Điều chế và kiểm tra chất lượng thuốc phóng xạ 90YDOTAnimotuzumab dùng trong điều trị ung thư đầu cổ

• Nghien cu'u - 1(9 thuOt

Dieu cht va kiem tra chAt luvng thulk
phong xa 90Y-DOTA-nimotuzumab
dung trong dieu tri ung thtr dau co
Nguyen Thi Thu'*, Vo Thi Cam Hoa', Nguyen Thi Khanh Giang'
Nguy& Thi Ng9c', Mai Trong Khoa2, TrOn Dinh Ha'
Nguy'Zn Linh Toa', HO Anh San'
hot Muhl (Da Lot)
Nah vier Bach Mai
'Hoc ri(M Quoit Y
*E-mail: ngthithu2006(myahoo.cam

Summary
With regard that the radioactive isotope Yttrium-90 ( 90Y) with the physical half-life of 64.14 hrs
and highly energetic beta-ray emission well serves as an ideal therapeutic radioisotope in clinical use,
90Y-DOTA-nimotuzumab radiopharmaceutical was prepared by conjugation of monoclonal antibody
with the bifunctional chelating agent p-NCS-Benzyl-DOTA. The chelating was covalently bonded to an
antibody using isothiocyanate (NCS) chemically chelated to the radioisotope Ytrium-90 by attachment
of carboxylate anions. The coupling of p-NCS-Benzyl-DOTA to nimotuzumab at molar ratio 25:1
accomplished at 37°C and pH 8.5, achieved the efficiency of about 53.4 % after 12 hours. The coupling
labeled with 90Y isotope at 42°C and pH 6.0 attained a radiolabeling efficacy of 97.8±0.3 % after 60
minutes. The obtained Radiopharmaceutical 90Y-DOTA-nimotuzumab proved to satisfy the in vitro
stability and meet the quality requirements for use in the treatments of head and neck cancer.
Keywords: 90Y-DOTA-nimotuzumab, Radiopharmaceuticals, head and neck cancer therapy
Mee clAU
Trong nhOng nom gen day, thuoc phOng xa
khang the dun d6ng danh deu &rig vi phOng xa
da duvc nghien cOu dieu the va Ong dung trong
chAn doan va dieu tri lam sang. Khang the don
d6ng nimotuzumab duvc danh d5u vai ding vi

ph6ng xa 90Y to thanh thuoc ph6ng xa 90Y-DOTAnimotuzumab de nghien cipu dieu tri ung thu,
d'au co [1] . Nimotuzumab la khang the nhan h6a,
thuOc loai IgG1, thirc hien chipc nang nh5m dich
len thu the cac you to tang true:mg bleu bi EGFR
(Epidermal Growth Factor Receptor) tren cac to
bao ung thu' bieu mo. Ben c5nh ch(rc nang tieu
diet to bao ung thu' theo cac ca the sinh hoc,
90 Y-DOTA-nimotuzumab con diet to bao ung thu'
theo co' the bi:Pc xa ion h6a [21. V6i thei gian ban ra
64,14 gio,, nang luvng tia beta 2.280 KeV, quang
chay trong m6 4-11mm, 9°Y la &rig vi ph6ng )(ply
tuerng dOng trong dieu tri benh khi gen vitei phan
tiv khang the 131 . D6 danh d5u vitei (long vi ph6ng xa
TAP CHI DUQC HOC - 9/2015 (SO 473 NAM 55)

kim loai co hoa tri Ill nhu, 9°Y, khang the door gen
vai ph6,c trung gian la p-SCN-benzyl-DOTA to
thanh p-SCN-benzyl-DOTA-nimotuzumab. Ph5n
t6, p-SCN-benzyl-DOTA la ph& till, de t?o ph0,c
ben vOi nhieu nguyen to co h6a tri Ill nhu' 90Y3*,
do vay, 90 Y &roc gan vao theo co' the phan irng
the nucleophin SN tpo thanh phirc hap 90Y-DOTAnimotuzumab [41. Vi thuoc ph6ng xa co china khang
the va deng vi ph6ng xa 90Y co thOi gian ban r5
ngen, nen kh6ng the mua tO, nu,dec ngoai vi kh6
bao quen khi van chuyen, chu,a ke den la gia ce
rat det. Vi the, can thiet dieu the trong nu,dec de
ch0 citing nguOn nguyen lieu cung cep cho y te.
Bai bao nay trinh bay nghien cu'u danh dAu dieu
the thu6c phOng xa 90Y-DOTA-nimotuzumab de
dieu tri ung thu' dhu co ten lam sang.

Phu'oing phap nghien CCPU
Nguyen lieu, hoe chat, thiet bi
Deng vi ph6ng xa 9°Y clang 90Yttriumacetat (CH3COO)3 90Y, nOng d(f) ph6ng xa
23


• Nghien ciru - Kji thuOt
592 mCi/ml. Khang the nimotuzumab
50 mg/10 ml, h5ng CIMAB, Cu Ba. C6ng thac
phan tir C6566F110082N174602056S40 Cac hea chAt
2-ethylhexyl 2-ethylhexyl phosphoric acid
C16H3503P (PC88A), Daihatchi; p-NCS-BenzylDOTA
S-2-(4-lsothiocyanatobenzy1)-1,4,7,10
tetraazacyclododecan tetra acetic acid,
C24H33N508.2,5 HCI.2,5 H2O, Macrocyclic;
Sephadex G25 PD10, Hang GE Healthcare; Mang
th5m tich Slide A lyzer 10 K (dialysis cassettes),
MWCO 66453 Hang PIERCE; Te bao ung thu,
HEp-2 (Human Epithelial Type 2), CCL-23,
American Type Culture Collection (ATCC); Thiet
bj phOng xa to, chop Cyclone B431200, Perkin
Elmer, ph6 Ice gamma ORTEC, may do beta GM,
may do beta nhAp nhay long Aloka 6110. May do
PTS 100, Hang Charles River Laboratory.
Cac thi nghiem duvc tien hanh tai Vien Nghien
hAt nhan, DA Lat.
Khao sat ti le mol, nhiet do, thai gian va pH
dieu the DOTA-nimotuzumab
Thi nghiem thy'c hien vai n = 2 cho tat ca cac

diem khao sat va ket qual5y gia tri trung binh. Cho
vao cac chai phan Ong 50 ol bicarbonat 0,05 M,
met luong p-NCS-benzyl-DOTA (pha bang trong
DMSO den nong dd 10 mg/ml) va khang the
nimotuzumab theo cac ti le mol IA 5:1, 10:1, 25:1,
50:1 100:1 va 200:1 (dung 0,66 nmol khang the
trong m6i phan ang khao sat), dieu chinh pH 8,5
bAng 6 pi dung dich NaOH 0,1 M, ten nhe dung
dich h6n hap va 0 tai cac nhiet de 4°C, 24°C,
37°C. Khao sat thai gian phan Ong duvc thtyc
hien tai ti le mol 25:1, nhiet de 37°C, pH 8,5, thai
gian 10, 40, 120 , 180, 360, 720, 1080 va 1440
phOt. Khao sat pH tao phU'c thac hien tai cac pH
3, pH 4, pH 5, pH 6, pH 7, pH 8, pH 9 va pH 10,
dieu chinh bAng dung dich NaOH 1 M va HCI 1
M. Nhiet do ph& O'ng la 37°C, ti le mol 25:1, thai
gian 720 phOt.
Dieu the NY-DOTA-nimotuzumab va kiem
tra chat luvng

DOTA va khang the nimotuzumab theo ti le mol
IA 25:1 (230 og DOTA va 2000 og khang the),
dieu chinh pH 8,5 b5ng 6µl dung dich NaOH 1 M,
ten nhe dung dich h6n hop va 0 tai nhiet do 37°C
trong thai gian 12 gia. Hen hcyp duvc loc qua cot
gel Sephadex G25, dung ch5t raa giai IA dung
dich NaCl 0,9%, van toc rira giAi IA 30 cm3/gia,
thu DOTA-nimotuzumab vai the tich IA 3 ml tai
phan doAn 4-6 ml. Phirc hop &roc th5m tich
trong yang 24 gi6 trong dem phosphat 0,1 M

dung mang thArn tich dialysis cassettes Slide A
lyzer 10.000 Da, sau do ly tam va xac djnh ruing
do khang the tai baac song 280 nm.
Dieu the NY-DOTA-nimotuzumab M. D6
di6U the 30Y-DOTA-nimotuzumab co hoat do
rieng 0,37 GBq/mg, danh dAu 592 MBq 30Y (1000
ml) tai thai diem dieu the vai 1000 og (750 ol)
khang the DOTA-nimotuzumab trong dem acetat
0,5 M, pH 6. U hen hap 60 phOt a nhiet de 42°C,
sau de, phan tich lAng phaang phap sac k9 lap
mong TLC trong dung m6i amoniacetat 10% va
methanol theo ti le the tich 1:1, kiem tra hieu suet
danh d5u bAng phiyang phap loc gel Sephadex,
dung chAt raa giai IA dung dich NaCI 0,9%, vai
toc dd raa giai IA 30 cm3/gity.
Kiem tra chat
ng: Phac hap 30Y-DOTAnimotuzumab &roc kiem tra de tinh khiet h6a
phOng xa bAng phaang phap sec ly lap mong,
sec k9 loc gel (3.7] , Kiem tra de tinh khiet hat nh5n
bang phaang phap chiet ten giey Extraction
Paper Chromatography (EPC) 181, chAm PC88A
va mAu vao vi tri g6c, phat trien trong dung dich
NaCI 0,9%; Kiem tra do sach gamma bAng phe
Ice gamma 171; Kiem tra de on djnh b5ng phirang
phap 0 thuec trong huyet thanh ngt.rai va cac dieu
kien bao guar' nhir dem phosphat PBS 0,01 M,
NaCI 0,9%, phan tich, theo del den 7 ngay131. The,
ye khu5n va tha not do, c to vi khu5n theo TCCSDPPX-16/15-2010 191. Kiem tra hoat tinh mien dich
cue thuec ph6ng xa 30Y-DOTA-nimotuzumab, gen
in vitro tren to bao ung thy bieu me HEp-2, s6

luvng 0,5 x 106 to bao/gieng gen, phaang phap
Ap dung IA saturation binding assay (101.

Dieu the phirc khang the clan dong vai
chit tao pherc hai ch&c hang p-NCS-benzylDOTA-nimotuzumab: Thtyc hien phaang phap
gen khang the vai ch5t tao phac trung gian
p-NCS-benzyl-DOTA claa tren cac nghien cau
(55 ceng be [5 ' 6]. Cho vao chai phan O'ng 500 ol
bicarbonat 0,05 M, met luang p-NCS-benzyl-

Xtv ly s6
Cac ket qua nghien eau duvc xa
19 theo cac thu5t toan thong ke y hoc. Tinh gia tri
trung binh va de lech chu5n bAng Excel, tinh hoat
tinh mien dich bAng phAn mem GraphPad Prism 6.0.

24

TAP CHi DUOC HQC - 9/2015 (SO 473 NAM 55)


• Nghien cu'u - K9 thulat
Cac ket qua khao sat hieu suet gen theo ti
le mol, nhiet dO, thai gian va pH gen DOTA vai
khang the trong dem bicarbonat 0,05M tom tet
trong bang 1 nhu, sau:

Ket qua va ban luan
Ket qua dieu che phirc hap DOTAnimotuzumab

Bang 1: Ket qua khao sat cac lieu kin tao phut hop DOTA-nimotuzumab
Tile mot DOTA:
Khang the (nmol:nmol)
NhiOt dO 4°C
Hiiu suit gin (%)
Nhiet de, 24°C
Hiiu suit gin (%)

NhiOt c10 37°C
HiOu suit gin (%)
Then gian phan (mg (phtit)
Hi#u suit gin (%)
pH too phav
Hieu suit gin (%)

5:1

10:1

25:1

50:1

100:1

200:1

85,0

63,3

54,6

46,3

12,5

12,3

80,7

56,0

53,9

41,0

19,1

12,6

72,0

60,1

55,0

49,6

48,5

46,8

10
0,9
3
6,0

40
4,4
4
9,7

Ket qua er bang 1 cho they tai cac if le mol lb
5:1, 10:1, 25:1, 50:1 100:1 lib 200:1, hoet tinh gen
DOTA vai khang the dat ter 12 den 85%. Tai ti le
mol 5:1 ket qua gen det khoang 72-85%, kr do
tinh ra duvc so nhem DOTA trung binh gen ten
mei phgn ter khang the gen lb 3,9 (Hnatowich va
Ong sky) twang if le mol 25:1 so nhOm DOTA
trung binh gen ten mei phan ter khang the là 13,5.
Val ti le mol 50:1 b.& len, se nhern gen Ian hen
20,0 6 cac nhiet dO 0. Wet qua khao sat cho they
phU'c tao thbnh co hieu suet gen khac nhau, phan
tey p-NCS-benzyl-DOTA tham gia cang nhieu thi
hieu suet gen cua phOc vai 9°Y cang it, ljt do co
so canh tranh cua met luvng this DOTA chiem
ch6 met so !Leong c6 hen cua 'long vi ph6ng xa
90Y. Cac khdo sat nhiet de cho they nhiet de 0 4°C,
24°C va 37°C a gen DOTA vai nimotuzumab

khOng lam thay dei Ian ye hieu suet gen. Thai
gian phan Ong gen cua DOTA va nimotuzumab
10 phut phirc hcyp DOTA-nimotuzumab teo thanh
lb 0,9%. Thbi gian 360 phiit, phirc hap DOTAnimotuzumab tao thanh là 47,3%. Thbi gian phan
Ong 720 phut, phOc hap tao thanh lb 60,1%
va thai gian phan Ong 1440 phut, phirc hop

O
=N----C +

II

120
25,6
5
54,5

180
40,1
6
59,8

360
47,3
7
63,9

S

1080

53,2
9
77,0

1440
52,1
10
60,4

DOTA-nimotuzumab tao thanh lb 52,1%. Nho
vay, thai gian cang tang, hieu suet gen Gang tang,
deng hoc dm Olen Ong tao phOc hop p-SCN-BnDOTA-nimotuzumab tang theo thdi gian.
Anh hoeng dm pH len qua trinh gen cho they,
trong mien pH acid, hieu suet gen cua p-SCN-BnDOTA va nimotuzumab de tao phOc hap DOTAnimotuzumab dat 10,0 - 50,0%. Trong mien pH kiem,
hieu suet gen dat 64,7-77,0%. Co the do ph& Ong
gen p-SCN-Bn-DOTA len khang the nimotuzumab
lb phan Ong acid-base theo thuyet Lewis, trong de,
khang the dOng vai tro là base con p-SCN-Bn-DOTA
clang vai tr6 lb acid. Phan Ong gia,a nimotuzumab
va p-SCN-Bn-DOTA lb phan Ong giu,a acid yet, va
base yeu nen moi truing cang acid hay Gang base
thi san phem se de bj ph8n hay. Tern lei phOc hap
p-NCS-benzyl-DOTA-nimotuzumab doec dieu che
tai ti le mol la 25:1, nhiet de 37°C thai gian phan
Ong 720 phut va pH 8-9. Giai thich ca che, DOTA
lb tic nhen tao phOc hai chipc nang, lien ket Ong
hoe tri nhOm isothiocyanat vai nhom amin cua lysin
ten phan tot, khang the qua phan Ong the nucleofin
SN nhu, sau:

C
Protein-NH:

720
60,1
8
64,7

O

-N - C - NH-Protein
II 11
S H

Theo phan Ong tren, qua trinh tao phOc xay ra do
nguyen ter nits cua lysin co chrya del dien ter to do va
phan tir p-NCS-Bn-DOTA la chat nen chOa carbon

cation, vai hieu Ong Ong twang am (-M) bad \tong
benzen lam cho carbon mang dien tich &rang, Er
do lien ket isothiocyanat va amin tao thanh.

TAP CHI DUQC HQC - 9/2015 (S6 473 NAM 55)

25


▪

• Nghien aru - K9 thuOt

Phirc tao thanh va danh dau ph6ng xa vai 9°Y dupp bieu dien nhu' hinh 1 sau:
0
HOOC r—v-- COON
9try

S

)"

NH—C—NH

CH

N

(N soy

=---

N

)— 011

Pal—C —NH

Ss"

N

HOOC

or/

COON

O

Hinh 1: Phirc hop p-NCS-Benzyl-DOTA-nimotuzumab va 90Y-DOTA-nimotuzumab
Philp hgp p-NCS-Benzyl-DOTA-nimotuzumab
truirc khi tinh sach phirc qua cot, hiOu suet gen
vai 90Y dat duvc 53,4%, so phan tir DOTA ten
m6i phan tir khang the la 13,4 va 3,9. Tai ti la mol
5:1, hieu suet gen vai 90Y det duvc 85%, de dieu
the phirc p-NCS-benzyl-DOTA-90Y-nimotuzumab
dung trong nghien ciru dOng hoc va dieu tri, dkra
vao s6 nh6m gen can, ti le mol thay del cho phu
hap. SO nhOm gen DOTA ten phan tir khang the
can khoang 4-10 nh6m va hoat dO rieng la 0,37
GBq/mg. Phirc DOTA-nimotuzumab tao thanh c6
nong d9 khang the nimotuzumab &pop xac dinh
la 1066 mg/ml. Mang them tich MWCO 10K se

lopi cac phan tir nho va cac manh dirt gay cue
khang the c6 trong krong phan tir nho han 10.000
Da ra khoi mang theo ca the khuyech tan chon
19c qua mang. Bao quan DOTA-nimotuzumab
nhiet d9 -20°C de danh dau dieu the 90Y-DOTAnimotuzumab.

Ket qua dieu the 90Y-DOTA-nimotuzumab
va kiem tra chat luvng

Higu suet danh clAu: Philp hap 9°Y-DOTAnimotuzumab duvc dieu the vai hiau suet danh
dau 97,8 ± 0,3% (n = 6), (hinh 2a, 2b):

Hinh 2: Hieu suet danh dAu 90Y-DOTA-nimotuzumab (a): 97,5%; (b): 99,1%, TLC, radioautograph.
Hieu suet danh dau cua phirc hop mien dich
vai deng vi ph6ng xa dat tir 97,5% den 99,1%. Cac
dO thi 2a, 2b cho they DOTA-nimotuzumab sau khi
tinh sach, danh dau vai 90Y dat hreu suet cao, hoat
dO Hong 9°Y-DOTA-nimotuzumab dat 370 MBq/mg.

41lb

DO tinh khiet hem phong xa (RCP) va tinh

On dinh: RCP dat han 99,3 ± 0,4% (n = 10) (hinh
3a) va phirc On dinh trong huyet thanh ngtrol den
6 ngay, On dinh trong dem PBS 0,01 M va NaCI
0,9% cho den 7 ngay bao quan 6' 4°C (hinh 3b):
11.0
0300
t 90
ea I80

"Y-Nhnotezumab
99,9%

a ..
0

70

60
E- 50
*
§a 40
• 30
al 20
raa 10

/Ma

I
•

"Y

-

0

(b)

On Sink Iron, huyil Mani, ngun31

—II— BOO 009n Iron° PBS 0,011A
—16— Bin clan trong MCI UV.

1

3

4
Thing= (NOY)

6

7

Hinh 3: 06 tinh khiet h6a ph6ng Xp (a) va On dinh 9°Y-DOTA-nimotuzumab (b)

26

i

TAP CHI DUOC HOC - 9/2015 (SO 473 NAM 55)


• Nghien C01.1 - Ky mot
Dc) tinh khiet hcia phOng xa cua cltygc chat
phOng xa 90Y-DOTA-nimotuzumab dat duvc la
99,3%. Tren do thi cho thay vi tri cua 90Y-DOTAnimotuzumab Ityu tai diem gOc cua b5ng sac k9
TCC, Rf = 0,0 - 0,1 va R cua 90Y la 0,9 - 1,0.
90Y-DOTA-nimotuzumab On dinh trong dem PBS

0,01% hoac NaCI 0,9% cho thay co the bao quart
de sir dung va on dinh trong human serum cho
thay tinh ben cua thu& khi &pa vao trong ca the.
xa: DO tinh khiet
DO tinh khiet hat nhan

hat nhan ph6ng xa duvc kiem tra bang phtyang
phap sac k9 chiet tren gray EPC, (hinh 4a):

a

INDIC
no
Emmy, (.04
FNE$1 Ina 181007 lm, Tow MOO U15
Aepuew 03.13/1014 17 70 75
e. Or
ChM... 00
. I WV0H10,10POOHOGYlop clop 0vPx,6 ilanW 07
P.C. 40

Hinh 4: Ket qua ki6m tra dO tinh khiet hat nhan 90Y-DOTA-nimotuzumab, autoradiography (a)
va do sach gamma cua 90Y-DOTA-nimotuzumab, do bang ph6 ka gamma ORTEC (b)
Ket qua do tinh khiet hat nhan cua 90Y-DOTAnimotuzumab dat han 99,998%. Tai vi tri gOc,
chat chiet dac hieu PC88A
90Y-DOTAnimotuzumab, 90Sr trong san phem 0,002%
di chuyen ye tuyen ten cua dung mOi. Phirc
90Y-DOTA-nimotuzumab dpt kiem tra dO sach
gamma (hinh 4b), do bang phO ice gamma. PM
cho thay khong co dinh gamma nao trong dung
dich san phern. Thu& dpt chat luting ye do tinh
khiet hat nhan phOng xa.
K& qua kiem tra vo khuan va not di)c to vi
khuAn: Thu& phong xa 90Y-DOTA-nimotuzumab
dat do vo khuan, not dOc to vi khuan theo TCCSDPPX-16-2010, TCCS-DPPX-15-2010, IS017025.
Wet qua kiem tra hopt tinh mien dich: Kat

qua 90Y-DOTA-nimotuzumab gen ten to bao ung
thu, dirac phan tich bang phan mem Graphpad
Prism 6. DO thi dpt den gia tri cu'c dpi bao h6a,
hinh 5:
Saturation binding assay
3.5.

1.0
0.5
0.0
260

560

750 10130125014017.5020.002250

"Y-DOTA-nimotuzumab (nM/L)

Hinh 5: DO thi gan bac, hoa 9°Y-DOTAnimotuzumab vat t6 bao ung thu, HEp-2

12;11' CHI DUVC HQC - 9/2015 (SO 473 NAM 55)

Tren tryc hoanh la nOng dO tang den cua
Y-DOTA-nimotuzumab, phan gen dac hieu fmol/
cell duvc ye trail tryc tung la kat qua so phirc
On ten mOi to bao. Gia tri Kd la 3,2 nmol/L. Gia
tri 13m„ ten phan mem tinh duvc la 2,9 x 105.
Kat qua cho thay hopt tinh mien dich cua khang
the dam bac) sau khi clanh dau vOl chat ph6ng
xa. Cac ket qua kiem tra chat luting cho thay,

thu& phOng xa 90Y-DOTA-nimotuzumab dpt cac
Weil chi ye kiem tra chat luting thu& phong
On dinh in vitro, On dc hieu ten to bao ung thu,
bleu mo, co the sir dung trong cac nghien ciru
tien lam sang va nghien ciru lam sang.
90

Ket luen
Thu& phOng xa 90Y-DOTA-nimotuzumab
duvc nghien cu,u dieu the dy'a ten nguOn
nguyen lieu la khang the dan d6ng nimotuzumab
va clang A ph6ng xa 90Y. Phan Ong danh dau
phOng xa dieu che 9°Y-DOTA-nimotuzumab duvc
thy,c hien trong dam acetat 0,5 M, pH 6,0, dat
hieu suat cao 97,8 ± 0,3%. Thu& phOng xa thu
duvc dpt do tinh khiet hoa phOng xa han 99%,
dO tinh khiet hat nhan han 99%, On dinh in vitro
va apt cac chi tieu ve dc) vo khuan, nOi dOc to vi
khuan, dam bao hopt tinh mien dich gen ten to
bao ung thu, bieu ma c6 thy the EGFR. Duvc
chat ph6ng xa 90Y-DOTA-nimotuzumab dat tieu
chuan chat luting thu& phOng xa dung trong lam
sang dieu tri ung thu, du co.

27


• Nghien ciru - K9 thuOt
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Tecchnical Series Number 470.
8. Usha Pandey, Prem S. Dhami, Meera
Venkatesh, and M. R. A. Pillai. (2008), "Extraction
paper chromatography technique for the radionuclidic

purity evaluation of 90Y for Clinical Use", Anal. Chem.,
80 (3), pp 801-807.
9. Vin Nghien ciru hat nhan, (2010), Tieu chua'n
ca sO, DPPX-15/16-2010. ISO 17025.
10. Lindmo, T., Boven, E., and Cuttita, E. (1984),
"Determination of the immunoreactive fraction
of radiolabelled monoclonal antibody by linear
extrapolation to binding at infinite antigen excess", J.
Immunol. Methods, 27,77-89.

(Ngay nhan Oaf: 29/07/2015 - Ngay duyat clang: 04/09/2015)

Nghien cfru tong hcrp (S)-clopidogrel bisulfat
tur clopidogrel racemic
Nguyen Van Tai*, Phan Thi Trang, Nguyen Thi Thu Trang
Nguyen Thi Ha, WI Dtic Nam
Khoa H&J Time vat, Pen Dwaz. lieu
* E-mail: nguyenvantail 111@gmaiLcom

Summary
(S)-(+)-clopidogrel bisulfate was synthesized from clopidogrel racemic in high purity 99 %). The
process proved simple. (S)-clopidogrel was separated from clopidogrel racemic, then (S)-clopidogrel
bisulfate was synthesized in form I and form II. The form I exhibited better solubility and higher
bioavailability than form II. This suggested the form Ito be more suitable for medicinal use.
Keywords: (S)-(+)-Clopidogrel bisulfate, synthesis, chiral, form

DOA yen de
Tim mach dang la mdt trong nhOng can benh
gay chet ngutri hang dhu Van the giai cung nhu,
tai Viet Nam hien nay. (S)-Clopidogrel bisulfat (ten

Net du,gc Plavix), la thu6c chOng ngu'ng tap tieu
cAu, ngan ngiva cuc mau dong. (S)-Clopidogrel
bisulfat thip6ng sir dung de du' ph6ng nguyen
phat, du' ph6ng tho, phat va kiem soat cac roi loan
do nghen mach huyet khdi, xci vim Ong mach
28

nhu' nhOi mau co' tim, ddt quy va benh ddng much
vanh ngoai bran. Thu& co kha nang ngan ngiva
cuc mau thing manh hurl aspirin nhumg khong
gay tac dung phu Von da day giOng nhu' aspirin.
Clopidogrel (C16F116CINO2S), ton tai
2 dang clang phan enantiomer la (S)-(+)clopidogrel va (R)-(-)-clopidogrel. Clopidogrel
co clang dau, kem ben, kh6 tinh the va kh6 bao
the hom clang muiii dm no la clopidogrel bisulfat.
TAP CHI DUOC HQC - 9/2015 (SO 473 NAM 55)


• Nghien an' - Ky thuOt
Tac dung sinh hoc lai chi co 6' &rig phan (S)Vi vey, hoat chat trong
(+)-clopidogrel bisulfat
cac clang bao che chli yeu la (S)-(+)-clopidogrel
bisulfat, cho hieu qua chCra benh cao va tranh tac
dung phu. Tiou chuen trong cac Duvc dien M9,
Ch5u Au deu quy dinh t9.1e (long phan (R)-(-)clopidogrel bisulfat phai nho han hoc bang 1%.
Trong bai bao nay, chi:mg tei trinh bay phyang
phap tong hop (S)-(+)-clopidogrel bisulfat a clang
Bang I va clang II tiv clopidogrel racemic thOng
qua giai doan tao muOi vai acid (0-H-camphor10-sulfonic (10-L-CSA) de tach ley dOng phan
(S)-clopidogrel.

Nguyen vat lieu va phwang phap
nghien civu
Nguyen vet lieu va thiet bi
HOa chat duvc cung cep Nil cac hang Merck,

CI

Fisher, AK Scientific, hoc P.A. Trung Qu6c,
duvc sir dung kh6ng qua tinh che. Sac k9 lap
me:mg thiyc hien ten ban mong silica gel 60 GF25,
(Merck). Ph6 Ong huang tip hat nhan do tren
may Bruker Avance 500 MHz vai TMS lam chat
not chuen, ph6 hong ngoai do tren may Impact
410, ph6 khoi luting do tren may Auto Spec
Premier- Vien I-16a hoc, Vien Khoa hoc Viet Nam.
Phipang phap phan tich HPLC do ten may HPLC
Agilent 1200-Tnyang Dai hoc Duvc Ha N6i, cot
sec ky Chiral Ultrom ES-OVM + Guard cua Hang
Shinwa - Nhet Ban. Phirang phap xac dinh nhiet
d6 n6ng chay (DSC), kiem nghiem san pham
theo tieu chuan USP 34 duvc thus hien tai Vien
Kiem nghiem thu6c Trung 'yang.
Phtrang phap nghien cOu
(S)-Clopidogrel bisulfat &roc dieu che tip
clopidogrel racemic theo so' dO nhu, sau:

COOCH3
Nti\

.10-L-CSA

Dung mei

.10-L-CSA

(I)
Clopidogrel racemic

(III)
K61 Unit

Baia Dung mei

CI COOCH3

CI
.H2SO4

Dung mei
(1)

COOCH3

H2SO4

\
(S)

I

(IV)

(+)-Clopidogrel bisulfate

So, di) 1: Dieu che (S)-clopidogrel bisulfat ter h6n hop clopidogrel racemic

(S)-Clopidogrel bisulfat la san pham ciia qua
trinh tach dOng phan (S)-clopidogrel tir h6n hap
clopidogrel racemic, sau do teo muOi vai acid
sulfuric. Clopidogrel racemic phan Ong vai acid
(L)-(-)-camphor-10-sulfonic trong dung m6i thich
hap tao thanh h6n hap hai mu6i la hai dOng phan:
(S)-clopidogrel. (L)-Camphor-10-sulfonic va (R)clopidogrel. (L)-Camphor-10-sulfonic. Day la hai
dOng phanl5p the khong doi quang nen CO cac tinh

chat vet 19 khac nhau nhu, kha nang tao muOi, d6
tan trong dung m6i...; trong d6, muoi (S)-clopidogrel
va (L)-camphor-10-sulfonic de &pop tao thanh han,
CO dO tan kern han muOi cua (R)-clopidogrel va (L)camphor-10-sulfonic nen de clang tach ra kh6i Won
hop phan (Trig dirai clang tinh the ket tinh. Trong
qua trinh tao va ket tua muOi, main tinh the rat quan
trong, giitp cho qua trinh xay ra nhanh va chon loc
han. Neu khong có mem tinh the qua trinh nay rat
kh6 xay ra hoc can thai gian dai a nhiet d6 thap,
san pham thu &roc co dO tinh khiet thap. Loc, rira
kat ttia thu &roc (S)-clopidogrel. (L)-camphor-10sulfonic, sau do du,cyc chuyen thanh (S)-clopidogrel
bang cach trung hoa vai dung dich NaHCO3 5%
6' 0-5°C dr, pH 7. Y qua trinh nay CO the xay ra

TAP CHI DU VC HOC - 9/2015 (SO 473 NAM 55)

29

Thu'c nghiem, ket qua va ban luen
TOng hop S-clopidogel tip clopidogel
racemic (IV)
Khao sat qua trinh tach dong phan
S-clopidogel tip clopidogel racemic


• Nghien

- Ky thulat

hien tuvng racemic h6a (S)-clopidogrel khi nOng
de base cao hoac nhiat do, phan Ong cao. (S)Clopidogrel tao muoi vai acid H2SO4 de tao san
pham (S)-clopidogrel bisulfat. San pham duvc
kiem tra nhiat de nOng chay theo phoang phap
DSC de xac dinh la dang I hay clang II.

lai giam. Khi sir dung dung moi co the tich ghp
8 Ian khOi luvng clopidogrel racemic ban du thi
do tinh khiet cua san pham dat tren 99% va hiau
suht dat 29%.

Vai aux tiou thu duvc (S)-(+)-clopidogrel co
de tinh khiet quang hoc dat 99% chiing t6i tien
hanh nghien ciru cac yet, to anh hverng den qua
trinh ket tinh mu6i (S)-clopidogrel. (L)-camphor10-sulfonic [1-4] . Qua trinh ket tinh duvc kiem soat

bang hiau suht, de tinh khiet (tinh theo % dian
tich pic tren HPLC) cua (S)-clopidogrel.

Trong binh chu 1 L day ton co lap vai may
khuay tir gia nhiat, 96 g clopidogrel racemic
(1 du'ang luvng) duvc him tan trong dung mai
768 ml aceton (1:8) (w/v), sau dO 34,6 g 10-L-CSA
(0,5 &rang luvng) duvc them vao. H6n hop phan
Ong duvc dun h6i
trong 8 gi0, sau d6 lam
lanh ve nhiat do ph6ng. Cho mhm (S)-clopidogrel.
(L)-camphor-10-sulfonic vao va tiep tuc khuay
nhiat d0 phong 12 gitr. LQC va rira kat tila bang
aceton thu &pc (S)-clopidogrel. (L)-camphor-10sulfonic clang bet mau trong. Mu6i (S)-clopidogrel
camphor sulfonat nay duvc h6a tan trong 300
ml H2O, them 300 ml DCM, trung hoa dung dich
bang NaHCO3 5% den pH 7-8. Chiet dung dich vai
dichloromethan (3 Ihn x 300 ml), pha hiru ca duvc
rira vai NaCI bao hoa, lam khan bang Na2SO4, cat
loai dung moi thu duvc 27,84 g (S)-clopidogrel.
Hiau suht toan quy trinh dat 29% va de tinh khiet
quang hoc dat 99,08%.

Khao sat anh hu,deng cua logi dung moi den
hiau sub' t va d5 tinh khiet cua (S)-clopidogrel: Sir
dung cac dung mei ethyl acetat, aceton, toluen
vai tj/ la dung moi/clopidogrel racemic =10/1
(ml/g), tj/ le 10-L-CSA/clopidogrel =1/1 (mol/
mol). Ket qua cho thay dung moi ethyl acetat cho
hiau suht kha cao dat 44% nhu'ng de tinh khiet

cua san pham (S)-clopidogrel chi dat 65,68%,
con dung moi la aceton va toluen hiau suht thu
ducyc khong cao (37,5% va 37,0%), nhong do
tinh khiet cua san pham lai dat cao han (96,12%
va 96,25%). Tuy nhien, khi so sanh ye mat dec
hai va gia thanh thi aceton van dogc u'u the han
toluen.
Khdo sat anh hitting dm ti 15 acid (L)-camphor10-sulfonic tai hi6u suAt va dq tinh khiet caa (S)clopidogrel: Tien hanh phan u'ng trong dung mai
aceton vai cac tj/ le 10-L-CSA/clopidogrel là 0,5
va 1. Ket qua thu duvc nhu, sau: Khi ti la acid
10-L-CSA la 1 &rang luvng thi hiau suht phan
Ong dat 37,5%, tuy nhien de tinh khiet cua (S)clopidogrel lai chi dat 96,12%, nguyen nhan
co the do Itivng acid 10-L-CSA cao met phan
ding phan (R)-clopidogrel cung tham gia tao
thanh mu6i Nib cling ket tinh vai mu6i cua (S)clopidogrel. Khi ti la acid Om xuOng 0,5 doc/ng
locyng. mac d0 cho hiau suht thhp: dat 27,5%,
nhong de tinh khiet quang hoc dat 99,35%.

Ring hop S-clopidogel tir clopidogel
racemic

(S)-clopidogrel. (L) camphor-10-sulfonic
IR (cm-I, KBr): 3064 (C-H khOng no);
2965,04 (C-H no); 1756,13 (-C=O-), 1507,45
(-C=C), 752,45 (the ortho). ESI-MS: [M+H]* =
322,8 (clopidogrel M = 321,82), [M-H]- = 231,23
(10-L-CSA, M = 232,3). 'H-NMR (500 MHz,
CDC/3): .5 (ppm): 1,42 (m, 1 H); 1,58 (m, 1 H);
1,86 (d, 1 H); 2,04 (m, 2 H); 2,34 (m, 1 H); 2,70
(m, 1 H); 2,77 (d, 2 H); 3,75 (m, 1 H); 3,85 (s, 3

H, COOCH3); 4,83 (s, 1 H); 6,81 (d, 1 H);7,38 (d,
1 H); 7,52 (d, 1 H); 7,59 (m, 2 H); 7,64 (d, 1 H);
7,67 (d, 1 H) ppm. "C-NMR (125 MHz, CDCI3):
6(ppm): 20,14; 20,53, 23,18, 25,77, 27,79,
43,63; 44,13; 48,49; 54,70; 59,65; 67,38; 126,11;
126,85; 128,15; 128,28; 129,85; 131,40; 132,31;
132,90; 134,04; 136,71; 168,67; 218,32.
(S)-clopidogrel

Khao sat anh huang cua luceng dung moi den
hiau sugt va do tinh khiet cua (S)-clopidogrel:
Chon tj/ la acid 10-L-CSA/clopidogrel racemic la
0,5; t9 la the tich aceton/khOi luting clopidogrel
racemic la 5, 8,10 (ml/g). Kat qua cho thay khi
tang luring dung moi tir 5 Ian len 8, 10 Ian thi de
tinh khiet cua san pham tang nhong hiau suht

IR (cm-1, KBr) 3064,96 (C-H khong no),
2965,04 (C-H no); 1740,65 (-C=0); 1570,12
(-C=C-); 755,05 (the ortho yang benzen).
ESI-MS: [M+2H].= 323,99 (M = 321,82);
'H-NMR (500 MHz, CDC/3): 8 (ppm): 2,89-2,93
(m, 4H); 3,68 (d, 1 H); 3,75 (s, 3 H, -COOCH3);

30

TAP CHI DUQC HQC - 9/2015 (SO 473 NAM 55)


• Nghien

- Ky. thuot

3,80 (d, 1 H); 4,95 (s, 1 H); 6,68 (d, 1 H), 7,08 (d,
1 H), 7,27-7,33 (m, 2 H); 7,44 (d, 1 H); 7,74 (d, 1
H); "C-NMR (125 MHz, CDC/3); 6 (ppm): 25,62;
48,38; 50,76; 52,20; 67,97; 122,82; 125,31;
127,23; 129,50; 129,88; 130,07; 133,35; 133,42;
133,97; 134,79; 171,40.
Tong hop S-clopidogel bisulfat clang I ye
dang II
Kith() sat qua trinh tong hop S-clopidogel
bisulfat cl?ng I tea dang II
Cac tai lieu d5 cong be cho thAy cong met
dung mei (nhu' aceton) nhu'ng a cac dieu kien
CI COOCH3
)N

khac nhau c6 the cho cac clang tinh the khac nhau
cua (S)-clopidogrel bisulfat 151. Sty hinh thanh tinh
the c5u trim clang I duvc kiem soat bai qua trinh
dOng hoc (kinetic control) con dang II bol qua trinh
nhiet dOng hoc (thermodynamic control), nen de
c6 duvc cac ket qua On dinh CO dO lap lei cao can
phai tim duvc cac dieu kien ye dung mei, nhiet
do... rat chinh xac, neu kh6ng se thu duet dang
I thay vi dang II hoac ngtrac lei, hoac hon hap
cua ca 2 clang: clang I va dang II. Qua tham khao
cac cong trinh d5 duvc ding b6[4-71 chOng tOi ch9n
quy trinh tong hap (S)-clopidogrel bisulfat dang I

va dang II duvc m6 to trong sa dO 2 va sa do 3.
CI COOCH3

H2SO4

)
0-5°C
Aceton

.H2SO4

(S) - Clopidogrel bisulfat
dang I

So' dO 2: T6ng hop (S)-clopidogrel bisulfat clang I
(S)-clopidogrel bisulfat dang I duvc tong hap
trong dung mei aceton a 0-5°C, nho tir tir acid
sulfuric, khu5y b nhiet de thaang trong 12 gia.
Phan irng cho hieu suet tren 70%, san phArn
CI COOCH3

thu duvc la tinh the mau trong CO nhiet de n6ng
chay 184 ± 3°C (DSC), san ph5m da &vac kiem
tra do On dinh cho ket qua thei gian sir dung tren
2 nem.
CI COOCH3

H2SO4

0-5°C

MTBE

)L\

.H2SO4

(S) - Clopidogrel bisulfat
clang II

So' do 3: T6ng hop (S)-clopidogrel bisulfat dang II
(S)-clopidogrel bisulfat dang II thu duvc kha
kh6 khan. Chung tOi da thin rat nhieu dung m6i
(isopropanol, ethylacetat, methanol, aceton,
methyl tert butyl ether ...), phaang phap khac
nhau nhu'ng van chi thu duvc dang I hoac h6n
hap dm clang I va clang II (nhiet de n6ng chay
cua san phArn nem trong khoang 176°C-184°C.
Sau met thai gian dai nghien ciru, khi tier' hanh
ket tinh trong dung mei methyl tert butyl ether
(the tich dung m6i gap 10 len khOi luvng (S)clopidogrel ban d5u) lam lanh a 0-5 °C trong
2 tuen, they xuet hien ket tua, loc, rira ket tua
veri aceton duet (S)-clopidogrel bisulfat dang II
CO nhiet de nong chay 177 ± 3 °C. WA cac thi
nghiem sau, thvc hien phan Ong co mem tinh the
dang II thi san phArn tao ra de dang hen, sau

Khdo sat anh htreyng ceia dung m6i den hieu
suet va dO tinh khiet caa (S)-clopidogrel bisulfat:
Tien hanh cac phan Ong vai cong met luang ch5t
(feu, sir dung cac dung mai khac nhau. Wet qua

TAP CHI DOC HQC - 9/2015 (SO 473 NAM 55)

31

2 ngay lam lanh ir 0-5° thu &vac san ph5m (S)clopidogrel bisulfat clang II nhang hieu suet chi
dat 33%.
Hieu suet tao (S)-clopidogrel bisulfat dang I
cao hen clang II. Mat khac dang I lei c6 sinh kha
dung cao hen va ducyc san xuet cung nha sir
dung (Ong rai han, nen chOng tOi da ch9n Iva
nghien CCPU qua trinh ket tinh (S)-clopidogrel
bisulfat dang I kr (S)-clopidogrel de thu duvc
hieu suet cao nhet va net-1g cep len quy me 50
g/me, dinh hu&ng phat hien Crng dung trong san
xuet cong nghiep.


• Nghien ciru - K9 thu4t
cho they hai dung mOi aceton va isopropanol deu
cho san phem co do tinh khiet ten 99% (dat yeu
cau trong USP 34). Tuy nhien, aceton la dung mai
th6ng dung, de thu hoi, gia thanh hp oda duvc Iva
chon de ket tinh (S)-clopidogrel bisulfat clang I.
Bang 1: Anh huxing cua 100 dung mai
HiOu suet
(%)

Dijtinh
khiet (%)

1 Ethyl acetat

72,3

98,33

2 Ethyl acetat - methanol (1:1)

68,3

97,42

3 Aceton

65,5

99,03

4 Isopropanol

60,8

99,47

STT

Dung mai

Khao sat anh hirang cua ti le acid H2S0, dac

den hieu suet, do tinh khiet cua (S)-clopidogrel
bisulfat: Tien hanh phan Ong vai cac ti le so
mol acid H2S0, dac/(S)-clopidogrel khac nhau
trong dung mOi aceton (Vaceton = 20. moopidogrei).
Ket qua cho they, khi ti 16 acid H2SO4 dac/(S)clopidogrel la 1 thi phan Ong cho hieu suet cao
nhat 65,50% va de tinh khiet cao nhat la 99,35%.
Neu tang den ti le acid H2SO4 dac thi hieu suet
giarn den va de tinh khiet Om den va co phan
Ong bi oxy h6a manh kh6ng thu &pp san phem.
Bang 2: Anh Mrerng cern ti le acid H2SO4 dac
H SO dOc/
STT
2 4
(S)-clopidogrel

Hieu suet (%)

DQ tinh
khiet(%)

65,50

99,35

1

1

2

1,2

42,40

98,61

1,5

Kh6ng thu &roc
san phArn

0

3

Khao sat anh huifmg cua luring dung mdi den

hieu suet va dO tinh khiet cua (S)-clopidogrel
bisulfat: Tien hanh phan u'ng vai the tich aceton
Op 10, 15, 20 len khei luting (S)-clopidogrel
(ml/g). Trong qua trinh thu'c nghiem they khi dung
luting dung mai it, neng do acid sulfuric trong
dung dich cao, lam cho cac chat de bi oxi hoa va
co hien twang chuyen sang mau den den den de
tinh khiet cua san phern thap, hieu suet dm phan
Ong cung thap. Tuy nhien, khi tang dung mai qua
cao lai lam Om hieu suet cua phan Ong vi qua
trinh ket tinh kh6 khan hen. De san phem dat do
90%) va hieu suet phan
tinh khiet dam bao

Ong cao the tich dung mai Op 15 len khoi luting
chat ban deu la ph0 hap.
32

Bang 3: Anh huifmg cua ti re dung mdi
SIT Dung mai Hieu suet (%)

Da tinh khiet (%)

1

10

74,8

97,8

2

15

77,8

99,18

3

20

65,5

99,25

Quy trinh king hap (S)-clopidogrel bisulfat
Bang I &pp Chung t6i ap dung de tong hop 6,
quy m6 tren 50 g san phem/me. Qua trinh duvc
lap !pi nhieu len cho hieu suet 77,6 - 78,5%.
San phern duvc kiem nghiem dat tieu chuen cua
&pop dien M9 USP 34.
Tong hop (S)- clopidogrel bisulfat Ong I
Trong binh cau 2 L CO lap he thong lam lanh, may
khuay tO, china 72,50 g (S)- clopidogrel (1 &yang
locyng) hea tan trong 1088 ml aceton, lam lanh
h6n hap 6 nhiet de 0-5°C, nho tin tO, 11,23 ml acid
H2SO4 dac (1 throng luting) vao h6n hap phan Ong
de nhiet de luon a 0-5°C. Hon hap phan Ong sau
de duvc khuay 6, nhiet de 25- 30°C trong thai gian
24 gia. Ket th0c phan Ong, loc thu kat tua, riy'a tua
vai aceton lanh (2 x 10 ml), say san phem trong to
say char) kh6ng trong 5 gia thu ducyc 73,80 g (S)clopidogrel bisulfat (H = 78%). Nhiet do nOng chay:
184-186°C. [a]oi20 = 55,5°(C = 1 trong methanol). De
tinh khiet quang hoc: 99,53%
IR (cm-1, KBr): v = 3116,6 (CH khong no);
2996,46 (CH khong no); 1753,61 (CO ester );
1624,33; 1477,83; 1434,74 (-C=C- vong therm);
755 (the para-di cua wing benzen). ESI-MS:
[M1=322,1; 'H-NMR (500 MHz,CDC13) S(ppm):
3,20 (s, 2H); 3,60 (m, 5H); 4,10 (s, 1H); 4,30 (s,
1H); 5,70 (s, 1H); 6,60 (d, 1H); 7,20 (d, 1H); 7,37,5 (m, 3H); 7,5 ( s, 1H).

"C-NMR (125 MHz; CDCI 3) o(ppm): 21,60;
50,70; 54,54; 65,95; 125,03; 125,91; 126,0;
126,5; 128,75; 130,79; 131,14; 131,48,; 133,15;
135,14; 167,96.
T6ng hop (S)- clopidogrel bisulfat Ong II
Trong binh cau 50 ml c6 chCra 1,3 g (S)clopidogrel (1 &yang luting) hoe tan trong 13 ml
methyl tert butyl ether (MTBE), hen hop doge lam
lanh ye 0-5 °C, 0,21 ml acid H2SO4 dac (1 &yang
luting) duvc nho giot tO, tu, vao hen hap phan Ong.
Khuey hen hap phan Ong a nhiet dO pheng trong
24 gia, sau do lam lanh h6n hap phan Ong a 0 5°C trong 2 ngay. Ket thuc phan u'ng, lec ley ket
tua, riya tua bang aceton lanh (2 x 2 ml), say trong
TAP CHI DUQC HQC - 9/2015 (SO 473 NAM 55)


• Nghien CCPU - ic9 thuot
to say chan khOng 5 giO thu duvc 0,56 g san ph5rn
le (S)-clopidogrel bisulfat, la chAt ran mau tang
(H = 33%). Nhiet d6 nOng chay: 172 -175°C (DSC).

Ket luen
Chung t6i da tech dOng ph5n (S)-clopidogrel
co dO tinh khiet quang hoc 99% tir clopidogrel
racemic vai hiau suet 29%. Tong hop thanh cong
(S)-clopidogrel bisulfat d5ng I vai hieu suet 78%,
clang II vai hiOu suet 33%. Ben canh d6 mot s6
yeu to anh hi.reyng den qua trinh tech dOng phan
va te -1g hop (S)-clopidogrel bisulat dang I d5
&Pop khao sat.

Log cam an: Cong trinh nghien cap ducyc hoar)
thanh vai sytai tro, kinh phi cua Chuang trinh Haa
Dirac. Ma s6 de tai: CNHD.DT.024/11-13

Tai lieu tham khao
1. Alain Badorc, Daniel Frehel (1989), "Dextrorotatory enantiomer of methyl alpha-5(4,5,6,7-tetrahydro (3,2-C) thieno pyridyl)(2-chlorophenyl)-acetate

and the pharmaceutical compositions containing it",
US4847265.
2. Andre Bousquet, Serge Calet, Alain Heymes
(1992), "2-Thienylglycidic derivative, process for its
preparation and its use as synthesis intermediate",
US5132435.
3. Alain Heymes, Bertrand Castro, Maria Bakonyi
et al (2001), "Intermediates and process for the
preparation thereof", US6215005.
4. Alain Badorc, Daniel Frehel (1988), "Enantiomere
dextrogyre de l'alpha-(testrahydro-4,5,6,7 thiienso
(3,2-c)pyridyl-5)(chloro-2-phesnyl)-acetate de methyle,
son procede de preparation et les compositions
pharaceutiques le renfermant", EP0281459 81.
5. André Bousquet, Bertrand Castro, Jean
Saint Germain (1999), "Polymorphic clopidogrel
hydrogenesulfate form", WO/1999/065915.
6. Qiao-Gen Zou ang, et al (2011) "Study on
bioavailability difference between clopidogrel bisulfate
form I and form II using liquid chromatography/tandem
mass spectrometry", Arzneimittelforschung, 61(6),
353-357.
7. Revital Lifshitz-Liron et al, (2003), "Polymorphs

of clopidogrel hydrogensulfat", US20030225129A1.

(Ngay nhan bei: 25/04/2015 - Ngay duyOt dang: 04/09/2015)

Anh htgrng ctia cleeu kien chie't dtn ham
ltrcing polyphenol va danh gia hoat tinh
chong oxy hoe cua dich chitt tur la 6i
(Psidium guajava L .)
Ho Ba Viro'ng', Nguyen Xuan Duy2", Nguyen Anh TuAn2
'Cong tv TNHH MTV Vacxin va Sinh phdm Nha Trang, Khanh Hoa
C5ng nghC3 Thyv phcim, Trtrang Dai hac. Nha Trang, Khanh Hoa


2Khoa

Summary
Total polyphenols were extracted from the leaves of Vietnamese guava (Psidium guajava L.) with
ethanol. The extraction was optimized with respect to the ethanol concentration, solvent-material
ratio, temperature and extraction time. With 40° ethanol, the solvent-material ratio of 40/1 (ml/g), at
the temperature of 80 °C and extraction time of 60 min, the yield of the total polyphenols reached
approximately 189.73 mg GAE/g (calculated on the dried materials). The obtained extracts were valuated
antioxidant activities based on ABTS free radical scavenging ability and total reductive potential gave
the IC50 value of 1.95 and 5.08pg/ml, respectively. Moreover, the influence of the maturity and material
TAP CHI DU6C HOC - 9/2015 (SO 473 NAM 55)

33


• Nghien

- Ky thuOt

state of the guava leaves on the total polyphenol content (TPC) were investigated. The old leaves
possessed the highest content of the total polyphenols, followed by the matured and young leaves (p
< 0.05). Whereas, no significant differences were observed between the fresh and dried leaves in the
total phenol content (p > 0.05). These all suggest the guava leaves may serve as a promising source for
extraction of natural antioxidants.

Keywords: Extraction, antioxidant activity, guava leaf, guava leaf extract, Psidium guajava L.,
polyphenol.

DOt yen de
Nhieu nghien ciru chi ra rang trong la 6i co
china ham luvng polyphenol cao va co tiem n5ng
&rig dung trong cac nganh cong nghiep thU'c
pham, duvc pham va m9 pham
Trong duvc
hoc, polyphenol co nhieu tac dung quY nhu':
Chong lai stress oxy hoa, loai be cac gOc ty do,
ngan ngiya va han the met so benh tat lien quan
den tim mach, dot quy, tang huyet ap, dai thao
duang va ung thu, [4] .
Polyphenol trong la Oi co the bi hu, hong trong
qua trinh chiet tach do cac yeu to vat 19 va h6a
h9c gay nen. Do de, dieu kien chiet tach thich
hap can duvc thiet lap de dam bao tinh On dinh
va hieu qua thu nhen polyphenol 15] . Cho den
nay, co rat it nghien cipu ye dieu kien chiet tach
polyphenol *15 Oi ducyc cong bob Viet Nam. Vi
vey, nghien ciru khao sat anh hiyang cua dieu

kien chiet den ham luvng polyphenol tin là Oi la
rat can fillet.

Nguyen lieu va phu'o'ng phap
nghien aru
Nguyen lieu
La Oi (Psidium guajava L.) thuOc giOng Oi xa II.
Ten khoa hoc dircyc dinh danh bbi nho' Thy,c vat
hoc Pham DOT Duvng, cong tac tai Viyan quec
gia Cat Tien, Deng Nai. La Oi duvc thu hai trgc
tiep tai man dm nguai dan dia pi-Krung a thanh
phe Nha Trang, tinh Khanh Hoa trong thang
4/2014. Nguyen lieu duvc phai khO nhien de
dat de am khoang 10%, xay nho, sang qua mat
11_1,612 mm, bao gOi trong tui nilon but char) khOng
va bao quan b - 66°C cho den khi sir dung.
I-16a chat SG' dung trong nghien ciru deu
dat yeu cau phan tich
K3(Fe[CN]6),
AlC13, Na2CO3 NaNO2, folin ciocalteu,
ethanol, trichloacetic (TCA), NaOH mua tin
34

Hang Merck (arc). (±)-6-Hydroxy-2,5,7,8tetramethylchroman-2-carboxylic acid (Trolox),
2,2'-azino-bis(3-ethylbenzothiazolin-6-sulphonic
add) (ABTS) va add gallic mua cua Hang Sigma (M9).
Thiet bi
May cat (Super blender, MX-T2GN, Nhet
Ban), be On nhiet (Elma, S 300H, Din), may ly
tam (EBA 21, HETTICH, arc), may quang phO

ke (Cary 50, Varian, M9).
Phu'o'ng phap nghien avu
Chug') bi dich chiet the)

Nguyen lieu duvc chiet trong be On nhiet, sau
dO ly tam (5.000 yang/phut) trong 15 phut de thu
dich chiet the'. Cac phan tich du'o'c tien hanh tren
dich chiet th6.
Xac dinh anh huving cua giai doan trtreyng
thanh den s4r tich lay polyphenol

Là Oi difigc thu hai 0, ba giai doan: Non,
trubng thanh va gia (hinh 1). Tat ca nguyen lieu
duvc lam kh6, nghien nho va chiet trong dieu
kien giOng nhau de thu duvc dich chiet. Dieu kien
chiet la: 65°C, 60 phut, dung mai ethanol 50%,
tile dung moi/nguyen lieu 15/1 (ml/g). Qua trinh
chiet duvc lap lai ba len.
Xac dinh anh hurting cua dieu ki#n chiet
Nhiet de, thai gian, tile dung moi/nguyen lieu va

nOng de ethanol
Phu'ong phap be tri thi nghiem yeu to tiring
phan du,gc sir dung de nghien ciru anh Wang
cua cac nhan to chiet.
Anh hiyang cua nhiet de duvc khao sat trong
pham vi tip 30 den 90°C, vai buac nhay 10°C.
Cac thOng se) co dinh Om: Dung moi la ntrac,
thai gian 60 phut va ti le dung moi/nguyen lieu
40/1 (ml/g).

Anh huang cua thai gian duvc tien hanh tin
20 den 120 phut, vai tuft nhay 20 phut. Cac
th6ng so co dinh
NM* do do' duvc chon
TAP CHI DUQC HQC - 9/2015 (SO 473 NAM 55)


• Nghien an' - K9 thuOt
thi nghiem tardec, dung m6i la nuiac va ti le dung
moi/nguyen lieu 40/1 (ml/g).
Anh huieng cua ti le dung mai chiet so vai
nguyen lieu duvc Iva chon till, 20/1 den 70/1
(ml/g), vai btrac nhay 10 (ml/g). Cac thOng so co
Nhiet clO, theri gian da &roc chon & cac
dinh
thi nghiem triyac va dung m6i la nirac.
ket hop nuidec vai ethanol
Anh hue:1'N dm
duvc tien hanh trong pham vi tir 0 den 100%,
vai buidec nhay 20%. Cac thong so co dinh
Nhiet de, thoi gian, ti le dung moi/nguyen lieu d5
duvc chon b cac thi nghiem trvac.
Xac dinh ham luvng polyphenol tong
Ham luvng polyphenol tong dtrgc xac dinh
theo phuiong phap cua tai Wee). Kat qua ducyc
bao cao qui ye mg acid gallic Wang &yang
(GAE)/g chat kho.
Xdc dinh hoat tinh chOng oxy hoa du'a vao
kha nang kher gOc ty, do
Xac dinh kha nang khir goc tu' do ABTS theo

phucvng phap cua tai lieu Ket qua bao cao bei
gia tri IC50, la nOng do cua dich chiet khir duvc
50% gOc tty do ABTS trong dieu kien thi nghiem.
Xac clinh hoat tinh chOng oxy h6a diva vao
king nang Ivy kW ,
khir sat cluvc xac dinh theo
Tong nang
phirong phap cua tai lieu 18] . Ket qua &roc tinh

toan vai gia tri IC50 , la luvng mau lam tang de hap
thu quang hoc len 0,50 tai dieu kien thi nghiem.
Phtpang /Map

sei lieu

Cac phan tich duvc tien hanh lap lei it nhat ba
Ian de dam bao thu'c hien phan tich ANOVA. SO
lieu &roc phan tich ten phan mem SPSS 16.0
(SPSS Inc., Chicago, IL, M9). Kiem dinh Tukey
HSD cluvc thvc hien sau phan tich ANOVA de
danh gia str khac nhau cua cac gia tri vai mirc y
nghia p < 0,05. Cac hinh ye va do thi diycyc ye ten
phan mem Excel (Office 2007, Microsoft, M9).
Cac nghien ciru ten duVc thvc hien tai pricing
thi nghiem Khoa Cong nghe thvc pham, Truang
Dai hoc Nha Trang.
1(61 qua nghien cu'u
Anh htrevng cua su' truNlyng thanh den scr
tich luy polyphenol trong la Oi
Hinh 1 cho they su, tich luy polyphenol phy

thuec vao giai doan trueng thanh dm la Oi. Ham
luvng polyphenol tong (total polyphenol content =
TPC)trong la gia la 109,14, cua la trirong thanh la
86,57 va cua la non la 55,20 mg GAE/g chat kho.
TPC trong la gia cao hon clang ke so vai la trueng
thanh va la non (p < 0,05). TO, ket qua dat duvc,
chung toi chon la gia de tien hanh cac nghien a:1'u
tiep theo.

150
rq le°
3 L. r-- 50

-

I

g

a.—

0
La non

La tniong
thanh

La gia

Giai doan phattrien

Hinh 1: Anh huong cua su,trui&ng thanh den so, tich 10y polyphenol trong la 6i. Ch0 cal khac nhau
tren cot chi ra su' khac nhau cc) y nghia thong ke (p < 0,05).
Anh twang cua dieu kien chiet den ham
luvng polyphenol

cac nghien ciru trap theo.

Hinh 2A cho they khi tang nhiet de tir 30 len
80°C thi TPC tang len clang ke (p < 0,05), kr
53,59 len 118,96 mg GAE/g chat khO. TPC tai
nhiet de chiet 80°C va 90°C (119,59) kh6ng co
sir khac biet clang ke (p > 0,05). Diva vac) ket qua
dat duvc, chung toi chon nhiet de chiet 80°C cho

Kat qua dat &roc tir hinh 2B chi ra rang khi
tang thai gian chiet tiv 20 len 60 phut thi TPC tang
Bang ke (p < 0,05), tEr 95,17 len 120,25 mg GAE/g
chat khO. Tuy nhien, khi keo dai thoi gian chiet lau
han riCra thi polyphenol chiet duvc khOng tang
Bang ke (den 100 phut). Vi vay, chung toi chon 60
phUt de thyc hien cac nghien ciru tier) theo.

TAP CHI DOC HQC - 9/2015 (SO 473 NAM 55)

35


• c

• Nghien

- K9 thuOt

Khi tang ti le dung mOi/nguyen lieu chiet den
den ham luvng polyphenol tang (hinh 2C). ClY ti
le chiet 70 (ml/g) cho ham luting polyphenol cao
nhat (130,95 mg GAE/g chat kho). Tuy nhien,
met ti le cao han gripe dung mei chiet va nguyen
lieu khong duvc khuyen dung vi co the lam Om
hieu suet chiet cac hap chat phenolic cung nhu,
kho khan han trong viec tech b5 nguyen lieu khoi
dich
Vi vey, chung toi chgn ti le dung mei/
nguyen lieu chiet la 40/1 (ml/g) de thyc hien cac
nghien thu tiep theo.
Ket qua tir hinh 2D cho they ham luting
polyphenol tang len clang ke khi tang nOng de
ethanol tu, 0 den 60% (p < 0,05), twang &rig vai
•

140
, 120

4

,c

U40

t 2

an

tip 121,11 len 185,58 mg GAE/g chat kho. nOng
do ethanol ten 60% den den sv giam ham luting
polyphenol chiet duvc (p < 0,05). Ket qua cung
chi ra rang chiet polyphenol a dai nOng dd tip 40%
den 60% ethanol la thich hap. Ti' nhOng kat qua
dat duvc a ten, chung toi chon neng de ethanol
40% le nOng do dung m6i chiet thich hap nhet.
Nhu, yey, tong hap cac ket qua ten, chting
toi chon dieu kien chiet thich hap de thu nhen
polyphenol tir la Oi le: Wing de ethanol 40%,
nhiet do chiet 80°C, thol gian chiet 60 phOt va ti le
ethanol 40%/nguyen lieu 40/1 (ml/g) de thu'c hien
cac nghien cipu tiep theo.
110
00

C

40

0

r

20

9,1

0

10 40 50 60

10

80 90

7S0
CO 200
L
c

c

C

6x 1150

b
a

° 300

°E

50
0

20

Ti 4 4.14 m61/.004 14. ( 44)

40
60
80
N6ng d() ethanol (%)

100

Hinh 2: Anh /wrong cua diau kien chiet den ham lugng polyphenol
Hoot tinh chOng oxy hoe cua dich chiet la 6i

dich chiet là Oi the hien hoat tinh khi' gOc tv do
ABTS yeu han Trolox.

Nhin chung, kha nang khi' gOc tu' do ABTS
cua dich chiet le of phu thuec vao nong de hay
not cach khac khi nOng de dich chiet tang thi khe
nang khi' gOc tv do ABTS tang (hinh 3A). Gia tri
IC50 cua dich chiet le of la 1,95 pg/ml, trong khi
dO gia tri nay cue Trolox la 1,0 pg/ml. Nhu, vey,

TOng nang lyc khir cue dich chiet la of tang
len cung veyi sir tang cue nOng de dich chiet
(hinh 3B). Gia tri IC50 cua dich chiet le Oi le 5,08
pg/ml. Trong khi dO, gia tri nay cua Trolox le 0,52
pg/ml. Nhu, \fey, king nang
khi' cua dich chiet

la of thep han Trolox.
08

/0 -

01,

zei

02

12 60
01,

4 sa

05 o

;

-

5 0I

• mr-'

20 -

5 02 -

.1 10 -

0U

0

-

0

05

1

LS

,
II

7

2`3

N8f9 ay (14041)

A

Nene ed Wenn

Hinh 3: Kha nang ch6ng oxy hOe cua dich chief la of NO so yol Trolox (.)

36

TAP CHI

DUQC HQC - 9/2015 (SO 473 NAM 55)


• Nghien avu - K9 thOt
Ban luan

•

Ham luring polyphenol trong la of phu thuOc
vao tang thai truang thanh dm la 6i. Theo do,
la gib CO mirc dO tich by TPC cao nhat. Dieu nay
cOng ph0 hap vai su phat trien binh thuang cua
thu'c vat dO la khi thuc vat cang truang thanh thi
su tich lay cac chat cang nhieu. Mat khac, la 6i
gia thuteng duvet lopi 136 trong qua trinh them
soc cay Oi. Vi vay, dO se la mOt nguen dal dao, de
kiern va re ten de c6 the khai that cac polyphenol.
Cho den nay, chu'a co cong be) chinh thu,c nao
Viet Nam nghien cOu ve anh Wang cua giai doan
truang thanh cua la of den su tich lay TPC.
NhOng nhan to quan trong anh Wang den
dieu kien chiet polyphenol duvc khao sat bao
gem: Nhret de, thai gian, net-1g dO dung m6i va
ti 10 dung moUnguyen lieu. Ca bOn nhan to nay
anh Wang den ham luvng TPC thu duvc

Kr la 6i. Dieu kien chiet TPC tir la of thich hqp
duvc thiet 15p 6. 80°C, ethanol 40% va ti re dung
moUnguyen lieu 40/1 (ml/g). Tai dieu kien nay,
ham luvng TPC dat duvc 189,73 mg GAE/g chat
kilo. Met so tac gia cling dä cong be, ham luvng
polyphenol cua IA Oi. Tac gia Lai Thj Ngoc Ha [4J
d5 bao cao rang ham luvng polyphenol cua la
of thu dugc 6, dieu kien chiet t61 LIU la 147,69
mg GEA/g chat kho. Ket qua nghien
dm
You Dong-Hyun va CS. 1"lchl ra rang ham luvng
polyphenol trong Id of (Psidium guajava)treng tai
Han QuOc IA 128,48 mg GAE/g chat kh6 (chiet
bang ethanol, a nhiet dO ph6ng, trong 24 gia va
ti le dung moi/nguyen lieu IA 30/1, ml/g). Ket qua
nghien thu cua Nguyen Xuan Duy va CS [21 cho
they ham luvng TPC cua la of la 146,5 mg GAE/g
chat kh6. Nhu' vay, co the they rang la of &roc
chiet a dieu kiOn tOi
duvc thiet lap CO ham
luvng polyphenol cao han nhieu so vai cac ket
qua duvc cong bO truac day.

cua nhOm tac gia 1111 gan 9,5 Ian. Dieu nay cOng
cho phep du doan rang djch chiet fir la 6i co host
tinh chOng oxy h6a manh.
Dich chiet fir la of co hoot UM chOng oxy hoa
duvc danh gia th6ng qua kha nang khir g6c ty do
ABTS va tong nang luc kW,. Met s6 tac gia cOng
da bao cao ve hopt tinh chOng oxy hOa cua djch

chiet tO, la of 12 '12-141. Khi so sanh vai chat chOng
oxy hoa thuang mai Trolox (tuang ty vitamin E,
nhu'ng hoa tan duvc trong nuac), djch chiet tir la
of ca hopt tinh chOng oxy hite thep han. Dieu nay
CO the duvc l giai la do Trolox la chat a clang tinh
khiet trong khi dO djch chi& 6, dung the. Vi vey, de
lam tang hopt tfnh chOng oxy hOa thi djch chiet can
&Tic tinh sach han riCra. NhOng nghien thu tiep
theo nen tap trung vao viec tinh such djch chiet fir
16 of de CO the Ong dung nhu' la mOt chat chOng
oxy h6a manh dung trong thuc pham.
Kat luan
Qua trinh chiet xuet polyphenol fir la of chju
anh Wang Ian 136,i cac yeu to nhu': NhiOt de chiet,
thai gian chiet, ming dO ethanol va ti le dung m6i/
nguyen lieu. Nghien thu d5 xac djnh duvc dieu
kien chiet thfch hap polyphenol tir la of la: Dung
mai chiet ethanol 40%, nhiet dO 80°C, thai gian 60
phut, ti le dung mai/nguyen lieu 40/1 (ml/g). Tai dieu
kien chi& nay, ham luvng polyphenol thu duvc là
189,73 mg GAE/g chat kilo. Hopt tinh chOng oxy
hoa (1050) cua djch chiet thu duvc a dieu kien chiet
thfch hcyp dt.rgc danh gia dua vao kha nang kW,
gOc tu do ABTS va tong nang ly'c khir len luvt la
1,95 va 5,08 pg/ml. NhO,ng ket qua dat &Jac trong
nghien thu nay cho they tiem nang sir dung 16 of
nhu' mOt nguen thu nhan cac chat chOng oxy hOa
cO nguen g6c tu nhien. Cac nghien aru tie') theo
nen tap trung vao viec tOi u'u hOa dieu kien chiet
polyphenol cOng nhu' nhan dpng cac thanh phan

chinh &rig vai trO nhu' la nhirng chat chOng oxy
hda trong djch chiet thu duvc tir la Oi.

Marja va CS khi nghien dru ham luvng
polyphenol dm 92 lopi thuc vat an duvc va khong
an duvc de cho they rang ham luvng polyphenol
cua chUng dao Ong kha rang trong khoang fir
0,2 den 155,3 mg GAE/g chat kh6. Gang theo
nhom tac gia nay, nhO'ng loci thuc vet CO ham
lugng polyphenol Ian han 20 mg GAE/g chat kh6
thi cd• hopt tInh chOng oxy h6a manh. Ket qua
nghien ciru cua chung t6i cho they 16 6i &vac
chi& & dieu kien thich hop de durst thiet lap cei
ham luvng polyphenol cao han mirc khuyen cao

Tai lieu tham khalo
1. Nguyen Bin (2004), Cac qua trinh, thgt trong
cong ngh0 hoa ch.4t va thy phdm, Nha xu6t ban Khoa
hoc va K9 Thuat.

TAP CHI DU QC HQC - 9/2015 (SO 473 NAM 55)

37

Leri cam cm: Tac gia xin than thanh cam an
COng ty TNHH Thiet Khoa hoc Thien An, TP.
H6 Chi Minh (la h6 fro, mot phAn kinh phi c74 thac
hiOn nghien cifu nay.

• Nghien C CPU - K9 thuOt
2. Nguyen Xuan Duy va HO Ba Virang (2013), "Hoot
tinh chOng oxy hoa va t:fc the enzym polyphenoloxidase
ctia mOt so loaf thlyc vat an duvc a Viet Nam", Tap chi
Khoa hoc va Pilaf tridn, 11(3), 364-372.
4. Lai Thi Ngoc Hb (2011), "Polyphenol fir la of:
Ham lircing, kha nang khang oxy hOa va dieu kien tach
chief', Top chi Dinh during va Thyr pham, 8(4), 37-44.
i (2003), HOa hoc thy'c pham, Nha
5. Le Ngoc Ti
xuet ban Khoa hoc va K9 thubt Ha NOi.
6. Singleton, V. L., Orthofer, R., Lamuela-Raventos,
R. M. (1999), "Analysis of total phenol andother oxidation
substrates and antioxidants by means of Folin-Ciocalteu
reagent", Method Enzymol, 299, 152-78.
7. Richard, B. Walker and Jaced Everette (2009),
"Comparative reaction rates of various antioxidants
with ABTS radical cation", J. Agric. Food. Chem., 57,
1156-1161.
8. Oyaizu, M. (1986), "Antioxidative activity of
browning products of glucosamine fractionated by
organic solvent and thin-layer chroma-tography",
Nippon Shokuhin Kogyo Gakkaishi, 35, 771 - 775.
9. Vuong, Q. V., Golding, J. B, Stathopoulos, C.
E., Nguyen, M. H., Roach, P. D. (2011), "Optimizing
conditions for the extraction of catechins from green
tea using hot water", Journal of Separation Science,

34, 3099-106.
You Dong-Hyun, Park Ji-Won, Yuk Hyun10.

Gyun and Lee Seung-Cheol (2011), "Antioxidant
and tyrosinase inhibitory activities of different parts
of guava (Psidium guajava L.)", Food Sciene and
Biotechnology, 20(4), 1095-1100.
11. Marja, P. Kahkonen, Anu, I. H., Heikki, J. V.,
Jussi-Pekka, R., Kalevi, P., Tytti, S. K., Marina, H. (1999),
"Antioxidant activity of plant extracts containing phenolic
compounds", J. Agric. Food Chem., 47, 3954-3961.
12. Suganya Tachakittirungrod, Siriporn Okonogi
and Sombat Chowwanapoonpohn (2007), "Study
on antioxidant activity of certain plants in Thailand:
Mechanism of antioxidant action of guava leaf extract",
Food Chemistry, 103(2), 381-388.
13. Hui-Yin Chen va Gow-Chin Yen (2007),
"Antioxidant activity and free radical - scavenging
capacity of extracts from guava (Psidium guajava L.)
leaves", Food Chemistry, 101, 686-694.
14. Truang Tuyet Mai, Pharn Lan Anh, Truang
Hoang Kien, Nguyen Van S9, Nguyen Thi Phuring
Thiry va Nguyen Thi Lam (2012), "Xac dinh ham luvng
polyphenol town phen, kha nang triet tieu gOc tv' do va kha
nang CPC the men alpha-glucosidase fir la vOi, la of va la
sen", Top chi Dinh clueing va Myr pham, 8(1), 33-38.

(Ngay nha'n bai: 23/04/2015 - Ngay duyOt dang: 04/09/2015)

Dinh gia mot so hoat tinh sinh hoc
ciia chAt ent-halima-1(10),13E-dien715-oic acid
duvc phan lap tir cay quan dau khi
(Polyalthia simiarum Benth. & Hook. f.)

Nguy& Thi Minh Fling*, Cao Thi HO, Doan Thi Hu•o'ng
Vien Hoa sinh bier, - Vin Han lcim Khoa hoc va ("ring nglie. Viet Nam
E-mail:

Summary
Ent-halima-1(10),13E-dien-15-oic acid (1) was isolated from the n-hexane extracts of the barks
of Polyalthia simiarum. It's structure was confirmed by NMR and MS. The obtained products showed
significant cytotoxicity, with IC 50 values of 13,93-25,05 pg/mL against Lu-1, HepG2, LNCap, MCF7,
SW626, SW480 cancer cell lines.
Keywords: Polyalthia simiarum, "Benth. & Hook. f" ent-halima-1(10),13E-dien-15-oic acid.

DOt van de

Vi5t Nam, chi Polyalthia co ten là chi Quen

Chi Polyalthia gem co 120 loci duvc ph5n 136
(Ong rai tai cac yin-1g nhiet deyi va can nhi5t

deu (ho Na - Annonaceae) vai 28 loci ph5n bo

38

TAP CHI DOC HQC - 9/2015 (SO 473 NAM 55)

cha yeu a cac tinh mien Trung va mien Nam Mi.


• Nghien cu'u - K9 thUOt
Cay guen dau khi (Polyalthia simiarum
Benth.&Hook. f.) la cay dal mac nh6, cao 12 m,

cluvc phan be it mien Nam Viet Nam. 6' An DO,
vo cay duvc sit dung lam thuec tri bo cap clOt.
Trong qua trinh sang lac hoAt tinh sinh hoc cua
mat so loai thy,c vat Viet Nam, chimg tai de phat
hien can chiet ethyl acetat cua la cay quen dau
khi (P simiarum) CPC Ch6 100% ski, phat then cua
dong to bao ung thin tuyen thuvng then SW13.
Tuy nhien, cho den nay, tren the giai mai chi c6
duy nhat mat cong trinh ding be \re cac thanh
phan bisnor-clerodan diterpenoid cua v6 cay nay
6 Bangladesh [2]. Vai myc tieu tim kiem cac chat
CO hoat tinh sinh hoc tit mat so loai thtyc vet ho Na
chung va chi Quan dau (Polyalthia) n6i Hang,
chung tai da tier, hanh nghien dru thanh phan
h6a hoc cua vo cay quen dau khi (P simiarum).
Trong cong trinh nay, chimg tai th6ng bao ye
qua trinh phan lap va xac dinh cau trix cua mat
hop chat ditecpenoid khung ent-haliman IA enthalima-1(10),13E-dien-15-oic acid (1) cut-1g vai
nhitng danh gia ye hoat tinh gay dac te bao va
khang vi sinh vet kiem dinh cua hap chat nay.

Nguyen lieu va phu'eng phap
nghien citu
Mau thkrc vat
Mk v6 cay quen dau khi (P simiarum) duvc
ThS. Dao Dinh CtrO,ng thu hai tai xa So Pai,
huyen K'Bang, tinh Gia Lai, Viet Nam vao thang
9 nem 2013 va da duvc TS. Nguyen Oki& Binh
thuac Bao tang Thien nhien Viet Nam dinh ten.
Mau tieu ban mang ky hieu VN1751 hien clang

duvc luu gift tai Phong tieu ban cua Vien Sinh
thai va Tai nguyen sinh vet, Vien Han lam Khoa
hoc va Gong nghe Viet Nam.
Hoa chat, thiet bi
Sic ky 16,p mong (TLC): Sac ky I6,p mong
&roc thkrc hien tren ban mong tang se'n DCAlufolien 60 F254 (Merck). Phat hien chat bang
den to, ngoAi b bu,ac song 254 nm va dung thutic
thir la dung dich ceri sulphat.
Sgc ky cat (CC): Sec ky cot duvc tien hanh
vai chat hap phy la sillica gel pha thu,Ong va
Sephadex LH-20. Sillica gel pha thixong CO co'
hat la 0,040-0,063 mm (240-430 mesh).

Ph6 khoi luvng (ESI-MS va ACPI-MS):
&roc do tren he may AGILENT 6120.
Chiet xuAt va phan lap chat
Mau vo cay quen &au khi (P. simiarum) tuvi
duvc rim sach, barn nho va phai kh6 roi say
45°C -50°C trong 3 gib. Mk kho (1,8 kg) duvc
nghien thanh bat min rei ngam chiet trong Me0H
(4 len, 24 giolien, 5 Ulan). Gap cac dich chiet
Me0H lei vai nhau, cet loai dung mai xueng con
khoang 1/15 the tich ban dau. Pha bang dich
chiet con lei bang nip:5'c cet, chiet phan be len
Drat vai cac dung m6i n-hexan va ethyl acetat,
m6i dung mai chiet 3 Ian (500 ml/len). Gap dich
chiet cua 3 Ian va lam kho bang Na2SO4 khan.
Cat loai dung mai du,ai tip suet giam thu duvc
cac can chiet twang irng la n-hexan (7,5 g), ethyl
acetat (13,6 g). Phan dich Me0H-H2 0 con lei

&roc cet loai dung mai den kho thu &roc can
Me0H (16,5 g). Phan tech can n-hexan (7,5 g)
bang cat silica gel, rita giai theo phtrang phap
gradient he dung mai n-hexan/aceton thu duvc
6 phan doan, ki hieu F1-F5. Phan doan F1 tiep
tyc duvc tinh the bang cat silica gel pha thkrang,
ban du rota cat bang 100% n-hexan sau d6 rota
giai bang he dung mai n-hexan/aceton ( 98:2)
thu duvc 3 phan doan, ki hieu F1.1-F1.3. Phan
doan F1.3 sau khi de bay hai het dung m6i they
c6 xuet hien tinh the, rira phan doan nay bang
aceton va ket tinh lei trong hen hop dung m6i
n-hexan/aceton (7:3) thu &roc chat 1 (0,15 g)
dual clang tinh the hinh kim, kh6ng mau.
Phtnyng phap thin hopt tinh gay dt5c te Pao
Phu,crng phap nuoi cay to bao in vitro
Cac (Jong te bao ung thu, duvc nuai cey duel
Bang don lap trong mai trirerng nuai cey DMEM
vai thanh phan kern theo gem 2 mM L-glutamin,
10 mM HEPES, va 1,0 mM natri pyruvat, ngoai ra
be sung 10% fetal bovine serum - FBS (GIBCO).
Te bao (bac cey chuyen sau 3-5 ngay vai ti
le (1:3) va nuOi trong to em CO2 6' dieu kien 37°C,
5% CO2.
Phep them sinh hoc xac dinh tinh clOc to bao
(cytotoxic assay)

Phi; cong tuning to hat nhan (NMR):
duvc ghi bang may Bruker AM500 FT-NMR
Spectrometer vai chat chuen not IA TMS.

Phuvng phap this do dec te bao in vitro duvc
Vien Ung thu, Oki& gia Hoa Ky (National Cancer
Institute — NCI) chap nhen. Phep thin nay &roc
thtyc hien theo phkrang phap cua Monks 13-71.
Phep the, tien hanh xac dinh ham luting protein
te bao tong so dkra vao mat do quang hoc (OD —

TAP CHI DUVC HOC - 9/2015 (SO 473 NAM 55)

39


• Nghien ciru - K9 thuOt
Optical Density) do duvc khi thanh phan protein
cua to bao &vac nhuern bang Sulforhodamine B
(SRB). Gia trj OD may do duvc ti le thuan vai
luvng SRB gAn vai phan tir protein, do dO luvng
to bao tang nhieu (luting protein tang nhieu) thi
gia trj OD cang Ian. Phep thin drat thkrc hien
trong dieu kien cu the nhu, sau:
Chet thin (10 pi) pha trong DMSO 10% duvc
dua vao cac gieng cua khay 96 gieng de co nOng
(30 sang log la 20 µg/ml. Chet thin CO hoat tinh
duvc xac dinh IC50 nher dai nOng TO 20 µg/ml; 4
ug/m1; 0,8 lig/m1; 0,16 µg/ml.
Trypsin hea to bao thi nghiem a lam rai to
bao va dem trong buong dem de dieu chinh mat
de cho phu hap vai thi nghiem.
Them vao cac gieng thi nghiem luvng to bao

ph0 hap (trong 190 ul mei twang) va de chUng
phat trien trong yang tir 3-5 ngay.
Met khay 96 gieng khac khong CO chat thin nhung
co TBUT (180 ul) se duvc sir dung lam clei chirng
ngay 0. Sau 1 gitr, dia dei chi:mg ngay 0 se direc co
dinh to bao bang Trichloracetic acid — TCA.
Sau giai doan phat trien trong to em CO2, to bao
duvc co dinh vao day gieng bang TCA trong 30
phut, duvc nhuem bang SRB trong 1 gia & 37°C.
DO loo SRB va cac gieng thi nghiem duvc rira 3
len bang 5% acetic acid rOi de kho trong khong
khi b nhiet do phong.
Cu6i cOng, sir dung 10 mM unbuffered Tris
base de Ma tan luting SRB da barn va nhuem
cac phan tir protein, dua len may lac dia lac
nhe trong 10 phut va sir dung may ELISA Plate
Reader (Bio-Rad) de doc ket qua ye ham luting
mau cua chat nhuem SRB qua ph6 hap thu 6,
bu,ac song 515 nm. Kha nang song set cua to
bao khi c6 mat chat thin se (bac xac dinh thOng
qua cong thirc sau:

nang Crc the su, phat trien hoac diet TBUT.
Phtrang phap thir hoat tinh khang vi sinh
vat kiem (firth

Hopt tinh khang vi sinh vat kiem dinh duvc
thu,c hien tren phien vi luvng 96 gieng theo
phuvng phap dm Vander Bergher va Vlietlinck
va McKane L. & Kandel. Cac chi:mg vi sinh vat

kiem djnh duvc hoat h6a va pha bang tai nOng
do khoang 0,5 dan vi Mc Fland. Cac phien thi
nghiem de trong to em a 37°C/24 gia cho vi
khuan va 30°C/48 gia cho nam. Cac mau &vac
pha bang theo cat thang nOng OO thap den, to,
5-10 thang nOng TO de tinh gia tri nOng do tOi
thieu ma 6, d6 vi sinh vat bi Crc the phat trien gAn
nhu, hoar) toan.
Cac thkrc nghiem duvc tien hanh tai Vien Han
lam Khoa het va C6ng nghe Viet Nam.

Ket qua va that:, luan
Ket qua phan lap

ch6 = 100% - % song sot
Cac phep thin duvc lap V 3 lAn de dam bao
tinh chinh xac. Ellipticine (Sigma) lam &vac SC1'
dung nhu, la chat clOi chirrig &rang va &vac thir
nghiem a cac nOng do 10 ug/mL; 2 ug/mL; 0,4
ug/mL; 0,08 ug/mL.
DMSO 10% ILI& &rev sir dung nhu, el& ch&rig
am. Gia tri IC50 (nOng de tic the 50% ski, phat trien)
se dirge xac djnh nha vao phan mem may tinh
TableCurve. Chat thir nao co IC < 20 ug/mL
„
&rev xem la cO hopt tinh gay doc to bao va co kha

Chat 1 duvc phan lap dual clang tinh the hinh
kim, khong mau, nOng chay a 123-124°C. l'alD25
+67,5 (Me0H, c 0,62). 1H-NMR va 13C-NMR: xem

bang 1. ESI-MS m/z: 305 [M+I-1]..
Then phO 'H-NMR xuat hien tin hieu tong Wang
cua met nhOm methyl clang doublet tai 0 0,81 (3H,
d, J = 7,0 Hz) ding vai 4 nhOm methyl clang singlet
tai 0 0,84 (s), 8 0,98 (s), S 0,91 (s) va S 2,15 (s), hai
proton clang olephin xuat hien tai 8. 5,33 (1H, t, J
= 3,5 Hz) va 8 5,66 (1H, br s). Ngoai ra, tren phO
'H-NMR con xuet hien tin hieu cua proton thuec cac
nhOm methylen va methin trong vOng 0 1,11-2,05.
Phan tich phO "C-NMR va DEPT dm 1 cho they
tin hieu cua 20 carbon trong dO CO hai nhOm methin
Bang olephin tai 8 120,32 va 114,55, hai nhOm
methin tai 0 39,16 va 43,51, sau nhOm methylen
twang irng vai S 23,12; 33,25; 23,63; 29,10; 37,20
va 36,29, nam nhOm methyl tai 0 15,65, 19,46,
22,29; 26,01; 28,23 cOng vai ba carbon bac bOn tai
0 31,44, 141,18 va 164,91 va met nhOm carboxyl
tai 0 172,0. Ph6 khoi luting ESI-MS xuet hien pic
305 [M+H]' (positive). Qua cat du, lieu phO ten co
the nhan dinh SP1 la met hap chat acid thuec lap
chat diterpenoid va co khung ent-haliman co cong
thin phan tir la C2O H3202. TO, cac phan tich ten,
sO lieu phO 'H-NMR va ' 3C-NMR cua chat 1 &rev
so sanh vai cat gia tri twang ung da duvc cong
136 cua hap chat ent-halima-1(10),13E-dien-15-oic
acid PI. Skr phO hap hoan toan ye sO lieu phO giCra
hai hap chat (bang 1) cho phap khang dinh chat 1
la ent-halima-1(10),13E-dien-15-oic acid.

40

TAP CHI DOC HOC - 9/2015 (SO 473 NAM 55)

% song sot % we

[OD (chat thir) - OD (ngay 0)] x 100
OD (clei chirng am) - OD (ngay o)


• Nghien aru - K9 thuat
Bang 1: SO Wu oho NMR ceia 1

•

14

C

6c"

OH"

.6c84'

'8„•4

1

120,32

5,33 (t, 3,5)

120,3

5,33 (t, 3,8)

2

23,12

23,1

3

33,25

33,2

4

31,44

31,4

5

43,51

43,5

OH

6

23,63

23,6

7

29,10

29,1

8
9

39,16

39,2

43,0

43,0

Hoot tinh gay dc)c to bao

10

141,18

141,1

Ent-halima-1(10),13E-dien-15-oic acid (1) de

11

37,20

37,2

12

36,29

36,3

13

164,91

14

114,55

15

171,81

16

19,46

2,15 (s)

19,5

2,16 (s)

17

15,65

0,82 (d, 6,5)

15,6

0,82 (d, 6,4)

18

28,23

0,89 (s)

0,89 (s)

19

26,01

0,84 (s)

28,2
26,0

0,84 (s)

&roc danh gia hoot tinh gay dee to bao tren 7
clang to bao ung this & ngu,o1
ung this phOi
(Lu-1), ung this gan (HepG2), ung this tier' liOt
tuyen (LNCap), ung this vu (MCF7), ung this
buOng tru,ng (SW626), ung this dai trang (SW480)
va ung this bleu me mieng (KB). m6i thi nghiem
duvc 10p !pi 3 Ihn de dam bao tinh chinh xac cua
thi nghiem va dip lieu.

20

22,29

0,91 (s)

22,3

0,91 (s)

Hinh 1: C6u tritc has hoc cOa 1

164,8
5,66 (s)

115,1

5,67 (s)

172,4

trong CDC/3, b125 MHz, c500 MHz,
ent-halima-1(10),13E-dien-15-oic acid

cOa

Bang 2: Kat qua tha, hoot tinh gay cffic to bao mia 1
% tic obi

Ming B¢ (pg/ml)

Lu-1

HepG2

LNCap

MCF7

1

Ellipticine

1

Ellipticine

1

Ellipticine

1

Ellipticine

100

80,14

92,80

98,30

90,22

91,35

89,53

86,23

88,75

20

58,40

78,73

53,23

71,74

63,28

70,68

50,64

75,14

4

13,83

56,70

27,08

50,77

12,51

59,67

10,50

53,54

0.8

3,39

10,47

4,89

10,90

2,03

11,23

3,72

22,32

IC50

19,74

0,50

16,16

0,51

16,35

0,54

25,05

0,44

% Cfc the

Along diS Gig/ml)

SW626

SW480

1

Ellipticine

88,54

92,65

20

53,35

4

16,09

0.8
IC50

100

KB

1
88,47

Ellipticine
92,85

1

Ellipticine

98,35

99,77

78,04

58,45

78,38

49,35

76,43

51,19

26,88

57,19

1,86

40,58

7,21

17,53

6,03

18,83

0,87

12,78

21,18

0,48

13,93

0,41

19,95

0,57

Chat doi chirng durrng Ellipticine hoot Ong on dinh trong thi nghiam. Cac kit qua tren là chinh xac veri r2 a 0,99

TAP CHI DUVC HQC - 9/2015 (S6 473 NAM 55)

41


• Nghien ctvu - KS(' thuOt
Dieu clang chu y la chat ent-halima-1(10),13Edien-15-oic acid (1) co tac dung clang ke dal vai
tat ca 7 clang to bao ung thu' this nghiem, gia tri
IC50 nAm trong khoang 13,93-25,05 pg/mL. Trong
7 d6ng to bao th0, nghiem, 1 co tac dung tot nhat
clOi vai cac dong to bao ung thu, bung tru,ng
SW480 vai IC50 la 13,93 pg/mL. Day la nhErng kat
qua bu,ac clau cho thay kha n5ng c6 the nghien
aru sau han ve hoat tinh gay Otic to bao cub hap
chat nay theo dinh hu,ang tao the pham h6 tra
ph6ng chOng benh ung thu,.

Hoat tinh khang vi sinh vat kiem dinh
Chat ent-halima-1(10),13E-dien-15-oic acid
(1) duvc thin hoat tinh khang vi sinh vat kiem
dinh tron 2 chung vi khan Gram (-) la E. coli va
P aeruginosa, 2 chung vi khuen Gram (+) la B.
subtilis va S.aureus, 2 chung nOrn mOc la A. niger
va F. oxysporum cOng vai 2 chung nOrn men la
S. cerevisiae va C.albicans. Tuy nhien, chat nay
455 kh6ng the hien hoat tinh ton chung vi sinh vat
thin nghiem nao.

Ket luan
Hap chat ent-halima-1(10),13E-dien-15-oic
acid (1) cla duvc phan lap tb, v6 cay quAn dau khi
(P simiarum). Hap chat nay the hien hoat tinh
gay dOc to bao clang ke clOi vai 07 to bao ung
thu, nguai Lu-1, FlepG2, LNCap, MCF7, SW626,
SW480 va KB vai gia tri IC50 narn trong khoang
13,93-25,05 pg/mL. Trong so cac dOng to bao ung
thu, thin nghiem, chat nay co tac dung manh nhat
clai vai citing to bao ung thu, bung tru,ng SW480.
Tuy nhien, chat 1 da kh6ng the hien hoat tinh tren
cac chung vi sinh vat kiem dinh thin nghiem. Day
la lAn dhu tien chat ent-halima-1(10),13E-dien15-oic acid duvc danh gia ve hoat tinh gay (Ric
to bao va khang vi sinh vat kiem dinh.
Nghien cOu nay (Ivor tai tro obi Quy phat frian
khoa hoc va tong ngha qu6c gia (NAFOSTED) trong
de tai ma s6 104.01-2012.23.

Tai lieu tham khan

1. Pham Hoang HO (2000), C5y co Viet Nam, Nha
xuat ban Tre, Quyen I, trang 256-263.
2. S. Kabir, M. S. Rahman, A. M. Sarwaruddin
Chowdhury, C. M. Hasan and M. A. Rashid (2010), "An
unusual bisnor-clerodane diterpenoid from Polyalthia
simiarum", Natural Product Communications, 5(10),
1543-1546.
3.A. Monks, D. Scudiero, P. Skehan, R. Shoemake,
K. Paull, D. Vistica, C. Hose, J. Langley, P. Cronise, H.
Campbell, J. Mayo, M. Boyd (1991), "Feasibility of a
high-flux anticancer drug screen using a diverse panel
of cultured human tumor cell lines", Journal of National
Cancer Institute, 83(11), 757-766.
4. D. A. Scudiero, R. H. Shoemaker, K. D. Paull,
A. Monks, S. Tierney, T. H. Nofziger, M. J. Currens,
D. Seniff, M. R. Boyd. (1988), "Evaluation of a soluble
Tetrazolium/Formazan assay for cell growth and drug
sensivity in culture using human and other tumor cell
lines", Cancer Research, No.48, 4827-4833.
5. L. B. S. Kardono, C. K. Angerhofer, S. Tsauri, K.
Padmawinata, J. M. Pezzuto, A. D. Kinghorn. (1991),
"Cytotoxic and antimalarial constituents of the roots of
Eutycoma longifolia", J. Nat. Prod., 54(5), 1360-1367.
6. M. C. Alley, D. A. scudiero, A. Monks, M. L.
Hursey, M. J. Czerwinski, D. L. Fine, B. J. Abbott,
J. G. Mayo, R. H. Shoemaker, M. R. Boyd. (1998),
"Feasibility of drug screening with panels of human
tumor cell lines using a microculture tetrazolium
assay", Cancer Research, No. 48, 589-601.
7. R. H. Shoemaker, D. A. Scudiero, E. A. Suasville

(2002), "Application of high-throughput, molecular —
targeted screening to anticancer drug discovery", Curr.
Top. Med. Chem., 3(2), 229-246.
8. Noriyuki Hara, Hitomi Asaki, Yoshinori Fujimoto,
Yogesh Kumar Gupta, Ashish Kumar Singh and
Mahendra Sahai (1995), "Clerodane and ent-halimane
diterpenes from Polyalthia longifolia", Phytochemistry,
38(1), 189-194.

(Ngay nhan bai: 10/03/2015 - Ngay duyat clang: 04/09/2015)

42

TAP CHI DUQC HOC - 9/2015 (SO 473 NAM 55)


• Nghien ciru - K9 thulat

Tong hop va tic dung sinh hoc dm mot so
dan chit N-alkyl hoa cua 5-arylidenrhodanin
va 5-arylidenthiazolidin-2,4-dion
n

Nguyen Due Hong, Ming Cao Dai, Nguyen Cong Tru*ng
Ngo Xuan Hoang, Dinh Thi Thanh Hai"
Bp mon Hoa Hiru ea, Tiatang Dai hoc Denyc. Ha Nai
E-mail:

Summary
By condensation of aromatic aldehydes with rhodanine or thiazolidin-2,4-dion, the compounds la 4a were obtained. These compounds then underwent N-alkylation reaction by application of microwave

technology, to give 4 corresponding derivatives 1 b - 4b. The structures of the synthesized compounds
were confirmed by IR, 'H-NMR, 13C-NMR and MS. The obtained compounds were tested for some bioactivities including antifungal, antibacterial and cytotoxic activity.
Keywords: 5-(4-fluorobenzyliden) rhodanine, 5-(2, 4-diclorobenzyliden)rhodanine, 5-(4-fluorobenzyliden)
thiazolidin-2,4-dion, 5-(2,4-diclorobenzyliden)thiazolidin-2,4-dion, N-alkylation reaction, biological activity

DOA yen de
Thiazolidin-2,4-dion va dar n chat da duvc cac
nha khoa hoc trong va ngoai nu,O'c nghien ciru mat
cach co he thong ve tong hap hoa hoc va tac dung
sinh hoc. Nhieu cong trinh nghien tiro cho thay
cac clan chat dm thiazolidin-2,4-dion c6 hoat tinh
khang khuan, khang n6m, khang to bao ung thin,
chong tieu cluong. MOt s6 d6n chat dm thiazolidin2,4-dion da duvc sip dung trong y hoc lam thuOc
dieu tri dai thao duting nhu' pioglitazon (bet
duvc: Actos, Dopili) va rosiglitazon (brat duvc:
Avandia) [2 '34]. Ben canh d6, rhodanin (2-thioxo-4thiazolidinon) va cac dan chat da duvc nhieu nha
khoa hoc nghien ciru ve tong hap h6a hoc va tac
dung sinh hoc. Cac cong trinh nghien ciru cua cac
tac gia trong va ngoai nultec da cong bo cho thay
cac an chat rhodanin co hoat tinh sinh hoc da

Bang nhu' khang khuan, khang n6m, chang ung
thiy, khang virus 11 '2'5]. Trap tuc phat then htrang
nghien ciru ve thiazolidin-2,4-dion, rhodanin trong
bai bao nay chUng toi trinh bay kat qua "TOng

h9p va tac dung sinh hoc coa mot so can
chit N-alkyl hem cOa 5-arylidenrhodanin va
5-atylidenthiazolidin-2,4-dion".
Nguyen lieu va phvang phap

nghien civu

Nguyen lieu:
Cac h6a chat, dung mai si), dung trong nghien
ciru deu nhap by cac hang Merck, Sigma -Aldrich
va tip Trung Quoc.

Phwang Oar) nghien
So, dO tong h9p hod h9c: Tien hanh cac phan
&rig tong hap theo so, do phan (mg chung sau day:
131

CHO •

CH2CI
K,CO,, MW
H

lb -4b

TAP CHt Du'cic HOC - 9/2015 (SO 473 NAM 55)

43


• Nghien ciru - Ky thuast
Trong dO:
ChAt
la, lb
2a, 2b

3a, 3b
4a, 4b

X
S
S
0
0

R1
F
Cl
F
Cl

phan Ong ngu'ng tu cua cac aldehyd therm
(p-fluorobenzaldehyd va 2,4-diclorobenzaldehyd)
vao vi tri s6 5 dm nha'n rhodanin va thiazolidin2,4-dion trong mOi trubrig AcOH vai xOc tac base
la AcONa.

R2

H
CI
H
Cl

Tong hop haa hoc: Sir dung cac phan Ong
hipu co, thOng thiyang, ket hop vai kyr thuat vi
song de tong hop cac chht du, kien. Xu, 19 h6n

hap phan Ong bAng phuang phap phO hap de
thu san phhm th6 sau dO tinh the bang phiyang
phap ket tinh lai vai cac dung m6i khac nhau tOy
theo tO,ng san phern.
DOng sac kk lop mong (SKLM) de theo doi
phan tYng, xac dinh thol gian chieu xa vi song
thich hop va sa be xac dinh dO tinh khiet cua san
phhm.
Xac dinh ca'u trUc caa cac chOt tong hop duipp
b'hng phe Wong ngoai (IR), phO khoi luting (MS),
phO cOng hu,deng tip hat nhan ('H - NMR va 13C NMR).
Sa"c Irk lap mong (SKLM) diroc thu,c hien tren
ban mong silicagel Kieselgel 60 F254. Diem chay
duvc ghi tren may EZ-Melt. L6 phan 0,ng vi song
model Sineo uwave-1000. Phe hong ngoai (IR)
duvc ghi tren may Perkin Elmer. PhO khoi luting
(MS) ghi Wen may LC-MSD-Trap SL. PhO cOng
hu,ang tip hat nh5n ('H - NMR va 13C - NMR) ghi
ten may Bruker - AV500.
ThO, tac dung khang khuhn, khang nem tai
Ph6ng Sinh hoc thiyc nghiem - Vien Hoa hoc
cac chht thien nhien (Vien Han lam Khoa hoc
va Cong nghe Viet Nam) theo phiyang phap cua
Vlietlink A. J. va Vanden Berger V. A (8] . Thu, tac
dung khang to bao ung thu' ngu,61 duvc tien hanh
tai Phang Sinh hoc thu'c nghiem - Vien Hoa hoc
cac chht thien nhien (Vien Han lam Khoa hoc va
COng nghe Viet Nam) theo phiyang phap SRB (6]
hien dang
hanh va tip dung tai ViOn Nghien

ung
thu'
qu6c
gia My (NCI).
civu

TOng hop 5-(p-fluorobenzyliden)rhodanin
(la): Cho vao binh chu 3 co 1,06 g (0,008 mol)
rhodanin, 0,99 g (0,008 mol) p-fluorobenzaldehyd,
1,97 g (0,024 mol) natri acetat khan, 25 ml acid
acetic bang. Khuhy bhng khuhy tip cho tan, deng
thoi tang nhiet dO tip tip. Hen hop soi va hoi
117 - 121°C thi bet deu tinh gia. Theo dal
phan Ong bAng sac lq/ lap mong vai he dung mai
CHCI3 : (CH3)2CHOH (40 : 1). Xac dinh thbi gian
phan Ong thich hop la 5 gia. KM thCic phan Cyng,
dO hon hop phan Ong ra coc, de yen a nhiet do
ph6ng trong 24 gia, tao tua. LQC hut, riva tua cho
den het acid, say kh6 thu duvc tua the). KM tinh
!pi trong dung m6i EtOH. Hieu suet: 59%. Nhiet
do ruing chair: 224 - 226°C. Rf = 0,66 (CHCI3 :
(CH3)2CHOH 40 : 1). IR (KBr, cm-1): 3100(vNH),
1724 (vc,,0), 1594 (vc.c), 1594, 1447 (vc.caron,),
1229 (vc,,$), 1185 (vcerm_F). ESI-MS (m/z): 239
(NV; 38,9), 180 (M - NHCS; 3,5), 152 (180 - CO;
100), 107(152 - CS; 14,2). 'H-NMR ( 500 MHz,
DMSO-d6), 8 (ppm): 13,8 (s, 1H, NH); 7,68-7,66
(m, 2H, H-2'; H-6'); 7,65 (s, 1H, -CH=); 7,407,36 (m, 2H, H-3'; H-5'). "C-NMR (125 MHz,
DMSO-d6), 8 (ppm): 195,47 (C-2); 169,25 (C-4);
164,00 (C-4'); 132,92 (C-3', C-5'); 130,47 (-CH=);

129,63 (C-5); 125,20 (C-1'); 116,68 (C-2', C-6').

Du'a vao kha nang phan Ong cua nhOm
methylen (>CH2) a vi tri 5 trong phan tO, rhodanin
va thiazolidin-2,4-dion, chOng to' tien hanh

TOng hgp 5-(2,4-diclorobenzyliden)
rhodanin (2a): Tuvng tu' nhu, vai Ch6l la, chi
thay hop phAn aldehyd la 1,4 g (0,008 mol)
2,4-diclorobenzaldehyd. Xac dinh thai gian phan
Ong thich hap la 6 gib. Ket tinh lei trong dung moi
EtOH. Hieu suet 68,5%. Nhffit do nong chair:
237-239°C. Rf= 0,62 (CHCI3 : (CH3)2CHOH 40 : 1).
IR (KBr, cm-1): 3114 (v„), 1734 (vc„,0), 1578 (vc=c),
1578, 1467 (vc,cerorn), 1224 (vc=s), 1196 (vc,rom_c1).
ESI-MS (m/z): 287 (Mt; 100); 250 (M.-Cl; 3,2);
195 (250-NHCS;9); 167 (289-CONHCS-Cl; 40).
'H-NMR ( 500 MHz, DMSO-d6), .5 (ppm): 14 (s,
1H, NH); 7,83 (d, 1H, J=2Hz, H-6'); 7,67 (s, 1H,
-CH=); 7,61-7,59 (m, 2H, H-3'; H-5'). 13C-NMR
(125 MHz, DMSO-d6), .5 (ppm): 195,18 (C-2);
169,02 (C-4); 135,69 (C-3'; C-5') 130,36 (C-4');
129, 96 (C-2', C-6'); 129,68 (C-1'); 128,42 (C-5);
124,80 (-CH=).

44

TAP CHI DOC HQC - 9/2015 (SO 473 NAM 55)

Thu'c nghiem, k6t qua va ban luan

TOng hcyp 5-arylidenrhodanin
5-arylidenthiazolidin-2,4-dion (la - 4a)

va


• Nghien ciru - K9 thuOt
Ting hop 5-(p-fluorobenzyliden)thiazolidin2,4-dion (3a): Cho vao binh cau ba c6 dung tich
500 ml 12,4 g (0,1 mol) p-fluorobenzaldehyd;
100 ml AcOH bang, khuay deu b nhiat do phong.
Them 13,3 g (0,1 mol)AcONa va 11,7 g (0,1 mol)
thiazolidin-2,4-dion r6i khuay cho tan hoan toan
nhiat de phong, sau dO nang nhiat d0 cua hon
hop phan Ong den soi thi bat du tinh gia, duy
tri nhiat do la 118 - 123°C. Theo dal phan Ong
bang sec k9 lap mong vai dung moi khai trien
la CHCI3:CH3OH (30:1). Xac Binh thai gian phan
Ong thich hop là 5 gib.. 'Ma tgo thanh, them 20 ml
nivac cat, khuay a nhiat de phong 10 phut. LQc
hilt qua pheu chan khong, rira tua nhieu Ian bang
nuac cat sau dO rim bap bang ethanol. Say kh6
nhiet de 70°C. Hieu suet: 71,0%. Nhiat dQ ming
chay: 222 - 224°C. R,= 0,57 (CHCI3 : CH3OH
30:1). IR (KBr, cm-1): 3155 (vNH), 1729, 1700
(vc=0), 1593 (vc.c), 1593, 1506, 1411 (vc.,carorn),
1154 (vcarm_F ). ESI-MS (m/z): 223 (M*;36); 180
(M*-NHCO;3,8); 152 (180-CO; 28,5); 108 (152CS;21,5). 'H-NMR ( 500 MHz, DMSO-d6), 5
(ppm): 12,5 (s, 1H, NH); 7,79 (s, 1H,-CH=); 7,667,64 (dd, 2H, J=5,5Hz va J=5,0Hz; H-2'; H-6');
7,38-7,35 (t, 2H, H-3'; H-5'). "C-NMR (125 MHz,
DMSO-d6), 8 (ppm): 167,68 (C-2); 167,20 (C-4);

163,80 (C-4'); 161,82 (C-5); 132,4 (C-3', C-5');
130,67 (C-1);129,68 (-CH=); 116,51 (C-2', C-6').
Ting hop 5-(2,4-diclorobenzyliden)
thiazolidin-2,4-dion (4a): Tureng to, nhu' &Si vai
chat 3a nhu'ng thay hop phan aldehyd là 17,50 g
(0,1 mol) 2,4-diclorobenzaldehyd; Thai gian phan
Ong thich hop la 4 gia. Hieu swat: 73,0%. Nhiat
dc) :gong char 216 - 218°C. !if = 0,75 (CHCI3 :
CH3OH 30:1). IR (KBr, cm-1): 3182 (vNH), 1754,
1708 (vc.0), 1597 (vc.c), 1579, 1467 (vc.cm,),
1164 (vc,rm_ci). ESI-MS (m/z): 274 (M.;60); 230
(M-NHCO;10); 203 (230-00;50); 168(203-CI;6);
133 (168-CI;9). 1H-NMR ( 500 MHz, DMSO-d6), 6
(ppm): 12,8 (s, 1H, NH); 7,83 (s, 1H, H-3'); 7,80
(d, 1H, J=2Hz, -CH=); 7,60-7,53 (m, 2H, H-5' va
H-6'). 13C-NMR (125 MHz, DMSO-d6), 5 (ppm):
167,26 (C-2); 166,79 (C-4); 135,39 (C-3', C-5');
130,00 (C-4'); 129,91 (C-2', C-6'); 128,24 (C-1');
127,82 (C-5); 125,47 (-CH=).

Tien hanh phan Ong: Dina tren phu,ang phap
cua Maria Sol Shmidt va Ong so, 17] sir dung k9
thuat vi song de tong hop. Cho vao binh cau 0,03
mol chat 1a d6i vai tong hgp chat 1b (tu,ang to, la
2a-4a doi vai Ring hop chat 2b-4b), 3,5 ml (0,03
mol) benzyl clorid, 0,03 mol K2003 va 15 ml DMF.
Day binh cau bang nut nhy'a, cai dat the de phan
Ong vi song va Wen hanh phan Ong.
XLO /)% hit) hop Olen &rig: Sau khi phan Ong
ket thOc, d6 hi& hgp ra cOc CO m6 dung tich 100

ml. De nguei cho ket tinh, sau dO dem loc hid
chan khong, rira tOa bang nulac lanh. Ket tinh !pi
trong EtOH. Say kho thu duvc san phem.
Ting hop chit lb: Tinh the hinh kim mau
yang, ket tinh Igi trong EtOH, hiau suet: 84,9%.
Nhiat di, nong chair: 92 - 94°C. IR (KBr, cm1): 2943 (vcH2), 1700 (vc.0), 1592 (vc.c), 1509,
1451, 1374 (vc.cmrn), 1235 (vc.6), 1161 (vc.r..6).
1592 (vc.N). ESI-MS (m/z): 329 [Ml. 'H-NMR
( 500 MHz, Aceton-d6), 5 (ppm): 7,83 (s, 1H,
-CH=); 7,80-7,73 (m, 4H, H-2', 11-6', H-2", H-6");
7,54-7,53 (d, 2H, J=5Hz, H-3", H-5"); 7,427,38 (dd, 2H, J=2Hz, H-3', H-5'); 7,37-7,29 (m,
1H, H-4"); 4,76 (s, 2H, -CH2-). ). 13C-NMR (125
MHz, Aceton-d6), 5 (ppm): 206,49 (C-2); 167,65
(C-4); 166,42 (-CH=); 165,85 (C-1"); 163,82 (C2'); 136,32 (C-4'); 133,43 (C-3'); 132,82 (C-5');
130,76 (C-2", C-6"); 129,71 (C-1'); 128,82 (C-6');
122,43 (C-3"; C-5"); 117,34 (C-4"); 117,16 (C-5);
45,77(-CH2-).

Ung hop m.,t s6 can chAt N-alkyl hoa cua
5-arylidenrhodanin va 5-arylidenthiazolidin2,4-dion (lb- 4b)

Ting hop ch‘t2b: Tinh the hinh kim mau do,
ket tinh lei trong EtOH, hiau suet 82,0 %. Nhiet dO
ming ashy 86 - 88°C. IR (KBr, cm-1): 2930 (vcH2),
1699 (vc.0), 1580 (vc.c), 1580, 1450 (vc.carm),
1200 (vc.6), 1165 (vc,rom_c,), 1580 (vc_0. ESI-MS
(m/z): 389 [Ml. 'H-NMR ( 500 MHz, Aceton-d6), 5
(ppm): 8,03 (s, 1H, -CH=); 7,97 (s, 1H, H-3'); 7,72
(d, 2H, J=5 Hz, H-3", H-5"); 7,70 (d, 2H, J=5Hz,
H-2", H-6"); 7,67-7,56 (m, 1H, H-4"); 7,54 (d, 1H,

J=5Hz, H-5'); 7,43 (m, 1H, J=7,5Hz, H-6'); 4,77
(s, 2H, -CH2-). 13C-NMR (125 MHz, Aceton-d6), 8
(ppm): 206,58 (C-2); 167,37 (C-4); 166,13 (-CH=);
137,19 (C-1"); 136,73 (C-2'); 136,37 (C-4'); 131,33
(C-3'); 131,16 (C-5'); 130,27 (C-1'); 129,47 (C-2",
C-6"); 129,13 (C-6'); 129,09 (C-3", C-5"); 128,74
(C-4"); 128,27 (C-5); 45,97 (-CH2-).
Ting hop ch4t 3b: Tinh the hinh kim mau
yang nhat, kat tinh Igi trong EtOH, hiau suet

TAP CHI DUVC HQC - 9/2015 (SO 473 NAM 55)

45


• Nghien cipu - K9 thulat
90,3%. NhiOst do n6ng chay 136 - 138°C. IR
(KBr, cm-1): 2941 (vcH2), 1746, 1671 (vc=0), 1598
(vc.c), 1503, 1470, 1387 (vc.c6rm), 1150 (vc,rom„),
1598 (vc_N). ESI-MS (m/z): 313 [Ml. 'H-NMR
( 500MHz, Aceton-d6), 6 (ppm): 7,92 (s, 1H,
-CH=); 7,74-7,70 (m, 2H, H-2', H-6'); 7,40 (d, 2H,
J=1,5Hz, H-2", H-6"); 7,39-7,36 (m, 1H, H-4");
7,35-7,29 (m, 4H, H-3', H-5', H-3", H-5"); 4,91
(s, 2H, -CH2-). "C-NMR (125 MHz, Aceton-d6),
(ppm): 206,19 (C-2); 167,86 (C-4); 166,49
(-CH=); 165,45 (C-1"); 163,45 (C-2'); 136,65 (C4'); 133,48 (C-3'); 132,92 (C-5'); 130,85 (C-2",
C-6"); 129,46 (C-1'); 128,77 (C-6'); 122,31 (C-3";
C-5"); 117,36 (C-4"); 117,14 (C-5); 45,77(-CH2-).
TOng hap chht 4b: Tinh the hinh kim mau trong,

Wet tinh lei trong EtOH, hieu suet 87,5%. Nhiet do
ming chay 155 -157°C. IR (KBr, cm-1): 2953 (vcH2),
1739, 1687 (vc.0), 1576 (vc.c), 1500, 1462, 1376
(vc.0,r0,6), 1200 (vc„6,,c,), 1576 (vc_14 ). ESI-MS (m/z):
364 [M1.1H-NMR ( 500 MHz, Aceton-d6), .5 (ppm):
8,09 (s, 1H, -CH=); 7,71 (d, 1H, J=2Hz, H-5');
7,659 (s, 1H, H-3'); 7,58 (d, 1H, J=2Hz, H-6');
7,42 (d, 2H, J=1,5Hz, H-3", H-5"); 7,38-7,35 (m,
2H, H-2", H-6"); 7,33-7,31 (m, 1H, H=4"); 4,92
(s, 2H, -CH2-). "C-NMR (125 MHz, Aceton-d6), 8
(ppm): 206,16 (C-2); 167,59 (C-4); 166,11 (-CH=);
137,41 (C-1"); 136,80 (C-2'); 136,47 (C-4'); 131,37
(C-3'); 131,03 (C-5'); 130,91 (C-1'); 129,49 (C-2",
C-6"); 129,13 (C-6'); 129,01 (C-3", C-5"); 128,85
(C-4"); 128,27 (C-5); 45,97 (-CH2-).

Ban luan
Ve tong h913 hoa h9c: Phan Crng ngung ty
p-fluorobenzaldehyd va 2,4-diclorobenzaldehyd
vai rhodanin va thiazolidin-2,4-dion chrec thyp
hien trong dung mei acid acetic bang va x0c tac
natri acetat khan, dat hieu suet - 58-70%. Ve thdri
gian phan irng ngung ty: Thai gian phan u'ng
ngung ty cua rhodanin keo dai han (5-6 gia =
gia) so vai thiazolidin-2,4-dion (4-5 gia) do nhOm
methylen (›CH2) trong nhan thiazolidin-2,4-dion
co hydro linh Ong han so vai nguyen VI, hydro
trong nhOm methylen cua rhodanin. Deng thai,
hieu suet phan Ong ngung ty cua cac aldehyd
therm vai thiazolidin-2,4-dion (3a: 71,0%; 4a:

73,0%) cao han so vai rhodanin (1a: 59,0 %; 2a:
68,5%). Phan irng N-alkyl hoa &roc thy,c hien
bang k9 thuet vi song vai hiOu suet kha cao: tir
82,0 - 90,0%. Thai gian phan u'ng del vai cac
46

hop chet rhodanin (2-5 phut) ngen han so vai
cac thiazolidin-2,4-dion (10 phut) do cac den chet
ngung ty cua cac aldehyd tham vai rhodanin de
bi nhy,a h6a trong qua trinh phan irng. Dieu nay
cOng den den hiOu suet cua phan Crng N-alkyl
h6a cac rhodanin (lb: 84,9 %; 2b: 82,0%) the')
han so vai thiazolidin-2,4-dion (3b: 90,3%; 4b:
87,5%).
Ve xac dinh cau trim cua cac chAt tang
hop duvc
Ve phO hOng ngoai (IR): Tam chet tang hop
der duet ghi ph6 hong ngoai. Qua nghien CCM phO
de cua cac chet, chi:mg tai nhan biet duvc cac dai
hep thy d'ec tru•ng cua cac nhOm chin va cac lien
ket cua cac chet tang hop duvc. PhO IR dm cac
chet (la - 4a) CO dai hep thy dm -NH amid dm
nhan rhodanin va thiazolidin-2,4-dion trong vung
3182 - 3100cm-1. PhO IR cua 4 chet (la, 2a, lb,
2b) deu co dai hep thy cua nhOm >C=0 cua nhan
rhodanin trong vung 1734-1699cm-1, dai hep thy
cua nhOm >C=S trong vung tin 1235-1200 cm-1.
Ph6 IR cua 4 san phem N-benzyl h6a cua cac chet
tir 1a - 4a deu xuet hien dao (Ring h6a tri cua nhOm
methylen (-CH2-) trong vung tir 2953 - 2930 cm-1.

Ph6 IR cua 4 chet 3a, 4a va 3b, 4b deu co china
yang thiazolidin-2,4-dion nen deu they xuet hien
dai hep thy dec trong cho dao dOng hoa tri cua
nhOm >C=0 amid va nhOm >0=0 imid trong cac
vung twang irng tir 1754 - 1729 cm-1 va 1708 1671 cm-1. Phe IR cua 8 chet deu co dai hep thy
dec tru'ng cho dao dOng hoa tri dm lien ket trong
vung 1594 - 1500cm-1 va 1467 - 1376cm-1. Ca
8 chet duvc ghi phO hOng ngoai deu cho phep
nhan biet sy' CO met cua lien ket C.rorn-X vai dao
Ong hoa tri dec trong cua lien ket Carom-F trong
vung tir 1185-1150 cm-1 (chet la, 3a, lb, 3b) va
dao Ong hoa tri dec trong cua lien 1(61 C6rom-CI
trong vung till, 1200-1164 cm-1.
Ve ph6 khOi luvng (MS): Tam chet tong hap
duvc de duvc ghi phO khOi luvng. Cac chat du,gc
ghi phe deu c6 pic phan tir co so khoi dung !Deng
khoi luvng phan tin tinh theo cong thirc chet du,
kien va ph0 hap vai guy tec nita.
Ve phO cong hirtfrng ter proton CH-NMR) va
13C (13C-NMR)
Ve ph6 'H-NMR
Ph6 cong hirang tir proton dm 4 chet (la 4a) deu CO tin hieu proton cua nhom -NH vai do
TAP CHI DUVC HOC - 9/2015 (SO 473 NAM 55)


• Nghien

CCPU - K9 thu at

dich chuyen 12,5-14 ppm. Ph6 cua ca 8 chat deu

co tin hieu proton cua nhom -CH= vai do dich
chuyen 7,65 - 8,09 ppm. Ph6 cua 4 chat (lb - 4b)
c6 tin hieu proton dm nh6m -CH2- vai dO dich
chuyen 4,76-4,92 ppm.
I/6 ph6 13C-NMR

Ket qua phan tich ph6 cho they chat CO se
carbon va de) dich chuyen hoe hoc S cua cac vi tri
carbon phi) hop vai tong tht:rc cky kien. Ph6 cua
ca 8 chat deu co tin hieu nhom -CH= vai do dich
chuyen 125,47 - 166,49 ppm. Ph6 cua 4 chat (lb
- 4b) c6 tin hieu nh6m -CH2- vai
dich chuyen
45,77-45,97 ppm. Ph6 DEPT90 va DEPT135 cua
chat la va 4a cho they su' CO mat cua 5 nhOm CH
6, cac vi tri 2', 3', 5', 6' (6' nhan benzen) va nhom CH= (cluvc tao ra phan ung ngu'ng tu). C6n del
vai chat 2a va 4a they su, CO mat cua 4 nhOm CH
a cac vi tri 3', 5', 6' (nhan benzen) va nhOm -CH=
(duvc tao ra tu, phan Ong ngung tu).
73

Ket qua phan tich ph6 IR, MS, 'H- NMR va
C- NMR cho phep xac dinh ceu trim cua cac

chat tong hop duvc dung nhu, du, kien.
Ve tac dyng sinh hoc
Chung toi der tien hanh th0, hoat tinh khang
khuan, khang nem dm 4 chat (1a-4a) va hoat
tinh khang to bao ung thu, cua ca 8 chat (1a-4a
va lb-4b).

Ve hoat tinh khang khuAn, khang nem
- Ket qua th6, hoat tinh khang khuan dm 4
chat (la - 4a) cho they Ca 4 chat deu CO hoat tinh
tren cac chung vi khuan khac nhau vai mup do
khac nhau. Chat 2a, 3a, 4a co hoat tinh khang vi
khuan B. subtilis. ChAt la, 2a, 4a bieu hien hopt
tinh khang vi khuan E. coll. Ngoai ra, chat la con
co hopt tinh khang vi khuan S. aureus.
- Ket qua thu, hoat tinh khang nem dm cac
chat la - 4a cho they ca 4 chat deu CO tac dung
tren chung nem moc Fusarium oxysporum va
chung nem men Candida albicans. TCF ket qua
CO the nhan they tac dung cua chat 2a va 4a tren
chung nem mOc Fusarium oxysporum manh hop
so vai chat la va 3a. Nhu' vay, 2 nhom the do
trong nhan benzen lam tang hoat tinh khang nem
cua cac chat.

4a co hoat tinh khang to bao ung thu, Hep-G2
vai mik de khac nhau. Nhu' vay, 2 nh6m the clo
trong nhan benzen co anh Wang den hoat tinh
khang to bao ung thu' Hep-G2 dm chat 2a va 4a.
- Kat qua thii, hoat tinh khang to bao ung thu,
cua 4 chat (lb - 4b) tren 4 d6ng to bao ung thu'
ngu'ai cho they chat lb co hoat tinh khang to bao
ung thu, tren 3 d6ng: to bao ung thu' ph& A549,
to bao ung thu' t6, cung Hela va to bao ung thu,
buOng tru'ng OVCAR-8. Chat 2b chi co hoat tinh
khang to bao ung thu' bueng tru,ng OVCAR-8.

Hai chat 3b va 4b kh6ng the hien hopt tinh khang
to bao ung thu' tren 4 d6ng to bao kiem dinh.
vay, khi tren nhan therm dm hap phan aldehyd
tham chtira nguyen tu, fluor (6' chat lb) thi hoat
tinh khang to bao ung thu' tot hen so vai khi thay
the nguyen tu, fluoro bai 2 nguyen by clo vi tri
ortho va para (6, chat 2b). Cu the la chat lb tac
dung tren 3 d6ng to bao ung thu', con chat 2b chi
tac dung tren 1 d6ng to bao ung thu'. Han ne,a,
del vai dong to bao OVCAR-8, chat lb cOng the
hien tac dung khang manh hen so vai chat 2b.

Ket IWO
- Da tong hap duvc 8 chat (la - 4a, lb - 4b),
trong dO co 4 chat (lb, 2b, 3b, 4b) chu'a they
cOng b6 trong cac tai lieu tham khao &roc.
- De thu, tac dung khang khuan va xac dinh
nOng de 6,c the toi thieu (MIC) cua 4 chat tong
hop duvc (la - 4a) vai 4 chung vi khuan kiem dinh
gem: Escherichia coli, Pseudomonas aeruginosa,
Bacillus subtillis, Staphylococcus aureus. Ket qua
cho they 3 chat (2a - 4a) ca hoat tinh manh doi vai
chung Bacillus subtillis (vai MIC = 25 ug/m1), 3 chat
(la, 3a, 4a) co tac dung tren chung vi khuan kiem
dinh Escherichia coli (veil MIC Ian krat là 25, 50,
12,5 Kim!), ngoai ra chat la co hopt tinh khang
Staphylococcus aureus (vai MIC = 50 ug/m1). Da
thin tac dung khang nem va xac dinh nOng do Crc
the toi thieu (MIC) dm 4 chat (la - 4a) vai 4 chung
vi nem kiem dinh gem: Aspergillus niger, Fusarium

oxysporum, Saccharomyces cerevisiae, Candida
albicans. Ket qua cho they ca 4 chat deu CO hoat
tinh khang 2 chung vi nem Fusarium oxysporum
(MIC = 25 - 50 ug/m1)va Candida albicans (vai MIC

- Ket qua thi' hoat tinh khang to bao ung thu,
cua 4 chat (la - 4a) cho they chi 2 chat 2a va

= 50 ug/m1). De thin tac dung khang met d6ng to
bao ung thu, gan ngoai (Hep-G2) cua 4 chat (la 4a). Ket qua cho they 2 chat 2a va 4a co hoat tinh
khang to bao ung thu' Hep-G2 vai IC50 In luvt la

TAP CHI DU (7C HQC - 9/2015 (SO 473 NAM 55)

47

IM hog tinh khang to bao ung thu,