Synthesis and pharmacological evaluation of thiazole and isothiazole derived apomorphines

Article abstract of DOI:10.1002/ardp.200900100

We have presented the synthesis of novel thiazolo- and isothiazolo- apomorphines 12-17 resulting - in part - from an unexpected isomerization step occurred during the acid-catalyzed rearrangement of precursor thiazolo-morphinandienes 3-5. These 2,3-disubs

Full text of DOI:10.1002/ardp.200900100

Arch. Pharm. Chem. Life Sci. 2009, 342, 557–568
A. Sipos et al.
557
Full Paper
Synthesis and Pharmacological Evaluation of Thiazole and
Isothiazole Derived Apomorphines
Attila Sipos1, Franziska K. U. Mueller2, Jochen Lehmann2, Sꢀndor Berꢁnyi1, and Sꢀndor Antus1, 3
1 Department of Organic Chemistry, University of Debrecen, Debrecen, Hungary
2 Institut fꢀr Pharmazie, Lehrstuhl fꢀr Pharmazeutische / Medizinische Chemie, Friedrich-Schiller-Universitꢁt
Jena, Jena, Germany
3 Research Group for Carbohydrates of the Hungarian Academy of Sciences, Debrecen, Hungary
We have presented the synthesis of novel thiazolo- and isothiazolo-apomorphines 12–17 result-
ing–in part–from an unexpected isomerization step occurred during the acid-catalyzed rear-
rangement of precursor thiazolo-morphinandienes 3–5. These 2,3-disubstituted apomorphines
represent a new group of A-ring substituted aporphines. The receptor binding studies revealed
that with the exception of two derivatives all the tested compounds have limited affinity for
dopamine-receptor subtypes. Functional calcium assay for the most active isothiazolo-apomor-
phine showed higher affinities for D1 and D2L subtypes. The docking of these ligands has been
modelled to human D2 and D3 receptors. On the basis of the predicted models, we identified an
important cation-p interaction for the binding of isothiazolo-apomorphine 16.
Keywords: Benzothiazole-benzisothiazole isomerization / Docking models / Dopamine receptor activity / Thiazole moi-
ety /
Received: May 5, 2009; accepted: June 15, 2009
DOI 10.1002/ardp.200900100
been summarized in a recent review [6]. It was shown
that the absolute configuration of C-6a is a critical factor
for the dopaminergic properties, since R-(–)-enantiomers
are known to be dopamine (DA) agonist and S-(+)-counter-
parts have antagonist features. Moreover, it was also
reported that C2- and C3-substituents on the A-ring have
an important role in the DA-receptor binding ability.
This conclusion was based on the results revealing the
existence of a lipophilic cleft on DA receptors. Sønder-
gaard et al. [7] reported results which revealed that a com-
pound having large substituents such as a 4-hydroxy-
phenyl group in the 2-position had also high affinity for
the D2 receptor subtype. Our concept for the development
of selective and potent D2 receptor agonists was based on
the fact that the 2-aminothiazole functionality was
shown to be a heterocyclic bioisostere of the phenol moi-
ety in case of the dopamine agonist pramipexole [8] and
an aminothiazole-containing catechol aporphine [9]
(Fig. 1).
Introduction
Thiazole ring systems are widely used structural ele-
ments in medicinal chemistry. This structure, especially
in case of the 2-amino substitution, has been applied in
the development of drugs for the treatment of allergies,
hypertension, inflammation, schizophrenia, and bacte-
rial and HIV infections [1]. It is known to be a ligand of
estrogen receptors [2] as well as of a novel class of adeno-
sine receptors [3]. Gꢀrlitzer et al. prepared aminothiazole-
fused morphinans in order to test the effect of this novel
moiety on the affinity for opioid receptors [4]. Recently,
Neumeyer's group [5] synthesized 29-aminothiazole deriv-
atives of morphans and morphinans to study their opioid
agonist feature.
The efforts of several research groups to explore the
structure-activity relationships (SAR) of apomorphine
derivatives to different dopamine receptor subtypes have
A procedure has been elaborated for the synthesis of
2,3-thiazole-fused apomorphine derivatives. The synthe-
sis was based on naturally occurring thebaine 1 which
Correspondence: Attila Sipos, Department of Organic Chemistry, Uni-
versity of Debrecen, P. O. Box 20, H-4010, Debrecen, Hungary
E-mail: asipos@puma.unideb.hu
Fax: +36 52 512-836
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Arch. Pharm. Chem. Life Sci. 2009, 342, 557–568
Figure 1. Structural similarity of two D2 receptor agonists.
ensured the R absolute configuration of C-6a. In deter-
mining the C2 and C3 positions, the phenol-bioisostere 2-
aminothiazole moiety has been inserted to mimic the 2-
(4-hydroxyphenyl)-apomorphine structure (Fig. 2). The
synthesis was extended for the preparation of thiazolo-
aporphines containing other pharmacophore groups in
the 29-position.
Zhang et al. [10] recently reported the synthesis and
pharmacological evaluation of a series of apomorphine
derivatives privileged with a 2-aminothiazole functional-
ity. The design of these compounds was based on funda-
mentally the same rationales as presented above. The 29-
aminothiazolo-apomorphine, presented in Fig. 2, was
synthesized in a completely different approach involving
the insertion of a 2-amino function to the protected apor-
phine backbone.
Figure 2. Bioisostere structures of apomorphine derivatives.
nandienes 3 to 5, we observed a major difference in the
behaviour of 29-aminothiazolo-morphinandiene 3 and its
29-methyl- (4) and phenyl- (5) congeners. This type of mor-
phinandiene rearrangement has been studied in our lab-
oratory for over 20 years [15] and it has been concluded
that this reaction produces apocodeine derivatives
almost quantitatively.
Our observations for the amino-type starting material
3 were in accordance with this general experience, how-
ever, in case of the methyl- and phenyl-derivatives 4, 5 we
found two products in approximately 1:1 ratio (Scheme
2). After isolation and full characterization of the prod-
ucts it was revealed that isothiazole-type compounds 9,
10 were formed beside the previously expected thiazolo-
aporphines 7, 8.
Results and discussion
Synthesis
The explanation for the occurrence of isothiazolo-apor-
phines 9, 10 is an isomerization reaction which happens
simultaneous with the acid-catalyzed rearrangement.
Sharp et al. presented the same phenomenon for ziprasi-
done [16]. They adopted a reversible photo-/thermal-
induced isomerization mechanism as the rationalization
The synthesis of morphinan derivatives bearing an ami-
nothiazole moiety at the C-ring of the morphinan skele-
ton was first reported by Neumeyer and his co-workers
[11]. In one of our recent publications, we have reported
a different approach for the synthesis of 29-substituted
thiazolo-morphinandienes [12]. In the first step, the-
baine 1 is converted into 14b-bromo-codeinone 2 in a
well-known reaction [13]. A Hantzsch-type thiazole syn-
thesis is accomplished on compound 2 exploiting its
ability to react as an a-haloketone [14] in the presence
of an appropriate nucleophile partner (Scheme 1).
The morphinandiene structure of products 3 to 5 offers
the possibility of rearrangement into aporphinoids. In
the acid-catalyzed rearrangement of thiazolo-morphi-
for
benzisothiazole-benzothiazole
rearrangement
(Scheme 3).
We applied 90–958C heat, a polar solvent, and no pro-
tection from light as conditions for acid-catalyzed rear-
rangements. These conditions are in agreement with
those used by Sharp in isomerization tests. The acid-cata-
lyzed rearrangement was also performed in inert atmos-
phere (nitrogen gas) with the use of protection from light
but there was no significant change observed in the ratio
Scheme 1. Synthesis route to 29-sub-
stituted thiazolo-morphianandienes.
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Arch. Pharm. Chem. Life Sci. 2009, 342, 557–568
Thiazole and Isothiazole Derived Apomorphines
559
Scheme 2. Formation of thiazolo- and isothiazolo-apocodeines 6–10.
Scheme 3. Proposed mechanism for the isomerisation.
of thiazolo and isothiazolo products. A photochallenge
[17] was also conducted with compounds 7 and 8 using
the same solvents and concentrations as Sharp et al. On
the basis of these results, we concluded that in this case
the heat was the most important factor in the activation
of the isomerization in comparison with ziprasidone [16].
D'Auria discussed the isomerization of some thiazoles
into isothiazoles presenting examples for the photo-
induced isomerization as well as for photo and thermal
dependence of this type of rearrangements [18]. A mecha-
nism was described for the photo- and thermal-induced
Scheme 4. Tautomer forms of 29-aminothiazolo-apocodeine (6).
HMBC correlations of 29-methylthiazolo- (7) and 29-methyl-
isothiazolo-derivatives (9) are presented in Fig. 3.
In order to expand the range of pharmacophores in
the 29-position and to facilitate further functionalization
(e.g. by palladium-catalyzed cross-coupling), the 29-amino
group was replaced in product 6 with a chloro substitu-
ent in a one-pot method using isoamyl-nitrite-effected (i-
Am-ONO) diazotation and halogenations with CuCl2 in
acetonitrile (Scheme 5) [19].
The conversion of 10-methoxyaporphines 6–11 into
catechol-aporphines 12–17 was carried out by means of
the methanesulfonic acid/methionine reagent-combina-
tion which was successfully used earlier by our labora-
tory (Scheme 6) [20].
As discussed above, the isomerization from thiazole
derivatives to isothiazoles was primarily induced by ther-
mal effects. To inhibit further isomerization of apoco-
deines 7–10 during the O-demethylation step, the classic
conditions were modified and a microwave-assisted pro-
cedure was established on the basis of previous studies
isomerization of 2-Ph-thiazoles involving
2-Ph-thiazole structure as an intermediate.
a Dewar
The reason for the difference in behavior of the amino-
derivative and its methyl- and phenyl-congeners could be
justified by the significant willingness of the 2-amino-
benzothiazole moiety for tautomerization (Scheme 4).
The appearance of this imine-type tautomer means an
extra stabilization for the benzothiazole structure
against benzisothiazole, and may be an inhibitory effect
of the isomerization.
The structure of the isothiazoloaporphines was
unequivocally established byNMR spectral studies. Full 1H
and13C resonance assignments were obtained from COSY,
TOCSY, HSQC, and HMBC spectra. The main differences in
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Scheme 5. Synthesis of 29-chlorothiazolo-apocodeine 11.
Figure 3. Important HMBC correlations for benzothiazole- and
benzisothiazole-type compounds.
Table 1. Affinities for dopamine receptor subtypes measured by
radioligand-binding studies.
Ki (nM)
Compounds
Average l SD or SEM (experi-
ments in triplicate)
hD1
101
hD2L
32
Apomorphi-
ne N HCl*
2113 € 203 (2) 2883 l 664 (2)
12
13
14
15
16
17
Scheme 6. Formation of apomorphine derivatives 12–17.
2753l 61 (2)
1812 l 49 (2)
>10 000
5192 l 684 (2)
2548 l 544 (2)
>10 000
on the application of microwave-promoted rearrange-
ments of morphinans [21]. The temperature was
decreased from 90–958C to 758C, and inert atmosphere
was applied. The reaction time was sharply reduced from
an average of 120 min to 5 min. No significant isomeriza-
tion was observed during the O-demethylation of benzo-
thiazolo- and benzisothiazolo-type apocodeines 6–11.
Apomorphine derivatives 12–17 were precipitated from
ether in HCl salt form. These salts were found to be appro-
priately water-soluble for pharmacological testing.
1027 l 137 (2) 197 l 83 (2)
Pharmacology
All compounds were screened for their binding affinities
for the human cloned D1 and D2L receptor subtypes by in
vitro radioligand-binding studies. The results are sum-
marized in Table 1. With the exception of 15, all of the
compounds display low affinities reaching the maxi-
mum for the isothiazole derivative 16 at the D2 binding
site. It could also be concluded from the presented data
that the binding activities for both D1 and D2L subtypes
>10 000
5362 l 2172 (2)
SD = Standard deviation; SEM = Standard error of the mean; the
SEM was used, when the number of values was less than three; *
data from Ref. [7].
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Arch. Pharm. Chem. Life Sci. 2009, 342, 557–568
Thiazole and Isothiazole Derived Apomorphines
561
Table 2. Affinities for dopamine receptor subtypes measured by
radioligand-binding studies.
were largely affected by the spatial size and lipophilicity
of the substitutent at the 29-position of the thiazole ring.
The affinities of compound 14, containing a large and a
lipophilic phenyl moiety at 29-position, were found to be
superior in comparison to ligands 12, 13, and 15 bearing
either a small and lipophilic subtituent (compound 13, R
= CH3) or small and hydrophilic moieties (compounds 13
and 15, R = NH3+ and Cl). In the case of compound 12, the
obtained binding affinities for the human cloned D1 and
D2L were found to be in good agreement with the trends
observed by Zhang et al. [10].
In the case of isothiazole-derived apomorphines, the
spatial size of the 39-subtituent was found to be determin-
ing. For isothiazole-fused backbone it was observed that
it tolerated much smaller deviation in spatial size in the
proximity of the 2-position of the aporphine skeleton.
This observation contradicts the generally accepted view
that the binding affinity of 2-substituted apomorphines
are not predominantly determined by the size of the moi-
ety introduced at the 2-position (referring to the high
affinities of both 2-methoxy- and 2-(4-hydroxyphenyl)-
apomorphines) [6, 7]. This phenomenon was associated
with the additional electrostatic effects of the isothiazole
moiety being introduced on the aporphine backbone.
However, it was only possible to draw further conclu-
sions after the establishment of binding models for the
studied dopamine-receptor subtypes (see Section 2.3:
Binding models and predicting ligand interactions).
In order to obtain a detailed pharmacological profile
Compounds
Ki (nM)
hD2L/hDx
Average l SD or SEM
specificity
(experiments in triplicate) fold
Apomorphine N HCl* hD1
101
32
26
3.2
–
0.8
hD2L
hD3
hD4.4
hD5
2.6
10
0.08
0.3
hD1
hD2L
hD3
hD4.4
hD5
1027l 137 (2)
197l 83 (2)
1874 (1)
1142l 603 (2)
2361 (1)
5.2
–
9.5
5.8
12.0
16
SD = Standard deviation; SEM = Standard error of the mean;
SEM was used, when the number of values was less than three;
*data from Ref. [7].
Table 3. Functional calcium assay results for compound 16.
Functional Ca2+ assay
Compound
Change% of the agonist in-
Ki-values (nM)
duced fluorescence by a 10-lM Average € SEM
solution of the compound
16
D1 88.1%
2L 79.2%
D1 62.8 l 2.3
D2L 124 l 36
D
for the most active compound, the binding affinities of for a majority of recombinant GPCRs. Assay of the pro-
compound 16 were extended to all five human dopa- duced [Ca2+] can be performed using Oregon Green 488
mine-receptor subtypes and the results are summarized BAPTA-1/AM and a microplate reader. Dopamine agonists
in Table 2.
or antagonists are expected to alter the response to the
These tests revealed that 39-methylisothiazolo-apomor- standard agonists. The microplate reader-based calcium
phine 16 had a significantly stronger affinity for the D2L assay is a very useful method for simply and quickly
subtype referring to the calculated specificities. It is also selecting the active compounds. Comparing radioligand-
remarkable that there is a considerable difference in the and functional-study data, it can be seen that there are
binding activity compared to the two subtypes of the D2- differences in the Ki values obtained, as the calcium assay
like receptor family.
monitors a fast calcium signal, these results represent
For functional studies, hD1 and hD2L receptors were non-equilibrium data, whereas radioligand-binding stud-
chosen as characteristic representatives of each of the ies were performed under equilibrium conditions.
two dopamine-receptor subtype groups allowing a com-
parison of functional and binding data. The inhibition by
Binding models and predicting ligand interactions
compound 16 of agonist-induced changes in intracellular As a part of a larger project devoted to the modelling and
[Ca2+] in intact HEK293 cells was estimated (Table 3 and understand of highly dopamine-active aporphinoids, our
Fig. 4). In the functional assay, compound 16 was of ago- group established models for the binding of substituted
nistic activity and it was almost equally active on the D1 apomorphines to human D1-D5 receptor subtypes. To
and D2. The functional assay is dependent on the fact that have a further insight into the binding characteristics of
stimulation of the dopamine receptors by agonists, for above-described thiazolo- and isothiazolo-apomorphines
example, quinpirole for D2-like receptors and SKF 38393 12–17, we applied the available models for these ligands
for D1 will cause an increase in intracellular [Ca2+] which with special emphasis on D2-like receptors and com-
appears to represent a universal second messenger signal pound 16. The reason for this focusing on D2-like recep-
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Arch. Pharm. Chem. Life Sci. 2009, 342, 557–568
AM6 is the in-house code for compound 16. Data are shown as average l SEM (n = 4).
Figure 4. Functional characterization of the affinity of compound 16 for D1 and D2L subtypes.
Table 4. Determining hD2L residues in the formation of binding complex with compound 16 upon predicted model.
Interaction
Pharmacophore of D2 residue
compound 16
Helix
Type of interaction
Distance (ꢁ)
1
2
3
4
5
6
N17
Asp 114
Phe 198
Ser 197
Ser 194
Ser 197
Tyr 387
TM3
TM5
TM6
TM6
TM6
TM7
Salt bridge
2.11
3.75
2.56
1.37
3.72
2.68
Ring D
C10-OH
C10-OH
C11-OH
N29
Hydrophobic (p-p)
Hydrogen bonding
Hydrogen bonding
Hydrogen bonding
Cation-p
Table 5. Determining hD3 residues in the formation of binding complex with compound 16 upon predicted model.
Interaction
Pharmacophore of D3 residue
compound 16
Helix
Type of interaction
Distance (ꢁ)
1
2
3
4
5
6
N17
Asp 110
Ser 192
Ser 196
Ser 196
Phe 346
Tyr 373
TM3
TM5
TM5
TM5
TM6
TM7
Salt bridge
2.61
2.65
2.70
3.34
2.49
2.82
C10-OH
C10-OH
C11-OH
Ring D
N29
Hydrogen bonding
Hydrogen bonding
Hydrogen bonding
Hydrophobic (p-p)
Cation-p
tors is that they are generally considered as main phar- mated to be less important. Our D2 docking studies sug-
macological targets of A-ring-substituted apomorphines. gest that there are two determining features in the phar-
The predicted D2L and D3 binding models for this com- macophore: a salt bridge with N17, a cation-p interaction
pound are shown in Fig. 5. In the case of the D2 receptor and there are three further important factors: a hydro-
ligand, 16 occupies the binding site formed by TM3, TM5, phobic p-p interaction and donors/acceptors for two
and TM6 [22]. Residues Asp 114, Ser 194, Ser 197, and Tyr hydrogen bonds (Table 3). This model suggests that Tyr
387 in the receptor were found to interact with ligand 16 387 has a significant impact on the properties of the
and with isothiazolo-apomorphine 17. These residues receptor-ligand complexes. The cation-p interaction
interact also with thiazolo-aporphines 12–15, however, formed with the protonated isothiazole ring proved
the role of Tyr 387 changes dramatically. Residues Phe more significant than the weaker hydrophobic (-CH3 –Ar
198, Trp 160, and Trp 357 make contact with some but in the binding of compound 13) or aromatic p-p interac-
not all of the ligands examined and their function is esti- tions (-Ph–Ar in the binding of compound 14). Interest-
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Arch. Pharm. Chem. Life Sci. 2009, 342, 557–568
Thiazole and Isothiazole Derived Apomorphines
563
Electrostatic surface calculations were extended for the major residues of the binding site to highlight interactions.
Figure 5. Conformations and significant interactions of the D2 (Panels A & B) and the D3 (Panels C & D) binding site complexes with
compound 16.
ingly, we found the formation of a stable hydrogen and with isothiazolo-apomorphine 17 (Table 4). Residues
bridge between 29-chloro substituent of compound 15 Cys 114, Val 117, Phe 345, and Hys 349 were found to inter-
and Tyr 387 incompatible with maintaining the position actwith all the ligandsexamined buttheir function isesti-
of higher priority interactions (i.e. salt bridge between mated to be less important. The same function of Tyr 373
N17 and Asp 114 and hydrogen bridges of catecholic ofhelix7wasidentifiedinthebindingcomplexesofrecep-
hydroxyls as it is described in Section 4.3.5: Computa- torD3asTyr387inD2receptorbinding.
tional background).
Comparing the predicted interactions for ligand 16
Regarding the model for D3 receptor binding, it is (Tables 3 and 4) it can be concluded that it was possible
important thatligand 16occupies the binding site formed to position this compound in a more favorable position
by TM3, TM5, and TM6 [23]. Residues Asp 110, Ser 192, Ser at the hD2L binding site with respect to the predicted dis-
196, Phe 346, and Tyr 373 in the receptor were identified tances of the most important complex-forming interac-
as the most significant ones interacting with ligand 16 tions.
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Arch. Pharm. Chem. Life Sci. 2009, 342, 557–568
tion, USA. Controlled temperature, power, pressure, and time
settings were used for all reactions.
Conclusion
We hereby present the synthesis of novel thiazolo- and
isothiazolo-apomorphines 12–17. During the synthesis
of the aimed thiazolo derivatives, an unexpected isomer-
ization step was identified allowing the preparation and
characterization of isothiazolo-apomorphines. With the
exception of two derivatives, all the studied ligands
showed low affinities for dopamine-receptor subtypes in
comparison to apomorphine. Compound 16 was found
to possess moderate affinity to D2L subtype with approxi-
mately 10-fold selectivity over D3 affinity. This derivative
was studied in a conventional functional assay in order
to determine its agonistic properties to D1-like and D2-
like subtypes. These tests revealed similar agonistic prop-
erties to both receptor families and suggest higher affin-
ities in comparison to the radioligand-binding assays. To
be able to give a detailed interpretation, binding models
were established for D2-like receptors which were gener-
ally considered as main pharmacological targets of A-
ring-substituted apomorphines. On the basis of the pre-
dicted models, we identified an important cation-p inter-
action for the binding of isothiazolo-apomorphine 16.
Chemistry
Acid-catalyzed rearrangement of morphinandienes
A mixture of the diene (1 g) and methanesulfonic acid (5 mL) was
stirred for 20 min at 08C. Then, the reaction mixture was added
dropwise, with stirring and external ice-cooling, to a solution of
potassium hydrogen carbonate (10 g) in water (50 mL). After
extraction with chloroform (3615 mL), the combined extracts
were washed with saturated brine, dried (MgSO4), and concen-
trated in vacuo. In case of necessity, the residue was submitted to
purification by means of column chromatography (Kieselgel 40,
chloroform/methanol, 1:1) to yield the appropriate apocodeines.
(–)-R-29-Amino-11-hydroxy-10-methoxy-2,3:49,59-
thiazolo-aporphine 6
Pale yellow, cubic crystals; m.p.: 109–1118C; yield: 488 mg
(89%); [a]D25 = –203 (c = 0.1, methanol); Rf (CH2Cl2/CH3OH, 8:2):
1
0.34; H-NMR (360 MHz, DMSO-d6) d: 8.87 (s, 1H, 11-OH), 8.29 (s,
1H, H1), 7.41 (s, 2H, 29-NH2), 6.82 (dd, J8,9 = 8.4 Hz, 2H, H8-9), 3.84
(s, 3H, 10-OCH3), 3.31-2.78 (m, 4H, H4a, H5a, H5b, H6a), 2.47 (s, 3H,
NCH3), 2.66–2.09 (m, 3H, H4b, H7a, H7b); 13C-NMR (90 MHz,
DMSO-d6) d: 162.87 (C29), 148.32 (C2), 148.10 (C10), 143.41 (C11),
135.21–114.51 (Ar, 9C), 60.33 (C6a), 56.41 (OCH3), 52.41 (C5),
42.41 (=NCH3), 35.75 (C7), 28.82 (C4); HRMS (ESI) m/z (%) found:
354.1261 [M+ + 1] (100); calculated: 354.1274 [M+ + 1].
The authors are grateful for the financial support to the
National Science Foundation (Grant OTKA reg. No. NI61336).
We thank the Pꢀter Pazmꢁny Programme (NKTH, RET006/
2004) to allow us the use of microwave reactor and Szabolcs
Fekete for valuable technical help. A. S. is indebted to the Eꢂtvꢂs
Scholarship of the Hungarian State. We thank Bꢃrbel Schmal-
wasser and Petra Wiecha for skilful technical assistance.
(–)-R-11-Hydroxy-29-methyl-10-methoxy-2,3:49,59-
thiazolo-aporphine 7
Column chromatography was applied to separate compounds 7
and 9. Compound 7 was the first eluted fraction. Grey, cubic crys-
tals; m.p.: 170–1738C; yield: 216 mg (44%); [a]D25 = –109 (c = 0.1,
chloroform); Rf (CH2Cl2/CH3OH, 8:2): 0.47; 1H-NMR (360 MHz,
CDCl3) d: 8.95 (s, 1H, H1), 6.77 (dd, J8,9 = 7.2 Hz, 2H, H8-9), 6.71 (s,
1H, 11-OH), 3.92 (s, 3H, 10-OCH3), 3.52–2.92 (m, 4H, H4a, H5a, H5b,
H6a), 2.85 (s, 3H, 29-CH3), 2.81–2.51 (m, 6H, H4b, H7a, H7b, NCH3);
13C-NMR (90 MHz, CDCl3) d: 169.60 (C29), 152.87 (C2), 148.48
(C10), 142.60 (C11), 136.72–114.38 (Ar, 9C), 60.41 (OCH3), 58.63
(C6a), 53.10 (C5), 40.72 (=NCH3), 35.11 (C7), 24.21 (C4), 22.22 (C-
CH3); HRMS (ESI) m/z (%) found: 353.1297 [M+ + 1] (100); calcu-
lated: 353.1304 [M+ + 1].
The authors have declared no conflict of interest.
Experimental
General
Melting points were determined with a Kofler hot-stage appara-
tus (C. Reichert, Vienna, Austria) and are uncorrected. Thin layer
chromatography was performed on precoated Merck 5554 Kie-
selgel 60 F254 foils (Merck, Germany) using chloroform/methanol,
8:2 mobile phase. The spots were visualized with Dragendorff's
reagent. 1H- and 13C-NMR spectra were recorded on a Bruker AM
360 spectrometer (Bruker Bioscience,USA), chemical shifts are
reported in ppm (d) from internal TMS and coupling constants (J)
are measured in Hz. 1H- and 13C-NMR assignments were based on
2D-NMR studies of the compounds. High resolution mass spec-
tral measurements were performed with a Bruker micrOTOF-Q
instrument (Bruker) in the ESI mode. Optical rotation was deter-
mined with a Perkin Elmer Model 241 polarimeter (Perkin-
Elmer, Norwalk, CT, USA).
(–)-R-11-Hydroxy-10-methoxy-29-phenyl-2,3:49,59-
thiazolo-aporphine 8
Column chromatography was applied to separate compounds 8
and 10. Compound 8 was the first eluted fraction. Off-white,
cubic crystals, m.p.: 162–1648C; yield: 188 mg (39%); [a]D25 = –206
(c = 0.1, chloroform); Rf (CH2Cl2/CH3OH, 8:2) 0.56; 1H-NMR (360
MHz, CDCl3) d: 7.91 (s, 1H, H1), 7.77–7.19 (m, 5H, 29-Ph), 6.68 (dd,
J8,9 = 7.7 Hz, 2H, H8-9), 6.12 (br s, 1H, 11-OH), 3.83 (s, 3H, 10-OCH3),
3.09–2.41 (m, 4H, H4a, H5a, H5b, H6a), 2.38 (s, 3H, NCH3), 2.30–
2.07 (m, 3H, H4b, H7a, H7b); 13C-NMR (90 MHz, CDCl3) d: 166.66
(C29), 154.24 (C2), 149.14 (C10), 142.69 (C11), 135.42–112.11 (Ar,
15C), 59.01 (OCH3), 56.53 (C6a), 52.79 (C5), 41.21 (=NCH3), 35.10
(C7), 27.44 (C4); HRMS (ESI) m/z (%) found: 415.1459 [M+ + 1] (100);
calculated: 415.1484 [M+ + 1].
The microwave-induced reactions were carried out in a Dis-
cover model microwave reactor manufactured by CEM Corpora-
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Arch. Pharm. Chem. Life Sci. 2009, 342, 557–568
Thiazole and Isothiazole Derived Apomorphines
565
collected, washed with saturated NaCl solution, dried over anhy-
drous MgSO4, and evaporated. The residue was subjected to silica
gel column chromatography. Elution with chloroform/metha-
nol, 1:1 gave the corresponding apomorphines. The crystalline
product is precipitated by addition of abs. diethylether and con-
verted into hydrochloride salt with 1 M HCl/ethanol.
(–)-R-11-Hydroxy-39-methyl-10-methoxy-2,3:49,59-
isothiazolo-aporphine 9
Column chromatography was applied to separate compounds 7
and 9. Compound 9 was the second eluted fraction. Grey, cubic
crystals, m.p.: 142–1478C; yield: 167 mg (44%); [a]D25 = +16 (c = 0.1,
chloroform); Rf (CH2Cl2/CH3OH, 8:2) 0.22; 1H-NMR (360 MHz,
CDCl3) d: 7.98 (s, 1H, H1), 6.77 (dd, J8,9 = 7.9 Hz, 2H, H8-9), 6.44 (s,
1H, 11-OH), 3.92 (s, 3H, 10-OCH3), 3.52–2.83 (m, 5H, H4a, H4b, H5b,
H6a, H7b,), 2.60 (s, 3H, 39-CH3), 2.55 (m, 1H, H7a), 2.51 (s, 3H, NCH3),
2.41 (m, 1H, H5a); 13C-NMR (90 MHz, CDCl3) d: 158.69 (C29), 147.91
(C10), 142.88 (C11), 142.71 (C3a), 138.21 (C3), 133.80–114.58 (Ar,
8C), 60.63 (C6a), 56.38 (OCH3), 52.07 (C5), 41.21 (=NCH3), 33.98
(C7), 23.80 (C4), 19.47 (C-CH3); HRMS (ESI) m/z (%) found:
353.1315 [M+ + 1] (100); calculated: 353.1321 [M+ + 1].
(–)-R-29-Amino-10,11-dihydroxy-2,3:49,59-thiazolo-
aporphine trihydrochloride 12 N 3 HCl
Yellow, plate shape crystals; m.p.: 194–1968C; yield: 750 mg
(92%); [a]D25 = –119 (c = 0.1, DMSO); Rf (CH2Cl2/CH3OH, 8:2): 0.27;
1H-NMR (360 MHz, DMSO-d6) d: 8.20 (s, 1H, H1), 6.76 (dd, J8,9 = 7.9
Hz, 2H, H8-9), 6.34 (br s, 2H, 10-OH, 11-OH), 3.11–2.51 (m, 3H,
H4a, H5b, H6a), 2,47 (s, 3H, NCH3), 2.40–2.09 (m, 4H, H4b, H5a, H7a,
H7b), no NH2 sign was observed; 13C-NMR (90 MHz, DMSO-d6) d:
160.68 (C29), 145.10 (C10), 144.62 (C11), 140.32 (C2), 132.79–
116.11 (Ar, 9C), 59.93 (C6a), 51.14 (C5), 39.92 (=NCH3), 36.64 (C7),
27.44 (C4); HRMS (ESI) m/z (%) found: 340.1121 [M+ + 1] for free
base (100); calculated: 340.1114 [M+ + 1].
(–)-R-11-Hydroxy-10-methoxy-39-phenyl-2,3:49,59-
isothiazolo-aporphine 10
Column chromatography was applied to separate compounds 8
and 10. Compound 10 was the second eluted fraction. White,
cubic crystals; m.p.: 179–1838C; yield: 159 mg (33%); [a]D25 = +15
1
(c = 0.1, chloroform); Rf (CH2Cl2/CH3OH, 8:2): 0.37; H-NMR (360
MHz, CDCl3) d: 9.23 (s, 1H, H1), 8.11–7.43 (m, 5H, 39-Ph), 6.70 (dd,
J8,9 = 7.9 Hz, 2H, H8-9), 3.59 (s, 3H, 10-OCH3), 3.31–2.77 (m, 5H,
H4a, H4b, H5b, H6a, H7b), 2.63–2.51 (m, 5H, H5b, H7a, NCH3); 13C-
NMR (90 MHz, CDCl3) d: 157.01 (C29), 149.21 (C10), 144.42 (C3),
142.36 (C11), 141.20 (C3a), 133.81–113.34 (Ar, 14C), 60.23 (OCH3),
56.93 (C6a), 52.09 (C5), 40.87 (=NCH3), 35.42 (C7), 29.31 (C4);
HRMS (ESI) m/z (%) found: 415.1499 [M+ + 1] (100); calculated:
415.1485 [M+ + 1].
(–)-R-10,11-Dihydroxy-29-methyl-2,3:49,59-thiazolo-
aporphine dihydrochloride 13 N 2 HCl
Off-white, plate shape crystals; m.p.: A2508C; yield: 199 mg
(79%); [a]D25 = –93 (c = 0.025, DMSO); Rf (CH2Cl2/CH3OH, 8:2) 0.21);
1H-NMR (360 MHz, CDCl3) d: 7.68 (s, 1H, H1), 6.88 (dd, J8,9 = 7.6 Hz,
2H, H8-9), 6.61 (br s, 2H, 10-OH, 11-OH), 3.44–2.87 (m, 3H, H4a,
H5b, H6a), 2.85 (s, 3H, 29-CH3), 2.74–2.37 (m, 7H, H4b, H5a, H7a, H7b,
NCH3); 13C-NMR (90 MHz, CDCl3) d: 166.42 (C29), 145.48 (C10),
144.22 (C11), 142.63 (C2), 132.11–116.32 (Ar, 9C), 58.63 (C6a),
51.18 (C5), 39.82 (=NCH3), 36.42 (C7), 26.58 (C4), 24.37 (C-CH3);
HRMS (ESI) m/z (%) found: 339.1149 [M+ + 1] (100); calculated:
339.1162 [M+ + 1].
(–)-R-29-Chloro-11-hydroxy-10-methoxy-2,3:49,59-
thiazolo-aporphine 11
100 mg (0.28 mmol) of compound 6 were dissolved and mixed in
2 mL of acetonitrile and cooled to 08C. 45 mg (0.34 mmol) CuCl2
were added followed by a dropwise addition of isoamyl nitrite
(49 mg, 0.41 mmol). The mixture was stirred for 2 h. The solvent
was removed by evaporation under reduced pressure and the
residue was purified by flash chromatography (EtOAc/MeOH,
8:2). Pale brown powder, m.p.: 111–1148C; yield: 63 mg (69%);
[a]D25 = –217 (c = 0.1, chloroform); Rf (CH2Cl2/CH3OH, 8:2) 0.31; 1H-
NMR (360 MHz, CDCl3) d: 7.82 (s, 1H, H1), 6.62 (dd, J8,9 = 7.9 Hz,
2H, H8-9), 3.91 (s, 3H, 10-OCH3), 3.17–2.52 (m, 3H, H4a, H5b, H6a),
2.39 (s, 3H, NCH3), 2.44–2.03 (m, 4H, H4b, H5a, H7a, H7b); 13 C-NMR
(90 MHz, CDCl3) d: 154.77 (C29), 150.32 (C2), 149.52 (C10), 142.61
(C11), 135.55–113.38 (Ar, 9C), 59.73 (C6a), 56.22 (OCH3), 53.32
(C5), 40.88 (=NCH3), 35.10 (C7), 24.52 (C4); HRMS (ESI) m/z (%)
found: 373.0802 [M+ + 1] (100); calculated: 373.0782 [M+ + 1].
(–)-R-10,11-Dihydroxy-29-phenyl-2,3:49,59-thiazolo-
aporphine dihydrochloride 14 N 2 HCl
Off-white, plate shaped crystals, m.p.: A2508C; yield: 176 mg
(82%); [a]D25 = –188 (c = 0.1, DMSO); Rf (CH2Cl2/CH3OH, 8:2) 0.19;1H-
NMR (360 MHz, CDCl3) d: 8.02 (s, 1H, H1), 7.82–7.29 (m, 5H, 29-
Ph), 6.72 (dd, J8,9 = 7.6 Hz, 2H, H8-9), 6.54 (br s, 2H, 10-OH, 11-OH),
3.14–2.37 (m, 6H, H4a, H5b, H6a, NCH3), 2.23–1.99 (m, 4H, H4b,
H5a, H7a, H7b); 13C-NMR (90 MHz, CDCl3) d: 160.36 (C29), 145.21
(C10), 145.63 (C11), 140.28 (C2), 135.42–112.11 (Ar, 15C), 56.53
(C6a), 52.79 (C5), 41.21 (=NCH3), 36.42 (C7), 27.01 (C4); HRMS (ESI)
m/z (%) found: 401.1342 [M+ + 1] (100); calculated: 401.1333
[M+ + 1].
O-Demethylation of apocodeines by MW irradiation
(–)-R-29-Chloro-10,11-dihydroxy-2,3:49,59-thiazolo-
aporphine dihydrochloride 15 N 2 HCl
A mixture of the apocodeine (4.56 mmol), methionine (1 g, 6.70
mmol) and methanesulfonic acid (4 mL) was stirred in the pres-
surized glass vial, equipped with a magnetic stirring bar, for 20
min at 08C carefully protected from sunlight under nitrogen
atmosphere. The vial was inserted into the microwave cavity of
the microwave reactor, irradiated at the target temperature
758C for 5 min hold time and subsequently cooled by rapid gas-
jet cooling. The product mixture was allowed to cool to room
temperature in the microwave cavity. After cooling, the pH of
the mixture was set to 10 by concentrated NH3 solution and
extracted with chloroform (3615 mL). The organic layers were
Greenish yellow, shiny, plate shape crystals, m.p.: 203–2068C;
yield: 64 mg (87%); [a]D25 = –198 (c = 0.1, DMSO); Rf (CH2Cl2/CH3OH,
8:2) 0.20; 1H-NMR (360 MHz, CDCl3) d: 7.92 (s, 1H, H1), 6.68 (dd, J8,9
= 8.0 Hz, 2H, H8-9), 6.56 (br s, 2H, 10-OH, 11-OH), 3.24–2.32 (m,
6H, H4a, H5b, H6a, NCH3), 2.30–1.92 (m, 4H, H4b, H5a, H7a, H7b);
13C-NMR (90 MHz, CDCl3) d: 158.37 (C29), 145.32 (C10), 144.69
(C11), 140.31 (C2), 132.30–116.25 (Ar, 9C), 59.82 (C6a), 51.19 (C5),
39.98 (=NCH3), 36.33 (C7), 26.15 (C4); HRMS (ESI) m/z (%) found:
359.0610 [M+ + 1] (100); calculated: 359.0616 [M+ + 1].
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566
A. Sipos et al.
Arch. Pharm. Chem. Life Sci. 2009, 342, 557–568
(–)-R-10,11-Dihydroxy-39-methyl-2,3:49,59-isothiazolo-
aporphine dihydrochloride 16 N 2 HCl
Radioligand-binding assay
The binding studies were performed following the protocol pre-
viously described but in 96- well format [24]. The assays with the
whole-cell-suspension were carried out in triplicate in a volume
of 550 lL (final concentration): TRIS-Mg2+-buffer (345 lL), [3H]-
ligand (50 lL), whole-cell-suspension (100 lL), and appropriate
drugs (55 lL). Non-specific binding was determined using flu-
phenazine (100 lM) in hD1 tests and haloperidol (10 lM) in hD2L
tests. The incubation was initiated by addition of the radioli-
gand [3H]SCH23390 for hD1-like receptors and [3H]spiperone for
hD2L-like receptors (both Amersham Biosiences, Little Chalfront,
UK). It was carried out in 96-deep well plates (Greiner Bio-One)
using a Thermocycler (Eppendorf, Wessling, Germany) at 278C.
The incubation was terminated after 90 min by rapid filtration
with a PerkinElmer Mach III HarvesterTM using a PerkinElmer Fil-
termat A, previously treated with a 0.25% polyethyleneimine-sol-
ution (Sigma-Aldrich) and washed once with water. The filter-
mat was dried for 3 min with 400 W using a microwave (MW 21,
Clatronic, Kempen, Germany). The dry filtermat was placed in a
filter plate (Omni filter plates, PerkinElmer Life Sciences) and
each field of the filtermat moistened with 50 lL Microscint 20TM
scintillation cocktail. The radioactivity retained on the filters
was counted using a Top Count NXTTM microplate scintillation
counter (Packard Instruments, USA). For determining the Ki val-
ues at least two independent experiments each in triplicate
were performed.
Off-white, plate shape crystals, m.p.: 208–2108C; yield: 175 mg
(90%); [a]D25 = +33 (c = 0.1, DMSO); Rf (CH2Cl2/CH3OH, 8:2) 0.24; 1H-
NMR (360 MHz, CDCl3) d: 8.02 (s, 1H, H1), 6.77 (dd, J8,9 = 7.9 Hz,
2H, H8-9), 6.44 (s, 1H, 11-OH), 3.92 (s, 3H, 10-OCH3), 3.52–2.92 (m,
4H, H4a, H5a, H5b, H6a), 2.85 (s, 3H, 39-CH3), 2.81–2.51 (m, 6H, H4b,
H7a, H7b, NCH3); 13C-NMR (90 MHz, CDCl3) d: 159.12 (C39), 145.81
(C10), 144.78 (C11), 140.33 (C2), 132.24–116.04 (Ar, 9C), 59.80
(C6a), 51.69 (C5), 40.09 (=NCH3), 36.33 (C7), 26.82 (C4), 18.98 (C-
CH3); HRMS (ESI) m/z (%) found: 339.1181 [M+ + 1] (100); calcu-
lated: 339.1162 [M+ + 1].
(–)-R-10,11-Dihydroxy-39-phenyl-2,3:49,59-isothiazolo-
aporphine dihydrochloride 17 N 2 HCl
White, plate shape crystals; m.p.: 179–1838C; yield: 160 mg
(88%); [a]D25 = +23 (c = 0.1, DMSO); Rf (CH2Cl2/CH3OH, 8:2) 0.31; 1H-
NMR (360 MHz, CDCl3) d: 7.91 (s, 1H, H1), 7.77–7.19 (m, 5H, 39-
Ph), 6.68 (dd, J8,9 = 7.7 Hz, 2H, H8-9), 3.83 (s, 3H, 10-OCH3), 3.09–
2.41 (m, 4H, H4a, H5a, H5b, H6a), 2.38 (s, 3H, NCH3), 2.30–2.07 (m,
3H, H4b, H7a, H7b); 13C-NMR (90 MHz, CDCl3) d: 159.58 (C3'),
145.72 (C10), 144.63 (C11), 140. 27 (C2), 132.87–116.21 (Ar, 15C),
59.75 (C6a), 51.30 (C5), 39.97 (=NCH3), 37.29 (C7), 26.44 (C4);
HRMS (ESI) m/z (%) found: 401.1338 [M+ + 1] (100); calculated:
401.1318 [M+ + 1].
The competition binding data were analysed by the software
GraphPad PrismTM using nonlinear least squares fit. For calculat-
ing the mean, standard deviation and standard error of the
mean the software Microsoft ExcelTM was used. Ki values were cal-
culated from IC50 values applying the equation of Cheng and
Prusoff [25].
Pharmacology
Cell culture
Human D1, D2L, or D5 receptors were stably expressed in human
embryonic kidney cells (HEK293). Stable cell lines of HEK293
cells were generated by transfecting the plasmids coding for hD3
using Polyfectm transfection reagent (Qiagen, Hilden, Germany)
according to manufacturer's instructions and were selected
using G-418 (400 lg/mL medium). The human D4.4 receptor was
stably expressed in CHO cells. The density of D1-like receptors
measured with [3H]-SCH23390 was 3139 fmol/mg protein. For D2-
like receptors the density of receptors was 186.53 fmol/mg pro-
tein measured with [3H]-spiperone. Cells were grown at 378C
under a humidified atmosphere of 5% CO2/95% air in Dulbecco's
modified Eagles Medium Nutrient mixture F-12 Ham, supple-
mented with 10% fetal bovine serum, 1 mM L-glutamine and 0.2
lg/mL G 418 (all by Sigma-Aldrich, Germany).
Measurement of changes in intracellular [Ca2+] in HEK293
cells
The functional calcium assay based on dopamine-receptor ago-
nist induced intracellular calcium decrease. Intracellular cal-
cium is measured using the calcium binding fluorescent dye
Oregon Green [26]. Human embryonic kidney cells (HEK293) sta-
bly expressing human D1 or D2L receptors were grown at 378C
under a humidified atmosphere of 5% CO2, 95% air in Dulbecco's
modified Eagles Medium Nutrient mixture F-12 Ham, supple-
mented with 10% fetal bovine serum, 1 mM L-glutamine and 0.2
lg/mL G 418 (all by Sigma-Aldrich). Human D1 and D2L receptor
cell lines were grown on T 175 culture dishes (Greiner Bio-One)
to 85–90% confluence. The medium was removed and cells
rinsed twice with 6 mL Krebs-HEPES buffer (118 mM NaCl, 4.7
mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 4.2 mM NaHCO3, 11.7
mM D-Glucose, 1.3 mM CaCl2, 10 mM HEPES, pH 7.4) each time.
After two washes, cells were loaded with 3 lL of a 0.5 M Oregon
Green 488 BAPTA-1/AM-solution (Molecular Probes, Eugene, OR,
USA) (in DMSO) in 6 mL of the same buffer containing 3 lL of a
20% Pluronic F-127-solution (Sigma Aldrich) (in DMSO) for 45
min at 378C. After 35 min incubation, the culture dish was
rapped slightly in order to remove all cells from the dish for fur-
ther incubation. Then, 5 mL of Krebs-HEPES buffer were added
and cells were suspended. The resulting suspension was sepa-
rated in ten vials (1.5 mL) and centrifuged (10 000 rpm, 10 s), the
pellets were resuspended in 1 mL Krebs-HEPES buffer twice per
five pellets and centrifuged again. The pellets were resuspended
18 mL Krebs-HEPES buffer. And plated into 96-well plates (Opti-
Plate HTRF-96, Packard Instruments, Meriden, CT, USA; Cellstar,
Preparation of whole-cell-suspension
Human D1-like and D2-like receptor cell lines were grown on T
175 culture dishes (Greiner Bio-One, Frickenhausen, Germany)
to 85% confluency, the medium was removed and the cells were
incubated with 3 mL trypsine-EDTA-solution (Sigma-Aldrich) to
remove the cells from the culture dish. After incubation cells
were suspended in 3–6 mL added medium in order to stop the
effect of trypsine-EDTA-solution. The resulting suspension was
centrifuged (1800–2400 rpm, 48C, 4 min), the pellet resus-
pended in 10 mL PBS (ice-cooled, calcium- and magnesium-free),
pelleted, and this procedure repeated. The resulting pellet was
then resuspended in 12 mL buffer (5 mM magnesium chloride,
50 mM TRIS-HCl, pH 7.4) and the resulting suspension was
directly used for the radioligand-binding assay.
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Arch. Pharm. Chem. Life Sci. 2009, 342, 557–568
Thiazole and Isothiazole Derived Apomorphines
567
Tissue Culture Plate, 96W, Greiner Bio-One) Microplates were
kept at 378C under a humidified atmosphere of 5% CO2, 95% air
for 30 min. Screening for agonistic and antagonistic activity was
performed using a NOVOstar microplate reader (BMG LabTech-
nologies) with a pipettor system. All compounds were dissolved
in Krebs-HEPES buffer and experiments were performed at 378C.
SKF 38393 was used as standard agonist for D1 receptors and
Quinpirole for D2L receptors (final concentration: 1 lM). Dose
response curves in presence of an agonist were received after 30
min incubation of the plated cell suspension, by injecting 20 lL
of different compound dilutions (final concentrations: 50, 10, 5,
1 lM, 500 nM, 100, 50, 10, 1, 0.1 nM) into separate wells. To get
control values for every experiment, 20 lL of buffer alone and
standard agonist were injected.
Determination of antagonistic activity was conducted by pre-
incubating the cells with 20 lL of the compound dilutions (final
concentrations: 50, 10, 5, 1 lM, 500 nM, 100, 50, 10, 1, 0.1 nM) 30
min prior to injection of 20 lL standard agonist. Starting with
injection of buffer or agonist, fluorescence intensity was meas-
ured separately for each well during 30 s at 0.4 s intervals. Con-
centration-response curves were obtained by determination of
the maximum fluorescence intensity of each data set and non-
linear regression on GraphPadPrismTM 3.0. Ki values were then
calculated applying a modified Cheng-Prusoff equation [25]:
Grid maps of 90690690 points with a grid-point spacing of
0.375 ꢁ were generated using the AutoGrid tool. 250 Genetic
Algorithm (GA) runs were performed with the following param-
eters: population size of 50, maximum number of 2.56105
energy evaluations, maximum number of 27 000 generations,
an elitism of 1, a mutation rate of 0.02, and a crossover rate of
0.8.
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