In vitro structure-activity relationships of aplysinopsin analogs and their in vivo evaluation in the chick anxiety-depression model

Article abstract of DOI:10.1016/j.bmc.2013.09.011

Aplysinopsins are tryptophan-derived natural products that have been isolated from a variety of marine organisms and have been shown to possess a range of biological activities. In vitro receptor binding assays showed that of the 12 serotonin receptor subtypes, analogues showed a high affinity for the 5-HT_2B and 5-HT_2C receptor subtypes, with selectivity for 5-HT_2B over 5-HT_2C. While no conclusions could be drawn about the number and position of N-methylations, bromination at C-4 and C-5 of the indole ring resulted in greater binding affinities, with K_i's as low as 35 nM. This data, combined with previous knowledge of the CNS activity of aplysinopsin analogs, suggested that these compounds may have potential as leads for antidepressant drugs. Compounds 3c, 3u, and 3x were evaluated in the chick anxiety-depression model to assess their in vivo efficacy. Compound 3c showed a modest antidepressant effect at a dose of 30 nM/kg in the animal model.

Full text of DOI:10.1016/j.bmc.2013.09.011

Accepted Manuscript
In vitro structure-activity relationships of aplysinopsin analogs and their in vivo
evaluation in the chick anxiety-depression model
Kevin Lewellyn, Dobroslawa Bialonska, Melissa J. Loria, Stephen W. White,
Kenneth J. Sufka, Jordan K. Zjawiony
PII:
S0968-0896(13)00782-7
DOI:
http://dx.doi.org/10.1016/j.bmc.2013.09.011
BMC 11095
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
2 July 2013
28 August 2013
5 September 2013
Please cite this article as: Lewellyn, K., Bialonska, D., Loria, M.J., White, S.W., Sufka, K.J., Zjawiony, J.K., In
vitro structure-activity relationships of aplysinopsin analogs and their in vivo evaluation in the chick anxiety-
depression model, Bioorganic & Medicinal Chemistry (2013), doi: http://dx.doi.org/10.1016/j.bmc.2013.09.011
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1 In vitro structure-activity relationships of aplysinopsin analogs
2 and their in vivo evaluation in the chick anxiety-depression
3 model
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Kevin Lewellyna, Dobroslawa Bialonskaa,e, Melissa J. Loriab, Stephen W. Whiteb, Kenneth J.
Sufkab,c,d and Jordan K. Zjawionya,d,*
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Departments of Pharmacognosya, Psychologyb, Pharmacologyc; Research Institute of
Pharmaceutical Sciencesd, University of Mississippi, University, MS 38677 USA
Institute of Environmental Sciences, Jagiellonian University, 30-387, Krakow, Polande
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Corresponding author:
Prof. Jordan K. Zjawiony, PhD
Department of Pharmacognosy and Research Institute of Pharmaceutical Sciences
School of Pharmacy, University of Mississippi
PO Box 1848
University, MS 38677-1848, USA
Tel +662 915 7026
Fax +662 915 6975
E-mail: jordan@olemiss.edu
Abstract: Aplysinopsins are tryptophan-derived natural products that have been isolated from a
variety of marine organisms and have been shown to possess a range of biological activities. In
vitro receptor binding assays showed that of the 12 serotonin receptor subtypes, analogues
showed a high affinity for the 5-HT2B and 5-HT2C receptor subtypes, with selectivity for 5-HT2B
over 5-HT2C. While no conclusions could be drawn about the number and position of N-
methylations, bromination at C-4 and C-5 of the indole ring resulted in greater binding affinities,
with Ki’s as low as 35 nM. This data, combined with previous knowledge of the CNS activity of
aplysinopsin analogs, suggested that these compounds may have potential as leads for
antidepressant drugs. Compounds 3c, 3u, and 3x were evaluated in the chick anxiety-depression
model to assess their in vivo efficacy. Compound 3c showed a modest antidepressant effect at a
dose of 30 nM/kg in the animal model.
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Keywords: aplysinopsin, serotonin, monoamine oxidase, natural products, depression
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1. Introduction
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As part of our effort to find new antidepressant leads from natural products, we continued our
work with the indole alkaloids, aplysinopsins. Aplysinopsins are tryptophan-derived natural
products isolated from a wide variety of marine organisms such as sponges1, corals2, mollusks3,
and sea anemones.4 Natural and synthetic aplysinopsins have been shown to possess antitumor5,
antimalarial6, and antimicrobial7 activities.
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Aplysinopsins have also received considerable research attention as promising
neuromodulators.8 Previously, we reported several synthetic aplysinopsin analogs possessed
significant monoamine oxidase inhibitory activity.9 After their discovery in the 1950s,
monoamine oxidase inhibitors (MAOIs) were used as first line treatment for depression for
several decades. However, their extensive side effects and food and drug interactions led to a
decline in use. The discovery of two isoforms of MAO, MAO-A and MAO-B, led to a renewed
interest in the development of MAOIs, especially ligands which were able to selectively bind to
one isoform.10 The two isoforms have been found to vary in their distribution in the body, but
both are found in various regions in the brain. They also have different preferred substrates.
MAO-A’s preferred substrates are serotonin and norepinepherine, while MAO-B preferentially
breaks down phenylethylamine and histamine. Dopamine and tyramine are substrates for both
isoforms. In our previous work we found that in general, aplysinopsins possessed a high
selectivity for MAO-A over MAO-B. The role of MAO-A levels in depression have been well
documented.11
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Serotonin receptor modulation has also been linked to antidepressant action; therefore
aplysinopsins represent potentially useful candidates for antidepressant drug development. The
serotonin receptor family is made up of seven classes (5-HT1 – 5-HT7) with 15 subtypes.12
Dysfunction of the serotonin receptor systems can lead to a wide array of psychiatric disorders
including anxiety, depression and schizophrenia.13 The role of serotonin in mood control,
appetite, sleep, thermoregulation and pain has also been extensively studied.14 In addition to
CNS disorders, the 5-HT receptor system has also been implicated in disorders of the
cardiovascular and pulmonary systems.15 With the increased understanding of how serotonin
and its receptors function in multiple systems throughout the body, we must take into account the
potentially wide ranging side effects of drugs on systems other than the CNS. This problem
further underscores the need to develop drug leads which are potent, but also highly selective
among the serotonin receptor subtypes.
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A group of aplysinopsins isolated from Jamaican sponge Smenospongia aurea exhibited an
affinity for human serotonin 5-HT2 receptor subtypes 5-HT2A and 5-HT2C expressed in a
mammalian cell line.6 These subtypes were of interest as possible targets in the treatment of
schizophrenia, depression, anxiety and obesity.16, 17 It has been shown that 5-HT2C receptor
knockout mice exhibit obesity, epilepsy and cognitive disorders.18, 19 The three receptor
subtypes of the 5-HT2 family (2A, 2B, 2C) share highly conserved transmembrane sequences
(80%), thus designing ligands which are potent and selective remains a challenge.
A major problem in the development of 5-HT2C agonists has been a lack of selectivity over 5-
HT2A and 5-HT2B subtypes. Activation of 5-HT2A receptors may lead to hallucinations and
activation of 5-HT2B to cardiac insufficiency and pulmonary hypertension (fenfluramine case).20
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Thus, discovery of highly selective 5-HT2C agonists could provide new and safer drugs. A good
example of this is lorcaserin (Belviq), a recently approved anti-obesity drug.21 It was developed
based on the potent 5-HT2C agonist dexfenfluramine, which was approved as a weight loss agent
in 1996. However, its cardiovascular side effects stemming from its 5-HT2B agonist activity led
to its withdrawal from the market just one year later.22 Using a medicinal chemistry approach,
lorcaserin was developed to retain its potent 5-HT2C agonism, but also be selective for 5-HT2C
over 5-HT2B. Lorcaserin has high affinity (Ki = 15 nM) to human 5-HT2C receptors and exhibits
18- and 104-fold selectivity over 5-HT2A and 5-HT2B, respectively.23
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To date there has been little published regarding the structure activity relationship between
aplysinopsin analogs and their serotonin binding affinities and selectivity. A preliminary SAR
analysis of naturally occurring aplysinopsins pointed to significance of functional groups at
positions 6, 2’ and 3’ in binding to human 5-HT2 receptors.6 Bromination at C-6 of the indole
ring and the increased length of the alkyl chain at position 3’ enhanced affinity. In addition, the
bromination enhanced the selectivity to 5-HT2C over the 5-HT2A subtype. On the other hand, the
methylation at C-2’ increased affinity to the 5-HT2A receptor. In another study of a synthetic
library of aplysinopsin analogs it was shown that the aplysinopsin scaffold can be manipulated
such that selection between 5-HT2A and 5-HT2C can be achieved.24 Cummings et al. found that
alkylation at C-2’ and C-4’ coupled with various halogenation patterns can yield highly selective
ligands. More specifically, substitution of the indole ring at C-6 with a chlorine or bromine
resulted in a preference for 5-HT2C binding. However, if C-6 is left unsubstituted or substituted
with a fluorine there was a preference for 5-HT2A. It is proposed that the size of the halogen
plays a key role in this selectivity. Both of these studies are promising results that suggest a
potential therapeutic value for aplysinopsin analogs. However, both also use a limited number of
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analogs and only evaluate affinity at 5-HT2A and 5-HT2C, ignoring potential binding at other
serotonin receptor subtypes. The small library size also limits the amount of SAR information
that is able to be gathered from the study. We hope to further add to these promising reports by
working with a larger library of compounds and evaluating their activity on a larger number of
serotonin receptor subtypes.
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In the present study, 50 aplysinopsin analogs have been obtained synthetically and screened
for affinity to twelve human serotonin receptors. Tested analogs varied in the substitution
pattern of the indole ring and the imidazolidinone moiety. The indole ring was either non-
brominated or brominated in the 4, 5, 6, or 7 positions. The imidazolidinone was not substituted,
or methylated in one (2’, 3’, 4’), two (2’,3’; 2’,4’; and 3’,4’), or three positions (2’, 3’, and 4’).
There have been a limited number of studies examining the biological activities of
aplysinopsins in vivo. The number of neuromodulatory studies is even less. One early study did
use an ex-vivo system to examine MAO inhibition and perform preliminary pharmacokinetic
studies.25 They found that the MAO activity was short-term and reversible and that the
biological activity correlated with the disappearance of methylaplysinopsin from the plasma. In
a more recent study, aplysinopsin was tested in the Porsolt forced swim test but did not exhibit
any antidepressant activity.26 Considering these results, following in vitro evaluation of our
library, we wanted to evaluate several lead compounds in an appropriate animal model to
determine if their in vitro efficacy translated to an in vivo model. Three compounds (3c, 3u, and
3x) were screened in the chick anxiety-depression model.27 This hybrid social isolation stress
paradigm is capable of screening for both anxiolytic and anti-depressant activity.
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2 Results and discussion
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2.1. Chemistry
Aplysinopsins consist of an indole moiety coupled with a corresponding imidazolidinone
(Scheme 1). There have been several papers published on the various synthetic strategies to
obtain aplysinopsin analogs.28 Most synthetic strategies involve the synthesis of the desired
indole aldehyde followed by condensation with the appropriate imidazolidinone. In order to
obtain an adequate structure activity relationship picture in regards to serotonin receptor subtype
affinity and selectivity, we sought to explore the effects of halogenations and N-methylations on
the aplysinopsin scaffold.
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The general scheme for the synthesis of aplysinopsin analogs 3a-ii is shown below in Scheme
1. Briefly, the condensation of indole-3-carboxyaldehydes with the appropriate imidazolidinones
was carried out according to the procedure from Djura and Faulkner.29 The indole aldehydes 1a-
e were commercially available and the imidazolidinones 2a-h were obtained according to
published procedures.30-32
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Compounds 7a-e were obtained via condensation of the indole aldehydes (1a-e) with
thiohydantoin (4) as shown in Scheme 2. This was followed by methylation of the intermediate
thiones 5a-e, and substitution of the thioethers 6a-e with methylamine.
The E or Z configuration of analogs was determined using 1H,13C heteronuclear coupling
constants between H-8 and C-5’. The E isomer possess a larger H-8,C-5’ heteronuclear coupling
constant (9-11 Hz) compared to the Z isomer (4-5.5 Hz).33 The substitution pattern of the
imidazolidinone moiety determines the resulting configuration after the condensation. If N-2’ is
substituted with a methyl group, then the resulting double bond will occur in predominantly the
E configuration. If the N-2’ is non-methylated, the result is a Z configuration.34
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2.2. In vitro evaluation
A series of 50 synthesized compounds (compounds 3a-ii, 5a-e, 6a-e, 7a-e) were evaluated by
the NIMH Psychoactive Drug Screening Program (PDSP) at the University of North Carolina-
Chapel Hill. Briefly, compounds were first evaluated in a primary radioligand binding assay for
twelve serotonin receptor subtypes. A “hit” is defined as an inhibition > 50 % in the primary
assay. Secondary assays are used to determine the affinity constants (Ki) of hits at the specified
serotonin receptor subtypes. Ki’s are calculated using the Cheng-Prusoff equation.35
A cursory overview of the in vitro binding data reveals that the majority of the activity among
the twelve receptor subtypes lies in the 5-HT2 family, specifically at 5-HT2B and 5-HT2C.
Selected secondary binding highlights are shown in Table 1. Complete in vitro secondary assay
results for all compounds and receptors can be found in the supplementary material.
The library of compounds can be organized into eight groups of analogs, based on the
substitution pattern of their imidazolidinone moiety. In each of those eight groups there are five
analogs, one non-brominated and four analogs brominated at positions 4, 5, 6, and 7 of the indole
ring. As shown in Table 1, the “A group”, with no N-methylations present on the
imidazolidinone moiety, showed the lowest Ki values, but with varying degrees of selectivity
among the 5-HT2 family of receptor subtypes. Compound 3c showed the highest affinity overall,
with a Ki = 35nM at 5-HT2C, however, it lacked selectivity among the 5-HT2 family. The same
can be said for compounds 3a and 3d, both of which possess nanomolar level affinities for 5-
HT2B and 5-HT2C, but were not notably selective for either one over the other.
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A general trend seen across all sub-groups of the library was that among each series of
compounds it was shown that brominated compounds possessed lower Ki values than their non-
brominated counterparts. This was in line with previous reports that various halogenations
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improved Ki values.6, 24 More specifically, we found that compounds brominated at C-4 and C-5
possess higher binding affinities in general. This is intriguing as there have been no reports of
aplysinopsin analogs, synthetic or natural, halogenated at C-4. All of the naturally occurring
aplysinopsin derivatives are either halogenated at C-6 or are di-brominated at C-5 and C-6.
Concerning selectivity, across the entire library there was a noticeable selectivity for 5-HT2B
over 5-HT2C. The average relative selectivity index (SI) for 5-HT2B over 5-HT2C was 4.32. A Ki
value of 10,000 nM was assumed for compounds which were not tested in secondary binding
assays to determine the relative SI. As mentioned earlier, the previous aplysinopsin SAR
studies6, 24 examining 5-HT receptor activity have failed to evaluate binding affinity for the 5-
HT2B receptor subtype, therefore it is difficult to make comparisons between the two data sets
based on selectivity among 5-HT2 receptor subtypes.
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Concerning the effects of N-methylations of the imidazolidinone moiety, our results were in
contrast to previous reports that pointed to an importance of N-alkylation in selectivity among 5-
HT2 receptor subtypes.24 We discovered no clear pattern of an increase in potency or selectivity
among serotonin receptor subtypes based on variation in N-methylations. In fact, our most active
compound subset was the group which had no alkylations of the imidazolidinone moiety. Again,
comparison to previous SAR studies6, 24 regarding imidazolidinone substitution patterns is
difficult, as they did not evaluate any analogs which were unsubstituted on the imidazolidinone
moiety.
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Concerning our previous work with aplysinopsins, we found that the most potent and selective
MAO-A inhibitors (3u and 3x, see Table 2) did not possess significant affinities for any
serotonin receptor subtypes. In addition, we found that compounds with the lowest Ki values for
serotonin receptor subtypes (3c, 3m, and 3q) did not possess significant MAO-A or MAO-B
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inhibitory activity. For a more detailed description of the MAO inhibitory properties of
aplysinopsins, and full experimental methodology, please see our previous work.9
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2.3. In vivo evaluation
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The previous study that assessed in vivo efficacy of aplysinopsins found that despite potent in
vitro CNS activity, they had diminished or non-existent in vivo activity.26 Kochanowska et al.
evaluated the parent compound aplysinopsin (3u) in a Porsolt forced swim test. It did not
display any significant antidepressant activity. Based on these findings we sought to evaluate
our compounds in a different behavioral model. We chose the chick anxiety-depression model.
It is different from the aforementioned forced swim test in that it acts as both a panic-model to
simulate anxiolytic conditions27 and as a behavioral despair model to evaluate a depression-like
state.36 Further, the model shows solid face37, construct38, and predictive validity.38, 39 The chick
anxiety-depression model entails isolation of socially raised chicks isolated at 5-6 days post-
hatch. Chicks tested with conspecifics show low rates of vocalization throughout the session
whereas chicks tested in isolation show high rates of distress vocalizations (DVocs) during the
first 5 minutes (i.e., the anxiety-like phase; see 27) which decline over the next 20 minutes by 40-
50% of the initial rate and remain steady throughout the remaining isolation period (i.e.,
depression-like phase). The reduced DVoc rates during this latter phase mirror the pattern seen
in traditional behavioral despair depression models.
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In addition to using a new behavioral model, we wanted to assess whether key structural
changes (e.g. halogenations and N-methylations) would result in increased in vivo efficacy
profiles. Based on our in vitro results, we chose three lead compounds (3c, 3u, 3x) to be
evaluated in the in vivo anxiety-depression model.27 Compounds 3u and 3x showed nanomolar
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level affinity for MAO-A and both had a high selectivity index for MAO-A over MAO-B.9
Despite its limited selectivity, compound 3c was chosen due to its potency and ability to serve a
good primary representative of the general aplysinopsin pharmacophore in initial in vivo
screening to assess bioavailability.
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Compounds were screened in the chick anxiety-depression model at three concentrations (10,
30, and 100 nM/kg). Of the three compounds tested, 3c displayed the most promising activity.
The pattern of distress vocalization rates across the isolation stressor for the various treatment
conditions are summarized in Figure 1 for compound 3c. Socially tested chicks given vehicle
displayed few DVocs across the test session. Vehicle-isolated chicks exhibited high DVoc rates
during the first five minute block (anxiety-like phase) that declined over the next 25-30 minute
period to approximately 50% of the initial rate and remained relatively stable for the remaining
of the test session (depression-like phase). During the anxiety-like phase, none of the tested
compounds altered DVocs. Imipramine attenuated the decline in DVocs during the depression-
like phase (i.e., antidepressant effect). Further, the 30 nM/kg dose of 3c produced modest
attenuation of the depression-like phase. Consistent with these observations, a two-way ANOVA
revealed a significant drug treatment effect F(5,726)=23.44, p<0.0001, a significant isolation
time effect F(11,726)=35.43, p<0.0001, and a significant drug x isolation interaction
F(55,726)=2.05, p<0.0001.
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These findings prompted an additional set of analyses to evaluate drug treatment effects
during the anxiety-like phase, the first half (30-45 m) of the depression-like phase, and the
second half (45-60 m) of the depression-like phase. These analyses for compound 3c are
summarized in Figures 2 A-C. A one-way ANOVA of the DVoc data from the anxiety-like
phase (0-5 min block; see Fig. 2A) revealed a significant treatment effect F(5,66)=32.86,
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p<0.0001. Fisher’s post-hoc analyses revealed that the mean DVoc rate of the isolated-vehicle
group was higher than mean DVoc rate of the social-vehicle group, p<0.0001. All other relevant
comparisons failed to reach statistical significance.
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A one-way ANOVA of the DVoc data from the first half of the depression-like phase (see
Fig. 2B) revealed a significant treatment effect F(5,66)=17.605, p<0.0001. Post-hoc analyses
(paired t-test) revealed that in the isolated-vehicle group the mean DVoc rate for the depression-
like phase was lower than in the anxiety-like phase, p<0.0001 and that Fisher’s demonstrated
imipramine attenuated this decline, p<0.0001. All other relevant comparisons failed to reach
statistical significance.
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Analysis of the second half of the depression-like phase (see Fig. 2C) showed a significant
treatment effect F(5,66)=14.344, p<0.0001. A paired t-test revealed that in the isolated-vehicle
group the mean DVoc rate for the depression-like phase was lower than in the anxiety-like phase,
p<0.0001. Fisher’s least significant difference (LSD) revealed that the means DVoc rate of the
imipramine group (p=0.0002) and 30 nM/kg 3c (p=0.017) was higher than the mean DVoc rate
of the isolated-vehicle group.
Data from the in vivo screening demonstrated that isolation stress produced sequential
anxiety-like and depression-like phases and that the positive control imipramine produced
antidepressant effects in the model. A modest antidepressant effect was seen with compound 3c
at the intermediate dose during the latter half of the depression phase. We believe this late onset
of action may reflect the interaction of this compound at serotonin receptors and that an isolation
test session beyond the 60 minute may reveal more robust effects.37
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3. Conclusion
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In conclusion, we report that among fifty analogs evaluated at twelve serotonin receptor
subtypes, bromination of the indole moiety increased binding affinity for the 5-HT2 subfamily of
receptors. More specifically we found that bromination at positions 4 and 5 of the indole moiety
increased affinity the most. It was shown that in general, analogs showed a selectivity for 5-
HT2B over 5-HT2A and 5-HT2C. This finding is important, as aplysinopsins have previously not
been evaluated for the 5-HT2B affinity, and increased affinity at this receptor can often lead to
undesirable side effects. As more information becomes available, such as the recent report of a
5-HT2B crystal structure with ergotamine bound40, we should be able to better understand the
binding behavior of 5-HT2B selective ligands and design more selective ligands.
Based on in vitro screening findings, combined with previous MAO inhibitory studies,
we chose to evaluate three compounds in an in vivo model. Similarly to previous in vivo studies
of aplysinopsins using different depression models, we were unable to reproduce the in vitro
efficacy in an animal model. However, one compound, 3c, did exhibit a modest antidepressant
effect in the later stages of the evaluation period. It is clear that the aplysinopsin scaffold has the
potential to be a valuable scaffold to generate leads to potentially treat CNS disorders or serve as
molecular probes to further investigate the underlying selectivity issues among 5-HT receptor
subtypes.
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Acknowledgements
The authors would like to thank the following for their contributions:
This publication was made possible by Grant Number P20GM104931 from the National
Institute of General Medical Sciences (NIGMS), a component of the National Institutes of Health
(NIH). Its contents are solely the responsibility of the authors and do not necessarily represent
the official view of NIGMS or NIH.
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This investigation was conducted in a facility constructed with support from research facilities
improvement program C06 RR-14503-01 from the NIH National Center for Research Resources.
Ki determinations were generously provided by the National Institute of Mental Health's
Psychoactive Drug Screening Program (NIMH PDSP). The NIMH PDSP is directed by Bryan L.
Roth MD, PhD at the University of North Carolina at Chapel Hill and Project Officer Jamie
Driscol at NIMH, Bethesda MD, USA. Contract # HHSN-271-2008-025C.
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4. Experimental
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4.1. Chemistry
Unless specified all reagents were purchased from commercial sources and used without
further purification. Compounds were purified via column chromatography using Sorbtech
silica gel, 60A, 40-63um from Sorbent Technologies. Whatman silica gel F254 polyester backed
plates were used for TLC and visualized with UV light and/or ninhydrin, vanillin, and
anisaldehyde. NMR spectra were recorded on a 400 MHz Bruker instrument (400 UltraShield,
54 mm standard magnet bore, Billerica, MA) with 3mm direct carbon probe. H-NMR and 13C-
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NMR spectra were recorded at 400 MHz and 100 MHz, respectively. Chemical shifts were
standardized to TMS and solvent signals. All biologically evaluated compounds were found to
possess ≥ 95% purity by HPLC (UV detection at 210 and 254 nm). Purified samples were
analyzed by LC-MS (Bruker Daltonik microTOF, Leipzig Germany) using a 150 x 4.6 mm C8
column (Luna Phenomenex). HR-ESI-MS analysis was done in positive ionization mode.
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General procedures for the synthesis of aplysinopsin analogs and full characterizations of
three compounds evaluated in vivo are provided below. Analytical data for all other compounds
can be found in the Supplementary data.
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4.1.1. General procedure for the synthesis of aplysinopsin analogs 3a-ii (GP1)
Equimolar amounts of the appropriate indole aldehydes 1a-e and imidazolidinones 2a-h
were mixed under nitrogen and heated over an open flame until the reaction mixture began
effervescing. Several minutes after the mixture stopped effervescing it was cooled to room
temperature. The crude mixture was then extracted with MeOH and insoluble materials filtered
off. The solution was then concentrated and loaded on to silica gel for purification using
hexane/EtOAc 1:1 as eluent.
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4.1.2. General procedure for the synthesis of aplysinopsin analogs 5a-e (GP2)
Equimolar amounts of the appropriate indole aldehydes 1a-e and thiohydantoin 4 were
dissolved in abs. EtOH and 1 mL of ethanolamine was added. The mixture was heated at reflux
for 1 h. After cooling the precipitate was filtered off and dried to yield compounds 5a-e, which
were used without further purification.
4.1.3. General procedure for the synthesis of aplysinopsin analogs 6a-e (GP3)
To a solution of appropriate intermediates 5a-e (1.5 mmol) and NaOH (1.5 mmol) in 5
mL of MeOH and 0.5 mL water was added MeI (1.5 mmol). The mixture was stirred at room
temperature for 18h. The resulting precipitant was filtered and dried to yield compounds 6a-e,
which were used without further purification.
14

1
2
4.1.4. General procedure for the synthesis of aplysinopsin analogs 7a-e (GP4)
Compounds 6a-e were dissolved in abs. EtOH and 2x molar equivalents of CH3NH2 was
added. The mixture was heated to 70ºC in a sealed vessel for 24h. After cooling the crude
mixture was purified on silica gel using heanxe/EtOAc 1:1 as eluent.
3
4
5
6
4.1.5. (Z)-2-amino-5-((5-bromo-1H-indol-3-yl)methylene)-1H-imidazol-4(5H)-one (3c)
7
5-Bromoindole-3-carboxaldehyde (448 mg, 2 mmol) and demethylcreatinine (198 mg, 2
1
8
mmol) were reacted according to GP1 to yield 3c (380 mg, 62% yield). H NMR (400 MHz,
9
DMSO) δ 11.72 (bs, 1H), 8.41 (s, 1H), 8.06 (s, 1H), 7.38 (d, J = 8.5 Hz, 1H), 7.24 (d, J = 8.5 Hz,
1H), 6.59 (s, 1H). 13C NMR (101 MHz, DMSO) δ 168.36, 164.54, 137.31, 135.03, 128.95,
125.35, 124.79, 121.68, 119.28, 114.19, 112.78, 102.97. HRMS m/z calcd 305.0038 (M.+ +H);
found: 305.0040
10
11
12
13
14
15
16
17
18
19
20
21
22
23
4.1.6. (E)-5-((1H-indol-3-yl)methylene)-2-imino-1,3-dimethylimidazolidin-4-one (3u):
Indole-3-carboxaldehyde (290 mg, 2 mmol) and 1,3-dimethyl-2-imino-4-imidazolidinone
1
(254 mg, 2 mmol) were reacted according to the above GP1 to yield 3u (365 mg, 72% yield). H
NMR (400 MHz, DMSO) δ 11.46 (bs, 1H), 8.70 (s, 1H), 7.86 (d, J = 7.7 Hz, 1H), 7.42 (d, J =
7.8 Hz, 1H), 7.12 (dt, J = 14.5, 7.0 Hz, 2H), 6.42 (s, 1H), 3.25 (s, 3H), 3.06 (s, 3H). 13C NMR
(101 MHz, DMSO) δ 160.53, 152.24, 136.22, 131.67, 128.14, 123.24, 122.25, 121.20, 118.95,
116.21, 112.80, 108.94, 29.30, 26.65. HRMS m/z calcd 255.1246 (M.+ +H); found: 255.1290
4.1.7. (E)-5-((6-bromo-1H-indol-3-yl)methylene)-2-imino-1,3-dimethylimidazolidin-4-one
(3x):
15

1
2
5-Bromoindole-3-carboxaldehyde (448 mg, 2 mmol) and 1,3-dimethyl-2-imino-4-
imidazolidinone (254 mg, 2 mmol) were reacted according to the above GP1 to yield 3u (312
1
3
mg, 47% yield). H NMR (400 MHz, DMSO) δ 12.07 (bs, 1H), 8.93 (s, 1H), 8.04 (d, J = 8.5 Hz,
4
1H), 7.69 (s, 1H), 7.34 (d, J = 8.4 Hz, 1H), 7.23 (s, 1H), 3.48 (s, 3H), 3.19 (s, 3H). 13C NMR
(101 MHz, DMSO) δ 160.53, 152.49, 137.09, 132.25, 127.21, 123.90, 122.92, 120.94, 115.84,
115.47, 115.35, 109.00, 29.37, 26.70. HRMS m/z calcd 333.0351 (M.+ +H); found: 333.0352
5
6
7
8
4.2. Bioassays
9
4.2.1. In vitro evaluation of serotonin binding affinity
10
11
12
13
14
15
16
17
18
19
Ki determinations were generously provided by the National Institute of Mental Health's
Psychoactive Drug Screening Program. Briefly, compounds are initially evaluated in a primary
binding assay, in which they are tested at a final concentration of 10 μM. They are tested in
quadruplicate and compounds that show > 50% inhibition are deemed hits and advance to
secondary radioligand binding assays. Here compounds are tested at eleven concentrations (0.1,
0.3, 1, 3, 10, 30, 100, 300 nM, 1, 3, 10 μM) to determine the Ki values at specific receptors. For
complete experimental details please refer to the PDSP web site http://pdsp.med.unc.edu/ and
click on "Binding Assay" on the menu bar.
4.2.2. In vivo evaluation of aplysinopsin analogs
20
21
22
23
All procedures involving animals were performed as approved by the Institutional Animal
Care and Use Committee of The University of Mississippi. Cockerels (Production Red, Ideal
Poultry, Cameron, TX, USA) were received into the laboratory at 2 days post hatch and housed
in 34 x 57 x 40 cm cages with 12 chicks per cage. Food and water are available ad libitium via
16

1
2
3
4
gravity feeders. Daily maintenance that entails the replacement of tray liners and filling food and
water gravity feeders is conducted during the hour that precedes the animal’s dark cycle. Lights
are operated on a 12:12 light dark cycle. Supplemental heating sources are provided to maintain
appropriate housing temperatures in the range of 32 +/- 1oC.
5
4.2.2. Testing Equipment
6
7
A six unit testing apparatus containing Plexiglas chambers (25 x 25 x 22 cm) surrounded by
sound attenuating media is used to record separation-induced vocalizations. Each unit is lined
with acoustical fiber media, illuminated by a 25-W light bulb, and ventilated by an 8-cm-
diameter rotary fan (Model FP-108AX S1, Commonwealth Industrial Corp., Taipei, Taiwan).
Miniature video cameras (Model PC60XP, SuperCircuits, Inc., Liberty Hill, TX) mounted in the
sound-attenuating enclosures at floor level and routed through a multiplexor (Model PC47MC,
SuperCircuits, Inc.) provided televised display of the chicks for behavioral observation. To
record DVocs, microphones (Radio Shack Omnidirectional Model 33-3013 modified for AC
current) are mounted at the top of the Plexiglas chamber. These vocalizations are routed to a
computer equipped with custom designed software for data collection.
8
9
10
11
12
13
14
15
16
4.2.3. Methods
17
18
19
20
21
Squads of six chicks were taken from their home cage and placed within a lidded plastic
transport container. To track subject assignment to treatment conditions, chicks are marked
using colored felt pens and body weight is determined for each chick to determine dosing and
identify outliers (i.e., low body weight). Drugs were administered IP 15 minutes prior to
behavioral testing. Chicks were placed into individual testing units for a 60 minute session.
17

1
2
Following the completion of the session chicks were removed from the testing apparatus and
returned to their home cage.
3
4
5
Supplementary Information
Complete secondary in vitro binding data, spectral data for all compounds, and approval letter
for animal studies can be found in the online version of this article.
6
References:
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9
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29
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19

Figure(s)
Compd R1
Compd R2 R3 R4
H
H
Me
H
H
H
H
H
1a
1b
1c
1d
1e
2a
2b
2c
2d
2e
2f
4-Br
5-Br
6-Br
7-Br
Me
H
H
H
Me
H
Me Me
Me
H
H
Me
Me Me
2g
2h
Me Me Me
Compd R1 R2 R3 R4
Compd R1 R2 R3 R4
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
5-Br Me Me
6-Br Me Me
7-Br Me Me
H
3a
3b
3c
3d
3e
3f
3r
3s
4-Br
5-Br
6-Br
7-Br
H
H
H
H
3t
H
H
Me
H
H
H
H
H
Me
Me
Me
Me
Me
3u
3v
3w
3x
3y
3z
H
4-Br Me
5-Br Me
6-Br Me
7-Br Me
Me
4-Br Me
5-Br Me
6-Br Me
3g
3h
3i
H
H
Me Me

7-Br Me
H
H
H
H
H
H
H
4-Br
5-Br
6-Br
7-Br
H
H
H
H
H
Me Me
Me Me
Me Me
Me Me
3j
3k
3l
3aa
3bb
3cc
3dd
3ee
3ff
H
H
H
H
H
H
Me
Me
Me
Me
Me
H
4-Br
5-Br
6-Br
7-Br
H
3m
3n
3o
3p
3q
Me Me Me
4-Br Me Me Me
5-Br Me Me Me
6-Br Me Me Me
7-Br Me Me Me
Me Me
3gg
3hh
3ii
4-Br Me Me
H
Scheme 1. Synthesis of aplysinopsin analogs 3a-ii.

Scheme 2. The synthesis of C-3’ N-methylamine aplysinopsin analogs (7a-e). Reagents and
conditions: (a) ETA, EtOH, reflux, 1h; (b) CH3I, NaOH, MeOH, RT, 18h; (c) CH3NH2, EtOH,
sealed vessel, 70ºC, 24h.

Table 1. Selected serotonin receptor subtype binding affinities
Binding affinity (Ki nM ± S.D.)
Cmpd
Structure
5-HT2A
5-HT2B
5-HT2C
1169 ± 158
232 ± 22
349 ± 44
3a
58 ± 6
51 ± 7
35 ± 4
3c
3128 ± 317
269 ± 34
201 ± 24
3d
279 ± 21
662 ± 92
94 ± 9
1271 ± 168
320 ± 61
3m
3q
99 ± 10

Table 2. Aplysinopsin analogs tested for inhibition (IC50)a of human MAO-A and MAO-B
activities in vitro.
Cmpd
Structure
S.I.b
MAO-A IC50 (nM)
MAO-B IC50 (nM)
35 ± 1
10,017 ± 76
290.34
3u
5.6 ± 0.2
447 ± 35
80.24
3x
a The values are IC50s computed from dose response curves and are mean ± S.D. of at least
triplicate observations.
bThe relative selectivity for MAO-A is defined by the ratio of IC50 (MAO-B)/IC50 (MAO-A) for
each compound.

140
120
100
80
60
40
20
0
1-5
6-10
11-15
16-20
21-25
26-30
31-35
36-40
41-45
46-50
51-55
56-60
Time (5 Minute Intervals)
Vehicle - Isolated
Vehicle - Social
3c - 10 nM/kg
Imipramine - 5mg/kg
3c - 100 nM/kg
3c - 30 nM/kg
Figure 1. Mean distress vocalizations as a rate/minute function (+/- SEM) for time in five
minute intervals. Sample sizes were n=10-12.

140
120
100
80
*
60
40
20
0
Vehicle -
Social
Vehicle - Imipramine 3c - 10
3c - 30
nM/kg
3c - 100
nM/kg
Isolated
- 5mg/kg
nM/kg
Treatment Group
Figure 2A. The effects of social separation on mean DVocs (+/- SEM) in each drug treatment
condition under the anxiety-like phase (minutes 1-5). Sample sizes were n=10-12. * indicates a
significant increase compared to the social-vehicle condition.

120
100
80
60
40
20
0
**
*
Vehicle -
Social
Vehicle - Imipramine 3c - 10
Isolated - 5mg/kg nM/kg
Treatment Group
3c - 30
nM/kg
3c - 100
nM/kg
Figure 2B. The effects of social separation on mean DVocs (+/- SEM) in each drug treatment
condition under the first depression-like phase (minutes 31-45). Sample sizes were n=10-12. *
indicates a significant decrease compared to the anxiety-like phase. ** indicates a significant
increase compared to the isolated-vehicle condition

120
100
80
60
40
20
0
**
**
*
Vehicle -
Social
Vehicle - Imipramine - 3c - 10
Isolated 5mg/kg nM/kg
3c - 30
nM/kg
3c - 100
nM/kg
Treatment Group
Figure 2C. The effects of social separation on mean DVocs (+/- SEM) in each drug treatment
condition under the second depression-like phase (minutes 46-60). Sample sizes were n=10-12. *
indicates a significant increase compared to the social-vehicle condition. ** indicates a
significant increase compared to the isolated-vehicle condition

Graphical Abstract
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In vitro structure-activity relationships of
aplysinopsin analogs and their in vivo
evaluation in the chick anxiety-depression
model
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Kevin Lewellyn, Dobroslawa Bialonska, Melissa J. Loria, Stephen White, Kenneth J. Sufka and
Jordan K. Zjawiony