TAP CHI HOA HOC
DOI: 10.15625/0866-7144.2014-0058
T. 52(6) 702-705 THANG 12 NAM 2014
HOAT T I N H G A Y O O C TE BAO CClA CAC HOT C H A T B R O M O T Y R O S I N
ANCALOIT PHAN LAP TLP LOAI HAI MIEN lantheHa basta
Hoang Le Tuin Anh', Ngo Van Quang^, Pham Hai Vln', Nguyin Xuan Nhiem', Dan Thi Thuy King', Dirong Thi Hai Yen', Chau Van Minh', D6 Thi Thao^ Phan Van Kiem'*
' Vien Hda sinh biin, Vien Han lam Khoa hgc vd Cdng nghi Viet Nam
^ Vien Hoa hoc, Vien Han Idm Khoa hgc vd Cdng nghe Viet Nam
^Viin Cdng ngh? sinh hgc, Vien Hdn lam Khoa hgc va Cdng nghe Viet Nam Din Tda soan 25-6-2014; Chip nhan dang 4-12-2014
Abstract
Two known bromotyrosine alkaloids were isolated for the first time from the methanol extract of Vietnamese marine sponge lanthella basta. Their structures were identified as purealidin R (1) and fistularin-3 (2) by spectroscopic methods and m comparison with the previously reported data. Compound 1 showed strong cytotoxicity against 8 human cancer cell lines as HepG2, KB, LU-1, MCF7, LNCaP, SW480, MKN7, and HL-60 with the IC50 values ranging from 3.27 to 6.08 |ig/mL, whereas 2 had moderate to weak or no cytotoxicity against the human cancer cell lines.
Keywords. lanthella basta, marine sponge, bromotyrosine alkaloid, cytotoxicity.
1. MO DAU
Hien nay, cac hgp chat dang bromotyrosin alkaloid phan lap tir nhieu loai hai mien thugc hg Verongida da dugc nghien cuu nhieu do chiing the hien nhiing hoat tinh sinh hgc ly thu, dac biet la hoat tinh gay dgc te bao [1-4],
Tuy vay, cho den nay van chua cd nghien ciiu nao dugc cdng bd vl thanh phan hda hgc va hoat tinh sinh hgc cua cac hgp chat bromotyrosin alkaloid tir cac loai hai mien ciia Viet Nam. Bai bao nay iin dau tien thdng bao ket qua phan lap, xac dinh cau triic hda hgc va hoat ti'nh gay dgc te bao ciia hai hgp chat purealidin R (1) va fistularin-3 (2) phan lap duoc tir loai Hal mien lanthella basta thu tai Cat Ba, Hai Phdng.
2. THI/C NGHIEM 2.1. Phutfng phap tach chiet
Sac ky Idp mdng (TLC) dugc thuc hien tren ban mdng trang san DC-Alufolien 60 F254 va RPjg F254 (Merck-Dire). Cac vlt chat dugc phat hien bing den tir ngoai d hai budc sdng 254 va 365 nm hoac diing thudc thii la dung dich H2SO4 10 % phun deu len ban mdng rdi siy d nhiet do cao cho din khi hien mau,
sic ky cot (CC) dugc tien hanh vdi chit hip phu pha thudng (Silica gel 240-430 mesh, Merck) hoac pha dao (ODS-60-14/63, Fujisilisa-Nhat Ban).
2.2. Cac phirong phap pho
Phd khdi lugng (MS) dugc do tren may LC- MSD Agilent 1200 Series (USA), Vien Hda hgc cac hgp chit thien nhien, Vien Han lam Khoa hgc v^
Cdng nghe Viet Nam.
Phd cdng hudng tir hat nhan (NMR) dugc do tren may Bruker AM500 FT-NMR Spectrometer, Vien Hda hgc, Vien Han lam Khoa hgc va Cong nghe Viet Nam.
2.3. Phuong phap thu hoat tinh gay doc tl bao Phuong phap thii ddc tl bao in vitro dugc Vien Ung thu Qudc gia Hoa Ky (National Cancer Institute - NCI) xac nhan la phep thir do ddc tl bao chi'in nham sang lgc, phat hien cac chit cd kha nang kim ham su phat triln hoac diet TBUT d dieu kien in vitro. Phep thii nay dugc thuc hien theo phuang phap cua Monks (1991). Hai hgp chit dugc thir nghiem hoat tinh gay ddc tl bao in vitro tren 8 ddng te bao ung thu ngudi thuc nghiem: ung thu gan (HepG2); ung thu tl bao bilu md (KB); ung thu phii ngudi (LUI); ung thu vti (MCF7); ung thu tuylt tiln Met (LNCaP); ung thu rugt kit (SW480), ung diu da day (MKN7) va ung thu bach ciu (HL60) vdi chit ddi chiing duong la ellipticine.
702
TCHH, T. 52(6), 2014 2.4. Mau sinh vat WID
Mau hai mien lanthella basta dugc thu vao thang 8 nam 2011 tai Cat Ba, Quang Ninh. Ten khoa hgc dugc PCS. TS. Dd Cdng Thung, Vien Tai nguyen va Mdi trudng bien giam dinh. Mau tieu ban dugc luu giii tai Vien Tai nguyen va Mdi trudng bien va Vien Hda sinh bien.
2.5. Phan l$p cac hop chat
Mau hai mien lanthella basta tuoi (4 kg) dugc thai nhd, phoi khd, nghien vun va chiet sieu am bang metanol (3x3h, 50°C). Dich chilt dugc Igc va loai bd dung mdi dudi ap suit giam thu dugc 28 g can chiet metanol. Can chilt nay dugc hda tan vao 1 L nudc cit va tien hanh chiet phan bd vdi cac dung mdi cd dg phan cue tang dan: n-hexan, clorofoc, etyl axetat va butanol (3x1 L). Cac phan dich chiet dugc tien hanh loai bd dung mdi bang he thdng cd quay ap suit giam thu dugc 5 g can hexan, 12 g can clorofoc, 6 g can etyl axetat va 2 g can butanol.
Can clorofoc (12 g) dugc phan tach thd bang sac ky cot silica gel pha thudng vol he dung mdi rua giai gradient clorofoc/metanol cd do phan cue tang dan (100/0-0/100, v/v) thu dugc 6 phan doan (1B1-IB6).
Phan doan IBS dugc phan tach tren cot sac ky pha dao vdi he dung mdi rira giai axeton/nudc: 3/1 (v/v), thu dugc 3 phan doan (IB3A-IB3C).
Phan doan IB3C dugc tinh ehe tren cot sac ky pha thudng vdi he dung mdi riia giai n-hexan/axeton: 3/2 (v/v); ket hgp vdi sic ky cot pha dao sir dung he dung mdi axeton/nudc: 2/1 (v/v) thu dugc hgp chit 1(12 mg).
Phan doan IB3B dugc tinh chi tren cot sic ky pha dao vdi hp dung mdi riia giai axeton/nudc: 2/1 (v/v); kit hgp vdi sic ky cot pha thudng sii dung he dung mdi rira giai clorofoc/etyl axetat: 2/3 (v/v) thu dugc hgp chat 2 (8 mg).
Purealidin R (1): chat dau khdng mau.
Do quay cue [ajo^": +86 (c = 0,19, MeOH).
ESI-MS: Cac pic m/z 380, 382 va 384 cd ti le 1:2:1, cdng thirc phan tu CioHioBr2N204 (M = 382).
' H - N M R (500 MHz, CDCI3) 5 (ppm): 4,41 (IH, s, H-1), 6,33 (IH, s, H-5), 3,00 (IH, d, J= 18,5 Hz, Ha-7), 3,94 (IH, d,J= 18,5 Hz, Hb-7), 3,76 (3H, s, OCH3), 6,33 (IH, s,l-OH), 5,69 (IH, br s, 9-NH2) va 6,57(lH,brs,9-NH2).
'^C-NMR (125 MHz, CDCI3) 5 (ppm): 73,95 (C- 1), 112,44 (C-2), 148,15 (C-3), 121,33 (C-4), 130,95 (C-5), 92,16 (C-6), 38,38 (C-7), 153,60 (C-8), 160,90 (C-9) va 60,17 (OCH3).
Fistularin-3 (2): Bdt vd dinh hinh, mau trang.
Phan Vdn Kiem vd congsu ' H - N M R (500 MHz, mASO-ds) va '^C-NMR (125 MHz, DMSO-di), xem bang 1.
Bdng I: Sd lieu phd NMR ciia 2 va chit tham khao
c
1 2 3 4 5 6 78 9 10 11 12 13 14 14' 15 15' 16 17 18 2'
r
3' 4' 5' 6' 7'
8' 9' 3- OCH3 3'-0CH 9-NH2 9'-NH;
'6c 73,6 121,1 147,2 113,2 131,3 90,4 39,4
154,5 159,2 42,6 68,1 75,4 151,3 117,3 117,3 130,5 130,5 142,7 69,4 46,4 73,6 121,1 147,2 113,2 131,2 90,4 39,4
154,5 159,1 59,7 [3 59,7
ca,b 0 C
73,53 120,87 147,10 113,03 131,17 90,25 39,33
154,40 159,04 42,53 67,98 75,31 151,20 117,10 117,10 130,41 130,41 142,60 69,33 46,27 73,48 120,81 147,10 113,03 131,10 90,25 39,33
154,32 158,97 59,57
59,57
6"„(J=Hz) 3,93 (s)
-
6,59 (s)-
3,23 (d, 18,5) 3,62 (d, 18,5)-
3,26*3,48 (m) 4,07 3,89*
3,82 (m)
7,57 (s) 7,57 (s)
-
4,67 (brs) 3,31 * 3,91 (s)6,57 (s)
-
3,22 (d, 18,5) 3,62 (d, 18,5)-
3,65 (s) 3,65 (s) 8,41 (t, 6,0) 8,37 (t, 6,0)HMBC 3,6
1,3,4,6,7 1,5,6,8
9,11 10,12 11,10
13,14,17 13, 14', 15 15, 15', 18 3', 6'
l ' , 3 ' , 4 ' , 6 ' 1', 5', 6', 8'
3 3' 9,10 9', 18
*6 ciia Fistularin-3 do trong DMSO-dg [6], ^Do trong DMSO-dfi, ''125 MHz, '500 MHz, *tin hieu chap, 3. KET QUA VA THAO LUAN 3.1. Xac dinh cau triic cac hop chat
TCHH, T. 52(6), 2014
Phd khdi lugng cua hgp chit 1 xuat hien cum pic ddng vi tai m/z 380, 382 va 384 vdi ti le cudng do la 1:2:1 cho phep i.)x doan trong phan tii cd 2 nguyen tu brdm.
Tren phd 'H-NMR thiy xuit hien tin hieu hai proton ciia mdt nhdm NH2 tai 5H 5,69 (br s)/6,57 (br s); mdt proton OH tai 5H 6,33 (s); mdt proton olefin tai 6H 6,33 (s) va mdt nhdm metoxi tai 5H 3,76 (s, OCH3).
Pho "C-NMR va DEPT xuit hien tin hieu cua 10 nguyen tii cacbon, trong do cd 7 nguyen tir cacbon khdng ndi vdi nguyen tii hidro, mdt cacbon metin, mdt cacbon metjjien va mdt nhdm metoxi.
Phan tich cac dii ki$n phd thay xuat hien cac tin hieu d^c trung ciia mdt cau true vdng splrocyclohexadienylisoxazoline, gdm: tin hieu cOa mgt spirocacbon [5] tai 6c 92,16 (C-6); mgt cacbon olefin bac bdn gan vdi nito tai 6c 153,60 (C-8); hai tin hieu cacbon lien ket vdi brom t^i 6c 112,44 (C- 2), 121,33 (C-4); cimg vdi tin hleu cua bdn cacbon tai 5c 73,95 (C-1), 148,15 (C-3), 130,95 (C-5), 38,38 (C-7). Ddng thdi tren phd cdn thay tin hieu ciia mgt nhom amit tai 5c 160,90 (C-9). Kit qua phan tich phd tren ciing vdi tra ciiu ngudn gdc cac hgp chit tir hai mien cd thi du doan hgp chit 1 la purealidin R [5]. Cac dir kien phd ciia 1 va purealidin R la hoan toan phii hgp tai tit ca cac vi tri tuong iing, ddng thdi ket qua phd khdi vdi vice xuat hien cac pic ion ddng vi cua Br neu tren hoan toan phii hgp vdi cdng thirc C,oHioBr2N204 (M = 382) cua purealidin R.
Hgp chit nay da dugc phan lap tir loai Psammaplysilla pure ciia Nhat Ban, nhung day la
Hogt tinh gay dgc ti hao cua...
Iin diu tien dugc phan lap tu loai Hai mi6n lanthella basta cua Viet Nam.
Hgp chat 2 thu dugc d dang bgt vd djnh hinh, mau tring. Tren phd 'H-NMR xuit hien tin hl8u ciia hai proton vdng tham tgi 5H 7,57 (s, H-15, H-IS*);
tin hleu ciia hai nhdm metoxi tai 5H 3,65 (s, 3-OCH3;
3'-0CH3); tin hieu cua hai nhdm NH tai 6H 8,37 ( t , /
= 6,0 Hz), 8,41 (t, J = 6,0 Hz) va hai proton olefin tai 6„ 6,59 (s, H-5) va 6,57 (s, H-5').
Pho '^C-NMR va DEPT xuit hien tin hieu cua 31 nguyen tir cacbon bao gdm 16 cacbon khdng noi vdi nguyen tii hidro, 8 cacbon metin, 5 cacbon metylen va 2 nhdm metoxi. Tuong tu hgp chat 1, dga vao ket qua phd NMR, cau true hai vdng splrocyclohexadienylisoxazollne cua hgp chat 2 ciing dugc xac dinh. Vdng thii nhat, gdm: tin hieu mdt spirocacbon tai 5c 90,25 (C-6), mdt cacbon olefin gan vdi nito tai 6c 154,40 (C-8), tin hieu ciia hai cacbon lien ket vdi brom tai 6c 120,87 (C-2), 113,03 (C-4), ciing vdi tin hieu ciia 4 cacbon ciia vdng thii nhit tgi 6c 73,53 (C-1), 147,10 (C-3), 131,17 (C-5), 39,22 (C-7); nhdm tin hieu ciia vong thir hai tai 6c 73,48 (C-1'), 120,81 (C-2'), 147,10 (C- 3'), 113,03 (C-4'), 131,10 (C-5'), 90,25 (C-6'), 39,33 (C-7'), 154,32 (C-8'); sau tin hieu dac trung cho mgt vdng phenolic bj the d cac vi tri 1, 3, 4, 5 [5c 151,20 (C-13), 117,10 (C-14), 117,10 (C-14'), 130,41 (C- 15), 130,41 (C-15'), 142,60 (C-16)]; ngoai ra con xuat hien tin hleu ciia hai nhdm amit kha dac trung tai 6c 159,04 (C-9) va 158,97 (C-9') [6].
Hinh I: Cau tnic hda hgc va cac tugng tac HMBC chinh ciia hgp chit 1 va 2
TCHH, T. 52(6), 2014
Phan tich chi tilt cac tuong tac tren phd HSQC, cac proton dugc gan vdi cac cacbon tuong img (bang 1). Tren phd HMBC, cau true ciia 2 vdng spirocyclohexadienylisoxazoline mdt lan niia dugc khang djnh do xuit hien cac tuong tac giira H-1 (5H 3,93) vdi C-3 (6c 147,10)/C-6 (5c 90,25); giira H-5 (6H 6,59) vdi C-I (5c 73,53)/C-3 (5c 147,10)/C-4 (5c 113,03)/C-6 (6C 90,25)/C-7 (5c 39,33); giifa H^-l (5H 3,23)/Hb-7 (5H 3,62) vdi C-1 (6c 73,53)/C-5 (6c 131,17)/C-6 (5c 90,25)/C-8 (5, 154,40); hrong tu, tuong tac cua giiia H-l' (5H 3,91) vdi C-3' (5c 147,10)/C-6" (5c90,25); giiia H-5' (5H 6,59) vdi C-1' (Sc 73,48)/ C-3' (6C 147,10)/C-4' (6c 113,03)/ C-6' (5c 90,25); giifa H^-?' (5H 3,23)/Hb-7' (5H 3,62) vdi C-r (5C 73,48)/C-5' (5C 131,10)/C-6' (5c 90,25)/C-8' (6c 154,32); Vi tri ciia hai nhdm NH ciing dugc xac djnh thdng qua cac tuong tac giira Ha-10 (5H 3,26)/Hb-7 (6H 3,48) vdi C-9 (6c 159,04)/C-1 l(6c 67,98); giua proton cua 9-NH (6H 8,41) vdi C-9 (5c 159,04)/C-10 (5c 42,53); va tuong tu H-18 (2H, 5H 3,26) vdi C-9' (6c 158,97)/C-16 (5c 142,60)/C-17 (5c 69,33); glQ-a proton cua 9'-NH (5H 8,41) vdi C-9' (5C 158,97)/C-18 (5c 46,27). Dua vao cac phan tich tren ciing vdi ket qua so sanh sd lieu phd NMR vdi tai lieu da cdng bd [6] hgp chat 2 dugc khang dinh la ilstularin-3, mdt hgp chit da dugc phan lap tir loai Aiolochroia crassa nhung day la lan dau tien dugc phan lap tii loai Hai mien lanthella basta ciia Viet Nam.
3.2. Ket qua thu* hoait tinh gay ddc te bao Kit qua danh gia boat tinh gay ddc te bao in vitro ciia hgp chit 1 vi 2 tren 8 ddng te bao ung thu thii nghiem (bang 2) cho thiy, hgp chat 1 the hien hoat tinh gay ddc t l bao manh tren ca 8 ddng t l bao thtr nghiem vdi gia trj IC50 trong khoang tir 3,27- 6,08 pg/mL. Hgp chit 2 dil hien boat tinh ddi vdi ddng tl bao ung thu gan (HepG2); ung thu bieu md (KB); ung thu phdi (LU-1); ung thu vu (MCF7) d mire trung binh ylu.
Loi cam on. Cdng trinh duac hoan thanh vdi su tdi tra kinh phi ciia Nhi?m vu nghien ciru ca bdn dinh hudng img dung (02/2011/HB-NCCBUD). Tap thi
Phan Vdn Kiem vd cdng su tdc gid xin cam an PGS. TS. Bd Cong Thung Vien Tdi nguyen vd Mdi truang biin da giiip gidm dinh ten khoa hgc mau hdi miin vd ThS. Bang VU Lirang, Vien Hda hoc dd giup do cdc pho NMR.
Bdng 2: Kit qua thii boat tinh gay ddc te bao DoDg te bao
HepG2 KB LUI MCF7 LNCaP SW480 MKN7 HL60
ICjoC^g/mL) 1 4,25 3,27 6,08 4,06 4,92 4,77 4,24 3,94
2 41,89 31,12 53,01 48,31
>50
>50
>50
>50
Ellipticine 1,06 0,99 0,87 0,92 0,70 0,84 0,95 0,62.
TAI LIEU THAM KHAO
1. Cimmiello P., DelrAversano C, Fattorusso E., Magno S., and Pansini M. Chemistry of Verongida Sponges . 9. Secondary Metabolite Composition of the Caribbean Sponge Aplysina cauliformis, J. Nat.
Prod., 62, 590 (1999)
2. Lee Y. J., Han S., Lee H. S., Kang J. S., Yun J., Sim C. J., Shin H. J., Lee J. S. Cytotoxic Psammaplysin Analogues from a Suberea sp Marine Sponge and the Role of the Spirooxepinisoxazoline in their Activity, J. Nat. Prod,, 76(9), 1731-6 (2013).
3. Kunze K., Niemann H., Ueberlein S., Schulze R., Ehrlich H., Brunner E., Proksch P., Van P6e K. H.
Brominated skeletal components of the marine demosponges. Aplysina cavernicola and lanthella basta: analytical and biochemical investigations.
Marine Dmgs, 11(4), 1271-12816 (2013).
4 Tran T. D., Pham N. B., Fechner G., Hooper J. N., Quinn R. J. Bromotyrosine alkaloids fi-om the Australian marine sponge Pseudoceratina verrucosa J. Nat. Prod., 76(4), 516-523 (2013).
5 J. Kobayashi, K. Honma, T. Sasaki, and M. Tsuda.
Purealidins J-R, New bromotyrosine Alkaloids from the Okinawan marine sponge Psammaplysilla purea, Chem. Phar. Bull, 43(3), 403^07 (1995).
6. Hongfeng Gao, Michelle Kelly, and Mark T.
Hamann Bromotyrosine-derived metabolites from the sponge Aiolochroia crassa. Tetrahedron, 55, 9717-9726(1999).
Liin h?: Phan Van Ki|m
Vien Hda sinh biln, Vien Han lam Khoa hgc va Cdng nghe Viet Nam Sd 18 Hoang Qudc Viet, Ciu Giiy, Ha Ngi
E-mail; [email protected] Dien thogi: 0983555031.
