1-substituted apomorphines as potent dopamine agonists

Article abstract of DOI:10.1016/j.bmc.2013.05.014

A novel set of 1-substituted apomorphines as dopaminergic agonists were synthesized according to our new strategy employing the acid-catalyzed rearrangement of diversely functionalized 5β-substituted-6- demethoxythebaines. The activities of new compounds for dopamine receptors subtypes were evaluated using HEK293 based stable cell lines expressing D ₁, D_2L or D₃ receptor subtypes. All studied compounds had affinities in nanomolar range for D_2L and D₃ receptors and the change of the nature of substituent in position 1 had only moderate effect. D₁ receptors were sensitive to the introduction of the 4-OH-benzyl function resulting in an increased affinity. The small hydrophilic group (hydroxymethyl) highly reduced the agonist affinity and potency thereby increasing subtype selectivity. This strategy for selective modulation of affinities and potencies of 1-substituted apomorphines gives essential hints for future design of subtype selective dopaminergic ligands.

Full text of DOI:10.1016/j.bmc.2013.05.014

Bioorganic & Medicinal Chemistry 21 (2013) 4143–4150
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
1-substituted apomorphines as potent dopamine agonists
a,b
Reet Reinart-Okugbeni a,b, Argo Vonk a, Ain Uustare a, Zsuzsanna Gyulai c, Attila Sipos d, , Ago Rinken
⇑
a Institute of Chemistry, University of Tartu, Ravila 14a, 50411 Tartu, Estonia
b Competence Centre on Reproductive Medicine and Biology, Tiigi 61b, 50410 Tartu, Estonia
c Department of Organic Chemistry, University of Debrecen, PO Box 20, H-4010 Debrecen, Hungary
d Department of Pharmaceutical Chemistry, University of Debrecen, PO Box 70, H-4010 Debrecen, Hungary
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 17 February 2013
Revised 5 May 2013
Accepted 8 May 2013
Available online 16 May 2013
A novel set of 1-substituted apomorphines as dopaminergic agonists were synthesized according to our
new strategy employing the acid-catalyzed rearrangement of diversely functionalized 5b-substituted-6-
demethoxythebaines. The activities of new compounds for dopamine receptors subtypes were evaluated
using HEK293 based stable cell lines expressing D1, D2L or D3 receptor subtypes. All studied compounds
had affinities in nanomolar range for D2L and D3 receptors and the change of the nature of substituent in
position 1 had only moderate effect. D1 receptors were sensitive to the introduction of the 4-OH-benzyl
function resulting in an increased affinity. The small hydrophilic group (hydroxymethyl) highly reduced
the agonist affinity and potency thereby increasing subtype selectivity. This strategy for selective modu-
lation of affinities and potencies of 1-substituted apomorphines gives essential hints for future design of
subtype selective dopaminergic ligands.
Keywords:
Apomorphine
Acid-catalyzed rearrangement
Dopamine agonist
Dopamine D1 and D2 receptors
Morphinandiene
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
These examples suggest that aporphines modified in position 2
are the most important derivatives of this family of alkaloids, how-
Dopamine plays crucial regulatory functions in the brain. Mal-
functions in the dopaminergic system, and especially disturbances
related to the dopamine D2 receptor function, are connected to
many different neurological and psychiatric disorders, including
Parkinson’s disease, Huntington’s chorea, schizophrenia, attention
deficit-hyperactivity disorder, Tourette’s syndrome, restless leg
syndrome, erectile dysfunction and addiction.1,2 Therefore diverse
approaches towards the synthesis and characterization of selective
and potent D2 agonists are considered very important.3,4 Dopamine
D1 and D2 receptors are both ubiquitous in the brain and are con-
nected to different effector systems. The D2 receptor is highly
homologous to the dopamine D3 receptor, which is present at low-
er densities and has more restricted distribution in the brain, and
also a different function.5 Most dopamine D2 receptor ligands have
similar affinity also for D3 receptors, which has to be considered
when developing subtype selective ligands.
ever it has been already confirmed that other substitution patterns
in ring A may also lead to potent ligands.10–12 On the basis of these
results and the classic model for the interactions of aporphinoids
with D2 receptor binding site identifying a lipophilic cleft in the
proximity of position 2 of the aporphine skeleton, the synthesis
and pharmacological characterization of a novel set of 1-substi-
tuted apomorphines was targeted.13,14 It was presumed that re-
sults on these uniquely substituted aporphines might remarkably
contribute to the existing structure–activity relationships and help
further explore the electronic and steric interactions in the region
of ring A of this scaffold.
2. Results and discussion
The synthesis of the aimed 1-substituted apomorphine deriva-
tives was based on the previous observation that 6-demethoxyt-
hebaine (5) can be selectively alkylated in the 5b-position to
yield precursors to ring A derivatized aporphines (Scheme 1).15,16
The preparation of compound 5 was achieved in accordance
with the original procedure published by Berényi et al.,17 however
there were other alternative routes reported to this morphinandi-
ene.18 In brief, neopine, a minor opium alkaloid, was mesylated in
position 6 and the mesyloxy group was eliminated in the presence
of tetrabutylammonium fluoride. The strategy for versatile 5b-sub-
stitution of thebaine was elaborated by Gates et al.19,20 and was
successfully extended by many other groups.18 The procedure
The chemical modification of the substitution pattern of ring A
of apomorphine (1, Fig. 1), the non-selective dopamine agonist
clinically used for the management of Parkinson’s disease and
erectile dysfunction, led to several potent and selective D2 agonist
derivatives, for example 2-fluoroapomorphine (2), 2-hydroxyapo-
morphine (3) and 2-(4-hydroxyphenyl)apomorphine (4).6–9
⇑
Corresponding author. Tel.: +36 52 512 900x22478; fax: +36 52 512 586.
E-mail address: sipos.attila@pharm.unideb.hu (A. Sipos).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.05.014

4144
R. Reinart-Okugbeni et al. / Bioorg. Med. Chem. 21 (2013) 4143–4150
under such harsh conditions. As it was reported previously,16 the
4
3
R
5
2
1
R
reaction between the morphinandiene anion and electrophiles re-
sults two main products, namely the 5b- and 7-substituted mor-
phinans. In the course of the preparation of compounds 8–11 the
crude product mixtures were found to be more complex. The isola-
tion of the targeted compounds was based on the observation that
the first eluted fraction resulted by column chromatography was
the 5b-derivatized morphinandiene. Unfortunately, it was not pos-
sible to isolate 7-substituted morphinandines in pure form; there-
fore we were unable to access to 3-substituted aporphines in this
way. The yields for 5b-substituted morphinans were in the range
of 15–30%. The next step of the synthesis was the acid-catalyzed
rearrangement of compounds 6–11 into apocodeines 12–17
(Scheme 3).
C
D
N 6
N
H
6a
2
3
4
F
H3C
H3C
OH
H
B
OH
OH
OH
OH
7
OH
11
10
A
8
2-4
1
Figure 1. Apomorphine (1) and some of its potent and selective ring A modified
congeners.
was performed in accordance with the modified methodology of
Gates et al. described earlier for the benzylation of 6-demethoxyt-
hebaine (5).20 The carefully dried compound 5 was dissolved in
anhydrous THF, cooled to ꢀ78 °C and treated with t-BuLi affording
the corresponding anion. This intermediate was reacted with the
electrophile partner. In order to be able to extensively examine
the targeted structure–activity relationships, the electrophile part-
ners were chosen in a wide range with respect to their spatial and
electronic properties (Scheme 2).
It was decided to insert also hydroxyl functions in the proximity
of ring A of the aporphine skeleton to be able to test the effect of
this moiety and to compare it with the superior binding activity
and agonist efficacy of reference compound 4. According to the
nature of the 5b-substitution of morphinandiene 5 in the presence
of t-BuLi, it was critical to choose adequately protected alcohols
and phenols. On the other hand, it was also an important aspect
to choose protective groups that could be removed as a one-pot
procedure during the closing step of our strategy (10-O-demethyl-
ation of the intermediate apocodeines) without the need for addi-
tional synthesis steps. Therefore phenolic OH groups were
introduced in the form of methyl ethers and alcoholic hydroxyls
were protected with benzyl group, because it is known to be stable
The conventional explanation21,22 for the acid-catalyzed rear-
rangement of the thebaine-like molecules involves the proton-pro-
moted cleavage of the C5–O ether bond as the first step, resulting
in a cation at ring C. Meanwhile the bridgehead bond of ring N at
the fusion of rings B and C relocates from 13-position to 14-posi-
tion. As the next steps of the mechanism, a series of electron pair
shifts take place at ring C in order to form the aromatic structure;
these rearrangements are terminated with a further relocation of
ring N from 14-position to 8-position of the late morphinan back-
bone. As a results of the configuration of C9 of the morphinan
structure, the configuration of the only resided chiral centre
(C6a) is predetermined. The rearrangement was achieved by
methanesulfonic acid (99.5% purity) and yielded the aimed com-
pounds in good yields (67–77%). The closing step of this synthetic
route was the BCl3-mediated 10-O-demethylation at ꢀ10 °C in
CH2Cl2 (Scheme 4).
This procedure was applied successfully for the efficient prepa-
ration of derivatized apomorphines.23 In the case of apocodeines
14, 15 and 17 it was aimed to perform a double deprotection of
both 10-O-methyl function and the substituent in position 1.
N
H
R
N
H
N
N
H3C
R
H3C
R
H3C
H3C
O
O
OH
OH
CH3
CH3
CH3
O
OH
O
O
5
Scheme 1. Retrosynthetic analysis of our route to 1-substituted apomorphines.
OH
OMs
MsCl
N
N
TBAF
H3C
H3C
O
O
CH3
CH3
O
O
R
neopine
6
Me
Bn
7
8
6
7
Li
t-BuLi/
14
5
R
9
R-Cl
8
Bn-O-CH2
4-OMe-Bn
N
N
anh. THF
N
H3C
H3C
H3C
O
O
O
9
10
10
11
4-(N,N-dimethylamino)-Bn
3
CH3
CH3
1
CH3
O
O
O
4-MeO-naphthyl-CH2
5
6-11
Scheme 2. Synthesis of 5b-substituted-6-demethoxythebaines 6–11.

R. Reinart-Okugbeni et al. / Bioorg. Med. Chem. 21 (2013) 4143–4150
4145
R
6, 12
7, 13
Me
Bn
R
N
N
CH3SO2OH
R
8, 14
9, 15
Bn-O-CH2
4-OMe-Bn
H3C
H3C
O
H
OH
CH3
CH3
10, 16 4-(N,N-dimethylamino)-Bn
O
O
6-11
12-17
11, 17
4-MeO-naphthyl-CH2
Scheme 3. Synthesis of apocodeines 12–17.
R
R'
Me
12
Me
Bn
18
19
20
21
22
23
N
N
13
Bn
BCl3 / CH2Cl2
R'
R
H3C
H3C
H
H
14
15
16
17
Bn-O-CH2
OH-CH2
OH
OH
4-OMe-Bn
4-OH-Bn
CH3
OH
O
4-(N,N-dimethylamino)-Bn
4-MeO-naphthyl-CH2
4-(N,N-dimethylamino)-Bn
4-OH-naphthyl-CH2
18-23
12-17
Scheme 4. Synthesis of targeted apomorphines 18–23.
According to the literature, it was found that the benzyl deprotec-
tion of 1-hydroxymethyl function was also possible with this re-
agent.24 The one-pot deprotections of compounds 14, 15 and 17
gave rise to the desired apomorphines in lower yields (36%, 41%
and 51%, respectively) in comparison to the simple 10-O-demethy-
lated compounds 12, 13 and 16 (66%, 70% and 54%, respectively).
The apomorphines 18–23 were isolated in the stable hydrochloride
salt form.
teristic of a drug. Most direct approach is to follow activation of
receptor-coupled G proteins by measuring its GTPase activity or
[
35S]GTP
c
S binding, what we also used for characterization of li-
gands’ potencies with D2 receptors.30 An alternative approach is
to measure modulation of the level of second messengers (Ca2+
,
IP3, cAMP). In the case of dopamine receptors, the inhibition and
activation of adenylate cyclase and measuring the corresponding
change in cAMP levels are widely used for the characterization of
receptor activation.31 The latter method is widely applied for Gi-
coupled receptors, but for other G-protein subtypes the results
are contradictory.32 The reason for choosing the cAMP accumula-
tion assay for comparing the properties of ligands at different
receptor subtypes was the fact that it allows the measurement of
the Gs and Gi protein coupled receptor signaling at the same down-
stream effector level.
With the generated cell lines the cyclic AMP accumulation assay
was successfully established for the dopamine D1 and D2L recep-
tors, while several problems were encountered in obtaining reli-
able and consistent results with the cells expressing the
dopamine D3 receptors. To confirm the existence of a functional
receptor-G protein complex which is required for effective signal
transduction, the extent of GTP-dependent high affinity agonist
binding33 was measured in membrane preparations of the gener-
ated cell lines. With both D1 and D2L receptors a clear effect of
nucleotides on dopamine affinity was achieved, but for the D3
receptors only a negligible effect was detected (data not shown).
The guanine nucleotide insensitivity on agonist binding to the
dopamine D3 receptors has been reported several times earlier
for various cell lines.34,35 It has been, however, under debate
whether the observation is connected with the lack of appropriate
subtypes of Gi/o proteins or their regulators in these cells or even
with the probability of the preference of D3 receptor to a different
signaling pathway. Zaworski et al. have shown that the major fac-
tors limiting proper D3 receptor signaling in HEK293 cells are the
lack of AC-V isoform and also N-type Ca2+ channels, whereas Go
proteins necessary for signaling are present in sufficient amount.36
As the results of cAMP accumulation assay for the D3 receptors
were controversial, only the data for the dopamine D1 and D2L
For testing the new compounds three stable cell lines express-
ing the human dopamine receptor subtypes D1, D2L and D3 were
generated in human embryonic kidney cell line (HEK293). The
HEK293 cell line is easy to transfect has high protein expression
levels as well as a proper set of the signaling pathway components
of many GPCRs.25 The obtained stable lines were characterized by
radioligand binding and compared to tritium-labeled control com-
pounds. The dopamine D1 receptor expression was determined by
the specific binding of [3H]SCH23390 with its KD = 0.48 0.06 nM
and Bmax = 570 20 fmol/106 cells. The expression of dopamine
D2L and D3 receptors was determined by the specific binding of a
non-selective D2-family radioligand, [3H]raclopride with
KD = 0.66 0.07 nM, cells, and
Bmax = 164 4 fmol/106
a
KD = 0.83 0.13 nM, Bmax = 111 4 fmol/106 cells, for D2L and D3
receptors, respectively. These affinities are in agreement with data
published earlier for these radioligands and receptor subtypes.26–28
Subtype identity was further confirmed by displacement exper-
iments with different dopaminergic ligands. A representative
example for the selectivity of ligands for the D1, D2L and D3 recep-
tors is their endogenous ligand, dopamine, which reportedly has
the highest affinity for the D3 subtype and the lowest for the D1
subtype.29 The potency of dopamine to displace the binding of
radioligand was characterized with Ki values of 12 lM, 2.1 lM
and 15 nM for the D1R, D2R and D3R, respectively. Additionally
more than 200-fold binding selectivity for the D3 over the D2 sub-
type was recorded with known dopaminergic compounds, 7-OH-
PIPAT and NGB2904, confirming the distinct characteristics of the
receptor subtypes (data not shown).
In addition to the receptor binding affinity, the ability to mod-
ulate downstream signaling cascade is also an important charac-

4146
R. Reinart-Okugbeni et al. / Bioorg. Med. Chem. 21 (2013) 4143–4150
Table 1
Binding affinities of synthesized compounds for human dopamine D1, D2L and D3 receptors and their subtype selectivities
Ki (nM)
Selectivity
D1/D2L
D1 versus [3H]SCH23390
D2L versus [3H]raclopride
D3 versus [3H]raclopride
15
D2L/D3
Dopamine
1
12,000
2100
5.7
9.3
3.4
2.2
22
0.8
1.7
2.6
140
3.2
2.4
1.4
2.6
1.7
1.5
1.3
492 49
187 46
168 34
980 160
40 11
53 15
54 10
77 20
43 13
17
23
55
17
28
3
5
9
4
6
18
19
20
21
22
23
47
9
135 39
437 83
79 31
170 64
53 12
130 30
Ki characterizes the ability of a compound to inhibit [3H]SCH23390 (D1) or [3H]raclopride (D2 and D3) binding to the corresponding human dopamine receptor subtypes in
membrane preparations of HEK293 cells. Values are represented as mean SEM of the apparent inhibition constant (Ki) from at least three independent experiments carried
out in duplicate or triplicate. Selectivity toward corresponding subtype is the ratio of the compound’s affinity constants.
binding affinity.3,4 However, the subtype selectivity of the com-
pound was completely lost by this 4-hydroxybenzyl substitution
(Tables 1 and 2). It seems likely that the 1-methyl and 1-benzyl
Table 2
Potencies and efficacies of synthesized compounds in cAMP accumulation assay using
HEK293 cells expressing the human dopamine D1 and D2L receptors
EC50, nM (efficacy, %)
D1
Selectivity
D1/D2L
substituents, as for example in compounds 18 and 19, form a cer-
tain scaffold for a better interaction with the D1 receptor, whereas
addition of the hydroxyl to the benzyl group, as in compound 21,
fixes the compound into an active conformation, which results in
a superior binding affinity and increased potency compared to apo-
morphine (1). The largest difference in the potency and binding
affinity to D1 receptors appeared with compound 22, in which case
the addition of a basic dimethylaminobenzylic moiety decreased
the potency in activation of cAMP synthesis up to 30-fold, while
its binding affinity, on the contrary, was found to be slightly in-
creased (Tables 1 and 2). Introducing and modifying basic moieties
in position 1 might therefore be useful for developing more sub-
type selective ligands. The bicyclic 1-(4-hydroxynaphthyl) function
of compound 23 reduced potency for the D1 receptors and it also
reduced the affinity and potency for the D2-type receptors. Loss
in receptor function with no or minor loss in ligand affinity has
been related to the loss of interaction with the Ser198 in TM5 re-
gion,38,39 which would be the reason for the observed decrease in
potencies of compounds 19, 22 and 23.
D2L
1
25.7 4.8 (100 5)
21.9 6.7 (109 9)
78 17 (97 6)
117 20 (102 8)
10.5 3.7 (95 11)
741 138 (106 7)
316 44 (101 5)
4.0 0.9 (100 8)
5.1 1.2 (97 8)
49 11 (98 8)
3.4 0.9 (95 8)
2.7 0.5 (102 13)
174 33 (104 12)
76 15 (100 12)
6.5
4.3
1.6
34.7
3.9
4.3
18
19
20
21
22
23
4.2
EC50 characterizes the concentration of a compound to generate 50% of the recep-
tor-dependent change in cAMP concentration. Efficacy represents the compound’s
maximal response in comparison with the effect of apomorphine (1). Values are
represented as mean SEM from at least three independent experiments carried
out in duplicate or triplicate. Selectivity toward the D2L receptors is the ratio of the
compound’s D1 and D2L potency values.
receptor activation has been reported herein for the new com-
pounds (Table 2).
All 1-substituted apomorphines 18–23 tested had at least sub-
micromolar affinity for the three abovementioned dopamine
receptor subtypes (Table 1). The compounds 18–23 behaved as full
agonists in our model systems (Table 2). The substitutions made at
position 1 had only a slight effect on the affinities for the D2 and D3
receptors and the main changes were connected with the binding
to D1 receptors. The highest subtype selectivity was achieved with
compound 20, having a 1-hydroxymethyl function, as the selectiv-
ities for D2 and D3 over D1 receptors were more than 20- and 50-
fold, respectively. The increase in selectivity was connected with
the loss of affinity for D1 receptors, while the affinity for the D2
and D3 receptors remained comparable to the reference compound,
apomorphine (1). Small hydrophilic substituent in position 1, as in
compound 20, might be disrupting the hydrogen bonding of cate-
chol hydroxyl groups with serine residues in TM5 region that are
shown to be important for ligand binding to the D1, but affects less
the binding to the D2-type receptors.23,37 The above is supported
also by the results from functional assays—the potency for the D1
receptor decreased fivefold with practically no difference on the
potency for D2 receptors in comparison to the data of compound
1. The presence of 4-hydroxybenzyl moiety (compound 21) caused
significant (10-fold) increase in the affinity for the D1 receptor
compared to reference 1. It can be proposed that this group no sig-
nificant effect on the interactions between the TM5 serines
(Ser198, Ser199 and Ser202) of the receptor and the catechol moi-
ety, and the additional hydroxyl group generates hydrogen bonds
with neighboring amino acid residues. Besides that it has also been
proposed that the presence of hydrophobic substituents in the
proximity of position 2 of the aporphine skeleton leads increased
Generally it is difficult to draw exact conclusions from the com-
parison of binding and cellular profile data; however major trends
with the change of the substitution pattern could be properly fol-
lowed up. It was found in previous studies that, in contrast to
dopamine antagonists, for the agonist compounds, both efficacy
and affinity drive the functional effects.40 In fact, the affinity, an
intrinsic property of the molecule, can easily be determined by
binding assays, but the efficacy depends on the environment of
the receptor, expression level of receptors and coupling proteins
and the method of detection. It was confirmed that the cellular re-
sponse to a pharmacological agonists depended on the efficacy of
the agonist and the sensitivity of the system.41
3. Conclusions
In summary, a set of novel 1-substituted apomorphines was
synthesized and characterized for their activity toward human
dopamine D1, D2L and D3 receptors. Newly developed HEK293
based stable cell lines expressing the dopamine D1, D2L and D3
receptors enabled to assess binding and functional activity in the
same cell type. The substitution in position 1 of the apomorphine
backbone mainly decreased the interactions with the dopamine
D1 receptor and thus increased selectivity for the D2 and D3 recep-
tors. The most characteristic effect was noticed in case of hydroxy-
methyl group, which probably disrupts the interactions between
the catechol moiety and the Ser202 of the receptor TM5 region.
4-OH-benzyl group in the same position, on the other hand, in-

R. Reinart-Okugbeni et al. / Bioorg. Med. Chem. 21 (2013) 4143–4150
4147
creased the affinity for the D1 receptors and led to the loss of the
4.4. 5b-(Benzyloxymethyl)-6-demethoxythebaine (8)
subtype selectivity of the compounds. The basic function (e.g.,
dimethylaminobenzyl) caused significant loss in the ligand po-
tency in modulation of AC for both subtypes studied, but only a
slight influence on the affinity. In summary, the results show that
substitutions at position 1 of apomorphine remarkably may affect
the affinity for D1 receptor subtype, while the other subtypes stud-
ied were only slightly affected. The modulation in affinities and
potencies connected with 1-substituted apomorphines suggests
possibilities for future design of selective dopaminergic ligands in
the aporphine series.
Yield: 27%. Off-white solid. Mp: 99–101 °C; calculated for free
base C26H27NO3: C, 77.78; H, 6.78; N, 3.49; found: C, 77.69; H,
6.91; N, 3.52; 1H NMR (CDCl3) d = 7.21–7.42 (m, 5H, Ph), 6.69 (d,
1H, H1, J1–2 = 7.8), 6.63 (d, 1H, H2, J1–2 = 7.8), 5.91–5.74 (m, 3H,
H6, H7, H8), 4.88 (s, 2H, OCH2-Ph), 3.87 (s, 3H, OCH3), 3.88–3.61
(m, 2H, C5-CH2), 3.31 (1H, d, H10
3H, H10b, H16 , H16b), 2.33 (s, 3H, NCH3), 2.21 (ddd, 1H, H15b,
J = 5.4, >2, >2), 1.79 (ddd, 1H, H15
J = 5.4, >2, >2). 13C NMR (CDCl3)
a, J10 –10b = 16.4), 2.82–2.52 (m,
a
a
a
d = 144.6, 141.4, 138.6, 136.9, 135.4, 131.9, 128.6, 128.1, 127.0,
126.6, 126.4, 126.3, 125.1, 121.3, 114.7, 108.7, 90.4, 74.8, 73.4,
63.5, 57.1, 48.9, 47.6, 42.7, 37.2, 29.1.
4. Experimental
4.5. 5b-(4-Methoxybenzyl)-6-demethoxythebaine (9)
Fine chemicals used in the synthesis were purchased from Sig-
ma–Aldrich and UkrOrgSynthesis Ltd. and were used without any
purification. Melting points were determined with a Kofler hot-
stage apparatus and are uncorrected. Thin layer chromatography
was performed on pre-coated Merck 5554 Kieselgel 60 F254 foils.
The spots were visualized with Dragendorff’s reagent. 1H NMR
and 13C NMR spectra were recorded on a Bruker Avance 360 spec-
trometer, at 360 MHz and 90 MHz, respectively; using CDCl3 as a
solvent for morphinans and apocodeines and DMSO-d6 for apo-
morphine salts. Chemical shifts are reported in parts per million
(d) from internal TMS and coupling constants (J) are measured in
hertz. LC–MS spectra were recorded on an Agilent 1100 Series
LC/MSD instrument, including quaternary gradient pump, auto-
matic injector, column oven, DAD detector and MSD Trap XCT
ion trap mass spectrometer (equipped with electrospray ionization
source). Applied chromatographic parameters—sample size:
Yield: 30%. Pale yellow foam. Calculated for free base
C
26H27NO3: C, 77.78; H, 6.78; N, 3.49; found: C, 77.48; H, 6.83; N,
3.50; 1H NMR (CDCl3) d = 7.04 (d, 2H, Ar, J = 6.1), 6.81 (d, 2H, Ar,
J1–2 = 6.1), 6.61 (d, 1H, H1, J1–2 = 8.0), 6.59 (d, 1H, H2, J1–2 = 8.0),
5.81–5.69 (m, 3H, H6, H7, H8), 3.88 (s, 3H, OCH3), 3.81 (s, 3H,
OCH3), 3.29 (1H, d, H10
H10b, H16 , H16b, C5-CH2), 2.28 (s, 3H, NCH3), 2.14 (ddd, 1H,
H15b, J = 5.4, >2, >2), 1.87 (ddd, 1H, H15
J = 5.2, >2, >2). 13C
a, J10 –10b = 16.1), 2.84–2.37 (m, 5H,
a
a
a
NMR (CDCl3) d = 158.4, 144.0, 142.7, 136.7, 132.6, 132.4, 129.7,
129.4, 127.5, 125.4, 124.6, 121.3, 120.4, 114.9, 114.7, 113.9, 91.4,
63.8, 59.7, 56.3, 50.8, 44.1, 42.9, 35.9, 28.1.
4.6. 5b-[4-(N,N-Dimethylamino)-benzyl]-6-demethoxythebaine
(10)
100
l
L; column: Agilent TC-C18 (4.6 mm ꢁ 250 mm, 5
lm); col-
Yield: 15%. Pale brown foam. Calculated for free base
umn temperature: 30 °C; rate: 1.0 mL minꢀ1; detection wave-
length: 272 nm; mobile phase: acetonitrile (A)—0.01% aqueous
formic acid (B). Gradient elution: eluent A was 2% in 0–10 min
and increased from 2% to 16% in 10–40 min. Applied detector set-
tings—MS: ESI, inspection method: positive ion; atomization pres-
sure: 40 kPa; flow rate of dry gas: 10 L minꢀ1; temperature of dry
gas: 350 °C; spray voltage: 4 kV; multi-level scanning collision
gas: nitrogen. Optical rotation was determined with a Perkin Elmer
Model 241 polarimeter. Elemental analyses (C, H and N) were ob-
tained on a Carlo Erba EA1108 analyzer.
C
27H30N2O2: C, 78.23; H, 7.29; N, 6.76; found: C, 78.11; H, 7.41;
N, 6.89; 1H NMR (CDCl3) d = 6.70 (d, 1H, H1, J1–2 = 7.9), 6.63–6.51
(m, 5H, H2, Ar), 5.72-5.54 (m, 3H, H6, H7, H8), 3.90 (s, 3H,
OCH3), 3.28 (1H, m, H10
5H, H10b, H16 , H16b, C5-CH2), 2.31 (s, 3H, NCH3), 2.07 (ddd,
1H, H15b, J = 5.4, >2, >2), 1.76 (ddd, 1H, H15
J = 5.2, >2, >2). 13C
a), 3.12 (s, 6H, 2 ꢁ NCH3), 3.04–2.55 (m,
a
a
NMR (CDCl3) d = 148.9, 143.9, 142.6, 136.2, 134.6, 132.1, 130.0,
129.7, 127.5, 126.7, 125.0, 124.9, 124.7, 124.6, 114.3, 110.3, 91.6,
63.4, 56.1, 50.3, 48.6, 43.7, 42.4, 40.7, 35.9, 28.1.
4.7. 5b-(4-Methoxynaphthylmethyl)-6-demethoxythebaine (11)
4.1. 5b-Substitution of 6-demethoxythebaine (5)
Yield: 17%. Pale brown solid. Mp: 123–126 °C, calculated for
free base C30H29NO3: C, 79.80; H, 6.47; N, 3.10; found: C, 79.59;
H, 6.58; N, 3.20; 1H NMR (CDCl3) d = 8.06–7.98 (m, 2H, Ar), 7.56–
7.43 (m, 2H, Ar), 6.78–6.49 (m, 4H, H1, H2, Ar), 5.68–5.45 (m, 3H,
H6, H7, H8), 3.94 (s, 3H, OCH3), 3.81 (s, 3H, OCH3), 3.29–3.12
To a stirred solution of 1 g (3 mmol) of 6-demethoxythebaine
(5)19 in 80 mL of dry THF, cooled to ꢀ78 °C, was added 1.7 M
tert-BuLi (in pentane, 1.2 equiv). The mixture was stirred for
30 min before the corresponding chloride (1.2 equiv) was added.
The solution was stirred at ꢀ78 °C for 1 h before it was allowed
to come to room temperature over 2 h and stirred at ambient tem-
perature for further 2 h. The workup was performed in line with
Gates’ method,20 products were separated by means of column
(3H, m, H10
2.37 (s, 3H, NCH3), 2.04 (ddd, 1H, H15b, J = 5.6, >2, >2), 1.80 (ddd,
1H, H15
J = 5.6, >2, >2). 13C NMR (CDCl3) d = 156.2, 145.0, 143.3,
a, C5-CH2), 2.88–2.39 (m, 3H, H10b, H16a, H16b),
a
136.7, 135.5, 134.6, 133.5, 133.0, 128.9, 127.5, 126.8, 125.5,
124.9, 124.7, 124.0, 123.9, 121.1, 114.3, 112.2, 104.9, 92.2, 63.3,
56.9, 55.9, 52.6, 48.7, 42.3, 40.9, 35.3, 28.2.
chromatography
(dichloromethane:methanol:concentrated
ammonium hydroxide 90:9:1).
4.8. Acid-catalyzed rearrangement of 5b-substituted-6-
demethoxythebaines (6–11) into 1-substituted-apocodeines
(12–17)
4.2. 5b-Methyl-6-demethoxythebaine (6)
Yield: 30%. The physical and spectral characteristics of the prod-
uct were in accordance with previously published data.15
A mixture of the diene (250 mg) and 99.5% methanesulfonic
acid ( mL) was stirred for min at 0 °C and then for 30 min at 90–
95 °C (the progress of the reaction was followed by TLC). After
completion the reaction mixture was added dropwise, with stirring
and external ice cooling, to a solution of potassium hydrogen car-
bonate (2 g) in water (12.5 mL). After extraction with chloroform
4.3. 5b-Benzyl-6-demethoxythebaine (7)
Yield: 27%. The physical and spectral characteristics of the prod-
uct were in accordance with previously published data.16

4148
R. Reinart-Okugbeni et al. / Bioorg. Med. Chem. 21 (2013) 4143–4150
(3 ꢁ 15 mL), the combined organic extracts were washed with sat-
urated brine, dried (MgSO4) and concentrated under vacuum to
yield crude apocodeine. Purification was realized by means of col-
umn chromatography (dichloromethane:methanol 8:2).
found: C, 79.61; H, 6.55; N, 3.23; 1H NMR (CDCl3) d = 8.28–8.20
(m, 2H, Ar), 7.65–7.59 (m, 2H, Ar), 7.09 (d, 1H, H3, J2–3 = 7.6),
7.04 (d, 1H, H2, J2–3 = 7.6), 6.90–6.78 (m, 4H, H8, H9, Ar), 4.21 (s,
2H, C6-CH2), 3.90–3.87 (m, 4H, H6
a
, OCH3), 3.82 (s, 3H, OCH3),
, H7b), 2.36 (s, 3H,
3.13–2.39 (6H, m, H4 , H4b, H5 , H5b, H7a
a
a
4.9. 1-Methylapocodeine (12)
NCH3). 13C NMR (CDCl3) d = 155.1, 147.3, 143.9, 142.7, 141.6,
137.8, 135.5, 131.7, 130.9, 129.3, 128.4, 127.5, 126.8, 126.7,
126.5, 125.6, 124.3, 123.9, 123.5, 120.3, 111.3, 108.9, 65.8, 56.9,
56.7, 54.6, 45.7, 43.7, 30.2, 23.1.
Yield: 77%. Off-white solid. ½a D25
ꢀ 76 (c 0.10, dichloromethane);
ꢂ
mp: 123 °C (dec.); calculated for free base C19H21NO2: C, 77.27; H,
7.17; N, 4.74; found: C, 77.01; H, 7.28; N, 4.80; 1H NMR (CDCl3)
d = 7.09 (dd, 2H, H2, H3, J2–3 = 7.4), 6.78 (d, 1H, H8, J8–9 = 7.6),
4.15. O-Deprotection of aporphines 12–17
6.70 (d, 1H, H9, J8–9 = 7.6), 3.94–3.92 (m, 1H, H6
a), 3.88 (s, 3H,
OCH3), 3.02–2.44 (6H, m, H4
a
, H4b, H5
a
, H5b, H7
a
, H7b), 2.40 (s,
A suspension of the apocodeine (0.5 mmol) in 10 mL of abs
CH2Cl2 was cooled to ꢀ10 °C and treated with cooled solution of
BCl3 (1 mL, 1 M in CH2Cl2) by stirring for 6 h under argon atmo-
sphere. The reaction mixture was quenched with 2 mL of metha-
nol. After evaporation of the solvent, the residue was treated
with aqueous NaHCO3 and extracted with CH2Cl2 (3 ꢁ 15 mL).
The extract was dried over Na2SO4, filtered and treated with meth-
anol saturated with HCl and then with ether to give rise to stable
HCl salt.
3H, NCH3), 2.29 (s, 3H, C1-CH3). 13C NMR (CDCl3) d = 148.1,
144.3, 138.4, 135.4, 133.2, 130.0, 128.5, 127.9, 127.1, 126.7,
121.6, 119.4, 66.7, 57.1, 54.9, 43.2, 30.5, 27.1, 23.9.
4.10. 1-Benzylapocodeine (13)
Yield: 69%. The physical and spectral characteristics of the prod-
uct were in accordance with previously published data.16
4.11. 1-(Benzyloxymethyl)apocodeine (14)
4.16. 1-MethylapomorphineꢃHCl (18)
Yield: 71%. Off-white foam. ½a D25
ꢀ 80 (c 0.10, dichloromethane);
ꢂ
Yield: 66%. Off-white solid. Mp: 149–150 °C; calculated for salt
Calculated for free base C26H27NO3: C, 77.78; H, 6.78; N, 3.49; found:
C, 77.61; H, 6.89; N, 3.50; 1H NMR (CDCl3) d = 7.39–7.29 (m, 5H, Ar),
7.24 (d, 1H, H2, J2–3 = 6.8), 7.21 (d, 1H, H3, J2–3 = 6.8), 6.89 (d, 1H, H8,
J8–9 = 7.4), 6.81 (d, 1H, H9, J8–9=7.3), 4.87 (s, 2H, C5-CH2), 4.33 (s, 2H,
C
18H20ClNO2: C, 68.03; H, 6.34; N, 4.41; found: C, 68.17; H, 6.48; N,
4.50; HPLC–MS (m/z) for free base: 282.2 [M+1+] base peak. HPLC
purity: >97.4% (rt 14.14 min). 1H NMR (DMSO-d6) d = 10.91 (br s,
1H, NH+), 9.73 (br s, 2H, OH), 7.10 (d, 1H, H2, J2–3 = 7.9), 7.12 (d,
1H, H3, J2–3 = 7.9), 6.76 (d, 1H, H9, J8–9 = 7.4), 6.72 (d, 1H, H8, J8–
O-CH2-Ph), 3.99–3.97 (m, 1H, H6
a
a
), 3.83 (s, 3H, OCH3), 2.84–2.31
(9H, m, H4 , H4b, H5 , H5b, H7
a
a
, H7b, NCH3). 13C NMR (CDCl3)
9 = 7.4), 3.98–3.96 (m, 1H, H6
a), 2.89–2.52 (9H, m, H4a, H4b,
H5 , H5b, H7
a
a
, H7b, NCH3), 2.49 (s, 3H, C1-CH3). 13C NMR
d = 147.8, 143.9, 136.4, 136.2, 133.1, 131.3, 130.7, 130.4, 129.4,
128.6, 128.4, 128.3, 127.8, 126.1, 124.9, 124.7, 120.8, 111.6, 73.9,
72.7, 65.7, 56.6, 56.4, 43.7, 30.1, 23.7.
(DMSO-d6) d = 144.7, 141.8, 132.1, 131.5, 128.7, 127.8, 127.4,
126.7, 124.9, 124.5, 120.9, 113.4, 66.3, 54.3, 44.5, 29.1, 26.1, 22.8.
4.12. 1-(4-Methoxybenzyl)apocodeine (15)
4.17. 1-BenzylapomorphineꢃHCl (19)
Yield: 67%. Pale yellow solid. ½a D25
ꢀ 69 (c 0.10, dichlorometh-
ꢂ
Yield: 70%. Pale grey solid. Mp: 137–139 °C; calculated for salt
ane); Mp: 114 °C (dec.); calculated for free base C26H27NO3: C,
77.68; H, 6.78; N, 3.49; found: C, 77.80; H, 6.66; N, 3.41; 1H NMR
(CDCl3) d = 7.07 (d, 1H, H2, J2–3 = 7.2), 7.02 (d, 1H, H3, J2–3 = 7.2),
7.00–6.85 (m, 4H, Ar), 6.78 (d, 1H, H8, J8–9 = 7.8), 6.74 (d, 1H, H9,
C
24H24ClNO2: C, 73.18; H, 6.14; N, 3.56; found: C, 73.32; H, 6.31;
N, 3.43; HPLC–MS (m/z) for free base: 358.2 [M+1+] base peak.
HPLC purity: >96.3% (rt 12.23 min). 1H NMR (DMSO-d6) d = 11.13
(br s, 1H, NH+), 9.66 (br s, 2H, OH), 7.28–7.21 (m, 5H, Ar), 7.08
(dd, 2H, H2, H3, J2–3 = 7.8), 6.82 (d, 1H, H9, J8–9 = 7.6), 6.77 (d, 1H,
J8–9 = 7.8), 3.99 (s, 2H, C6-CH2), 3.95–3.92 (m, 1H, H6
3H, OCH3), 3.81(s, 3H, OCH3), 2.91–2.44 (6H, m, H4 , H4b, H5
H5b, H7
, H7b), 2.38 (s, 3H, NCH3). 13C NMR (CDCl3) d = 159.8,
a), 3.87 (s,
a
a,
H8, J8–9 = 7.6), 3.95–3.92 (m, 1H, H6
a
), 3.91 (s, 2H, C5-CH2),
2.86–2.44 (9H, m, H4 , H4b, H5 , H5b, H7a
a
a
, H7b, NCH3). 13C
a
148.1, 144.9, 143.7, 141.5, 137.6, 135.5, 134.6, 132.7, 130.5,
128.5, 128.3, 126.9, 126.4, 122.4, 117.8, 112.4, 112.3, 64.9, 57.3,
53.8, 43.4, 39.9, 36.7, 30.5, 23.9.
NMR (DMSO-d6) d = 145.0, 144.0, 142.8, 137.1, 135.1, 131.1,
130.7, 129.4, 129.2, 128.3, 128.1, 127.5, 126.7, 126.4, 126.3,
124.5, 121.2, 114.0, 65.4, 57.1, 43.5, 38.9, 29.7, 23.4.
4.13. 1-[4-(N,N-Dimethylamino)-benzyl]-apocodeine (16)
4.18. 1-(Hydroxymethyl)-apomorphineꢃHCl (20)
Yield: 70%. Pale yellow foam. [½a D25
ꢀ 73 (c 0.10, dichlorometh-
ꢂ
Yield: 36%. Off-white solid. Mp: 121–123 °C; calculated for salt
ane); Calculated for free base C27H30N2O2: C, 78.23; H, 7.29; N,
6.76; found: C, 78.12; H, 7.40; N, 6.89; 1H NMR (CDCl3) d = 7.10–
7.01 (m, 6H, H2, H3, Ar), 6.78 (d, 1H, H8, J8–9 = 8.0), 6.69 (d, 1H,
H9, J8–9 = 8.0), 3.99 (s, 2H, C6-CH2), 3.90 (s, 3H, OCH3), 3.89–3.83
C
18H20ClNO3: C, 64.77; H, 6.04; N, 4.20; found: C, 64.61; H, 6.27; N,
4.29; HPLC–MS (m/z) for free base: 298.1 [M+1+] base peak. HPLC
purity: >96.0% (rt 12.20 min). 1H NMR (DMSO-d6) d = 11.31 (br s,
1H, NH+), 9.88 (br s, 2H, OH), 7.21 (d, 1H, H2, J2–3 = 8.0), 7.12 (d,
1H, H3, J2–3 = 8.0), 6.82 (d, 1H, H9, J8–9 = 7.7), 6.80 (d, 1H, H8, J8–
(m, 1H, H6
(6H, m, H4
a
a
), 3.03 (s, 3H, N-CH3), 3.00 (s, 3H, N-CH3), 2.84–2.44
, H4b, H5 , H5b, H7
a
a
, H7b), 2.36 (s, 3H, NCH3). 13C
9 = 7.7), 4.51 (s, 2H, C5-CH2), 4.08–4.05 (m, 1H, H6
a), 3.12–2.69
(9H, m, H4 , H4b, H5 , H5b, H7
a
a
a
, H7b, NCH3). 13C NMR (DMSO-
NMR (CDCl3) d = 149.8, 147.8, 144.4, 144.3, 142.1, 141.3, 137.5,
135.7, 133.2, 130.9, 129.9, 129.6, 128.9, 125.7, 120.5, 113.6,
113.4, 111.7, 65.3, 56.9, 56.8, 44.2, 41.4, 37.8, 33.2, 29.4, 23.7.
d6) d = 144.3, 142.9, 137.4, 135.1, 131.5, 128.7, 127.8, 127.4,
124.9, 124.5, 121.6, 115.0, 66.1, 55.8, 44.1, 29.7, 24.7.
4.14. 1-(4-Methoxynaphthylmethyl)-apocodeine (17)
4.19. 1-(4-Hydroxybenzyl)-apomorphineꢃHCl (21)
Yield: 66%. Yellow foam. ½a D25
ꢀ 87 (c 0.10, dichloromethane);
ꢂ
Yield: 41%. Pale green solid. Mp: 133–135 °C; calculated for salt
24H24ClNO3: C, 70.32; H, 5.90; N, 3.42; found: C, 70.44; H, 6.01; N,
Calculated for free base C30H29NO3: C, 79.80; H, 6.47; N, 3.10;
C

R. Reinart-Okugbeni et al. / Bioorg. Med. Chem. 21 (2013) 4143–4150
4149
3.37; HPLC–MS (m/z) for free base: 374.2 [M+1+] base peak. HPLC
purity: >95.4% (rt 14.23 min). 1H NMR (DMSO-d6) d = 10.98 (br s,
1H, NH+), 9.47 (br s, 3H, OH), 7.14–7.04 (m, 4H, H2, H3, Ar), 6.75
(d, 1H, H9, J8–9 = 8.0), 6.72 (d, 1H, H8, J8–9 = 8.0), 6.59 (d, 2H, Ar,
[3H]raclopride were from PerkinElmer, MA, USA, as well as all
the equipment and materials used for counting radioactivity.
6. Generation of stably transfected HEK293 cells expressing
dopamine D1, D2L and D3 receptors
J = 7.2), 4.21–4.18 (m, 1H, H6
a
), 3.91 (s, 2H, C5-CH2), 3.19–2.44
(9H, m, H4 , H4b, H5 , H5b, H7a
a
a
, H7b, NCH3). 13C NMR (DMSO-
d6) d = 153.6, 144.4, 142.8, 142.4, 137.2, 135.7, 134.2, 131.9,
130.7, 129.3, 128.1, 127.5, 126.7, 126.4, 125.8, 124.5, 122.3,
114.7, 65.7, 56.8, 43.2, 39.7, 29.1, 23.7.
HEK293 cells were cultured on Petri dishes in DMEM supple-
mented with 10% FBS, 100 lg/mL streptomycin, 100 U/mL penicil-
lin and maintained at 37 °C and 5% CO2 in a humidified incubator.
For transfection cells were seeded on 6-well plates, cultured 24 h
4.20. 1-[4-(N,N-Dimethylamino)-benzyl]-apomorphineꢃ2HCl
to reach ꢄ90% confluence and transfected with 4
lg of DNA
(22)
(pcDNA3.1.(+) with a gene encoding for either hD1, hD2L or hD3
receptors) per well using Lipofectamine™ 2000 according to man-
ufacturer’s instructions. To obtain stable lines the cells were main-
Yield: 54%. Pale green solid. Mp: 148 °C (dec); calculated for salt
C
26H30Cl2N2O2: C, 65.96; H, 6.36; N, 5.92; found: C, 66.09; H, 6.43;
tained and passed for 2 weeks in the presence of 800 lg/mL G418.
N, 5.81; HPLC–MS (m/z) for free base: 401.2 [M+1+] base peak. c 1H
NMR (DMSO-d6) d = 10.40 (br s, 1H, NH+), 10.01 (br s, 1H, NH+),
9.25 (br s, 2H, OH), 7.80–7.62 (m, 4H, Ar), 7.10 (d, 1H, H3, J2–
3 = 7.7), 7.13 (d, 1H, H2, J2–3 = 7.7), 6.85 (d, 1H, H9, J8–9 = 8.0),
Four to six G418 resistant colonies per each receptor subtype were
selected and after five further passages the clonal cultures were
tested for receptor expression by radioligand binding. Henceforth
the cells were maintained in the presence of 400 lg/mL of G418.
6.80 (d, 1H, H8, J8–9 = 8.0), 4.19–4.09 (m, 1H, H6
C5-CH2), 3.22 (s, 6H, 2 ꢁ NCH3), 3.21–2.36 (9H, m, H4
H5 , H5b, H7
, H7b, NCH3). 13C NMR (DMSO-d6) d = 150.6, 144.9,
a
), 3.87 (s, 2H,
The clones with sufficient receptor densities were further tested
for their binding of known dopaminergic ligands to confirm their
identity (data not shown) and then used in experiments with the
novel test compounds.
a, H4b,
a
a
142.9, 142.6, 142.5, 137.6, 135.1, 131.9, 129.3, 128.3, 128.2,
126.7, 126.4, 125.8, 124.5, 121.6, 121.3, 114.9, 66.0, 57.4, 47.9,
43.1, 37.8, 29.7, 23.6.
7. Membrane preparation
4.21. 1-(4-Hydroxynaphthylmethyl)-apomorphineꢃHCl (23)
Yield: 51%. Grey solid. Mp: 135 °C (dec.); Calculated for salt
All ligand binding experiments were performed on membrane
suspensions, prepared as follows. The cells were centrifuged at
800g at room temperature and the pellet stored at ꢀ80 °C. The fro-
zen pellets were melted on ice and washed by homogenization
with a tissue homogenizer (Coleparmer Labgen 125) for 30 s in
ice-cold DPBS and centrifugation at 800g for 5 min at 4 °C. The pel-
let was re-homogenized in 50 mM Tris–HCl buffer, (pH 7.4) and
centrifuged at 30,000g for 20 min followed by a second resuspen-
sion and homogenization step. The latter homogenization and cen-
trifugation steps were repeated once more and the final pellet was
homogenized in incubation buffer (IB: 50 mM Tris–HCl, 120 mM
NaCl, 5 mM KCl, 5 mM MgCl2, 1 mM EDTA, pH 7.4) typically at a
concentration of 1 ꢁ 107 cells/mL (corresponding to ꢄ0.5 mg pro-
tein/mL quantified using Bio-Rad protein assay Kit (Bio-Rad Labo-
ratories) with BSA as a standard. The membrane preparations were
stored at ꢀ80 °C until further testing.
C
28H26ClNO3: C, 73.11; H, 5.70; N, 3.05; found: C, 73.31; H, 5.83;
N, 3.20; HPLC–MS (m/z) for free base: 424.2 [M+1+] base peak.
HPLC purity: >96.8% (rt 13.29 min). 1H NMR (DMSO-d6) d = 10.32
(br s, 1H, NH+), 9.64 (br s, 2H, 2 ꢁ OH), 8.20–8.15 (m, 2H, Ar),
7.51–7.47 (m, 2H, Ar), 7.06 (d, 1H, H3, J2–3 = 8.0), 7.01 (d, 1H, H2,
J2–3 = 8.0), 6.80–6.61 (m, 4H, H8, H9, Ar), 4.67 (s, 2H, C6-CH2),
4.14–4.06 (m, 1H, H6
a), 3.23–2.56 (9H, m, H4a, H4b, H5a, H5b,
H7a
, H7b, NCH3). 13C NMR (DMSO-d6) d = 151.4, 149.7, 142.9,
142.0, 141.3, 137.1, 135.0, 132.2, 131.4, 129.8, 128.5, 127.2,
126.4, 126.7, 125.7, 124.4, 124.0, 123.5, 123.0, 122.0, 113.8,
113.7, 65.3, 56.8, 44.9, 43.5, 30.1, 23.4.
5. Detailed description of pharmacological methods
The human embryonic kidney cell line (HEK293) used as a host
for dopamine receptors in pharmacological assays was obtained
from American Type Culture Collection, Boras, Sweden. The
pcDNA3.1+ expression vectors (Invitrogen) containing the desired
gene of human wild type dopamine receptor (hD1, hD2L and hD3)
was purchased from the Missouri S&T cDNA Resource Center
(http://www.cdna.org). Lipofectamine™ 2000, fetal bovine serum
(FBS, GibcoÒ) were from Invitrogen Life Technologies, Paisley, UK.
Dulbecco’s Modified Eagle’s Medium (DMEM), Dulbecco’s Phos-
phate Buffered Saline (DPBS), geneticin (G418) and penicillin/
streptomycin were from PAA Laboratories GmbH, Pasching, Aus-
tria. Cell culture plates (Nunc™) were from Thermo Fisher Scien-
tific, Roskilde, Denmark. Dopamine, (+)-butaclamol and 3-
isobutyl-1-methylxanthine (IBMX) were from Sigma–Aldrich Co.,
Helsinki, Finland. Tris(hydroxymethyl)aminomethane (Tris),
MgCl2, KOH, NaCl, KCl, dithiotreitol (DTT), potassium phosphate
were from AppliChem GmbH, Darmstadt, Germany. Perchloric acid
was from Reakhim, Moscow, Russia and 2,20,200,2000-(ethane-1,2-
diyldinitrilo)tetraacetic acid (EDTA) from Merck, Munich, Ger-
many. Cyclic adenosine monophosphate (cAMP) was from Tocris
(http://www.tocris.com) and tritium labeled cAMP from BIO-
TREND Chemikalien GmbH, Köln, Germany. [3H]SCH23390 and
8. Radioligand competition experiments with 1-substituted
apomorphines
Receptor expression and affinity of radioligands was deter-
mined as described earlier39,42 with some modifications. Reactions
were carried out in U-bottom 96-well plates in a final volume of
250
150
l
l
L in IB and incubated at 25 °C for 90 min. In all assays
L of membrane suspension was added to wells containing
50 lL of radioligand and 50 lL of competing unlabeled ligand (or
buffer) at their 5ꢁ final concentration. Receptor-positive clones
were identified by one-point assay at a saturating concentration
of [3H]SCH23390 or [3H]raclopride with or without an unlabeled
dopaminergic antagonist, 1 lM (+)-butaclamol (data not shown).
In saturation binding experiments the concentration of radioli-
gands was varied from 0.2 to 10 nM and non-specific binding
was determined in the presence of 1 lM (+)-butaclamol. For 8-
point competition assay the concentration of radioligand was fixed
to near Kd value (0.9 0.2 nM) and concentration of the test com-
pounds was varied from 0.1 nM to 100 lM. Reactions were initi-
ated by addition of membrane suspension (app 5 ꢁ 104–
5 ꢁ 105 cells/assay for different receptor subtypes).

4150
R. Reinart-Okugbeni et al. / Bioorg. Med. Chem. 21 (2013) 4143–4150
4. Booth, R. G.; Neumeyer, J. L.; Baldessarini, R. J. Therapeutic and diagnostic
All the reactions were stopped by fast filtration through thick
agents for the Parkinson’s disease Burger’s Medicinal Chemistry and Drug
Discovery, 7th edition; John Wiley and Sons, Inc., 2012.
5. Cho, D. I.; Zheng, M.; Kim, K.-M. Arch. Pharm. Res. 2010, 33, 1521.
6. Zhang, A.; Zhang, Y.; Branfman, A. R.; Baldessarini, R. J.; Neumeyer, J. L. J. Med.
Chem. 2007, 50, 171.
7. Søndergaard, K.; Kristensen, J. L.; Palner, M.; Gillings, N.; Knudsen, G. M.; Roth,
B. L.; Begtrup, M. Org. Biomol. Chem. 2005, 3, 4077.
8. Sipos, A.; Kiss, B.; Schmidt, E.; Greiner, I.; Berényi, S. Bioorg. Med. Chem. 2008,
16, 3773.
9. Herm, L.; Berényi, S.; Vonk, A.; Rinken, A.; Sipos, A. Bioorg. Med. Chem. 2009, 17,
4756.
10. Liu, Z.; Chen, X.; Yu, L.; Zhen, X.; Zhang, A. Bioorg. Med. Chem. 2008, 16,
6675.
11. Sipos, A.; Mueller, F. K. U.; Lehmann, J.; Berényi, S.; Antus, S. Arch. Pharm. 2009,
342, 557.
12. Sipos, A.; Tóth, M.; Mueller, F.; Lehmann, J.; Berényi, S. Monatsh. Chem. 2009,
140, 473.
GF/B glass fiber filters (Whatham) using a FilterMate Harvester
(Model D961962, Perkin Elmer). After five washes with ice-cold
phosphate buffer (20 mM K-phosphate, 100 mM NaCl, pH 7.4), fil-
ter mats were dried in a microwave oven and impregnated with a
MeltiLex™ B/HS scintillant using a MeltiLexÒ Heatsealer. Filter-
bound radioactivity was counted using a PerkinElmer/Wallac
MicroBeta TriLux 1450 LSC Luminescence Counter.
All pharmacological data were analyzed by means of non-linear
least squares regression analysis using the commercial program
GRAPHPAD PRISM™ 4.03 (GraphPad Software Inc., CA). Data were
fit to one-site binding curve and inhibition constant values (Ki) cal-
culated according to the Cheng–Prusoff equation.43
13. Kalani, M. Y.; Vaidehi, N.; Hall, S. E.; Trabanino, R. J.; Freddolino, P. L.; Kalani, M.
A.; Floriano, W. B.; Kam, V. W.; Goddard, W. A. Proc. Natl. Acad. Sci. U.S.A. 2004,
101, 3815.
9. cAMP accumulation assay
14. Neumeyer, J. L. Synthesis and structure–activity relationships of aporphines as
dopamine receptor agonists and antagonists The Chemistry and Biology of
Isoquinoline Alkaloids; Spinger-Verlag, 1985. pp. 146–170.
15. Woudenberg, R. H.; Piet, D. P.; Sinnema, A.; Lie, T. S.; Maat, L. Rec. Trav. Chim.
Pays-Bas 1991, 110, 405.
16. Sipos, A.; Berényi, S. Tetrahedron 2008, 64, 5851.
17. Berényi, S.; Makleit, S.; Hosztafi, S.; Szeifert, I. Acta Chim. Acad. Sci. Hung. 1982,
110, 363.
Two to three days before the assay cells were seeded on 96-well
cell culture plates at a density of 2–5 ꢁ 104 cells/well in 100
lL of
DMEM without G418. On the day of assay DMEM was replaced by
50
50
l
l
L DPBS w/o Ca2+ and Mg2+ prewarmed to 37 °C. Thereafter
L of increasing concentrations of 2ꢁ ligand solutions in DPBS
with 2 mM MgCl2 and 200 lM IBMX, also prewarmed to 37 °C,
18. Berényi, S.; Csutorás, Cs.; Sipos, A. Curr. Med. Chem. 2009, 16, 3215.
19. Boden, R. M.; Gates, M.; Ho, S. P.; Sundararaman, P. J. Org. Chem. 1982, 47, 1347.
20. Gates, M.; Boden, R. M.; Sundararaman, P. J. Org. Chem. 1989, 54, 972.
21. Grancelli, F. E.; Filer, C. N.; Soloway, A. H.; Neumeyer, J. L. J. Org. Chem. 1980, 45,
2275.
22. Berényi, S.; Czirják, M.; Makleit, S. J. Chem. Soc., Perkin Trans. 1 1993, 2137.
23. Reinart, R.; Gyulai, Zs.; Berényi, S.; Antus, S.; Vonk, A.; Rinken, A.; Sipos, A. Eur.
J. Med. Chem. 2011, 46, 2992.
were added to cells and incubated for 15 min at 37 °C. In case of
the D2L and D3 receptors, the added mixture contained also For-
skolin at a final concentration of 24
Reaction was stopped by addition of 50
chloric acid and incubation on ice for 1 h. The mixture in wells was
neutralized by addition of 50 L of 1.2 M KOH and the plates were
lM.
lL of ice cold 1.2 M per-
l
24. Williams, D. R.; Brown, D. L.; Benbow, J. W. J. Am. Chem. Soc. 1923, 1989, 111.
25. Thomas, P.; Smart, T. G. J. Pharmacol. Toxicol. Meth. 2005, 51, 187.
26. Cao, Y.; Sun, W.-C.; Jin, L.; Xie, K.-Q.; Zhu, X.-Z. Eur. J. Pharmacol. 2006, 548, 29.
27. Häidkind, R.; Eller, M.; Harro, M.; Kask, A.; Rinken, A.; Oreland, L.; Harro, J. Eur.
Neuropsychopharmacol. 2003, 13, 19.
28. Hidaka, K.; Tada, S.; Matsumoto, M.; Ohmori, J.; Tasaki, Y.; Nomura, T.; Usuda,
S.; Yamaguchi, T. Br. J. Pharmacol. 1996, 117, 1625.
29. Freedman, S. B.; Patel, S.; Marwood, R.; Emms, F.; Seabrook, G. R.; Knowles, M.
R.; McAllister, G. J. Pharmacol. Exp. Ther. 1994, 268, 417.
30. Sipos, A.; Csutorás, C.; Berényi, S.; Uustare, A.; Rinken, A. Bioorg. Med. Chem.
2008, 16, 4563.
then stored at ꢀ20 °C until the cAMP assay.
cAMP concentration was assayed as described earlier by Vonk
et al.44 with modifications. In brief, the samples were thawed
and diluted 2- to 10-fold, depending on the amount of cAMP in
samples, in Brown’s Buffer containing 50 mM Tris–HCl (pH 7.4 at
4 °C), 8 mM theophyllin, 10 mM EDTA, 100 mM NaCl and 6 mM
DTT. To a 50
lL of diluted sample a 50
l
L of 30 nM tritium labeled
cAMP (specific activity 25 Ci/mmol) and 50
lL of cAMP-binding
protein (stock solution diluted 10-fold), prepared as described by
Brown et al.,45 were added and incubated at 2–8 °C for 1 h. After
incubation the samples were filtrated and further treated in es-
sence as described above under radioligand binding experiments.
31. McAllister, G.; Knowles, M. R.; Ward-Booth, S. M.; Sinclair, H. A.; Patel, S.;
Marwood, R.; Emms, F.; Patel, S.; Smith, A.; Seabrook, G. R.; Freedman, S. B. J.
Recept. Signal. Transd. 1995, 15, 267.
32. Milligan, G. Trends Pharmacol. Sci. 2003, 24, 87.
33. Rinken, A.; Terasmaa, A.; Raidaru, G.; Fuxe, K. Neurosci. Lett. 2001, 302, 5.
34. Vanhauwe, J. F.; Fraeyman, N.; Francken, B. J.; Luyten, W. H.; Leysen, J. E. J.
Pharmacol. Exp. Ther. 1999, 290, 908.
35. Skinbjerg, M.; Namkung, Y.; Halldin, C.; Innis, R. B.; Sibley, D. R. Synapse (New
York, N.Y.) 2009, 63, 462.
Acknowledgments
The authors are grateful to the National Science Foundation
(Grant OTKA Reg. No. K81701), Estonian Science Foundation
(8314, 8011), Estonian Ministry of Education and Science
(SF0180032s12) and the European Regional Development Fund
(TK114, 30020) for the financial supports.
36. Zaworski, P. G.; Alberts, G. L.; Pregenzer, J. F.; Im, W. B.; Slightom, J. L.; Gill, G. S.
Br. J. Pharmacol. 1999, 128, 1181.
37. Chemel, B. R.; Bonner, L. A.; Watts, V. J.; Nichols, D. E. Mol. Pharmacol. 2012, 81,
729.
38. Hwa, J.; Perez, D. M. J. Biol. Chem. 1996, 271, 6322.
39. Harro, J.; Terasmaa, A.; Eller, M.; Rinken, A. Brain Res. 2003, 976, 209.
40. Fouchaq, B.; Wang, S. R.; Piwinca, D.; Coussy, L.; Jolas, T.; Néliat, G. Comparison
of Binding and Cellular profiles of Multi-target G Protein-Coupled Receptors.
In: 12th Society for Biomolecular Sciences Annual Conference and Exhibition—
Seattle, WA (USA), 17–21/09/2006.
References and notes
1. Bozzi, Y.; Borelli, E. Trends Neurosci. 2006, 29, 167.
2. Marsden, C. A. Br. J. Pharmacol. 2006, 147, S136.
3. (a) Zhang, A.; Neumeyer, J. L.; Baldessarini, R. J. Chem. Rev. 2007, 107, 274; (b)
Finnema, S. F.; Bang-Andersen, B.; Wikstrom, H. V.; Halldin, C. Curr. Top. Med.
Chem. 2010, 10, 1477.
41. Kenakin, T. Nat. Rev. Drug Disc. 2003, 2, 429.
42. Uustare, A.; Reinart, R.; Rinken, A. Neurosci. Lett. 2006, 406, 169.
43. Cheng, Y.-C.; Prusoff, W. H.; Yung-Chi, C. Biochem. Pharmacol. 1973, 22, 3099.
44. Vonk, A.; Reinart, R.; Rinken, A. J. Pharm. Sci. 2008, 108, 63.
45. Brown, D. L.; Albano, J. D.; Ekins, R. P.; Sgherzi, A. M. Biochem. J. 1971, 121, 561.