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  BY TTRƯỜNG ĐẠI HC DƯỢC HÀ NI ĐỖ THMAI DUNG TNG HP VÀ THHOT TÍNH SINH HC CA 1 SACID HYDROXAMIC MANG KHUNG 1,3,4-THIADIAZOL KHÓA LUN TT NGHIP DƯỢC SĨ HÀ NI - 2013

Tổng hợp và thử hoạt tính sinh học của một số acid Hydroxamic mang khung 1,3,4 - Thiadiazol.pdf

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  • B Y T

    TRNG I HC DC H NI

    TH MAI DUNG

    TNG HP V TH HOT TNH

    SINH HC CA 1 S ACID

    HYDROXAMIC MANG KHUNG

    1,3,4-THIADIAZOL

    KHA LUN TT NGHIP DC S

    H NI - 2013

  • B Y T

    TRNG I HC DC H NI

    TH MAI DUNG

    TNG HP V TH HOT TNH

    SINH HC CA 1 S ACID

    HYDROXAMIC MANG KHUNG

    1,3,4-THIADIAZOL

    KHA LUN TT NGHIP DC S

    Ngi hng dn :

    1.PGS.TS. Nguyn Hi Nam

    2.DS. Nguyn Xun Phong

    Ni thc hin :

    1. B mn Ha Dc

    H NI - 2013

  • LI CM N

    Sau mt thi gian thc hin ti vi nhiu n lc v c gng, thi im

    hon thnh kha lun l lc ti xin php c by t lng bit n chn thnh vi

    nhng ngi dy d, hng dn v gip ti trong sut thi gian qua.

    Trc ht vi lng knh trng v bit n su sc nht ti xin by t li cm

    n chn thnh n PGS.TS. Nguyn Hi Nam, DS. Nguyn Xun Phong - B mn

    Ha Dc - trng i hc Dc H Ni, nhng ngi thy trc tip hng dn

    v tn tnh ch bo ti trong thi gian thc hin kha lun ny.

    Ti cng xin gi li cm n n cc thy gio, c gio v cc anh ch k

    thut vin ca B mn Ha Dc - trng i hc Dc H Ni, Khoa Ha - i

    hc Khoa hc t nhin H Ni, Vin Khoa hc v Cng ngh Vit Nam, Khoa

    Dc - i hc Quc gia Chungbuk - Hn Quc lun gip to iu kin

    thun li cho ti thc hin kha lun tt nghip.

    Cui cng ti xin c gi li cm n su sc n b m, gia nh v bn b

    lun ng vin khch l ti trong sut qu trnh hc tp v nghin cu.

    H Ni, ngy 21 thng 5 nm 2013

    Sinh vin

    Th Mai Dung

  • MC LC

    Trang

    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 (HDAC) .............................................................. 2

    1.1.1. Khi nim v histon deacetylase .................................................................. 2

    1.1.2. Phn loi cc HDAC .................................................................................... 3

    1.1.3. Mi lin quan gia ung th v hot ng bt thng ca HDAC ................. 4

    1.2. CC CHT C CH HDAC......................................................................... 6

    1.2.1. Phn loi cc cht c ch HDAC ................................................................. 6

    1.2.2. Cu trc ca cc cht c ch HDAC ............................................................ 8

    1.3. MT S HNG NGHIN CU TNG HP CC ACID HYDROXAMIC

    C CH HDAC TRN TH GII ....................................................................... 9

    1.3.1. Lin quan cu trc tc dng ca cc acid hydroxamic c ch HDAC ........... 9

    1.3.2. Mt s hng thit k nghin cu v tng hp trn th gii ......................... 10

    1.4. PHNG PHP TO LIN KT AMID V TNG HP ACID

    HYDROXAMIC ................................................................................................... 13

    1.4.1. Tng hp amid vi tc nhn acyl ha l acid carboxylic .............................. 13

    1.4.2. Tng hp acid hydroxamic t ester .............................................................. 14

    Chng 2. NGUYN LIU, THIT B, NI DUNG V PHNG PHP

    NGHIN CU ..................................................................................................... 15

    2.1. NGUYN VT LIU, THIT B .................................................................. 15

    2.1.1. Ha cht ...................................................................................................... 15

    2.1.2. Thit b, dng c .......................................................................................... 15

    2.2. NI DUNG NGHIN CU ........................................................................... 16

  • 2.2.1. Tng hp ha hc ........................................................................................ 16

    2.2.2. Th tc dng sinh hc ca cc cht tng hp c ...................................... 16

    2.3. PHNG PHP NGHIN CU ................................................................... 16

    2.3.1. Tng hp ha hc ........................................................................................ 16

    2.3.2. Th tc dng sinh hc .................................................................................. 17

    2.3.3. nh gi mc ging thuc ca cc cht tng hp c ........................... 19

    Chng 3. THC NGHIM, KT QU V BN LUN ............................... 20

    3.1. HA HC ...................................................................................................... 20

    3.1.1. Nghin cu Docking....................................................................................... 20

    3.1.2. Tng hp ha hc ........................................................................................ 21

    3.1.3. Kim tra tinh khit .................................................................................. 34

    3.1.4. Xc nh cu trc ......................................................................................... 34

    3.2. TH HOT TNH SINH HC ...................................................................... 40

    3.2.1. Th hot tnh sinh hc ................................................................................. 40

    3.2.2. nh gi mc ging thuc ...................................................................... 40

    3.3. BN LUN ................................................................................................... 41

    3.3.1. Tng hp ha hc ........................................................................................ 41

    3.3.2. Tc dng sinh hc ........................................................................................ 41

    KT LUN V KIN NGH ............................................................................. 44

    TI LIU THAM KHO

    PH LC

  • DANH MC CC K HIU, CC CH VIT TT ALL : Bnh ung th nguyn bo lympho cp tnh

    AML : Bnh ung th bch cu dng ty cp tnh

    APL

    CDI

    CLL

    CTCL

    :

    :

    :

    :

    Bnh ung th bch cu ty bo cp tnh

    Carbonyldiimidazol

    Bnh lympho mn tnh (Chronic lymphocytic leukemia)

    T bo lymphoT di da (Cutaneous T cell lymphoma) 13C-NMR : Cng hng t ht nhn 13C

    DCM : Dicloromethan

    DMF : Dimethyl formamid

    DMSO : Dimethyl sulfoxid

    EtOH : Ethanol

    HAT : Histon acetyltranferase

    HDAC : Histon deacetylase 1H-NMR : Cng hng t ht nhn 1H

    IC50 : Nng c ch hot t bo xung mt na

    IR

    MTT

    :

    :

    Phng php ph t ngoi

    3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromid

    MeOH : Methanol

    MS

    NSCLC

    :

    :

    Ph khi lng

    Ung th phi t bo khng nh (Non-smali lung cell)

    NST : Nhim sc th

    SAHA : Acid suberoylanilid hydroxamic

    T0nc : Nhit nng chy

    TLC : Phng php sc k lp mng

    TMS : Tetramethylsilan

    TSA : Trichostatin A

  • DANH MC CC BNG

    STT Tn bng Trang

    1 Bng 1.1: Cc cht c ch HDAC ang th nghim trn lm

    sng 8

    2 Bng 1.2: Tc dng c ch HDAC ca cc acid isoxazol-

    hydroxamic 13

    3 Bng 3.1: Kt qu docking ca cc cht 5a-f vi HDAC8 20

    4 Bng 3.2: Ch s l ha v hiu sut tng hp cc acid

    hydroxamic t ester 33

    5 Bng 3.3: Gi tr Rf v nhit nng chy ca cc cht 5a-f 34

    6 Bng 3.4: Kt qu phn tch ph MS ca cc cht 4a-f 35

    7 Bng 3.5: Kt qu phn tch ph IR ca cc cht 5a-f 36

    8 Bng 3.6: Kt qu phn tch ph MS ca cc cht 5a-f 37

    9 Bng 3.7: Kt qu phn tch ph 1H-NMR ca cc cht 5a-f 37

    10 Bng 3.8: Kt qu phn tch ph 13C-NMR ca cc cht 5a-f 39

    11 Bng 3.8: nh gi mc ging thuc ca cc cht 5a-f theo

    quy tc Lipinsky 40

    12 Bng 3.10: Kt qu th tc dng c ch HDAC v hot tnh

    khng t bo ung th 42

  • DANH MC CC HNH V

    STT Tn hnh Trang

    1 Hnh 1.1: Cu trc ca histon trong nucleosom 2

    2 Hnh 1.2: M t c im ca cc loi HDAC 4

    3 Hnh 1.3: Vai tr sinh hc ca cc HDAC trong sinh l t bo

    ung th 6

    4 Hnh 1.4: Mt s cht c ch HDAC ang th nghim trn lm

    sng 7

    5 Hnh 1.5: Cu trc ca SAHA v trung tm hot ng ca

    HDAC 9

    6 Hnh 1.6: Cu trc ca TSA, oxamflatin v cc dn cht N-

    hydroxy-2-propenamid 10

    7 Hnh 1.7: Cc dn cht amid ngc ca SAHA 11

    8 Hnh 1.8: Cc dn cht -alkoxy ca SAHA 11

    9 Hnh 1.9: Cc acid phenylthiazol-hydroxamic tng t SAHA 11

    10 Hnh 1.10: Cc dn cht aicd biphenyl-hydroxamic 12

    11 Hnh 1.11: Cng thc cu to ca WR301849 12

    12 Hnh 3.1: M hnh tng tc ca 6 dn cht 5a-f vi HDAC8 21

  • DANH MC CC S

    STT Tn s Trang

    1 S 1.1: C ch xc tc ca CDI 14

    2 S 3.1: Quy trnh tng hp chung 21

    3 S 3.2: Quy trnh tng hp cht 2a 22

    4 S 3.3: Quy trnh tng hp cht 3a 23

    5 S 3.4: Quy trnh tng hp cht 4a 24

    6 S 3.5: Quy trnh tng hp cht 5a 25

    7 S 3.6: Quy trnh tng hp cht 5b 26

    8 S 3.7: Quy trnh tng hp cht 3b 27

  • 1

    T VN

    Acid suberoylanilid hydroxamic (Zolinza, 2006) l cht c ch histon

    deacetylase u tin c cc qun l thc phm v dc phm M (US-FDA) ph

    duyt trong iu tr u lympho da t bo T. Sau , nm 2009 depsipeptid

    (Romidepsin) mt cht c ch HDAC khc cng c cp php trong iu tr ung

    th ti M. Nh vy c th thy, HDAC l mt mc tiu phn t quan trng trong

    iu tr ung th v cc cht c ch HDAC l cc tc nhn chng ung th y trin

    vng.

    Nhm nghin cu ti b mn Ha Dc - i hc Dc H Ni cng thit

    k, tng hp v cng b nhiu dy cht vi nh hng c ch HDAC c hot tnh

    khng t bo ung th tt [40,41]. Cc nghin cu gn y ti b mn v cc acid

    hydroxamic c tc dng c ch HDAC cho thy c th thay th nhm nhn din b

    mt ca SAHA l nhn phenyl bng vng thm khc nh benzothiazol cho hot tnh

    in vitro rt kh quan [3]. Tip theo , trong lun vn thc s ca Th nh Tuyt

    [4] khung 5-phenyl-1,3,4-thiadiazol c s dng lm nhm nhn din b mt

    thay cho benzothiazol. Tuy nhin nghin cu trn mi ch tng hp c 5 dn

    cht, cha nh gi kh nng tng tc ca nhm nhn din b mt mi vi

    HDAC, cng nh cha tm c cht c hot tnh khng t bo ung th in vitro tt

    tin hnh cc th nghim su hn. Trn c s thay i cc nhm th vng

    thm s lm thay i tng tc ca cht vi mc tiu phn t, chng ti thit k cc

    nhm th mi trn khung 5-phenyl-1,3,4-thiadiazol v tin hnh ti Tng hp

    v th hot tnh sinh hc ca mt s acid hydroxamic mang khung 1,3,4-

    thiadiazol vi hai mc tiu:

    1. Tng hp N1-hydroxy-N8-(5-phenyl-1,3,4-thiadiazol-2-yl)octandiamid v 5

    dn cht.

    2. Th c tnh t bo v tc dng c ch HDAC ca cc cht tng hp c.

  • 2

    Chng 1. TNG QUAN

    1.1. HISTON DEACETYLASE (HDAC)

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

    i phn t protein-ADN. Nhim sc th c cu trc ng, c t chc cao, bao

    gm: ADN, cc protein histon v protein khng phi histon. n v cu trc c bn

    ca NST l nucleosom. Mt 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 [1,48] (hnh 1.1):

    Hnh 1.1: Cu trc ca histon trong nucleosom

    Khi u amin ca histon tch in dng s tng tc vi phn phosphat mang

    in m trn phn t ADN lm ng xon NST gy c ch dch m v tng hp

    protein, lm c ch s bin hin gen. Ngc li khi tng tc in tch ny yu

    NST tho xon , qu trnh tng hp protein din ra, c tnh ca gen c biu hin

    thng qua cc tnh trng. Vic tch in dng ca histon mnh hay yu thng qua

    qu trnh acetyl ha u amin phn ui ca histon lin quan n hot ng ca 2

    emzym histon deacetylase (HDAC) v histon acetyltransferase (HAT).

    1.1.1. Khi nim v histon deacetylase

  • 3

    Histon deacetylase l mt nhm cc enzym xc tc qu trnh loi b nhm

    acetyl t -N acetyl lysine amino acid ca histon. HDAC c tc dng i lp vi

    histon acetyltransferase (HAT) - enzym xc tc chuyn nhm acetyl t acetyl

    coenzym A n -amino ca lysin u N ca histon [49].

    Histons ngng t cc nhim sc th

    ngn cn phin m

    Acetyl-histons ko gin cc nhim sc th

    kch thch phin m

    1.1.2. Phn loi cc HDAC

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

    trc ca chng ln lt vi Rpd3, Hdal v Sir2 trong nm men [22,43] (hnh 1.2):

    Nhm I: HDAC1, HDAC2, HDAC3, HDAC8.

    Nhm II: HDAC4, HDAC5, HDAC6, HDAC7, HDAC9, HDAC10.

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

    - Cht ng ng ca Sir2 trong nm men Saccharomyces cerevisiae.

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

    SIRT5, SIRT6, SIRT7).

    Nhm IV: HDAC11.

    Cc HDAC nhm I, II, IV c coi l cc HDAC kinh in gm 11 thnh

    vin, trong khi cc thnh vin nhm III c gi l cc sirtuin [22]. Cc HDAC

    kinh in v sirtuin c c ch xc tc khc nhau. Cc HDAC kinh in l nhng

    enzym ph thuc Zn2+, chng c cha 1 ti xc tc vi 1 ion Zn2+ y ti. Do

    nhng enzym ny c th b c ch bi cc hp cht to phc vi Zn2+ nh cc acid

    hydroxamic, cc thiol Ngc li cc sirtuin khng b c ch bi cc hp cht to

    chelat vi Zn2+ v c ch hot ng ca chng ph thuc vo NAD+ nh 1 cofactor

    thit yu [43]. Thut ng cc cht c ch HDAC thng c s dng cho nhng

    hp cht nhm mc tiu vo cc HDAC kinh in v nhng hp cht ny ang

    c nh gi da trn cc th nghim lm sng cc giai on khc nhau.

    HATs

    HDACs

  • 4

    Ghi ch: Vng xc tc Vng tn hiu nhn din nhn t bo

    Hnh 1.2: M t c im ca cc loi HDAC

    1.1.3. Mi lin quan gia ung th v hot ng bt thng ca HDAC

    HDAC xc tc loi b nhm acetyl t histon v protein khng phi histon, lm

    tng s tch in dng trn u N ca histon v ng xon NST, kt qu l c ch

    qu trnh phin m do ngn cn cc nhn t sao m tin n ch ca chng trn

    ADN. Ngc li, HAT acetyl ha histon lm trung ha cc dng trn lysin v gip

    tho xon cu trc ca nucleosom, do gip cho cc yu t sao m d dng tip

    cn ADN. Nh vy s acetyl ha v deacetyl ha NST ng vai tr quan trng

    trong iu ha qu trnh biu hin gen. Vic mt cn bng hot ng gia HAT v

    HDAC c th dn n nhng bt thng biu hin gen v do dn n ung th

    [18,27,30,35,39].

  • 5

    HDAC c xc nh b ri lon iu ha trong ung th. S huy ng bt

    thng ca cc HDAC vo chui iu ha ca gen ch c th xy ra thng qua

    tng tc bin i ca chng vi vic tng cng vai tr ca cc protein gn kt

    ADN gy ung th. V d nh receptor -RAR gn kt gen PML (promyelocytic

    leukemia gen) hoc PLZF (promyelocytic leukemia zinc finger) trong bnh bch

    cu tin ty bo cp (APL). Trong iu kin sinh l, -RAR l nhn t sao m hot

    ha acid retinoic gip gii phng phc hp ng c ch cha HDAC v lm gia

    tng cc cht ng hot ha sao m (bao gm c HAT). iu ny dn n s acetyl

    ha histon v c ch gen y mnh s bit ha t bo. Trong bnh bch cu tin ty

    bo cp (APL), protein gn kt -RAR/PML hoc -RAR/PLZF gi HDAC v phi

    hp vi phc hp ng c ch. Do tng methyl transferase histon v ADN dn

    n ngn cn s phin m [18,21,27]. V vy, t bo ung th khng tri qua giai

    on bit ha v pht trin qu mc. S gia tng bt thng ca HDAC cn c

    quan st khi gen t bin gy ung th Bcl6 c biu th qu mc trong bnh u

    lympho t bo B [7]. Tng t, protein gn kt AML1-ETO tm thy trong bnh

    bch cu ty bo cp (AML) c chc nng nh cht c ch sao m ch yu thng

    qua ETO, nn c vai tr nh v tr gn kt cho phc hp ng c ch N-

    Cor/Sin3/HDAC1. Vic chuyn i AML1 t cht hot ha phin m thnh cht c

    ch c iu khin bi protein gn kt CBFb-SMMHC thng qua s gia tng phc

    hp ng c ch mSin3A/HDAC. Cui cng, biu th qu mc nhn t sao m

    SCL/Tal1 trong bnh u lympho t bo T cp c nguyn nhn l do gia tng bt

    thng HDAC1 nm trong phc hp ng c ch, dn n ngn cn gen ch iu

    ha E47/HEB [14]. Biu th qu mc HDAC1 v/hoc HDAC2 v/hoc HDAC6

    cn gp trong mt s bnh ung th tng c nh ung th tin lit tuyn, ung th d

    dy, trc trng, ung th v v ung th no [23,45,47] cng nh trong cc bnh l c

    tnh v mu (bnh bch cu ty bo cp, bch cu t bo B, bnh u lympho t bo T

    ngoi vi, bnh u lympho t bo B v bnh u lympho da t bo T) [37]. Hn na,

    vic tm ra cc c cht ca HDAC l cc protein nh p53, E2F, Rb, Bcl6, Gli1 lin

    quan n xu hng gy ung th v tin trin bnh ung th khng nh vai tr ca

  • 6

    HDAC trong ung th [12,36]. Tm li, cc bin i sau phin m ca HDAC c th

    lm thay i tng tc ca chng vi phc hp ng c ch m cc phc hp ny

    lin quan n qu trnh phin m ca cc gen gy ung th.

    Cc nghin cu c tnh thng k cn ch ra rng cc HDAC lin quan n

    nhiu giai on iu ha c bn ca qu trnh sinh hc trong t bo ung th nh chu

    trnh t bo, s bit ha, s cht t bo theo chng trnh k c s xm ln, s di

    chuyn v s to mch. Vai tr chc nng ca cc HDAC trong qu trnh sinh hc

    ca t bo ung th c tm tt trong hnh 1.3 [9,20,28,44].

    Nh vy c phin m c iu ha bi s ra tng HDAC v c th kim sot

    ung th bng cch c ch hot ng ca HDAC. Cc cht c ch HDAC v ang

    c nhiu nh khoa hc trn th gii nghin cu nhm tm ra cht c tc dng c

    ch chn lc tng loi HDAC ng dng trong iu tr ung th.

    Hnh 1.3: Vai tr sinh hc ca cc HDAC trong sinh l t bo ung th

    1.2. CC CHT C CH HDAC

    1.2.1. Phn loi cc cht c ch HDAC

  • 7

    T nhng pht hin u tin v tc dng c ch HDAC ca natri butyrat, cho

    ti nay cc nh nghin cu tm ra rt nhiu hp cht mi c tc dng c ch

    HDAC. Trong trichostatin A (TSA), mt sn phm ln men ca Streptomyces, l

    cht c tc dng c ch mnh nht tuy nhin vic sn xut n li khng kh thi [38].

    Hin nay, c 2 cht c tc dng c ch HDAC c FDA cp php lu hnh trn

    th trng iu tr u lympho t bo T di da l SAHA (vorinostat, Zolinza) v

    Romidepsin (Istodax). Ngoi ra, cn khong 15 cht c ch HDAC khc cng

    ang c th nghim tc dng iu tr ung th trn lm sng cc giai on khc

    nhau (hnh 1.4) v c chia lm 4 nhm da theo cu trc (bng 1.1).

    Hnh 1.4: Mt s cht c ch HDAC ang c th nghim lm sng

    Mi nhm dn cht ny u c nhng hn ch ring nh: cc acid hydroxamic

    b chuyn ha nhanh, c ch khng chn lc trn cc loi enzym HDAC; cc

    benzamid v cc 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

    acid hydroxamic c ch mnh HDAC nhm I v II, cc acid carboxylic v peptid

    vng c ch mnh HDAC nhm I.

  • 8

    Bng 1.1: Cc cht c ch HDAC ang th nghim trn lm sng Nhm Hp cht Pha Loi ung th

    Acid carboxylic

    Butyrat AN-9 (tin thuc) Acid valproic Phenyl butyrat

    I, II I, II I, II I

    Ung th i trng Tng c, NSCLC Tng c, ung th mu, AML, MDS, CTCL, u trung biu m Tng c, AML/MDS

    Acid hydroxamic

    SAHA PXD101 NVP-LAQ824 LBH-589 ITF-2357 SB-939 CRA 024781 JNJ-16241199

    I, II II I II, III II I I I

    Tng c, ung th mu Ung th mu Tng c, ung th mu Tng c, AML, MDS, ALL U lympho Hodgkin Tng c, ung th mu

    Cc benzamid

    SNDX-275 CI-994 MGCD-0103

    I, II I, II II

    Tng c, u lympho, AML Tng c, NSCLC, t bo thn,ty Tng c, ung th bch cu, MDS

    Peptid vng Depsipeptid (FK228)

    I, II Tng c, CLL, AML, u a ty xng, NHL t bo T ngoi vi, RAI khng thyroid, ung th i trng tin trin

    Ghi ch: NSCLC: carcinom t bo phi khng nh; AML: ung th bch cu ty bo cp; MDS: hi chng lon sn ty; CTCL: u lympho da t bo T; ALL: ung th bch cu cp; CLL: ung th bch cu mn.

    1.2.2. Cu trc ca cc cht c ch HDAC

    Cu trc ca cc cht c ch HDAC rt a dng nhng nhn chung u gm 3

    phn chnh:

    - Nhm kha hot ng (capping group) hay vng nhn din b mt (surface

    recognition group): l cc vng thm hoc peptid vng, thng nm trn b mt

    enzym.

    - Vng cu ni s nc: thng l nhng hydrocacbon thn du mch thng

    hay vng, c th no hoc khng no to cc lin kt Van der Waals vi knh

    enzym.

  • 9

    - Nhm kt thc gn vi km (Zinc binding group - ZBG): tng tc vi ion

    Zn2+ ti trung tm hot ng ca cc HDAC nh acid hydroxamic, cc thiol, nhm

    o-aminoanilin ca benzamid, mercaptoceton...

    Cu trc tinh th kt tinh ca cc HDAC cho thy nhm kt thc, cu ni v

    mt phn ca nhm kha hot ng nm trong ti enzym lm lp y khong trng

    trong lng knh enzym. Phn cn li ca nhm kha hot ng tng tc vi phn

    vnh trn b mt ming ti enzym. Nhm nhn din b mt c th lin kt vi phn

    cu ni thng qua mt s lin kt peptid lm tng kh nng phn cc v gp phn

    ci thin dc ng hc cho cc cht c ch HDAC. Vic nghin cu thit k cu

    trc cc cht mi u da trn cu trc c in ny.

    1.3. MT S HNG NGHIN CU TNG HP CC ACID

    HYDROXAMIC C CH HDAC TRN TH GII

    1.3.1. Lin quan cu trc tc dng ca cc acid hydroxamic c ch HDAC

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

    bit n nhiu, v d nh SAHA (hnh 1.5).

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

    Cu trc ca nhm cht ny cng gm 3 phn chnh c lin quan n tc dng

    nh sau [38]:

    - Nhm kha hot ng: lin quan n tnh c hiu v hiu lc c ch enzym

    HDAC, thng l cc aryl, cc nghin cu v cc cht tng hp c cho thy kch

    thc vng nhn thm ln s cho tc dng tt hn vng nh.

  • 10

    - Cu ni s nc: lin quan n kh nng c ch enzym, quyt nh s ph

    hp v cu trc ca cc cht vi chiu di ca knh enzym. Phn ny thng c cu

    trc amid - alkyl, l cc hydrocarbon mch h hoc cc vng thm c kch thc

    nh. Trong cu ni lin kt amid l quan trng nhng khng phi l then cht,

    di mch carbon ti u l 5 - 6C.

    - Nhm lin kt vi Zn2+: acid hydroxamic, l phn khng th thiu c tc

    dng c ch HDAC.

    1.3.2. Mt s hng thit k nghin cu v tng hp trn th gii

    1.3.2.1. Thay i cu ni

    - Cc N-hydroxy-2-propenamid, cc acid hydroxamic tng t TSA

    Nhm cc N-hydroxy-2-propenamid (hnh 1.6) c thit k v tng hp da

    trn c s nghin cu cu trc ca TSA v oxamflatin (hnh 1.6). Cc cht ny c

    tc dng c ch HDAC yu hn TSA song tng t oxamflatin [29].

    Hnh 1.6: Cu trc ca TSA, oxamflatin v cc dn cht N-hydroxy-2-propenamid

    - Cc amid ngc ca SAHA

    Nhm nghin cu thuc cng ty Topo Target (Anh) thit k v tng hp

    gn 40 dn cht amid ngc ca SAHA (hnh 1.7) [6]. Nhiu cht trong s ny c

    hot tnh c ch HDAC tng ng thm ch mnh hn SAHA. Trong c 2

    cht c th nghim m hnh ung th in vivo trn chut cho kt qu kh quan,

    nh hng cho cc nghin cu tip theo.

  • 11

    Hnh 1.7: Cc dn cht amid ngc ca SAHA

    - Cc dn cht -alkoxy ca SAHA

    Nhm nghin cu thuc trung tm nghin cu Sigma-Tau (Pomezia, )

    thit k v tng hp dy dn cht -alkoxy ca SAHA (hnh 1.8) [24]:

    Hnh 1.8: Cc dn cht -alkoxy ca SAHA

    Kt qu sng lc hot tnh c ch HDAC2 cho thy tt c cc dn xut -

    alkoxy ca SAHA u cho gi tr IC50 mc rt thp (0,05 - 0,5 M). c tnh ca

    cc cht vi t bo ung th th hin mnh hn so vi SAHA trn c 3 dng t bo

    NB4 (ung th bch cu tin ty bo cp), H460 (ung th phi ngi), HCT-116

    (ung th i trng).

    1.3.2.2. Thay i nhm kha hot ng

    - Cc acid phenylthiazol-hydroxamic tng t SAHA

    Vin nghin cu Walter Reed (M) thit k v tng hp hng trm dn cht

    acid hydroxamic mang hp phn phenylthiazol thay th vo v tr phenyl ca SAHA

    (hnh 1.9). Trong c nhiu cht c tc dng c ch HDAC tng ng hoc

    mnh hn SAHA [19].

    Hnh 1.9: Cc acid phenylthiazol - hydroxamic tng t SAHA

    - Cc acid biphenyl-hydroxamic tng t SAHA

    Nhm nghin cu ca Alan P. Kozikowski thit k v tng hp mt dy

    cc dn cht acid biphenyl-hydroxamic tng t SAHA (hnh 1.10) [31]. Kt qu

  • 12

    cc dn cht ny c ch HDAC mnh hn SAHA trn 6 loi HDAC (HDAC1, 2, 3,

    6, 8, 10). Mt s acid biphenyl-hydroxamic cng c c tnh trn 5 dng t bo ung

    th ty th nghim nhng tc dng khng tt bng cc acid phenylthiazol-

    hydroxamic.

    Hnh 1.10: Cc dn cht acid biphenyl-hydroxamic

    - Cc acid isoxazol-hydroxamic tng t SAHA

    Trong qu trnh nghin cu cc dn cht ca acid phenylthiazol-hydroxamic,

    nhm nghin cu ca Alan P. Kozikowski thuc i hc Illinois (Chicago, M)

    tng hp dn cht acid phenylisoxazol-hydroxamic WR301849 (hnh 1.11) [32].

    Dn cht isoxazol ny c tc dng c ch HDAC1, 3 v 6 rt mnh vi IC50 thp

    n nng nanomol (0,002 nM).

    Hnh 1.11: Cng thc cu to ca WR301849

    Tip tc mch nghin cu ny, mt s dn cht trong vng isoxazol c

    a vo v tr ngay st nhm acid hydroxamic c thit k v tng hp. Kt

    qu th hot tnh c ch HDAC cho thy 4 dn cht I-IV c ch c 5 loi HDAC1,

    2, 3, 6 v 10 vi IC50 trung bnh khong 150 nM, thp nht l 25 nM (bng 1.2).

  • 13

    Bng 1.2: Tc dng c ch HDAC ca cc acid isoxazol-hydroxamic

    Cht Ar IC50 (nM)/HDAC

    HDAC1 HDAC2 HDAC3 HDAC6 HDAC10

    I

    303 430 30 68 254

    II

    139 164 25 82 250

    III

    328 469 102 51 471

    IV

    885 3750 7410 885 #

    SAHA 96 282 17 14 72 Ghi ch: # : Khng th hot tnh

    Nhn chung, cc nghin cu u cho thy acid hydroxamic l nhm cht c

    kh nng c ch HDAC tt, mt s cht c tc dng c tnh vi t bo ung th

    nng rt thp. Tng hp cc acid hydroxamic hng c ch HDAC ang l mt

    hng nghin cu mi v c nhiu trin vng trong vic tm kim cc hp cht c

    tc dng khng t bo ung th chn lc, c hiu qu iu tr cao v t gy tc dng

    khng mong mun.

    1.4. PHNG PHP TO LIN KT AMID V TNG HP ACID

    HYDROXAMIC

    Cc cht tng hp trong ti ny u qua 4 bc phn ng. Trong , c 2

    phn ng quan trng l phn ng to lin kt amid v phn ng tng hp acid

    hydroxamic. Qua tham kho ti liu chng ti la chn cc phng php tng

    hp ph hp vi iu kin phng th nghim nh sau:

    1.4.1. Tng hp amid vi tc nhn acyl ha l acid carboxylic

    Acid carboxylic l tc nhn acyl ha yu, acyl ha amin cn c cht xc

    tc. Cc tc nhn hot ha acid carboxylic l imidazolium t ra kh hu hiu trong

    vic to thnh lin kt amid, trong carbonyldiimidazol (CDI) l cht hay c s

  • 14

    dng nht trong nhm. Victor Andrianov v cng s dng CDI xc tc phn

    ng to amid cho hiu sut cao [6], phng trnh phn ng tng qut nh sau:

    RCOOH + R1NH2 RCONHR1

    C ch xc tc ca CDI c biu din trong s 1.1:

    S 1.1: C ch xc tc ca CDI

    1.4.2. Tng hp acid hydroxamic t ester

    C nhiu cch khc nhau tng hp acid hydroxamic t ester nhng hay

    dng nht l s dng KCN hoc NaOH lm xc tc. Cc phn ng c phng trnh

    nh sau [5,13,16,24]:

    Vi iu kin ha cht, dung mi ti phng th nghim v tham kho ti liu,

    chng ti la chn xc tc NaOH v dung mi MeOH tin hnh phn ng ny.

  • 15

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

    PHNG PHP NGHIN CU

    2.1. NGUYN VT LIU, THIT B

    2.1.1. Ha cht

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

    tng hp c nhp t cng ty Merck hoc Sigma-Aldrich. Cc ha cht ny c

    s dng trc tip khng qua tinh ch thm. Bao gm:

    Cc benzaldehyd:

    - Benzaldehyd

    - 2,6-Diclorobenzaldehyd

    - 4-Bromobenzaldehyd

    - 4-Flourobenzaldehyd

    - 4-Methylbenzaldehyd

    - 4-Dimethylaminobenzaldehyd

    Thiosemicarbazid

    FeCl3.6H2O

    Acid monomethyl suberic

    Carbonyldiimidazol (CDI)

    Hydroxylamin hydroclorid

    Cc ha cht v dung mi khc: DMF, aceton, acid acetic bng,

    dicloromethan, ethanol, methanol, NaOH, HCl, nc ct.

    2.1.2. Thit b, dng c

    - Dng c thy tinh: bnh cu y trn dung tch 50 ml c nt mi, sinh hn

    hi lu, pipet, bnh chit, phu thy tinh, bnh chy sc k lp mng (TLC).

    - My khuy t gia nhit.

    - My ct quay chn khng Buchi R-210.

    - Cn phn tch, cn k thut Shimazu.

    - T lnh, t sy, my siu m.

    - Bn mng silicagel Merck 70-230 mesh chy sc k lp mng.

    - My o nhit nng chy nhit in (Electrothermal digital) xc nh

    nhit nng chy.

    - My Perkin Elmer xc nh ph IR.

    - My khi ph HP 5989B-MS ghi ph MS.

    - My cng hng t Bruker AV-500 ghi ph 1H-NMR, 13C-NMR.

  • 16

    2.2. NI DUNG NGHIN CU

    2.2.1. Tng hp ha hc

    * Nghin cu Docking vi 6 acid hydroxamic d kin tng hp.

    * Tng hp 6 acid hydroxamic:

    - N1-hydroxy-N8-(5-phenyl-1,3,4-thiadiazol-2-yl)octandiamid (5a).

    - N1-{5-[4-(dimethylamino)phenyl]-1,3,4-thiadiazol-2-yl}-N8-

    hydroxyoctandiamid (5b).

    - N1-[5-(4-flourophenyl)-1,3,4-thiadiazol-2-yl]-N8-hydroxyoctandiamid (5c).

    - N1-[5-(4-bromophenyl)-1,3,4-thiadiazol-2-yl]-N8-hydroxyoctandiamid (5d).

    - N1-hydroxy-N8-[5-(4-methylphenyl)-1,3,4-thiadiazol-2-yl]octandiamid (5e).

    - N1-[5-(2,6-diclorophenyl)-1,3,4-thiadiazol-2-yl]-N8-hydroxyoctandiamid

    (5f).

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

    - Th tc dng c ch HDAC.

    - Th c tnh trn t bo ung th i trng (SW620).

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

    2.3. PHNG PHP NGHIN CU

    2.3.1. Tng hp ha hc

    2.3.1.1. Nghin cu Docking

    - Vic nghin cu s b tnh tng tc ca cc cht vi HDAC (docking) c

    thc hin ti Phng Nghin cu cu trc i hc Quc gia Seul, Hn Quc.

    - tm hiu s b tng tc ca cc cht vi HDAC, chng ti tin hnh

    docking cho cc cht tng hp c da trn c s mng li ca HDAC8 tng

    tc vi SAHA [26]. Cu trc ca HDAC8 c im tng ng cao vi HDAC4 vi

    im DALI Z (Z-score im nh gi ging) = 40,4 v im r.m.s.d (root-mean-

    square deviation, lch) = 2,1, th t acid amin ging nhau (46%) v l HDAC

    ca ng vt c v u tin c cu trc khng gian c nghin cu k nht. Chng

    ti thc hin kim sot th nghim lp ghp ca cc cht vo HDAC8 bng chng

  • 17

    trnh AutoDock Vina [42]. Tip theo chng ti d on nng lng ca s tng

    tc lin kt ca cc cht tng hp c vi HDAC8 t s lp ghp trn.

    Kt qu c minh ha bng hnh nh m hnh v s liu d on tng tc.

    2.3.1.2. Tng hp

    - Da trn nhng nguyn tc v phng php c bn ca ha hc hu c

    tng hp cc dn cht theo thit k.

    - Theo di qu trnh phn ng bng sc k lp mng (TLC).

    2.3.1.3. Kim tra tinh khit

    - Kim tra tinh khit ca sn phm bng chy sc k lp mng v o nhit

    nng chy.

    2.3.1.4. Xc nh cu trc cc cht tng hp c

    Cc cht tng hp c c xc nh cu trc bng cc ph: ph hng ngoi,

    ph khi, ph cng hng t ht nhn 1H-NMR v 13C-NMR.

    - Ph hng ngoi (IR): c ghi ti khoa Ha, trng i hc Khoa hc t

    nhin H Ni trn my Perkin Elmer vi k thut vin nn KBr trong vng 4000-

    600 cm-1. Cc mu rn c phn tn trong KBr sy kh vi t l khong 1: 200

    ri p di dng film mng di p lc cao c ht chn khng loi b hi m.

    - Ph khi lng (MS): c o bng my khi ph HP 5989B-MS ca Vin

    Ha hc Vit Nam, ch o LC-MS, dung mi methanol.

    - Ph cng hng t ht nhn proton 1H-NMR v carbon 13C-NMR c ghi

    trn my Bruker AV-500 ti Trung tm Khoa hc v Cng ngh Vit Nam, dung

    mi DMSO-d6. 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).

    2.3.2. Th tc dng sinh hc

    2.3.2.1. 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

  • 18

    mu th lm tng hoc gim s acetyl ha histon H3, H4 khi so snh vi mu trng.

    Chi tit cch tin hnh da trn phng php c cng b trc y [4].

    2.3.2.2. Th c tnh t bo

    * 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 Quc gia Chungbuk, Cheongju, Hn Quc theo

    phng php MTT v gi tr IC50 c tnh trn phn mm GraphPad Prism [8,34].

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

    Cc 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 (KRIBB) v c nui cy trong mi

    trng RPMI b sung 10% FBS (huyt thanh bo thai b). c tnh t bo ca cc

    cht c th bng phng php MTT 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% FBS 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 37oC trong 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 48 gi, thm vo mi ging 20 l thuc nhum MTT

    (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromid) vi nng MTT l

    5 mg/mL trong mui m phosphat - PBS. Cc a c thm 3 gi 37oC trong

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

    pht cho MTT formazan c ha tan. hp th c c bc sng 510 nm.

    * Tnh kt qu

    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

  • 19

    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.

    2.3.3. nh gi mc ging thuc ca cc cht tng hp c

    Tnh gi tr logP ca cc cht tng hp bng phn mn EPTsuite cung cp bi

    US Environmental Protection Agency's Office of Pollution Prevention and Toxics

    and Syracuse Research Corporation (SRC). Quy trnh bao gm v cu trc 2D, to

    Smile notation bng phn mm ChemSketch 4.0 ca ACD labs sau a Smile

    notation vo phn mm EPIsuite tnh cc gi tr logP.

    nh gi mc ging thuc ca cc cht da trn quy tc Lipinsky [33]:

    + Khi lng phn t ca cht khng ln hn 500 g/mol.

    + S trung tm nhn lin kt hydro (N, O) khng ln hn 10.

    + S trung tm cho lin kt hydro (NH, OH) khng ln hn 5.

    + Gi tr logP ca cht khng ln hn 5.

    + S lin kt linh ng khng ln hn 15.

  • 20

    Chng 3: THC NGHIM, KT QU V BN LUN

    3.1. HA HC

    3.1.1. Nghin cu Docking

    tm hiu s b v kh nng tng tc ca cc cht vi mc tiu phn t l

    cc HDAC, chng ti nghin cu v d on nng lng lin kt cc cht trong

    thit k vi trung tm hot ng ca HDAC8. Kt qu c minh ha bng m hnh

    tng tc ca cc cht vi HDAC8 (hnh 3.1), nng lng lin kt d on c

    trnh by trong bng 3.1

    Bng 3.1: Kt qu docking ca cc cht 5a-f vi HDAC8

    Cht 5a 5b 5c 5d 5e 5f

    SAHA R H 4-N(CH3)2 4-F 4-Br 4-CH3 2,6-Cl2

    Nng lng tng tc (kcal/mol)

    -6,7 -7,1 -6,9 -6,6 -7,4 -6,8 -4,4

  • 21

    Hnh 3.1: M hnh tng tc ca 6 dn cht 5a-f vi HDAC8

    3.1.2. Tng hp ha hc

    Qu trnh tng hp 6 cht trong kha lun c thc hin theo s chung

    nh sau (s 3.1):

    S 3.1. Quy trnh tng hp chung

    Tc nhn v iu kin: i) Thiosemicarbazid, ethanol, acid acetic bng, hi lu, 4 h; ii) FeCl3.6H2O, ethanol, hi lu, 3 h; iii) Acid monomethyl suberic, carbonyldiimidazol, DMF, 60oC, 24 h; iv) Hydroxylamin hydroclorid, NaOH, methanol, -10oC, 1 h.

  • 22

    3.1.2.1. Tng hp N1-hydroxy-N8-(5-phenyl-1,3,4-thiadiazol-2-yl)octandiamid

    (5a)

    a. Tng hp cht 2a

    Cht 2-benzylidenhydrazincarbothioamid (2a) c tng hp t benzaldehyd

    (1a) qua phn ng ngng t vi thiosemicarbazid c xc tc l acid hu c theo s

    3.2:

    S 3.2: Quy trnh tng hp cht 2a

    Tin hnh phn ng:

    + Ly 0,2 ml (2 mmol) benzaldehyd cho vo bnh cu y trn dung tch 50

    ml, thm 20 ml ethanol khan.

    + Thm 0,218 g (2,4 mmol) thiosemicarbazid v 3-4 git acid acetic bng vo

    bnh phn ng.

    + un hi lu kt hp khuy t 300 vng/pht, tin hnh phn ng trong 4 h.

    X l phn ng:

    + Ct loi dung mi bng my ct quay chn khng trong 5 pht nhit

    40oC.

    + Thm 20 ml nc ct ta sn phm.

    + Lc, ra ta 3 ln bng 15 ml nc ct lnh.

    + Sy kh ta 60oC v thu c 0,32 g sn phm.

    Kt qu

    + Cm quan: bt kt tinh mu trng ng.

    + Hiu sut: 90%.

    + Tonc: 164-165oC.

    + Rf = 0,63 (DCM/MeOH = 9/1).

    b. Tng hp cht 3a

  • 23

    Tng hp cht 5-phenyl-1,3,4-thiadiazol-2-amin (3a) t 2a bng phn ng

    ng vng ni phn t theo s 3.3 nh sau:

    S 3.3: Quy trnh tng hp cht 3a

    Tin hnh phn ng:

    + Ha tan 1,22 g (4,5 mmol) FeCl3.6H2O vo 3 ml ethanol trong bnh cu y

    trn dung tch 50 ml.

    + Ha tan 0,268 g (1,5 mmol) cht 2a vo 20 ml ethanol trong cc c m, rt

    vo bnh phn ng.

    + un hi lu kt hp khuy t 300 vng/pht, tin hnh phn ng trong 3 h.

    X l phn ng:

    + Ct loi dung mi bng my ct quay trong 5 pht nhit 40oC.

    + Thm 20 ml nc ct lnh vo hn hp phn ng.

    + Kim ha hn hp bng dung dch NaOH 30% n pH = 8-9.

    + Chit thu sn phm bng DCM (chit 3 ln mi ln 20 ml DCM), gp cc

    dch chit v lc vi nc mui bo ha, thu v lm khan dch chit bng Na2SO4

    khan, ct quay chn khng 40oC n khi ht dung mi v sn phm kt tinh

    dng rn.

    + Sy sn phm 60oC trong 4 h, thu c 0,19 g.

    Kt qu

    + Cm quan: bt kt tinh mu vng nht.

    + Hiu sut: 72%.

    + Tonc: 192-194oC.

    + Rf = 0,54 (DCM/MeOH = 9/1).

    c. Tng hp cht 4a

  • 24

    Cht methyl 8-oxo-8-(5-phenyl-1,3,4-thiadiazol-2-ylamino)octanoat (4a) c

    tng hp bng phn ng acyl ha hp cht 3a vi acid monomethyl suberic theo s

    3.4 nh sau:

    S 3.4: Quy trnh tng hp cht 4a

    Tin hnh phn ng:

    + Ha tan 0,162 g (1 mmol) CDI v 0,17 ml (1 mmol) acid monomethyl

    suberic vo 1 ml DMF trong bnh phn ng, hot ha nhit phng trong 1 h.

    + Tip tc cho 0,177 g (1 mmol) cht 3a vo bnh phn ng.

    + Duy tr nhit phn ng 60oC kt hp khuy t 300 vng/pht, tin hnh

    phn ng trong 24 h.

    X l phn ng:

    + Lm lnh bnh phn ng, thm t t 20 ml dung dch HCl 5% lnh vo bnh,

    va thm va lc u. lnh 30 pht ta sn phm.

    + Lc ly ta, ra ta bng nc ct n khi dch lc trung tnh.

    + Sy kh ta 60oC, thu c 0,22 g sn phm.

    Kt qu

    + Cm quan: bt kt tinh mu trng hi hng.

    + Hiu sut: 65%.

    + Tonc: 236-238oC.

    + Rf = 0,81 (DCM/MeOH = 9/1).

    d. Tng hp cht 5a

    Cht N1-hydroxy-N8-(5-phenyl-1,3,4-thiadiazol-2-yl)octandiamid (5a) c

    tng hp bng phn ng to acid hydroxamic gia cht 4a vi hydroxylamin

    hydroclorid theo s 3.5 nh sau:

  • 25

    S 3.5: Quy trnh tng hp cht 5a

    Tin hnh phn ng:

    + Cho 0,174 g (0,5 mmol) cht 4a v 0,35 g (5 mmol) hydroxylamin

    hydroclorid vo bnh cu dung tch 50 ml, thm 5 ml methanol, em siu m 5 pht

    to hn dch ng nht.

    + Ha tan 0,8 g (20 mmol) NaOH vo 3 ml nc ct trong ng nghim.

    + ng thi lm lnh bnh phn ng v dung dch NaOH xung di 0oC

    bng hn hp nc mui. Thm t t dung dch NaOH vo bnh phn ng. Duy

    tr nhit phn ng -10oC kt hp khuy t 300 vng/ pht, phn ng kt thc

    sau 1 h. Theo di tin trnh phn ng v xc nh thi im kt thc bng cch dng

    dung dch FeCl3 5% v TLC, c th lm nh sau:

    - Acid ha khong 0,1 ml hn hp phn ng bng dung dch HCl long trn

    khay s, sau nh 1 git dung dch FeCl3 5%, nu xut hin mu vang chng

    t c acid hydroxamic c to ra.

    - Acid ha khong 0,1 ml hn hp phn ng trong vial, thm methanol

    ha tan ta, kim tra bng TLC vi pha ng DCM/MeOH/CH3COOH = 90/10/1.

    Kt thc phn ng khi kt qu cho thy phn ng ht ester ban u.

    X l phn ng:

    + Acid ha hn hp phn ng bng dung dch HCl 5% lnh n pH = 6-7,

    lnh 15 pht ta sn phm.

    + Lc ly ta, ra ta bng nc ct lnh.

    + Sy kh ta 60oC, thu c 0,09 g sn phm.

    Kt qu

    + Cm quan: bt kt tinh mu trng.

    + Hiu sut: 52%.

  • 26

    + Nhit nng chy v Rf c xc nh sau khi kt tinh li trong hn hp

    dung mi methanol - nc, kt qu c th c trnh by trong bng 3.2.

    + Xc nh cu trc bng ph IR, MS, 1H-NMR, 13C-NMR kt qu c th

    c trnh by trong phn 3.1.4.

    3.1.2.2. Tng hp N1-{5-[4-(dimethylamino)pheny]-1,3,4-thiadiazol-2-yl}-N8-

    hydroxyoctandiamid (5b)

    Cht 5b c tng hp theo cc phn ng trnh by trong s 3.6:

    S 3.6: Quy trnh tng hp 5b

    Tc nhn v iu kin: i) Thiosemicarbazid, ethanol, acid acetic bng, hi lu, 4 h; ii) FeCl3.6H2O, HCl 5%, ethanol, hi lu, 3 h; iii) Acid monomethyl suberic, carbonyldiimidazol, DMF, 60oC, 24 h; iv) Hydroxylamin hydroclorid, NaOH, methanol, -10oC, 1 h. a. Tng hp cht 2b

    tng hp cht 5b cn tng hp cht 2-[4-(dimethylamino)benzyliden]

    hydrazincarbothioamid (2b). Qu trnh tng hp cht 2b t 0,298 g (2 mmol) cht

    4-dimethylaminobenzaldehyd (1b) c thc hin tng t nh tng hp cht 2a.

    Kt qu nh sau:

    + Cm quan: bt kt tinh mu trng hi vng.

    + Hiu sut: 88% (0,391 g).

    + Tonc: 169-170oC.

    + Rf = 0,65 (DCM/MeOH = 9/1).

    b. Tng hp cht 3b

    Tng hp cht 5-[4-(dimethylamino)phenyl]-1,3,4-thiadiazol-2-amin (3b) t

    2b bng phn ng ng vng ni phn t theo s 3.7 nh sau:

  • 27

    S 3.7: Quy trnh tng hp 3b

    Tin hnh phn ng:

    + Ha tan 1,22 g (4,5 mmol) FeCl3.6H2O vo 5 ml ethanol trong bnh cu

    dung tch 50 ml.

    + Ha tan 0,333 g (1,5 mmol) cht 2b vo 20 ml HCl 5% trong cc c m, rt

    vo bnh phn.

    + un hi lu kt hp khuy t 300 vng/pht, tin hnh phn ng trong 3 h.

    X l phn ng:

    + Ct loi dung mi bng my ct quay trong 1 pht nhit 40oC.

    + Thm 10 ml nc ct lnh vo hn hp phn ng.

    + Kim ha hn hp bng dung dch NaOH 30% n pH = 8-9.

    + Chit thu sn phm bng DCM, chit 3 ln mi ln 20 ml DCM, gp cc

    dch chit v lc vi nc mui bo ha, thu v lm khan dch chit bng Na2SO4

    khan, ct quay chn khng 40oC n khi ht dung mi v sn phm kt tinh

    dng rn.

    + Sy sn phm 60oC trong 4 h, thu c 0,23 g.

    Kt qu

    + Cm quan: bt kt tinh mu vng nht.

    + Hiu sut: 70%.

    + Tonc: 190-191oC.

    + Rf = 0,50 (DCM/MeOH = 9/1).

    c. Tng hp cht 4b

    Tng hp cht methyl 8-{5-[4-(dimethylamino)phenyl]-1,3,4-thiadiazol-2-

    ylamino}-8-oxooctanoat (4b) t 0,220 g (1 mmol) cht 3b c tin hnh tng t

    nh tng hp cht 4a. Kt qu ca qu trnh nh sau:

    + Cm quan: bt kt tinh mu trng hi hng.

  • 28

    + Hiu sut: 60% (0,23 g).

    + Tonc: 206-208oC.

    + Rf = 0,83 (DCM/MeOH = 9/1).

    d. Tng hp cht 5b

    Tng hp cht N1-{5-[4-(dimethylamino)phenyl]-1,3,4-thiadiazol-2-yl}-N8-

    hydroxyoctandiamid (5b) t 0,195 g (0,5 mmol) cht 4b c tin hnh tng t

    nh tng hp cht 5a. Kt qu nh sau:

    + Cm quan: bt kt tinh mu vng nu nht.

    + Hiu sut: 62% (0,12 g).

    + Nhit nng chy v Rf c xc nh sau khi kt tinh li trong hn hp

    dung mi methanol - nc, kt qu c th c trnh by trong bng 3.2.

    + Xc nh cu trc bng ph IR, MS, 1H-NMR, 13C-NMR kt qu c th

    c trnh by trong phn 3.1.4.

    3.1.2.3. Tng hp N1-[5-(4-fluorophenyl)-1,3,4-thiadiazol-2-yl]-N8-hydroxy

    octandiamid (5c)

    a. Tng hp cht 2c

    tng hp cht 5c cn tng hp cht 2-(4-fluorobenzyliden)

    hydrazincarbothioamid (2c). Qu trnh tng hp cht 2c t 0,21 ml (2 mmol) cht

    4-fluorobenzaldehyd (1c) c thc hin tng t nh tng hp cht 2a. Kt qu

    nh sau:

    + Cm quan: bt kt tinh mu trng.

    + Hiu sut: 90% (0,354 g).

    + Tonc: 166-167oC.

    + Rf = 0,60 (DCM/ MeOH = 9/1).

    b. Tng hp cht 3c

    Tng hp cht 5-(4-fluorophenyl)-1,3,4-thiadiazol-2-amin (3c) t 0,289 g (1,5

    mmol) cht 2c bng phn ng ng vng ni phn t tng t nh tng hp cht

    3a. Kt qu nh sau:

    + Cm quan: bt kt tinh mu xanh en.

  • 29

    + Hiu sut: 71% (0,21 g).

    + Tonc: 185-186oC.

    + Rf = 0,55 (DCM/ MeOH = 9/1).

    c. Tng hp cht 4c

    Tng hp cht methyl 8-[5-(4-fluorophenyl)-1,3,4-thiadiazol-2-ylamino]-8-

    oxooctanoat (4c) t 0,195 g (1 mmol) cht 3c c tin hnh tng t nh tng hp

    cht 4a. Kt qu ca qu trnh nh sau:

    + Cm quan: bt kt tinh mu trng.

    + Hiu sut: 62% (0,23 g).

    + Tonc: 196-198oC.

    + Rf = 0,75 (DCM/MeOH = 9/1).

    d. Tng hp cht 5c

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

    hydroxyoctandiamid (5c) t 0,182 g (0,5 mmol) cht 4c c tin hnh tng t

    nh tng hp cht 5a. Kt qu nh sau:

    + Cm quan: bt kt tinh mu trng.

    + Hiu sut: 60% (0,11 g).

    + Nhit nng chy v Rf c xc nh sau khi kt tinh li trong hn hp

    dung mi methanol - nc, kt qu c th c trnh by trong bng 3.2.

    + Xc nh cu trc bng ph IR, MS, 1H-NMR, 13C-NMR kt qu c th

    c trnh by trong phn 3.1.4.

    3.1.2.4. Tng hp N1-[5-(4-bromophenyl)-1,3,4-thiadiazol-2-yl]-N8-hydroxy

    octandiamid (5d)

    a. Tng hp cht 2d

    tng hp cht 5d cn tng hp cht 2-(4-

    bromobenzyliden)hydrazincarbothioamid (2d). Qu trnh tng hp cht 2d t 0,370

    g ( 2 mmol) cht 4-bromobenzaldehyd (1d) c thc hin tng t nh tng hp

    cht 2a. Kt qu nh sau:

    + Cm quan: bt kt tinh mu trng.

  • 30

    + Hiu sut: 85% (0,44 g).

    + Tonc: 171-172oC.

    + Rf = 0,67 (DCM/MeOH = 9/1).

    b. Tng hp cht 3d

    Tng hp cht 5-(4-bromophenyl)-1,3,4-thiadiazol-2-amin (3d) t 0,387 g

    (1,5 mmol) cht 2d bng phn ng ng vng ni phn t tng t nh tng hp

    cht 3a. Kt qu nh sau:

    + Cm quan: bt kt tinh mu vng nht.

    + Hiu sut: 75% (0,28 g).

    + Tonc: 192-194oC.

    + Rf = 0,50 (DCM/MeOH = 9/1).

    c. Tng hp cht 4d

    Tng hp cht methyl 8-(5-(4-bromophenyl)-1,3,4-thiadiazol-2-ylamino)-8-

    oxooctanoat (4d) t 0,254 g (1 mmol) cht 3d c tin hnh tng t nh tng

    hp cht 4a. Kt qu nh sau:

    + Cm quan: bt kt tinh mu trng hng.

    + Hiu sut: 65% (0,28 g).

    + Tonc: 216-218oC.

    + Rf = 0,80 (DCM/MeOH = 9/1).

    d. Tng hp cht 5d

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

    hydroxyoctandiamid (5d) t 0,214 g (0,5 mmol) cht 4d c tin hnh tng t

    nh tng hp cht 5a. Kt qu nh sau:

    + Cm quan: bt kt tinh mu trng.

    + Hiu sut: 60% (0,12 g).

    + Nhit nng chy v Rf c xc nh sau khi kt tinh li trong hn hp

    dung mi methanol - nc, kt qu c th c trnh by trong bng 3.2.

    + Xc nh cu trc bng ph IR, MS, 1H-NMR, 13C-NMR, kt qu c th

    c trnh by trong phn 3.1.4.

  • 31

    3.1.2.5. Tng hp N1-hydroxy-N8-[5-(4-methylphenyl)-1,3,4-thiadiazol-2-yl]

    octandiamid (5e)

    a. Tng hp cht 2e

    tng hp cht 5e cn tng hp cht 2-(4-methylbenzyliden)

    hydrazincarbothioamid (2e). Qu trnh tng hp cht 2e t 0,24 ml (2 mmol) cht

    4-methylbenzaldehyd (1e) c thc hin tng t nh tng hp cht 2a. Kt qu

    nh sau:

    + Cm quan: bt kt tinh mu trng.

    + Hiu sut: 87% (0,335 g).

    + Tonc: 170-172oC.

    + Rf = 0,63 (DCM/MeOH = 9/1).

    b. Tng hp cht 3e

    Tng hp cht 5-(4-methylphenyl)-1,3,4-thiadiazol-2-amin (3e) t 0,289 g

    (1,5 mmol) cht 2e bng phn ng ng vng ni phn t tng t nh tng hp

    cht 3a. Kt qu nh sau:

    + Cm quan: bt kt tinh mu trng.

    + Hiu sut: 75% (0,21 g).

    + Tonc: 182-184oC.

    + Rf = 0,50 (DCM/MeOH = 9/1).

    c. Tng hp cht 4e

    Tng hp cht methyl 8-[5-(4-methylphenyl)-1,3,4-thiadiazol-2-ylamino]-8-

    oxooctanoat (4e) t 0,191 g (1 mmol) cht 3e c tin hnh tng t nh tng hp

    cht 4a. Kt qu nh sau:

    + Cm quan: bt kt tinh mu trng.

    + Hiu sut: 60% (0,22 g).

    + Tonc: 196-198oC.

    + Rf = 0,77 (DCM/MeOH = 9/1).

    d. Tng hp cht 5e

  • 32

    Tng hp cht N1-hydroxy-N8-[5-(4-methylphenyl)-1,3,4-thiadiazol-2-

    yl]octandiamid (5e) t 0,180 g (0,5 mmol) cht 4e c tin hnh tng t nh

    tng hp cht 5a. Kt qu nh sau:

    + Cm quan: bt kt tinh mu vng nht.

    + Hiu sut: 65% (0,11 g).

    + Nhit nng chy v Rf c xc nh sau khi kt tinh li trong hn hp

    dung mi methanol - nc, kt qu c th c trnh by trong bng 3.2.

    + Xc nh cu trc bng ph IR, MS, 1H-NMR, 13C-NMR kt qu c th

    c trnh by trong phn 3.1.3.

    3.1.2.6. Tng hp N1-[5-(2,6-diclorophenyl)-1,3,4-thiadiazol-2-yl]-N8-hydroxy

    octandiamid (5f)

    a. Tng hp cht 2f

    tng hp cht 5f cn tng hp cht 2-(2,6-diclorobenzyliden)

    hydrazincarbothioamid (2f). Qu trnh tng hp cht 2f t 0,35 g (2 mmol) cht

    2,6-diclorobenzaldehyd (1f) c thc hin tng t nh tng hp cht 2a. Kt qu

    nh sau:

    + Cm quan: bt kt tinh mu trng.

    + Hiu sut: 85% (0,42 g).

    + Tonc: 170-172oC.

    + Rf = 0,66 (DCM/MeOH = 9/1).

    b. Tng hp cht 3f

    Tng hp cht 5-(2,6-diclorolphenyl)-1,3,4-thiadiazol-2-amin (3f) t 0,372 g

    (1,5 mmol) cht 2f bng phn ng ng vng ni phn t tng t nh tng hp

    cht 3a. Kt qu nh sau:

    + Cm quan: bt kt tinh mu trng.

    + Hiu sut: 70% (0,26 g).

    + Tonc: 192-194oC.

    + Rf = 0,50 (DCM/MeOH = 9/1).

    c. Tng hp cht 4f

  • 33

    Tng hp cht methyl 8-[5-(2,6-diclorophenyl)-1,3,4-thiadiazol-2-ylamino]-8-

    oxooctanoat (4f) t 0,246 g (1 mmol) cht 3f c tin hnh tng t nh tng hp

    cht 4a. Kt qu nh sau:

    + Cm quan: bt kt tinh mu trng.

    + Hiu sut: 62% (0,26 g).

    + Tonc: 212-213oC.

    + Rf = 0,73 (DCM/MeOH = 9/1).

    d. Tng hp cht 5f

    Tng hp cht N1-[5-(2,6-diclorophenyl)-1,3,4-thiadiazol-2-yl]-N8-

    hydroxyoctandiamid (5f) t 0,209 g (0,5 mmol) cht 4f c tin hnh tng t

    nh tng hp cht 5a. Kt qu nh sau:

    + Cm quan: bt kt tinh mu vng nht.

    + Hiu sut: 61% (0,13 g).

    + Nhit nng chy v Rf c xc nh sau khi kt tinh li trong hn hp

    dung mi methanol - nc, kt qu c th c trnh by trong bng 3.2.

    + Xc nh cu trc bng ph IR, MS, 1H-NMR, 13C-NMR kt qu c th

    c trnh by trong phn 3.1.4.

    * Sau khi tng hp c 6 cht 5a-f, chng ti tin hnh kt tinh li cc cht

    bng hn hp dung mi methanol nc. Di y l bng tng hp cc ch s l

    ha v hiu sut tng hp ca cc cht 5a-f:

    Bng 3.2: Ch s l ha v hiu sut tng hp cc acid hydroxamic t ester

    Cht R CTPT KLPT Mu H(%)

    5a H C16H20N4O3S 348 Trng 52 5b 4-N(CH3)2 C18H25N5O3S 391 Vng nu nht 62 5c 4-F C16H19FN403S 366 Trng 60 5d 4-Br C16H19BrN403S 427 Trng 60 5e 4-CH3 C17H22N4O3S 362 Vng nht 65 5f 2,6-Cl2 C16H18Cl2N4O3S 417 Vng nht 61

  • 34

    3.1.3. Kim tra tinh khit

    * Sc k lp mng (TLC)

    Sc k lp mng c tin hnh trn bn nhm trng sn silicagel Merck 70-

    230 mesh, vi 3 h dung mi khc nhau l DCM/MeOH (9/1), DCM/MeOH (5/1),

    DCM/MeOH/AcOH (90/10/1), quan st di n t ngoi bc sng 254 nm. Kt

    qu cho thy cc cht u c vt gn, r, khng c vt ph. Ch s Rf ca cc dn

    cht 5a-f khi s dng 1 h dung mi c th c trnh by trong bng 3.2.

    * o nhit nng chy

    Cc cht sau khi c kt tinh li u c dng tinh th rn, c th tin hnh o

    c nhit nng chy bng my o nhit nng chy nhit in. Kt qu trnh

    by trong bng 3.2 cho thy cc cht 5a-f u c nhit nng chy xc nh

    (khong dao ng nhit hp t 1-2oC).

    Nh vy t kt qu chy sc k lp mng v o nhit nng chy ca 6 dn

    cht 5a-f, chng ti khng nh cc cht tng hp c l tinh khit v c th s

    dng o ph IR, MS, 1H-NMR, 13C-NMR.

    Bng 3.3: Gi tr Rf v nhit nng chy (tonc) ca cc cht 5a-f

    Cht R H dung mi Rf T

    onc (

    oC) 5a H DCM:MeOH:AcOH=90:10:1 0,32 202-204

    5b 4-N(CH3)2 DCM:MeOH:AcOH=90:10:1 0,40 197-198

    5c 4-F DCM:MeOH:AcOH=90:10:1 0,35 223-225

    5d 4-Br DCM:MeOH:AcOH=90:10:1 0,37 216-217

    5e 4-CH3 DCM:MeOH:AcOH=90:10:1 0,40 194-196

    5f 2,6-Cl2 DCM:MeOH:AcOH=90:10:1 0,32 206-207

    3.1.4. Xc nh cu trc

    khng nh cu trc ca cc hp cht tng hp c, chng ti tin hnh

    ghi ph khi lng (MS) ca dy cht 4a-f; ph khi lng (MS), ph hng ngoi

    (IR) v ph cng hng t ht nhn proton (1H-NMR) v carbon (13C-NMR) ca

  • 35

    dy cht 5a-f. Kt qu ghi ph ca cc cht c th c trnh by trong phn phn

    tch ph v ph lc.

    3.1.4.1. Kt qu phn tch ph khi lng ca dy cht 4a-f

    Bng 3.4: Kt qu phn tch ph MS ca cc cht 4a-f

    Cht R KLPT m/z (ESI-MS)

    4a H 347 347 [M]-

    4b 4-N(CH3)2 390 388 [M-2H]-

    4c 4-F 365 363 [M-2H]-

    4d 4-Br 426 425 [M-H]-

    4e 4-CH3 361 359 [M-2H]-

    4f 2,6-Cl2 416 415 [M-H]-

    Nhn xt: Qua kt qu phn tch ph bng 3.3 v ph ca 6 cht 4a-f

    (ph lc 7-12), chng ti thy trn tt c cc ph u c pc phn t c s khi

    ng bng s khi d kin ca cht tng ng vi cng mnh. Ph t c pc

    phn mnh, cc pc phn mnh c cng nh. V vy c th khng nh ph

    l ph hp vi cng thc ca 6 cht d kin 4a-f [25].

    3.1.4.2. Kt qu phn tch ph ca dy cht 5a-f

    a. Ph hng ngoi (IR)

    Kt qu phn tch ph hng ngoi c trnh by trong bng 3.4:

  • 36

    Bng 3.5: Kt qu phn tch ph IR ca cc cht 5a-f

    Cht R S sng (cm-1) ng vi cc dao ng

    N-H CH

    (benzen) CH

    (CH2) C=O

    C=C (benzen)

    5a H 3251 3050 2927 2865

    1629 1558

    5b 4-N(CH3)2 3227 2926 2853

    1652 1609

    1570 1539

    5c 4-F 3239 3054 2939 2853

    1687 1628

    1597 1557

    5d 4-Br 3163 3049 2923 2850

    1691 1616

    1571 1557

    5e 4-CH3 3162 3023 2929 2863

    1702 1615

    1561

    5f 2,6-Cl2 3155 2927 2856

    1701 1629

    1534

    Ghi ch: l dao ng ha tr.

    Nhn xt: Ph IR ca cc cht 5a- f khng c nhiu tn hiu khng nh

    chc chn cu trc ca cc cht. Nhng da trn ph (ph lc 1-6) v tham kho

    mt s ti liu [10,11,15], cng c th nhn dng s b s hin din ca 1 s nhm

    chc nh NH, CO... thng qua cc gi tr ca di hp thu, cng pic, hnh dng

    pic. C th, cc cht u c nh hp thu ca nhm NH trong khong 3140-3250

    cm-1, c nh hp thu ca C=O amid t 1600-1700 cm-1. Bn cnh , cc di hp

    thu c trng cho vng benzen cng c ghi nhn nh di ko cng Caren-H xut

    hin gia 3020-3060 cm-1 v di ko cng ca C=C trong vng thm hp thu trong

    vng 1500-1600 cm-1. Trn ph cn c cc nh hp thu trong khong 2850-

    2950 cm-1 c trng cho dao ng ko cng C-H ca cc nhm methylen. Nh vy

    c th khng nh d liu ph IR ph hp vi cng thc cu to d kin ca 6 cht

    5a-f.

    b. Ph khi lng (MS)

  • 37

    Bng 3.6: Kt qu phn tch ph MS ca cc cht 5a-f

    Cht R KLPT m/z (ESI-MS)

    5a H 348 347 [M-H]-

    5b 4-N(CH3)2 391 389 [M-2H]-, 374 [M-OH]-

    5c 4-F 366 364 [M-2H]-

    5d 4-Br 427 426 [M-H]-

    5e 4-CH3 362 360 [M-2H]-, 345 [M-OH]-

    5f 2,6-Cl2 417 416 [M-H]-

    Nhn xt: Qua kt qu phn tch ph bng 3.5 v ph (ph lc 13-18)

    chng ti thy cc ph u c pc phn t c s khi ng bng s khi d kin vi

    cng mnh nht, ph t pc phn mnh, cc pc phn mnh u c cng

    nh. V vy c th khng nh ph l ph hp vi cng thc phn t d kin ca

    6 cht 5a-f [25].

    c. Ph cng hng t ht nhn 1H (1H-NMR)

    Bng 3.7: Kt qu phn tch ph 1H-NMR ca cc cht 5a-f

    Cht R S liu phn tch 1H-NMR

    5a H

    1H-NMR (500 MHz, DMSO-d6, ppm): 1,26 (4H, m,

    CH2); 1,47-1,49 (2H, m, CH2); 1,59-1,61 (2H, m, CH2);

    1,92-1,95 (2H, m, CH2); 2,47-2,50 (2H, m, CH2); 7,51-7,52 (3H, m, H-2,H-4,H-6); 7,92-7,93 (2H, m, H-3, H-5);

    8,65 (1H, s, OH); 10,36 (1H, s, NH); 12,61 (1H, s, NH).

    5b 4-N(CH3)2

    1H-NMR (500 MHz, DMSO-d6, ppm): 1,27 (4H, m,

    CH2); 1,47-1,50 (2H, m, CH2); 1,58-1,61 (2H, m, CH2);

    1,95 (2H, t, CH2); 2,46-2,50 (2H, m, CH2); 2,98 (6H, s, -CH3); 6,79 (2H, d, H-3, H-5); 7,71 (2H, d, H-2, H-6);

    10,37 (1H, s, NH); 12,41 (1H, s, NH).

  • 38

    5c 4-F

    1H-NMR (500 MHz, DMSO-d6, ppm): 1,25-1,26 (4H, m, CH2); 1,46-1,50 (2H, m, CH2); 1,56-1,60 (2H, m, CH2);

    1,93-1,96 (2H, m, CH2); 2,47-2,50 (2H, m, CH2); 7,34

    (2H, t, H-3, H-5); 7,95-7,98 (2H, m, H-2, H-6); 10,42(1H,

    s, NH); 12,63 (1H, s, NH).

    5d 4-Br

    1H-NMR (500 MHz, DMSO-d6, ppm): 1,27-1,29 (4H, m,

    CH2); 1,45-1,49 (2H, m, CH2); 1,59-1,61 (2H, m, CH2);

    1,94 (1H, t, CH2); 2,19 (1H, t, CH2); 2,48-2,50 (2H, m,

    CH2); 7,71 (2H, d, H-3, H-5); 7,86-7,88 (2H, m, H-2, H-6); 10,37(1H, s, NH); 12,68 (1H, s, NH).

    5e 4-CH3

    1H-NMR (500 MHz, DMSO-d6, ppm): 1,27-1,29 (4H, m,

    CH2); 1,47-1,50 (2H, m, CH2); 1,58-1,61 (2H, m, CH2);

    1,94 (1H, t, CH2a); 2,19 (1H, t, CH2b); 2,35 (3H, s, -CH3); 2,47-2,50 (2H, m, CH2); 7,32 (2H, d, H-3, H-5); 7,80 (2H,

    d, H-2, H-6); 10,36 (1H, s, NH); 12,59 (1H, s, NH).

    5f 2,6-Cl2

    1H-NMR (500 MHz, DMSO-d6, ppm): 1,22-1,29 (4H, m,

    CH2); 1,47-1,49 (2H, m, CH2); 1,59-1,61 (2H, m, CH2);

    1,95 (2H, t, CH2); 2,50-2,54 (2H, m, CH2); 7,61 (1H, t, H-4); 7,67 (2H, d, H-3, H-5); 8,75 (1H, s ,OH); 10,46 (1H,

    s, NH). Ghi ch: : chuyn dch ha hc (ppm); s: singlet, vch n; d: doublet, vch i; t: triplet, vch ba; m: multiplet, a vch.

    Nhn xt: T ph 1H-NMR ca cc cht tng hp c c th thy s lng

    proton, dch chuyn, bi tn hiu l ph hp vi cng thc cu to d kin

    ca c 6 cht 5a-f [2]. C th, cc cht u c cc proton ca vng benzen nm

    trong vng dch chuyn t 6,8-7,9 ppm v 12 proton ca phn cu ni nm trong

    vng dch chuyn t 1,2-2,5 ppm. Hai proton ca 2 nhm -NH-CO- u xut hin

    r, di dng 2 vch n khong 10,3-12,7 ppm. Tn hiu ca proton nhm OH

    xut hin dng vch n nhng b gin rng khong 8,6-8,8 ppm trn ph

    ca cht 5a v 5f, nhng ph ca cht 5b-e khng c tn hiu ny. C th do

    proton ca nhm OH linh ng d b trao i hay h bin nn cho tn hiu rt yu

    hoc khng cho tn hiu trn ph . Ngoi ra, hai dn cht 5b v 5e c nhm th

    cha proton trn vng benzen tng ng l -N(CH3)2 v -CH3 u cho tn hiu ca

    cc proton trong nhm th rt r rng.

  • 39

    d. Ph cng hng t ht nhn13C (13C-NMR)

    Bng 3.8: Kt qu phn tch ph 13C-NMR ca cc cht 5a-f

    Cht R S liu phn tch 13C-NMR

    5a H

    13C-NMR (125 MHz, DMSO-d6, ppm): 171,65 (C5);

    169,22 (C1); 161,74 (C8); 158,37 (C2); 130,59 (C1); 130,26 (C4); 129,40 (C2-C6); 126,92 (C3-C5); 34,87 (C2);

    30,25 (C7); 28,30 (C4); 28,25 (C5); 24,99 (C3); 24,50

    (C6).

    5b 4-N(CH3)2

    13C-NMR (125 MHz, DMSO-d6, ppm): 171,26 (C5); 169,09 (C1); 162,25 (C8); 156,64 (C2); 151,52 (C4);

    127,89 (C2-C6); 117,52 (C1); 112,06 (C3-C5); 34,78 (C2);

    32,16 (C7); 28,23 (C4); 28,18 (C5); 24.91 (C3); 24,48

    (C6).

    5c 4-F

    13C-NMR (125 MHz, DMSO-d6, ppm): 171,62 (C5);

    169,21 (C1); 162,30 (C4); 160,60 (C8); 158,39 (C2);

    129,22 (C2); 129,15 (C6); 126,88 (C1); 116,47 (C3); 116,30

    (C5); 34,83 (C2); 32,22 (C7); 28,28 (C4); 28,23 (C5); 24,97 (C3); 24,47 (C6).

    5d 4-Br

    13C-NMR (125 MHz, DMSO-d6, ppm): 174,37 (C5);

    171,63 (C1); 160,58 (C8); 158,56 (C2); 132,26 (C3-C5);

    129,34 (C1); 128,71 (C2-C6); 123,76 (C4); 34,80 (C2);

    33,59 (C7); 32,17 (C4); 28,18 (C5); 24,91 (C3); 24,42 (C6).

    5e 4-CH3

    13C-NMR (125 MHz, DMSO-d6, ppm): 174,40 (C5);

    171,53 (C1); 161,71 (C8); 157,98 (C2); 140,39 (C4);

    129,85 (C3-C5); 127,53 (C1); 126,78 (C2-C6); 34.80 (C2); 33,61 (C7); 28,18 (C4-C5); 24.94 (C6); 24,43 (C3);

    20,93 (C, CH3).

    5f 2,6-Cl2

    13C-NMR (125 MHz, DMSO-d6, ppm): 172,04 (C5);

    169,07 (C1); 160,46 (C8); 155,32 (C2); 135,05 (C2-C6); 132,86 (C1); 128,66 (C3-C5); 128,34 (C4); 34,82 (C2);

    32,16 (C7); 28,24 (C4-C5); 24,92 (C3); 24,42 (C4). Ghi ch: : chuyn dch ha hc (ppm)

  • 40

    Nhn xt: Qua kt qu phn tch ph bng 3.7 v ph (ph lc 25-30)

    chng ti nhn thy: s lng carbon, dch chuyn ha hc ca cc carbon l

    ph hp vi cng thc cu to d kin [2]. Cc cht u c 2 pc ca 2 nhm C=O,

    2 pc ca vng thiadiazol, cc pc ca nhn benzen v cc pc ca 6 nhm CH2.

    * Nh vy, kt hp kt qu phn tch cc ph IR, MS, 1H-NMR v 13C-NMR

    chng ti c th khng nh 6 cht 5a-f tng hp c c cng thc cu to ng

    nh d kin. Trn c s , cc acid hydroxamic ny tip tc c tin hnh th

    hot tnh sinh hc.

    3.2. TH HOT TNH SINH HC

    3.2.1. Th hot tnh sinh hc

    3.2.1.1. Tc dng c ch HDAC

    Cc acid hydroxamic 5a-f c th nghim tc dng c ch HDAC ti khoa

    Dc, i hc Chungbuk (Cheongju, Hn Quc). Kt qu c trnh by bng

    3.10 trong phn bn lun.

    3.2.1.2. Hot tnh khng t bo ung th in vitro

    Cc cht 5b-f c nh gi hot tnh khng t bo ung th ngi trn dng

    t bo ung th i trng (SW620). Kt qu hot tnh ca cht 5a trch dn t lun

    vn thc s ca Th nh Tuyt [4]. Kt qu gi tr IC50 c th ca cc cht c

    trnh by bng 3.10 trong phn bn lun.

    3.2.2. nh gi mc ging thuc

    Bng 3.9: nh gi mc ging thuc ca cc cht 5a-f theo quy tc Lipinsky

    Cht R LogP KLPT S NH,

    OH S N, O

    S lin kt linh ng

    5a H 1,35 348 3 7 3 5b 4-N(CH3)2 1,53 391 3 8 3 5c 4-F 1,55 366 3 7 3 5d 4-Br 2,24 427 3 7 3 5e 4-CH3 1,90 362 3 7 3 5f 2,6-Cl2 2,64 417 3 7 3

  • 41

    Nhn xt :Tt c cc cht u tha mn c 5 yu cu ca quy tc Lipinsky

    v ging thuc. Cng vi kt qu hot tnh sinh hc in vitro tt, cc cht c

    nhiu trin vng cho vic nghin cu tc dng in vivo.

    3.3. BN LUN

    3.3.1. Tng hp ha hc

    Cc cht 5a-f v cc cht trung gian m chng ti tng hp nhn chung l cc

    cht c cu to phc tp, c mch C di, c cc nhm chc nhy cm vi cc yu t

    ca mi trng phn ng. V vy khi thc hin phn ng chng ti cn m bo

    nghim ngt v iu kin phn ng. C th:

    - Vi phn ng tng hp cc ester 4a-f, cn m bo dng c phi kh hon

    ton, cc ha cht v dung mi phi khan nc. V CDI d b phn hy v khng

    cn kh nng hot ha acid monomethyl suberic nu tip xc vi nc.

    - Vi phn ng tng hp acid hydroxamic 5a-f: trnh phn hy ester thnh

    acid carboxylic bi dung dch NaOH m c, cn lm lnh hn dch cht 4a-f v

    dung dch NaOH trc khi cho vo bnh phn ng. Phn ng cng cn tin hnh

    nhit thp vi thi gian phn ng ngn trnh phn hy nhm amid. Lng

    NaOH phi dng d chuyn ht NH2OH.HCl thnh NH2OH tan v ng thi

    m bo cc acid hydroxamic to thnh tan ht trong dung dch phn ng.

    3.3.2. Tc dng sinh hc

    h tr trong qu trnh thit k cu trc v tm hiu kh nng gn vo trung

    tm hot ng trn enzym HDAC ca cc cht, chng ti tin hnh nghin cu

    docking vi 6 cht 5a-f. So snh m hnh tng tc vi HDAC8 ca cc cht 5a-f

    v SAHA c th thy phn cu ni gia nhm kha hot ng v nhm gn vi

    Zn2+ ca 6 cht cng nh ca SAHA c cu trc tng i ging nhau. Nh vy cu

    ni vi di 6C ca cc cht tng hp c c nhiu kh nng a c nhm -

    NHOH tin gn ion Zn2+ ging nh SAHA. Bn cnh , nng lng lin kt ca

    cc cht 5a-f vi trung tm hot ng ca HDAC8 c d on t -7,4 n -6,6

    kcal/mol, ngha l u ln hn so vi SAHA (-4,4 kcal/mol), chng t cc cht c

    i lc vi HDAC8 mnh hn SAHA. C th gii thch do trong nhm nhn din b

  • 42

    mt ca c 6 cht ny u c nguyn t S v N lm tng tng tc Van der Waals

    ca cc cht vi cc acid amin trn ming ti ca enzym HDAC8. Tm li, kt qu

    docking cho thy cc cht 5a-f ph hp vi hng thit k cu trc nhm tm kim

    nhng cht c kh nng c ch HDAC. Trn c s chng ti tip tc tin

    hnh tng hp ha hc v th tc dng sinh hc.

    Bng 3.10: Kt qu th tc dng c ch HDAC v hot tnh khng t bo ung th

    Cht R Tc dng c ch HDAC1

    c tnh t bo (IC50) 2

    (g/ml) (M) 5a H +3 4,690 13,477 5b 4-N(CH3)2 + 1,070 2,737 5c 4-F + 0,394 1,077 5d 4-Br + 0,857 2,007 5e 4-CH3 + 1,878 5,188 5f 2,6-Cl2 + 3,296 7,904

    SAHA + 0,976 3,697 Ghi ch: 1Hot tnh ca enzym b c ch hon ton khi th ti 10g/mL bng phng php Western Blot; 2Nng c ch 50% s pht trin ca t bo ung th SW620; 3Hin tng deacetyl

    ha khng xy ra (hot tnh ca enzyme b c ch hon ton).

    Kt qu th tc dng trn enzym HDAC cho thy c 6 cht 5a-f u lm cho

    hot tnh enzym b c ch hon ton khi th ti nng 10 g/ml bng phng

    php Western Blot. T kt qu ny c th kt lun c 6 cht tng hp c u c

    tc dng c ch HDAC.

    Tc dng c ch HDAC quyt nh tc dng khng t bo ung th, tuy nhin

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

    trong t bo tip cn vi HDAC. Do chng ti tip tc tin hnh th c tnh t

    bo in vitro nhm tm ra nhng cht thc s c tc dng sinh hc tt. Th nghim

    tin hnh trn dng t bo ung th i trng SW620 cho thy IC50 ca cc u nh

    hn 20 g/ml, tc l c 6 cht u c hot tnh khng t bo ung th SW620. Do

    cc cht tc dng theo c ch phn t nn so snh hot tnh gia cc cht chng

  • 43

    ti quy i gi tr IC50 t g/ml sang M. Khi gi tr IC50 ca 5a-f l t 1,007

    M n 13,477 M, so vi SAHA (IC50= 3,697 M) chng ti c 3 cht 5b, 5c, 5d

    c tc dng tt hn, cc cht cn li nng c ch 50% s pht trin ca t bo

    ung th u tng i thp. Kt qu th c tnh t bo cng cho thy s thay i

    nhm th trn khung 5-phenyl-1,3,4-thiadiazol mang li hot tnh khng t bo ung

    th khc nhau r rt. C th, cht 5a khng mang nhm th trn nhn phenyl c

    hot tnh thp nht (IC50= 13,477 M), nhng khi c nhm th d l nhm ht hay

    y in t th hot tnh cng tng ln ng k. So snh hot tnh ca cc cht vi

    5a c th thy, cht 5b c thm 1 nhm y in t ( -N(CH3)2) trn nhn phenyl

    th IC50 gim khong 5 ln, cht 5e cng mang nhm y in t -CH3 trn nhn

    phenyl c IC50 gim 2,5 ln. Vi cht 5c, 5d c nhm th ht in t (-F, -Br) gi tr

    IC50 cn thp hn rt nhiu v thp hn c SAHA. So snh vi cht c nhm th 4-

    Cl cng b trc y trong lun vn thc s ca Th nh Tuyt th 5c cng c

    c tnh trn dng t bo SW620 cao hn (IC50 = 1,007 M vi 1,63 M). Tuy

    nhin, cht 5f c 2 nhm th trn nhn phenyl tc dng c tnh t bo li gim so

    vi cc cht c 1 nhm th, mc d vn cao hn cht khng c nhm th. C th do

    2 nhm th ca cht 5f lm mt phng ca vng benzen xoay i nhiu so vi vng

    thiadiazol v cu trc ny khng ph hp vi trung tm hot ng ca HDAC. Tm

    li, s c mt ca 1 nhm th trn nhn phenyl l cn thit i vi cc dn cht N1-

    hydroxy-N8-(5-phenyl-1,3,4-thiadiazol-2-yl)octandiamid c hot tnh sinh hc tt

    hn SAHA.

    Vi nhng kt qu thu c c th khng nh cu ni gia khung 5-phenyl-

    1,3,4-thiadiazol v nhm NHOH c di 6C l ph hp em hot tnh c ch

    HDAC cng nh c tnh trn t bo ung th v khung 5-phenyl-1,3,4-thiadiazol c

    nhiu trin vng thay th nhn phenyl ca SAHA nhm tm ra cc cc cht c ch

    HDAC mi.

    Thm vo , cc cht ch 5a-f tng hp c khng ch c hot tnh sinh

    hc cao m cn tha mn c 5 yu cu ca quy tc Lipinsky, iu ny m ra trin

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

  • 44

    KT LUN V KIN NGH

    I. KT LUN

    T cc kt qu nghin cu trnh by c th rt ra mt s kt lun sau:

    1.1. V tng hp v khng nh cu trc

    * tng c 6 cht nh d kin, trong 5 cht 5b-f cha tng c cng

    b trong bt k ti liu no:

    - N1-hydroxy-N8-(5-phenyl-1,3,4-thiadiazol-2-yl)octandiamid (5a).

    - N1-{5-[4-(dimethylamino)phenyl]-1,3,4-thiadiazol-2-yl}-N8-

    hydroxyoctandiamid (5b).

    - N1-[5-(4-flourophenyl)-1,3,4-thiadiazol-2-yl]-N8-hydroxyoctandiamid (5c).

    - N1-[5-(4-bromophenyl)-1,3,4-thiadiazol-2-yl]-N8-hydroxyoctandiamid (5d).

    - N1-hydroxy-N8-[5-(4-methylphenyl)-1,3,4-thiadiazol-2-yl]octandiamid (5e).

    - N1-[5-(2,6-diclorophenyl)-1,3,4-thiadiazol-2-yl]-N8-hydroxyoctandiamid

    (5f).

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

    ph IR, MS, 1H-NMR, 13C-NMR.

    1.2. V hot tnh sinh hc

    * th tc dng c ch HDAC v hot tnh khng t bo ung th ca cc

    cht tng hp c, kt qu cho thy:

    - V tc dng c ch HDAC: c 6 cht u c tc dng c ch HDAC.

    - V tc dng khng t bo ung th: c 6 cht u c hot tnh c ch mnh

    vi dng t bo ung th i trng SW620. Trong cht 5c (IC50 = 1,007 M) c

    tc dng mnh nht, mnh gp 3 ln so vi SAHA (IC50 = 3,697 M).

    II. KIN NGH

    - Tin hnh th hot tnh khng t bo ung th in vitro ca cc cht tng hp

    c trn cc dng t bo ung th khc, tm ra cht c tc dng mnh lm c s cho

    vic th tc dng in vivo.

    - Nghin cu tng hp dy cht thay th khung 5-phenyl-1,3,4-thiadiazol bng

    cc nhm ng cu in t v cc vng thm khc.

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  • PH LC

    Ph lc 1 : Ph hng ngoi IR ca 5a

    Ph lc 2 : Ph hng ngoi IR ca 5b

    Ph lc 3 : Ph hng ngoi IR ca 5c

    Ph lc 4 : Ph hng ngoi IR ca 5d

    Ph lc 5 : Ph hng ngoi IR ca 5e

    Ph lc 6 : Ph hng ngoi IR ca 5f

    Ph lc 7 : Ph khi MS ca 4a

    Ph lc 8 : Ph khi MS ca 4b

    Ph lc 9 : Ph khi MS ca 4c

    Ph lc 10 : Ph khi MS ca 4d

    Ph lc 11 : Ph khi MS ca 4e

    Ph lc 12 : Ph khi MS ca 4f

    Ph lc 13 : Ph khi MS ca 5a

    Ph lc 14 : Ph khi MS ca 5b

    Ph lc 15 : Ph khi MS ca 5c

    Ph lc 16 : Ph khi MS ca 5d

    Ph lc 17 : Ph khi MS ca 5e

    Ph lc 18 : Ph khi MS ca 5f

    Ph lc 19: Ph 1H-NMR ca 5a

    Ph lc 20: Ph 1H-NMR ca 5b

    Ph lc 21: Ph 1H-NMR ca 5c

    Ph lc 22: Ph 1H-NMR ca 5d

    Ph lc 23: Ph 1H-NMR ca 5e

    Ph lc 24: Ph 1H-NMR ca 5f

    Ph lc 25: Ph 13C-NMR ca 5a

    Ph lc 26: Ph 13C-NMR ca 5b

    Ph lc 27: Ph 13C-NMR ca 5c

    Ph lc 28: Ph 13C-NMR ca 5d

    Ph lc 29: Ph 13C-NMR ca 5e

    Ph lc 30: Ph 13C-NMR ca 5f

  • Ph lc 1: Ph hng ngoi IR ca 5a

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

    BO MON HOA VAT LIEU-KHOA HOA-TRUONG DHKHTN

    Nguoi do: Phan Thi Tuyet MaiTen mau: T1Date: 3/5/2012

    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

    100.0

    cm-1

    %T

    3251

    3050

    2927

    2865

    1629

    1558

    1384

    1339

    1311

    1179

    1094

    974

    841

    759

    689

    668

  • Ph lc 2: Ph hng ngoi IR ca 5b

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

    BO MON HOA VAT LIEU-KHOA HOA-TRUONG DHKHTN

    Nguoi do: Phan Thi Tuyet MaiTen mau: 3IDate: 11/26/2012

    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

    80.0

    cm-1

    %T

    3227

    2926

    2853 1652

    16091570

    1539

    1459

    1439

    1424

    1371

    1344

    1305

    1234

    1189

    1121

    1095

    1059

    1040

    974

    946

    890

    870

    851

    833

    809

    758

    741

    723

    704

    686

    663N N

    SHN

    O

    O

    NH

    OH

    N

  • Ph lc 3: Ph hng ngoi IR ca 5c

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

    BO MON HOA VAT LIEU-KHOA HOA-TRUONG DHKHTN

    Nguoi do: Phan Thi Tuyet MaiTen mau: 3EDate: 11/26/2012

    4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 400.0

    0.0

    5

    10

    15

    20

    25

    30

    35

    40

    45

    50

    55

    60

    65

    70

    75

    80

    85

    90

    95

    100.0

    cm-1

    %T

    3239

    3054

    2939

    2853 1687

    1628

    1597

    1557

    1515

    1465

    14451416

    1383

    1342

    1312

    1301

    1272

    1232

    1178

    1159

    1110

    1098 991

    974

    841

    728

    660

    606

    584

    517

  • Ph lc 4: Ph hng ngoi IR ca 5d

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

    BO MON HOA VAT LIEU-KHOA HOA-TRUONG DHKHTN

    Nguoi do: Phan Thi Tuyet Maiten mau: 3FDate: 11/26/2012

    4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 400.0

    0.0

    5

    10

    15

    20

    25

    30

    35

    40

    45

    50

    55

    60

    65

    70

    75

    80

    85

    90

    95

    100.0

    cm-1

    %T

    3163

    3049

    2923

    2850

    1691

    1616

    1571

    1557

    1496

    1443

    1409

    1394

    1340

    1317

    1302

    1252

    1230

    1175

    1116

    1103

    1069

    1041

    1011

    985

    964

    829

    792

    727

    705

    637

    619

    603

    500

  • Ph lc 5: Ph hng ngoi IR ca 5e

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

    BO MON HOA VAT LIEU-KHOA HOA-TRUONG DHKHTN

    Nguoi do: Phan Thi Tuyet MaiTen mau: 3GDate: 12/7/2012

    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

    100.0

    cm-1

    %T

    3162

    3023

    2929

    2863

    1702

    1615

    1561

    1463

    1442

    1410

    1385

    1335

    1306

    1270

    1242

    1177

    1101

    985

    966

    883

    819

    792 709

    658

    630

    606

  • Ph lc 6: Ph hng ngoi IR ca 5f

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

    BO MON HOA VAT LIEU-KHOA HOA-TRUONG DHKHTN

    Nguoi do: Phan Thi Tuyet MaiTen mau: 12lDate: 10/15/2012

    4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 400.0

    0.0

    5

    10

    15

    20

    25

    30

    35

    40

    45

    50

    55

    60

    65

    70

    75

    80

    85

    90.0

    cm-1

    %T 3155

    2927

    2856 1701

    1629

    1534

    1431

    1383

    1302

    1242

    1192

    1166

    1130

    1091 982

    787

    734

    494

    409

  • Ph lc 7: Ph khi MS ca 4a Ph lc 8: Ph khi MS ca 4b

    S

    NN

    NH

    O

    OCH3

    O

  • Ph lc 9: Ph khi MS ca 4c Ph lc 10: Ph khi MS ca 4d

  • Ph lc 11: Ph khi MS ca 4e Ph lc 12: Ph khi MS ca 4f

  • Ph lc 13: Ph khi MS ca 5a Ph lc 14: Ph khi MS ca 5b

  • Ph lc 15: Ph khi MS ca 5c Ph lc 16: Ph khi MS ca 5d

  • Ph lc 17: Ph khi MS ca 5e Ph lc 18: Ph khi MS ca 5f

  • Ph lc 19: Ph 1H-NMR ca 5a

  • Ph lc 19: Ph 1H-NMR ca 5a (m rng)

  • Ph lc 20: Ph 1H-NMR ca 5b

  • Ph lc 20: Ph 1H-NMR ca 5b (m rng)

  • Ph lc 21: Ph 1H-NMR ca 5c

  • Ph lc 21: Ph 1H-NMR ca 5c (m rng)

  • Ph lc 22: Ph 1H-NMR ca 5d

  • Ph lc 22: Ph 1H-NMR ca 5d (m rng)

  • Ph lc 23: Ph 1H-NMR ca 5e

  • Ph lc 23: Ph 1H-NMR ca 5e (m rng)

  • Ph lc 24: Ph 1H-NMR ca 5f

  • Ph lc 24: Ph 1H-NMR ca 5f (m rng)

  • Ph lc 25: Ph 13C-NMR ca 5a

  • Ph lc 26: Ph 13C-NMR ca 5b

  • Ph lc 27: Ph 13C-NMR ca 5c

  • Ph lc 28: Ph 13C-NMR ca 5d

  • Ph lc 29: Ph 13C-NMR ca 5e

  • Ph lc 30: Ph 13C-NMR ca 5f