CAO HC HU C K20 TNG HP THUC

ACETYLCHOLINE V CHIN LC THIT K THUC

I. M U Thuc tc ng ln h dn truyn thn kinh cholinergic.
O
H HO OH NH2

Cl
N(CH3)3 O

H3C

HO

Acetylcholine
H HO

R(-)-Norepinephrine
H N

H H H3C O

Cl
N(CH3)3
N CH3

(+)-Muscarine choride

S(+)-Nicotine

II. ACETYLCHOLINE 1. Acetylcholine l g? Tng t nh muscarin v nicotine acetylcholine l cht dn truyn thn kinh, nhng n c tnh cht tr liu yu do cha nhm ester v mui amoni bc bn trong phn t. 2. Tnh cht - Kh bn trng thi rn nhng b thy phn nhanh chng trong dung dch, c xc tc bi axit hoc base, chnh v vt acetylcholine khng c iu ch di dng ung hoc dng dch truyn do d b thy phn trong ng rut hoc nhanh chng b ph hy bi cc enzim trong huyt tng v cc esterase t bo. - Mui amoni bc bn tan tt trong nc nhng hp th rt t qua mng lipit t bo. 3. Acetylcholine c u trong c th (Tch tr) Hu ht acetylcholine c to ra t qu trnh sinh tng hp (mt qu trnh tng i phc tp c s kt hp gia cholin v coenzym A) c chuyn vn mt cch hot ng vo khu vc tch tr trong tin synapse, v cho n khi c tit ra nh mt cht chuyn vn thn kinh. 4. Qu trnh tit - Acetylcholine c tit ra t ni d tr di tc ng ca th, qu trnh ny m ca cho ion Ca2+ i vo ni bo, lm cm ng s gn kt nhng ti d tr acetylcholine vi mng tin synapse trc khi s tit cht chuyn vn thn kinh xy ra. - Mi ti d tr cha khong 12.000 n 60.000 phn t acetylcholine. V mi tc ng ca th c th tit ra hng trm ti d tr acetylcholine i vo synapse. 5. S chuyn ha - Acetylcholine trong synapse c th gn kt vi th th cm trn mng trc hoc sau synapse to ra p ng. Cc phn t acetylcholine khng gn kt vi mng s b thy phn bi acetylcholinesterase, qu trnh ny l c ch sinh l chm dt tc ng ca acetylcholine.
| Ti liu lu hnh ni b

CAO HC HU C K20 TNG HP THUC - C kh nhiu acetylcholinesterase trong synaspe thy phn khong 300 triu phn t acetylcholine trong mi mili giy (msec). iu ny chng t rng hot tnh enzym thy phn tt c acetylcholine (3 triu phn t) c tit ra trong mt th tc ng. - Nhiu thuc iu tr bt chc acetylcholine c to ra trn c s c ch acetylcholinesterase. II. CHIN LC THIT K THUC - Da theo mi quan h cu trc - hot tnh acetylcholine. - Tng hp cht c ch acetylcholinesterase (AchEIs). 1. Da theo mi quan h cu trc - hot tnh acetylcholine Thc s l rt cn thit tng hp nhng hp cht thay th trong tr liu c tnh nng ging vi acetylcholine. Nhng nghin cu v mi quan h gia cu trc hot tnh ch ra rng s sa i ha hc ca acetylcholine c th thc hin trong ba phn ring bit ca phn t, nh ch ra di y
O

Nhm etylen

Cl
N(CH3)3

H3C

Nhm acyloxy

Nhm amonium bc bn

a) Sa i nhm amonium bc bn - Tng t nh acetylcholine, khi thay th nguyn t N bi As, P hoc S cho cc hp cht c hot tnh yu hn v khng s dng c trn lm sng. Kt lun: ch c cc hp cht c nhm mang in tch dng trn nguyn t N mi c hot tnh. - Khi thay th ba nhm metyl bng cc nhm ankyl ln hn hoc H cng khng c hot tnh. b) Sa cu ni etylen - Khi thay th phn alcohol vi chiu di ln hn, nhanh chng cng gim hot tnh theo Qui tc Ing 5 (S khng qu 5 nguyn t gia nguyn t N v H tn cng ca nhm CH3 cho hot tnh cao nht). - Khi thay th nguyn t H trn cu ni etylen bng cc nhm alkyl ln hn cho hp cht t c hot tnh hn acetylcholine. in hnh nh khi thay nhm CH3 vo cacbon cho acetyl--metylcholine cho hp cht c hot tnh tng t nh acetylcholine v n bn hn nn c dng lm thuc xt qua ng h hp tr hen suyn rt hiu qu, cn khi thay th v tr cacbon th cho hp cht acetyl- -metylcholine t hot tnh hn.
O CH3

Cl
N(CH3)3
H3C

O N(CH3)3 O CH3

H3C

acetyl--metylcholine c) Sa i nhm acyloxy

acetyl- -metylcholine

| Ti liu lu hnh ni b

CAO HC HU C K20 TNG HP THUC - S gim hot tnh dc hc cng nh km bn v mt ha hc ca acetylcholine l do s thy phn, chnh v vy s thay th cc nhm chc chu c s thy phn s ci thin c nhng nhc im ny. - Este carbamat ca axit carbamit v choline c ngh v bn hn este carboxylate nhiu (do cacbonyl ca carbamat t electrophile hn) + Carbachol (este ca choline v acid carbamic) t b thy phn trong ng rut v enzym acetylcholinesterase hn l acetylcholine nn c th ung c. + Tng hp tng t nh acetylmetylcholine, ta cng c th tng hp carbamat este bethanechol c th ung hoc tim di da tr hu phu ng rut hay ng niu.
O

Cl
N(CH3)3

CH3

Cl
N(CH3)3

H2N

H2N
CH3

Carbachol

Bethanechol

2. Tng hp cht c ch enzym acetyesterase (AChEIs) - Acetylcholine b thy phn khi c s can thip ca enzym Acetylcholinesterase (AchE), iu ny c li xong mt mt cng rt c hi do AchE s thy phn hon ton Acetylcholine gy mt kh nng dn truyn thn kinh. - S c ch AchE lm tng nng acetylcholine trong synapse v kt qu l tng p ng c muscarine v nicotine. p dng trong tr liu - Nhng cht c ch AchE hay anticholinesterase c phn loi nh cht bt chc choline mt cch gin tip do c ch tc ng ch yu cu chng khng lin quan th th cm cholinergic. - Cc cht ny c s dng ci thin sc co c, glaucome gc m gim p sut ni nhn, Alzhemeir,nhng bnh sinh ra do thiu cholinergic trong no. - Ngoi ra chng cn c s dng nh thuc tr su, v tng c s dng nh v kh ha hc. a) C ch thy phn Acetylcholine O OH Trong mi trng acid ' + H ' R R
R O R O

- H2O
O

+ H2O

OH

3
R

' H + ROH
R O R O H H O

'

| Ti liu lu hnh ni b

CAO HC HU C K20 TNG HP THUC

Trong mi trng base

O R R O O

'

+ OH
R OH O

'

' + ROH
R O R O

' +

' RO

Thy phn acetylcholine bi AchE

Serine
O H C HN CH2
NH2

O H H3C C+ H3C H3C N+

3
COOH

N H

H2 C C H2

O H

Tryptophan

COO-CH

CH2 H N C O C H NH

Histidine

| Ti liu lu hnh ni b

CAO HC HU C K20 TNG HP THUC - Nito bc bn gn vo v tr anion tryptophan ca enzym, l s tng tc cation-. - Oxy carbonyl b proton ha bi proton trong gc imidazol ca hitstidine. - Cacbon electrophile carbonyl b tn cng bi cc nhm hydroxy ca gc serine. - Phn cn li ca c ch c gii thch theo s sau:
O AchE-Ser-OH H3C O A N(CH3)3 H3C AchE-Ser-O OH O B

N(CH3)3

O H3C OH AchE-Ser-OH D H2 O AchE-Ser-O

O CH3 HO N(CH3)3

C (Khng hot ng)

+ Trng thi chuyn tip B khng bn, phn hy cho choline v AchE b acetyl ha C. + Dng C ny xem nh l enzym b acetyl ha, n khng th gn vi phn t acetylcholine khc, enzym ny trng thi khng hot ng. ( y chnh l vn : i vi ngi b
bnh Glaucome gc m, nu enzym C ny nhanh chng b thy phn v lng AchE nhiu th bnh s nng thm, nhng nu chm thy phn th bnh s gim i, v nu khng b thy phn th s nng thm, y l vn th v hiu nhng vn tip theo)

+ Enzym b acetyl ha nhanh chng tri qua s thy phn ti sinh dng hot ng ban u ca AchE (D) v mt phn t acetic acid. b) m mu (chin lc) thit k thuc khng AchE - Nh trnh by trn, giai on sau trong c ch ti sinh l rt quan trng trong vic thit k thuc c ch enzym AchE. Nu enzym b acyl ha bi mt nhm chc (nh carbamyl hoc phosphat,) bn thy phn hn este carboxylate, th enzym vn gi trng thi khng hot ng trong mt thi gian di hn, hoc thm ch khng hot ng mi mi (ng ngha vi ngm). - Nguyn l ha hc lin quan n tc thy phn ny dn n vic khm ph v to ra hai loi AchEI: cht c ch thun nghch v cht c ch bt thun nghch. S c ch thun nghch Axetylcholinesterase - Cc cht AcheI l nhng hp cht: + C cht phn ng vi AchE to enzym b acyl ha m chng bn hn enzym b acetyl nhng vn x kh nng thy phn ti sinh AchE. + Gn kt vi AchE vi i lc ln hn acetylcholine nhng khng phn ng vi enzym nh c cht.

| Ti liu lu hnh ni b

CAO HC HU C K20 TNG HP THUC - Cc cht c ch u tin c s dng lm sng thuc loi c ch thun nghch bao gm cc carbamate (este ca carbamit acid v ancol hoc este ca carbamit acid v phenol nh physostigmine). + Cc alkyl carbamate nh carbachol v bethanechol c cu trc tng t acetylcholine cng l c cht cho v c ch cnh tranh AchE bi v chng rt kh thy phn hn acetylcholine di xc tc ca AchE. + Cc aryl carbamate nh physostigmine v nhng dn xut tng t gn kt vo v tr xc tc ca AchE, lm cho enzym ny b carbamyl ha. Tc carbamyl ha nh sau: carbamic acid ester > methylcarbamic acid ester > imethylcarbamic acid ester.(tc l b thy phn
bi enzym ri nhng cht C kh ti to lai AchE).

R2N

Ser

AChE

Carbamyl AChE

Aryl carbamate tt hn ankyl carbamate v c hai u c i lc mnh hn AchE. Physostigmine v cc aryl carbamate c nng c ch vo khong 10-9M n 10-8M nh hn hng trm, hng nghn ln so vi cc ankyl carbamate c nng c ch vo khong 10-5M. Nguyn nhn l do ion phenoxide th bn do s cng hng ca vng thm v l nhm ri tt hn ion alkoxide. Chnh v vy, cc cht c ch carbamate c hiu qu tr liu l cc dn xut ca phenol. - S ti sinh AchE hot tnh bi s thy phn ca enzym b carbamyl ha th chm hn rt nhiu so vi s thy phn enzym b acetyl ha. Tc ti sinh ca enzym b carbamyl ha th c o trong hng pht, th d + Vi metylcarbamate enzym khong 15 pht. + Vi Acetylated AchE khong 0.2 msc. - Mc d i hi thi gian lu hn ti sinh AchE t carbamylate enzym, nhng AchE vn c ti sinh, do vy cht c ch thuc loi ny l cht c ch thun nghch.

O O

R2N

Ser

AChE

H2O

R2N

OH

+ AChE

Ser

OH

NHR2

+ CO2
O N(CH3)2

Cc cht c ch thun nghch

| Ti Oliu lu hnh ni b
N(CH3)3

Br

CAO HC HU C K20 TNG HP THUC

CH3 H3C O

O N CH3 N CH3

Physostigmine
O N(CH3)2

Neostigmine bromide
O

O
O N CH3

NHCH3

Br

Pyridostigmine bromide

Carbaryl

S c ch bt thun nghch ca Acetylcholinesterase - Chng ta thy rng enzym b carbamyl ha th thy phn chm v mt hng pht, tuy vy cc phosphate este th bn hn na, chnh v l do ny cc dn xut ca phosphoric, pyrophosphoric , v phosphonic acid l nhng cht c ch AchE hiu qu. - Tc thy phn ti sinh ca phosphorylate enzyme th rt chm v c o trong hng gi (v d thi gian bn hy ca iethyl phosphat vo khong 8 gi). Do thi gian ti to qua di v vy chng c xem l cc cht c ch bt thun nghch. - Cc cht ny tc ng c ch vi c ch ging nh carbamate este ngoi tr chng ri enzym b este ha di dng mt phosphat este. - Mt s khc nhau quan trng gia AchEI thun nghch v bt thun nghch l AchEI bt thun nghch (dn xut phosphoeste) c th tri qua qu trnh lo ha (Aging). (y cng l mt
iu th v).

| Ti liu lu hnh ni b

CAO HC HU C K20 TNG HP THUC

O O OR

AchE

Ser

P OR

aging

AchE

Ser

P OR

O-

aging

AchE

Ser

P O-

O-

Qu trnh lo ha v c tnh ca cht c ch bt thun nghch - Qu trnh lo ha ng mt vai tr quan trng trong c tnh ca cht c ch bt thun nghch. - Aging l qu trnh phn ct mt hoc nhiu lin kt phosphoeste trong khi cc AchE khc vn b phosphoryl ha. Phn ng ny to ra anion phosphate c nguyn t phospho t electrophile hn nhiu v do rt kh b thy phn so vi phosphoeste ban u. Do , phosphorylate enzym b lo ha khng b nucleophile tn cng v ti sinh bi thuc gii. - Thi gian xy ra qu trnh lo ha ty thuc vo tc phn ct lin kt P-O. Trong sut thi gian ny thuc gii c i vi ng c phosphoester vn c hiu qu, nhng nu qu trnh lo ha xy ra th khng th gii c c. Vic ny c ng dng trong vic to ra cc cht c ha hc cht ngi hng lot, mt trong s l sarin.
H3C O H3C O P CH3 F

sarin - Khi hp cht ny phosphoryl ha AchE ch c mt phn ng lo ha xy ra, v ri enzym hon ton b chai l i vi qu trnh ti sinh bi cc cht gii c hin ti. Thuc gii c cho cht c ch bt thun nghch Tnh c ca cht c ch bt thun nghch c ng dng trong thuc tr su v v kh ha hc, gy ra nhng hu qu nng n, do vic iu ch thuc gii c cho cc hp cht loi ny l rt cn thit. Loi thuc c kh nng thy phn nhanh chng phosphatester ti sinh AchE v phi an ton trong tr liu. Nc l mt cht thn hch c th thy phn acetylated AchE v ti sinh AchE mt cch nhanh chng, tuy vy i vi phosphateste, bng
| Ti liu lu hnh ni b

CAO HC HU C K20 TNG HP THUC nhng nghin cu ng hc ngi ta thy rng n thy phn rt chm phosphateste hu c v nc c tnh thn hch yu. Cn mt cht thn hch mnh hn, v Hydroxylamine (NH2OH) c ngh tuy nhin tc thy phn ch hiu qu nng gy c ca cht ny, c tnh ny c trong tt c cc dn xut hydroxylamine cho nn cn phi cc tiu ha liu lng c th s dng v gii c. Yu cu c ra: - Thit k loi thuc c chn lc cao, i lc gn kt mnh vi AchE. - Mang nucleophine ging hydroxylamine n gn gc serine b phosphoryl ha. iu ny c th t c bng cch tng hp dn xut hydroxylamine hu c c mang mt nhm mang in tch dng. - Ba ng phn pyridine aldoxime methiodide c tng hp v u c hot tnh sinh hc, trong dn xut t 2-pyridinealdehyde l c hiu qu nht. Hp cht ny c bit nh 2-pyridine methyl chloride (pralidoxime, 2-PAM) l cht duy nht hin nay chng minh c hiu qu lm sng nh cht gii c ng c phosphate ester AchEI.
OH N
N N+ OH H

R'

Cl
CH3

Oxime

Pralidoxime chloride (2-PAM)

O OR

OR

AChE

Ser

P OR

HO

PAM

AChE

Ser

P OR

O N H O N+ CH3

OR

AChE

Ser

OH

+ PAM

P OR

- C ch cho s ti sinh AchE bi 2-PAM: Giai on u lin quan n s gn kt ca nito amoni bc bn ca 2-PAM vo v tr gn kt anion, oxi mang in tch m tn cng P+, kt qu

| Ti liu lu hnh ni b

CAO HC HU C K20 TNG HP THUC b gy lin kt gia nguyn t oxi ca serine v nguyn t phospho. Sn phm cui cng l AchE hot tnh c ti sinh v phosphorylate 2-PAM. Ch khi ng c phosphat este: 2-PAM phi c cho ung trong thi gian ngn ngay khi b ng c, nu lu th khng hiu qu do qu trnh lo ha phosphorylate enzyme xy ra lm thuc khng c tc dng. Cng v l do , cc phosphate este AchEI c kh nng lo ha nhanh chng ang c pht trin nh thuc tr su v v kh ha hc, v song song cc nh khoa hc cng nghin cu to ra cc cht mi tt hn v thay th 2-PAM c th xuyn qua hng ro chn mu no ti sinh AchE trong no.

10

| Ti liu lu hnh ni b