Identification of fluorinated (R)-(?)-aporphine derivatives as potent and selective ligands at serotonin 5-HT2C receptor

Article abstract of DOI:10.1016/j.bmcl.2018.11.050

A series of novel aporphine derivatives were synthesized for initial screening at the 5-HT₂ receptor subtypes. Among them, Compounds 11a and 11b were identified as potent 5-HT_2C hit ligands with high selectivity over other 5-HT₂ receptor subtypes. Molecular docking study revealed that compounds 11a and 11b formed two key interactions with the binding site of 5-HT_2C receptor, including a salt-bridge to D3.32 and a H-bond interaction with N6.55.

Full text of DOI:10.1016/j.bmcl.2018.11.050

Accepted Manuscript
Identification of Fluorinated (R)-(-)-Aporphine Derivatives as Potent and Se-
lective Ligands at Serotonin 5-HT2C Receptor
Yulong Xu, Anna W. Sromek, John L. Neumeyer
PII:
S0960-894X(18)30926-0
https://doi.org/10.1016/j.bmcl.2018.11.050
BMCL 26161
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
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Accepted Date:
26 October 2018
23 November 2018
Please cite this article as: Xu, Y., Sromek, A.W., Neumeyer, J.L., Identification of Fluorinated (R)-(-)-Aporphine
Derivatives as Potent and Selective Ligands at Serotonin 5-HT2C Receptor, Bioorganic & Medicinal Chemistry
Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.11.050
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Identification of Fluorinated (R)-(-)-Aporphine Derivatives as
Potent and Selective Ligands at Serotonin 5-HT2C Receptor
Yulong Xu a,b, Anna W. Sromek a,b and John L. Neumeyer *,a,b
a
Division of Basic Neuroscience, Medicinal Chemistry Program, Mclean Hospital,
Belmont, Massachusetts 02478, United States
b
Department of Psychiatry, Harvard Medical School, Boston, Massachusetts 02115,
United States
 Corresponding author. Tel.: 617-855-3388; Fax: 617-855-2519; E-mail:
jneumeyer@mclean.harvard.edu (J. L. Neumeyer)
Abstract: A series of novel aporphine derivatives were synthesized for initial
screening at the 5-HT2 receptor subtypes. Among them, Compounds 11a and 11b
were identified as potent 5-HT2C hit ligands with highly selectivity over other 5-HT2
receptor subtypes. Molecular docking study revealed that compounds 11a and 11b
formed two key interactions with the binding site of 5-HT2C receptor, including a salt-
bridge to D3.32 and a H-bond interaction with N6.55.
Keywords: Aporphine derivatives; 5-HT2C receptor; Hit ligands; Molecular docking
The serotonin (5-hydroxytryptamine, 5-HT) system is known to play a critical
role in the regulation of numerous neurological functions.1,2 The current classification
of the serotonin receptor family includes at least fourteen receptor subtypes classified
into seven major classes (5-HT1-7) based upon the conjunction of genetic and

molecular structure, intracellular transduction mechanisms, and pharmacological
criteria.3 All of these serotonin receptor subtypes are G-protein-coupled receptors
(GPCR) with the exception of the ligand-gated ion channel 5-HT3.4 The 5-HT2
receptor subtype family is composed of three GPCR members (5-HT2A, 5-HT2B, 5-
HT2C) which share a high level of amino acid sequence homology within
transmembrane regions.5 In particular, the 5-HT2C receptor is predominantly
expressed in the central nervous system (CNS), and has been identified as a promising
therapeutic target for the treatment of obesity and other CNS disorders, such as
depression, schizophrenia and drug addiction.6-9 To date, a number of 5-HT2C ligands
have been disclosed (Fig. 1). Among them, lorcaserin was approved by the FDA as a
first-in-class antiobesity drug in 2012.10 Its efficacy in treatment of nicotine addiction
is currently being evaluated in clinical trials.11 Despite dramatic advancements in 5-
HT2C ligands development, attaining high 5-HT2C receptor selectivity remains an
attractive research area, as non-specific interaction with the other two 5-HT2 receptors,
5-HT2A and 5-HT2B receptors, results in hallucinations and potentially fatal fibrotic
cardiac valvulopathy, respectively.12-14 A major challenge to develop selective 5-HT2C
ligands involves the highly conserved ligand recognition transmembrane region of the
5-HT2 receptor subtypes.4 Therefore, it is difficult to design a ligand exhibiting high
specificity for the 5-HT2C receptor.
HN
N
NH
N
Cl
N
NH
O
N
N
Cl
N
NH
NH
Vabicaserin
Lorcaserin
CP-809101
PF-4479745
Fig. 1. Structures of some representative 5-HT2C receptor ligands.

Aporphines are a group of tetrahydroisoquinoline alkaloids which exhibit a wide
range of pharmacological activities.15-20 In the CNS, aporphines have been extensively
explored as ligands for serotonin, adrenergic and dopamine receptors.21-24 Recently,
Liu and co-workers identified several aporphine derivatives as 5-HT2C receptor hit
ligands by screening a focused target-specific natural compound library, indicating
that aporphines can be a viable source of novel ligands for 5-HT2C receptor.25
Previously reported structure-activity relationship (SAR) studies on aporphines
indicated that introducing a more lipophilic group at C11 position leads to the
compound possessing high affinity toward the serotonin receptors with a complete
loss of affinity for the dopamine receptors.26 Inspired by these valuable results and
aiming to develop potent 5-HT2C ligands with highly selectivity, a series of novel
aporphine derivatives with fluorinated alkyl, benzyl or benzoyl groups attached to
C11 through an O-linkage were synthesized for initial screening at the 5-HT2 receptor
subtypes (Fig. 2). Generally, introduction of a fluorine atom into bioactive molecules
is well-established strategy for improving the target affinity.27,28 Herein, we wish to
report the synthetic and pharmacological experiments of these novel aporphine
derivatives.
MeO
MeO
MeO
MeO
N
N
F
N
F
N
R
R
R
R
H
H
H
H
O
F
O
O
O
O
11
11
11
11
F
8a:
8b:
9a:
9b:
10a:
10b:
11a:
11b:
R = Me
R = n-Pr
R = Me
R = n-Pr
R = Me
R = n-Pr
R = Me
R = n-Pr
Fig. 2. Fluorinated aporphine derivatives for initial screening at the 5-HT2 receptor

subtypes.
Aporphine derivatives 8a-11a and 8b-11b were synthesized from natural
alkaloid thebaine (1) as depicted in Scheme 1, and the detailed protocols are found in
Supporting Information. (R)-(-)-2-Methoxy-11-hydroxyaporphine (7a) and (R)-(-)-N-
propyl-2-methoxy-11-hydroxynoraporphine (7b) were used as key intermediates, and
were obtained as we have detailed in previous publication.29 Deprotonation of
intermediate 7a or 7b with potassium carbonate in dry DMF followed by alkylation
with 1-fluoro-2-iodoethane, 1-fluoro-3-iodopropane or 4-fluorobenzyl bromide
provided aporphine derivatives 8a-10a or 8b-10b. In addition, esters 11a and 11b
were prepared by facile coupling 4-fluorobenzoyl chloride with intermediates 7a and
7b in the presence of 4-(Dimethylamino)pyridine, respectively. Spectral data (1H and
13C NMR) and elemental analysis for these target compounds were consistent with
their proposed structures.
Scheme 1. Synthesis of novel aporphine derivatives 8a-11a and 8b-11ba

R
R
MeO
Me
N
N
N
N
iv
v
iii
R
H
HO
O
O
O
MeO
OMe
HO
OMe
TfO
OMe
TfO
1,
Thebaine
4a:
4b:
5a:
5b:
R = Me, Oripavine
R = n-Pr
R = Me
R = n-Pr
6a:
6b:
R = Me
R = n-Pr
i
vi
iii
MeO
n-Pr
NH
HCl
N
N
ii
R
H
HO
O
O
MeO
2,
OMe
MeO
OMe
7a:
R = Me
Northebaine
3
7b:
R = n-Pr
ix
vii
viii
MeO
MeO
MeO
N
F
N
F
N
H
R
R
R
H
H
F
O
n
O
O
O
8a:
8b:
9a:
9b:
10a:
10b:
11a:
11b:
R = Me, n = 2
R = n-Pr, n = 2
R = Me, n = 3
R = n-Pr, n = 3
R = Me
R = n-Pr
R = Me
R = n-Pr
a
Reagents and conditions: (i): 1. DIAD, Benzene, reflux; 2. Pyridine hydrochloride,
MeOH, rt, yield 90%. (ii): 1-Iodopropane, K2CO3, EtOH, reflux, yield 65%. (iii): L-
Selectride, THF, reflux, yields 22-35%. (iv): N-Phenyl-
bis(trifluoromethanesulfonimide), Et3N, CH2Cl2, rt, yields 71-72%. (v): MeSO3H, 90
°C, yields 55-59%. (vi): Pd/C, Mg, NH4OAc, MeOH, rt, yields 62-87%. (vii): 1-
Fluoro-2-iodoethane or 1-fluoro-3-iodopropane, K2CO3, DMF, 80 °C, yields 56-89%.
(viii): 4-Fluorobenzyl bromide, K2CO3, DMF, 80 °C, yields 74-76%. (ix): 4-
Fluorobenzoyl chloride, 4-(Dimethylamino)pyridine, Et3N, THF, rt, yields 77-86%.
The binding affinities of aporphine derivatives 8a-11a and 8b-11b for the 5-HT2

receptor subtypes were examined by the National Institute of Mental Health (NIMH)
Psychoactive Drug Screening Program (PDSP), using competitive binding assays with
membrane preparations obtained from transiently transfected HEK293 T cells (for 5-
HT2A) and stably transfected HEK293 (for 5-HT2B), HEK293 T cells (for 5-HT2C).30
Data from these evaluations are presented in Table 1.
Among these newly synthesized aporphine derivatives, compounds 8a, 9a, 11a
and 11b were identified as potential hits for 5-HT2C receptor. Compounds 11a and
11b showed more significant binding property to 5-HT2C receptor, with inhibitory
constant (Ki) value of 32 nM and 24 nM, respectively. The other two aporphine
derivatives, 8a and 9a, exhibited moderate binding affinity (Ki = 343 and 338 nM,
respectively). These results indicated that the carbonyl moiety of C11 fluorinated
benzoyl group in compounds 11a and 11b might form a strong interaction with the
binding site of 5-HT2C receptor, while N- substituent might not play a large role here.
To our delight, compounds 11a and 11b also possessed good 5-HT2 receptor subtype
selectivity. Compound 11a showed 14-fold selectivity over the 5-HT2A receptor (Ki =
450 nM) and 3.2-fold selectivity versus the 5-HT2B receptor (Ki = 102 nM).
Compound 11b showed 17-fold selectivity over the 5-HT2A receptor (Ki = 414 nM)
and 4.4-fold selectivity versus the 5-HT2B receptor (Ki = 106 nM). Notably, in
comparison with the reference drug lorcaserin, compounds 11a and 11b exhibited
slightly lower affinity at the 5-HT2C receptor [11a (Ki = 32 nM) and 11b (Ki = 24 nM)
vs. Lorcaserin (Ki = 15 nM)] and lower selectivity over the 5-HT2B receptor, but

improved selectivity over the 5-HT2A receptor.
Table 1. Binding affinities of aporphine derivatives 8a-11a and 8b-11b and the
reference for the 5-HT2 receptor subtypesa
Ki ± SEM (nM)b
5-HT2B
Selectivity
Compound
5-HT2A
1138 ± 205
434 ± 62
2313 ± 389
450 ± 61
>10000
5-HT2C
343 ± 41
338 ± 36
>10000
32 ± 3
5-HT2A/5-HT2C 5-HT2B/5-HT2C
8a
9a
552 ± 95
422 ± 67
2681 ± 451
102 ± 13
3044 ± 589
>10000
3.3
1.3
-
1.6
1.2
-
10a
11a
14
-
3.2
-
8b
>10000
>10000
>10000
24 ± 3
9b
>10000
-
-
10b
>10000
>10000
-
-
11b
414 ± 51
112c
106 ± 13
174c
17
7.5
4.4
12
Lorcaserin
15c
a
All compounds were tested as their HCl salts. The following tritiated radioligands
were used: [3H]Ketanserin (5-HT2A), [3H]LSD (5-HT2B), [3H]Mesulergine (5-HT2C). b
The Ki values are means ± standard errors of 2-3 experiments. c Data from ref. 31.
To explain the higher binding affinity of compounds 11a and 11b at the 5-HT2C
receptor and explore a potential binding mode, molecular docking study on compound
11b using the GoldSuite was performed based on 5-HT2C receptor model which was
disclosed very recently (PDB 6BQG).32 As shown in Fig. 3, compound 11b is

properly stabilized into the 5-HT2C receptor binding site through a key salt-bridge
between the positively charged nitrogen atom of compound 11b and the carboxylate
of D3.32, which is fully conserved in 5-HT and other monoamine receptors.33
Compound 11b also forms a H-bond interaction with N6.55 through the carbonyl
oxygen atom of C11 fluorinated benzoyl group. This observation provided a potential
explanation as to why compounds 11a and 11b possessed higher binding affinity than
other newly synthesized aporphine derivatives, and supported our previous prediction.
N-n-Propyl substituent of compound 11b orients toward the vacant binding site of 5-
HT2C receptor, thereby explained the comparable binding affinity between compounds
11a and 11b at the 5-HT2C receptor. In addition, the diphenyl moiety of aporphine
structure may elicit π-alkyl interactions with some residues on TMH3, TMH6 and
TMH7 (transmembrane helix) to further stabilize the ligand binding.
D3.32
O
H
F
N
n-Pr
H
O
O
Salt Bridge
Hydrogen Bond
N6.55
Fig. 3. Binding mode of compound 11b in the binding site of 5-HT2C receptor. Left is
3D view of the receptor-ligand interactions. Amino acid residues engaged in ligand
binding are shown as sticks. Dotted yellow lines represent H-bonds with polar
residues. Right is 2D view of the receptor-ligand interactions.

In summary, a series of novel aporphine derivatives were synthesized for initial
screening at the 5-HT2 receptor subtypes. Compounds 11a and 11b were identified as
potent 5-HT2C hit ligands with highly selectivity over other 5-HT2 receptor subtypes.
Molecular docking study revealed that compounds 11a and 11b formed two key
interactions with the binding site of 5-HT2C receptor, including a salt-bridge to D3.32
and a H-bond interaction with N6.55, which result in higher binding affinity than
other newly synthesized aporphine derivatives. Compounds 11a and 11b are useful
starting points for further SAR exploration and optimization studies. We are
continuing in this vein and will report our findings in due course.
Acknowledgments
This work was supported by grants from Branfman Family Foundation (J.L.N.),
NIH R21-MH103718 (A.W.S.) and International Postdoctoral Exchange Fellowship
of China 2016[53] (Y.X.). The Ki determinations of novel fluorinated aporphines
were generously provided by the National Institute of Mental Health's Psychoactive
Drug Screening Program, Contract #HHSN-271-2013-00017-C (NIMH PDSP). The
NIMH PDSP is directed by Bryan L. Roth, M.D., Ph.D., at the University of North
Carolina at Chapel Hill and Project Officer Jamie Driscoll at NIMH, Bethesda MD,
USA.
Supplementary data
Supplementary data associated with this article can be found, in the online

version.
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