In vitro functional evaluation of isolaureline, dicentrine and glaucine enantiomers at 5-HT2 and α1 receptors

Article abstract of DOI:10.1111/cbdd.13390

Compounds with activity at serotonin (5-hydroxytryptamine) 5-HT₂ and α₁ adrenergic receptors have potential for the treatment of central nervous system disorders, drug addiction or overdose. Isolaureline, dicentrine and glaucine enantiomers were synthesized, and their in vitro functional activities at human 5-HT₂ and adrenergic α₁ receptor subtypes were evaluated. The enantiomers of isolaureline and dicentrine acted as antagonists at 5-HT₂ and α₁ receptors with (R)-isolaureline showing the greatest potency (pK_b?=?8.14 at the 5-HT_2C receptor). Both (R)- and (S)-glaucine also antagonized α₁ receptors, but they behaved very differently to the other compounds at 5-HT₂ receptors: (S)-glaucine acted as a partial agonist at all three 5-HT₂ receptor subtypes, whereas (R)-glaucine appeared to act as a positive allosteric modulator at the 5-HT_2A receptor.

Full text of DOI:10.1111/cbdd.13390

DR MICHAEL BUCKLE (Orcid ID : 0000-0003-2438-2376)
Article type : Research Article
In vitro functional evaluation of isolaureline, dicentrine and glaucine enantiomers at
5-HT2 and 1 receptors
Hui Li Heng1, Chin Fei Chee1,2, Chun Keng Thy2, Jia Ti Tee2,4, Sek Peng Chin1,2, Deron R.
Herr3, Michael J. C. Buckle1,*, Ian C. Paterson4, Stephen W. Doughty5, Noorsaadah Abd.
Rahman2, Lip Yong Chung1,*
1 Department of Pharmacy, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur,
Malaysia. Email: mbuckle8@gmail.com; chungly@um.edu.my
2 Department of Chemistry, Faculty of Science, University of Malaya, 50603 Kuala Lumpur,
Malaysia
3 Department of Pharmacology, Yong Loo Lin School of Medicine, National University of
Singapore, Singapore 117597
4 Department of Oral Biology and Craniofacial Sciences and Oral Cancer Research and
Coordinating Centre, Faculty of Dentistry, University of Malaya, 50603 Kuala Lumpur,
Malaysia
5 Penang Medical College, 4 Jalan Sepoy Lines, 10450 George Town, Pulau Pinang,
Malaysia
Compounds with activity at serotonin (5-hydroxytryptamine) 5-HT2 and 1 adrenergic
receptors have potential for the treatment of central nervous system (CNS) disorders, drug
addiction or overdose. Isolaureline, dicentrine and glaucine enantiomers were synthesised
and their in vitro functional activities at human 5-HT2 and adrenergic 1 receptor subtypes
were evaluated. The enantiomers of isolaureline and dicentrine acted as antagonists at 5-
HT2 and 1 receptors with (R)-isolaureline showing the greatest potency (pKb = 8.14 at the 5-
HT2C receptor). Both (R)- and (S)-glaucine also antagonised 1 receptors, but they behaved
very differently to the other compounds at 5-HT2 receptors: (S)-glaucine acted as a partial
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agonist at all three 5-HT2 receptor subtypes, whereas (R)-glaucine appeared to act as a
positive allosteric modulator at the 5-HT2A receptor.
KEYWORDS
aporphines; enantiomers; functional studies; G protein-coupled receptors, serotonin 5-HT2
receptors; adrenergic 1 receptors; antagonist; partial agonist; allosteric modulator
1 INTRODUCTION
The serotonin (5-hydroxytryptamine) 5-HT2 (5-HT2A, 5-HT2B and 5-HT2C) and 1 (1A, 1B and
1D) adrenergic receptors are two families of G protein-coupled receptors (GPCRs) with
significant clinical interest. In particular, the action of second generation (atypical)
antipsychotics at these two sets of receptors is thought to contribute to the suppression of
positive symptoms of schizophrenia and the reduction of extrapyramidal side effects
compared to first generation (typical) antipsychotics.[1,2] Furthermore, compounds with 5-HT2
and/or 1 receptor activity have potential for the treatment of psychostimulant addiction[3,4]
and as antidotes for 3,4-methylenedioxy-methamphetamine (MDMA, ecstasy) overdose.[5-7]
Aporphines are tetrahydroisoquinoline alkaloids which contain a chiral centre at
position C6a and display a wide range of pharmacological properties.[8,9] In particular, there
have been a number of reports on aporphines acting at dopamine[8] and 5-HT1A[10] receptors
and these have indicated that enantiomers tend to show contrasting functional activities, with
(R)- and (S)-isomers acting as agonists and antagonists, respectively. However, in the first
reported study on the actions of the individual enantiomers of an aporphine at 5-HT2A
receptors, both isomers of nantenine (1) (Figure 1) were found to act as competitive
antagonists.[11] This pattern of behaviour has also been observed in studies carried out in our
laboratory on the individual enantiomers of roemerine (2a) and nuciferine (3a).[12] Similarly,
both enantiomers of these three compounds, as well as apomorphine (4a) and N-n-
propylnorapomorphine (4b), have been found to antagonise 1 receptors.[11-13] We therefore
decided to investigate whether the structurally-related compounds, isolaureline (2b),
dicentrine (2c) and glaucine (3b) follow the same pattern. Since (S)-glaucine is the only one
of these compounds that is commercially-available with sufficient purity, we first performed a
stereoselective synthesis of each enantiomer of the compounds prior to evaluating their
functional activities at human 5-HT2 and 1 receptors.
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2 MATERIALS AND METHODS
2.1 Chemicals
(R)-isolaureline (2b), (R)-dicentrine (2c) and (R)-glaucine (3b) were prepared via a synthetic
route (Scheme 1) that is analogous to that previously reported by us[12] using (S,S)-8 as the
catalyst for the asymmetric hydrogenation step (see Additional Supporting Information). (S)-
isolaureline and (S)-dicentrine were prepared in the same way, but using (R,R)-8 as the
catalyst. (S)-glaucine was obtained from Santa Cruz Biotechnology (Dallas, TX, U.S.A.) and
its identity and purity were confirmed by 1H NMR spectroscopy, polarimetry and chiral HPLC
analysis. All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) or
Merck (Darmstadt, Germany).
2.2 Functional assays at 5-HT2 and α1 receptors
Agonism and antagonism testing at human 5-HT2 (5-HT2A, 5-HT2B and 5-HT2C) and 1 (1A,
1B and 1D) receptors was carried out using a transforming growth factor- shedding assay
to assess GPCR activation in HEK293 cells (ATCC #CRL-1573) transiently transfected with
the relevant receptor, as previously described.[12,14] For agonism testing, the cells were
incubated for 1 hour at 37°C with varying concentrations (0.1 nM to 60 μM final
concentration) of test compound or endogenous agonist (serotonin for 5-HT2 receptors,
noradrenaline for 1 receptors) in stimulation buffer. For antagonism testing, the cells were
pre-treated with varying concentrations (0.1 nM to 60 μM final concentration) of test
compound or positive control (ketanserin for 5-HT2 receptors, prazosin for 1 receptors) in
stimulation buffer for 15 minutes, followed by addition of a fixed concentration of serotonin (1
μM, 0.1 μM or 0.1 μM final concentration for 5-HT2A, 5-HT2B and 5-HT2C, respectively) or
noradrenaline (0.1 μM, 1 μM or 1 μM final concentration for 1A, 1B and 1D receptors,
respectively) and the mixture was incubated for 1 hour at 37°C. Alkaline phosphatase
activity in the cells and in the supernatant was determined by measuring the absorbance of
p-nitrophenol (from hydrolysis of p-nitrophenyl phosphate) at 405 nm using a Cytation 3 cell
imaging multi-mode microplate reader (Biotek Instruments, Inc., Winooski, VT, USA). Each
assay was performed in triplicate. Receptor response was defined as the alkaline
phosphatase activity of the supernatant/total alkaline phosphatase activity (cells +
supernatant) and normalised against the maximal response obtained with the endogenous
agonist. In order to obtain dose-response curves for serotonin at 5-HT2A receptors in the
presence of different concentrations of (R)-glaucine, a similar procedure was followed as for
antagonism testing, except that varying concentrations of serotonin (10 pM to 60 μM final
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concentration) were added after pre-treatment of 5-HT2A receptor-expressing cells with (R)-
glaucine.
Data processing was performed using Prism 5 (GraphPad Software., San Diego, CA,
USA) to generate relative Emax and EC50 (for agonists) or IC50 (for antagonists). Antagonist
dissociation constant (Kb) values for the test compounds were calculated from pIC50 curves
using a modified form of the Gaddum equation: Kb = IC50/([A]/EC50 – 1) where IC50 = the IC50
value of the antagonist, [A] = the concentration of the agonist and EC50 = the EC50 value of
the agonist.[15]
2.3 Molecular modelling
The 3D structures of the test compounds were downloaded from the PubChem substances
and compound database[16] and were prepared using LigPrep 2.3 (Schrödinger LLC, New
York, USA), including formation of the protonated nitrogen species, which is expected to
predominate under physiological conditions. The 5-HT2A, 5-HT2B and 5-HT2C receptor
structures previously generated by us were used for docking studies.[12] Receptor and ligand
coordinate files were prepared using AutoDockTools 1.5.6.[17] Docking was performed with
]
AutoDock Vina 1.0.2 with an exhaustiveness of 100.[18 The grid box was set to cover the
extracellular half of the receptor with 1Å grid spacing. Images of the ligand-receptor
complexes obtained were generated using PyMOL (Schrödinger LLC).
3 RESULTS AND DISCUSSION
3.1 Synthesis
The (R)-enantiomers of the test compounds were prepared using (S,S)-8 as the catalyst for
the asymmetric hydrogenation step (see Scheme 1).[12,19] Coupling of 2-(2-bromo-5-
methoxyphenyl)acetic acid (5a) or 2-(2-bromo-4,5-dimethoxyphenyl)acetic acid (5b) with 2-
(3,4-methylenedioxyphenyl)ethylamine (6a) or 2-(3,4-dimethoxyphenyl)ethylamine (6b) gave
the corresponding amides (7aa–7bb). Bischler-Napieralski cyclisation, followed by
asymmetric hydrogenation of the resulting imines, gave the (R)-1-benzyl-1,2,3,4-
tetrahydroisoquinolines (9aa–9bb). Protection of the amino group as the methylcarbamate,
followed by oxidative cyclisation and then reduction of the methylcarbonate group gave (R)-
isolaureline (2b), (R)-dicentrine (2c) and (R)-glaucine (3b), in approximately 30% overall
yield and with 95%, 98% and 90%, e.e., respectively, determined by chiral HPLC. In a similar
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manner, but this time using (R,R)-8 as the catalyst for the asymmetric hydrogenation step,
(S)-isolaureline and (S)-dicentrine were obtained with 93% and 96% e.e.
3.2 Pharmacological evaluation
The functional activities of each enantiomer of the test compounds were evaluated at human
5-HT2 and 1 receptors in HEK293 cells transiently transfected with the relevant
receptor.[12,14] The results are summarised in Tables 1–3.
The enantiomers of isolaureline (2b) and dicentrine (2c) all antagonised the response
obtained at both 5-HT2 and 1 receptors in the presence of the corresponding endogenous
agonist (serotonin and noradrenaline, respectively). The potency of (R)-isolaureline at the
5-HT2C receptor (pKb = 8.14) was even greater than that previously determined for (R)-
roemerine (pKb = 7.82).[12] Its selectivity ranged from 4- to 5-fold compared to the 5-HT2A and
5-HT2B receptors and from 25- to 63-fold compared to the three 1 receptor subtypes. The
results for (S)-dicentrine at 1 receptors are consistent with previous studies.[20-24]
Both (R)- and (S)-glaucine (3b) were also observed to act as antagonists at 1
receptors, in line with previous studies on (S)-glaucine.[13,25-28] However, they behaved very
differently to the other test compounds at 5-HT2 receptors: (S)-glaucine acted as a partial
agonist (Figure 2A), whereas (R)-glaucine appeared to enhance the response obtained in
the presence of serotonin (in antagonism testing), particularly at the 5-HT2A receptor,
suggesting that it may act as a positive allosteric modulator (Figure 2B). The former result is
in agreement with a recent report of a radioligand binding study on (S)-glaucine that showed
that it can displace [3H]-ketanserin from 5-HT2A receptors (Ki = 966 nM).[29] Interestingly,
some analogues of nantenine with cycloalkyloxy substituents at position C1 of the A ring
have also been reported to have weak (partial) agonist activities.[30]
In order to investigate further the positive allosteric modulatory behaviour of (R)-
glaucine at the 5-HT2A receptor, dose-response curves for serotonin in the presence of
different concentrations of (R)-glaucine were obtained (Figure 3). The observed upward shift
of the serotonin response (with minimal horizontal shift) is consistent with (R)-glaucine being
able to increase the efficacy of serotonin. Although allosteric modulation of other GPCRs is
relatively well known, to date, there have been very few reports of such behaviour at 5-HT2
receptors.[31-33]
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With regards to stereoselectivity, the most striking observation is the aforementioned
contrast in the functional behaviour of (R)- and (S)-glaucine at 5-HT2 receptors. Isolaureline
also exhibited some stereoselectivity, albeit more moderate, with the (R)-enantiomer
displaying 4-fold selectivity over the (S)-enantiomer at the 5-HT2C receptor and the (S)-
enantiomer displaying 5- and 3-fold selectivity over the (R)-enantiomer at the 1B and 1D
receptors, respectively. However, the two enantiomers of dicentrine were observed to have
very similar activities at all 5-HT2 and 1 receptor subtypes.
3.3 Molecular modelling
An attempt was made to rationalise the difference in behaviour of (R)-glaucine at the 5-HT2
receptors compared to the other test compounds by docking the compounds into structures
generated from a crystal structure of the 5-HT2B receptor in complex with ergotamine (PDB
code: 4IB4).[12,34] For all the test compounds, the majority of the top-ranked binding poses at
the 5-HT2A receptor were at the orthosteric site as expected (results not shown). However,
some of the top-ranked binding poses were at a possible allosteric site located adjacent to
the extracellular ends of transmembrane (TM) helices IV and V. In particular, (R)-glaucine
exhibited a binding pose which enabled the A ring to engage in - stacking interactions with
Phe213, the protonated nitrogen atom to form a salt bridge with Asp232 and the D ring to
engage in hydrophobic interactions with Leu215 and other residues in extracellular loop 2
(ECL2) (Figure 4). ECL2 links the ends of TM helices IV and V and has been found to be
involved in the binding of allosteric ligands to other class A GPCRs.[35] However, there was
no clear indication from the current study why only (R)-glaucine should preferentially bind to
the proposed allosteric site of the 5-HT2A receptor rather than to the orthosteric site.
Interestingly, Phe213 and Asp232 are not present in either 5-HT2B or 5-HT2C
receptors – the equivalent residues are Lys193 and Gly215 in the 5-HT2B receptor and
Ile192 and Pro212 in the 5-HT2C receptor – and neither (R)-glaucine nor any of the other
compounds were observed to bind to the corresponding regions of these receptor subtypes.
This might account for (R)-glaucine producing its most noticeable positive allosteric effect at
the 5-HT2A receptor.
4 CONCLUSIONS
The results described in this study confirm our previous observations that the presence of a
methylenedioxy group at positions C1 and C2 in the A ring of aporphines increases
antagonist activity at 5-HT2A and 5-HT2C receptors and that the configuration of the chiral
centre at position C6a may confer stereoselectivity in their action at these two receptors.
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Furthermore, it appears that the presence of alkoxy substituents at position C10 in the D ring
of aporphines decreases antagonist activity at 5-HT2 receptors and may even cause
compounds to act as partial agonists or allosteric modulators. Overall, the results underline
the potential of aporphines as pharmacological probes or agents for the treatment of
psychotic disorders, drug addiction or overdose.
ACKNOWLEDGEMENTS
This study was funded by grants from the Ministry of Science, Technology and Innovation,
Malaysia (02-01-03-SF0937) and the Ministry of Higher Education, Malaysia, High Impact
Research
Grant
scheme
(HIR-MoHE:
UM.C/625/1/HIR/MOHE/MED/17
&
UM.C/625/1/HIR/MOHE/MED/33).
CONFLICTS OF INTEREST
There are no conflicts of interest to declare.
LIST OF FIGURE LEGENDS
FIGURE 1 The structures of aporphines previously tested as individual enantiomers at 5-HT2 and 1
receptors and the aporphines used in this study
SCHEME 1 Aporphine synthesis. Reagents and conditions: a) (COCl)2, CH2Cl2; b) 6, Et3N, CH2Cl2;
c) POCl3, CH2Cl2, reflux; d) (S,S)-8 (3 mol-%), HCO2H/NEt3, DMF, rt; e) MeOCOCl, DIPEA, CH2Cl2, rt;
f) Pd(OAc)2 (5 mol-%), DavePhos 11 (10 mol-%), KOAc (2 eq.), DMA, 130 C; g) LiAlH4, Et2O, 0 C
FIGURE 2 A. Dose-response curves for (S)-glaucine and serotonin at the 5-HT2A receptor. B. Dose-
response curves from antagonism testing of (R)-glaucine and ketanserin at the 5-HT2A receptor in the
presence of 1 μM serotonin. Data points are normalised and shown as the mean ± SD from triplicate
readings.
FIGURE 3 Dose-response curves for serotonin at the 5-HT2A receptor in the presence of different
concentrations of (R)-glaucine. Data points are normalised and shown as the mean ± SD from
triplicate readings.
FIGURE 4 The docking poses of serotonin (green space-filling representation) at the orthosteric site
of the 5-HT2A receptor and (R)-glaucine (grey stick representation) at the proposed allosteric site. A.
Global view showing the locations of the orthosteric and allosteric sites. B. Close-up view showing
(R)-glaucine interacting with residues (yellow stick representation) in the allosteric site.
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SUPPORTING INFORMATION
Additional Supporting Information may be found online in the supporting information tab for
this article.
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TABLE 1 Antagonist potencies and selectivities of aporphine enantiomers at 5-HT2 receptor
subtypes
a
pKb
Kb (nM)
Subtype selectivityb
5-HT2A/ 5-HT2A/ 5-HT2C
/
Compound
5-HT2A
5-HT2B
5-HT2C
5-HT2A
5-HT2B
5-HT2C
5-HT2B
5-HT2C
5-HT2B
(R)-isolaureline
(S)-isolaureline
(R)-dicentrine
(S)-dicentrine
(R)-glaucine
7.48 + 0.03
7.64 + 0.01
7.12 + 0.07
7.08 + 0.11
n.d.c
7.50 + 0.09
7.44 + 0.10
6.68 + 0.03
6.35 + 0.12
n.d.c
8.14 + 0.02
7.53 + 0.16
6.11 + 0.06
6.27 + 0.23
n.d.c
33.3
22.7
75.8
83.9
-
31.7
36.0
208
444
-
7.2
29.8
776
536
-
1
0.2
1
4
2
1
3
10
6
0.3
0.8
-
5
-
-
(S)-glaucine
n.d.c
n.d.c
n.d.c
-
-
-
-
-
-
(R)-roemerined
(S)-roemerined
(R)-nuciferined
(S)-nuciferined
Ketanserind,e
10.8
42.9
65.3
84.3
2.0
84.0
92.7
31.0
41.7
1980
15.1
76.9
36.4
41.7
112
8
1
6
7.97  0.18
7.37  0.10
7.18  0.03
7.07  0.17
8.69  0.03
7.08  0.43
7.03  0.33
7.51  0.21
7.38  0.01
5.70  0.04
7.82  0.08
7.12  0.13
7.44  0.01
7.51  0.22
6.95  0.08
2
2
1
0.5
0.5
1000
0.6
0.4
60
1
1
20
a Values are expressed as mean  SEM from at least 2 separate experiments. b Selectivities for receptor X/receptor Y were
calculated as the ratio of Kb receptor Y/Kb receptor X. c Not determined - (R)-glaucine appeared to act as a positive allosteric
modulator; (S)-glaucine is a partial agonist. d Data from ref.12 e Positive control.
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TABLE 2 Antagonist potencies and selectivities of aporphine enantiomers at 1 receptor subtypes
a
pKb
Kb (nM)
1A
Subtype selectivityb
Compound
1A
1B
1D
1B
1D
1A/1B
1A/1D
1B/1D
(R)-isolaureline 6.73 + 0.16
(S)-isolaureline 6.88 + 0.04
6.34 + 0.01
7.02 + 0.04
6.69 + 0.01
6.81 + 0.03
6.00 + 0.14
6.34 + 0.02
6.57  0.05
6.81  0.03
7.22  0.01
6.50  0.01
11.41  0.09
6.71 + 0.01
7.20 + 0.06
6.66 + 0.07
6.74 + 0.06
6.42 + 0.11
6.38 + 0.01
7.34  0.15
7.04  0.07
6.78  0.01
6.94  0.01
10.60  0.02
185
453
96.4
202
155
1010
454
266
154
60.6
316
0.004
193
62.5
218
181
383
418
46.0
92.2
165
116
0.025
2
1
2
132
0.7
2
0.5
2
2
(R)-dicentrine
(S)-dicentrine
(R)-glaucine
(S)-glaucine
(R)-roemerinec
(S)-roemerinec
(R)-nuciferinec
(S)-nuciferinec
Prazosinc,d
6.96 + 0.03
6.79 + 0.02
7.00 + 0.05
6.64 + 0.03
6.47  0.08
7.07  0.01
7.42  0.07
7.17  0.08
9.69  0.07
109
1
163
1
1
1
100
10
2
4
3
230
2
1
338
0.8
2
0.1
1
0.2
0.6
3
86.0
37.8
67.1
0.20
2
4
5
2
0.4
6
0.02
0.1
a Values are expressed as mean  SEM from at least 2 separate experiments. b Selectivities for receptor X/receptor Y were
calculated as the ratio of Kb receptor Y/Kb receptor X. c Data from ref.12 d Positive control.
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TABLE 3 Agonist potencies and relative efficacies of (S)-glaucine at 5-HT2 receptor subtypes
5-HT2A
5-HT2B
5-HT2C
a
a
a
a
a
a
Compound
(S)-glaucine
Serotoninc
pEC50
Emax
pEC50
Emax
pEC50
Emax
6.18 ± 0.01
7.51 ± 0.09
42 + 1
n.d.b
n.d. b
6.35 ± 0.11
8.35 ± 0.04
52 ± 1
100 ± 1
8.16 ± 0.01
100 ± 1
100 ± 1
a Values expressed as mean ± SEM from at least 2 separate experiments. b Not determined - % of AP-T α a < 3.[14]
c Positive control.
This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.