N-Propylnoraporphin-11-O-yl carboxylic esters as potent dopamine D2 and serotonin 5-HT1A receptor dual ligands

Article abstract of DOI:10.1016/j.bmc.2008.08.056

A small series of N-propylnoraporphin-11-O-yl carboxylic esters with variant ester lengths were synthesized and their binding potencies at dopamine receptors (D₁, D₂) and serotonin receptors (5-HT_1A, 5-HT_2A) were evaluated. Monoesters 3a-f showed binding potency of 100 nM or less for the D₂ receptor, and potency of 10-30 nM for the 5-HT_1A receptor. Butyryl ester 3d was found to be the best compound possessing the highest potency for both receptors, with K_i values of 55 and 12 nM for D₂ and 5-HT_1A receptors, respectively. There is no correlation between the binding potency and the length of the monoesters, but the diesters 9 and 10 were inactive for the D₂ receptor. The dual binding profile of these monoesters for the D₂ and 5-HT_1A receptors may be useful for the treatment of neuropsychiatric disorders.

Full text of DOI:10.1016/j.bmc.2008.08.056

Bioorganic & Medicinal Chemistry 16 (2008) 8335–8338
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
N-Propylnoraporphin-11-O-yl carboxylic esters as potent dopamine D2
and serotonin 5-HT1A receptor dual ligands
a,
Zhili Liu a, , Xuetao Chen b, , Peihua Sun b, Leiping Yu b, Xuechu Zhen b, , Ao Zhang
*
*
a
Synthetic Organic & Medicinal Chemistry Laboratory (SOMCL), Shanghai Institute of Materia Medica (SIMM), Chinese Academy of Sciences, Shanghai 201203, China
b Neuropharmacology Laboratory, Shanghai Institute of Materia Medica (SIMM), Chinese Academy of Sciences, Shanghai 201203, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A small series of N-propylnoraporphin-11-O-yl carboxylic esters with variant ester lengths were synthe-
sized and their binding potencies at dopamine receptors (D1, D2) and serotonin receptors (5-HT1A, 5-
HT2A) were evaluated. Monoesters 3a–f showed binding potency of 100 nM or less for the D2 receptor,
and potency of 10–30 nM for the 5-HT1A receptor. Butyryl ester 3d was found to be the best compound
possessing the highest potency for both receptors, with Ki values of 55 and 12 nM for D2 and 5-HT1A
receptors, respectively. There is no correlation between the binding potency and the length of the mono-
esters, but the diesters 9 and 10 were inactive for the D2 receptor. The dual binding profile of these mono-
esters for the D2 and 5-HT1A receptors may be useful for the treatment of neuropsychiatric disorders.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 25 June 2008
Revised 22 August 2008
Accepted 23 August 2008
Available online 28 August 2008
Keywords:
Apomorphine
Aporphine analog
Dopamine receptor
Serotonin receptor
Monoester
1. Introduction
found that without any substitution at C-10, a single alkyl group at
C-11 (compounds 7, 8) was sufficient for high binding potency and
The tetracyclic skeleton of aporphine analogs is a long-standing
scaffold for the dopamine (DA) receptor agonists.1–3 The prototypic
compound, R-(À)-Apomorphine (APO, 1, Fig. 1), is a well-docu-
mented D2 receptor tool drug, and has been marketed for the treat-
ment of Parkinson’s disease.4–6 11-Hydroxy-N-propylnoraporphine
(27), with deletion of the 10-OH of 1, possesses compatible D2
receptor activity and enhanced bioavailability.8 The pharmacoki-
netic properties were further improved by esterification of the
11-OH of compound 2 without significant decrease in D2 receptor
binding, for example, 11-valeryloxynoraporphine (3a)8 is only
slightly less potent than 2 but has longer duration of action and
better bioavailability.
Interestingly, subtle structural modifications on the aporphine
core can also lead to serotonin 5-HT1A receptor selective ligands.2
In the early 1990s, Cannon et al.9,10 reported that replacing the
10-OH moiety in 1 with Me-group resulted in compound 4 possess-
ing extremely high potency for the 5-HT1A receptor, and almost
complete loss of potency for the D2 receptor. Hedberg and co-work-
ers11,12 further explored the hypothesized ‘methyl pocket’ in the
5-HT1A receptor binding site, and found that other 10-alkyl substit-
uents, for example, Et- (5), were also tolerated. Furthermore, it was
selectivity for the 5-HT1A receptor.13 Very recently, Si et al.14
reported that replacing Cannon’s initial 10-Me-11-OH substitution
in the aporphine core with a combination of 10-HOCH2-11-OH
(compound 6) retains nanomolar binding for the 5-HT1A receptor
but micromolar range of potency for the D2 receptor.
Although a large number of aporphine analogs have been
reported with good binding potency either for the D2 or for the
5-HT1A receptors,1–3 ligands with dual potencies for both D2 and
5-HT1A receptors are rare. In 1996, Hedberg et al.13 reported that
compound 2 was a dual binder with potent agonism for both
receptors. Its valeryl (n-pentanoyl) ester 3a has been reported
showing potent D2 receptor potency in our previous report,8 but
the binding potency for the 5-HT1A receptor of this compound
and other ester analogs has not been explored. In this regard, we
decided to resynthesize N-propyl-aporphin-11-O-yl esters 3a8,
3b8, and expand to other monoesters 3c–f as well as diesters 9
and 10. The binding potencies of these esters for both D2 and
5-HT1A receptors were evaluated. Such a dual pharmacological
profile may have potential for the treatment of schizophrenia
and Parkinson’s disease.15–17
2. Chemistry
* Corresponding authors. Tel./fax: +86 21 50806750 (X.Z.); tel.: +86 21 50806035;
fax: +86 21 50806600 (A.Z.).
The synthesis of esters 3a–f is very straightforward and demon-
strated in Scheme 1. 11-Hydroxy-N-propylnoraporphine (27) was
prepared in six steps from morphine by using a procedure we re-
E-mail addresses: xczhen@mail.shcnc.ac.cn (X. Zhen), aozhang@mail.shcnc.ac.cn
(A. Zhang).
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.08.056

8336
Z. Liu et al. / Bioorg. Med. Chem. 16 (2008) 8335–8338
2
1
6
N
N
H
N
N
Me
Pr
Pr
Me
H
H
H
HO
R
11
R
RO
7
HO
HO
10
2, R = H
3a, R = valeryl
4, R = Me
5, R = Et
6, R = HOCH2
7, R = Et
8, R = Ph
apomorphine
(1, APO)
Figure 1. Apomorphine and its analogs.
Me
N
H
N
H
N
6 steps
Pr
Pr
acid, EDCI
H
O
ref 8
HO
R
DMAP,CH2Cl2
O
HO
OH
O
morphine
2
3e, n-C5H11
3a, R = n-C4H9
3f, n-C6H13
3g, CBZ-NH-CH2
3b, R = CH3
3c, R = C2H5
3d, n-C3H7
HOOC(CH2)nCOOH
EDCI
DMAP
H2
Pd/C
3h, NH2CH2
N
Pr
H
O
(H2C)n
O
2
9, n = 2; 10, n = 3
Scheme 1.
ported previously.8 Esterification of phenol 2 with an appropriate
carboxylic acid under EDCI/DMAP condensation conditions8,18 gave
valeryl (n-pentanoyl) ester 3a8 (80%), acetyl ester 3b8 (67%), propi-
onyl ester 3c (88%), n-butyryl ester 3d (77%), n-hexanoyl ester 3e
(90%), and n-heptanoyl ester 3f (90%). Esterification of 11-hydro-
xy-aporphine 2 with N-CBZ-protected glycine under the same con-
dition yielded the aminoacid ester 3g in 70% yield (Scheme 1).
However, the following hydrogenation with Pd/C did not afford
the expected CBZ-removed aporphine 3h except a complete recov-
ery of the starting phenol 2. Diesters 9 and 10 were synthesized in
88% and 94% yield, respectively, by reacting two equivalents of
phenol 2 with one equivalent of succinic acid or glutaric acid,
and a catalytic amount of DMAP in CH2Cl2 in the presence of EDCI
as the condensation agent.18
3. Results and discussion
The previously reported esters 3a8 and 3b,8 together with our
newly prepared monoesters 3c–3g and diesters 9, 10 were sub-
jected to the competitive binding assays for DA receptors (D1, D2)
and serotonin receptors (5-HT1A, 5-HT2A), respectively, using mem-
brane preparations obtained from stable transfected HEK293 or
CHO cells with individual receptor. These procedures are similar
to those reported previously by us.8,19 [3H]SCH23390, [3H]Spipe-
rone, [3H]8-OH-DPAT, and [3H]Ketanserin were used as the stan-
dard radioligands for DA D1, D2 and serotonin 5-HT1A, 5-HT2A
receptors, respectively. 11-Hydroxy-N-propylnorapophine (2) was
also tested for comparison. Data for compound 3b was directly
taken from Ref. 8.
Table 1
Binding affinity of aporphine esters for DA (D1, D2) and 5-HT (5-HT1A, 5-HT2A) receptors from HEK293 or CHO cellsa
Compound
Ki (nM)
D1 ([3H]SCH23390)
D2 ([3H]Spiperone)
5-HT1A ([3H]8-OH-DPAT)
45 28
5-HT2A ([3H]Ketanserin)
2
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
114 83
92 18
>10,000
>10,000
ND
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
3a
3bb
3c
3d
3e
3f
3g
9
10
23
ND
18
12
10
9
72
7
109 38
56 13
109 72
95 85
>10,000
>10,000
>10,000
1
3
4
31 18
ND
ND
ND
a
Values are means of five to six experiments.19 ND denotes that the activity was not determined.
From Ref. 8.
b

Z. Liu et al. / Bioorg. Med. Chem. 16 (2008) 8335–8338
8337
As expected, compound 2 as well as most of the esters showed
good binding potency for both D2 and 5-HT1A receptors (Table 1).
Compound 2, previously reported by Hedberg13 with dual binding
potentials for both D2 and 5-HT1A receptors, displayed Ki values of
114 and 45 nM for both receptors, respectively, in our current as-
say. In agreement with our previous report,8 compound 3a showed
good potency for the D2 receptor. It was four fold more potent for
the 5-HT1A receptor with a Ki value of 23 nM. Esters 3b–3f with
variant ester lengths displayed similarly high potency for the D2
receptor (ꢀ100 nM) with butyryl ester 3d possessing the highest
potency (Ki, 55 nM). These esters also displayed excellent binding
potency for the 5-HT1A receptor with Ki values of 10–30 nM.
Although there is no significant correlation between the length of
the ester and the binding potency for the D2 and 5-HT1A receptors,
compound 3d was found to be the most potent compound with
highest binding potency for both receptors (Ki, 55 and 12 nM for
D2 and 5-HT1A receptors, respectively). It was of note that N-CBZ-
protected aminoacetate 3g was inactive for any of the receptors
tested. It was also intriguing that diesters 9 and 10 did not show
appreciable binding for the D2 receptor, therefore their potency
for the 5-HT1A receptor was not tested. All these esters together
with the phenol 2 did not show binding potency for D1 and 5-
HT2A receptors, which is consistent with the results reported in
the literature.2
Na2SO4, and evaporated. The residue was purified by silica gel
chromatography (petroleum/ethyl acetate = 3:1, 1% Et3N) to give
a pure oily product, which was then converted into the hydrochlo-
ride salt with HCl-ether (1 M). Spectroscopic data for compounds
3a and 3b were same as that we reported previously.8
5.2.1. 11-Propionyloxy-N-n-propylnoraporphine (3c)
This compound was prepared as pale solid in 88% yield from
propionic acid. MS (EI-LR) 335 (M+); 1H NMR (300 MHz, CDC13) d
7.75 (d, 1H, J = 7.8 Hz), 7.21 (m, 3H), 7.07 (d, 1H, J = 7.5 Hz), 7.01
(d, 1H, J = 7.2 Hz), 3.43 (dd, 1H, J = 3.0, 13.5 Hz), 3.14 (m, 3H),
2.90 (m, 1H), 2.77 (dd, 1H, J = 16.5, 4.2 Hz), 2.48 (m, 5H), 1.65 (m,
2H), 1.22 (t, 3H, J = 7.8 Hz), 0.98 (t, 3H, J = 7.2 Hz); 13C NMR
(75 MHz, CDC13) d 172.6, 147.3, 138.6, 135.8, 133.6, 130.7, 128.1,
127.7, 127.3, 125.9, 125.8, 124.7, 122.0, 59.1, 56.5, 48.8, 35.0,
29.3, 28.0, 19.5, 12.1, 8.9; Anal. (C22H25N2Á3/4HClÁ3/4H2O) Calcd:
C, 70.22; H, 7.30; N, 3.72. Found: C, 69.91; H, 7.30; N, 4.22.
5.2.2. 11-Butyryloxy-N-n-propylnoraporphine (3d)
This compound was prepared as pale solid in 77% yield from
butanoic acid. MS (EI-LR) 349 (M+); 1H NMR (300 MHz, CDC13) d
7.73 (d, 1H, J = 7.5 Hz), 7.21 (m, 3H), 7.06 (d, 1H, J = 7.5 Hz), 7.01
(dd, 1H, J = 8.1, 1.2 Hz), 3.42 (dd, 1H, J = 12.0, 3.6 Hz), 3.15 (m,
3H), 2.90 (m, 1H), 2.75 (dd, 1H, J = 16.2, 4.5 Hz), 2.51 (m, 5H),
1.74 (m, 2H), 1.62 (m, 2H), 0.98 (m, 6H); 13C NMR (75 MHz,
CDC13) d 171.7, 147.3, 138.6, 135.8, 133.6, 130.7, 128.1, 127.6,
127.3, 125.9, 125.8, 124.7, 122.1, 59.1, 56.5, 48.8, 36.4, 35.0, 29.3,
19.5, 18.1, 13.7, 12.0; Anal. (C23H27NO2ÁHClÁ1/2H2O) Calcd: C,
69.95; H, 7.40; N, 3.55. Found: C, 69.87; H, 7.46; N, 3.55.
4. Conclusions
In summary, we synthesized a small series of N-propylnorapor-
phin-11-O-yl carboxylic esters with variant ester lengths. Most of
these compounds were potent for both D2 and 5-HT1A receptors.
Compounds 3a–f showed binding potency of 100 nM or less for
the D2 receptor, and potency of 10–30 nM for the 5-HT1A receptor.
Butyryl ester 3d was found to be the most potent compound pos-
sessing the highest binding potency for both receptors, with Ki val-
ues of 55 and 12 nM for D2 and 5-HT1A receptors, respectively.
There is no significant correlation between the binding potency
and the length of the monoesters, but the diesters 9 and 10 were
inactive for the D2 receptors. The dual binding profile of these
monoesters for the D2 and 5-HT1A receptors may be useful for
the treatment of neuropsychiatric disorders.
5.2.3. 11-Hexanoyloxy-N-n-propylnoraporphine (3e)
This compound was prepared in 90% yield from hexanoic acid.
MS (EI) 377 (M+), 1H NMR (300 MHz, CDC13) d 7.73 (d, 1H,
J = 7.5 Hz), 7.21 (m, 3H), 7.05 (d, 1H, J = 7.5 Hz), 6.99 (d, 1H,
J = 7.5 Hz), 3.42 (dd, 1H, J = 16.2, 2.7 Hz), 3.15 (m, 3H), 2.90 (m,
1H), 2.75 (dd, 1H, J = 16.2, 4.5 Hz), 2.51 (m, 5H), 1.65 (m, 4H), 1.30
(m, 4H), 0.98 (m, 6H); 13C NMR (75 MHz, CDC13) d 172.0, 147.3,
138.6, 135.8, 133.6, 130.7, 128.1, 127.6, 127.3, 125.9, 125.8, 124.7,
122.1, 59.1, 56.5, 48.8, 35.0, 34.5, 29.3, 24.3, 22.3, 19.5, 13.9, 12.0;
HR-MS Calcd for C25H31NO2 (M+) 377.2355. Found: 377.2348.
5.2.4. 11-Heptanoyloxy-N-n-propylnoraporphine (3f)
5. Experimental
5.1. Chemistry
This compound was prepared in 90% yield as pale solid from
heptanoic acid. MS (EI) 391 (M+), 1H NMR (300 MHz, CDC13) d
7.73 (d, 1H, J = 7.5 Hz), 7.21 (m, 3H), 7.05 (d, 1H, J = 7.5 Hz), 6.99
(d, 1H, J = 7.5 Hz), 3.41 (dd, 1H, J = 16.2, 2.7 Hz), 3.16 (m, 3H),
2.90 (m, 1H), 2.76 (d, 1H, J = 16.2 Hz), 2.51 (m, 5H), 1.65 (m, 4H),
1.30 (m, 6H), 0.98 (m, 6H); 13C NMR (75 MHz, CDC13) d 172.0,
147.3, 138.6, 135.8, 133.6, 130.6, 128.1, 127.6, 127.3, 125.9,
125.8, 124.7, 122.1, 59.1, 56.5, 48.8, 35.0, 34.6, 31.4, 29.3, 28.7,
24.6, 22.4, 19.5, 13.9, 12.0; HR-MS Calcd for C26H33NO2 (M+)
391.2511. Found: 391.2514.
Melting points were determined on a Thomas–Hoover capillary
tube apparatus and are reported uncorrected. 1H and 13C NMR
spectra were recorded on a Brucker AC300 spectrometer using tet-
ramethylsilane as an internal reference. Element analyses, per-
formed by the Analytic Laboratory, SIMM, were within 0.4% of
theoretical values. Analytical thin-layer chromatography (TLC)
was carried out on 0.2-mm Kieselgel 60F254 silica gel plastic sheets
(EM Science, Newark). Flash chromatography was used for the rou-
tine purification of reaction products. The column output was
monitored with TLC. Yields of all the reactions were not optimized.
5.2.5. 11-[2-(Benzyloxycarbonylamino)acetyloxy]-N-n-
propylnoraporphine (3g)
This compound was prepared in 70% yield as a yellow solid from
2-(benzyloxy carbonylamino)acetic acid. MS (EI) 470 (M+), 1H NMR
(300 MHz, CDC13) d 7.65 (d, 1H, J = 7.8 Hz), 7.34 (m, 5H), 7.22 (m,
3H), 7.05 (m, 1H), 5.30 (br s, 1H), 5,13 (s, 2H), 4.42 (m, 2H), 3.40
(dd, 1H, J = 13.5, 3.0 Hz), 3.14 (m, 3H), 2.90 (m, 1H), 2.77 (d, 1H,
J = 13.8 Hz), 2.48 (m, 3H), 1.65 (m, 2H), 0.97 (t, 3H, J = 7.2 Hz).
5.2. General procedure for the synthesis of 11-hydroxy-N-n-
propylnoraporphine carboxylic esters (3a–g)
To
a
solution of 11-hydroxy-N-n-propylnoraporphine
2
(0.5 mmol), an appropriate acid (1 mmol) and a catalytic amount
of DMAP in anhydrous CH2Cl2 (10 mL) under N2, EDCI (1 mmol)
was added at rt. The reaction mixture was stirred overnight, and
then diluted with CH2Cl2 (30 mL) and H2O (20 mL). The organic
layer was separated, washed with brine, dried over anhydrous
5.3. General procedure for the synthesis of diesters 9 and 10
To
a
solution of 11-hydroxy-N-n-propylnoraporphine
2
(0.5 mmol), succinic acid or glutaric acid (0.24 mmol) and a cata-

8338
Z. Liu et al. / Bioorg. Med. Chem. 16 (2008) 8335–8338
lytic amount of DMAP in anhydrous CH2Cl2 (10 mL) under N2, EDCI
(0.6 mmol) was added at rt. The reaction mixture was stirred over-
night, and diluted with CH2Cl2 (30 mL) and H2O (20 mL). The or-
ganic layer was separated, washed with brine, dried over
anhydrous Na2SO4, and evaporated. The residue was subjected to
silica gel chromatography (petroleum/ethyl acetate = 2:1, 1%
Et3N) to yield the pure products.
mine receptors), or [3H]Spiperone (for dopamine D2 receptor) in
a final volume of 200 L binding buffer containing 50 mM Tris,
4 mM MgCl2, pH 7.4. Nonspecific binding was determined by par-
allel incubations with either 10 M WAY100635 for 5-HT1A, Ketan-
serin for 5-HT2A, SCH23390 for D1 or Spiperone for D2 dopamine
receptors, respectively. The reaction was started by addition of
membranes (15 ng/tube) and stopped by rapid filtration through
Whatman GF/B glass fiber filter and subsequent washing with cold
buffer (50 mM Tris, 5 mM EDTA, pH 7.4) using a Brandel 24-well
cell harvester. Scintillation cocktail was added and the radioactiv-
ity was determined in a MicroBeta liquid scintillation counter. The
IC50 and Ki values were calculated by nonlinear regression (PRISM,
Graphpad, San Diego, CA) using a sigmoidal function.
l
l
5.3.1. Bis[N-propylnoraporphin-11-O-yl]succinate (9)
This compound was prepared as green solid in 94% yield from
succinic acid. MS (EI) 640 (M+), 1H NMR (300 MHz, CDC13) d 7.71
(d, 2H, J = 7.5 Hz), 7.17 (m, 6H), 7.04 (d, 2H, J = 7.8 Hz), 6.97 (m,
2H), 3.42 (dd, 2H, J = 13.2, 2.4 Hz), 2.95 (m, 12H), 2.74 (d, 2H,
J = 16.2 Hz), 2.51 (m, 6H), 1.62 (m, 4H), 0.98 (t, 3H, J = 7.2 Hz);
13C NMR (75 MHz, CDC13) d 170.3, 147.1, 138.6, 135.9, 133.7,
130.6, 128.2, 127.7, 127.2, 126.1, 124.6, 122.0, 59.2, 56.5, 48.8,
35.0, 29.4, 29.3, 19.6, 12.1; HR-MS Calcd for C42H44N2O4 (M+)
640.3301. Found: 640.3278.
Acknowledgments
This work was financially supported by grants from Chinese Na-
tional Science Foundation (30672517 to A.Z.), Shanghai Commis-
sion of Science and Technology (07pj14104 to A.Z. and X.Z.), and
grant from Ministry of Science and Technology (2007AA022163
to X.Z.). Support from Chinese Academy of Sciences, and Shanghai
Institute of Materia Medica was also appreciated. We also thank
Professors John L. Neumeyer and Ross J. Baldessarini for their
instructive discussion during this work.
5.3.2. Bis(N-propylnoraporphin-11-O-yl) glutamate (10)
This compound was prepared in 88% yield as a yellow solid
from glutaric acid. MS (EI) 654 (M+), 1H NMR (300 MHz, CDC13) d
7.71 (d, 2H, J = 8.1 Hz), 7.20 (m, 6H), 7.06 (d, 2H, J = 7.2 Hz), 7.00
(d, 2H, J = 7.2 Hz), 3.42 (d, 2H, J = 15.3 Hz), 3.14 (m, 6H), 2.90 (m,
2H), 2.60 (m, 12H), 2.10 (m, 2H), 1.60 (m, 4H), 0.98 (t, 3H,
J = 6.6 Hz); 13C NMR (75 MHz, CDC13) d 171.1, 147.1, 138.6,
135.7, 133.7, 130.6, 128.2, 127.7, 127.3, 126.0, 124.6, 122.0, 59.1,
56.5, 48.8, 34.9, 33.5, 29.2, 19.6, 19.4, 12.1; HR-MS Calcd for
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2008.08.056.
C
43H46N2O4 (M+) 654.3458; Found: 654.3448.
5.4. Established stable expression of cell lines
References and notes
1. Neumeyer, J. L. In The Chemistry and Biology of Isoquinoline Alkaloids; Phillipson,
J. D., Roberts, M. F., Zenk, M. H., Eds.; Springer-Verlag: Berlin, Germany, 1985; p
146.
2. Zhang, A.; Zhang, Y.; Branfman, A. R.; Baldessarini, R. J.; Neumeyer, J. L. J. Med.
Chem. 2007, 50, 171.
3. Zhang, A.; Neumeyer, J. L.; Baldessarini, R. J. Chem. Rev. 2007, 107, 274.
4. Neumeyer, J. L.; Baldessarini, R. J.; Booth, R. G. In Burger’s Medicinal Chemistry
and Drug Discovery, 6th ed.; Abraham, D. J., Ed.; John Wiley & Sons: New York,
2003; Vol. 6, Chapter 12, pp 711–714.
5. Kolls, B. J.; Stacy, M. Clin. Neuropharmacol. 2006, 29, 292.
6. Subramony, J. A. Mol. Pharm. 2006, 3, 380.
7. Gao, Y.; Zong, R.; Campbell, A.; Kula, N. S.; Baldessarini, R. J.; Neumeyer, J . L.
J. Med. Chem. 1988, 31, 1392.
8. Csutoras, C.; Zhang, A.; Zhang, K.; Kula, N. S.; Baldessarini, R. J.; Neumeyer, J. L.
Bioorg. Med. Chem. 2004, 12, 3553.
9. Cannon, J. G.; Mohan, P.; Bojarski, J.; Long, J. P.; Bhatnagar, R. K.; Leonard, P. A.;
Flynn, J. R.; Chatterjee, T. K. J. Med. Chem. 1988, 31, 313.
10. Cannon, J. G.; Moe, S. T.; Long, J. P. Chirality 1991, 3, 19.
11. Hedberg, M. H.; Johansson, A. M.; Hacksell, U. J. Chem. Soc., Chem. Commun.
1992, 845.
12. Hedberg, M. H.; Johansson, A. M.; Nordvall, G.; Yliniemela, A.; Li, H.-B.;
Martin, A. R.; Hjorth, S.; Unelius, L.; Sundell, S.; Hacksell, U. J. Med. Chem.
1995, 38, 647.
The rat 5-HT1A receptor gene, human 5-HT2A receptor gene,
human D1 and human D2 receptor genes were individually cloned
into pcDNA3.0 vector. The 5-HT1A and 5-HT2A construct was then
transfected into CHO cells. The 5-HT2A receptor, D1 and D2 recep-
tors were transfected to HEK293 cells, respectively. G418 at
800 lg/ml was used for selection. Monoclonal transfected cells
were isolated and maintained in medium containing Ham’s F12
nutrient mixture (for 5-HT1A-CHO) or DMEM (for 5-HT2A-, D1- or
D2-HEK293) (Gibco), 10% fetal bovine serum, 100 U/ml penicillin,
100 U/ml streptomycin, and 200 lg/ml G418 at 37 °C and 5% CO2.
To confirm the success of transfection, the saturation binding
experiment that the expression of 5-HT1A receptor in the CHO cell
line is 1.5531 0.2803 nmol/g protein with
1.2058 nM, the Kd for 5-HT2A is 0.80 nM. The expression for the
D1 receptor is 10.67 nmol/g protein with Kd value of
a Kd value of
a
1.31 0.16 nM. The Kd for the D2 receptor is 0.06 nM.
5.5. Radioligand binding assays
13. Hedberg, M. H.; Jansen, J. M.; Nordvall, G.; Hjorth, S.; Unelius, L.; Johansson, A.
M. J. Med. Chem. 1996, 39, 3491.
14. Si, Y.-G.; Gardner, M. P.; Tarazi, F. I.; Baldessarini, R. J.; Neumeyer, J. L. Bioorg.
Med. Chem. Lett. 2007, 17, 4128.
15. Lacivita, E.; Leopoldo, M. J. Med. Chem. 2008, 51, 1492.
16. Newman-Tancredi, A.; Cussac, D.; Depoortere, R. Curr. Opin. Invest. Drugs 2007,
8, 539.
17. Wolf, W. A. Solvay. Curr. Opin. Invest. Drugs 2003, 4, 878.
18. Neumeyer, J. L.; Zhang, A.; Xiong, W.; Gu, X.-H.; Hilbert, J. E.; Knapp, B. I.;
Negus, S. S.; Mello, N. K.; Bidlack, J. M. J. Med. Chem. 2003, 46, 5162.
19. Liu, Z.; Chen, X.; Yu, L.; Zhen, X.; Zhang, A. Bioorg. Med. Chem. 2008, 16,
6675.
The affinity of the aporphine compounds to the D1 and D2 dopa-
mine receptors, and the 5-HT1A, 5-HT2A receptor was determined
by competition binding assays. Membrane homogenates of 5-
HT1A-CHO, 5-HT2A-293, cells, D1- or D2-HEK293 cells were pre-
pared as described previously.8,19 Duplicated tubes were incubated
at 30 °C for 50 min with increasing concentrations of respective
compound and with 0.7 nM [3H]8-OH-DPAT (for 5-HT1A receptor),
[3H]Ketanserin (for 5-HT2A receptor), [3H]SCH23390 (for D1 dopa-