• Ngiiien cii'u - Ky thuat
•? tv r
Tong hop dan chat 8,9-dimethoxy-5- methylbenzo[c] [l]phenanthrolinium va
12- ethoxy-8,9-diniethoxy-5-
methylbenzo [c] [1,9] phenanthrolinium tiem nang khang ung thir
Huynh Thj Nggc Phuong', Jean-Jacques Helesbreux^ Olivier Duval^
Sp mon Hoa Dufrc - Khoa Du(rc - Dgi HQC Y DUVC Thdnh Pho Ho Chi Minh
^SONAS (UPRES-EA 921). Faculte de Pharmacie. Universited'Angers, France
Oat van de
Topoisomerase-ADN (TOP-ADN) - enzym cin thiet cho cdc hogt dgng sao chep, phien md vd tdi td hpp ADN eiia t i bdo - hign trd thdnh mpt trong nhu'ng mue tidu day hua hgn cho vide tim kiem vd phdt triin eac thudc khdng ung thu ^^'. Dac bi#t, topoisomerase-1 (TOPI) dugc biet Id mye tidu duy nhit cua camptothecin vd din chit, hidn dang duoc sir dyng trong dieu tr[ ung thu nhu irinoteean vd topoteean'^'. Nhieu din chit non-camptothecin dang dugc nghien ciru hiu tim ra nhung tdc nhdn khdng ung thu mgnh va it tac dyng phy, trong sd dd cd cdc chit tuong ddng tdng hop ben20[c]phenanthriclin (BOP), dgc bigt cdc BOP bdc 4 (quaternary) - chira trong ciu triic nhdm chirc iminlum; nhdm chuc niy dugc cho id quan trpng vd ein thiit cho tdc ddng khdng TOP-1 vd ddc tfnh t i bdo f^"^'. Hai trong sd cdc BCP bdc 4 ngudn gdc ting hgp dupe xem Id ed tiim nang trd thdnh nhu'ng thudc khdng ung thu tuong lai id NK-
H3C0 ^ ^ * CH3
fagaronne (1) ethoxidine (2)
Hinh 1: Mot sd BCP b$c 4 (BCP quaternary)
109'^' vd ethoxidin. Ethoxidin mgt din chit 12- ethoxy BOP iminium t h i hign dpc tinh te bao ed y nghta trong thir nghlgm in vitro trdn nhung ddng te bdo leukemia ngudi ^. Nhu-ng thir nghidm sinh hda eijng cho thiy ethoxidin cd di iyc gin kit vdi ADN va kha ndng ire che TOPI ddng k i , trong dd vai trd cua nhdm 12- ethoxy dugc cho Id ein thiit trong tdc dpng ire chi TOPI ndy '^""l Nhim gdp phin tim kiim nhi>ng ciu tnJc tuong ddng BCP mdi vdi hogt tinh khdng ung thu cd y nghTa, chiing toi tien hdnh tdng hop cdc din chit 8,9- dimethoxy-S-methylbenzo [c][1,9]phenanthroliniurTi vd 12- ethoxy-8,9^imethoxy-5-methylbenzo[(^[1,9j phenanthrolinium, trong nhung dan chit iminium ndy chijng tdi dua mgt nguydn ti> nitro vdo vong A vdi myc dich Idm tdng didn tfch dm cua khung BCP, ciing vdi (ho$c khdng) nhdm the 12-ethoxy.
Cdc chit ting hgp dugc dugc thd nghidm so bp die tfnh trdn ddng t i bdo L1210 (murine leukemia cells) cho thiy tit cd diu cd tdc dgng mgnh hon fagaronin, mdt BOP ty nhiin.
TiM* CHi Dirpc HQC - 05/2012 (SO 433 NAM S^
' Nghien CLPU - Ky thuat
N g u y e n l i e u v a p h u ^ a n g p h a p Nguyen ligu
Hda ehit diing trong nghien cdu co dupc ttr cde nhd eung c i p Acros, Aldrich, Aifei Aesar, Avocado va Fisher Chemicals. Biem chdy dugc xdc djnh trdn mdy Eiectrothennal 8100, pho IR duge do tren mdy Bmker FT IR spectrophotometer Vector 22, p h i ^H- NMR vd ^^0-NMR dupc do tren mdy GSX 270 WB spectrometer vd Bnjker Avance DRX 50
spectrometer, phd khoi LRMS dugc xdc djnh trgn mdy Esquire 3000 Plus Boiker Daltonics spectrometer.
P h u o n g phdp Tdng hgp hda hgc
Cde d i n e h i t 8,9-dimethoxy-5-methyibenzo [c][1,9]phenanfrirDiinium vd 12-ethoxy-8,9-dimethoxy- 5methylbenzo[c][1,9]phenanthrolinium d u p c t i n g hgp theo quy trinh ghl trong so d i 1.
I T
(6) R = H, (87%) (7) R = OEt, (84%)
HgCO- HjCO'
(a) NBS. con. HjSO*. -22°C: KNO3, con H2SO4, -1CPC i t 68%, thu C\SOc (4) (b) (4), NaCaHsOH, Cu*. DMF-CjHsOH, rt, 95%, Ihu diJOc (5) (c) Pd/C 10%, NH2-NH2, ethanol, reflux; (d) SOCIj, DMF(cat), CHCI3, reflux;
(e) NaH, DMF, CH3I. rt (f) Pd(0Ac)2, P(o-Tol)3, Ag2C03, DMF, reflux { g ) L i A I H / r H F ; { h ) a i r
So* do 1 : Tiin trinh tdng hop cic din chit BCP b$c 4 Thir nghiim sinh hgc
Thir nghlgm dpc tinh t i bao in vitro cua cdc c h i t tdng h y p d u g c d u p c t h y c hign tren dong te bao L1210 (murine leukemia cells) theo phuong phdp "microcuiture tetrazolium assay"^^^.
Ket qua
T o n g h g p hda hgc
Cac dan c h i t aminoisoquinolin (6) vd (7) c i n thiet cd d u p c bang s y khu* cac dan c h i t nitro (4) va (5) t u o n g irng d u d i tac dyng ciJa hydrazin va Pd/C. T i i n trinh chung de cd d u g c cdc amid tir phdn U'ng giira cde amin thom vd acid clorid tao thanh ngay trong phan ung d u p c mo ta r i t
nhieu trong ede tai ligu. Theo dd, vigc them vao mpt base amin nhu triethylamin, DBU, diisopropylamin d u o c eho Id c i n thiet d i nang cao higu s u i t phan ij'ng vi cdc base amin d u p c thdm vdo ed nhigm vy ' b i t giO'' HOI sinh ra trong qud trinh phan irng Tuy nhien trong trudng hpp ciia chiing toi, k i t qud thir nghiem cho t h i y phdn irng xay ra rat de dang, higu s u i t cdc amid (8) vd (9) r i t cao (^ 90%) ma khdng can phdi them b i t ky mpt base amin nao. Ket qud ndy theo chimg tdi cd t h i gidi thich Id do nhifng amid tgo thanh tLr cdc d i n x u i t amin thom cua isoquinolin vd phtalazin, ban than chiing cd the thay the vai trd eiia cac base amin
T^P CHi DirqfC HOC - 05/2012 (S6 433 NAM 52)
• NgKiien cieu — Ky thiuat
trong viec 'bat giu'' HCI 3k phcin Crng d6 dcing dat d^n mijc g^n nhLF djnh lu'p'ng (Scy d6 1). Sau khi metiiyi ii6a c^c amid tgo thdnin, s y d6ng v6ng ngi phan tCf xCic t^c Pd dyi?c thi^c hl$n trSn c6c d j n ch^t (10) vd (11) dLcAi di^u ki$n oi^ia phdn U'ng Heck '^^l Chijng toi thu du'Q'c hai dUn chlit bsnzolc][1,9]phenanthroiin-6(5H)-on (12) v4 (13) vM hieu sullt An iuqrt Id 42%, 4 1 % . RiSng trong phdn ling d6ng vdng thyc hi$n trSn amid (11), ngodi dSn ch^t chinh id h(?p chSt (13), chCing t6i cbn thu du-p-c mOt lu-tp-ng nhd (khodng 7%) hydroxyisoquinobenzoazepinon (16) {Sa dfi 2).
S y tgo thdnh sdn p h i m phy cii c i u trOc naphtobenzazepinon trong qud trinh ddng vdng n§u tren cung d y o c ghi nh§n trong nhung nghiSn cu-u cua iHamaraya vd cOng s y '^''"^®'.
Bang cdch khtl hda cdc amid iactam v6i tdc
nhdn LiAiHVTHF, t i l p theo dd id s y oxy hoa n g i u nhiSn khi d l sdn p h i m khCr hda tiep xuc vf^i khdng khi, chi:ing tdi thu dyg-c 8,9-dimethoxy- 5-methyibenzo [c][1,9]phenanthroiinium (14) vd 12- ethoxy-8,9-dimethoxy-5-methylbenzo [c][1,9]
phenanthrolinium (15) vdi hi^u s u i t l i n ly(?t id 60% vd 61 %. TrSn p h i ^H-NMR cua (14) vd (15) cho t h i y cd hai tin hi^u d|ic tryng: 1 singiet i>ng vdi nhdm CH3 g i n trdn N5 vd mdt singlet iing vdi proton H6 (Bdng 1).
Bdng 1 : Vii du' ll^u phd 'H NMR cua (U) vi
m
Htjipchit 'HNIIIIR(2T0MHi:DMSO),lppm) 5,01 (s, 3H, N'CHs) I
10,32(s, 1H, H6) J (14)
(15) 6,02 (s, 3H, N'CHa) 10,25 (s,1H,H6)
(d) Pd{0Ac}2, P(o-Tol)3, Ag2C03, DMF, reflux
S c do 2: Phan ung dong vdng amid (11) tao sin phim chinh (13) vi sin pham phu (16) Duf lieu ve diem chdy va pho cua (12),
(13), (14) vd (15)
8,9-Dimethoxy-5-methylbenzo[c][1,9lphe nanthrolin-e(5H)-on (CigHisNzOa = 320,1) (12):
C h i t ran vang nau, mp 228-230°C; IR v 1652, 1605, 1280, 1032 c m ' ; 'H-NMR (500 MHz;
DIVISO) 10,06 (s, 1H); 9,12 (d, J = 9,0 Hz, 1H);
8,66 (d, J = 6,5 Hz, 1H); 8,36 (d, J = 6,0 Hz, 1H); 8,09 (d, J = 9,0 Hz, 1H); 8,05 (s, 1H); 7,79 (s, 1H); 4,08 (s, 3H); 4,07 (s, 3H); 3,96 (s, 3H);
"C-NMR (125 MHz; DIVISO) 162,0; 153,8;
150,5; 146,2; 138,3; 136,0; 129,7; 129,6; 127,4;
123,3; 121,6; 118,9; 118,9; 118,3; 107,8; 104,5;
56,3; 55,8; 40,2; LRMS [M*Hf theo tinh todn ly thuyet C u H u N z O j : 321,12, thyc t l do dyp'c:
321,10.
12-Ethoxy-8,9-dlmethoxy-S-methylbenzolc]
[1,9]phenanthrolin-e(5H)-on (CaiHaoNzO, = 364,1) (13): Chat rdn vdng nau, mp 160-165°C; IR v 1646, 1609, 1229, 1030 c m ' ; 'H-NMR (270 MHz; CDCis) 9,90 (s, 1H); 8,68 (d, J = 7,5 Hz, 1H); 8,43 (d, J = 7,5 Hz, 1H); 8,00 (s, 1H); 7,95
(s, 1H); 7,54 (s, 1H); 4,48 (q, J = 7,0 Hz, 2H);
4,16 (s, 3H); 4,10 (s, 3H); 4,09 (s, 3H); 1,7 (t, J = 7,0 Hz, 3H); "C-NMR (67,5 MHz, CDCI3) 162,8;
153,6; 150,4; 147,6; 147,0; 140,1; 130,1; 129,5;
127,7; 120,0; 118,1; 116,0; 108,8; 108,6; 103,3;
102,7; 64,7; 66,3; 56,3; 4 1 , 1 ; 14,8; LRUS [M*Hf theo tinh todn ly t h u y l t CjiHjiNzOt:
365,14, t h y c t l do dyiyc: 365,10.
8,9-Dimethoxy-S-methylbertzo[cl[1,91 pfienan;
throlinlum ( C S H I B N J O Z = 304,1) (14): C h t e r i n vdng ndu vfi djnh hinh; IR v cm"' 1 6 1 ^ 1517, 1428, 1384, 1215, 1033; ^H-NMR (270 MHz; DMSO) 10,32 (s, 1H); 10,09 (s, 1H);
9,37 (d, J = 8,5 Hz, 1H); 8,93 (d, J = 6,5 Hz, 1H); 8,6 (m, 2H); 8,29 (d, J = 5,5 Hz, 1H); 8,0 (s, 1H); 5,01 (s, 3H); 4,27 (s, 3H); 4,08 (s,
"C-NMR (67,5 MHz; DMSO) 158,6;.
3H); DMSO) 158,6;.,
152,0; 151,9; 149,3; 145,7; 137,7; 135,4;
132,0; 131,7; 129,1; 125,7; 125,3; 120,4;
108,9; 104,1; 103,6; 57,5; 66,4; 62,2; LRMS [M-mf theo tinh todn ly t h u y l t C i g H n N j O ! * : 306,12, t h y c t l do dyp'c: 305,0.
T . ^ CHi D i r p c HQC - 05/2012 (SO 433 N A J M 52) I
• Ngiiien ciJu - Ky tiiuat
12-Ethoi!y-S,9-dlmethoxy-5-methylbenzo [ol[1,9]phenanthrolinium (C2,H2oN203 = 348,1) (15): c h i t ran vdng nau vd djnh hinh; IR v cm'' 1609, 1516, 1433, 1379, 1216, 1025; 'H-NMR (270 MHz; DMSO) 10,25 (s, 1H); 9,87 (s, 1H);
8,95 (d, J = 5,5 Hz, 1H); 8,34 (m, 2H); 8,26 (s, 1H); 7,77 (s, 1H); 5,02 (s, 3H); 4,65 (q, J = 7,0 Hz, 2H); 3,96 (s, 3H); 3,90 (s, 3H); 1,62 (t, J = 7,0 Hz, 3H); " C - N M R (67,6 MHz; DMSO) 158,0;
153,5; 162,0; 149,3; 149,0; 146,9; 131,3; 130,6;
127,0; 126,7; 120,2; 120,1; 114,7; 108,8; 103.6;
101.6; 66,3; 57.3; 56,2; 51.8; 14.1; LRMS /M+H7* theo tInh todn ly t h u y l t CjiHjiNzOs*:
349,15, t h y c t l do d y o c : 349,10.
Thip n g h i e m s i n h hoc
K i t qua t h y nghiem s o bfi dfic tinh t l bdo in vitro cua cac chat tu-ong dong BCP tong hgp (12), (13), (14), (15), t h y c hiSn trSn dfing t l bao L1210 (murine leukemia cells) theo p h y a n g phap "microcuiture tetrazolium assay"'"^ dyg-c ghi trong Bang 2. K i t qua cho t h i y cac d i n c h i t (12), (13), (15) deu cfi IC50 t h i p ban fagaronin.
dac biSt Id d i n c h i t (13) cfi tac dung manh n h i t vdi IC50 nhfi hon 5 ian so vdi fagaronin. Cac dan c h i t iminium deu co iCso tren dfing t l bdo L1210 cao hon so vdi cdc amid lactam tifong Lpng.
Bang 2; Ket qui doc tinh te bio in vitro cua cic BCP tSng hap
Hap chit L1210 ICaipM)
Fagaronin
(12) (13) (14) (15)
6.40 5.17 1.61 10.04 5.79
B a n l u a n v a k e t l u a n
Trong t h d nghiem s o bd ddc tinh t i bao tren ddng te bdo ung thu- L1210, hai c h i t tu-ong ddng BCP tdng hpp du'Q'c chira trong c i u triic N bdc 4 (14) va (15) the hien boat tinh dpc tinh te bdo kdm hon cac e h i t tu-ong ddng BCP amid iactam tu-ong irng. Dieu nay co ve nhu' phii hpp vdi nhgn djnh ciia mot sd tac gia la si^ hien dien cua nhdm iminium cd the can t r d s y h i p thu qua mang te bao cua nhdng tdc nhdn nay d i n den boat tinh kem hay t h i t t h o d n g '^^^^l mac dii cung cd nhijng quan diem cho r i n g nhdm iminium trong cdc c h i t t u o n g ddng BCP c i n t h i i t cho hoat tinh ^^.
Hpp chit 12-ethoxy-8,9-dimethoxy-&^ethylbenzo [c][1,9]phenanthroltn-6(5H)-on (13) t h i hi^n dgc tinh t i bdo ro rgt vd mgnh n h i t trong thir nghi$m trdn so vdi fagaronin vd cdc c h i t tuong ddng BCP (12), (14), (15). Phdn tfch c i u triic (13) chiing tdi nhgn t h i y : voi khung da vdng dj vdng thom p h i n g hpp c h i t (13) ed dtrpe dieu ki0n tidn q u y i t d i Idn vdo ADN '^"'i chirc amid iactam trong hpp c h i t ndy cd t h i c h u y i n thanh amin Idm gia tdng tinh tan trong n u d c '^^"^^, tir dd cd t h i Idm tang sinh khd dgng, cd t h i ddng gdp vdo vi$e gia tang d$e tfnh t i bdo; nhdm 12 ethoxy ciia hpp c h i t (13) cung cd the gdp p h i n Idm thudn Ipi hon khd ndng (yc c h i TOP-1 cCia c h i t ndy b i n g cdch ngan chdn TOP-1 tien tdi vj trf ddc hi§u eiia enzyme ndy trdn ADN ^^' d i n d i n dpc tfnh te bdo tang len ddng ke.
Tdm Igi cdc dan c h i t 8,9-dimethoxy-5- methylbenzo[c][1]phenanthroiinium va 12- ethoxy-8,9-dimethoxy-5-methylbenzo[c][1,9]
phenanthrolinium tong hpp dupe trong nghien ciru nay the hien dpc tfnh te bdo khdng nhu' mong dpi, trai lai cdc c h i t trung gian amid lactam tuong irng eua chung, trong do hpp c h i t 12-ethoxy-8,9-dimethcxy-5-methyl benzo[c][1,9]
phenanthrolin-6(5H)-on (13) cd t h i trd thanh hpp chit khdi nguon (lead compound), tir do cd the t h i i t k i vd t i n g hpp nhirng c h i t tu'ong ddng BCP khde ed tiem nang ddc tfnh te bao va ire c h i TOP-1 cao hon.
S u m m a r y
Promoted by the fact that many a non- camptothecin derivatives have been investigated for new antitumor agents targeting topoisomerase- 1 possessing higher potent activity and less side effects, and fijriher taken tocus on benzoic]
phenanthridine analogues, this study aimed to synthesize two t>enzo[cJphenanthridine quaternary compounds: 8,9-dimethoxy-5 methylbenzo[c][1,9]
f^enanOini^inium (14) and 12-ettioxy-8,9-dimettioxy- 5-methylbenzo[c][1,9]phenanthrolinium (15) from the amide iactam derivatives (12) and (13) respectively. In vitro cytoxicity was tested in L1210 cell line showing the iminium derivatives were less potent than the corresponding lactam compounds.
T a i l i e u t h a m k h a o 1. Holden, J. A.: DNA topoisomerases as anticancer daig targets: from Itie laboratory to the clinic, Curr. Med.
Chem. AnScancer Agents, (2001), 1(1), p. 1-25.
TAP CHI DirgfC HQC-05/2012 (S6433 NAM52)
• Nghien CIFU - Ky thuat
2. Pommier, Y.: Topoisomerase I inhibitors:
camptothecins and beyond, Reviews/Cancer, (2006), 6.
p. 789-802
3. Stermitz, F. R., K. A. Larson, and D. K, Kim:
Some structural relationships among cytotoxic and antitumor benzophenanthridine alkaloid derivatives, J.
Med. Chem.. (1973), 16. p. 939-940.
4. Bai, L. P., et al.: DNA-binding affinities and sequence selectivity of quaternary benzophenanthridine alkaloids sangulnarine, chelerythrine, and nitidine, Bioorg. Med. Chem..
(2006), 14. p. 5439-5445.
5. Qin. Y,. et ai.: Synthesis. DNA-bindIng affinities, and binding mode of berberine dimers, Bioorg. Med.
Chem,,(2006). 14, p. 25-32.
6. Kanzawa, F., et al,: Anti-tumour activities of a new benzo[c]phenanthridine agent, 2,3-(mathylenedioxy) -5-methyl-7-hydroxy-8-methoxytDenzo[c] phenanthridinium hydrogensulphate dihydrale (NK109), against several drug-resistant human tumour cell lines, Br. J. Cancer, (1997), 76(5), p. 571-581.
7. MacKay, S. P., et al.: The effect of 12-alkoxy modification on the in vivo antileukaemic activity of N- methyl 2,3,8,9-tetramethoxybenzo[c]phenanthridinium salts, Anticancer Drug Design, (1998), 13, p. 797-813.
8. Clark, R. L., et al.: Explonng DNA topoisomerase I inhibition by the benzo[c] phenanthridines fagaronine and ethoxidine using steered molecular dynamics, Bioorg. Med Chem., (2007), 15, p 4741-4752.
9. Fleury, F., et al., Proceedings of AACR conference " Molecular Determinants of Sensitivity to Antitumor Agents", (1999), p. 12.
10. Lanoui, A.: DNA Binding by Fagaronine and Ethoxidine, Inhibitors of Human DNA Topoisomerases I and II, Probed by SERS and Flow Linear Dichroism Spectroscopy, J. Phys. Chem. B, (1999), 103(11), p. 2008-2013.
11. Lynch, M. A., et al.: Synthesis, biological activity and comparative analysis of DNA binding affinities and human DNA topoisomerase I inhibitory activities of novel 12-alkoxy-benzo[c]phenanthridintum salts, Bioorganic and Medicinal Chemistry Letters, (2001), 11 (19), p. 2643-2646.
12. Smith, M. B. and J. March: Advanced organic chemistry, NewYork: Wiley-lnterscience, (2001), p, 506-507.
13. Rucheiman, A. L., et al.: 5H-Dibenzo[c,h]1,6- naphthyridin-6-ones: novel topoisomerase l-Targeting anticancer agents with potent cytotoxic activity, Bioorganic and Medicinal Chemistry. (2003), 11 (9), p. 2061-2073.
14. Harayama, T, et al.: Synthesis of Benzo[c]Phenanthridine Alkaloids, Using a Novel Palladium-Phosphine Ck)mbination System Pd(OAc)zDPPP, and BuaP, Synthesis. (2002), p. 237-241.
15. Harayama, T., et al.: Total synthesis of benzo[c]phenanthridine alkaloids, chelerythrine and 12-methoxydihydrochelerythrine, by a palladium- assisted internal biaryl coupling reaction, Synthesis, (2001), 3, p. 444-450.
16. Harayama, T., et al.; Novel Synthesis of Naphthobenzazepines from N-Bromobenzylnaphthy lamines by Regioselective C-H Activation Utilizing the Intramolecular Coordination of an Amine to Pd, Synlett, (2003), 8, p. 1141-1144.
17. Pierre, A., et al.: Preclinical Antitumor Activity of a New Vinca Alkaloid Denvative, S 12363, Cancer research, (1991), 51, p. 2312-2318.
18. Prado, S., et al: Synthesis and cytotoxic activity of benzo[c][1,7] and [1,8]phenanthrolines analogues of nitidine and fagaronine, Bioorganic and Medicinal Chemistry. (2004), 12(14), p. 3943-3953.
19. Janin, Y. L, et ai.: Synthesis and evaluation of new 6-amino- substituted benzo[c]phenanthridine derivatives, J. Med. Chem.. (1993), 36(23), p. 3686-3692
20. Avendano, C. and J.C: Menendez, Medicinal chemistry of anticancer dnjgs. Amsterdam, TheNetheriands: Elsevier, (2008), p. 199-220.
21. Cho, W. J , et ai., Molecular modeling of 3- arylisoquinoline antitumor agents active against A- 549. A comparative molecular field analysis study, Bioorg. Med. Chem.. (2002), 10, p. 2953-2961.
22. Cho, W. J., et al.: Synthesis of new 3- aryiisoquinolinamines: Effects on Topoisomerase I inhibition and cytotoxicity, Bioorg. Med Chem., (2003), 13, p. 4415-4454.
M o t k h a n g S'lnh... (Tiip theo trang 57}
Raymond Poincare de Garches (Phdp), s y phat hign ndy Id mpt hy vpng thyc s y vi vdch t i bao la yeu td bao v$ chii yeu cua vi k h u i n .
K i t luan
Chat BTZ 043 ed the du'pc t h d nghigm Idm sang phase I trong nam tdi. Sau dd c i n 5-6 nam
d i du-a ra thj t n r d n g . Cd t h i k i t hpp vdi cdc thudc khang lao khdc de hgn che s y xuat hien tryc k h u i n khdng thuoc.
