Nghien CCFU - Ky thuat
Nghien cuu dieu clie piiirc hop 3-(2-cloropyridin 3-yl)-2-methyl-4(J^-quiiiazoUnon(NL197)
v612-hydroxylpropyl-beta-cycIodextrin
Huynh Ngoc Duy'*, Nguyen Dang Khoa^
Nguyin Ngoc Vinh', Truong Thg Ky^
' Vien Kiem nghiem thuoc TP. Ho Chi Minh 'Dgi hoc YDuac TP. Ho Chi Minh
'E-mail: [email protected]
Summary
HPPCD had increased the water-solubility of NL197 (the complexity rate of ca. 4%). The molar ratio of NL197 to HPPCD was found to be 0.4/1.5, high level of NL197 underthe optimmized conditions: water wetting, evaporating temperature 60 °C, stirring temperature at 60°C, stining time 30 minutes. The method was validated to meet all the requirements for phamnaceutical control analysis.
Keywords: Cyclodextrin, quinazolinon, NL197, solubility.
Dat van de
NL197 CO c i u triic (2-cleropyridin-3-yl)-2- metyl-4(3/-/)-quinazolinen duae nhom nghien ciru cua PGS.TS. Nguyin Ngpe Vinh va GS.TS.
Truang T h i Ky ting hap eo tac dung an thin gay ngu, giam dau, khang khuan, ching ung thu...i'i. Tuy nhien, NL197 rit kho tan va cac dan chit dang muli da duo'c ting hop cung khOng eai thien dp tan trong nuac, dan din rit kho pha ehl dang dung dieh uIng va dung djch tiem diing cho thir nghiem sinh hpc. Do do, nghien ciru huang d i n kha nang tao phirc giira NL197 va 2-hydroxyl-beta-cyelodextrin nhim cai thien dp tan va dp hip thu ciia NL197 va gop phin treng viec nghien ciru sinh kha dung cua heat chit sau nay.
Nguyen lieu va phu'O'ng phap Ddi tu>o>ng nghien cu'u
Hap chit 3-(2-cloropyridin-3-yl)-2-methyl- 4(3/-/)-quinazelinon (NL197) nguyen lieu, ham luang 99,41%, dp i m 0,12% dat theo TCCS PI.
Hydrexylpropyl-p-cyclodextrin ham lupng 96,0%, dp i m 3,1% dat thep tieu chuin NSX Roquette Pharma.
- Xay dung quy trinh tae phirc NL197-HPBCD.
- Xay dung va tham djnh quy trinh djnh luang phirc hap NL197-HPBCD bang HPLC vai diu do PDA.
Dung moi, hoa chit
Ethanol, methylen clorid, amoni format, acid
formic, ameniac, acid sulfuric dat PA; methanol, aeetonitril dat HPLC.
Trang thilt bi
May c6 quay chan khong Yamato RE 801, may khuiy tir gia nhiet IKA C-MAG HS7, may lie Certemat MO, may do pH Mettler S20K Seven Easy, be sieu am Elma Transsenic T700/H, can phan tich 5 so le AB 265S, may sac ky long hieu nang eao Shimadzu-LC-20AD, may phan tieh nhiet DSC-20, may quang phi hong ngoai iS50FT- IR, may de dilm chay MP 90, may de dp hoa tan Erweka, 16 nung Heraeus D6450.
Phu'ong phap nghien ciru '^•''•^' Cac buvv khao sat sir tao phirc
- Tiin hanh vai phuang phap trpn kh6, phuang phap trpn u61 vol nuac, phuang phap trpn up't vol ethanpl hpae vai hin hap ethanol va methylen clorid.
- Khae sat cac diiu kien tao phirc nhu ty le tae phirc, thai gian c6 quay nhiet dp co quay, thai gian khuiy.
- Oanh gia phirc tap thanh bang dp tan, dp hOa tan, phi hing ngoai va quet nhiet vi sai.
Dieu kien sac ky HPLC ™
- Cpt sae ky Phenpmex C18, 150 x 4,6 mm, kieh thuac hat 5 pm.
- Chuin bj dung djch dem pH 4,5: Can 3,1 g ampni format hoa tan trong 1000 ml nuac cit, chinh pH 4,5 bing acid formic heac ameniac.
- Pha dpng: dung djeh dem amoni format pH 4,5 - aeetonitril (60:40).
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Nghien CIFU - Ky thuat
- T i c dp ddng: 1 ml/phiit.
- Deteetpr: UV 265 nm.
- Nhiet dp cpt: 40 °C.
- T h i tich tiem: 20 pi.
Banh gia phuxmg phap
Quy trinh djnh luang d u a c kiem tra tinh t u o n g thieh he t h i n g va tham djnh theo ACEAN bao g i m cae yeu c i u v l tinh dac hieu, tinh t u y i n tinh, dp dung, dp chinh xac trong ngay va khae ngay dp I n djnh ciia san p h i m .
K i t qua va banluan
T h y c nghiem t a o phiJc Phuvng phap frgn kho
Trpn d i u NL197 va HPBCD trong e l l thiiy tinh trpng 30 phiit theo ty le s l moi 1 :1P1.
Phuvng phap tron uxrt voi dung moi ethanol T i i n hanh trpn kh6 NL197 va HPBCD theo ty le s l moi 1:1, sau do them tir tir ethanol vae va nghiin trpn d i u thanh bpt nhao treng 30 phut''', khi nao t h i y h i n hap ran kho lai thi t i l p tue them ethanol.
Sau giai dean nghiin trpn thanh bpt nhao hoan t i t , d l met thai gian ehe dung moi bay hoi thi n g h i i n k h i i c h i t r i n thanh bpt min. Bpt d u a c dem di say a 55 °C, ap s u i t 80 hPa, thai gian 120 phiit.
Bang 1 : Kit qua tao phire eiia
Phuong phap frgn uxrt vai nuvrc
Hoa tan HPBCD trong mpt lup'ng t i l thieu nuoc cat b i n g may k h u i y tir gia nhiet. D u n nong d i n 50 "C. Them met lup'ng NL197 theo ty le s l moi 1:1 vao dung djeh tren, v i n duy tri nhiet dp 50 °CW.
K h u i y trong 30 phut, Ipc qua mang Ipc 0,45 pm, bo tiia, thu l i y djch dem bay hai n u a c va s i y kho san pham tren may co quay 6' 55 °C, 80 hPa, thoi gian 120 phut
Phuvng phap dong bay hoi ethanol va methylen clorid
Hoa tan HPBCD trong ethanol o' 50 °C, NL197 trong methylen clorid. Them tir tir dung djch NL197 thee ty le s l moi 1:1 vae dung djch HPBCD. Dupc dung djeh trong, khong eo tiia. K h u i y 30 phut.
D e m CO quay a 55 °C, 120 hPa, 100 v/p. San phim thu duo'c sau do d u a c n g h i i n mjn, eho mpt luang n u o c vira dii de hoa tan, Ipc qua mang Ipc 0.45 pm, bo tua, thu l i y djch dem bay hoi n u a c va s i y kh6 san pham tren may c6 quay a 55 °C, 80 hPa, thai gian 120 phiit.
Trong cae p h u a n g phap d l l u c h i da khao sat, chpn p h u a n g phap trpn u a t v o l n u o c vi co ty le tao phirc cao n h i t (bang 1) va san p h i m tan hoan toan, khOng sir dung dung m6l hCVu c a . Danh gia k i t qua b i n g ANOVA.
cac phuang phap khac nhau
Phuvngphap Phuxyng phap tron Phuvng phap tron Phuvng phap dong bay hoi v&i tron kho uvi v&i ethanol u^v&inum: ethanol va methylen clorid Phan tram ^NL197 trong
phifc chit (%) 1% 1,36% 3,54% 3,52%
Khao sat ty le tao phirc giO'a NL197 va HPBCD
Bang 2: Ket qua ty le tao phiec Nil97 v&i HPBCD
Ham iLfcmg Lan 1 NL197 trong , phii'c(%) ' - ^ " ^ Hieu suat ^^ phirc chit thu dugc (%)
0,2.-1,5 2,26 2,31 74,2%
Tv le moi NL197/HPBCD 0,4:1,5 0,8:1,5 1:1,5
3,67 3,57 3,62 3,58 3,63 3,50 78,2% 68,8% 64,3%
1,5:1,5 3,54 3,61 69,7%
Chpn ty le NL197: HPBCD la 0,4:1,5 la ty le d l bao ehl che phim phirc NL197 tren.
Khao sat nhiet do co quay, nhiet do khuay, thoi gian khuay tao phirc NL197 v&i HPBCD (phuKmg phap fron ir&t voi nu&c)
- Khae sat khoang nhiet dp cc quay (tir 50 d i n 80 °C), nhiet dp khuiy (tir 30 d i n 80 °C), thai gian khuiy (tir 15 phut din 60 phut) va lam thee quy trlnh che thiy: nhiet dp e6 quay va nhiet dp khuiy 6' 60 °C, thai gian khuiy 30 phiit la phu hpp nhit.
Tom tit quy trinh tao phirc NL197-HPbCD (phucmg phap tron u&t vai nteac)
Nghiin mjn NL197, hoa tan HPpCD trong mot luang t i l thilu nuac c i t bing may khuiy tir gia nhiet. Dun nong 60 °C, them met luang NL197 thee ty le moi NL197/HPBCD la 0,4:1,5 vae dung djch tren, khuiy trong 30 phut, duy tri nhiet dp 60
°C. Lpe qua mang lpc 0,45 pm, bo tua, thu liy djch dem bay hoi nuac tren may e6 quay 6' 60 °C, 80 hPa trong 120 phut San phim duae nghiin mjn, bao quan trong binh hiit i m .
Danh gia phiFC tao thanh
Xac dJnh do hoa tan cua NL197 nguyen lieu va ciia phirc tao thanh
T h i l t bj k i l u eanh khuiy, t i c dp k h u i y 50 vpng/
phut, nhiet dp: 37 + 0,5 °C, m6i truang hoa tan la n u a c c i t Can khoang 50 mg NL197 hoac luang phirc NL197 t u o n g irng vol khoang 50 mg NL197 d u a e rac d i u tren b l mat moi truang. Rut 10 ml
26 TAP CHI DirOC HOC - 4/2016 (SO 480 NAM 56)
Nghien CIFU - Ky thuat
m i u tai cac tho'i diem 3, 5, 10, 15, 20, 30, 40, 50 phut. Ojnh lup'ng b i n g HPLC.
Do hoa tan
Z ^
1—•— r^uyen lieu M.197- • — RiLTC M.197
Hinh 1 : Oo thi biiu diin do hda tan eiia NL197 nguyen lieu va phirc NL197
0 6 hoa tan cua phirc NL197 nhanh va cao hon n h i l u so vai dp hpa tan eiia N i l 97 nguyen lieu tai cac thai d i l m t u o n g irng. Phirc NL197 tai thai diem 3 phut h i u n h u da hoa tan hoan toan.
Pho hong ngoai
Se sanh p h i IR cua phirc tao thanh vai p h i IR eua NL197 va HPpCD eho t h i y co s u djch ehuyen cue dai ciia viing h i p thu ciia nhom - O H v l 3340,1 c m ^ Nhieu dinh dac trung cua NL197 trong viing d i u van tay 1300 - 910 cm-^ h i u h i t da bj bien m i t , dinh tai 1336,2 c m ' eua HP|3CD eung b i i n m i t . Chirng to co s u t u a n g tac giO'a NL197 va HPBCD (hinh 2)
h'iii7iun>i»H unn-mon
-\ .^.,.
Nhiet vi sai
Hinh 3: Biiu dd nhiet eiia NL197 ehuan
Hinh 4: Biiu do nhiet eiia phirc chit P h i phan tich nhiet cua phirc NL197/HPBCD khOng t h i y s u x u i t hien dinh npi nhiet ciia NL197 tai nhiet dp kheang 178,6 °C, s u bien m i t nay chirng to eo s u tao phirc, eae phan tir NL197 da n i m hoan toan trong khung ciia HPpCD.
T h a m d j n h q u y t r i n h d m h l u ' o n g NL197 t r o n g phiFC tao t h a n h
Hinh 2: Phd IR cua phirc NL197-HPBCD
Bang 3; Bang tdm tit kit qua thim dinh phuang phap dinh lugng Tinh tu<mg thi'ch he thong
(RSD < 2,0%) ' Tinh dac hieu Tinh tuyen tinh Gioi han djnh lumg Do chlnh xac ngay 1 Do chinh xac ngay 2 Do chinh xac ngay 3 Do dung
(tLr5-50iig/ml) (LOQ) (RSD < 2,0 %) (RSD < 2,0%) (RSD < 2,0%) (100 + 2 %)
Thoi gian lu-u Dien tich pic Ket qua 0,075% 1,518% Dat
Phu'O'ng phap co tinh dac hieu Phu'ong trinh hoi quy: y = 44235x + 20691
R2 = 0,9999 1,9ng/ml
1,07% Dat 0,43 % Dat 0,34 % Dat 100,69% Oat Do on djnh
Bao quan
dd 0,31% trong nu'oc, dong ong tiem 2 ml, sau khi pha che Dd trong, khong mau
010 °C/sau 6 thang Dd trong, khong mau 100,58%
C„H„0N3C1 100,33%
C„H.„ON,CI Oat
Dat
TAP CHi DirOfC HOC - 4/2016 (SO 480 NAM 56) 27
Nghien CLFU - Ky thuat
Ket qua kiem nghiem phiic NL197-HPbCD theo tieu chuan de nghj Bang 4: Kit qua kiim nghiem chi phim phirc NL197-HPpCD
Chi tieu M(K chat luang Ket qua .,.. , ,.,, Chit bot min, mau trang, d i hut am, kich img nhe. D i tan trong nu'oc, kho tan trong ethanol va
Tmh chat „,^^^,„{^„ ,:\M 3 . 3 . a £, methylen clond.
-A. Pho hing ngoai ciia che pham phai co dinh dactnrng a 3340 c m ' , 2926 c m ' , 1079 c m ' , 1021 cm-'.
_ . , , , . - B. Sac ky long hieu nang cao: Thoi gian lu'u tren sac ky do che pham trung vol thoi qian lu'u
Dmh tmh , . 4 . , , i , i ' ; ° '^ " " Dunq
tren sac ky do mau chuan » - C. Sac ky lop mong: Cac vet tren sac ky do cua dung djch thif cd vj tri, mau sac tu'ong ty cac
vet tren sac ky d6 ciia dung djch doi chieu.
Dung
Dung 06 trong va mau sac Dung dich S phai trong va khong mau
Tro sulfat Khong dugc qua 1 % IVlat khoi lu'ong do lam kho Khong qua 5 %
DjnhlTOng 3,8-4,2%C„H,„ON,CI
Dat Oat (0,12%) Oat (1,56%) Dat (41%) Ket luan cac dSn chit mo'i cua d| vong 4(3H)-Quinazolinon co
„ - . . , * - u . l . - K l l . . r ^ , tac dung sinh hpc",/.uan an r/Sn s;" Duoe hoc, Dal hoc Da xay dung d u a c quy tnnh tao phuc NL197- y Duac TR H6 Chi Minh • • ' • • HPf5CD tan hoan toan trong n u a c vol cac dieu 3. E. M. Matrin Del Valle (2003), "Cyclodextrins and kien thee phuang phap trpn u a t vai nuoc, ty le tao (heir use: a review", Process biochemistry, p. 1-15 phue la 0,4:1,5; nhiet dp c6 quay 60 °C; nhiet dp 3 p^^.g ^^^ t r u y i n , Le Huu Phuc, Dang Van khuay 60 °C; thai gian khuay^30 phut Oa xay dung Tjnh, Huynh Van Hoa (2011), "Bao o h i vien nen chira d u p c tieu ehuan eo s o va t h i m djnh quy trinh djnh phirc rutin va 2-hydroxylpropyl-p-cyclodextrin cd do hda luong NL197 trong phirc. Viec tae phirc da cai tan oao". Tap chi Yhoc TP. Hd Chi Minh, 15(1), 106.
thien duoe dp tan ciia NL197 g i p 101,5 l i n so vai 4. Tarong Ngoc Tuyin, Nguyen Ly HuyIn (2011), nguyen lieu NL197. "Nghien ciru kha nang tao phirc cua risperidon va T- • I - 11. . . . . P-cyclodextrin", Tap chi Yhoc TP Hd Chi Minh, 15(1),
Tai lieu tham khao ^51 ^'
1. Nguyin Ngoc Vinh (2006), "Nghien ciru tong ha'p 5. U.S.P 35/N.F.29, Hydroxylpropyl Betadex.(Ngay nhan bai: 12/01/2016 - Ngay duyet dang: 04/04/2016)
Phat Men gen ma hoa hop chdt polyketid moi tir loai bot bien Theonella swinhoei
Nguyen Tii Anh Khoa Duac - Dgi hoc YDuac TP. Ho Chi Minh E-mail: [email protected]
Summary
The onn gene cluster coding onnamide was isolated from the Theonella swinhoei Also, the other PKS gene cluster was discovered Particulariy this PKS showed responsible for biosynthesis of a new unchracterized l^olyketide. The complete sequence comprised ~ 54.8 kb, including ~ 45.5 kb of the PKS genes. The position of ZnlnnH^ ^"f'l ^^l P'^"'"''"'' °^^^^ f>olyketide stmcture only by their KS sequences without requinng the sequences of other PKS domains.
Keywords: Theonella swinhoei, metagenomic DNA, polyketide, polyketide synthase, ketosynthase.
D a t v i n d e
dung treng dieu tri cac benh ly n h u n h i i m khuin, Hpp chat polyketid - nenribosemal peptid (PK n h i l m virus hay ire c h i s u phat t r i i n eua cac kh6i - NRP) CO nhieu heat tinh sinh hpc, eO t h i u n g u,... m. Trpng t u nhien, sinh t i n g h g p PK - NRP
