Synthesis and pharmacological investigation of novel 2-aminothiazole-privileged aporphines

Article abstract of DOI:10.1016/j.bmc.2008.05.077

A series of apomorphine ((-)-1, APO)-derived analogues ((±)-3, (-)-4-(-)-6) were designed and synthesized by hybridizing APO with a privileged 2-aminothiazole functionality which was lent from the orally available anti-parkinsonian drug, pramipexole (2). Among these hybridized compounds, catecholic aporphine (-)-6 shows good affinity at the D₂ receptor with K_i of 328 nM, slightly less potent (3-fold), but more selective against the D₁ receptor than that of the parent compound, APO. Although possessing reduced affinity at the D₂ receptor, aporphines 15 and 18 show significant potency at both the D₁ and 5-HT_1A receptors. The former compound is equipotent at both receptors (K_i: 116 and 151 nM, respectively), while the latter is 8-fold more potent at the D₁ (K_i: 78 nM) than at the 5-HT_1A receptors (K_i: 640 nM). These results indicate that the catechol fragment is critical for the D₂ receptor binding of the anti-parkinsonian drug, APO ((-)-1), but not necessary for binding at the D₁ and 5-HT_1A receptors.

Full text of DOI:10.1016/j.bmc.2008.05.077

Bioorganic & Medicinal Chemistry 16 (2008) 6675–6681
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and pharmacological investigation of novel
2-aminothiazole-privileged aporphines q
a,
Zhili Liu a, , Xuetao Chen 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, Building 3, Room 426,
555 Zuchongzhi Road, Pudong, Shanghai 201203, China
b Neuropharmacological 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 series of apomorphine ((ꢀ)-1, APO)-derived analogues (( )-3, (ꢀ)-4-(ꢀ)-6) were designed and synthe-
sized by hybridizing APO with a privileged 2-aminothiazole functionality which was lent from the orally
available anti-parkinsonian drug, pramipexole (2). Among these hybridized compounds, catecholic apor-
phine (ꢀ)-6 shows good affinity at the D2 receptor with Ki of 328 nM, slightly less potent (3-fold), but
more selective against the D1 receptor than that of the parent compound, APO. Although possessing
reduced affinity at the D2 receptor, aporphines 15 and 18 show significant potency at both the D1 and
5-HT1A receptors. The former compound is equipotent at both receptors (Ki: 116 and 151 nM, respec-
tively), while the latter is 8-fold more potent at the D1 (Ki: 78 nM) than at the 5-HT1A receptors (Ki:
640 nM). These results indicate that the catechol fragment is critical for the D2 receptor binding of the
anti-parkinsonian drug, APO ((ꢀ)-1), but not necessary for binding at the D1 and 5-HT1A receptors.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 23 April 2008
Revised 29 May 2008
Accepted 30 May 2008
Available online 5 June 2008
Keywords:
Aminothiazole
Apomorphine
Dopamine receptor
Hybridize
Serotonin receptor
1. Introduction
and oxidative potential contribute to APO’s poor bioavailability
and short duration of action. In this regard, it would be possible
Naturally occurring aporphine alkaloids and their synthetic
derivatives have served as leads for the development of therapeu-
tic agents for decades.1–3 R-(ꢀ)-Apomorphine (APO, (ꢀ)-1), the
semisynthetic4,5 or total synthetic6 prototypical aporphine, is a
well-documented dopamine (DA) receptor agonist, and has been
marketed for the treatment of Parkinson’s disease in Europe since
the 1990s and in US since 2004.7,8 However, despite its high intrin-
sic agonism activity and fast onset of anti-parkinsonian effects,
APO suffers from poor bioavailability, short duration of action
and potential central emetic side-effects.7–9 As part of our drug dis-
covery program, we recently initiated an approach to developing
novel aporphinoids by modification of the catechol moiety in this
molecule. The rationale for this approach is on the basis of the gen-
erally accepted hypothesis that the catechol moiety influences the
pharmacological profiles of APO in several ways.1–3,10–12 Firstly, the
catecholic function contributes to the high dopaminergic activity
by H-bonding with DA receptors; on the other hand, the catechol
fragment is not physico-chemically stable. Its high water solubility
to improve APO’s pharmacological profile by bioisosterically
replacing the catecholic component with a more stable function
group.2,3 Our first effort was directed to the integration of a 2-ami-
nothiazole function into the structure of APO (Fig. 1). The 2-amino-
thiazole fragment is a key pharmacophoric fragment of the widely
prescribed orally stable anti-parkinsonian drug, pramipexole
(2).13–15 It is a widely accepted privileged structure with unique
pharmacological property and has been extensively applied as a
heterocyclic bioisostere of the phenol moiety.16–18 We envisioned
that replacement of the catechol component of APO with a 2-ami-
nothiazole moiety would substantially retain the H-bonding ability
and the aromaticity of the catechol moiety. Therefore, a series of
aminothiazole-privileged structures (( )-3, (ꢀ)-4-(ꢀ)-6) were de-
signed and synthesized as shown in Figure 1.
Compound ( )-3 contains a 2-aminothiazole that completely re-
places the catechol moiety of APO ((ꢀ)-1). Compounds (ꢀ)-4 and
(ꢀ)-5 represent the replacement of the catecholic hydroxy func-
tions of APO by a thiazole moiety. In addition, a C2-methoxy, or
a C2,C3-fused aminothiazole moiety is included to explore the
hypothesized accessory binding sites around C2 or C3.2,10,11 Com-
pound (ꢀ)-6 is prepared as a control to explore the effect of a
C2,C3-fused aminothiazole moiety without changing the catechol
group of APO. All compounds in this series possess the pharmaco-
phoric fragments of both APO ((ꢀ)-1) and pramipexole (2), and
thus can be viewed as hybrids of APO and pramipexole. In this
q
Part of the results in this manuscript has been presented at the Gordon Research
Conferences, Salve Regina University, USA, July 2, 2006, Poster 280.
* Corresponding authors. Tel./fax: +86 21 50806750 (X.Z.); tel.: +86 21 50806035;
fax: +86 21 50806600 (A.Z.).
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.2008.05.077

6676
Z. Liu et al. / Bioorg. Med. Chem. 16 (2008) 6675–6681
2
1
N6
H
6a
Me
N
S
NH2
HO 11
N
H
10
HO
2 (Pramipexole)
(-)-1 (Apomorphine, APO)
Hybridization
H2N
N
H2N
N
S
S
MeO
N
H
Pr
N
H
Me
N
N
H
Me
Me
N
N
S
H
HO
HO
S
N
S
H2N
H2N
H2N
(+)-3
(-)-4
(-)-6
(-)-5
Figure 1. Apomorphine, pramipexole, and proposed 2-aminothiazole-privileged aporphines.
report, we present our synthetic efforts to these aminothiazole-
privileged compounds and the results from our preliminary phar-
macological investigation.
compound 12.26,27 Treating triflate 12 with MeSO3H gave the rear-
ranged aporphinic skeleton 13.26,27 Reduction of triflate 13 using
Pd/C and Mg metal gave 11-hydroxy-aporphine 1410 in 41% yield
following a literature procedure reported recently.27,28 Aporphine
14 was reacted with Tf2O and pyridine to afford triflate 16, which
was subsequently converted to imine 17 and then amine 18 by
using a similar C–N coupling reaction as described before in 28%
yield (2 steps).16,17 Mono-hydroxyaporphine 14 was esterified29
with valeric acid to yield valeroyl ester 15. Treating amine 18 with
Br2 and KSCN yielded a dark complex, and the expected aminothi-
azole (ꢀ)-4 was isolated in 20% yield.
2. Chemistry
Synthesis of ( )-3 was started from N-propyl-1-methyl-3,4-
dihydro-isoquinoline iodide 7, which was prepared according to
a literature procedure19–22 (Scheme 1). Treating salt 7 with 10%
NaOH, followed by alkylation with ethyl bromoacetate yielded 9
in quantitative yield. NaBH4 reduction followed by cyclization with
polyphosphoric acid (PPA) gave the key intermediate ketone 10.19–
In addition, compound 14 was reacted with BBr3 (1 M) to give
O-demethylated 2,11-dihydroxyaporphine 19 in 89% yield. Com-
pound 19 was then converted to diamine 21 in 38% yield (3 steps)
by triflating both hydroxy groups to yield bistriflate 20 followed by
a C–N coupling reaction using a similar procedure described
above.16–18,30,31 Treating 2,11-diaminoaporphine 21 with Br2 and
KSCN gave the much polar bisaminothiazole (ꢀ)-5 as the sole prod-
uct in 28% yield, and no other regioisomer was observed. The
chemical shift of H-1 in this compound is 7.83 ppm as a singlet
(300 M 1H NMR). The down-field of the chemical shift of H-1 could
be explained by the effect of the N atom in the C10,C11-fused thi-
azole moiety. Such an effect was also observed in compounds 13–
21 where the chemical shift of H-1 is in much down-field than that
of H-3 (ꢁ1 ppm). The regioselective formation of the aminothia-
zole component at C2,C3 instead of C2,C1 in this reaction can be
rationalized by the relatively less steric effect at C3 than that at
C1 in amine 21, similar to our observation in preparation of amino-
thiazole-derived opioids.16–18
22
Aminothiazole ( )-3 was prepared by bromination of ketone 10
and then treating the resulting bromo-intermediate with thiourea
in AcOH.17,23 A similar preparative strategy had been reported in
1982 by Berney20 and Schneider21,22 who reported the preparation
of a series of 5-membered heterocycle analogues of APO ((ꢀ)-1),
including ( )-3 and its N-methyl analogue. In their report, they sta-
ted that dopaminergic properties were observed from some of
their compounds, but the detail was not disclosed, especially the
property at each of the DA receptor subtypes. In our current report,
we synthesized the N-propyl aminothiazole ( )-3 as a racemate
and evaluated its activity at both DA and serotonin receptors.
Using thebaine as starting material, aminothiazoles (ꢀ)-4 and
(ꢀ)-5 were prepared in multiple steps (Scheme 2). The R-(ꢀ)-abso-
lute configuration of C6a in these compounds is derived from nat-
ural alkaloid thebaine. Thus, treating thebaine with L-selectride
(1 M)24,25 selectively gave the 3-O-demethylated compound 11
(oripavine), which was triflated with Tf2O and pyridine to yield
N
H
+
N
Pr
+
Pr
Br-
v
i
ii
N
H
iii,iv
N Pr
N Pr
CH2
I-
Pr
N
CH3
S
O
COOEt
H2N
7
8
9
10
(+)-3
Scheme 1. Synthesis of ( )-3. Reagents and conditions: (i) NaOH (10% NaOH); (ii) ethyl bromoacetate; (iii) NaBH4; (iv) PPA, 100 °C; (v) Br2, AcOH, then (NH2)2C@S, reflux.

Z. Liu et al. / Bioorg. Med. Chem. 16 (2008) 6675–6681
6677
N CH3
MeO
N CH3
MeO
v( for 15)
1
N
H
N
H
iii
i
CH3
CH3
2
vi (for 16)
Y
3
HO
O
OMe
X
ii
O
OMe
MeO
11 (X = OH)
12 (X = OTf)
thebaine
X
iv
15 (Y = OCOC4H9)
16 (Y = OTf)
13 (X = OTf)
14 (X = H)
vii
17 (Y = N=CPh2)
18 (Y = NH2)
viii
x
H2N
ix
S
N
X
X
MeO
HO
vi
N
H
N
H
6a
N
H
6a
N
H
ix
CH3
CH3
H2N
CH3
CH3
N
S
N
S
HO
H2N
20 (X = OTf)
21 (X = NH2)
vii,viii
(-)-5
(-)-4
19
Scheme 2. Synthesis of aminothiazoles (ꢀ)-4 and (ꢀ)-5. Reagents and conditions: (i) L-selectride (1 M), rt; (ii) Tf2O, Py, THF; (iii) MeSO3H, 90 °C; (iv) Pd/C, Mg, MeOH; (v)
C4H9COOH, DCC, DMAP; (vi) Tf2O, Py, DCM; (vii) Ph2C@NH, Pd(PPh3)4, BINAP, DMF; (viii) NH2OH, NaOAc; (ix) Br2, AcOH, then KSCN; (x) BBr3 (1 M), CH2Cl2.
2
N CH3
HO
HO
TfO
N6 CH3
H
N CH3
H
1
1
N CH3
H
i
iii
ii
2
11
HO
HO
O
O
O
O
3
O
OMe
MeO
10
thebaine
24
22
23
H2N
H2N
S
S
H2N
N
N
vi
vii
N CH3
H
iv, v
N CH3
H
N CH3
H
O
O
O
HO
HO
O
25
(-)-6
26
Scheme 3. Synthesis of aminothiazoles (ꢀ)-6. Reagents and conditions: (i) MeSO3H, 90 °C, then HBr (48%), reflux; (ii) BrCH2Br, THF, reflux; (iii) Tf2O, Py; (iv) Ph2C@NH,
Pd(PPh3)4, BINAP, DMF; (v) NH2OH, NaOAc; (vi) Br2, HOAc, then KSCN, rt; (vii) BBr3, CH2Cl2, ꢀ78 °C.
Similarly, starting from thebaine, aminothiazole (ꢀ)-6 was pre-
pared as described in Scheme 3. MeSO3H-catalyzed rearrangement
of thebaine followed by O-demethylation yielded 2,10,11-trihy-
droxy-aporphine 22.32,33 After protection of the catecholic hydrox-
yls, the resulting 2-hydroxy-10,11-methylene dioxoaporphine
2332,33 was subjected to triflation yielding triflate 24, followed by
a C–N coupling reaction to give amine 25. The subsequent amino-
thiazole formation reaction was conducted using a similar proce-
dure described above, to yield aminothiazole 26, which was O-
deprotected by reacting with BBr3 at ꢀ78 °C to give the expected
highly polar aminothiazoloaporphine (ꢀ)-6 in 7% overall yield (6
steps from 22). Again, the regioisomer containing a C2,C1-fused
aminothiazole was not identified during aminothiazole formation
step. This was confirmed by the significant down-field of chemical
shift of H-1 which is at 8.71 ppm (300 M 1H NMR) indicating a
remarkable effect of the C11–OH on this proton in compound
(ꢀ)-6. Same as that of thiazole (ꢀ)-5, the regiochemical selectivity
of the aminothiazole formation can be rationalized by the rela-
tively less steric effect at C3 compared to that at C1 in amine 25.
3. Results and discussion
The synthesized aminothiazole-privileged aporphines (( )-3,
(ꢀ)-4-(ꢀ)-6), valeroyl ester 15, and related intermediates (18, 21)
were subjected to the competitive binding assays for DA receptors
(D1, D2) and serotonin receptor (5-HT1A), respectively, using mem-
brane preparation obtained from stable transfected HEK293 or
CHO cells with individual receptor. First, the ability at 10 lM con-
centration to inhibit the binding of a tritiated radioligand to the
corresponding receptor was tested. Compounds with binding
inhibited by more than 80% were further assayed at six or more
concentrations, ranging above and below IC50. The Ki SE was then
derived from the equation Ki = IC50/1 + [C/Kd]. These procedures
are similar to those reported previously10–12,26,27,29 by us or others.

6678
Z. Liu et al. / Bioorg. Med. Chem. 16 (2008) 6675–6681
Table 1
Competitive binding assay of novel aporphinesa
Compound
D1 [3H]SCH23390
D2 [3H]spiperone
5-HT1A [3H]8-OH-DPAT
Ki (nM)
%
Ki (nM)
%
Ki (nM)
%
( )-3
ꢀ1.7
27.8
34.3
82.0
—
—
—
26.6
14.0
6.3
82.0
—
24.0
32.7
24.0
83.5
ND
—
—
—
ND
48.1
0.0
ND
—
—
(ꢀ)-4
(ꢀ)-5
(ꢀ)-6
14
15
18
2520 313
46.0 2.8b
116 13
78 20
2920 583
290 60
0.8 0.1
ND
328 110
235 32b
—
—
—
98 20
ND
0.44 0.1
ND
64.4
—
94.9
95.0
53.4
89
100
ND
100
94.0
48.1
ND
ND
ND
151
6
640 210
2300
ND
ND
ND
21
Apomorphine
SCH-23390
Spiperone
5-HT
100
ND
ND
ND
100
3.0 1.1
a
Dashed lines indicate that the compound has Kis of higher than 20 lM.
From Ref. [10]. ND denotes that the activity was not determined.
b
[3H]SCH23390, [3H]spiperone, and [3H]8-OH-DPAT were used as
the standard radioligands for DA D1, D2, and serotonin 5-HT1A
receptors, respectively. APO ((ꢀ)-1) was also tested for compari-
son. Data for compound 14 were taken from Ref. 10.
good D2 receptor affinity in our previous report.29 However, a sim-
ilar result was reported by Neumeyer et al.10 recently that 2-meth-
oxy-11-hydroxy-aporphine 14, the precusor of compound 15,
displays good binding affinity at the D1 receptor but moderate
affinity at the D2 receptor. Interestingly, compounds 15 and 18 also
show good inhibition of [3H]8-OH-DAPT binding at the serotonin
5-HT1A receptor, with Ki values of 151 and 640 nM, respectively.
Thus, compound 15 is equally potent at both D1 and 5-HT1A recep-
tors, whereas compound 18 is 8-fold more potent for the D1 recep-
tor against the 5-HT1A receptor. 2,11-Bisamino-aporphine 21 does
not show appreciable affinity at either D1 or 5-HT1A receptors.
Again, the loss of D2 receptor binding activity of these three com-
pounds can be attributed to the absence of the catecholic function.
The binding inhibition of the radioligands is described in Table
1. To our surprise, compound ( )-3, in which the catechol fragment
of APO is replaced directly by the aminothiazole moiety, shows
only 27% binding inhibition (Ki > 20 l
M) of [3H]spiperone (D2
receptor) in the competitive binding assays, whereas an inhibition
of 84% is observed from the parent compound, APO ((ꢀ)-1). Com-
pound ( )-3 also has poor inhibition ability on [3H]SCH23390 bind-
ing to the D1 receptor indicating that ( )-3 is inactive at this
receptor. Although dopaminergic property of this compound was
described early in Berney and Schneider’s report,20–22 our results
suggest that this compound is inactive at both D1 and D2 DA recep-
tor subtypes.
4. Conclusions
Aminobenzothiazoles (ꢀ)-4 and (ꢀ)-5 also show poor binding
affinity at both D1 and D2 DA receptor sites. Since a C2-methoxy
and C6a R-configuration in aporphine derivatives are supposed to
contribute to DA receptor binding of these compounds,1–3 the poor
binding of (ꢀ)-4 and (ꢀ)-5 at the D2 receptor clearly demonstrates
that replacement of the catechol moiety of APO by an aminothia-
zole functionality results in a complete loss of DA receptor binding.
These two compounds also show poor binding at the serotonin 5-
HT1A receptor. To rule out the possible effect that the aminothia-
zole moiety at C2,C3 has on the loss of DA receptor binding activity
of(ꢀ)-5 (both sterically and electronically), catecholic aporphine
(ꢀ)-6 was examined in the same assays. To our surprise, aminothi-
azole (ꢀ)-6 shows a good Ki binding value of 328 nM at the D2
In summary, we have designed and synthesized a series of apo-
morphine derivatives (( )-3, (ꢀ)-4-(ꢀ)-6) with a privileged 2-ami-
nothiazole functionality which is lent from the orally available
anti-parkinsonian drug, pramipexole (2). Compound ( )-3 was ob-
tained by total synthesis in racemic form. Aminothiazoles (ꢀ)-4-
(ꢀ)-6, 11-valeroyl ester 15, and the intermediates 18, and 21 were
prepared in R-(ꢀ)-configuration from alkaloid thebaine. All these
compounds were screened for their binding ability to dopamine
D1, D2, and serotonin 5-HT1A receptors. Among these compounds,
only catecholic aporphine (ꢀ)-6 shows a good affinity at the D2
receptor with Ki of 328 nM, slightly less potent (3-fold), but more
selective against the D1 receptor than that of the parent compound,
APO ((ꢀ)-1). Although possessing reduced affinity at the D2 recep-
tor, aporphines 15 and 18 show significant affinity at both D1 and
5-HT1A receptors. The former compound is equipotent at both
receptors (Ki: 116 and 151 nM, respectively), whereas the latter
is 8-fold more potent at the D1 (Ki, 78 nM) than at the 5-HT1A
receptor (Ki, 640 nM). These results indicate that the catechol frag-
ment is critical for the D2 receptor binding of apomorphine (APO,
(ꢀ)-1), but not necessary for binding at the D1 and 5-HT1A
receptors.
receptor but the affinity at the D1 receptor is poor (2.5 lM). There-
fore, (ꢀ)-6 is slightly (3-fold) less potent than APO ((ꢀ)-1) at the D2
receptor, but has improved binding selectivity (7.6-fold) than APO
(3.4-fold) for the D2 over D1 receptors. This result indicates the
importance of the catecholic function for the D2 receptor binding
in APO and its derivatives, and that a relative large C2/C3 substitu-
ent, for example, aminothiazole, is tolerated. This is an important
appendage to the early observations by Neumeyer and oth-
ers2,3,32,33 that a relative by small C2-substituent in APO, such as
MeO-, OH-, NH2-, and F-, is beneficial to the DA receptor binding.
It is of note that 11-O-valeroyl-(15), and 11-amino-(18) apor-
phines show remarkable inhibition of [3H]SCH23390 binding at
the D1 receptor, but not of [3H]spiperone binding at the D2 recep-
tor. Both compounds produce a same level of binding inhibition of
95% with Ki values of 116 and 78 nM, respectively, at the D1 recep-
tor. The good affinity at the D1 and poor affinity at the D2 receptors
of valeroyl ester 15 is intriguing since a similar 11-valeroyl ester
without the C2-MeO substituent has been reported possessing
5. Experimental
Chemistry. Melting points were determined on a Thomas–Hoo-
ver capillary tube apparatus and are reported uncorrected. 1H
and 13C NMR spectra were recorded on a Brucker AC300 spectrom-
eter using tetramethylsilane as an internal reference. Element
analyses, performed by the Analytic Lab, SIMM, were within
0.4% of theoretical values. Analytical thin-layer chromatography
(TLC) was carried out on 0.2-mm Kieselgel 60F 254 silica gel plastic

Z. Liu et al. / Bioorg. Med. Chem. 16 (2008) 6675–6681
6679
sheets (EM Science, Newark). Flash chromatography was used for
the routine purification of reaction products. The column output
was monitored with TLC. Yields of all the reactions were not
optimized.
(5 ꢂ 50 mL). The combined organic layer was washed with brine
and dried over anhydrous Na2SO4. The solvent was evaporated.
The residue was purified with silica gel column chromatography
eluting with CHCl3/MeOH = 20:1 (1% Et3N) to afford the title com-
pound 14 as yellow oil (519 mg, 41%). 1H NMR (300 MHz, CDCl3) d
7.67 (d, 1H, J = 2.4 Hz), 7.06 (dd, 1H, J = 7.5, 7.8 Hz), 6.82 (d, 1H,
J = 7.5 Hz), 6.74 (d, 1H, J = 8.1 Hz), 6.59 (d, 1H, J = 2.4 Hz), 3.78 (s,
3H), 3.16 (m, 4H), 2.62 (m, 6H).
5.1. N-Propyl-2,3,9,9a-tetrahydro-1H-benzo[de]quinolin-
7(8H)-one (10)
This compound was prepared from phenylethylamine in 7 steps
using a slightly modified procedure reported by Berney20,21 or
Dijkstra.19 MS (EI) 229 (M+); 1H NMR (300 MHz, CDCl3) d 7.86
(m, 1H), 7.27 (m, 2H), 3.56 (m, 1H), 3.15 (m, 2H), 2.83 (m, 3H),
2.54 (m, 4H), 1.80 (m, 1H), 1.57 (m, 2H), 0.94 (m, 3H).
5.6. (R)-2-Methoxyaporphin-11-yl pentanoate (15)
Phenol 14 (70 mg, 0.25 mmol), valeric acid (36 lL, 0.33 mmol),
and a catalytic amount of DMAP were dissolved in 15 mL of anhy-
drous CH2Cl2 under nitrogen. To the stirred mixture, a solution of
N,N-dicyclohexylcarbodiimide (DCC, 72 mg, 0.35 mmol) in 5 mL
of anhydrous CH2Cl2 was added at rt. After stirring for 4 h, the reac-
tion mixture was filtered and evaporated to dryness. Purification
by silica gel chromatography (petroleum/EtOAc = 2:1, 1% Et3N)
yielded the title compound 15 as oil (33 mg 36%).1H NMR
(300 MHz, CDCl3) d 7.39 (d, 1H, J = 2.7 Hz), 7.20 (m, 2H), 6.99 (d,
1H, J = 7.8 Hz), 6.61 (d, 1H, J = 2.7 Hz) 3.80 (s, 3H), 3.14 (m, 3H),
3.03 (m, 1H), 2.60 (m, 8H), 1.68 (m, 2H), 1.40 (m, 2H), 0.93 (t,
3H). 13C NMR (75 MHz, CDCl3) d 171.9, 157.9, 147.4, 138.5, 134.3,
131.6, 127.8, 127.6, 127.1, 126.0, 122.2, 112.3, 111.9, 61.4, 55.2,
52.9, 43.8, 35.2, 34.4, 29.4, 26.7, 22.2, 13.7. MS (EI) 337 (M+). HRMS
(EI) calcd for C19H19N3OS 337.1249; found 337.1251.
5.2. N-Propyl-5,6,6a,7-tetrahydro-4H-benzo[de]thiazolo[4,5-
g]quinolin-9-amine (( )-3)
To a solution of 10 (0.215 g, 0.9 mmol) in AcOH (2 mL), 4 drops
of 40% HBr solution were added dropwise, followed by Br2 (0.162 g,
1.01 mmol). The reaction was heated to 60 °C overnight. The mix-
ture was cooled and then evaporated in vacuo. The residue was
basified (pH 8–9) with NH4OH, and extracted with CH2C12 (3ꢂ
30 mL). The combined organic layer was washed with brine, dried
over anhyd Na2SO4, and evaporated in vacuo. The residue was sus-
pended in 5 mL of abs EtOH, and thiourea (30 mg, 0.39 mmol) was
added in one portion. The mixture was refluxed for 12 h and cooled
to rt. The solution was evaporated in vacuo, basified with NH4OH,
and extracted with CH2Cl2 (3ꢂ 30 mL). The combined organic
phase was washed with brine, dried over anhyd Na2SO4, and evap-
orated in vacuo. The residue was purified by silica gel chromatog-
raphy (petroleum/EtOAc = 2:1) to give the title compound ( )-3
5.7. (R)-2-Methoxy-11[(trifluoromethyl)sulfonyl]-aporphine (16)
This compound was prepared from phenol 14 in 89% yield using
a same procedure as preparation of triflate 12. 1H NMR (300 MHz,
CDCl3) d 7.32 (d, 1H, J = 2.4 Hz), 7.26 (m, 3H), 6.79 (d, 1H,
J = 2.4 Hz), 3.83 (s, 3H), 3.12 (m, 5H), 2.75 (1H, dd, J = 3.3,
16.5 Hz), 2.16 (s, 3H), 2.15 (m, 1H).
(22 mg, 22%). 1H NMR (300 MHz, CDC13)
d 7.51 (d, 1H,
J = 7.2 Hz), 7.18 (dd, 1H, J = 7.5, 7.5 Hz), 6.99 (d, 1H, J = 7.8 Hz),
4.96 (s, 2H), 3.81 (m, 1H), 3.15 (m, 3H), 2.74 (m, 5H), 1.62 (m,
2H), 0.95 (m, 3H). 13C NMR (75 MHz, CDC13-CD3OD) d 167.8,
143.5, 133.2, 129.9, 129.6, 126.8, 126.5, 119.9, 116.2, 59.0, 55.4,
48.8, 27.8, 25.3, 17.2, 11.0. MS (EI-LR) 285 (M+). HRMS calcd for
5.8. (R)-11-Amino-2-methoxyaporphine (18)
C
16H19N3S (M+) 285.1300; found 285.1278.
To a solution of triflate 16 (0.102 g, 0.24 mmol) in THF (10 mL)
were added Pd(OAc)2 (20 mg), rac-2,2-bis (diphenyl- phosphino)-
1,1-binaphthyl (25 mg), benzophenone imine (0.070 mL,
0.42 mmol), Cs2CO3 (250 mg), and 4,7,13,16,21,24-hexaoxa-1,10-
diazabicyclo[8.8.8]hexacosane (20 mg) under nitrogen. The mix-
ture was heated to 65–70 °C with stirring overnight. The solvent
was removed. The residue was diluted with CH2Cl2, washed with
brine, dried, and concentrated. The crude product was purified by
column chromatography eluting with petroleum/EtOAc = 1:2 (with
1% of Et3N), to yield imine 17 as yellow oil (72 mg, 66%).
5.3. 3-O-((Trifluoromethyl)sulfonyl)oripavine (12)24–27
This compound was prepared using a similar procedure re-
ported by Neumeyer et al. from oripavine 1124,25 in 90% yield.
5.4. (R)-2-Methoxy-10[(trifluoromethyl)sulfonyl]-11-
hydroxyaporphine (13)26,27
Compound 12 (0.160 g, 0.47 mmol) was dissolved in 1 mL of
98% MeSO3H at rt. The mixture was stirred for 25 min at 95–
100 °C, and then cooled to rt, and quenched with ice. The mixture
was basified with NH4OH, and extracted with CH2Cl2 (4ꢂ 20 mL).
The organic layer was combined and washed with brine, and dried
over anhydrous Na2SO4. The solvent was evaporated yielding the
title compound 13 in quantitative yield. 1H NMR (300 MHz,
DMSO-d6) d 10.7 (br s, 1H), 7.42 (d, 1H, J = 1.5 Hz), 7.22 (d, 1H,
J = 8.7 Hz), 6.95 (dd, 1H, J = 8.4, 3.2 Hz), 6.70 (dd, 1H, J = 14.1,
2.1 Hz), 3.74 (s, 3H), 3.10 (m, 5H), 2.43 (s, 3H), 2.29 (m, 2H). MS
(EI-LR) 429 (M+).
To a solution of the intermediate 17 (72 mg, 0.16 mmol) in
10 mL of MeOH were added NH2OH.HCl (78 mg, 1.1 mmol) and
anhydrous NaOAc (120 mg, 1.46 mmol). The mixture was stirred
overnight at rt. The solvent was removed. The residue was diluted
with 0.1 M NaOH solution, and extracted with CH2Cl2 (5ꢂ 20 mL).
The combined organic layer was washed with brine, dried over
anhydrous Na2SO4, and the solvent was evaporated. The residue
was purified by column chromatography eluting with CHCl3/
MeOH = 30:1 to give 2-methoxy-11-aminoaporphine 18 as yellow
solid (32 mg, 70%). 1H NMR (300 MHz, CDCl3) d 7.54 (d, 1H,
J = 2.4 Hz), 7.03 (t, 1H), 6.70 (t, 2H), 6.60 (d, 1H, J = 2.4 Hz), 4.08
(br s, 2H), 3.82 (s, 3H), 3.05 (m, 4H), 2.75 (m, 1H), 2.52 (m, 5H).
MS (EI-LR) 280 (M+).
5.5. (R)-2-Methoxy-11-hydroxyaporphine (14)26,27
To a mixture of triflate 13 (1.33 g, crude) and 10% Pd/C (0.3 g) in
anhydrous MeOH (100 mL) at rt was added Mg metal (freshly pol-
ished, 1.33 g) and NH4OAc (1.5 g). The mixture was stirred at rt for
2 days and then filtered, and the filtrate was evaporated. The resi-
due was diluted with NH4OH (100 mL) and extracted with CH2Cl2
5.9. (R)-10,11:[5,4-m]-20-Aminothiazolo-2-methoxyaporphine
((ꢀ)-4)
Amine 18 (17 mg, 0.06 mmol) and KSCN (25 mg, 1.36 mmol)
were mixed in a solution of AcOH (8 mL). A solution of Br2

6680
Z. Liu et al. / Bioorg. Med. Chem. 16 (2008) 6675–6681
(65 mg, 0.41 mmol) in AcOH (0.4 mL) was added dropwise. The
reaction mixture was stirred overnight at rt and then the solvent
was evaporated. The residue was diluted with 15 mL of NH4OH
(8%), and extracted with CH2Cl2 (4ꢂ 20 mL). The combined organic
layer was washed with brine, dried over anhydrous Na2SO4, and
then evaporated. The residue was subjected to chromatography
(CHCl3/MeOH) to yield the title compound (ꢀ)-4 as yellow solid
(15 mg, 91%). 1H NMR (300 MHz, CDCl3) d 7.43 (d, 1H, J = 1.8 Hz),
7.36 (d, 1H, J = 8.7 Hz), 6.70 (d, 1H, J = 8.7 Hz), 6.64 (d, 1H,
J = 1.2 Hz), 4.40 (br s, 2H), 3.81 (s, 3H), 3.70 (m, 1H), 3.10 (m,
3H), 2.73 (m, 1H), 2.55 (m, 4H), 2.38 (m, 1H); 13C NMR (75 MHz,
CDCl3) d 157.7, 145.7, 141.3, 134.7, 134.3, 132.4, 127.4, 121.5,
116.1, 111.5, 111.3, 109.6, 109.2, 60.9, 55.0, 52.5, 43.7, 33.1, 29.4.
MS (EI) 337 (M+). HRMS: calcd for C19H19N3OS (M+) 337.1249;
found 337.1251.
65%). 1H NMR (300 MHz, CDCl3) d 7.31 (dd, 1H, J = 2.4 Hz), 7.01
(m, 1H), 6.69 (m, 2H), 6.41 (d, 1H, J = 2.1 Hz), 4.05 (br s, 2H), 3.60
(br s, 2H), 3.10 (m, 4H), 2.69 (m, 1H), 2.53 (m, 5H). MS (EI-LR)
265 (M+).
5.13. (R)-2,3:[4,5-b]-10,11:[5,4-m]-Bis(20-aminothiazolo)-
aporphine ((ꢀ)-5)
Diamine 21 (42 mg, 0.16 mmol) and KSCN (92 mg, 0.90 mmol)
were mixed in a solution of AcOH (5 mL). A solution of Br2
(43 mg, 0.28 mmol) in AcOH (4.2 mL) was added dropwise. The
resulting mixture was stirred overnight at rt and then evapo-
rated. The residue was diluted with 28 mL of NH4OH, and ex-
tracted with CH2Cl2 (5ꢂ 20 mL). The combined organic layer
was washed with brine, dried over anhydrous Na2SO4, and then
evaporated. The residue was purified by column chromatography
(CHCl3/CH3OH = 25:1 with 1% Et3N) to yield bisaminothiazole
(ꢀ)-5 as a white solid (17 mg, 28%). 1H NMR (300 MHz, CDCl3)
d 8.02 (s, 1H), 7.36 (d, 1H, J = 8.7 Hz), 6.69 (d, 1H, J = 8.4 Hz),
5.37 (br s, 2H), 4.47 (s, 2H), 3.69 (q, 1H), 3.10 (m, 3H), 2.60
5.10. (R)-2,11-Dihydroxyaporphine (19)
To a solution of 2-methoxy-11-hydroxy-aporphine 14 (127 mg)
in 10 mL of CH2Cl2, cooled to ꢀ78 °C was added dropwise a solu-
tion of BBr3 (1 M in CH2Cl2, 10 mL). The mixture was stirred at
ꢀ78 °C for 2 h, then at rt overnight. After cooling to ꢀ78 °C again,
a solution of MeOH (10 mL) was added dropwise. The mixture
was stirred at rt for 2 h, and then evaporated. The residue was dis-
solved in MeOH, evaporated again. After repeating this procedure
twice, the title compound 19 was obtained as a pale solid
(140 mg, 89%). This compound was used for the next step without
further purification.
(m, 5H), 2.38 (t, 1H); 13C NMR (75 MHz, CDC13-CD3OD)
d
167.8, 149.5, 146.7, 140.2, 134.3, 129.6, 128.6, 128.0, 125.7,
121.2, 116.2, 112.7, 111.7, 107.6, 61.3, 51.8, 43.0, 32.6, 27.8.
MS (EI) 379 (M+). HRMS calcd for C19H17N5S2 (M+) 379.0925;
found 379.0920.
5.14. (R)-2-Hydroxy-10,11-(methylenedioxy)apomorphine (23)
Finely ground NaOH (0.452 g, 11.3 mmol) was added to a solu-
tion of 2232,33 (1.23 g, 3.2 mmol) in 30 mL of dry DMSO at rt under
N2 and stirred for 1 h. Methylene dibromide (0.290 mL, 4.2 mmol)
was added, and the mixture was heated at 80 °C overnight. After
cooling, the solution was diluted with ice water and extracted with
EtOAc (5ꢂ 100 mL). The organic phase was combined, washed with
brine, dried over anhydrous Na2SO4, and evaporated in vacuo. The
residue was purified by column chromatography (CHCl3/
MeOH = 20:1) to yield the title compound 2332,33 (0.344 g, 36%).
1H NMR (300 MHz, CDC13) d 7.30 (d, 1H, J = 2.4 Hz), 6.67 (d, 1H,
J = 8.1 Hz), 6.61 (d, 1H, J = 7.8 Hz), 6.38 (d, 1H, J = 2.4 Hz), 5.84 (d,
1H, J = 1.5 Hz), 5.79 (d, 1H, J = 1.2 Hz), 3.12 (m, 4H), 2.69 (t, 1H),
2.54 (m, 5H).
5.11. (R)-2,11-Di[(trifluoromethyl)sulfonyl]aporphine (20)
To
a solution of 19 (53 mg, 0.15 mmol) and pyridine
(0.42 mmol) in CH2Cl2 (10 mL), cooled to ꢀ30 °C to 40 °C, Tf2O
(110 mL, 0.66 mmol) was added dropwise. The reaction mixture
was allowed to reach rt and stirred for 2 h. The reaction was
quenched with cold water. The organic layer was separated,
washed with brine and dried (Na2SO4). After removal of the sol-
vent, the residue was subjected to column chromatography (petro-
leum/EtOAc = 5:1, with 1% Et3N) to yield bistriflate 20 as light
yellow oil (80 mg, 98%).1H NMR (300 MHz, CDCl3) d 7.67(d, 1H,
J = 2.4 Hz), 7.35 (m, 1H), 7.09 (d, 1H, J = 2.4 Hz), 6.74 (d, 1H,
J = 8.1 Hz), 6.59 (d, 1H, J = 2.4 Hz), 3.78 (s, 3H), 3.16 (m, 4H),
2.81(dd, 1H, J = 3.0, 16.5 Hz), 2.56 (m, 5H). MS(EI-LR): 531(M+).
5.15. (R)-2-(Trifluromethyl)sulfonyl-10,11-
(methylenedioxy)apomorphine (24)
5.12. (R)-2,11-Diaminoaporphine (21)
A solution of 23 (181 mg, 0.61 mmol) and Et3N (0.15 mL,
1.06 mmol) in 20 mL of anhydrous CH2Cl2 was cooled to ꢀ30 °C.
Tf2O (0.125 mL, 0.75 mmol) in 0.5 mL of anhydrous CH2Cl2 was
added dropwise. The reaction mixture was allowed to reach rt
and stirred for 2 h. The reaction was quenched with cold water
and the organic layer was separated, washed with brine, dried over
anhydrous Na2SO4 and evaporated in vacuo. Purification by silica
gel chromatography (petroleum/EtOAc = 2:1, 1% Et3N) afforded
the title compound 24 (0.247 g, 95%). 1H NMR (300 MHz, CDC13)
d 7.85 (d, 1H, J = 2.4 Hz), 6.97 (d, 1H, J = 2.4 Hz), 6.76 (s, 2H), 6.15
(d, 1H, J = 1.2 Hz), 6.01 (d, 1H, J = 1.2 Hz), 3.14 (m, 4H), 2.77 (dd,
1H, J = 16.5, 2.7 Hz), 2.58 (m, 5H).
To a solution of triflate 20 (71 mg, 0.13 mmol) in THF (10 mL)
were added Pd(OAc)2 (20 mg), rac-2,2-bis (diphenylphosphino)-
1,1-binaphthyl (BINAP, 25 mg), benzophenone imine (0.100 mL,
0.60 mmol), Cs2CO3 (250 mg), and 4,7,13,16,21,24-hexaoxa-1,10-
diazabicyclo[8.8.8]hexacosane (20 mg) under nitrogen. The mix-
ture was heated to 65–70 °C with stirring overnight, and then the
solvent was removed. The residue was diluted with CH2Cl2,
washed with brine, dried, and concentrated. The crude product
was purified by column chromatography (petroleum/EtOAc = 1:1,
with 1% Et3N) to yield the imine intermediate as a yellow solid
(45 mg, 61%).
To a solution of the imine intermediate (45 mg, 0.075 mmol) in
MeOH (10 mL) were added NH2OHꢃHCl (78 mg, 1.1 mmol) and
NaOAc (120 mg, 1.46 mmol). The reaction mixture was stirred
overnight at rt, and the solvent was removed. The residue was di-
luted with 0.1 N NaOH solution, and extracted with CH2Cl2 (5ꢂ
20 mL). The combined organic layer was washed with brine, dried
over anhydrous Na2SO4, and then evaporated. The residue was
purified by column chromatography (CHCl3/MeOH = 20:1 with 1%
Et3N), to yield diaminoaporphine 21 as a yellow solid (13 mg,
5.16. (R)-2-Amino-10,11-(methylenedioxy)apomorphine (25)
This compound was prepared from 24 by using a similar proce-
dure as that of preparation of 18 in 74% yield (CHCl3/MeOH = 20:1,
1% Et3N). 1H NMR (300 MHz, CDCl3) d 7.33 (d, J = 2.4 Hz, 1H), 6.73
(d, J = 7.8 Hz, 1H), 6.69 (d, J = 7.8 Hz, 1H), 6.41 (d, 1H, J = 2.1 Hz),
6.08 (d, J = 1.5 Hz, 1H), 5.95 (d, J = 1.5 Hz, 1H), 3.50 (br s, 2H),
3.10 (m, 4H), 2.50 (m, 6H).

Z. Liu et al. / Bioorg. Med. Chem. 16 (2008) 6675–6681
6681
5.17. (R)-2,3:[4,5-b]-20-Aminothiazolo-10,11-
Acknowledgments
(methylenedioxy)aporphine (26)
This work was financially supported by grants from Chinese
National Science Foundation (30672517, to A.Z.), Shanghai Com-
mission 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 Sci-
ences and Shanghai Institute of Materia Medica was also appre-
ciated. We also thank Professors John L. Neumeyer and Ross J.
Baldessarini for their instructive discussion during this work
and reevaluation of some of the synthetic compounds. Thebaine
and morphine alkaloid were kindly provided by the neurophar-
macological laboratory, SIMM.
This compound was prepared from amine 25 in 91% yield by
using a similar procedure as that of preparation of (ꢀ)-4. 1H
NMR (300 MHz, CDCl3) d 8.19 (s, 1H), 6.75 (d, 1H, J = 7.8 Hz), 6.68
(d, 1H, J = 8.1 Hz), 6.09 (d, 1H, J = 0.9 Hz), 5.97 (d, 1H, J = 1.2 Hz),
5.41 (br s, 2H), 3.18 (m, 4H), 2.60 (m, 6H).
5.18. (R)-2,3:[4,5-b]-20-Aminothiazolo-10,11-
dihydroxyaporphine ((ꢀ)-6)
To a solution of 26 (100 mg, 0.29 mmol) in 10 mL of anhydrous
CH2Cl2, cooled to ꢀ78 °C, was added dropwise a solution of BBr3
(1 M in CH2Cl2, 5 mL). The mixture was stirred at ꢀ78 °C for 2 h,
then at rt overnight. After cooling to ꢀ78 °C again, 10 mL MeOH
was added dropwise. The mixture was stirred at rt for 2 h, and
evaporated. The residue was redissolved in MeOH, and evaporated
again. After repeating this procedure twice, the residue was recrys-
tallized from anhydrous MeOH to give the title compound (ꢀ)-6 as
pale yellow solid (45.2 mg, 32%). MS (EI) 339 (M+). 1H NMR
(300 MHz, CD3OD-d4) d 8.71 (s, 1H), 6.82 (d, 1H, J = 8.1 Hz), 6.77
(d, 1H, J = 7.8 Hz), 3.18 (m, 4H), 2.60 (m, 6H). Anal. Calcd for
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2008.05.077.
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;
pp 146–170.
2. Zhang, A.; Zhang, Y.; Branfman, A. R.; Baldessarini, R. J.; Neumeyer, J. L. J. Med.
Chem. 2007, 50, 171–181.
C18H17N3O2S. 2.5HBr: C, 39.91; H, 3.63; N, 15.48; found: C, 39.79;
H, 4.03; N, 15.59.
3. Zhang, A.; Neumeyer, J. L.; Baldessarini, R. J. Chem. Rev. 2007, 107, 274–302.
4. Mathiessen, A.; Wright, C. R. A. Proc. R. Soc., Ser. B. 1869, 17, 455–460.
5. Mathiessen, A.; Wright, C. R. A. Proc. R. Soc., Ser. B. 1869, 17, 460–462.
6. Neumeyer, J. L.; Neustadt, B. R.; Weinhardt, K. K.; Aporphines, V. J. Pharm. Sci.
1970, 59, 1850–1852.
7. Neumeyer, J. L.; Baldessarini, R. J.; Booth, R. G., 6th ed.. In Burger’s Medicinal
Chemistry and Drug Discovery; Abraham, D. J., Ed.; John Wiley & Sons: New York,
2003; vol. 6, pp 711–714. Chapter 12.
8. Kolls, B. J.; Stacy, M. Clin. Neuropharmacol. 2006, 29, 292–301.
9. Subramony, J. A. Mol. Pharm. 2006, 3, 380–385.
10. Si, Y.; Gardner, M. P.; Tarazi, F. I.; Baldessarini, R. J.; Neumeyer, J. L. J. Med. Chem.
2008, 51, 983–987.
11. Zhang, A.; Csutoras, C.; Zong, R.; Neumeyer, J. L. Org. Lett. 2005, 7, 3239–3242.
12. Garrido, J. M.; Delerue-Matos, C.; Borges, M. F. M.; Macedo, T. R. A.; Oliveira-
Brett, A. M. Bioelectrochemistry 2002, 55, 113–114.
13. Vernier, V. G., 5th ed.. In Burger’s Medicinal Chemistry and Drug Discovery;
Wolff, M. E., Ed.; John Wiley & Sons: New York, 1996; vol. 3, pp 43–98.
Chapter 37.
5.19. Established stable expression of cell lines
The rat 5-HT1A receptor gene, human D1 receptor gene, and hu-
man D2 receptor gene were individually cloned into pcDNA3.0 vec-
tor. The 5-HT1A construct was then transfected into CHO cells. The
D1 and D2 receptors 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 (Gibco), 10% fetal bovine serum, 100 U/ml
penicillin, 100 U/ml streptomycin, and 200
and 5% CO2.
lg/ml G418 at 37 °C
To confirm the success of transfection, the saturation binding
experiment that the expression of 5-HT1A receptor in the CHO cell
14. Moller, J. C.; Oertel, W. H. Expert. Rev. Neurother. 2005, 5, 581–586.
15. Reichmann, H.; Brecht, M. H.; Koster, J.; Kraus, P. H.; Lemke, M. R. CNS Drugs
2003, 17, 965–973.
16. Zhang, A.; van Vliet, L. A.; Neumeyer, J. L. Tetrahedron Lett. 2003, 44, 6459–
6462.
line is 1.5531 0.2803 nmol/g protein with
1.2058 nM, expression for D1 is 10.67 nmol/g protein with a Kd va-
lue of 1.31 0.16 nM. The Kd for D2 is 0.06 nM.
a Kd value of
17. Zhang, A.; Xiong, W.; Hilbert, J. E.; DeVita, E. K.; Bidlack, J. M.; Neumeyer, J. L. J.
Med. Chem. 2004, 47, 1886–1888.
5.20. Radioligand binding assays
18. van Vliet, L. A.; Rodenhuis, N.; Wikstrom, H. J. Med. Chem. 2000, 43, 3549–3557.
19. Liu, D.; Venhuis, B. J.; Wikstrom, H. V.; Dijkstra, D. Tetrahedron 2007, 63, 7264–
7270.
20. Berney, D.; Schuh, K. Helv. Chim. Acta 1982, 65, 1305–1308; Schneider, C. S.;
Mierau, J. J. Med. Chem. 1987, 30, 494–498.
21. Schneider, F.; Gerold, M.; Bernauer, K. Helv. Chim. Acta 1973, 56, 759–773.
22. Schneider, C.; Merz, H.; Sobotta, R.; Bauer, R.; Mierau, J.; Schingnitz, G. Ger.
Offen. DE 3624607 A1, 1988, 13pp.
23. Zhang, A.; Li, F.; Ding, C.; Yao, Q.; Knapp, B. I.; Bidlack, J. M.; Neumeyer, J. L. J.
Med. Chem. 2007, 50, 2747–2751.
24. Coop, A.; Janetka, J. W.; Lewis, J. W.; Rice, K. C. J. Org. Chem. 1998, 63, 4392–
4396.
25. Coop, A.; Lewis, J. W.; Rice, K. C. . J.Org. Chem. 1996, 61, 6774.
26. Si, Y.; Gardner, M. P.; Tarazi, F.; Baldessarini, R. J.; Neumeyer, J. L. . Bioorg. Med.
Chem. Lett. 2007, 17, 4128–4130.
27. Si, Y.; Neumeyer, J. L. Synthesis 2007, 24, 3787–3790.
28. Sajiki, H.; Mori, A.; Mizusaki, T.; Ikawa, T.; Maegawa, T.; Hirota, K. Org. Lett.
2006, 8, 987–990.
29. Csutoras, C.; Zhang, A.; Zhang, K.; Kula, N. S.; Baldessarini, R. J.; Neumeyer, J. L.
Bioorg. Med. Chem. 2004, 12, 3553–3559.
The affinity of compounds to the D1 and D2 dopamine recep-
tors, and the 5-HT1A receptor was determined by competition
binding assays. Membrane homogenates of 5-HT1A-CHO cells,
D1- or D2-HEK293 cells were prepared as described previ-
ously.26,29 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]SCH23390 (for
D1 dopamine receptors), or [3H]spiperone (for dopamine D2
receptor) in a final volume of 200
50 mM Tris, 4 mM MgCl2, pH 7.4. Nonspecific binding was deter-
mined by parallel incubations with either 10 M WAY100635 for
lL binding buffer containing
l
5-HT1A, SCH23390 for D1, or spiperone for D2 dopamine recep-
tors, respectively. The reaction was started by addition of mem-
branes (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
radioactivity 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.
30. Hedberg, M. H.; Jansen, J. M.; Nordvall, G.; Hjorth, S.; Unelius, L.; Johansson, A.
M. J. Med. Chem. 1996, 39, 3491–3502.
31. Hedberg, M. H.; Linnanen, T.; Jansen, J. M.; Nordvall, G.; Hjorth, S.; Unelius, L.;
Johansson, A. M. J. Med. Chem. 1996, 39, 3503–3513.
32. Gao, Y.; Baldessarini, R. J.; Kula, N. S.; Neumeyer, J. L. J. Med. Chem. 1990, 33,
1800–1805.
33. Neumeyer, J. L.; Gao, Y.; Kula, N. S.; Baldessarini, R. J. J. Med. Chem. 1990, 33,
3122–3124.