JP6233929B2 - Compound, standard substance for quantitative analysis using the same, and method for quantifying desmosines - Google Patents
Compound, standard substance for quantitative analysis using the same, and method for quantifying desmosines Download PDFInfo
- Publication number
- JP6233929B2 JP6233929B2 JP2014055337A JP2014055337A JP6233929B2 JP 6233929 B2 JP6233929 B2 JP 6233929B2 JP 2014055337 A JP2014055337 A JP 2014055337A JP 2014055337 A JP2014055337 A JP 2014055337A JP 6233929 B2 JP6233929 B2 JP 6233929B2
- Authority
- JP
- Japan
- Prior art keywords
- compound
- desmosine
- group
- general formula
- desmosines
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 150000001875 compounds Chemical class 0.000 title claims description 51
- 239000000126 substance Substances 0.000 title claims description 19
- 238000000034 method Methods 0.000 title claims description 16
- 125000000798 desmosine group Chemical group 0.000 title claims description 15
- 238000004445 quantitative analysis Methods 0.000 title claims description 12
- VEVRNHHLCPGNDU-MUGJNUQGSA-O desmosine Chemical compound OC(=O)[C@@H](N)CCCC[N+]1=CC(CC[C@H](N)C(O)=O)=C(CCC[C@H](N)C(O)=O)C(CC[C@H](N)C(O)=O)=C1 VEVRNHHLCPGNDU-MUGJNUQGSA-O 0.000 claims description 23
- 125000004432 carbon atom Chemical group C* 0.000 claims description 17
- 125000002947 alkylene group Chemical group 0.000 claims description 16
- -1 dimethylene group Chemical group 0.000 claims description 10
- 125000000383 tetramethylene group Chemical group [H]C([H])([*:1])C([H])([H])C([H])([H])C([H])([H])[*:2] 0.000 claims description 8
- 125000003258 trimethylene group Chemical group [H]C([H])([*:2])C([H])([H])C([H])([H])[*:1] 0.000 claims description 4
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 38
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 30
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 27
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 27
- 239000000243 solution Substances 0.000 description 20
- 238000005259 measurement Methods 0.000 description 19
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 18
- 235000019439 ethyl acetate Nutrition 0.000 description 17
- 238000004458 analytical method Methods 0.000 description 15
- 208000006545 Chronic Obstructive Pulmonary Disease Diseases 0.000 description 9
- 239000002904 solvent Substances 0.000 description 9
- 238000003756 stirring Methods 0.000 description 9
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 8
- 238000011088 calibration curve Methods 0.000 description 8
- 239000011734 sodium Substances 0.000 description 8
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical class C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 7
- 239000000203 mixture Substances 0.000 description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- 238000010586 diagram Methods 0.000 description 6
- 238000013467 fragmentation Methods 0.000 description 6
- 238000006062 fragmentation reaction Methods 0.000 description 6
- KWGKDLIKAYFUFQ-UHFFFAOYSA-M lithium chloride Chemical compound [Li+].[Cl-] KWGKDLIKAYFUFQ-UHFFFAOYSA-M 0.000 description 6
- YPJUNDFVDDCYIH-UHFFFAOYSA-N perfluorobutyric acid Chemical compound OC(=O)C(F)(F)C(F)(F)C(F)(F)F YPJUNDFVDDCYIH-UHFFFAOYSA-N 0.000 description 6
- 239000011541 reaction mixture Substances 0.000 description 6
- SZUVGFMDDVSKSI-WIFOCOSTSA-N (1s,2s,3s,5r)-1-(carboxymethyl)-3,5-bis[(4-phenoxyphenyl)methyl-propylcarbamoyl]cyclopentane-1,2-dicarboxylic acid Chemical compound O=C([C@@H]1[C@@H]([C@](CC(O)=O)([C@H](C(=O)N(CCC)CC=2C=CC(OC=3C=CC=CC=3)=CC=2)C1)C(O)=O)C(O)=O)N(CCC)CC(C=C1)=CC=C1OC1=CC=CC=C1 SZUVGFMDDVSKSI-WIFOCOSTSA-N 0.000 description 5
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical group C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 5
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 5
- 229940126543 compound 14 Drugs 0.000 description 5
- 229910052736 halogen Inorganic materials 0.000 description 5
- 150000002367 halogens Chemical class 0.000 description 5
- 238000010898 silica gel chromatography Methods 0.000 description 5
- LNUFLCYMSVYYNW-ZPJMAFJPSA-N [(2r,3r,4s,5r,6r)-2-[(2r,3r,4s,5r,6r)-6-[(2r,3r,4s,5r,6r)-6-[(2r,3r,4s,5r,6r)-6-[[(3s,5s,8r,9s,10s,13r,14s,17r)-10,13-dimethyl-17-[(2r)-6-methylheptan-2-yl]-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1h-cyclopenta[a]phenanthren-3-yl]oxy]-4,5-disulfo Chemical compound O([C@@H]1[C@@H](COS(O)(=O)=O)O[C@@H]([C@@H]([C@H]1OS(O)(=O)=O)OS(O)(=O)=O)O[C@@H]1[C@@H](COS(O)(=O)=O)O[C@@H]([C@@H]([C@H]1OS(O)(=O)=O)OS(O)(=O)=O)O[C@@H]1[C@@H](COS(O)(=O)=O)O[C@H]([C@@H]([C@H]1OS(O)(=O)=O)OS(O)(=O)=O)O[C@@H]1C[C@@H]2CC[C@H]3[C@@H]4CC[C@@H]([C@]4(CC[C@@H]3[C@@]2(C)CC1)C)[C@H](C)CCCC(C)C)[C@H]1O[C@H](COS(O)(=O)=O)[C@@H](OS(O)(=O)=O)[C@H](OS(O)(=O)=O)[C@H]1OS(O)(=O)=O LNUFLCYMSVYYNW-ZPJMAFJPSA-N 0.000 description 4
- 239000000090 biomarker Substances 0.000 description 4
- 239000012267 brine Substances 0.000 description 4
- IJOOHPMOJXWVHK-UHFFFAOYSA-N chlorotrimethylsilane Chemical compound C[Si](C)(C)Cl IJOOHPMOJXWVHK-UHFFFAOYSA-N 0.000 description 4
- 239000012043 crude product Substances 0.000 description 4
- 125000000623 heterocyclic group Chemical group 0.000 description 4
- 239000010410 layer Substances 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 238000004949 mass spectrometry Methods 0.000 description 4
- 230000007935 neutral effect Effects 0.000 description 4
- 239000012044 organic layer Substances 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 229920006395 saturated elastomer Polymers 0.000 description 4
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 4
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 4
- FPGGTKZVZWFYPV-UHFFFAOYSA-M tetrabutylammonium fluoride Chemical compound [F-].CCCC[N+](CCCC)(CCCC)CCCC FPGGTKZVZWFYPV-UHFFFAOYSA-M 0.000 description 4
- 238000004809 thin layer chromatography Methods 0.000 description 4
- 239000011701 zinc Substances 0.000 description 4
- YSUIQYOGTINQIN-UZFYAQMZSA-N 2-amino-9-[(1S,6R,8R,9S,10R,15R,17R,18R)-8-(6-aminopurin-9-yl)-9,18-difluoro-3,12-dihydroxy-3,12-bis(sulfanylidene)-2,4,7,11,13,16-hexaoxa-3lambda5,12lambda5-diphosphatricyclo[13.2.1.06,10]octadecan-17-yl]-1H-purin-6-one Chemical compound NC1=NC2=C(N=CN2[C@@H]2O[C@@H]3COP(S)(=O)O[C@@H]4[C@@H](COP(S)(=O)O[C@@H]2[C@@H]3F)O[C@H]([C@H]4F)N2C=NC3=C2N=CN=C3N)C(=O)N1 YSUIQYOGTINQIN-UZFYAQMZSA-N 0.000 description 3
- OPFJDXRVMFKJJO-ZHHKINOHSA-N N-{[3-(2-benzamido-4-methyl-1,3-thiazol-5-yl)-pyrazol-5-yl]carbonyl}-G-dR-G-dD-dD-dD-NH2 Chemical compound S1C(C=2NN=C(C=2)C(=O)NCC(=O)N[C@H](CCCN=C(N)N)C(=O)NCC(=O)N[C@H](CC(O)=O)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](CC(O)=O)C(N)=O)=C(C)N=C1NC(=O)C1=CC=CC=C1 OPFJDXRVMFKJJO-ZHHKINOHSA-N 0.000 description 3
- MXZNUGFCDVAXLG-CHWSQXEVSA-N [(2S)-1-[(2R)-3-methyl-2-(pyridine-4-carbonylamino)butanoyl]pyrrolidin-2-yl]boronic acid Chemical compound CC(C)[C@@H](NC(=O)c1ccncc1)C(=O)N1CCC[C@@H]1B(O)O MXZNUGFCDVAXLG-CHWSQXEVSA-N 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 229940125810 compound 20 Drugs 0.000 description 3
- 229940126086 compound 21 Drugs 0.000 description 3
- 229940126208 compound 22 Drugs 0.000 description 3
- DOBRDRYODQBAMW-UHFFFAOYSA-N copper(i) cyanide Chemical compound [Cu+].N#[C-] DOBRDRYODQBAMW-UHFFFAOYSA-N 0.000 description 3
- 238000006880 cross-coupling reaction Methods 0.000 description 3
- 239000000706 filtrate Substances 0.000 description 3
- JAXFJECJQZDFJS-XHEPKHHKSA-N gtpl8555 Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C(C)C)C(=O)N1CCC[C@@H]1C(=O)N[C@H](B1O[C@@]2(C)[C@H]3C[C@H](C3(C)C)C[C@H]2O1)CCC1=CC=C(F)C=C1 JAXFJECJQZDFJS-XHEPKHHKSA-N 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 125000006239 protecting group Chemical group 0.000 description 3
- 238000003908 quality control method Methods 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 239000000741 silica gel Substances 0.000 description 3
- 229910002027 silica gel Inorganic materials 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 238000001228 spectrum Methods 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 3
- 238000006257 total synthesis reaction Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- CYPYTURSJDMMMP-WVCUSYJESA-N (1e,4e)-1,5-diphenylpenta-1,4-dien-3-one;palladium Chemical compound [Pd].[Pd].C=1C=CC=CC=1\C=C\C(=O)\C=C\C1=CC=CC=C1.C=1C=CC=CC=1\C=C\C(=O)\C=C\C1=CC=CC=C1.C=1C=CC=CC=1\C=C\C(=O)\C=C\C1=CC=CC=C1 CYPYTURSJDMMMP-WVCUSYJESA-N 0.000 description 2
- QEGNZPFIIRTNBJ-UHFFFAOYSA-N 3,5-dibromo-4-iodopyridine Chemical compound BrC1=CN=CC(Br)=C1I QEGNZPFIIRTNBJ-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 0 COCCCc1c*(CCCCC(*)*)cc(CCCO)c1CCCC[C@](*)OC Chemical compound COCCCc1c*(CCCCC(*)*)cc(CCCO)c1CCCC[C@](*)OC 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 102000016942 Elastin Human genes 0.000 description 2
- 108010014258 Elastin Proteins 0.000 description 2
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 2
- 238000003477 Sonogashira cross-coupling reaction Methods 0.000 description 2
- 125000003277 amino group Chemical group 0.000 description 2
- 125000000051 benzyloxy group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])O* 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- IJKVHSBPTUYDLN-UHFFFAOYSA-N dihydroxy(oxo)silane Chemical compound O[Si](O)=O IJKVHSBPTUYDLN-UHFFFAOYSA-N 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 229920002549 elastin Polymers 0.000 description 2
- 238000004992 fast atom bombardment mass spectroscopy Methods 0.000 description 2
- 238000003818 flash chromatography Methods 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 239000002198 insoluble material Substances 0.000 description 2
- RGXCTRIQQODGIZ-UHFFFAOYSA-O isodesmosine Chemical compound OC(=O)C(N)CCCC[N+]1=CC(CCC(N)C(O)=O)=CC(CCC(N)C(O)=O)=C1CCCC(N)C(O)=O RGXCTRIQQODGIZ-UHFFFAOYSA-O 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical class [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 2
- 125000006633 tert-butoxycarbonylamino group Chemical group 0.000 description 2
- GHYOCDFICYLMRF-UTIIJYGPSA-N (2S,3R)-N-[(2S)-3-(cyclopenten-1-yl)-1-[(2R)-2-methyloxiran-2-yl]-1-oxopropan-2-yl]-3-hydroxy-3-(4-methoxyphenyl)-2-[[(2S)-2-[(2-morpholin-4-ylacetyl)amino]propanoyl]amino]propanamide Chemical compound C1(=CCCC1)C[C@@H](C(=O)[C@@]1(OC1)C)NC([C@H]([C@@H](C1=CC=C(C=C1)OC)O)NC([C@H](C)NC(CN1CCOCC1)=O)=O)=O GHYOCDFICYLMRF-UTIIJYGPSA-N 0.000 description 1
- IWZSHWBGHQBIML-ZGGLMWTQSA-N (3S,8S,10R,13S,14S,17S)-17-isoquinolin-7-yl-N,N,10,13-tetramethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-amine Chemical compound CN(C)[C@H]1CC[C@]2(C)C3CC[C@@]4(C)[C@@H](CC[C@@H]4c4ccc5ccncc5c4)[C@@H]3CC=C2C1 IWZSHWBGHQBIML-ZGGLMWTQSA-N 0.000 description 1
- YRBAQEQNCDDFKM-UHFFFAOYSA-N 2-bromoethynyl(trimethyl)silane Chemical compound C[Si](C)(C)C#CBr YRBAQEQNCDDFKM-UHFFFAOYSA-N 0.000 description 1
- QBWKPGNFQQJGFY-QLFBSQMISA-N 3-[(1r)-1-[(2r,6s)-2,6-dimethylmorpholin-4-yl]ethyl]-n-[6-methyl-3-(1h-pyrazol-4-yl)imidazo[1,2-a]pyrazin-8-yl]-1,2-thiazol-5-amine Chemical compound N1([C@H](C)C2=NSC(NC=3C4=NC=C(N4C=C(C)N=3)C3=CNN=C3)=C2)C[C@H](C)O[C@H](C)C1 QBWKPGNFQQJGFY-QLFBSQMISA-N 0.000 description 1
- YJTLZFAEISQPFZ-FQEVSTJZSA-N C(C)(C)(C)OC(=O)N[C@H](C(=O)OCC1=CC=CC=C1)CCC#CC1=C(C=NC=C1Br)Br Chemical compound C(C)(C)(C)OC(=O)N[C@H](C(=O)OCC1=CC=CC=C1)CCC#CC1=C(C=NC=C1Br)Br YJTLZFAEISQPFZ-FQEVSTJZSA-N 0.000 description 1
- 206010014561 Emphysema Diseases 0.000 description 1
- 208000033962 Fontaine progeroid syndrome Diseases 0.000 description 1
- 238000005033 Fourier transform infrared spectroscopy Methods 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 206010036790 Productive cough Diseases 0.000 description 1
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 238000003915 air pollution Methods 0.000 description 1
- 125000000217 alkyl group Chemical group 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- FDPQLDYNTFSKFS-HNNXBMFYSA-N benzyl (2S)-2-[(2-methylpropan-2-yl)oxycarbonylamino]hex-5-ynoate Chemical compound C(C)(C)(C)OC(=O)N[C@H](C(=O)OCC1=CC=CC=C1)CCC#C FDPQLDYNTFSKFS-HNNXBMFYSA-N 0.000 description 1
- ADTJUAMOILOVQV-HNNXBMFYSA-N benzyl (2s)-6-iodo-2-[(2-methylpropan-2-yl)oxycarbonylamino]hexanoate Chemical compound CC(C)(C)OC(=O)N[C@@H](CCCCI)C(=O)OCC1=CC=CC=C1 ADTJUAMOILOVQV-HNNXBMFYSA-N 0.000 description 1
- FDPQLDYNTFSKFS-UHFFFAOYSA-N benzyl 2-[(2-methylpropan-2-yl)oxycarbonylamino]hex-5-ynoate Chemical compound CC(C)(C)OC(=O)NC(CCC#C)C(=O)OCC1=CC=CC=C1 FDPQLDYNTFSKFS-UHFFFAOYSA-N 0.000 description 1
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 206010006451 bronchitis Diseases 0.000 description 1
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 150000001721 carbon Chemical group 0.000 description 1
- 239000011203 carbon fibre reinforced carbon Substances 0.000 description 1
- 238000001460 carbon-13 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 229940125904 compound 1 Drugs 0.000 description 1
- 229940125797 compound 12 Drugs 0.000 description 1
- 229940125782 compound 2 Drugs 0.000 description 1
- 229940125846 compound 25 Drugs 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 230000008602 contraction Effects 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 210000004177 elastic tissue Anatomy 0.000 description 1
- 238000002451 electron ionisation mass spectrometry Methods 0.000 description 1
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 1
- 238000000119 electrospray ionisation mass spectrum Methods 0.000 description 1
- 125000005677 ethinylene group Chemical group [*:2]C#C[*:1] 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 239000010422 internal standard material Substances 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 238000001948 isotopic labelling Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 125000001570 methylene group Chemical group [H]C([H])([*:1])[*:2] 0.000 description 1
- COCAUCFPFHUGAA-MGNBDDOMSA-N n-[3-[(1s,7s)-5-amino-4-thia-6-azabicyclo[5.1.0]oct-5-en-7-yl]-4-fluorophenyl]-5-chloropyridine-2-carboxamide Chemical compound C=1C=C(F)C([C@@]23N=C(SCC[C@@H]2C3)N)=CC=1NC(=O)C1=CC=C(Cl)C=N1 COCAUCFPFHUGAA-MGNBDDOMSA-N 0.000 description 1
- 239000012299 nitrogen atmosphere Substances 0.000 description 1
- 125000004433 nitrogen atom Chemical group N* 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 239000012041 precatalyst Substances 0.000 description 1
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- BJDYCCHRZIFCGN-UHFFFAOYSA-N pyridin-1-ium;iodide Chemical compound I.C1=CC=NC=C1 BJDYCCHRZIFCGN-UHFFFAOYSA-N 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000006722 reduction reaction Methods 0.000 description 1
- 238000002553 single reaction monitoring Methods 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 208000024794 sputum Diseases 0.000 description 1
- 210000003802 sputum Anatomy 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 239000010421 standard material Substances 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- WROMPOXWARCANT-UHFFFAOYSA-N tfa trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F.OC(=O)C(F)(F)F WROMPOXWARCANT-UHFFFAOYSA-N 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
- DLQYXUGCCKQSRJ-UHFFFAOYSA-N tris(furan-2-yl)phosphane Chemical compound C1=COC(P(C=2OC=CC=2)C=2OC=CC=2)=C1 DLQYXUGCCKQSRJ-UHFFFAOYSA-N 0.000 description 1
- 238000002211 ultraviolet spectrum Methods 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
Images
Landscapes
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
- Pyridine Compounds (AREA)
Description
本発明は、化合物、これを用いた定量分析用標準物質およびデスモシン類の定量方法に関する。 The present invention relates to a compound, a standard substance for quantitative analysis using the compound, and a method for quantifying desmosines.
慢性閉塞性肺疾患（Chronic Obstructive Pulmonary Disease：ＣＯＰＤ）は、気管支炎や肺気腫などの病気の総称である。世界保健機関（World Health Organization：ＷＨＯ）によると、現在死亡原因の第４位を占めており、２０２０年までに第３位に浮上すると警告している。ＣＯＰＤについては、そもそもの病態が極めて複雑で未知の部分が多く、根本的治療薬すら存在しない。今世紀、発展途上国での喫煙者の増加や産業発展による大気汚染により、世界規模でのＣＯＰＤ患者の急増が危惧されているため、迅速かつ簡便な検査法の確立が至上命題となっている。 Chronic obstructive pulmonary disease (COPD) is a general term for diseases such as bronchitis and emphysema. According to the World Health Organization (WHO), it is currently the fourth leading cause of death and warns that it will rise to third by 2020. As for COPD, the pathological condition is extremely complicated and there are many unknown parts, and there is no fundamental therapeutic drug. In this century, the rapid increase in the number of COPD patients around the world is feared due to the increase in smokers in developing countries and air pollution due to industrial development, so the establishment of a rapid and simple test method has become a top priority.
ＣＯＰＤ患者の痰・血液・尿を加水分解処理し、高速液体クロマトグラフ−質量分析計（Liquid Chromatography-Mass Spectrometry：ＬＣ−ＭＳ）で分析すると、肺胞の伸縮を司る弾性繊維エラスチンの架橋アミノ酸であり下記式に示されるデスモシン（化合物１）およびその異性体であり下記式に示されるイソデスモシン（化合物２）が観測される。健常者と比べて、ＣＯＰＤ患者におけるそれらの存在量が異なることから、デスモシン類はＣＯＰＤのバイオマーカーとして有望視されている。 Hydrolysis of sputum, blood, and urine from patients with COPD and analysis with a high-performance liquid chromatograph-mass spectrometer (LC-MS) reveals that the cross-linked amino acid of elastic fiber elastin is responsible for the expansion and contraction of alveoli. There is observed desmosine (compound 1) represented by the following formula and isodesmosine (compound 2) which is an isomer thereof and represented by the following formula. Desmosines are promising as COPD biomarkers because their abundance in COPD patients is different from that in healthy individuals.
しかし、生体内に含まれるデスモシン類の絶対量が微量であるため、ＬＣ−ＭＳ／ＭＳ（Liquid Chromatography−tandem Mass Spectrometry）等による厳密な定量分析のためには、内部標準物質の添加が必要となっている。このため、デスモシン類の標準物質として用いられる化合物が容易に合成できるとよい。デスモシンの全合成に関する技術として、非特許文献１および２に記載のものがある。
However, since the absolute amount of desmosine contained in the living body is very small, it is necessary to add an internal standard substance for strict quantitative analysis by LC-MS / MS (Liquid Chromatography-tandem Mass Spectrometry) or the like. It has become. For this reason, it is desirable that a compound used as a standard substance for desmosine can be easily synthesized. Non-patent
本発明は、上記事情に鑑みてなされたものであり、新規なデスモシン類似体を提供するものである。 The present invention has been made in view of the above circumstances, and provides a novel desmosine analog.
本発明によれば、
下記一般式（Ｉ）に示される化合物が提供される。
A compound represented by the following general formula (I) is provided.
また、本発明によれば、前記本発明における化合物を含む、定量分析用内部標準物質等の定量分析用標準物質が提供される。
また、本発明によれば、測定対象の試料に前記本発明における化合物を添加するステップを含む、デスモシン類の定量方法が提供される。
なお、本明細書において、デスモシンおよびイソデスモシンをあわせて「デスモシン類」とも呼ぶ。
In addition, according to the present invention, there is provided a standard material for quantitative analysis such as an internal standard material for quantitative analysis containing the compound of the present invention.
Moreover, according to this invention, the determination method of desmosines including the step which adds the compound in this invention to the sample of a measuring object is provided.
In the present specification, desmosine and isodesmosine are also collectively referred to as “desmosines”.
なお、これらの各構成の任意の組み合わせや、本発明の表現を方法、装置などの間で変換したものもまた本発明の態様として有効である。 It should be noted that any combination of these components, or a conversion of the expression of the present invention between a method, an apparatus, and the like is also effective as an aspect of the present invention.
たとえば、本発明によれば、前記本発明における化合物を内部標準物質等の標準物質として使用する定量分析方法が提供される。
また、本発明によれば、前記本発明における化合物を内部標準物質等の標準物質として使用するデスモシン類の分析または定量方法が提供される。
For example, according to the present invention, there is provided a quantitative analysis method using the compound of the present invention as a standard substance such as an internal standard substance.
The present invention also provides a method for analyzing or quantifying desmosines using the compound of the present invention as a standard substance such as an internal standard substance.
本発明によれば、新規なデスモシン類似体を提供することができる。 According to the present invention, a novel desmosine analog can be provided.
以下、本発明の実施形態を具体例に基づいて説明する。以下の実施形態に記載の複数の態様を組み合わせて用いることもできる。 Hereinafter, embodiments of the present invention will be described based on specific examples. A plurality of aspects described in the following embodiments can also be used in combination.
本実施形態における化合物は、デスモシンの類似化合物であって、下記一般式（Ｉ）に示される。 The compound in this embodiment is a desmosine analog and is represented by the following general formula (I).
（上記一般式（Ｉ）中、Ｒ1は、炭素数２以上６以下のアルキレン基であり、Ｒ2は炭素数１以上４以下のアルキレン基であり、Ｒ3は、炭素数２以上６以下のアルキレン基である。ここで、Ｒ1がトリメチレン基であり、Ｒ2がジメチレン基であり、かつ、Ｒ3がテトラメチレン基であることはない。） (In the general formula (I), R 1 is an alkylene group having 2 to 6 carbon atoms, R 2 is an alkylene group having 1 to 4 carbon atoms, and R 3 is 2 to 6 carbon atoms. Where R 1 is a trimethylene group, R 2 is a dimethylene group, and R 3 is not a tetramethylene group.)
デスモシンは、一般式（Ｉ）において、Ｒ1がトリメチレン基であり、Ｒ2がジメチレン基であり、かつ、Ｒ3がテトラメチレン基である化合物である。そして、本実施形態における化合物は、デスモシンの類似体であって、複素環に結合する一または二以上の側鎖のアルキル鎖長が、デスモシンと異なるものである。 Desmosine is a compound in which R 1 is a trimethylene group, R 2 is a dimethylene group, and R 3 is a tetramethylene group in the general formula (I). And the compound in this embodiment is an analog of desmosine, Comprising: The alkyl chain length of the 1 or 2 or more side chain couple | bonded with a heterocyclic ring differs from a desmosine.
一般式（Ｉ）において、複素環の４位の炭素原子に結合する側鎖における炭素数が、具体的には当該側鎖におけるアルキレン鎖長がデスモシンと異なるとき、Ｒ1は、炭素数２以上６以下のアルキレン基である。このとき、本実施形態における化合物とデスモシンとの分子量差を−１４以上４２以下の範囲で調整することができる。 In the general formula (I), when the carbon number in the side chain bonded to the carbon atom at the 4-position of the heterocyclic ring, specifically, the alkylene chain length in the side chain is different from desmosine, R 1 is 2 or more carbon atoms. 6 or less alkylene group. At this time, the molecular weight difference between the compound and desmosine in the present embodiment can be adjusted in the range of −14 to 42.
一般式（Ｉ）において、複素環の３位および５位の炭素原子に結合する側鎖における炭素数が、具体的には当該側鎖におけるアルキレン鎖長がデスモシンと異なるとき、２つのＲ2は、炭素数１以上４以下のアルキレン基である。このとき、本実施形態における化合物とデスモシンとの分子量差を−２８以上５６以下の範囲で調整することができる。 In the general formula (I), when the number of carbon atoms in the side chain bonded to the 3-position and 5-position carbon atoms of the heterocyclic ring, specifically, the alkylene chain length in the side chain is different from that of desmosine, two R 2 are And an alkylene group having 1 to 4 carbon atoms. At this time, the molecular weight difference between the compound and desmosine in the present embodiment can be adjusted in the range of −28 to 56.
また、一般式（Ｉ）において、複素環の１位の窒素原子に結合する側鎖における炭素数が、具体的には当該側鎖におけるアルキレン鎖長がデスモシンと異なるとき、Ｒ3は、炭素数２以上６以下のアルキレン基である。このとき、本実施形態における化合物とデスモシンとの分子量差を−２８以上２８以下の範囲で調整することができる。 In the general formula (I), when the carbon number in the side chain bonded to the nitrogen atom at the 1-position of the heterocyclic ring, specifically, the alkylene chain length in the side chain is different from that of desmosine, R 3 is the number of carbon atoms. 2 or more and 6 or less alkylene group. At this time, the molecular weight difference between the compound and desmosine in the present embodiment can be adjusted in the range of −28 to 28.
一般式（Ｉ）に示した化合物は、デスモシンの定量分析において標準物質として用いる観点から、デスモシンとの分子量差が−２８以上であり、好ましくは−１４以上である。同様の観点から、一般式（Ｉ）に示した化合物は、デスモシンとの分子量差が５６以下であり、好ましくは４２以下、さらに好ましくは２８以下である。これにより、たとえば一般式（Ｉ）に示した化合物をデスモシンと適度な質量差を有する質量分析用の標準物質として用いることができる。 From the viewpoint of using the compound represented by the general formula (I) as a standard substance in the quantitative analysis of desmosine, the molecular weight difference from desmosine is −28 or more, preferably −14 or more. From the same viewpoint, the compound represented by the general formula (I) has a molecular weight difference with desmosine of 56 or less, preferably 42 or less, more preferably 28 or less. Thereby, for example, the compound represented by the general formula (I) can be used as a standard substance for mass spectrometry having an appropriate mass difference from desmosine.
また、一般式（Ｉ）に示した化合物の安定性の観点からは、一般式（Ｉ）中、Ｒ3がテトラメチレン基であることが好ましい。また、化合物の安定性および分子量の制御性をさらに高める観点からは、Ｒ2がジメチレン基であり、Ｒ3がテトラメチレン基である構造とすることが好ましい。このとき、Ｒ1は炭素数２または４以上６以下のアルキレン基である。 Further, from the viewpoint of the stability of the compound represented by the general formula (I), in the general formula (I), R 3 is preferably a tetramethylene group. From the viewpoint of further improving the stability of the compound and the controllability of the molecular weight, it is preferable to have a structure in which R 2 is a dimethylene group and R 3 is a tetramethylene group. At this time, R 1 is an alkylene group having 2 or 4 to 6 carbon atoms.
次に、本実施形態における化合物の製造方法を説明する。本実施形態における化合物の製造方法に制限はないが、たとえば非特許文献１または２に記載の方法を用いることができる。さらに具体的には、以下のスキーム１に示す手順とすることができる。
なお、スキーム１においては、アミノ基の保護基をｔ−ブトキシカルボニル（Ｂｏｃ）基とし、カルボキシル基の保護基をベンジル（Ｂｎ）基とする場合を例に挙げて説明するが、アミノ基およびカルボキシル基のそれぞれについて、保護基の有無、数および種類に制限はなく、適宜選択することができる。
Next, the manufacturing method of the compound in this embodiment is demonstrated. Although there is no restriction | limiting in the manufacturing method of the compound in this embodiment, For example, the method of a
In
スキーム１中、一般式（ＩＩ）および（ＩＩＩ）において、Ｒ11基は、単結合または炭素数１以上４以下のアルキレン基である。すなわち、Ｒ11基のアルキレン鎖長は、一般式（Ｉ）におけるＲ1基のアルキレン鎖長より２少ない。また、Ｘ1はハロゲンである。
一般式（ＩＶ）中、Ｘ4およびＸ5は、それぞれ異なるハロゲンである。一般式（ＩＶ）に示した化合物として、たとえば３，５−ジブロモ−４−ヨードピリジンが挙げられる。
一般式（Ｖ）中、Ｒ2は一般式（Ｉ）におけるＲ2と同じであり、Ｘ2はハロゲンである。
また、式（ＶＩ）中、Ｒ3は一般式（Ｉ）におけるＲ3と同じであり、Ｘ3はハロゲンである。
なお、スキーム１において、Ｒ11がメチレン基であり、Ｒ2がジメチレン基であり、かつ、Ｒ3がテトラメチレン基であることはない。
In
In the general formula (IV), X 4 and X 5 are different halogens. Examples of the compound represented by the general formula (IV) include 3,5-dibromo-4-iodopyridine.
In general formula (V), R 2 is the same as R 2 in general formula (I), and X 2 is halogen.
In the formula (VI), R 3 is the same as R 3 in the general formula (I), and X 3 is halogen.
In
スキーム１に示した手順では、まず、一般式（ＩＩ）に示した化合物を一般式（ＩＩＩ）に示した化合物に変換する。そして、薗頭クロスカップリングにより、一般式（ＩＶ）に示した化合物のピリジン環の４位に、一般式（ＩＩＩ）に示した化合物の−Ｃ≡Ｃ−Ｒ11−Ｃ（ＮＨＢｏｃ）ＣＯ2Ｂｎ基を導入する。
また、根岸クロスカップリングにより、ピリジン環の３位および５位に、一般式（Ｖ）に示した化合物の−Ｒ2−Ｃ（ＮＨＢｏｃ）ＣＯ2Ｂｎ基を導入する。ここで、Ｒ2は一般式（Ｉ）におけるＲ2と同じであり、Ｘ2はハロゲンである。
なお、薗頭クロスカップリングによる反応と根岸クロスカップリングによる反応の順序に制限はない。
In the procedure shown in
In addition, the —R 2 —C (NHBoc) CO 2 Bn group of the compound represented by the general formula (V) is introduced into the 3rd and 5th positions of the pyridine ring by Negishi cross coupling. Here, R 2 is the same as R 2 in formula (I), and X 2 is halogen.
In addition, there is no restriction | limiting in the order of the reaction by Sonogashira cross coupling and the reaction by Negishi cross coupling.
さらに、ピリジン環の１位に、一般式（ＶＩ）に示した化合物の−Ｒ3−Ｃ（ＮＨＢｏｃ）ＣＯ2Ｂｎ基を導入する。
その後、ピリジン環の４位の側鎖に含まれるエチニレン基の還元反応をおこない、炭素−炭素三重結合を一重結合とする。そして、各側鎖のアミノ基およびカルボキシル基を脱保護する。
以上の手順により、一般式（Ｉ）に示した化合物が得られる。
各手順における詳細な反応条件については、実施例の項において後述する。
Furthermore, the —R 3 —C (NHBoc) CO 2 Bn group of the compound represented by the general formula (VI) is introduced at the 1-position of the pyridine ring.
Thereafter, a reduction reaction of the ethynylene group contained in the side chain at the 4-position of the pyridine ring is carried out to convert the carbon-carbon triple bond into a single bond. Then, the amino group and carboxyl group of each side chain are deprotected.
By the above procedure, the compound represented by the general formula (I) is obtained.
Detailed reaction conditions in each procedure will be described later in the Examples section.
次に、本実施形態の作用効果を説明する。
本実施形態における化合物は、デスモシン類との構造の類似性が高く、かつ、デスモシン類に対して分子量の絶対値を１４ｍ／ｚ以上シフトさせることができる。このため、デスモシン類の定量分析用標準物質として好適に用いられる。さらに具体的には、ＬＣ−ＭＳやＬＣ−ＭＳ／ＭＳ法等の質量分析を含む方法によりデスモシン類を定量分析する際の内部標準物質として好適である。このとき、デスモシン類の定量方法は、たとえば測定対象の試料に本実施形態における化合物を添加するステップを含む。また、本実施形態における化合物は、たとえばＣＯＰＤバイオマーカー分析における内部標準物質として用いることができる。
また、本実施形態の化合物は、製造工程中に同位体標識工程を必要としないため、デスモシン類の同位体標識化合物と比べて合成が容易である。このため、本実施形態の化合物は、大量生産に好適である。
また、質量分析において異なる質量数であっても同一の構造の化合物が同時に存在すると、お互いに干渉し合い、厳密性が損なわれる場合がある。これに対し、本実施形態の化合物を質量分析の内部標準物質として用いることにより、たとえばデスモシン類との干渉を抑制することも可能となるため、より厳密な定量分析に寄与しうる。
Next, the effect of this embodiment is demonstrated.
The compound in the present embodiment has high structural similarity to desmosines, and can shift the absolute value of molecular weight by 14 m / z or more with respect to desmosines. Therefore, it is suitably used as a standard substance for quantitative analysis of desmosines. More specifically, it is suitable as an internal standard substance for quantitatively analyzing desmosines by a method including mass spectrometry such as LC-MS and LC-MS / MS. At this time, the method for quantifying desmosines includes, for example, a step of adding the compound in the present embodiment to a sample to be measured. In addition, the compound in the present embodiment can be used as an internal standard substance in COPD biomarker analysis, for example.
In addition, since the compound of this embodiment does not require an isotope labeling step during the production process, it is easier to synthesize than the isotope labeled compound of desmosines. For this reason, the compound of this embodiment is suitable for mass production.
In addition, if compounds having the same structure are present at the same time, even if the mass numbers are different in mass spectrometry, they may interfere with each other and impair strictness. In contrast, by using the compound of the present embodiment as an internal standard substance for mass spectrometry, for example, it is possible to suppress interference with desmosines, which can contribute to stricter quantitative analysis.
（実施例１）
本実施例では、スキーム２に示す手順にてデスモシン−ＣＨ2（化合物１８）の合成をおこなった。また、デスモシン−ＣＨ2（化合物１８）およびこれに関連する化合物の炭素原子の番号付けは以下の通りである。
Example 1
In this example, desmosine-CH 2 (compound 18) was synthesized according to the procedure shown in
本実施例にて用いた試薬、分析装置等は以下の通りである。
（試薬）
非水系の反応は、すべて、窒素雰囲気下、マグネチックスターラーを用いておこない、特に記載のない場合には蒸留した新鮮な溶媒を用いた。Diisopropylethylamine (iPr2NEt) および trimethylsilyl chloride (TMSCl) は蒸留により乾燥した。Dimethylformamide (DMF) はMgSO4 により蒸留し、活性化したモレキュラーシーブ上で保存した。脱水したtetrahydrofuran (THF)、methanol (MeOH) および ethanol (EtOH) は関東化学社より購入し、活性化したモレキュラーシーブ上で保存した。すべての試薬を製造業者より入手し、特に記載のない場合にはさらに精製することなく用いた。
The reagents, analyzers, etc. used in this example are as follows.
(reagent)
All non-aqueous reactions were carried out using a magnetic stirrer under a nitrogen atmosphere, and fresh distilled solvent was used unless otherwise specified. Diisopropylethylamine (iPr 2 NEt) and trimethylsilyl chloride (TMSCl) were dried by distillation. Dimethylformamide (DMF) was distilled over MgSO 4 and stored over activated molecular sieves. Dehydrated tetrahydrofuran (THF), methanol (MeOH), and ethanol (EtOH) were purchased from Kanto Chemical Co. and stored on activated molecular sieves. All reagents were obtained from the manufacturer and used without further purification unless otherwise noted.
（分析）
薄層クロマトグラフィー（Thin Layer Chromatography：ＴＬＣ）分析にはメルク社製のSilica gel 60 F254プレートを用いた。
カラムクロマトグラフィーには、酸性 Silica gel 60 (spherical, 40-50 μm) または中性Silica gel 60N (spherical, 40-50 μm) を用いた（いずれも関東化学社製）。
逆相シリカゲルクロマトグラフィーは、Wako gel 100 C18（和光純薬工業社製）を用いておこなった。
融点の測定には、ATM-01装置（アズワン社製）を用いた。
旋光度の測定には、JASCO P-2200 デジタル旋光計およびナトリウムランプ (λ= 589 nm) Ｄ線を用いた。測定結果を「[α]D T (c g/100 mL, solvent)」と表記する。
ＵＶスペクトル測定にはJASCO V-560 UV/VIS分光光度計を用いた。
赤外線（ＩＲ）スペクトル測定には、JASCO FT-IR 4100分光計を用いた。測定結果を波数(cm-1) で示す。
1H および 13C NMRスペクトル測定には、JEOL JNM-EXC 300 スペクトロメータ (300 MHz) または JEOL JNM-ECA 500 スペクトロメータ (500 MHz) を用いた。
1H NMRデータは、以下のように示す：化学シフト (δ, ppm)、積分値、多重度 (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet), カップリング定数 (J) in Hz、帰属。
13C NMR データは、化学シフト (δ, ppm)により示す。
EI-MS スペクトル測定は、Shimadzu GCMS QP-5050 装置またはJEOL JMS-700 装置によりおこなった。FAB-MS スペクトル測定にもJEOL JMS-700を用いた。ESI-MSスペクトル測定は、JEOL JMS-T100LC 装置またはThermo Exactive スペクトロメータを用いた。
(analysis)
For thin layer chromatography (TLC) analysis, a Merca Silica gel 60 F254 plate was used.
For column chromatography, acidic Silica gel 60 (spherical, 40-50 μm) or neutral Silica gel 60N (spherical, 40-50 μm) was used (both manufactured by Kanto Chemical Co., Inc.).
Reversed phase silica gel chromatography was performed using Wako gel 100 C 18 (manufactured by Wako Pure Chemical Industries, Ltd.).
An ATM-01 apparatus (manufactured by ASONE) was used for measuring the melting point.
A JASCO P-2200 digital polarimeter and a sodium lamp (λ = 589 nm) D-ray were used to measure the optical rotation. The measurement result is expressed as “[α] D T (cg / 100 mL, solvent)”.
JASCO V-560 UV / VIS spectrophotometer was used for UV spectrum measurement.
A JASCO FT-IR 4100 spectrometer was used for infrared (IR) spectrum measurement. The measurement result is shown by wave number (cm −1 ).
JEOL JNM-
1 H NMR data is shown as follows: chemical shift (δ, ppm), integral, multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet), coupling constant (J) in Hz, attribution.
13 C NMR data is indicated by chemical shift (δ, ppm).
EI-MS spectrum measurement was performed with Shimadzu GCMS QP-5050 instrument or JEOL JMS-700 instrument. JEOL JMS-700 was also used for FAB-MS spectrum measurement. For the ESI-MS spectrum measurement, a JEOL JMS-T100LC apparatus or a Thermo Exactive spectrometer was used.
Benzyl 2-(S)-[(tert-butoxycarbonyl)amino]-hex-5-ynoate（化合物２０）の合成 Synthesis of Benzyl 2- (S)-[(tert-butoxycarbonyl) amino] -hex-5-ynoate (Compound 20)
CuCN (322.5 mg, 3.58 mmol, 1.0 eq)およびLiCl (309.3 mg, 7.16 mmol, 2.0 eq) を１つのフラスコに合わせ入れ、減圧下で150℃にて２時間加熱乾燥した。CuCNおよびLiClの乾燥中に、ヒートガンを用いて亜鉛粉末(1.45 g, 22.2 mmol, 6.2 eq)を減圧下で５分間加熱乾燥した。そして、亜鉛粉末の入ったフラスコにDMF (1.5 mL)およびTMSCl (0.14 mL, 1.1 mmol, 5 mol% to Zn)を室温（２５℃、以下同じ。）で加えた。室温にて３０分撹拌後の懸濁液に、2-(S)-2-(benzyloxycarbonylamino)-4-iodobutyric acid benzyl ester （化合物１４）(1.5 g, 3.58 mmol, 1.0 eq)のDMF溶液(1.0 mL, washed with 0.5 mL x 2)を加えた。化合物１４にＺｎが導入されたことをＴＬＣにて確認した。その後、別のフラスコ中で−５℃に冷却しておいたCuCNおよびLiClのDMF (7.5 mL)溶液を、Ｚｎが導入された化合物１４の溶液に３０分かけて添加した。
−５℃にて１５分撹拌後、−２０℃に冷却し、化合物１４の反応混合物に、新たに調製した(2-bromoethynyl)trimethylsilane (1.0 mL, 7.16 mmol, 2.0 eq)を５分かけて滴下した。その後、混合液を徐々に室温まで温め、１７時間撹拌を続けた。得られた混合物をEtOAcで希釈し、飽和NH4Cl溶液でクエンチした。続いて、EtOAcにより水層を抽出した。有機層をあわせて食塩水で洗浄し、Na2SO4上で乾燥させ、さらに真空中で濃縮した。これをシリカゲルカラムクロマトグラフィー(hexane/EtOAc = 20/1→10/1)により精製し、化合物19を非分離副生物とともに得た(870.4 mg)。
得られた粗生成物のTHF (30.0 mL)およびEtOH (654μL)溶液を0 ℃に冷却し、そこにtetrabutylammonium fluoride (TBAF) (607μL, 1.0 M solution in THF)を加えた。0 ℃にて1.5時間撹拌した後、反応混合物をEtOAcにて希釈し、飽和NH4Cl溶液でクエンチした。そして、水層をEtOAcにて抽出した。有機層をあわせて食塩水で洗浄し、Na2SO4上で乾燥させ、さらに真空中で濃縮した。これをシリカゲルカラムクロマトグラフィー(hexane/EtOAc = 15/1)により精製し、化合物20を白色固体として得た(496.4 mg, 1.18 mmol, 44% (2 steps)); Rf 0.29 (hexane/EtOAc = 8/1); [α]D 20 +5.7 (c 0.1, CHCl3); mp 61-62 ℃; IR (ATR, cm-1) 3400, 3326, 1755, 1683, 1514, 1451, 1368, 1295, 1254, 1213, 1160, 1052, 959, 755, 695, 638; 1H NMR (300 MHz, CDCl3) δ7.37-7.33 (5H, m, Bn), 5.22-5.17 (2H, m, Bn), 5.13 (1H, s, 17NH), 4.43 (1H, m, H17), 2.29-2.23 (2H, m, H15), 2.13-2.04 (1H, m, H16), 1.96 (1H, t, J = 2.84 Hz, H13), 1.92-1.79 (1H, m, H16), 1.43 (9H, s, t-Bu); 13C NMR (75 MHz, CDCl3) δ171.8, 155.0, 135.0, 128.3, 128.2, 128.0, 82.5, 79.7, 69.0, 66.9, 52.6, 31.1, 28.0, 14.5; FAB-MS (m/z) calcd for C18H24NO4 [M+H]+ 318.17, found 318.25。
CuCN (322.5 mg, 3.58 mmol, 1.0 eq) and LiCl (309.3 mg, 7.16 mmol, 2.0 eq) were put into one flask and heat-dried at 150 ° C. for 2 hours under reduced pressure. During the drying of CuCN and LiCl, zinc powder (1.45 g, 22.2 mmol, 6.2 eq) was heat-dried under reduced pressure for 5 minutes using a heat gun. Then, DMF (1.5 mL) and TMSCl (0.14 mL, 1.1 mmol, 5 mol% to Zn) were added to the flask containing zinc powder at room temperature (25 ° C., the same applies hereinafter). To the suspension after stirring at room temperature for 30 minutes, a DMF solution (1.0 g) of 2- (S) -2- (benzyloxycarbonylamino) -4-iodobutyric acid benzyl ester (compound 14) (1.5 g, 3.58 mmol, 1.0 eq) was added. mL, washed with 0.5 mL x 2) was added. It was confirmed by TLC that Zn was introduced into
After stirring at −5 ° C. for 15 minutes, the solution was cooled to −20 ° C., and newly prepared (2-bromoethynyl) trimethylsilane (1.0 mL, 7.16 mmol, 2.0 eq) was added dropwise to the reaction mixture of
A solution of the obtained crude product in THF (30.0 mL) and EtOH (654 μL) was cooled to 0 ° C., and tetrabutylammonium fluoride (TBAF) (607 μL, 1.0 M solution in THF) was added thereto. After stirring at 0 ° C. for 1.5 hours, the reaction mixture was diluted with EtOAc and quenched with saturated NH 4 Cl solution. The aqueous layer was extracted with EtOAc. The organic layers were combined, washed with brine, dried over Na 2 SO 4 and further concentrated in vacuo. This was purified by silica gel column chromatography (hexane / EtOAc = 15/1) to give compound 20 as a white solid (496.4 mg, 1.18 mmol, 44% (2 steps)); R f 0.29 (hexane / EtOAc = 8/1); [α] D 20 +5.7 (c 0.1, CHCl 3 ); mp 61-62 ° C; IR (ATR, cm -1 ) 3400, 3326, 1755, 1683, 1514, 1451, 1368, 1295, 1254, 1213, 1160, 1052, 959, 755, 695, 638; 1 H NMR (300 MHz, CDCl 3 ) δ7.37-7.33 (5H, m, Bn), 5.22-5.17 (2H, m, Bn), 5.13 (1H, s, 17NH), 4.43 (1H, m, H17), 2.29-2.23 (2H, m, H15), 2.13-2.04 (1H, m, H16), 1.96 (1H, t, J = 2.84 Hz , H13), 1.92-1.79 (1H, m, H16), 1.43 (9H, s, t-Bu); 13 C NMR (75 MHz, CDCl 3 ) δ171.8, 155.0, 135.0, 128.3, 128.2, 128.0, 82.5, 79.7, 69.0, 66.9, 52.6, 31.1, 28.0, 14.5; FAB-MS (m / z) calcd for C 18 H 24 NO 4 [M + H] + 318.17, found 318.25.
(S)-Benzyl 2-(tert-butoxycarbonylamino)-6-(3,5-dibromo-pyridin-4-yl)-hex-5-ynoate (化合物21)の合成 Synthesis of (S) -Benzyl 2- (tert-butoxycarbonylamino) -6- (3,5-dibromo-pyridin-4-yl) -hex-5-ynoate (Compound 21)
3,5-dibromo-4-iodopyridine (化合物１２：249.1 mg, 0.69 mmol, 1.0 eq)、2-tert-butoxycarbonylamino-hex-5-ynoic acid benzyl ester (化合物２０：327.1 mg, 1.03 mmol, 1.5 eq)、tris(dibenzylideneacetone)dipalladium (0) (Pd2dba3) (64.2 mg, 70.0 μmol, 10 mol%)、P(2-furyl)3 (64.9 mg, 0.28 mmol, 40 mol%)およびCuI (55.7 mg, 0.28 mmol, 40 mol%)のDMF (34.5 mL)溶液を、freeze/pump/thawサイクルにより脱気し、得られた溶液にiPr2NEt (6.9 mL)を加えた。４０℃にて１７時間撹拌後、反応混合物をEtOAcで希釈し、飽和NH4Cl溶液でクエンチした。続いて、EtOAcにより水層を抽出した。有機層をあわせて食塩水で洗浄し、Na2SO4上で乾燥させ、さらに真空中で濃縮した。これをフラッシュカラムクロマトグラフィー(hexane/ethyl acetate = 5/1)により精製し、化合物２１を白色固体として得た(276.3 mg, 0.50 mmol, 73%)； Rf 0.27 (hexane/EtOAc = 5/1); [α]D 20 +9.9 (c 0.1, CHCl3); mp 114-115 ℃; IR (ATR, cm-1) 3348, 1753, 1682, 1518, 1448, 1360, 1303, 1215, 1166, 1055, 959, 752, 694, 606; 1H NMR (300 MHz, CDCl3) δ8.61 (2H, s, H2/6), 7.37-7.36 (5H, m, Bn), 5.24-5.18 (2H, m, Bn), 5.14 (1H, m, 17NH), 4.52 (1H, m, H17), 2.67-2.61 (2H, m, H15), 2.32-2.21 (1H, m, H16), 2.08-1.96 (1H, m, H16), 1.42 (9H, s, t-Bu); 13C NMR (75 MHz, CDCl3) δ172.0, 155.5, 149.7, 135.3, 134.7, 128.8, 128.6, 128.4, 123.5, 104.3, 80.2, 78.1, 67.4, 53.1, 31.3, 28.4, 16.7; ESI-HRMS (m/z) calcd for C23H24Br2N2NaO4 [M+Na]+ 574.9980, found 574.9980。 3,5-dibromo-4-iodopyridine (Compound 12: 249.1 mg, 0.69 mmol, 1.0 eq), 2-tert-butoxycarbonylamino-hex-5-ynoic acid benzyl ester (Compound 20: 327.1 mg, 1.03 mmol, 1.5 eq) , Tris (dibenzylideneacetone) dipalladium (0) (Pd 2 dba 3 ) (64.2 mg, 70.0 μmol, 10 mol%), P (2-furyl) 3 (64.9 mg, 0.28 mmol, 40 mol%) and CuI (55.7 mg , 0.28 mmol, 40 mol%) in DMF (34.5 mL) was degassed by freeze / pump / thaw cycle, and iPr 2 NEt (6.9 mL) was added to the resulting solution. After stirring at 40 ° C. for 17 hours, the reaction mixture was diluted with EtOAc and quenched with saturated NH 4 Cl solution. Subsequently, the aqueous layer was extracted with EtOAc. The organic layers were combined, washed with brine, dried over Na 2 SO 4 and further concentrated in vacuo. This was purified by flash column chromatography (hexane / ethyl acetate = 5/1) to obtain Compound 21 as a white solid (276.3 mg, 0.50 mmol, 73%); R f 0.27 (hexane / EtOAc = 5/1 ); [α] D 20 +9.9 (c 0.1, CHCl 3 ); mp 114-115 ° C; IR (ATR, cm -1 ) 3348, 1753, 1682, 1518, 1448, 1360, 1303, 1215, 1166, 1055 , 959, 752, 694, 606; 1 H NMR (300 MHz, CDCl 3 ) δ8.61 (2H, s, H2 / 6), 7.37-7.36 (5H, m, Bn), 5.24-5.18 (2H, m , Bn), 5.14 (1H, m, 17NH), 4.52 (1H, m, H17), 2.67-2.61 (2H, m, H15), 2.32-2.21 (1H, m, H16), 2.08-1.96 (1H, m, H16), 1.42 (9H, s, t-Bu); 13 C NMR (75 MHz, CDCl 3 ) δ172.0, 155.5, 149.7, 135.3, 134.7, 128.8, 128.6, 128.4, 123.5, 104.3, 80.2, 78.1, 67.4, 53.1, 31.3, 28.4, 16.7; ESI-HRMS (m / z) calcd for C 23 H 24 Br 2 N 2 NaO 4 [M + Na] + 574.9980, found 574.9980.
((2S,2'S)-benzyl 4,4'-(4-((S)-6-(benzyloxy)-5-(tert-butoxycarbonyl amino)-6-oxopent-1-ynyl)pyridine-3,5-diyl)bis(2-(tert-butoxycarbonylamino)butanoate))(化合物22)の合成
((2S, 2'S) -
亜鉛末(198 mg, 3.0 mmol) を窒素パージした1.5 mLマイクロチューブに入れた。これに乾燥DMF (150 μL) およびTMSCl (60.0 μL, 0.47 mmol) を加え、得られた混合物を室温にて１５分間強く撹拌した。撹拌を停止し、マイクロシリンジを用いて溶液を除去した。残った固体を減圧下で熱空気銃を用いて乾燥させた。得られた活性亜鉛を室温に冷却し、これに2-(S)-2-(benzyloxycarbonylamino)-4-iodobutyric acid tert-butyl ester (化合物１４：217.5 mg, 0.5 mmol, 5.0 eq)の乾燥DMF (150 μL and rinsed with 100 μL DMF) 溶液を加えた。反応混合物を室温にて１時間撹拌した。ＴＬＣ分析(hexane/ethyl acetate = 5/1) にて出発物質が残存していないことを確認した後、撹拌を停止し、遠心分離機を用いて亜鉛末を沈澱させた。マイクロシリンジおよび200 μL のDMFにより亜鉛末中の溶液を取り出し、Pd-pyridine-enhanced precatalyst preparation stabilization and initiation (PEPPSI（商標）)-IPr (13.2 mg, 20 mol%)および化合物21 (55.2 mg, 0.1 mmol, 1.0 eq) の入った10 mLのフラスコに加えた。６０℃にて１．５時間撹拌を続け、反応混合物をEtOAcで希釈し、飽和NH4Cl溶液でクエンチした。EtOAcにより水層を抽出した。有機層をあわせて食塩水で洗浄し、Na2SO4上で乾燥させ、さらに真空中で濃縮して黄色油状の粗生成物を得た。これをフラッシュカラムクロマトグラフィー(hexane/ethyl acetate = 2/1→1/1)により精製し、化合物２２の精製物を黄色油状物質として得た (77.8 mg, 0.83 mmol, 83%) ；Rf 0.42 (hexane/EtOAc = 1/1); [α]D 20 +23.5 (c 0.1, CHCl3); IR (ATR, cm-1) 3783, 3465, 3084, 2976, 2361, 1713, 1514, 1452, 1367, 1254, 1168, 1053, 859, 748, 697; 1H NMR (300 MHz, CDCl3) δ8.18 (2H, s, H2/6), 7.33-7.30 (15H, m, Bn), 5.38 (1H, s, 17NH), 5.32-5.30 (2H, m, 21NH/21’NH), 5.19-5.09 (6H, m, Bn), 4.43 (1H, m, H17), 4.38-4.36 (3H, m, H11/21/21’), 2.77-2.68 (4H, m, H19/19’), 2.58-2.54 (2H, m, H16), 2.20-1.84 (6H, m, H16/20/20’), 1.43-1.40 (27H, m, t-Bu); 13C NMR (75 MHz, CDCl3) δ172.3, 172.1, 155.5, 149.4, 147.7, 135.4, 135.3, 128.7, 128.6, 128.4, 128.3, 80.0, 67.3, 67.1, 53.5, 53.0, 33.0, 28.4, 28.1, 16.5; ESI-MS (m/z) calcd for C55H68N4NaO12 [M+Na]+ 999.47, found 979.44.; ESI-HRMS (m/z) calcd for C55H68N4NaO12 [M+Na]+ 999.4731, found 999.4744。 Zinc dust (198 mg, 3.0 mmol) was placed in a 1.5 mL microtube purged with nitrogen. To this was added dry DMF (150 μL) and TMSCl (60.0 μL, 0.47 mmol) and the resulting mixture was stirred vigorously at room temperature for 15 minutes. Agitation was stopped and the solution was removed using a microsyringe. The remaining solid was dried using a hot air gun under reduced pressure. The obtained active zinc was cooled to room temperature, and then dried DMF (2- (S) -2- (benzyloxycarbonylamino) -4-iodobutyric acid tert-butyl ester (compound 14: 217.5 mg, 0.5 mmol, 5.0 eq)) 150 μL and rinsed with 100 μL DMF) solution was added. The reaction mixture was stirred at room temperature for 1 hour. After confirming that no starting material remained by TLC analysis (hexane / ethyl acetate = 5/1), stirring was stopped and zinc powder was precipitated using a centrifuge. Remove the solution in zinc dust with a microsyringe and 200 μL of DMF, Pd-pyridine-enhanced precatalyst preparation stabilization and initiation (PEPPSI ™) -IPr (13.2 mg, 20 mol%) and compound 21 (55.2 mg, 0.1 mmol, 1.0 eq) in a 10 mL flask. Stirring was continued at 60 ° C. for 1.5 hours, the reaction mixture was diluted with EtOAc and quenched with saturated NH 4 Cl solution. The aqueous layer was extracted with EtOAc. The organic layers were combined, washed with brine, dried over Na 2 SO 4 and further concentrated in vacuo to give a crude product as a yellow oil. This was purified by flash column chromatography (hexane / ethyl acetate = 2/1 → 1/1) to obtain a purified product of compound 22 as a yellow oily substance (77.8 mg, 0.83 mmol, 83%); R f 0.42 (hexane / EtOAc = 1/1); [α] D 20 +23.5 (c 0.1, CHCl 3 ); IR (ATR, cm -1 ) 3783, 3465, 3084, 2976, 2361, 1713, 1514, 1452, 1367 , 1254, 1168, 1053, 859, 748, 697; 1 H NMR (300 MHz, CDCl 3 ) δ8.18 (2H, s, H2 / 6), 7.33-7.30 (15H, m, Bn), 5.38 (1H , s, 17NH), 5.32-5.30 (2H, m, 21NH / 21'NH), 5.19-5.09 (6H, m, Bn), 4.43 (1H, m, H17), 4.38-4.36 (3H, m, H11 / 21/21 '), 2.77-2.68 (4H, m, H19 / 19'), 2.58-2.54 (2H, m, H16), 2.20-1.84 (6H, m, H16 / 20/20 '), 1.43- 1.40 (27H, m, t-Bu); 13 C NMR (75 MHz, CDCl 3 ) δ172.3, 172.1, 155.5, 149.4, 147.7, 135.4, 135.3, 128.7, 128.6, 128.4, 128.3, 80.0, 67.3, 67.1 , 53.5, 53.0, 33.0, 28.4, 28.1, 16.5; ESI-MS (m / z) calcd for C 55 H 68 N 4 NaO 12 [M + Na] + 999.47, found 979.44 .; ESI-HRMS (m / z ) calcd for C 55 H 68 N 4 NaO 12 [M + Na] + 999.4731, found 999.4744
(3,5-bis((S)-4-(Benzyloxy)-3-(tert-butoxycarbonylamino)-4-oxobutyl)-4-((S)-6-(benzyloxy)-5-(tert-butoxycarbonylamino)-6-oxopent-1-ynyl)-1-((S)-6-(benzyloxy)-5-(tert-butoxycarbonylamino)-6-oxohexyl)pyridinium iodide) (化合物26) の合成 (3,5-bis ((S) -4- (Benzyloxy) -3- (tert-butoxycarbonylamino) -4-oxobutyl) -4-((S) -6- (benzyloxy) -5- (tert-butoxycarbonylamino) -6-oxopent-1-ynyl) -1-((S) -6- (benzyloxy) -5- (tert-butoxycarbonylamino) -6-oxohexyl) pyridinium iodide) (Compound 26)
化合物２２(13.3 mg, 13.6 μmol, 1.0 eq)およびbenzyl 2-(S)-((tert-butoxycarbonyl)amino)-6-iodohexanoate (化合物２５：18.3 mg, 40.8 μmol, 3.0 eq) をCH3NO2 (1.0 mL) 中の混合物とし、６０℃で２３時間加熱した。その後、８０℃に昇温して２５時間反応させた。反応混合物を真空中で濃縮した。中性シリカゲルクロマトグラフィー (Hexane/Ethyl acetate = 1/1→CH2Cl2/MeOH = 10/1) により精製し、化合物２６(15.9 mg, 11.2 μmol, 82%) を黄色油状物質として得た； Rf 0.32 (CH2Cl2/MeOH = 10/1); [α]D 25 +10.9 (c 0.1, CHCl3); IR (ATR, cm-1) 3862, 3740, 3360, 2976, 2363, 2224, 1711, 1628, 1513, 1370, 1252, 1167, 1053, 861, 749, 698; 1H NMR (300 MHz, CDCl3) δ8.95 (2H, s, H2/6), 7.34-7.31 (20H, m, Bn), 5.62-5.59 (2H, m, 21NH/21’NH), 5.33 (1H, s, 17NH), 5.24 (1H, s, 11NH), 5.21-5.11 (8H, m, Bn), 4.73-4.66 (1H, m, H7), 4.57-4.53 (1H, m, H7), 4.40-4.39 (1H, m, H17), 2.97 (4H, t, J = 7.35 Hz, H19/19’), 2.64 (2H, t, J = 6.15 Hz, H15), 2.27-1.94 (8H, m, H8/16/20/20’), 1.91-1.65 (4H, m, H9/10), 1.42-1.39 (36H, m, t-Bu); 13C NMR (75 MHz, CDCl3) δ172.2, 155.7, 142.9, 135.4, 135.3, 135.2, 128.7, 128.5, 80.2, 67.5, 67.4, 67.2, 53.1, 29.7, 28.4, 17.1; ESI-HRMS (m/z) calcd for C73H94N5O16 [M]+ 1296.6673, found 1296.6696。 Compound 22 (13.3 mg, 13.6 μmol, 1.0 eq) and benzyl 2- (S)-((tert-butoxycarbonyl) amino) -6-iodohexanoate (compound 25: 18.3 mg, 40.8 μmol, 3.0 eq) were converted to CH 3 NO 2 (1.0 mL) and heated at 60 ° C. for 23 hours. Then, it heated up at 80 degreeC and made it react for 25 hours. The reaction mixture was concentrated in vacuo. Purification by neutral silica gel chromatography (Hexane / Ethyl acetate = 1/1 → CH 2 Cl 2 / MeOH = 10/1) gave compound 26 (15.9 mg, 11.2 μmol, 82%) as a yellow oil; R f 0.32 (CH 2 Cl 2 / MeOH = 10/1); [α] D 25 +10.9 (c 0.1, CHCl 3 ); IR (ATR, cm -1 ) 3862, 3740, 3360, 2976, 2363, 2224 , 1711, 1628, 1513, 1370, 1252, 1167, 1053, 861, 749, 698; 1 H NMR (300 MHz, CDCl 3 ) δ8.95 (2H, s, H2 / 6), 7.34-7.31 (20H, m, Bn), 5.62-5.59 (2H, m, 21NH / 21'NH), 5.33 (1H, s, 17NH), 5.24 (1H, s, 11NH), 5.21-5.11 (8H, m, Bn), 4.73 -4.66 (1H, m, H7), 4.57-4.53 (1H, m, H7), 4.40-4.39 (1H, m, H17), 2.97 (4H, t, J = 7.35 Hz, H19 / 19 '), 2.64 (2H, t, J = 6.15 Hz, H15), 2.27-1.94 (8H, m, H8 / 16/20/20 '), 1.91-1.65 (4H, m, H9 / 10), 1.42-1.39 (36H, m, t-Bu); 13 C NMR (75 MHz, CDCl 3 ) δ172.2, 155.7, 142.9, 135.4, 135.3, 135.2, 128.7, 128.5, 80.2, 67.5, 67.4, 67.2, 53.1, 29.7, 28.4, 17.1 ESI-HRMS (m / z) calcd for C 73 H 94 N 5 O 16 [M] + 1296.6673, found 1296.6696.
Desmosine-CH2 (化合物18) の合成 Synthesis of Desmosine-CH 2 (Compound 18)
化合物２６(33.3 mg, 23.4 μmol, 1.0 eq) のMeOH (1.0 mL) 溶液を10% Pd/C (125.6 mg, 116.9 μmol, 5.0 eq) で処理し、バルーン圧にて水素添加した。４０℃にて４日間撹拌後、中性シリカゲル上のセライトパッドで濾過し、ＭｅＯＨで溶出することにより不溶物を分離した。濾液を濃縮して得られた粗生成物(19.0 mg) にMeOH (0.6 mL) を加え、10% Pd/C (71.6 mg) で処理し、バルーン圧、４０℃にて水素添加した。２日間撹拌後、中性シリカゲル上のセライトパッドで濾過し、ＭｅＯＨで溶出することにより不溶物を分離し、濾液を真空中で濃縮した。得られた粗生成物 (13.6 mg) をさらに精製することなく次の反応に用いた。
トリフルオロ酢酸（ＴＦＡ）および蒸留水の混合物 (2.9 mL, TFA/water = 95/5) を濾液に加え、室温で３時間撹拌した。その後、溶媒を真空中で除去した。Ｃ１８シリカゲルカラムクロマトグラフィー (0.1% TFA in distilled water) により精製し、化合物１８ (6.7 mg, 10.3 μmol, 44% (2 steps)) を黄色油状物質として得た； Rf 0.22 [MeOH (0.1% TFA)/H2O (0.1% TFA)]; [α]D 20 +16.4 (c 0.1, H2O); 1H NMR (500 MHz, D2O) δ8.55 (2H, s, H2/6),4.1 (2H, t, J = 7.6 Hz, H7), 4.10 (2H, t, J = 6.1 Hz,H21/21’), 5.14 (1H, m, NH), 4.04-3.97 (2H, m, H11/17), 3.10-3.04 and 2.97-2.89 (4H, m, H19/19’), 2.97-2.89 (2H, m, H13), 2.26-2.21 (4H, m, H20/20’), 2.07-1.94 (8H, m, H8/10/15/16), 1.68-1.55 (4H, m, H9/14); 13C NMR (75 MHz, D2O) δ173.2, 173.1, 172.8 (C12/18/22/22’), 160.1 (C4), 142.3 (C2/6), 140.3 (C3/5), 61.2 (C7), 53.7, 53.6 (C11/17/21/21’), 31.0, 30.5, 30.1, 29.8 (C8/10/15/20/20’), 29.1 (C13), 26.1, 25.2 (C9/14), 21.6 (C16); ESI-HRMS (m/z) calcd for C25H42N4O8 [M]+ 540.3033, found 540.3031。
A solution of compound 26 (33.3 mg, 23.4 μmol, 1.0 eq) in MeOH (1.0 mL) was treated with 10% Pd / C (125.6 mg, 116.9 μmol, 5.0 eq) and hydrogenated with balloon pressure. After stirring at 40 ° C. for 4 days, the mixture was filtered through a celite pad on neutral silica gel and eluted with MeOH to separate insoluble materials. MeOH (0.6 mL) was added to the crude product (19.0 mg) obtained by concentrating the filtrate, treated with 10% Pd / C (71.6 mg), and hydrogenated at 40 ° C. under balloon pressure. After stirring for 2 days, the mixture was filtered through a celite pad on neutral silica gel, eluted with MeOH to separate insoluble materials, and the filtrate was concentrated in vacuo. The obtained crude product (13.6 mg) was used in the next reaction without further purification.
A mixture of trifluoroacetic acid (TFA) and distilled water (2.9 mL, TFA / water = 95/5) was added to the filtrate, and the mixture was stirred at room temperature for 3 hours. The solvent was then removed in vacuo. Purification by C18 silica gel column chromatography (0.1% TFA in distilled water) gave Compound 18 (6.7 mg, 10.3 μmol, 44% (2 steps)) as a yellow oil; R f 0.22 [MeOH (0.1% TFA ) / H 2 O (0.1% TFA)]; [α] D 20 +16.4 (c 0.1, H 2 O); 1 H NMR (500 MHz, D 2 O) δ8.55 (2H, s, H2 / 6 ), 4.1 (2H, t, J = 7.6 Hz, H7), 4.10 (2H, t, J = 6.1 Hz, H21 / 21 '), 5.14 (1H, m, NH), 4.04-3.97 (2H, m, H11 / 17), 3.10-3.04 and 2.97-2.89 (4H, m, H19 / 19 '), 2.97-2.89 (2H, m, H13), 2.26-2.21 (4H, m, H20 / 20'), 2.07- 1.94 (8H, m, H8 / 10/15/16), 1.68-1.55 (4H, m, H9 / 14); 13 C NMR (75 MHz, D 2 O) δ 173.2, 173.1, 172.8 (C12 / 18 / 22/22 '), 160.1 (C4), 142.3 (C2 / 6), 140.3 (C3 / 5), 61.2 (C7), 53.7, 53.6 (C11 / 17/21/21'), 31.0, 30.5, 30.1 , 29.8 (C8 / 10/15/20/20 '), 29.1 (C13), 26.1, 25.2 (C9 / 14), 21.6 (C16); ESI-HRMS (m / z) calcd for C 25 H 42 N 4 O 8 [M] + 540.3033, found 540.3031.
（実施例２）
本例では、実施例１で得られたdesmosine-CH2（「des-CH2」とも呼ぶ。）のLC-MSによる分析をおこなった。
desmosine-CH2の溶液（3.4 mg/ml in 1mM HFBA、3 mM NH4Ac aq）を希釈してLC-MS用のサンプル（100 μg/ml）を調製し、以下の条件にて分析をおこなった。
・LC-MS（Waters社製）
・カラム温度：30 ℃
・オートサンプラー内温度：18 ℃
・インジェクション量：10 μL
・流速：0.2 mL/min
・移動相溶媒：A…7 mM heptafluorobutyric acid (HFBA) および 5 mM NH4Ac aq
B…7 mM HFBA および 5 mM NH4Ac in 80% Acetonitrile
・溶媒比率 (A:B)：時間（分） A B
0-6 100 0
6-7 95 5
7-8 10 90
・MS分析時間：1-8 min
・Cone voltage：45 V
(Example 2)
In this example, desmosine-CH 2 (also referred to as “des-CH 2 ”) obtained in Example 1 was analyzed by LC-MS.
Prepare a sample (100 μg / ml) for LC-MS by diluting the desmosine-CH 2 solution (3.4 mg / ml in 1 mM HFBA, 3 mM NH 4 Ac aq), and perform the analysis under the following conditions. It was.
・ LC-MS (manufactured by Waters)
-Column temperature: 30 ° C
・ Autosampler temperature: 18 ℃
・ Injection volume: 10 μL
・ Flow rate: 0.2 mL / min
・ Mobile phase solvent: A… 7 mM heptafluorobutyric acid (HFBA) and 5 mM NH 4 Ac aq
B… 7 mM HFBA and 5 mM NH 4 Ac in 80% Acetonitrile
・ Solvent ratio (A: B): Time (min) AB
0-6 100 0
6-7 95 5
7-8 10 90
・ MS analysis time: 1-8 min
・ Cone voltage: 45 V
分析の結果、保持時間は6.1 minでありピークが分析時間内に下がりきることが確認された。
また、すべての化合物のMSを検出するfull scan modeにてdesmosine-CH2溶液を分析した結果、desmosine-CH2は540.5 m/zで検出された。そこで、MSで特定の質量の化合物のみを選択して検出するSelected Ion Monioring (SIM)では540.5 m/zを追跡することとした。これによりその他の質量の化合物は検出されなくなるため、より低濃度のサンプルであってもdesmosine-CH2を検出することができるようになる。
図１は、SIM modeおよびFull scan modeでのLC-MS測定結果を示す図である。
As a result of the analysis, the retention time was 6.1 min, and it was confirmed that the peak fell within the analysis time.
Moreover, as a result of analyzing the desmosine-CH 2 solution in the full scan mode for detecting MS of all the compounds, desmosine-CH 2 was detected at 540.5 m / z. Therefore, it was decided to track 540.5 m / z in Selected Ion Monioring (SIM), which selects and detects only compounds with a specific mass by MS. As a result, other mass compounds are not detected, and desmosine-CH 2 can be detected even in a sample having a lower concentration.
FIG. 1 is a diagram showing LC-MS measurement results in SIM mode and Full scan mode.
検量線の作成
様々な濃度のサンプル（200, 1000, 2000, 5000, 10000, 15000, 20000 ng/mL）を調製し、SIMモードにて測定をおこなった。各サンプルについて測定されたピーク面積値から検量線を作成したところ、以下の式が得られた。
y = 4.5928x - 1631.2 (R2 = 0.9978)
上記式中、ｘはdesmosine-CH2の濃度であり、ｙはピーク面積値である。
測定結果を表１および図２に示す。
Preparation of calibration curve Samples of various concentrations (200, 1000, 2000, 5000, 10000, 15000, 20000 ng / mL) were prepared and measured in SIM mode. When a calibration curve was created from the peak area values measured for each sample, the following formula was obtained.
y = 4.5928x-1631.2 (R 2 = 0.9978)
In the above formula, x is the concentration of desmosine-CH 2 and y is the peak area value.
The measurement results are shown in Table 1 and FIG.
Desmosine-CH2の定量分析
検量線の信頼性を確認するため、QC (Quality Control)サンプルを用いて定量を行った。
QCサンプルとして既知濃度のdesmosine-CH2溶液（entry 1：2600 ng/mL、entry 2：16700 ng/mL）を新たに調製し、これをLC-MSによって分析した。そして得られたピーク面積値を式のyに代入することで濃度xを算出した。結果を表２に示す。
Quantitative analysis of Desmosine-CH 2 In order to confirm the reliability of the calibration curve, quantification was performed using a QC (Quality Control) sample.
As a QC sample, a desmosine-CH 2 solution (entry 1: 2600 ng / mL, entry 2: 16700 ng / mL) having a known concentration was newly prepared and analyzed by LC-MS. Then, the concentration x was calculated by substituting the obtained peak area value into y in the equation. The results are shown in Table 2.
理論値との誤差は±15%までが許容範囲であるとされている。表２より、entry 1および2における誤差はそれぞれ-10.2％、-4.0％であり、ともに許容範囲内であった。よって、得られた検量線は定量分析に用いることができる正確性を有することがわかる。
The tolerance for the theoretical value is up to ± 15%. From Table 2, the errors in
さらに、SIMモードでの分析において、移動相の比率、溶媒比を変える速度(curve)、cone voltageといった条件を1つずつ変えることで、さらなる条件の最適化をおこなった。その結果、desmosine-CH2のLC-MS分析の観点からは、
・溶媒比率（A:B）：85:15
・Curve：6
・Cone voltage：45 V
とすることが好ましい条件であった。
Furthermore, in the SIM mode analysis, the conditions were further optimized by changing the mobile phase ratio, the speed of changing the solvent ratio (curve), and the cone voltage one by one. As a result, from the viewpoint of LC-MS analysis of desmosine-CH 2 ,
・ Solvent ratio (A: B): 85:15
-Curve: 6
・ Cone voltage: 45 V
It was a preferable condition.
（実施例３）
本例では、実施例１で得られたdesmosine-CH2のLC-MS/MSによる分析をおこなった。
Full Scan Modeにおけるフラグメンテーションの確認
LC-MS/MS分析では、SIMモードで選択した分子を開裂させることで得られるフラグメンテーションを選択的に検出できる(Selected Reaction Monitoring = SRM )。そのため、SIMモードよりもさらに高感度となり、より低濃度の化合物を分析することが可能となる。
そこでまずはdesmosine-CH2のLC-MS/MS分析をおこなうために、すべての化合物を検出するfull scanにおいて測定し、化合物を開裂させるためのcollision energyを調節することでdesmosine-CH2から得られるフラグメンテーションを確認した。図３は、desmosine-CH2のLC-MS/MS測定で得られたフラグメンテーションを示す図である。
比較的高濃度である10000 ng/mLのdesmosine-CH2の溶液を、シリンジから一定の流速で直接MS/MSにインジェクトした。その後、測定画面を確認しながらcollision energyを調節し、生成するフラグメンテーションを確認したところ、40 Vのcollision energyにおいて495.14 m/zのピークが最も強く検出された。そこでdesmosine-CH2をSRMモードで分析する際には、collision energyを40 Vとし、追跡するフラグメンテーションを495.14 m/zとした。
(Example 3)
In this example, desmosine-CH 2 obtained in Example 1 was analyzed by LC-MS / MS.
Confirmation of fragmentation in Full Scan Mode
In LC-MS / MS analysis, fragmentation obtained by cleaving a molecule selected in SIM mode can be selectively detected (Selected Reaction Monitoring = SRM). Therefore, the sensitivity is further higher than in the SIM mode, and it becomes possible to analyze a compound at a lower concentration.
Therefore First To perform LC-MS / MS analysis of desmosine-CH 2, measured in full scan to detect all compounds obtained from desmosine-CH 2 by adjusting the collision energy for cleaving a compound Confirmed fragmentation. FIG. 3 is a diagram showing fragmentation obtained by LC-MS / MS measurement of desmosine-CH 2 .
A relatively high concentration solution of 10000 ng / mL desmosine-CH 2 was directly injected into the MS / MS from a syringe at a constant flow rate. Thereafter, the collision energy was adjusted while confirming the measurement screen, and the fragmentation to be generated was confirmed. As a result, the peak of 495.14 m / z was detected most strongly at 40 V collision energy. Therefore, when analyzing desmosine-CH 2 in SRM mode, the collision energy was set to 40 V, and the fragmentation to be tracked was set to 495.14 m / z.
分析条件の最適化、SRM モードにおける感度確認
LC-MS/MSにおけるdesmosine-CH2の分析条件は以下の通りである。
・LC-MS/MS（Thermo社製）
HPLC: FINNIGAN Surveyor
MS/MS: FINNIGAN Quantum discovery
・カラム：N-dc18-2x150 mm
・インジェクション量：10 μL
・流速：0.2 mL/min
・移動相溶媒：A…7 mM HFBA および 5 mM NH4Ac aq
B…7 mM HFBA および 5 mM NH4Ac in 80% Acetonitrile
・溶媒比率（A:B）：時間（分） A B
0-10 100 0
10-11 75 25
11-14 10 90
14-17 10 90
17-30 100 0
Optimization of analysis conditions, sensitivity check in SRM mode
Analysis conditions for desmosine-CH 2 in LC-MS / MS are as follows.
・ LC-MS / MS (manufactured by Thermo)
HPLC: FINNIGAN Surveyor
MS / MS: FINNIGAN Quantum discovery
・ Column: N-dc18-2x150 mm
・ Injection volume: 10 μL
・ Flow rate: 0.2 mL / min
・ Mobile phase solvent: A… 7 mM HFBA and 5 mM NH 4 Ac aq
B… 7 mM HFBA and 5 mM NH 4 Ac in 80% Acetonitrile
・ Solvent ratio (A: B): Time (min) AB
0-10 100 0
10-11 75 25
11-14 10 90
14-17 10 90
17-30 100 0
上記の条件において、100 ng/mlのdesmosine-CH2溶液のSRMモードによる分析について感度確認をおこなった結果、desmosine-CH2のピークが明確に確認された。測定結果を図４に示す。また、SRMモードではSIMモードよりもさらに低濃度のサンプルについても分析できることがわかった。 Under the above conditions, the sensitivity of the 100 ng / ml desmosine-CH 2 solution was analyzed using the SRM mode. As a result, the desmosine-CH 2 peak was clearly confirmed. The measurement results are shown in FIG. It was also found that the SRM mode can analyze even lower concentration samples than the SIM mode.
検量線の作成
各濃度のサンプル(2, 10, 20, 50, 100, 150, 200 ng/ml)をSRMモードにて分析し、ピーク面積値から検量線を作成したところ、以下の式が得られた。
y = 415.17x - 5379.2 (R2 = 0.967)
上記式中、ｘはdesmosine-CH2の濃度であり、ｙはピーク面積値である。
測定結果を表３および図５に示す。
Preparation of calibration curve Samples of each concentration (2, 10, 20, 50, 100, 150, 200 ng / ml) were analyzed in SRM mode, and a calibration curve was created from the peak area values. It was.
y = 415.17x-5379.2 (R 2 = 0.967)
In the above formula, x is the concentration of desmosine-CH 2 and y is the peak area value.
The measurement results are shown in Table 3 and FIG.
本明細書において、以下の略語を用いた。
Ｍｅ：メチル
Ｅｔ：エチル
Ｐｒ：プロピル
Ｂｕ：ブチル
Ａｃ：アセチル
ｒｔ：室温
ｍｉｎ：分
ｈ：時間
ｄ：日
In this specification, the following abbreviations were used.
Me: methyl Et: ethyl Pr: propyl Bu: butyl Ac: acetyl rt: room temperature min: minutes h: time d: day
Claims (5)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2014055337A JP6233929B2 (en) | 2014-03-18 | 2014-03-18 | Compound, standard substance for quantitative analysis using the same, and method for quantifying desmosines |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2014055337A JP6233929B2 (en) | 2014-03-18 | 2014-03-18 | Compound, standard substance for quantitative analysis using the same, and method for quantifying desmosines |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2015178957A JP2015178957A (en) | 2015-10-08 |
| JP6233929B2 true JP6233929B2 (en) | 2017-11-22 |
Family
ID=54263113
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2014055337A Active JP6233929B2 (en) | 2014-03-18 | 2014-03-18 | Compound, standard substance for quantitative analysis using the same, and method for quantifying desmosines |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP6233929B2 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP7195568B2 (en) * | 2018-03-22 | 2022-12-26 | 学校法人上智学院 | complex |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE10027794A1 (en) * | 2000-06-07 | 2001-12-13 | Basf Ag | Analyzing enzyme-catalyzed reactions of small compounds, useful e.g. in screening enzymatic activities, by matrix-assisted, laser desorption and ionization mass spectrometry |
| US7268117B2 (en) * | 2003-07-11 | 2007-09-11 | Praecis Pharmaceuticals Incorporated | Compositions, kits, and methods for identification, assessment, prevention, and therapy of endometriosis |
| JP2005249493A (en) * | 2004-03-02 | 2005-09-15 | Ono Pharmaceut Co Ltd | Method for quantifying desmosine and isodesmosine |
| JP2010210564A (en) * | 2009-03-12 | 2010-09-24 | Sumika Chemical Analysis Service Ltd | Method for measuring desmosine and isodesmosine |
| WO2011094343A1 (en) * | 2010-01-26 | 2011-08-04 | The Trustees Of Columbia University In The City Of New York | Methods of validating candidate compounds for use in treating copd and other diseases |
-
2014
- 2014-03-18 JP JP2014055337A patent/JP6233929B2/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| JP2015178957A (en) | 2015-10-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US9714260B2 (en) | Asymmetrical Si rhodamine and rhodol synthesis | |
| US9506929B2 (en) | Selective detection of amino group containing thiols | |
| US10261090B2 (en) | Glutathione-detecting fluorescent probe | |
| JP5223391B2 (en) | Sensitization assay method using fluorescent cysteine derivative and fluorescent cysteine derivative | |
| Wei et al. | A two-step responsive colorimetric probe for fast detection of formaldehyde in weakly acidic environment | |
| Khreit et al. | Synthesis, full chemical characterisation and development of validated methods for the quantification of the components found in the evolved “legal high” NRG-2 | |
| Renault et al. | Deeper insight into protease-sensitive “covalent-assembly” fluorescent probes for practical biosensing applications | |
| JP2023516154A (en) | Intercellular and intracellular proximity-based labeling compositions and systems | |
| CN112358414A (en) | Unnatural amino acids and their use in protein site-directed modification and protein interactions | |
| JP6233929B2 (en) | Compound, standard substance for quantitative analysis using the same, and method for quantifying desmosines | |
| Murakami et al. | Synthesis and LC-MS/MS analysis of desmosine-CH 2, a potential internal standard for the degraded elastin biomarker desmosine | |
| CN110603059B (en) | Chemiluminescent androstenedione conjugates | |
| CN102603756B (en) | Rhodamine B sulfide compound and preparation method and applications of Rhodamine B sulfide compound | |
| JP5219144B2 (en) | Novel affinity labeling method and screening method using the labeling method | |
| Payne et al. | Synthesis and protein conjugation studies of vitamin K analogues | |
| WO2017154997A1 (en) | Indole structure-selective crosslinking agent and composite in which same is used | |
| JP2018025399A (en) | Hydro-polysulfide detection fluorescence probe | |
| CN110156815A (en) | 12a hydrogen-benzo[f]pyridine[1,2-d][1,4]thiazepine and its preparation method | |
| JP6670502B2 (en) | Development of ligand screening system for neurotransmitter receptor | |
| FI90410B (en) | Optically active reagent and method for the determination of enantiomeric primary and secondary amines by HPLC | |
| JPWO2006019105A1 (en) | Fluorescent labeling agent | |
| JP5267096B2 (en) | N, S-di-tert-butoxycarbonylglutathione fluorescent derivative and method for producing glutathione fluorescent derivative using the same as an intermediate | |
| Rosen et al. | Discovery of potent cholecystokinin-2 receptor antagonists: Elucidation of key pharmacophore elements by X-ray crystallographic and NMR conformational analysis | |
| JP4174588B2 (en) | Heterocyclic compounds with active chlorine | |
| CN116987495B (en) | Construction and application of a protein membrane macromolecular probe for biological detection |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20170221 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20171017 |
|
| A977 | Report on retrieval |
Free format text: JAPANESE INTERMEDIATE CODE: A971007 Effective date: 20171020 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20171020 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 6233929 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |