Tổng hợp và thử tác dụng sinh học của một số acid hydroxamic mang khung 5 -aryl-1,3,4- Thiadiazol.pdf

B Y T

TRNG I HC DC H NI

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LNG XUN HUY

TNG HP V TH TC DNG SINH HC

CA MT S ACID HYDROXAMIC MANG

KHUNG 5-ARYL-1,3,4-THIADIAZOL

KHA LUN TT NGHIP DC S

H NI - 2014

B Y T

TRNG I HC DC H NI

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LNG XUN HUY

TNG HP V TH TC DNG SINH HC

CA MT S ACID HYDROXAMIC MANG

KHUNG 5-ARYL-1,3,4-THIADIAZOL

KHA LUN TT NGHIP DC S

Ngi hng dn:

1. TS. o Th Kim Oanh

2. DS. Trn Th Bch Lan

Ni thc hin:

B mn Ha Dc

H NI-2014

Li cm n

u tin ti xin c gi li cm n chn thnh v su sc n ngi

thy v ngi c ng knh ca ti: PGS.TS. Nguyn Hi Nam v TS. o

Th Kim Oanh, ging vin b mn Ha Dc, trng i hc Dc H Ni.

Thy c khng ch to nhng iu kin thun li nht gip ti hon thnh

kha lun m lun c nhng ch dn chnh xc, kp thi v ng vin ti

nhng lc kh khn.

Ti xin gi li cm n n DS. Trn Th Bch Lan v DS. Th Mai

Dung nhit tnh gip ti trong qu trnh lm thc nghim ti b mn

Ha Dc, trng i hc Dc H Ni.

Ti cng xin gi li cm n chn thnh ti cc thy c gio, cc anh

ch k thut vin ca b mn Ha Dc trng i hc Dc H Ni trong

sut thi gian qua to iu kin ti thc hin kha lun ti b mn.

Cui cng, ti xin gi li cm n ti gia nh, cc anh ch, cc bn v

cc em trong nhm thc nghim ti b mn Ha Dc ng hnh cng ti

chia s vui bun trong sut thi gian qua.

H Ni, ngy 1 thng 5 nm 2014

Ngi vit

Lng Xun Huy

MC LC

Danh mc cc k hiu, cc ch vit tt

Danh mc cc bng

Danh mc cc hnh v

Danh mc cc s

T VN 1

CHNG 1. TNG QUAN 2

1.1. Histon deacetylase 2

1.1.1. nh ngha histon deacetylase (HDAC) 3

1.1.2. Phn loi HDAC 3

1.2. Cht c ch HDAC trong iu tr ung th 4

1.2.1. Histon deacetylase (HDAC) v ung th 4

1.2.2. C ch tc dng ca cc cht c ch HDAC 5

1.2.3. Phn loi cc cht c ch HDAC 6

1.2.4. Lin quan cu trc v tc dng ca cc cht c ch HDAC 8

1.3. Mt s nghin cu trn th gii v trong nc v cc acid

hydroxamic hng c ch HDAC cng b gn y 9

CHNG 2. NGUYN LIU, THIT B, NI DUNG V

PHNG PHP NGHIN CU 16

2.1. Nguyn liu 16

2.1.1. Ha cht chnh 16

2.1.2. Dung mi v ha cht khc 16

2.2. Thit b, dng c 16

2.3. Ni dung nghin cu 17

2.3.1. Tng hp ha hc 17

2.3.2. Th tc dng sinh hc ca cc cht tng hp c 17

2.4. Phng php nghin cu 18

2.4.1. Tng hp ha hc v kim tra tinh khit 18

2.4.2. Xc nh cu trc 18

2.4.3. Th tc dng c ch HDAC 18

2.4.4. Th tc dng khng t bo ung th in vitro 19

2.4.5. Gi tr LogP v nh gi mc ging thuc ca cc dn cht

tng hp c

20

CHNG 3. THC NGHIM, KT QU V BN LUN 21

3.1. Tng hp ha hc 21

3.1.1. Tng hp cht N1-[5-(furan-2-yl)-1,3,4-thiadiazol-2-yl]-N8-

hydroxyoctandiamid (Va)

21

3.1.2. Tng hp cht N1-hydroxy-N8-[5-(thiophen-2-yl)-1,3,4-

thiadiazol-2-yl]octandiamid (Vb)

25

3.1.3. Tng hp cht N1-[5-(3-bromofuran-2-yl)-1,3,4-thiadiazol-2-yl]-

N8-hydroxyoctandiamid (Vc)

27

3.1.4. Tng hp cht N1-hydroxy-N8-[5-(3-methylfuran-2-yl)-1,3,4-

thiadiazol-2-yl]octanediamid (Vd)

28

3.2. Kim tra tinh khit 30

3.3. Xc nh cu trc 31

3.3.1. Ph hng ngoi (IR) 31

3.3.2. Ph khi lng (MS) 32

3.3.3. Ph cng hng t ht nhn 1H-N v 13C-NMR 33

3.4. Hot tnh sinh hc v ging thuc 35

3.4.1. Hot tnh sinh hc 35

3.4.2. nh gi mc ging thuc 36

3.5. Bn lun 36

3.5.2. Ha hc 36

3.5.2. Hot tnh sinh hc 38

KT LUN V KIN NGH 41

Kt lun 41

Kin ngh 41

PH LC

TI LIU THAM KHO

DANH MC CC K HIU, CC CH VIT TT

ADN : Acid deoxyribonucleic

AsPC-1 : T bo ung th ty

DCM : Dicloromethan

DMF : Dimethylformamid

DMSO : Dimethylsulfoxid

EtOH : Ethanol

HAT : Histon acetyltranferase

HDAC : Histon deacetylase

HDACi : Cht c ch histon deacetylase

IC50 : Nng c ch 50% s pht trin ca t bo

IR : Phng php ph hng ngoi

MCF-7 : T bo ung th v

MeOH : Methanol

MS : Ph khi lng

NCI-H460 : T bo ung th phi

NMR : Phng php ph cng hng t ht nhn

PC3 : T bo ung th tin lit tuyn

SAHA : Acid suberoylanilid hydroxamic

SW620 : T bo ung th i trng

TLC : Phng php sc k lp mng

TSA : Trichostatin A

DANH MC CC BNG

STT Tn bng Trang

Bng 1.1 Cc cht c ch HDAC ang th nghim trn lm sng 7

Bng 1.2 Tc dng c ch HDAC ca cc dn xut 1,3,4-

thiadiazol

10

Bng 1.3 Tc dng ca acid aryl ether/aryl sulfon hydroxamic 11

Bng 1.4 Hot tnh sinh hc ca mt s dn cht alkyl piperidin 12

Bng 1.5 c tnh ca cc cht c ch HDAC tng hp c 14

Bng 3.1 Gi tr Rf v T0

nc ca cc cht Va-d 31

Bng 3.2 S liu phn tch ph IR ca Va-d 31

Bng 3.3 S liu phn tch ph khi lng ca cc cht 32

Bng 3.4 S liu phn tch ph 1H-NMR 33

Bng 3.5 S liu phn tch ph 13C-NMR 34

Bng 3.6 Kt qu th hot tnh v tc dng c ch HDAC 35

Bng 3.7 nh gi mc ging thuc theo quy tc Lipinsky 36

Bng 3.8 So snh kt qu ca Va-d vi SAHA v mt s cht 38

DANH MC CC HNH V

STT Tn hnh Trang

Hnh 1.1 Cu to nucleosom 2

Hnh 1.2 M t hot ng ca HDAC 3

Hnh 1.3 Phn loi cc HDAC ph thuc Zn2+ 4

Hnh 1.4 M t hot ng ca cc HDAC v cc cht c ch

HDAC

6

Hnh 1.5 Cu trc ca SAHA v trung tm hot ng ca HDAC 8

Hnh 1.6 Dn xut acid 1,3,4-oxadiazol/thiadiazol hydroxamic 9

Hnh 1.7 Dn xut acid 1,3,4-thiadiazol hydroxamic 10

Hnh 1.8 Cng thc acid aryl ether/aryl sulfon hydroxamic 11

Hnh 1.9 Cng thc chung cc 4-alkyl piperidin v 4-alkyl

piperazin

12

Hnh 1.10 Thit k cht c ch kp da trn cu trc SAHA v

Lovastatin 13

DANH MC CC S

STT Tn S Trang

S 2.1 Quy trnh tng hp chung Va-d 18

S 3.1 Quy trnh tng hp cht Va 21

S 3.2 Tng hp cht IIa 21

S 3.3 Tng hp cht IIIa 22

S 3.4 Tng hp cht IVa 23

S 3.5 Tng hp cht Va 24

S 3.6 Quy trnh tng hp cht Vb 25

S 3.7 Quy trnh tng hp cht Vc 27

S 3.8 Quy trnh tng hp cht Vd 29

S 3.9 C ch phn ng ng vng tng hp 1,3,4-

thiadiazol

37

S 3.10 C ch xc tc ca CDI 37

1

t vn

Cc histon deacetylase (HDAC) l mt nhm cc enzym c vai tr

quan trng trong qu trnh phin m gen. Nhiu bng chng cho thy nhng

enzym ny khng ch lin quan n cu trc ca chromatin v s biu hin

ca gen m cn tc ng n chu trnh pht trin ca t bo v tin trnh ung

th bao gm s hnh thnh ca cc khi u c tnh [12,16]. Chnh v vy, cc

enzym ny ang l mt trong nhng ch quan trng trong vic nghin cu

pht trin cc thuc iu tr ung th mi [7,22].

Cc nh nghin cu t c nhiu thnh qu trong vic tm ra

nhiu cht c ch HDAC, nh l Trichosatin A, SAHA (Vorinostat), MS-27-

275 (Entinostat), LBH-589 (Panobinostat), PDX-101 v Oxamflatin. Trong s

ny, acid suberoylanilid hydroxamic (SAHA, Zolinza) c ph duyt bi

Cc qun l thc phm v dc phm M (FDA) vo nm 2006 trong iu tr

u lympho t bo T di da. Ti nay, t nht 24 cht c ch HDAC ang trong

qu trnh th nghim lm sng cc cp khc nhau [10].

Nhm nghin cu ti b mn Ha Dc trng i hc Dc H Ni

cng thit k, tng hp v cng b nhiu dy cht nh hng c ch

HDAC cho kt qu hot tnh sinh hc rt kh quan, c bit l khi thay nhn

phenyl ca SAHA bng vng thm khc nh benzothiazol, 5-phenyl-1,3,4-

thiadiazol. Tip tc hng nghin cu ny, chng ti tin hnh ti Tng

hp v th tc dng sinh hc ca mt s acid hydroxamic mang khung 5-

aryl-1,3,4-thiadiazol vi hai mc tiu:

1. Tng hp 4 acid hydroxamic mang khung 5-aryl-1,3,4-thiadiazol.

2. Th hot tnh khng t bo ung th in vitro v tc dng c ch HDAC

ca cc cht tng hp c.

2

CHNG 1. TNG QUAN

1.1. Histon deacetylase

Tt c b gen ca ngi c gi trong nhim sc th (NST), mt

phc hp i phn t protein-ADN. n v cu trc c bn ca NST l

nucleosom.

Hnh 1.1. Cu to nucleosom

Mi nucleosom in hnh bao gm mt octamer hnh a ca 4 cp

histon (2 cp ca H2A vi H2B v 2 cp ca H3 vi H4) c qun quanh

bi 146 cp nucleotid. u amin ca histon mang nhiu in tch dng nn

tng tc mnh vi u phosphat mang in m ca ADN to nn cu trc

ca nucleosom v cu trc bc cao ca NST quy nh qu trnh biu hin gen.

Khi u amin ca histon tch in dng cng ln tng tc ny cng mnh,

NST ng xon cng cht c ch qu trnh phin m. Ngc li th qu trnh

phin m din ra v gen c biu hin. Mc tch in dng ca histon

ph thuc vo qu trnh acetyl ha u amin ca histon. S acetyl ha lm

trung ha bt in tch dng u amin ca histon. Trong t bo c 2 enzym

ng vai tr chnh trong qu trnh acetyl ha l HDAC v HAT. Hai enzym

ny c vai tr tri ngc nhau. S cn bng trong hot ng ca chng m

bo mc tho xon ca nhim sc th din ra bnh thng.

3

1.1.1. nh ngha histon deacetylase (HDAC)

Histon deacetylase (HDAC) l mt nhm cc enzym xc tc qu trnh

loi b nhm acetyl t -N acetyl lysine amino acid ca histon. N c tc

dng i lp vi histon acetyltransferase (HAT) [23].

Hnh 1.2. M t hot ng ca HDAC

1.1.2. Phn loi HDAC

C 18 HDAC ngi c chia thnh 4 nhm da trn s tng ng

cu trc v chc nng ca chng [16]:

Nhm I: HDAC1, HDAC2, HDAC3, HDAC8.

Nhm IIa: HDAC4, HDAC5, HDAC7, HDAC9.

Nhm IIb: HDAC6, HDAC10.

Nhm III: Cc protein iu ha chui thng tin bao gm:

- Cht ng ng ca sir2 trong men Saccharomyces cerevisiae.

- Sirtuin trong ng vt c v (SIRT1, SIRT2, SIRT3, SIRT4, SIRT5,

SIRT6, SIRT7).

Nhm IV: HDAC11.

Nhm I, II v IV c coi nh nhng HDAC c in v gm 11

thnh vin l nhng enzym ph thuc Zn2+, chng c cha mt ti xc tc vi

4

mt ion Zn2+ y ca ti. Nhng enzym ny c th b c ch bi cc hp

cht to chelat vi Zn2+ nh cc acid hydroxamic, thiol,Trong khi , cc

thnh vin nhm III c gi l nhng sirtuin c c ch ph thuc vo NAD+

nh mt cofactor thit yu [20]. Thut ng cc cht c ch HDAC thng

c s dng cho nhng hp cht c ch cc HDAC thuc nhm I, II v IV

v nhng hp cht ny hin nay ang c tin hnh cc th nghim lm

sng.

Hnh 1.3. Phn loi cc HDAC ph thuc Zn2+

1.2. Cht c ch HDAC trong iu tr ung th

1.2.1. Histon deacetylase (HDAC) v ung th

5

Ung th xy ra do t bin trong ADN, dn n t bo tng sinh v hn

, v t chc v khng tun theo cc c ch kim sot v pht trin ca c

th. Qu trnh deacetyl ha histon ca HDAC nh hng ln s biu hin ca

gen qua tc ng ti s khi u v tin trin ca ung th.

Cc HDAC gy ra s khi u ca ung th bng cch [15]:

- Gim c ch chu trnh t bo.

- c ch bit ha t bo.

- Trnh s cht t bo theo chng trnh (apoptosis).

Cc HDAC thc y s tin trin ca ung th bng cch [15]:

- Thc y s hnh thnh mch (angiogenesis).

- Tng cng xm ln (invastion).

- Gim bm dnh (adhesion).

- Tng di chuyn (migration).

1.2.2. C ch tc dng ca cc cht c ch HDAC

Kh nng chng ung th ca cc cht c ch HDAC (HDACi) bt

ngun t kh nng tc ng ln nhiu giai on chu trnh t bo b bin i

cc t bo ung th.

Cc cht c ch HDAC tc ng ti t bo ung th theo ba c ch ch

yu [15]:

- Gip hot ha cc CDKI (cht c ch kinase ph thuc cyclin) gy c

ch s sinh si ca t bo.

- Hot ha cc yu t bit ha (differentiation factors) lm tng bit ha

thay v tng s lng t bo.

- HDACi hot ha cc yu t cht t bo theo chng trnh.

Hnh 1.4 di y m t hot ng ca HDAC v cht c ch HDAC.

6

Hot ng ca HDAC Hot ng ca cht c ch HDAC (HDACi)

Hnh 1.4. M t hot ng ca cc HDAC v cc cht c ch HDAC

1.2.3. Phn loi cc cht c ch HDAC

Cc cht c ch HDAC ang c th nghim lm sng s dng

trong iu tr ung th c th c chia lm 4 nhm da trn cu trc [6]:

- Cc hydroxamat: TSA, SAHA, CBHA,...

- Cc peptid vng: depsipeptid, CHAPs,

- Cc acid bo mch ngn: butyrat, phenylbutyrat, valproic acid,

- Cc benzamid: N-acetyldinalin, MS-275,...

Mi nhm trn u c nhng hn ch nht nh nh: cc acid

hydroxamic b chuyn ha nhanh, c ch khng chn lc ln cc loi enzym

HDAC; cc benzamid v acid bo c hiu lc km; cc peptid vng kh to

thnh v mt ha hc. Cc nhm khc nhau c tc dng c ch cc loi

HDAC khc nhau: cc hydroxamic acid c ch mnh HDAC nhm I v II,

cc acid carboxylic v peptid vng c ch mnh HDAC nhm I. Hin nay c

khong 24 cht c ch HDAC thuc 4 nhm ang c th nghim trn lm

sng iu tr ung th (c th trnh by trong bng 1.1) [10].

7

Bng 1.1. Cc cht c ch HDAC ang th nghim trn lm sng

Nhm Cht Nng HDAC c hiu Th nghim

Acid bo

mch ngn

Butyrat mM Nhm I, IIa121

Pha I, II

Valproic acid (VPA) mM Nhm I, IIa121

Pha I, II

AN-9 (tin thuc) M N/A* Pha I, II

Hydroxamat

Trichostatin A (TSA) nM Nhm I, IIa121

N/A

SAHA, Vorinostat M Nhm I, IIa121

Pha I, II, III

PDX101 M Nhm I, IIa121

Pha I

Oxamflatin M N/A N/A

LAQ824 nM Nhm I, IIa121

Pha I

LBH589 nM Nhm I, IIa121

Pha I

m-carboxycinamic acid

bis-hydroxamid (CBHA) M N/A

N/A

Scriptaid M N/A N/A

Pyroxamid M Nhm I Pha I

Suberic bishydroxamic

acid (SBHA) M N/A N/A

Azelaic bishydroxamic

acid (ABHA) M N/A N/A

SK-7041 nM HDAC 1 v 2177

N/A

SK-7068 nM HDAC 1 v 2177

N/A

CG-1521 M N/A N/A

Tubacin M Nhm IIb39

N/A

Benzamid MS-275 M HDAC 1, 2, 3, 8 (nh)34 Pha I, II

CI-994 (tacedinaline) M N/A Pha I, II, III

Cyclic

tetrapeptid

Depsipeptid nM Nhm IIb36

Pha I, II

Trapoxin A nM Nhm I, IIa121

N/A

Apicidin nM HDAC1, 3, khng

HDAC8170

N/A

CHAPs nM Nhm I178

N/A

*Cha c s liu

8

1.2.4. Lin quan cu trc v tc dng ca cc cht c ch HDAC

Cc cht c ch HDAC da trn cu trc acid amid-alkyl-hydroxamic

c bit n nhiu, v d nh SAHA. Cu trc ca chng bao gi cng

bao gm 3 phn chnh A - B - C: Phn nhm nhn din b mt, phn cu ni

v nhm gn kt vi ion Zn2+.

Hnh 1.5. Cu trc ca SAHA v trung tm hot ng ca HDAC

Ni chung hiu qu tc dng ca cc cht c ch HDAC (HDACi) da

trn s c mt ca 3 yu t chnh [14]:

- Nhm kt thc c th gn kt vi Zn2+ trn v tr tc dng ca cc

enzym HDAC (zinc-binding group-ZBG): acid hydroxamic, cc thiol,

benzamid, mercaptoceton,...y l phn khng th thiu c tc dng c

ch HDAC.

- Cu ni s nc (hydrophobic linker): thng c cu trc amid-alkyl,

nm trong lng enzym. Cu ni ny lin quan n kh nng c ch, quyt

nh s ph hp v cu trc ca cc cht vi chiu di ca knh enzym. Lin

kt amid quan trng nhng khng phi then cht, di mch ti u l 5-6C.

9

- Nhm kha hot ng (capping group): thng l vng thm hoc

peptid vng v thng nm trn b mt enzym. Nhm ny lin quan n tnh

c hiu v hiu lc c ch HDAC.

Cho n nay phn ln cc nghin cu lin quan cu trc tc dng u

tp trung tm hiu vai tr ca nhm gn vi ion Zn2+, c gng nghin cu thay

th acid hydroxamic v mt vi cu ni.

1.3. Mt s nghin cu trn th gii v trong nc v cc acid

hydroxamic hng c ch HDAC cng b gn y

Thit k acid 1,3,4-oxadiazol/thiadiazol hydroxamic

Hai dy 1,3,4-oxadiazol/thiadiazol c tng hp vi nhm gn kt

ion Zn2+

(C), cu ni (B), v nhm nhn din b mt (A). Cc nh gi kh

nng c ch ung th, TT, c ch t bo Ehrlich ung th c trng chut

bch tng Thy S cho kt qu y ha hn [9].

Hnh 1.6. Dn xut acid 1,3,4-oxadiazol/thiadiazol hydroxamic

Kt qu nghin cu in vitro cho thy trong dy oxadiazol, cht 1a (R =

H, X = O) c ch HDAC-1 mnh nht vi IC50 = 0,006 M v HCT-116 vi

IC50 = 0,09 M. Trong dy thiadiazol, cht 2a (R = H, X = S) c ch HDAC-

1 mnh nht vi IC50 = 0,008 M v HCT-116 vi IC50 = 0,08 M.

Cc dn xut acid 1,3,4-thiadiazol hydroxamic

Mt nhm cc nh khoa hc Trung Quc thit k v tng hp mt

dy cc acid 1,3,4-thiadiazol hydroxamic c ch HDAC vi cc cu ni v

10

nhm th trn vng 1,3,4-thiadiazol khc nhau. Kh nng c ch enzym ca

cc cht 3a-g cho thy cu ni 5-6 cacbon gia nhm gn Zn2+ v vng

1,3,4-thiadiazol l ti u cho hot tnh.

Hnh 1.7. Dn xut acid 1,3,4- thiadiazol hydroxamic

Mt trong s cc dn cht ny 3b (n=5, R = -C6H5) cho IC50 = 0,089

c kh nng c ch tt hn SAHA (IC50 = 0,15 ) [18].

Bng 1.2. Tc dng c ch HDAC ca cc dn xut 1,3,4-thiadiazol

Cht R n IC50 ca HDAC* (M)

3a

3 0,16 0,03

3b

5 0,089 0,005

3c

5 0,22 0,04

3d

5 0,33 0,05

3e

6 0,27 0,004

3f

6 0,26 0,05

3g

6 0,32 0,05

*: Mi cht c th c lp ba ln.

Thit k acid aryl ether/aryl sulfone hydroxamic tng t TSA

Ln u tin cc dn cht acid hydroxamic vi nhm kha hot ng l

aryl ether v aryl sulfon cng cu ni c 5C khng bo ha c tng hp

v nh gi. Mt s hp cht cha nhm th meta v para trn vng aryl cho

11

thy kh nng c ch mnh HDAC nng nanomol [4].

Hnh 1.8. Cng thc acid aryl ether/aryl sulfon hydroxamic

Hu ht cc dn xut acid aryl ether hydroxamic cho thy kt qu ha

hn. Trong cht u tin nghin cu 4a cho thy kh nng c ch HDAC

mc trung bnh (IC50 = 188 n ), c bit 4d cho kh nng c ch mnh nht

vi IC50 = 18 nM. Kt qu thu c ch ra rng nhm th ti v tr meta

v/hoc para trn vng aryl cho tc dng c ch tt. Ngoi ra kh nng c ch

HDAC cng ph thuc ch yu vo nhm th trn vng aryl v rt t ph

thuc vo vic c hay khng nhm -CH3 v tr R.

i vi dn xut acid aryl sulfon hydroxamic, kt qu khng nh mong

i, tt c u c ch HDAC vi IC50 > 1 M.

Bng 1.3. Tc dng ca acid aryl ether/aryl sulfon hydroxamic

Cht Ar

Tc dng c ch HDAC IC50 (M)

R = H R = CH3

4a

0,188 -

4b

0,041 0,055

4c

0,020 -

4d

0,018 -

4e

0,068 -

TSA

- 0,003

12

Cc alkyl piperidin v piperazin

Cc nh khoa hc Italia cng b kt qu nghin cu mt dy cc

acid 4-alkyl piperidin v 4-alkyl piperazin hydroxamic hng c ch HDAC

da trn cu trc ca SAHA. Cc cht ny c thay i nhm kha hot

ng v cu ni so vi SAHA [19].

Hnh 1.9. Cng thc chung cc 4-alkyl piperidin v 4-alkyl piperazin

Sau khi tin hnh th kh nng c ch HDAC v dng t bo ung th

HTC-116, h xc nh c mt nhm cc cht 5a-f c hot tnh. Tt c

u l dn cht 4-alkyl piperidin vi n=3, trong 5f cho hot tnh mnh nht

v c hai cht 5e, 5f u c th so snh c vi SAHA.

Cc dn cht alkyl piperazin cho kt qu khng nh mong i.

Bng 1.4. Hot tnh sinh hc ca mt s dn cht alkyl piperidin

Cht X n IC50 (M)

HDAC HCT-116

5a

3 0,84 3,2

5b

3 0,54 2,7

5c

3 1,39 17,2

5d

3 0,26 5,1

5e

3 0,10 0,7

5f

3 0,09 0,3

SAHA 0,079 0,6

13

Kt hp cht c ch kp HDAC v HMGR

Mt dy cc cht c ch HDAC v 3-hydroxy-3-methylglutaryl

coenzym A reductase (HMGR) c to ra bng cch s dng nhm

hydroxamat v cu trc c bn ca statin kt ni hai protein. Nhng cht

ny cho thy tim nng c ch HDAC v HMGR vi gi tr IC50 mc nM.

Nhng cht ny cng c hiu qu c ch H G cng nh thc y acetyl

ha histon v tubulin trong t bo ung th, nhng khng gy c cho t bo

lnh [11].

Hnh 1.10. Thit k cht c ch kp da trn cu trc SAHA v Lovastatin

Cc cht c ch HDAC do nhm nghin cu b mn Ha Dc,

trng i hc Dc H Ni thit k v nghin cu.

Tip ni cc nghin cu pht trin thuc mi theo hng tc dng ln

ch l cc enzym HDAC, nhm nghin cu ti b mn Ha Dc trng i

hc Dc H Ni thit k v pht trin cc dn cht hydroxamic acid

mang khung 5-phenyl-1,3,4-thiadiazol mi. Kt qu cng b trn tp ch

Journal of Enzyme Inhibition and Medicinal Chemistry thng 10/2013 cho

thy mt s cht trong dy, v d 7b-d cho kt qu kh quan v kh nng c

ch HDAC cng nh c tnh trn nhiu dng t bo [17].

14

Bng 1.5. c tnh ca cc cht c ch HDAC tng hp c

Cht R c tnh trn cc dng t bo (IC50,

* M) Log

p SW620 MCF-7 PC3 AsPC-1 NCI-H460

7a H 0,70 1,80 0,88 2,71 1,07 1,35

7b 2-Cl 0,34 1,23 1,42 0,63 0,11 1,99

7c 3-Cl 0,45 1,23 1,76 1,10 1,94 1,99

7d 4-Cl 1,62 0,73 0,84 1,34 1,50 1,99

7e 4-F 3,58 8,05 2,92 1,88 4,79 1,55

7f 4-Br 0,72 1,27 0,83 0,08 1,42 2,24

7g 4-CH3 11,52 20,78 14,90 6,38 13,16 1,90

7h 4-OCH3 1,46 2,46 1,63 0,80 1,97 1,43

7i 4-N(CH3)2 6,61 6,67 7,35 7,16 8,18 1,53

7j 2-NO2 19,23 31,18 14,69 33,47 16,69 1,17

7k 4-NO2 26,71 76,25 25,57 76,25 76,25 1,17

7l 2,6-Cl2 16,21 16,29 15,18 14,95 14,38 2,64

7m 3,4-CH2OCH2- 1,89 2,15 2,54 3,04 3,34 1,41

7n 2,3,4-(OCH3)3 2,29 4,15 1,77 2,11 3,21 0,86

7o 3,4,5-(OCH3)3 4,31 3,50 1,89 6,02 10,20 0,86

SAHA 3,70 6,82 4,31 3,66 2,77 1,44

*Nng ca cht gy ra s gim 50% s lng t bo, s liu c ly trung

bnh ba kt qu vi lch di 10%.

c lng bng phn mm Kowin v1.67 US EPA.

SAHA: acid suberoylanilid hydroxamic.

D liu t bng trn cho thy cht 7a c c tnh mnh trn c nm

dng t bo. c bit trn hai dng SW620 v PC3, hiu lc ca 7a gp gn 5

ln SAHA. iu ny cho thy cc cht c cu trc cha d vng 1,3,4-

thiadiazol gia vng phenyl v nhm amid cho kt qu tt hn SAHA.

15

S c mt ca nhm halogen trn vng phenyl ti mt trong ba v tr

2,3,4 c th gi li c hoc tng nh tc dng (7b-d, 7f). Kt qu cho thy

v tr s 2 dng nh cho tc dng mnh nht. Tuy nhin khi thm mt nhm

Cl vo v tr s 6 (cht 7l) th tc dng gim r rt.

Ngoi ra, hai cht c cha nhm NO2 (7j v 7k) l nhng cht c hot

tnh thp nht trong s cc cht kho st.

Cc cht 7m-o cho c tnh thp hn 7a nhiu ln nhng so vi SAHA

th tng ng hoc thm ch mnh hn trn c 5 dng t bo.

Cc kt qu trn to nn tng cho tc gi pht trin hng nghin

cu trong thit k cng thc, tng hp. Bng vic gi nguyn cu trc acid

hydroxamic vi d vng 5 cnh 1,3,4-thiadiazol gia vng thm v nhm

amid, v thay vng phenyl bng d vng cha O hoc S, cc cht thu c

cho nhng kt qu kh quan v c c tnh trn cc dng t bo v tc dng

c ch HDAC. Kt qu c th s c trnh by trong cc chng tip theo

ca kha lun ny.

16

CHNG 2. NGUYN LIU, THIT B, NI

DUNG V PHNG PHP NGHIN CU

2.1. Nguyn liu

Cc ha cht, dung mi s dng trong qu trnh thc nghim l loi

dnh cho tng hp c nhp t cng ty Merck hoc Sigma-Aldrich v c

s dng trc tip khng c tinh ch g thm.

2.1.1. Ha cht chnh

Thiosemicarbazid

FeCl3.12H2O

Acid suberic monomethyl ester

Carbonyldiimidazol

Hydroxylamin

Cc benzaldehyd:

Furan-2-carbaldehyd

Thiophen-2-carbaldehyd

5-bromo-furan-2-carbaldehyd

5-methyl-furan-2-carbaldehyd

2.1.2. Dung mi v ha cht khc

DMF Aceton MeOH Acid acetic bng n-Hexan

HCl EtOH NaOH Dicloromethan Nc ct

2.2. Thit b, dng c

- Bnh cu y trn dung tch 50 ml c nt mi, my khuy t gia nhit,

sinh hn hi lu, my ct quay chn khng, t lnh, t sy, my siu m,

pipet, bnh chit, phu thy tinh, giy lc, cn phn tch, cn k thut, bnh

chy sc k lp mng (TLC).

- TLC tin hnh trn bn mng silicagel Merck 70-230 mesh.

- Nhit nng chy (Tonc) c xc nh bng my o nhit nng

chy nhit in (Electrothermal digital).

- Ph hng ngoi (I ) c ghi trn my Perkin Elmer, s dng k

17

thut vin nn KBr, ghi vng 4000-600 cm-1 ti phng th nghim b mn

ha vt liu i hc Khoa hc t nhin.

- Ph khi lng ( S) c ghi bng my khi ph Agilent 6310 Ion

Trap MS ca vin Ha hc cc hp cht t nhin Vit Nam.

- Ph cng hng t ht nhn proton 1H-N c ghi bng my

Bruker AV-500, dng DMSO-d6 lm dung mi. chuyn dch ha hc ()

c biu th bng n v phn triu (parts per million - ppm), ly mc l pic

ca cht chun ni tetramethylsilan (TMS).

2.3. Ni dung nghin cu

2.3.1. Tng hp ha hc

Tng hp 4 acid hydroxamic Va-d vi cng thc chung nh sau:

STT Cht R Tn

1 Va

N1-[5-(furan-2-yl)-1,3,4-thiadiazol-2-yl]-N

8-

hydroxyoctanediamid.

2 Vb

N1-hydroxy-N

8-[5-(thiophen-2-yl)-1,3,4-

thiadiazol-2-yl]octanediamid.

3 Vc

N1-[5-(3-bromofuran-2-yl)-1,3,4-thiadiazol-2-

yl]-N8-hydroxyoctanediamid.

4 Vd

N1-hydroxy-N

8-[5-(3-methylfuran-2-yl)-1,3,4-

thiadiazol-2-yl]octanediamid.

2.3.2. Th tc dng sinh hc ca cc cht tng hp c

- Th tc dng c ch HDAC bng phng php Western blot.

- Th tc dng khng t bo ung th i trng (SW620) in vitro.

- S b nh gi tnh ging thuc ca cc cht tng hp c.

18

2.4. Phng php nghin cu

2.4.1. Tng hp ha hc v kim tra tinh khit

Nghin cu tng hp 4 cht mc tiu bng phng php ha hc.

Dng TLC theo di qu trnh tin trin ca phn ng.

Kim tra tinh khit ca sn phm bng TLC v o nhit nng

chy.

Tng hp 4 cht trong kha lun ny c tin hnh theo s sau:

S 2.1. Quy trnh tng hp chung Va-d

Tc nhn v iu kin: i) thiosemicarbazid, ethanol, acid acetic bng; ii)

FeCl3.12H2O, ethanol; iii) acid monomethyl suberic, carbonyl diimidazol, DMF; iv)

hydroxylamin, NaOH, methanol.

2.4.2. Xc nh cu trc

Xc nh cu trc ca cc cht tng hp c da trn kt qu phn

tch cc ph IR, MS , 1H-N v 13C-NMR.

2.4.3. Th tc dng c ch HDAC

Tc dng c ch HDAC ca cc dn cht tng hp c nh gi gin

tip thng qua xc nh mc acetyl ha histon H3, H4 trong t bo ung th

i trng (SW620). Phn tch da trn Western blot c th khng nh c

tc dng ca cc mu th lm tng hoc gim s acetyl ha histon H3, H4 khi

so snh vi mu trng.

19

2.4.4. Th tc dng khng t bo ung th in vitro

a) Nguyn tc th

Th hot tnh khng t bo ung th (th c tnh t bo) in vitro c

thc hin ti Khoa Dc, Trng i hc uc gia Chungbuk, Cheong u,

Hn Quc theo phng php TT v gi tr IC50 c tnh trn phn mm

GraphPad Prism.

Dng t bo th nghim: t bo ung th i trng (SW620).

Dng t bo ung th c ly t Ngn hng t bo ung th ca Vin

nghin cu sinh hc v cng ngh sinh hc Hn Quc (K IBB) v c nui

cy trong mi trng RPMI b sung 10 BS (huyt thanh bo thai b).

c tnh t bo ca cc cht c th bng phng php TT theo

cc bc sau:

Chun b: Cc t bo pha logarit c trypsin ha v phn tn vo

hn dch n t bo trong mi trng RPMI b sung 10 BS v iu chnh

n nng khong 1,5.104 n 3,5.104 t bo, sau chia u vo cc ging

ca a 96 ging, mi ging 200 L. Cc a sau c 370Ctrong iu

kin 5% CO2. Sau 24 gi , cc mu th c chun b trong 20 L mi

trng RPMI b sung 10% FBS t dung dch gc 10 mg/mL trong dimethyl

sulfoxid (DMSO) ri thm 2 L mu th vo cc ging nhiu nng khc

nhau, cc a ny sau c thm 48 gi. Tt c cc mu c chun b

sao cho nng cui cng ca DMSO khng qu 0,1 . Tin hnh th: Sau

khi 4 gi, thm vo mi ging 20 L thuc nhum TT (nng TT 5

mg mL trong mui m phosphat - PBS). Cc a c thm 3 gi 370C

trong iu kin 5% CO2. Tip theo, mi ging c cho 100 L dung dch

D S , 5 pht cho TT formazan c ha tan. hp th c c

bc sng 510 nm.

b) Tnh kt qu

20

Gi tr IC50 l nng ca mu th m , hp th gim i 50

so vi nhm chng (trng m tnh l ging ch thm mi trng nui cy).

Kt qu cui cng l gi tr trung bnh ca 4 ln o c lp vi gi tr hp

th khc nhau khng qu 5 .

Gi tr IC50 c tnh da trn phn mm GraphPad Prism. Trong

phng php th ny, IC50 20 g/mL c coi l c hot tnh.

2.4.5. Gi tr LogP v nh gi mc ging thuc ca cc dn cht tng

hp c

Tnh cc gi tr logP bng phn mm EPIsuite cung cp bi US

Environmental Protection Agencys office of Pollution Prevention and Toxics

and Syracuse Research Corporation (SRC).

nh gi mc ging thuc ca cc dn cht da vo quy tc

Lipinsky [13]:

- Khi lng mol phn t ca cht < 500 g/mol.

- S trung tm nhn lin kt hydro (N, O) phi

21

CHNG 3. THC NGHIM, KT QU V

BN LUN

3.1. Tng hp ha hc

3.1.1. Tng hp cht N1-[5-(furan-2-yl)-1,3,4-thiadiazol-2-yl]-N8-

hydroxyoctandiamid (Va)

S 3.1. Quy trnh tng hp cht Va

3.1.1.1. Tng hp cht IIa

Tng hp 2-(furan-2-ylmethyliden)hydrazincarbothioamid (IIa) t

furan-2-carbaldehyd (Ia) c thc hin bng phn ng ngng t vi xc tc

l acid hu c, tin hnh theo s 3.2.

S 3.2. Tng hp cht IIa

Tin hnh phn ng:

Cho 0,20 ml (2 mmol) furan-2-carbaldehyd (Ia) v 0,2180g (2,4 mmol)

thiosemicarbazid vo bnh cu 50 mL. Thm 20 ml dung mi EtOH tuyt i

v 2 git acid acetic bng lm xc tc. un hi lu v khuy 300 vng/pht

trong 4h.

22

Thu sn phm:

Ct thu hi dung mi bng my ct quay nhit 400C sau thm

15 mL nc ct ta sn phm. Lc v ra ta 3 ln bng 15 mL nc ct.

Sy kh ta 600C (trong khong 24h).

Kt qu:

Cm quan: Bt tinh th mu trng .

Hiu sut: 91% (0,3070g).

T0nc: 165-1660C.

Kim tra bng TLC vi pha ng DCM : MeOH (9:1) cho Rf = 0,66.

Ton b sn phm IIa c dng thc hin bc tip theo ca qu

trnh tng hp cht Va.

3.1.1.2. Tng hp cht IIIa

Chng ti thc hin vic tng hp cht 5-(furan-2-yl)-1,3,4-thiadiazol-

2-amin (IIIa) t IIa bng phn ng ng vng ni phn t. Qu trnh tng

hp c thc hin theo s 3.3.

S 3.3. Tng hp cht IIIa

Tin hnh phn ng:

Cn 0,81g (3 mmol) mui st FeCl3.12H2O, ha tan bng 3 mL dung

mi EtOH trong bnh cu 50 mL, gia nhit n hi lu. ng thi cn

0,1690g (1 mmol) cht IIa, ha tan bng 20 mL dung mi EtOH tuyt i

trong cc c m, un nng khong 70C.

dung dch cht IIa t cc c m vo bnh phn ng, tip tc un hi

lu v khuy 300 vng/pht. Kt thc phn ng sau 3 gi.

Thu sn phm:

23

Ct thu hi bt dung mi bng my ct quay nhit khong 400C

trong 2 pht sau pha long hn hp phn ng bng nc ct. Kim ha

hn hp bng khong 10 mL dung dch NaOH 30% (kim tra bng giy qu).

Chit 3 ln bng cloroform (mi ln 5-10 mL), thu lp di. Gp dch

chit ca 3 ln chit li vi nc mui bo ha. Ct thu hi dung mi ca dch

chit bng my ct quay nhit 400C n khi ht dung mi, thu sn phm

IIIa. Sy sn phm 600C trong 4h.

Kt qu:

Cm quan: Bt kt tinh mu vng nht.

Hiu sut: 72% (0,1200g).

T0nc: 196-1970C.


3.1.1.3. Tng hp cht IVa

Cht methyl 8-{[5-(furan-2-yl)-1,3,4-thiadiazol-2-yl]amino}-8-

oxooctanoat (IVa) c tng hp t cht IIIa bng phn ng acyl ha ca

cht IIIa vi acid monomethyl suberic. Quy trnh tng hp c thc hin

nh sau:

S 3.4. Tng hp cht IVa

Tin hnh phn ng:

Cho 0,1620g (1 mmol) 1,1-carbonyldiimidazol (CDI) v 1mL (1

mmol) acid monomethyl suberic vo bnh cu 50mL. Thm 3mL dung mi

DMF. Hot ha trong 5 pht nhit phng. Sau khi hn hp trong bnh

phn ng hot ha thi gian, cho 0,1670g (1 mmol) cht IIIa vo bnh

phn ng, gia nhit ln 600C, khuy 300 vng/pht trong vng 24h.

24

Thu sn phm:

Lm lnh bnh phn ng bng nc . t t 15 mL dung dch HCl

long, lnh vo bnh phn ng, va va lc. yn 15 pht.

Lc v ra ta 3 ln bng 15 mL dung dch acid HCl long, lnh. Sy

ta 600C trong 24h.

Kt qu:

Cm quan: Bt kt tinh mu trng hi hng.

Hiu sut: 57% (0,1920g).

T0nc: 236-2380C.


3.1.1.4. Tng hp cht Va

Cht N1-[5-(furan-2-yl)-1,3,4-thiadiazol-2-yl]-N8-hydroxyoctandiamid

(Va) c tng hp bng phn ng gia cht IVa vi hydroxylamin

hydroclorid. Tng hp cht Va c thc hin nh sau:

S 3.5. Tng hp cht Va

Tin hnh phn ng:

Cho 0,1690g (0,5 mmol) cht IVa v 0,33g (5 mmol) hydroxylamin

hydroclorid vo bnh cu 50 mL, thm 20 mL dung mi MeOH, dng siu m

to hn dch ng nht. t bnh phn ng trong hn hp nc v mui

n, duy tr nhit di 00C, khuy 300 vng/pht.

Ha tan 0,40g (10mmol) NaOH trong 5 ml nc ct trong ng nghim

v lm lnh xung di 00C trong hn hp nc mui . dung dch

NaOH vo bnh phn ng, gi nhit di 00C, phn ng kt thc sau 1,5

gi. Theo di tin trnh phn ng v xc nh thi im kt thc bng cch

dng dung dch FeCl3 v TLC, c th lm nh sau:

25

- Acid ha khong 0,1 mL hn hp phn ng bng 1 git dung dch HCl

long trn khay s, sau nh 1 git dung dch FeCl3 5%, xut hin mu

vang chng t c acid hydroxamic c to ra.

- Acid ha khong 0,1 mL hn hp phn ng, kim tra TLC vi pha

ng DCM : MeOH (9:1). Kt thc phn ng khi ester ban u ht.

Thu sn phm:

Acid ha hn hp phn ng bng dung dch 20 mL dung dch HCl

long, lnh iu chnh pH v khong 4-5 (kim tra bng giy qu vn nng).

qua m sau lc v ra ta 3 ln bng 15 mL dung dch HCl long. Sy

ta 600C trong 24h.

Kt qu:

Cm quan: Bt kt tinh mu trng.

Hiu sut: 51,0% (0,0860g).

Kim tra bng TLC v xc nh cu trc (kt qu c th c trnh by

trong bng 3.1 v phn 3.3).

T0nc: 202-2040C.

3.1.2. Tng hp cht N1-hydroxy-N8-[5-(thiophen-2-yl)-1,3,4-thiadiazol-

2-yl]octandiamid (Vb)

Cht Vb c tng hp theo s 3.6.

S 3.6. Quy trnh tng hp cht Vb

26

3.1.2.1. Tng hp cht IIb

Tng hp cht IIb t 0,24mL (2mmol) cht thiophen-2-carbaldehyd

(Ib) c thc hin tng t nh tng hp cht IIa. Kt qu nh sau:


Hiu sut: 86% (0,3180g).

T0nc: 170-1720C.


3.1.2.2. Tng hp cht IIIb

Tng hp cht 5-(thiophen-2-yl)-1,3,4-thiadiazol-2-amin (IIIb) t

0,185g (1 mmol) cht IIb c tin hnh tng t nh tng hp cht IIIa.

Kt qu nh sau:

Cm quan: Bt kt tinh mu vng nu.

Hiu sut: 70% (0,1300g).

T0nc: 184-1820C.


3.1.2.3. Tng hp cht IVb

Tng hp cht methyl 8-oxo-8-{[5-(thiophen-2-yl)-1,3,4-thiadiazol-2-

yl]amino}octanoat (IVb) c tin hnh tng t nh tng hp cht IVa.

Kt qu ca qu trnh nh sau:

Cm quan: Bt kt tinh mu trng .

Hiu sut: 60% (0,2130g).

T0nc: 196-1980C.


3.1.2.4. Tng hp cht Vb

Tng hp cht ch th hai N1-hydroxy-N8-[5-(thiophen-2-yl)-1,3,4-

thiadiazol-2-yl]octanediamid (Vb) t 0,1775g ( 0,5 mmol) cht IVb c

tin hnh tng t nh tng hp cht Va. Kt qu nh sau:

27


Hiu sut phn ng: 55% (0,9790g).

T0nc: 177-1780C.

Kim tra bng TLC v xc nh cu trc (kt qu c th c trnh by

trong bng 3.1 v phn 3.3).

3.1.3. Tng hp cht N1-[5-(5-bromofuran-2-yl)-1,3,4-thiadiazol-2-yl]-N8-

hydroxyoctandiamid (Vc)

Cht Vc c tng hp theo s 3.7.

S 3.7. Quy trnh tng hp cht Vc

3.1.3.1. Tng hp cht IIc

Tng hp cht IIc t 0,3500g (2 mmol) cht 5-bromofuran-2-

carbaldehyd (Ic) c thc hin tng t nh tng hp cht IIa. Kt qu

nh sau:

Cm quan: Sn phm thu c l bt kt tinh mu trng.

Hiu sut: 85% (0,4199g).

T0nc: 171-1720C.


3.1.3.2. Tng hp cht IIIc

Tng hp cht 5-(5-bromofuran-2-yl)-1,3,4-thiadiazol-2-amin (IIIc) t

0,2480g (1 mmol) cht IIc c tin hnh tng t nh tng hp cht IIIa.

28

Kt qu nh sau:


Hiu sut: 70% (0,1722g).

T0nc: 192-1940C.


3.1.3.3. Tng hp cht IVc

Tng hp cht methyl 8-{[5-(5-bromofuran-2-yl)-1,3,4-thiadiazol-2-

yl]amino}-8-oxooctanoat (IVc) t 0,2460g (1 mmol) cht IIIc c tin hnh

tng t nh tng hp cht IVa. Kt qu ca qu trnh nh sau:

Cm quan: Bt kt tinh mu trng hi hng.

Hiu sut: 64% (0,2662g).

T0nc: 210-2120C.


3.1.3.4. Tng hp cht Vc

Tng hp cht ch th ba N1-[5-(5-bromofuran-2-yl)-1,3,4-thiadiazol-

2-yl]-N8-hydroxyoctandiamid (Vc) t 0,2080g (0,5 mmol) cht IVc c tin

hnh tng t nh tng hp cht Va. Kt qu ca qu trnh nh sau:



T0nc: 202-2040C.

Kim tra bng TLC v xc nh cng thc (kt qu c th c trnh

by trong bng 3.1 v phn 3.3).

3.1.4. Tng hp cht N1-hydroxy-N8-[5-(5-methylfuran-2-yl)-1,3,4-

thiadiazol-2-yl]octandiamid (Vd)

Cht Vd c tng hp theo s 3.8.

29

S 3.8. Quy trnh tng hp cht Vd

3.1.4.1. Tng hp cht IId

Qu trnh tng hp cht IId t 0,24 mL (2 mmol) cht 5-methylfuran-

2-carbaldehyd (Id) c thc hin tng t cht IIa. Kt qu nh sau:

Cm quan: Bt kt tinh mu trng hi vng.

Hiu sut: 86% (0,3148g).

T0nc: 170-1720C.


3.1.4.2. Tng hp cht IIId

Tng hp cht 5-(5-methylfuran-2-yl)-1,3,4-thiadiazol-2-amin (IIId) t

0,1830g (1 mmol) cht IId c tin hnh tng t nh tng hp cht IIIa.

Kt qu nh sau:


Hiu sut: 71% (0,1285g).

T0nc: 182-1840C.


3.1.4.3. Tng hp cht IVd

Tng hp cht methyl 8-{[5-(5-methylfuran-2-yl)-1,3,4-thiadiazol-2-

yl]amino}-8-oxooctanoat (IVd) t 0,1810g (1 mmol) cht IIId c tin

hnh tng t nh tng hp cht IVa. Kt qu nh sau:

30

Cm quan: Bt mu trng hi hng.

Hiu sut: 58% (0,2036g).

T0nc: 196-1980C.


3.1.4.4. Tng hp cht Vd

Tng hp cht ch th t N1-hydroxy-N8-[5-(5-methylfuran-2-yl)-

1,3,4-thiadiazol-2-yl]octandiamid (Vd) t 0,1755g (0,5 mmol) cht IVd c

tin hnh tng t nh tng hp cht Va. Kt qu nh sau:



T0nc: 170-1720C.

Kim tra bng TLC v xc nh cng thc (kt qu c th c trnh

by trong bng 3.1 v phn 3.3).

3.2. Kim tra tinh khit

tinh khit cc cht Va-d c kim tra bng TLC v o nhit

nng chy (Tonc) ca chng.

TLC c tin hnh trn bn nhm trng sn silicagel Merck 70-230

mesh, quan st di n t ngoi bc sng 254 nm. Ha tan trong dung

mi D v sau kho st vi cc h dung mi trin khai. Kt qu thu c

trn cc sc k u thy mt vt r, gn khi soi di nh sng n t

ngoi.

o nhit nng chy: 4 cht sau khi c tinh ch bng cch kt tinh

li u c dng tinh th rn c o nhit nng chy bng my o nhit

nng chy nhit in (Electrothermal digital).

Nhit nng chy v gi tr Rf c tm tt trong bng 3.1.

31

Bng 3.1. Gi tr Rf v T0

nc ca cc cht Va-d

STT Cht R Hqu trnh T

0nc

(0C)

Rf

H DCM : MeOH (9:1)

1 Va

19% 202-204 0,47

2 Vb

20% 177-178 0,45

3 Vc

20% 202-204 0,45

4 Vd

20% 170-172 0,46

3.3. Xc nh cu trc

khng nh cu trc ha hc ca cc cht tng hp c, chng ti

cn c vo vic phn tch d liu ph hng ngoi (IR), ph khi lng (MS)

v ph cng hng t ht nhn (1H-NMR v 13C-NMR) [1,2].

3.3.1. Ph hng ngoi (IR)

Ti phng th nghim khoa Ha trng i hc Khoa hc t nhin, cc

cht c ghi ph hng ngoi (IR) trn my Perkin Elmer vi k thut lm

vin nn KBr ghi trong vng 4000-600 cm-1

. Cc ph c ph lc.

Bng 3.2. S liu phn tch ph IR ca Va-d

STT Cht R

S sng ng vi dao ng ha tr (cm-1)

C=C C=O C-H

(benzen)

C-H

(CH3)

O-H N-H

1 Va

1567

1504

1465

1637

1673

2923

2865

2853

3350 3152

32

2 Vb

1563

1461

1436

1674

1637

2931

2854 3316 3154

3 Vc

1561

1499

1464

1690

1638 3025

2931

2858 3245 3158

4 Vd

1567

1552

1464

1718

1694

1651

3032 2925

2856 3416 3158

Nhn xt: S liu phn tch ph IR hon ton ph hp vi cc ti liu

tham kho cng nh cu trc d kin ca cc cht Va-d c th nh sau:

Cc cht ch Va-d u cho d liu ph ph hp vi cc nhm chc d

kin trong cng thc: Vng 1436-1567 cm-1 c trng cho dao ng C=C ca

vng thm, vng 1637-1718 cm-1 c trng cho dao ng C=O ca nhm

carbonyl, vng 3400-3200 cm-1

c trng cho O-H v 3150 cm-1 cho N-H.

3.3.2. Ph khi lng (MS)

Bn cnh phng php o ph IR, phng php o ph khi lng

ch khng phn mnh CI-MS v ESI gip khng nh thm cu trc ca

cc cht tng hp c. Kt qu phn tch ph MS trnh by trong bng 3.3.

Bng 3.3. S liu phn tch ph khi lng ca cc cht

Cht R M m/z

Va

338 337 [M-H]-

Vb

354 353 [M-H]-

Vc

417 416 [M-H]-

Vd

352 351 [M-H]-

33

Nhn xt: S liu ph MS bng 3.3 cho thy rng cc cht kho st

u c pic phn t ch ra s khi ng bng s khi d kin.

3.3.3. Ph cng hng t ht nhn 1H-NMR v 13C-NMR

khng nh chc chn hn cu trc, chng ti tin hnh ghi ph 1H-

NMR ca 4 cht ti phng Phn tch ph - Vin Ha hc - Vin Khoa hc v

Cng ngh Vit Nam, dng DMSO lm dung mi, chun ni tetramethylsilan

(TMS). Kt qu ph 1H-N c trnh by trong bng 3.4 v trong ph lc.

Bng 3.4. S liu phn tch ph 1H-NMR

Cht R (ppm) (500 MHz, DMSO-d6)

Va

10,35 (1H, s, NH); 7,93 (1H, s, H5); 7,18 (1H, d, J = 3,0 Hz,

H4); 6,72 (1H, s, H3); 2,47-2,49 (2H, m, CH2); 1,92-1,95 (2H,

m, CH2); 1,58-1,61 (2H, m, CH2); 1,47-1,49 (2H, m, CH2); 1,26-

1,28 (4H, m, CH2).

Vb

12,65 (H, s, NH); 10,34 (1H, s, NH); 8,67 (1H, s, OH); 7,75

(1H, d, J = 4,5 Hz, H5); 7,69 (1H, d, J = 2,5 Hz, H4); 7,19 (H,

s, H3); 2,47-2,48 (2H, m, CH2); 1,92-1,95 (2H, m, CH2); 1,58-

1,60 (2H, m, CH2); 1,47-1,49 (2H, m, CH2); 1,26 (4H, m, CH2).

Vc

12,70 (1H, s, NH); 10,33 (1H, s, NH); 7,23 (1H, d, J = 3,5 Hz,

H4); 6,83 (1H, d, J = 3,5 Hz, H3); 2,47-2,49 (2H, m, CH2);

1,92-1,95 (2H, m, CH2); 1,57-1,61 (2H, m, CH2); 1,45-1,51 (2H,

m, CH2); 1,26-1,28 (4H, m, CH2).

Vd

12,64 (1H, s, NH); 10,36 (1H, s, NH); 7,04 (1H, d, J = 3 Hz,

H3); 6,32 (1H, d, J = 2,5 Hz, H4); 2,47-2,48 (2H, m, CH2);

2,36 (3H, s, CH3); 2,17-2,20 (1H, m, CH2); 1,92-1,95 (1H, m,

CH2); 1,58-1,60 (2H, m, CH2); 1,46-1,48 (2H, m, CH2); 1,20-

1,34 (4H, m, CH2);

34

Nhn xt: S c mt ca cc proton ph hp vi cng thc ca 4 cht

Va-d. Trn ph c cc pic c trng ca cc proton trong c phn khung

5-aryl-1,3,4-thiadiazol v proton ca cc nhm chc, nhm th. Tuy nhin i

vi cht Va tn hiu ca proton nhm NH yu; Va, Vc v Vd khng c nh

tn hiu proton -H. Nguyn nhn l do khi o b sung thm dung mi

e D ha tan cht v cc H linh ng ny trao i vi D trong dung

mi MeOD.

Ph 13C-N c trnh by trong bng 3.5 v trong ph lc.

Bng 3.5. S liu phn tch ph 13C-NMR

Cht Cng thc (ppm) (125 MHz, DMSO-d6)

Va

171,66 (C5); 169,10 (C8); 157,66 (C1); 152,38 (C2);

145,17-145,31 (C5-C2); 112,52 (C3); 110,75 (C4);

34,80 (C7); 32,19 (C2); 28,21-28,25 (C3-C6); 24,40-24,93

(C4-C5).

Vb

171,61 (C5); 169,10 (C8); 157,79 (C1); 156,10 (C2);

132,32 (C2); 129,00-129,11 (C5-C3); 128,39 (C4);

34,80 (C7); 32,21 (C2); 28,20-28,28 (C3-C6); 24,45-24,96

(C4-C5).

Vc

171,71 (C5); 169,13 (C8); 157,82 (C1); 151,33 (C2);

147,07 (C2); 124,45 (C5); 114,59 (C3); 113,57 (C4);

34,81 (C7); 32,21 (C2); 28,25-28,21 (C3-C6); 24,94 (C4);

24,41 (C5).

Vd

171,64 (C5); 169,21 (C8); 157,15 (C1); 154,60 (C5);

152,54 (C2); 143,57 (C2); 108,85-112,03 (C3-C4);

34,81 (C7); 33,63 (C2); 32,21 (C3); 28,19 (C6); 24,31-24,95

(C4-C5); 13,38 (CH3).

35

Nhn xt: S c mt ca cc tn hiu cng hng ht nhn carbon ph

hp vi cng thc cu to ca 4 cht Va-d. Trn ph , cc pic c trng

ca cc carbon trong c phn khung 5-aryl-1,3,4-thiadiazol v carbon ca cc

nhm chc, nhm th.

3.4. Hot tnh sinh hc v ging thuc

3.4.1. Hot tnh sinh hc

Th hot tnh khng t bo ung th (th c tnh t bo) in vitro c

thc hin ti Khoa Dc, Trng i hc uc gia Chungbuk, Cheong u,

Hn Quc theo phng php TT v gi tr IC50 c tnh trn phn mm

GraphPad Prism. Chng ti s b kho st hot tnh ca cc cht Va-d

trn dng t bo ung th SW620. c th so snh y , chng ti cng

kho st hot tnh ca Vorinostat (SAHA) trn dng t bo ny.

Th tc dng c ch HDACcng c thc hin ti Khoa Dc,

Trng i hc uc gia Chungbuk, Cheong u, Hn uc.

Bng 3.6. Kt qu th hot tnh v tc dng c ch HDAC

Cht

KLPT

Tc dng

c ch

HDAC*

Tc dng khng t bo ung th SW6201

IC50 (g/mL)2 IC50 (M)

2

Va

33 + 0,09 0,29

Vb

354 + 0,099 0.2

Vc

427 + 0,107 0,25

Vd

352 + 0,22 0,65

SAHA 264 + 0.976 3,70

36

Ghi ch: *Hot tnh ca enzym b c ch hon ton khi th ti 10 g/mL bng phng php

Western Blot. 1Dng t bo SW620: ung th rut kt.

2Nng (g/mL hoc M) lm c ch 50% s pht trin ca t bo. Cc kt qu

c ly trung bnh qua 3 ln th nghim vi sai lch khng qu 10%.

3.4.2. nh gi mc ging thuc

Di y l bng nh gi da theo Lipinsky ca cc cht Va-d.

Bng 3.7. nh gi mc ging thuc theo quy tc Lipinsky

Cht R X LogP KLPT S NH, OH S N, O S lin kt

linh ng

Va H O 0,72 338 3 8 3

Vb H S 1,17 292 3 8 3

Vc Br O 1,61 417 3 8 3

Vd CH3 O 1,27 352 3 8 3

Yu cu < 5 < 500 < 5 < 10 < 15

Nhn xt: Cc cht tng hp c u tha mn quy tc Lipinsky.

3.5. Bn lun

3.5.1. Ha hc

Cc acid hydroxamic mang khung 5-aryl-1,3,4-thiadiazol c tng

hp theo quy trnh 4 bc.

Trong bc u tin, cc thiosermicarbazon IIa-d c to nn bng

cch ngng t cc benzaldehyd Ia-d vi thiosemicarbazid. Phn ng xy ra

d dng v t hiu sut cao.

Bc th 2, phn ng to vng ni phn t ca cc cht IIa-d vi tc

nhn FeCl3 to thnh cc dn xut 2-amino-5-aryl-1,3,4-thiadiazol (IIIa-d).

37

Trong phn ng ny, tc nhn oxi ha (v d cc acid Lewis nh FeCl3,

NH4Fe(SO4)2,...) ng vai tr quan trng.

S 3.9. C ch phn ng ng vng tng hp 1,3,4-thiadiazol

Bc th 3, ghp ni IIIa-d vi suberic acid monomethyl ester s dng

CDI nh mt tc nhn hot ha to ra IVa-d [5].

S 3.10. C ch hot ha ca CDI

Thc hin bc ny dng c phi kh hon ton, cc ha cht v dung

mi phi khan nc do CDI d phn hy dn n khng cn kh nng hot

ha acid monomethyl suberic nu tip xc vi nc.

Bc cui cng, phn ng gia ester IVa-d vi hydroxylamin trong

mi trng kim cho ra sn phm cui cng Va-d. trnh phn hy ester

38

thnh acid carboxylic bi dung dch Na H m c, cn lm lnh hn dch

IVa-d v Na H trc khi cho vo bnh phn ng. Phn ng cng cn thc

hin nhit thp v thi gian ngn trnh phn hy nhm amid. Lng

NaOH phi d chuyn ht NH2OH.HCl thnh NH2 H tan v m bo acid

hydroxamic to thnh tan ht trong dung dch phn ng.

Cu trc ca cc cht thu c xc nh trc tip bng cc php o

quang ph, bao gm IR, MS, 1H-NMR, 13C-NMR.

3.5.2. Hot tnh sinh hc

Kt qu th hot tnh cc cht c th hin trong bng 3.7 di y.

Bng 3.7. So snh kt qu ca Va-d vi SAHA v mt s cht

Tn

KLPT

Tc dng

c ch

HDAC*

Tc dng khng t bo ung th

SW6201

IC50 (g/mL)2 IC50 (M)

Va

33 + 0,09 0,29

Vb

354 + 0,099 0,2

Vc

427 + 0,107 0,25

Vd

352 + 0,22 0,65

7a

3 4 + 0,269 0,70

7f

427 + 0,307 0,72

7g

362 + 4,170 11,52

SAHA 264 + 0.976 3,70

Ghi ch: *Hot tnh ca enzym b c ch hon ton khi th ti 10g/mL bng phng php

Western Blot.

39

1Dng t bo SW620: dng t bo ung th i trng.

2Nng (g/mL hoc M) lm c ch 50% s pht trin ca t bo. Cc kt qu

c ly trung bnh qua 3 ln th nghim vi sai lch khng qu 10%.

V tc dng c ch HDAC, bng trn cho thy tt c cc cht ny u

c tc dng c ch hon ton HDAC nng 10 g mL. Tuy nhin c

th gy ra c tnh vi t bo, cc cht phi c kh nng thm qua mng sinh

hc v tip cn c vi HDAC. Do chng ti tip tc hnh th c tnh t

bo in vitro.

Th nghim trn dng t bo ung th i trng SW620, hot tnh sinh

hc ca cc cht u gp t 5-15 ln SAHA (gi tr IC50 ca Va-d t 0,25 M

n 0,65 M so vi 3,70 M), trong cht cho tc dng tt nht l Vc (IC50

= 0,25 M) c kh nng khng li loi t bo ung th ny vi hot tnh gp

gn 15 ln so vi SAHA. iu ny cho thy vic s dng khung 5-aryl-1,3,4-

thiadiazol m c th l vic s dng d vng (furan, thiophen) cho kt qu rt

kh quan.

Thm ch, so vi kt qu th hot tnh ca mt s hp cht mang khung

5-phenyl-1,3,4-thiadiazol m nhm nghin cu ti b mn Ha Dc trng

i hc Dc H Ni cng b trc y (cc cht 7a, 7f, 7g), c th thy

cc cht ny cho hot tnh sinh hc mnh hn hn. Hai cht Va v Vb s

dng vng furan v thiophen thay v vng phenyl trong 7a u cho hot tnh

gp hn 2 ln. Tng t, hot tnh ca Vc cao hn 7f v Vd cao hn 7g.

Ngoi ra cng c th thy cht Vb (s dng vng thiophen) cho hot

tnh vi IC50 = 0,28 M tt hn Va (s dng vng furan) c IC50 = 0,29 M.

V trn cng vng furan, nhm th -CH3 cho hp cht Vd c IC50 = 0,65 M

gp hn 2,5 ln Vc s dng nhm th -Br (IC50 = 0,25 M). C th gii thch

iu nay l do nhm th methyl lm cho mt phng vng furan xoay i

nhiu so vi vng thiadiazol v khng ph hp vi trung tm hot ng ca

HDAC.

40

T nhng kt qu thu c, c th khng nh cc acid hydroxamic c

khung 5-aryl-1,3,4-thiadiazol vi cu ni 6C nh trnh by trn ph hp

em li hot tnh c ch HDAC cng nh khng t bo ung th.

Thm vo , cc cht mi tng hp c khng ch c hot tnh sinh

hc cao m cn tha mn cc yu cu ca quy tc Lipinsky, iu ny m ra

trin vng cho vic nghin cu lm sng v cn lm sng ca cc cht sau

ny. Tm li, khung 5-aryl-1,3,4-thiadiazol c nhiu trin vng thay th vng

phenyl ca SAHA nhm tm ra cc thuc c ch HDAC mi.

41

KT LUN V KIN NGH

1. Kt lun

T nhng kt qu nghin cu trnh by trn y c th rt ra mt s kt

lun nh sau:

Tng hp v khng nh cu trc

tng hp c 4 cht:

N-[5-(furan-2-yl)-1,3,4-thiadiazol-2-yl]-N'-hydroxyoctandiamid (Va).

N-hydroxy-N'-[5-(thiophen-2-yl)-1,3,4-thiadiazol-2-yl]octandiamid (Vb).

N-[5-(3-bromofuran-2-yl)-1,3,4-thiadiazol-2-yl]-N'-hydroxyoctandiamid (Vc).

N-hydroxy-N'-[5-(3-methylfuran-2-yl)-1,3,4-thiadiazol-2-yl]octandiamid (Vd).

khng nh cu trc cc cht tng hp c nh phn tch cc d

liu ph IR, MS, 1H-NMR, 13C-NMR. C 4 cht Va-d u cha thy cng b

trong cc ti liu tham kho.

Th hot tnh sinh hc

V tc dng c ch HDAC, c 4 cht u c tc dng c ch HDAC.

Tc dng khng t bo ung th in vitro th c 4 cht Va-d u c hot tnh c

ch mnh vi dng t bo SW620. Tt c u cho hot tnh gp nhiu ln

SAHA, trong Vc c hot tnh mnh nht vi IC50 = 0,25 M.

2. Kin ngh

Vi cc kt qu t c, chng ti kin ngh:

Tin hnh th c tnh trn t bo ung th v hot tnh c ch histon

deacetylase ca cc dn cht Va-d trn cc dng t bo thng v cc dng

t bo ung th khc. ua nh gi cc kt qu thu c lm c s cho

vic th tc dng in vivo.

Nghin cu tng hp v th tc dng dy cht thay th khung 5-aryl-

1,3,4-thiadiazol bng cc nhm ng cu in t v vng thm khc.

42

Ph lc

Ph lc 1 : Ph hng ngoi (IR) ca cht Va

Ph lc 2 : Ph khi lng (MS) ca cht Va

Ph lc 3 : Ph 1H-NMR ca cht Va

Ph lc 4 : Ph 13

C-NMR ca cht Va

Ph lc 5 : Ph hng ngoi (IR) ca cht Vb

Ph lc 6 : Ph khi lng (MS) ca cht Vb

Ph lc 7 : Ph 1H-NMR ca cht Vb

Ph lc 8 : Ph 13

C-NMR ca cht Vb

Ph lc 9 : Ph hng ngoi (IR) ca cht Vc

Ph lc 10 : Ph khi lng (MS) ca cht Vc

Ph lc 11 : Ph 1H-NMR ca cht Vc

Ph lc 12 : Ph 13

C-NMR ca cht Vc

Ph lc 13 : Ph hng ngoi (IR) ca cht Vd

Ph lc 14 : Ph khi lng (MS) ca cht Vd

Ph lc 15 : Ph 1H-NMR ca cht Vd

Ph lc 16 : Ph 13

C-NMR ca cht Vd

TI LIU THAM KHO

Ting Vit Nam

1. Trn Mnh Bnh, Nguyn uang t, Ha hc hu c tp 1-2, Nxb Y

hc, 2007, H Ni.

2. Nguyn Hi Nam, Lin quan gia cu trc v tc dng sinh hc, Ti liu

sau i hc, 2011, Nh xut bn y hc.

3. o Th Kim Oanh, Tng hp v th hot tnh sinh hc ca mt s n

cht acid hydroxamic hng c ch enzym histon deacetylase, Lun n

tin s Dc hc, Th vin i hc Dc H Ni, 2013, H Ni.

Ting Anh

4. Chittari P, Brian G, et al. Design and synthesis of aryl ether and sulfone

hydroxamic acids as potent histone deacetylase (HDAC) inhibitors.

Bioorganic and Medicinal Chemistry, 2011; pp 324-328.

5. Christian A. G. N. Montalbettiand Virginie Falque. Amide bond

formation and peptide coupling. Tetrahedron, 2005; 61: 10827-10852.

6. Drummond DC, Noble CO, Kirpotin DB, Guo Z, Scott GK, Benz CC.

Clinical development of histone deacetylase inhibitors as anticancer

agents. Annu. Rev. Pharmacol. Toxicol, 2005; 45:495528.

7. Glaser KB. HDAC inhibitors: clinical update and mechanism-based

potential. Biochem Pharmacol, 2007; 74: 659-871.

8. Gray SG and Esktrom TJ. The human histone deacetylase family. Exp

Cell Res, 2001; 262: 75-83.

9. Harish R, Avantika A, et al. 2,5-disubstituted-1,3,4-

oxadiazole/thiadiazole hydroxamic acid as surface recognition moiety:

Design and synthesis of novel hydroxamic acid based histone

deacetylase inhibitors.Bioorganic and Medicinal Chemistry, 2011, 21:

5735-5738.

10. Jessica E. Bolden, Melissa J. Peart and Ricky W. Johnstone. Anticancer

activities of histone deacetylase inhibitors. Nature Publishing Group,

2006; 5: 769-784.

11. Jhih-Bin C, Ting-Rong C, et al. Design and Synthesis of Dual-Action

Inhibitors Targeting Histone Deacetylases and 3-Hydroxy-3-

methylglutaryl Coenzyme A Reductase for Cancer Treatment. J. Med.

Chem., 2013, 56 (9), 36453655.

12. Johnstone RW. Histone-deacetylase inhibitor: novel drugs for the

treatment of cancer. Nature Rev Drug Disc, 2002;1: 287-99.

13. Lipinski C.A., et al. Experimental and computational approaches to

estimate solubility and permeability in drug discovery and development

settings. Advanced Drug Delivery Reviews, 1997; 46: 3-26.

14. Lu A, Luo H, Shi M, Wu G, Yuan Y, Liu J. Design, synthesis and

docking studies on benzamide derivatives as histone deacetylase

inhibitors.Bioorg Med Chem Lett 21, 2011, pp. 49247.

15. MA Glozak and E Seto. Histone deacetylase and cancer. Oncogene,

2006; 26: 5420-5432.

16. Marks PA, Rifkind RA, Richon VM, et al. Histone deacetylases and

cancer: causes and therapies. Nature Rev Cancer 2001; 1: 194-202.

17. Nam NH, Huong TL, Dung DTM, et al. Synthesis, bioevaluation and

docking study of 5-substitutedphenyl-1,3,4-thiadiazole-based

hydroxamic acids as histone deacetylase inhibitors and antitumor agents.

J Enzyme Inhib Med Chem,2013.

18. Peng Guan, enge Sun, et al. Design, synthesis and preliminary

bioactivity studies of 1,3,4-thiadiazole hydroxamic acid derivatives as

novel histone deacetylase inhibitors. Bioorganic and Medicinal

Chemistry 20, 2012, pp 3865-3872.

19. Rossi C, Porcelloni M, et al. Alkyl piperidine and alkyl piperazine

hydroxamic acids as HDAC inhibitors. Bioorganic and Medicinal

Chemistry, 2011, 21(8): 2305-2308.

20. Ruijter de A.J el al. HDACs: Characterization of classical HDAC family

J. Biol. Chem, 2003; 370: 737-49.

21. Verdin E, Dequiedt F and Kasler HG. Class II histone deacetylase:

versatile regulator. Trend Genet, 2003; 19: 286-293.

22. Witt O, Deubzer HE, Milde T, Oehme I. HDAC family: what are the

cancer relevant targets? Cancer Lett, 2009; 277: 8-21.

23. Youngson Robert M.Collins Dictionary of Human Biology,

HarperCollins,London, 2006.

Ph lc 1. Ph hng ngoi (IR) cht Va

Ten may: GX-PerkinElmer-USA Nguoi do: Nguy en Thi Son DT: 0912140352

Mail: [email protected]: 4cm-1

BO MON HOA VAT LIEU-KHOA HOA-TRUONG DHKHTN

MAU 16ADate: 3/13/2013

4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600.0

0.0

10

20

30

40

50

60

70

80

90

101.6

cm-1

%T

3350

3152

2923

2865

2850

1673

1637

1567

1504

1465

1444

1428

1384

1342

1318

1306

1225

1210

1191

1156

1094

1034

1004

978

899

845

813

745

731

Ph lc 2. Ph kh lng (MS) ca cht Va

Display Report - All Windows Selected Analysis

Analysis Name: 16A.d Instrument: LC-MSD-Trap-SL Print Date: 2/1/2013 11:14:22 AM Method:

Sample Name:

Analysis Info:

DEF_LCMS.m

16A

16A

Operator: LEANH Acq. Date: 2/1/2013 11:10:37 AM

MSD Trap Report v 4 (A4-Opt2) Page 1 of 1

Ph lc 3. Ph 1H-NMR ca cht Va

0.725

10.348

1.002

7.931

7.184

7.178

6.715

1.000

1.011

3.565

3.379

3.368

2.500

2.484

2.469

2.200

2.185

2.171

1.948

1.934

1.919

1.608

1.595

1.582

1.493

1.479

1.466

1.278

1.264

2.134

0.352

1.566

2.111

2.164

4.346

16ADMSO1H

Current Data Parameters NAME DUNG_16A

EXPNO 1

PROCNO 1

F2 Acquisition Parameter Date_ 20130308

Time 16.29 INSTRUM spect PROBHD 5 mm Multinucl PULPROG zg30 TD 65536 SOLVENT DMSO NS 16 DS 0 SWH 10000.000 Hz FIDRES 0.152588 Hz AQ 3.2769001 se

RG 45.3 DW 50.000 us DE 6.00 us TE 0.0 K D1 1.00000000 se MCREST 0.00000000 se

MCWRK 0.01500000 se

======== CHANNEL f1 ====== NUC1 1H

P1 8.50 us PL1 3.00 dB

SFO1 500.1335009 MH

F2 Processing parameters SI 32768 SF 500.1300039 MH WDW EM

SSB 0 LB 0.30 Hz GB 0

PC 1.00

14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 ppm

Ph lc 4. Ph 13C-NMR ca cht Va

174.456

171.655

169.093

157.658

152.381

145.313

145.173

112.521

110.752

40.000

39.832

39.666

39.499

39.332

39.165

38.998

34.804

33.613

32.190

28.247

28.206

24.926

24.400

24.296

16ADMSOC13CPD

Current Data Parameters NAME DUNG_16A

EXPNO 2

PROCNO 1

F2 Acquisition Paramet Date_ 20130308 Time 16.35 INSTRUM spect PROBHD 5 mm Multinucl PULPROG zgpg30 TD 65536 SOLVENT DMSO NS 256 DS 2 SWH 31446.541 FIDRES 0.479836 AQ 1.0420883 RG 32768 DW 15.900 DE 6.00

TE 0.0 D1 2.00000000 d11 0.03000000 DELTA 1.89999998 MCREST 0.00000000

MCWRK 0.01500000

======== CHANNEL f1 ==== NUC1 13C

P1 11.40 PL1 4.00 SFO1 125.7716224

======== CHANNEL f2 ==== CPDPRG2 waltz16 NUC2 1H PCPD2 80.00 PL2 3.00 PL12 22.47 PL13 22.00

SFO2 500.1320005

F2 Processing paramete SI 32768 SF 125.7578499 WDW EM SSB 0 LB 1.00

GB 0

200 180 160 140 120 100 80 60 40 20 ppm

Ph lc 5. Ph hng ngoi (IR) ca cht Vb

Ten may: GX-PerkinElmer-USA Nguoi do: Nguyen Thi Son DT: 0912140352



16BDate: 5/3/2013

4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 500.0

0.0

10

20

30

40

50

60

70

80

90

100.0

cm-1

%T

3361

3154

2931

2854

1674

1637

1563

1461

1436

1376

1343

1301

1229

1194

1094

1060

979 833

808

719

700

Ph lc 6. Ph khi lng (MS) ca cht Vb

Analysis Info: Intens.

x105 -MS, 1.4min #53

353.0 1.0

0.8

0.6

0.4

0.2

0.0

Display Report - Selected Window Selected Analysis

Analysis Name: 13041502.d Instrument: Agilent 6310 Ion Trap Print Date: 4/15/2013 12:09:10

Method:

Sample Name:

GERANIIN.m

16B

Operator: Phong PTHC, Vien Hoa HCTN Acq. Date: 4/15/2013 1A2M:04:18 AM

200 220 240 260 280 300 320 340 m/z


Ph lc 7. Ph 1H-NMR ca cht Vb

1.000

12.645

0.899

10.339

0.789

8.671

7.753

7.744

7.691

7.686

7.193

2.082

1.062

3.366

2.499

2.482

2.468

1.947

1.933

1.919

1.596

1.584

1.493

1.480

1.468

1.262

2.079

1.783

2.047

2.082

4.221

16BDMSO1H

Current Data Parameters NAME DUNG_16B

EXPNO 1

PROCNO 1


Time 15.55 INSTRUM spect PROBHD 5 mm Multinucl PULPROG zg30 TD 65536 SOLVENT DMSO NS 16 DS 0 SWH 10000.000 Hz FIDRES 0.152588 Hz AQ 3.2769001 se

RG 50.8 DW 50.000 us DE 6.00 us TE 0.0 K D1 1.00000000 se MCREST 0.00000000 se

MCWRK 0.01500000 se

======== CHANNEL f1 ====== NUC1 1H

P1 8.50 us PL1 3.00 dB

SFO1 500.1335009 MH



PC 1.00

16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 1 ppm

Ph lc 8. Ph 13C-NMR ca cht Vb

171.605

169.100

157.786

156.101

132.323

129.111

128.997

128.394

40.000

39.833

39.666

39.500

39.333

39.166

38.998

34.802

32.209

28.275

28.223

24.956

24.445

B16DMSOC13CPD

Current Data Parameters NAME DUNG_16B

EXPNO 2

PROCNO 1

F2 Acquisition Paramet Date_ 20130409 Time 15.51 INSTRUM spect PROBHD 5 mm Multinucl PULPROG zgpg30 TD 65536 SOLVENT DMSO NS 256 DS 2 SWH 31446.541 FIDRES 0.479836 AQ 1.0420883 RG 32768 DW 15.900 DE 6.00

TE 0.0 D1 2.00000000 d11 0.03000000 DELTA 1.89999998 MCREST 0.00000000

MCWRK 0.01500000

======== CHANNEL f1 ==== NUC1 13C

P1 11.40 PL1 4.00 SFO1 125.7716224


SFO2 500.1320005


GB 0

200 180 160 140 120 100 80 60 40 20 ppm

Ph lc 9. Ph hng ngoi (IR) ca Vc

Ten may: GX-PerkinElmer-USA Nguoi do: Nguyen Thi Son DT: 0912140352



TEN MAU: 16HDate: 10/21/2013

4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600.0

0.0

10

20

30

40

50

60

70

80

90

100.0

cm-1

%T

3158

3025

2931

2858

2735

1690

1638

1561

1499

1464

1448

1425

1387

1340

1317

1178

1129

1087

1035

1008

975

929

793

727

701

3245

Ph lc 10. Ph khi lng (MS) ca cht Vc


x104 -MS, 0.4min #16

416.0 8

6

4

2

391.3

453.0

0


Analysis Name: 13080501.d Instrument: Agilent 6310 Ion Trap Print Date: 8/5/2013 5:08:42 PM

Method:

Sample Name:

Acarbose.m

16H

Operator: Phong PTHC, Vien Hoa HCTN Acq. Date: 8/5/2013 11:04:31 AM

100 200 300 400 500 600 m/z


Ph lc 11. Ph1H-NMR ca cht Vc

1.172

12.702

1.000

10.326

7.233

7.226

6.838

6.831

1.203

1.195

3.371

2.500

2.486

2.471

1.947

1.933

1.918

1.611

1.597

1.583

1.568

1.508

1.493

1.479

1.465

1.451

1.280

1.267

1.262

2.508

2.007

2.358

2.371

4.781

16HDMSO1H

Current Data Parameters NAME DUNG_16H

EXPNO 1

PROCNO 1


Time 14.49 INSTRUM spect PROBHD 5 mm Multinucl PULPROG zg30 TD 65536 SOLVENT CDCl3 NS 16

DS 0 SWH 10000.000 Hz FIDRES 0.152588 Hz AQ 3.2769001 se RG 80.6 DW 50.000 us DE 6.00 us TE 300.0 K D1 1.00000000 se MCREST 0.00000000 se

MCWRK 0.01500000 se

======== CHANNEL f1 ====== NUC1 1H

P1 10.50 us

PL1 1.00 dB SFO1 500.1335009 MH



PC 1.00

14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 ppm

171.714

169.131

157.820

151.326

147.069

124.448

114.589

113.568

40.001

39.924

39.834

39.756

39.667

39.500

39.333

39.166

38.999

34.806

32.205

28.252

28.207

24.941

24.406

16HDMSOC13CPD

Current Data Parameters NAME DUNG_16H

EXPNO 2

PROCNO 1

F2 Acquisition Paramet Date_ 20130817 Time 15.02 INSTRUM spect PROBHD 5 mm Multinucl PULPROG zgpg30 TD 65536 SOLVENT DMSO NS 256 DS 2 SWH 31446.541 FIDRES 0.479836 AQ 1.0420883 RG 32768 DW 15.900 DE 6.00 TE 300.0 D1 2.00000000 d11 0.03000000 DELTA 1.89999998 MCREST 0.00000000

MCWRK 0.01500000

======== CHANNEL f1 ==== NUC1 13C

P1 5.00 PL1 1.00 SFO1 125.7716224

======== CHANNEL f2 ====

CPDPRG2 waltz16 NUC2 1H

PCPD2 80.00 PL2 1.00 PL12 16.64 PL13 22.00

SFO2 500.1320005

F2 Processing paramete SI 32768 SF 125.7578480 WDW EM

SSB 0 LB 1.00

GB 0

200 180 160 140 120 100 80 60 40 20 0 ppm

Ph lc 12. Ph 13C-NMR ca cht Vc

Ph lc 13. Ph hng ngoi (IR) ca cht Vd

Ten may: GX-PerkinElmer-USA Resolution: 4cm-1


Nguoi do: Phan Thi Tuyet MaiTen mau 16IDate: 11/25/2013

4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600.0

0.0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100.4

cm-1

%T

3158

3032

2925

28561718

1694

1651

1567

1552

1464

1443

1415

1383

1340

1321

1303

1248

1229

1206

1128

1106

1081

1053

1018

970

953

907

813

790

732

703

658

3416

2759

Ph lc 14. Ph khi lng (MS) ca cht Vd


x105 -MS, 1.8min #201

5

351.4

4

3

2

1

0


Analysis Name: 13102502.d Instrument: Agilent 6310 Ion Trap Print Date: 10/25/201 11:17:12

Method:

Sample Name:

Flavonoids.m

61I

Operator: Phong PTHC, Vien Hoa HCTN Acq. Date: 130/25/2013A1M0:53:20

AM

300 310 320 330 340 350 360 370 380 m/z


Ph lc 15. Ph 1H-NMR ca cht Vd

1.000

12.636

10.416

10.364

10.112

2.313

0.573

7.045

7.039

6.326

6.321

1.065

1.065

3.559

3.427

2.500

2.498

2.482

2.468

2.358

2.197

2.182

2.168

1.952

1.938

1.923

1.599

1.588

1.576

1.479

1.471

1.458

1.337

1.274

1.266

1.204

1.906

3.269

0.849

1.196

2.170

2.175

4.636

16IDMSO1H

Current Data Parameters NAME DUNG_16I

EXPNO 1

PROCNO 1


Time 14.42 INSTRUM spect PROBHD 5 mm Multinucl PULPROG zg30 TD 65536 SOLVENT DMSO NS 16 DS 0 SWH 10000.000 Hz

FIDRES 0.152588 Hz AQ 3.2769001 se RG 71.8 DW 50.000 us DE 6.00 us TE 300.0 K D1 1.00000000 se MCREST 0.00000000 se

MCWRK 0.01500000 se

======== CHANNEL f1 ====== NUC1 1H

P1 10.30 us PL1 1.00 dB SFO1 500.1335009 MH



PC 1.00

13 12 11 10 9 8 7 6 5 4 3 2 1 0 ppm

Ph lc 16. Ph 13C-NMR ca cht Vd

174.471

171.637

169.206

157.146

154.599

152.538

143.566

112.034

108.845

39.998

39.831

39.664

39.497

39.330

39.163

38.996

34.807

33.628

32.205

28.191

24.952

24.460

24.426

24.306

13.381

16IDMSOC13CPD

Current Data Parameters NAME DUNG_16I

EXPNO 2

PROCNO 1

F2 Acquisition Paramet Date_ 20131027 Time 14.49 INSTRUM spect PROBHD 5 mm Multinucl PULPROG zgpg30 TD 65536 SOLVENT CDCl3 NS 128 DS 2 SWH 31446.541 FIDRES 0.479836 AQ 1.0420883 RG 32768

DW 15.900 DE 6.00 TE 300.0 D1 2.00000000 d11 0.03000000 DELTA 1.89999998 MCREST 0.00000000

MCWRK 0.01500000

======== CHANNEL f1 ==== NUC1 13C P1 5.20 PL1 0.00

SFO1 125.7716224


SFO2 500.1320005


GB 0

200 180 160 140 120 100 80 60 40 20 ppm

Documents

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