CVv14V49S62011725.pdf

TAP CHI HOA HOC. T. 49(6) 725-732. THANG 12 NAM 2011. T 6 N G Hgp VA NGHIEN CLfU A N H HU'6'NG HIEU LfNG KHONG GIAN D6I vai PHAN L^NG NHUOM ESTERAZA DAC HIEU re BAO BACH CAU NGUai C O A C A C N-THe-3-CACBAIVldYLNAPHTHALEN-2-YL a-CLOCACBOXYLAT Tran Van Tinh'*, Phan Thj Trang', Nguyen Anh T^i^ Luu Van Boi' 'Tnrdng Dgi hpc KHTN. BHQGHN ^Vi4n Huyet hpc vd Truyin mdu Trung irong Den Tda sogn 13-6-2011. Abstract By direct reaction between 3-hydroxy-2-naphtoic acid and the corresponding amines and phosphorous trichloride there have been synthesized four N-substituted-3-hydroxy-naphthalene-2-carboxamides that were utilized as starting materials for the preparation of eight N-substituted-3-carbamoylnaphlhalene-2-yl a-chlorocarbocylates, including seven new substances. The laters were used as substrates for specific esterase staining of human leukocyte cells. The results showed that the substrale conlaining the substituent of medium-size (naphthyl) in the amide group have a higher sensitivity than those containing the small (phenyl, hexyl) or the large size (antraxyl) substituent. 1. DAT VAN DE 2. THUC NGHIEM Hipn nay mac dii di cd mpt sd phuong phip xic dinh vi phan logi te bao bgch ciu cd dg chinh xic cao hon, nhu phuang phap dinh diu [1] vi miln dich [2], nhung phuong phip nhudm hda hgc vin dugc dung rpng rii trong cic bpnh vien vi phdng thi nghiem nghien ciiu ve huyet hpc [3, 4]. Cd nhieu nguyen nhan thuc day irng dung phuang phip niy. Thii nhit la ky thugt don giin, de xii ly vi cho ket qua nhanh. Thti hai li gii thinh re, de tilp can ddi vdi mpi benh nhan. Vi vay, viec nghien ciiu tim kiem co chit mdi, khao sat mdi lien quan giiJa cau true vdi hoat tinh ciia co chit vi cii tien ky thugt tien hinh phin ling nhupm te bio vin li de tai thu hut sy chii y cija nhilu nhdm nghien ciru tren the gidi. Thyc nghipm cho thiy do chinh xic ciia phuang phap nhudm hda hpc chu yeu phy thudc vao ciu triic phan tii" ca chit. Trong cdng trinh cdng bd trudc day [5, 6], chiing tdi da chi ra rang, thii nhat, cac nhdm the day electron d vj tri ado ciia nhin phenyl lien ket vdi nhdm amit lim ting dg nhgy cua ca chit so vdi cic nhdm hut electron; thii hai, cimg logi nhdm thi thi anh hudng ciia vj tri acta ldn hon vj tri para. Trong cdng trinh niy chiing tdi tien hanh nghien ciru anh hudng yeu td khdng gian (kich thudc) cic nhdm thi ddi vdi dp nhay cua phin litig nhudm mau enzym esteraza die hieu bgch cau ngudi.. 2.1. Hoa chat va thiet bi PCI3 hda chat tinh khiet ciia Anh. Axit 3-hydroxynaphtoic hang Sigma. 2-cIoropropionyI chloride hang Sigma. Cic amin: cyclohexylamine 99,5%, anilin 99,5%, 1-naphthylamin, 1-aminoanthracene 90% ciia hang Sigma. Cic dung mdi: etanol 98%t ciia Trung Qudc, DMF logi P ciia Labosi, formandehyt 37% ACS 99,5% hing Sigma-Aldrich. Muoi diazo: Fast Blue RR Salt ciia hang Acros. KH2PO4 cua hang Kanto Chemical Co., INC Nhit Bin. Na2HP04 ciia hang P. P. H Polskie Odczynniki Chemiczne Gliwice Ba Lan. Nuclear fast red cua hang Sigma-Aldrich. Cic hda chit tren dugc dimg khdng tinh che lai. Miy u nhiet (Stat-Fax 2200, hang Awareness Technology INC, USA) O-SOT, do chinh xic ±0,rC, miy do pH (inoLab pH 730 ciia Diic) dp chinh xic din 0,001, cin phan tich dp chinh xic 0,0001, kinh hiln vi vdi dd phdng dgi 1.000 lin. Dilm ndng chay do tren miy Stuard STP 3 (Anh). Phd hdng ngogi do tren miy GX ghep ndi kinh hiln vi hdng ngoai ciia hang Perkin-Elmer trong khoang 400-4000 cm"' bing ep vien KBr d phdng thi nghiem Hda vat lieu, Khoa hda DHKHTN, DHQGHN.. Trdn Van Tinh vd cpng s{. TCHH, T. 49(6), 2011 Pho cdng hudng tir 'H-NMR, ghi tren miy Bruker AVANCE 500, 500 MHz, Vipn Khoa hgc va Cdng nghp Vipt Nam. Phd khoi lugng ghi tren may GC/MS phan giai cao AUTOSPEC Premier ciia Micromass Water, phdng thi nghipm Hda vgt lipu, Khoa Hda hpc, DHKHTN, DHQGHN. Dieu che cac N-phcnyl-3-hydroxynaphthalen-2cacboxamit Hda tan 0,01 moi axit 3-hydroxy-2-naphtoic va 0,01 moi anilin vio 16 ml clobenzcn. Hdn hgp dugc khuiy, lam Ignh den khoing lO^C. Nhd tir tir dung dich 0,003 moi PCb trong 3,5 ml clobenzcn vio hdn hgp phin irng. giii nhipt dg hdn hgp trong khoing tir 10 den 25*'C. Khoing 5 phut sau khi nhd gipt hit PCI3, dun hoi luu hdn hgp d HO^C khoang 2,5 gid. Trong suot thdi gian dd HCI bay ra va nhipt dp phin irng ting len. Khi HCI bay hit, nhipt dp phin irng len tdi 142^. Sau khi phin irng kit thiic, cit logi bdt clobenzcn, lim Ignh tir tir den nhicl dp phdng. Lpe ket tiia, ket tinh trong cdn. Tuong ty da dieu che cac N-the-3hydroxynaphthaIen-2-cacboxamit Ib-d khic. Hipu suit va mpt sd hang sd hda ly va cic sd lieu phd dugc dua ra trong bang I vi 2. Dieu che cac dan xuat N-phenyl-3cacbamoylnaphthaIen-2-yl a-cloaxetat Nhd tir tir 0,0011 moi NaOH 5 N lanh vio dung djch 0,001 moi N-the-3-hydroxynaphthalen-2cacboxamit trong THE, khuiy vi lim Ignh bing di mudi din 0°C. Sau khi cho het NaOH, tiep tuc nhd tir tir 0,0011 moi axit a-cloaxetyl clorua vio hdn hgp phin img. Khuiy diu 60 phut, de yen binh phin irng d nhiet dp phdng trong 1,5-2 gid. Dd hdn hgp vio nudc lanh, khuay, Igc ket tua. Sin phim dugc kit tinh trong hdn hc^ cdn-axeton va bio quin d nhipt dp thap. Tuong ty da dieu chl cic N-thl-3cacbamoyInaphthalen-2-yI a-clo-cac boxy lat llb-h khic. Hieu suit vi mgt so hang sd hda iy vi cic so lieu phd dugc dua ra trong bing 3 vi 4. Nhuom te bio bach cau bang cac ca chat dicu che dirge Pha che ihudc thu:. Dung djch cd djnh tieu bin: etanol-formol ty lp thi tich 9:1. Dung djch nhudm:. car: OH. + Co chat (cic este Ila-h tong hgp dugc d tren 0,480 mg. + Muoi diazo: 0,459 mg. + DMF: 43,5 ^1. + DMSO: 44 nl. + Dpm photphat pH = 7,42 (Sorensen ): 1.411,87 fil Dung djch nhugm nhan: Nuclear fast red 0, g/100 ml. B^nh phdm: mau mau ngogi vi ciia ngudi dii^. dan thinh 30 tieu bin dl khd trong dilu kipn l| nhien. Kp thugt tien hdnh nhupm: Co djnh tieu bin trong thdi gian 1 phut. Saiid( gdi ciic tieu bin vi dua vio hgp chong am bio quai d nhipt dg 4-8''C, thdi gian bio quin cd the keo dai; thing [9]. Phil dung djch nhupm chuan bj d tren len tiei bin dl trong binh cich thuy 37''C va thdi gian la It phiit. Sau do rira tieu bin dudi vdi nudc 5 phut. Nhupm nin bing dung djch nuclear fast rec trong 15 phiit d 3TC, nia nudc vi dl khd. Dgc tieu bin dudi vat kinh dau vdi dp phong dai 1.000 lan. Kit qui: Cic viing cd chira enzym esteraza b^cli cau trung tinh se xuit hien cac hgt bit mau xanh den (xem inh 1). Dinh gii mire dg duong tinh, tong so diem vi hieu suit bing phucmg phip phin dp tren kinh hien vi cd dg phdng dgi 1.000 Ian theo quy trinh ky thuat ciia hang Sigma-Aldrich [10]. 3. KET QUA VA THAO LUAN 3.1. Tong hgp cacboxamit. N-the-3-hydroxynaphthaleiht. Nham myc dich nghien ciiru inh hudng hieu ung khdng gian ciia nhdm amit the, cic amin dupe lita chgn la xiclohexyiamin, anilin, 1-naphtyl vi 1antraxyl. Cic chat dau N-thl-3-hydroxynaphthaIel^ 2-cacboxamit (la-d) dugc tong hgp theo phiBBig phip tryc tilp, nhu md ta trong tii lieu [7, 8], tfic la cho hdn hgp axit 3-hydroxy-2-naphtoic vi amin tucmg ling tic dyng vdi mgt lugng PCI3 xic djnh. Hdn hgp dugc dun trong dung mdi cd nhipt dp sfii cao la clobenzcn. Phucmg trinh cac phan imgdirpc md ti nhu tren so dd 1. Ket qua d i tong hgp dugc bon N-thfr-3hydroxynaphthalen-2-cacboxamit (la-d), hipu sual phan ling va mgt so hing sd hda ly dugc md ti tronj bing 1 v i b i n g 2 .. PCIj/Clobenzen R,NH2. 130 C/120' Saddl. Trong do: R| = xiclohexyl, phenyl, a-naphtyl, 1-antraxyl. 726. TCHH, T. 49(6)77071. Tdng hgp vd nghiin cuu dnh hudng... Cic chat tdng hgp dugc nhu sau:. „^?=:7. (IG). Bdng 1 • K i t q u i tdng hgp, mdt so h i n g so hda ly v i c i c du- kipn pho IR ciia cac N-the-3-hydroxynaphthalen-2-cacboxamit (la-d). Hi?u Tnc, °C suat, %. H<?p chat. Rl. Moi, g. la. -QHii. 269,3. 47. lb. -C.Hs. 263,3. SI. IR (u, cm"', KBr). 160162. 3441 (OH naphtol), 3030 (Ar-H), 2930 (CH), 1623 (C=0 amit), 1604 (8N„), 1513 (C=C, aren), 1122 (C-N).. 246247. 3440 (OH naphtol), 3298 (VNH), 3049 (Ar-H), 1636 (C=0 amit), 3458 (OH naphtol), 3245 (VNH), 3059 (Ar-H), 1648 (C=0 amit), 1648 (6NH), 1559 (C=C, aren), 1176(C-N). 3445 (OH naphtol), 3301 (VNH), 3053 (Ar-H), 1642 (C=0 amit), 1624(6NH), 1598, 1567, 1551 (C=C, aren), I174(C-N). lc. -C10H7. 313,3. 67. 214215. Id. -C14H9. 363,4. 58. 242244. 1622 (6NH), 1594, 1562, 1498 (C=C, aren), 1175 (C-N). Bang 2: Phd ' H - N M R vi MS ciia cic N-the-3-hydroxynaphthalen-2-cacboxamit (la-d) dieu che dugc Ki hieu. 'H-NMR (DMSO-dsA ppm, J, Hz). MS(ITMS, + ESI) m/z [Itd](%). la. 1,16 (t, IH, Ha-4', 'J„=I0,88); 1,31-1,43 (m, 4H, Ha-2', Ha-6', Ha-3', Ha-5'); 1,61 (d, IH, He-4', X . = I 3 ) ; 1,74 (dd, 2H, He-3', He-5', 'J=9,75); 1,88 (d, 2H, He320,22 2', He-6', 'J„=9,5); 3,84-3,90 (m, IH, Ha-l'); 7,25 (s, IH, H-4); 7,31 (t, IH, H-7, [M-H] (100) 'J=7,38); 7,47 (t, IH, H-6, =J=7,0); 7,72 (d, IH, H-5, 'J=8,5); 7,84 (d, IH, H-8, 'J=8,0); 8,53 (d, IH, H-l, 'J=4,5); 8,77 (d, IH, NH, 'j=7,5); 12,10 (s, IH, OH).. lb. 7,13 (t, IH, H-4', 'J=7,25); 7,33 (s, IH, H-4); 7,35-7,41 (m, 3H, H-3', H-6, H-7); 262,22 7,50 (t, IH, H-5', 'J=7,5); 7,76 (d, 3H, H-5, H-6', H-2'; 'j=7,25); 7,92 (d, IH, H-8, [M-H] (100) 'J=8,0); 8,50 (s, IH, H-l); 10,59 (s, IH, NH), 11,33 (s, IH, OH).. lc. 7,38 (t, IH, H-3', 'J=8,0); 7,42 (s, IH, H-4); 7,53 (t, IH, H-7, 'j=7,75); 7,58-7,64 268,19 (m, 3H, H-6, H-6', H-7'); 7,80 (d, IH, H-4', 'j=8,5); 7,84 (d, IH, H-2', 'j=8,5); 7,98-8,0 (ra, 2H, H-5, H-5'); 8,09 (d, IH, H-8', 'j=7,5); 8,12 (d, IH, H-8, 'J=8,5); [M-H]-(100) 8,75(s, IH, H-l); 11,11 (s, IH,NH), 11,93 (s, 1H,0H).. Id. 7,40 (d, IH, H-3', 'J=7,5); 7,47 (s, IH, H-4); 7,54-7,60 (m, 4H, H-8', H-7', H-6, H312,6 7); 7,82 (d, IH, H-2', 'j=8,0); 8,00 (dd, 2H, H-S, H-6', 'j=8,0); 8,11 (t, 3H, H-8. H10' H-4' 'J=7,25); 8,67 (s, IH, H-l); 8,77 (s, IH, H-5'); 8,82 (s, IH, H-9'); 11,27 [M-H]'(100) (s, iH,NH), 12,0(s, lH,OH). 727. TCHH,T. 49(6), 2011 Cd till chd dgi hieu suit ciia cic N-the-3hydroxynaphthalen-2-cacboxamit (la-d) ty le nghjch vdi kich thudc ciia cac phan tii amin, tiic la cic amit thu dugc tu xiclohexyl vi anilin phai cd hipu suit cao nhit, den naphtylamit vi cuoi cimg la antraxylamit. Tuy nhien quy tic dd chi tuan thii doi vdi the tich cic amin thorn. Hipu suat ciia la thap nhit, cd le do tinh bazo cao nhat nen trong dung djch nhdm amin ciia xiclohexyiamin bj proton hda mgnh nhat, do dd phin ling amit hda \ay ra khd khan han. c i u triic ciia cic N-lhe-3-hydroxynaphthalen-2cacboxamit (la-d) da dugc khang djnh bang cic so lieu phd IR. ' H - N M R vi pho MS, Tren phd hdng ngogi xuat hicn cic dao dgng hda trj ciia nhdm VQH d 3440-3458, VNH d 3245-3301, vc.o amit d 1623-1648 vi V(.N d 1122-1175 cm'' (bing 1). Tren phd ' H - N M R , tin hipu cgng hudng. Trdn Vdn Tinh vd cpngsu ciia nhdm OH xuat hipn d 11,33-12,10; NH ciia amit d 8,11-11,27, proton nhdm CH va CH2 ciia v6ng naphlyl, phenyl, antraxyl, xycio hexyl Ian iiryt 6 7,25-8,75; 7,13-7,76; 7,39-8,11; 1,16-3,84 ppm. Ph^ MS (ESI) cho thay khoi lugng pic ion phin tir cua cic chal dieu che dugc diing bing khoi lugng ph3n tir dy kien vi deu cd cudng dg mgnh [M-H]" (100%) (bing 2). Tong hgp N-thc-3-cacbamoylnaphthalen-2-y| aclocacboxylat N-thc-3-cacbamoyInaphthalen-2-yl a-clocacboxylat dugc tong hgp theo quy trinh cai tiln dugc chiing tdi md ti trong tai lipu [9], bing each cho N-th 1-3-hydroxynaphthaIen-2-cacboxamit trong THE tic dyng vdi dung dich NaOH 5N, sau do vdi clorua axit (xem phan thyc nghipm). Qua trinh phin irng dugc md ti tren so do 2.. NaOH, THF/0 C. Rj = -CH,CI; R,= -CftH,, (Ila), -C^Hi (Mb), -C|oH7(IIc), -C,4H9(IId). R2 - -CHjCHCl; R,= -C^H,, (Me), -C^Hs (Ilf), -C,oH,(ng), -CMH,(IIh). Ket qua da dilu che dugc tim N-thl-3cacbamoylnaphthalen-2-yl a-clocacboxylat (la-h), trong dd cd bay chit mdi. Hieu suat, mgt so hing sd hda iy, sd lieu phd IR, ' H - N M R vi MS dugc md ta yong cicbing 3 vi 4. So vdi naphtol AS-D hieu suit CLia cic din xuit N-thl-3-cacbamoylnaphthalen-2-yl aclocacboxylat (la-h) thu dugc cao hon, dgt tir 52-61 %. Ciu true cua cic N-thl-3-cacbamoylnaphthalen2-yl a-clocacboxylat (la-h) dugc xic dinh bing cic so lieu phd IR, ' H - N M R vi MS. Tren pho hong ngogi cd cac dao dgng hda trj ciia nhdm VNnd 3239-3347, vc<.. (este) d 1753-1775, Vco (amit) 1600-1663, Vc<w:cr 1200-1217 vivc-Nd 1161-1172 cm"' (bing 3). Tren phd ' H - N M R , tin hipu cdng hudng proton nhdm NH ciia amit ddi vdi cic Ri li vdng therm va xyclohejQ'l lan lugt d va 10,53-10,74, 8,33-8,77, proton nhom CH ciia vdng naphtyl, phenyl, antraxyl va CH2 cua xyclohexyl lin lugt d 7,52-8,68; 7,10-8,72; 7,33-8,81; 1,13-3,70 ppm. Phd MS (ESI) cho thly khdi lupng pic ion phan tii cua cic chat dilu che dugc diing bSng vdi klidi lugng phin tii dy kiln vi diu cd cudng dO mgnh [M-H]" (100%) (bing 4). y. Bdng 3: Ket qua tdng hgp, mgt so hing so hda ly vi cac sd lipu phd IR cua cac N-the-3-cacbamoylnaphthalen-2-yl a-clocacboxylat dilu che dugc chat. Rl. R2. Moi, 8. HS,. (4). (5). (1). (2). Ila. -CsH,,. -CH2CI. 345,8. lib. -CsHs. -CH2CI. 339,8. (3). Tnc, °C IR (u, cm-1, KBr). % (6). (7). 53. 160-161. 3301 (VNH amit); 2933 (CH=); 2856 (VCH2); 1775 (vc.o este); 1639 (vco amit); 1625 (Sm, amit); 1534, 1447(VCK:); 1218 (VCO^Ceste), 1164(C-N).. 52. 186-188. 3308 (Vm), 3021 (Ar-H), 2941 (CH), 1773 (C=0 este), 1647 (0=0 amit), 1626, 1596, 1515, 1500, 1439 (C=C, aren), 1200 (C-O-C), 1161 (C-N).. TCHH, T. 49(6), 2011. Tong hap vd nghien ciru anh hir&ng.... Cl). (2). (3). lie. -C10H7. -CH,C1. 389,8. 61. lid. -C14H9. -CH2C1. 439,9. 3248 (VNII amit); 3052 (VA,.II), 2851 (CH=); 1769 52 265-266,5 (vc.o este); 1663 (vc-o amit); 1597, 1547 (vc.c); 1261 (vc-o-c este), 1172 (C-N).. lie. -CeH,,. (4). (5). (6). (7). 204-206. 3347 (VNI, amit); 3204 (VA,.H), 3060 (CH=); 1753 (vc.o este); 1645 (vc-o amit); 1628 (SN,, amit); 1558, 1504 (vc.c); 1206 (vc-o-c este), 1169 (C-N).. -CH3CHC1 359,8. 63. 146-148. 3335 (NH), 3062 (Ar-H), 2937, 2854 (CH), 1765 (C=0 este), 1641 (C=0 esic), 1600 (C=0 amit), 1536, 1499 (C=C, aren), 1217 (C-O-C), 1168 (C-N).. Ilf. -C6H5. -CHjCHCI. 353,8. 55. 189-191. 3346 (Nil), 3057, 3003 (Ar-H), 2930 (CH), 1764 (C=0 eslc), 1657 (C=0 amit), 1597, 1525, 1476 (C=C, aren), 1203 (C-O-C), 1165 (C-N).. Ilg. -Ci„H, -CHiCHCI. 403,9. 63. 174-176. 3239 (VNH amit); 3058 (vci,,); 1753 (vc.o este); 1647 (Vc.o amit); 1601-1556-1503 (vc.c); 1206 (vc-o-c este); 1165 (C-N).. Ilh. -CMH,. -CH3CHCI. 455,9. 46. 186-188. 3244 (VNI, amit); 3051 (vai); 1759 (vc.o este); 1643 (Vc.o amit); 1627 (6N,I amit); 1600-1451 (vc-c); 1218 (VC-O-C este); 1164 (C-N).. Bang 4: Sd lieu phd ' H - N M R vi MS cua cic N-the-3-cacbamoylnaphthalen-2-yl a-clocacboxylat dieu che dugc Ki hieu. 'H-NMR (DMSO-ds, 8, ppm, J, Hz). MS(1TMS, + ESI) m/z [ltd](%). (1). (2). (3). Ila. 1,13 (t, IH, Ha-4', 'J...=10); 1,25 (q, 4H, Ha-2', Ha-6', Ha-3', Ha-5', 'J.-.=9,9); 1,59 (d, IH, He-4', 'j...=12,5); 1,73 (s, 2H, He-3', He-5'); 1,85 (d, 2H, He-2', He-6', 'j,..=12,5); 3,7 (t, IH, Ha-l', 'J„=3,75); 4,64 (s, 2H, CH;); 7,58-7,64 (m, 2H, H-6, H-7); 7,77 (s, 1H, H-4); 7,96 (d, 1H, H-5, 'j=7,5); 8,04 (d, 1H, H-8, 'J=8,0); 8,16 (s, 1H, H-1); 8,33 (d, IH, HN, 'j-8,0).. lib. 5,00 (s, 2H, CH,CI ); 7,11 (t, IH, H-4', 'j=7,5); 7,36 (t, 2H, H-3', H-5', 'J=7,75); 7,62-7,69 (m, 2H, H-6, H-7); 7,75 (d, 2H, H-2', H-6', 'j=7,5); 7,86 (s, IH, H-4); 8,02 (d, IH, H-5,'j=8,0); 8,11 (d, 1H, H-8,'j=8,0); 8,38 (s, IH, H-l); 10,53 (s, IH.NH).. 362,17 [M+Na]* (100). lie. 4,66 (s, 2H, CH2); 7,56-7,60 (m, 3H, H-3', H-?', H-6'); 7,65-7,71 (m, 3H, H-6, H-7, H-4'); 7,86 (d, IH, H-2', 'J=8,0); 7,89 (s, IH, H-4); 7,98 (d, IH, H-5, 'J=6,5); 8,03 (d, IH, H-5', 'j=8,0); 8,12 (d, IH, H-8, ^J=8,13); 8,17 (d, IH, H8'. 'J=7,5); 8,6 (s, 1H, H-1); 10,64 (s, 1H, NH).. 390,02 [M+H]' (100). lid. 4,65 (s, 2H, CH2); 7,53-7,59 (m, 3H, H-6', H-7', H-5'); 7,67-7,73 (m, 3H, H-6, H-7, H-8', 'J=7,31); 7,92 (s, IH, H-4); 8,04 (t, 2H, H-3, H-5, 'j=9,0); 8,11 (dd, IH, H-8, J„„=8,0, J„.„=l,5); 8,16 (d, IH, H-4', 'j=8,0); 8,21 (d, IH, H-2', 'J=7,5); 8,66 (s, IH, H-I); 8,71 (s, IH, H-10'); 8,79 (s, IH, H-9'); 10,74 (s, 1H,NH).. 440,33 [M+H]* (100). lie. 1,15-1,23 (m, IH, Ha-4'); 1,28-1,43 (m, 4H, Ha-2', Ha-6', Ha-3', Ha-5'); 1,58 (t, IH, He-4', 'J,.,=I3,0); 1,74-1,78 (m, 5H, He-3', He-5', CHj); 1,86 (t, 2H, H'e-2' He-6','j..=10,38);3,85-3,90(m, IH, Ha-l'); 7,25 (s, IH, H-4); 7,32 (t, IH, H-7, '1 =7,5); 7,47 (t, IH, H-6, ' j =7,5); 7,72 (d, IH, H-5, 'j=8,5); 7,84 (d, IH, H-8, 'J=8,5); 8,53 (s, IH, H-l); 8,77 (d, IH, HN, 'j=7,5).. 368,27 [M+Na]* (100). 382,25 [M+Na]* (100). T C H H , T. 49(6), 2011 esteraza dgc hi^u te bdo bach cdu ngudi, Tgp chi Hoa hoc 49(4), 479-488 (2011). ^. an ngu' khdng gian nhd (phenyl). TAI LIEU T H A M K H A O 1.. Leucocyte activation markers in clinical practice, Clin Chem Lab Med 1999 Jun; 37(6), 607-622.. 2. Horwilz D. A.. D.AIiinson P , Ward&Nancy Kight. Identification of human mononuclear leucocyte populations hv. Clin. exp. Immunol.. 30. 289-298 (1977). 3.. C. Y. Li, K. W. Lam. L. T. Yam. Estera.ses m human leucocytes, h Histochem. Cylocheni.. 21, 1-12(1973).. 4.. Nguyln Trieu Van. Le Xuan Hai. Thilu Thj M^ng Bitdc dau img dung ky thuql flow cytometry de xep loai hrxemi cap tqi I 'n'li Huyet hpc-7'niyen mdu Irung iro-ng. Tap chi Y hgc Viet Nam. 02. 237-243 (2010).. 5.. Trin VSn Tinh, Phgm Hoii Thu. Nguyln Anh Trf, Luu VSn Boi. Tong hgp v(i nghien cim anh hudng ciia hieu ung electron doi vdi phan ung nhupm. 6.. Trin Vin Tinh, Phgm Hoii Thu, Nguyln Anh "frf) Luu van B6i. Phuang phdp mdi tong hgp naptol^ D ldm tdc nhdn dieu che ca chat nhupm esteraza die hi4u tc bao bgch cau ngudi. Tgp chi H6a hpc, 48(4A). 736-741 (2010).. 7.. William C. Moloney, Kenneth Mcpherson, Ljla Filcgelman. Esterase activity in leulocytes demonstrated by the use of naphthol AS-D chloroacetate substrate. Hematology Laboratoiy, Bosion City Hospital, Boston, Massachusetts (1959).. 8.. Burstone, M. S. Esterase activity of developing bones andtecth. A. M. A ArchPalh,60, 164-167(1957).. y.. D. Catovsky. Practical Haematology, Churchill Livingstone , Hong Kong, 151 -159 (1994).. 10. Sigma-Aldrich Catalougue. Naphthol AS-D chloracefafe esterase and a-Naphlhyl acetate esterase (Procedure 9\), Sigma-Aldrich, 1-2(2003),. Lien he: T r a n V a n T i n h Khoa Hda hgc T r u d n g Dgi hpc Khoa hgc T y nhien, D H Q G H i Ngi 19 Le T h i n h Tdng, Hoan Kiem, H i Ngi Email, tranvantinh@yahoo.com. TAP Chl n\j/\ Hyt^. CAC. 1.49(6)733-738. THANG 12 NAM 2011. H g p C H A T F L A V O N O I T G L Y C O S I T P H A N L A P TIJ. CAY. TETRADIUM GLABRIFOLIUM Truong Thi Thu H i c n ' \ Nguyen Van Thanh^ Chau Van Minh', Phan Van Ki§m'\ Nguyen Van Tuyen^, Dan Thi Thuy Hang', Ninh Khic Ban', Nguyen Xuan Cirdng' Vi4n Hoa sinh biin, Vi^n Khoa hpc vd Cdng ngh^ Vi^t Nam Tnrdng Bgi hgc Mo Bia chdi, Bp Gido due vd Ddo tgo Vi^n Hda hpc. Vi^n Khoa hpc va Cong ngh? Viit Nam Den Tda soan 14-6-2011 Abstract Fiveflavonoidglycosides, phellamurin (I), epimedoside C (2), astragalin (3), nicotiflorin (4), and trifolin (5) were isolated from the methanol extract of the Tetradium glabrifolium leaves by various chromatographic experiments. The structure elucidation of isolated compounds was done by spectroscopic methods including ESI-MS, ID- and 2D-NMR in comparison with the literature values. This is thefirstreport of these compounds from T. glabrifolium. Keywords: Tetradium glabrifolium, Rutaceae, flavonoid glycoside. 1. MO DAU Cay dau dau li nhan cd ten khoa hgc li Tetradium glabrifolium (Benth.) Hartl., thugc hg Rutaceae. Day la loai ciy gd Idn, cao 6-7 m; vd it nut, tring tring; cinh non cd long. Li mang 9-19 li phy thon, diy khdng ddi xiing, bia nguyen, khdng ldng, gin phu 12-15 cap, cudng phu 3-5 mm; cudng cd long [1]. Mdt sd nghien cdu tren the gidi cho thiy thanh phin hda hpc chinh cua loii niy la cic hgp chit phenol, limonoit, alcanoit, lignan vi courmarin [2]. Tuy nhien d trong nudc van chua cd cdng trinh khoa hgc nio nghien ciiu ve thanh phan hda hgc cua loii T. glabrifolium. Bai bao nay thdng bio ket qui phan Igp vi xic djnh cau true cua nam hgp chit flavonoit glycosit tir djch chiet metanol ciia ciia la cay T. glabrifolium. Cau true hda hgc ciia cac hgp chit dugc xic dinh li phellamurin (1), epimedoside C (2), astragalin (3), nicotiflorin (4) vi trifolin (5) bing cic pho khoi lugng (ESI-MS), phd cgng hudng tir hat nhan mdt chi6u va hai chieu (ID vi 2D NMR). 2. THUC NGHIEM VA PHLTONG PHAP NGHIEN CUU. diing thudc thu li dung djch H2S04 10% phun d6u len ban mdng rdi say d nhiet dg cao cho den khi hien mau. - Sic ky cgt (CC) dugc tien hinh vdi chit hip phy pha thudng (Silica gel 240-430 mesh, Merck) hoic pha dio (ODS-60-14/63, Fujisilisa-Nhgt Bin). 2. Cac phiromg phap phd - Phd khdi lugng ESI-MS dugc do tren may Agilent 1200 LC-MSD Trap, Vien Hda hgc, Vi?n Khoa hgc vi Cdng nghe Viet Nam. - Phd cdng hudng tir nhin (NMR) dugc do tren miy Bruker AM500 FT-NMR Specfrometer, Vien Hda hgc, Vien Khoa hgc vi Cdng nghe Viet Nam. 3. Mau thirc v^t Miu cay Tetradium glabrifolium dugc thu hii vio thing 6 nam 2010 tgi Tay Thien, VTnh Phiic. Ten khoa hpc dugc Nguyen The Cudng, Vien Sinh thai vi Tai nguyen sinh vat giim dinh. Mau tieu bin dugc Itm tra tgi Vien Sinh thii va Tii nguy6n sinh vgt 4. Phan l^p cac hgp chat. 2.1. Phuong phap tach chiet - Sic ky Idp mdng (TLC) dugc thyc hien tren bin mdng tring sin DC-AIufolien 60 F254 va RPig F254 (Merck-Dirc). Cic vet chit dugc phat hi?n bing den tu" ngoai d hai bude sdng 254 vi 368 nm hoac. Bgt la Tetradium glabrifolium khd (3,5 kg) dugc chi6t 3 lin bing metanol ndng tren thiet bi chiet sieu am (d 50°C, mdi lin I gid). Cac djch chiet dugc Ipc qua giiy Igc, gom lai va logi bd dung mdi dudi ap. TCHH, T. 49(6), 2011. rnan. suat giam thu dupe 180 g c$n chiet metanol. C?n ehiet nay duge hoa tan vao 3 lit nirdc cat va tien hanh chiet phan bo lan Iir<p1 vai H-hexan va etyl axetat (3x31) thu dirge cac cgn dicji n-hexan (H, 50 g), etyl axetat (E, 40 g) va lap nu*c (N). CJn chiet E (40 g) dugc phan tach thiinh chin phan doan, EI-E9, bang sac ky cgt silica gel pha thucrng ma gifii gradient cloroform/metanol 25/1 -> l/I. Hgp chat 4 (20 mg) dugc tinh che tir phan doan E5 (5 g) biing sac ky cgt silica gel pha thucrng rua giai bang cloroform/axelon/nuac 1/3/0,1 ket hgp voi sSc ky cgt silica gel pha tliuofng su dung he dung m6i rira giai cloroform/metanol 3/1. Lap nucrc dugc chay tren cgt sac ky trao doi ion vai chat hap phy la Dianion HP-20 rCra giai tang dfin n6ng dg metanol trong nuoc (0, 25, 50, 75 va 100%) thu dugc bon phan doan N1-N4 Phan doan N3 (15 g) tiep tyc dugc phan tach bang s^c ky cgt silica gel pha thuang. run l\.llfrri vu ^yn^ a,. rua giai gradient cloroform/metanol 20/1 -> 1/1 thi dugc nSm phan doan nho, N3A-N3E. Tilp tuc phat tach phan doan N3C (2 g) bang sac ky cgt silica ge pha thuang riia giai bang cloroform/metanol 3/1 thi dugc cac hgp chat 3 (30 mg) va 5 (15 mg). Cac hgp chat I (8 mg) va 2 (10 mg) dugc tinh che tir phan doan N3D (2,5 g) bang sac ky cgt silica gel pha thucrng su dyng h? dung moi rua giai cloroform/metanol/nuijc 4/1/0,1. Phellamurin (1): Tinh the hinh kim mau vang, diem chay 205''C. ESI-MS m/z 519 [M+H]*, cong thiic phan tir C2iIIj„Oii,M = 518. 'il-NMR (500 MHz, DMSO-r/j) va "C-NMR (125 MHz, DMSO-</«),xem bang I. Epimedoside C (2): Tinh the hinh kim mau vang; diem chay mp. 289''C.. Hmh P. cau true hoa hgc cua 1-5 ESI-MS m/z 517 [M+H]*, cong thuc phan tir C26H2,0|,(M = 516). 'H-NMR (500 MHz, DMSO-rf<) va "C-NMR (125 MHz, DMSO-rfj), xem bang 1. Astragalin (3): Tinh the hinh kim mau vang; dilm chay mp. 178°C; [a]D^' +20 (c 0,5, MeOH). ESI-MS: m/z 447 [M-H]^, cong thire phan tit C2iH2oO,,(M = 448). 'H-NMR (500 MHz, DMSO-rf.) va "C-NMR (125 MHz, DMSO-c/(),em bang 2. Nicotinorin (4): Tinh the hinh kim mau vang; diem chay mp. I9I°C; [«]„" -50 (c 0,5, MeOH). ESI-MS m/z 595 [M+H]*, cong thii'c phan tir C2,H3oO,5(M = 594). 'H-NMR (500 MHz, DMSO-rfj) va "C-NMR (125 MHz, DMSO-rf«),xem bang 2. Trifolin (5): Tinh thi hinh kim mau vang; dilm chay mp. 235°C; ESI-MS: m/z 471 [M+Na]*, cong thirc phan tir C2iH2oO|i(M = 448).. 'H-NMR (500 MHz, DMSO-rf«) va "C-NMR (125 MHz, DMSO-rf,),xem bang 2. 3. KET QUA VA THAO LUAN Hgp chat 1 thu dugc dual d^ing tinh thi mau vang CO nhijt dg nong ehay 205°C. Pho NMR cua 1 khang djnh sy c6 m§t cua mgt vong benzen the para t?i6„7,31 (2H,d, y = 8 , 5 H z ) , 7,90(2H,d, ,7=8,5 Hz) ciing vai hai tin hi$u. CH CO cuong dg eao gap doi cac tin hieu CH khac tji 5c 129,18 va 114,84. Cac tin hi$u tai 8H 5,05 (IH, d, J= 11,0 Hz, H-2), 4,57 (IH, d, 7 = 11,0 Hz, H-3) va tji Sc 82,85 (CH, C-2), 71,79 (CH, C-3) chiing to sy t6n tai mgt hgp chdt flavanol. Tin hieu nhom cacbonyl dugc xac djnh tai Sc 198,97. Tin hi?u cua mgt proton dieh chuyin rat manh vl tnrcmg thap tgi 5H 11,80 chiing to mOt nhom hydroxyl g^n vcri C-S. Tuang tac HMBC cua proton CH tai h 6,29 (H-6) v6i cac cacbon bac bon tai 160,96 (C-5),. 734.