Nghien CLFU - Ky thuat
•7 ^ A
Nghien ciru tong hop thuoc dieu tri ung thu*
altretamin su* dung ky thuat vi song
Dinh Thj Thanh Hai, Le Dinh Manh Bo mon Hod Hiru Ca, Trudng dgi hoc Duac Hd Noi
Dat van de
Altretamin (Hexamethylmelamin) Id thuoc chong ung thy dd dygc sy dgng trong y hpe de diiu trj ung thy phii, ung thu buong triing, ung thy vu. Id cdc loai ung thy pho biin nhit Loai thuoc ndy hidn dang dugc su dgng trong dieu trj ung thu vdi cdc biet dygc nhy hexastat (Phdp), hexinawas (Tdy Ban Nha), altretamin (My), hexalen (US Bioseienee - My) v.v... '^"^' ^. Quy trinh tong hgp altretamin dd dygc cdng bo bd'i Donal W. Kaiser vd cdng sy ndm 1951 '^l Trong bdo cdo tryde ehung tdi dd cong bo kit qiia nghien cuu eai tiin quy trinh tong hgp altretamin eiia Donal W. Kaiser phii hgp vdi diiu kien thyc tiin Viet Nam, trong de tdi ndy vdi mong muon tiip tgc nghien cd'u eai tiin quy trinh tong hgp altretamin, tiin tdi c6 the ty san xuat nguon nguyen lieu hod dygc ddp iing cho nhu cau san xuit thuoc dieu trj trong nude theo tinh thin eua chuang trinh phdt then cdng nghd Hod Dygc Viet Nam din ndm 2010 do Thu tud-ng Chinh phu phd duyet '^'. Chung tdi dd tien hdnh nghidn ciiu tong hgp altretamin sii dung ky thudt vi sdng, m6t ky thuat dygc su dgng khd rpng rdi trong tong hgp huu ea hien nay vi tinh lyng dgng cao do ky thuat tiin hdnh dan gian, rut ngdn ddng k i thdi gian tien hdnh phan u'ng, it phan O'ng phu vd hieu suit phan yng tong hgp eao '^'.
Trong qud trinh nghien eiiu ehung tdi dd tien hdnh khao sdt cdc yiu to anh hud'ng din hidu suit ciia phan Ccng nhy thdi gian chieu xa, ty Id mol giiia cdc hgp phin tham gia phan iing nhy ty Id: eyanurie elorid/NaOH; ty Id mol giiya eyanuric elorid/dimethylamin. So sdnh kit qua khao sdt gilJa phuang phdp truyen thong vd phuang phdp sy dung ky thuat vi song. San pham altretamin tong hgp dugc se dugc xdc djnh ciu tnic dya trdn eae phyang phdp phdn tieh pho IR, MS vd
^H-NMR, ^^C-NMR vd ddnh gid tieu chuan theo tieu chuan dugc dien My USP30 '^°'.
Ddi tu'O'ng va Phu'O'ng phap nghien
CLFU
Nguyen lieu, hoa chit
Cyanuric clorid, dimethylamin, aceton, con tuyet doi, natri hydroxyd khan, natri earbonat, ethyl acetat, natri clorid khan, altretamin (hexamethylmelamin) chuan.
Thilt bj
- Thiet bj tien hdnh phan yng tong hgp: Binh ba CO dung tfch 250ml, sinh hdn hoi luu, nhiet ke cc mdi, mdy khuay ty gia nhidt, binh cau ddy trdn dung tieh 250ml, mdy cat quay, binh gan cd niit mdi dung tich 500ml, phiu Ipe hiit chdn khdng Buehner, tii say Memmert, tii siy hiit chdn khdng Shellab, ede ong nghiem, pipet djnh md'c, pipet Pasteur, cdn dien ty Shimadzu EL - 300.
- Ld vi sdng SHARP microwave oven model R-218 L (s), 220V, 216H, dung tieh 275cm^
- Sde ky ldp mong (SKLM) dygc tien hdnh trdn ban mong silieagel Kieselgel 60 F254 (Merck).
- Nhiet d6 ndng chay do trdn mdy Electrothermal Digital tai Bp m6n Hpd HOu ca - Trudng dai hpc Dygc Hd N6i.
- Pho tCf ngoai (UV) dygc ghi tren mdy Cary IE UV - Visible Spectrophptometer Varian tai Ph6ng thi nghiem trung tdm, Trydng dai hpc Duge Hd Npi.
- Pho hing ngoai (IR) dygc ghi tren mdy Perkin - Elmer, tai Phdng thi nghidm trung tdm, Trydng dai hpc Dygc Hd Npi.
- Phe khii lygng (MS) ghi tren mdy AutpSpec Premier tai Ph6ng thi nghidm Hda vdt lidu, khpa H6a hpc, Try6'ng dai hpe Khea hpc ty nhien, dai hpc quoc gia Ha Npi.
Pho epng huang tu hat nhan (^H-NWIR vd
C-NMR) ghi tren mdy Bruker - AV500 taiphdng Phdn tich cau triic - Vien Hod hpc (Vidn Khoa hpc vd cdng nghe Viet Nam).
13,
34
TAP CHi DUQC HQC - 6/2010 (SO 410 NAM 50)Nghien CLFU - Ky thuat
Phu'O'ng phap nghien cii'u
- Ap dgng phuang phdp thyc nghidm trong hda hpe hiyg ea vd ky thudt vi sdng de tong hgp san phim dy kiin.
- Theo d5i tiin trinh phan iing tong hgp bdng SKLM.
- Sa b6 kiem tra d6 tinh khiit ciia ede san phim bdng SKLM vd do nhiet dp ndng chay.
- Xdc dinh ciu tnic eiia altretamin tong hgp dugc dya trdn kit qua phdn tich pho ty ngoai (UV), pho hong ngoai (IR) vd pho khoi lygng (MS), pho cdng hudng tu' hat nhdn (^H - NMR vd " C - NMR).
- Kiim nghiem kiem nghiem altretamin tong hgp dugc theo tieu chuin eiia Dygc diin My USP30 (2007) '^°'.
K§t qua t h y c nghiem
K i t qua tdng hap hoa hoc
Quy trinh tong hgp hexamethylmelamin duge Donald W. Kaiser vd cpng sy e6ng bo lin diu tien nam 1951'°' di tu nguyen lieu diu Id eyanuric clorid vd dimethylamin vdi xiie tde Id natri hydroxyd khan trong dung mdi aceton. Quy trinh nay cd met so nhugc diem sau ddy:
Sa do phan u'ng:
Ci - N .
Y Y
Cl
Cl
-I-
3(CH3)2NH- NaOHthdi gian phan iing kdo ddi, viee su dgng dung m6i methanol kit tinh lai vda dpc, vua khdng kinh t i so vdi viec su dung ethanol. Hieu suit eiia phan ung tong hgp thip, ehi dat 37%).
Do vdy, nhu da trinh bay trong bao eao trudc, chiing toi dd eai tiin quy trinh tong hgp altretamin nhu sau: Tien hdnh phan yng khdng qua giai dean cit quay loai dung mdi aceton vd nude. Vi qua kit qua khao sat thyc nghiem eho thiy vide cit loai dung mdi phan ung vd loai hodn todn nude ra khdi hin hgp phan yng khdng anh hydng nhieu tdi hieu suit phan u'ng.
Xy ly hon hgp sau phan yng bdng each diing dung mdi hlyu ea chiit san phim th6, nia san phim tho vdi dung djeh natri clorid bao hda, lam khan bdng Na2S04, lpc, c i t quay loai dung mdi, thay thi dung mdi kit tinh MeOH bdng ede dung mdi thich hgp khde it ddc vd dat hidu suit kit tinh cao. Thdi gian phan yng Id 2h, hieu suit dat 70%.
Cdn cii vdo cdc kit qua dd cdng bo tren ddy, nhdm tiip tgc eai tiin quy trinh tong hgp altretamin ung dgng ky thudt vi sdng trong cdng trinh ndy ehung t6i da tiin hdnh nghien cdu tong hgp altretamin bdng ky thudt vi sdng.
H^C^ ^Ctlj N
Aceton, MW
H i C
-f 3 NaCl + 3 H 2 O
N CHl I
N N CH, I
,CH,
Trong qud trinh nghien cdu ehiing tdi da tien hdnh khao sdt cae yiu to anh hud'ng din hidu suit ciia phan iing nhy thdi gian chiiu xa, ty Id mol giiia: eyanuric clorid/NaOH vd ty Id mol giOa eyanuric elorid/dimethylamin.
Kit qua khao sdt nhy sau:
Khao sat hieu suit phan irng thay doi theo thai gian chieu xa
Cdn cie vdo quy trinh tong hgp altretamin dd eai tiin theo tdi lieu tham khao so '^' trong nghien cd'u ndy ehiing tdi SCP dung ty Id mol cdc chit tham gia phan yng cyanuric clorid/NaOH
/dimethylamin Id 1: 3,3 : 3. Khao sdt 7 moc thdi gian chiiu xa vi sdng khae nhau Id 5, 10, 15, 20, 30, 45, 60 phiiL (Sy dgng chi dp chiiu xa Medium Low, 6' ehe dp nay nhiet dp hdn hgp phan ling Id khpang 55°C phii hgp vdi nhiet dO phan U'ng tong hgp theo phyang phdp truyin thong md ehiing tdi da khao sat vd eong bo trong th6ng bdo tryde). Mii moc thdi gian chiiu xa tiin hdnh 5 thi nghidm liy kit qua trung binh.
Kit qua khao sdt anh hudng thdi gian chiiu xa den hidu suit cua phan iing tong hgp thu dygc ghi d bang 1.
Bgng 1: Hidu suit phan ung khi thay ddi theo th&i gian chiiu xa
Thai gian (phiit) 5 10 15 20Hieu suat (%) 57,5 69,7 82,6 82,6 30
82,5
45
82,7
60 82,8
TAP CHi DUQC H Q C - 6 / 2 0 1 0 (SO 410 NAM 50) 35
Nghien CLFU - Ky thuat
K i t qua khao sdt trinh bay a bang 1 cho thay thdi gian c h i i u xa phii hgp Id 15 phiit vdi hieu s u i t phan iing dat 82,6%, khi tdng thdi gian chieu xa len thi hieu s u i t cCia phan u'ng tong hgp cung khdng thay doi ddng ke.
Khao sat hieu suit phan irng thay doi theo ty ie mol cyanuric clorid/NaOH
Cdn cu vdo thdi gian chieu xa vi sdng phii hgp da xdc djnh dugc a trdn Id 15 phut, chiing toi tiip tgc khao sdt ty Id mol tham gia phan iing giiia cyanuric clorid/NaOH /dimethylamin. Vdi ty Id mol
ede chat tham gia phan yng thay doi khde nhau, vdi mci ty Id tiin hdnh 3 thi nghidm lay k i t qua tmng binh. Ty Id mpi giiya eyanurie cIcrid/NaOH thay doi nhy sau: 1: 3, 1 : 3,3, 1: 3,6, 1: 3,9 (trong qud trinh khao sdt ty Id mol giiia eyanuric clorid/NaOH thi co djnh ty Id eyanurie/dimethylamin). Tyang ty, ty Id mol giiJ'a cyanuric elorid/dimethylamin thay doi nhy sau:
1 : 3 , 1 : 3 , 3 , 1 : 3 , 6 , 1 : 3,9 (trong qud trinh khao sdt ty le mol giOa cyanuric elorid/dimethylamin thi co djnh ty Id cyanuric clorid/NaOH). K i t qua thu dugc ghi d bdng 2.
Bang 2:
DKKS Tyle Hieu suat
Hieu suit phdi n irng khi thay ddi theo ty Id mol chit tham Ty Ig mol: cyanuric clorid/NaOH
1 :3,0 82,6
1 : 3,3 82,4
1 : 3,6 82,7
1 :3,9 82,6
gia phdn dng
Ty Ig mol: cyanuric elorid/dimethylamin 1 :3,0
80,4
1 :3,3 82,6
1 :3,6 82,7
1 :3,9 82,5 K i t qua khao sdt anh hud'ng cda ty Id mol
ede hgp phan tham gia phan ung d i n hieu s u i t eiia phan yng d y g c trinh bdy a bang 2 cho t h i y ty Id mol giCra eac hgp phan tham gia phan u'ng phu hgp Id: eyanuric clorid/NaOH/dimethylamine Id 1:3,0: 3,3
ThiKC nghiem tdng hg-p altretamin
Cho 5,52 g cyanuric clorid (0,03 mol) vdo binh c i u dung tich 250 ml, them 40 ml aceton vao binh roi khuiy eho tan. Them tu' tu' 20,0 ml dimethylamin 33%) (0,099 mol) vdo binh, h i n hgp phan iing toa nhidt manh. Tiep theo, cho 1,8 g (0,09 mol) natri hydroxyd, ddy binh phan ling bdng n i p nhya. Ddt binh phan y n g vdo Id vi sdng, s y dgng c h i dp c h i i u xa vi sdng c' t i n s6 216 H, trpng thdi gian Id 15 phiiL Sau dd t i i n hanh c i t quay loai dung mdi.
Hda tan cdn thd trong 600ml h i n hgp dung m6i ethyl acetat: nude c i t , ti Id 1 : 1. Diing binh
gan dung tich 1000 ml chiet lay Id'p ethyl acetat (ldp trdn). R y a ldp h y u c a vdi 120 ml dung djeh natri clorid bdo h6a de loai hd't nyde, chiet lay ldp ethyl acetat. Ldm khan djeh chiit bing Na2S04 khan, Ipe l i y djeh. Cat quay leai dung mdi thu d y g c khpang 5,6 g tiia thp. K i t tinh lai trpng cen tuyet d i i , Ipc hiit thu d y g c tCia. Dem say khe a nhidt dp 70°C thu d u g c 5,2g san pham k i t tinh hinh kim, mdu trdng. Hidu suit dat 82,6%. Nhiet do ndng chdy 172,8 - 173,5°C.
Xac dinh cdu true cua altretamin tong hap du'gc
De k h i n g dinh cau triic cua altretamin tong hgp d y g c chung toi dd tien hdnh phdn tieh pho UV, IR, MS, • ' H - N M R , " C - N M R ciia altretannin tcng hgp d y g c sc sdnh vd'i k i t qua phdn tich
pho UV, IR, MS, • ' H - N M R , ^ X - N M R cua
altretamin chuan.
Bang 3: Kit qua phdn tich phd UV, IR, MS, H-NMR, ^^C-NMR
So lieu phan tich pho Hexamethylmelamin chuan Hexamethylmelamin tong hgp dirgfc
i ^ ^ i l .
226 226IR (KBr), Vmax cm
6s max c m "
2858 (Carom-N-CHs); 2778 (C-H/CH3- N), 1528 (C = N); 1211 (C - N); 1385 (CH3)
2866 (Carom-N-CHs); 2780 (C-H/CH3-N), 1527 (C = N); 1222 (C - N); 1386 (CH3) El MS (m/z) 210(M*); 195[M*-CH3]; 167 [M*-
N(CH3)2]: 152 [195 - N(CH3)2]; 96[167 • [NH=C-N(CH3)2]; 81 [152- NH-C- N(CH3)2]
210(M*); 195[M*-CH3]; 167 [M*- N(CH3)2]; 152 [195 - N(CH3)2]; 96[167 - [NH=C-N(CH3)2]; 81 [152- NH= C-N(CH3)2]
^H-NMR(ppm) 3,08(3, 18H,6-CH3) 3,09 (s, 18H,6-CH3)
^^C-NIVIR(ppm) 165,66 (s, 3C, Ctriazin), 35,65 (s, 6C, 6- CH3)
165,66 (s, 3C, Criazin), 35,65 (s, 6C, 6-CH3) T y k i t qua phdn tich pho UV, IR, MS, ' ' H - H M M
NMR, "C-NMR eiia altretamin tong hgp d y g c diing va altretamin chuan che phep chiing tci k i t ludn
ting hgp dygc tinh khiet vd cd cau tnic nhy dy kien.
36 TAP CHI DUQC HQC - 6/2010 (SO 410 NAM 50)
• Nghien CLFU - Ky thuat
K i t qua kilm nghiem altretamin theo tieu chudn du'gc diin My USP 30
Sau khi thy dp tinh khiit vd xde djnh c i u true ciia HMM tong hgp d y g c chiing tdi dd tien hdnh kiem nghiem HMM tong hgp d y g c theo tidu chuin ciia Dygc dien My USP30 (2007) ^^°\ tai phdng Hod ly - Vidn kiem nghidm thuoc trung yang - Bp Y t i .
Chit chuin: Altretamin (hexamethylmelamin) CLJa hang Aldrieh (USA)
Kit qua kiim nghem: K i t qua kiem nghiem tai Vien kiem nghidm thuoc trung yang - B6 Y Te theo phiiu kiem nghiem 40G174, ngdy 13/1/2010 cho thiy altretamin tong hgp dygc dat yeu c i u ehi tieu theo Dygc dien My USP 30 (2007).
Ban luan
Lfng dgng ky thudt vi sdng trong phan u'ng tong hgp altretamin dd giiip giam thdi gian phan iing tong hgp tu' 2h theo phyang phdp tong hgp thdng thydng xuong edn 15 phiiL Ky thudt thao tde phan U'ng sy dgng ky thudt vi sdng cung dan gian han so vdi phuang phdp thdng thudng: t i t ea ede hgp phin tham gia phan ung dugc eho vdo binh phan U'ng eung luc, kit thuc phan u'ng sau 15 phut; n i u su dgng ky thudt thdng thudng trong qud phai thdm lygng NaOH Idm xue tde thdnh 2 lan m i i lan cdch nhau Ih. K i t qua nghien cd'u cung eho thiy khi tien hdnh phan u'ng tong hgp vdi ciing ty Id mol cdc chit tham gia phan iing, chieu xa vi sdng 6' thdi gian 15 phiit thi hidu suit ciia phan iing tong hgp tdng ldn rd ret la 82,6% (so vdi hieu suit 70%) ciia phuang phdp tong hgp thdng thydng). Trong thdi gian tdi, de nghien ciiu sdu han ed t h i tiin hdnh nghien cdu phan yng tong hgp altretamin bdng phyang phdp tong hgp pha r i n sy dgng xiic tde zeolit vd U'ng dung ky thudt vi sdng de eai tiin phyang phdp tong hgp altretamin vdi hidu suit eao vd gid thdnh ha nhdm hudng tdi san xuat phue vu nhu eau dieu trj trong nyde.
K§t luan
- Od nghidn ciiu cai tiin quy trinh tong hgp altretamin (hexamethylmelamin) u'ng dgng ky thudt vi sdng vdi thdi gian phan u'ng nit ngdn c6n Id 15 phiit, dd xde djnh dugc ty Id mol chit phan iing phii hgp Id: cyanuric clorid /NaOH/dimethylamin Id 1:
3,0 : 3,3 vd tong hgp dugc altretamin dat tieu chuan dugc dung theo Dygc dien My 2007 (USP 30) vdi hidu suit Id 82,6%.
- Dd xdc dinh cag triie cCia altretamin tong hgp dygc bing phan tich pho t y ngoai (UV),
pho hong ngoai (IR), pho khoi lygng (MS), pho e6ng hydng liy proton ^H (''H - NMR) vd pho c6ng hydng t d hat nhdn " c (^'C - NMR).
S u m m a r y
Altretamine (hexamethylmelamine), known as a potent antitumour compound used in the treatment of lung, breast, ovarian cancer, was synthesized by the method by Donal W. Kaiser et al. (1951), with our some modification.
Following our previous report on the improvement of Donal W. Kaiser's procedure to obtain altretamin in the yield of 73%, this report described a new synthesis method for altretamin using microwave technique to symplify, accelerate the reaction and reduce the reaction time. The structure of the obtained altretamin was confirmed by IR, ^H-NMR, "C-/\//WR and MS. The quality of synthesized altretamin was controlled showing to meet the requirements of USP 30
Keyword: Altretamine, hexamethylmelamine, synthesis, microwave technology.
Tai lieu tham khao
1. Dd Trung Dam, Thuoc chu'a ung thu, NXB Y hgc Ha Ngi. (1995), trang 137 - 138.
2. Nguyin Ba Diic, Cham soc va dieu trj trieu chiing cho benh nhan ung thu', NXB Y hgc. Ha Ngi.
(2003), trang 5.
3. Nguyen Ba Diic, Hda chat dieu trj ung thu', NXB Yhge, Ha Ngi. (2003), trang 130 -137.
4. Trinh Vdn Quang, Bach khoa thu' ung thu- hpc, NXB Yhge, Ha Ngi. (2002), trang 69 - 70.
5. Hoang Thu Trang, Nghien ciiu tong hgp thuoc dieu tri ung thu hexamethylmelamin, khoa lu$n tot nghigp Duge sT d^i hge. khoa 58 - DH Du-gc Ha Npi, (2008).
6. Thii tu'dng chinh phii, Chu'ccng trinh nghien ciiu khoa hpc cdng nghp trpng diem quoc gia phat trien cdng nghiep Hda du'gc den nam 2020, Van phong ehinh phu. Ha Ngi. (2007).
7. Goodman and Gilman's, The Pharmaeologycal basis of therapeutics, ^0'*^ ed, Mc Grow Hill Inc. New York, London, Seoul, Tokyo, Singapore, Sydney, .Toronto, (2001), Vol II, chapter 52.
8. Donald W.Kaiser et al., Cyanuric cloride derivatives, Joumal of American Chemistry Society.
(1951),Vol.73, pp. 2984-2986.
9. Laura Parrettes, Fundamentals of Microwave Organic Synthesis, Miletones Inc., Shelton, USA.
10. USP 30, The United States Pharmacopeia, The United States Phanvacopeial convention. Inc., (2007),volumll, p.1330.
TAP CHi DUQC HQC - 6/2010 (SO 410 NAM 50) 37