You are on page 1of 10

Xc nh cc hng s phn ly (pK) ca cc cht c ch acetylcholinesterase c bn bng

sc k lng giai on o ngc


Bnh Alzheimer (AD) l mt ri lon thoi ha thn kinh c trng bi s gim dn ca cc
chc nng nhn thc. Thm ht ng k trong cc du hiu cholinergic tin khp thn kinh
quan st thy trong no ca bnh nhn AD dn n vic xy dng cc gi thuyt
cholinergic ca AD [1]. Cho n nay, ch c vi cht c ch acetylcholinesterase ca
(AChE), chng hn nh tacrine, donepezil v rivastigmine, c s dng trong y hc
iu tr cc triu chng AD bng cch tng acetylcholine (ACh) ni dung trong khp thn
kinh [2-4]. Hn na, mt s bng chng cho thy v tr lin kt bin AChE c th ng mt
vai tr quan trng trong s pht trin ca cc mng lo suy, tng tc b-amyloid peptide lng
ng [5].
to cc phi t mi trn c s mt trc chnh Poliamine c hot ng c ch cho c hai
mt lin kt hot ng (Chnh) v ngoi vi ca AchE, caproctamine c tm ra lm mu
cho mt lp mi ca diamine diamide nh cc cht c ch AChE khng cng ha tr, cho
thy cng l s i lp cnh tranh ng k i vi cc th th gy ng c muscarin M2
Trong nghin cu trc, s gii quyt s nh gi s nh hng ln i lc ca nhm th
ln c hai vng thm v ln cc nguyn t Nito c bn, ch ra rng s a vo 2 nhm 2-
CH3O v 1 nhm ethyl dn n kt qu hot ng tt nht. Mt nghin cu m hnh phn t
s b cho thy diamides diamine diprotonated c yu cu phi thit lp cc tng tc
thun li c th vi c hai Trp 84 trong mt hot ng v Trp 279 trong cc trang mt ngoi
vi ca AChE [6,8].
Do n c mc nhin cng nhn rng bn cht ca cc nhm th 2-benzyl c th
nh hng n km ca Benzylamine ca nhm amin bc 3. thit lp vai tr ca cc
nhm th vng thm ln kim ca nhm chc benzylamine c coi l mt cha kha
quan tnh cht l ha ci thin i lc ln AChE, xc nh hng s axit pKa ca mt dy
dn xut ca Caprotamine c thc hin bng phng php HPLC
nhm 2 -methoxy c thay th vi mt s nhm khc xc nh nh hng ca steric
v cc hiu ng in t trn s c ch AChE . Thay th ca h vi cc nguyn t halogen ,
clo nh vy ( XXI) hoc brom ( XXXII ) , khng lm tng cc mi quan h i vi AChE gi
rng cc bin th ca mt in t trn vng thm khng phi l quan trng trong s
tng tc vi AChE . xc nh kh nng thit lp H- lin kt vi enzyme , nhm methoxy
c chuyn t 2 - v tr 3 - v 4 - v tr dn u vi phi sinh XXXIII, XXXIV tng ng .
Hp cht XXXV , m khng c bt k nhm th vo vng phenyl l hi nhiu hot ng hn
XXXIII v XXXIV .
iu tra vai tr ca cc nhm th metyl trn hai chc nng c bn , h c thay th
bng cc nhm ethyl c c mt lot cc dn xut ethyl ( XXXVII - XLI ), trong mnh
nht dn n XLI . Cc c s tng i t do XL c chn l hp cht dn nh dc
ng hc tt hn ca n .
Nghin cu ti u ha hn na c thc hin trn XL bng cch thay th cc nhm gia
bi mt nguyn t oxy , dn n XLII v XLIII rng t mnh i vi AChE . Hn na , s thay
th ca nhm ethyl trn hai nguyn t nit amin c bn vi mt d lng i- propyl khng
nh hng n hot ng c ch AChE ( XLIV ) .
Cui cng, xc minh xem cc chc nng 2 -methoxy ca XL c mt vai tr trong s
tng tc vi enzyme , chng c thay th bi cc nhm c la chn c gi tr khc
nhau ca cc thng s v da trn liphophilic ( ) v in t ( ) c trng ca nhm
th , tng ng
. Mt ln na cc hp cht mnh nht kt qu XL, cho thy vai tr quan trng ca hai nhm
th thm ci thin kim ca hai chc nng gia. Khch sn ny cho php proton
trong hai chc nng c bn pH sinh l, cho s tng tc ti u vi AChE, c xc nhn
ca mt mi tng quan gia o AChE hot ng c ch v pK a ca cc hp cht thay
th khc nhau. Cc nhm th tt hn c tm thy l 2-methoxy, c l bi v n lm
tng t l protonat amin pH sinh l bng cch tng kim nit thng qua hiu ng
mesomeric v quy np. Tht vy, mt mi tng quan trc tip gia pK a v hot ng
AChE-c ch c tm thy cho cc hp cht XLV-LI
Vic xc nh cc hng s phn ly (pK) cc loi thuc c bn hoc cc hp cht hot ng
l rt quan trng d on trng thi ion ho pH sinh l v ha hc vt l tham s ny c
th quan trng i vi nghin cu SAR [9]. Do , vic xc nh tham s ny ha hc vt l
l iu cn thit trong vic tm hiu cc hot ng sinh hc ca thuc, cho php mt bc
tranh r rng hn v s tng tc lin kt phn t phi t. Hn na, h thng th nghim
sinh hc c th c nhiu gi tr pH khc nhau, do , cn bng ion ho c th nh hng
n nng ca thuc c sn cho s tng tc vi cc trang mt hot ng enzyme.
Mt cch tip cn phng php c la chn l pKa c tnh ca isocratic giai on o
ngc HPLC [10 - 14]. Trong thc t, nh hu ht cc hp cht hu c c xu hng km
ha tan trong nc, cc k thut tiometric tim c in nghin cu (protolytic) cn bng
acido-c bn l khng thc t. Mt u im khc ca phng php HPLC l mt lng nh
mu, m khng cn phi c tinh khit cao, c yu cu.
S proton ha ca cc bazo yu trong HPLC c coi nh l mt cn bng ha hc th
cp, u tin l s cn bng phn b ca cht tan i vi pha ng v pha tnh. S cn
bng th cp c iu khin bi c pH ca pha ng v t l ca cht iu chnh hu c.
L thuyt c bn ca cc cn bng c tm ra vo nhng nm 1970 v 1980 [15,16].
Vic lu gi cht phn tch trong sc k pha o miu t, th hin cho mnh phn t c khi
lng trung bnh ca s lu gi cc dng phn ly v khng phn ly.
Biu thc ca cc h s lu gi c kho st ( , trong tr l thi gian
lu gi ca cht phn tch v t0 l th tch ch trng.) c th t c di dng

x
+
HB
l phn mol ph thuc vo pH ca hp cht bazo proton HB
+
, 1 - x
+
HB
l pH ph
thuc phn mol ca hp cht B khng b proton ha.
Hng s cn bng :

Biu thc ca h s lu gi kho st t c dng

Do s ph thuc pH ca cc h s dung lng theo ng cong phn ly
y n c bo co mt nghin cu HPLC xc nh pKa ca mt lot cc hp cht
c bn lin quan n caproctamine [6], mt dibenzylamine-diamide cht c ch o ngc
ca AChE. Cc cht tng t 2 th, nhm th mang vi c tnh khc nhau c phn tch
bi RP-HPLC bng cch s dng C4 v C18 pha c nh vi mt pha ng bao gm hn
hp acetonitrile v trietylamine m phosphate (pH bao gm gia 4 v 10). Mt cch gii
thch s bin i ca cc gi tr pK l mt php ton th, bng cch xc nh cc mi tng
quan v cu trc tnh cht trong dy cng nh l php phn tch cc c tnh cu dng ca
mi cht.
2.1 Ha cht v phng php
2.1.1 Vt liu
2-Methoxy benzylamine (MBA) - Aldrich (Milan, Italy)
Hp cht nghin cu c tng hp theo cc bo co

Dung dch ca chng (nng 1mM) c chun b trong acetonitrile v c pha long vi
pha ng trc khi bm.
acetonitrile tinh khit HPLC ((Romil Ltd, Cambridge) v nc t c t thit b Milli-RX
(Millipore, USA) c s dng chun b dung dch v pha ng. Phosphoric acid (85%)
and triethyl amine cht lng phn tch (Carlo Erba Reagenti) c s dng chun b
dung dch m triethylamine- phosphate (50 mM) trong di pH t 4-10
Dung dch m c lc qua mng lc 0,45 m v loi b kh trc khi em s dng cho
HPLC.
2. Thit b
H thng cung cp dung mi l bm Jasco PU-90Intelligent HPLC c trang b kim tim
Reodyne Model 7125 vi vng mu l 20l. Dung mi gii hp c gim st bi tector
a bc song Jasco MD 910 Multiwavelength Detector kt ni vi my tnh. thng thng
phn tch bc song c ci t l 220 260nm. Tc dng 1ml/pht.
2.1.3. Xc nh hng s axit biu kin (pKa)
S tch sc k c thc hin trn ct 5mm Phenomenex Prodigy C18 100m (150x4.6
mm I.D.) v ct 5 mm Phenomenex Jupiter C4 300m (15034.6 mm I.D.) 25
0
C. Tc
dng 1ml/pht
Pha ng bao gm acetonitrile(50 mM) m triethylammonium phosphate 50:50 (v/v v th
tch) cc gi tr pH khc nhau t 4-10
Cc dung dch ca cc hp cht em kim tra (1 mM trong acetonitrile) cpha long vi
dung dch m n nng em bm (300M). Mi hp cht c bm vo 20l c
chun b thnh ba ln vo trong h thng sc k cn bng vi pha ng gi tr pH xc nh
Th tch cht ca h thng c o nh s bin dng u tin ca ng c s sau khi
bm mu acetonitrile tinh khit. mi thnh phn pha ng, h s lu k c tnh theo
k = (tr-t0)/t0 trong tr v t0 l cc thi gian lu ca cht phn tch v cht khng b gi li
H s lu c kho st vi pH ca cc pha ng c ph hp vi phng trnh sau
Boltzmann sigmoid Y = bottom + (top- bottom) / [1+exp((V50 - X) /slope)] s dng mt
chng trnh t vung khng tuyn tnh. Cc biu ca gi tr k v pH t my tnh c tm
ra v gi tr pH im un V50 c ly nh mt ch s gi tr ca pKa (bng 1)

2.1.4. Xc nh gi tr pKa (Phng php Poly Cractic)
Da theo dng iu ng sc k, cc thi gian lu ca cht tan (2, 7 v MBA) nh l mt
hm ca pH vi pha ng c xc nh 4 hn hp m acetonitrilephosphate khc
nhau c khong t 35-55% v th tch acetonitrile. Gi tr pKa(w) (pka 100% pha ng
nc) c tm ra t phn b chn y ca cc biu gia pKa vi % ca acetonitrile trong
pha ng (Bng 2). Nghin cu s tng quan s dng mt chng trnh thng k.

2.1.5 QSRP v mu phn t
Cc thng s v in t v khng gian cho cc nhm th c ly t c s d liu ca
chng trnh QSAR, cc hi quy n bin c tnh bi chng trnh tng t.
Cc mu 3D ca phn t c xy dng bng chng trnh SYbYL, v cc nghin cu v
cu dng c tm ra trn mi phn t d on xu hng thit lp tng tc lin kt
H ni phn t. Dng proton ha ca cc hp cht c nghin cu bng s phn tch cu
dng Monte Carlo, nh c b sung trong gi MarcroModel (phin bn 5.5). tnh ton
hiu ng ca dung mi ln cc hp cht trong qu trnh phn tch cu dng mu solvate ha
c s dng. phn loi cc cu dng tm ra cho mi phn t s phn tch tp hp
hnh hc c thc hin trn u ra ca cc tm kim cu dng Monte Carlo v cu dng
thng dng c pht hin
2.2 Kt qu v tho lun
2.2.1. Xc nh hng s axit biu kin
x nh pKa ca cc dn xut Caproctamone trn hnh , nhn mnh s khc nhau v
tnh bazo sinh ra bi nhm th benzyl v tr ortho, cc hp cht c phn tch bng RP-
HPLC s dng ct C4 v C18 lm pha tnh v pha ng l hn hp trn ln t l phn trm
ca axetonitril (50%) v m triethylamine phosphate (c khong pH t 4-10). Dung dch
m ny c chn v n c th bao ph mt khong pH rng t 3-11 v bi v TEA c th
che cc Silanol, trnh pic tha v cc tng tc th cp vi pha tnh. Hai nguyn t c tnh
bazo Nito c gi thit c hng s phn ly ging nhau mang cc nhm nh nhau v cch
xa nhau.
Cc ng cong sigmoid thu c hnh 2 ch ra s ph thuc v h s dung lng cht
phn tch vi pH ca pha ng. Nhn chung, s lu gi ca cc cht c kho st tng vi
s tng ca pH (hnh 3). Cc gi tr k thc nghim c xc nh t 3 ln bm khc nhau
hu ht cc thnh phn ca pha ng v mi gi tr pH c xem xt. lch chun
tng i c gi tr thp hn 2%.


Hnh 2. S bin thin ca h s lu (k) ca mt s hp cht c kim tra vi hm pH 50
v th tch cht cht hiu chnh.

Hnh 3. Cc sc xen ph ch ra tnh cht lu ca hp cht 7 vi hm pH vi cht hiu
chnh t l 50% trn pha tnh C4, hp th UV 260nm tc dng 1ml/pht.
im un (V50) ca s ph thuc theo ng un vo pH c s dng xc nh hng
s phn ly t l 50% axetonitril (bng 1)
S khc nhau gia cc gi tr k trn v k di ca ng xicma c tm ra mi lin h ti
tnh khng no ca phn t: v d, cc gi tr pH ca bazo,hp cht 1 nhm th CF3, b lu
gi nhiu hn 3 (-Cl) ci m c gi tr k cao hn 7 (-OCH3)
C th thy rng tnh a cht bo ca pha tnh khng nh hng n s xc nh pKa,
nhng ch nh hng n thi gian lu ca cht phn tch. Do phn tch cc hp cht
cng t l (thnh phn %) acetonitrile trong pha ng v vi s lu tr hp l (k), cc cht
phn tch cho kt qu lu tr lu hn, ngha l nhng cht c tnh a cht bo cao hn (1,
2, 3 v 5) c phn tch trn pha tnh ct C4 v cc cht khc (4, 6, 7) trn ct C18 pha
tnh.
2.2.2 QSRP v mu phn t
nh ch ra trong bng 1, cc dn xut cha nhm th CF -, NO -, and Cl cho gi tr pKa
thp hn hp cht khng c nhm th ban u hp cht 4, dn xut methoxy v n-
propyloxyl cho kt qu bazo hn. Cc hp cht tng t th v tr otho ca caroctamine
mang cc nhm th trn vng phenyl biu th cc tnh cht v in khc nhau, v theo l
thuyt cc nguyn t hoc nhm nh th c th cho hoc nhn electron cho hiu ng cm
ng hoc hiu ng mezzo. Do , n c gi thuyt rng tnh bazo ca bezylamin bc 3 b
nh hng bi nhm th ortho ln cn. Tuy nhin s nh hng ny c th cng l do bn
cht khng gian, khi xem xt kh nng nhm chc v tr ortho c th cn tr hoc to iu
kin (qua s to thnh lin kt H) cho s proton ha amine
gii thch cho mi quan h gia bazo c kho st bng sc k v tnh cht ca cc
nhm th v tr ortho chng ta tm kim mi quan h (i chiu) (QSPR) gia gi tr pKa v
cc h s khng gian v h s in ca cc dn xut Caprotacmin
Tt c cc hng s c nhm th khng gian v nhm th mang in t ca cc hp cht t
1-7 c em vo xem xt trong s tm kim mi quan h c ngha c tnh thng k, v kt
qa tt nht t c l hng s c in Hammet (Bng 1). S tng quan c m t bi
phng trnh sau:

l thng s in t, n l s hp cht, s l lch chun r2 v q2 l bnh phng h
s tng quan n gin v crossvalidate ca s hi quy. Cng cn phi lu l rng ch ,
khng phi l
ortho
,
0
, F hoc R, gii thch hu ht 95% s bin i gi tr
pKa.Cc hng s khc c cp n c nhim v gii thch hon ton
cho cc hiu ng in t ortho, tc ng ln cc trung tm phn ng c lp,
hon ton hiu ng cm ng hoc hiu ng cng hng.S gii thch ca
mi tng quan biu th phng trnh (1) c th l cc nhm th trong
cc hp cht t 1-7 tc ng n s protion ha N qua s trn ln ca hiu
ng cm ng v hiu ng mezzo, cng nh cch m chng nh hng n
s ion ha trong phn ng mu, ngha l s ion ha ca axit benzoic. iu
ny l kh ngc nhin nu ngh n cc v tr tng i khc nhau ca cc
nhm th v cc trung tm phn ng trong cc axit benzoic v Benzyl
amine, nhng n c th cgii thch nh l mt h qu ca c hai s
bin thin cc tnh cht in t v ca s ging nhau v cng tnh gia cc
hng s (V d r
2
(/
0
) = 0,986) trong qu trnh nghin cu. Hn na cn
phi nh rng cc gi tr
ortho
khng c thc cho nhiu nhm th v n th
hin mt khong kh rng cc gi tr thng s sn c ca mt vi trong s
chng.
S xem xt cc thuc tnh v khng gian ca cc nhm th c tham
s ha bi cc gi tr Es hoc bi cc thng s Sterimol khng cho phng
trnh tt hn phng trnh 1. V vy, dng nh l khng c s nh hng
v khng gian ca cc nhm th ln phn ng proton ha. lm r kha
cnh ny, chng ti tin hnh phn tch cu dng trn cc dng c
proton ha ca cc hp cht, v trong mi tp hp ca cc ng phn
chng ti tnh ton t l phn trm ca cc lin kt Hidro trong ni phn t.
Cc t l phn trm c ch ra bng 1. C v nh kh nng to mt lin
kt hidro ni phn t trong dng proton ha th ph thuc vo bn cht
ca nhm th, nhng, d sao tnh cht ny khng vt qua hiu ng in
t vic xc nh gi tr pKa. minh ha, cc hp cht 2 v 7 (cc dn
xut c nhm th 2-nitro- v 2-methoxy- tng ng) cho t l ging nhau
ca cc ng phn c lin kt H, nhng gi tr pKa vn khc nhau, c gi
tr cao hn i vi dn xut mang nhm th c kh nng cho electron,
ngha l OCH3 (c gi tr m). Tm li c th a ra gi thuyt rng,
trong mt dy cc hp cht ny s proton ha ca amin bc 3 chu nh
hng bi cc tnh cht in t ca nhm th v tr ortho, trong khi
kh nng to thnh lin kt H tc ng n hnh thi cu dng ca cc phn
t proton ha, bng cch lm cho ph hp dng lin kt ni phn t
2.2.3. Xc nh gi tr pKa(w)
Sau khi xc nh tnh bazo ca cht phn tch c lin quan, n c coi l
th v khi xc nh hng s axit pKa(w), ngha l ngoi suy n 100%
nc. Hai hp cht c gi tr pKa cao nht v thp nht c chn vi mc
ch thnh lp cc gi tr thang o pKa(w) cao hn v thp hn. MBA gi
tr pKa ca n c tnh bng phng php in th (pKa = 9.77) c
chn l hp cht tham kho.
Cc gi tr pKa biu kin do t c 4 t l phn trm acetonitril
trong khong 55-35% cho 7 v 2 (hnh 4) v 30-5% cho MBA

Hnh 4. Cc h s dung dng c kho st (k) vi pH ca hp cht 2 trong cc hn hp
m v axetonitril.
Cc gi tr pKa c tm ra tng bi s gim ca hm lng acetonitrile trong pha ng. Do
, khi dng th pKa vi % ca acetonitrile trong pha ng, s hi quy tuyn tnh vi cc
dc t c: phn b chn y cho gi tr pKa(w) trong pha nc. Trong bng 2 cc gi tr
pKa(w) c bo co. Cc hp cht c kim tra ch ra cc gi tr Bazo cao hn MBA
v tng ng vi gi tr pKa tng i. Cc kt qu ny thch hp vi nhm amin bc 3 ca
chng, c tnh bazow mnh hn nhm chc amin s cp ca MBA.
Tuy nhin, MBA hp cht tham kho c gi tr pKa(w) c tm thy gi tr pKa=8,2 thp
hn gi tr tm ra bi phng php in th. iu ny c th l do mt phn thc t l gi tr
pKa(w) t c bng cch ngoi suy v 0% acetonitrile, ngha l 100% dung dch nc vi
lc ion khc vi phng php in th (0,5M KNO3), thm ch nu ch c s khc nhau v
lc ion c th khng tnh c s khc nhau ca pKa n 1,5. Hn na trong cc bi bo
trc cc gi tr pKa xc nh bng HPLC khng tm ra c s thng nht hon ton vi
gi tr tm c bng phng php in th. V vy, iu ny c th suy din rng cc gi tr
c tm ra cho cc hp cht c kim tra ca chng ti c nh gi thp.
Mt vn ny sinh i vi hp cht 7, mt bazo mnh hn l gi tr pKa tng bi s gim
ca t l phn trm axetonitril, c gi tr pH t n s lu gi n nh (bng phng) (ngha
l s ph bin ca dng khng b proton ha) nm ngoi vng kho st, c th s ph hp
vi h thng HPLC c s dng. Ngoi ra, tnh a cht bo tng i cao ca cc hp
cht c kim tra khng cho php lm vic vi t l% axetonitril thp hn 35%. Trong cc
iu kin ny, php ngoi suy t c c s dng cc gi tr pKa thc nghim trong
khong hp
3. Kt lun
Phng php HPCL c bo co xc nh hng s axit pKa ca cc cht c ch AchE
c tnh bazo cho thy c ch thu c cc ch s tng i bazo. S c lng gi tr
pKa bng phng php HPLC cho thy c ch nhn mnh s khc nhau v tnh bazo ca
benzyl amin sinh ra bi cc nhm th v tr ortho ca nhn thm.
S thun li chnh ca h thng HPLC, s dng t l % hn hp axetonitril, chnh l s
sonvat ha c u th trong hn hp acetonitrile nc cho gi tr pKa thp hn mong i,
do h s dung lng bng phng ca ng cong ch S cc gi tr pH thp hn 12.
Gi tr pH ny l gii hn c s dng vi pha tnh trong HPLC .
S bin thin ca pka trong dy cc hp cht c lin h vi cc tnh cht v in t ca
cc nhm th v tr ortho qua thng s Hammmett , kh nng ca cc nhm
th chp nhn lin kt H c thy l c vai tr trong vic xc nh trng
thi cu dng ca cc nguyn t.
S c tnh ca cc hng s pKa(w) tuyt i, t c bng ngoi suy cc
gi tr tng i pKa c xc nh cc t l % c thc nghim cho
hai hp cht i din (2,7). Tuy nhin chng ti tm c mt gi tr
pKa(w) thp hn gi tr t c bng phng php in th cho MPA
c chn lm cht tham kho
Thc t rng phng php HPLC c th cho cc gi tr pKa(w) khc vi
phng php in th c in c thc nghim bi cc nghin cu vin khc trong
cc bi bo trc . Hn na cch xc nh kh kh khn v n khng them mt thng tin
no hn cho hot ng v cc nghin cu ha l tng ng. cho cc gi tr pKa tng
i miu t mt cch y s bin i tnh bazo trong dy.
Phng php ngoi suy pKa(w), theo quan im ca chng ti, dng nh c ng dng tt
hn cho cc hn cht bazo c gi tr pKa thp hn 10 v c trng bi cc gi tr a cht
bo trung bnh

You might also like