Derivatives of (R)-1,11-methyleneaporphine: Synthesis, structure, and interactions with G-protein coupled receptors

Article abstract of DOI:10.1021/jm9911433

The design and synthesis of a well-characterized novel ring system, (R)- λ1,11-methyleneaporphine [(R)-4], and 15 derivatives thereof are presented. The addition of various nucleophiles to (R)-λ1,11-carbonylaporphine [(R)-11] or to the 1,11-hydroxymethyleneaporphine epimers gave separable mixtures of epimers. The epimeric ratios obtained in most reactions seem to be a result of steric factors directing the nucleophilic attack. The structure of the epimers was determined by a combination of X-ray crystallography (5 derivatives), NMR spectroscopy, and chemical correlation. Interesting and diverse pharmacological profiles of the derivatives were revealed through binding studies at serotonin 5-HT₇ and 5-HT₁(A) receptors as well as at dopamine D₂(A) receptors. Two derivatives appeared to be selective 5-HT₇ receptor antagonists. It is evident from our results that the novel ring system [(R)-4] provides a useful complement to other scaffolds available to medicinal chemists involved in studies of GPC receptors.

Full text of DOI:10.1021/jm9911433

J . Med. Chem. 2000, 43, 1339-1349
1339
Der iva tives of (R)-1,11-Meth ylen ea p or p h in e: Syn th esis, Str u ctu r e, a n d
In ter a ction s w ith G-P r otein Cou p led Recep tor s
Tero Linnanen,† Magnus Brisander,‡ Lena Unelius,§,# Go¨ran Sundholm,| Uli Hacksell, and
Anette M. J ohansson*,†
Organic Pharmaceutical Chemistry, Uppsala University, Uppsala Biomedical Center, Box 574, SE-751 23 Uppsala, Sweden,
Department of Structural Chemistry, Arrhenius Laboratory, Stockholm University, SE-106 91 Stockholm, Sweden,
Department of Lead Generation, Preclinical R&D, and Pharmaceutical and Analytical R&D, AstraZeneca R&D So¨derta¨lje,
SE-151 85 So¨derta¨lje, Sweden, and ACADIA Pharmaceuticals, 3911 Sorrento Valley Boulevard, San Diego, California 92121
Received August 9, 1999
The design and synthesis of a well-characterized novel ring system, (R)-λ1,11-methyleneapor-
phine [(R)-4], and 15 derivatives thereof are presented. The addition of various nucleophiles
to (R)-λ1,11-carbonylaporphine [(R)-11] or to the 1,11-hydroxymethyleneaporphine epimers gave
separable mixtures of epimers. The epimeric ratios obtained in most reactions seem to be a
result of steric factors directing the nucleophilic attack. The structure of the epimers was
determined by a combination of X-ray crystallography (5 derivatives), NMR spectroscopy, and
chemical correlation. Interesting and diverse pharmacological profiles of the derivatives were
revealed through binding studies at serotonin 5-HT7 and 5-HT1A receptors as well as at
dopamine D2A receptors. Two derivatives appeared to be selective 5-HT7 receptor antagonists.
It is evident from our results that the novel ring system [(R)-4] provides a useful complement
to other scaffolds available to medicinal chemists involved in studies of GPC receptors.
In tr od u ction
which can be readily functionalized and which allow the
chemist to explore the properties of the receptor site in
three dimensions.
Dopamine (DA) and serotonin (5-HT) are neurotrans-
mitters with important central as well as peripheral
functions. In the central nervous system (CNS), they
are, for example, involved in the regulation of mood,
perception, reward mechanisms, memory, behavior,
appetite, and sexual function.1 Hence, molecules which
modify dopaminergic or serotonergic brain function may
be of interest as drugs in the therapy of diseases related
to CNS dysfunction,1a,2 and the receptors for DA and
5-HT represent targets for novel CNS drugs. Currently,
5 and 14 subtypes have been identified within the
DA1a,b,2a,3 and 5-HT4 receptor families, respectively.
With one exception, the 5-HT3 receptor, a ligand gated
ion channel, these receptors belong to the G-protein
coupled (GPC) receptor superfamily.5
The 2-aminotetralin [(S)-1],7 3-aminochroman [(R)-
2],8 and aporphine [(R)-3]9 moieties have served as
useful templates for the mapping of the ligand binding
sites of certain receptors for DA and 5-HT, primarily
the D1, D2, D3, D4, and 5-HT1A receptors. The aromatic
rings of these structures can be readily functionalized
allowing well-defined structural extensions in the plane
of the ring system.
Detailed structural information providing atomic
resolution about these and other GPC receptors is still
lacking.6 Hence, ligands to DA and 5-HT receptors
cannot be designed using (direct) structure-based meth-
ods, but such efforts have to rely on established indirect
methods such as receptor mapping and quantitative
structure-activity relationships (QSAR). Alternatively,
ligands with suitable properties may be discovered by
use of a combination of combinatorial methods and
serendipity (screening). The above approaches require
access to fairly rigid template moieties which have the
potential to give derivatives with diverse pharmacology,
We have now designed a novel ring system, (R)-1,11-
methyleneaporphine [(R)-4], which can be functionalized
in space below and above the plane of the ring system,
thereby allowing for structural extension into previously
unexplored areas of the ligand binding site of the DA
and 5-HT receptors. Herein, we present our syntheses
of (R)-4 and derivatives thereof, the structures of these
derivatives as determined by a combination of X-ray
crystallography, NMR spectroscopy, and chemical cor-
relation, and their affinities for selected DA and 5-HT
receptors. The compounds are rigid, are feasible to
synthesize, and display diverse and interesting phar-
macology. This provides support for the use of (R)-4 as
* To whom correspondence should be addressed. Tel: +46 18-
4714336. Fax: +46 18-4714024. E-mail: anette@bmc.uu.se.
† Uppsala University.
‡ Stockholm University.
§ Preclinical R&D, AstraZeneca R&D So¨derta¨lje.
| Pharmaceutical and Analytical R&D, AstraZeneca R&D So¨derta¨lje.
ACADIA Pharmaceuticals.
# Present address: Regulatory Affairs, AstraZeneca R&D So¨derta¨lje,
SE-151 85 So¨derta¨lje, Sweden.
10.1021/jm9911433 CCC: $19.00 © 2000 American Chemical Society
Published on Web 03/10/2000

1340 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7
Linnanen et al.
Ta ble 1. Reaction of Ketone (R)-11 with Nucleophiles
Sch em e 1a
T
yield epimeric
(%)a ratioa,b
entry reagents (°C)
products
1
2
4
NaBH4
rt (6aR,12R)-12/(6aR,12S)-12 90
45:55
35:65
35:65
LiAlH4 -70 (6aR,12R)-12/(6aR,12S)-12 88
MeLi
-70 (6aR,12R)-13/(6aR,12S)-13 95
a
b
The mean of 2-4 experiments. The epimeric ratio was
determined by HPLC of the crude reaction mixture.
a
Reagents: (a) (CF3SO2)2NPh, Et3N, K2CO3, CH2Cl2; (b) Bu3N,
HCO2H, (PPh3)2PdCl2, dppp, DMF, N2; (c) MeSO3H, N2.
Sch em e 2a
F igu r e 1. Stereoscopic representation of the lowest energy
MM3 conformation of (R)-11 showing the slightly vaulted
structure of the 1,11-methyleneaporphine structure. For clarity
the psuedoaxial nitrogen lone pair has been added and the
hydrogens have been omitted.
in this reaction, and several reaction conditions were
tried in order to minimize the formation of the byprod-
uct. By using N,N,N′,N′-tetramethylethylenediamine
(TMEDA) as base in DMF at 70 °C, the amount of the
reduced product was suppressed to <1% as indicated
by 1H NMR. The benzyl ester was easily converted into
the corresponding amino acid by hydrogenolysis (Pd/C,
H2, 1 atm, room temperature). The crude amino acid
was treated with polyphosphoric acid (PPA)11 to give the
novel ketone (R)-11 in 72% yield from (R)-9.
Ad d ition of Nu cleop h iles to (R)-11 (Ta ble 1).
Ketone (R)-11 was used as starting material for the
synthesis of several novel derivatives as outlined in
Scheme 2. The methylene derivative (R)-4 was obtained
by using classical conditions for hydrogenolysis of
benzylic ketones (Pd/C, HCl, H2).12
Reduction of (R)-11 with NaBH4 consistently gave a
45:55 mixture of the epimeric alcohols (6aR,12R)-12 and
(6aR,12S)-12 (Table 1). Several other reducing agents
(LiAlH4, K-selectride, Bu4NBH4, i-Bu3BHK, i-Bu2AlH)
were tried in attempts to increase the stereoselectivity
of the reduction. A slight increase in the stereoselectivity
and a high yield were obtained by use of LiAlH4 in THF
at -70 °C. Attempts to form the alcohols by catalytic
hydrogenation of (R)-11 failed; either no reduction
[Ru(C)] or complete reduction [PtO2 and Pd(C)] to (R)-4
was observed. Addition of MeLi in cumene/THF (9:1)
to (R)-11 afforded isomers (6aR,12R)-13 and (6aR,12S)-
13 in a ratio of 35:65.13
Nucleophilic addition of a hydride or a methyl anion
to (R)-11 consistently gave a higher ratio of the (6aR,-
12S)-isomer. The diastereoselective formation of (6aR,-
12S)-12 and (6aR,12S)-13 might be due to a preferred
nucleophilic attack at the least hindered re-face of the
prochiral carbonyl carbon of (R)-11; the structure of (R)-
11 is slightly vaulted in the direction of the pseudoaxial
lone pair of the nitrogen (Figure 1). Hence, the confor-
mation of (R)-11 may make the opposite face of the
a
Reagents: (a) BnOH, Pd(OAc)2, dppp, TMEDA, CO, DMF; (b)
i. Pd/C (10%), 1,4-dioxane, H2O, H2, ii. PPA; (c) Pd/C (10%), HCl
(1 M), EtOH, H2; (d) NaBH4, MeOH/H2O (1:1); (e) MeCN, H2SO4
(concd); (f) HCl (12 M); (g) MeLi, THF; (h) NaBH4, TFA; (i)
(MeO)3CH, H2SO4 (concd), MeOH; (j) EtMgBr, THF; (k) (EtO)3CH,
p-TsOH, EtOH.
a scaffold in medicinal chemistry studies of interactions
between ligands and GPC receptors.
Syn th esis
Syn th esis of Key In ter m ed ia te (R)-11. The syn-
thesis of the (R)-1,11-methyleneaporphine derivatives
is outlined in Schemes 1 and 2. Our synthetic strategy
utilizes the known triflate (R)-99t to introduce a car-
boxylic acid function at C11 of (R)-aporphine. A subse-
quent intramolecular electrophilic addition of the car-
boxylic acid function to the C1 position gave the key
intermediate (R)-11.
Triflate (R)-9 was synthesized from natural morphine
in four steps (Scheme 1).9q,t Conversion of (R)-9 into the
benzyl ester (R)-10 was accomplished by a palladium-
catalyzed carbonylative coupling reaction.10 Various
amounts of the reduced byproduct (R)-3 were formed

Derivatives of (R)-1,11-Methyleneaporphine
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7 1341
Ta ble 2. Acid-Catalyzed Addition of Nucleophiles to Alcohols 12 and 13
entry
1
alcohol
reagent
MeCN/H2SO4
products
yield (%)a
73
epimeric ratioa,b
65:35
(6aR,12R)-12/(6aR,12S)-12
(45:55)
(6aR,12R)-12
(6aR,12S)-12
(6aR,12R)-13/(6aR,12S)-13
(40:60)
(6aR,12R)-14/(6aR,12S)-14
2
3
4
MeCN/H2SO4
MeCN/H2SO4
NaBH4/TFA
(6aR,12R)-14/(6aR,12S)-14
(6aR,12R)-14/(6aR,12S)-14
(6aR,12R)-16/(6aR,12S)-16
ndc
nd
46
65:35
65:35
35:65
5
6
7
(6aR,12R)-13/(6aR,12S)-13
(40:60)
(6aR,12R)-13/(6aR,12S)-13
(35:65)
(6aR,12R)-13/(6aR,12S)-13
(40:60)
Et3SiH/TFA
(6aR,12R)-16/(6aR,12S)-16
(6aR,12R)-16/(6aR,12S)-16
(6aR,12R)-17/(6aR,12S)-17
23
90
70
30:70
2:98
H2/Pd(C)/HCl/EtOH
(MeO)3CH/MeOH/H2SO4
55:45
8
9
(6aR,12R)-13
(MeO)3CH/MeOH/H2SO4
(MeO)3CH/MeOH/H2SO4
(6aR,12R)-17/(6aR,12S)-17
(6aR,12R)-17/(6aR,12S)-17
nd
nd
50:50
65:35
(6aR,12S)-13d
a
b
The mean of 2-4 experiments. The epimeric ratio was determined by HPLC of the crude reaction mixture. c nd ) not determined.
d
Contains <5% of (6aR,12R)-13.
molecule more accessible for nucleophilic attack. The
mixture in agreement with the corresponding methoxy
methyl derivatives (17) indicating a similar reaction
mechanism. Acetamides (6aR,12R)-14 and (6aR,12S)-
14 were converted into the corresponding amines (6aR,-
12R)-15 and (6aR,12S)-15 by acid hydrolysis.
The separation of the epimeric mixtures was in most
cases performed by flash column chromatography. The
isomers of 17 were, however, separated by straight-
phase preparative HPLC using a silica gel column (see
Experimental Section). The separation of the epimers
of 16 and 18 was difficult and could not be accomplished
by the above-mentioned procedures. Instead semiprepar-
ative HPLC was performed. Both pairs of epimers were
readily chromatographed on a Hypercarb column in the
straight-phase mode. Excellent separation was achieved
with R-values of 2.89 and 3.88 for (6aR,12R)-18/(6aR,-
12S)-18 and (6aR,12R)-16/(6aR,12S)-16, respectively.
small but consistent difference in stereoselectivity ob-
tained with the two nucleophiles, a hydrid and a methyl
anion, is probably related to the size of the nucleophile.
Acid -Ca ta lyzed Nu cleop h ilic Ad d ition to Alco-
h ols 12 a n d 13 (Ta ble 2). The epimeric acetamides 14
were synthesized from a mixture of (6aR,12R)-12/(6aR,-
12S)-12 (45:55) by a Ritter reaction14 to give a 65:35
ratio of (6aR,12R)-14 and (6aR,12S)-14. This reaction
is believed to proceed via a carbocation intermediate,14
and experiments with the pure isomers (6aR,12R)-12
and (6aR,12S)-12 as substrates produced a 65:35 mix-
ture of (6aR,12R)-12 and (6aR,12S)-12, respectively,
supporting the involvment of a common carbocation
intermediate. Such an intermediate is probably also
involved in the reduction of the mixture of the tertiary
alcohols (6aR,12R)-13 and (6aR,12S)-13 (40:60) by
NaBH4/TFA which gave a 35:65 mixture of (6aR,12R)-
16 and (6aR,12S)-16, i.e., the same ratio (35:65) as the
Ritter reaction.15 Indeed, this reaction is also presumed
to proceed via a carbocation intermediate.16 A similar
diastereoselectivity was obtained when (6aR,12R)-13/
(6aR,12S)-1315 (40:60) was reduced with triethylsilane
in TFA. The epimeric ratio obtained in these reactions
seems to be a result of steric factors directing the
nucleophilic attack (vide supra), the presumed inter-
mediate cation being more accessible from the re-face
of C12.
Hydrogenolysis (Pd/C, H2, HCl, EtOH, 1 atm, room
temperature) of (6aR,12R)-13/(6aR,12S)-13 (35:65) pro-
ceeded stereoselectively to give a mixture of (6aR,12R)-
16/(6aR,12S)-16 in a 2:98 ratio. Addition of sulfuric acid
and trimethyl orthoformate to (6aR,12R)-13/(6aR,12S)-
13 (40:60) in methanol gave (6aR,12R)-17/(6aR,12S)-
17 in a ratio of 55:45. When the pure isomers (6aR,12R)-
13 and (6aR,12S)-1317 were reacted under the same
conditions, 50:50 and 65:35 mixtures of (6aR,12R)-17/
(6aR,12S)-17 were formed, respectively. The outcome
of these reactions indicates that (6aR,12S)-13 probably
reacts via a carbocation intermediate while the reaction
path of (6aR,12R)-13 is different.
Str u ctu r a l Stu d ies
The stereochemistry of derivatives 12-18 was estab-
lished by a combination of X-ray crystallography, 13C
NMR spectroscopy, and chemical correlation. The rela-
tive stereochemistry of (6aR,12R)-12‚HCl, (6aR,12S)-
12‚HCl, (6aR,12S)-15‚2HCl‚H2O, (6aR,12S)-16‚HCl‚
MeCN, and (6aR,12R)-17‚HCl was determined by X-ray
crystallography. 13C NMR chemical shift correlation was
used in order to assign the structure of the epimers of
13 and 18. The stereochemistry of the epimers of 14 was
indirectly assigned from the epimers of 15. The CD
spectra of compound 4 and epimers 12-17 were also
studied in an attempt to determine the stereochemistry
Treatment of (R)-11 with EtMgBr in ether gave a 50:
50 mixture of unstable epimeric ethyl-substituted alco-
hols, which were converted into a 50:50 mixture of the
ethyl-substituted ethoxy derivatives (6aR,12R)-18/(6aR,-
12S)-18 by an acid-catalyzed reaction with triethyl
orthoformate in ethanol. The conversion of the ethyl
alcohols into the ethoxy ethyl derivatives gave a product

1342 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7
Linnanen et al.
Ta ble 3. Crystal Data and Details of the Final Structure Refinement Calculation for (6aR,12R)-12‚HCl, (6aR,12S)-12‚HCl,
(6aR,12S)-15‚2HCl‚H2O, (6aR,12S)-16‚HCl‚MeCN, and (6aR,12R)-17‚HCl
(6aR,12R)-12‚HCl (6aR,12S)-12‚HCl (6aR,12S)-15‚2HCl‚H2O (6aR,12S)-16‚HCl‚MeCN (6aR,12R)-17‚HCl
formula
C
18H18ClNO
C18H18ClNO
299.78
orthorhombic
P212121
8.310(4)
11.833(3)
14.881(3)
90
1463.2(9)
4
1.361(1)
150(2)
2478
C18H22Cl2N2O
353.28
orthorhombic
P212121
7.071(1)
10.893(1)
23.004(2)
90
1771.9(3)
4
1.324(1)
100(2)
14016
3436
3006
275
0.040
0.105
C21H23ClN2
338.86
orthorhombic
P212121
6.993(1)
14.988(3)
17.269(4)
90
1810.0(6)
4
1.244(1)
150(2)
3861
C20H22ClNO
327.84
monoclinic
P21
10.329(9)
7.109(3)
11.813(7)
101.58(9)
849.7(10)
2
1.281(2)
100(2)
6520
3112
2089
formula weight
crystal system
space group
a, Å
b, Å
c, Å
299.78
orthorhombic
P212121
9.965(3)
12.101(5)
12.209(4)
90
1472.3(8)
4
1.352(1)
150(2)
2483
â (deg)
Vc, Å3
Z
Dc, g‚cm-1
T, K
N (measured)
N (unique)
N [Fo > 4σ(Fo)]
no. of parameters
R [Fo > 4σ(Fo)]
wR (F2) for all
unique data
goodness-of-fit for all
unique data
2458
1679
262
0.033
2453
1788
262
0.031
3645
2158
288
0.066
276
0.041
0.091
0.081
0.079
0.186
1.04
1.06
1.05
1.05
0.87
at C12. However, no consistent effect of diagnostic value
was obtained.
The solid-state conformation and the relative stereo-
chemistry at C12 in the five-membered ring of five
epimers were determined using single-crystal X-ray
diffraction techniques. Thermal ellipsoid plots of the
solid-state conformations are shown in Figure 2, and
selected experimental data are collected in Table 3. The
solid-state conformations of the studied pentacyclic
structures are, as expected, very similar (Figure 2).
Derivatives (6aR,12R)-12 and (6aR,12S)-16 gave the
best fit with a root-mean-square (rms) deviation of 0.037
Å between the carbon atoms and the nitrogen atom in
the pentacyclic skeleton, while the largest rms devia-
tion, 0.120 Å, was observed between molecules (6aR,-
12S)-15 and (6aR,12R)-17. The formation of the five-
membered ring makes the molecules more rigid and
planar as compared to (R)-3. The two aromatic rings
are almost coplanar with a torsion angel τ(C11-C11a-
C11b-C1) of about -2° to -3° in the crystal structures
(Table 4).
For comparison, a database search for aporphine
derivatives, substituted with hydrogen at either C1 or
C11, in the Cambridge Structural Database18 (CSD) was
performed. A total number of 13 structures (17 confor-
mations) was found. These structures have noncoplanar
aromatic rings and an average τ(C11-C11a-C11b-C1)
value of 24(4)°,19 which is in agreement with results
obtained from (R)-3. A fit between the average structure
of the solid-state conformations of (6aR,12R)-12‚HCl,
(6aR,12S)-12‚HCl, (6aR,12S)-15‚2HCl‚H2O, (6aR,12S)-
16‚HCl‚MeCN, and (6aR,12R)-17‚HCl and an average
structure of (R)-3 calculated from the structures ob-
tained in the CSD search clearly shows the difference
in the 3D-structure between these two classes of apor-
phine derivatives (Figure 3).
The conformational distributions of the nonprotonated
(6aR)-isomers of 11-18 and 4 were studied by molecular
mechanics MM3 calculations.20 The results indicate that
the 1,11-methyleneaporphine structure mainly adopts
four conformations: the most stable conformation has
a pseudoequatorial N-methyl group and adopts a half-
chair conformation of the tetrahydropyridine ring with
F igu r e 2. Perspective view (ORTEP) of the solid-state
structure of (6aR,12R)-12‚HCl (A), (6aR,12S)-12‚HCl (B),
(6aR,12S)-15‚2HCl (C), (6aR,12S)-16‚HCl (D), and (6aR,12R)-
17‚HCl (E). The cocystallized CH3CN in (6aR,12S)-16‚HCl‚
CH3CN and H2O in (6aR,12S)-15‚2HCl‚H2O are left out for
clarity. Displacement ellipsoids are represented at the 50%
probability level.
P-helicity. It constitutes about 90%21 of the conforma-
tions. The conformational distribution of the epimers
of 12-18 was found to be the same.
Minimization (MM3) of the solid-state conformation
of (6aR,12R)-12‚HCl, (6aR,12S)-12‚HCl, (6aR,12S)-15‚
2HCl‚H2O, (6aR,12S)-16‚HCl‚MeCN, and (6aR,12R)-17‚
HCl in their nonprotonated form resulted in the most

Derivatives of (R)-1,11-Methyleneaporphine
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7 1343
Ta ble 4. Observed and Calculated Values of τ(C11-C11a-C11b-C1) in (6aR,12R)-12‚HCl, (6aR,12S)-12‚HCl,
(6aR,12S)-15‚2HCl‚H2O, (6aR,12S)-16‚HCl‚MeCN, (6aR,12R)-17‚HCl, and the (R)-Aporphine Skeleton
(R)-aporphine
skeleton
(6aR,12S)-15‚
2HCl‚H2O
(6aR,12S)-16‚
HCl‚MeCN
method
(6aR,12R)-12‚HCl
(6aR,12S)-13‚HCl
(6aR,12R)-17‚HCl
crystal structure
ab initiob
-24(4)a
-24.4
-23.5
-2.5(3)
-3.2
-4.1
-2.8(3)
-3.1
-4.2
-1.8(5)
-2.9
-4.2
-1.8(3)
-3.1
-4.2
-2.9(3)
-2.9
-4.2
MM3c
a
Average value, calculated for crystal structures of aporphine derivatives substituted with hydrogen at either the C1 or C11 position
b
and included in the CSD (see Supporting Information). Ab initio geometry optimization at the RHF/6-31* level of the derivatives in
their nonprotonated state. c Molecular mechanics MM3 minimization of the derivatives in their nonprotonated state.
of 13 and 18 was used to assign their respective
stereochemistry.
In ter a ction s w ith GP C Recep tor s
The compounds were examined in vitro for their
ability to displace [3H]5-HT, [3H]8-OH-DPAT, and [3H]-
raclopride binding to cloned rat 5-HT7 receptors and to
F igu r e 3. Stereoscopic representation of a rms fit between
the average solid-state structure of (6aR,12R)-12‚HCl, (6aR,-
cloned human 5-HT1A and D2A receptors, respectively,
12S)-12‚HCl, (6aR,12S)-15‚2HCl‚H2O, (6aR,12S)-16‚HCl‚CH3-
as described in the Experimental Section. The results
CN, and (6aR,12R)-17‚HCl (solid lines) and the average
are presented in Table 6, and the affinities of (R)-3, (R)-
aporphine structure calculated from the solid-state structures
19, (R)-apomorphine, (R)-8-OH-DPAT, (S)-UH301, and
5-CT to some of these receptors are also included.
found in the CSD search (dashed lines). The carbon atoms C7a,
C8, C9, C10, C11, and C11a and the nitrogen atom are fitted
with unit weight for all atoms.
Ta ble 5. Selected 13C NMR Spectral Dataa of Diagnostic Value
for Assignment of the Stereochemistry at C12 of the
1,11-Methyleneaporphine Derivatives
δ (ppm)
compd
R
R1
C1
C11
(6aR,12R)-12
(6aR,12S)-12
(6aR,12R)-13
(6aR,12S)-13
(6aR,12R)-14
(6aR,12S)-14
(6aR,12R)-15
(6aR,12S)-15
(6aR,12R)-16
(6aR,12S)-16
(6aR,12R)-17
(6aR,12S)-17
(6aR,12R)-18
(6aR,12S)-18
(R)-4
OH
H
H
139.5
140.6
143.8b
144.6b
138.1
139.3
141.0
142.1
143.3
143.6
140.0
141.2
139.7b
141.2b
139.1
143.3
144.2
147.4b
148.6b
141.9
142.4
144.6
145.3
146.6
146.9
143.9
144.2
143.6b
144.2b
141.0
OH
Me
OH
H
NHAc
H
NH2
H
Me
Me
OMe
Et
OH
Me
NHAc
H
NH2
H
Me
H
OMe
Me
OEt
Et
The methylene derivative (R)-4 and the unsubstituted
aporphine (R)-3 exhibit similar affinities to 5-HT1A and
D2A receptors, (R)-4 being slightly more potent (Table
6). At 5-HT7 receptors, however, the affinity of (R)-4 is
about 12 times higher than that of (R)-3. Hence, the
added bulk of the methylene group in (R)-4 and the
geometrical difference between the two ring systems,
which is a consequence of the ring strain imposed by
the fusion of a five-membered ring onto (R)-4, do not
appear to lead to a major difference in the interaction
with the 5-HT1A and D2A receptors. However, it seems
to be beneficial for the interaction with 5-HT7 receptors.
The introduction of substituents at C12 in (R)-4, i.e.,
above and/or below the plane of the ring system,
produces ligands with great variations in pharmacologi-
cal profiles: a C12-acetamido group [(6aR,12S)-14]
reduces affinity to 5-HT7 receptors 190-fold whereas the
simultaneous introduction of methyl and methoxy groups
[(6aR,12R)-17 and (6aR,12S)-17] leads to a 6-fold in-
crease in 5-HT7 receptor affinity. Similarly, the affinities
for 5-HT1A and D2A receptors are affected by the
OEt
H
H
a
Unambiguously assigned by 1H-1H, 1H-13C, and COLOC
correlation experiments. Assigned by 13C NMR chemical shift
b
correlation.
stable conformation as identified by the molecular
mechanics MM3 calculations. Ab initio geometry opti-
mization at the RHF/6-31* level confirmed the MM3
minimizations. Consequently, both experimental and
computational methods give the same preferred confor-
mation of these rigid structures (Table 4).
The 13C NMR chemical shifts of the pentacyclic
derivatives 12 and 14-17 have unambiguously been
assigned by a combination of 1H-1H, 1H-13C, and long-
range 1H-13C (COLOC22) correlation experiments.
Throughout, a downfield shift of C1 and C11 was
obtained for the (12S)-isomer (Table 5). Therefore, the
observed chemical shift difference between the epimers

1344 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7
Linnanen et al.
Ta ble 6. Affinities of 1,11-Methyleneaporphine Derivatives for Cloned Rat 5-HT7 and Cloned Human 5-HT1A and D2A Receptors
Labeled by [3H]5-HT, [3H]8-OH-DPAT, and [3H]Raclopride, Respectively
Ki (nM)a
compd
R
R1
[3H]5-HT (5-HT7)
[3H]8-OH-DPAT (5-HT1A
)
[3H]raclopride (D2A
)
(R)-4
(R)-11
H
H
H
OH
Me
OH
H
NHAc
H
NH2
H
6.9 ( 0.3
291 ( 110
13.5 ( 3.2
103 ( 16
40.7 ( 12
140 ( 33b
31.0 ( 3.5b
1210 ( 91b
315 ( 110b
61.5 ( 9.9b
204 ( 12
716 ( 12
142 ( 72
355 ( 32
5.05 ( 1.2
31.0 ( 8.0
17.3 ( 2.3
16.9 ( 6.9
1.84 ( 0.2
19.1 ( 0.1
80.0 ( 2.3b,c
14.4 ( 1.5b,c
296 ( 15b,d
1.30
83.2 ( 9.9
317 ( 170
23.8 ( 1.7
215 ( 12
182 ( 6.0
26.0 ( 2.0
3650 ( 660
7980 ( 1200
261 ( 53
2250 ( 550
19.4 (n ) 1)
39.2 ( 1.6
7.1 ( 0.3
71.0 ( 7.5
70.8 (n ) 1)
24.5 ( 0.8
527 ( 296c
201 ( 91c
41.6 ( 4.7d
819
dO
(6aR,12R)-12
(6aR,12S)-12
(6aR,12R)-13
(6aR,12S)-13
(6aR,12R)-14
(6aR,12S)-14
(6aR,12R)-15
(6aR,12S)-15
(6aR,12R)-16
(6aR,12S)-16
(6aR,12R)-17
(6aR,12S)-17
(6aR,12R)-18
(6aR,12S)-18
(R)-3
OH
H
OH
Me
NHAc
H
NH2
H
Me
H
27.7 ( 2.9
4.3 ( 0.9
538 ( 8.5
1320 ( 31
13.4 ( 2.9
18.0 ( 1.2
3.36 ( 1.1
5.6 ( 1.0
1.1 ( 0.03
1.1 ( 0.04
25.7 ( 0.66
3.05 ( 0.7
88.0 ( 3.7
38.3 ( 0.03
188 ( 17
Me
Me
OMe
Et
OMe
Me
OEt
Et
OEt
(R)-19
(R)-apomorphine
(R)-8-OH-DPAT
(S)-UH301
8.81 ( 0.02
3180 ( 50
0.029 ( 0.01
87
<0.3
3760
5-CT
a
b
The Ki values are means ( standard errors of 2-3 experiments. Data obtained from competition experiments with rat brain 5-HT1A
receptor recognition site. c From ref 9t. From ref 9q.
d
introduction of C12 substituents in (R)-4: the affinity
to D2A receptors can be decreased 95-fold [(6aR,12S)-
14] or increased almost 12-fold [(6aR,12R)-17] and the
affinity to 5-HT1A receptors can be reduced 30-fold
[(6aR,12S)-12] or increased 22-fold [(6aR,12R)-18] by
the introduction of the proper substituents.
tivity of interaction of the epimers with the 5-HT7
receptor was smaller than that observed with the other
two receptors.
The observed preferences in affinity of the novel
derivatives at 5-HT7, 5-HT1A, and D2A receptors are
interesting. The selectivity of compounds (6aR,12S)-15
and (6aR,12S)-17 for 5-HT7 receptors is especially
intriguing since only two ligands with selectivity for
5-HT7 receptors, 20 and 21, have been published so
far.23 These two ligands are antagonists and preliminary
data obtained in second-messenger studies indicate that
also (6aR,12S)-15 and (6aR,12S)-17 are 5-HT7 receptor
antagonists. In a preliminary profiling effort of (6aR,-
12S)-15 the following affinities were obtained: serotonin
5-HT2A 469 ( 102 nM ([3H]ketanserine, n ) 3); adren-
ergic R1 373 ( 25 nM ([3H]prazocin, n ) 2); adrenergic
R2 92.8 ( 3 nM ([3H]RX821002, n ) 2); adrenergic â
>1000 nM ([3H]DHA, n ) 1); muscarinic M >1000 nM
([3H]QNB, n ) 2); and dopamine D1 >1000 nM ([3H]-
SCH23390, n ) 2). Hence, (6aR,12S)-15 in particular
and also (6aR,12S)-17 may be considered as valuable
As mentioned above, the unsubstituted (R)-4 has a
higher affinity for 5-HT7 receptors than for 5-HT1A and
D
2A receptors. This receptor selectivity may be increased
by introducing an amino group at C12 [(6aR,12S)-15]
or partially reversed by substituting C12 with ethyl and
ethoxy groups [(6aR,12R)-18], the latter compound
exhibiting selectivity for 5-HT1A receptors. Whereas
(6aR,12S)-15 and (6aR,12R)-18 displayed selectivity for
5-HT7 and 5-HT1A receptors, respectively, no derivative
within the present series of compounds displayed D2A
receptor selectivity. However, several of the derivatives
exhibit selectivity for two of the three receptor subtypes
studied: for example, (6aR,12S)-12 and (6aR,12R)-17
have higher affinity for the 5-HT7 and D2A receptors
than for the 5-HT1A receptor. Only one epimer, (6aR,-
12R)-12, has a similar affinity for the three receptors
studied.
Throughout, the epimers generated by the introduc-
tion of substituents at C12 in the 1,11-methyleneapor-
phine ring system interacted with at least one of the
three receptors in a stereoselective fashion. The highest
stereoselectivity and the largest variation in stereose-
lectivity were observed in the interaction with the
5-HT1A receptor: the affinity of the epimers of 12
differed 39-fold whereas the epimers of 17 had equal
affinity at this receptor. The most pronounced stereo-
selectivity at the D2A receptor was displayed by the
epimers of 12, 15, and 17. In general, the stereoselec-

Derivatives of (R)-1,11-Methyleneaporphine
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7 1345
1,4-dioxane (40 mL) and water (8 mL) was vigorously stirred
under an atmosphere of H2 at room temperature for 5 h. The
catalyst was filtered off and the volatiles were evaporated.
Polyphosphoric acid (25 g) was added to the residue and the
slurry was mechanically stirred at 90 °C for 30 min. H2O (300
mL) was added and the pH of the mixture was brought to pH
8-9 by addition of NaOH(s). The aqueous phase was extracted
with CH2Cl2. The combined organic layers was dried (MgSO4),
filtered and concentrated. The residue was chromatographed
[silica; CHCl3/MeOH (95:5)]. The amine was converted into the
hydrochloride salt, which was recrystallized (MeOH/Et2O) to
give 2.64 g (83%) of pure (R)-11‚HCl: IR (KBr) 1712 cm-1
structural leads in future efforts to obtain selective
5-HT7 receptor drugs.
It is evident that the pharmacology displayed by the
1,11-methyleneaporphine derivatives exhibits a large
variation and, hence, a large information content. The
pharmacology of the present series has not been suf-
ficiently studied, and the series is not large enough to
allow us to derive final conclusions regarding SAR.
Nevertheless, it is apparent that the novel ring system
presented herein provides a useful complement to the
arsenal of scaffolds used in medicinal chemistry studies
of GPC receptors. Its well-defined structure and relative
ease of synthesis add to its potential value in this
context.
23
(CdO); mp 231-234 °C; [R]D -98° [c 1.0, MeOH/H2O (1:1)].
Anal. (C18H15NO‚HCl) C, H, N.
(R)-11: 1H NMR δ 2.52 (s, 3H), 2.65 (ddd, J ) 12, 12, 4 Hz,
1H), 2.73-2.92 (m, 2H), 3.08 (ddd, J ) 17.5, 12, 6 Hz, 1H),
3.18 (dd, J ) 12, 6 Hz, 1H), 3.30 (dd, J ) 16, 7 Hz, 1H), 3.57
(dd, J ) 13, 7 Hz, 1H), 6.94 (d, J ) 7.5 Hz, 1H), 7.15 (dd, J )
7.5, 7 Hz, 1H), 7.23 (d, J ) 7.6 Hz, 1H), 7.31 (d, J ) 7.5 Hz,
1H), 7.35 (d, J ) 7 Hz, 1H); 13C NMR δ 28.0, 31.0, 43.3, 55.1,
60.9, 123.0, 123.5, 126.7, 128.5, 129.2, 129.3, 130.1, 132.2,
133.3, 140.8, 141.2, 194.4. One 13C-signal is missing due to
overlapping resonances.
Exp er im en ta l Section
Ch em istr y. Gen er a l Com m en ts. Melting points (uncor-
rected) were determined in open glass capillaries using an
Electrothermal melting point apparatus. Optical rotation
measurements were obtained on a polarimeter. Elemental
analyses (C, H, N) were performed at Mikrokemi AB, Uppsala,
Sweden, and were within (0.4% of the theoretical values.
Accurate mass determination was perfomed on a quadrupole
time-of-flight hybrid instrument equipped with electrospray
ionization. The 1H and 13C NMR spectra of the amines were
recorded at 399.8 and 100.5 MHz, respectively, and were
referenced to internal tetramethylsilane. Spectra were re-
corded using a mixture of CDCl3 and CD3OD (90:10 v/v) at 25
°C. Infrared (IR) spectra were recorded on a FTIR spectro-
photometer. Thin-layer chromatography (TLC) was performed
by using aluminum sheets precoated (0.2 mm) with either
silica gel 60 F254 (Riedel-de Hae¨n) or aluminum oxide 60 F254
neutral (E. Merck). Column chromatography was performed
on silica gel 60 (0.040-0.063 mm; Riedel-de Hae¨n or E. Merck)
or aluminum oxide 90 (0.063-0.2 mm, deactivated with 5%
water; E. Merck). In preparative HPLC a Dynamax 60-A
column (21.4 × 250 mm, 8-µm silica gel, flow 13 mL/min) was
used, and in semipreparative HPLC a Hypercarb S column
(100 × 10 mm, 7-µm particles, flow 1.5 mL/min) guarded with
a Hypercarb S stationary-phase precolumn was used. Diaste-
reoselectivities were determined by analytical HPLC using
LiChrospher C18 (4 × 250 mm, 5-µm particles, flow 0.7 mL/
min) or Nucleosil C18 (4.6 × 100 mm, 5-µm particles, flow 0.7
mL/min) columns. The eluent was either 0.1 M phosphate
buffer (pH 3.2)/MeOH/acetonitrile/N,N-dimethyloctylamine
(DMOA) (500:350:150:1) or 0.05 M phosphate buffer (pH 6.0)/
acetonitrile/DMOA (400:600:1 or 600:400:1).
(-)-(R)-1,11-Meth ylen ea p or p h in e [(R)-4]. A mixture of
(R)-11 (116 mg, 0.44 mmol), Pd/C (10%, 56 mg, 0.053 mmol
Pd), aqueous 1 M HCl (10 mL) and EtOH (20 mL) was
vigorously stirred under an atmosphere of H2 at room tem-
perature for 8 h. The catalyst was filtered off and the volatiles
were evaporated. Aqueous saturated NaHCO3 was added to
the residue and the mixture was extracted with CH2Cl2. The
combined organic layers was dried (K2CO3), filtered and
concentrated. The amine was converted into the hydrochloride
salt, which was recrystallized (acetonitrile/Et2O) to give 93 mg
22
(84%) of (R)-4‚HCl: mp 246-249 °C dec; [R]D -25° [c 1.0,
MeOH/H2O (1:1)]. Anal. (C18H17N‚HCl‚1/4H2O) C, H, N.
(R)-4: 1H NMR δ 2.56 (s, 3H), 2.66-2.86 (m, 2H), 2.93 (dd,
J ) 14.5, 14.5 Hz, 1H), 3.12-3.25 (m, 2H), 3.41 (dd, J ) 15.5,
6.5 Hz, 1H), 3.77 (dd, J ) 13, 6.5 Hz, 1H), 3.95 (d, J ) 21 Hz,
1H), 3.83 (d, J ) 21 Hz, 1H), 6.99 (d, J ) 7.5 Hz, 1H), 7.14 (d,
J ) 7.5 Hz, 1H), 7.20 (dd, J ) 7.5, 7.5 Hz, 1H), 7.30 (d, J )
7.5 Hz, 1H), 7.36 (d, J ) 7.5 Hz, 1H); 13C NMR δ 27.4, 32.1,
37.9, 43.6, 55.8, 62.4, 123.4, 123.6, 125.2 126.4 (2C), 127.5,
129.1, 131.5, 137.6, 137.9, 139.1, 141.0.
(-)-(6a R ,12R )-1,11-(H yd r oxym e t h yle n e )a p or p h in e
[(6a R,12R)-12] a n d (-)-(6a R,12S)-1,11-(Hyd r oxym eth yl-
en e)a p or p h in e [(6a R,12S)-12]. A mixture of (R)-11‚HCl (168
mg, 0.56 mmol), NaBH4 (250 mg, 6.6 mmol) and 50% aqueous
MeOH (10 mL) was stirred at room temperature for 5 min.
Aqueous HCl (5 M) was added until no more H2 evolution
occurred. The mixture was alkalinized with aqueous saturated
NaHCO3. The aqueous phase was extracted with CHCl3 and
the combined organic layers was dried (K2CO3), filtered and
concentrated to give a mixture of (6aR,12R)-12 and (6aR,12S)-
12 (45:55). The epimers (6aR,12R)-12 and (6aR,12S)-12 were
separated by chromatography [Al2O3; EtOAc/CHCl3/EtOH (90:
10:0 followed by 89:10:1)]. The amines were converted into the
hydrochloride salts and recrystallized from MeOH to give 75
mg (44%) of (6aR,12S)-12‚HCl and 54 mg (32%) of (6aR,12R)-
12‚HCl.
(-)-(R)-11-Ben zyloxycar bon ylapor ph in e [(R)-10]. A mix-
ture of Pd(OAc)2 (171 mg, 0.76 mmol) and 1,3-bis(diphen-
ylphosphine)propane (348 mg, 0.84 mmol) in DMF (25 mL)
was stirred under N2 for 30 min. Half of the catalyst-ligand
mixture was added to a solution of (R)-99t (2.92 g, 7.6 mmol),
TMEDA (3.4 mL, 20 mmol) and benzyl alcohol (10 mL) in DMF
(25 mL). The reaction mixture was heated at 70 °C and CO
was bubbled through the mixture. After 2 h the other half of
the catalyst-ligand mixture was added to the reaction mix-
ture. After another 2 h at 70 °C the volatiles were evaporated
and the oily residue was chromatographed [Al2O3; EtOAc
followed by Al2O3; EtOAc/hexanes (1:4)]. The amine was
converted into the hydrochloride salt to give 2.45 g (79%) of
pure (R)-10‚HCl: IR (KBr) 1718 cm-1 (CdO); mp 132-134 °C;
23
(6a R,12R)-12‚HCl: mp 274-277 °C dec; [R]D -21° [c 0.2,
MeOH/H2O (1:1)]. Anal. (C18H17NO‚HCl) C, H, N.
(6a R,12R)-12: 1H NMR δ 2.44 (s, 3H), 2.39-2.78 (m, 3H),
2.96-3.13 (m, 2H), 3.18 (dd, J ) 15.5, 6.5 Hz, 1H), 3.51 (dd, J
) 13, 6.5 Hz, 1H), 5.62 (s, 1H), 6.91 (d, J ) 7.5 Hz, 1H), 7.05
(d, J ) 7.5 Hz, 1H), 7.12 (dd, J ) 7.5, 7 Hz, 1H), 7.29 (d, J )
7.5 Hz, 1H), 7.35 (d, J ) 7.0 Hz, 1H); 13C NMR δ 27.2, 31.3,
43.3, 55.6, 62.1, 77.1, 123.4, 123.7, 126.0 127.1, 127.3, 128.3,
128.9, 133.7, 136.1, 137.0, 139.5, 143.3.
22
[R]D -164° [c 1.0, MeOH/H2O (1:1)]. Anal. (C21H23NO2‚HCl)
H, C, N.
(R)-10: 1H NMR δ 2.49-2.70 (m, 2H), 2.58 (s, 3H), 2.80
(dd, J ) 16, 3.5 Hz, 1H), 3.03-3.33 (m, 4H), 5.18, (d, J ) 12
Hz, 1H), 5.34 (d, J ) 12 Hz, 1H), 6.96-7.11 (m, 3H), 7.20-
7.35 (m, 6H), 7.39 (ddd, J ) 7.5, 1.0, 1.0 Hz, 1H), 7.55 (ddd, J
) 7.5, 1.0, 1.0 Hz, 1H); 13C NMR δ 28.6, 34.5, 43.9, 52.9, 61.6,
67.3, 125.8, 126.4, 127.1 128.3, 128.4, 128.5, 128.6,128.7, 130.1,
130.7, 131.8, 132.8, 134.0, 134.3, 135.3, 137.0, 170.7.
(-)-(R)-1,11-Ca r bon yla p or p h in e [(R)-11]. A mixture of
(R)-10 (4.34 g, 10.7 mmol), Pd/C (10%, 1.23 g, 1.2 mmol Pd),
23
(6a R,12S)-12‚HCl: mp 310-311 °C dec; [R]D -44° [c 0.2,
MeOH/H2O (1:1)]. Anal. (C18H17NO‚HCl) C, H, N.
(6a R,12S)-12: 1H NMR δ 0.99 (dd, J ) 14, 14.5 Hz, 1H),
2.26 (s, 3H), 2.35-2.59 (m, 2H), 2.65 (dd, J ) 16, 7 Hz, 1H),
2.88-3.07 (m, 2H), 3.27 (dd, J ) 13.5, 7 Hz, 1H), 5.62 (s, 1H),
6.62 (d, J ) 7.5 Hz, 1H), 6.81 (d, J ) 7.5 Hz, 1H), 7.13 (dd, J

1346 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7
Linnanen et al.
) 7.5, 7.5 Hz, 1H), 7.20 (d, J ) 7.5 Hz, 1H), 7.38 (d, J ) 7.5
Hz, 1H); 13C NMR δ 27.0, 30.3, 43.0, 55.4, 61.0, 77.8, 122.4,
122.7, 125.4 127.1, 127.2, 128.0 129.2, 132.9, 135.0, 137.0
140.6, 144.2.
2.84 (m, 3H), 3.02-3.32 (m, 2H), 3.35 (dd, J ) 15.5, 6.5 Hz,
1H), 3.69 (dd, J ) 13, 6.5 Hz, 1H), 6.28 (s, 1H), 6.95 (d, J )
7.5 Hz, 1H), 7.05 (app d, 0.3H), 7.12 (d, J ) 7.5 Hz, 1H), 7.18
(dd, J ) 7.5, 7 Hz, 1H), 7.25 (d, J ) 7.5 Hz, 1H), 7.31 (d, J )
7 Hz, 1H); 13C NMR δ 22.9, 27.4, 31.9, 43.6, 55.6, 57.3 (2C),
61.7, 123.2, 123.4, 126.4, 126.9, 127.1, 128.4, 129.0, 133.5,
136.5, 137.9, 139.3, 142.4, 171.7 (2C).
(-)-(6aR,12R)-1,11-(Am in om eth ylen e)apor ph in e [(6a R,-
12R)-15]. A solution of (6aR,12R)-14 (95.7 mg, 0.32 mmol) in
concentrated aqueous HCl (15 mL) was heated in a closed vial
at 120 °C for 80 h. Aqueous saturated NaHCO3 was added
and the aqueous phase was extracted with CHCl3. The
combined organic layers was dried (K2CO3), filtered and
concentrated. The residue was chromatographed [silica; CHCl3/
MeOH/aqueous NH3 (900:95:5)] and the diamine was converted
into the dihydrochloride salt, which was recrystallized [aceto-
nitrile/EtOH (1:1)] to give 43 mg (52%) of (6aR,12R)-15‚2HCl:
mp 239-242 °C dec; [R]D23 -16° [c 1.0, MeOH/H2O (1:1)]. Anal.
(C18H17N2‚2HCl‚1/2H2O) C, H, N.
(-)-(6a R,12R)-1,11-(1-H yd r oxyet h ylid en e)a p or p h in e
[(6a R,12R)-13] a n d (-)-(6a R,12S)-1,11-(1-H yd r oxyet h -
ylid en e)a p or p h in e [(6a R,12S)-13]. A solution of (R)-11 (189
mg, 0.72 mmol) in dry THF (20 mL) under an atmosphere of
N2 was cooled to -70 °C. MeLi (0.80 M,24 0.94 mL, 0.75 mmol)
in THF/cumene (1:9) was added during 5 min. The reaction
temperature was allowed to rise to room temperature and
aqueous saturated NaHCO3 was added. The aqueous phase
was extracted with CH2Cl2 and the combined organic layers
was dried (Na2SO4), filtered and concentrated to give a mixture
of (6aR,12R)-13 and (6aR,12S)-13 (35:65). The epimers were
separated by column chromatography [silica; isohexanes/
CHCl3/MeOH/Et2NH (200:189:10:1)]. The amines were con-
verted into the oxalate salts to give 77 mg (38%) of (6aR,12S)-
13‚oxalate and 40 mg (20%) of (6aR,12R)-13‚oxalate. The
isomer (6aR,12R)-13‚oxalate contains <5% of (6aR,12S)-13 as
(6a R,12R)-15: 1H NMR δ 2.55 (s, 3H), 2.70 (ddd, J ) 11.5,
10.5, 4.5 Hz, 1H), 2.80 (dd, J ) 17, 4 Hz, 1H), 2.84 (dd, J )
14.5, 14.5 Hz, 1H), 3.08-3.24 (m, 2H), 3.36-3.40 (m, 1H), 3.72
(dd, J ) 13.5, 6.5 Hz, 1H), 4.98 (s, 1H), 7.02, (d, J ) 7.5 Hz,
1H), 7.16, (d, J ) 7.5 Hz, 1H), 7.22 (dd, J ) 7.5, 7 Hz, 1H),
7.32-7.38 (m, 1H), 7.40 (d, J ) 7 Hz, 1H); 13C NMR δ 27.3,
31.6, 43.5, 55.7, 60.3, 62.3, 122.8, 123.2, 126.4, 127.0, 127.3,
128.3, 129.2, 133.4, 136.2, 137.2, 141.0, 144.6.
(-)-(6aR,12S)-1,11-(Am in om eth ylen e)apor ph in e [(6aR,-
12S)-15]. Compound (6aR,12S)-15 was synthesized from
(6aR,12S)-14 (60.3 mg, 0.18 mmol) using the same method as
described for the synthesis of (6aR,12R)-15 to give 52 mg (88%)
of (6aR,12S)-15‚2HCl: mp 244-248 °C dec; [R]D23 -36° [c 1.0,
MeOH/H2O (1:1)]. Anal. (C18H17N2‚2HCl‚1H2O) C, H, N.
(6a R,12S)-15: 1H NMR δ 2.55 (s, 3H), 2.70 (ddd, J ) 12,
11, 4 Hz, 1H), 2.80 (dd, J ) 16, 4.5 Hz, 1H), 2.88 (dd, J )
14.5, 14.5 Hz, 1H), 3.09-3.23 (m, 2H), 3.39-3.49 (m, 1H), 3.73
(dd, J ) 13, 6.5 Hz, 1H), 5.09 (s, 1H), 7.01, (d, J ) 7.5 Hz,
1H), 7.15, (d, J ) 7.5 Hz, 1H), 7.23 (dd, J ) 7.5, 7.5 Hz, 1H),
7.31-7.37 (m, 1H), 7.39 (d, J ) 7 Hz, 1H); 13C NMR δ 27.4,
32.1, 43.6, 55.6, 60.6, 61.8, 122.5, 122.9, 126.5, 126.7, 127.1,
128.5, 129.2, 133.3, 136.1, 137.5, 142.1, 145.3.
(-)-(6a R,12S)-1,11-E t h ylid en ea p or p h in e [(6a R,12S)-
16]. A mixture of (6aR,12R)-13‚oxalate/(6aR,12S)-13‚oxalate
(40:60) was synthesized using the same method as described
above for the synthesis of (6aR,12R)-13 and (6aR,12S)-13. Pd/C
(10%, 62 mg, 0.057 mmol Pd) and aqueous 1 M HCl (15 mL)
was added to (6aR,12R)-13/(6aR,12S)-13‚oxalate [(40:60), 213
mg, 0.58 mmol] in EtOH (30 mL). The reaction mixture was
vigorously stirred under an atmosphere of H2 for 8 h. The
catalyst was filtered off and the volatiles were evaporated.
Aqueous saturated NaHCO3 was added to the residue and the
mixture was extracted with CH2Cl2. The combined organic
layers was dried (K2CO3), filtered through Al2O3 and concen-
trated. The amine was converted into the hydrochloride salt
and recrystallized (acetonitrile/Et2O) to give 155 mg (90%) of
(6aR,12S)-16‚HCl: mp 198-200 °C; [R]D23 -18° [c 1.0, MeOH/
H2O (1:1)]. Anal. (C19H19N‚HCl‚1/4H2O) C, H, N.
1
determined by H NMR.
(6a R,12R)-13‚oxa la te: 95% pure; mp 178-182 °C dec;
[R]D23 -11° [c 1.0, MeOH/H2O (1:1)]. Anal. (C19H19NO‚C2H2O4‚
1/2H2O) C, H, N.
1
(6a R,12R)-13: 95% pure; H NMR δ 1.60 (s, 3H), 2.30 (s,
3H), 2.37-2.67 (m, 3H), 2.83-3.03 (m, 2H), 3.08 (dd, J ) 16,
7 Hz, 1H), 3.41 (dd, J ) 13, 6 Hz,1H), 6.82 (d, J ) 7.5 Hz,
1H), 6.93 (d, J ) 7.5 Hz, 1H), 7.02 (dd, J ) 7.5, 7 Hz, 1H),
7.11 (d, J ) 7.5 Hz, 1H), 7.16 (d, J ) 7 Hz, 1H); 13C NMR δ
25.4, 27,1, 31.3, 43.2, 55.5, 61.9, 83.1, 121.4, 121.7, 126.0, 127.2,
127.3, 128.5, 128.9, 133.5, 134.8, 135.8, 143.8, 147.4.
23
(6a R,12S)-13‚oxa la te: mp 232-234 °C dec; [R]D -18° [c
1.0, MeOH/H2O (1:1)]. Anal. (C19H19NO‚C2H2O4) C, H, N.
(6a R,12S)-13: 1H NMR δ 0.75 (dd, J ) 14.5, 14.5 Hz, 1H),
1.68 (s, 3H), 2.21 (s, 3H), 2.31-2.63 (m, 3H), 2.82-3.05 (m,
2H), 3.24 (dd, J ) 13, 7 Hz, 1H), 6.59 (d, J ) 7.5 Hz, 1H), 6.78
(d, J ) 7.5 Hz, 1H), 7.10 (d, J ) 7.5 Hz, 1H), 7.13 (dd, J ) 7.5,
7.5 Hz, 1H), 7.30 (d, J ) 7.5 Hz, 1H); 13C NMR δ 26.0, 27.1,
29.9, 43.1, 55.4, 61.1, 83.0, 120.5 120.9, 125.4 127.1, 127.1,
128.2, 129.4, 132.7, 133.6, 135.8, 144.6, 148.6.
(+)-(6a R,12R)-1,11-(Acet a m id om et h ylen e)a p or p h in e
[(6a R,12R)-14] a n d (-)-(6a R,12S)-1,11-(Aceta m id om eth -
ylen e)a p or p h in e [(6a R,12S)-14]. A mixture of (6aR,12R)-
12/(6aR,12S)-12 (45:55) was synthesized using the same
method as described for the synthesis of (6aR,12R)-12 and
(6aR,12S)-12. Acetonitrile (2 mL, 49 mmol) and concentrated
H2SO4 (0.7 mL, 10 mmol) were added to (6aR,12R)-12/(6aR,-
12S)-12 [(45:55) 336 mg, 1.3 mmol] and the reaction mixture
was stirred at 70 °C for 5 min. Aqueous saturated NaHCO3
was added and the mixture was extracted with CH2Cl2. The
combined organic layers was dried (K2CO3), filtered and
concentrated to give a mixture of (6aR,12R)-14 and (6aR,12S)-
14 (65:35). The epimers were separated by chromatography
[silica; CHCl3/MeOH (100:0 followed by 90:10) and silica;
CHCl3/isohexanes/MeOH (45:45:10)]. The amines were con-
verted into the hydrochloride salts and recrystallized (EtOH/
Et2O) to give 91 mg (19%) of (6aR,12S)-14‚HCl and 189 mg
(40%) of (6aR,12R)-14‚HCl.
(6a R,12S)-16: 1H NMR δ 1.48 (d, J ) 7.5 Hz, 3H), 2.56 (s,
3H), 2.64-2.84 (m, 2H), 2.92 (dd, J ) 14.5, 14.5 Hz, 1H), 3.09-
3.24 (m, 2H), 3.40 (dd, J ) 15.5, 6.5 Hz, 1H), 3.76, (dd, J )
13, 6.5 Hz, 1H), 4.11 (q, J ) 7.5 Hz, 1H), 6.99 (d, J ) 7.5 Hz,
1H), 7.12 (d, J ) 7.5 Hz, 1H), 7.21 (dd. J ) 7.5, 7.5 Hz, 1H),
7.25 (d, J ) 7.5 Hz, 1H), 7.30 (d, J ) 7.5 Hz, 1H); 13C NMR δ
17.1, 27.4, 32.2, 43.6, 44.9, 55.8, 62.2, 122.3, 122.5, 125.4 126.3,
126.5, 127.8 129.0, 131.6, 136.6, 137.9, 143.6, 146.9.
(-)-(6a R,12R)-1,11-E t h ylid en ea p or p h in e [(6a R,12R)-
16]. An ice-cold solution of trifluoroacetic acid (2 mL) and
NaBH4 (81 mg, 2.1 mmol) was added to (6aR,12R)-13‚oxalate/
(6aR,12S)-13‚oxalate [(40:60), 137 mg, 0.37 mmol]. The reac-
tion mixture was stirred at 0 °C for 5 min. Aqueous saturated
NaHCO3 was added and the aqueous phase was extracted with
CH2Cl2. The combined organic layers was dried (K2CO3),
filtered and concentrated. The amine was converted into the
hydrochloride salt, which was recrystallized (acetonitrile/Et2O)
(6a R,12R)-14‚HCl: IR (KBr) 1635 cm-1 (CdO); mp 234-
23
237 °C; [R]D +19° (c 1.0, MeOH). Anal. (C20H20N2O‚HCl‚5/
4H2O) C, H, N.
(6a R,12R)-14: 1H NMR δ 2.05 (s, 3H), 2.51 (s, 3H), 2.61
(ddd, J ) 11, 9.5, 4 Hz, 1H), 2.74 (dd, J ) 15.5, 5 Hz, 1H),
2.85 (dd, J ) 13, 13 Hz, 1H) 3.04-3.23 (m, 2H), 3.32 (dd, J )
15.5, 7 Hz, 1H), 3.62 (dd, J ) 13, 7 Hz, 1H), 6.10 (app.d, 1H),
6.85 (app d, 0.6H), 6.97 (d, J ) 7.5 Hz, 1H), 7.11-7.21 (m,
2H), 7.28 (d, J ) 7.5 Hz, 1H), 7.31-7.37 (m, 1.4H); 13C NMR
δ 22.9, 27.4, 31.6, 43.5, 55.6, 56.9 (2C), 62.2, 123.6, 123.9, 126.3,
127.2, 127.3, 128.4, 129.1, 133.6, 136.7, 137.6, 138.1, 141.9,
171.6 (2C).
(6a R,12S)-14‚HCl: IR (KBr) 1636 cm-1 (CdO); mp 222-
23
226 °C dec; [R]D -37° (c 1.0, MeOH). Anal. (C20H20N2O‚HCl‚
5/4H2O) C, H, N.
(6a R,12S)-14: 1H NMR δ 2.04 (s, 3H), 2.52 (s, 3H), 2.62-

Derivatives of (R)-1,11-Methyleneaporphine
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7 1347
to give 51 mg (46%) of a 35:65 mixture of (6aR,12R)-16‚HCl/
(6aR,12S)-16‚HCl. The epimeric amines (6aR,12R)-16‚HCl and
(6aR,12S)-16‚HCl (39 mg) were separated by semipreparative
HPLC [Hybercarb S; MeOH/CH2Cl2/Et2NH (2000:8000:1)]. The
amines were converted into the hydrochloride salts to give 10
mg of (6aR,12R)-16‚HCl and 20 mg of (6aR,12S)-16‚HCl.
amines was converted into the hydrochloride salt and recrys-
tallized (acetonitrile/Et2O) to give 89 mg (23%) of a 50:50
mixture of (6aR,12R)-18‚HCl/(6aR,12S)-18‚HCl. The epimeric
amines (6aR,12R)-18‚HCl and (6aR,12S)-18‚HCl (40 mg) were
separated by semipreparative HPLC [Hypercarb S; MeOH/
CH2Cl2/Et2NH (8000:2000:1)]. The amines were converted into
the hydrochloride salts to give 16 mg of (6aR,12R)-18‚HCl and
12 mg of (6aR,12S)-18‚HCl.
23
(6a R,12R)-16‚HCl: mp 206-213 °C dec; [R]D -3° [c 1.0,
MeOH/H2O 1.1)]; HRMS m/z (M+ + 1) calcd 262.1596, obsd
262.1591; 1H NMR (CD3OD) δ 1.53 (d, J ) 7.5 Hz, 3H), 3.00-
3.28 (m, 2H), 3.20 (s, 3H), 3.29-3.51 (m, 1H) 3.68-3.82 (m,
2H), 3.96 (ddd, J ) 12.5, 6, 1.5 Hz, 1H) 4.04-4.16 (m, 1H),
5.00 (dd, J ) 13, 6.5 Hz, 1H), 7.16 (d, J ) 7.5 Hz, 1H), 7.24 (d,
J ) 7.5 Hz, 1H), 7.28 (dd, J ) 7.5, 7.5 Hz, 1H),7.40 (d, J ) 7.5
Hz, 1H), 7.45 (d, J ) 7.5 Hz, 1H); 13C NMR δ 18.0, 26.4, 30.2,
41.7, 46.6, 56.8, 64.0, 121.7, 124.4, 126.2, 126.8, 127.8, 128.1,
130.3, 130.5, 137.6, 138.2, 146.0, 148.4.
22
(6a R,12R)-18‚HCl: mp 188-192 °C; [R]D -1° [c 1.0,
MeOH/H2O (50:50)]; HRMS m/z (M+ + 1) calcd 320.2014, obsd
320.2012.
(6a R,12R)-18: 1H NMR δ 0.58 (t, J ) 7.5 Hz, 3H), 1.06 (t,
J ) 7 Hz, 3H), 2.01-2.18 (m, 2H), 2.59 (s, 3H), 2.73-2.97 (m,
2H), 3.01-3.31 (m, 3H), 3.36 (dd, J ) 15, 6.5 Hz, 1H), 3.66 (q,
J ) 7 Hz, 2H), 3.82 (dd, J ) 13, 6.5 Hz, 1H), 7.00 (d, J ) 7.5
Hz, 1H), 7.14 (d, J ) 7.5 Hz, 1H), 7.16-7.22 (m, 2H), 7.24 (d,
J ) 7.5 Hz, 1H); 13C NMR δ 8.2, 15.7, 27.2, 31.4, 32.0, 43.1,
55.6, 60.0, 62.0, 92.7, 122.7, 123.2, 125.6, 127.3, 127.3, 128.4,
128.6, 133.5, 136.5, 137.2, 139.7, 143.6.
(-)-(6a R,12R)-1,11-(1-Met h oxyet h ylid en e)a p or p h in e
[(6a R,12R)-17] a n d (-)-(6a R,12S)-1,11-(1-Met h oxyet h -
ylid en e)a p or p h in e [(6a R,12S)-17]. Concentrated H2SO4
(520 mg, 5.3 mmol) in MeOH (2 mL) was added dropwise to a
solution of (6aR,12R)-13‚oxalate/(6aR,12S)-13‚oxalate [(40:60),
301 mg, 0.82 mmol] and (MeO)3CH (2 mL) in MeOH (4 mL).
The reaction mixture was stirred at room temperature for 2
min, aqueous saturated NaHCO3 was added and the volatiles
were evaporated. The aqueous phase was extracted with
CHCl3 and the combined organic layers was dried (K2CO3),
filtered and concentrated to give a mixture of (6aR,12R)-17
and (6aR,12S)-17 (55:45). The epimeric mixture was chro-
matographed [silica; CHCl3/MeOH (95:5)] and the epimers
were separated by preparative HPLC [silica; isohexanes/EtOH/
Et3N (898:100:2)]. The amines were converted into the hydro-
chloride salts and recrystallized (acetonitrile/Et2O) to give 72
mg (27%) of (6aR,12S)-17‚HCl and 80 mg (30%) of (6aR,12R)-
17‚HCl.
22
(6a R,12S)-18‚HCl: mp 221-223 °C dec; [R]D -12° [c 1.0,
MeOH/H2O (1:1)]; HRMS m/z (M+ + 1) calcd 320.2014, obsd
320.2016.
(6a R,12S)-18: 1H NMR δ 0.82 (t, J ) 7.5 Hz, 3H), 1.01 (t,
J ) 7 Hz, 3H), 2.11 (q, J ) 7.5 Hz, 2H), 2.60 (s, 3H), 2.72-
2.97 (m, 3H), 3.10-3.31 (m, 2H), 3.39 (dd, J ) 14.5, 6.5 Hz,
1H), 3.47 (q, J ) 7 Hz, 2H), 3.82 (dd, J ) 12.5, 6.5 Hz, 1H),
6.98 (d, J ) 7.5 Hz, 1H), 7.13 (d, J ) 7.5 Hz, 1H), 7.16-7.22
(m, 2H), 7.24 (d, J ) 7.5 Hz, 1H); 13C NMR δ 8.6, 15.6, 27.2,
31.6, 32.3, 43.2, 55.5, 59.9, 61.7, 93.2, 122.6, 123.2, 125.7, 127.0,
127.1, 128.4, 128.7, 133.4, 136.2, 137.4, 141.2, 144.2.
X-r a y Cr ysta llogr a p h y. The reflection intensities for
(6aR,12R)-12‚HCl, (6aR,12S)-12‚HCl, and (6aR,12S)-16‚HCl‚
CH3CN were measured with a STOE/AED2 diffractometer and
ω - 2θ scan technique, and the intensity data for (6aR,12S)-
15‚2HCl‚H2O and (6aR,12R)-17‚HCl were collected using a
STOE image plate detector system and φ oscillation technique.
Data reductions included corrections for background and
Lorentz and polarization effects. The structures were solved
by application of direct methods (SHELXS25) and refined with
full-matrix least-squares technique based on F2 for all observa-
tions (SHELXL-9326). The non-hydrogen atoms were refined
as vibrating anisotropic. The hydrogen atoms were located in
difference electron density maps and their positions were
refined, whereas their isotropic vibration parameters Uiso were
set to 1.2‚Ueq (methyl groups 1.5‚Ueq) of the parent non-
hydrogen atom. The geometric calculations were performed
using the programs SHELXL-9326 and PLATON.27 Crystal
data and selected details of the refinement calculations are
listed in Table 3.
Com p u ta tion a l Meth od s. An automated conformational
analysis (MM3) of the novel pentacyclic derivatives in their
nonprotonated state was performed using the BatchMin
program included in the MacroModel v5.5 package.28 A 2000-
step Monte Carlo search was performed on each compound.
Geometry optimizations of the solid-state conformations of the
nonprotonated (6aR,12R)-12, (6aR,12S)-12, (6aR,12S)-15, (6aR,-
12S)-16, and (6aR,12R)-17 and of the (R)-aporphine skeleton
were performed in SPARTAN29 at the RHF/6-31G* level with
a net charge of zero and by molecular mechanics calculations
using the MM3 force field included in MacroModel.
P h a r m a cology: Recep tor Bin d in g Assa ys. The DA D2A
receptor binding assay was performed as described previously30
by measuring the ability of the compounds to displace [3H]-
raclopride binding from cloned human D2A receptors expressed
in mouse fibroblast (Ltk-) cells. The 5-HT1A receptor binding
assay was performed using cloned human 5-HT1A receptors
expressed in Chinese hamster ovary (CHO) cells or as de-
scribed earlier using rat hippocampus.31 The same Kd-value
of the radioligand [3H]8-OH-DPAT was obtained both in
hippocampus and in the cell line. The CHO cells expressing
human 5-HT1A receptors were obtained from Dr. P. G. Strange
(University of Reading, Reading, U.K.). Membranes were
prepared as described previously,30 resuspended in binding
buffer (in mM: 50 Tris-HCl, 4 MgCl2, pH 7.4) and stored in
23
(6a R,12R)-17‚HCl: mp 246-250 °C dec; [R]D -14° [c 1.0,
MeOH/H2O (1:1)]. Anal. (C20H21NO‚HCl) C, H, N.
(6a R,12R)-17: 1H NMR δ 1.68 (s, 3H), 2.55 (s, 3H), 2.67-
2.90 (m, 3H), 2.94 (s, 3H), 3.07-3.23 (m, 2H), 3.36 (dd, J )
15.5, 6.5 Hz, 1H), 3.75 (dd, J ) 13, 6.5 Hz, 1H), 7.00 (d, J )
7.5 Hz, 1H), 7.14 (d, J ) 7.5 Hz, 1H), 7.20 (dd, J ) 7.5, 7.5 Hz,
1H), 7.20 (d, J ) 7.5 Hz, 1H), 7.26 (d, J ) 7.5 Hz, 1H); 13C
NMR δ 25.3, 27.5, 31.7 43.5, 52.3, 55.6, 62.0, 89.2, 122.3, 122.5,
126.2, 127.4, 127.5, 128.6, 129.1, 134.0, 135.9, 136.9, 140.0
143.9.
23
(6a R,12S)-17‚HCl: mp 235-237 °C dec; [R]D -19° [c 1.0,
MeOH/H2O (1:1)]. Anal. (C20H21NO‚HCl) C, H, N.
(6a R,12S)-17: 1H NMR δ 1.76 (s, 3H), 2.55 (s, 3H), 2.65-
2.85 (m, 2H), 2.76 (s, 3H), 2.90 (dd, J ) 14, 14 Hz, 1H), 3.08-
3.23 (m, 2H), 3.37 (dd, J ) 15.5, 7 Hz, 1H), 3.71 (dd, J ) 13,
7 Hz, 1H), 6.99 (d, J ) 7.5 Hz, 1H), 7.14 (d, J ) 7 Hz, 1H),
7.20 (dd, J ) 7.5, 8 Hz, 1H), 7.20 (d, J ) 7.5 Hz, 1H), 7.25 (d,
J ) 7 Hz, 1H); 13C NMR δ 25.6, 27.5, 31.7 43.5, 51.2, 55.6,
61.8, 89.9, 121.9, 122.4, 126.5, 127.2, 127.3, 128.5, 129.0, 134.0,
135.8, 137.0, 141.2, 144.2.
(-)-(6a R,12R)-1,11-(1-E t h oxyp r op ylid en e)a p or p h in e
[(6a R,12R)-18] a n d (-)-(6a R,12S)-1,11-(1-Eth oxyp r op y-
lid en e)a p or p h in e [(6a R,12S)-18]. EtMgBr in THF (5.5 M,
0.40 mL, 2.2 mmol) was added to a solution of (R)-11 (284 mg,
1.1 mmol) in dry THF (10 mL) kept at -70 °C and under an
atmosphere of N2. After one min at -70 °C, H2O (2 mL) was
added and the reaction temperature was allowed to rise to 0
°C. Aqueous saturated NaHCO3 was added and the mixture
was extracted with CH2Cl2. The combined organic layers was
filtered through a pad of K2CO3 and concentrated to give a
mixture of epimeric ethyl substituted alcohols (50:50). (EtO)3CH
(1.5 mL), EtOH (4.5 mL) and p-toluenesulfonic acid (120 mg,
0.70 mmol) were added to the residue and after stirring for 1
h at room temperature additional p-toluenesulfonic acid (120
mg, 0.70 mmol) and (EtO)3CH (1.5 mL) were added. After 5 h
aqueous saturated NaHCO3 was added and the mixture was
extracted with CH2Cl2 The combined organic layers was dried
(K2CO3), filtered and concentrated to give a mixture of (6aR,-
12R)-18 and (6aR,12S)-18 (50:50). The residue was chromato-
graphed [silica; CHCl3/MeOH (98:2)] and the mixture of

1348 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7
Linnanen et al.
Smith, G.; DeKosky, S. T.; Pollock, B. G.; Mathis, C. A.; Moore,
R. Y.; Kupfer, D. J .; Reynolds, C. F., III. Serotonin in Aging,
Late-Life Depression, and Alzheimer’s Disease: The Emerging
Role of Functional Imaging. Neuropsychopharmacology 1998, 18,
407-430. (n) Barnes, N. M.; Sharp, T. A review of central 5-HT
receptors and their function. Neuropharmacology 1999, 38,
1083-1152.
aliquots at -70 °C. The frozen cell membranes were thawed,
homogenized with Ultra Turrax, and suspended in binding
buffer. The binding assays were performed in a total volume
of 0.5 mL with a receptor concentration of about 40 pM. 1 nM
[3H]8-OH-DPAT (127 Ci/mmol) was incubated with nonlabeled
ligand for 60 min at 30 °C. Binding in the presence of WAY-
100635 (10 µM) was defined as nonspecific. The incubations
were terminated by rapid filtration through Whatman GF/B
glass fiber filters (Maidstone, England) using a Tomtec cell
harvester. The filters were washed with ice-cold buffer (5 mM
Tris-HCl, pH 7.4), and the radioactivity was determined in a
Packard 2500TR liquid scintillation analyzer or in a Wallac
Betaplate counter. The serotonin 5-HT7 receptor binding assay
was performed using cloned rat 5-HT7 receptors expressed in
CHO cells or Sf9 cells. The same Kd-value of the radioligand,
[3H]5-HT, was obtained in both cell lines. The CHO cell
membranes were prepared as described previously30 and above
for the 5-HT1A receptor binding. The Sf9 cell membranes were
used as obtained from BioSignal (Montreal, Canada). The
5-HT7 receptor binding assay was performed essentially as the
5-HT1A receptor binding assay described above, with the
following modifications: Membranes were suspended in bind-
ing buffer (in mM: 50 Tris-HCl, 4 MgCl2, 1 EDTA, pH 7.4).
The binding assays were performed in a total volume of 1 mL
with a receptor concentration of 30-40 pM. [3H]5-HT (0.8 nM,
103 Ci/mmol) was incubated with nonlabeled ligand for 60 min
at 30 °C. Binding in the presence of methiothepin (10 µM) was
defined as nonspecific. The binding data were analyzed by
nonlinear regression using the LIGAND program.32
(2) For DA receptors as drug targets, see for example: (a) Hartman,
D. S.; Civelli, O. Dopamine Receptor Diversity: Molecular and
Pharmacological Perspectives. Prog. Drug Res. 1997, 48, 173-
194. For 5-HT receptors as drug targets, see for example: (b)
Hamon, M. Neuropharmacology of Anxiety: Perspectives and
Prospects. Trends Pharmacol. Sci. 1994, 15, 36-39. (c) Dourish,
C. T. Multiple Serotonin Receptors: Opportunities for New
Treatments for Obesity? Obes. Res. 1995, 3, 229S-462S. (d)
Stahl, S. M. Mixed Depression and Anxiety: Serotonin1A Recep-
tors as a Common Pharmacologic Link. J . Clin. Psychiatry 1997,
58 (Suppl. 8), 20-26. (e) Goadsby, P. J . Serotonin Receptors and
the Acute Attack of Migraine. Clin. Neurosci. 1998, 5, 18-23.
(3) (a) J ackson, D. M.; Westlind-Danielsson, A. Dopamine Recep-
tors: Molecular Biology, Biochemistry and Behavioural Aspects.
Pharmacol. Ther. 1994, 64, 291-370. (b) Lachowicz, J . E.; Sibley,
D. R. Molecular Characteristics of Mammalian Dopamine Re-
ceptors. Pharmacol. Toxicol. 1997, 81, 105-113.
(4) (a) Peroutka, S. J . Molecular Biology of Serotonin (5-HT)
Receptors. Synapse 1994, 18, 241-260. (b) Boess, F. G.; Martin,
I. L. Molecular Biology of 5-HT Receptors. Neuropharmacology
1994, 33, 275-317. (c) Hoyer, D.; Martin, G. 5-HT Receptor
Classification and Nomenclature: Towards a Harmonization
with the Human Genome. Neuropharmacology 1997, 36, 419-
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Binding of 2-Aminotetralins to Human Dopamine D2A and D3
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Dopamine D2, D3, and D4 Receptors of 2-Aminotetralins. Rel-
evance of D2 Agonist Binding for Determination of Receptor
Subtype Selectivity. J . Med. Chem. 1996, 39, 4233-4237.
(8) For recent studies, see: (a) Comoy, C.; Marot, C.; Podona, T.;
Baudin, M.-L.; Morin-Allory, L.; Guillamet, G.; Pfeiffer, B.;
Caignard, D.-H.; Renard, P.; Rettori, M.-C.; Adam, G.; Guardiola-
Lemaitre, B. 3-Amino-3,4-dihydro-2H-1-Benzopyran Derivatives
as 5-HT1A Receptor Ligands and Potential Anxiolytic Agents. 2.
Synthesis and Quantitative Structure-Activity Relationship
Studies of Spiro[pyrrolidine- and piperidine-2,3′(2′H)-benzopy-
rans]. J . Med. Chem. 1996, 39, 4285-4298. (b) J ohansson, L.;
Sohn, D.; Thorberg, S.-O.; J ackson, D. M.; Kelder, D.; Larsson,
L.-G.; Re´nyi, L.; Ross, S. B.; Wallsten, C.; Eriksson, H.; Hu, P.-
Ack n ow led gm en t. We gratefully acknowledge Dr.
Magnus J o¨rnte´n-Karlsson for providing the HRMS data,
Dr. Ingeborg Cso¨regh for valuable discussions concern-
ing X-ray crystallography, Prof. J an Sandstro¨m for help
with the CD spectroscopy, and the skillful technical
assistance by Anne-Marie Eriksson, Gun Torell-Svant-
esson, and Susanne Rosqvist in the binding assays.
Financial support was obtained from Astra Arcus AB.
Su p p or tin g In for m a tion Ava ila ble: Tables of crystal
data, structure solution and refinement, atomic coordinates,
and anisotropic displacement parameters for (6aR,12R)-12‚
HCl, (6aR,12S)-12‚HCl, (6aR,12S)-15‚2HCl‚H2O, (6aR,12S)-
16‚HCl‚MeCN, and (6aR,12R)-17‚HCl; also conformational
features of selected aporphine derivatives included in the CSD.
This material is available free of charge via the Internet at
http://pubs.acs.org.
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