82 (m, 2H), 0.83 (t, J=7.2 Hz, 3H); .sup.13C NMR (100 MHz, DMSO-d.sub.6): 170.1, 167.9, 138.0, 134.1, 130.5, 129.9, 127.6, 126.89, 126.90, 124.3, 123.0, 122.9, 120.2, 118.8, 111.5, 110.7, 46.3, 42.7, 31.4, 25.7, 25.5, 23.1, 10.9; IR (ZnSe-ATR) 2961 (w), 1812 (w), 1783 (w), 1733 (s), 1460 (w), 1424 (w), 1348 (m), 1201 (m), 1065 (m), 1045 (m), 908 (w), 810 (w), 744 (vs) cm.sup.1; Anal. Calcd for C.sub.25H.sub.24N.sub.2O.sub.4S.sub.2: C, 62.48; H, 5.03; N, 5.83; S, 13.34. Found: C, 62.42; H, 5.03; N, 5.82 S, 13.40.

##STR00025##

[0073] 2,5-Dioxopyrrolidin-1-yl 3-((11-butyl-6,11-dihydrothiochromeno[4,3-b]indol-6-yl)thio)propanoate (3d): 36% (yield synthesized from compound 6d using Methods A1 and B); mp=76-81 C. dec; R.sub.f=0.35 (EtOAc:hexane=1:1); .sup.1H NMR (400 MHz, DMSO-d.sub.6): 7.85 (d, J=8.0 Hz, 1H), 7.70 (d, J=8.0 Hz, 1H), 7.58-7.60 (m, 2H), 7.43 (dd, J=8.0, 8.0 Hz, 1H), 7.32 (dd, J=8.0, 7.2 Hz, 1H), 7.26 (dd, J=7.6, 7.2 Hz, 1H), 7.15 (dd, J=7.6, 7.2 Hz, 1H), 6.21 (s, 1H), 4.40-4.46 (m, 2H), 3.08-3.21 (m, 2H), 2.96-3.03 (m, 1H), 2.77-2.82 (m, 5H), 1.65-1.75 (m, 2H), 1.21-1.29 (m, 2H), 0.86 (t, J=7.0 Hz, 3H); .sup.13C NMR (100 MHz, DMSO-d.sub.6): 170.1, 167.9, 137.9, 134.2, 130.5, 129.9, 127.6, 126.9, 126.8, 124.3, 123.03, 122.96, 120.2, 118.8, 111.5, 110.6, 44.5, 42.8, 31.8, 31.4, 25.7, 25.5, 19.3, 13.5; IR (ZnSe-ATR) 2954 (w), 2925 (w), 1811 (w), 1783 (w), 1735 (s), 1459 (w), 1422 (w), 1359 (w), 1200 (m), 1066 (m), 1042 (w), 810 (w), 745 (s), 669 (m) cm.sup.1; Anal. Calcd for C.sub.26H.sub.26N.sub.2O.sub.4S.sub.2: C, 63.14; H, 5.30; N, 5.66; S, 12.96. Found: C, 63.14; H, 5.30; N, 5.69 S, 12.90.

Example 2: Synthesis of Mass Tag (II)

Synthesis of sodium 1-((3-((11-methyl-6,11-dihydrothiochromeno[4,3-b]indol-6-yl)thio)propanoyl)oxy)-2,5-dioxopyrrolidine-3-sulfonate (9)

[0074] ##STR00026##

[0075] 3-((11-Methyl-6,11-dihydrothiochromeno[4,3-b]indol-6-yl)thio)propanoic acid (8a, 0.015 g, 0.042 mmol), N-hydroxysulfosuccinimide sodium salt (0.009 g, 0.04 mmol), and N,N-dimethylpyridinium p-toluenesulfonate (DPTS) (0.001 g, 0.004 mmol) were dissolved in anhydrous dimethylformamide (0.3 mL) in a nitrogen atmosphere and then cooled to 0 C. The resulting reaction mixture was slowly added with a solution of N,N-dicyclohexylcarbodiimide (0.010 g, 0.051 mmol) separately dissolved in anhydrous dichloromethane (0.3 mL) in a nitrogen atmosphere. The temperature of the reaction mixture was gradually elevated to room temperature and stirred. After 6 hr, the reaction mixture was cooled to 0 C. and the precipitated solid was filtered under reduced pressure and washed with cold dichloromethane. The solvent of the filtrate was completely removed under reduced pressure, thus obtaining compound 9 as a yellow solid. (45%): .sup.1H NMR (400 MHz, DMSO-d.sub.6): 7.98 (d, J=7.6 Hz, 1H), 7.10 (d, J=7.6 Hz, 1H), 7.60 (d, J=7.6 Hz, 1H), 7.55 (d, J=8.4 Hz, 1H), 7.41 (dd, J=7.6, 7.6 Hz, 1H), 7.33 (dd, J=8.4, 7.2 Hz, 1H), 7.27 (dd, J=7.6, 7.2 Hz, 1H), 7.16 (dd, J=7.6, 7.2 Hz, 1H), 6.24 (s, 1H), 4.00 (s, 3H), 3.13-3.19 (m, 4H), 2.94-3.02 (m, 1H), 2.76-2.83 (m, 1H).

Example 3: Synthesis of Mass Tag (III)

Synthesis of perfluorophenyl 3-((11-methyl-6,11-dihydrothiochromeno[4,3-b]indol-6-yl)thio)propanoate (10)

[0076] ##STR00027##

[0077] 3-((11-Methyl-6,11-dihydrothiochromeno[4,3-b]indol-6-yl)thio)propanoic acid (8a, 0.020 g, 0.056 mmol) and pentafluorophenol (0.011 mg, 0.062 mmol) were dissolved in anhydrous dichloromethane (0.7 mL) in a nitrogen atmosphere. The resulting reaction mixture was added with a solution of N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (0.032 mg, 0.017 mmol) separately dissolved in anhydrous dichloromethane (0.3 mL). The resulting reaction mixture was stirred at room temperature for 2.5 hr and then diluted with dichloromethane (3 mL). The resulting solution was washed two times with water (3 mL), added with anhydrous sodium sulfate and filtered under reduced pressure. The solvent of the filtrate was completely removed under reduced pressure, followed by purification through column chromatography (hexane:ethyl acetate=9:1), thus obtaining compound 10 as a yellow solid. (75%): .sup.1H NMR (400 MHz, DMSO-d.sub.6): 7.98 (d, J=8.0 Hz, 1H), 7.67 (d, J=8.4 Hz, 1H), 7.55-7.59 (m, 2H), 7.41 (dd, J=6.8, 6.8 Hz, 1H), 7.33 (dd, J=7.2, 7.2 Hz, 1H), 7.27 (dd, J=8.4, 7.2 Hz, 1H), 7.14 (dd, J=7.2, 6.8 Hz, 1H), 6.25 (s, 1H), 4.00 (s, 3H), 3.27-3.33 (m, 2H), 2.99-3.02 (m, 1H), 2.83-2.88 (m, 1H).

Example 4: Synthesis of Mass Tag (IV)

Synthesis of sodium 2,3,5,6-tetrafluoro-4-((3-((11-methyl-6,11-dihydrothiochromeno[4,3-b]indol-6-yl)thio)propanoyl)oxy)benzenesulfonate (11)

[0078] ##STR00028##

[0079] 3-((11-Methyl-6,11-dihydrothiochromeno[4,3-b]indol-6-yl)thio)propanoic acid (8a, 0.050 g, 0.14 mmol), sodium 2,3,5,6-tetrafluoro-4-hydroxybenzenesulfonate (0.038 g, 0.14 mmol), and N,N-dimethylpyridiniump-toluenesulfonate (DPTS) (0.002 g, 0.007 mmol) were dissolved in anhydrous acetone/anhydrous dimethylformamide (v:v=15:1, 3.0 mL) in a nitrogen atmosphere and then cooled to 0 C. The resulting reaction mixture was slowly added with a solution of N,N-dicyclohexylcarbodiimide (0.035 g, 0.17 mmol) dissolved in anhydrous acetone (1.0 mL) in a nitrogen atmosphere. The temperature of the reaction mixture was gradually elevated to room temperature and stirred for 4.5 hr. The reaction mixture was cooled to 0 C., after which the precipitated solid was filtered under reduced pressure and washed with cold dichloromethane. The solvent of the filtrate was completely removed under reduced pressure, followed by purification through column chromatography (dichloromethane:acetone=3:7), thus obtaining compound 11 as a yellow solid. (37%): .sup.1H NMR (400 MHz, DMSO-d.sub.6): 7.98 (d, J=8.0 Hz, 1H), 7.67 (d, J=7.2 Hz, 1H), 7.55-7.60 (m, 2H), 7.41 (dd, J=7.2, 7.2 Hz, 1H), 7.32 (dd, J=8.0, 7.2 Hz, 1H), 7.27 (dd, J=7.6, 7.2 Hz, 1H), 7.14 (dd, J=7.6, 7.2 Hz, 1H), 6.25 (s, 1H), 4.00 (s, 3H), 3.20-3.34 (m, 2H), 2.96-3.03 (m, 1H), 2.80-2.89 (m, 1H).

Example 5: Solubility Comparison of Compounds 1, 2, 3a

[0080] ##STR00029##

[0081] As shown in FIGS. 5 and 6, indole-introduced derivative 3a exhibited high absorbance at 355 nm compared to compounds 1 and 2, resulting in very effective ionization through laser irradiation.

[0082] Indole-introduced derivative 3 was remarkably improved in solubility compared to existing ferrocene derivative mass tags. For example, the ferrocene derivative formed a cloud point when 10% water was added upon dissolution in DMSO, whereas indole-introduced 2-alkylthio-2H-thiochromene derivative 3a did not form a cloud point until 30% water was added.

Example 6: Matrix-Less LDI MS Spectrum

A) Matrix-Less LDI-TOF Test of Photocleavable Mass Tag or Biomaterial Tagged Therewith

[0083] A photocleavable mass tag or a biomaterial tagged therewith was dissolved in tetrahydrofuran, and 1.04 thereof was dropped on each spot of a plate and exposed in air at room temperature, thereby drying the solvent. The dried sample on the plate was analyzed using a MALDI-TOF mass spectrometer (Autoflex Speed series, Bruker Daltonics, Leipzig, Germany). All spectra were measured in a positive reflectron mode. The mass range was set to 0-800 Da, and data analysis was performed using flex Analysis software.

(B) Matrix-Less LDI MS Spectrum of Mass Tag 3a-3d

[0084] The matrix-less LDI MS spectra of mass tags 3a-3d are shown in FIG. 7. In matrix-less LDI-TOF MS, a desired photocleavage reaction occurred and thus peaks corresponding to respective cations were detected at high intensity. Here, a peak at about 235 m/z was observed in common, which means that some mass-changing group was detached during the photocleavage. The detached proportion thereof is very low and thus it is not expected to cause a big problem when actually applied to marker detection.

Example 7: Matrix-Less LDI MS Sensitivity Comparison

[0085] The sensitivity at which the novel photocleavable mass tag is detected in matrix-less LDI-TOF MS was compared with that of the conventionally known tag.

[0086] A trityl tag (trityl-SPh), which is one of the photocleavable tags reported in existing documents, and compound 3a of the present invention were added in the same amount and analyzed. The results are shown in FIG. 8. As shown in FIG. 8, the tag 3a exhibited sensitivity and a signal-to-noise ratio as high as about 110 times those of the trityl tag.

[0087] Furthermore, the results of matrix-less LDI MS sensitivity comparison thereof with the ferrocene-based tag disclosed in the related patent (Korean Patent Application No. 10-2014-0050964) are shown in FIG. 9. As shown in FIG. 9, the tag according to the present invention exhibited higher detection intensity by about 4 times. Therefore, the tag of the present invention can be concluded to be a mass tag having remarkably high sensitivity compared to conventionally known tags.

Example 8: Detection Limit of Mass Tag

[0088] In order to evaluate the minimum detection limit of the novel mass tag, a test for determining the minimum sample content in matrix-less LDI-TOF MS was performed. The tags of 100/50/10/5/2/1 pmol were loaded on MALDI measurement plates and spectrum results thereof were compared. Based on the results in which the peak area and the signal-to-noise ratio (S/N ratio) were represented depending on molar amounts, linear graphs were obtained in all of tags 3a, 3b, 3c, 3d, as shown in FIG. 10 (typically only posted data for 3d).

[0089] This suggests that the present analysis method is applicable to the quantitative analysis of biomarkers. The minimum amount of the tag that may be reproducibly detected was measured to be 1 pmol, at which the signal-to-noise ratio was measured to be about 100. The cation was detected even in lower molar amounts, but the difference in detection intensity was found to be significant depending on the measurement position on the spot of the plate. This is interpreted as a phenomenon that occurs because the tag does not spread evenly and is crystallized and agglomerated only in a portion thereof owing to the high crystallinity of the tag. However, conjugation of the tag to the antibody may weaken the crystallinity so that the tag may be evenly distributed within the spot, and therefore the minimum detection limit thereof is deemed to be sufficiently improved.