Nghien CLFU - Ky thuat
"^Nghien ciru xay dirng quy trinh san xuat thuoc dieu trj ung thu altretamin
Phan Dinh Chau', Vu Binh Duong^', Nguyin Thj Thu Hang^ Bui Thi Thu Ha^
' Truang Dai hoc Bdch khoa Hd Noi
-Hoc vien Quan Y, Bo Quoc phong ' E-mail: [email protected]Summary
To synthesize altretamine (hexamethylmelamine), a potent antitumour agent mainly for treatment of lung, breast, ovarian cancers, which was previously synthesized by D. W. Kaiser et al (1951) with the yield of 37%, and later, by improving this method, by D.T.T. Hai (2010) raising the yield to 70%, In this work, by further improvement of both D. W. Kaiser's and D. T. T.Hai's procedure, the yield was raised to 89.40%. The structure of the obtained hexamethylmelamine was confirmed by IR, MS, ^H-NMR and
"C-NMR. The quality control showed the product to meet the requirements by USP-34.
Keywords: Altretamine, synthesis, antitumour
Cat van de
Altretamin hay hexamethylmelamin cd ten khoa hpc 2,4,6-tris(dimethylamino)-s-triazin hay N,N,N',N',N",N"-hexamethyl[1,3,5]tria2in-2,4,6- tnamin la c h i t khang ung thu d u a c s u dung trong dieu trj cac loai ung t h u nhu: ung t h u phoi, ung thu vu, ung thu budng trung (la cac loai ung t h u pho b i l n ) , dac biet d u g c dung trong d i l u tri ung thu budng trung tien trien sau khi phuang phap tri lieu dau tien that bai ''""i Altretamin hien dang d u g c s u dyng trong d i l u tri vdi cac biet d u a c Hexalen (U.S. Bioscience), Hexastat (Roger Bellon), Hexamethylmelamin (Rhone-Poulenc)...
la thudc d u a c coi la it dpc han so vdi cac thudc hda tri lieu khac s u dung de d i l u tn ung thu va la thuoc cd cdng thuc dan gian, qua trinh dieu c h i it giai doan, tinh kha thi trong san x u l t la kha cao.
0 nude ta nhu c l u v l thudc d i l u trj ung t h u la kha Idn va cung nhu cac thudc ung thu khac d i u phai nhap ngoai vdi gia thanh qua dat, chi phi cho d i l u trj qua cao. Chinh vi t h i chung toi dat vdn de nghien cuu xay dung quy trinh san xuat altretamin dat tieu chuan d u a c dung, thich hop vdi dieu kien san xuat cua Viet Nam. nhdm t i l n tdi t u san xuat trong nude cung cdp cho dieu tn vdi gia thanh re han thudc nhap ngoai.
Doi tu'O'ng va phiFO'ng phap nghien CLFU
Doi t u a n g nghien c u u
Xay dung quy trinh cai t i l n san xuat altretamin dat tieu chuan d u a c dung t u cyanuric clorid va dimethylamin trong s u cd mat cua natri hydroxyd.
Nguyen lieu, hoa chat
Cyanuric clorid, dimethylamin, aceton, ethanol 96%, natn hydroxyd, altretamin (hexamethylmelamin) chuan.
Thiet bj nghien c u u
- Can dien t u Electronic Balance WT-CF; Can phan tich Metller, dp chinh xac 0,1mg, Tu say Medda, may k h u l y t u gia nhiet IKA RH Basic 2 (Due), may cdt quay Rotavapor R.200, bam hut Chan khong, p h l u Igc hut chan khong Buchner.
- Dung cy thuy tinh dat tieu chuan dung cho phdng thi nghiem tong h a p h u u c a (binh ba co 250 ml, 2000 ml, sinh han hoi luu, nhigt ke, binh c l u 250 ml, binh gan cd nut mai 250 ml, binh djnh muc, pipet c a c o n g nghiem...).
- S i c ky Idp mdng d u p e t i l n hanh tren bdn mdng silicagel Kieselgel 60 F^^^ (Merck), den soi sdc ky UV hai b u d c sdng 254 va 360 nm.
2
TAP CHi DUOC HQC-10/2014 {S6 462 NAM 54)
• Nghien ciru - Ky thuat
- Nhipt do ndng chay do tren mdy do nhiet dp ndng ehdy Stuart, SMP-10.
- Pho hong ngogi (IR): d u g e ghi tren may GX- P e r k i n E l m e r - U S A .
- Pho khdi lupng (MS): dugc ghi tren may AuloSpee Primer t^i Phdng Phdn tich cdu truc-Vien Hda hpc (Vien Khoa hgc vd cdng nghe Viet Nam).
- Phd cgng hudng t d hat nhdn (^H - NMR vd " C - N M R ) : ghi tren mdy Bruker - AV500 tai Phdng Phan tieh cau tnJe - Vien Hda hgc (Vien Khoa hgc va Cdng nghd Vidt Nam).
P h u o n g phdp nghien c u u
- Ap dung phuang phap thuc nghiem trong hda hgc huu ca de tong hgp san pham d u kidn.
- Theo do! tien trinh phan ung tong hgp bang SKLM.
- Sa bg kiem tra dp tinh khidt cua cac san pham bdng SKLM va do nhiet dp nong chay,
- Xac dinh cau true cua altretamin tong hap d u g e dp'a tren ket qua phan tich phd t d ngoai (UV), pho hdng ngogi (IR) va pho khoi lugng (MS), pho cpng hudng t u hat nhan (^H - NMR va '^C - NMR).
- Kiem nghiem altretamin tong hgp d u g c theo tidu chuan Dugc dien My 2010 {USP 34).
C a s d cua p h u a n g phdp t o n g hg'p D y a tren ket qua cdng bo cua cac tac gia tren the gidi vd trong nude ve tong hgp altretamin, chung tdi nghien cuu phan tich ve cac u u , nhuac diem cOa cac phuang phap cdng bo do de qua do phat huy nhung uu diem, khdc phuc nhung ton tgi va tien hanh khao sat toi uu hda cac thdng so, nhdm dua ra mdt quy trinh san xuat phu hap, dan gidn cd cac thdng so ky thudt dn djnh, cd tinh khd thi cao, hieu suat san pham dat d u g c la cao nhat, kinh te nhat vd san pham altretamin dat tieu chuan Dugc dien My,
Cd nhieu cdng trinh tong hgp altretamin d u g c cdng bo i^'^' di t u nhieu ngudn nguyen lieu
khdc nhau (melamin'^'.cyanuric cloridi^"'', N,N- dimethylamino-cyanamidi^'-'^, acid cyanurid^^O- thdm chi cung xudt phat t u mdt loai nguyen lieu nhu nhau nhung bdng cac tac nhdn, xuc tac, dung mdi, giai phap trien khai phan ung hoac x u ly hdn hgp phan dng cd khac nhau thi cung dua ra cac quy trinh tong hap cd hieu suat cung khdc xa nhau ^^-^"K Day ta chua ke ddn cdc phuang thuc trien khai thuc hien phan ung cung khdc nhau (thuc hien phan ung theo each thdng thudng d nhiet do cao-ap sudt Idn, s u dyng phan ung trong Id vi sdng d nhiet dp cao {180°C)'^'^^, dung sdng sieu am'^°i, dung dung moi ion Idng w...
T u cac cdng bo nay, sau khi can nhdc ca vd tinh kinh te (gia thanh) cac nguyen lieu s u dung trong quy trinh, trang thiSt bi dung cu cdn tdi cho tung quy trinh cung nhu hieu sudt va kha nang chat lugng san pham dat duac, chung toi cho la phuang phap di t u nguyen lieu cyanuric va dimethylamin tren ca sd cdng bo dau tien cua D.
W. KaiseH^ivaonam 1951 la thuc te va co tinh kha thi cao nhat trong san xudt, nhung phai dugc cai tien quy trinh nay de khong phai thuc hien d nhiet dp cao va x u ly cau ky, phuc tap, hieu suat thap, ton kem ca ve cong sue, thdi gian Ian dung mdi'^.
Mat khac theo chung toi ty le moi giua cac chat tham gia phan dng s u dyng trong cac cdng trinh neu tren cung nhu phuang phap x u ly tinh che cung chua hoan toan toi uu nen can phai d u a c nghien cdu, khao sat lai mgt each ky lugng va khoa hpc han.
K§t qua chung tdi da dua ra d u g c mpt quy trinh cai tien cd nhiiu diem uu viet han hdn so vdi cac tai lieu da cdng bo'^^i; quy trinh thuc hien kha dan gian, hieu suat cua ca qua trinh cao han han so vdi cac tai lieu da cdng bo (dat 86- 92%), ma san pham altretamin cOng dat tieu chuan Dugc dien My 2010 (USP 34).
Sa dd phan dng cua quy trinh san xuat nay cd the thdy trong so dd phan dng sau:
a\^'^Y°
Cl Cyanuric chtorid
H 3 C - NaOH
H3C CH3 Altretamin T * P C H i DUOC H p c - 10/2014 (SO 462 S V M 54)
• Nghien CLPU - Ky thuat
Trong dd chung tdi di sau vao nghien c u u tdi uu hda va cai ti§n cac ydu td:
- Cai tidn each thuc hien phan ung va cdeh x u ly, phan lap, tinh chd san pham,
- Khao sat dung mdi tinh che san pham, - Khao sat tdi uu hda ty ie moi giua cac chdt tham gia phan dng,
- Khao sat anh hudng cua nhiet do phan ung ddn hieu sudt.
ThiFC nghiem, l<et qua va ban iuan
Khao sat tdi u u hda each t h y c hien phan d n g va tach, t i n h che san pham
Chung tdi thuc hien lai phan ung theo md ta cua D.W. Kaiser ^^l theo cai tidn cua D.T.T. Hai ^ va theo cai tidn cua chiing tdi (ca ba thi nghiem ddu tien hanh d ty le cyanuric: dimethylamin:
natri hydroxyd = 1 : 3,3 : 3) nhu trong quy trinh '^i- - D. W. Kaiser '^i {Phuang phap 1): Thuc hien phan ung trong 2 budc; cd cdt loai nude ra trong qua trinh phan ung bang cdt ddng phi vdi xylen, sau do tidn hanh phan ung d nhiet dd 100°C, khi phan ung k§t thuc cat loai xylen trong chan khdng de loai toluen, can kdt tinh phan doan trong methanol, thu dugc altretamin, diem chay 170- 173-C, hieu sudt 36,94% va 25,36% 2-chloro^,6- P/s(dimethylamino)-S-tria2in. Tdng thdi gian thuc hien la 9 gid 20 phut (chua ke thdi gian sdy kho tinh the).
- D.T.T Hai f^i (Phuang phap 2): Ti6n hanh phan dng trong hai budc nhung khong cat loai nude, khdng dun trong xylen, khi phan ung ket thuc cat loai het dung mdi va x u ly tach san phdm thd bang each chiet vdi ethyl acetat, cdt loai dung moi, can k§t tinh lai trong ethanol tuyet ddi, thu d u g c altretamin, diem chay 171-173°C, hieu sudt 67,12%. Tong thdi gian thuc hien la 7 gid 30 phut (chua ke thdi gian s l y khd tinh the).
- Chung toi (Phuang phap 3): Tien hanh phan dng trong mot b u d c bang each cho 1 duang lugng (dl) cyanunc clond (CYA, 9,39 g (0,05 moi) hoa vao luang aceton tdi thidu Bang 1: Anh huang cua tile moi cyanunc clorid
(40 ml), tidp dd v d a khudy v u a cho thdm 3,3 dl dimetylamin (DMA), sau dd cho t d t u cho 3,0 dl natri hydroxyd (NaOH) vao vd dun hoi luu hdn hgp phan dng dgn khi kdt thuc (2 gid, theo doi tren sac ky ban mdng, he dung mdi then khai la toluen: AcOEl:CHCl3:MeOH = 6:1:4:3, hien thj d u d i den UV 254 nm). Khi phan dng k^tthucttii x u ly vdi n u d e de thu san pham thd, ket tinh Ip trong ethanol 96%, thu d u g e altretamin, dem chay 171-173''C, hieu sudt 7 7 , 8 1 % . Tong thoi gian thuc hien la 5 gid 30 phut (chua ke thdi gian sdy khd tinh the).
Whan xet: T u ket qua khao sat tren cho thay tien hanh phan ung vd x u ly hon hap sau phan ung theo p h u a n g phap 3 dan gian han, nhanh han. It ton kem dung mdi han ma hieu sudt thu d u g c lai cao han p h u a n g phap 1 va phu-ang phdp 2 (77,81% so vdi 36,94% va 67,12%).
Vi vay, chung toi lua chgn each tien hanh phan dng va x d ly hdn hgp phan ung theo phu'cng phdp 3 de tidp tuc cac khao sat tidp trong nghien cuu ndy
Khao sat anh h u d n g eua d u n g mdi ket tinh T h u k i t tinh lai altretamin thd trong mot so dung mdi id methanol, ethanol TD, ethanol 96%, ethanol 90%, ket qua cho thdy giua methanol, ethanol TD, ethanol 9 6 % khong cd gi khac bietve chat lugng (diem chay) Idn hieu suat san thu hoi pham (93-95%) nhung vi ddc tinh va gia thanh nen chung tdi chgn ethanol 96% lam dung mfii tinh chd san pham altretamin trong de tai nay.
K h a o sat t y le m o i g i u a cac c h a t tham gia t r o n g p h a n u n g
Khao sat anh hwang cua ti le moi cyanunc clorid va NaOH toi hieu suit phan O'ng
Tidn hanh phan ung tong hgp altretamin theo p h u a n g phap 3 da neu tren, khdo sat anh hipang cua tl le moi giua CYA: NaOH thay doi t u 1:3,0- 1 : 4 , 5 v d i t y l e C Y A : D M A khdng thay doi la 1:3,3;
k i t tinh san pham trong ethanol 96%. Ket qua d u g c trinh bay d bang 1.
va NaOH tai hieu suit phan u-ng (n = 2-3) STT
1 2 3 4 5 6
Ti le moi CYA: DMA: NaOH 1:3,3:3,0 1:3,3:3,3 1:3,3. 3,6 1:3,3-3.9 1 3,3 4,2 1 3.3-4.5
KL truttg binh SP (gj 7.69 8,55 8.51 8,30 8,15 8.16
Nhiet do nong chay ^C) 172-174 172-174 172-174 171-173 172-174 171-173
Hieu suat trung binit fl^
77,23 _ 81,42 81,09 __
79.04 __
77,62 _ 77,71 _
TAP CHi Dirpc HOC - 10/2014 (S6 462 NAMM
• Nghien ciru - Ky thuat
Nh$n x4t: Ket qua bdng 1 eho thay khi tdng ty Id moi CYA: NaOH t u 1 : 3 d i n 1 : 3.3 thi hieu sudt dd d u g c cai thien dang ke tang t u 77,23%
len 81,42%. Tuy nhien, neu tiep tuc tang Idn tdi ty Id 1 : 3,6 thi hieu suat tdng khdng dang ke, tiep tyc tdng NaOH thi hidu suat phan dng lai giam.
Dieu nay cd the la do khi tang lugng NaOH qua mdc can thiet lam cho pH mdi trudng tang len phan nao gay pha huy san pham. N h u vay vdi ty le moi CYA: NaOH la 1 : 3,3 la phu hgp cho phan Bang 2: Kit qua khao sat anh huang ti le moi
ung tong hap altretamin (me sd 2).
Khao sat anh hipcmg cua ti le moi cyanuric clorid va DMA toi hieu suit phan O'ng
Tidn hanh phan ung tong hgp altretamin theo phuang phap 3 da neu tren, khao sat anh hudng vdi ti le moi gida CYA: DMA thay doi t u 1 : 3,3 - 1 • 4,5 vdi ty le CYA:NaOH khdng thay doi la 1 : 3,3; k i t tinh san pham trong ethanol 96%. K i t qua d^rgc trinh bay d bang 2.
cyanuric chloric: DMA (n = 2-3) STT
1 2 3 4 5
Ti lo moi CYA : DMA:
1:3,3:3,3 1:3,6:3,3 1:3,9.3.3 1.4.2:3,3 1:4,5-3,3
NaOH Kl trung binh SP (gj 8,55 9,36 9,28 9,39 9,42
Nhiet (?6 nong chay Hieu suat trung binh (%) 171-173
172-174 171-173 172-174 172-174
81.42 89,14 88.38 89,42 89,71 Nhan xet: Theo phuang trinh phan ung
thi ty le giua cac chat tham gia phan dng la CYA:DMA:NAOH 1 : 3:3 nhung vi thanh phan DMA cd dp soi thap, d§ bay hai nen d l mat mat trong qua trinh thao tac vi t h i khi tang ty Ip DMA len thi hieu suat phan ung tang len, do dd cdn phai cho d u DMA. Tuy nhien, tang ty le CYA:DMA tdi 1 : 3,9 den 1 : 4,5 thi hieu suat phan ung thay doi khdng dang k l so vdi ty 1^ 1 : 3,6. N h u vay khdng can thilt phai tang ty le moi DMA qua nhieu, lang phi va cd the gay bat tien trong qua trinh tinh che san pham. Nhu vdy ty le
Bang 3: Anh huang cua nhiet do tien hanh phan
CYA. DMA: NaOH thich hap cho phan ung tong hap altretamin la 1 : 3,6. 3,3, luc nay hieu suat altretamin dat d u a c la 89,14% (me so 2)
Khao sat anh h u d n g cua nhiet do phan u n g den t h d i gian phan u n g va hieu suat
T i l n hanh phan ung tong hap altretamin theo phuang phap 3 neu tren, chi thay doi nhiet dp thuc hien phan dng, vdi cac nhiet dd SS-SS^C, 30-35°C, 20-25''C, theo ddi diem ket thuc phan dng bdng s^c ky Idp mong. Ket qua d u g c trinh bay d bang 3.
yng din thai gian phan ung va hieu suit Nhi^t tfp phan img
fC) 5 5 - 5 8 30-35 (miia he) 20-23 (mua (Jong)
Thoi gian phan img (giai 2giCT 5 gia 6,5 gia
Khoi luvng sai ' (g)
9,36 9.22 8.95
1 pham Nhiet do nong chay
rc)
172-174 171-173 172-174
Hieu suat
'(%)
89,14 87,81 85,24 Nhfin xet: Phan ung tao altretamin t u
cyanuric clorid vd dimethylamin vdi s u co mat cua NaOH cd the thuc hign d u g c t d 20''C trd len, nhi^t dp cdng cao thi thdi gian thyc hi^n phan dng cdng gidm, nhung nhiet dp thuan igi, toi uu Id t h y c hi^n d nhigt do soi cua dung moi aceton 55-58°C, luc ndy thdi gian la khoang 2 gid.
Quy trinh s a n suat
Cho 94,0 g (0,5 moi) cyanuric clorid vdo binh ba c6 dung tich 2 lit, cd Idp sinh hdn. nhi^t ke, ph§u nhd gigt. ndi each thuy, mdy khuay t u , eho
vao do 400 ml aceton, khudy 10 phut, tiep do cho them vao dung djch nay 230 ml dimetylamin 40%
(1,80 moi). Lam lanh ngoai bang nude da, vua khudy vua cho 66,2 g (1,65 moi) natn hydroxyd vao, khuay 10 phut, sau do dun hoi luu I g i d 45 phut. Sau khi phan ung ket thuc, vua khuay vua cho vao hon hgp phan dng tren 1,2 lit nude cat - da, tua tao ra d u g c khuay tiep 30 phut, sau dd d u g c Ipc qua phdu Buchner, rua nude lanh, hut kiet, thu d u o c 121,4 g san pham thd. Ket tinh lai toan bp san pham thd trong ethanol 96", say khd
I .Al'CHJ DUgfC H p c - 10/2014 (SO 462 ^ . \ M 54)
Nghien CLFU - Ky thuat
san pham d 70°C trong tu hut chan khdng 3 gid, thu d u g c 93,87 g san pham altretamin, tinh the hinh kim trdng, nhiet dp ndng chay 172-174°C.
Hieu sudt 89,40%.
Ddnh gia chat l u a n g s a n pham altretamin s a n xuat ra
Do diem chay
Nhiet dp chay cua san pham altretamin cac me khac nhau dao dpng t u 171-174 °C.
sic ky ban mong
Sa bp kiem tra bang SKLM so vdi chat chudn vdi he dung mdi khai trien la (toluen:
AcOEt : CHCI3: MeOH ) (6:1:4:3) cho 1 vdtggn len ngang dung vdt altretamin chuan, hien thj ro d u d i dnh sdng den t u ngoai b u d c sdng 254 nm (R, = 0,769).
So lieu phan tich pho
Loai pho Hexamethylmelamin chuan Hexamethylmelamin tong hop tfiroc
UV, >.^^_(nm) 226
IR (KBr), v^^^cm-^ 2858 (vC^^^^^^^-N-CHj); 2278 (S^C-H/N-CH,), 1528 (vC=N),"l385 (S^CHj), 1211 (uC-N).
2866 (vC^,„„,,,-N-CH3); 2278 (5^C-H/N- CH3), 1527 (vC=N), 1386 (S^CHg); 1211 (vC-N).
210 (M*); 195 [M^-CHg]; 167 [M*-N(CH3)J;
152[195-N(CH3)J;96[167-NH=C-N(CH3)2];
81[152-NH=C-N{CH3)J
210 (M*); 195 [M^-CHJ; 167 [M*-N(CH3)J;
152[195-N(CH3)J;96[167-NH=C-N(CH3)J;
81[152-NH=C-N(CH3)^1 3,08 (s, 18H, 6-CH3) 3,09 (s, 18H, 6-CH3)
'=C-NMR (ppm) 165,66(S,3C,C,„^^,J; 35,65(s,6C,6CH3) 165,66(s,3C,C,,,^^,J;35,65(s,6C,6CH3) T u ket qua do diem chay, chay sac ky ban
mdng va phan tich pho IR, MS, ''H-NMR va
^^C - N M R cua hgp chdt tong hgp d u g c vd chdt chuan cho phep chung tdi k i t luan altretamin tdng hgp d u g c tinh khiet va cd c l u true dung nhu d u kien.
Chat Iwang cua altretamin tong hgp dwac K i l m nghiem theo Dugc d i l n My 2010 {USP 34). K i t qua m l u gdi kiem nghiem dat yeu cau c h i t luong theo Dugc dien My 2010 (USP 34), philu kiem nghiem sd 44G237 ngay 25 thang 4 nam 2014 cua Vien Kiem nghiem thudc Tmng uang).
Ket luan
Da nghien cdu cai tien thanh cong quy trinh cua Donald W. Kaiser i^i va Dmh Thj Thanh Hai"^!
de tong hap altretamin. Vdi quy trinh cai t i l n nay vu'a cd thao tac don gian han, tieu ton hda chdt, dung mdi it hon, de trien khai d quy md Idn, hieu suat thu duac lai vua cao han nhilu so vdi hai cdng trinh da neu (89,40% so vdi 36,94% vd 68,12%). San pham altretamin san xudt ra dat tieu chuan Duac dien My 2010 (USP 34), vdi p h i l u kiem nghiem so 44G237 ngay 25 thang 4 ndm 2014 cua Vien Kiem nghiem thudc Trung u a n g .
Tai lieu tliam khao
1. D6 Trung Dam (1995), Thuoc chus ung thw, NXB YhgcHaNoi, tr-137-138.
2. Nguyin Ba Bdc (2003), Hoa chat diiu tn ung thif, NXB Y hgc HN,tr. 130-137
3. Trinh Van Quang (2002), Bach khoa toan thw. ung thwhoc, NXB Y hoc, tr. 69-70,
4 Goodman and Gilman's (2001), The Pharmacological basis of Therapeutics, 10*" Ed. MC Grow Hill Inc. New York, London, Seol, Tokyo, Singapore, Sydney, Toronto; vol. II, Ctiapter 52.
5 Hofmann A. W.(1855); Ber 18, 2755, 6. Kaiser D. W. et al (1952); J. Am. Chem. Soc. 73, 2984-2986.
7. Dmh Thi Thanh Hal, Hoang Thu Trang, (2010);
Tap chi Duxyc hQC, 407 (3-2010), 25-27.
8. Kapnl A. and Anshu, D (2007); Bioorganic and Medicial Chemistry Letters. 17, 3298-3304.
9. Dmh Thj Thanh Hai, Le Dinh Manh, (2010); T9P chi Duxyc hoc, 410 (6-2010), 34-37.
10 Dinh Thi Thanh Hal, Dinh Th; Hai, Tran Tu5n Hiep (2011); TapchiDum:hQcA2\ (5-2011), 41-44.
11. Antonio H., et al. (2004): Synthesis. 4, 503-505.
12. Dornan P. et al (2008): Chem. Comm., 44, 3645-3647,
13. Baudgar D. (2006). Synthetic Comm.. 37 (7).
859-864
{Ngay nhan bar 27/08/2014 - Ngay duyet dang: 06/10/2014)
