4-Hydroxy-1-[2-(4-hydroxyphenoxy)ethyl]-4-(4-methylbenzyl)piperidine: A

Novel, Potent, and Selective NR1/2B NMDA Receptor Antagonist

Zhang-Li n Zhou,*
,
Sui Xi ong Cai ,

Edward R. Whi ttemore,

Chri stopher S. Konkoy,

Stephen A. Espi ti a,

Mi nhtam Tran,

Davi d M. Rock,

Li nda L. Coughenour,

Jon E. Hawki nson,

Peter A. Boxer,

Chri stopher F. Bi gge,

Lawrence D. Wi se,

Eckard Weber,

Ri chard M. Woodward,

and John F. W. Keana

CoCensys, I nc., 213 Technology Drive, I rvine, California 92618, Department of Chemistry, Parke-Davis Pharmaceutical
Research, Division of Warner-Lambert Company, Ann Arbor, Michigan 48105, and Department of Chemistry, University of
Oregon, Eugene, Oregon 97403
Received May 18, 1999
A structure-based search and screen of our compound l i brary i denti fi ed N-(2-phenoxyethyl )-
4-benzyl pi peri di ne (8) as a novel N-methyl -D-aspartate (NMDA) receptor antagoni st that has
hi gh sel ecti vi ty for the NR1/2B subuni t combi nati on (I C
50
) 0.63 M). We report on the
opti mi zati on of thi s l ead compound i n terms of potency, si de effect l i abi l i ty, and i n vi vo acti vi ty.
Potency was assayed by el ectri cal recordi ngs i n Xenopus oocytes expressi ng cl oned rat NMDA
receptors. Si de effect l i abi l i ty was assessed by measuri ng affi ni ty for R
1
-adrenergi c receptors
and i nhi bi ti on of neuronal K
+
channel s. Central bi oavai l abi l i ty was gauged i ndi rectl y by
determi ni ng anti convul sant acti vi ty i n a mouse maxi mal el ectroshock (MES) assay. Maki ng
progressi ve modi fi cati ons to 8, a hydroxyl substi tuent on the phenyl ri ng para to the oxyethyl
tether (10a) resul ted i n a 25-fol d i ncrease i n NR1A/2B potency (I C
50
) 0.025 M). p-Methyl
substi tuti on on the benzyl ri ng (10b) produced a 3-fol d i ncrease i n MES acti vi ty (ED
50
) 0.7
mg/kg i v). I ntroducti on of a second hydroxyl group i nto the C-4 posi ti on on the pi peri di ne ri ng
(10e) resul ted i n a substanti al decrease i n affi ni ty for R
1
receptors and reducti on i n i nhi bi ti on
of K
+
channel s wi th onl y a modest decrease i n NR1A/2B and MES potenci es. Among the
compounds descri bed, 10e (4-hydroxy-N-[2-(4-hydroxyphenoxy)ethyl ]-4-(4-methyl benzyl )pi p-
eri di ne, Co 101244/PD 174494) had the opti mum pharmacol ogi cal profi l e and was sel ected for
further bi ol ogi cal eval uati on.
Introduction
Over acti vati on of N-methyl -D-aspar tate (NMDA)
receptors by gl utamate i s bel i eved to pl ay a rol e i n
numerous acute and chroni c neurodegenerati ve di s-
orders.
1,2
I n the search for cl i ni cal l y effecti ve neuro-
protectants, a l arge vari ety of NMDA receptor
antagoni sts have been i denti fi ed and characteri zed.
3
Thus far, however, many NMDA antagoni sts tested
cl i ni cal l y have been compromi sed by dose-l i mi ti ng car-
di ovascul ar, behavi oral , or neurotoxi c si de effects.
4
Nati ve NMDA receptors are heterool i gomeri c as-
sembl i es composed of two di fferent types of subuni ts
termed NMDA receptor 1 (NR1) and NR2.
5
NR1 sub-
uni ts have ei ght di fferent i soforms generated by al ter-
nati ve RNA spl i ci ng, and NR2 subuni ts have four
di sti nct subtypes.
5
NMDA receptor subuni ts are di ffer-
enti al l y di stri buted across brai n regi ons.
6
Thi s rai ses
the possi bi l i ty that subtype-sel ecti ve NMDA receptor
antagoni sts coul d have therapeuti c potenti al as neuro-
protectants wi thout the cardi ovascul ar, behavi oral , and
neurotoxi c si de effects often associ ated wi th broad-
spectrum antagoni sts.
I fenprodi l (1) was the fi rst NMDA receptor antagoni st
shown to have pronounced subtype sel ecti vi ty. Sel ecti v-
i ty for thi s cl ass of antagoni st i s di rected toward the
NR2B subuni t.
7
A number of structural l y rel ated com-
pounds have subsequentl y been i denti fi ed as NR1/2B-
sel ecti ve NMDA receptor antagoni sts. Exempl ary com-
pounds (Chart 1) i ncl ude el i prodi l (2),
7b,8
hal operi dol
(3),
7b,9
CP-101,606 (4),
10
and Ro 25-6981 (5).
11
Paral l el
experi ments have i nvesti gated the compl ex mechani sm
of al l osteri c i nhi bi ti on and the acti ons of thi s cl ass of
antagoni sts on neuronal NMDA currents i n si tu.
11a,12,13
Herei n we report on a novel seri es of NR2B-sel ecti ve
antagoni sts. Structure-acti vi ty rel ati onshi p (SAR) stud-
i es reveal how three si mpl e structural modi fi cati ons can

CoCensys, I nc.

Parke-Davi s Pharmaceuti cal Research.

Uni versi ty of Oregon.

Chart 1
2993 J . Med. Chem. 1999, 42, 2993-3000
10.1021/jm990246i CCC: $18.00 1999 Ameri can Chemi cal Soci ety
Publ i shed on Web 07/02/1999
transform the screeni ng l ead i nto a potent and speci fi c
i nhi bi tor.
14
Chemistry
Compound 8was prepared by N-al kyl ati on of 4-ben-
zyl pi peri di ne (6b) wi th 2-phenoxyethyl bromi de (7a).
Compounds 10a-10ewere prepared by N-al kyl ati on of
the requi si te pi peri di nes 6a-6e wi th bromi de 7b fol -
l owed by debenzyl ati on of the benzyl oxy anal ogues 9a-
9e as shown i n Scheme 1.
Those substi tuted 4-benzyl pi peri di nes that were not
commerci al l y avai l abl e were prepared from the ap-
propri atel y substi tuted benzyl bromi des through a
Wi tti g reacti on.
15
Bri efl y, reacti on of substi tuted benzyl
bromi de 11 wi th tri phenyl phosphi ne afforded the cor-
respondi ng phosphoni um sal t 12. Treatment of 12wi th
sodi um methyl sul fi nyl carbani on i n di methyl sul foxi de
at 80 C gave the yl i de whi ch reacted wi th 1-benzyl -4-
pi peri done to form the ol efi n 13. Reducti on of 13 wi th
H
2
i n the presence of PtO
2
gave compound 14. Further
debenzyl ati on of 14 by hydrogenati on i n the presence
of 10% Pd/C gave the desi red pi peri di nes 6c and 6d i n
quanti tati ve yi el d (Scheme 2).
4-Hydroxy-4-(4-methyl benzyl )pi peri di ne (6e) was pre-
pared accordi ng to Scheme 3. Reacti on of 4-methyl ben-
zyl magnesi um chl ori de, whi ch was made i n si tu from
4-methyl benzyl chl ori de (15) and magnesi um, wi th
1-benzyl -4-pi peri done i n THF afforded the correspond-
i ng al cohol 16i n 93%yi el d. Debenzyl ati on of compound
16by hydrogenati on i n the presence of 10% Pd/C gave
the desi red pi peri di ne 6e i n excel l ent yi el d.
Unsubsti tuted 2-phenoxyethyl bromi de (7a) and 2-(4-
benzyl oxyphenoxy)ethyl bromi de (7b) were synthesi zed
from the reacti on of the correspondi ng phenol wi th 1,2-
di bromoethane.
Pharmacology
Compounds were tested for antagoni sm of cl oned
NMDA receptors expressed i n Xenopus oocytes usi ng
standard two-el ectrode vol tage cl amp techni ques.
16,17
I C
50
val ues for i nhi bi ti on of NMDA responses were
determi ned by curve fi tti ng.
18
Sampl e data for com-
pound 10ei l l ustrati ng sel ecti vi ty for NR1A/2B are gi ven
i n Fi gure 1. Affi ni ty for R
1
-adrengeri c receptors was
deteri med by [
3
H]prazoci n bi ndi ng assays.
19
I nhi bi ti on
of vol tage-gated K
+
currents was measured by whol e-
cel l vol tage cl amp recordi ngs from di ssoci ated rat
superi or cervi cal gangl i on neurons.
20
Mouse MES assays
were done as reported previ ousl y.
21,22
Results and Discussion
SARs of N-(2-phenoxyethyl )-4-benzyl pi peri di ne (8)
and rel ated anal ogues are gi ven i n Fi gure 2 and Tabl e
1. The screeni ng l ead 8had an I C
50
val ue of 0.63 M at
NR1A/2B receptors: roughl y comparabl e i n potency to
the prototype compound el i prodi l and 6-fol d l ess acti ve
than i fenprodi l . Li ke el i prodi l and i fenprodi l , 8 was
>100-fol d sel ecti ve for NR1A/2B as compared to NR1A/
Scheme 1
a
a
(i ) K2CO3/CH3CN/refl ux; (i i ) 1 M HCl /MeOH; (i i i ) 10% Pd/C or 20% Pd(OH)2/MeOH/H2.
Scheme 2
a
a
(i ) Ph3P/ether/rt; (i i ) NaH/DMSO/80 C, then 1-benzyl -4-
pi peri done; (i i i ) PtO2/MeOH/H2, then 1 M HCl /MeOH; (i v) 10%Pd/
C, 95% EtOH/H2.
Scheme 3
a
a
(i ) Mg/THF, then 1-benzyl -4-pi peri done/-78 C to rt; (i i ) 1 M
HCl /MeOH; (i i i ) 10% Pd/C, 95% EtOH/H2.
2994 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15 Zhou et al.
2A or NR1A/2C. I n terms of potenti al si de effects, 8had
submi cromol ar affi ni ty for R
1
-adrenergi c receptors (I C
50
) 0.82 M), presenti ng a l i abi l i ty to produce hypoten-
si on. I n addi ti on, 8i nhi bi ted neuronal vol tage-gated K
+
channel s by 65% at 10 M, suggesti ng a potenti al for
prol ongati on of cardi ac QT i nterval , a dose-l i mi ti ng
cl i ni cal si de effect for el i prodi l (i n our assay el i prodi l
i nhi bi ted neuronal K
+
channel s by 67%at 10 M). Li ke
el i prodi l , 8 was a potent anti convul sant when admi n-
i stered i ntravenousl y (i v) to mi ce (ED
50
) 2 mg/kg). Our
goal was to modi fy 8 to i mprove NR2B potency whi l e
at the same ti me reduci ng R
1
affi ni ty and K
+
channel
i nhi bi ti on whi l e retai ni ng i n vi vo acti vi ty.
I ntroducti on of a hydroxyl group on the 4-posi ti on of
the phenyl ri ng, whi ch i s attached to the tether con-
necti ng to the ni trogen atom of the pi peri di ne ri ng, l ed
to compound 10a and i ncreased NR2B potency by 25
ti mes. As descri bed i n other seri es of compounds,
10,18,23
thi s emphasi zes the i mportance of havi ng a hydrogen-
bond-donati ng group at thi s posi ti on for i nteracti on wi th
the NMDA receptor. Though NR2B potency was i n-
creased i n 10a, the R
1
acti vi ty remai ned l ess than 1 M.
Removi ng the methyl ene group from the spacer
connecti ng the C-4 phenyl group to the pi peri di ne ri ng
gave compound 10dand unexpectedl y resul ted i n a 200-
fol d drop i n potency. I n thi s i nstance, the SAR di d not
fol l ow that descri bed previ ousl y for compounds such as
CP-101,606 where phenyl pi peri di nes are opti mal .
10
Si mpl e substi tuti on on the benzyl ri ng of 10a, such
as 4-methyl (10b) and 4-fl uoro (10c), had l i ttl e effect
on NR1A/2B potency or on the potenti al si de effects
profi l e. However, the i n vi vo potency of 10b was
i ncreased roughl y 3 ti mes (MES ED
50
) 0.7 mg/kg)
compared to that of 10a, whi l e the i n vi vo potency of
10c was sl i ghtl y reduced (MES ED
50
) 3 mg/kg). Thi s
Figure 1. I nhi bi ti on of i nward currents medi ated by cl oned
NMDA receptors expressed i n Xenopus oocytes by 10e i l -
l ustrati ng the effect of varyi ng the NR2 subuni t. I nhi bi ti on i s
pl otted as a fracti on (FR) of control responses el i ci ted by
saturati ng, or near-saturati ng, concentrati ons of agoni sts:
NR1A/2B and NR1A/2C, gl utamate (100 M) and gl yci ne (1
M); NR1A/2A, gl utamate (100 M) and gl yci ne (10 M). Curve
fi tti ng i s as descri bed i n the Experi mental Secti on. Data are
taken from three i ndi vi dual oocytes for NR1A/2B and two
oocytes for NR1A/2A and NR1A/2C.
Figure 2. SAR of the substi tuted benzyl pi peri di nes at the NR1A/2B subtype.
Novel, Potent, SelectiveNMDA Receptor Antagonist J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15 2995
suggests that substi tuti ons on the benzyl ri ng can have
si gni fi cant effects on i n vi vo potency.
I ntroducti on of a hydroxyl group i nto the C-4 posi ti on
of the pi peri di ne ri ng of 10b l ed to 10eand resul ted i n
a modest reducti on i n NR2B potency (I C
50
) 0.043 M).
However, thi s substi tuti on greatl y i mproved the si de
effects profi l e: R
1
acti vi ty was reduced by 15-fol d (I C
50
) 27 M), and K
+
channel i nhi bi ti on was reduced to
onl y 23% at 10 M. A si mi l ar reducti on i n R
1
acti vi ty
has been reported wi th 4-hydroxy substi tuti on i nto
phenyl pi peri di nes such as CP-101,606.
10
The i n vi vo
acti vi ty of 10ewas not greatl y compromi sed (MES ED
50
) 1.5 mg/kg) gi vi ng a mol ecul e wi th the desi red phar-
macol ogi cal properti es.
I n concl usi on, 10e (4-hydroxy-N-[2-(4-hydroxyphe-
noxy)ethyl ]-4-(4-methyl benzyl )pi peri di ne, Co 101244/PD
174494) has the best overal l profi l e of the compounds
descri bed, one that ri val s or exceeds that of previ ousl y
reported NR2B antagoni sts. I n addi ti on, 10e has the
advantage of achi evi ng the desi red profi l e wi thout
i ntroducti on of stereocenters. The SAR l eadi ng to 10e
i l l ustrates some of the key features necessary for
desi gni ng potent, hi ghl y speci fi c NR2B antagoni sts.
Compound 10ei s currentl y undergoi ng pharmacol ogi cal
eval uati on for a vari ety of therapeuti c appl i cati ons
i ncl udi ng stroke and Parki nsons di sease.
24
Experimental Section
Mel ti ng poi nts were determi ned i n open capi l l ary tubes on
a Mel -Temp apparatus and are uncorrected. The
1
H NMR
spectra were recorded at 300 MHz. Chemi cal shi fts are
reported i n ppm (), and J coupl i ng constants are reported i n
Hz. El emental anal yses were performed by Desert Anal yti cs,
Tucson, AZ. Mass spectra (MS) were obtai ned wi th a VG 12-
250 or VG ZAB-2FHF mass spectrometer. Reagent grade
sol vents were used wi thout further puri fi cati on unl ess other-
wi se speci fi ed. TLC was performed usi ng TLC pl ates GF
254.
Col umn chromatography was performed on si l i ca gel (230-
400 mesh). Reverse-phase HPLC anal yses were obtai ned at
254 nm on a 4.6- 250-mm mi crosorb-MV C18 col umn, usi ng
0.1% tri fl uoroaceti c aci d i n water (A) and 0.1% tri fl uoroaceti c
aci d i n acetoni tri l e (B) as sol vents. The l i near gradi ent was
20% B i n A to 95% B i n A wi th a fl ow rate of 1 mL/mi n.
4-Benzyl-1-(2-phenoxyethyl)piperidineHydrochloride
(8). A mi xture of 4-benzyl pi peri di ne (35 g, 0.20 mol ), 1-bromo-
2-phenoxyethane (42 g, 0.21 mol ), and potassi um carbonate
(111 g, 0.80 mol ) i n 400 mL of methyl ethyl ketone was heated
at refl ux wi th vi gorous sti rri ng for 12 h. The reacti on mi xture
was fi l tered, and the fi l trate was evaporated. The resi due was
extracted wi th water/ether, and the ether l ayer was washed
wi th 2 N hydrochl ori c aci d. The ami ne hydrochl ori de sal t
separated from the aqueous l ayer was col l ected by fi l trati on.
The sal t was treated wi th 2 N aqueous NaOH sol uti on (150
mL) and extracted wi th ether. The extract was dri ed over
MgSO4 and treated wi th Nori te. After fi l trati on, the ether was
removed, and the product was vacuum-di sti l l ed (45.6 g, bp )
159-162 C at 0.1 mmHg, 75% yi el d). The hydrochl ori de sal t
was formed i n 2-propanol /HCl and recrystal l i zed from ethyl
acetate to gi ve 8 as col orl ess crystal s: mp 170-172 C;
1
H
NMR (300 MHz, CDCl 3) 1.62 (m, 3 H), 1.82 (m, 2 H), 2.10
(m, 2 H), 2.80 (m, 2 H), 3.39 (m, 2 H), 3.65 (m, 2 H), 4.54 (m,
2 H), 6.85-7.31 (m, 10 H), 12.50 (brs, 1 H). Anal . (C20H26Cl NO)
C, H, N.
2-(4-Benzyloxyphenoxy)ethyl Bromide(7b). A mi xture
of 4-benzyl oxyphenol (10 g, 0.05 mol ) and potassi um carbonate
(17.3 g, 0.125 mol ) i n 50 mL of acetoni tri l e and 21.6 mL of
1,2-di bromoethane was refl uxed wi th sti rri ng for 24 h. The
i norgani c sal t was removed by fi l trati on through a short
col umn of si l i ca gel whi ch was washed wi th ethyl acetate (3
25 mL). The combi ned fi l trate was evaporated i n vacuo, and
the resi due was further puri fi ed by fl ash chromatography (5%
EtOAc i n hexane) to gi ve 12 g (79%) of the ti tl e product as a
whi te sol i d: mp 75-77 C;
1
H NMR (300 MHz, CDCl 3) 3.61
(t, J ) 6.2 Hz, 2 H), 4.24 (t, J ) 6.2 Hz, 2 H), 5.02 (s, 2 H),
6.87 (m, 4 H), 7.38 (m, 5 H).
4-Benzyl-1-[2-(4-benzyloxyphenyl)ethoxy]piperidine
Hydrochloride (9a). A mi xture of 2-(4-benzyl oxyphenoxy)-
ethyl bromi de (7b) (1.44 g, 4.7 mmol ), 4-benzyl pi peri di ne (0.876
g, 5.0 mmol ), potassi um carbonate (1.725 g, 12.5 mmol ), and
50 mL of acetoni tri l e was refl uxed for 24 h. The i norgani c sal t
was removed by fi l trati on through a short col umn of si l i ca gel
whi ch was washed wi th ethyl acetate (3 25 mL). The
combi ned fi l trate was evaporated i n vacuo, and the resi due
was further puri fi ed by fl ash chromatography (50% EtOAc i n
hexane), gi vi ng 1.62 g (86%) of the free ami ne. The ami ne was
di ssol ved i n 50 mL of methanol and treated wi th 1 N HCl i n
methanol (6 mL). The sol uti on was evaporated i n vacuo and
ti tratuted wi th ether (100 mL). The whi te sol i d was col l ected
by fi l trati on and dri ed i n vacuo, gi vi ng the ti tl e HCl sal t i n
100% yi el d: mp 164-166 C;
1
H NMR (CDCl 3, 300 MHz)
1.51 (m, 1 H), 1.68 (d, J ) 12.3 Hz, 2 H), 2.46 (m, 4 H), 2.94
(m, 2 H), 3.35 (m, 2 H), 3.45 (d, J ) 11.7 Hz, 2 H), 4.26 (s, 2
H), 5.01 (s, 2 H), 6.89 (m, 4 H), 7.18-7.40 (m, 10 H), 10.2 (brs,
2 H); EI MS m/e311 (M
+
, 5), 202 (10), 188 (100), 91 (30).
4-Benzyl-1-[2-(4-hydroxyphenyl)ethoxy]piperidineHy-
drochloride (10a). To a sol uti on of 4-benzyl -1-[2-(4-benzyl -
oxyphenyl )ethoxy]pi peri di ne (9a) (401 mg, 1.0 mmol ) i n 25 mL
of ethanol were added 1.0 mL of 1 M HCl i n methanol and
100 mg of 10%Pd/C. The resul ti ng mi xture was hydrogenated
at 30 psi of H
2 for 2 h. The catal yst was removed by fi l trati on
through a short col umn of Cel i te (5 g) and washed wi th
methanol (3 15 mL). The combi ned fi l trate was evaporated
i n vacuo to gi ve an oi l , and then ether (30 mL) was added to
the resi due. The resul ti ng mi xture was sti rred at room
temperature overni ght. The whi te sol i d was col l ected by
fi l trati on and dri ed i n vacuo, gi vi ng 330 mg (100%) of the ti tl e
compound: mp 212-215 C;
1
H NMR (300 MHz, CDCl 3 +
DMSO-d6) 1.66 (m, 3 H), 1.83 (m, 2 H), 2.43 (s, 2 H), 2.63
(m, 2 H), 3.19 (m, 2 H), 3.40 (brs, 1 H), 4.25 (s, 2 H), 6.55 (m,
4 H), 6.94-7.09 (m, 5 H), 12.0 (brs, 1 H); EI MS m/e311 (M
+
,
5), 202 (10), 188 (100), 91 (30). Anal . (C20H26Cl NO2) C, H, N.
Table 1. I n Vi tro and i n Vi vo Profi l es of Substi tuted Benzyl pi peri di nes
a
cl oned NMDA receptors expressed i n oocytes: I C50 (M)
no. NR1A/2A NR1A/2B NR1A/2C R1: I C50 (M)
K
+
channel s:
% i nhi b at 10 M
MES:
ED50 (mg/kg)
1 20 ( 6 0.11 ( 0.01 >100 0.22 54 ( 7.7 7.0
2 100.0 1.40 100.0 3.30 67 ( 6.2 1.0
8 >100 0.63 ( 0.10 >100 0.82 65 ( 4.3 2.0
10a >100 0.025 ( 0.009 >100 0.47 46 ( 2.9 2.0
10b >100 0.028 ( 0.004 >100 1.80 67 ( 5.2 0.7
10c 21 ( 4 0.05 ( 0.017 >100 1.60 53 ( 8.9 3.0
10d 110 ( 19 7.7 ( 1.5 >100 nd nd nd
10e >100 0.043 ( 0.004 >100 27 23 ( 5.6 1.5
a
I C50 val ues for i nhi bi ti on of NMDA responses at cl oned NMDA receptors expressed i n Xenopus oocytes, as i l l ustrated i n Fi gure 1.
Data are presented as mean ( SEM. Val ues were obtai ned from at l east three oocytes for NR1A/2B and at l east two oocytes for the other
subuni ts. nd, not determi ned.
2996 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15 Zhou et al.
4-MethylbenzyltriphenylphosphoniumBromide(12a).
To a sol uti on of tri phenyl phosphi ne (35.45 g, 0.135 mol ) i n 100
mL of ether was added 4-methyl benzyl bromi de (11a) (25 g,
0.135 mol ). The resul ti ng sol uti on was sti rred at room tem-
perature overni ght. The whi te sol i d was col l ected by fi l trati on
and dri ed to gi ve 60.0 g (98%) of the ti tl e product as a whi te
sol i d: mp 256-258 C;
1
H NMR (CDCl 3, 300 MHz) 2.38 (s,
3 H), 5.27 (d, J ) 14.4 Hz, 2 H), 6.93 (m, 4 H), 7.59-7.76 (m,
15 H).
1-Benzyl-4-(4-methylbenzylidene)piperidine (13a). A
suspensi on of sodi um hydri de (1.20 g, 0.03 mol , 60%i n mi neral
oi l ) i n 20 mL of dry DMSO was heated at 80 C under N2 for
1 h. The resul ti ng sol uti on was cool ed i n an i ce-water bath
and treated wi th a suspensi on of 4-methyl benzyl tri phenyl phos-
phoni um bromi de (12a) (13.41 g, 0.03 mol ) i n 120 mL of warm
DMSO. The resul ti ng sol uti on was sti rred at 0 C for 10 mi n
and then at room temperature for 15 mi n, to whi ch 1-benzyl -
4-pi peri done (5.67 g, 0.03 mol ) was then added dropwi se under
N2. The resul ti ng mi xture was sti rred at 80 C overni ght, then
poured over i ce (400 g), and extracted wi th ether (3 200 mL).
The combi ned extracts were dri ed over sodi um sul fate. The
sol vent was evaporated i n vacuo and further puri fi ed by fl ash
chromatography (el uent 5% EtOAc i n hexanes), gi vi ng 7.0 g
(84%) of the ti tl e compound as a pal e-yel l ow oi l :
1
H NMR
(CDCl 3, 300 MHz) 2.26 (s, 3 H), 2.32 (m, 4 H), 2.43 (m, 4 H),
3.45 (s, 2 H), 6.16 (s, 1 H), 7.03 (m, 4 H), 7.18-7.26 (m, 5 H).
1-Benzyl-4-(4-methylbenzyl)piperidineHydrochloride
(14a). To a sol uti on of 1-benzyl -4-(4-methyl benzyl i dene)-
pi peri di ne (13a) (4.16 g, 15 mmol ) i n 100 mL of methanol was
added 200 mg of PtO2. The resul ti ng mi xture was hydroge-
nated at 40 psi for 8 h. The catal yst was removed by fi l trati on
through a short col umn of Cel i te (10 g) and washed wi th
methanol (3 20 mL). The fi l trate was evaporated i n vacuo,
redi ssol ved i nto 20 mL of methanol , and treated wi th 30 mL
of 1 M sol uti on of HCl i n methanol . The resul ti ng sol uti on was
evaporated i n vacuo and tri turated wi th 60 mL of ether. An
off-whi te sol i d was col l ected by fi l trati on and dri ed to gi ve 4.6
g (97%) of the ti tl e product: mp 190-192 C;
1
H NMR (CDCl 3,
300 MHz) 1.60 (m, 1 H), 1.75 (m, 2 H), 2.07 (m, 2 H), 2.29 (s,
3 H), 2.54 (m, 4 H), 3.40 (d, J ) 10.5 Hz, 2 H), 4.09 (d, J ) 4.8
Hz, 2 H), 6.99 (d, J ) 7.8 Hz, 2 H), 7.05 (d, J ) 7.8 Hz, 2 H),
7.42 (m, 3 H), 7.60 (m, 2 H), 12.40 (brs, 1 H).
4-(4-Methylbenzyl)piperidine Hydrochloride (6c). A
mi xture of 1-benzyl -4-(4-methyl benzyl )pi peri di ne hydrochl o-
ri de (14a) (4.1 g, 13 mmol ) and 1.02 g of 10% Pd/C i n 100 mL
of 95% ethanol was hydrogenated at 60 psi for 12 h. The
catal yst was removed by fi l tati on through a short col umn of
Cel i te (10 g) and washed wi th methanol (3 20 mL). The
fi l trate was evaporated i n vacuo and tri turated wi th ether (50
mL). A whi te sol i d was col l ected by fi l trati on and dri ed i n
vacuo, gi vi ng 2.8 g (98%) of the ti tl e product: mp 209-211
C;
1
H NMR (CDCl 3, 300 MHz) 1.70 (m, 3 H), 1.82 (m, 2 H),
2.32 (s, 3 H), 2.55 (m, 2 H), 2.78 (m, 2 H), 3.44 (d, J ) 8.7 Hz,
2 H), 6.99 (d, J ) 7.8 Hz, 2 H), 7.05 (d, J ) 7.8 Hz, 2 H), 9.30
(brs, 1 H), 9.60 (brs, 1 H).
1-[2-(4-Benzyloxyphenyl)ethoxy]-4-(4-methylbenzyl)-
piperidine Hydrochloride (9b). A mi xture of 2-(4-benzyl -
oxyphenoxy)ethyl bromi de (7b) (0.61 g, 2.0 mmol ), 4-(4-
methyl benzyl )pi peri di ne hydrochl ori de (6c) (0.45 g, 2.0 mmol ),
and potassi um carbonate (0.69 g, 5.0 mmol ) i n 20 mL of
acetoni tri l e was refl uxed for 24 h. The i norgani c sal t was
removed through a short col umn of si l i ca gel and washed wi th
ethyl acetate (3 25 mL). The combi ned fi l trate was evapo-
rated i n vacuo to gi ve a crude mi xture, whi ch was puri fi ed by
fl ash chromatography (20%methanol i n ethyl acetate), gi vi ng
a pal e-yel l ow oi l . A sol uti on of thi s oi l i n 10 mL of methanol
was treated wi th 3 mL of 1 M HCl i n methanol . The resul ti ng
sol uti on was evaporated i n vacuo and tri turated wi th 50 mL
of ether. A whi te sol i d was col l ected by fi l trati on and dri ed i n
vacuo, gi vi ng 650 mg (72%) of the ti tl e product: mp 194-196
C;
1
H NMR (300 MHz, CDCl 3) 1.70 (m, 1 H), 1.80 (m, 2 H),
2.02 (m, 2 H), 2.31 (s, 3 H), 2.57 (d, J ) 6.9 Hz, 2 H), 2.72 (m,
2 H), 3.34 (m, 2 H), 3.63 (d, J ) 11.4 Hz, 2 H), 4.49 (s, 2 H),
5.01 (s, 2 H), 6.81 (d, J ) 10.8 Hz, 2 H), 6.88 (d, J ) 10.8 Hz,
2 H), 6.99 (d, J ) 7.8 Hz, 2 H), 7.08 (d, J ) 7.8 Hz, 2 H), 7.32-
7.39 (m, 5 H), 12.51 (brs, 1 H).
1-[2-(4-Hydroxyphenyl)ethoxy]-4-(4-methylbenzyl)-
piperidine Hydrochloride (10b). To a sol uti on of 1-[2-(4-
benzyl oxyphenyl )ethoxy]-4-(3-methyl benzyl )pi peri di ne hydro-
chl ori de (9b) (250 mg, 0.55 mmol ) i n 25 mL of ethanol was
added 60 mg of 20% Pd(OH)2. The resul ti ng mi xture was
hydrogenated at 30 psi of hydrogen for 2 h. The catal yst was
removed through a short col umn of Cel i te (5 g) and washed
wi th methanol (3 15 mL). The combi ned fi l trate was
evaporated i n vacuo and tri turated wi th 30 mL of ether. The
whi te sol i d was col l ected by fi l trati on and dri ed i n vacuo, gi vi ng
140 mg (88%) of the ti tl e product: mp 198-200 C;
1
H NMR
(300 MHz, CD3OD) 1.60 (m, 2 H), 1.88-1.92 (m, 3 H), 2.29
(s, 3 H), 2.57 (d, J ) 6.6 Hz, 2 H), 3.06 (m, 2 H), 3.47 (m, 2 H),
3.61 (m, 2 H), 4.24 (t, J ) 5.1 Hz, 2 H), 6.71 (dd, J 1 ) 2.4 Hz,
J 2 ) 6.6 Hz, 2 H), 6.83 (dd, J 1 ) 2.4 Hz, J 2 ) 6.6 Hz, 2 H),
7.70 (m, 4 H). Anal . (C21H28Cl NO2) C, H, N.
4-FluorobenzyltriphenylphosphoniumBromide(12b).
To a sol uti on of tri phenyl phosphi ne (26.2 g, 0.1 mol ) i n 100
mL of ether was added 4-fl uorobenzyl bromi de (12b) (18.9 g,
0.1 mol ). The resul ti ng sol uti on was sti rred at room temper-
ature overni ght. The whi te sol i d was col l ected by fi l trati on and
dri ed to gi ve 37.0 g (82%) of the product as a whi te sol i d: mp
280-282 C;
1
H NMR (CDCl 3, 300 MHz) 5.49 (d, J ) 14.4
Hz, 2 H), 6.77 (d, J ) 8.7 Hz, 2 H), 7.12 (m, 2 H), 7.60 (m, 6
H), 7.75 (m, 9 H).
1-Benzyl-4-(4-fluorobenzylidene)piperidine (13b). A
suspensi on of sodi um hydri de (1.44 g, 0.036 mol , 60% i n
mi neral oi l ) i n 20 mL of dry DMSO was heated at 80 C under
N
2 for 1 h. The resul ti ng sol uti on was cool ed i n an i ce-water
bath and treated wi th a suspensi on of 4-fl uorobenzyl tri ph-
enyl phosphoni um bromi de (12b) (16.2 g, 0.036 mol ) i n 120 mL
of warm DMSO. The resul ti ng sol uti on was sti rred at 0 C for
10 mi n and then at room temperature for 15 mi n, to whi ch
1-benzyl -4-pi peri done (5.67 g, 0.03 mol ) was then added
dropwi se under N
2. The resul ti ng mi xture was sti rred at 80
C overni ght and then poured over i ce (400 g) and extracted
wi th ether (3 200 mL). The combi ned extracts were dri ed
over sodi um sul fate. The sol vent was evaporated i n vacuo and
further puri fi ed by fl ash chromatography (el uent 5% EtOAc
i n hexanes), gi vi ng 7.0 g (83%) of the ti tl e compound as a pal e-
yel l ow oi l :
1
H NMR (CDCl 3, 300 MHz) 2.36-2.54 (m, 8 H),
3.53 (s, 2 H), 6.22 (s, 1 H), 6.98 (m, 2 H), 7.14 (m, 2 H), 7.26-
7.34 (m, 5 H).
1-Benzyl-4-(4-fluorobenzyl)piperidineHydrochloride
(14b). To a sol uti on of 1-benzyl -4-(4-fl uorobenzyl i dene)pi peri -
di ne (13b) (4.22 g, 15 mmol ) i n 100 mL of methanol was added
200 mg of PtO
2. The resul ti ng mi xture was hydrogenated at
40 psi for 8 h. The catal yst was removed through a short
col umn of Cel i te (10 g) and washed wi th methanol (3 20
mL). The fi l trate was treated wi th 30 mL of 1 M HCl i n
methanol . The resul ti ng sol uti on was evaporated i n vacuo and
tri turated wi th 100 mL of ether. An off-whi te sol i d was
col l ected by fi l trati on and dri ed to gi ve 4.6 g (96%) of the ti tl e
product: mp 168-170 C;
1
H NMR (CDCl 3, 300 MHz) 1.63
(m, 2 H), 1.73 (m, 2 H), 2.09 (q, J ) 12.3 Hz, 2 H), 2.56 (m, 3
H), 3.41 (d, J ) 11.1 Hz, 2 H), 4.10 (d, J ) 5.1 Hz, 2 H), 6.94
(m, 2 H), 7.05 (m, 2 H), 7.43 (m, 3 H), 7.61 (m, 2 H), 12.41 (s,
1 H).
4-(4-Fluorobenzyl)piperidine Hydrochloride (6d). A
mi xture of 1-benzyl -4-(4-fl uorobenzyl )pi peri di ne hydrochl ori de
(14b) (4.5 g, 14 mmol ) and 1.93 g of 10% Pd/C i n 100 mL of
95%ethanol was hydrogenated at 60 psi for 12 h. The catal yst
was removed through a short col umn of Cel i te (10 g) and
washed wi th methanol (3 20 mL). The fi l trate was evapo-
rated i n vacuo and tri turated wi th 80 mL of ether. A whi te
sol i d was col l ected by fi l trati on and dri ed i n vacuo, gi vi ng 3.2
g (98%) of the ti tl e product: mp 158-160 C;
1
H NMR (CDCl 3,
300 MHz) 1.70-1.81 (m, 5 H), 2.57 (m, 2 H), 2.79 (m, 2 H),
3.45 (m, 2 H), 6.98 (m, 2 H), 7.05 (m, 2 H), 9.45 (brs, 2 H).
1-[2-(4-Benzyloxyphenyl)ethoxy]-4-(4-fluorobenzyl)-
piperidine (9c). A mi xture of 2-(4-benzyl oxyphenoxy)ethyl
bromi de (7b) (3.50 g, 11.4 mmol ), 4-fl uorobenzyl pi peri di ne
Novel, Potent, SelectiveNMDA Receptor Antagonist J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15 2997
hydrochl ori de (6c) (2.6 g, 11.4 mmol ), and potassi um carbonate
(3.91 g, 28 mmol ) i n 60 mL of acetoni tri l e was refl uxed for 12
h. The i norgani c sal t was removed by fi l trati on through a short
col umn of si l i ca gel and washed wi th ethyl acetate (3 25
mL). The combi ned fi l trate was evaporated i n vacuo to gi ve a
crude mi xture, whi ch was puri fi ed by fl ash chromatography
(20%methanol i n ethyl acetate), gi vi ng 4.0 g (84%) of the ti tl e
product as a pal e-yel l ow sol i d: mp 73-75 C;
1
H NMR (300
MHz, CDCl 3) 1.26 (m, 3 H), 1.62 (m, 1 H), 2.01 (m, 2 H),
2.47 (d, J ) 6.9 Hz, 2 H), 2.73 (t, J ) 6.0 Hz, 2 H), 2.93 (m, 2
H), 4.02 (t, J ) 5.7 Hz, 2 H), 4.99 (s, 2 H), 6.78-6.96 (m, 7 H),
7.07 (m, 2 H), 7.29-7.41 (m, 4 H).
4-(4-Fluorobenzyl)-1-[2-(4-hydroxyphenyl)ethoxy]-
piperidine Hydrochloride (10c). To a sol uti on of 1-[2-(4-
benzyl oxyphenyl )ethoxy]-4-(4-fl uorobenzyl )pi peri di ne (9c) (4.0
g, 9.5 mmol ) i n 100 mL of methanol was added 1.0 g of 5%
Pd/C. The resul ti ng mi xture was hydrogenated at 35 psi of
hydrogen for 4 h. The catal yst was removed through a short
col umn of Cel i te (5 g) and washed wi th methanol (3 15 mL).
The combi ned fi l trate was treated wi th 15 mL of 1 M HCl i n
methanol . The sol uti on was evaporated i n vacuo and tri turated
wi th 100 mL of ether. An off-whi te sol i d was col l ected by
fi l trati on and dri ed i n vacuo, gi vi ng 3.2 g (95%) of the ti tl e
product: mp 196-198 C;
1
H NMR (300 MHz, CD3OD) 1.58
(m, 2 H), 1.89 (m, 3 H), 2.60 (d, J ) 6.3 Hz, 2 H), 3.08 (m, 2
H), 3.49 (t, J ) 5.1 Hz, 2 H), 3.62 (m, 2 H), 4.25 (t, J ) 5.1 Hz,
2 H), 6.77 (d, J ) 9.3 Hz, 2 H), 7.02 (m, 2 H), 7.20 (m, 2 H).
Anal . (C20H25Cl FNO2) C, H, N.
1-[2-(4-Benzyloxyphenyl)ethoxy]-4-phenylpiperidine
Hydrochloride (9d). A mi xture of 2-(4-benzyl oxyphenoxy)-
ethyl bromi de (7b) (0.377 g, 1.23 mmol ), 4-phenyl pi peri di ne
hydrochl ori de (6a) (0.20 g, 1.23 mmol ), and potassi um carbon-
ate (0.42 g, 3.07 mmol ) i n 20 mL of acetoni tri l e was refl uxed
for 12 h. The i norgani c sal t was removed through a short
col umn of si l i ca gel and washed wi th ethyl acetate (3 25
mL). The combi ned fi l trate was evaporated i n vacuo to gi ve a
crude mi xture, whi ch was puri fi ed by fl ash chromatography
(20% methanol i n ethyl acetate) to gi ve a resi due, a sol uti on
of whi ch i n 10 mL of methanol was treated wi th 3 mL of 1 M
HCl i n methanol . The resul ti ng sol uti on was evaporated i n
vacuo and tri turated wi th 50 mL of ether. A whi te sol i d was
col l ected by fi l trati on and dri ed i n vacuo, gi vi ng 455 mg (88%)
of the ti tl e product: mp 163-165 C;
1
H NMR (300 MHz,
CDCl 3) 2.01 (d, J ) 13.5 Hz, 3 H), 2.62 (m, 3 H), 2.98 (m, 2
H), 3.44 (m, 2 H), 3.79 (m, 2 H), 4.54 (m, 2 H), 5.02 (s, 2 H),
6.83 (d, J ) 8.7 Hz, 2 H), 6.90 (d, J ) 8.7 Hz, 2 H), 7.32 (m, 10
H), 12.80 (brs, 1 H).
1-[2-(4-Hydroxyphenyl)ethoxy]-4-phenylpiperidineHy-
drochloride(10d). To a sol uti on of 1-[2-(4-benzyl oxyphenyl )-
ethoxy]-4-phenyl pi peri di ne hydrochl ori de (9d) (254 mg, 0.60
mmol ) i n 25 mL of methanol was added 85 mg of 20% Pd-
(OH)2. The resul ti ng mi xture was hydrogenated at 20 psi of
hydrogen for 2 h. The catal yst was removed through a short
col umn of Cel i te (5 g) and washed wi th methanol (3 15 mL).
The combi ned fi l trate was evaporated i n vacuo and tri turated
wi th 30 mL of ether. The whi te sol i d was col l ected by fi l trati on
and dri ed i n vacuo, gi vi ng 180 mg (90%) of the ti tl e product:
mp 198-200 C;
1
H NMR (300 MHz, CD3OD) 2.11 (m, 4 H),
2.85 (m, 1 H), 3.30 (m, 2 H), 3.59 (m, 2 H), 3.77 (d, J ) 10.5
Hz, 2 H), 4.31 (d, J ) 5.1 Hz, 2 H), 6.73 (d, J ) 8.7 Hz, 2 H),
6.87 (d, J ) 8.7 Hz, 2 H), 7.29(m, 5 H). Anal . (C19H24Cl NO2
0.3H2O) C, H, N.
1-Benzyl-4-hydroxy-4-(4-methylbenzyl)piperidine(16).
To a suspensi on of 2.31 g (0.095 mol ) of Mg turni ngs i n 15 mL
of anhydrous THF was added a sol uti on of 1,2-di bromoethane
(0.489 g, 2.65 mmol ) i n 5 mL of THF dropwi se at room
temperature under N2. After the addi ti on, the THF was
removed and the resi due was ri nsed wi th THF (2 5 mL). To
thi s resi due was added a sol uti on of 4-methyl benzyl chl ori de
(15) (13.0 g, 92.6 mmol ) i n 50 mL of THF dropwi se at 0 C.
After the addi ti on, the sol uti on was sti rred at room temper-
ature for 2 h and another 50 mL of THF was added. After the
mi xture cool ed to -35 to -40 C, a sol uti on of 4-benzyl pi peri -
done (5.0 g, 26.5 mmol ) i n 20 mL of THF was added dropwi se.
After the addi ti on was compl ete, the reacti on mi xture was
sti rred at room temperature for 3 h and stood overni ght. The
reacti on mi xture was treated wi th 100 mL of saturated
aqueous NH4Cl sol uti on at 0 C and then extracted wi th
di chl oromethane (2 50 mL). The combi ned organi c phases
were evaporated i n vacuo to gi ve an oi l , whi ch was redi ssol ved
i n 200 mL of di chl oromethane, washed wi th saturated aqueous
NH4Cl sol uti on (2 30 mL) and bri ne (50 mL), and then dri ed
over sodi um sul fate. Evaporati on of sol vent fol l owed by fl ash
chromatography (EtOAc, Rf ) 0.25) gave 7.5 g (96%) of the
product as a pal e-yel l ow oi l :
1
H NMR (300 MHz, CDCl 3) 1.48
(m, 2 H), 1.73 (m, 2 H), 2.05 (s, 1 H), 2.32 (m, 5 H), 2.61 (m, 2
H), 2.71 (s, 2 H), 3.51 (s, 2 H), 7.09 (m, 4 H), 7.30 (m, 5 H).
4-Hydroxy-4-(4-methylbenzyl)piperidine Hydrochlo-
ride(6e). A mi xture of 1-benzyl -4-hydroxy-4-(4-methyl benzyl )-
pi peri di ne (16) (2.8 g, 9.5 mmol ) and 700 mg of 10% Pd/C i n
100 mL of 95% ethanol was hydrogenated at 50 psi for
overni ght. The catal yst was removed by fi l trati on through a
short col umn of Cel i te (10 g) and washed wi th methanol (3
15 mL). The fi l trate was treated wi th 12 mL of 1 M HCl i n
methanol . The resul ti ng sol uti on was evaporated i n vacuo and
tri turated wi th 30 mL of ether. A whi te sol i d was col l ected by
fi l trati on, gi vi ng 2.1 g (92%) of the ti tl e product: mp 183-
185 C;
1
H NMR (300 MHz, CDCl 3) 1.68 (m, 2 H), 2.10 (m,
2 H), 2.34 (s, 3 H), 2.78 (s, 2 H), 3.24 (m, 5 H), 7.05 (d, J ) 7.5
Hz, 2 H), 7.14 (d, J ) 7.5 Hz, 2 H), 9.30 (brs, 1 H), 9.52 (brs,
1 H).
1-[2-(4-Benzyloxyphenoxy)ethyl]-4-hydroxy-4-(4-meth-
ylbenzyl)piperidineHydrochloride(9e). A mi xture of 2-(4-
benzyl oxyphenoxy)ethyl bromi de (7b) (368 mg, 1.2 mmol ),
4-hydroxy-4-(4-methyl benzyl )pi peri di ne hydrochl ori de (6e) (290
mg, 1.2 mmol ), and potassi um carbonate (414 mg, 3 mmol ) i n
30 mL of acetoni tri l e was refl uxed for 12 h. The i norgani c sal t
was removed through a short col umn of si l i ca gel and washed
wi th ethyl acetate (3 25 mL). The combi ned fi l trate was
evaporated i n vacuo to gi ve a crude mi xture, whi ch was
puri fi ed by fl ash chromatography (5% methanol i n ethyl
acetate), gi vi ng a pal e-yel l ow oi l , a sol uti on of whi ch i n 10 mL
of methanol was treated wi th 4 mL of 1 M HCl i n methanol .
The resul ti ng sol uti on was evaporated i n vacuo and tri turated
wi th 50 mL of ether. A whi te sol i d was col l ected by fi l trati on
and dri ed i n vacuo, gi vi ng 420 mg (75%) of the ti tl e product:
mp 179-181 C;
1
H NMR (300 MHz, CDCl 3) 1.61 (s, 2 H),
1.73 (d, J ) 14.1 Hz, 2 H), 2.33 (s, 3 H), 2.45 (m, 2 H), 2.81 (s,
2 H), 3.22 (m, 2 H), 3.36 (s, 1 H), 3.46 (d, J ) 8.4 Hz, 2 H),
4.49 (s, 2 H), 5.01 (s, 2 H), 6.82 (d, J ) 9.0 Hz, 2 H), 6.90 (d,
J ) 9.0 Hz, 2 H), 7.08 (d, J ) 7.5 Hz, 2 H), 7.17 (d, J ) 7.5 Hz,
2 H), 7.38 (m, 5 H), 12.40 (brs, 1 H).
4-Hydroxy-1-[2-(4-hydroxyphenoxy)ethyl]-4-(4-methyl-
benzyl)piperidine Hydrochloride (10e). To a sol uti on of
1-[2-(4-benzyl oxyphenoxy)ethyl ]-4-hydroxy-4-(4-methyl benzyl )-
pi peri di ne (9e) (0.25 g, 0.53 mmol ) i n 30 mL of methanol was
added 62.5 mg of 20% Pd(OH)2. The resul ti ng mi xture was
hydrogenated at 20 psi of hydrogen for 3 h. The catal yst was
removed through a short col umn of Cel i te (5 g) and washed
wi th methanol (3 15 mL). The fi l trate was evaporated i n
vacuo and tri turated wi th 30 mL of ether. A whi te sol i d was
col l ected by fi l trati on and dri ed i n vacuo, gi vi ng 200 mg (100%)
of the ti tl e product: mp 133-135 C;
1
H NMR (300 MHz, CD3-
OD) 1.58 (m, 2 H), 1.75 (m, 2 H), 2.12 (s, 3 H), 2.62 (s, 2 H),
3.20-3.30 (m, 6 H), 4.06 (m, 2 H), 6.53 (d, J ) 9.0 Hz, 2 H),
6.65 (d, J ) 9.0 Hz, 2 H), 6.94 (s, 4 H). Anal . (C21H28Cl NO3) C,
H, N.
Oocyte Electrophysiology. Oocytes were obtai ned from
mature femal e Xenopus l aevi s and were prepared as descri bed
previ ousl y.
16
I ndi vi dual oocytes were mi croi njected wi th a
mi xture of NMDA receptor-encodi ng cRNAs, provi ded by Dr.
P. H. Seeburg (Hei del berg Uni versi ty, Hei del berg, Germany).
5d
NR1A and NR2A were i njected at a 1:4 rati o; al l other bi nary
subuni t combi nati ons were i njected 1:1 (1-10 ng of each
subuni t). Oocytes were stored i n Barths medi um contai ni ng
(i n mM): NaCl , 88; KCl , 1; CaCl 2, 0.41; Ca(NO3)2, 0.33; MgSO4,
0.82; NaHCO3, 2.4; Hepes, 5; pH 7.4, wi th 0.1 mg/mL genta-
myci n sul fate. Standard vol tage cl amp recordi ngs were made
2998 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15 Zhou et al.
at -70 mV i n nomi nal l y Ca
2+
-free Ri nger sol uti on (i n mM):
NaCl , 115; KCl , 2; BaCl 2, 1.8; Hepes, 5; pH 7.4.
17
Drugs were
di l uted i n Ca
2+
-free Ri nger sol uti on and appl i ed by bath
perfusi on (7-10 mL/mi n) i n a conventi onal fl ow-through
chamber (vol ume 0.2 mL). Test drugs were di ssol ved i n
DMSO and di l uted i nto Ri nger just pri or to appl i cati on (fi nal
[DMSO] ) 0.1-1%). I C50 val ues were obtai ned from parti al
(3-5 poi nts) concentrati on-i nhi bi ti on curves usi ng the equa-
ti on:
where I
control i s the current i n the absence of antagoni st, mi n
(mi ni mum) i s the resi dual fracti onal response at a saturati ng
concentrati on of antagoni st, n i s the sl ope factor, and I C50 i s
the concentrati on of drug that produces one-hal f thi s l evel of
i nhi bi ti on. To fi t the curves for NR1A/2B, mi n was fi xed at
0.15.
17
Data i n the text are mean ( standard error (SE).
r1-Adrenergic Receptor Binding. Test compounds were
eval uated at ni ne concentrati ons i n dupl i cate added i n 5-mL
al i quots (1% DMSO fi nal ) to 96-wel l , 1.0-mL vol ume assay
pl ates and i ncubated i n a total vol ume of 500 mL for 60 mi n
at room temperature as descri bed bel ow. Assays were termi -
nated by fi l trati on through GF/B fi l ter pl ates (Packard, Meri -
den, CT), and the fi l ter pl ates were ri nsed three ti mes wi th
0.8 mL of assay buffer/wel l . Mi crosci nt-20 sci nti l l ati on
cocktai l (50 mL/wel l ; Packard) was added to the dri ed fi l ter
pl ates, whi ch were then counted on a TopCount (Packard)
sci nti l l ati on counter for 8 mi n/wel l . I C50 val ues were deter-
mi ned by fi tti ng the data to the si gmoi dal equati on usi ng
Pri sm (GraphPad, San Di ego, CA). The [
3
H]prazosi n bi ndi ng
assay was modi fi ed from previ ousl y descri bed methods.
19
Frozen Sprague-Dawl ey rat corti ces obtai ned from ABS
(Wi l mi ngton, DE) were thawed, homogeni zed i n 10 vol umes
of i ce-col d 0.25 M sucrose/10 mM Tri s-HCl (pH 7.4) buffer, and
centri fuged at 1000gfor 10 mi n at 4 C. The supernatant was
centri fuged at 40000gfor 30 mi n; the pel l et was resuspended
i n 10 vol umes of i ce-col d 140 mM NaCl /5 mM MgCl
2/50 mM
Tri s-HCl (pH 7.4) buffer (prazosi n bi ndi ng buffer) and centri -
fuged at 40000g for 30 mi n. The pel l et was resuspended i n
prazosi n bi ndi ng buffer and centri fuged twi ce more for a total
of three wash steps, and the fi nal pel l et was stored at -80 C.
On the day of the bi ndi ng assay, the membrane pel l ets were
thawed and resuspended i n prazosi n bi ndi ng buffer, and 200
mg of membrane protei n was i ncubated wi th 0.8 nM [
3
H]-
prazosi n (80 Ci /mmol ; NEN, Boston, MA). Nonspeci fi c bi nd-
i ng was determi ned i n the presence of 10 mM phentol ami ne.
K
+
Channel Electrophysiology. Superi or cervi cal gan-
gl i on (SCG) neurons from 1-4-day-ol d rat pups were di ssoci -
ated and pl ated i nto 35-mm di shes usi ng standard techni ques.
Whol e-cel l vol tage cl amp recordi ngs of K
+
channel currents
were made 24-48 h l ater.
20
The external sol uti on contai ned
NaCl (150 mM), KCl (5 mM), MgCl 2 (1.1 mM), CaCl 2 (2.6 mM),
Hepes (10 mM), and gl ucose (10 mM), wi th pH adjusted to
7.4 wi th NaOH. The i nternal sol uti on contai ned KCl (80 mM),
potassi um aspartate (50 mM), EGTA (10 mM), and Hepes (10
mM), wi th pH adjusted to 7.3 wi th KOH. Test compounds were
di ssol ved i n DMSO at a concentrati on of 10 mM wi th fi nal
concentrati ons obtai ned by seri al di l uti on i n the external
sol uti on. Neurons were vol tage-cl amped at a potenti al of -60
mV, and 30-ms steps to +50 mV el i ci ted K
+
channel currents.
Control responses were obtai ned before appl i cati on of drug to
neurons by l ocal perfusi on. Data were expressed as a percent
i nhi bi ti on of sustai ned K
+
current.
MES Assays. Procedures for the mouse MES assay were
as reported previ ousl y.
21,22
Compounds were di ssol ved i n 0.05
M Tri s and tested for anti convul sant effect at the peak of
acti vi ty whi ch occurred 2 mi n after i v admi nstrati on. ED50
val ues were determi ned by Li tchfi el d and Wi l coxon anal ysi s.
Acknowledgment. The cDNAs encodi ng the rat
NR1A, NR2A, NR2B, and NR2C subuni ts were a gener-
ous gi ft from Dr. P. H. Seeburg (Uni veri sty of Hei del -
burg, Hei l el burg, Germany).
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